Bispecific polypeptide molecule based on T cell receptor and application thereof

Abstract

The invention relates to bispecific polypeptide molecules based on T cell receptors and uses thereof. The invention also provides nucleic acids encoding the polypeptide molecules of the invention, vectors or vector systems comprising the nucleic acids of the invention, host cells comprising the polypeptide molecules of the invention, conjugates and pharmaceutical compositions comprising the polypeptide molecules of the invention, and methods of using the polypeptide molecules of the invention to prevent or treat diseases in subjects.

Description

T cell receptor-based bispecific polypeptide molecules and their uses

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

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

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

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

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

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

[0007] Summary of the Invention

[0008] To enhance the in vivo efficacy of TCEs, in addition to increasing the affinity of the TCR for pMHC, the potency of the TCE against target cell targets and the potency of the TCE against molecules on the surface of effector cells can also be increased to enhance T cell cytotoxicity and proliferation. Furthermore, to maintain the ability of TCEs to enter tumors and maintain an appropriate half-life in vivo, the molecular size of TCEs must be kept within a certain range. Furthermore, for protein drugs, the glomerular filtration limit is generally around 60 kDa. This means that small molecular weight antibody fragments or derivatives, such as nanobodies (15 kDa), scFvs (28 kDa), and even Fabs (50 kDa), are cleared from the body through glomerular filtration, resulting in a short half-life. Among them, nanobodies have the smallest molecular weight of all antibody types, with a half-life of often only tens of minutes. Therefore, to enhance the efficacy of protein drugs, it is necessary to improve them to extend their half-life and increase their activity. Appropriately adding other functional domains to the polypeptide molecule, such as the hinge region of an antibody, the Fc domain of an antibody or its dimerization portion and / or the functional domain of albumin (Alb), can not only prolong the half-life of the polypeptide molecule, but also enhance the killing activity of the polypeptide molecule.

[0009] Accordingly, the present invention provides a TCR-based bispecific polypeptide molecule. The bispecific polypeptide molecule of the present invention utilizes a specific antigen-binding region connection method and is combined with the TCR constant region (TRAC and TRBC). This combination with the TCR constant region significantly improves the stability of the molecule. This specific combination of domains achieves efficient immune cell activation and has significantly better effects than using the constant region of an antibody (CH3, hinge region-CH2-CH3, or CH1 / CL).

[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 the Fab (antigen binding fragment). The Fc region, also known as the Fc domain, contains two or three heavy chain constant regions (CH2, CH3, CH4) in each heavy chain. In IgG class antibodies, the Fc region contains the CH2 and CH3 domains. In the native structure of immunoglobulins, the Fc regions of the two heavy chains dimerize to form a dimerized portion. The Fc fragment is responsible for immunoglobulin effector functions, including, for example, complement fixation and binding to cognate Fc receptors on effector cells.

[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: 7), which, when dimerized through disulfide bonds, generates a cyclic octapeptide that is believed to act as a pivot, conferring flexibility. The conformational changes allowed by the structure and flexibility of the immunoglobulin hinge region polypeptide sequence can influence the effector functions of the antibody Fc portion.

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

[0027] 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.

[0028] 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.

[0029] Examples of antigen-binding fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region; (iii) a Fab' fragment, which is essentially a Fab but has a portion of the hinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul ed., 3.sup.rd ed. 1993); (iv) a Fd fragment consisting of the VH and CH1 domains; (v) a Fd' fragment having the VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (vi) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (vii) a dAb fragment (Ward et al. (1989) Nature 557(5)). 341: 544-546), which consists of a VH domain; (viii) a single complementarity determining region (CDR); and (ix) a nanobody, which is a heavy chain variable region comprising a single variable domain and two constant domains. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked using recombinant methods by a synthetic linker that enables them to form a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called single-chain Fv (ScFv); see, e.g., Bird et al. (1988) Science 242, 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. In addition, the term also includes "linear antibodies" containing a pair of tandem Fd segments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides and modified versions of any of the foregoing segments that retain antigen-binding activity form an antigen-binding region.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] As used herein, the term "viral antigen" refers to a substance in a virus that can induce an immune response in the body.

[0035] 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.

[0036] "Immune cell surface molecules" may include, for example, immune cell surface antigens (eg, T cell antigens) and co-stimulatory molecules.

[0037] 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.

[0038] 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.

[0039] >gi|4502027|ref|NP_000468.1|Serum albumin preproprotein [Homo sapiens]

[0040] The signal peptide is underlined, the propeptide is bold, and mature human serum albumin is italicized).

[0041] 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.

[0042] 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).

[0043] 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.

[0044] 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.

[0045] 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.

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

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

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

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

[0050] As used herein, the term "effective amount" means an amount of a therapeutic agent that, when administered to a subject for the treatment or prevention of a disease, is sufficient to achieve such treatment or prevention. The "effective amount" may vary depending on the compound, the disease and its severity, and the age, weight, etc. of the subject to be treated. A "therapeutically effective amount" refers to an effective amount for therapeutic treatment. A "prophylactically effective amount" refers to an effective amount for preventive treatment.

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

[0052] Preferred embodiments of the present invention

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

[0054] In one aspect, the present invention provides a bispecific polypeptide molecule comprising a first binding region that binds a first antigen and a second binding region that binds a second antigen on one or more polypeptide chains.

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

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

[0057] wherein the TRAV, TRBV, VH and VL are distributed on the one or more polypeptide chains such that when the one or more polypeptide chains are folded, the TRAV and TRBV are spatially close to form the first binding region, and the VH and VL are spatially close to form the second binding region;

[0058] The first antigen is selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, and a self-antigen, and the second antigen is an immune cell surface molecule.

[0059] In some embodiments, the TRAV, TRBV, VH and VL are distributed on the one or more polypeptide chains and adjacent variable regions on the same chain are separated by a linker sequence.

[0060] In some embodiments, the bispecific polypeptide molecule comprises a first binding region that binds a first antigen and a second binding region that binds a second antigen on two polypeptide chains.

[0061] wherein the first binding region comprises an α chain variable region (TRAV) and a β chain variable region (TRBV) derived from a T cell receptor (TCR) that binds to the first antigen-MHC complex;

[0062] wherein the second binding region comprises a heavy chain variable region VH and a light chain variable region VL derived from an antibody that binds to the second antigen;

[0063] 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;

[0064] The two polypeptide chains of the bispecific polypeptide molecule respectively comprise:

[0065] TRAV-VH and VL-TRBV;

[0066] TRAV-VL and VH-TRBV;

[0067] TRBV-VH and VL-TRAV; or

[0068] TRBV-VL and VH-TRAV;

[0069] Adjacent variable regions in the two polypeptide chains are connected by a linker; and

[0070] The bispecific polypeptide molecule further comprises a TCR constant region or fragment thereof connected to the C-terminus of each polypeptide chain via an optional linker.

[0071] In a preferred embodiment, the two polypeptide chains of the bispecific polypeptide molecule comprise: TRAV-VH and VL-TRBV, respectively.

[0072] In a preferred embodiment, the two polypeptide chains of the bispecific polypeptide molecule comprise: TRAV-VH and VL-TRBV, respectively, and the adjacent variable regions in the two polypeptide chains are connected by a linker; and the bispecific polypeptide molecule further comprises a TCR constant region or a fragment thereof connected to the C-terminus of each polypeptide chain via an optional linker.

[0073] In some embodiments, the TCR constant region is selected from the group consisting of a TCR alpha chain constant region (TRAC) and a TCR beta chain constant region (TRBC). In a preferred embodiment, one polypeptide chain of the bispecific polypeptide molecule comprises TRAC and the other polypeptide chain comprises TRBC.

[0074] In some embodiments, the N-terminal 1-5 amino acids in TRAC may be substituted.

[0075] In some embodiments, 1-5 amino acids at the N-terminus of TRBC may be substituted.

[0076] TRAC and TRBC can be human or murine. TRAC and TRBC can be wild-type or variants thereof. For example, a TRAC variant can include one or more of T48C, N113K, PESS deletion mutation, or FFPSPESS deletion mutation relative to the wild-type sequence. For example, a TRBC variant can include one or more of S57C, C187A, N210D, or FG loop deletion mutation relative to the wild-type sequence. In a preferred embodiment, TRAC and / or TRBC include at least one cysteine ​​mutation relative to the wild-type sequence to form a disulfide bond between TRAC and TRBC. More preferably, the cysteine ​​mutation is at position 48 of the wild-type TCR α chain constant region and position 57 of the wild-type TCR β chain constant region.

[0077] In some embodiments, the polypeptide molecule comprises TRAC or a fragment thereof, which is not connected to TRAV via a linker. Preferably, 1-5 amino acids at the N-terminus of TRAC can be substituted.

[0078] In some embodiments, the polypeptide molecule comprises TRBC or a fragment thereof, which is not connected to TRBV via a linker. Preferably, 1-5 amino acids at the N-terminus of TRBC may be substituted.

[0079] Unless otherwise indicated, the term "cis-linked" refers to the linkage between the TCR constant region and the variable region in the natural state.

[0080] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV-linker-VH-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRBC (format 22-2).

[0081] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises from N-terminus to C-terminus: TRAV-linker-VH-linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV-linker-TRBC.

[0082] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV-linker-VH-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRAC.

[0083] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV-linker-VL-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRBV-optional linker-TRBC.

[0084] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV-linker-VL-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRBV-optional linker-TRAC.

[0085] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV-linker-VH-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRAV-optional linker-TRBC.

[0086] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV-linker-VH-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRAV-optional linker-TRAC.

[0087] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV-linker-VL-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRBC.

[0088] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV-linker-VL-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRAC.

[0089] In some embodiments, the bispecific polypeptide molecule further comprises at least one of the following functional domains:

[0090] 1) Hinge region derived from an antibody;

[0091] 2) Fc domain or its dimerization portion derived from an antibody;

[0092] 3) Albumin (Alb).

[0093] The introduction of the functional domain can not only prolong the half-life of the polypeptide molecule, but also enhance the killing activity of the polypeptide molecule.

[0094] In some embodiments, the functional domain is connected to the C-terminus of one or both polypeptide chains of the bispecific polypeptide molecule via an optional linker. In some embodiments, the functional domain is connected to the C-terminus of either polypeptide chain of the bispecific polypeptide molecule via an optional linker. In some embodiments, the functional domain is connected to the C-terminus of both polypeptide chains of the bispecific polypeptide molecule via an optional linker.

[0095] In some embodiments, the functional domain is derived from albumin or a fragment thereof. In some embodiments, albumin or a fragment thereof is linked to the C-terminus of one or both polypeptide chains of the bispecific polypeptide molecule via an optional linker. In preferred embodiments, albumin or a fragment thereof is linked to the C-terminus of either polypeptide chain of the bispecific polypeptide molecule via an optional linker. In some embodiments, the albumin can be any mammalian-derived albumin, such as human albumin, bovine albumin, mouse albumin, etc. In some embodiments, the albumin is a serum albumin, such as human serum albumin or bovine serum albumin. In some embodiments, the functional domain may be a fragment of albumin, for example, a fragment having a length of 100-550, 150-550, 200-550, 250-550, 300-550, 350-550, 400-550, 450-550, 500-550, 100-500, 150-500, 200-500, 250-500, 300-500, 350-500, 400-500, 450-500, 100-450, 150-450, 200-450, 250-450, In a preferred embodiment, the albumin is human serum albumin having an amino acid sequence as shown in SEQ ID NO: 9.

[0096] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV-linker-VH-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRBC-optional linker-Alb (Format64).

[0097] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV-linker-VH-linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-linker-TRBC-linker-Alb.

[0098] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV-linker-VH-optional linker-TRAC-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRBC.

[0099] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV-linker-VH-optional linker-TRBC-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRAC.

[0100] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV-linker-VH-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRAC-optional linker-Alb.

[0101] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV-linker-VL-optional linker-TRAC-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRBV-optional linker-TRBC.

[0102] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV-linker-VL-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRBV-optional linker-TRBC-optional linker-Alb.

[0103] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV-linker-VL-optional linker-TRBC-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRBV-optional linker-TRAC.

[0104] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV-linker-VL-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRBV-optional linker-TRAC-optional linker-Alb.

[0105] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV-linker-VH-optional linker-TRAC-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRAV-optional linker-TRBC.

[0106] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV-linker-VH-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRAV-optional linker-TRBC-optional linker-Alb.

[0107] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV-linker-VH-optional linker-TRBC-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRAV-optional linker-TRAC.

[0108] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV-linker-VH-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRAV-optional linker-TRAC-optional linker-Alb.

[0109] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV-linker-VL-optional linker-TRAC-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRBC.

[0110] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV-linker-VL-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRBC-optional linker-Alb.

[0111] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV-linker-VL-optional linker-TRBC-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRAC.

[0112] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV-linker-VL-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRAC-optional linker-Alb.

[0113] In some embodiments, the functional domain is derived from the hinge region of an antibody and / or the Fc domain of an antibody or a dimerization portion thereof; and the functional domain is connected to the C-termini of the two polypeptide chains of the bispecific polypeptide molecule via an optional linker.

[0114] The hinge region may comprise part or all of the wild-type hinge sequence of an antibody or a variant thereof having one or more substitutions. In some embodiments, the hinge region of the antibody is the hinge region of a human antibody. The hinge region of the antibody may be of any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4. For example, the hinge region of the antibody may be derived from the hinge domain of human IgG1, IgG2, or IgG4, or a portion thereof. In some embodiments, the hinge region may comprise one of the following amino acid sequences: EPKSSDKTHTCPPCPAPPVAGP (SEQ ID NO:69), EPKSSDKTHTCPPCP (SEQ ID NO:70), PKSSDKTHTCPPCPAPPVAGP (SEQ ID NO:71), KSSDKTHTCPPCPAPPVAGP (SEQ ID NO:72), SSDKTHTCPPCPAPPVAGP (SEQ ID NO:73), SDKTHTCPPCPAPPVAGP (SEQ ID NO:74), DKTHTCPPCPAPPVAGP (SEQ ID NO:75), KTHTCPPCPAPPVAGP (SEQ ID NO:76), THTCPPCPAPPVAGP (SEQ ID NO:77), HTCPPCPAPPVAGP (SEQ ID NO:78), TCPPCPAPPVAGP (SEQ ID NO:79), CPPCPAPPVAGP (SEQ ID NO:80), PPCPAPPVAGP (SEQ ID NO:81), CPAPPVAGP (SEQ ID NO:82). NO:82), or PAPPVAGP (SEQ ID NO:83); or a variant thereof having one or more substitutions (e.g., 1-6 substitutions, such as 1-5, 1-4, 1, 2, 3, 4, 5, or 6 substitutions).

[0115] The Fc domain of an antibody comprises CH2 and CH3. In some embodiments, the Fc domain of an antibody or its dimerization portion is the Fc domain of a human antibody or its dimerization portion. The Fc domain of an antibody can be of any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and can comprise one or more mutations or modifications. In some embodiments, the Fc domain of an antibody or its dimerization portion is derived from the Fc domain of human IgG1, IgG2, or IgG4, or a portion thereof, and preferably comprises at least two cysteine ​​residue mutations, such as S354C and Y349C or L242C and K334C. In one embodiment, the Fc domain is an IgG1 isotype or derived therefrom, optionally with one or more mutations or modifications. In another embodiment, the Fc domain is an IgG4 isotype or derived therefrom, optionally with one or more mutations or modifications. In one embodiment, the Fc domain is human IgG1Fc or human IgG4Fc.

[0116] In some embodiments, the functional domain comprises the hinge region - CH2-CH3 of an antibody. In some embodiments, both polypeptide chains of the bispecific polypeptide molecule comprise the hinge region - CH2-CH3. In some embodiments, the CH3 in both polypeptide chains of the bispecific polypeptide molecule comprises at least one mutation that promotes heterodimer formation of the polypeptide molecule. In some embodiments, the CH3 in each of the two polypeptide chains independently comprises a mutation at one or more positions selected from amino acid positions 366, 368, 405, and 407, and the two CH3s do not comprise the same mutation. Preferably, one of the CH3s comprises a T366W mutation and the other comprises a T366S, L368A, and Y407V mutation. In a preferred embodiment, the amino acid sequence of the hinge region - CH2-CH3 is as shown in SEQ ID NO: 33 or 34.

[0117] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV-linker-VH-optional linker-TRAC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRBC-optional linker-hinge region-CH2-CH3 (format 63).

[0118] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV-linker-VH-linker-TRAC-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-linker-TRBC-hinge region-CH2-CH3.

[0119] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV-linker-VH-optional linker-TRBC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRAC-optional linker-hinge region-CH2-CH3.

[0120] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV-linker-VL-optional linker-TRAC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRBV-optional linker-TRBC-optional linker-hinge region-CH2-CH3.

[0121] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV-linker-VL-optional linker-TRBC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRBV-optional linker-TRAC-optional linker-hinge region-CH2-CH3.

[0122] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV-linker-VH-optional linker-TRAC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRAV-optional linker-TRBC-optional linker-hinge region-CH2-CH3.

[0123] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV-linker-VH-optional linker-TRBC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRAV-optional linker-TRAC-optional linker-hinge region-CH2-CH3.

[0124] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV-linker-VL-optional linker-TRAC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRBC-optional linker-hinge region-CH2-CH3.

[0125] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV-linker-VL-optional linker-TRBC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRAC-optional linker-hinge region-CH2-CH3.

[0126] In embodiments of the bispecific polypeptide molecules of the present invention, when the polypeptide molecules comprise more than one linker, the linkers may be the same or different. In some embodiments, the linkers in the polypeptide molecules are each independently selected from linkers consisting of 1-35 amino acids.

[0127] In some embodiments, the linkers are each independently selected from the group consisting of S (SEQ ID NO:41), GGGS (SEQ ID NO:62), GGGGS (SEQ ID NO:42), GGGSGGGG (SEQ ID NO:50), GGSGGS (SEQ ID NO:47), GGSGGSGGS (SEQ ID NO:48), GGGGSGGGGS (SEQ ID NO:46), GGGGSGGGGSGGGGS (SEQ ID NO:44), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:43), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:45), GQPKAAP (SEQ ID NO:49), TVLRT (SEQ ID NO:53), TVSSAS (SEQ ID NO:54), GGEGG (SEQ ID NO:55), GSEGGGS (SEQ ID NO:56). NO: 56), RTSGPGDGGKGGPGKGPGGEGTKGTGPGG (SEQ ID NO: 57), GKGPGGEGTKGTGPGG (SEQ ID NO: 58), TVLSSAS (SEQ ID NO: 59), EDLKN (SEQ ID NO: 63) or a variant thereof, EDLNK (SEQ ID NO: 64) or a variant thereof, and ANIQK (SEQ ID NO: 65) or a variant thereof, or any combination thereof.

[0128] In some embodiments, the variant of EDLKN is an amino acid sequence formed by substituting, deleting or adding one or more amino acids to EDLKN; and / or the variant of EDLNK is an amino acid sequence formed by substituting, deleting or adding one or more amino acids to EDLNK; and / or the variant of ANIQK is an amino acid sequence formed by substituting, deleting or adding one or more amino acids to ANIQK.

[0129] Preferably, the variant of EDLKN is an amino acid sequence formed by replacing one or several amino acids of EDLKN; and / or the variant of EDLNK is an amino acid sequence formed by replacing one or several amino acids of EDLNK; and / or the variant of ANIQK is an amino acid sequence formed by replacing one or several amino acids of ANIQK.

[0130] Preferably, the variant of EDLKN is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or several amino acids from EDLKN; and / or the variant of EDLNK is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or several amino acids from EDLNK; and / or the variant of ANIQK is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or several amino acids from ANIQK.

[0131] Preferably, the variant of EDLKN is EDL, DL, L, ED, E, D; and / or the variant of ANIQK is ANI, NI, I, AN, N, A, PNI, P, PN.

[0132] Preferably, the variant of EDLKN is an amino acid sequence consisting of 6-35 amino acids formed by adding one or several amino acids to EDLKN (preferably at the N-terminus); and / or the variant of EDLNK is an amino acid sequence consisting of 6-35 amino acids formed by adding one or several amino acids to EDLNK (preferably at the N-terminus); and / or the variant of ANIQK is an amino acid sequence consisting of 6-35 amino acids formed by adding one or several amino acids to ANIQK (preferably at the N-terminus).

[0133] In some embodiments, the linkers in the polypeptide molecule are each independently selected from a linker consisting of no more than 12 amino acids. Preferably, the linkers are each independently selected from S, GGGS, GGGGS, GGGSGGGG, GGSGGS, GGSGGSGGS, GGGGSGGGGS, EDLKN or a variant thereof, EDLNK or a variant thereof, and ANIQK or a variant thereof. In some embodiments, a variant of EDLKN is an amino acid sequence formed by substitution, deletion, or addition of one or more amino acids to EDLKN; and / or a variant of EDLNK is an amino acid sequence formed by substitution, deletion, or addition of one or more amino acids to EDLNK; and / or a variant of ANIQK is an amino acid sequence formed by substitution, deletion, or addition of one or more amino acids to ANIQK. Preferably, the variant of EDLKN is an amino acid sequence formed by substituting one or more amino acids in EDLKN; and / or the variant of EDLNK is an amino acid sequence formed by substituting one or more amino acids in EDLNK; and / or the variant of ANIQK is an amino acid sequence formed by substituting one or more amino acids in ANIQK. Preferably, the variant of EDLKN is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or more amino acids in EDLKN; and / or the variant of EDLNK is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or more amino acids in EDLNK; and / or the variant of ANIQK is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or more amino acids in ANIQK. Preferably, the variant of EDLKN is an amino acid sequence consisting of 6-12 amino acids formed by adding one or more amino acids to EDLKN (preferably at the N-terminus); and / or the variant of EDLNK is an amino acid sequence consisting of 6-12 amino acids formed by adding one or more amino acids to EDLNK (preferably at the N-terminus); and / or the variant of ANIQK is an amino acid sequence consisting of 6-12 amino acids formed by adding one or more amino acids to ANIQK (preferably at the N-terminus). Preferably, the variants of EDLKN are EDL, DL, L, ED, E, D; and / or the variants of ANIQK are ANI, NI, I, AN, N, A, PNI, P, PN.

[0134] In some embodiments, the linkers in the polypeptide molecule are each independently one or a combination of 2-3 or more linkers. In some embodiments, the linkers in the polypeptide molecule are each independently one or more linkers. In some embodiments, the linkers in the polypeptide molecule are each independently two or more linkers. In some embodiments, the linkers in the polypeptide molecule are each independently three or more linkers.

[0135] In some embodiments, the C-terminus of TRAV is connected to VH or VL via two linkers, and the proximal linker of TRAV is ANIQK or a variant thereof. In some embodiments, the variant of ANIQK is an amino acid sequence formed by replacing, deleting, or adding one or more amino acids to ANIQK. Preferably, the variant of ANIQK is an amino acid sequence formed by replacing one or more amino acids in ANIQK. Preferably, the variant of ANIQK is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or more amino acids from ANIQK, for example, ANI, NI, I, AN, N, A, PNI, P, PN.

[0136] In some embodiments, the C-terminus of TRBV is connected to VH or VL via two linkers, and the proximal linker of TRBV is EDLKN or a variant thereof, EDLNK or a variant thereof, or ANIQK or a variant thereof. In some embodiments, a variant of EDLKN is an amino acid sequence formed by substitution, deletion, or addition of one or more amino acids to EDLKN; and / or a variant of EDLNK is an amino acid sequence formed by substitution, deletion, or addition of one or more amino acids to EDLNK. Preferably, a variant of EDLKN is an amino acid sequence formed by substitution of one or more amino acids to EDLKN; and / or a variant of EDLNK is an amino acid sequence formed by substitution of one or more amino acids to EDLNK. Preferably, a variant of EDLKN is an amino acid sequence consisting of 1-3 amino acids formed by deletion of one or more amino acids to EDLKN; and / or a variant of EDLNK is an amino acid sequence consisting of 1-3 amino acids formed by deletion of one or more amino acids to EDLNK, for example, EDL, DL, L, ED, E, D.

[0137] In some embodiments, the C-terminus of TRAV is connected to TRAC via a linker, wherein the linker is ANIQK or a variant thereof. In some embodiments, the variant of ANIQK is an amino acid sequence formed by replacing, deleting, or adding one or more amino acids to ANIQK. Preferably, the variant of ANIQK is an amino acid sequence formed by replacing one or more amino acids to ANIQK. The variant of ANIQK is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or more amino acids from ANIQK, for example, ANI, NI, I, AN, N, A, PNI, P, PN.

[0138] In some embodiments, the C-terminus of TRBV is connected to TRBC via a linker, wherein the linker is EDLKN or a variant thereof, or EDLNK or a variant thereof. In some embodiments, a variant of EDLKN is an amino acid sequence formed by substitution, deletion, or addition of one or more amino acids to EDLKN; and / or a variant of EDLNK is an amino acid sequence formed by substitution, deletion, or addition of one or more amino acids to EDLNK. Preferably, a variant of EDLKN is an amino acid sequence formed by substitution of one or more amino acids to EDLKN; and / or a variant of EDLNK is an amino acid sequence formed by substitution of one or more amino acids to EDLNK. Preferably, a variant of EDLKN is an amino acid sequence consisting of 1-3 amino acids formed by deletion of one or more amino acids to EDLKN; and / or a variant of EDLNK is an amino acid sequence consisting of 1-3 amino acids formed by deletion of one or more amino acids to EDLNK, for example, EDL, DL, L, ED, E, D.

[0139] In some embodiments, VH and VL contain at least one cysteine ​​mutation to form a disulfide bond between VH and VL, and the cysteine ​​is introduced into FR4 in the case of VL and into FR2 in the case of VH. In a preferred embodiment, the cysteine ​​mutation is at the following positions: position 44 of VH and position 100 of VL.

[0140] In some embodiments, the TRAV and VL comprise at least one cysteine ​​mutation to form a disulfide bond between TRAV and VL, preferably, the cysteine ​​mutation is at the following positions: 104 of TRAV and 80 of VL. In some embodiments, the TRBV and VL comprise at least one cysteine ​​mutation to form a disulfide bond between TRBV and VL, preferably, the cysteine ​​mutation is at the following positions: 84 of TRBV and 80 of VL. In some embodiments, the TRAV and VH comprise at least one cysteine ​​mutation to form a disulfide bond between TRAV and VH, preferably, the cysteine ​​mutation is at the following positions: 104 of TRAV and 89 of VH. In some embodiments, the TRBV and VH comprise at least one cysteine ​​mutation to form a disulfide bond between TRBV and VH, preferably, the cysteine ​​mutation is at the following positions: 104 of TRBV and 89 of VH.

[0141] In some embodiments, TRAV can be native TRAV or a functional variant thereof, as long as the functional variant retains the ability to bind to the target antigen. For example, TRAV can be a truncated variant of native TRAV, such as a variant with 8 amino acids truncated at the N-terminus or C-terminus. In some embodiments, TRBV can be native TRBV or a functional variant thereof, as long as the functional variant retains the ability to bind to the target antigen. In some embodiments, TRAV and TRBV are TRAV and TRBV of αβ TCR.

[0142] In some embodiments, the polypeptide molecule further comprises one or more amino acid mutations, insertions, or deletions that can enhance its affinity or biological activity (e.g., enhance its solubility, enhance its aggregation, enhance its stability, extend its half-life, reduce its immunogenicity) or reduce its post-translational modification (e.g., glycosylation modification), such as 1, 2, 3, 4, 5, or more amino acid mutations, insertions, or deletions. The mutated, inserted, or deleted amino acids include, but are not limited to: (a) neutral amino acids: Ser, Gln; (b) acidic amino acids: Asp; (c) long-chain aliphatic amino acids: Ile; (d) short aliphatic amino acids: Ala, Gly. In some embodiments, the amino acid mutations, insertions, or deletions are located at the following amino acid positions: the beginning of TRAV, TRBV, VL, VH, the boundary amino acids of TRAV / TRAC, the boundary amino acids of TRBV / TRBC, the hinge region and its adjacent amino acids, the boundary amino acids of CH2 / CH3, the C-terminus of CH3, and amino acids near the linker. In some embodiments, the amino acid mutations include mutations that enhance binding to FcRn and / or effector function silencing mutations. In some embodiments, mutations that enhance binding to FcRn include, for example, one or more of the following mutations: L234A, L235A, M252Y, S254T, T256E, M428L, N434S, T250R, M428L, N434A in the CH2 and CH3 domains, preferably L234A L235A, M252Y S254T T256E, M428L N434S and T250R M428L N434A. In some embodiments, the effector function silent mutation is at one or more of the following positions of the CH2-CH3 domain: 233, 234, 235, 236, 297, and 331; preferably, the effector function silent mutation is generated by replacing at least one residue at positions 233, 234, 235, 236, and 331 with the corresponding residue derived from IgG2 or IgG4. In some embodiments, amino acid mutations that can reduce post-translational modification of the polypeptide molecule include, for example, N113K in TRAC and N210D in TRBC.

[0143] In some embodiments, the polypeptide molecule further comprises a signal peptide sequence at the N-terminus of one or both polypeptide chains, preferably a signal peptide sequence at the N-terminus of each chain, such as a signal peptide sequence derived from albumin or immunoglobulin, preferably MGWSCIILFLVATATGVHS.

[0144] In some embodiments, the first antigen is a TAA selected from the group consisting of melanoma-associated antigens (e.g., gp100, MAGEA1, MAGEA3, MAGEA6, MAGEA4, MAGEA2, MAGEA12, MAGEA2B, MAGEA9B, MAGEA10, MAGEA11, MAGEB2, MAGEC1, MAGEC2), IGF2BP1, GNGT1, PI4K2B, CCR8, NPSR1, COX7B2, ONECUT3, SMC1B, FOXI3, GAGE2A, FBXO43, BRDT, PAGE2, GAGE13, POU5F1B, CTAG1A, and an endogenous reverse transcriptase antigen.

[0145] In some embodiments, the first antigen is a TSA selected from the group consisting of KRAS (e.g., G12D, G12V, G12C, G12R, G12A, G13D, Q61H, G125), TP53 (e.g., R175H, R173H, R273C, R248W, R248Q, R282W, Y220C, V157F, G245S, Y163C, R249S), PIK3CA (e.g., E542K, E545K, H1047R), CTNNB1 (e.g., S45P, T41A), EGFR (e.g., L858R, T790M), BRAF (e.g., V600E), and GNAS (e.g., R201C, R201H).

[0146] In some embodiments, the first antigen is a viral antigen selected from the group consisting of: HPV E6 or E7 antigens, CMV antigens, HBV antigens, EBV antigens, herpes virus antigens, human immunodeficiency virus (HIV) antigens, influenza virus antigens, and coronavirus antigens.

[0147] In some embodiments, the first antigen is an autoantigen selected from the group consisting of AFP, CEA, CD19, CD20, BCMA, CD22, CD30, SLAM, CLDN18.2, GD2, mesothelin, CD38, Her2, GPC3, MUC1, Ro52, Ro60, La, Jo-1, SRP, IFIH1, CENPA, CENPB, SNRPA1, SNRNP70, SNR-PD3, RNAP3, TOPO1, Insulin, GAD65, IA2, Znt8, PL7, TARS, ARS, MI2, topoisomerase 1, EXOSC9, EXOSC107, POLR3A, POLR3K, PTRN, GAD2, SLC30A8, AchR, MUSK, LRP4, PLA2R, THSD7A, TSHR, IFN-γ, CHRNA1, MUSK, LRP4, AQP4, MOG, GRIN1, COL4A3, PLA2R, GM-SCF, PR3, and MPO.

[0148] In some embodiments, the second antigen is selected from CD3 (e.g., CD3γ, CD3δ, and CD3ε chain), CD4, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD25, CD32a, CD32b, CD41, CD41b, CD42a, CD42b, CD44, CD45RA, CD49, CD61, CD64, CD68, CD94, CD90, CD117, Nkp46, NKG2D, FcεR I. TCRα / β, TCRγ / δ, HLA-DR, CD28, 4-1BB(CD137), OX40(CD134), ICOS(CD278), 2B4(CD244), HVEM, LAG3, DAP10, DAP12, CD27, CD40, GITR, LFA-1, MyD88, CD2, CD7, LIGHT, B7-H3, CTLA-4, PD-1, CD80, BTLA, TIM3, TIGIT and LAG-3.

[0149] In some embodiments, the first antigen is a TAA selected from the group consisting of melanoma-associated antigens (e.g., gp100, MAGEA1, MAGEA3, MAGEA6, MAGEA4, MAGEA2, MAGEA12, MAGEA2B, MAGEA9B, MAGEA10, MAGEA11, MAGEB2, MAGEC1, MAGEC2), IGF2BP1, GNGT1, PI4K2B, CCR8, NPSR1, COX7B2, ONECUT3, SMC1B, FOXI3, GAGE2A, FBXO43, BRDT, PAGE2, GAGE13, POU5F1B, CTAG1A, and an endogenous reverse transcriptase antigen; and the second antigen is selected from the group consisting of CD3 (e.g., CD3γ, CD3δ, and CD3ε chains), CD4, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD25, CD32a, CD32b, CD41, CD41b, CD42 a, CD42b, CD44, CD45RA, CD49, CD61, CD64, CD68, CD94, CD90, CD117, Nkp46, NKG2D, FcεR I. TCRα / β, TCRγ / δ, HLA-DR, CD28, 4-1BB(CD137), OX40(CD134), ICOS(CD278), 2B4(CD244), HVEM, LAG3, DAP10, DAP12, CD27, CD40, GITR, LFA-1, MyD88, CD2, CD7, LIGHT and B7-H3.

[0150] In some embodiments, the first antigen is a TSA selected from the group consisting of KRAS (e.g., G12D, G12V, G12C, G12R, G12A, G13D, Q61H, G125), TP53 (e.g., R175H, R173H, R273C, R248W, R248Q, R282W, Y220C, V157F, G245S, Y163C, R249S), PIK3CA (e.g., E542K, E545K, H1047R), CTNNB1 (e.g., S45P, T41A), EGFR (e.g., L858R, T790M), BRAF (e.g., V600E), and GNAS (e.g., R201C, R201H); and the second antigen is selected from the group consisting of CD3 (e.g., CD3γ, CD3δ, and CD3ε chains), CD4 , CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD25, CD32a, CD32b, CD41, CD41b, CD42a, CD42b, CD44, CD45RA, CD49, CD61, CD64, CD68, CD94, CD90, CD117, Nkp46, NKG2D, FcεRI, TCRα / β, TCRγ / δ, HLA-DR, CD28, 4-1BB(CD137), OX40(CD134), ICOS(CD278), 2B4(CD244), HVEM, LAG3, DAP10, DAP12, CD27, CD40, GITR, LFA-1, MyD88, CD2, CD7, LIGHT and B7-H3.

[0151] In some embodiments, the first antigen is a viral antigen selected from the group consisting of HPV E6 or E7 antigens, CMV antigens, HBV antigens, EBV antigens, herpes virus antigens, human immunodeficiency virus (HIV) antigens, influenza virus antigens, and coronavirus antigens; and the second antigen is selected from the group consisting of CD3 (e.g., CD3γ, CD3δ, and CD3ε chains), CD4, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD25, CD32a, CD32b, CD41, CD41b, CD42a, CD42b, CD44, CD45RA, CD49 , CD61, CD64, CD68, CD94, CD90, CD117, Nkp46, NKG2D, FcεRI, TCRα / β, TCRγ / δ, HLA-DR, CD28, 4-1BB(CD137), OX40(CD 134), ICOS (CD278), 2B4 (CD244), HVEM, LAG3, DAP10, DAP12, CD27, CD40, GITR, LFA-1, MyD88, CD2, CD7, LIGHT and B7-H3.

[0152] In some embodiments, the first antigen is an autoantigen selected from the group consisting of AFP, CEA, CD19, CD20, BCMA, CD22, CD30, SLAM, CLDN18.2, GD2, mesothelin, CD38, Her2, GPC3, MUC1, Ro52, Ro60, La, Jo-1, SRP, IFIH1, CENPA, CENPB, SNRPA1, SNRNP70, SNR-PD3, RNAP3, TOPO1, Insulin, GAD65, IA2, Znt8, PL7, TARS, ARS, MI2, topoisomerase 1, EXOSC9. , EXOSC107, POLR3A, POLR3K, PTRN, GAD2, SLC30A8, AchR, MUSK, LRP4, PLA2R, THSD7A, TSHR, IFN-γ, CHRNA1, MUSK, LRP4, AQP4, MOG, GRIN1, COL4A3, PLA2R, GM-SCF, PR3 and MPO; and the second antigen is selected from CTLA-4, PD-1, CD80, BTLA, TIM3, TIGIT and LAG-3.

[0153] In some embodiments, the first antigen is selected from gp100, MAGEA1, KRAS, and HPVE7.

[0154] In some embodiments, the second antigen is selected from CD3, CD28 and 4-1BB (CD137). In some embodiments, the second antigen is CD3. In some embodiments, the second antigen is CD28. In some embodiments, the second antigen is 4-1BB (CD137).

[0155] In some embodiments, the second antigen includes but is not limited to OKT3, UCHT-1, BMA031, and 12F6.

[0156] In some embodiments, the first polypeptide chain comprises from N-terminus to C-terminus: TRAV-first linker-second linker-VH-linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV-linker TRBC.

[0157] In some embodiments, the first polypeptide chain comprises from N-terminus to C-terminus: TRAV-first linker-second linker-VH-linker-TRAC-hinge region-CH2-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV-linker TRBC-hinge region-CH2-CH3.

[0158] In some embodiments, the first polypeptide chain comprises from N-terminus to C-terminus: TRAV-first linker-second linker-VH-linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV-linker TRBC-linker-ALB.

[0159] In some embodiments, the first antigen is gp100.

[0160] In some embodiments, the polypeptide molecule comprises: a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 2, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 2. In a preferred embodiment, the polypeptide molecule comprises: a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 1, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 2.

[0161] SEQ ID NO: 1

[0162] Format 22-2 (GPA2_TCD3_B22-2_M0) first chain:

[0163] SEQ ID NO:2

[0164] Format 22-2 (GPA2_TCD3_B22-2_M0) Second Chain:

[0165] In some embodiments, the polypeptide molecule comprises: a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 3, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 3, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 4, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 4. In a preferred embodiment, the polypeptide molecule comprises: a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 3, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 4.

[0166] SEQ ID NO:3

[0167] Format 64 (GPA2_TCD3_B64) first chain:

[0168] SEQ ID NO:4

[0169] Format 64 (GPA2_TCD3_B64) second chain:

[0170] In some embodiments, the polypeptide molecule comprises: a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 5, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 5, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 6. In a preferred embodiment, the polypeptide molecule comprises: a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 5, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 6.

[0171] SEQ ID NO:5

[0172] Format 63 (GPA2_TCD3_B63) first chain:

[0173] SEQ ID NO:6

[0174] Format 63 (GPA2_TCD3_B63) Second Chain:

[0175] In some embodiments, the first antigen is KRAS. In some embodiments, in the polypeptide molecule, TRAV comprises the CDR1 set forth by NSASQS (SEQ ID NO:89), the CDR2 set forth by VYSSGN (SEQ ID NO:90), and the CDR3 set forth by VVPGGTGGGNKLT (SEQ ID NO:91); and TRBV comprises the CDR1 set forth by LGHDT (SEQ ID NO:92), the CDR2 set forth by YNNKEL (SEQ ID NO:93), and the CDR3 set forth by ASSHWGAQETQY (SEQ ID NO:94).

[0176] In some embodiments, TRAV comprises RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVPGGTGGGNKLTFGTGTQLKVEL (SEQ ID NO: 95). In some embodiments, TRAV comprises RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVPGGTGGGNKLTFGTGTQLPVPL (SEQ ID NO: 96). In some embodiments, TRAV comprises RKIVEQDPGPFEVPEGATVAFICTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQDIHLLIRDSKLSDSATYLCVVPGGTGGGNKLTFGTGTQLPVPL (SEQ ID NO: 97).

[0177] In some embodiments, TRBV comprises DTAVSQTPKYLVTQMGNDKSIKCEQNLGHDTMDWYKQDSKKFLKIMFSYNNKELIINETVPNRFSPKSPDKAHLNLHINSLELGDSAVYFCASSHWGAQETQYFGPGTRLLVL (SEQ ID NO: 98).

[0178] In some embodiments, the polypeptide molecule comprises: a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 99, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 99, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 100, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 100. In a preferred embodiment, the polypeptide molecule comprises: a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 99, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 100.

[0179] SEQ ID NO:99

[0180] Format 22-2 (KVA11-N03_B22-2_WT) first chain:

[0181] SEQ ID NO: 100

[0182] Format 22-2 (KVA11-N03_B22-2_WT) Second Chain:

[0183] In some embodiments, the polypeptide molecule comprises: a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 101, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 101, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 102, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 102. In a preferred embodiment, the polypeptide molecule comprises: a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 101, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 102.

[0184] SEQ ID NO: 101

[0185] Format 22-2 (KVA11-N03_B22-2_Mut1) first chain:

[0186] SEQ ID NO:102

[0187] Format 22-2 (KVA11-N03_B22-2_Mut1) Second Chain:

[0188] In some embodiments, the polypeptide molecule comprises: a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 103, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 103, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 104, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 104. In a preferred embodiment, the polypeptide molecule comprises: a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 103, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 104.

[0189] SEQ ID NO:103

[0190] Format 22-2 (KVA11-N03_B22-2_Mut2) first chain:

[0191] SEQ ID NO:104

[0192] Format 22-2 (KVA11-N03_B22-2_Mut2) Second Chain:

[0193] In some embodiments, the polypeptide molecule comprises: a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 105, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 105, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 106, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 106. In a preferred embodiment, the polypeptide molecule comprises: a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 105, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 106.

[0194] SEQ ID NO:105

[0195] Format 22-2 (KVA11-N03_B22-2_Mut3) first chain:

[0196] SEQ ID NO:106

[0197] Format 22-2 (KVA11-N03_B22-2_Mut3) Second Chain:

[0198] In some embodiments, the first antigen is MAGE-A1.

[0199] In some embodiments, in the polypeptide molecule, TRAV comprises RGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAGSGGGTDKLIFGTGTRLQVFPN (SEQ ID NO: 107). In some embodiments, in the polypeptide molecule, TRAV comprises RGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYTWPHMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAGSGGGTDKLIFGTGTRLQVFPN (SEQ ID NO: 108).

[0200] In some embodiments, in the polypeptide molecule, TRBV comprises GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSDYQTCVTYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSAREPGQGPFEQYFGPGTRLTVTE (SEQ ID NO: 109). In some embodiments, in the polypeptide molecule, TRBV comprises GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSAREPGQGPYEQYFGPGTRLTVTE (SEQ ID NO: 110).

[0201] In some embodiments, the first antigen is an HPV antigen, such as HPV E7.

[0202] In some embodiments, in some embodiments, in the polypeptide molecule, TRAV comprises the CDR1 shown by NSASQS (SEQ ID NO:111), the CDR2 shown by VYSSGN (SEQ ID NO:112), and the CDR3 shown by AVISAGTALI (SEQ ID NO:113); TRBV comprises the CDR1 shown by SGHDT (SEQ ID NO:114), the CDR2 shown by YYEEEE (SEQ ID NO:115), and the CDR3 shown by ASSLGWRGGLYTEAF (SEQ ID NO:116).

[0203] In some embodiments, TRAV comprises RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDG RFTAQLNRASQYISLLIRDSKLSDSATYLCAVISAGTALIFGKGTTLSVSS (SEQ ID NO: 117).

[0204] In some embodiments, TRBV comprises DAGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSLGWRGGLYTEAFFGQGTRLTVV (SEQ ID NO: 118).

[0205] On the other hand, the present invention provides a T cell receptor (TCR) comprising a TCR α chain variable region (TRAV) and a TCR β chain variable region (TRBV), wherein the TRAV comprises CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91, respectively, or functional variants formed by inserting, deleting or substituting one or several amino acids; and / or the TRBV comprises β chain CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NO: 92, SEQ ID NO: 93 and SEQ ID NO: 94, respectively, or functional variants formed by inserting, deleting or substituting one or several amino acids.

[0206] In some embodiments, the TRAV comprises CDR1, CDR2 and CDR3 having the amino acid sequences shown as SEQ ID NO:89, SEQ ID NO:90 and SEQ ID NO:91, respectively; and / or the TRBV comprises β chain CDR1, CDR2 and CDR3 having the amino acid sequences shown as SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94, respectively.

[0207] In some embodiments, the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:95, and / or the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:98.

[0208] In some embodiments, the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:96, and / or the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:98.

[0209] In some embodiments, the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:97, and / or the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:98.

[0210] In another aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding each chain of the bispecific polypeptide molecule of the present invention. The present invention also provides a nucleic acid comprising a nucleotide sequence encoding the TCR of the present invention.

[0211] In another aspect, the present invention provides a vector comprising the nucleic acid of the present invention.

[0212] Any suitable vector can be used in the present invention.The example of vector includes but is not limited to plasmid, viral vector (including retroviral vector, lentiviral vector, adenoviral vector, vaccinia virus vector, polyoma virus vector and adenovirus-associated vector (AAV)), phage, phagemid, cosmid and artificial chromosome (including BAC and YAC).The vector itself is usually a nucleotide sequence, usually a DNA sequence comprising an insert (transgene) and a larger sequence as a vector "skeleton". The engineered vector is usually included in the starting point (if stable expression of polynucleotides is required) of autonomous replication in the host cell, a selective marker and a restriction enzyme cleavage site (such as a multiple cloning site, MCS). The vector can additionally include a promoter, a genetic marker, a reporter gene, a targeting sequence and / or a protein purification tag. As known to those skilled in the art, a large amount of suitable vectors are known to those skilled in the art, and many are commercially available. In J.Sambrook et al., Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2012), an example of a suitable vector is provided, which is incorporated herein by reference in its entirety.

[0213] In some embodiments, the vector is preferably selected from the group consisting of a lentiviral vector, a retroviral vector, a plasmid, a DNA vector, an mRNA vector, a transposon-based vector, and an artificial chromosome.

[0214] In yet another aspect, the present invention provides a vector system comprising, on one or more vectors, a nucleotide sequence encoding each chain of a bispecific polypeptide molecule of the present invention.

[0215] In another aspect, the present invention provides a host cell comprising the nucleic acid, vector or vector system of the present invention.

[0216] Any cell can be used as a host cell for nucleic acid or vector of the present invention. The cell can be a eukaryotic cell, for example, a plant (not having the potential to develop into a plant), an animal, a fungus or an algae, or can be a prokaryotic cell, for example, a bacterium or a protozoan. The cell can be a cultured cell or a primary cell, i.e., directly isolated from an organism, such as a human. The cell can be an adherent cell or a suspension cell, i.e., a cell grown in suspension. Suitable host cells are known in the art and include, for example, DH5α Escherichia coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For the purpose of producing polypeptide molecules of the present invention, the cell is preferably a mammalian cell. Most preferably, the host cell is a human cell.

[0217] In some embodiments, the cell is selected from lymphocytes (e.g., T cells, NK cells), monocytes (e.g., PBMCs), and stem cells. For example, stem cells can be lymphocyte progenitor cells, induced pluripotent stem cells (iPSCs), or hematopoietic stem cells (HSCs). In some embodiments, stem cells do not include embryonic stem cells obtained by destroying human embryos, and / or do not include totipotent stem cells for developing and forming individual animals.

[0218] In another aspect, the present invention provides a conjugate comprising a bispecific polypeptide molecule of the present invention and a chemical moiety conjugated to the bispecific polypeptide molecule. The present invention also provides a conjugate comprising a TCR of the present invention and a chemical moiety conjugated to the TCR.

[0219] In some embodiments, the chemical moiety is selected from the group consisting of a therapeutic agent, an immunostimulatory molecule, and a detectable label.

[0220] In some embodiments, therapeutic agents include but are not limited to immunomodulators, radioactive compounds, enzymes (e.g., perforin), chemotherapeutic agents (e.g., cisplatin), or toxins. In some embodiments, therapeutic agents can be, for example, maytansine, geldanamycin, tubulin inhibitors such as tubulin binders (e.g., auristatins), or minor groove binders such as calicheamicin.

[0221] Other suitable therapeutic agents include, for example, small molecule cytotoxic agents, i.e., compounds with a molecular weight of less than 700 Daltons that have the ability to kill mammalian cells. Such compounds may also contain toxic metals that can have cytotoxic effects. Furthermore, it should be understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that decompose or transform under physiological conditions to release the cytotoxic agent. Examples of such agents include cisplatin, maytansine derivatives, raclethines, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer porphyrin sodium II, temozolomide, topotecan, metformin, auristatin E, vinca alkaloids, and doxorubicin; peptide cytotoxins, i.e., proteins or fragments thereof that have the ability to kill mammalian cells, such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNA enzymes, and RNA enzymes; radionuclides, i.e., unstable isotopes of elements that decay with the simultaneous emission of one or more alpha or beta particles or gamma rays, such as iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210, bismuth-213, actinium-225, and astatine-213; and chelating agents, which can be used to facilitate the binding of these radionuclides to molecules or polymers thereof.

[0222] In some embodiments, the immunostimulatory molecule is an immune effector molecule that stimulates an immune response. For example, the immunostimulatory molecule can be a cytokine such as IL-2 and IFN-γ, a chemokine such as IL-8, platelet factor 4, melanoma growth stimulating protein, a complement activator; a viral / bacterial protein domain, or a viral / bacterial peptide. In a preferred embodiment, the immunostimulatory molecule is selected from a cytokine, a chemokine, a platelet factor, and a complement initiator.

[0223] In some embodiments, the detectable label can be selected from biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, or a fluorescent, phosphorescent, or chemiluminescent molecule. Detectable moieties for diagnostic purposes include, for example, fluorescent labels, radioactive labels, enzymes, nucleic acid probes, and contrast agents.

[0224] In another aspect, the present invention provides a pharmaceutical composition comprising the bispecific polypeptide molecule, nucleic acid, vector, vector system, host cell, or conjugate of the present invention, and a pharmaceutically acceptable excipient.

[0225] The pharmaceutical compositions of the present invention are particularly suitable for administration to humans; however, they are also suitable for administration to non-human animals. The compositions and their components (i.e., the active agent and optional excipients) are preferably pharmaceutically acceptable, i.e., capable of eliciting the desired therapeutic effect in the recipient without causing any undesirable local or systemic effects. The pharmaceutical compositions of the present invention can, for example, be sterile.

[0226] The example of excipient includes but is not limited to filler, binding agent, disintegrant, coating agent, adsorbent, antiadherent, glidant, preservative, antioxidant, flavoring, coloring agent, sweetener, solvent, cosolvent, buffer, chelating agent, viscosity imparting agent, surfactant, diluent, wetting agent, carrier, diluent, preservative, emulsifying agent, stabilizer and tension regulator. It is known to those skilled in the art that suitable excipient is selected to prepare pharmaceutical composition of the present invention. The exemplary carrier for pharmaceutical composition of the present invention comprises saline, buffered saline, glucose and water. Usually, the selection of suitable excipient especially depends on the desired dosage form of employed activating agent, disease to be treated and composition.

[0227] Depending on the active agent employed, the pharmaceutical composition of the present invention can be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, and in particular can be in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tinctures or fluid extracts, or in a form particularly suitable for the desired method of administration. Known processes for producing pharmaceuticals are shown in Remington's Pharmaceutical Sciences, 22nd edition (Ed. Maack Publishing Co, Easton, Pa., 2012), and may include, for example, conventional mixing, dissolving, granulating, sugar-coating, grinding, emulsifying, encapsulating, embedding or lyophilizing processes.

[0228] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent, preferably, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, and a small molecule drug.

[0229] Preferred examples of the second therapeutic agent include known anticancer drugs such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, auristatin E, E), vincristine and doxorubicin; and peptide cytotoxins, such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNA enzymes and RNA enzymes; radionuclides, such as iodine-131, rhenium-186, indium-111, iridium-90, bismuth-210 and 213, actinium-225 and astatine-213; prodrugs, such as antibody-directed enzyme prodrugs; immunostimulants, such as IL-2, chemokines such as IL-8, platelet factor 4; antibodies or fragments thereof, such as anti-CD3 antibodies or fragments thereof; complement activators; viral / bacterial protein domains and viral / bacterial peptides.

[0230] In another aspect, the present invention provides a method for preventing or treating a disease in a subject, comprising administering to the subject an effective amount of the bispecific polypeptide molecule, TCR, nucleic acid, vector, vector system, host cell, conjugate, or pharmaceutical composition of the present invention, wherein the disease is selected from cancer, infectious disease, autoimmune disease, and inflammatory disease.

[0231] The cancer can be any cancer, such as cancers of the hematological system, cancers of the central and peripheral nervous systems, cancers of the lymphoid lineage, cancers of the myeloid lineage, cancers of mesenchymal origin, solid tumors, and the like. Examples of cancer include, but are not limited to, acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer, vaginal cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumors, glioma, Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal cancer, omental cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, ureter cancer, and bladder cancer. In some embodiments, the cancer is a KRAS expression-associated cancer. In some embodiments, the cancer is a MAGE-A1 expression-associated cancer.

[0232] Infectious diseases are diseases caused by infection with pathogens, including infectious diseases and non-infectious diseases. Pathogens that cause infectious diseases include viruses, bacteria, mycoplasmas, chlamydia, rickettsiae, prions, fungi, spirochetes, and parasites. Examples of viruses include, but are not limited to, HPV, CMV, HBV, EBV, herpes viruses, human immunodeficiency virus (HIV), influenza viruses, and coronaviruses.

[0233] Common autoimmune and inflammatory diseases include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, psoriatic arthritis, autoimmune destruction of pancreatic islets leading to diabetes, Sjögren's syndrome, Hashimoto's thyroiditis, Graves' thyroiditis, toxic diffuse goiter, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease (such as ulcerative colitis and Crohn's disease), psoriasis, glomerulonephritis, nephrotic syndrome, anti-glomerular basement membrane Disease, membranous nephropathy, systemic sclerosis, polymyositis, myasthenia gravis, psoriasis, pemphigus, vitiligo, autoimmune diseases of the central nervous system, celiac disease, autoimmune gastritis, primary biliary cholangitis, autoimmune hepatitis, Goodpasture's syndrome, autoimmune oophoritis, autoimmune orchitis, idiopathic leukopenia, primary adrenal cortical atrophy, antiphospholipid antibody syndrome, post-infectious fibrosis, post-myocardial infarction fibrosis, liver fibrosis, and scar hyperplasia.

[0234] In some embodiments, the dosage administered to a subject may vary with the embodiment, the drug used, the method of administration, and the site and subject being treated. However, the dosage should be sufficient to provide a therapeutic response. A clinician can determine an effective amount to administer to a person or other subject for the treatment of a medical condition. The precise amount required for effective treatment may depend on many factors, such as the activity of the active agent and the route of administration.

[0235] The dosage of the polypeptide molecule, conjugate or pharmaceutical composition of the present invention can be administered to a mammal at once or in a series of sub-doses over an appropriate time period, for example, daily, semi-weekly, weekly, bi-weekly, semi-monthly, bi-monthly, bi-monthly or annually as needed. A dosage unit comprising an effective amount of the polypeptide molecule, conjugate or pharmaceutical composition can be administered as a single daily dose, or the total daily dose can be administered as two, three, four or more divided doses administered daily as needed.

[0236] Suitable modes of administration can be selected by a physician. The route of administration can be parenteral administration, for example, by injection, nasal administration, pulmonary administration or transdermal administration. Systemic or local administration can be performed by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. In some embodiments, the polypeptide molecule, conjugate or pharmaceutical composition is selected for parenteral delivery, inhalation or delivery through the digestive tract, for example, orally. The dosage and method of administration can vary according to the weight, age, condition, etc. of the subject and can be appropriately selected.

[0237] In some embodiments, the method further comprises administering a second therapeutic agent to the subject. In certain embodiments, the polypeptide molecule, conjugate, or pharmaceutical composition of the present invention is administered before, substantially simultaneously with, or after the administration of the second therapeutic agent. Preferably, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, and a small molecule drug. Preferred examples of the second therapeutic agent are described above.

[0238] In another aspect, the present disclosure provides a method for detecting (e.g., diagnosing) a disease in a subject, wherein the method comprises (i) contacting a sample obtained from the subject with a polypeptide molecule, TCR or conjugate of the invention; and (ii) detecting the presence of a target antigen in the sample, wherein the presence of the target antigen indicates that the subject has the corresponding disease.

[0239] The target antigen can be selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, and an autoantigen. The disease can be selected from a cancer, an infectious disease, an autoimmune disease, and an inflammatory disease. In some embodiments, the target antigen is KRAS. In some embodiments, the antigen is MGAE-A1. The cancer, infectious disease, autoimmune disease, and inflammatory disease are as defined above.

[0240] In some embodiments, the sample obtained from the subject can be a blood sample, a urine sample, a tissue sample, or a cell sample. In certain embodiments, the method is performed in vitro. In some embodiments, the method comprises (i) contacting a sample obtained from the subject with a conjugate of the present invention, wherein the conjugate comprises a detectable label; and (ii) detecting the presence of the target antigen in the sample by detecting the detectable label. Examples of detectable labels include, but are not limited to, 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; preferably an enzyme or a catalytically active fragment thereof, a radionuclide, a fluorescent, phosphorescent, or chemiluminescent molecule.

[0241] In yet another aspect, the present disclosure provides a kit comprising the polypeptide molecule, TCR or conjugate of the present disclosure, wherein the kit is used to detect the presence of a target antigen in a test sample.

[0242] In some embodiments, the kit is a kit for detecting (e.g., diagnosing) a disease in a subject, comprising a polypeptide molecule or conjugate of the present disclosure. The target antigen can be selected from tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), viral antigens, and autoantigens. The disease can be selected from cancer, infectious diseases, autoimmune diseases, and inflammatory diseases. The cancer, infectious diseases, autoimmune diseases, and inflammatory diseases are as defined above.

[0243] In some embodiments, the conjugate comprises a detectable label. Examples of detectable labels include, but are not limited to, biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, a nucleic acid probe, a contrast agent, and a fluorescent, phosphorescent, or chemiluminescent molecule; preferably, an enzyme or catalytically active fragment thereof, a radionuclide, a fluorescent, phosphorescent, or chemiluminescent molecule. In some embodiments, the kit may further comprise instructions for use of the kit.

[0244] In another aspect, the present disclosure provides use of a bispecific polypeptide molecule, TCR, nucleic acid, vector, vector system, host cell, conjugate or pharmaceutical composition of the invention in the preparation of a medicament for treating or preventing a disease in a subject.

[0245] In yet another aspect, the disclosure provides a bispecific polypeptide molecule, TCR, nucleic acid, vector, vector system, host cell, conjugate, or pharmaceutical composition of the invention for use in treating or preventing a disease in a subject.

[0246] In another aspect, the present disclosure provides use of the bispecific polypeptide molecule, TCR or conjugate of the present invention in preparing a kit for detecting the presence of a target antigen in a test sample.

[0247] In some embodiments, the present disclosure provides use of a bispecific polypeptide molecule, TCR, or conjugate of the invention in the preparation of a kit for detecting (eg, diagnosing) a disease in a subject.

[0248] In yet another aspect, the present disclosure provides a bispecific polypeptide molecule, TCR, or conjugate of the present invention for use in detecting the presence of a target antigen in a test sample.

[0249] In some embodiments of the use of the present disclosure, the target antigen can be selected from tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), viral antigens, and autoantigens. The disease can be selected from cancer, infectious diseases, autoimmune diseases, and inflammatory diseases. The cancer, infectious diseases, autoimmune diseases, and inflammatory diseases are as defined above. BRIEF DESCRIPTION OF THE DRAWINGS

[0250] Figure 1 shows a structural schematic diagram of a specific form of format 22-2 of the present invention, an AlphaFold2 diagram, an immunoblotting result diagram, and an ELISPOT assay result diagram.

[0251] FIG2 shows a schematic structural diagram of a specific form of format 64 of the present invention, a diagram of immunoblotting results, and a diagram of ELISPOT assay results.

[0252] FIG3 shows a schematic structural diagram of a specific form of the format 63 of the present invention.

[0253] FIG4 shows a schematic diagram of the structure of a specific form of control format 17, protein staining and immunoblotting results, and ELISPOT assay results.

[0254] FIG5 shows a schematic diagram of the structure of a specific form of control format 22, protein staining and immunoblotting results, and ELISPOT assay results.

[0255] FIG6 shows the results of ELISPOT assay of format 22-2 of the present invention using KRAS as the target peptide. DETAILED DESCRIPTION

[0256] The present invention is further described by the following specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, were carried out according to conventional conditions in the art, for example, the conditions described in Sambrook and Russeii et al., Molecular Cloning: A Laboratory Manual (3rd Edition) (2001), CSHL Press, or according to the conditions recommended by the manufacturer. Unless otherwise stated, the experimental materials and reagents used in the following examples are all commercially available.

[0257] Example 1 Construction of TCR-based multispecific polypeptide molecules

[0258] 1.1 Construction of multispecific peptide molecules targeting gp100

[0259] Various TCR-based bispecific polypeptide molecules, format 22-2 (a first chain as shown in SEQ ID NO: 1 and a second chain as shown in SEQ ID NO: 2), format 64 (a first chain as shown in SEQ ID NO: 3 and a second chain as shown in SEQ ID NO: 4), and format 63 (a first chain as shown in SEQ ID NO: 5 and a second chain as shown in SEQ ID NO: 6), were designed and constructed. The structures of these formats are shown in Figures 1-3. Three-dimensional structural diagrams of these formats are also displayed using AlphaFold2.

[0260] In addition, to demonstrate the advantages of using TRAC and TRBC in the formats of the present invention over other constant regions (e.g., CH3 or hinge region (CH2-CH3) of an antibody), formats 17 and 22 were also constructed as controls. Format 22-2 of the present invention replaces TRAC and TRBC with hinge region (CH2-CH3) to create Format 17; format 22-2 of the present invention replaces TRAC and TRBC with CH3 to create Format 22. The structure of Format 17 is as follows: the first polypeptide chain, from N-terminus to C-terminus, comprises: TRAV-linker-VH-optional linker-hinge region-CH2-CH3; and the second polypeptide chain, from N-terminus to C-terminus, comprises: VL-linker-TRBV-optional linker-hinge region-CH2-CH3. The structure of Format 22 is as follows: the first polypeptide chain, from N-terminus to C-terminus, comprises: TRAV-linker-VH-optional linker-CH3; and the second polypeptide chain, from N-terminus to C-terminus, comprises: VL-linker-TRBV-optional linker-CH3. The structural diagrams of Format 17 and Format 22 are shown in Figure 4 and Figure 5 respectively.

[0261] The amino acid sequence components used in these formats are as follows:

[0262] TRAV

[0263] >TRAV of gp100-specific TCR

[0264] >TRAV of gp100-specific TCR containing the A104C mutation

[0265] TRAC

[0266] >TRAC contains T48C, N113K, FFPSPESS deletion mutations

[0267] >TRAC contains T48C, N113K, PESS deletion mutations

[0268] >TRAC contains T48C and N113K mutations

[0269] >TRAC

[0270] TRBV

[0271] >TRBV of gp100-specific TCR

[0272] >TRBV of gp100-specific TCR containing the N84C mutation

[0273] TRBC

[0274] >TRBC2, including S57C, C187A, and N210D mutations

[0275] >TRBC2, including S57C, C187A, N210D, and FG loop deletion mutations

[0276] >TRBC1, including S57C, C187A, and N210D mutations

[0277] >TRBC1, including S57C, C187A, N210D, and FG loop deletion mutations

[0278] >TRBC

[0279] VH

[0280] >VH1 (anti-CD3 antibody huUCHT1, containing the G44C mutation)

[0281] >VH1 (anti-CD3 antibody huUCHT1, containing G44C and E89C mutations)

[0282] >VH2 (anti-CD28 antibody, containing the C50Y mutation)

[0283] >VH2 (anti-CD28 antibody, containing C50Y and D89C mutations)

[0284] >VH-BB1 (anti-4-1BB antibody, monovalent)

[0285] >VH-BB2 (anti-4-1BB antibody, bivalent)

[0286] VL

[0287] >VL1 (anti-CD3 antibody huUCHT1, containing the E100C mutation)

[0288] >VL1 (anti-CD3 antibody huUCHT1, containing E80C and E100C mutations)

[0289] >VL2 (anti-CD28 antibody)

[0290] >VL2 (anti-CD28 antibody containing the E80C mutation)

[0291] >VL-BB1 (anti-4-1BB antibody, monovalent)

[0292] >VL-BB2 (anti-4-1BB antibody, bivalent)

[0293] Hinge-CH2-CH3

[0294] >Hinge-CH2-CH3 hole containing a disulfide bond (including T366S, L368A and Y407V hole mutations and Y349C mutation)

[0295] >Hinge containing a disulfide bond - CH2-CH3 Knob (containing T366W knob mutation and S354C mutation)

[0296] >Hinge without disulfide bond -CH2-CH3 hole (including T366S, L368A and Y407V hole mutations and Y349C mutation)

[0297] >Hinge without disulfide bond - CH2-CH3 Knob (containing T366W knob mutation and S354C mutation)

[0298] CH2-CH3

[0299] CH2-CH3 Hole (including T366S, L368A, Y407V and Y349C mutations)

[0300] CH2-CH3 Knob (including T366W knob mutation and S354C mutation)

[0301] CH3

[0302] >CH3 Hole (including T366S, L368A, and Y407V hole mutations and Y349C mutation)

[0303] >CH3 Knob (containing T366W knob mutation and S354C mutation)

[0304] Alb

[0305] connector

[0306] S (SEQ ID NO: 41)

[0307] GGGGS (SEQ ID NO:42)

[0308] GGGGSGGGGSGGGGSGGGGSGGGS(SEQ ID NO:43)

[0309] GGGGSGGGGSGGGGS (SEQ ID NO: 44)

[0310] GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO:45)

[0311] GGGGSGGGGS (SEQ ID NO: 46)

[0312] GGSGGS (SEQ ID NO: 47)

[0313] GGSGGSGGS (SEQ ID NO: 48)

[0314] GQPKAAP (SEQ ID NO: 49)

[0315] GGGSGGGG (SEQ ID NO: 50)

[0316] RTSGPGDGGKGGPGKGPGGEGTKGTGPGG(SEQ ID NO:51)

[0317] GGEGGGSEGGGS (SEQ ID NO: 52)

[0318] TVLRT (SEQ ID NO: 53)

[0319] TVSSAS (SEQ ID NO: 54)

[0320] GGEGG (SEQ ID NO: 55)

[0321] GSEGGGS (SEQ ID NO: 56)

[0322] RTSGPGDGGKGGPGKGPGGEGTKGTGPGG(SEQ ID NO:57)

[0323] GKGPGGEGTKGTGPGG(SEQ ID NO:58)

[0324] TVLSSAS (SEQ ID NO: 59)

[0325] EDL (SEQ ID NO: 60)

[0326] ANI (SEQ ID NO: 61)

[0327] PNI (SEQ ID NO: 88)

[0328] The exemplary amino acid sequences of the two polypeptide chains of control format 17 are shown below:

[0329] First Chain

[0330] Second Chain

[0331] Exemplary amino acid sequences of the two polypeptide chains of control format 22 are shown below:

[0332] First Chain

[0333] Second Chain

[0334] The signal peptide sequence MGWSCIILFLVATATGVHS (SEQ ID NO:66) was added to the N-terminus of each polypeptide chain of these formats to enhance secretion of the polypeptide molecules, and a His6 tag was added to the C-terminus of each chain of the format for purification. The formats in this example are capable of binding to the complex of the gp100 epitope YLEPGPVTA (SEQ ID NO:67) or its mutant form (YLEPGPVTV, SEQ ID NO:68) with HLA-A*02.

[0335] 1.2 Construction of multispecific peptide molecules targeting KRAS

[0336] A multispecific polypeptide molecule format targeting KRAS was constructed as described in Example 1.1.

[0337] As an example, based on these sequences, the foramt 22-2 molecules targeting KRAS, KVA11-N03_B22-2_WT (the amino acid sequences of its two chains are shown in SEQ ID NOs: 99 and 100, respectively), KVA11-N03_B22-2_Mut1 (the amino acid sequences of its two chains are shown in SEQ ID NOs: 101 and 102, respectively), KVA11-N03_B22-2_Mut2 (the amino acid sequences of its two chains are shown in SEQ ID NOs: 103 and 104, respectively), and KVA11-N03_B22-2_Mut3 (the amino acid sequences of its two chains are shown in SEQ ID NOs: 105 and 106, respectively) were constructed.

[0338] The format in this example can bind to the complex of KRAS antigen epitope VVGAVGVGK (SEQ ID NO: 119) and HLA-A*11.

[0339] 1.3 Construction of multispecific peptide molecules targeting MAGEA1

[0340] A multispecific polypeptide molecule format targeting MAGEA1 was constructed as described in Example 1.1. The structure of the format was the same as that in Example 1.1, except that TRAV and TRBV of the gp100-specific TCR in Example 1.1 were replaced by TRAV and TRBV of the MAGEA1-specific TCR.

[0341] The amino acid sequences of TRAV and TRBV of the MAGEA1-specific TCR are shown below:

[0342] TRAV

[0343] TRBV

[0344] The format in this example can bind to the complex of MAGEA1 antigen epitope KVLEYVIKV (SEQ ID NO: 120) and HLA-A*02.

[0345] Example 2 Expression and purification of TCR-based multispecific polypeptide molecules

[0346] 1. Vector Construction and Plasmid Extraction

[0347] The nucleotide sequence encoding one or more polypeptide chains of the format in Example 1 was directly cloned into the expression vector pTT5. The obtained vector clones were confirmed by sequencing and then amplified and cultured, and plasmids were extracted.

[0348] 2. Transfection

[0349] Adjust the CHO cell density to 1×10 6 Cells were diluted to 40 mL per bottle, and the volume of each cell solution was 40 mL. The bottle cap was then tightened and placed on a shaker for continued culture. After 2-4 hours of culture at 36.5°C, 175 rpm, and 5% CO2, the plasmid was transfected. Prepare the transfection solution (1 mL): Dilute 10 μg of DNA with approximately 800 μL of 150 mM sterile NaCl solution, mix thoroughly, and let it sit on the bench for 5 minutes. Add approximately 50 μL of transfection reagent to the DNA dilution and mix thoroughly, for a final total volume of 1 mL. After 10 minutes on the bench, the transfection solution was added dropwise to the cell culture medium. After shaking, the bottle cap was tightened and placed back on a shaker (36.5°C, 5% CO2 off, 175 rpm). 20-24 hours after transfection, SMS 293-I feed solution (0.7 mL / bottle) was added. The culture was then fed every other day for 6-10 days, and the culture supernatant was collected.

[0350] 3. Purification

[0351] The collected culture supernatant was subjected to nickel column purification. A 1 ml packed nickel column was prepared, rinsed with 10 ml sterile water, and balanced with 10 ml 1X Bind Buffer. The culture supernatant was centrifuged at 3000 rpm for 5 min, then filtered through 450 nm chlorine gas and loaded. After loading, the column was washed with 10 ml 1X Bind Buffer, 10 ml 1X Bind Buffer, and 10 ml 1X Wash Buffer (collecting 10 tubes of eluent at a flow rate of about 0.3-0.4 ml / min). The protein was eluted with 1.2 ml 1X Elution Buffer. The collected eluent was the purified protein and stored at -80°C after aliquoting.

[0352] 4. Protein Staining and Immunoblotting

[0353] The purified protein was subjected to denaturing (R) or non-denaturing (NR) treatment followed by SDS-PAGE gel electrophoresis and Coomassie blue staining to assess protein expression purity and aggregation. Another set of parallel samples was subjected to gel electrophoresis and then subjected to western blot. Mouse anti-His6 was used as the primary antibody to detect the His6 tag of the purified protein to confirm the reliability of the Coomassie blue staining. The results demonstrated that the format provided by the present invention could be successfully expressed.

[0354] Exemplary western blot results are shown in Figures 1 and 2. The concentration of the target protein was estimated based on the harvested target protein yield, and the purity was determined by SDS-PAGE Coomassie blue staining. This demonstrates that the format provided by the present invention can be successfully expressed.

[0355] Example 3 ELISPOT assay

[0356] 3.1 ELISPOT assay of culture supernatant

[0357] The culture supernatant collected in Example 2 of the present invention is gradiently diluted (usually 2-fold dilution and 5-fold dilution are used). The corresponding target peptide and non-related peptide are then dissolved in DMSO and diluted with water to a working concentration of 10-4M. T2 cells are loaded with 10-6M target peptide and non-related peptide respectively. 1640 complete culture medium containing 10% FBS is added to the ELISPOT plate and blocked at room temperature for 30 minutes. The culture medium is discarded, and 5x105 cells / mL of PBMC (100μL / well), 5x105 cells / mL of T2 cells loaded with polypeptide (100μL / well), and different concentrations of format (2-fold dilution, 5-fold dilution) are added to the plate. The negative control is added with T2 cells and PBMC that are not loaded with polypeptide, and the concentration of the candidate polypeptide molecule is consistent with that of the experimental group; the positive control is PBMC with 10μL of the anti-CD3-2 mAb that comes with the kit as a positive control. After adding all the samples, cover the plate and place it on After culturing in a 5% CO 2 incubator for 20-24 hours, the secretion of IFN-γ was determined by ELISPOT assay to evaluate the immune cell activation induced by the target antigen peptide-MHC complex of the format of the present invention.

[0358] The results showed that the format of the present invention can activate immune cells in the presence of target antigens, but cannot activate immune cells in the negative control group with irrelevant target peptides or without adding polypeptides.

[0359] Exemplary results of the formats of the present invention are shown in FIG6 , wherein the culture supernatants of format 22-2 with different amino acid sequences can activate immune cells at 2-fold and 5-fold dilutions.

[0360] 3.2 ELISPOT assay of purified protein

[0361] The purified format of Example 1 of the present invention was diluted in gradients (10 -7 to 10 -13 M). gp100 polypeptide, KRAS polypeptide, and MAGE-A1 polypeptide were dissolved in DMSO, and then diluted with water to a working concentration of 10-4 M. were loaded with T2 cells 10 -6 M gp100 peptide, HPV E7 peptide, MAGE-A1 peptide. Add 1640 complete medium containing 10% FBS to the ELISPOT plate and block for 30 minutes at room temperature. Discard the medium and add 5x10 5 cells / mL of PBMC (100 μL / well), 5x10 5 T2 cells loaded with peptides (100 μL / well) and different concentrations of format (10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 M). Negative controls included unloaded T2 cells and PBMCs, with the candidate peptide concentration consistent with that in the experimental group. A positive control consisted of PBMCs supplemented with 10 μL of the anti-CD3-2 mAb provided with the kit. After all samples were added, the plate was covered and incubated in a 37°C, 5% CO2 incubator for 20-24 hours. IFN-γ secretion was then determined by ELISPOT assay to assess immune cell activation induced by the target antigen peptide-MHC complex.

[0362] The results showed that the format of the present invention can activate immune cells in the presence of target antigens.

[0363] Exemplary results of the formats of the present invention are shown in Figures 1 and 2 (the reference symbol "+" indicates an experimental group to which a gp100 polypeptide was added, "-" indicates a negative control group to which no gp100 polypeptide was added, "irrelevant" indicates a control group to which an irrelevant polypeptide was added, and "no target" indicates a control group to which no target peptide was added). The results of control formats 17 and 22 are shown in Figures 4 and 5, respectively (the reference symbol "+" indicates an experimental group to which a gp100 polypeptide was added, and "-" indicates a negative control group to which no gp100 polypeptide was added).

[0364] The results show that the format 22-2 and format 64 of the present invention are respectively 10 -7 -10 -10 M and 10 -7 -10 -9 M concentration range can achieve effective activation of immune cells, especially format 22-2 at 10 -10 M can effectively activate immune cells (Figure 1), while the control formats 17 and 22 need 10 -8M is required to activate immune cells (Figures 4 and 5), indicating that the format of the present invention has a significantly better immune cell activation effect than the control formats 17 and 22. This suggests that TRAC and TRAC used in the format of the present invention have a significantly better effect than using the CH3 or hinge region-CH2-CH3 of an antibody when combined with specific first and second antigen-binding regions.

[0365] Example 4 Target cell killing assay

[0366] The format of Example 1 of the present invention was diluted in gradients (10 -7 to 10 -13 M). gp100 polypeptide, KRAS polypeptide, and MAGE-A1 polypeptide were dissolved in DMSO, and then diluted with water to a working concentration of 10 -4 M. Loaded with T2 cells 10 -6 M target peptide. Add 5x10 5 cells / mL of PBMC (100 μL / well), 5x10 4 T2 cells loaded with peptides (100 μL / well) and different concentrations of format (10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 M). For negative controls, T2 cells and PBMCs without gp100 peptide loading were added. After all samples were added, the plate was covered and incubated at 37°C, 5% CO2 for 20-24 hours. The supernatant was discarded and 100 μL of the detection solution from the Luciferase Reporter Gene Quantification Assay Kit was added to assess target cell cytotoxicity by measuring luciferase activity in the target cells. Based on the target cell cytotoxicity data, the EC50 for each format was calculated using nonlinear regression using GraphPad software.

[0367] The results show that the format provided by the present invention is 10 -7 -10 -10 Killing of target cells can be achieved within the concentration range of M or even lower.

[0368] Example 5 Protein expression, refolding and purification

[0369] The prepared expression plasmids containing the TCR α-chain and β-chain were transformed into the Escherichia coli strain Rosetta (DE3) pLysS, protein expression was induced, inclusion bodies were harvested, solubilized with a denaturant, and then dialyzed for renaturation and purification. This yielded the purified TCR-based multispecific polypeptide molecules provided by the present invention.

[0370] Example 6 Determination of affinity and kinetic parameters of TCR-based multispecific polypeptide molecules and their ligands

[0371] Surface plasmon resonance (SPR) biosensors detect refractive index changes expressed in response units (RU) near the sensor surface within a small flow chamber, which can be used to monitor receptor-ligand interactions and analyze their affinity and kinetic parameters. The TCR-based multispecific polypeptide molecules provided by the present invention bind to MHC-peptide complexes with a KD of less than or equal to 1 μM and / or a koff of 1×10⁻³ s⁻¹ or slower, and bind to T cell surface molecules with a KD of less than or equal to 1 μM and / or a koff of 1×10⁻³ s⁻¹ or slower.

[0372] Example 7 In vivo antitumor efficacy determination

[0373] Anti-tumor efficacy was studied in immunodeficient mice using a target cell xenograft model. The killing of transplanted tumors by the TCR-based multispecific polypeptide molecules of the present invention was measured to assess their in vivo tumor inhibition effects. The TCR-based multispecific polypeptide molecules provided by the present invention exhibited in vivo anti-tumor efficacy.

[0374] This application makes reference to various issued patents, published patent applications, journal articles and other publications, all of which are incorporated herein by reference. If any incorporated reference conflicts with this specification, this specification shall prevail. In addition, any specific embodiment of the present invention that falls within the scope of the prior art may be explicitly excluded from any one or more claims. Because the embodiments are considered to be known to those skilled in the art, they may be excluded, even if the exclusion is not explicitly listed in this application. Any specific embodiment of the present invention may be excluded from any claim for any reason, regardless of whether it is related to the existence of the prior art.

[0375] Although the present invention has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition, method, or process step to the objective, spirit, and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

Claims

1. A bispecific polypeptide molecule comprising a first binding region that binds a first antigen and a second binding region that binds a second antigen on two polypeptide chains, wherein the first binding region comprises an α chain variable region (TRAV) and a β chain variable region (TRBV) derived from a T cell receptor (TCR) that binds to the first antigen-MHC complex; wherein the second binding region comprises a heavy chain variable region VH and a light chain variable region VL derived from an antibody that binds to the second antigen; 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; The two polypeptide chains of the bispecific polypeptide molecule respectively comprise: TRAV-VH and VL-TRBV; TRAV-VL and VH-TRBV; TRBV-VH and VL-TRAV; or TRBV-VL and VH-TRAV; Adjacent variable regions in the two polypeptide chains are connected by a linker; and The bispecific polypeptide molecule further comprises a TCR constant region or fragment thereof connected to the C-terminus of each polypeptide chain via an optional linker.

2. The bispecific polypeptide molecule according to claim 1, wherein the TCR constant region is selected from the group consisting of a TCR α chain constant region (TRAC) and a TCR β chain constant region (TRBC), preferably, one polypeptide chain of the bispecific polypeptide molecule comprises TRAC and the other polypeptide chain comprises 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 TCRα chain constant region and position 57 of the wild-type TCRβ chain constant region.

3. The bispecific polypeptide molecule according to claim 2, wherein the two polypeptide chains of the bispecific polypeptide molecule comprise: TRAV-VH and VL-TRBV, respectively.

4. The bispecific polypeptide molecule according to any one of claims 1 to 3, wherein the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: The first polypeptide chain comprises from N-terminus to C-terminus: TRAV-linker-VH-optional linker-TRAC, and The second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRBC.

5. A bispecific polypeptide molecule comprising a first binding region that binds a first antigen and a second binding region that binds a second antigen on one or more polypeptide chains, wherein the first binding region comprises an α chain variable region (TRAV) and a β chain variable region (TRBV) derived from a T cell receptor (TCR) that binds to the first antigen-MHC complex; wherein the second binding region comprises a heavy chain variable region VH and a light chain variable region VL derived from an antibody that binds to the second antigen; in, The TRAV, TRBV, VH and VL are distributed on the one or more polypeptide chains so that when the one or more polypeptide chains are folded, the TRAV and TRBV are spatially close to form the first binding region, and the VH and VL are spatially close to form a second binding region; wherein the first antigen is selected from tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), viral antigens and autoantigens, and the second antigen is an immune cell surface molecule, The bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: The first polypeptide chain comprises from N-terminus to C-terminus: TRAV-linker-VH-optional linker-TRAC, and The second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRBC.

6. The bispecific polypeptide molecule according to any one of claims 1 to 5, wherein the bispecific polypeptide molecule further comprises at least one of the following functional domains: 1) Hinge region derived from an antibody; 2) Fc domain or its dimerization portion derived from an antibody; 3) Albumin (Alb).

7. The bispecific polypeptide molecule according to claim 6, wherein the functional domain is connected to the C-terminus of one or both polypeptide chains of the bispecific polypeptide molecule via an optional linker.

8. The bispecific polypeptide molecule according to claim 6 or 7, wherein the functional domain is derived from albumin, preferably serum albumin, more preferably human serum albumin.

9. The bispecific polypeptide molecule according to claim 8, wherein the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: The first polypeptide chain comprises from N-terminus to C-terminus: TRAV-linker-VH-optional linker-TRAC, and The second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRBC-optional linker-Alb.

10. The bispecific polypeptide molecule according to claim 6 or 7, wherein the functional domain is derived from the hinge region of an antibody and / or the Fc domain of an antibody or a dimerization portion thereof; and the functional domain is connected to the C-termini of the two polypeptide chains of the bispecific polypeptide molecule via an optional linker.

11. The bispecific polypeptide molecule according to claim 10, wherein 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; and / or The Fc domain of the antibody or the dimerization part thereof is human, for example, the Fc domain or the part thereof derived from human IgG1, IgG2 or IgG4, and preferably comprises at least two cysteine ​​residue mutations, for example S354C and Y349C or L242C and K334C.

12. The bispecific polypeptide molecule according to claim 10 or 11, wherein the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: The first polypeptide chain comprises from N-terminus to C-terminus: TRAV-linker-VH-optional linker-TRAC-optional linker-hinge region-CH2-CH3, and The second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRBC-optional linker-hinge region-CH2-CH3.

13. The bispecific polypeptide molecule according to any one of claims 1 to 12, wherein the linkers in the polypeptide molecule are each independently selected from linkers consisting of 1 to 35 amino acids; Preferably, the linkers are each independently selected from S, GGGS, GGGGS, GGGSGGGG, GGSGGS, GGSGGSGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGS, GGGGSGGGGSGGGGSGGGGSGGGGS, GQPKAAP, TVLRT, TVSSAS, GGEGG, GSEGGGS, RTSGPGDGGKGGPGKGPGGEGTKGTGPGG, 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.

14. The bispecific polypeptide molecule according to any one of claims 1 to 13, wherein the linkers in the polypeptide molecule are each independently selected from linkers consisting of no more than 12 amino acids; Preferably, the linkers are each independently selected from S, GGGS, GGGGS, GGGSGGGG, GGSGGS, GGSGGSGGS, GGGGSGGGGS, EDLKN or a variant thereof, EDLNK or a variant thereof and ANIQK or a variant thereof; Preferably, the variant of EDLKN is an amino acid sequence formed by substitution, deletion or addition of one or several amino acids of EDLKN; and / or the variant of EDLNK is an amino acid sequence formed by substitution, deletion or addition of one or several amino acids of EDLNK; and / or the variant of ANIQK is an amino acid sequence formed by substitution, deletion or addition of one or several amino acids of ANIQK.

15. The bispecific polypeptide molecule according to any one of claims 1 to 14, wherein the VH and VL comprise at least one cysteine ​​mutation to form a disulfide bond between VH and VL, preferably, the cysteine ​​mutation is at position 44 of VH and position 100 of VL.

16. The bispecific polypeptide molecule according to any one of claims 1 to 15, 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.

17. The bispecific polypeptide molecule according to any one of claims 1 to 16, wherein the polypeptide molecule further comprises a signal peptide sequence at the N-terminus of one or both polypeptide chains, preferably a signal peptide sequence at the N-terminus of each chain, such as a signal peptide sequence derived from albumin or immunoglobulin, preferably MGWSCIILFLVATATGVHS.

18. The bispecific polypeptide molecule of any one of claims 1 to 17, wherein the first antigen is a TAA selected from the group consisting of melanoma-associated antigens (e.g., gp100, MAGEA1, MAGEA3, MAGEA6, MAGEA4, MAGEA2, MAGEA12, MAGEA2B, MAGEA9B, MAGEA10, MAGEA11, MAGEB2, MAGEC1, MAGEC2), IGF2BP1, GNGT1, PI4K2B, CCR8, NPSR1, COX7B2, ONECUT3, SMC1B, FOXI3, GAGE2A, FBXO43, BRDT, PAGE2, GAGE13, POU5F1B, CTAG1A, and an endogenous reverse transcriptase antigen; or wherein the first antigen is a TSA selected from the group consisting of KRAS, TP53, PIK3CA, CTNNB1, EGFR, BRAF, and GNAS; or wherein the first antigen is a viral antigen selected from the group consisting of HPV E6 or E7 antigen, CMV antigen, HBV antigen, EBV antigen, herpes virus antigen, human immunodeficiency virus (HIV) antigen, influenza virus antigen, and coronavirus antigen; or The first antigen is an autoantigen selected from the group consisting of AFP, CEA, CD19, CD20, BCMA, CD22, CD30, SLAM, CLDN18.2, GD2, mesothelin, CD38, Her2, GPC3, MUC1, Ro52, Ro60, La, Jo-1, SRP, IFIH1, CENPA, CENPB, SNRPA1, SNRNP70, SNR-PD3, RNAP3, TOPO1, Insulin, and GAD65. , IA2, Znt8, PL7, TARS, ARS, MI2, topoisomerase 1, EXOSC9, EXOSC107, POLR3A, POLR3K, PTRN, GAD2, SLC30A8, AchR, MUSK, LRP4, PLA2R, THSD7A, TSHR, IFN-γ, CHRNA1, MUSK, LRP4, AQP4, MOG, GRIN1, COL4A3, PLA2R, GM-SCF, PR3, and MPO.

19. The bispecific polypeptide molecule according to any one of claims 1 to 18, wherein the second antigen is selected from the group consisting of CD3 (e.g., CD3γ, CD3δ, and CD3ε chain), CD4, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD25, CD32a, CD32b, CD41, CD41b, CD42a, CD42b, CD44, CD45RA, CD49, CD61, CD64, CD68, CD94, CD90, CD117, Nkp46, NKG2D, FcεRI, TCRα / β, TCRγ / δ, HLA-DR, CD28, 4-1BB(CD137), OX40(CD134), ICOS(CD278), 2B4(CD244), HVEM, LAG3, D AP10, DAP12, CD27, CD40, GITR, LFA-1, MyD88, CD2, CD7, LIGHT, B7-H3, CTLA-4, PD-1, CD80, BTLA, TIM3, TIGIT and LAG-3.

20. The bispecific polypeptide molecule according to any one of claims 1 to 19, wherein the first antigen is selected from gp100, MAGEA1, KRAS and HPVE7, and / or the second antigen is selected from CD3, CD28 or 4-1BB (CD137).

21. The bispecific polypeptide molecule according to any one of claims 1 to 20, wherein the polypeptide molecule comprises: a first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 1, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 2; a first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 3, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 4; a first polypeptide chain having the amino acid sequence shown in SEQ ID NO:5, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO:6; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:99, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:100; a first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 101, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 102; A first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 103, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 104; or A first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 105, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO:

106.

22. A nucleic acid comprising a nucleotide sequence encoding each chain of the bispecific polypeptide molecule of any one of claims 1-21.

23. A vector comprising the nucleic acid of claim 22.

24. A vector system comprising, on one or more vectors, a nucleotide sequence encoding each chain of the bispecific polypeptide molecule of any one of claims 1 to 21.

25. A host cell comprising the nucleic acid of claim 22, the vector of claim 23 or the vector system of claim 24.

26. A conjugate comprising the bispecific polypeptide molecule of any one of claims 1-21 and a chemical moiety conjugated to the bispecific polypeptide molecule.

27. The conjugate according to claim 26, wherein the chemical moiety is selected from the group consisting of a therapeutic agent, an immunostimulatory molecule, and a detectable label; Preferably, the therapeutic agent is selected from the group consisting of an immunomodulator, a radioactive compound, an enzyme, a chemotherapeutic agent, and a toxin. Preferably, the immunostimulatory molecules are selected from cytokines, chemokines, platelet factors and complement initiators; Preferably, the detectable label is selected from the group consisting of biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, a nucleic acid probe, a contrast agent, and a fluorescent, phosphorescent or chemiluminescent molecule.

28. A pharmaceutical composition comprising the bispecific polypeptide molecule of any one of claims 1 to 21, the nucleic acid of claim 22, the vector of claim 23, the vector system of claim 24, the host cell of claim 25, the conjugate of claim 26 or 27, and a pharmaceutically acceptable excipient.

29. The pharmaceutical composition according to claim 28, wherein the pharmaceutical composition further comprises a second therapeutic agent, preferably, the second therapeutic agent is selected from the group consisting of antibodies, chemotherapeutic agents and small molecule drugs.

30. A method for preventing or treating a disease in a subject, comprising administering to the subject an effective amount of the bispecific polypeptide molecule of any one of claims 1-21, the nucleic acid of claim 22, the vector of claim 23, the vector system of claim 24, the host cell of claim 25, the conjugate of claim 26 or 27, or the pharmaceutical composition of claim 28 or 29, wherein the disease is selected from cancer, an infectious disease, an autoimmune disease, and an inflammatory disease.

31. The method according to claim 30, further comprising administering to the subject a second therapeutic agent, preferably, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

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