T cell receptor-based multispecific polypeptide molecules, compositions thereof, and uses thereof

Abstract

Disclosed herein are T cell receptor (TCR)-based multispecific polypeptide molecules, nucleic acids encoding the polypeptide molecules, vectors or vector systems comprising the nucleic acids, host cells comprising the polypeptide molecules, conjugates and pharmaceutical compositions comprising the polypeptide molecules, and methods of using the polypeptide molecules to prevent or treat a disease in a subject.

Description

Multispecific polypeptide molecules based on T cell receptors and compositions and uses thereof

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

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

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

[0004] Bispecific antibodies (BsAbs) are antibody molecules that simultaneously target two antigens or two different antigenic epitopes of one antigen. Compared with conventional antibodies, bispecific antibodies have stronger specificity, can more accurately target tumor cells, and reduce off-target toxicity. With the development of recombinant protein expression technology and antibody engineering technology, many different antibody formats have been produced. Multispecific antibodies are used for various purposes, including (1) receptor activation (2) blocking (3) internalization (4) aggregation, (5) binding of membrane-associated proteins, or (6) directed targeting of cytotoxic effector cells.

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

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

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

[0008] Summary of the Invention

[0009] In order to increase the in vivo effect of TcE, in addition to improving the affinity of TCR for pMHC, the titer of TcE against target cell targets can also be increased, and the titer of TcE against effector cell surface molecules can also be increased to enhance T cell killing activity and proliferation capacity. At the same time, in order to maintain the ability of TcE to enter tumors while maintaining a suitable half-life of TcE in vivo, it is necessary to maintain the molecular size of TcE within a certain range and appropriately add other functional domains to the polypeptide molecule, such as functional domains derived from the hinge region of an antibody, the Fc domain of an antibody or its dimerization portion and / or albumin (Alb) or its binding portion. The addition of these functional domains not only prolongs the half-life of the polypeptide molecule, but also maintains the killing activity of the polypeptide molecule. Accordingly, the present invention provides multispecific polypeptide molecules based on TCR, such as bispecific polypeptide molecules, trispecific polypeptide molecules or tetraspecific polypeptide molecules based on TCR.

[0010] the term

[0011] Unless otherwise specified or defined, all terms used herein have the common meanings in the art known to those skilled in the art. For example, the terms used herein are as defined in Janeway CA Jr, Travers P, Walport M et al., Immunobiology, Fifth Edition, New York: Garland Science (2001) and "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and Definitions as described in H., ed. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0012] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, the terms "a," "an," "one or more," and "at least one" can be used interchangeably. Similarly, the terms "comprising," "including," and "having" can be used interchangeably.

[0013] Unless otherwise stated or defined, the term "comprise" and variations thereof such as "comprises" and "comprising" should be understood to mean the inclusion of stated elements or steps but not the exclusion of any other elements or steps. For the purposes of the present invention, the term "consisting of" is considered a preferred embodiment of the term "comprising." If a group is defined hereinafter as comprising or containing at least a certain number of embodiments, it should also be understood to disclose a group that preferably consists only of these embodiments.

[0014] As used herein, the term "T cell receptor" or "TCR" includes native TCRs as well as TCR variants, fragments, and constructs. The term thus includes heterodimers, as well as multimers and single-chain constructs comprising a TCR alpha chain and a TCR beta chain; optionally comprising other domains and / or portions, so long as the TCR retains its ability to recognize an antigen target.

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

[0016] Antigen specificity is conferred by the variable regions of the α and β chains (TRAV and TRBV). Both variable regions of the TCR α and β chains contain three hypervariable or complementarity-determining regions (CDR1α / β, CDR2α / β, and CDR3α / β) surrounded by framework (FR) regions. CDR3 is the primary determinant of antigen recognition and specificity (i.e., the ability to recognize and interact with a specific antigen), while CDR1 and CDR2 primarily interact with the MHC molecules that present the antigenic peptide.

[0017] TCR recognizes antigenic peptides that are bound to major histocompatibility complex (MHC) molecules at the surface of antigen-presenting cells ("presented / displayed on MHC molecules"). Antigenic peptides presented on MHC molecules are also referred to herein as "complexes of epitopes and MHC molecules," "epitope-MHC complexes," "antigen-MHC complexes," or "target antigen peptide-MHC complexes." There are two different classes of MHC molecules: MHC I and MHC II, which present peptides from different cell compartments. MHC class I molecules are expressed on the surface of all nucleated cells in the human body and display peptides or protein fragments from intracellular compartments to cytotoxic T cells. In humans, MHC is also known as human leukocyte antigen (HLA). There are three main types of MHC class I: HLA-A, HLA-B, and HLA-C. Once the TCR binds to its specific epitope-MHC complex, the T cell is activated and exerts biological effector functions.

[0018] As used herein, the term "TCR constant region" includes the TCR alpha chain constant region (TRAC) and beta chain constant region (TRBC), which may be human constant regions or derived from another species, such as mouse.

[0019] It has been reported that the addition of disulfide bonds in the constant region can promote the correct pairing of TCRα and β chains (Kuball J et al. Blood. 2007 Mar 15; 109(6): 2331-8). Therefore, the present invention also contemplates the addition of one or more cysteine ​​modifications in the constant region to form a disulfide bond between the TCRα chain and the TCRβ chain.

[0020] The sequence of the wild-type TCR constant region can be found in the public database of the International Immunogenetics Information System (IMGT), such as the constant domain sequence of the TCR molecule α chain is "TRAC*01", and the constant domain sequence of the TCR molecule β chain is "TRBC1*01" or "TRBC2*01".

[0021] The positions of the amino acid sequences of wild-type TCRs in the present invention are numbered according to the nomenclature of the International Immunogenetics Information System (IMGT). For example, if an amino acid in the TCRα chain variable region (TRAV) is numbered as position 104 in IMGT, it will be described as the 104th amino acid of TRAV in the present invention; if an amino acid in the TCRβ chain variable region (TRBV) is numbered as position 84 in IMGT, it will be described as the 84th amino acid of TRBV in the present invention; and so on. For example, if an amino acid in the TCRα chain constant region (TRAC) is numbered as position 48 in IMGT, it will be described as the 48th amino acid of TRAC in this article; if an amino acid in the TCRβ chain constant region (TRBC) is numbered as position 57 in IMGT, it will be described as the 57th amino acid of TRBC in this article; and so on. In the present invention, if the sequence position numbering of other amino acids is specially specified, it shall be in accordance with the special instructions.

[0022] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a specific antigen. Such molecules typically comprise two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (or domain) (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (or domain) (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The variable regions of the heavy and light chains of an antibody contain a binding domain that interacts with an antigen. The constant region of an antibody can mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system, such as C1q (the first component in the classical pathway of complement activation).

[0023] The heavy chain of an immunoglobulin can be divided into three functional regions: the Fd region, the hinge region, and the Fc region (crystallizable fragment). The Fd region contains the VH and CH1 domains and combines with the light chain to form a 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 immunoglobulin effector functions, including, for example, complement fixation and binding to cognate 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 can promote the formation of Fc dimers is called the "dimerization portion of the Fc domain," which typically includes a portion of the CH2 domain and the CH3 domain.

[0024] The hinge region of IgG class antibodies refers to the short amino acid sequence region between the heavy chain CH1 and CH2 parts, which is relatively flexible in the native state of the antibody. The hinge region found in IgG, IgA and IgD immunoglobulin classes acts as a flexible spacer, allowing the Fab part to move freely in space relative to the Fc region. The hinge domain is structurally diverse, with different sequences and lengths between immunoglobulin classes and subclasses. According to crystallographic studies, the immunoglobulin hinge region can be further divided into three regions in structure and function: the upper hinge, the core hinge and the lower hinge (Shin et al., Immunological Reviews 130:87, 1992). The upper hinge includes the amino acid residues from the carboxyl terminus of CH1 to the first residue in the hinge that restricts movement, typically the first cysteine ​​residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region is related to the flexibility of the antibody fragment. The core hinge region contains an inter-heavy chain disulfide bond. The lower hinge region connects the amino terminus of the CH2 domain and includes residues in the CH2 domain. The core hinge region of human IgG1 contains the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 1), which, when dimerized through disulfide bonds, generates a cyclic octapeptide. This cyclic octapeptide is believed to act as a pivot, conferring flexibility. The conformational changes allowed by the structure and flexibility of the immunoglobulin hinge region polypeptide sequence can influence the effector functions of the antibody Fc portion.

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

[0026] Single-domain antibodies (sdAb), also known as nanobodies, are composed of a heavy chain variable domain. The heavy chain variable domain was originally discovered in a heavy chain antibody (HCAb) isolated from the serum of camelids and sharks. The heavy chain variable domain was amplified by genetic means. This variable domain present in naturally occurring heavy chain antibodies is called "VHH" to distinguish it from the heavy chain variable domain (VH) present in conventional 4-chain antibodies and the light chain variable domain (VL) present in conventional 4-chain antibodies. The VHH cloned and expressed separately has a structural stability and equivalent antigen binding activity to the original heavy chain antibody. VHH is the smallest unit currently known to bind to the target antigen.

[0027] VHH domains have many unique structural features and functional properties that make isolated VHH domains (and nanobodies based on VHH domains that share these structural features and functional properties with naturally occurring VHH domains) and proteins containing isolated VHH domains very advantageous for use as functional antigen-binding domains or proteins. In particular, but not limited to, VHH domains (which have essentially been "engineered" to functionally bind to antigens in the absence of, and without any interaction with, light chain variable domains) and nanobodies can act as single, relatively small, functional antigen-binding structural units, domains or proteins. This distinguishes VHH domains from the VH and VL domains of conventional four-chain antibodies, which are generally not suitable for practical application as single antigen-binding proteins or domains by themselves, 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 a VH domain covalently linked to a VL domain).

[0028] The amino acid assignments for each VL, VH, and VHH domain follow any conventional definition of CDRs. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)); the Chothia definition (Chothia & Lesk, J. Mol. Biol. 196: 901-917, 1987; Chothia et al., Nature 342: 878-883, 1989); a composite of the Chothia Kabat CDRs, in which CDR-H1 is a composite of the Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular's antibody modeling software; and the CONTACT definition of Martin et al. (World Wide Web bioinfo.org.uk / abs). Kabat provides a widely used numbering convention (the Kabat numbering system), in which corresponding residues between different heavy chains or between different light chains are given the same number. The present disclosure may use CDRs defined according to any of these numbering systems, but preferred embodiments use the Kabat defined CDRs.

[0029] The term "antibody" as used herein should be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) comprising at least two antigen-binding regions. Antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to the antigen recognition site, as long as these antibodies exhibit the desired biological activity.

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

[0031] Examples of antigen-binding fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region; (iii) a Fab' fragment, which is essentially a Fab but has a portion of the hinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul ed., 3.sup.rd ed. 1993); (iv) a Fd fragment consisting of the VH and CH1 domains; (v) a Fd' fragment having the VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (vi) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (vii) a dAb fragment (Ward et al. (1989) Nature 557(5)). 341: 544-546), which consists of a VH domain; (viii) a separate complementarity determining region (CDR). In addition, although the two domains of the Fv fragment, VL and VH, are encoded by independent genes, they can be linked using recombinant methods by a synthetic linker that enables them to form a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called single-chain Fv (ScFv); see, 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 encompassed within the term "antigen-binding fragment" of an antibody. In addition, the term also includes "linear antibodies" containing a pair of tandem Fd fragments (VH-CH1-VH-CH1), which together form an antigen-binding region with a complementary light chain polypeptide and modified versions of any of the aforementioned fragments that retain antigen-binding activity.

[0032] As used herein, the term "antigen" refers to any substance capable of inducing an immune response in the body. Specifically, it refers to a substance that can be specifically recognized and bound by antigen receptors (TCR / BCR) on the surface of T / B lymphocytes, activating T / B cells, causing them to proliferate and differentiate, and produce immune response products (sensitized lymphocytes or antibodies), and that can specifically bind to corresponding products in vivo or in vitro.

[0033] Herein, the antigen may be a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, a self-antigen, and an immune cell surface molecule.

[0034] As used herein, the term "tumor-associated antigen (TAA)" refers to an antigen that is differentially expressed in tumor cells (eg, has significantly increased expression on tumor cells) compared to normal cells.

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

[0036] As used herein, the term "viral antigen" refers to a substance in a virus that can induce an immune response in the body.

[0037] As used herein, the term "autoantigen" refers to an antigenic substance that causes changes in the structure and composition of one's own tissue cells caused by biological, physical, and chemical factors, thereby causing the body's immune system to produce an immune response to these self-tissue cell components. The release of hidden antigens can produce an immune response. Changes in one's own components may produce autoantigens. For example, denatured IgG can stimulate the body to produce anti-denatured IgG antibodies (rheumatoid factor), causing rheumatoid arthritis. After clinical use of certain drugs, the surface antigenicity of blood cells can be changed, causing autoimmune hemolytic anemia or granulocytopenia. The cross-reaction caused by common antigens can produce an immune response. Certain bacteria, viruses and certain tissue cells of the normal human body have similar antigenic determinants. The autoantibodies and sensitized lymphocytes produced for these bacteria and virus antigenic determinants can cross-react with one's own tissue cells, causing autoimmune diseases.

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

[0039] The site on the antigen that is bound by TCR or antibody is called "epitope". The epitope can be formed by continuous amino acids or non-continuous amino acids juxtaposed by the tertiary folding of one or more proteins. The epitope formed by continuous amino acids (also referred to as linear epitope) is usually retained after exposure to denaturing solvents, while the epitope formed by tertiary folding (also referred to as conformational epitope) is usually lost in the treatment of denaturing solvents. The epitope usually comprises at least 3, more generally at least 5 or 8-10 amino acids in a unique spatial conformation. The epitope defines the minimum binding site of TCR or antibody and is therefore the specific target of TCR or antibody or its antigen-binding fragment.

[0040] As used herein, the term "albumin" encompasses any natural albumin of any vertebrate origin, including mammals such as primates (e.g., humans and monkeys) and rodents (e.g., mice and rats). Albumin also encompasses full-length, unprocessed albumin preproproteins and any form of albumin produced by processing in cells. Albumin also encompasses naturally occurring variants of albumin, such as splice variants or allelic variants. The term also encompasses any recombinant form of albumin. Albumin sequences are known in the art. For example, information about the human serum albumin gene (including genomic DNA sequences) can be found at NCBI. The amino acid sequence of an exemplary full-length human serum albumin preproprotein can be found under NCBI accession number NP_000468.1. Human serum albumin is synthesized in the form of prealbumin. Mature albumin is a single-chain polypeptide of 585 amino acid residues with a molecular weight of 66,458, 17 disulfide bonds in the molecule, and no sugar components. In the environment of body fluid pH 7.4, albumin is a negative ion, and each molecule can carry more than 200 negative charges. After 2 hours, about 10% of exogenously infused albumin enters the extravascular tissue space, and equilibrium is reached after 20 days. The half-life of albumin in plasma is 15-19 days.

[0041] >gi|4502027|ref|NP_000468.1|Serum albumin preproprotein [Homo sapiens]

[0042] (Signal peptide is underlined, propeptide is bold, and mature human serum albumin (SEQ ID NO: 2) is italicized).

[0043] As used herein, the term "albumin-binding moiety" refers to a fragment that is non-covalently linked, or covalently linked to albumin by conjugation or direct genetic fusion, and that retains the minimum functional unit for binding to albumin. In some embodiments, the albumin-binding moiety includes any known moiety capable of binding to albumin, for example, an endogenous or exogenous ligand that specifically binds to albumin, a small organic molecule, a fatty acid, a protein, a short peptide (e.g., QRLMEDICLPRWGCLWEDDF (SEQ ID NO: 3)), an antibody, an antibody fragment, an antibody-like scaffold, a Streptococcal protein G protein, and the like.

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

[0045] Computer programs are typically used to perform such calculations. Exemplary programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

[0046] In addition, when determining the degree of sequence identity between two amino acid sequences, the skilled person may consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of a similar chemical structure, which has little or substantially no effect on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example WO 04 / 037999, GB-A-2 357 768, WO 98 / 49185, WO 00 / 46383 and WO 01 / 09300; and (preferably) the type and / or combination of these substitutions can be selected according to the relevant teachings from WO 04 / 037999 and WO 98 / 49185 and the further references cited therein.

[0047] Such conservative substitutions are preferably substitutions in which an amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0048] 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 segment" refers to a polypeptide segment containing cysteine ​​residues that is capable of forming a disulfide bond between two polypeptide chains comprising the peptide segment, and has a length of 3-10 amino acids.

[0050] The term "vector" as used herein is a nucleic acid molecule used as a vehicle for transferring (exogenous) genetic material into a host cell where it can, for example, be replicated and / or expressed.

[0051] As used herein, the term "host cell" refers to any type of cell into which an expression vector has been introduced so as to be capable of expressing foreign genetic material.

[0052] The term "pharmaceutically acceptable" means that the carrier or adjuvant is compatible with the other ingredients of the composition and not substantially deleterious to the recipient thereof, and / or that the carrier or adjuvant is approved or acceptable for inclusion in pharmaceutical compositions for parenteral administration to humans.

[0053] As used herein, the terms "treat", "treat", and the like refer to the administration of an agent or the performance of a procedure in order to obtain an effect. These effects may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of affecting a partial or complete cure of a disease and / or disease symptoms. As used herein, "treatment" may include the treatment of a disease or condition (e.g., cancer, infectious diseases, autoimmune diseases, or inflammatory diseases) in mammals, particularly humans, and includes: (a) preventing the occurrence of the disease or disease symptoms in a subject susceptible to the disease but not yet diagnosed as having the disease (e.g., including diseases that may be associated with or caused by the primary disease); (b) inhibiting the disease, i.e., preventing its development; (c) alleviating the disease, i.e., causing regression of the disease. Treatment may refer to any reference to success in treating, ameliorating, or preventing cancer, including any objective or subjective parameter, such as elimination; alleviation; reducing symptoms or making the disease condition more tolerable for the patient; slowing the rate of deterioration or decline; or reducing the end point of deterioration. Treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a doctor's examination. Thus, the term "treatment" includes administering a multispecific polypeptide molecule, pharmaceutical composition, or conjugate disclosed herein to prevent, delay, alleviate, arrest, or inhibit the development of symptoms or conditions associated with a disease (e.g., cancer). The term "therapeutic effect" refers to the reduction, elimination, or prevention of a disease, disease symptom, or disease side effect in a subject.

[0054] As used herein, the term "effective amount" means an amount of a therapeutic agent that, when administered to a subject for the treatment or prevention of a disease, is sufficient to achieve such treatment or prevention. The "effective amount" may vary depending on the compound, the disease and its severity, and the age, weight, etc. of the subject to be treated. A "therapeutically effective amount" refers to an effective amount for therapeutic treatment. A "prophylactically effective amount" refers to an effective amount for preventive treatment.

[0055] As used herein, the term "subject" refers to any mammalian subject for whom diagnosis, treatment, or therapy is desired. "Mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, livestock, and laboratory, zoo, sport, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, and the like.

[0056] Preferred embodiments of the present invention

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

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

[0059] wherein the first binding region comprises an α chain variable region (TRAV1) and a β chain variable region (TRBV1) derived from a T cell receptor (TCR) that binds to the first antigen-MHC complex;

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

[0061] wherein the first antigen is selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, and an autoantigen, and the second antigen is an immune cell surface molecule;

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

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

[0064] 1) Hinge region derived from an antibody;

[0065] 2) Fc domain or its dimerization portion derived from an antibody;

[0066] 3) CH3 domain derived from an antibody;

[0067] 4) albumin (Alb) or its binding portion;

[0068] 5) TCR-derived constant region or fragment thereof.

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

[0070] 1) Hinge region derived from an antibody;

[0071] 2) Fc domain or its dimerization portion derived from an antibody;

[0072] 3) CH3 domain derived from an antibody;

[0073] 4) albumin (Alb) or its binding portion;

[0074] 5) TCR-derived constant region or fragment thereof.

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

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

[0077] In some embodiments, the multispecific polypeptide molecules provided herein do not comprise the C-terminus of the second binding region amino acid chain connected to the N-terminus of the first binding region amino acid chain.

[0078] In some embodiments, the multispecific polypeptide molecules provided by the present invention do not comprise a second binding region connected to the N-terminus of one or more polypeptide chains of the first binding region via the C-terminus of any one polypeptide chain in a single chain (VH1 and VL1 are distributed on one polypeptide chain, or the antibody comprises VHH1) or a double chain (VH1 and VL1 are distributed on two polypeptide chains).

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

[0080] VL1 and VH1 are distributed on one polypeptide chain to form a second binding region single chain, or the second binding region single chain comprises VHH1, TRAV1 and TRBV1 are distributed on one polypeptide chain to form a first binding region single chain, and the C-terminus of the second binding region single chain is connected 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 comprises VHH1, TRAV and TRBV1 are distributed on two polypeptide chains to form an α chain and a β chain, respectively, and the C-terminus of the second binding region single chain is connected to the N-terminus of the α chain or the β chain;

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

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

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

[0085] From N-terminus to C-terminus: 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 embodiments, VL1 and VH1 are distributed on a single polypeptide chain, the C-terminus of which is linked to the N-terminus of the TCR.

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

[0088] In some embodiments, the TRAV1 and TRBV1 are distributed on two different polypeptide chains (ie, the TCR is a heterodimeric αβ TCR).

[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, the TRAV1 and TRBV1 are distributed on two different polypeptide chains, the VHH1 or the VH1 and VL1 are distributed on one polypeptide chain (for example, forming scFv1), and their N-termini are connected to the C-terminus of the polypeptide chain where the TRAV1 or TRBV1 is located, or the N-terminus of one of the polypeptide chains where the VH1 and VL1 are located is connected to the C-terminus of the polypeptide chain where the TRAV1 is located, and the N-terminus of the other polypeptide chain where the VH1 and VL1 are located is connected to the C-terminus of the polypeptide chain where the TRAV1 is located.

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

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

[0094] In a second aspect, the present invention provides a multispecific polypeptide molecule comprising a first binding region that binds a first antigen and a second binding region that binds a second antigen on two polypeptide chains,

[0095] wherein the first binding region comprises an α chain variable region (TRAV1) and a β chain variable region (TRBV1) derived from a T cell receptor (TCR) that binds to the first antigen-MHC complex;

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

[0097] wherein the first antigen is selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, and an autoantigen, and the second antigen is an immune cell surface molecule;

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

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

[0100] 1) Hinge region derived from an antibody;

[0101] 2) Fc domain or its dimerization portion derived from an antibody;

[0102] 3) CH3 domain derived from an antibody;

[0103] 4) albumin (Alb) or its binding portion;

[0104] 5) TCR-derived constant region or fragment thereof.

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

[0106] 1) Hinge region derived from an antibody;

[0107] 2) Fc domain or its dimerization portion derived from an antibody;

[0108] 3) CH3 domain derived from an antibody;

[0109] 4) albumin (Alb) or its binding portion;

[0110] 5) TCR-derived constant region or fragment thereof.

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

[0112] In some embodiments, the TRAV1 and TRBV1 are located on two different polypeptide chains (ie, the TCR is a heterodimeric αβ TCR).

[0113] In some embodiments, VH1 and VL1 are linked into one polypeptide chain, e.g., to form scFv1 or Fab1, and attached 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 connected to one polypeptide chain, and their N-termini are connected to the C-terminus of the polypeptide chain where TRAV1 is located, or the C-terminus of the polypeptide chain where TRBV1 is located.

[0115] In some embodiments, the VH1 and VL1 are connected by a linker.

[0116] In an embodiment of the multispecific polypeptide molecule of the second aspect of the present invention, the VH1 and VL1 form scFv1, and the N-terminus of scFv1 is linked 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 comprise: TRBV1-scFv1 and TRAV1; or TRAV1-scFv1 and TRBV1, respectively.

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

[0118] In some embodiments, the second binding region comprises a single heavy chain variable region (VHH1) derived from a single-domain antibody or nanobody that binds to the second antigen, wherein the VHH1 is attached 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 attached 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: TRBV1-VHH1 and TRAV1; or TRAV1-VHH1 and TRBV1, respectively.

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

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

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

[0122] In some embodiments, the multispecific polypeptide molecule further comprises a constant domain selected from the group consisting of:

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

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

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

[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 that binds 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 a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, and an autoantigen, and the third antigen is the same as or different from the first antigen. In some embodiments, TRAV2 is the same as TRAV1. In some embodiments, TRBV2 is the same as TRBV1.

[0127] In some embodiments, the TRAV2 and the TRBV2 are connected to the same polypeptide chain via a linker.

[0128] In some embodiments, the VH1 and VL1 are distributed on one polypeptide chain and connected to the C-terminus of the TRAV1 or the TRBV1, and the TRAV2 and the TRBV2 are connected on the same polypeptide chain via a linker and connected 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 group consisting of:

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

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

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

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

[0143] In some embodiments, the VHH2 or the scFv2 formed by the VH2 and VL2 is linked to the N-terminus of the TRAV1 or the TRBV1, or one of the VH2 and VL2 is linked to the N-terminus of the TRAV1 and the other is linked to the N-terminus of the TRBV1.

[0144] In some embodiments, the multispecific polypeptide molecule comprises the first binding region, the second binding region, and the fourth binding region, and the VH2 and VL2 form a scFv2 that is linked to the N-terminus of the TRAV1 or TRBV1. In some embodiments, the VH2 and VL2 are linked via a linker to form a 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 comprises the first binding region, the second binding region, and the fourth binding region, and the VHH2 is linked to the N-terminus of the TRAV1 or TRBV1. In some embodiments, the two polypeptide chains of the multispecific polypeptide molecule respectively 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 comprises the first binding region, the second binding region, and the fourth binding region, and one of the VH2 and VL2 is linked to the N-terminus of the TRAV1 and the other is linked to the N-terminus of the TRBV1. In some embodiments, the two polypeptide chains of the multispecific polypeptide molecule respectively comprise:

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

[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 comprises the first binding region, the second binding region, the third binding region, and the fourth binding region, and the VHH2 is linked to the N-terminus of the TRAV1 or TRBV1. In some embodiments, the two polypeptide chains of the multispecific polypeptide molecule respectively comprise:

[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 comprises the first binding region, the second binding region, the third binding region, and the fourth binding region, and one of the VH2 and VL2 is linked to the N-terminus of the TRAV1 and the other is linked to the N-terminus of the TRBV1. In some embodiments, the two polypeptide chains of the multispecific polypeptide molecule respectively comprise:

[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 a binding portion thereof and optionally the hinge region of an antibody; and the functional domain is connected to the C-terminus or N-terminus, preferably the C-terminus, of any polypeptide chain of the multispecific polypeptide molecule via an optional linker.

[0236] In some embodiments, the functional domain is derived from albumin or a binding portion thereof, and the albumin or the binding portion thereof is connected 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 an antibody, the functional domain is connected to the C-terminus of any polypeptide chain of the multispecific polypeptide molecule via an optional linker, and albumin or its binding portion is connected to the C-terminus of any polypeptide chain of the hinge region of the antibody via an optional linker.

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

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

[0240] In some embodiments, the functional domain comprises two chains, which are respectively connected 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, preferably the C-terminus, of the TCR through an optional linker, and the other chain is connected to the C-terminus or N-terminus, preferably the C-terminus, of the antibody.

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

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

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

[0245] In some embodiments, TRAV1 and TRBV1 are located on two different polypeptide chains, and one of the VH1 and VL1 is connected to the C-terminus of the chain where TRAV1 is located, and the other is connected to the C-terminus of the chain where TRBV1 is located, wherein 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, preferably the C-terminus.

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

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

[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 or its dimerization portion and / or the CH3 domain of an antibody; and the functional domain is connected to the C-terminus or N-terminus, preferably the C-terminus, of the two polypeptide chains of the multispecific polypeptide molecule via an optional linker. In some embodiments, the functional domain is derived from the hinge region of an antibody and / or the Fc domain or its dimerization portion and / or the CH3 domain of an antibody; and the two chains of the functional domain are connected to the C-terminus or N-terminus, preferably the C-terminus, of the two polypeptide chains of the multispecific polypeptide molecule via an optional linker.

[0249] 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 dimerization portion and / or the CH3 domain of an antibody, and the two chains of the multispecific polypeptide molecule are connected by covalent or non-covalent binding between the two chains of the functional domain.

[0250] In some embodiments, the functional domain is derived from the Fc domain of an antibody or its dimerization portion and optionally the hinge region of an antibody. In some embodiments, the functional domain is derived from the hinge region of an antibody and the Fc domain of an antibody or its dimerization portion. In some embodiments, the functional domain is derived from the Fc domain of an antibody. In some embodiments, the functional domain is the Fc domain of an 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 an optional linker, preferably the C-terminus.

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

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

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

[0254] In one embodiment, the constant domain is a TCR constant region or a fragment thereof, the TCR constant region, for example, comprising the TCR alpha chain constant region (TRAC) and / or beta chain constant region (TRBC).

[0255] In one embodiment, the constant domain is CH3, which is derived from the Fc domain of an antibody. Preferably, the CH3 in any one polypeptide chain or both polypeptide chains contains at least one mutation that can promote the polypeptide molecule to form a heterodimer.

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

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

[0258] In some embodiments, the multispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, and comprises the first binding region and the second binding region, and a functional domain derived from albumin or a binding portion thereof and optionally a hinge region of an antibody, respectively, connected 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 comprises the first binding region and the second binding region, and a functional domain derived from an antibody Fc domain or a dimerization portion thereof, and optionally an antibody hinge region, connected to the C-termini of the first polypeptide chain and the second polypeptide chain, respectively; 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 comprises the first binding region, the second binding region, and the third binding region, and a functional domain derived from albumin or a binding portion thereof and optionally an antibody hinge region connected to the C-terminus of any one of the polypeptide chains, and the VH1 and VL1 are connected to the C-terminus of the TRAV1 or TRBV1 on one polypeptide chain, and the TRAV2 and TRBV2 are connected to the same polypeptide chain via a linker and connected between VH1 and VL1; wherein:

[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 the first binding region, the second binding region, and the third binding region, and a functional domain derived from an antibody Fc domain or a dimerization portion thereof, and optionally an antibody hinge region, 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 comprises the first, second, and fourth binding regions and a functional domain derived from albumin or a binding portion thereof and optionally a hinge region of an antibody, and the multispecific polypeptide molecule comprises 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-linker-TRAV1-optional linker-TRAC-linker-VL1-optional linker-optional hinge region, 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-Alb.

[0389] In some embodiments, the multispecific polypeptide molecule comprises the first, second, and fourth binding regions and a functional domain derived from an antibody Fc domain or a dimerization portion thereof and optionally an antibody hinge region, 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 comprises the first, second, third, and fourth binding regions, and a functional domain derived from albumin or a binding portion thereof and optionally a hinge region of an antibody, and the multispecific polypeptide molecule comprises 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 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, and the second polypeptide chain comprises, from N-terminus to C-terminus: 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 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, and the second polypeptide chain comprises, from N-terminus to C-terminus: 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 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, and the second polypeptide chain comprises, from N-terminus to C-terminus: 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 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, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0435] The first polypeptide chain comprises, from N-terminus to C-terminus: 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 comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0436] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-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

[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 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, and the second polypeptide chain comprises, from N-terminus to C-terminus: 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 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, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0441] The first polypeptide chain comprises, from N-terminus to C-terminus: 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 comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region; or

[0442] The first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-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

[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 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: 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 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: 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 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: 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 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: 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 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, and the second polypeptide chain comprises, from N-terminus to C-terminus: 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 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, and the second polypeptide chain comprises, from N-terminus to C-terminus: 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 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, and the second polypeptide chain comprises, from N-terminus to C-terminus: 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 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: 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-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: VH2-linker-TRAV1-optional linker-TRAC-optional linker-optional hinge region; or

[0492] 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, 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

[0493] 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-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

[0494] 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, 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

[0495] 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-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

[0496] 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, 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

[0497] 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-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

[0498] 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, 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

[0499] 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-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

[0500] 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, 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

[0501] 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-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

[0502] 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, 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

[0503] 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-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

[0504] 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, 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

[0505] 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-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-linker-optional linker-TRAC-optional linker-optional hinge region; or

[0506] 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, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-linker-optional linker-TRAC-optional linker-optional hinge region-Alb; or

[0507] 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-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

[0508] 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, 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

[0509] 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-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

[0510] 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, 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

[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-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: VL2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb; or

[0513] 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-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

[0514] 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, 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

[0515] 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-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

[0516] 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, 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

[0517] 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-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

[0518] 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, 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

[0519] 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-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

[0520] 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, 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

[0521] 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-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

[0522] 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, and the second polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-optional linker-optional hinge region-Alb.

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

[0524] 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-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

[0525] 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-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

[0526] 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-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

[0527] 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-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

[0528] The first polypeptide chain comprises, from N-terminus to C-terminus: 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 comprises, from N-terminus to C-terminus: 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 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-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

[0531] The first polypeptide chain comprises, from N-terminus to C-terminus: 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 comprises, from N-terminus to C-terminus: 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 embodiments, the scFv1 consists of the following from N-terminus to C-terminus: VH1-linker VL1 or VL1-linker-VH1.

[0573] In some embodiments, the scFv2 consists of the following from N-terminus to C-terminus: VH2-linker VL2 or VL2-linker-VH2.

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

[0575] In some embodiments, the multispecific polypeptide molecule comprises CL and CH1 connected to the C-termini of TRAV1 and TRBV1 via an optional linker. 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, which are connected to the C-termini of TRAV1 and TRBV1, respectively, in the first and second polypeptide chains of the multispecific polypeptide molecule, are replaced with CL and CH1, or with CH1 and CL, respectively.

[0576] In some embodiments, the multispecific polypeptide molecule does not comprise a constant region, e.g., a TCR constant region or fragment thereof, CH3, or CL and CH1, connected to the C-terminus of TRAV1 and / or TRBV1 via an optional linker. In this case, TRAC and TRBC, which are connected to the C-terminus of TRAV1 and TRBV1, respectively, are removed from the first and second polypeptide chains of the multispecific polypeptide molecule. 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 a CH1 and a CL derived from an antibody, connected to the VH1 and VL1, respectively, wherein the VH1, VL1, CH1, and CL form a single-chain Fab and are connected to the C-terminus of the TRAV1 or 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 that binds to a fourth antigen, wherein the fourth binding region comprises a heavy chain variable region VH2 and a light chain variable region VL2 derived from an antibody that binds to the fourth antigen, and CH1 and CL derived from the antibody.

[0583] In some embodiments, the VH2, VL2, CH1 and CL form a single-chain Fab2 connected to the N-terminus of the TRAV1 or TRBV1. In some embodiments, the two polypeptide chains of the multispecific polypeptide molecule respectively 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 comprises the first binding region, the second binding region, the third binding region and the fourth binding region, the fourth binding region comprises the heavy chain variable region VH2 and the light chain variable region VL2 derived from the antibody that binds to the fourth antigen, and CH1 and CL derived from the antibody, wherein the VH2, VL2, CH1 and CL form a single-chain Fab2 connected 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 embodiments, the Fab2 is selected from the group consisting of VL2-CL-VH2-CH1, VH2-CH1-VL2-CL, VL2-CL-VH2-CH1 and VH2-CH1-VL2-CL from N-terminus to C-terminus.

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

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

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

[0624] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-linker-Alb, and the second polypeptide chain comprises from N-terminus to C-terminus: 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 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 (format 58-2); or

[0631] 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 (format 72); or

[0632] 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 (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 comprising a first binding region that binds to a first antigen and a second binding region that binds to a second antigen on three polypeptide chains.

[0636] wherein the first binding region comprises an α chain variable region (TRAV1) and a β chain variable region (TRBV1) derived from a T cell receptor (TCR) that binds to the first antigen-MHC complex;

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

[0638] wherein the first antigen is selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, and an autoantigen, and the second antigen is an immune cell surface molecule;

[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 extending the half-life of the multispecific polypeptide molecule. In some embodiments, the functional domain that extends the half-life is selected from any one of the following functional domains:

[0641] 1) Hinge region derived from an antibody;

[0642] 2) Fc domain or its dimerization portion derived from an antibody;

[0643] 3) CH3 domain derived from an antibody;

[0644] 4) albumin (Alb) or its binding portion;

[0645] 5) TCR-derived constant region or fragment thereof.

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

[0647] 1) Hinge region derived from an antibody;

[0648] 2) Fc domain or its dimerization portion derived from an antibody;

[0649] 3) CH3 domain derived from an antibody;

[0650] 4) albumin (Alb) or its binding portion;

[0651] 5) TCR-derived constant region or fragment thereof.

[0652] In some embodiments, the second binding region is not directly linked to the first binding region. In some embodiments, the second binding region is not connected to the first binding region via a linker. The phrase "not directly linked" 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 in any manner on a single polypeptide chain.

[0653] In some embodiments, the VH1 and VL1 are linked in series on one polypeptide chain (e.g., forming scFv1 or Fab1), and the TRAV1 and TRBV1 are located on different polypeptide chains. In some embodiments, the VH1 and VL1 are linked in series on one polypeptide chain (e.g., forming scFv1 or Fab1), and the TRAV1 and TRBV1 are located on two separate polypeptide chains. In some embodiments, the VH1 and VL1 are linked in series on one 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 consisting of:

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

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

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

[0662] In some embodiments, the multispecific polypeptide molecule comprises a TCR constant region or fragment thereof connected to the C-terminus of TRAV1 and TRBV1 via an optional linker. In some embodiments, the multispecific polypeptide molecule comprises TRAC and TRBC connected to the C-terminus of TRAV1 and TRBV1, respectively, via an optional linker. In some embodiments, the three polypeptide chains of the multispecific polypeptide molecule respectively 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 present invention, the multispecific polypeptide molecule further comprises antibody-derived CH1 and CL, linked to the VH1 and VL1, respectively, wherein the VH1, VL1, CH1, and CL form a single-chain Fab1 and are linked to the N-terminus of the functional domain via an optional linker. In some embodiments, the three polypeptide chains of the 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 that binds 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 a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, and an autoantigen, and the third antigen is the same as or different from the first antigen. In some embodiments, TRAV2 is the same as TRAV1, and / or TRBV2 is the same as TRBV1.

[0669] In some embodiments, the TRAV2 and the TRBV2 are connected to the same polypeptide chain via a linker.

[0670] In some embodiments, the TRAV2 and the TRBV2 are connected on the same polypeptide chain via a linker and are connected 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 consisting of:

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

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

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

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

[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 invention, the multispecific polypeptide molecule further comprises a fourth binding region that binds 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 that binds to the fourth antigen, or a single heavy chain variable region (VHH2) derived from a single domain antibody or nanobody that binds to the fourth antigen; wherein the fourth antigen is an immune cell surface molecule, and the fourth antigen is the same as or different from the second antigen. In some embodiments, the VH2 is the same as the VH1. In some embodiments, the VL2 is the same as the VL1. In some embodiments, the VHH2 is the same as the VHH1.

[0681] In some embodiments, the VHH2 or the VH2 and VL2 form an scFv2 and are connected to the N-terminus or C-terminus of the TRAV1 or TRBV1, or one of the VH2 and VL2 is connected to the N-terminus or C-terminus of the TRAV1 and the other is connected to the N-terminus or C-terminus of the TRBV1. In some embodiments, the VHH2 or the VH2 and VL2 form an scFv2 and are connected to the N-terminus of the TRAV1 or 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. In some embodiments, the 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 present invention, the multispecific polypeptide molecule further comprises antibody-derived CH1 and CL linked to the VH2 and VL2, respectively. In some embodiments, the VH2, VL2, CH1, and CL form a Fab2 and are linked to the N-terminus of TRBV1 or TRAV1 via an optional linker. In some embodiments, the 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 comprises a first binding region, a second binding region, a third binding region, and a fourth binding region, and the three polypeptide chains of the polypeptide molecule respectively comprise:

[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, which are respectively 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 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.

[0709] In some embodiments, the functional domain contains two chains, which are respectively 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, and 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.

[0710] In some embodiments, the first binding region and the second binding region of the multi-specific 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, preferably the C-terminus, of the peptide chain where the antibody is located in the second binding region, and the other chain is connected to the C-terminus or N-terminus, preferably the C-terminus, of the peptide chain where TRAV1 or TRBV1 is located in the first binding region.

[0711] In some embodiments of the multispecific polypeptide molecule of the third aspect of the invention, the functional domain is derived from the hinge region of an antibody and / or the Fc domain of an antibody or its dimerization portion; and 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 invention, the functional domain is derived from the CH3 domain 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 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.

[0714] In some embodiments, the constant region of the TCR or a fragment thereof is selected from the group consisting of a TCR alpha chain constant region (TRAC) and a TCR beta 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 connected in series to form an scFv, forming a third polypeptide chain; and the functional domain is derived from the hinge region and the Fc domain of an antibody or its dimerization portion; wherein one chain of the functional domain is connected to the C-terminus of the scFv via an optional linker, and the other chain is connected 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 comprise a variable region, for example, the α chain comprises TRAV1 and the β chain comprises TRBV1. In some embodiments, the α chain and β chain each independently comprise a variable region and a constant region, for example, the α chain comprises TRAV1 and TRAC, and the β chain comprises TRBV1 and TRBC.

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

[0717] 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: 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-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 comprising a first binding region that binds a first antigen and a second binding region that binds a second antigen on three polypeptide chains,

[0724] wherein the first binding region comprises an α chain variable region (TRAV1) and a β chain variable region (TRBV1) derived from a T cell receptor (TCR) that binds to the first antigen-MHC complex;

[0725] wherein the second binding region comprises a heavy chain variable region VH1 and a light chain variable region VL1 derived from an antibody that binds to the second antigen;

[0726] wherein the first antigen is selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, and an autoantigen, and the second antigen is an immune cell surface molecule;

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

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

[0729] 1) Hinge region derived from an antibody;

[0730] 2) Fc domain or its dimerization portion derived from an antibody;

[0731] 3) CH3 domain derived from an antibody;

[0732] 4) albumin (Alb) or its binding portion;

[0733] 5) TCR-derived constant region or fragment thereof.

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

[0735] 1) Hinge region derived from an antibody;

[0736] 2) Fc domain or its dimerization portion derived from an antibody;

[0737] 3) CH3 domain derived from an antibody;

[0738] 4) albumin (Alb) or its binding portion;

[0739] 5) TCR-derived constant region or fragment thereof.

[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 linker. 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 linker.

[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 consisting of:

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

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

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

[0751] In some embodiments, the multispecific polypeptide molecule comprises a TCR constant region or fragment thereof connected to the C-terminus of TRAV1 and TRBV1 via an optional linker. In some embodiments, the multispecific polypeptide molecule comprises TRAC and TRBC connected to the C-terminus of TRAV1 and TRBV1, respectively, via an optional linker. In some embodiments, the three polypeptide chains of the multispecific polypeptide molecule respectively 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 a CH1 and a CL derived from an antibody, connected to the VH1 and VL1, respectively. 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 invention, the multispecific polypeptide molecule further comprises a third binding region that binds 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 a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, and an autoantigen, and the third antigen is the same as or different from the first antigen. In 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-terminus of VH1 or VL1 via an optional linker. In some embodiments, the TRAV2 and TRBV2 are tandemly linked on a single chain and connected to the C-terminus of VH1 or VL1 via an optional linker. In some embodiments, one of the TRAV2 and TRBV2 is connected to the C-terminus of VH1 and the other is connected to the C-terminus of VL1.

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

[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:

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

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

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

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

[0793] In some embodiments, the VHH or VH2 and VL2 form an scFv that is linked to the N-terminus or C-terminus of TRAV1 or TRBV1, or one of the VH2 and VL2 is linked to the N-terminus or C-terminus of TRAV1 and the other is linked to the N-terminus or C-terminus of TRBV1. In some embodiments, the VHH or VH2 and VL2 form an scFv that is linked to the N-terminus of TRAV1 or TRBV1, or one of the VH2 and VL2 is linked to the N-terminus of TRAV1 and the other is linked to the N-terminus of 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 a CH1 and a CL derived from an antibody, connected to the VH2 and VL2, respectively, wherein the VH2, VL2, CH1, and CL form a single-chain Fab and are connected to the N-terminus of the functional domain via an optional linker. 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 invention comprises a first binding region, a second binding region, a third binding region, and a fourth binding region, and the three polypeptide chains of the polypeptide molecule respectively comprise:

[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 a binding portion thereof and an optional hinge region, and is connected to the C-terminus or N-terminus, preferably the C-terminus, of any one polypeptide chain via an optional linker.

[0918] In some embodiments of the multispecific polypeptide molecule of the fourth aspect of the invention, the functional domain is derived from the hinge region of an antibody and / or the Fc domain of an antibody or its dimerization portion; and the functional domain is connected to the C-terminus or N-terminus of any two polypeptide chains of the multispecific polypeptide molecule through an optional linker, preferably the C-terminus.

[0919] In some embodiments of the multispecific polypeptide molecule of the fourth aspect of the invention, the functional domain is derived from the CH3 domain 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 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 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.

[0921] In some embodiments, the constant region of the TCR or a fragment thereof is selected from the group consisting of a TCR alpha chain constant region (TRAC) and a TCR beta chain constant region (TRBC).

[0922] In some embodiments, the functional domain comprises two peptide chains, and any two chains in the multispecific polypeptide molecule are connected by covalent or non-covalent binding 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 binding between the two chains of the functional domain.

[0923] In some embodiments of the multispecific polypeptide molecule of the fourth aspect of the invention, the polypeptide molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; wherein:

[0924] 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-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 (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 comprises from N-terminus to C-terminus: TRBV1-optional linker-TRBC-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: TRAV1-optional linker-TRAC.

[0929] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-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: 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 comprises from N-terminus to C-terminus: TRBV1-optional linker-TRBC-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: TRAV1-optional linker-TRAC-optional linker-optional hinge region.

[0934] 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, and the third polypeptide chain comprises 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 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, and the third polypeptide chain comprises, from N-terminus to C-terminus: 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 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, and the third polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-optional hinge region-optional linker-Alb.

[0944] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-optional linker-optional hinge region-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: TRAV1-optional linker-TRAC.

[0945] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-optional linker-optional hinge region, 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.

[0946] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-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: TRAV1-optional linker-TRAC-optional linker-Alb.

[0947] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-optional linker-optional hinge region-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.

[0948] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-optional linker-optional hinge region, 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.

[0949] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-optional linker-optional hinge region, 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-Alb.

[0950] 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-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.

[0951] 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-optional linker-Alb, and the third polypeptide chain comprises, from N-terminus to C-terminus: 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 comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-optional linker-optional hinge region-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.

[0954] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-optional linker-optional hinge region, 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.

[0955] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH1-optional linker-optional hinge region, 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-Alb.

[0956] In a fifth aspect, the present invention provides a multispecific polypeptide molecule comprising a first binding region that binds to a first antigen and a second binding region that binds to a second antigen on two polypeptide chains,

[0957] wherein the first binding region comprises an α chain variable region (TRAV1) and a β chain variable region (TRBV1) derived from a T cell receptor (TCR) that binds to the first antigen-MHC complex;

[0958] wherein the second binding region comprises a heavy chain variable region VH1 and a light chain variable region VL1 derived from an antibody that binds to the second antigen;

[0959] wherein the first antigen is selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, and an autoantigen, and the second antigen is an immune cell surface molecule;

[0960] wherein VH1 and VL1 are respectively distributed on two different polypeptide chains of the multispecific polypeptide molecule, and the C-terminus of the polypeptide chain where VH1 or VL1 is located 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) Hinge region derived from an antibody;

[0963] 2) Fc domain or its dimerization portion derived from an antibody;

[0964] 3) CH3 domain derived from an antibody;

[0965] 4) albumin (Alb) or its binding portion;

[0966] 5) TCR-derived constant region or fragment thereof.

[0967] In some embodiments, TRAV1 and TRBV1 are linked in series on a single polypeptide chain. In some embodiments, VH1 and VL1 are located on two different polypeptide chains of the multispecific polypeptide molecule, and TRAV1 and TRBV1 are linked in series on a single polypeptide chain, with the N-terminus of the polypeptide chain linked to the C-terminus of the VH1 or VL1 polypeptide chain.

[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 consisting of:

[0975] (1) a TCR constant region or a fragment thereof, wherein the TCR constant region comprises, for example, a TCR α chain constant region (TRAC) and / or a β chain constant region (TRBC);

[0976] (2) CH3, wherein the CH3 is derived from the Fc domain of an antibody, and preferably the CH3 in any one or both polypeptide chains contains at least one mutation that can promote the formation of heterodimers of the polypeptide molecule;

[0977] (3) CL and CH1, wherein the CL is derived from the antibody light chain CL domain, and the CH1 is derived from the antibody heavy chain CH1 domain.

[0978] In some embodiments of the multispecific polypeptide molecule of the fifth aspect of the invention, the multispecific polypeptide molecule further comprises antibody-derived CH1 and CL linked to the VH1 and VL1, respectively.

[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 that binds 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 a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, and an autoantigen, and the third antigen is the same as or different from the first antigen. In 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, one of which is linked to the N-terminus of VH1 and the other to the N-terminus of VL1.

[0981] In some embodiments of the multispecific polypeptide molecule of the fifth aspect of the 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 invention, the multispecific polypeptide molecule further comprises a constant domain selected from the group consisting of:

[0999] (1) a TCR constant region or a fragment thereof, wherein the TCR constant region comprises, for example, a TCR α chain constant region (TRAC) and / or a β chain constant region (TRBC);

[1000] (2) CH3, wherein the CH3 is derived from the Fc domain of an antibody, and preferably the CH3 in any one or both polypeptide chains contains at least one mutation that can promote the formation of heterodimers of the polypeptide molecule;

[1001] (3) CL and CH1, wherein the CL is derived from the antibody light chain CL domain, and the CH1 is derived from the antibody heavy chain CH1 domain.

[1002] In some embodiments of the multispecific polypeptide molecule of the fifth aspect of the invention, the multispecific polypeptide molecule further comprises a fourth binding region that binds 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 that binds to the fourth antigen, or a single heavy chain variable region (VHH) derived from a single domain antibody or nanobody that binds to the fourth antigen; wherein the fourth antigen is an immune cell surface molecule, and the fourth antigen is the same as or different from the second antigen. In some embodiments, the VH2 is the same as the VH1. In some embodiments, the VL2 is the same as the VL1.

[1003] In some embodiments, the VHH or the VH2 and VL2 form an scFv2 connected to the N-terminus of the VH1 or the VL1 through a linker, or connected between TRAV1 and TRBV1, or one of the VH2 and VL2 is connected to the N-terminus of the VH1 through a linker, and the other is connected to the N-terminus of the VL1 through a linker.

[1004] In some embodiments of the multispecific polypeptide molecule of the fifth aspect of the 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 linked by covalent or non-covalent binding between the two chains of the functional domain.

[1038] In some embodiments, the functional domain comprises two chains, and the first binding region and the second binding region of the multispecific polypeptide molecule are linked 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 connected to the C-termini of the two polypeptide chains of the multispecific polypeptide molecule.

[1040] In some embodiments, the functional domain comprises two chains and is respectively connected to the N-termini of the two polypeptide chains of the multispecific polypeptide molecule.

[1041] In some embodiments, VH1 and VL1 are respectively distributed on two different polypeptide chains of the multispecific polypeptide molecule, and TRAV1 and TRBV1 are connected in series on one polypeptide chain, and the N-terminus of the peptide chain is connected to the C-terminus of the peptide chain where VH1 or VL1 is located, the functional domain comprises two chains, and the first binding region and the second binding region of the multispecific polypeptide molecule are connected through covalent or non-covalent binding between the two chains of the functional domain, for example, the C-terminus of the peptide 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 a binding portion thereof and optionally the hinge region of an antibody; and the functional domain is connected to the C-terminus or N-terminus, preferably the C-terminus, of any polypeptide chain of the multispecific polypeptide molecule via an optional linker. In some embodiments, the functional domain is derived from albumin or a binding portion thereof, and the albumin or a binding portion thereof is connected to the C-terminus or N-terminus, preferably the C-terminus, of any polypeptide chain of the multispecific polypeptide molecule via an optional linker. In some embodiments, the functional domain is derived from albumin or a binding portion thereof and the hinge region of an antibody, and the functional domain is connected to the C-terminus of any polypeptide chain of the multispecific polypeptide molecule via an optional linker, and the albumin or a binding portion thereof is connected to the C-terminus of any chain of the hinge region of the antibody via an optional linker.

[1043] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain; wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRBV1-linker-TRAV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-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-linker-TRBV1-linker-TRAV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-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-linker-TRAV1-linker-TRBV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-optional hinge region.

[1048] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRAV1-linker-TRBV1-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.

[1049] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-optional hinge region.

[1050] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: 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.

[1051] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VH1-linker-TRBV1-linker-TRAV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-optional hinge region.

[1052] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-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.

[1053] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VL1-linker-TRBV1-linker-TRAV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-optional hinge region.

[1054] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-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.

[1055] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VH1-linker-TRAV1-linker-TRBV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-optional hinge region.

[1056] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VH1-linker-TRAV1-linker-TRBV1-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.

[1057] 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-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-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-linker-TRBV1-linker-TRAV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VL1-optional linker-optional hinge region.

[1060] 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: scFv2-linker-VL1-optional linker-optional hinge region-optional linker-Alb.

[1061] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-linker-TRAV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VH1-optional linker-optional hinge region.

[1062] 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: scFv2-linker-VH1-optional linker-optional hinge region-optional linker-Alb.

[1063] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRAV1-linker-TRBV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VL1-optional linker-optional hinge region.

[1064] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRAV1-linker-TRBV1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VL1-optional linker-optional hinge region-optional linker-Alb.

[1065] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VH1-optional linker-optional hinge region.

[1066] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: scFv2-linker-VH1-optional linker-optional hinge region-optional linker-Alb.

[1067] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VH1-linker-TRBV1-linker-TRAV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-optional hinge region.

[1068] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-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.

[1069] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VL1-linker-TRBV1-linker-TRAV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-optional hinge region.

[1070] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-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.

[1071] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VH1-linker-TRAV1-linker-TRBV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-optional hinge region.

[1072] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VH1-linker-TRAV1-linker-TRBV1-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.

[1073] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VL1-linker-TRAV1-linker-TRBV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-optional linker-optional hinge region.

[1074] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VHH-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.

[1075] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRBV1-linker-TRAV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VL1-optional linker-optional hinge region.

[1076] 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: VHH-linker-VL1-optional linker-optional hinge region-optional linker-Alb.

[1077] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-linker-TRAV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VH1-optional linker-optional hinge region.

[1078] 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: VHH-linker-VH1-optional linker-optional hinge region-optional linker-Alb.

[1079] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRAV1-linker-TRBV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VL1-optional linker-optional hinge region.

[1080] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRAV1-linker-TRBV1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VL1-optional linker-optional hinge region-optional linker-Alb.

[1081] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VH1-optional linker-optional hinge region.

[1082] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VHH-linker-VH1-optional linker-optional hinge region-optional linker-Alb.

[1083] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-linker-TRBV1-linker-TRAV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VL1-optional linker-optional hinge region.

[1084] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-linker-TRBV1-linker-TRAV1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VL1-optional linker-optional hinge region-optional linker-Alb.

[1085] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VH1-linker-TRBV1-linker-TRAV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VL1-optional linker-optional hinge region.

[1086] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VH1-linker-TRBV1-linker-TRAV1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VL1-optional linker-optional hinge region-optional linker-Alb.

[1087] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VL1-linker-TRBV1-linker-TRAV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VH1-optional linker-optional hinge region.

[1088] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VL1-linker-TRBV1-linker-TRAV1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VH1-optional linker-optional hinge region-optional linker-Alb.

[1089] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VL1-linker-TRBV1-linker-TRAV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VH1-optional linker-optional hinge region.

[1090] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VL1-linker-TRBV1-linker-TRAV1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VH1-optional linker-optional hinge region-optional linker-Alb.

[1091] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-linker-TRAV1-linker-TRBV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VL1-optional linker-optional hinge region.

[1092] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-linker-TRAV1-linker-TRBV1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VL1-optional linker-optional hinge region-optional linker-Alb.

[1093] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VH1-linker-TRAV1-linker-TRBV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VL1-optional linker-optional hinge region.

[1094] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VH1-linker-TRAV1-linker-TRBV1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VL1-optional linker-optional hinge region-optional linker-Alb.

[1095] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VL1-linker-TRAV1-linker-TRBV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VH1-optional linker-optional hinge region.

[1096] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VL1-linker-TRAV1-linker-TRBV1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VH1-optional linker-optional hinge region-optional linker-Alb.

[1097] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VL1-linker-TRAV1-linker-TRBV1-optional linker-optional hinge region-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VH1-optional linker-optional hinge region.

[1098] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VL1-linker-TRAV1-linker-TRBV1-optional linker-optional hinge region, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VH1-optional linker-optional hinge region-optional linker-Alb.

[1099] In some embodiments of the multispecific polypeptide molecule of the fifth aspect of the invention, the functional domain is derived from the hinge region of an antibody and / or the Fc domain of an antibody or its dimerization portion and / or the CH3 domain of an antibody; and 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 embodiments, the functional domain is derived from the hinge region of an antibody and the Fc domain of an antibody or a dimerizing portion thereof.

[1101] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain; wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-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 (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 N-terminus to C-terminus: CH3-CH2-hinge region-optional linker-VH1-linker-TRBV1-linker-TRAV1, and the second polypeptide chain comprises, from N-terminus to 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 N-terminus to C-terminus: CH3-CH2-hinge region-optional linker-VH1-linker-TRBV1-linker-TRAV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: CH3-CH2-hinge region-optional linker-VL1-optional linker.

[1107] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: CH3-CH2-hinge region-optional linker-VL1-linker-TRBV1-linker-TRAV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: CH3-CH2-hinge region-optional linker-VH1.

[1108] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: CH3-CH2-hinge region-optional linker-VH1-linker-TRAV1-linker-TRBV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: CH3-CH2-hinge region-optional linker-VL1.

[1109] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: CH3-CH2-hinge region-optional linker-VL1-linker-TRAV1-linker-TRBV1, and the second polypeptide chain comprises, from N-terminus to 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-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.

[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-linker-VL1-linker-TRBV1-linker-TRAV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VH1-optional linker-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-linker-VL1-linker-TRAV1-linker-TRBV1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VH1-optional linker-hinge region-CH2-CH3.

[1134] In some embodiments, the functional domain is derived from the CH3 domain of an 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 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: 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 N-terminus to C-terminus: CH3-optional linker-VH1-linker-TRBV1-linker-TRAV1, and the second polypeptide chain comprises, from N-terminus to 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 N-terminus to C-terminus: CH3-optional linker-VH1-linker-TRBV1-linker-TRAV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: CH3-optional linker-VL1-optional linker.

[1141] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: CH3-optional linker-VL1-linker-TRBV1-linker-TRAV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: CH3-optional linker-VH1.

[1142] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: CH3-optional linker-VH1-linker-TRAV1-linker-TRBV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: CH3-optional linker-VL1.

[1143] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: CH3-optional linker-VL1-linker-TRAV1-linker-TRBV1, and the second polypeptide chain comprises, from N-terminus to 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 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.

[1169] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain; wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRBV1-linker-TRAV1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to 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 N-terminus to C-terminus: TRAC-optional linker-VH1-linker-TRBV1-linker-TRAV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBC-optional linker-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 comprises, from N-terminus to C-terminus: VH2-Linker-VL1-Linker-TRBV1-Linker-TRAV1-Optional Linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VH1-Optional Linker-TRBC.

[1193] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-Linker-VL1-Linker-TRBV1-Linker-TRAV1-Optional Linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VH1-Optional Linker-TRBC.

[1194] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-Linker-VH1-Linker-TRAV1-Linker-TRBV1-Optional Linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VL1-Optional Linker-TRAC.

[1195] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-Linker-VH1-Linker-TRAV1-Linker-TRBV1-Optional Linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VL1-Optional Linker-TRAC.

[1196] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-Linker-VL1-Linker-TRAV1-Linker-TRBV1-Optional Linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-VH1-Optional Linker-TRAC.

[1197] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL2-Linker-VL1-Linker-TRAV1-Linker-TRBV1-Optional Linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-VH1-Optional Linker-TRAC.

[1198] In a sixth aspect, the present invention provides a multispecific polypeptide molecule comprising a first binding region that binds to a first antigen and a second binding region that binds to a second antigen on three polypeptide chains,

[1199] wherein the first binding region comprises an α chain variable region (TRAV1) and a β chain variable region (TRBV1) derived from a T cell receptor (TCR) that binds to the first antigen-MHC complex;

[1200] wherein the second binding region comprises a heavy chain variable region VH1 and a light chain variable region VL1 derived from an antibody that binds to the second antigen;

[1201] wherein the first antigen is selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, and an autoantigen, and the second antigen is an immune cell surface molecule;

[1202] And the C-terminus of the peptide chain where VH1 or VL1 is located is connected to the N-terminus of the TCR.

[1203] In some embodiments, the multispecific polypeptide molecule further comprises at least one functional domain capable of extending the half-life of the multispecific polypeptide molecule. In some embodiments, the functional domain that extends the half-life is selected from a...

Claims

1. A multispecific polypeptide molecule comprising a first binding region that binds a first antigen and a second binding region that binds a second antigen, wherein the first binding region comprises an alpha chain variable region (TRAV1) and a beta chain variable region (TRBV1) derived from a T cell receptor (TCR) that binds to the first antigen-MHC complex; wherein the second binding region comprises a heavy chain variable region VH1 and a light chain variable region VL1 derived from an antibody that binds to the second antigen or a single heavy chain variable region (VHH1) derived from a single domain antibody or nanobody that binds to the second antigen; wherein the first antigen is selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen and an autoantigen, and the second antigen is an immune cell surface molecule; wherein the N-terminus of the antibody is connected to the C-terminus of the TCR; The multispecific polypeptide molecule further comprises at least one of the following functional domains: 1) Hinge region derived from antibodies; 2) Fc domain or dimerization portion thereof derived from an antibody; 3) CH3 domain derived from an antibody; 4) albumin (Alb) or its binding portion; 5) The constant region or fragment thereof derived from TCR.

2. The multispecific polypeptide molecule of claim 1, wherein TRAV1 and TRBV1 are distributed on different polypeptide chains; and / or The VH1 and VL1 are distributed on one polypeptide chain (eg, forming scFv1); and 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 that binds to a first antigen and a second binding region that binds to a second antigen on two polypeptide chains; Preferably, TRAV1 and TRBV1 are distributed on different polypeptide chains, the VHH1 or the VH1 and VL1 are distributed on one polypeptide chain (for example, forming scFv1) and connected to the C-terminus of the TCR; or TRAV1 and TRBV1 are distributed on different polypeptide chains, the VH1 and VL1 are distributed on different polypeptide chains, and one of the VH1 and VL1 polypeptide chains is connected to the C-terminus of the polypeptide chain where the TRAV1 is located, and the other polypeptide chain is connected to the C-terminus of the polypeptide chain where the TRBV1 is located; or, The multispecific polypeptide molecule comprises a first binding region that binds to a first antigen and a second binding region that binds to a second antigen on three polypeptide chains; Preferably, TRAV1 and TRBV1 are distributed on different polypeptide chains, and VH1 and VL1 are distributed on different polypeptide chains, and the polypeptide chain where VH1 or VL1 is located is connected to the C-terminus of the polypeptide chain where TRBV1 or TRAV1 is located.

4. The multispecific polypeptide molecule according to any one of claims 1 to 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 that binds 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 a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen and an autoantigen, and the third antigen is the same as or different from the first antigen.

6. The multispecific polypeptide molecule of claim 5, wherein the TRAV2 is identical to the TRAV1, and / or the TRBV2 is identical to the TRBV1.

7. The multispecific polypeptide molecule according to claim 5 or 6, wherein the TRAV2 and the TRBV2 are connected to the same polypeptide chain via a linker, and are preferably connected between VH1 and VL1.

8. The multispecific polypeptide molecule of claim 7, wherein the multispecific polypeptide molecule comprises two polypeptide chains, wherein 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. A multispecific polypeptide molecule according to any one of claims 1-8, wherein the multispecific polypeptide molecule further comprises a fourth binding region that binds 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 that binds to the fourth antigen or a single heavy chain variable region (VHH2) derived from a single domain antibody or nanobody that binds to the fourth antigen; wherein the fourth antigen is an immune cell surface molecule, and the fourth antigen is the same as or different from the second antigen.

10. The multispecific polypeptide molecule of claim 9, wherein the VH2 is identical to the VH1, and / or the VL2 is identical to the VL1, and / or the VHH2 is identical to the VHH1.

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 (for example, to form scFv2 or Fab2) and connected to the N-terminus of the TRAV1 or the TRBV1, or one of the VH2 and VL2 is connected to the N-terminus of the TRAV1 and the other is connected to the N-terminus of the TRBV1.

12. The multispecific polypeptide molecule of claim 11, wherein the multispecific polypeptide molecule comprises two polypeptide chains, wherein the two polypeptide chains respectively 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 of 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, wherein the two polypeptide chains respectively comprise: 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 through an optional linker, preferably the C-terminus.

15. The multispecific polypeptide molecule of claim 14, wherein the albumin is serum albumin, preferably human serum albumin; and / or The hinge region of the antibody is of human origin, for example, a hinge domain derived from human IgG1, IgG2 or IgG4 or a portion thereof.

16. The multispecific polypeptide molecule according to any one of claims 1 to 13, wherein the functional domain is derived from the hinge region of an antibody and / or the Fc domain of an antibody or its dimerization portion and / or the CH3 domain of an antibody; and any two polypeptide chains of the multispecific 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 multi-specific polypeptide molecule, preferably the C-terminus, respectively, through an optional linker; or The first binding region and the second binding region are connected by covalent or non-covalent binding between the two chains of the functional domain.

17. The multispecific polypeptide molecule according to claim 16, wherein the hinge region of the antibody is of human origin, for example, a hinge domain or a portion thereof derived from human IgG1, IgG2 or IgG4; and / or The Fc domain of the antibody or the dimerization part thereof is of human origin, for example, an Fc domain or a part thereof derived from human IgG1, IgG2 or IgG4.

18. The multispecific polypeptide molecule of claim 16 or 17, wherein the hinge region of the antibody comprises at least one amino acid mutation that can reduce or eliminate the binding of the multispecific molecule to FcR receptor and / or C1q, for example, the mutation site is selected from positions 233, 234, 235 and 236; and / or The hinge region of the antibody comprises at least one mutation that can reduce or eliminate the formation of a disulfide bond between the hinge region and the non-hinge region, for example, the mutation site is 220; and / or The CH2 domain in the Fc domain comprises at least one amino acid mutation that can reduce or eliminate the binding of the multispecific molecule to the FcR receptor and / or C1q; for example, the mutation site is selected from positions 237, 250, 252, 254, 256, 270, 297, 322, 327, 330 and 331; and / or The CH3 domain in the Fc domain comprises at least one amino acid mutation that can reduce or eliminate the binding of the multispecific molecule to the FcR receptor and / or C1q; for example, the mutation site is selected from positions 428 and 434; The CH3 in the Fc domain comprises at least one of the following mutations that can promote the polypeptide molecule to form a heterodimer: a) comprises at least two cysteine ​​residues mutated to form an interchain disulfide bond, such as 354C and 349C or 242C and 334C; b) comprising a mutation that enables two CH3 to form a knob-in-hole structure, for example, each of the CH3 independently comprises a mutation at one or more positions selected from amino acid positions 366, 368, 405 and 407, preferably, one of the CH3 comprises a 366W mutation and the other comprises 366S, 368A and 407V mutations; c) comprising a mutation that can change the charge properties of the amino acids in the CH3 region interaction interface to promote heterodimer formation, for example, the mutation sites of the two CH3 chains are each independently selected from at least one of Y391, K392, T393, T394, P395, P396, V397, L398, D399, S400, Q347, V348, Y349, T350, S354, R355, E356, E357, F405, Y407 and K409.

19. The multispecific polypeptide molecule according to claim 16 or 17, wherein the CH3 domain of the antibody is of human origin, for example, a CH3 domain derived from human IgG1, IgG2 or IgG4; and / or The CH3 domain comprises at least one of the following mutations that can promote the polypeptide molecule to form a heterodimer: a) comprises at least two cysteine ​​residues mutated to form an interchain disulfide bond, such as 354C and 349C or 242C and 334C; b) comprising a mutation that enables two CH3 to form a knob-in-hole structure, for example, each of the CH3 independently comprises a mutation at one or more positions selected from amino acid positions 366, 368, 405 and 407, preferably, one of the CH3 comprises a 366W mutation and the other comprises 366S, 368A and 407V mutations; c) comprising a mutation that can change the charge properties of the amino acids in the CH3 region interaction interface to promote heterodimer formation, for example, the mutation sites of the two CH3 chains are each independently selected from at least one of Y391, K392, T393, T394, P395, P396, V397, L398, D399, S400, Q347, V348, Y349, T350, S354, R355, D356, E357, K370, F405, Y407, K409 and K439.

20. The multispecific polypeptide molecule according to any one of claims 1 to 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 binding 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 multi-specific polypeptide molecule, preferably the C-terminus, respectively, through an optional linker; or The first binding region and the second binding region are connected by covalent or non-covalent binding between the two chains of the functional domain.

21. The multispecific polypeptide molecule according to claim 20, wherein the hinge region of the antibody is of human origin, for example, a hinge domain or a portion thereof derived from human IgG1, IgG2 or IgG4; and / or The constant region of the TCR or a fragment thereof is of human or murine origin and is selected from a TCR α chain constant region (TRAC) and a TCR 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 TRAC and TRBC, more preferably, the cysteine ​​mutation is at the following positions: position 48 in the wild-type TCR α chain constant region and position 57 in the wild-type TCR β chain constant region.

22. The multispecific polypeptide molecule of any one of claims 1-21, wherein the multispecific polypeptide molecule further comprises a constant domain selected from the group consisting of: (1) a TCR constant region or a fragment thereof, wherein the TCR constant region comprises, for example, a TCR α chain constant region (TRAC) and / or a β chain constant region (TRBC); Preferably, the TRAC and / or TRBC comprises at least one cysteine ​​mutation relative to the wild-type sequence to form a disulfide bond between TRAC and TRBC, more preferably, the cysteine ​​mutation is at the following positions: position 48 of the wild-type TCR α chain constant region and position 57 of the wild-type TCR β chain constant region; (2) CH3, wherein the CH3 is derived from the Fc domain of an antibody, and preferably the CH3 in any one or both polypeptide chains contains at least one mutation that can promote the polypeptide molecule to form a heterodimer; Preferably, each of the CH3s independently comprises a mutation at one or more positions selected from amino acid positions 366, 368, 405 and 407 and the two CH3s do not comprise the same mutation, more preferably, one of the CH3s comprises a T366W mutation and the other comprises T366S, L368A and Y407V mutations; (3) CL and CH1, wherein the CL is derived from the antibody light chain CL domain, and the CH1 is derived from the antibody heavy chain CH1 domain.

23. The multispecific polypeptide molecule of claim 22, wherein the multispecific polypeptide molecule comprises TRAC and TRBC linked to the C-termini of TRAV1 and TRBV1, respectively, via an optional linker.

24. The multispecific polypeptide molecule according to any one of claims 1-23, wherein the multispecific polypeptide molecule further comprises CH1 and CL derived from an antibody connected to the VH1 and VL1, respectively, wherein the VH1, VL1, CH1 and CL form a single-chain Fab and are connected to the C-terminus of the TRAV1 or the TRBV1 via a linker.

25. The multispecific polypeptide molecule according to any one of claims 1 to 4 and 14 to 24, wherein the polypeptide chain of the polypeptide molecule comprises, from N-terminus to C-terminus: 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: TRBV1-optional linker-TRBC-linker-VL1-CL-linker-VH1-CH1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3; The first polypeptide chain: TRBV1-optional linker-TRBC-linker-VL1, the second polypeptide chain: VH1-optional linker-hinge region-CH2-CH3, and the third polypeptide chain: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3; The first polypeptide chain: TRBV1-optional linker-TRBC-linker-VH1, the second polypeptide chain: VL1-optional linker-hinge region-CH2-CH3, and the third polypeptide chain: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3; The first polypeptide chain: TRAV1-optional linker-TRAC-linker-VL1, the second polypeptide chain: VH1-optional linker-hinge region-CH2-CH3, and the third polypeptide chain: TRBV1-optional linker-TRBC-optional linker-hinge region-CH2-CH3. The first polypeptide chain: TRAV1-optional linker-TRAC-linker-VH1, the second polypeptide chain: VL1-optional linker-hinge region-CH2-CH3, and the third polypeptide chain: TRBV1-optional linker-TRBC-optional linker-hinge region-CH2-CH3; The first polypeptide chain: TRBV1-optional linker-TRBC-linker-VL1-optional linker-hinge region-CH2-CH3, the second polypeptide chain: VH1-optional linker-hinge region-CH2-CH3, and the third polypeptide chain: TRAV1-optional linker-TRAC; The first polypeptide chain: TRBV1-optional linker-TRBC-linker-VH1-optional linker-hinge region-CH2-CH3, the second polypeptide chain: VL1-optional linker-hinge region-CH2-CH3, and the third polypeptide: TRAV1-optional linker-TRAC; The first polypeptide chain: TRAV1-optional linker-TRAC-linker-VL1-optional linker-hinge region-CH2-CH3, the second polypeptide chain: VH1-optional linker-hinge region-CH2-CH3, and the third polypeptide chain: TRBV1-optional linker-TRBC; First polypeptide chain: TRAV1-optional linker-TRAC-linker-VH1-optional linker-hinge region-CH2-CH3, second polypeptide chain: VL1-optional linker-hinge region-CH2-CH3, and third polypeptide chain: TRBV1-optional linker-TRBC.

26. The multispecific polypeptide molecule of 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 of 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 of 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-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.

29. The multispecific polypeptide molecule of any one of claims 1-28, wherein The VH1 and VL1 comprise at least one cysteine ​​mutation to form a disulfide bond between VH1 and VL1, preferably, the cysteine ​​mutation is at the following positions: position 44 of VH1 and position 100 of VL1; and / or The VH2 and VL2 comprise at least one cysteine ​​mutation to form a disulfide bond between VH2 and VL2, preferably, the cysteine ​​mutation is at the following positions: position 44 of VH2 and position 100 of VL2; and / or The TRAV1 and VL1 or VL2 comprise at least one cysteine ​​mutation to form a disulfide bond between TRAV1 and VL1 or VL2, preferably, the cysteine ​​mutation is at the following positions: position 104 of TRAV1 and position 80 of VL1 or VL2; and / or The TRAV2 and VL1 or VL2 comprise at least one cysteine ​​mutation to form a disulfide bond between TRAV2 and VL1 or VL2, preferably, the cysteine ​​mutation is at the following positions: position 104 of TRAV2 and position 80 of VL1 or VL2; and / or The TRBV1 and VL1 or VL2 comprise at least one cysteine ​​mutation to form a disulfide bond between TRBV1 and VL1 or VL2, preferably, the cysteine ​​mutation is at the following positions: position 84 of TRBV1 and position 80 of VL1 or VL2; and / or The TRBV2 and VL1 or VL2 comprise at least one cysteine ​​mutation to form a disulfide bond between TRBV2 and VL1 or VL2, preferably, the cysteine ​​mutation is at the following positions: position 84 of TRBV2 and position 80 of VL1 or VL2; and / or The TRAV1 and VH1 or VH2 comprise at least one cysteine ​​mutation to form a disulfide bond between TRAV1 and VH1 or VH2, preferably, the cysteine ​​mutation is at the following positions: position 104 of TRAV1 and position 89 of VH1 or VH2; and / or The TRAV2 and VH1 or VH2 comprise at least one cysteine ​​mutation to form a disulfide bond between TRAV2 and VH1 or VH2, preferably, the cysteine ​​mutation is at the following positions: 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, extend its half-life, reduce its immunogenicity or reduce its post-translational modification; for example, the amino acid mutations include mutations that can enhance binding to FcRn and / or effector function silencing mutations.

31. The multispecific polypeptide molecule of any one of claims 1-30, wherein the linkers in the polypeptide molecule are each independently selected from linkers consisting of 1-35 amino acids; Preferably, the linkers are each independently selected from S, GS, GGGS, GGGGS, GGGSGGGG, GGSGGS, GGSGGSGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGS, GGGGSGGGGSGGGGSGGGGSGGGGS, GQPKAAP, TVLRT, TVSSAS, GGEGG, GSEGGGS, RTSGPGDGGKGGPGGKGPGGEGTKGTGPGG, GKGPGGEGTKGTGPGG, TVLSSAS, EDLKN or a variant thereof, EDLNK or a variant thereof, and ANIQK or a variant thereof, or any combination thereof; Preferably, the variant of EDLKN is an amino acid sequence formed by substitution, deletion or addition of one or several amino acids of EDLKN; and / or the variant of EDLNK is an amino acid sequence formed by substitution, deletion or addition of one or several amino acids of EDLNK; and / or the variant of ANIQK is an amino acid sequence formed by substitution, deletion or addition of one or several amino acids of ANIQK.

32. The multispecific polypeptide molecule of any one of claims 1-31, wherein each linker in the polypeptide molecule is independently selected from a linker consisting of no more than 15 amino acids; Preferably, the linkers are each independently selected from S, GS, GGGS, GGGGS, GGGSGGGG, GGSGGS, GGSGGSGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, EDLKN or a variant thereof, EDLNK or a variant thereof, and ANIQK or a variant thereof; Preferably, the variant of EDLKN is an amino acid sequence formed by substitution, deletion or addition of one or several amino acids of EDLKN; and / or the variant of EDLNK is an amino acid sequence formed by substitution, deletion or addition of one or several amino acids of EDLNK; and / or the variant of ANIQK is an amino acid sequence formed by substitution, deletion or addition of one or several amino acids of ANIQK.

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. The multispecific polypeptide molecule of any one of claims 1-33, wherein the first antigen and the third antigen are each independently a TAA selected from the group consisting of melanoma-associated antigens (e.g., gp100, MAGEA1, MAGEA3, MAGEA6, MAGEA4, MAGEA2, MAGEA12, MAGEA2B, MAGEA9B, MAGEA10, MAGEA11, MAGEB2, MAGEC1, MAGEC2), IGF2BP1, GNGT1, PI4K2B, CCR8, NPSR1, COX7B2, ONECUT3, SMC1B, FOXI3, GAGE2A, FBXO43, BRDT, PAGE2, GAGE13, POU5F1B, CTAG1A, and an endogenous reverse transcriptase antigen; or wherein the first antigen and the third antigen are each independently a TSA selected from the following: KRAS antigen (e.g., KRAS G12 mutant antigen), TP53, PIK3CA, CTNNB1, EGFR, BRAF and GNAS; or wherein the first antigen and the third antigen are each independently a viral antigen selected from the following: HPV antigen (e.g., HPV E6 or E7 antigen), CMV antigen, HBV antigen, EBV antigen, herpes virus antigen, human immunodeficiency virus (HIV) antigen, influenza virus antigen, and coronavirus antigen; or The first antigen and the third antigen are each independently an autoantigen selected from the following: AFP, CEA, CD19, CD20, BCMA, CD22, CD30, SLAM, CLDN18.2, GD2, mesothelin, CD38, Her2, GPC3, MUC1, Ro52, Ro60, La, Jo-1, SRP, IFIH1, CENPA, CENPB, SNRPA1, SNRNP70, SNR-PD3, RNAP3, TOPO1, Insulin , GAD65, IA2, Znt8, PL7, TARS, ARS, MI2, topoisomerase 1, EXOSC9, EXOSC107, POLR3A, POLR3K, PTRN, GAD2, SLC30A8, AchR, MUSK, LRP4, PLA2R, THSD7A, TSHR, IFN-γ, CHRNA1, MUSK, LRP4, AQP4, MOG, GRIN1, COL4A3, PLA2R, GM-SCF, PR3, and MPO.

35. The multispecific polypeptide molecule of 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. A 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 antigens) and HPV antigens (e.g., HPV E7 antigens), 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 chain of the multispecific polypeptide molecule of 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 chain of the multispecific polypeptide molecule of 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 the multispecific polypeptide molecule of any one of claims 1-36, and a chemical moiety conjugated to the multispecific polypeptide molecule.

42. The conjugate of claim 41, wherein the chemical moiety is selected from a therapeutic agent, an immunostimulatory molecule, and a detectable label; Preferably, the therapeutic agent is selected from the group consisting of an immunomodulator, an agonist, a radioactive compound, an enzyme, a chemotherapeutic agent, and a toxin. Preferably, the immunostimulatory molecules are selected from cytokines, chemokines, platelet factors and complement initiators; Preferably, the detectable label is selected from biotin, streptavidin, an enzyme or a catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, a nucleic acid probe, a contrast agent, and a fluorescent, phosphorescent or chemiluminescent molecule.

43. A pharmaceutical composition comprising the multispecific polypeptide molecule of any one of claims 1-36, the nucleic acid of claim 37, the vector of claim 38, the vector system of claim 39, the host cell of claim 40, the conjugate of claim 41 or 42, and a pharmaceutically acceptable excipient.

44. The pharmaceutical composition according to claim 43, wherein the pharmaceutical composition further comprises a second therapeutic agent, preferably, the second therapeutic agent is 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 the multispecific polypeptide molecule of any one of claims 1-36, the nucleic acid of claim 37, the vector of claim 38, the vector 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, an infectious disease, an autoimmune disease, and an inflammatory disease.

46. ​​The method of claim 45, further comprising administering to the subject a second therapeutic agent, preferably, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, and a small molecule drug.

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