T-cell receptor-based multi-specific polypeptide molecule, and composition and use thereof

Abstract

The present invention relates to a T-cell receptor-based multi-specific polypeptide molecule. Also provided are a nucleic acid encoding the polypeptide molecule, a vector or a vector system comprising the nucleic acid, a host cell comprising the polypeptide molecule, a conjugate and a pharmaceutical composition comprising the polypeptide molecule, and a method for preventing or treating a disease in a subject by using the polypeptide molecule.

Description

Multispecific polypeptide molecules based on T cell receptors, their compositions and applications

[0001] This international patent application claims priority to Chinese patent application CN202311415371.1, filed on October 27, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to multispecific polypeptide molecules based on T-cell receptors and their uses, particularly in the treatment of cancer, infectious diseases, autoimmune diseases and / or inflammatory diseases. Background Technology

[0003] T cells are a crucial component of the vertebrate adaptive immune system, playing a vital role in viral infections, cancer, and autoimmunity. The TCR recognizes the major histocompatibility complex (MHC)-antigen peptide complex (pMHC), a key step in T cell-mediated immune responses. The TCR non-covalently binds to the CD3 signaling apparatus, composed of γ, δ, ε, and ζ subunits, forming the TCR–CD3 complex. The binding of pMHC to the TCR activates LCK-induced phosphorylation of the intracellular immune receptor tyrosine activation motif (ITAM) in the TCR CD3ζ subunit. CD3ζ phosphorylation then triggers a series of phosphorylation signal transduction pathways involving ζ chain-associated protein kinase 70 (ZAP70) and the activated T cell connectosome (LAT) in T cells, leading to the recruitment of multiple downstream adapters and signaling molecules and the formation of the LAT signaling body. The assembled LAT signaling body activates various signaling pathways involving transcription factors, such as activator protein 1 (AP-1), nuclear factor-κB (NF-κB), and activated T cell nuclear factor (NFAT). Activated transcription factors can induce subsequent T cell activation, proliferation, cytokine production, and effector function.

[0004] Bispecific antibodies (BsAbs) are antibody molecule that simultaneously targets two antigens or two different epitopes of a single antigen. Compared to ordinary antibodies, bispecific antibodies are more specific, more accurately target tumor cells, and have reduced off-target toxicity. With the development of recombinant protein expression technology and antibody engineering technology, many different antibody forms have been produced. Multispecific antibodies are used for various purposes, including (1) receptor activation, (2) blockade, (3) internalization, (4) aggregation, (5) binding to membrane-associated proteins, or (6) targeted targeting of cytotoxic effector cells.

[0005] T-cell repositioning antibodies (bsAbs, also known as T-cell engagers, TcEs) are important forms of cytotoxic effector cell repositioning and are a central pillar of current cancer immunotherapy. These bsAbs recognize targets on the surface of tumor cells and simultaneously recognize molecules on the surface of T cells (most commonly CD3). This allows the bsAb to couple tumor cells with cytotoxic T cells, activating downstream signaling pathways of the T-cell TCR and resulting in the cytotoxic killing of tumor cells by the T cells. Blincyto (blinatumomab) is a CD19 / CD3 bispecific antibody that induces complete remission in 69% of patients with relapsed / refractory B-prednisolone acute lymphoblastic leukemia (ALL). Currently, many new forms of T-cell repositioning antibodies have emerged, such as BITE, BITE-Fc, DART-Fc, and TriTAC.

[0006] bsAbs, which rely on antibodies to recognize target cell surface molecules, only recognize tumor cell surface antigens and cannot recognize nearly 90% of intracellular proteins. TCRs, on the other hand, can recognize antigenic peptides of MHC molecules on the cell surface after processing of intracellular and cell surface tumor-specific antigens, thus having a broader recognition range. In TCR-based T cell repositioning bispecific antibodies, the target cell recognition region is changed from that of traditional antibodies to the TCR itself, allowing for increased target selectivity by leveraging the broad recognition characteristics of the TCR.

[0007] For protein drugs, the glomerular filtration limit is generally around 60 kDa. This means that small molecular weight antibody fragments or derivatives, such as nanobodies (15 kDa), scFv (28 kDa), and even Fab (50 kDa), are cleared from the body through glomerular filtration, resulting in a short half-life. Nanobodies, in particular, have the smallest molecular weight among all antibody types, with half-lives often only a few tens of minutes. Therefore, to increase the efficacy of protein drugs, it is necessary to improve them to extend their half-life.

[0008] Summary of the Invention

[0009] To enhance the in vivo efficacy of TcE, in addition to increasing the affinity of TCR for pMHC, the titer of TcE against TCR targets on target cells can be increased, as can the titer of TcE against effector cell surface molecules, thereby improving T cell cytotoxic activity and proliferative capacity. Simultaneously, to maintain the ability of TcE to enter tumors and preserve its appropriate half-life in vivo, the molecular size of TcE needs to be maintained within a certain range, and other functional domains need to be appropriately added to the peptide molecule. These domains can be derived from the hinge region of the antibody, the Fc domain of the antibody or its dimerized portion, and / or functional domains derived from albumin (Alb) or its binding portion. The addition of these functional domains not only prolongs the half-life of the peptide molecule but also maintains its cytotoxic activity. Accordingly, this invention provides TCR-based multispecific peptide molecules, such as TCR-based bispecific, trispecific, or tetraspecific peptide molecules.

[0010] the term

[0011] Unless otherwise stated or defined, all terms used herein have their common meaning in the art as is well known to those skilled in the art. For example, terms used herein include those in Janeway CA Jr, Travers P, Walport M, et al., *Immunobiology*, 5th edition, New York: Garland Science (2001) and “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Leuenberger, HGW, Nagel, B. and The definition is given in H. (ed., 1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0012] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. Therefore, the terms “a,” “an,” “one or more,” and “at least one” are used interchangeably. Similarly, the terms “comprising,” “including,” and “having” are used interchangeably.

[0013] Unless otherwise stated or defined, the term "comprising" and its variations such as "including" and "containing" should be understood to mean including the stated elements or steps, but not excluding any other elements or steps. For the purposes of this invention, the term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined below as including or containing at least a certain number of embodiments, it should also be understood to disclose a group preferably consisting only of these embodiments.

[0014] As used herein, the term "T cell receptor" or "TCR" includes the natural TCR as well as TCR variants, fragments, and constructs. Therefore, the term includes heterodimers, multimers, and single-chain constructs comprising TCRα and TCRβ chains; optionally, it may include other domains and / or portions, provided that the TCR retains its ability to recognize antigen targets.

[0015] In its natural form, the TCR exists as a complex of several proteins on the surface of T cells. The T cell receptor consists of two (separate) protein chains, produced by independent T cell receptor α and β (TCRα and TCRβ) genes, and are referred to as the α chain and β chain. Each TCR chain has an N-terminal immunoglobulin-like (Ig)-variable (V) region / domain, an Ig-constant (C) region / domain, a transmembrane / cell membrane-crossing region anchoring the chain in the plasma membrane, and a short cytoplasmic tail at the C-terminus.

[0016] Antigen specificity is conferred by the variable regions (TRAV and TRBV) of the α and β chains. 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 a framework (FR) region. CDR3 is the major determinant of antigen recognition and specificity (i.e., the ability to recognize and interact with specific antigens), while CDR1 and CDR2 primarily interact with the MHC molecules that present the antigenic peptide.

[0017] The TCR recognizes an antigenic peptide that binds to a major histocompatibility complex (MHC) molecule on the surface of the antigen-presenting cell (“presentation / display on an MHC molecule”). Antigenic peptides presented on MHC molecules are also referred to herein as “epitope-MHC molecule complexes,” “epitope-MHC complexes,” “antigen-MHC complexes,” or “target antigen peptide-MHC complexes.” Two distinct classes of MHC molecules exist: MHC I and MHC II, which present peptides from different cellular compartments. MHC class I molecules are expressed on the surface of all nucleated cells in the human body and display peptide 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 performs its biological effector functions.

[0018] As used in this article, the term “TCR constant region” includes the TCR α-chain constant region (TRAC) and β-chain constant region (TRBC), which can be human constant regions or derived from another species, such as mice.

[0019] It has been reported that the addition of disulfide bonds in the constant region promotes proper pairing of the TCRα and β chains (Kuball J et al. Blood. 2007 Mar 15; 109(6): 2331-8). Therefore, this paper also envisions the addition of one or more cysteine ​​modifications in the constant region to form disulfide bonds between the TCRα and TCRβ chains.

[0020] The sequences of the constant regions of wild-type TCRs can be found in the public database of the International Immunogenetic Information System (IMGT). For example, the constant domain sequence of the α chain of the TCR molecule is “TRAC*01”, and the constant domain sequence of the β chain of the TCR molecule is “TRBC1*01” or “TRBC2*01”.

[0021] In this invention, the amino acid sequences of wild-type TCRs are numbered according to the nomenclature rules of the International Immunogenetic Information System (IMGT). For example, an amino acid in the TCRα chain variable region (TRAV) whose position number listed in IMGT is 104 is described as the 104th amino acid of the TRAV in this invention; an amino acid in the TCRβ chain variable region (TRBV) whose position number listed in IMGT is 84 is described as the 84th amino acid of the TRBV in this invention; and so on. For example, an amino acid in the TCRα chain constant region (TRAC) whose position number listed in IMGT is 48 is described as the 48th amino acid of the TRAC in this invention; an amino acid in the TCRβ chain constant region (TRBC) whose position number listed in IMGT is 57 is described as the 57th amino acid of the TRBC in this invention; and so on. Where the sequence position numbers of other amino acids are specifically specified in this invention, those specific specifications shall apply.

[0022] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a particular antigen. Such molecules typically consist of two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (or domain) (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (or domain) (abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain: CL. The variable regions of both the antibody heavy and light chains contain binding domains that interact with the antigen. The antibody constant regions mediate the binding of the 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 complement activation pathway).

[0023] The heavy chain of immunoglobulins 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 binds to 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 antibodies, the Fc region contains the CH2 and CH3 domains. The Fc fragment is responsible for the effector functions of immunoglobulins, including, for example, complement binding and binding to homologous Fc receptors on effector cells. In the native structure of immunoglobulins, the Fc domains of the two heavy chains dimerize to form an Fc dimer. The portion of the Fc domain that promotes the formation of the Fc dimer is called the "dimerized portion of the Fc domain," which typically includes a portion of the CH2 domain and the CH3 domain.

[0024] The hinge region of IgG antibodies refers to the short amino acid sequence region between the CH1 and CH2 portions of the heavy chain, which is relatively flexible in the native state of the antibody. Hinge regions found in IgG, IgA, and IgD immunoglobulin classes act as flexible spacers, allowing the Fab portion to move freely in space relative to the Fc region. Hinge domains are structurally diverse, varying in sequence and length across immunoglobulin classes and subclasses. Based on crystallographic studies, immunoglobulin hinge regions can be further subdivided structurally and functionally into three regions: 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 from the carboxyl terminus of CH1 to the first residue in the hinge that restricts movement; typically, it is 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 fragmental flexibility of the antibody. The core hinge region contains the interchain disulfide bond. The lower hinge region connects to the amino terminus of the CH2 domain and includes residues within the CH2 domain. The core hinge region of human IgG1 contains the sequence Cys-Pro-Pro-Cys (SEQ ID NO:1), which forms a cyclic octapeptide upon dimerization via disulfide bonds. This cyclic octapeptide is believed to act as a pivot, thereby imparting flexibility. The conformational changes allowed by the structure and flexibility of the polypeptide sequence in the immunoglobulin hinge region can affect the effector function of the antibody Fc moiety.

[0025] The "light chain variable region (VL)" or "heavy chain variable region (VH)" consists of a "framework" region interspersed with three "complementarity-determining regions" or "CDRs". The framework region modulates the CDR for specific binding to antigenic epitopes. The CDR contains the amino acid residues in the antibody that are primarily responsible for antigen binding. From the amino terminus to the carboxyl terminus, both the VL and VH domains contain the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0026] Single-domain antibodies (sdAbs), also known as nanobodies, consist of a single heavy-chain variable domain. This heavy-chain variable domain was initially discovered in a heavy-chain antibody (HCAb) isolated from the serum of camels and sharks. Through genetic engineering, the heavy-chain variable domain was amplified. This variable domain, present in naturally occurring heavy-chain antibodies, is called "VHH" to distinguish it from the heavy-chain variable domain (VH) and light-chain variable domain (VL) found in conventional four-chain antibodies. Individually cloned and expressed VHHs possess structural stability and antigen-binding activity comparable to the original heavy-chain antibody. VHHs are currently the smallest known unit capable of binding target antigens.

[0027] The VHH domain possesses numerous unique structural and functional features that make isolated VHH domains (as well as VHH-based nanobodies that share these features with naturally occurring VHH domains) and proteins containing isolated VHH domains highly advantageous for use as functional antigen-binding domains or proteins. In particular, but not limited to, VHH domains (which are essentially “designed” to functionally bind to antigens in the absence of light chain variable domains and without any interaction with them) and nanobodies can serve as single, relatively small, functional antigen-binding structural units, domains, or proteins. This distinguishes VHH domains from the VH and VL domains of conventional 4-chain antibodies, which are generally unsuitable on their own for practical application as single antigen-binding proteins or domains, but rather need to be combined in some form to provide a functional antigen-binding unit (e.g., in conventional antibody fragments such as Fab fragments; in ScFv fragments consisting of VH domains covalently linked to VL domains).

[0028] The amino acid assignment of each VL, VH, and VHH domain follows any conventional definition of the CDR. 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); the complex of the Chothia and Kabat CDRs, where CDR-H1 is a complex of the Chothia and Kabat CDRs; the AbM definition used by the Oxford Molecular antibody modeling software; and the CONTACT definition by Martin et al. (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 assigned the same number. This disclosure may use CDRs defined according to any of these numbering systems, but the preferred embodiment uses the CDRs defined by Kabat.

[0029] As used herein, the term "antibody" 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) containing 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 at glycosylation sites. Antibodies also include post-translational modified antibodies, fusion proteins containing antibody antigenic determinants, and immunoglobulin molecules containing any other modifications to antigen recognition sites, provided that these antibodies exhibit the intended biological activity.

[0030] 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 antigens. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0031] Examples of antigen-binding fragments include, but are not limited to: (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide bonds in a hinge region; (iii) Fab' fragments, which are essentially Fab fragments but have a partial hinge region (see Fundamental Immunology (Paul ed., 3.sup.rd ed. 1993)); (iv) Fd fragments consisting of VH and CH1 domains; (v) Fd' fragments having VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (vi) Fv fragments consisting of VL and VH domains of an antibody single arm; (vii) dAb fragments (Ward et al. (1989) Nature). (viii) A separate complementarity-determining region (CDR). Furthermore, although the two domains VL and VH of the Fv fragment are encoded by independent genes, they can be linked by a synthetic linker using recombination methods, enabling them to form a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (ScFv); see, for example, 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 included within the term "antigen-binding fragment" of antibody. Additionally, the term also includes "linear antibodies" containing a pair of tandem Fd fragments (VH-CH1-VH-CH1), which together with a complementary light chain polypeptide and any modified version of the aforementioned fragment that retains antigen-binding activity to form the antigen-binding region.

[0032] As used in this article, the term "antigen" refers to any substance that can induce an immune response in the body. That is, 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, produce immune response products (sensitized lymphocytes or antibodies), and that can specifically bind to the corresponding products in vivo and in vitro.

[0033] In this article, antigens can be tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), viral antigens, autoantigens, and immune cell surface molecules.

[0034] As used in this article, the term "tumor-associated antigen (TAA)" refers to an antigen that is differentially expressed in tumor cells compared to normal cells (e.g., significantly increased expression in tumor cells).

[0035] As used in this article, the term "tumor-specific antigen (TSA)" refers to an antigen that is unique to tumor cells and is not present in normal tissues and cells.

[0036] As used in this article, the term "viral antigen" refers to substances in viruses that can induce an immune response in the body.

[0037] As used in this article, "autoantigen" refers to antigenic substances that, due to biological, physical, or chemical factors, alter the structure and composition of one's own tissues and cells, thereby triggering an immune response against these components. The release of occult antigens can generate an immune response. Alterations 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. Clinically, the use of certain drugs can alter the antigenicity of blood cell surfaces, causing autoimmune hemolytic anemia or granulocytopenia. Cross-reactions triggered by common antigens can generate an immune response. Some bacteria and viruses have similar antigenic determinants to certain tissues and cells in the normal human body. Autoantibodies and sensitized lymphocytes produced against these bacterial and viral antigenic determinants can cross-react with one's own tissues and cells, causing autoimmune diseases.

[0038] "Immune cell surface molecules" may include, for example, immune cell surface antigens (such as T cell antigens) and co-stimulatory molecules.

[0039] The site on an antigen that is bound by a TCR or antibody is called an "epitaph." Epitopes can be formed from consecutive amino acids or from discontinuous amino acids juxtaposed through the ternary folding of one or more proteins. Epitopes formed from consecutive amino acids (also known as linear epitopes) are generally retained after exposure to denaturing solvents, while epitopes formed through ternary folding (also known as conformational epitopes) are generally lost during treatment with denaturing solvents. Epitopes typically contain at least three, more usually at least five, or eight to ten amino acids in a unique spatial conformation. Epitopes define the minimum binding site for a TCR or antibody and are therefore specific targets for the TCR or antibody or its antigen-binding fragments.

[0040] In this document, the term "albumin" encompasses any naturally occurring 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 precursor protein and any form of albumin produced by cellular processing. Albumin also encompasses variants of naturally occurring albumin, such as splice variants or allelic variants. The term also encompasses any recombinant form of albumin. Albumin sequences are known in the art. Information on the human serum albumin gene (including the genomic DNA sequence) can be found, for example, in NCBI. An exemplary amino acid sequence of full-length human serum albumin precursor protein 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, containing 17 disulfide bonds and no sugar components. In a bodily fluid environment of pH 7.4, albumin is an ionic protein, with each molecule potentially carrying more than 200 negative charges. Approximately 10% of exogenously infused albumin enters the extravascular interstitial space within 2 hours, reaching equilibrium after 20 days. The half-life of albumin in plasma is 15-19 days.

[0041] >gi|4502027|ref|NP_000468.1|Serve Albumin Pre-Origin [Homo sapiens]

[0042] (Underlined indicates signal peptide, bold indicates propeptide, italic indicates mature human serum albumin (SEQ ID NO:2)).

[0043] In this document, the term "albumin-binding moiety" refers to a fragment that is non-covalently linked, or covalently linked to albumin through conjugation or direct genetic fusion, and retains the minimum functional unit for albumin binding. In some embodiments, the albumin-binding moiety includes any known portion capable of binding to albumin, such as endogenous or exogenous ligands that specifically bind to albumin, small organic molecules, fatty acids, proteins, short peptides (e.g., QRLMEDICLPRWGCLWEDDF (SEQ ID NO:3)), antibodies, antibody fragments and antibody-like scaffolds, streptococcal G protein, etc.

[0044] As used in this invention, the term "sequence identity" refers to the degree to which two sequences (amino acids) have identical residues at the same positions when aligned. For example, "the amino acid sequence is X% identical to SEQ ID NO: Y" refers to the percentage of identity between the amino acid sequence and SEQ ID NO: Y, and is described as X% of the residues in the amino acid sequence being identical to the sequence residues disclosed in SEQ ID NO: Y.

[0045] Such calculations are typically performed using computer programs. 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).

[0046] Furthermore, in determining the degree of sequence identity between two amino acid sequences, those skilled in the art may consider so-called “conserved” amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, having little or no effect on the function, activity, or other biological properties of the polypeptide. Such conserved 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 based on the relevant teachings from WO 04 / 037999 and WO 98 / 49185 and other references cited therein.

[0047] Such conservative substitution is preferably a substitution in which one 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.

[0048] The 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.

[0049] As used herein, the term "cysteine-containing peptide" refers to a polypeptide segment containing cysteine ​​residues, which is capable of forming a disulfide bond between two polypeptide chains containing the peptide segment, and has a length of 3-10 amino acids.

[0050] As used herein, the term "vector" refers to a nucleic acid molecule that serves as a medium for transferring (exogenous) genetic material into a host cell, where the nucleic acid molecule, acting as a vector, can, for example, be replicated and / or expressed.

[0051] As used in this article, “host cell” refers to any type of cell that has been introduced with an expression vector so that it can express exogenous genetic material.

[0052] The term “pharmaceuticalally acceptable” means that the carrier or adjuvant is compatible with the other components of the composition and does not cause significant toxicity to the recipient, and / or that such carrier or adjuvant is approved or may be used in pharmaceutical compositions for parenteral administration to humans.

[0053] As used herein, the terms “treatment,” “management,” etc., refer to the administration of a drug or the performance of a procedure to achieve an effect. These effects may be preventative in terms of the complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of the partial or complete cure of a disease and / or its symptoms. As used herein, “treatment” may include treating diseases or conditions in mammals, particularly humans (e.g., cancer, infectious diseases, autoimmune diseases, or inflammatory diseases), and includes: (a) preventing the occurrence of a disease or its symptoms in subjects susceptible to the disease but not yet diagnosed with it (e.g., including diseases that may be related to or caused by the primary disease); (b) suppressing the disease, i.e., halting its development; and (c) alleviating the disease, i.e., causing its remission. Treatment may refer to any indication of success in treating or improving or preventing cancer, including any objective or subjective parameter, such as elimination; relief; reduction of symptoms or making the disease condition more tolerable for the patient; slowing the rate of deterioration or decline; or weakening or reducing the endpoint of deterioration. Treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a physician’s examination. Therefore, the term "treatment" includes the administration of the multispecific polypeptide molecules or pharmaceutical compositions or conjugates disclosed in this invention to prevent or delay, alleviate or stop 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 disease, disease symptoms or disease side effects in a subject.

[0054] As used herein, the term "effective amount" refers to the amount of a therapeutic agent sufficient to achieve such treatment or prevention when administered to a subject for the treatment or prevention of a disease. "Effective amount" can vary depending on the compound, the disease and its severity, and the age and weight of the subject to be treated. "Therapeutic effective amount" refers to an effective amount for therapeutic treatment. "Prophylactic effective amount" refers to an effective amount for prophylactic treatment.

[0055] As used herein, the term “subject” refers to any mammalian subject for diagnosis, treatment, or therapy. For therapeutic purposes, “mammal” means any animal classified as a mammal, including humans, livestock, and laboratory animals, zoo animals, sporting animals, or pet animals, such as dogs, horses, cats, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.

[0056] Preferred embodiments of the present invention

[0057] The features and advantages of the invention, as well as its additional features and advantages, will become more clearly understood below through a detailed description of the following embodiments in conjunction with the accompanying drawings. The embodiments described herein with reference to the accompanying drawings are illustrative and explanatory, and are intended for a general understanding of the invention. The embodiments should not be construed as limiting the scope of the invention. Identical or similar elements and elements having the same or similar functions are denoted using the same reference numerals throughout the description.

[0058] In a first aspect, the present invention provides a multispecific polypeptide molecule, the multispecific polypeptide molecule comprising a first binding region for binding a first antigen and a second binding region for binding a second antigen.

[0059] The first binding region includes the α-chain variable region (TRAV1) and the β-chain variable region (TRBV1) derived from the T-cell receptor (TCR) that binds to the first antigen-MHC complex.

[0060] The second binding region includes a heavy chain variable region VH1 and a light chain variable region VL1 derived from an antibody that binds to the second antigen, or a single heavy chain variable region (VHH1) derived from a single-domain antibody or nanobody that binds to the second antigen.

[0061] The first antigen is selected from tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), viral antigens, and autoantigens, and the second antigen is an immune cell surface molecule.

[0062] The N-terminus of the antibody is attached to the C-terminus of the TCR.

[0063] In some embodiments, the multispecific polypeptide molecule further comprises at least one functional domain capable of prolonging the half-life of the multispecific polypeptide molecule. In some embodiments, the functional domain capable of prolonging the half-life of the multispecific polypeptide molecule is selected from at least one of the following:

[0064] 1) Derived from the hinge region of the antibody;

[0065] 2) Derived from the Fc domain of the antibody or its dimerized portion;

[0066] 3) Derived from the CH3 domain of the antibody;

[0067] 4) Albumin (Alb) or its bound fraction;

[0068] 5) Originating from the constant region of TCR or its fragments.

[0069] In some embodiments, the multispecific polypeptide molecule further comprises at least one of the following functional domains:

[0070] 1) Derived from the hinge region of the antibody;

[0071] 2) Derived from the Fc domain of the antibody or its dimerized portion;

[0072] 3) Derived from the CH3 domain of the antibody;

[0073] 4) Albumin (Alb) or its bound fraction;

[0074] 5) Originating from the constant region of TCR or its fragments.

[0075] In some embodiments, the N-terminus of the antibody is attached to the C-terminus of the TCR.

[0076] In some embodiments, the N-terminus of the second binding region amino acid chain is attached to the C-terminus of the first binding region amino acid chain.

[0077] In some embodiments, the multispecific polypeptide molecule provided by the present invention does not contain a C-terminus of the second binding region amino acid chain linked to the N-terminus of the first binding region amino acid chain.

[0078] In some embodiments, the multispecific polypeptide molecule provided by the present invention does not contain a second binding region connected to the N-terminus of one or more polypeptide chains of the first binding region by the C-terminus of either a single-chain (VH1 and VL1 are distributed on one polypeptide chain, or the antibody contains VHH1) or a double-chain (VH1 and VL1 are distributed on two polypeptide chains).

[0079] In some embodiments, the multispecific polypeptide molecules provided by this invention do not include the following:

[0080] VL1 and VH1 are distributed on a polypeptide chain to form a second binding region single chain or the second binding region single chain contains VHH1, TRAV1 and TRBV1 are distributed on a polypeptide chain to form a first binding region single chain, and the C-terminus of the second binding region single chain is attached to the N-terminus of the first binding region single chain.

[0081] VL1 and VH1 are distributed on one polypeptide chain to form a second binding region single chain, or the second binding region single chain contains VHH1, TRAV and TRBV1 are distributed on two polypeptide chains to form α chain and β chain respectively, and the C-terminus of the second binding region single chain is attached to the N-terminus of the α chain or β chain.

[0082] VL1 and VH1 are distributed on different polypeptide chains to form L chains and H chains respectively, while TRAV1 and TRBV1 are distributed on one polypeptide chain to form a first binding region single chain. The C-terminus of the L chain or H chain is attached to the N-terminus of the first binding region single chain.

[0083] VL1 and VH1 are distributed on different polypeptide chains, forming L chains and H chains respectively. TRAV1 and TRBV1 are distributed on two polypeptide chains, forming α chains and β chains respectively. The C-terminus of the L chain or H chain is attached to the N-terminus of the α chain or β chain.

[0084] In some embodiments, the multispecific polypeptide molecules provided by this invention do not include the following:

[0085] From N-terminal to C-terminal: scFv1-TRBV1, scFv1-TRAV1, scFv1-scTCR, VHH1-TRBV1, VHH1-TRAV1, VHH1-scTCR, VH1-TRBV1, VH1-TRAV1, VH1-scTCR, VL1-TRBV1, VL1-TRAV1, VL1-scTCR, Fab1-TRBV1, Fab1-TRAV1, Fab1-scTCR.

[0086] In some implementations, VL1 and VH1 are distributed on a single polypeptide chain whose C-terminus is linked to the N-terminus of the TCR.

[0087] In some implementations, VL1 and VH1 are distributed on different polypeptide chains, and the C-terminus of the polypeptide chain containing VL1 or VH1 is connected to the N-terminus of the TCR.

[0088] In some implementations, the TRAV1 and TRBV1 are distributed on two different polypeptide chains (i.e., the TCR is a heterodimer αβTCR).

[0089] In some embodiments, VL1 and VH1 are distributed on one polypeptide chain, or VL1 and VH1 are distributed on two different polypeptide chains, and TRAV1 and TRBV1 are distributed on two different polypeptide chains.

[0090] In some embodiments, the multispecific polypeptide molecule comprises two polypeptide chains.

[0091] In some embodiments, the multispecific polypeptide molecule comprises two polypeptide chains, with TRAV1 and TRBV1 distributed on two different polypeptide chains, and VHH1 or VH1 and VL1 distributed on one polypeptide chain (e.g., forming scFv1), with their N-terminus attached to the C-terminus of the polypeptide chain containing TRAV1 or TRBV1, or the N-terminus of one of the polypeptide chains containing VH1 and VL1 attached to the C-terminus of the polypeptide chain containing TRAV1, and the N-terminus of the other polypeptide chain attached to the C-terminus of the polypeptide chain containing TRBV1.

[0092] In some embodiments, the multispecific polypeptide molecule comprises three polypeptide chains.

[0093] In some embodiments, the multispecific polypeptide molecule comprises three polypeptide chains, with VH1 and VL1 distributed on different polypeptide chains, and TRAV1 and TRBV1 distributed on different polypeptide chains, wherein the N-terminus of the polypeptide chain containing VH1 or VL1 is attached to the C-terminus of the polypeptide chain containing TRBV1 or TRAV1.

[0094] In a second aspect, the present invention provides a multispecific polypeptide molecule, wherein the multispecific polypeptide molecule includes a first binding region for binding a first antigen and a second binding region for binding a second antigen on both polypeptide chains.

[0095] The first binding region includes the α-chain variable region (TRAV1) and the β-chain variable region (TRBV1) derived from the T-cell receptor (TCR) that binds to the first antigen-MHC complex.

[0096] The second binding region includes a heavy chain variable region VH1 and a light chain variable region VL1 derived from an antibody that binds to the second antigen, or a single heavy chain variable region (VHH1) derived from a single-domain antibody or nanobody that binds to the second antigen.

[0097] The first antigen is selected from tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), viral antigens, and autoantigens, and the second antigen is an immune cell surface molecule.

[0098] The N-terminus of the antibody is attached to the C-terminus of the TCR.

[0099] In some embodiments, the multispecific polypeptide molecule further comprises at least one functional domain capable of prolonging the half-life of the multispecific polypeptide molecule. In some embodiments, the functional domain capable of prolonging the half-life of the multispecific polypeptide molecule is selected from at least one of the following:

[0100] 1) Derived from the hinge region of the antibody;

[0101] 2) Derived from the Fc domain of the antibody or its dimerized portion;

[0102] 3) Derived from the CH3 domain of the antibody;

[0103] 4) Albumin (Alb) or its bound fraction;

[0104] 5) Originating from the constant region of TCR or its fragments.

[0105] In some embodiments, the multispecific polypeptide molecule further comprises at least one of the following functional domains:

[0106] 1) Derived from the hinge region of the antibody;

[0107] 2) Derived from the Fc domain of the antibody or its dimerized portion;

[0108] 3) Derived from the CH3 domain of the antibody;

[0109] 4) Albumin (Alb) or its bound fraction;

[0110] 5) Originating from the constant region of TCR or its fragments.

[0111] In some embodiments, the N-terminus of the antibody is attached to the C-terminus of the TCR.

[0112] In some implementations, the TRAV1 and TRBV1 are located on two different polypeptide chains (i.e., the TCR is a heterodimer αβTCR).

[0113] In some implementations, VH1 and VL1 are linked to a single polypeptide chain, for example, forming scFv1 or Fab1, and are linked to the C-terminus of the TCR.

[0114] In some embodiments, TRAV1 and TRBV1 are located on two different polypeptide chains, and VH1 and VL1 are linked to one polypeptide chain, with their N-terminus linked to the C-terminus of either the polypeptide chain containing TRAV1 or the polypeptide chain containing TRBV1.

[0115] In some implementations, VH1 and VL1 are connected by a connector.

[0116] In an embodiment of the multispecific polypeptide molecule of the second aspect of the present invention, VH1 and VL1 form scFv1, and the N-terminus of scFv1 is attached to the C-terminus of the polypeptide chain containing TRAV1 or the polypeptide chain containing TRBV1. In some embodiments, the two polypeptide chains of the multispecific polypeptide molecule respectively comprise: TRBV1-scFv1 and TRAV1; or TRAV1-scFv1 and TRBV1.

[0117] In some embodiments, the two polypeptide chains of the multispecific polypeptide molecule respectively comprise: TRBV1-VL1-VH1 and TRAV1; or TRBV1-VH1-VL1 and TRAV1; or TRAV1-VL1-VH1 and TRBV1; or TRAV1-VH1-VL1 and TRBV1.

[0118] In some embodiments, the second binding region comprises a single-chain variable region (VHH1) derived from a single-domain antibody or nanobody that binds to the second antigen, the VHH1 being linked to the C-terminus of the TCR. In some embodiments, TRAV1 and TRBV1 are located on two different polypeptide chains, and the second binding region comprises VHH1, wherein the N-terminus of the VHH1 is linked to the C-terminus of the polypeptide chain containing TRAV1 or TRBV1. In some embodiments, the two polypeptide chains of the multispecific polypeptide molecule comprise, respectively: TRBV1-VHH1 and TRAV1; or TRAV1-VHH1 and TRBV1.

[0119] In some embodiments, the second binding region comprises VH1 and VL1, and VH1 and VL1 are located on two different polypeptide chains. In some embodiments, TRAV1 and TRBV1 are located on two different polypeptide chains, and VH1 and VL1 are located on two different polypeptide chains, wherein one of the polypeptide chains containing VH1 and VL1 is attached to the C-terminus of the polypeptide chain containing TRAV1, and the other polypeptide chain is attached to the C-terminus of the polypeptide chain containing TRBV1.

[0120] In some embodiments, the two polypeptide chains of the multispecific polypeptide molecule comprise TRBV1-VL1 and TRAV1-VH1, respectively; or TRAV1-VL1 and TRBV1-VH1.

[0121] In some embodiments, the TRAV1 and TRBV1 are located on two different polypeptide chains, and the VHH1 or the VH1 and VL1 are attached to the C-terminus of the TRAV1 or the TRBV1 on one polypeptide chain (e.g., forming scFv1), or one of the VH1 and VL1 is attached to the C-terminus of the TRAV1 and the other is attached to the C-terminus of the TRBV1.

[0122] In some embodiments, the multispecific polypeptide molecule further comprises a constant domain selected from the group consisting of the following, which is optionally linked to the C-terminus of TRAV1 and / or TRBV1 via a linker:

[0123] (1) A TCR constant region or a fragment thereof, the TCR constant region including, for example, a TCR α chain constant region (TRAC) and / or a β chain constant region (TRBC);

[0124] (2) CH3, wherein the CH3 is derived from the Fc domain of the antibody, preferably any one or two polypeptide chains contain at least one mutation in CH3 that can promote the formation of heterodimers of the polypeptide molecule.

[0125] (3) CL and CH1, wherein CL is derived from the CL domain of the antibody light chain and CH1 is derived from the CH1 domain of the antibody heavy chain.

[0126] In an embodiment of the multispecific polypeptide molecule of the second aspect of the present invention, the multispecific polypeptide molecule further comprises a third binding region for binding to a third antigen, the third binding region comprising an α-chain variable region (TRAV2) and a β-chain variable region (TRBV2) derived from a TCR that binds to the third antigen-MHC complex; wherein the third antigen is selected from tumor-associated antigens (TAAs), tumor-specific antigens (TSA), viral antigens, and autoantigens, and the third antigen may be the same as or different from the first antigen. In some embodiments, TRAV2 is the same as TRAV1. In some embodiments, TRBV2 is the same as TRBV1.

[0127] In some implementations, the TRAV2 and the TRBV2 are linked to the same polypeptide chain via a connector.

[0128] In some embodiments, VH1 and VL1 are distributed on a single polypeptide chain and are attached to the C-terminus of TRAV1 or TRBV1, and TRAV2 and TRBV2 are attached to the same polypeptide chain via a linker and are attached between VH1 and VL1.

[0129] In some embodiments, the two polypeptide chains of the multispecific polypeptide molecule each comprise:

[0130] TRBV1-VL1-TRAV2-TRBV2-VH1 and TRAV1; or

[0131] TRBV1-VL1-TRBV2-TRAV2-VH1 and TRAV1; or

[0132] TRBV1-VH1-TRBV2-TRAV2-VL1 and TRAV1; or

[0133] TRBV1-VH1-TRAV2-TRBV2-VL1 and TRAV1; or

[0134] TRAV1-VL1-TRBV2-TRAV2-VH1 and TRBV1; or

[0135] TRAV1-VL1-TRAV2-TRBV2-VH1 and TRBV1; or

[0136] TRAV1-VH1-TRAV2-TRBV2-VL1 and TRBV1; or

[0137] TRAV1-VH1-TRBV2-TRAV2-VL1 and TRBV1.

[0138] In some embodiments, the multispecific polypeptide molecule further comprises a constant domain selected from the C-terminus of TRAV2 and / or TRBV2, which is optionally linked to a linker:

[0139] (1) A TCR constant region or a fragment thereof, the TCR constant region including, for example, a TCR α chain constant region (TRAC) and / or a β chain constant region (TRBC);

[0140] (2) CH3, wherein the CH3 is derived from the Fc domain of the antibody, preferably any one or two polypeptide chains contain at least one mutation in CH3 that can promote the formation of heterodimers of the polypeptide molecule.

[0141] (3) CL and CH1, wherein CL is derived from the CL domain of the antibody light chain and CH1 is derived from the CH1 domain of the antibody heavy chain.

[0142] In an embodiment of the multispecific polypeptide molecule of the second aspect of the present invention, the multispecific polypeptide molecule further comprises a fourth binding region for binding to a fourth antigen, the fourth binding region comprising a heavy chain variable region VH2 and a light chain variable region VL2 derived from an antibody binding to the fourth antigen, or a single heavy chain variable region (VHH2) derived from a single-domain antibody or nanobody binding to the fourth antigen; wherein the fourth antigen is an immune cell surface molecule, and the fourth antigen may be the same as or different from the second antigen. In some embodiments, VH2 is the same as VH1. In some embodiments, VL2 is the same as VL1. In some embodiments, VHH2 is the same as VHH1.

[0143] In some implementations, VHH2 or VH2 and VL2 form scFv2 connected to the N end of TRAV1 or TRBV1, or one of VH2 and VL2 is connected to the N end of TRAV1 and the other is connected to the N end of TRBV1.

[0144] In some embodiments, the multispecific polypeptide molecule includes the first binding region, the second binding region, and the fourth binding region, and the VH2 and VL2 form scFv2 attached to the N-terminus of the TRAV1 or the TRBV1. In some embodiments, the VH2 and VL2 are connected via a linker to form scFv2.

[0145] In some embodiments, the two polypeptide chains of the multispecific polypeptide molecule each comprise:

[0146] scFv2-TRBV1-scFv1 and TRAV1; or

[0147] scFv2-TRAV1-scFv1 and TRBV1; or

[0148] scFv2-TRBV1-VHH1 and TRAV1; or

[0149] scFv2-TRAV1-VHH1 and TRBV1; or

[0150] scFv2-TRBV1-VL1 and TRAV1-VH1; or

[0151] scFv2-TRAV1-VL1 and TRBV1-VH1; or

[0152] TRBV1-scFv1 and scFv2-TRAV1; or

[0153] TRAV1-scFv1 and scFv2-TRBV1; or

[0154] TRBV1-VHH1 and scFv2-TRAV1; or

[0155] TRAV1-VHH1 and scFv2-TRBV1; or

[0156] TRBV1-VL1 and scFv2-TRAV1-VH1; or

[0157] TRAV1-VL1 and scFv2-TRBV1-VH1.

[0158] In some embodiments, the multispecific polypeptide molecule includes a first binding region, a second binding region, and a fourth binding region, and the VHH2 is linked to the N-terminus of the TRAV1 or the TRBV1. In some embodiments, the two polypeptide chains of the multispecific polypeptide molecule each comprise:

[0159] VHH2-TRBV1-scFv1 and TRAV1; or

[0160] VHH2-TRAV1-scFv1 and TRBV1; or

[0161] VHH2-TRBV1-VHH1 and TRAV1; or

[0162] VHH2-TRAV1-VHH1 and TRBV1; or

[0163] VHH2-TRBV1-VL1 and TRAV1-VH1; or

[0164] VHH2-TRAV1-VL1 and TRBV1-VH1; or

[0165] TRBV1-scFv1 and VHH2-TRAV1; or

[0166] TRAV1-scFv1 and VHH2-TRBV1; or

[0167] TRBV1-VHH and VHH2-TRAV1; or

[0168] TRAV1-VHH and VHH2-TRBV1; or

[0169] TRBV1-VL1 and VHH2-TRAV1-VH1; or

[0170] TRAV1-VL1 and VHH2-TRBV1-VH1.

[0171] In some embodiments, the multispecific polypeptide molecule includes the first binding region, the second binding region, and the fourth binding region, and one of VH2 and VL2 is attached to the N-terminus of TRAV1, and the other is attached to the N-terminus of TRBV1. In some embodiments, the two polypeptide chains of the multispecific polypeptide molecule each contain:

[0172] VL2-TRBV1-scFv1 and VH2-TRAV1; or

[0173] VH2-TRBV1-scFv1 and VL2-TRAV1; or

[0174] VL2-TRAV1-scFv1 and VH2-TRBV1; or

[0175] VH2-TRAV1-scFv1 and VL2-TRBV1; or

[0176] VL2-TRBV1-VHH and VH2-TRAV1; or

[0177] VH2-TRBV1-VHH and VL2-TRAV1; or

[0178] VL2-TRAV1-VHH and VH2-TRBV1; or

[0179] VH2-TRAV1-VHH and VL2-TRBV1; or

[0180] VL2-TRBV1-VL1 and VH2-TRAV1-VH1; or

[0181] VH2-TRBV1-VL1 and VL2-TRAV1-VH1; or

[0182] VL2-TRAV1-VL1 and VH2-TRBV1-VH1; or

[0183] VH2-TRAV1-VL1 and VL2-TRBV1-VH1.

[0184] In some embodiments, the multispecific polypeptide molecule includes a first binding region, a second binding region, a third binding region, and a fourth binding region, and VH2 and VL2 form scFv2 attached to the N-terminus of TRAV1 or TRBV1. In some embodiments, VH2 and VL2 are linked by a linker to form scFv2. In some embodiments, the two polypeptide chains of the multispecific polypeptide molecule each contain:

[0185] scFv2-TRBV1-VL1-TRAV2-TRBV2-VH1 and TRAV1; or

[0186] scFv2-TRBV1-VL1-TRBV2-TRAV2-VH1 and TRAV1; or

[0187] scFv2-TRBV1-VH1-TRBV2-TRAV2-VL1 and TRAV1; or

[0188] scFv2-TRBV1-VH1-TRAV2-TRBV2-VL1 and TRAV1; or

[0189] scFv2-TRAV1-VL1-TRBV2-TRAV2-VH1 and TRBV1; or

[0190] scFv2-TRAV1-VL1-TRAV2-TRBV2-VH1 and TRBV1; or

[0191] scFv2-TRAV1-VH1-TRAV2-TRBV2-VL1 and TRBV1; or

[0192] scFv2-TRAV1-VH1-TRBV2-TRAV2-VL1 and TRBV1; or

[0193] TRBV1-VL1-TRAV2-TRBV2-VH1 and scFv2-TRAV1; or

[0194] TRBV1-VL1-TRBV2-TRAV2-VH1 and scFv2-TRAV1; or

[0195] TRBV1-VH1-TRBV2-TRAV2-VL1 and scFv2-TRAV1; or

[0196] TRBV1-VH1-TRAV2-TRBV2-VL1 and scFv2-TRAV1; or

[0197] TRAV1-VL1-TRBV2-TRAV2-VH1 and scFv2-TRBV1; or

[0198] TRAV1-VL1-TRAV2-TRBV2-VH1 and scFv2-TRBV1; or

[0199] TRAV1-VH1-TRAV2-TRBV2-VL1 and scFv2-TRBV1; or

[0200] TRAV1-VH1-TRBV2-TRAV2-VL1 and scFv2-TRBV1.

[0201] In some embodiments, the multispecific polypeptide molecule includes a first binding region, a second binding region, a third binding region, and a fourth binding region, and the VHH2 is linked to the N-terminus of the TRAV1 or the TRBV1. In some embodiments, the two polypeptide chains of the multispecific polypeptide molecule each contain:

[0202] VHH2-TRBV1-VL1-TRAV2-TRBV2-VH1 and TRAV1; or

[0203] VHH2-TRBV1-VL1-TRBV2-TRAV2-VH1 and TRAV1; or

[0204] VHH2-TRBV1-VH1-TRBV2-TRAV2-VL1 and TRAV1; or

[0205] VHH2-TRBV1-VH1-TRAV2-TRBV2-VL1 and TRAV1; or

[0206] VHH2-TRAV1-VL1-TRBV2-TRAV2-VH1 and TRBV1; or

[0207] VHH2-TRAV1-VL1-TRAV2-TRBV2-VH1 and TRBV1; or

[0208] VHH2-TRAV1-VH1-TRAV2-TRBV2-VL1 and TRBV1; or

[0209] VHH2-TRAV1-VH1-TRBV2-TRAV2-VL1 and TRBV1; or

[0210] TRBV1-VL1-TRAV2-TRBV2-VH1 and VHH2-TRAV1; or

[0211] TRBV1-VL1-TRBV2-TRAV2-VH1 and VHH2-TRAV1; or

[0212] TRBV1-VH1-TRBV2-TRAV2-VL1 and VHH2-TRAV1; or

[0213] TRBV1-VH1-TRAV2-TRBV2-VL1 and VHH2-TRAV1; or

[0214] TRAV1-VL1-TRBV2-TRAV2-VH1 and VHH2-TRBV1; or

[0215] TRAV1-VL1-TRAV2-TRBV2-VH1 and VHH2-TRBV1; or

[0216] TRAV1-VH1-TRAV2-TRBV2-VL1 and VHH2-TRBV1; or

[0217] TRAV1-VH1-TRBV2-TRAV2-VL1 and VHH2-TRBV1.

[0218] In some embodiments, the multispecific polypeptide molecule includes a first binding region, a second binding region, a third binding region, and a fourth binding region, and one of VH2 and VL2 is attached to the N-terminus of TRAV1, and the other is attached to the N-terminus of TRBV1. In some embodiments, the two polypeptide chains of the multispecific polypeptide molecule each contain:

[0219] VL2-TRBV1-VL1-TRAV2-TRBV2-VH1 and VH2-TRAV1; or

[0220] VL2-TRBV1-VL1-TRBV2-TRAV2-VH1 and VH2-TRAV1; or

[0221] VH2-TRBV1-VL1-TRAV2-TRBV2-VH1 and VL2-TRAV1; or

[0222] VH2-TRBV1-VL1-TRBV2-TRAV2-VH1 and VL2-TRAV1; or

[0223] VL2-TRBV1-VH1-TRBV2-TRAV2-VL1 and VH2-TRAV1; or

[0224] VL2-TRBV1-VH1-TRAV2-TRBV2-VL1 and VH2-TRAV1; or

[0225] VH2-TRBV1-VH1-TRBV2-TRAV2-VL1 and VL2-TRAV1; or

[0226] VH2-TRBV1-VH1-TRAV2-TRBV2-VL1 and VL2-TRAV1; or

[0227] VL2-TRAV1-VL1-TRBV2-TRAV2-VH1 and VH2-TRBV1; or

[0228] VL2-TRAV1-VL1-TRAV2-TRBV2-VH1 and VH2-TRBV1; or

[0229] VH2-TRAV1-VL1-TRBV2-TRAV2-VH1 and VL2-TRBV1; or

[0230] VH2-TRAV1-VL1-TRAV2-TRBV2-VH1 and VL2-TRBV1; or

[0231] VL2-TRAV1-VH1-TRAV2-TRBV2-VL1 and VH2-TRBV1; or

[0232] VL2-TRAV1-VH1-TRBV2-TRAV2-VL1 and VH2-TRBV1; or

[0233] VH2-TRAV1-VH1-TRAV2-TRBV2-VL1 and VL2-TRBV1; or

[0234] VH2-TRAV1-VH1-TRBV2-TRAV2-VL1 and VL2-TRBV1.

[0235] In an embodiment of the multispecific polypeptide molecule of the second aspect of the invention, the functional domain is derived from albumin or its binding portion and optionally the hinge region of an antibody; and the functional domain is connected to the C-terminus or N-terminus of any polypeptide chain of the multispecific polypeptide molecule via an optional linker, preferably the C-terminus.

[0236] In some embodiments, the functional domain is derived from albumin or its binding moiety, and the albumin or its binding moiety is attached to the C-terminus of any polypeptide chain of the multispecific polypeptide molecule via a linker.

[0237] In some embodiments, the functional domain is derived from albumin or its binding portion and the hinge region of the antibody, the functional domain being connected to the C-terminus of any one chain of the multispecific polypeptide molecule via an optional adapter, and albumin or its binding portion being connected to the C-terminus of any one chain of the hinge region of the antibody via an optional adapter.

[0238] In some embodiments, the functional domain comprises two chains, and the two chains of the multispecific polypeptide molecule are connected by covalent or non-covalent bonding between the two chains of the functional domain.

[0239] In some implementations, the functional domain comprises two chains, and the first binding region and the second binding region are connected by covalent or non-covalent bonding between the two chains of the functional domain.

[0240] In some embodiments, the functional domain comprises two chains, respectively attached to the C-terminus or N-terminus of the two polypeptide chains of the multispecific polypeptide molecule, preferably the C-terminus.

[0241] In some embodiments, one chain of the functional domain is connected to the C-terminus or N-terminus of the TCR via an optional adapter, preferably the C-terminus, and the other chain is connected to the C-terminus or N-terminus of the antibody, preferably the C-terminus.

[0242] In some embodiments, one strand of the functional domain is attached to the C-terminus or N-terminus of the strand containing TRAV1 or TRBV1, preferably the C-terminus, and the other strand is attached to the C-terminus or N-terminus of the strand containing the antibody, preferably the C-terminus.

[0243] In some embodiments, TRAV1 and TRBV1 are located on two different polypeptide chains, with one chain of the functional domain attached to the C-terminus or N-terminus of the chain containing TRAV1 or TRBV1, preferably the C-terminus, and the other chain attached to the C-terminus or N-terminus of the chain containing the antibody, preferably the C-terminus.

[0244] In some embodiments, TRAV1 and TRBV1 are located on two different polypeptide chains, the second binding region contains VHH1, or contains VH1 and VL1 and said VH1 and VL1 are on one polypeptide chain, one chain of said functional domain is attached to the C-terminus or N-terminus of the chain containing TRAV1 or TRBV1, preferably the C-terminus, and the other chain is attached to the C-terminus or N-terminus of the chain containing the antibody, preferably the C-terminus.

[0245] In some embodiments, TRAV1 and TRBV1 are located on two different polypeptide chains, and one of VH1 and VL1 is attached to the C-terminus of the chain containing TRAV1, and the other is attached to the C-terminus of the chain containing TRBV1. The two chains of the functional domain are respectively attached to the C-terminus or N-terminus of the two polypeptide chains of the multispecific polypeptide molecule, preferably the C-terminus.

[0246] In some embodiments, TRAV1 and TRBV1 are located on two different polypeptide chains, with VH1 and VL1 on one polypeptide chain and the N-terminus of the polypeptide chain containing TRAV1 or TRBV1 connected to the C-terminus of the polypeptide chain containing TRAV1 or TRBV1. The C-terminus is connected to one chain of the functional domain, and the other chain of the functional domain is connected to the C-terminus of the polypeptide chain containing TRBV1 or TRAV1.

[0247] In some implementations, TRAV1 and TRBV1 are located on two different polypeptide chains, with VH1 and VL1 attached to the C-terminus of the polypeptide chain containing TRAV1 or TRBV1 on one polypeptide chain, and one chain of the functional domain attached to the N-terminus of the chain containing TRAV1 and the other chain attached to the N-terminus of the chain containing TRBV1.

[0248] In an embodiment of the multispecific polypeptide molecule of the second aspect of the present invention, the functional domain is derived from the hinge region of an antibody and / or the Fc domain of an antibody or its dimerized portion and / or the CH3 domain of an antibody; and the functional domain is optionally connected to the C-terminus or N-terminus of the two polypeptide chains of the multispecific polypeptide molecule via a linker, preferably the C-terminus. In some embodiments, the functional domain is derived from the hinge region of an antibody and / or the Fc domain of an antibody or its dimerized portion and / or the CH3 domain of an antibody; and the two chains of the functional domain are respectively connected to the C-terminus or N-terminus of the two polypeptide chains of the multispecific polypeptide molecule via optional linkers, preferably the C-terminus.

[0249] In some embodiments, the functional domain is derived from the hinge region of the antibody and / or the Fc domain of the antibody or its dimerized portion and / or the CH3 domain of the antibody, and the two chains of the multispecific polypeptide molecule are connected by covalent or non-covalent bonding between the two chains of the functional domain.

[0250] In some embodiments, the functional domain is derived from the Fc domain of the antibody or its dimerized portion and optionally the hinge region of the antibody. In some embodiments, the functional domain is derived from the hinge region of the antibody and the Fc domain of the antibody or its dimerized portion. In some embodiments, the functional domain is derived from the Fc domain of the antibody. In some embodiments, the functional domain is the Fc domain of the antibody. In some embodiments, the functional domain is connected to the C-terminus or N-terminus of the two polypeptide chains of the multispecific polypeptide molecule via optional linkers, preferably the C-terminus.

[0251] In some embodiments, the functional domain is derived from the CH3 domain of the antibody and optionally the hinge region of the antibody. In some embodiments, the functional domain is derived from the hinge region of the antibody and the CH3 domain of the antibody. In some embodiments, the functional domain is optionally linked to the C-terminus or N-terminus of the two polypeptide chains of the multispecific polypeptide molecule via a linker, preferably the C-terminus.

[0252] In some embodiments, the functional domain is derived from the constant region of the TCR or a fragment thereof and optionally the hinge region of the antibody. In some embodiments, the functional domain is derived from the hinge region of the antibody and the constant region of the TCR or a fragment thereof. In some embodiments, the functional domain is optionally linked to the C-terminus or N-terminus of the two polypeptide chains of the multispecific polypeptide molecule via a linker, preferably the C-terminus. In some embodiments, the constant region or a fragment thereof comprises a TCRα chain constant region (TRAC) and a TCRβ chain constant region (TRBC), and the TRAC and TRBC are respectively linked to the C-terminus or N-terminus of the two polypeptide chains of the multispecific polypeptide molecule via an optional linker, preferably the C-terminus.

[0253] In an embodiment of the multispecific polypeptide molecule of the second aspect of the invention, the multispecific polypeptide molecule further comprises a constant structural domain connected to the C-terminus of TRAV1 and / or TRBV1 via an optional linker.

[0254] In one embodiment, the constant structural domain is a TCR constant region or a fragment thereof, the TCR constant region including, for example, the TCR α chain constant region (TRAC) and / or the β chain constant region (TRBC).

[0255] In one embodiment, the constant structural domain is CH3, which is derived from the Fc domain of the antibody. Preferably, the CH3 in any one or both polypeptide chains contains at least one mutation that can promote the formation of heterodimers of the polypeptide molecule.

[0256] In one embodiment, the constant domains are CL and CH1, wherein CL is derived from the CL domain of the antibody light chain and CH1 is derived from the CH1 domain of the antibody heavy chain.

[0257] In some embodiments, the multispecific polypeptide molecule includes a TCR constant region or a fragment thereof connected to the C-terminus of TRAV1 and TRBV1 via optional linkers. In some embodiments, the multispecific polypeptide molecule includes TRAC and TRBC connected to the C-terminus of TRAV1 and TRBV1, respectively, via optional linkers.

[0258] In some embodiments, the multispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, and includes a first binding region and a second binding region, as well as functional domains of hinge regions derived from albumin or its binding portion and optionally an antibody, respectively linked to the C-terminus of either polypeptide chain; wherein:

[0259] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - linker - scFv1 - optional linker - optional hinge region - Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC - optional linker - optional hinge region; or

[0260] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - linker - scFv1 - optional linker - optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC - optional linker - optional hinge region - Alb; or

[0261] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-scFv1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0262] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC - linker - scFv1 - optional linker - optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - optional linker - optional hinge region - Alb; or

[0263] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VHH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0264] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - linker - VHH1 - optional linker - optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC - optional linker - optional hinge region - Alb; or

[0265] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VHH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0266] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VHH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0267] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-optional linker-optional hinge region; or

[0268] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - linker - VL1 - optional linker - optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC - linker - VH1 - optional linker - optional hinge region - Alb; or

[0269] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-optional linker-optional hinge region; or

[0270] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-optional linker-optional hinge region-Alb; or

[0271] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0272] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0273] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0274] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0275] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-linker-VH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0276] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0277] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-linker-VH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0278] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region.

[0279] In some embodiments, the multispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, and includes a first binding region and a second binding region, as well as functional domains respectively attached to the C-terminus of the first polypeptide chain and the second polypeptide chain, namely, an Fc domain derived from the antibody or a dimerized portion thereof, and optionally a hinge region of the antibody; wherein:

[0280] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-scFv1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0281] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-scFv1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0282] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VHH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0283] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VHH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0284] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-optional linker-optional hinge region-CH2-CH3; or

[0285] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-optional linker-optional hinge region-CH2-CH3; or

[0286] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0287] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0288] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0289] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3.

[0290] In some embodiments, the multispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, and includes a first binding region, a second binding region, and a third binding region, as well as a hinge region derived from albumin or its binding portion and optionally an antibody, attached to the C-terminus of either polypeptide chain. VH1 and VL1 are attached to the C-terminus of TRAV1 or TRBV1 on one polypeptide chain, and TRAV2 and TRBV2 are attached to the same polypeptide chain via a linker and are connected between VH1 and VL1.

[0291] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0292] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0293] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0294] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0295] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0296] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0297] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0298] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0299] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0300] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0301] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0302] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0303] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0304] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0305] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0306] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb.

[0307] In some embodiments, the multispecific polypeptide molecule comprises a first binding region, a second binding region, and a third binding region, as well as functional domains derived from an Fc domain or a dimerized portion thereof of the antibody and optionally a hinge region of the antibody, and the multispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain; wherein:

[0308] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0309] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0310] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0311] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0312] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0313] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0314] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0315] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3.

[0316] In some embodiments, the multispecific polypeptide molecule includes a first binding region, a second binding region, and a fourth binding region, as well as a functional domain derived from albumin or its binding moiety and optionally an antibody hinge region, and the multispecific polypeptide molecule includes a first polypeptide chain and a second polypeptide chain; wherein:

[0317] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-linker-scFv1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0318] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-linker-scFv1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0319] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-linker-scFv1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0320] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-linker-scFv1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0321] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-linker-VHH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0322] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-linker-VHH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0323] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-linker-VHH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0324] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-linker-VHH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0325] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-optional linker-optional hinge region; or

[0326] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-optional linker-optional hinge region-Alb; or

[0327] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-optional linker-optional hinge region; or

[0328] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-optional linker-optional hinge region-Alb; or

[0329] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-scFv1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0330] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - linker - scFv1 - optional linker - optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2 - linker - TRAV1 - optional linker - TRAC - optional linker - optional hinge region - Alb; or

[0331] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-scFv1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0332] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC - linker - scFv1 - optional linker - optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2 - linker - TRBV1 - optional linker - TRBC - optional linker - optional hinge region - Alb; or

[0333] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VHH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0334] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - linker - VHH1 - optional linker - optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2 - linker - TRAV1 - optional linker - TRAC - optional linker - optional hinge region - Alb; or

[0335] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VHH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0336] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VHH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0337] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-linker-VH1-optional linker-optional hinge region; or

[0338] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - linker - VL1 - optional linker - optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2 - linker - TRAV1 - optional linker - TRAC - linker - VH1 - optional linker - optional hinge region - Alb; or

[0339] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-linker-VH1-optional linker-optional hinge region; or

[0340] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-linker-VH1-optional linker-optional hinge region-Alb; or

[0341] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-linker-scFv1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0342] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-linker-scFv1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0343] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-linker-scFv1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0344] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-linker-scFv1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0345] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-linker-VHH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0346] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-linker-VHH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0347] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-linker-VHH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0348] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-linker-VHH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0349] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-optional linker-optional hinge region; or

[0350] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-optional linker-optional hinge region-Alb; or

[0351] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-optional linker-optional hinge region; or

[0352] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-optional linker-optional hinge region-Alb; or

[0353] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-scFv1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0354] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - linker - scFv1 - optional linker - optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2 - linker - TRAV1 - optional linker - TRAC - optional linker - optional hinge region - Alb; or

[0355] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-scFv1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0356] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-scFv1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0357] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VHH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0358] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - linker - VHH1 - optional linker - optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2 - linker - TRAV1 - optional linker - TRAC - optional linker - optional hinge region - Alb; or

[0359] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VHH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0360] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VHH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0361] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-linker-VH1-optional linker-optional hinge region; or

[0362] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-linker-VH1-optional linker-optional hinge region-Alb; or

[0363] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-linker-VH1-optional linker-optional hinge region; or

[0364] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-linker-VH1-optional linker-optional hinge region-Alb; or

[0365] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-linker-scFv1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0366] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-linker-scFv1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0367] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-linker-scFv1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0368] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-linker-scFv1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0369] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-linker-scFv1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0370] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-linker-scFv1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0371] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-linker-scFv1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0372] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-linker-scFv1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0373] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-linker-VHH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0374] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-linker-VHH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0375] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-linker-VHH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0376] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-linker-VHH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0377] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-linker-VHH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0378] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-linker-VHH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0379] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-linker-VHH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0380] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-linker-VHH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0381] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-linker-VH1-optional linker-optional hinge region; or

[0382] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-linker-VH1-optional linker-optional hinge region-Alb; or

[0383] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-linker-VH1-optional linker-optional hinge region; or

[0384] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-linker-VH1-optional linker-optional hinge region-Alb; or

[0385] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-linker-VH1-optional linker-optional hinge region; or

[0386] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-linker-VH1-optional linker-optional hinge region-Alb; or

[0387] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-linker-VH1-optional linker-optional hinge region; or

[0388] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRAV1-optional connector-TRAC-connector-VL1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRBV1-optional connector-TRBC-connector-VH1-optional connector-optional hinge region-Alb.

[0389] In some embodiments, the multispecific polypeptide molecule comprises a first binding region, a second binding region, and a fourth binding region, as well as functional domains derived from an Fc domain or a dimerized portion thereof derived from an antibody and optionally a hinge region of the antibody, and the multispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain; wherein:

[0390] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-linker-scFv1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0391] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-linker-scFv1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0392] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-linker-VHH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0393] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-linker-VHH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0394] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-optional linker-optional hinge region-CH2-CH3; or

[0395] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-optional linker-optional hinge region-CH2-CH3; or

[0396] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-scFv1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0397] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-scFv1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0398] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VHH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0399] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VHH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0400] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-linker-VH1-optional linker-optional hinge region-CH2-CH3; or

[0401] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-linker-VH1-optional linker-optional hinge region-CH2-CH3; or

[0402] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-linker-scFv1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0403] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-linker-scFv1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0404] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-linker-VHH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0405] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-linker-VHH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0406] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-optional linker-optional hinge region-CH2-CH3; or

[0407] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-optional linker-optional hinge region-CH2-CH3; or

[0408] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-scFv1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0409] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-scFv1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0410] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VHH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0411] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VHH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0412] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-linker-VH1-optional linker-optional hinge region-CH2-CH3; or

[0413] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-linker-VH1-optional linker-optional hinge region-CH2-CH3; or

[0414] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-linker-scFv1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0415] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-linker-scFv1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0416] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-linker-scFv1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0417] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-linker-scFv1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0418] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-linker-VHH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0419] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-linker-VHH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0420] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-linker-VHH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0421] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-linker-VHH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0422] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-linker-VH1-optional linker-optional hinge region-CH2-CH3; or

[0423] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-linker-VH1-optional linker-optional hinge region-CH2-CH3; or

[0424] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-linker-VH1-optional linker-optional hinge region-CH2-CH3; or

[0425] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-linker-VH1-optional linker-optional hinge region-CH2-CH3.

[0426] In some embodiments, the multispecific polypeptide molecule includes a first binding region, a second binding region, a third binding region, and a fourth binding region, as well as a functional domain derived from albumin or its binding moiety and optionally an antibody hinge region, and the multispecific polypeptide molecule includes a first polypeptide chain and a second polypeptide chain; wherein:

[0427] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0428] The first polypeptide chain, from N-terminus to C-terminus, comprises: scFv2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0429] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0430] The first polypeptide chain, from N-terminus to C-terminus, comprises: scFv2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0431] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0432] The first polypeptide chain, from N-terminus to C-terminus, comprises: scFv2-linker-TRBV1-optional linker-TRBC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0433] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0434] The first polypeptide chain, from N-terminus to C-terminus, comprises: scFv2-linker-TRBV1-optional linker-TRBC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0435] The first polypeptide chain, from N-terminus to C-terminus, comprises: scFv2-linker-TRAV1-optional linker-TRAC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-Alb, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0436] The first polypeptide chain, from N-terminus to C-terminus, comprises: scFv2-linker-TRAV1-optional linker-TRAC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0437] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0438] The first polypeptide chain, from N-terminus to C-terminus, comprises: scFv2-linker-TRAV1-optional linker-TRAC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0439] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0440] The first polypeptide chain, from N-terminus to C-terminus, comprises: scFv2-linker-TRAV1-optional linker-TRAC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0441] The first polypeptide chain, from N-terminus to C-terminus, comprises: scFv2-linker-TRAV1-optional linker-TRAC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0442] The first polypeptide chain, from N-terminus to C-terminus, comprises: scFv2-linker-TRAV1-optional linker-TRAC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0443] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0444] The first polypeptide chain, from N-terminus to C-terminus, comprises: TRBV1-optional linker-TRBC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region, and the second polypeptide chain, from N-terminus to C-terminus, comprises: scFv2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0445] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0446] The first polypeptide chain, from N-terminus to C-terminus, comprises: TRBV1-optional linker-TRBC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region, and the second polypeptide chain, from N-terminus to C-terminus, comprises: scFv2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0447] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0448] The first polypeptide chain, from N-terminus to C-terminus, comprises: TRBV1-optional linker-TRBC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain, from N-terminus to C-terminus, comprises: scFv2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0449] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0450] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0451] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0452] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0453] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0454] The first polypeptide chain, from N-terminus to C-terminus, comprises: TRAV1-optional linker-TRAC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region, and the second polypeptide chain, from N-terminus to C-terminus, comprises: scFv2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0455] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0456] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0457] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0458] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0459] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0460] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0461] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0462] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0463] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0464] The first polypeptide chain, from N-terminus to C-terminus, comprises: VHH2-linker-TRBV1-optional linker-TRBC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0465] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0466] The first polypeptide chain, from N-terminus to C-terminus, comprises: VHH2-linker-TRBV1-optional linker-TRBC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0467] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0468] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0469] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0470] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0471] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0472] The first polypeptide chain, from N-terminus to C-terminus, comprises: VHH2-linker-TRAV1-optional linker-TRAC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0473] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0474] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0475] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0476] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0477] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0478] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0479] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0480] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0481] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0482] The first polypeptide chain, from N-terminus to C-terminus, comprises: TRBV1-optional linker-TRBC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain, from N-terminus to C-terminus, comprises: VHH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0483] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0484] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0485] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0486] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0487] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0488] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0489] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0490] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0491] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRBV1-optional connector-TRBC-connector-VL1-connector-TRAV2-connector-TRBV2-connector-VH1-optional connector-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRAV1-optional connector-TRAC-optional connector-optional hinge region; or

[0492] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRBV1-optional connector-TRBC-connector-VL1-connector-TRAV2-connector-TRBV2-connector-VH1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRAV1-optional connector-TRAC-optional connector-optional hinge region-Alb; or

[0493] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRBV1-optional connector-TRBC-connector-VL1-connector-TRBV2-connector-TRAV2-connector-VH1-optional connector-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRAV1-optional connector-TRAC-optional connector-optional hinge region; or

[0494] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRBV1-optional connector-TRBC-connector-VL1-connector-TRBV2-connector-TRAV2-connector-VH1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRAV1-optional connector-TRAC-optional connector-optional hinge region-Alb; or

[0495] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRBV1-optional connector-TRBC-connector-VL1-connector-TRAV2-connector-TRBV2-connector-VH1-optional connector-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRAV1-optional connector-TRAC-optional connector-optional hinge region; or

[0496] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRBV1-optional connector-TRBC-connector-VL1-connector-TRAV2-connector-TRBV2-connector-VH1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRAV1-optional connector-TRAC-optional connector-optional hinge region-Alb; or

[0497] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRBV1-optional connector-TRBC-connector-VL1-connector-TRBV2-connector-TRAV2-connector-VH1-optional connector-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRAV1-optional connector-TRAC-optional connector-optional hinge region; or

[0498] The first polypeptide chain, from N-terminus to C-terminus, comprises: VH2-connector-TRBV1-optional connector-TRBC-connector-VL1-connector-TRBV2-connector-TRAV2-connector-VH1-optional connector-optional hinge region, and the second polypeptide chain, from N-terminus to C-terminus, comprises: VL2-connector-TRAV1-optional connector-TRAC-optional connector-optional hinge region-Alb; or

[0499] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRBV1-optional connector-TRBC-connector-VH1-connector-TRBV2-connector-TRAV2-connector-VL1-optional connector-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRAV1-optional connector-TRAC-optional connector-optional hinge region; or

[0500] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRBV1-optional connector-TRBC-connector-VH1-connector-TRBV2-connector-TRAV2-connector-VL1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRAV1-optional connector-TRAC-optional connector-optional hinge region-Alb; or

[0501] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRBV1-optional connector-TRBC-connector-VH1-connector-TRAV2-connector-TRBV2-connector-VL1-optional connector-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRAV1-optional connector-TRAC-optional connector-optional hinge region; or

[0502] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRBV1-optional connector-TRBC-connector-VH1-connector-TRAV2-connector-TRBV2-connector-VL1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRAV1-optional connector-TRAC-optional connector-optional hinge region-Alb; or

[0503] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRBV1-optional connector-TRBC-connector-VH1-connector-TRBV2-connector-TRAV2-connector-VL1-optional connector-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRAV1-optional connector-TRAC-optional connector-optional hinge region; or

[0504] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRBV1-optional connector-TRBC-connector-VH1-connector-TRBV2-connector-TRAV2-connector-VL1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRAV1-optional connector-TRAC-optional connector-optional hinge region-Alb; or

[0505] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRBV1-connector-optional-connector-TRBC-connector-VH1-connector-TRAV2-connector-TRBV2-connector-VL1-optional-connector-optional-hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRAV1-connector-optional-connector-TRAC-optional-connector-optional-hinge region; or

[0506] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRBV1-connector-optional-connector-TRBC-connector-VH1-connector-TRAV2-connector-TRBV2-connector-VL1-optional-connector-optional-hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRAV1-connector-optional-connector-TRAC-optional-connector-optional-hinge region-Alb; or

[0507] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRAV1-optional connector-TRAC-connector-VL1-connector-TRBV2-connector-TRAV2-connector-VH1-optional connector-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRBV1-optional connector-TRBC-optional connector-optional hinge region; or

[0508] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRAV1-optional connector-TRAC-connector-VL1-connector-TRBV2-connector-TRAV2-connector-VH1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRBV1-optional connector-TRBC-optional connector-optional hinge region-Alb; or

[0509] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRAV1-optional connector-TRAC-connector-VL1-connector-TRAV2-connector-TRBV2-connector-VH1-optional connector-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRBV1-optional connector-TRBC-optional connector-optional hinge region; or

[0510] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRAV1-optional connector-TRAC-connector-VL1-connector-TRAV2-connector-TRBV2-connector-VH1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRBV1-optional connector-TRBC-optional connector-optional hinge region-Alb; or

[0511] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0512] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRAV1-optional connector-TRAC-connector-VL1-connector-TRBV2-connector-TRAV2-connector-VH1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRBV1-optional connector-TRBC-optional connector-optional hinge region-Alb; or

[0513] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRAV1-optional connector-TRAC-connector-VL1-connector-TRAV2-connector-TRBV2-connector-VH1-optional connector-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRBV1-optional connector-TRBC-optional connector-optional hinge region; or

[0514] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRAV1-optional connector-TRAC-connector-VL1-connector-TRAV2-connector-TRBV2-connector-VH1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRBV1-optional connector-TRBC-optional connector-optional hinge region-Alb; or

[0515] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRAV1-optional connector-TRAC-connector-VH1-connector-TRAV2-connector-TRBV2-connector-VL1-optional connector-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRBV1-optional connector-TRBC-optional connector-optional hinge region; or

[0516] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRAV1-optional connector-TRAC-connector-VH1-connector-TRAV2-connector-TRBV2-connector-VL1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRBV1-optional connector-TRBC-optional connector-optional hinge region-Alb; or

[0517] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRAV1-optional connector-TRAC-connector-VH1-connector-TRBV2-connector-TRAV2-connector-VL1-optional connector-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRBV1-optional connector-TRBC-optional connector-optional hinge region; or

[0518] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRAV1-optional connector-TRAC-connector-VH1-connector-TRBV2-connector-TRAV2-connector-VL1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRBV1-optional connector-TRBC-optional connector-optional hinge region-Alb; or

[0519] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRAV1-optional connector-TRAC-connector-VH1-connector-TRAV2-connector-TRBV2-connector-VL1-optional connector-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRBV1-optional connector-TRBC-optional connector-optional hinge region; or

[0520] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRAV1-optional connector-TRAC-connector-VH1-connector-TRAV2-connector-TRBV2-connector-VL1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRBV1-optional connector-TRBC-optional connector-optional hinge region-Alb; or

[0521] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRAV1-optional connector-TRAC-connector-VH1-connector-TRBV2-connector-TRAV2-connector-VL1-optional connector-optional hinge region-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRBV1-optional connector-TRBC-optional connector-optional hinge region; or

[0522] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-TRAV1-optional connector-TRAC-connector-VH1-connector-TRBV2-connector-TRAV2-connector-VL1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-TRBV1-optional connector-TRBC-optional connector-optional hinge region-Alb.

[0523] In some embodiments, the multispecific polypeptide molecule comprises a first binding region, a second binding region, a third binding region, and a fourth binding region, as well as functional domains derived from an Fc domain or a dimerized portion thereof derived from an antibody and optionally a hinge region of the antibody, and the multispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain; wherein:

[0524] The first polypeptide chain, from N-terminus to C-terminus, comprises: scFv2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0525] The first polypeptide chain, from N-terminus to C-terminus, comprises: scFv2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0526] The first polypeptide chain, from N-terminus to C-terminus, comprises: scFv2-linker-TRBV1-optional linker-TRBC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0527] The first polypeptide chain, from N-terminus to C-terminus, comprises: scFv2-linker-TRBV1-optional linker-TRBC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0528] The first polypeptide chain, from N-terminus to C-terminus, comprises: scFv2-linker-TRAV1-optional linker-TRAC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0529] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0530] The first polypeptide chain, from N-terminus to C-terminus, comprises: scFv2-linker-TRAV1-optional linker-TRAC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0531] The first polypeptide chain, from N-terminus to C-terminus, comprises: scFv2-linker-TRAV1-optional linker-TRAC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0532] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0533] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0534] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0535] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0536] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0537] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0538] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0539] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0540] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0541] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0542] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0543] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0544] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0545] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0546] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0547] The first polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0548] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0549] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0550] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0551] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0552] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0553] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0554] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0555] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0556] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0557] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0558] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0559] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0560] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0561] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0562] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0563] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-linker-optional linker-TRBC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-linker-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3; or

[0564] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0565] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0566] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-linker-VL1-linker-TRBV2-linker-TRAV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0567] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0568] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0569] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-optional linker-TRAC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0570] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-linker-VH1-linker-TRAV2-linker-TRBV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3; or

[0571] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-linker-VH1-linker-TRBV2-linker-TRAV2-linker-VL1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-CH2-CH3.

[0572] In some implementations, the scFv1 consists of the following from the N end to the C end: VH1-connector VL1 or VL1-connector-VH1.

[0573] In some implementations, the scFv2 consists of the following from the N end to the C end: VH2-connector VL2 or VL2-connector-VH2.

[0574] In some embodiments, the multispecific polypeptide molecule includes CH3 molecules linked to the C-termini of TRAV1 and TRBV1 via optional linkers, respectively. In this case, the TRAC and TRBC molecules in the first and second polypeptide chains of the multispecific polypeptide molecule, respectively linked to the C-termini of TRAV1 and TRBV1, are replaced with CH3 molecules.

[0575] In some embodiments, the multispecific polypeptide molecule includes CL and CH1 connected to the C-termini of TRAV1 and TRBV1 via optional linkers. In some embodiments, CL is connected to the C-terminus of TRAV1, and CH1 is connected to the C-terminus of TRBV1. In some embodiments, CH1 is connected to the C-terminus of TRAV1, and CL is connected to the C-terminus of TRBV1. In this case, TRAC and TRBC, respectively connected to the C-termini of TRAV1 and TRBV1 in the first and second polypeptide chains of the multispecific polypeptide molecule, are replaced with CL and CH1, or CH1 and CL, respectively.

[0576] In some embodiments, the multispecific polypeptide molecule does not contain a constant region, such as a TCR constant region or a fragment thereof, CH3, or CL and CH1, that is optionally linked to the C-terminus of TRAV1 and / or TRBV1 via a linker. In this case, the TRAC and TRBC, respectively, linked to the C-terminus of TRAV1 and TRBV1 in the first and second polypeptide chains of the aforementioned multispecific polypeptide molecule are removed. For example, “The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-scFv1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-CH2-CH3” is adjusted to “The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-linker-scFv1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-optional hinge region-CH2-CH3”.

[0577] In an embodiment of the multispecific polypeptide molecule of the second aspect of the present invention, the multispecific polypeptide molecule further comprises antibody-derived CH1 and CL respectively linked to the VH1 and VL1, wherein the VH1, VL1, CH1, and CL form a single-chain Fab and are linked to the C-terminus of the TRAV1 or the TRBV1 via a linker. In some embodiments, the two polypeptide chains of the multispecific polypeptide molecule respectively comprise:

[0578] TRBV1-VL1-CL-VH1-CH1 and TRAV1; or

[0579] TRBV1-VH1-CH1-VL1-CL and TRAV1; or

[0580] TRAV1-VL1-CL-VH1-CH1 and TRBV1; or

[0581] TRAV1-VH1-CH1-VL1-CL and TRBV1.

[0582] In an embodiment of the multispecific polypeptide molecule of the second aspect of the present invention, the multispecific polypeptide molecule further comprises a fourth binding region for binding to a fourth antigen, the fourth binding region comprising a heavy chain variable region VH2 and a light chain variable region VL2 derived from an antibody binding to the fourth antigen, and CH1 and CL derived from the antibody.

[0583] In some embodiments, VH2, VL2, CH1, and CL form a single-chain Fab2 attached to the N-terminus of TRAV1 or TRBV1. In some embodiments, the two polypeptide chains of the multispecific polypeptide molecule each comprise:

[0584] Fab2-TRBV1-scFv1 and TRAV1; or

[0585] Fab2-TRAV1-scFv1 and TRBV1; or

[0586] Fab2-TRBV1-VHH1 and TRAV1; or

[0587] Fab2-TRAV1-VHH1 and TRBV1; or

[0588] Fab2-TRBV1-VL1 and TRAV1-VH1; or

[0589] Fab2-TRAV1-VL1 and TRBV1-VH1; or

[0590] TRBV1-scFv1 and Fab2-TRAV1; or

[0591] TRAV1-scFv1 and Fab2-TRBV1; or

[0592] TRBV1-VHH1 and Fab2-TRAV1; or

[0593] TRAV1-VHH1 and Fab2-TRBV1; or

[0594] TRBV1-VL1 and Fab2-TRAV1-VH1; or

[0595] TRAV1-VL1 and Fab2-TRBV1-VH1; or

[0596] In some embodiments, the multispecific polypeptide molecule includes a first binding region, a second binding region, a third binding region, and a fourth binding region, wherein the fourth binding region includes a heavy chain variable region VH2 and a light chain variable region VL2 derived from an antibody that binds to the fourth antigen, and CH1 and CL derived from the antibody, wherein VH2, VL2, CH1, and CL form a single-chain Fab2 attached to the N-terminus of the TRAV1 or the TRBV1.

[0597] In some embodiments, the two polypeptide chains of the multispecific polypeptide molecule each comprise:

[0598] Fab2-TRBV1-VL1-TRAV2-TRBV2-VH1 and TRAV1; or

[0599] Fab2-TRBV1-VL1-TRBV2-TRAV2-VH1 and TRAV1; or

[0600] Fab2-TRBV1-VH1-TRBV2-TRAV2-VL1 and TRAV1; or

[0601] Fab2-TRBV1-VH1-TRAV2-TRBV2-VL1 and TRAV1; or

[0602] Fab2-TRAV1-VL1-TRBV2-TRAV2-VH1 and TRBV1; or

[0603] Fab2-TRAV1-VL1-TRAV2-TRBV2-VH1 and TRBV1; or

[0604] Fab2-TRAV1-VH1-TRAV2-TRBV2-VL1 and TRBV1; or

[0605] Fab2-TRAV1-VH1-TRBV2-TRAV2-VL1 and TRBV1; or

[0606] TRBV1-VL1-TRAV2-TRBV2-VH1 and Fab2-TRAV1; or

[0607] TRBV1-VL1-TRBV2-TRAV2-VH1 and Fab2-TRAV1; or

[0608] TRBV1-VH1-TRBV2-TRAV2-VL1 and Fab2-TRAV1; or

[0609] TRBV1-VH1-TRAV2-TRBV2-VL1 and Fab2-TRAV1; or

[0610] TRAV1-VL1-TRBV2-TRAV2-VH1 and Fab2-TRBV1; or

[0611] TRAV1-VL1-TRAV2-TRBV2-VH1 and Fab2-TRBV1; or

[0612] TRAV1-VH1-TRAV2-TRBV2-VL1 and Fab2-TRBV1; or

[0613] TRAV1-VH1-TRBV2-TRAV2-VL1 and Fab2-TRBV1.

[0614] In some implementations, the Fab2 from the N end to the C end is selected from VL2-CL-VH2-CH1, VH2-CH1-VL2-CL, VL2-CL-VH2-CH1, and VH2-CH1-VL2-CL.

[0615] In a preferred embodiment of the multispecific polypeptide molecule of the second aspect of the present invention, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain; wherein:

[0616] The first polypeptide chain, from N-terminus to C-terminus, comprises: TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3 (format 62); or

[0617] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-linker-VH1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-hinge region-CH2-CH3 (format 62-LJH1); or

[0618] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-linker-VL1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3 (format 62-LJH2); or

[0619] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-linker-VL1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-hinge region-CH2-CH3 (format 62-LJH3); or

[0620] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-linker-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-CH2-CH3 (format 62-3); or

[0621] The first polypeptide chain, from N-terminus to C-terminus, comprises: TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-optional linker-hinge region-CH3, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRAV1-optional linker-TRAC-optional linker-hinge region-CH3 (format 62-4); or

[0622] The first polypeptide chain, from N-terminus to C-terminus, comprises: TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-optional linker-hinge region-TRBC, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRAV1-optional linker-TRAC-optional linker-hinge region-TRAC (format 62-5); or

[0623] The first polypeptide chain, from N-terminus to C-terminus, comprises: TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-optional linker-hinge region, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRAV1-optional linker-TRAC-optional linker-hinge region-Alb (format 62-6); or

[0624] The first polypeptide chain, from N-terminus to C-terminus, comprises: TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-linker-Alb, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRAV1-optional linker-TRAC (format 62-7); or

[0625] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-CH3-linker-VL1-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-CH3-optional linker-optional hinge region-CH2-CH3 (format 62-8); or

[0626] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-CH1-linker-VL1-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-CL-optional linker-optional hinge region-CH2-CH3; or

[0627] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-CL-linker-VL1-linker-VH1-optional linker-optional hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-CH1-optional linker-optional hinge region-CH2-CH3; or

[0628] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VHH1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3 (format 62-10); or

[0629] The antibody is an antibody Fab, and wherein the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-CL-linker-VH1-CH1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3 (format 62-12); or

[0630] The first polypeptide chain, from N-terminus to C-terminus, comprises: TRBV1-optional linker-TRBC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3 (format 58-2); or

[0631] The first polypeptide chain, from N-terminus to C-terminus, comprises: VL2-linker-VH2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain, from N-terminus to C-terminus, comprises: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3 (format 72); or

[0632] The first polypeptide chain, from N-terminus to C-terminus, comprises: VL2-linker-TRBV1-linker-VL1-linker-VH1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain, from N-terminus to C-terminus, comprises: VH2-linker-TRAV1-optional linker-hinge region-CH2-CH3 (format 72-2); or

[0633] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3 (format 72-3); or

[0634] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VH2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3 (format 50-2).

[0635] In a third aspect, the present invention provides a multispecific polypeptide molecule, wherein the multispecific polypeptide molecule includes a first binding region for binding a first antigen and a second binding region for binding a second antigen on three polypeptide chains.

[0636] The first binding region includes the α-chain variable region (TRAV1) and the β-chain variable region (TRBV1) derived from the T-cell receptor (TCR) that binds to the first antigen-MHC complex.

[0637] The second binding region includes a heavy chain variable region VH1 and a light chain variable region VL1 derived from an antibody that binds to the second antigen, or a single heavy chain variable region (VHH1) derived from a single-domain antibody or nanobody that binds to the second antigen.

[0638] The first antigen is selected from tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), viral antigens, and autoantigens, and the second antigen is an immune cell surface molecule.

[0639] The second bonding region is not connected to the first bonding region.

[0640] In some embodiments, the multispecific polypeptide molecule further comprises at least one functional domain capable of prolonging the half-life of the multispecific polypeptide molecule. In some embodiments, the functional domain for prolonging the half-life is selected from any of the following functional domains:

[0641] 1) Derived from the hinge region of the antibody;

[0642] 2) Derived from the Fc domain of the antibody or its dimerized portion;

[0643] 3) Derived from the CH3 domain of the antibody;

[0644] 4) Albumin (Alb) or its bound fraction;

[0645] 5) Originating from the constant region of TCR or its fragments.

[0646] In some embodiments, the multispecific polypeptide molecule further comprises at least one of the following functional domains:

[0647] 1) Derived from the hinge region of the antibody;

[0648] 2) Derived from the Fc domain of the antibody or its dimerized portion;

[0649] 3) Derived from the CH3 domain of the antibody;

[0650] 4) Albumin (Alb) or its bound fraction;

[0651] 5) Originating from the constant region of TCR or its fragments.

[0652] In some embodiments, the second binding region is not directly connected to the first binding region. In some embodiments, the second binding region is not connected to the first binding region via a linker. "Not directly connected" means that all or part of the amino acid sequence of the second binding region is not linked to all or part of the amino acid sequence of the first binding region on a polypeptide chain in any way.

[0653] In some embodiments, VH1 and VL1 are tandemly linked on a single polypeptide chain (e.g., forming scFv1 or Fab1), and TRAV1 and TRBV1 reside on different polypeptide chains. In some embodiments, VH1 and VL1 are tandemly linked on a single polypeptide chain (e.g., forming scFv1 or Fab1), and TRAV1 and TRBV1 reside on two separate polypeptide chains. In some embodiments, VH1 and VL1 are tandemly linked on a single polypeptide chain (e.g., forming scFv1 or Fab1) and are not directly connected to the polypeptide chains containing TRAV1 and TRBV1.

[0654] In some embodiments, the three polypeptide chains of the multispecific polypeptide molecule respectively comprise:

[0655] TRBV1, VL1-VH1, and TRAV1; or

[0656] TRBV1, VH1-VL1, and TRAV1; or

[0657] TRBV1, VHH1, and TRAV1.

[0658] In some embodiments of the multispecific polypeptide molecule of the third aspect of the invention, the multispecific polypeptide molecule further comprises a constant domain selected from the group selected and connected to the C-terminus of TRAV1 and / or TRBV1 via an optional linker:

[0659] (1) A TCR constant region or a fragment thereof, the TCR constant region including, for example, a TCR α chain constant region (TRAC) and / or a β chain constant region (TRBC);

[0660] (2) CH3, wherein the CH3 is derived from the Fc domain of the antibody, preferably any one or two polypeptide chains contain at least one mutation in CH3 that can promote the formation of heterodimers of the polypeptide molecule.

[0661] (3) CL and CH1, wherein CL is derived from the CL domain of the antibody light chain and CH1 is derived from the CH1 domain of the antibody heavy chain.

[0662] In some embodiments, the multispecific polypeptide molecule includes a TCR constant region or a fragment thereof connected to the C-terminus of TRAV1 and TRBV1 via optional linkers. In some embodiments, the multispecific polypeptide molecule includes TRAC and TRBC connected to the C-terminus of TRAV1 and TRBV1, respectively, via optional linkers. In some embodiments, the three polypeptide chains of the multispecific polypeptide molecule each comprise:

[0663] TRBV1-TRBC, VL1-VH1, and TRAV1-TRAC; or

[0664] TRBV1-TRBC, VH1-VL1, and TRAV1-TRAC; or

[0665] TRBV1-TRBC, scFv1, and TRAV1-TRAC; or

[0666] TRBV1-TRBC, VHH1, and TRAV1-TRAC.

[0667] In some embodiments of the multispecific polypeptide molecule of the third aspect of the invention, the multispecific polypeptide molecule further comprises antibody-derived CH1 and CL respectively linked to said VH1 and VL1, wherein said VH1, VL1, CH1 and CL form a single-chain Fab1 and are connected to the N-terminus of said functional domain via an optional linker. In some embodiments, the three polypeptide chains of said multispecific polypeptide molecule comprise: TRBV1-TRBC, Fab1, and TRAV1-TRAC respectively.

[0668] In an embodiment of the multispecific polypeptide molecule of the third aspect of the present invention, the multispecific polypeptide molecule further comprises a third binding region for binding to a third antigen, the third binding region comprising an α-chain variable region (TRAV2) and a β-chain variable region (TRBV2) derived from a TCR that binds to the third antigen-MHC complex; wherein the third antigen is selected from tumor-associated antigens (TAAs), tumor-specific antigens (TSA), viral antigens, and autoantigens, and the third antigen may be the same as or different from the first antigen. In some embodiments, the TRAV2 is the same as the TRAV1, and / or the TRBV2 is the same as the TRBV1.

[0669] In some implementations, the TRAV2 and the TRBV2 are linked to the same polypeptide chain via a connector.

[0670] In some implementations, the TRAV2 and TRBV2 are linked to the same polypeptide chain via a connector and are linked between VH1 and VL1.

[0671] In some embodiments of the multispecific polypeptide molecule of the third aspect of the invention, the multispecific polypeptide molecule further comprises a constant domain selected from the group selected and connected to the C-terminus of TRAV2 and / or TRBV2 via an optional linker:

[0672] (1) A TCR constant region or a fragment thereof, the TCR constant region including, for example, a TCR α chain constant region (TRAC) and / or a β chain constant region (TRBC);

[0673] (2) CH3, wherein the CH3 is derived from the Fc domain of the antibody, preferably any one or two polypeptide chains contain at least one mutation in CH3 that can promote the formation of heterodimers of the polypeptide molecule.

[0674] (3) CL and CH1, wherein CL is derived from the CL domain of the antibody light chain and CH1 is derived from the CH1 domain of the antibody heavy chain.

[0675] In some embodiments, the multispecific polypeptide molecule of the third aspect of the present invention includes a first binding region, a second binding region, and a third binding region, wherein the three polypeptide chains of the multispecific polypeptide molecule respectively include:

[0676] TRBV1-TRBC, VL1-TRBV2-TRAV2-VH1, and TRAV1-TRAC; or

[0677] TRBV1-TRBC, VL1-TRAV2-TRBV2-VH1, and TRAV1-TRAC; or

[0678] TRBV1-TRBC, VH1-TRBV2-TRAV2-VL1, and TRAV1-TRAC; or

[0679] TRBV1-TRBC, VH1-TRAV2-TRBV2-VL1, and TRAV1-TRAC.

[0680] In some embodiments of the multispecific polypeptide molecule of the third aspect of the present invention, the multispecific polypeptide molecule further comprises a fourth binding region for binding to a fourth antigen, the fourth binding region comprising a heavy chain variable region VH2 and a light chain variable region VL2 derived from an antibody binding to the fourth antigen, or a single heavy chain variable region (VHH2) derived from a single-domain antibody or nanobody binding to the fourth antigen; wherein the fourth antigen is an immune cell surface molecule, and the fourth antigen may be the same as or different from the second antigen. In some embodiments, VH2 is the same as VH1. In some embodiments, VL2 is the same as VL1. In some embodiments, VHH2 is the same as VHH1.

[0681] In some embodiments, VHH2, or VH2 and VL2, form an scFv2 connected to the N-terminal or C-terminal of TRAV1 or TRBV1, or one of VH2 and VL2 is connected to the N-terminal or C-terminal of TRAV1, and the other is connected to the N-terminal or C-terminal of TRBV1. In some embodiments, scFv2 comprises VH2-VL2 or VL2-VH2.

[0682] In some embodiments, the three polypeptide chains of the multispecific polypeptide molecule of the third aspect of the present invention respectively comprise:

[0683] scFv2-TRBV1-TRBC, VL1-VH1, and TRAV1-TRAC; or

[0684] TRBV1-TRBC, VL1-VH1, and scFv2-TRAV1-TRAC; or

[0685] scFv2-TRBV1-TRBC, VH1-VL1, and TRAV1-TRAC; or

[0686] TRBV1-TRBC, VH1-VL1, and scFv2-TRAV1-TRAC; or

[0687] scFv2-TRBV1-TRBC, scFv1, and TRAV1-TRAC; or

[0688] TRBV1-TRBC, scFv1, and scFv2-TRAV1-TRAC.

[0689] In some embodiments of the multispecific polypeptide molecule of the third aspect of the invention, the multispecific polypeptide molecule further comprises antibody-derived CH1 and CL respectively linked to said VH2 and VL2. In some embodiments, said VH2, VL2, CH1, and CL form Fab2 and are linked to the N-terminus of said TRBV1 or TRAV1 via optional linkers. In some embodiments, Fab2 comprises VL1-CL-VH1-CH1, VL1-CH1-VH1-CL, VH1-CH1-VL1-CL, or VH1-CL-VL1-CH1.

[0690] In some embodiments, the multispecific polypeptide molecule of the third aspect of the present invention includes a first binding region, a second binding region, a third binding region, and a fourth binding region, wherein the three polypeptide chains of the polypeptide molecule respectively include:

[0691] scFv2-TRBV1-TRBC, VL1-TRBV2-TRAV2-VH1, and TRAV1-TRAC; or

[0692] scFv2-TRBV1-TRBC, VH1-TRBV2-TRAV2-VL1, and TRAV1-TRAC; or

[0693] scFv2-TRBV1-TRBC, VL1-TRAV2-TRBV2-VH1, and TRAV1-TRAC; or

[0694] scFv2-TRBV1-TRBC, VH1-TRAV2-TRBV2-VL1, and TRAV1-TRAC; or

[0695] TRBV1-TRBC, VL1-TRBV2-TRAV2-VH1, and scFv2-TRAV1-TRAC; or

[0696] TRBV1-TRBC, VH1-TRBV2-TRAV2-VL1, and scFv2-TRAV1-TRAC; or

[0697] TRBV1-TRBC, VL1-TRAV2-TRBV2-VH1, and scFv2-TRAV1-TRAC; or

[0698] TRBV1-TRBC, VH1-TRAV2-TRBV2-VL1, and scFv2-TRAV1-TRAC; or

[0699] scFv2-TRBV1-TRBC, VH1-TRBV2-TRAV2-VL1, and TRAV1-TRAC; or

[0700] scFv2-TRBV1-TRBC, VL1-TRBV2-TRAV2-VH1, and TRAV1-TRAC; or

[0701] scFv2-TRBV1-TRBC, VH1-TRAV2-TRBV2-VL1, and TRAV1-TRAC; or

[0702] scFv2-TRBV1-TRBC, VL1-TRAV2-TRBV2-VH1, and TRAV1-TRAC; or

[0703] TRBV1-TRBC, VH1-TRBV2-TRAV2-VL1, and scFv2-TRAV1-TRAC; or

[0704] TRBV1-TRBC, VL1-TRBV2-TRAV2-VH1, and scFv2-TRAV1-TRAC; or

[0705] TRBV1-TRBC, VH1-TRAV2-TRBV2-VL1, and scFv2-TRAV1-TRAC; or

[0706] TRBV1-TRBC, VL1-TRAV2-TRBV2-VH1, and scFv2-TRAV1-TRAC.

[0707] In some embodiments, the functional domain contains two chains, each connected to the C-terminus or N-terminus of any two polypeptide chains of the multispecific polypeptide molecule of the third aspect of the present invention, preferably the C-terminus.

[0708] In some embodiments, the functional domain contains two chains, and the first and second binding regions of the multispecific polypeptide molecule are connected by covalent or non-covalent binding between the two chains of the functional domain.

[0709] In some embodiments, the functional domain contains two chains, each connected to the C-terminus or N-terminus, preferably the C-terminus, of any two polypeptide chains of the multispecific polypeptide molecule of the third aspect of the invention, and the first binding region and the second binding region of the multispecific polypeptide molecule are connected by covalent or non-covalent bonding between the two chains of the functional domain.

[0710] In some embodiments, the first binding region and the second binding region of the multispecific polypeptide molecule are connected by covalent or non-covalent binding between the two chains of the functional domain, and one chain of the functional domain is connected to the C-terminus or N-terminus of the peptide chain containing the antibody in the second binding region, preferably the C-terminus, and the other chain is connected to the C-terminus or N-terminus of the peptide chain containing TRAV1 or TRBV1 in the first binding region, preferably the C-terminus.

[0711] In some embodiments of the multispecific polypeptide molecule of the third aspect of the present invention, the functional domain is derived from the hinge region of an antibody and / or the Fc domain of an antibody or its dimerized portion; and the functional domain is connected to the C-terminus or N-terminus of any two polypeptide chains of the multispecific polypeptide molecule via an optional linker, preferably the C-terminus.

[0712] In some embodiments of the multispecific polypeptide molecule of the third aspect of the present invention, the functional domain is derived from the CH3 domain of an antibody and optionally the hinge region of an antibody; and the functional domain is connected to the C-terminus or N-terminus of any two polypeptide chains of the multispecific polypeptide molecule via an optional linker, preferably the C-terminus.

[0713] In some embodiments of the multispecific polypeptide molecule of the third aspect of the invention, the functional domain is derived from the constant region of a TCR or a fragment thereof and optionally the hinge region of an antibody; and the functional domain is connected via an optional linker to the C-terminus or N-terminus of any two polypeptide chains of the multispecific polypeptide molecule, preferably the C-terminus.

[0714] In some embodiments, the constant region of the TCR or a fragment thereof is selected from the TCRα chain constant region (TRAC) and the TCRβ chain constant region (TRBC).

[0715] In some embodiments of the multispecific polypeptide molecule of the third aspect of the present invention, the TCR is an αβTCR, forming two polypeptide chains, and VH1 and VL1 are tandemly connected to form an scFv, forming a third polypeptide chain; and the functional domain is derived from the hinge region of the antibody and the Fc domain of the antibody or its dimerized portion; wherein one chain of the functional domain is optionally linked to the C-terminus of the scFv via a linker, and the other chain is linked to the C-terminus of the α chain or β chain of the TCR. In some embodiments, the α chain and β chain each independently contain a variable region, for example, the α chain contains TRAV1 and the β chain contains TRBV1. In some embodiments, the α chain and β chain each independently contain a variable region and a constant region, for example, the α chain contains TRAV1 and TRAC, and the β chain contains TRBV1 and TRBC.

[0716] In some embodiments of the multispecific polypeptide molecule of the third aspect of the present invention, the polypeptide molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; wherein:

[0717] The first polypeptide chain contains, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC; the second polypeptide chain contains, from N-terminus to C-terminus: VL1 - linker - VH1 - optional linker - hinge region - CH2-CH3; and the third polypeptide chain contains, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC - optional linker - hinge region - CH2-CH3 (format 59).

[0718] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC, the second polypeptide chain comprises, from N-terminus to C-terminus: VH1 - linker - VL1 - optional linker - hinge region - CH2-CH3, and the third polypeptide chain comprises, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC - optional linker - hinge region - CH2-CH3.

[0719] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC, the second polypeptide chain comprises, from N-terminus to C-terminus: VHH1 - optional linker - hinge region - CH2-CH3, and the third polypeptide chain comprises, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC - optional linker - hinge region - CH2-CH3.

[0720] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - optional linker - hinge region - CH2-CH3, the second polypeptide chain comprises, from N-terminus to C-terminus: VL1 - linker - VH1 - optional linker - hinge region - CH2-CH3, and the third polypeptide chain comprises, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC.

[0721] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - optional linker - hinge region - CH2-CH3, the second polypeptide chain comprises, from N-terminus to C-terminus: VH1 - linker - VL1 - optional linker - hinge region - CH2-CH3, and the third polypeptide chain comprises, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC.

[0722] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - optional linker - hinge region - CH2-CH3, the second polypeptide chain comprises, from N-terminus to C-terminus: VHH1 - optional linker - hinge region - CH2-CH3, and the third polypeptide chain comprises, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC.

[0723] In a fourth aspect, the present invention provides a multispecific polypeptide molecule, wherein the multispecific polypeptide molecule includes a first binding region for binding a first antigen and a second binding region for binding a second antigen on three polypeptide chains.

[0724] The first binding region includes the α-chain variable region (TRAV1) and the β-chain variable region (TRBV1) derived from the T-cell receptor (TCR) that binds to the first antigen-MHC complex.

[0725] The second binding region includes a heavy chain variable region VH1 and a light chain variable region VL1 derived from the antibody that binds to the second antigen.

[0726] The first antigen is selected from tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), viral antigens, and autoantigens, and the second antigen is an immune cell surface molecule.

[0727] The TRAV1 and TRBV1 are distributed on two different polypeptide chains, the VH1 and VL1 are distributed on two different polypeptide chains, and the polypeptide chain containing VH1 or VL1 is connected to the C-terminus of the polypeptide chain containing TRAV1 or TRBV1.

[0728] In some embodiments, the multispecific polypeptide molecule further comprises at least one functional domain capable of prolonging the half-life of the multispecific polypeptide molecule. In some embodiments, the functional domain capable of prolonging the half-life of the multispecific polypeptide molecule is selected from at least one of the following:

[0729] 1) Derived from the hinge region of the antibody;

[0730] 2) Derived from the Fc domain of the antibody or its dimerized portion;

[0731] 3) Derived from the CH3 domain of the antibody;

[0732] 4) Albumin (Alb) or its bound fraction;

[0733] 5) Originating from the constant region of TCR or its fragments.

[0734] In some embodiments, the multispecific polypeptide molecule further comprises at least one of the following functional domains:

[0735] 1) Derived from the hinge region of the antibody;

[0736] 2) Derived from the Fc domain of the antibody or its dimerized portion;

[0737] 3) Derived from the CH3 domain of the antibody;

[0738] 4) Albumin (Alb) or its bound fraction;

[0739] 5) Originating from the constant region of TCR or its fragments.

[0740] In some embodiments, the N-terminus of the peptide chain containing VH1 or VL1 is connected to the C-terminus of the peptide chain containing TRAV1 or TRBV1. In some embodiments, the N-terminus of the peptide chain containing VH1 or VL1 is connected to the C-terminus of the peptide chain containing TRAV1 or TRBV1.

[0741] In some embodiments, the peptide chain containing VL1 or VH1 is connected to the C-terminus of the peptide chain containing TRBV1 or TRAV1 via a connector. In a preferred embodiment, the N-terminus of the peptide chain containing VL1 or VH1 is connected to the C-terminus of the peptide chain containing TRBV1 or TRAV1 via a connector.

[0742] In some embodiments, the three polypeptide chains of the multispecific polypeptide molecule respectively comprise:

[0743] TRBV1-VL1, VH1, and TRAV1; or

[0744] TRBV1-VH1, VL1, and TRAV1; or

[0745] TRAV1-VL1, VH1, and TRBV1; or

[0746] TRAV1-VH1, VL1, and TRBV1.

[0747] In some embodiments of the multispecific polypeptide molecule of the fourth aspect of the invention, the multispecific polypeptide molecule further comprises a constant domain selected from the group selected and connected to the C-terminus of TRAV1 and / or TRBV1 via an optional linker:

[0748] (1) A TCR constant region or a fragment thereof, the TCR constant region including, for example, a TCR α chain constant region (TRAC) and / or a β chain constant region (TRBC);

[0749] (2) CH3, wherein the CH3 is derived from the Fc domain of the antibody, preferably any one or two polypeptide chains contain at least one mutation in CH3 that can promote the formation of heterodimers of the polypeptide molecule.

[0750] (3) CL and CH1, wherein CL is derived from the CL domain of the antibody light chain and CH1 is derived from the CH1 domain of the antibody heavy chain.

[0751] In some embodiments, the multispecific polypeptide molecule includes a TCR constant region or a fragment thereof connected to the C-terminus of TRAV1 and TRBV1 via optional linkers. In some embodiments, the multispecific polypeptide molecule includes TRAC and TRBC connected to the C-terminus of TRAV1 and TRBV1, respectively, via optional linkers. In some embodiments, the three polypeptide chains of the multispecific polypeptide molecule each comprise:

[0752] TRBV1-TRBC-VL1, VH1, and TRAV1-TRAC; or

[0753] TRBV1-TRBC-VH1, VL1, and TRAV1-TRAC; or

[0754] TRAV1-TRAC-VL1, VH1, and TRBV1-TRBC; or

[0755] TRAV1-TRAC-VH1, VL1, and TRBV1-TRBC.

[0756] In an embodiment of the multispecific polypeptide molecule of the fourth aspect of the present invention, the multispecific polypeptide molecule further comprises antibody-derived CH1 and CL respectively linked to VH1 and VL1. In some embodiments, the three polypeptide chains of the multispecific polypeptide molecule respectively comprise:

[0757] TRBV1-TRBC-VL1-CL, VH1-CH, and TRAV1-TRAC; or

[0758] TRBV1-TRBC-VH1-CH, VL1-CL, and TRAV1-TRAC; or

[0759] TRAV1-TRAC-VL1-CL, VH1-CH, and TRBV1-TRBC; or

[0760] TRAV1-TRAC-VH1-CH, VL1-CL, and TRBV1-TRBC.

[0761] In an embodiment of the multispecific polypeptide molecule of the fourth aspect of the present invention, the multispecific polypeptide molecule further comprises a third binding region for binding to a third antigen, the third binding region comprising an α-chain variable region (TRAV2) and a β-chain variable region (TRBV2) derived from a TCR that binds to the third antigen-MHC complex; wherein the third antigen is selected from tumor-associated antigens (TAAs), tumor-specific antigens (TSA), viral antigens, and autoantigens, and the third antigen may be the same as or different from the first antigen. In some embodiments, TRAV2 is the same as TRAV1. In some embodiments, TRBV2 is the same as TRBV1.

[0762] In some embodiments, the TRAV2 or TRBV2 is connected to the C-end of VH1 or VL1 via an optional connector. In some embodiments, the TRAV2 and TRBV2 are connected in series on a chain and to the C-end of VH1 or VL1 via an optional connector. In some embodiments, one of the TRAV2 and TRBV2 is connected to the C-end of VH1, and the other is connected to the C-end of VL1.

[0763] In some embodiments, the multispecific polypeptide molecule of the third aspect of the present invention includes a first binding region, a second binding region, and a third binding region, wherein the three polypeptide chains of the multispecific polypeptide molecule respectively include:

[0764] TRBV1-TRBC-VL1-TRBV2-TRAV2, VH1, and TRAV1-TRAC; or

[0765] TRBV1-TRBC-VL1-TRAV2-TRBV2, VH1, and TRAV1-TRAC; or

[0766] TRBV1-TRBC-VL1, VH1-TRBV2-TRAV2, and TRAV1-TRAC; or

[0767] TRBV1-TRBC-VL1, VH1-TRAV2-TRBV2, and TRAV1-TRAC; or

[0768] TRBV1-TRBC-VH1-TRBV2-TRAV2, VL1, and TRAV1-TRAC; or

[0769] TRBV1-TRBC-VH1-TRAV2-TRBV2, VL1, and TRAV1-TRAC; or

[0770] TRBV1-TRBC-VH1, VL1-TRBV2-TRAV2, and TRAV1-TRAC; or

[0771] TRBV1-TRBC-VH1, VL1-TRAV2-TRBV2, and TRAV1-TRAC; or

[0772] TRAV1-TRAC-VL1-TRBV2-TRAV2, VH1, and TRBV1-TRBC; or

[0773] TRAV1-TRAC-VL1-TRAV2-TRBV2, VH1, and TRBV1-TRBC; or

[0774] TRAV1-TRAC-VL1, VH1-TRBV2-TRAV2, and TRBV1-TRBC; or

[0775] TRAV1-TRAC-VL1, VH1-TRAV2-TRBV2, and TRBV1-TRBC; or

[0776] TRAV1-TRAC-VH1-TRBV2-TRAV2, VL1, and TRBV1-TRBC; or

[0777] TRAV1-TRAC-VH1-TRAV2-TRBV2, VL1, and TRBV1-TRBC; or

[0778] TRAV1-TRAC-VH1, VL1-TRBV2-TRAV2, and TRBV1-TRBC; or

[0779] TRAV1-TRAC-VH1, VL1-TRAV2-TRBV2, and TRBV1-TRBC; or

[0780] TRBV1-TRBC-VL1-TRBV2, VH1-TRAV2, and TRAV1-TRAC; or

[0781] TRBV1-TRBC-VL1-TRAV2, VH1-TRBV2, and TRAV1-TRAC; or

[0782] TRBV1-TRBC-VH1-TRBV2, VL1-TRAV2, and TRAV1-TRAC; or

[0783] TRBV1-TRBC-VH1-TRAV2, VL1-TRBV2, and TRAV1-TRAC; or

[0784] TRAV1-TRAC-VL1-TRBV2, VH1-TRAV2, and TRBV1-TRBC; or

[0785] TRAV1-TRAC-VL1-TRAV2, VH1-TRBV2, and TRBV1-TRBC; or

[0786] TRAV1-TRAC-VH1-TRBV2, VL1-TRAV2, and TRBV1-TRBC; or

[0787] TRAV1-TRAC-VH1-TRAV2, VL1-TRBV2, and TRBV1-TRBC.

[0788] In some embodiments, the multispecific polypeptide molecule further comprises a constant domain selected from the group consisting of the following, which is optionally linked to the C-terminus of TRAV2 and / or TRBV2 via a linker:

[0789] (1) A TCR constant region or a fragment thereof, the TCR constant region including, for example, a TCR α chain constant region (TRAC) and / or a β chain constant region (TRBC);

[0790] (2) CH3, wherein the CH3 is derived from the Fc domain of the antibody, preferably any one or two polypeptide chains contain at least one mutation in CH3 that can promote the formation of heterodimers of the polypeptide molecule.

[0791] (3) CL and CH1, wherein CL is derived from the CL domain of the antibody light chain and CH1 is derived from the CH1 domain of the antibody heavy chain.

[0792] In some embodiments of the multispecific polypeptide molecule of the fourth aspect of the present invention, the multispecific polypeptide molecule further comprises a fourth binding region for binding to a fourth antigen, the fourth binding region comprising a heavy chain variable region VH2 and a light chain variable region VL2 derived from an antibody binding to the fourth antigen, or a single heavy chain variable region (VHH) derived from a single-domain antibody or nanobody binding to the fourth antigen; wherein the fourth antigen is an immune cell surface molecule, and the fourth antigen may be the same as or different from the second antigen. In some embodiments, VH2 is the same as VH1. In some embodiments, VL2 is the same as VL1.

[0793] In some embodiments, the VHH or the VH2 and VL2 form an scFv connected to the N-terminal or C-terminal of the TRAV1 or TRBV1, or one of the VH2 and VL2 is connected to the N-terminal or C-terminal of the TRAV1, and the other is connected to the N-terminal or C-terminal of the TRBV1. In some embodiments, the scFv comprises VH2-VL2 or VL2-VH2.

[0794] In some embodiments, the three polypeptide chains of the multispecific polypeptide molecule of the third aspect of the present invention respectively comprise:

[0795] scFv-TRBV1-TRBC-VL1, VH1, and TRAV1-TRAC; or

[0796] TRBV1-TRBC-VL1, VH1, and scFv-TRAV1-TRAC; or

[0797] VHH-TRBV1-TRBC-VL1, VH1, and TRAV1-TRAC; or

[0798] TRBV1-TRBC-VL1, VH1, and VHH-TRAV1-TRAC; or

[0799] VH2-TRBV1-TRBC-VL1, VH1, and VL2-TRAV1-TRAC; or

[0800] VL2-TRBV1-TRBC-VL1, VH1, and VH2-TRAV1-TRAC; or

[0801] scFv-TRBV1-TRBC-VH1, VL1, and TRAV1-TRAC; or

[0802] TRBV1-TRBC-VH1, VL1, and scFv-TRAV1-TRAC; or

[0803] VHH-TRBV1-TRBC-VH1, VL1, and TRAV1-TRAC; or

[0804] TRBV1-TRBC-VH1, VL1, and VHH-TRAV1-TRAC; or

[0805] VH2-TRBV1-TRBC-VH1, VL1, and VL2-TRAV1-TRAC; or

[0806] VL2-TRBV1-TRBC-VH1, VL1, and VH2-TRAV1-TRAC; or

[0807] scFv-TRAV1-TRAC-VL1, VH1, and TRBV1-TRBC; or

[0808] TRAV1-TRAC-VL1, VH1, and scFv-TRBV1-TRBC; or

[0809] VHH-TRAV1-TRAC-VL1, VH1, and TRBV1-TRBC; or

[0810] TRAV1-TRAC-VL1, VH1, and VHH-TRBV1-TRBC; or

[0811] VH2-TRAV1-TRAC-VL1, VH1, and VL2-TRBV1-TRBC; or

[0812] VL2-TRAV1-TRAC-VL1, VH1, and VH2-TRBV1-TRBC; or

[0813] scFv-TRAV1-TRAC-VH1, VL1, and TRBV1-TRBC; or

[0814] TRAV1-TRAC-VH1, VL1, and scFv-TRBV1-TRBC; or

[0815] VHH-TRAV1-TRAC-VH1, VL1, and TRBV1-TRBC; or

[0816] TRAV1-TRAC-VH1, VL1, and VHH-TRBV1-TRBC; or

[0817] VH2-TRAV1-TRAC-VH1, VL1, and VL2-TRBV1-TRBC; or

[0818] VL2-TRAV1-TRAC-VH1, VL1, and VH2-TRBV1-TRBC.

[0819] In some embodiments of the multispecific polypeptide molecule of the fourth aspect of the invention, the multispecific polypeptide molecule further comprises antibody-derived CH1 and CL respectively linked to said VH2 and VL2, wherein said VH2, VL2, CH1, and CL form a single-chain Fab and are connected to the N-terminus of said functional domain via optional linkers. In some embodiments, the Fab comprises VL1-CL-VH1-CH1, VL1-CH1-VH1-CL, VH1-CH1-VL1-CL, or VH1-CL-VL1-CH1.

[0820] In some embodiments, the multispecific polypeptide molecule of the fourth aspect of the present invention includes a first binding region, a second binding region, a third binding region, and a fourth binding region, wherein the three polypeptide chains of the polypeptide molecule respectively include:

[0821] scFv-TRBV1-TRBC-VL1-TRBV2-TRAV2, VH1, and TRAV1-TRAC; or

[0822] scFv-TRBV1-TRBC-VL1-TRAV2-TRBV2, VH1, and TRAV1-TRAC; or

[0823] scFv-TRBV1-TRBC-VL1, VH1-TRAV2-TRBV2, and TRAV1-TRAC; or

[0824] scFv-TRBV1-TRBC-VL1, VH1-TRAV2-TRBV2, and TRAV1-TRAC; or

[0825] TRBV1-TRBC-VL1-TRBV2-TRAV2, VH1, and scFv-TRAV1-TRAC; or

[0826] TRBV1-TRBC-VL1-TRAV2-TRBV2, VH1, and scFv-TRAV1-TRAC; or

[0827] TRBV1-TRBC-VL1, VH1-TRBV2-TRAV2, and scFv-TRAV1-TRAC; or

[0828] TRBV1-TRBC-VL1, VH1-TRAV2-TRBV2, and scFv-TRAV1-TRAC; or

[0829] VHH-TRBV1-TRBC-VL1-TRBV2-TRAV2, VH1, and TRAV1-TRAC; or

[0830] VHH-TRBV1-TRBC-VL1-TRAV2-TRBV2, VH1, and TRAV1-TRAC; or

[0831] VHH-TRBV1-TRBC-VL1, VH1-TRBV2-TRAV2, and TRAV1-TRAC; or

[0832] VHH-TRBV1-TRBC-VL1, VH1-TRAV2-TRBV2, and TRAV1-TRAC; or

[0833] TRBV1-TRBC-VL1-TRBV2-TRAV2, VH1, and VHH-TRAV1-TRAC; or

[0834] TRBV1-TRBC-VL1-TRAV2-TRBV2, VH1, and VHH-TRAV1-TRAC; or

[0835] TRBV1-TRBC-VL1, VH1-TRBV2-TRAV2, and VHH-TRAV1-TRAC; or

[0836] TRBV1-TRBC-VL1, VH1-TRAV2-TRBV2, and VHH-TRAV1-TRAC; or

[0837] VH2-TRBV1-TRBC-VL1-TRBV2-TRAV2, VH1, and VL2-TRAV1-TRAC; or

[0838] VH2-TRBV1-TRBC-VL1-TRAV2-TRBV2, VH1, and VL2-TRAV1-TRAC; or

[0839] VH2-TRBV1-TRBC-VL1, VH1-TRBV2-TRAV2, and VL2-TRAV1-TRAC; or

[0840] VH2-TRBV1-TRBC-VL1, VH1-TRAV2-TRBV2, and VL2-TRAV1-TRAC; or

[0841] VL2-TRBV1-TRBC-VL1-TRBV2-TRAV2, VH1, and VH2-TRAV1-TRAC; or

[0842] VL2-TRBV1-TRBC-VL1-TRAV2-TRBV2, VH1, and VH2-TRAV1-TRAC; or

[0843] VL2-TRBV1-TRBC-VL1, VH1-TRBV2-TRAV2, and VH2-TRAV1-TRAC; or

[0844] VL2-TRBV1-TRBC-VL1, VH1-TRAV2-TRBV2, and VH2-TRAV1-TRAC; or

[0845] scFv-TRBV1-TRBC-VH1-TRBV2-TRAV2, VL1, and TRAV1-TRAC; or

[0846] scFv-TRBV1-TRBC-VH1-TRAV2-TRBV2, VL1, and TRAV1-TRAC; or

[0847] scFv-TRBV1-TRBC-VH1, VL1-TRBV2-TRAV2, and TRAV1-TRAC; or

[0848] scFv-TRBV1-TRBC-VH1, VL1-TRAV2-TRBV2, and TRAV1-TRAC; or

[0849] TRBV1-TRBC-VH1-TRBV2-TRAV2, VL1, and scFv-TRAV1-TRAC; or

[0850] TRBV1-TRBC-VH1-TRAV2-TRBV2, VL1, and scFv-TRAV1-TRAC; or

[0851] TRBV1-TRBC-VH1, VL1-TRBV2-TRAV2, and scFv-TRAV1-TRAC; or

[0852] TRBV1-TRBC-VH1, VL1-TRAV2-TRBV2, and scFv-TRAV1-TRAC; or

[0853] VHH-TRBV1-TRBC-VH1-TRBV2-TRAV2, VL1, and TRAV1-TRAC; or

[0854] VHH-TRBV1-TRBC-VH1-TRAV2-TRBV2, VL1, and TRAV1-TRAC; or

[0855] VHH-TRBV1-TRBC-VH1, VL1-TRBV2-TRAV2, and TRAV1-TRAC; or

[0856] VHH-TRBV1-TRBC-VH1, VL1-TRAV2-TRBV2, and TRAV1-TRAC; or

[0857] TRBV1-TRBC-VH1-TRBV2-TRAV2, VL1, and VHH-TRAV1-TRAC; or

[0858] TRBV1-TRBC-VH1-TRAV2-TRBV2, VL1, and VHH-TRAV1-TRAC; or

[0859] TRBV1-TRBC-VH1, VL1-TRBV2-TRAV2, and VHH-TRAV1-TRAC; or

[0860] TRBV1-TRBC-VH1, VL1-TRAV2-TRBV2, and VHH-TRAV1-TRAC; or

[0861] VH2-TRBV1-TRBC-VH1-TRBV2-TRAV2, VL1, and VL2-TRAV1-TRAC; or

[0862] VH2-TRBV1-TRBC-VH1-TRAV2-TRBV2, VL1, and VL2-TRAV1-TRAC; or

[0863] VH2-TRBV1-TRBC-VH1, VL1-TRBV2-TRAV2, and VL2-TRAV1-TRAC; or

[0864] VH2-TRBV1-TRBC-VH1, VL1-TRAV2-TRBV2, and VL2-TRAV1-TRAC; or

[0865] VL2-TRBV1-TRBC-VH1-TRBV2-TRAV2, VL1, and VH2-TRAV1-TRAC; or

[0866] VL2-TRBV1-TRBC-VH1-TRAV2-TRBV2, VL1, and VH2-TRAV1-TRAC; or

[0867] VL2-TRBV1-TRBC-VH1, VL1-TRBV2-TRAV2, and VH2-TRAV1-TRAC; or

[0868] VL2-TRBV1-TRBC-VH1, VL1-TRAV2-TRBV2, and VH2-TRAV1-TRAC; or

[0869] scFv-TRAV1-TRAC-VL1-TRBV2-TRAV2, VH1, and TRBV1-TRBC; or

[0870] scFv-TRAV1-TRAC-VL1-TRAV2-TRBV2, VH1, and TRBV1-TRBC; or

[0871] scFv-TRAV1-TRAC-VL1, VH1-TRBV2-TRAV2, and TRBV1-TRBC; or

[0872] scFv-TRAV1-TRAC-VL1, VH1-TRAV2-TRBV2, and TRBV1-TRBC; or

[0873] TRAV1-TRAC-VL1-TRBV2-TRAV2, VH1, and scFv-TRBV1-TRBC; or

[0874] TRAV1-TRAC-VL1-TRAV2-TRBV2, VH1, and scFv-TRBV1-TRBC; or

[0875] TRAV1-TRAC-VL1, VH1-TRBV2-TRAV2, and scFv-TRBV1-TRBC; or

[0876] TRAV1-TRAC-VL1, VH1-TRAV2-TRBV2, and scFv-TRBV1-TRBC; or

[0877] VHH-TRAV1-TRAC-VL1-TRBV2-TRAV2, VH1, and TRBV1-TRBC; or

[0878] VHH-TRAV1-TRAC-VL1-TRAV2-TRBV2, VH1, and TRBV1-TRBC; or

[0879] VHH-TRAV1-TRAC-VL1, VH1-TRBV2-TRAV2, and TRBV1-TRBC; or

[0880] VHH-TRAV1-TRAC-VL1, VH1-TRAV2-TRBV2, and TRBV1-TRBC; or

[0881] TRAV1-TRAC-VL1-TRBV2-TRAV2, VH1, and VHH-TRBV1-TRBC; or

[0882] TRAV1-TRAC-VL1-TRAV2-TRBV2, VH1, and VHH-TRBV1-TRBC; or

[0883] TRAV1-TRAC-VL1, VH1-TRBV2-TRAV2, and VHH-TRBV1-TRBC; or

[0884] TRAV1-TRAC-VL1, VH1-TRAV2-TRBV2, and VHH-TRBV1-TRBC; or

[0885] VH2-TRAV1-TRAC-VL1-TRBV2-TRAV2, VH1, and VL2-TRBV1-TRBC; or

[0886] VH2-TRAV1-TRAC-VL1-TRAV2-TRBV2, VH1, and VL2-TRBV1-TRBC; or

[0887] VH2-TRAV1-TRAC-VL1, VH1-TRBV2-TRAV2, and VL2-TRBV1-TRBC; or

[0888] VH2-TRAV1-TRAC-VL1, VH1-TRAV2-TRBV2, and VL2-TRBV1-TRBC; or

[0889] VL2-TRAV1-TRAC-VL1-TRBV2-TRAV2, VH1, and VH2-TRBV1-TRBC; or

[0890] VL2-TRAV1-TRAC-VL1-TRAV2-TRBV2, VH1, and VH2-TRBV1-TRBC; or

[0891] VL2-TRAV1-TRAC-VL1, VH1-TRBV2-TRAV2, and VH2-TRBV1-TRBC; or

[0892] VL2-TRAV1-TRAC-VL1, VH1-TRAV2-TRBV2, and VH2-TRBV1-TRBC; or

[0893] scFv-TRAV1-TRAC-VH1-TRBV2-TRAV2, VL1, and TRBV1-TRBC; or

[0894] scFv-TRAV1-TRAC-VH1-TRAV2-TRBV2, VL1, and TRBV1-TRBC; or

[0895] scFv-TRAV1-TRAC-VH1, VL1-TRBV2-TRAV2, and TRBV1-TRBC; or

[0896] scFv-TRAV1-TRAC-VH1, VL1-TRAV2-TRBV2, and TRBV1-TRBC; or

[0897] TRAV1-TRAC-VH1-TRBV2-TRAV2, VL1, and scFv-TRBV1-TRBC; or

[0898] TRAV1-TRAC-VH1-TRAV2-TRBV2, VL1, and scFv-TRBV1-TRBC; or

[0899] TRAV1-TRAC-VH1, VL1-TRBV2-TRAV2, and scFv-TRBV1-TRBC; or

[0900] TRAV1-TRAC-VH1, VL1-TRAV2-TRBV2, and scFv-TRBV1-TRBC; or

[0901] VHH-TRAV1-TRAC-VH1-TRBV2-TRAV2, VL1, and TRBV1-TRBC; or

[0902] VHH-TRAV1-TRAC-VH1-TRAV2-TRBV2, VL1, and TRBV1-TRBC; or

[0903] VHH-TRAV1-TRAC-VH1, VL1-TRBV2-TRAV2, and TRBV1-TRBC; or

[0904] VHH-TRAV1-TRAC-VH1, VL1-TRAV2-TRBV2, and TRBV1-TRBC; or

[0905] TRAV1-TRAC-VH1-TRBV2-TRAV2, VL1, and VHH-TRBV1-TRBC; or

[0906] TRAV1-TRAC-VH1-TRAV2-TRBV2, VL1, and VHH-TRBV1-TRBC; or

[0907] TRAV1-TRAC-VH1, VL1-TRBV2-TRAV2, and VHH-TRBV1-TRBC; or

[0908] TRAV1-TRAC-VH1, VL1-TRAV2-TRBV2, and VHH-TRBV1-TRBC; or

[0909] VH2-TRAV1-TRAC-VH1-TRBV2-TRAV2, VL1, and VL2-TRBV1-TRBC; or

[0910] VH2-TRAV1-TRAC-VH1-TRAV2-TRBV2, VL1, and VL2-TRBV1-TRBC; or

[0911] VH2-TRAV1-TRAC-VH1, VL1-TRBV2-TRAV2, and VL2-TRBV1-TRBC; or

[0912] VH2-TRAV1-TRAC-VH1, VL1-TRAV2-TRBV2, and VL2-TRBV1-TRBC; or

[0913] VL2-TRAV1-TRAC-VH1-TRBV2-TRAV2, VL1, and VH2-TRBV1-TRBC; or

[0914] VL2-TRAV1-TRAC-VH1-TRAV2-TRBV2, VL1, and VH2-TRBV1-TRBC; or

[0915] VL2-TRAV1-TRAC-VH1, VL1-TRBV2-TRAV2, and VH2-TRBV1-TRBC; or

[0916] VL2-TRAV1-TRAC-VH1, VL1-TRAV2-TRBV2, and VH2-TRBV1-TRBC.

[0917] In some embodiments of the multispecific polypeptide molecule of the fourth aspect of the invention, the functional domain is derived from albumin or its binding portion and optionally a hinge region, and is connected to the C-terminus or N-terminus of any polypeptide chain via an optional linker, preferably the C-terminus.

[0918] In some embodiments of the multispecific polypeptide molecule of the fourth aspect of the present invention, the functional domain is derived from the hinge region of an antibody and / or the Fc domain of an antibody or its dimerized portion; and the functional domain is connected to the C-terminus or N-terminus of any two polypeptide chains of the multispecific polypeptide molecule via an optional linker, preferably the C-terminus.

[0919] In some embodiments of the multispecific polypeptide molecule of the fourth aspect of the present invention, the functional domain is derived from the CH3 domain of an antibody and optionally the hinge region of an antibody; and the functional domain is connected to the C-terminus or N-terminus of any two polypeptide chains of the multispecific polypeptide molecule via an optional linker, preferably the C-terminus.

[0920] In some embodiments of the multispecific polypeptide molecule of the fourth aspect of the present invention, the functional domain is derived from the constant region of a TCR or a fragment thereof and optionally the hinge region of an antibody; and the functional domain is connected via an optional linker to the C-terminus or N-terminus of any two polypeptide chains of the multispecific polypeptide molecule, preferably the C-terminus.

[0921] In some embodiments, the constant region of the TCR or a fragment thereof is selected from the TCRα chain constant region (TRAC) and the TCRβ chain constant region (TRBC).

[0922] In some embodiments, the functional domain comprises two peptide chains, and any two chains of the multispecific polypeptide molecule are connected by covalent or non-covalent bonding between the two chains of the functional domain. For example, the first binding region and the second binding region are connected by covalent or non-covalent bonding between the two chains of the functional domain.

[0923] In some embodiments of the multispecific polypeptide molecule of the fourth aspect of the present invention, the polypeptide molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; wherein:

[0924] The first polypeptide chain contains, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - linker - VL1; the second polypeptide chain contains, from N-terminus to C-terminus: VH1 - optional linker - hinge region - CH2 - CH3; and the third polypeptide chain contains, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC - optional linker - hinge region - CH2 - CH3 (format 62-1).

[0925] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1, the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-hinge region-CH2-CH3, and the third polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3.

[0926] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1, the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-hinge region-CH2-CH3, and the third polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-hinge region-CH2-CH3.

[0927] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1, the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-hinge region-CH2-CH3, and the third polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-hinge region-CH2-CH3.

[0928] The first polypeptide chain contains, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - linker - VL1 - optional linker - hinge region - CH2 - CH3, the second polypeptide chain contains, from N-terminus to C-terminus: VH1 - optional linker - hinge region - CH2 - CH3, and the third polypeptide chain contains, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC.

[0929] In some embodiments, the first polypeptide chain from N-terminus to C-terminus includes: TRBV1 - optional linker - TRBC - linker - VH1 - optional linker - hinge region - CH2-CH3, the second polypeptide chain from N-terminus to C-terminus includes: VL1 - optional linker - hinge region - CH2-CH3, and the third polypeptide chain from N-terminus to C-terminus includes: TRAV1 - optional linker - TRAC.

[0930] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-optional linker-hinge region-CH2-CH3, the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-hinge region-CH2-CH3, and the third polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC.

[0931] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-optional linker-hinge region-CH2-CH3, the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-hinge region-CH2-CH3, and the third polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC.

[0932] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - linker - VL1, the second polypeptide chain comprises, from N-terminus to C-terminus: VH1 - optional linker - optional hinge region - optional linker - Alb, and the third polypeptide chain comprises, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC - optional linker - optional hinge region.

[0933] The first polypeptide chain includes, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - linker - VL1 - optional linker - Alb; the second polypeptide chain includes, from N-terminus to C-terminus: VH1 - optional linker - optional hinge region; and the third polypeptide chain includes, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC - optional linker - optional hinge region.

[0934] The first polypeptide chain includes, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - linker - VL1; the second polypeptide chain includes, from N-terminus to C-terminus: VH1 - optional linker - optional hinge region; and the third polypeptide chain includes, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC - optional linker - optional hinge region - optional linker - Alb.

[0935] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - linker - VH1, the second polypeptide chain comprises, from N-terminus to C-terminus: VL1 - optional linker - optional hinge region - optional linker - Alb, and the third polypeptide chain comprises, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC - optional linker - optional hinge region.

[0936] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - linker - VH1 - optional linker - Alb, the second polypeptide chain comprises, from N-terminus to C-terminus: VL1 - optional linker - optional hinge region, and the third polypeptide chain comprises, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC - optional linker - optional hinge region.

[0937] In some embodiments, the first polypeptide chain from N-terminus to C-terminus includes: TRBV1 - optional linker - TRBC - linker - VH1, the second polypeptide chain from N-terminus to C-terminus includes: VL1 - optional linker - optional hinge region, and the third polypeptide chain from N-terminus to C-terminus includes: TRAV1 - optional linker - TRAC - optional linker - optional hinge region - optional linker - Alb.

[0938] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC - linker - VL1, the second polypeptide chain comprises, from N-terminus to C-terminus: VH1 - optional linker - optional hinge region - optional linker - Alb, and the third polypeptide chain comprises, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - optional linker - optional hinge region.

[0939] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-optional linker-Alb, the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-optional hinge region, and the third polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region.

[0940] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC - linker - VL1, the second polypeptide chain comprises, from N-terminus to C-terminus: VH1 - optional linker - optional hinge region, and the third polypeptide chain comprises, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - optional linker - optional hinge region - optional linker - Alb.

[0941] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1, the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-optional hinge region-optional linker-Alb, and the third polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region.

[0942] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-optional linker-Alb, the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-optional hinge region, and the third polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region.

[0943] In some embodiments, the first polypeptide chain from N-terminus to C-terminus includes: TRAV1 - optional linker - TRAC - linker - VH1, the second polypeptide chain from N-terminus to C-terminus includes: VL1 - optional linker - optional hinge region, and the third polypeptide chain from N-terminus to C-terminus includes: TRBV1 - optional linker - TRBC - optional linker - optional hinge region - optional linker - Alb.

[0944] The first polypeptide chain includes, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - linker - VL1 - optional linker - optional hinge region - optional linker - Alb; the second polypeptide chain includes, from N-terminus to C-terminus: VH1 - optional linker - optional hinge region; and the third polypeptide chain includes, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC.

[0945] The first polypeptide chain includes, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - linker - VL1 - optional linker - optional hinge region; the second polypeptide chain includes, from N-terminus to C-terminus: VH1 - optional linker - optional hinge region - optional linker - Alb; and the third polypeptide chain includes, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC.

[0946] The first polypeptide chain includes, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - linker - VL1 - optional linker - optional hinge region; the second polypeptide chain includes, from N-terminus to C-terminus: VH1 - optional linker - optional hinge region; and the third polypeptide chain includes, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC - optional linker - Alb.

[0947] In some embodiments, the first polypeptide chain from N-terminus to C-terminus includes: TRBV1 - optional linker - TRBC - linker - VH1 - optional linker - optional hinge region - optional linker - Alb, the second polypeptide chain from N-terminus to C-terminus includes: VL1 - optional linker - optional hinge region, and the third polypeptide chain from N-terminus to C-terminus includes: TRAV1 - optional linker - TRAC.

[0948] In some embodiments, the first polypeptide chain from N-terminus to C-terminus includes: TRBV1 - optional linker - TRBC - linker - VH1 - optional linker - optional hinge region, the second polypeptide chain from N-terminus to C-terminus includes: VL1 - optional linker - optional hinge region - optional linker - Alb, and the third polypeptide chain from N-terminus to C-terminus includes: TRAV1 - optional linker - TRAC.

[0949] In some embodiments, the first polypeptide chain from N-terminus to C-terminus includes: TRBV1 - optional linker - TRBC - linker - VH1 - optional linker - optional hinge region, the second polypeptide chain from N-terminus to C-terminus includes: VL1 - optional linker - optional hinge region, and the third polypeptide chain from N-terminus to C-terminus includes: TRAV1 - optional linker - TRAC - optional linker - Alb.

[0950] In some embodiments, the first polypeptide chain from N-terminus to C-terminus includes: TRAV1 - optional linker - TRAC - linker - VL1 - optional linker - optional hinge region - optional linker - Alb, the second polypeptide chain from N-terminus to C-terminus includes: VH1 - optional linker - optional hinge region, and the third polypeptide chain from N-terminus to C-terminus includes: TRBV1 - optional linker - TRBC.

[0951] In some embodiments, the first polypeptide chain from N-terminus to C-terminus includes: TRAV1 - optional linker - TRAC - linker - VL1 - optional linker - optional hinge region, the second polypeptide chain from N-terminus to C-terminus includes: VH1 - optional linker - optional hinge region - optional linker - Alb, and the third polypeptide chain from N-terminus to C-terminus includes: TRBV1 - optional linker - TRBC.

[0952] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1 - optional linker - TRAC - linker - VL1 - optional linker - optional hinge region, the second polypeptide chain comprises, from N-terminus to C-terminus: VH1 - optional linker - optional hinge region, and the third polypeptide chain comprises, from N-terminus to C-terminus: TRBV1 - optional linker - TRBC - optional linker - Alb.

[0953] In some embodiments, the first polypeptide chain from N-terminus to C-terminus includes: TRAV1 - optional linker - TRAC - linker - VH1 - optional linker - optional hinge region - optional linker - Alb, the second polypeptide chain from N-terminus to C-terminus includes: VL1 - optional linker - optional hinge region, and the third polypeptide chain from N-terminus to C-terminus includes: TRBV1 - optional linker - TRBC.

[0954] In some embodiments, the first polypeptide chain from N-terminus to C-terminus includes: TRAV1 - optional linker - TRAC - linker - VH1 - optional linker - optional hinge region, the second polypeptide chain from N-terminus to C-terminus includes: VL1 - optional linker - optional hinge region - optional linker - Alb, and the third polypeptide chain from N-terminus to C-terminus includes: TRBV1 - optional linker - TRBC.

[0955] In some embodiments, the first polypeptide chain from N-terminus to C-terminus includes: TRAV1 - optional linker - TRAC - linker - VH1 - optional linker - optional hinge region, the second polypeptide chain from N-terminus to C-terminus includes: VL1 - optional linker - optional hinge region, and the third polypeptide chain from N-terminus to C-terminus includes: TRBV1 - optional linker - TRBC - optional linker - Alb.

[0956] In a fifth aspect, the present invention provides a multispecific polypeptide molecule, wherein the multispecific polypeptide molecule includes a first binding region for binding a first antigen and a second binding region for binding a second antigen on both polypeptide chains.

[0957] The first binding region includes the α-chain variable region (TRAV1) and the β-chain variable region (TRBV1) derived from the T-cell receptor (TCR) that binds to the first antigen-MHC complex.

[0958] The second binding region includes a heavy chain variable region VH1 and a light chain variable region VL1 derived from the antibody that binds to the second antigen.

[0959] The first antigen is selected from tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), viral antigens, and autoantigens, and the second antigen is an immune cell surface molecule.

[0960] VH1 and VL1 are distributed on two different polypeptide chains of the multispecific polypeptide molecule, and the C-terminus of the polypeptide chain containing VH1 or VL1 is connected to the N-terminus of the TCR.

[0961] The multispecific polypeptide molecule further comprises at least one of the following functional domains:

[0962] 1) Derived from the hinge region of the antibody;

[0963] 2) Derived from the Fc domain of the antibody or its dimerized portion;

[0964] 3) Derived from the CH3 domain of the antibody;

[0965] 4) Albumin (Alb) or its bound fraction;

[0966] 5) Originating from the constant region of TCR or its fragments.

[0967] In some embodiments, TRAV1 and TRBV1 are tandemly linked on a single polypeptide chain. In some embodiments, VH1 and VL1 are distributed on two different polypeptide chains of the multispecific polypeptide molecule, respectively, and TRAV1 and TRBV1 are tandemly linked on a single polypeptide chain, with the N-terminus of this polypeptide chain attached to the C-terminus of the polypeptide chain containing VH1 or VL1.

[0968] In some embodiments, adjacent variable regions in the multispecific polypeptide molecule are connected by a linker.

[0969] In some embodiments, the multispecific polypeptide molecule comprises:

[0970] VH1-TRBV1-TRAV1 and VL1; or

[0971] VL1-TRBV1-TRAV1 and VH1; or

[0972] VH1-TRAV1-TRBV1 and VL1; or

[0973] VL1-TRAV1-TRBV1 and VH1.

[0974] In some embodiments of the multispecific polypeptide molecule of the fifth aspect of the invention, the multispecific polypeptide molecule further comprises a constant domain selected from the group selected and connected to the C-terminus of TRAV1 and / or TRBV1 via an optional linker:

[0975] (1) A TCR constant region or a fragment thereof, the TCR constant region including, for example, a TCR α chain constant region (TRAC) and / or a β chain constant region (TRBC);

[0976] (2) CH3, wherein the CH3 is derived from the Fc domain of the antibody, preferably any one or two polypeptide chains contain at least one mutation in CH3 that can promote the formation of heterodimers of the polypeptide molecule.

[0977] (3) CL and CH1, wherein CL is derived from the CL domain of the antibody light chain and CH1 is derived from the CH1 domain of the antibody heavy chain.

[0978] In some embodiments of the multispecific polypeptide molecule of the fifth aspect of the present invention, the multispecific polypeptide molecule further comprises antibody-derived CH1 and CL respectively linked to said VH1 and VL1.

[0979] In an embodiment of the multispecific polypeptide molecule of the fifth aspect of the present invention, the multispecific polypeptide molecule further comprises a third binding region for binding to a third antigen, the third binding region comprising an α-chain variable region (TRAV2) and a β-chain variable region (TRBV2) derived from a TCR that binds to the third antigen-MHC complex; wherein the third antigen is selected from tumor-associated antigens (TAAs), tumor-specific antigens (TSA), viral antigens, and autoantigens, and the third antigen may be the same as or different from the first antigen. In some embodiments, TRAV2 is the same as TRAV1. In some embodiments, TRBV2 is the same as TRBV1.

[0980] In some embodiments, TRAV2 and TRBV2 are tandemly linked on a single polypeptide chain. In some embodiments, TRAV2 and TRBV2 are tandemly linked on a single polypeptide chain and connected between TRAV1 and TRBV1. In some embodiments, TRAV2 and TRBV2 are distributed on two different polypeptide chains, with one linked to the N-terminus of VH1 and the other linked to the N-terminus of VL1.

[0981] In some embodiments of the multispecific polypeptide molecule of the fifth aspect of the present invention, the two polypeptide chains of the multispecific polypeptide molecule respectively comprise:

[0982] VH1-TRBV1-TRAV2-TRBV2-TRAV1 and VL1; or

[0983] VH1-TRBV1-TRBV2-TRAV2-TRAV1 and VL1; or

[0984] VL1-TRBV1-TRAV2-TRBV2-TRAV1 and VH1; or

[0985] VL1-TRBV1-TRBV2-TRAV2-TRAV1 and VH1; or

[0986] VH1-TRAV1-TRAV2-TRBV2-TRBV1 and VL1; or

[0987] VH1-TRAV1-TRBV2-TRAV2-TRBV1 and VL1; or

[0988] VL1-TRAV1-TRBV2-TRAV2-TRBV1 and VH1; or

[0989] VL1-TRAV1-TRAV2-TRBV2-TRBV1 and VH1; or

[0990] TRBV2-VH1-TRBV1-TRAV1 and TRAV2-VL1; or

[0991] TRBV2-VL1-TRBV1-TRAV1 and TRAV2-VH1; or

[0992] TRBV2-VH1-TRAV1-TRBV1 and TRAV2-VL1; or

[0993] TRBV2-VL1-TRAV1-TRBV1 and TRAV2-VH1; or

[0994] TRAV2-VH1-TRBV1-TRAV1 and TRBV2-VL1; or

[0995] TRAV2-VL1-TRBV1-TRAV1 and TRBV2-VH1; or

[0996] TRAV2-VH1-TRAV1-TRBV1 and TRBV2-VL1; or

[0997] TRAV2-VL1-TRAV1-TRBV1 and TRBV2-VH1.

[0998] In some embodiments of the multispecific polypeptide molecule of the fifth aspect of the present invention, the multispecific polypeptide molecule further comprises a constant domain selected from the group selected and connected to the C-terminus of TRAV2 and / or TRBV2 via an optional linker:

[0999] (1) A TCR constant region or a fragment thereof, the TCR constant region including, for example, a TCR α chain constant region (TRAC) and / or a β chain constant region (TRBC);

[1000] (2) CH3, wherein the CH3 is derived from the Fc domain of the antibody, preferably any one or two polypeptide chains contain at least one mutation in CH3 that can promote the formation of heterodimers of the polypeptide molecule.

[1001] (3) CL and CH1, wherein CL is derived from the CL domain of the antibody light chain and CH1 is derived from the CH1 domain of the antibody heavy chain.

[1002] In some embodiments of the multispecific polypeptide molecule of the fifth aspect of the present invention, the multispecific polypeptide molecule further comprises a fourth binding region for binding to a fourth antigen, the fourth binding region comprising a heavy chain variable region VH2 and a light chain variable region VL2 derived from an antibody binding to the fourth antigen, or a single heavy chain variable region (VHH) derived from a single-domain antibody or nanobody binding to the fourth antigen; wherein the fourth antigen is an immune cell surface molecule, and the fourth antigen may be the same as or different from the second antigen. In some embodiments, VH2 is the same as VH1. In some embodiments, VL2 is the same as VL1.

[1003] In some implementations, the VHH or the VH2 and VL2 form an scFv2 connected to the N end of the VH1 or VL1 via a connector, or connected between TRAV1 and TRBV1, or one of the VH2 and VL2 is connected to the N end of the VH1 via a connector, and the other is connected to the N end of the VL1 via a connector.

[1004] In some embodiments of the multispecific polypeptide molecule of the fifth aspect of the present invention, the two polypeptide chains of the multispecific polypeptide molecule respectively comprise:

[1005] scFv2-VH1-TRBV1-TRAV1 and VL1; or

[1006] scFv2-VL1-TRBV1-TRAV1 and VH1; or

[1007] scFv2-VH1-TRAV1-TRBV1 and VL1; or

[1008] scFv2-VL1-TRAV1-TRBV1 and VH1; or

[1009] VH1-TRBV1-scFv2-TRAV1 and VL1; or

[1010] VL1-TRBV1-scFv2-TRAV1 and VH1; or

[1011] VH1-TRAV1-scFv2-TRBV1 and VL1; or

[1012] VL1-TRAV1-scFv2-TRBV1 and VH1; or

[1013] VH1-TRBV1-VHH-TRAV1 and VL1; or

[1014] VL1-TRBV1-VHH-TRAV1 and VH1; or

[1015] VH1-TRAV1-VHH-TRBV1 and VL1; or

[1016] VL1-TRAV1-VHH-TRBV1 and VH1; or

[1017] VH1-TRBV1-TRAV1 and scFv2-VL1; or

[1018] VL1-TRBV1-TRAV1 and scFv2-VH1; or

[1019] VH1-TRAV1-TRBV1 and scFv2-VL1; or

[1020] VL1-TRAV1-TRBV1 and scFv2-VH1; or

[1021] VHH-VH1-TRBV1-TRAV1 and VL1; or

[1022] VHH-VL1-TRBV1-TRAV1 and VH1; or

[1023] VHH-VH1-TRAV1-TRBV1 and VL1; or

[1024] VHH-VL1-TRAV1-TRBV1 and VH1; or

[1025] VH1-TRBV1-TRAV1 and VHH-VL1; or

[1026] VL1-TRBV1-TRAV1 and VHH-VH1; or

[1027] VH1-TRAV1-TRBV1 and VHH-VL1; or

[1028] VL1-TRAV1-TRBV1 and VHH-VH1; or

[1029] VH2-VH1-TRBV1-TRAV1 and VL2-VL1; or

[1030] VL2-VH1-TRBV1-TRAV1 and VH2-VL1; or

[1031] VH2-VL1-TRBV1-TRAV1 and VL2-VH1; or

[1032] VL2-VL1-TRBV1-TRAV1 and VH2-VH1; or

[1033] VH2-VH1-TRAV1-TRBV1 and VL2-VL1; or

[1034] VL2-VH1-TRAV1-TRBV1 and VH2-VL1; or

[1035] VH2-VL1-TRAV1-TRBV1 and VL2-VH1; or

[1036] VL2-VL1-TRAV1-TRBV1 and VH2-VH1.

[1037] In some embodiments, the functional domain comprises two chains, and the two chains of the multispecific polypeptide molecule are connected by covalent or non-covalent bonding between the two chains of the functional domain.

[1038] In some embodiments, the functional domain comprises two chains, and the first and second binding regions of the multispecific polypeptide molecule are connected by covalent or non-covalent binding between the two chains of the functional domain.

[1039] In some embodiments, the functional domain comprises two chains and is respectively attached to the C-terminus of the two polypeptide chains of the multispecific polypeptide molecule.

[1040] In some embodiments, the functional domain comprises two chains, each attached to the N-terminus of one of the two polypeptide chains of the multispecific polypeptide molecule.

[1041] In some embodiments, VH1 and VL1 are distributed on two different polypeptide chains of the multispecific polypeptide molecule, and TRAV1 and TRBV1 are tandemly linked on one polypeptide chain, with the N-terminus of the polypeptide chain connected to the C-terminus of the polypeptide chain containing VH1 or VL1. The functional domain comprises two chains, and the first binding region and the second binding region of the multispecific polypeptide molecule are connected by covalent or non-covalent bonding between the two chains of the functional domain. For example, the C-terminus of the polypeptide chain is connected to one chain of the functional domain, and the C-terminus of the other polypeptide chain of the multispecific polypeptide molecule is connected to the other chain of the functional domain.

[1042] In some embodiments of the multispecific polypeptide molecule of the fifth aspect of the invention, the functional domain is derived from albumin or its binding moiety and optionally the hinge region of an antibody; and the functional domain is connected via an optional linker to the C-terminus or N-terminus of any polypeptide chain of the multispecific polypeptide molecule, preferably the C-terminus. In some embodiments, the functional domain is derived from albumin or its binding moiety, and the albumin or its binding moiety is connected via an optional linker to the C-terminus or N-terminus of any polypeptide chain of the multispecific polypeptide molecule, preferably the C-terminus. In some embodiments, the functional domain is derived from albumin or its binding moiety and the hinge region of an antibody, the functional domain is connected via an optional linker to the C-terminus of any polypeptide chain of the multispecific polypeptide molecule, and the albumin or its binding moiety is connected via an optional linker to the C-terminus of any chain of the hinge region of the antibody.

[1043] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain; wherein: the first polypeptide chain comprises, from the N-terminus to the C-terminus: VH1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from the N-terminus to the C-terminus: VL1-optional connector-optional hinge region.

[1044] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRBV1-linker-TRAV1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-optional hinge region-optional linker-Alb.

[1045] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional connector-optional hinge region.

[1046] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-linker-TRAV1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-optional hinge region-optional linker-Alb.

[1047] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional connector-optional hinge region.

[1048] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional connector-optional hinge region-optional connector-Alb.

[1049] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional connector-optional hinge region.

[1050] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional connector-optional hinge region-optional connector-Alb.

[1051] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-connector-VH1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional connector-optional hinge region.

[1052] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-connector-VH1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional connector-optional hinge region-optional connector-Alb.

[1053] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-connector-VL1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional connector-optional hinge region.

[1054] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-connector-VL1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional connector-optional hinge region-optional connector-Alb.

[1055] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-connector-VH1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional connector-optional hinge region.

[1056] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-connector-VH1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional connector-optional hinge region-optional connector-Alb.

[1057] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-connector-VL1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional connector-optional hinge region.

[1058] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VL1-linker-TRAV1-linker-TRBV1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-optional hinge region-optional linker-Alb.

[1059] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-connector-VL1-optional connector-optional hinge region.

[1060] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-connector-VL1-optional connector-optional hinge region-optional connector-Alb.

[1061] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-connector-VH1-optional connector-optional hinge region.

[1062] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-connector-VH1-optional connector-optional hinge region-optional connector-Alb.

[1063] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-connector-VL1-optional connector-optional hinge region.

[1064] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-connector-VL1-optional connector-optional hinge region-optional connector-Alb.

[1065] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-connector-VH1-optional connector-optional hinge region.

[1066] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-connector-VH1-optional connector-optional hinge region-optional connector-Alb.

[1067] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-connector-VH1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional connector-optional hinge region.

[1068] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-connector-VH1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional connector-optional hinge region-optional connector-Alb.

[1069] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-connector-VL1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional connector-optional hinge region.

[1070] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-connector-VL1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional connector-optional hinge region-optional connector-Alb.

[1071] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-connector-VH1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional connector-optional hinge region.

[1072] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-connector-VH1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional connector-optional hinge region-optional connector-Alb.

[1073] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-connector-VL1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional connector-optional hinge region.

[1074] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-connector-VL1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional connector-optional hinge region-optional connector-Alb.

[1075] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-connector-VL1-optional connector-optional hinge region.

[1076] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-connector-VL1-optional connector-optional hinge region-optional connector-Alb.

[1077] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-connector-VH1-optional connector-optional hinge region.

[1078] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-connector-VH1-optional connector-optional hinge region-optional connector-Alb.

[1079] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-connector-VL1-optional connector-optional hinge region.

[1080] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-connector-VL1-optional connector-optional hinge region-optional connector-Alb.

[1081] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-connector-VH1-optional connector-optional hinge region.

[1082] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-connector-VH1-optional connector-optional hinge region-optional connector-Alb.

[1083] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-VH1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VL1-optional connector-optional hinge region.

[1084] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-VH1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VL1-optional connector-optional hinge region-optional connector-Alb.

[1085] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-VH1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VL1-optional connector-optional hinge region.

[1086] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-VH1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VL1-optional connector-optional hinge region-optional connector-Alb.

[1087] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-VL1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VH1-optional connector-optional hinge region.

[1088] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-VL1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VH1-optional connector-optional hinge region-optional connector-Alb.

[1089] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-VL1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VH1-optional connector-optional hinge region.

[1090] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-VL1-connector-TRBV1-connector-TRAV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VH1-optional connector-optional hinge region-optional connector-Alb.

[1091] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-VH1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VL1-optional connector-optional hinge region.

[1092] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-VH1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VL1-optional connector-optional hinge region-optional connector-Alb.

[1093] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-VH1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VL1-optional connector-optional hinge region.

[1094] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-VH1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VL1-optional connector-optional hinge region-optional connector-Alb.

[1095] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-VL1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VH1-optional connector-optional hinge region.

[1096] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-connector-VL1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VH1-optional connector-optional hinge region-optional connector-Alb.

[1097] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-VL1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region-optional connector-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VH1-optional connector-optional hinge region.

[1098] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-VL1-connector-TRAV1-connector-TRBV1-optional connector-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VH1-optional connector-optional hinge region-optional connector-Alb.

[1099] In some embodiments of the multispecific polypeptide molecule of the fifth aspect of the present invention, the functional domain is derived from the hinge region of the antibody and / or the Fc domain of the antibody or its dimerized portion and / or the CH3 domain of the antibody; and the functional domain is connected to the C-terminus or N-terminus of the two polypeptide chains of the multispecific polypeptide molecule via an optional linker, preferably the C-terminus.

[1100] In some implementations, the functional domain is derived from the hinge region of the antibody and the Fc domain of the antibody or its dimerized portion.

[1101] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain; wherein: the first polypeptide chain comprises, from the N-terminus to the C-terminus: VH1-linker-TRBV1-linker-TRAV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from the N-terminus to the C-terminus: VL1-optional linker-hinge region-CH2-CH3 (format 62-2).

[1102] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain; wherein: the first polypeptide chain comprises, from the N-terminus to the C-terminus: CH3-CH2-hinge region-optional linker-VH1-linker-TRBV1-linker-TRAV1, and the second polypeptide chain comprises, from the N-terminus to the C-terminus: CH3-CH2-hinge region-optional linker-VL1.

[1103] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-linker-TRAV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-hinge region-CH2-CH3.

[1104] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRAV1-linker-TRBV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-hinge region-CH2-CH3.

[1105] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-hinge region-CH2-CH3.

[1106] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain; wherein: the first polypeptide chain comprises, from the N-terminus to the C-terminus: CH3-CH2-hinge region-optional linker-VH1-linker-TRBV1-linker-TRAV1, and the second polypeptide chain comprises, from the N-terminus to the C-terminus: CH3-CH2-hinge region-optional linker-VL1-optional linker.

[1107] In some embodiments, the first polypeptide chain comprises, from the N-terminus to the C-terminus: CH3-CH2-hinge region-optional linker-VL1-linker-TRBV1-linker-TRAV1, and the second polypeptide chain comprises, from the N-terminus to the C-terminus: CH3-CH2-hinge region-optional linker-VH1.

[1108] In some embodiments, the first polypeptide chain comprises, from the N-terminus to the C-terminus: CH3-CH2-hinge region-optional linker-VH1-linker-TRAV1-linker-TRBV1, and the second polypeptide chain comprises, from the N-terminus to the C-terminus: CH3-CH2-hinge region-optional linker-VL1.

[1109] In some embodiments, the first polypeptide chain comprises, from the N-terminus to the C-terminus: CH3-CH2-hinge region-optional linker-VL1-linker-TRAV1-linker-TRBV1, and the second polypeptide chain comprises, from the N-terminus to the C-terminus: CH3-CH2-hinge region-optional linker-VH1.

[1110] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VH1-linker-TRBV1-linker-TRAV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-hinge region-CH2-CH3.

[1111] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VL1-linker-TRBV1-linker-TRAV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-hinge region-CH2-CH3.

[1112] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VH1-linker-TRAV1-linker-TRBV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-hinge region-CH2-CH3.

[1113] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VL1-linker-TRAV1-linker-TRBV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-hinge region-CH2-CH3.

[1114] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRBV1-linker-TRAV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VL1-optional linker-hinge region-CH2-CH3 (format 72-1).

[1115] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-linker-TRAV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VH1-optional linker-hinge region-CH2-CH3.

[1116] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRAV1-linker-TRBV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VL1-optional linker-hinge region-CH2-CH3.

[1117] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VH1-optional linker-hinge region-CH2-CH3.

[1118] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VH1-linker-TRBV1-linker-TRAV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-hinge region-CH2-CH3.

[1119] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VL1-linker-TRBV1-linker-TRAV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-hinge region-CH2-CH3.

[1120] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-connector-VH1-connector-TRAV1-connector-TRBV1-optional connector-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional connector-hinge region-CH2-CH3.

[1121] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VL1-linker-TRAV1-linker-TRBV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-hinge region-CH2-CH3.

[1122] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRBV1-linker-TRAV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VL1-optional linker-hinge region-CH2-CH3.

[1123] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-linker-TRAV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VH1-optional linker-hinge region-CH2-CH3.

[1124] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRAV1-linker-TRBV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VL1-optional linker-hinge region-CH2-CH3.

[1125] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VH1-optional linker-hinge region-CH2-CH3.

[1126] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-linker-TRBV1-linker-TRAV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VL1-optional linker-hinge region-CH2-CH3.

[1127] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VH1-linker-TRBV1-linker-TRAV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VL1-optional linker-hinge region-CH2-CH3.

[1128] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VL1-linker-TRBV1-linker-TRAV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VH1-optional linker-hinge region-CH2-CH3.

[1129] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-VL1-connector-TRBV1-connector-TRAV1-optional connector-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VH1-optional connector-hinge region-CH2-CH3.

[1130] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-linker-TRAV1-linker-TRBV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VL1-optional linker-hinge region-CH2-CH3.

[1131] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VH1-linker-TRAV1-linker-TRBV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VL1-optional linker-hinge region-CH2-CH3.

[1132] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VL1-linker-TRAV1-linker-TRBV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VH1-optional linker-hinge region-CH2-CH3.

[1133] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-connector-VL1-connector-TRAV1-connector-TRBV1-optional connector-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VH1-optional connector-hinge region-CH2-CH3.

[1134] In some implementations, the functional domain is derived from the CH3 domain of the antibody.

[1135] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain; wherein: the first polypeptide chain comprises, from the N-terminus to the C-terminus: VH1-linker-TRBV1-linker-TRAV1-optional linker-CH3, and the second polypeptide chain comprises, from the N-terminus to the C-terminus: VL1-optional linker-CH3 (format 22-3).

[1136] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain; wherein: the first polypeptide chain comprises, from the N-terminus to the C-terminus: CH3-optional linker-VH1-linker-TRBV1-linker-TRAV1, and the second polypeptide chain comprises, from the N-terminus to the C-terminus: CH3-optional linker-VL1.

[1137] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-linker-TRAV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-CH3.

[1138] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRAV1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-CH3.

[1139] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-CH3.

[1140] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain; wherein: the first polypeptide chain comprises, from the N-terminus to the C-terminus: CH3-optional linker-VH1-linker-TRBV1-linker-TRAV1, and the second polypeptide chain comprises, from the N-terminus to the C-terminus: CH3-optional linker-VL1-optional linker.

[1141] In some embodiments, the first polypeptide chain comprises, from the N-terminus to the C-terminus: CH3-optional linker-VL1-linker-TRBV1-linker-TRAV1, and the second polypeptide chain comprises, from the N-terminus to the C-terminus: CH3-optional linker-VH1.

[1142] In some embodiments, the first polypeptide chain comprises, from the N-terminus to the C-terminus: CH3-optional linker-VH1-linker-TRAV1-linker-TRBV1, and the second polypeptide chain comprises, from the N-terminus to the C-terminus: CH3-optional linker-VL1.

[1143] In some embodiments, the first polypeptide chain comprises, from the N-terminus to the C-terminus: CH3-optional linker-VL1-linker-TRAV1-linker-TRBV1, and the second polypeptide chain comprises, from the N-terminus to the C-terminus: CH3-optional linker-VH1.

[1144] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VH1-linker-TRBV1-linker-TRAV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-CH3.

[1145] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VL1-linker-TRBV1-linker-TRAV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-CH3.

[1146] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VH1-linker-TRAV1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-CH3.

[1147] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VL1-linker-TRAV1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-CH3.

[1148] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRBV1-linker-TRAV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VL1-optional linker-CH3.

[1149] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-linker-TRAV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VH1-optional linker-CH3.

[1150] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRAV1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VL1-optional linker-CH3.

[1151] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VH1-optional linker-CH3.

[1152] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VH1-linker-TRBV1-linker-TRAV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-CH3.

[1153] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VL1-linker-TRBV1-linker-TRAV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-CH3.

[1154] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VH1-linker-TRAV1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-CH3.

[1155] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VL1-linker-TRAV1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-CH3.

[1156] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRBV1-linker-TRAV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VL1-optional linker-CH3.

[1157] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-linker-TRAV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VH1-optional linker-CH3.

[1158] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRAV1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VL1-optional linker-CH3.

[1159] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VH1-optional linker-CH3.

[1160] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-linker-TRBV1-linker-TRAV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VL1-optional linker-CH3.

[1161] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VH1-linker-TRBV1-linker-TRAV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VL1-optional linker-CH3.

[1162] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VL1-linker-TRBV1-linker-TRAV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VH1-optional linker-CH3.

[1163] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VL1-linker-TRBV1-linker-TRAV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VH1-optional linker-CH3.

[1164] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-linker-TRAV1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VL1-optional linker-CH3.

[1165] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VH1-linker-TRAV1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VL1-optional linker-CH3.

[1166] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VL1-linker-TRAV1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VH1-optional linker-CH3.

[1167] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VL1-linker-TRAV1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VH1-optional linker-CH3.

[1168] In some embodiments of the multispecific polypeptide molecule of the fifth aspect of the invention, the functional domain is derived from the constant region of a TCR or a fragment thereof and optionally the hinge region of an antibody; and the functional domain is connected via an optional linker to the C-terminus or N-terminus of the two polypeptide chains of the multispecific polypeptide molecule, preferably the C-terminus.

[1169] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain; wherein: the first polypeptide chain comprises, from the N-terminus to the C-terminus: VH1-linker-TRBV1-linker-TRAV1-optional linker-TRAC, and the second polypeptide chain comprises, from the N-terminus to the C-terminus: VL1-optional linker-TRBC (format 22-4).

[1170] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain; wherein: the first polypeptide chain comprises, from the N-terminus to the C-terminus: TRAC-optional adapter-VH1-adaptor-TRBV1-adaptor-TRAV1, and the second polypeptide chain comprises, from the N-terminus to the C-terminus: TRBC-optional adapter-VL1.

[1171] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-linker-TRAV1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-TRBC.

[1172] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRAV1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-TRAC.

[1173] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-TRAC.

[1174] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VH1-linker-TRBV1-linker-TRAV1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-TRBC.

[1175] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VL1-linker-TRBV1-linker-TRAV1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-TRBC.

[1176] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VH1-linker-TRAV1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-TRAC.

[1177] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VL1-linker-TRAV1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-TRAC.

[1178] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRBV1-linker-TRAV1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VL1-optional linker-TRBC.

[1179] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-linker-TRAV1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VH1-optional linker-TRBC.

[1180] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRAV1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VL1-optional linker-TRAC.

[1181] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VH1-optional linker-TRAC.

[1182] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VH1-linker-TRBV1-linker-TRAV1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-TRBC.

[1183] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VL1-linker-TRBV1-linker-TRAV1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-TRBC.

[1184] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VH1-linker-TRAV1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-TRAC.

[1185] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VL1-linker-TRAV1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-TRAC.

[1186] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRBV1-linker-TRAV1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VL1-optional linker-TRBC.

[1187] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-linker-TRAV1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VH1-optional linker-TRBC.

[1188] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRAV1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VL1-optional linker-TRAC.

[1189] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VH1-optional linker-TRAC.

[1190] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-linker-TRBV1-linker-TRAV1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VL1-optional linker-TRBC.

[1191] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VH1-linker-TRBV1-linker-TRAV1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VL1-optional linker-TRBC.

[1192] In some embodiments, the first polypeptide chain co...

Claims

1. A multispecific polypeptide molecule, said multispecific polypeptide molecule comprising a first binding region for binding a first antigen and a second binding region for binding a second antigen, The first binding region includes the α-chain variable region (TRAV1) and the β-chain variable region (TRBV1) derived from the T-cell receptor (TCR) that binds to the first antigen-MHC complex. The second binding region includes a heavy chain variable region VH1 and a light chain variable region VL1 derived from an antibody that binds to the second antigen, or a single heavy chain variable region (VHH1) derived from a single-domain antibody or nanobody that binds to the second antigen. The first antigen is selected from tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), viral antigens, and autoantigens, and the second antigen is an immune cell surface molecule. The N-terminus of the antibody is attached to the C-terminus of the TCR; The multispecific polypeptide molecule further comprises at least one of the following functional domains: 1) Derived from the hinge region of the antibody; 2) Derived from the Fc domain of the antibody or its dimerized portion; 3) Derived from the CH3 domain of the antibody; 4) Albumin (Alb) or its bound fraction; 5) Originating from the constant region of TCR or its fragments.

2. The multispecific polypeptide molecule according to claim 1, wherein TRAV1 and TRBV1 are distributed on different polypeptide chains; and / or The VH1 and VL1 are distributed on a single polypeptide chain (e.g., forming scFv1); while the TRAV1 and TRBV1 are distributed on different polypeptide chains.

3. The multispecific polypeptide molecule according to claim 1 or 2, wherein the multispecific polypeptide molecule comprises a first binding region for binding a first antigen and a second binding region for binding a second antigen on two polypeptide chains; Preferably, TRAV1 and TRBV1 are distributed on different polypeptide chains, and VHH1 or VH1 and VL1 are distributed on one polypeptide chain (e.g., forming scFv1) and attached to the C-terminus of the TCR; or TRAV1 and TRBV1 are distributed on different polypeptide chains, and VH1 and VL1 are distributed on different polypeptide chains, with one of the polypeptide chains containing VH1 and VL1 attached to the C-terminus of the polypeptide chain containing TRAV1, and the other polypeptide chain containing VH1 and VL1 attached to the C-terminus of the polypeptide chain containing TRBV1. or, The multispecific polypeptide molecule includes a first binding region for binding a first antigen and a second binding region for binding a second antigen on its three polypeptide chains; Preferably, TRAV1 and TRBV1 are distributed on different polypeptide chains, and VH1 and VL1 are distributed on different polypeptide chains, with the polypeptide chain containing VH1 or VL1 connected to the C-terminus of the polypeptide chain containing TRBV1 or TRAV1.

4. The multispecific polypeptide molecule according to any one of claims 1-3, wherein the polypeptide chains of the multispecific polypeptide molecule respectively comprise: TRBV1-scFv1 and TRAV1; or TRAV1-scFv1 and TRBV1; or TRBV1-VHH1 and TRAV1; or TRAV1-VHH1 and TRBV1; or TRBV1-VL1 and TRAV1-VH1; or TRAV1-VL1 and TRBV1-VH1; or TRBV1-VL1-VH1 and TRAV1; or TRBV1-VH1-VL1 and TRAV1; or TRAV1-VL1-VH1 and TRBV1; or TRAV1-VH1-VL1 and TRBV1; or TRBV1-VL1, VH1, and TRAV1; or TRBV1-VH1, VL1, and TRAV1; or TRAV1-VL1, VH1, and TRBV1; or TRAV1-VH1, VL1, and TRBV1.

5. The multispecific polypeptide molecule according to any one of claims 1-4, wherein the multispecific polypeptide molecule further comprises a third binding region for binding to a third antigen, the third binding region comprising an α-chain variable region (TRAV2) and a β-chain variable region (TRBV2) derived from a TCR that binds to the third antigen-MHC complex; wherein the third antigen is selected from tumor-associated antigens (TAAs), tumor-specific antigens (TSA), viral antigens, and autoantigens, and the third antigen may be the same as or different from the first antigen.

6. The multispecific polypeptide molecule according to claim 5, wherein the TRAV2 is the same as the TRAV1, and / or the TRBV2 is the same as the TRBV1.

7. The multispecific polypeptide molecule according to claim 5 or 6, wherein the TRAV2 and the TRBV2 are linked on the same polypeptide chain via a linker, and preferably linked between VH1 and VL1.

8. The multispecific polypeptide molecule according to claim 7, wherein the multispecific polypeptide molecule comprises two polypeptide chains, and the two polypeptide chains respectively comprise: TRBV1-VL1-TRAV2-TRBV2-VH1 and TRAV1; or TRBV1-VL1-TRBV2-TRAV2-VH1 and TRAV1; or TRBV1-VH1-TRBV2-TRAV2-VL1 and TRAV1; or TRBV1-VH1-TRAV2-TRBV2-VL1 and TRAV1; or TRAV1-VL1-TRBV2-TRAV2-VH1 and TRBV1; or TRAV1-VL1-TRAV2-TRBV2-VH1 and TRBV1; or TRAV1-VH1-TRAV2-TRBV2-VL1 and TRBV1; or TRAV1-VH1-TRBV2-TRAV2-VL1 and TRBV1.

9. The multispecific polypeptide molecule according to any one of claims 1-8, wherein the multispecific polypeptide molecule further comprises a fourth binding region for binding to a fourth antigen, the fourth binding region comprising a heavy chain variable region VH2 and a light chain variable region VL2 derived from an antibody binding to the fourth antigen or a single heavy chain variable region (VHH2) derived from a single-domain antibody or nanobody binding to the fourth antigen; wherein the fourth antigen is an immune cell surface molecule, and the fourth antigen is the same as or different from the second antigen.

10. The multispecific polypeptide molecule according to claim 9, wherein the VH2 is the same as the VH1, and / or the VL2 is the same as the VL1, and / or the VHH2 is the same as the VHH1.

11. The multispecific polypeptide molecule according to claim 9 or 10, wherein the VHH2 or the VH2 and VL2 are connected in series on a polypeptide chain (e.g., forming scFv2 or Fab2) to the N-terminus of the TRAV1 or the TRBV1, or one of the VH2 and VL2 is connected to the N-terminus of the TRAV1 and the other is connected to the N-terminus of the TRBV1.

12. The multispecific polypeptide molecule according to claim 11, wherein the multispecific polypeptide molecule comprises two polypeptide chains, and the two polypeptide chains each comprise: scFv2-TRBV1-scFv1 and TRAV1; or scFv2-TRAV1-scFv1 and TRBV1; or scFv2-TRBV1-VHH1 and TRAV1; or scFv2-TRAV1-VHH1 and TRBV1; or scFv2-TRBV1-VL1 and TRAV1-VH1; or scFv2-TRAV1-VL1 and TRBV1-VH1; or TRBV1-scFv1 and scFv2-TRAV1; or TRAV1-scFv1 and scFv2-TRBV1; or TRBV1-VHH1 and scFv2-TRAV1; or TRAV1-VHH1 and scFv2-TRBV1; or TRBV1-VL1 and scFv2-TRAV1-VH1; or TRAV1-VL1 and scFv2-TRBV1-VH1; or VHH2-TRBV1-scFv1 and TRAV1; or VHH2-TRAV1-scFv1 and TRBV1; or VHH2-TRBV1-VHH1 and TRAV1; or VHH2-TRAV1-VHH1 and TRBV1; or VHH2-TRBV1-VL1 and TRAV1-VH1; or VHH2-TRAV1-VL1 and TRBV1-VH1; or TRBV1-scFv1 and VHH2-TRAV1; or TRAV1-scFv1 and VHH2-TRBV1; or TRBV1-VHH1 and VHH2-TRAV1; or TRAV1-VHH1 and VHH2-TRBV1; or TRBV1-VL1 and VHH2-TRAV1-VH1; or TRAV1-VL1 and VHH2-TRBV1-VH1; or VL2-TRBV1-scFv1 and VH2-TRAV1; or VH2-TRBV1-scFv1 and VL2-TRAV1; or VL2-TRAV1-scFv1 and VH2-TRBV1; or VH2-TRAV1-scFv1 and VL2-TRBV1; or VL2-TRBV1-VHH1 and VH2-TRAV1; or VH2-TRBV1-VHH1 and VL2-TRAV1; or VL2-TRAV1-VHH1 and VH2-TRBV1; or VH2-TRAV1-VHH1 and VL2-TRBV1; or VL2-TRBV1-VL1 and VH2-TRAV1-VH1; or VH2-TRBV1-VL1 and VL2-TRAV1-VH1; or VL2-TRAV1-VL1 and VH2-TRBV1-VH1; or VH2-TRAV1-VL1 and VL2-TRBV1-VH1.

13. The multispecific polypeptide molecule according to claim 11, wherein the multispecific polypeptide molecule comprises a first binding region, a second binding region, a third binding region, and a fourth binding region, and the multispecific polypeptide molecule comprises two polypeptide chains, each of the two polypeptide chains comprising: scFv2-TRBV1-VL1-TRAV2-TRBV2-VH1 and TRAV1; or scFv2-TRBV1-VL1-TRBV2-TRAV2-VH1 and TRAV1; or scFv2-TRBV1-VH1-TRBV2-TRAV2-VL1 and TRAV1; or scFv2-TRBV1-VH1-TRAV2-TRBV2-VL1 and TRAV1; or scFv2-TRAV1-VL1-TRBV2-TRAV2-VH1 and TRBV1; or scFv2-TRAV1-VL1-TRAV2-TRBV2-VH1 and TRBV1; or scFv2-TRAV1-VH1-TRAV2-TRBV2-VL1 and TRBV1; or scFv2-TRAV1-VH1-TRBV2-TRAV2-VL1 and TRBV1; or TRBV1-VL1-TRAV2-TRBV2-VH1 and scFv2-TRAV1; or TRBV1-VL1-TRBV2-TRAV2-VH1 and scFv2-TRAV1; or TRBV1-VH1-TRBV2-TRAV2-VL1 and scFv2-TRAV1; or TRBV1-VH1-TRAV2-TRBV2-VL1 and scFv2-TRAV1; or TRAV1-VL1-TRBV2-TRAV2-VH1 and scFv2-TRBV1; or TRAV1-VL1-TRAV2-TRBV2-VH1 and scFv2-TRBV1; or TRAV1-VH1-TRAV2-TRBV2-VL1 and scFv2-TRBV1; or TRAV1-VH1-TRBV2-TRAV2-VL1 and scFv2-TRBV1; or VHH2-TRBV1-VL1-TRAV2-TRBV2-VH1 and TRAV1; or VHH2-TRBV1-VL1-TRBV2-TRAV2-VH1 and TRAV1; or VHH2-TRBV1-VH1-TRBV2-TRAV2-VL1 and TRAV1; or VHH2-TRBV1-VH1-TRAV2-TRBV2-VL1 and TRAV1; or VHH2-TRAV1-VL1-TRBV2-TRAV2-VH1 and TRBV1; or VHH2-TRAV1-VL1-TRAV2-TRBV2-VH1 and TRBV1; or VHH2-TRAV1-VH1-TRAV2-TRBV2-VL1 and TRBV1; or VHH2-TRAV1-VH1-TRBV2-TRAV2-VL1 and TRBV1; or TRBV1-VL1-TRAV2-TRBV2-VH1 and VHH2-TRAV1; or TRBV1-VL1-TRBV2-TRAV2-VH1 and VHH2-TRAV1; or TRBV1-VH1-TRBV2-TRAV2-VL1 and VHH2-TRAV1; or TRBV1-VH1-TRAV2-TRBV2-VL1 and VHH2-TRAV1; or TRAV1-VL1-TRBV2-TRAV2-VH1 and VHH2-TRBV1; or TRAV1-VL1-TRAV2-TRBV2-VH1 and VHH2-TRBV1; or TRAV1-VH1-TRAV2-TRBV2-VL1 and VHH2-TRBV1; or TRAV1-VH1-TRBV2-TRAV2-VL1 and VHH2-TRBV1; or VL2-TRBV1-VL1-TRAV2-TRBV2-VH1 and VH2-TRAV1; or VL2-TRBV1-VL1-TRBV2-TRAV2-VH1 and VH2-TRAV1; or VH2-TRBV1-VL1-TRAV2-TRBV2-VH1 and VL2-TRAV1; or VH2-TRBV1-VL1-TRBV2-TRAV2-VH1 and VL2-TRAV1; or VL2-TRBV1-VH1-TRBV2-TRAV2-VL1 and VH2-TRAV1; or VL2-TRBV1-VH1-TRAV2-TRBV2-VL1 and VH2-TRAV1; or VH2-TRBV1-VH1-TRBV2-TRAV2-VL1 and VL2-TRAV1; or VH2-TRBV1-VH1-TRAV2-TRBV2-VL1 and VL2-TRAV1; or VL2-TRAV1-VL1-TRBV2-TRAV2-VH1 and VH2-TRBV1; or VL2-TRAV1-VL1-TRAV2-TRBV2-VH1 and VH2-TRBV1; or VH2-TRAV1-VL1-TRBV2-TRAV2-VH1 and VL2-TRBV1; or VH2-TRAV1-VL1-TRAV2-TRBV2-VH1 and VL2-TRBV1; or VL2-TRAV1-VH1-TRAV2-TRBV2-VL1 and VH2-TRBV1; or VL2-TRAV1-VH1-TRBV2-TRAV2-VL1 and VH2-TRBV1; or VH2-TRAV1-VH1-TRAV2-TRBV2-VL1 and VL2-TRBV1; or VH2-TRAV1-VH1-TRBV2-TRAV2-VL1 and VL2-TRBV1.

14. The multispecific polypeptide molecule according to any one of claims 1-13, wherein the functional domain is derived from albumin or its binding portion and optionally the hinge region of an antibody; and the functional domain is connected to the C-terminus or N-terminus of any polypeptide chain of the multispecific polypeptide molecule via an optional linker, preferably the C-terminus.

15. The multispecific polypeptide molecule according to claim 14, wherein the albumin is serum albumin, preferably human serum albumin; and / or The hinge region of the antibody is human-derived, for example, a hinge domain or a portion thereof derived from human IgG1, IgG2, or IgG4.

16. The multispecific polypeptide molecule according to any one of claims 1-13, wherein the functional domain is derived from the hinge region of the antibody and / or the Fc domain of the antibody or its dimerized portion and / or the CH3 domain of the antibody; and any two polypeptide chains of the multispecific polypeptide molecule are connected by covalent or non-covalent bonding between the two chains of the functional domain. Preferably, the two chains of the functional domain are connected to the C-terminus or N-terminus of any two polypeptide chains of the multispecific polypeptide molecule via optional linkers, preferably the C-terminus; or The first binding region and the second binding region are connected by covalent or non-covalent bonding between the two chains of the functional structural domain.

17. The multispecific polypeptide molecule of claim 16, wherein the hinge region of the antibody is human-derived, for example, a hinge domain or portion thereof derived from human IgG1, IgG2, or IgG4; and / or The Fc domain of the antibody or its dimerized portion is human-derived, for example, the Fc domain or a portion thereof derived from human IgG1, IgG2 or IgG4.

18. The multispecific polypeptide molecule according to claim 16 or 17, wherein the hinge region of the antibody comprises at least one amino acid mutation capable of reducing or eliminating the binding of the multispecific molecule to the FcR receptor and / or C1q, for example, the mutation site being selected from positions 233, 234, 235, and 236; and / or The hinge region of the antibody contains at least one mutation capable of reducing or eliminating disulfide bond formation between the hinge region and the non-hinge region, for example, mutation site 220; and / or The CH2 domain of the Fc domain contains at least one amino acid mutation capable of reducing or eliminating the binding of the multispecific molecule to the FcR receptor and / or C1q; for example, the mutation site is selected from positions 237, 250, 252, 254, 256, 270, 297, 322, 327, 330, and 331; and / or The CH3 domain in the Fc domain contains at least one amino acid mutation that can reduce or eliminate the binding of the multispecific molecule to the FcR receptor and / or C1q; for example, the mutation site is selected from positions 428 and 434. The CH3 in the Fc domain contains at least one of the following mutations that can promote the formation of heterodimers of the polypeptide molecule: a) Contains mutations in at least two cysteine ​​residues to form interchain disulfide bonds, such as 354C and 349C or 242C and 334C; b) Contains mutations that enable two CH3 molecules to form a club-and-mortar structure, for example, each CH3 molecule independently contains a mutation located at one or more positions selected from amino acid positions 366, 368, 405 and 407, preferably, one of the CH3 molecules contains a 366W mutation and the other contains 366S, 368A and 407V mutations; c) Mutations that can alter the charge properties of amino acids at the CH3 region interaction interface to promote heterodimer formation, for example, mutation sites on the two CH3 chains are each independently selected from at least one of Y391, K392, T393, T394, P395, P396, V397, L398, D399, S400, Q347, V348, Y349, T350, S354, R355, E356, E357, F405, Y407, and K409.

19. The multispecific polypeptide molecule according to claim 16 or 17, wherein the CH3 domain of the antibody is human-derived, for example, a CH3 domain derived from human IgG1, IgG2, or IgG4; and / or The CH3 domain contains at least one of the following mutations that can promote the formation of heterodimers of the polypeptide molecule: a) Contains mutations in at least two cysteine ​​residues to form interchain disulfide bonds, such as 354C and 349C or 242C and 334C; b) Contains mutations that enable two CH3 molecules to form a club-and-mortar structure, for example, each CH3 molecule independently contains a mutation located at one or more positions selected from amino acid positions 366, 368, 405 and 407, preferably, one of the CH3 molecules contains a 366W mutation and the other contains 366S, 368A and 407V mutations; c) Mutations that can alter the charge properties of amino acids at the CH3 region interaction interface to promote heterodimer formation, for example, mutation sites on the two CH3 chains are each independently selected from at least one of Y391, K392, T393, T394, P395, P396, V397, L398, D399, S400, Q347, V348, Y349, T350, S354, R355, D356, E357, K370, F405, Y407, K409, and K439.

20. The multispecific polypeptide molecule according to any one of claims 1-13, wherein the functional domain is derived from the constant region of a TCR or a fragment thereof and optionally the hinge region of an antibody; and any two polypeptide chains of the multispecific polypeptide molecule are connected by covalent or non-covalent bonding between the two chains of the functional domain. Preferably, the two chains of the functional domain are connected to the C-terminus or N-terminus of any two polypeptide chains of the multispecific polypeptide molecule via optional linkers, preferably the C-terminus; or The first binding region and the second binding region are connected by covalent or non-covalent bonding between the two chains of the functional structural domain.

21. The multispecific polypeptide molecule of claim 20, wherein the hinge region of the antibody is human-derived, for example, a hinge domain or portion thereof derived from human IgG1, IgG2, or IgG4; and / or The constant region of the TCR or a fragment thereof is of human or mouse origin and is selected from the TCRα chain constant region (TRAC) and the TCRβ chain constant region (TRBC); preferably, the TRAC and / or TRBC contains at least one cysteine ​​mutation relative to the wild-type sequence to form a disulfide bond between the TRAC and TRBC, more preferably, the cysteine ​​mutation is located at position 48 of the wild-type TCRα chain constant region and position 57 of the wild-type TCRβ chain constant region.

22. The multispecific polypeptide molecule according to any one of claims 1-21, wherein the multispecific polypeptide molecule further comprises a constant domain selected from the group consisting of a C-terminus of TRAV1 and / or TRBV1, which is optionally linked to the C-terminus of TRAV1 and / or TRBV1 via a linker: (1) A TCR constant region or a fragment thereof, the TCR constant region including, for example, a TCR α chain constant region (TRAC) and / or a β chain constant region (TRBC); Preferably, the TRAC and / or TRBC contain at least one cysteine ​​mutation relative to the wild-type sequence to form a disulfide bond between the TRAC and TRBC; more preferably, the cysteine ​​mutation is located at position 48 of the constant region of the wild-type TCRα chain and position 57 of the constant region of the wild-type TCRβ chain. (2) CH3, wherein the CH3 is derived from the Fc domain of the antibody, preferably any one or two polypeptide chains contain at least one mutation in CH3 that can promote the formation of heterodimers of the polypeptide molecule. Preferably, each of the CH3 molecules independently contains a mutation located at one or more positions selected from amino acid positions 366, 368, 405, and 407, and the two CH3 molecules do not contain the same mutation. More preferably, one of the CH3 molecules contains the T366W mutation and the other contains the T366S, L368A, and Y407V mutations. (3) CL and CH1, wherein CL is derived from the CL domain of the antibody light chain and CH1 is derived from the CH1 domain of the antibody heavy chain.

23. The multispecific polypeptide molecule of claim 22, wherein the multispecific polypeptide molecule comprises TRAC and TRBC, which are optionally linked to the C-terminus of TRAV1 and TRBV1, respectively, via a linker.

24. The multispecific polypeptide molecule according to any one of claims 1-23, wherein the multispecific polypeptide molecule further comprises antibody-derived CH1 and CL respectively linked to the VH1 and VL1, wherein the VH1, VL1, CH1 and CL form a single-chain Fab and are connected to the C-terminus of the TRAV1 or the TRBV1 via a linker.

25. The multispecific polypeptide molecule according to any one of claims 1-4 and 14-24, wherein the polypeptide chain of said polypeptide molecule comprises, from the N-terminus to the C-terminus, respectively: First polypeptide chain: TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-optional linker-hinge region-CH2-CH3; and second polypeptide chain: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3; or First polypeptide chain: TRAV1-optional linker-TRAC-linker-VL1-linker-VH1-optional linker-hinge region-CH2-CH3; and second polypeptide chain: TRBV1-optional linker-TRBC-optional linker-hinge region-CH2-CH3; or First polypeptide chain: TRBV1-optional linker-TRBC-linker-VH1-linker-VL1-optional linker-hinge region-CH2-CH3; and second polypeptide chain: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3; or First polypeptide chain: TRAV1-optional linker-TRBC-linker-VH1-linker-VL1-optional linker-hinge region-CH2-CH3; and second polypeptide chain: TRBV1-optional linker-TRBC-optional linker-hinge region-CH2-CH3; or First polypeptide chain: TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-linker-CH2-CH3; and second polypeptide chain: TRAV1-optional linker-TRAC-linker-CH2-CH3; or First polypeptide chain: TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-optional linker-hinge region-CH3; and second polypeptide chain: TRAV1-optional linker-TRAC-optional linker-hinge region-CH3; or First polypeptide chain: TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-optional linker-hinge region-TRBC; and second polypeptide chain: TRAV1-optional linker-TRAC-optional linker-hinge region-TRAC; or First polypeptide chain: TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-optional linker-hinge region; and second polypeptide chain: TRAV1-optional linker-TRAC-optional linker-hinge region-Alb; or First polypeptide chain: TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-linker-Alb; and second polypeptide chain: TRAV1-optional linker-TRAC; or First polypeptide chain: TRAV1-optional linker-CH3-linker-VL1-linker-VH1-optional linker-optional hinge region-CH2-CH3; and second polypeptide chain: TRBV1-optional linker-CH3-optional linker-optional hinge region-CH2-CH3; or First polypeptide chain: TRAV1-optional linker-CH1-linker-VL1-linker-VH1-optional linker-optional hinge region-CH2-CH3; and second polypeptide chain: TRBV1-optional linker-CL-optional linker-optional hinge region-CH2-CH3; or First polypeptide chain: TRBV1-optional linker-TRBC-linker-VHH1-optional linker-hinge region-CH2-CH3, and second polypeptide chain: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3. The antibody is an antibody Fab, and wherein the first polypeptide chain is: TRBV1-optional linker-TRBC-linker-VL1-CL-linker-VH1-CH1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain is: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3. First polypeptide chain: TRBV1-optional linker-TRBC-linker-VL1, second polypeptide chain: VH1-optional linker-hinge region-CH2-CH3, and third polypeptide chain: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3. First polypeptide chain: TRBV1-optional linker-TRBC-linker-VH1, second polypeptide chain: VL1-optional linker-hinge region-CH2-CH3, and third polypeptide chain: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3. First polypeptide chain: TRAV1-optional linker-TRAC-linker-VL1, second polypeptide chain: VH1-optional linker-hinge region-CH2-CH3, and third polypeptide chain: TRBV1-optional linker-TRBC-optional linker-hinge region-CH2-CH3. First polypeptide chain: TRAV1-optional linker-TRAC-linker-VH1, second polypeptide chain: VL1-optional linker-hinge region-CH2-CH3, and third polypeptide chain: TRBV1-optional linker-TRBC-optional linker-hinge region-CH2-CH3. First polypeptide chain: TRBV1-optional linker-TRBC-linker-VL1-optional linker-hinge region-CH2-CH3, second polypeptide chain: VH1-optional linker-hinge region-CH2-CH3, and third polypeptide chain: TRAV1-optional linker-TRAC. First polypeptide chain: TRBV1-optional linker-TRBC-linker-VH1-optional linker-hinge region-CH2-CH3, second polypeptide chain: VL1-optional linker-hinge region-CH2-CH3, and third polypeptide chain: TRAV1-optional linker-TRAC. First polypeptide chain: TRAV1-optional linker-TRAC-linker-VL1-optional linker-hinge region-CH2-CH3, second polypeptide chain: VH1-optional linker-hinge region-CH2-CH3, and third polypeptide chain: TRBV1-optional linker-TRBC. First polypeptide chain: TRAV1-optional linker-TRAC-linker-VH1-optional linker-hinge region-CH2-CH3, second polypeptide chain: VL1-optional linker-hinge region-CH2-CH3, and third polypeptide chain: TRBV1-optional linker-TRBC.

26. The multispecific polypeptide molecule according to any one of claims 5-8 and 14-24, 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: TRBV1-optional linker-TRBC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3.

27. The multispecific polypeptide molecule according to any one of claims 9-12 and 14-24, 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: VL2-linker-VH2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3; or The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-linker-VL1-linker-VH1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-hinge region-CH2-CH3; or The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3.

28. The multispecific polypeptide molecule according to any one of claims 13-24, 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: VL2-connector-VH2-connector-TRBV1-optional-connector-TRBC-connector-VL1-connector-TRAV2-connector-TRBV2-connector-VH1-optional-connector-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional-connector-TRAC-optional-connector-hinge region-CH2-CH3.

29. The multispecific polypeptide molecule according to any one of claims 1-28, wherein... The VH1 and VL1 contain at least one cysteine ​​mutation to form a disulfide bond between VH1 and VL1, preferably at position 44 of VH1 and position 100 of VL1; and / or The VH2 and VL2 contain at least one cysteine ​​mutation to form a disulfide bond between VH2 and VL2, preferably at position 44 of VH2 and position 100 of VL2; and / or The TRAV1 and VL1 or VL2 contain at least one cysteine ​​mutation to form a disulfide bond between TRAV1 and VL1 or VL2, preferably at position 104 of TRAV1 and position 80 of VL1 or VL2; and / or The TRAV2 and VL1 or VL2 contain at least one cysteine ​​mutation to form a disulfide bond between TRAV2 and VL1 or VL2, preferably at position 104 of TRAV2 and position 80 of VL1 or VL2; and / or The TRBV1 and VL1 or VL2 contain at least one cysteine ​​mutation to form a disulfide bond between TRBV1 and VL1 or VL2, preferably at position 84 of TRBV1 and position 80 of VL1 or VL2; and / or The TRBV2 and VL1 or VL2 contain at least one cysteine ​​mutation to form a disulfide bond between TRBV2 and VL1 or VL2, preferably at position 84 of TRBV2 and position 80 of VL1 or VL2; and / or The TRAV1 and VH1 or VH2 contain at least one cysteine ​​mutation to form a disulfide bond between TRAV1 and VH1 or VH2, preferably at position 104 of TRAV1 and position 89 of VH1 or VH2; and / or The TRAV2 and VH1 or VH2 contain at least one cysteine ​​mutation to form a disulfide bond between TRAV2 and VH1 or VH2, preferably at position 104 of TRAV2 and position 89 of VH1 or VH2.

30. The multispecific polypeptide molecule according to any one of claims 1-29, 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, prolong its half-life, reduce its immunogenicity or reduce its post-translational modifications; for example, the amino acid mutations include mutations that can enhance binding to FcRn and / or effector function silencing mutations.

31. The multispecific polypeptide molecule according to any one of claims 1-30, wherein each of the linkers in the polypeptide molecule is independently selected from a linker composed of 1-35 amino acids; Preferably, each of the connectors is independently selected from S, GS, GGGS, GGGGS, GGGSGGGG, GGSGGSGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGQPKAAP, TVLRT, TVSSAS, GGEGG, GSEGGGS, RTSGPGDGGKGGPGKGPGGEGTKGTGPGG, GKGPGGEGTKGTGPGG, TVLSSAS, EDLKN or variants thereof, EDLNK or variants thereof, and ANIQK or variants thereof, or any combination thereof; Preferably, the variants of EDLKN are amino acid sequences formed by substituting, deleting, or adding one or more amino acids to EDLKN; and / or the variants of EDLNK are amino acid sequences formed by substituting, deleting, or adding one or more amino acids to EDLNK; and / or the variants of ANIQK are amino acid sequences formed by substituting, deleting, or adding one or more amino acids to ANIQK.

32. The multispecific polypeptide molecule according to any one of claims 1-31, wherein each of the linkers in the polypeptide molecule is independently selected from linkers composed of no more than 15 amino acids; Preferably, each of the connectors is independently selected from S, GS, GGGS, GGGGS, GGGSGGGG, GGSGGS, GGSGGSGGS, GGGGSGGGGS, GGGGSGGGGSGGGS, EDLKN or a variant thereof, EDLNK or a variant thereof, and ANIQK or a variant thereof. Preferably, the variants of EDLKN are amino acid sequences formed by substituting, deleting, or adding one or more amino acids to EDLKN; and / or the variants of EDLNK are amino acid sequences formed by substituting, deleting, or adding one or more amino acids to EDLNK; and / or the variants of ANIQK are amino acid sequences formed by substituting, deleting, or adding one or more amino acids to ANIQK.

33. The multispecific polypeptide molecule according to any one of claims 1-32, 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 MGWSLILLFLVAVATRVHS.

34. A multispecific polypeptide molecule according to any one of claims 1-33, wherein the first antigen and the third antigen are each independently selected from the group consisting of melanoma-associated antigens (e.g., gp100, MAGEA1, MAGEA3, MAGEA6, MAGEA4, MAGEA2, MAGEA12, MAGEA2B, MAGEA9B, MAGEA10, MAGEA11, MAGEB2, MAGEC1, MAGEC2), IGF2BP1, GNGT1, PI4K2B, CCR8, NPSR1, COX7B2, ONECUT3, SMC1B, FOXI3, GAGE2A, FBXO43, BRDT, PAGE2, GAGE13, POU5F1B, CTAG1A, and endogenous reverse transcriptase antigens; or The first antigen and the third antigen are each independently selected from the following TSAs: KRAS antigen (e.g., KRAS G12 mutant antigen), TP53, PIK3CA, CTNNB1, EGFR, BRAF, and GNAS; or The first antigen and the third antigen are each independently selected from the following viral antigens: HPV antigen (e.g., HPV E6 or E7 antigen), CMV antigen, HBV antigen, EBV antigen, herpesvirus antigen, human immunodeficiency virus (HIV) antigen, influenza virus antigen, and coronavirus antigen; or The first antigen and the third antigen are each independently selected from the following autoantigens: 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.

35. The multispecific polypeptide molecule according to any one of claims 1-34, wherein the second antigen and the fourth 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, Nk p46, NKG2D, FcεRI, TCRα / β, TCRγ / δ, HLA-DR, CD28, 4-1BB(CD137), OX40(CD134), ICOS(CD278), 2B4(CD244), HVEM, LAG3, DA P10, DAP12, CD27, CD40, GITR, LFA-1, MyD88, CD2, CD7, LIGHT, B7-H3, CTLA-4, PD-1, PD-L1, CD80, BTLA, TIM3, TIGIT and LAG-3.

36. The multispecific polypeptide molecule according to any one of claims 1-35, wherein the first antigen and the third antigen are each independently selected from melanoma-associated antigens (e.g., gp100, MAGEA1), KRAS antigens (e.g., KRAS G12 mutant antigen) and HPV antigens (e.g., HPV E7 antigen), and / or the second antigen and the fourth antigen are each independently selected from CD3, CD28, 4-1BB (CD137), PD-1 and PD-L1.

37. A nucleic acid comprising a nucleotide sequence encoding each strand of a multispecific polypeptide molecule as described in any one of claims 1-36.

38. A vector comprising the nucleic acid of claim 37.

39. A vector system comprising, on one or more vectors, a nucleotide sequence encoding each strand of a multispecific polypeptide molecule as described in any one of claims 1-36.

40. A host cell comprising the nucleic acid of claim 37, the vector of claim 38, or the vector system of claim 39.

41. A conjugate comprising a multispecific polypeptide molecule according to any one of claims 1-36, and a chemical moiety conjugated to said multispecific polypeptide molecule.

42. The conjugate of claim 41, wherein the chemical moiety is selected from therapeutic agents, immunostimulatory molecules, and detectable markers; Preferably, the therapeutic agent is selected from immunomodulators, agonists, radioactive compounds, enzymes, chemotherapeutic agents, and toxins. Preferably, the immunostimulatory molecules are selected from cytokines, chemokines, platelet factors, and complement initiators; Preferably, the detectable label is selected from biotin, streptavidin, enzymes or their catalytically active fragments, radionuclides, nanoparticles, paramagnetic metal ions, nucleic acid probes, contrast agents, and fluorescent, phosphorescent or chemiluminescent molecules.

43. A pharmaceutical composition comprising a multispecific polypeptide molecule of any one of claims 1-36, a nucleic acid of claim 37, a carrier of claim 38, a carrier system of claim 39, a host cell of claim 40, a conjugate of claim 41 or 42, and a pharmaceutically acceptable excipient.

44. The pharmaceutical composition of claim 43, wherein the pharmaceutical composition further comprises a second therapeutic agent, preferably selected from antibodies, chemotherapeutic agents, and small molecule drugs.

45. A method for preventing or treating a disease in a subject, comprising administering to the subject an effective amount of any one of claims 1-36, the nucleic acid of claim 37, the carrier of claim 38, the carrier system of claim 39, the host cell of claim 40, the conjugate of claim 41 or 42, or the pharmaceutical composition of claim 43 or 44, wherein the disease is selected from cancer, infectious diseases, autoimmune diseases, and inflammatory diseases.

46. ​​The method of claim 45, further comprising administering a second therapeutic agent to the subject, preferably, the second therapeutic agent being selected from antibodies, chemotherapeutic agents, and small molecule drugs.

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