Binding molecule specific for MAGE-A1 and fusion protein with anti-CD3

Abstract

The present invention relates to the field of immunology, particularly to a binding molecule specific for MAGE-A1 and a fusion protein against CD3. The specific binding molecule has the property of specifically binding to an epitope of MAGE-A1, and includes a variable region of the TCRα chain containing CDR1α, CDR2α, and CDR3α, and a variable region of the TCRβ chain containing CDR1β, CDR2β, and CDR3β. The fusion protein comprises a first polypeptide chain and a second polypeptide chain. The first polypeptide chain comprises, from the N-terminus to the C-terminus: TRAV-linker-VH-optional linker-TRAC. The second polypeptide chain comprises, from the N-terminus to the C-terminus: VL-linker-TRBV-optional linker-TRBC. The TCR provided by the present invention has the ability to bind to a target antigen peptide with high affinity, and the characteristics of the TCE product prepared therefrom have excellent tumor killing efficacy.

Description

Technical Field

[0001] The present invention relates to the field of immunology, in particular to a binding molecule specific for MAGE-A1 and a fusion protein against CD3. Background Art

[0002] A bispecific antibody (BsAb, abbreviated as "bispecific antibody") refers to an antibody molecule that targets two antigens simultaneously or two different antigenic epitopes of one antigen. Compared with ordinary antibodies, bispecific antibodies have stronger specificity, can more accurately target tumor cells, and reduce off-target toxicity. With the development of recombinant protein expression technology and antibody engineering technology, many different antibody forms have emerged. 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.

[0003] T cell redirected bsAb (also known as T cell engager, TCE) is an important form of cytotoxic effector cell redirection and is currently the central pillar of cancer immunotherapy. This bsAb can recognize a target on the surface of tumor cells and simultaneously recognize a molecule on the surface of T cells (which is mostly CD3 in most cases), so that the bsAb can couple tumor cells with cytotoxic T cells to activate the downstream signaling pathway of TCR in T cells and kill tumor cells through the cytotoxicity of T cells. Blincyto (blinatumomab) is a CD19 / CD3 bispecific antibody that results in complete remission in 69% of patients with relapsed / refractory B-precursor acute lymphoblastic leukemia (ALL). Currently, a large number of new T cell redirected antibody forms have emerged, such as BITE, BITE-Fc, DART-Fc, TriTAC, etc.

[0004] The bsAb based on antibody recognition of molecules on the surface of target cells only recognizes antigens on the surface of tumor cells and cannot recognize nearly 90% of intracellular proteins. While TCR can recognize antigen peptides presented on MHC molecules on the cell surface after processing of intracellular and cell surface tumor-specific antigens, with a wider recognition range. In the TCR-based T cell redirected bispecific antibody, the target cell recognition region is changed from a traditional antibody to TCR, so that the wide recognition property of TCR can be used to increase the selectivity of the target.

[0005] The melanoma-associated antigen gene family MAGE is the most thoroughly studied cancer-testis antigen to date, and MAGE-A1 is the first cancer-testis antigen gene to be identified. A large number of studies have shown that MAGE-A1 is expressed on the surface of various solid tumor cells such as melanoma, lung cancer, colorectal cancer, cervical cancer, and breast cancer. Therefore, the development of TCE products targeting MAGE-A1 may benefit more patients with these indications.

[0006] Previously, multiple HLA-A0201-restricted TCRs targeting MAGE-A1 have been developed, named "ht27" and "T1367" respectively (Obenaus, M., Leitão, C., Leisegang, M., Chen, X., Gavvovidis, I., van der Bruggen, P., Uckert, W. et al., Identification of human T‐cell receptors with optimal affinity to cancer antigens using antigen‐negative humanized mice. Nat. Biotechnol. 2015. 33: 402–407; Ottaviani, S., Zhang, Y., Boon, T. and van der Bruggen, P., A MAGE‐1 antigenic peptide recognized by human cytolytic T lymphocytes on HLA‐A2 tumor cells. Cancer Immunol. Immunother. 2005. 54: 1214–1220.). Among them, ht27 is a natural TCR sequence found in the human body, but the affinity of this TCR is relatively low and it cannot be prepared as a TCE product for use; while "T1367" is a murine TCR sequence targeting the human MAGE-A1 antigen found in transgenic mice. This TCR has a high affinity and can effectively kill tumor cells in in vitro experiments, but it may cause a strong autoimmune reaction when applied to human experiments, resulting in ineffective treatment.

[0007] Therefore, there is a need to develop TCR products that specifically target MAGE-A1 with high affinity. Summary of the Invention

[0008] The present invention uses the human TCR ht27 sequence as a template and mutates the complementarity-determining region (CDR) of ht27 by in vitro mutagenesis to obtain a mutant TCR sequence with high affinity, and further develops a TCE product targeting MAGE-A1, which has excellent tumor cell killing activity.

[0009] In a first aspect, the present invention provides a specific binding molecule, which has the property of specifically binding to a MAGE-A1 epitope and comprises a variable region of the TCR α chain (TRAV) and a variable region of the TCR β chain (TRBV). The variable region of the TCR α chain comprises CDR1α shown in SEQ ID No:1, CDR2α shown in SEQ ID No:2, and CDR3α shown in SEQ ID No:3. The variable region of the TCR β chain comprises CDR1β shown in SEQ ID No:4, CDR2β shown in SEQ ID No:5, and CDR3β shown in SEQ ID No:6.

[0010] In some preferred embodiments, the variable region of the α chain comprises the amino acid sequence shown in SEQ ID NO:7, and / or the variable region of the β chain comprises the amino acid sequence shown in SEQ ID NO:8.

[0011] In a second aspect, the present invention provides a specific binding molecule - anti-CD3 fusion protein, which comprises a first polypeptide chain and a second polypeptide chain. The first polypeptide chain comprises, from the N-terminus to the C-terminus: TRAV - linker - VH - optional linker - TRAC, and the second polypeptide chain comprises, from the N-terminus to the C-terminus: VL - linker - TRBV - optional linker - TRBC;

[0012] wherein the TRAV is the variable region of the α chain as described above, and the TRBV is the variable region of the β chain as described above.

[0013] In a third aspect, the present invention provides a nucleic acid molecule, which encodes the specific binding molecule as described above, or the specific binding molecule - anti-CD3 fusion protein as described above.

[0014] In a fourth aspect, the present invention provides an expression vector, wherein the vector comprises the nucleic acid molecule as described above.

[0015] In a fifth aspect, the present invention provides a cell, wherein the cell carries the nucleic acid molecule as described above or the expression vector as described above.

[0016] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the specific binding molecule as described above, the specific binding molecule - anti-CD3 fusion protein as described above, the nucleic acid molecule as described above, the expression vector as described above, or the cell as described above, and optionally one or more pharmaceutically acceptable carriers or excipients.

[0017] The TCR provided by the present invention has the ability to bind to the target antigen peptide with high affinity, reaching the level of mature TCE. Thus, the characteristics of the TCE product prepared therefrom have excellent tumor killing efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Shows the affinity of A1A2-M01-675 provided by the present invention, wherein, Figure 1 (A) shows the binding activity of A1A2-M01-675 to its target pMHC Monomer, Figure 1 (B) shows the activation of JKR9 reporter cells by A1A2-M01-675.

[0019] Figure 2 Shows the expression and purification results of CorEngaer provided by the present invention.

[0020] Figure 3 Shows the ability of CorEngaer provided by the present invention to specifically mediate T cell activation.

[0021] Figure 4 Shows the affinity of CorEngager provided by the present invention for the target pMHC complex.

[0022] Figure 5 Shows the ability of CorEngager provided by the present invention to specifically mediate T cell activation and kill target cells. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, the terms used herein such as those in Immunobiology by Janeway CA Jr, Travers P, Walport M, 5th Edition, New York: Garland Science (2001) and "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", edited by Leuenberger, H.G.W, Nagel, B. and Kölbl, H. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0024] 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" may be used interchangeably. Similarly, the terms "comprising", "including" and "having" may be used interchangeably.

[0025] When the term "comprising" is used herein and in the appended claims, it does not exclude other elements. 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 including or comprising at least a certain number of embodiments, it should also be understood that a group consisting only of these embodiments is also disclosed.

[0026] Specific binding molecule

[0027] The present invention provides a specific binding molecule having the property of specifically binding to a MAGE-A1 epitope and comprising a variable region of TCRα chain and a variable region of TCRβ chain, wherein the variable region of TCRα chain comprises CDR1α as shown in SEQ ID No:1, CDR2α as shown in SEQ ID No:2 and CDR3α as shown in SEQ ID No:3, and the variable region of TCRβ chain comprises CDR1β as shown in SEQ ID No:4, CDR2β as shown in SEQ ID No:5 and CDR3β as shown in SEQ ID No:6.

[0028] The TCR provided by the present invention uses ht27 as the parent and mutates the complementarity-determining region (CDR) of the TCR by in vitro mutagenesis to obtain a TCR clone that meets the TCE affinity level, that is, a TCR clone with an affinity at the pM level. Then, based on the obtained high-affinity TCR, TCE is prepared to verify its efficacy, and finally, the high-affinity TCR clone of the present invention is determined.

[0029] As used herein, the term "parental TCR" refers to the previously developed HLA-A0201-restricted human TCR ht27 targeting MAGE-A1. The specific sequence information can be found in the prior patent application WO 2023 / 232111 A1 of the applicant of the present invention, which is incorporated herein by reference. To avoid unnecessary repetition, it will not be described again in the present invention.

[0030] The TCR domain sequences defined in this application are described with reference to the IMGT nomenclature, which is well-known and accessible to those skilled in the TCR art. Briefly, the αβ TCR consists of two chains linked by a disulfide bond. Each chain (α and β) is generally considered to have two domains, namely, the variable region and the constant domain. A short linker region connects the variable region and the constant domain and is generally considered to be part of the α variable region. In addition, the β chain usually contains a short diversity region adjacent to the linker region, which is also generally considered to be part of the β variable region. The variable region of each chain is located at the N-terminus and contains three complementarity-determining regions (CDRs) embedded in the framework sequence (FR). The CDRs contain the recognition sites for peptide-MHC binding. There are several genes encoding the variable (Vα) region of the α chain and several genes encoding the variable (Vβ) region of the β chain, which differ in their framework, CDR1 and CDR2 sequences, and partially defined CDR3 sequences. The Vα and Vβ genes are denoted by the prefixes TRAV and TRBV, respectively, in the IMGT nomenclature. Similarly, there are several joining genes or J genes for the α and β chains, called TRAJ or TRBJ, respectively, and the diversity gene or D gene for the β chain is called TRBD. The huge diversity of T cell receptor chains comes from the combinatorial rearrangement between various V, J, and D genes, including allelic variants and junctional diversity. The constant regions or C regions of the TCR α and β chains are called TRAC and TRBC, respectively.

[0031] Antigen specificity is conferred by the variable regions of the α and β chains. The two variable regions of the TCR α and β chains (the variable region of the α chain (Vα) and the variable region of the β chain (Vβ)) each contain three hypervariable or complementarity-determining regions (CDR1α / β, CDR2α / β, and CDR3α / β) surrounded by four framework (FR) regions (FR1α / β, FR2α / β, FR3α / β, and FR4α / β). CDR3 is the major determinant of antigen recognition and specificity (i.e., the ability to recognize and interact with a specific antigen), while CDR1 and CDR2 mainly interact with MHC molecules presenting antigenic peptides.

[0032] In some embodiments, the framework sequences of the TCR variable domains of the present invention can be murine or human, preferably human. In some preferred embodiments, the framework region is derived from the framework region of a TCR against a MAGE epitope. In some more preferred embodiments, the framework region is derived from the framework region of a TCR against MAGE-A1. In some further preferred embodiments, the framework region is derived from the framework region of an HLA-A*02-restricted TCR against mutant KRAS G12V. In the most preferred embodiment, the framework region is derived from the framework region of ht27.

[0033] Therefore, after determining the 6 CDR domains of the TCR of the present invention, the variable region of the TCR α chain and the variable region of the TCR β chain are not unique. In some preferred embodiments of the present invention, the variable region of the TCR α chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:7. In some preferred embodiments of the present invention, the variable region of the TCR β chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:8.

[0034] As used herein, the term "sequence identity" refers to the degree to which two (nucleotide or amino acid) sequences have the same residues at the same positions in an alignment and is typically expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies with exactly the same sequence have 100% identity, but sequences with lower degrees of conservation and with deletions, additions or substitutions can have lower degrees of identity. Those skilled in the art will recognize that several algorithms can be used to determine sequence identity using standard parameters, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197) and ClustalW.

[0035] Thus, the amino acid sequence of SEQ ID NO: 7 or 8 can, for example, serve as the "subject sequence" or "reference sequence", while the amino acid sequence of the variable region of a TCR α-chain or β-chain that differs therefrom can serve as the "query sequence".

[0036] In some embodiments, the TCR α-chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 7. In some embodiments, the TCR β-chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 8. In some embodiments, the TCR α-chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 7 and the TCR β-chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 8.

[0037] As used herein, the term "specific binding molecule" refers to a molecule capable of binding to a target antigen. These molecules can take several different forms as discussed herein, for example, they can be fragments of the specific binding molecules of the invention. A fragment refers to a portion of a specific binding molecule that retains the ability to bind to the target antigen.

[0038] In some embodiments of the invention, the TCR provided herein is capable of specifically binding to an epitope comprising the amino acid sequence SEQ ID NO: 15 or a complex of said epitope with an MHC molecule.

[0039] The term "epitope" generally refers to a site on an antigen, usually a (poly)peptide recognized by a binding domain. The term "binding domain" in its broadest sense refers to an "antigen-binding site", i.e., a domain of a molecule that characterizes binding / interaction with a specific epitope on an antigen target. The antigen target can contain a single epitope, but usually contains at least two epitopes, and depending on the size, conformation, and type of the antigen, the antigen target can include any number of epitopes. The term "epitope" generally includes linear epitopes and conformational epitopes. A linear epitope is a continuous epitope contained within the primary amino acid sequence, and it usually includes at least 2 amino acids or more. A conformational epitope is formed by non-contiguous amino acids that are brought side by side through the folding of the target antigen, and particularly the target (poly)peptide.

[0040] In some embodiments, the MHC molecule is of the HLA-A*02 type, such as HLA-A*02:01 type, HLA-A*02:03 type, HLA-A*02:05 type, HLA-A*02:06 type, HLA-A*02:07 type, HLA-A*02:10 type, or HLA-A*02:11 type. In a preferred embodiment, the MHC molecule is of the HLA-A*02:01 type or HLA-A*02:05 type.

[0041] The TCR molecule provided by the present invention has a pM affinity, and thus can be used to prepare a TCE. In some embodiments of the present invention, the specific binding molecule comprises a first polypeptide chain that comprises the variable region TRAV of the α chain and a first binding region of the variable region of an antibody; and a second polypeptide chain that comprises the variable region TRBV of the β chain and a second binding region of the variable region of the antibody, wherein the first polypeptide chain and the second polypeptide chain bind and fold such that the specific binding molecule can simultaneously bind to the epitope of KVLEYVIKV (SEQ ID NO:15) or the complex of the epitope and an MHC molecule and the antigen of the antibody.

[0042] In the present invention, when the first polypeptide chain and the second polypeptide chain bind and fold, the TRAV and the TRBV will approach each other spatially to form a binding region that specifically binds to the MAGE-A1 epitope or the complex of the epitope and an MHC molecule, and the VH and the VL will approach each other spatially to form a binding region that specifically binds to the antigen.

[0043] In the present invention, the first binding region and the second binding region are each independently the variable heavy chain region (VH) or the variable light chain region (VL) of an antibody, and the first binding region and the second binding region are different. For example, when the first binding region is VL, the second binding region is VH; when the first binding region is VH, the second binding region is VL. Among them, the "variable light chain region (VL)" or "variable heavy chain region (VH)" is composed of "framework" regions interspersed with three "complementary determining regions" or "CDRs". The framework regions are used to adjust the CDRs for specific binding to antigenic epitopes. The CDRs contain the amino acid residues in the antibody that are mainly responsible for antigen binding. From the amino terminus to the carboxyl terminus, both the VL and VH domains contain the following framework (FR) regions and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0044] The amino acid assignment of each VL and VH domain follows 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); the composite of Chothia Kabat CDRs, where CDR-H1 is the composite of Chothia and Kabat CDRs; the AbM definition used by the antibody modeling software of Oxford Molecular; and the CONTACT definition of Martin et al. (world wide web bioinfo.org.uk / abs). Kabat provides a widely used numbering convention (Kabat numbering system), in which corresponding residues between different heavy chains or different light chains are given the same number. The present disclosure may use CDRs defined according to any one of these numbering systems, but the preferred embodiment uses CDRs defined by the Kabat definition.

[0045] As used herein, the term "antibody" should be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) containing at least two antigen-binding regions. Antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing the antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to the antigen recognition sites, provided that these antibodies exhibit the expected biological activity.

[0046] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more antibody fragments that retain the ability to specifically bind an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0047] As used herein, the term "antigen" refers to any substance that can induce an immune response in an organism. That is, a substance that can be specifically recognized and bound by the antigen receptors (TCR / BCR) on the surface of T / B lymphocytes, activate T / B cells, cause them to proliferate and differentiate, produce immune response products (sensitized lymphocytes or antibodies), and can specifically bind to the corresponding products in vivo and in vitro.

[0048] 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 selected from the positions at the 44th position of VH and the 100th position of VL.

[0049] In some embodiments, the antigen is selected from CD3, CD28, and 4-1BB (CD137). In some preferred embodiments, the antigen is CD3. In some more preferred embodiments, the antigen includes, but is not limited to, OKT3, UCHT-1, BMA031, and 12F6.

[0050] In some preferred embodiments, the antigen is UCHT-1, which contains VH and VL, and cysteine mutations are introduced at the 44th position of VH and the 100th position of VL. In a more preferred embodiment, the antigen has VH as shown in SEQ ID NO:9 and VL as shown in SEQ ID NO:10.

[0051] In some embodiments, the two polypeptide chains of the specific binding molecule respectively comprise TRAV-VH and VL-TRBV; TRAV-VL and VH-TRBV; TRBV-VH and VL-TRAV; or TRBV-VL and VH-TRAV; and adjacent variable regions in the two polypeptide chains are connected by a linker. In a preferred embodiment, the two polypeptide chains of the specific binding molecule respectively comprise: TRAV-VH and VL-TRBV.

[0052] In some embodiments, the specific binding molecule further comprises an alpha chain constant domain TRAC and a beta chain constant domain TRBC. In a preferred embodiment, one polypeptide chain of the specific binding molecule comprises TRAC and the other polypeptide chain comprises TRBC. The sequences of the wild-type TCR constant regions can be found in the public database of the International Immunogenetics Information System (IMGT). For example, the constant domain sequence of the alpha chain of the TCR molecule is "TRAC*01", and the constant domain sequences of the beta chain of the TCR molecule are "TRBC1*01" or "TRBC2*01".

[0053] TRAC and TRBC can be human or murine. TRAC and TRBC can be wild-type or variants thereof. For example, variant TRAC can contain one or more of T48C, N113K, PESS deletion mutation, FFPSPESS deletion mutation relative to the wild-type sequence. For example, variant TRBC can contain one or more of S57C, C187A, N210D, FG loop deletion mutation relative to the wild-type sequence. In a preferred embodiment, TRAC and / or TRBC contain at least one cysteine mutation relative to the wild-type sequence to form a disulfide bond between TRAC and TRBC. More preferably, the cysteine mutation is at the following positions: position 48 of the wild-type TCR alpha chain constant region and position 57 of the wild-type TCR beta chain constant region.

[0054] In a preferred embodiment, the TRAC contains a cysteine mutation at position 48, N113K and FFPSPESS deletion mutation, and has the amino acid sequence shown in SEQ ID NO:11. In some embodiments, the TRBC contains a cysteine mutation at position 57, C187A, N210D mutation, and has the amino acid sequence shown in SEQ ID NO:12.

[0055] In some embodiments, the polypeptide molecule comprises TRAC or a fragment thereof that is not covalently linked to TRAV through a linker. In some embodiments, the polypeptide molecule comprises TRBC or a fragment thereof that is not covalently linked to TRBV through a linker. Unless otherwise stated, the term "covalently linked" refers to the connection pattern of the TCR constant region and the variable region in the native state.

[0056] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain from the N-terminus to the C-terminus comprises: TRAV-linker-VH-optional linker-TRAC, and the second polypeptide chain from the N-terminus to the C-terminus comprises: VL-linker-TRBV-optional linker-TRBC.

[0057] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain from the N-terminus to the C-terminus comprises: TRAV-linker-VH-optional linker-TRBC, and the second polypeptide chain from the N-terminus to the C-terminus comprises: VL-linker-TRBV-optional linker-TRAC.

[0058] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain from the N-terminus to the C-terminus comprises: TRAV-linker-VL-optional linker-TRAC, and the second polypeptide chain from the N-terminus to the C-terminus comprises: VH-linker-TRBV-optional linker-TRBC.

[0059] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain from the N-terminus to the C-terminus comprises: TRAV-linker-VL-optional linker-TRBC, and the second polypeptide chain from the N-terminus to the C-terminus comprises: VH-linker-TRBV-optional linker-TRAC.

[0060] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain from the N-terminus to the C-terminus comprises: TRBV-linker-VH-optional linker-TRAC, and the second polypeptide chain from the N-terminus to the C-terminus comprises: VL-linker-TRAV-optional linker-TRBC.

[0061] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain from the N-terminus to the C-terminus comprises: TRBV-linker-VH-optional linker-TRBC, and the second polypeptide chain from the N-terminus to the C-terminus comprises: VL-linker-TRAV-optional linker-TRAC.

[0062] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain from the N-terminus to the C-terminus comprises: TRBV-linker-VL-optional linker-TRAC, and the second polypeptide chain from the N-terminus to the C-terminus comprises: VH-linker-TRAV-optional linker-TRBC.

[0063] In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from the N-terminus to the C-terminus: TRBV-linker-VL-optional linker-TRBC, and the second polypeptide chain comprises, from the N-terminus to the C-terminus: VH-linker-TRAV-optional linker-TRAC.

[0064] In some embodiments, the linkers are each independently selected from S, GGGS, GGGGS, GGGSGGGG, GGSGGS, GGSGGSGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGSGGGS, GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS, GQPKAAP, TVLRT, TVSSAS, GGEGG, GSEGGGS, RTSGPGDGGKGGPGKGPGGEGTKGTGPGG, GKGPGGEGTKGTGPGG, TVLSSAS.

[0065] Specific binding molecule - anti-CD3 fusion protein

[0066] The present invention provides a specific binding molecule - anti-CD3 fusion protein, which comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus: TRAV-linker-VH-optional linker-TRAC, and the second polypeptide chain comprises, from the N-terminus to the C-terminus: VL-linker-TRBV-optional linker-TRBC;

[0067] wherein, the TRAV is the variable region of the α chain as described above, and the TRBV is the variable region of the β chain as described above.

[0068] In a preferred embodiment, the specific binding molecule - anti-CD3 fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus: TRAV - first linker - VH - third linker - TRAC, and the second polypeptide chain comprises, from the N-terminus to the C-terminus: VL - second linker - TRBV - TRBC; wherein the first linker, the second linker and the third linker are the same or different. In a more preferred embodiment, the first linker and the third linker are each independently GGGSGGGG, and the second linker is S.

[0069] In some preferred embodiments, the fusion protein comprises: a first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO: 13, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO: 14. In a preferred embodiment, the polypeptide molecule comprises: a first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 13, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 14.

[0070] Nucleic acid

[0071] The present invention provides a coding nucleic acid molecule, wherein the nucleic acid molecule encodes the specific binding molecule as described above, or the specific binding molecule - anti-CD3 fusion protein as described above.

[0072] As is well known in the art, among the 20 different amino acids that make up proteins, except that Met (ATG) or Trp (TGG) are each encoded by a single codon, the other 18 amino acids are each encoded by 2 - 6 codons (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, Second Edition, 1989, see Appendix D on page 950). That is, due to the degeneracy of the genetic code, there are usually more than one codon that determines an amino acid, and the substitution of the third nucleotide in the triplet codon often does not change the amino acid composition. Therefore, the nucleotide sequences of genes encoding the same protein can be different. Those skilled in the art can completely deduce the nucleotide sequences of the genes capable of encoding them from the amino acid sequences disclosed in the present invention according to the well-known codon table, and obtain the nucleotide sequences by biological methods (such as PCR method, mutation method) or chemical synthesis methods. Therefore, these nucleotide sequences should all be included within the scope of the present invention.

[0073] In some preferred embodiments, the nucleic acid molecule encoding the fusion protein comprises: a first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO: 16, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO: 17.

[0074] Vector

[0075] The present invention provides a vector comprising the nucleic acid provided by the present invention.

[0076] As used herein, the term "vector" is a nucleic acid molecule that serves as a vehicle for transferring (exogenous) genetic material into a host cell, in which the nucleic acid molecule serving as the vector can, for example, replicate and / or be expressed. The term "vector" encompasses, but is not limited to, plasmids, viral vectors (including retroviral vectors, lentiviral vectors, adenoviral vectors, vaccinia virus vectors, polyomavirus vectors, and adeno-associated vectors (AAV)), phages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs). A vector is typically a nucleotide sequence, usually a DNA sequence containing an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. Engineered vectors typically contain an origin of replication that is autonomous in the host cell (if stable expression of the polynucleotide is desired), a selectable marker, and restriction enzyme cleavage sites (such as a multiple cloning site, MCS). Vectors may additionally contain promoters, genetic markers, reporter genes, targeting sequences, and / or protein purification tags. As is known to those skilled in the art, a large number of suitable vectors are known to those skilled in the art and many are commercially available. Examples of suitable vectors are provided in J. Sambrook et al., Molecular Cloning: A Laboratory Manual (4th ed.), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2012), which is incorporated herein by reference in its entirety.

[0077] In some embodiments, the vector is preferably selected from lentiviral vectors, retroviral vectors, plasmids, DNA vectors, mRNA vectors, transposon-based vectors, and artificial chromosomes.

[0078] Cell

[0079] The present invention provides a cell comprising a TCR, nucleic acid, or vector provided by the present invention.

[0080] As used herein, the term "cell" refers to any type of cell capable of expressing the TCR of the present invention. The cell can be a eukaryotic cell, for example, a plant (without the potential to develop into a plant), an animal, a fungus or an alga, or can be a prokaryotic cell, for example, a bacterium or a protozoan. The cell can be a cultured cell or a primary cell, that is, directly isolated from an organism, such as a human. The cell can be an adherent cell or a suspension cell, that is, a cell that grows 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 the TCR of the present invention, the cell is preferably a mammalian cell. Most preferably, the host cell is a human cell.

[0081] In some embodiments, the cell is selected from lymphocytes (such as T cells, NK cells), monocytes (such as PBMC) and stem cells. As used herein, the term "stem cell" is a stem cell for expressing the TCR (especially TCR) of the present invention. For example, the stem cell can be a lymphoid progenitor cell, an induced pluripotent stem cell (iPSC) or a hematopoietic stem cell (HSC). In some embodiments, the stem cell does not include embryonic stem cells obtained by destroying human embryos, and / or does not include totipotent stem cells used for developing and forming an animal individual. Transferring a gene to a stem cell generally does not result in the expression of TCR on the cell surface because CD3 molecules are not expressed on the surface of stem cells. However, when the stem cell differentiates into a lymphoid precursor that migrates to the thymus, the expression of CD3 molecules will initiate the expression of the introduced TCR molecule on the surface of thymocytes.

[0082] In some embodiments, the method for preparing the cell of the present invention includes the step of transducing or transfecting the cell with the vector of the present invention.

[0083] As used herein, the term "transfection" is the process of deliberately introducing a nucleic acid molecule or polynucleotide (including a vector) into a target cell. An example is RNA transfection, that is, the process of introducing RNA (such as in vitro transcribed RNA, ivtRNA) into a host cell. This term is mainly used for non-viral methods in eukaryotic cells. The term "transduction" is generally used to describe the virus-mediated transfer of nucleic acid molecules or polynucleotides. Transfection of animal cells usually involves opening transient pores or "holes" in the cell membrane to allow the uptake of materials. Transfection can be carried out using calcium phosphate, by electroporation, by cell squeezing or by mixing cationic lipids with the material to produce liposomes that fuse with the cell membrane and deposit their cargoes inside. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate / DNA co-precipitation), microinjection and electroporation.

[0084] In some embodiments, the method further comprises the step of amplifying and / or activating the cells before or after the transduction or transfection.

[0085] Pharmaceutical composition

[0086] In another aspect, the present invention provides a pharmaceutical composition comprising a specific binding molecule as described above, a specific binding molecule - anti-CD3 fusion protein as described above, a nucleic acid molecule as described above, an expression vector as described above, or a cell as described above, and optionally one or more pharmaceutically acceptable carriers or excipients.

[0087] The pharmaceutical composition of the present invention is particularly suitable for administration to humans; however, it is also suitable for administration to non-human animals. The composition and its components (i.e., the active agent and optionally the excipient) 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 composition of the present invention can be, for example, sterile.

[0088] Examples of excipients include, but are not limited to, fillers, binders, disintegrants, coating agents, adsorbents, anti-adhesives, glidants, preservatives, antioxidants, flavoring agents, coloring agents, sweetening agents, solvents, co-solvents, buffers, chelating agents, viscosity imparting agents, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifying agents, stabilizers and tonicity regulators. Those skilled in the art know how to select suitable excipients to prepare the pharmaceutical composition of the present invention. Exemplary carriers for use in the pharmaceutical composition of the present invention include saline, buffered saline, glucose and water. Generally, the selection of suitable excipients depends especially on the active agent used, the disease to be treated and the desired dosage form of the composition.

[0089] 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, especially 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.

[0090] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent, preferably, the second therapeutic agent is selected from antibodies, chemotherapeutic agents and small molecule drugs.

[0091] Preferred examples of the second therapeutic agent include known anti-cancer 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, vincristine and doxorubicin; and peptide cytotoxins such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase and RNase; radionuclides such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and 213, actinium 225 and astatine 213; prodrugs such as antibody-directed enzyme prodrugs; immunostimulants such as IL-2, chemokines such as IL-8, platelet factor 4; antibodies or fragments thereof such as anti-CD3 antibody or fragments thereof; complement activators; viral / bacterial protein domains and viral / bacterial peptides.

[0092] The pharmaceutical composition provided by the present invention can be used for the treatment of cancer, for example, MAGE-A1-related cancers, including but not limited to malignant tumors such as lung cancer, liver cancer, gastric cancer, esophageal cancer, melanoma and the like.

[0093] The present invention will be further illustrated by the following specific examples. It should be understood that these examples are only for illustrating the present invention and not for limiting the scope of the present invention. For the experimental methods without specific conditions noted in the following examples, they are carried out according to the conventional conditions in the art, for example, the conditions described in Molecular Cloning: A Laboratory Manual (Third Edition) (2001), Cold Spring Harbor Laboratory Press by Sambrook and Russel et al., or according to the conditions recommended by the manufacturer. Unless otherwise specified, the experimental materials and reagents used in the following examples are commercially available.

[0094] Example 1 Screening for Affinity-Enhanced TCRs

[0095] The inventors used in vitro substitution to replace the complementarity-determining region (CDR) of ht27 with the human TCR ht27 sequence as a template, and obtained high-affinity substituted clones by in vitro positive screening of the substituted TCRs. By this method, the preferred TCRs screened were subjected to in vitro positive screening to obtain high-affinity substituted clones. By this method, the sequence information of the preferred TCR sequences screened is as follows:

[0096] The amino acid sequences of CDR1α, CDR2α and CDR3α are shown in SEQ ID NO: 1-3 respectively;

[0097] The amino acid sequences of CDR1β, CDR2β, and CDR3β are shown in SEQ ID NO: 4 - 6, respectively;

[0098] The amino acid sequence of the variable region of the TCR α chain is shown in SEQ ID NO: 7; the amino acid sequence of the variable region of the TCR β chain is shown in SEQ ID NO: 8; the TCR with improved affinity thus obtained is named A1A2 - M01 - 675.

[0099] The affinity of the TCR provided by the present invention is determined by the following method:

[0100] The parental TCR and the TCR sequence provided by the present invention are respectively cloned into a TCR tandem expression lentiviral vector, the lentivirus is packaged, the reporter cells JKR9 are transduced, and then the TCR - expressing positive cells are sorted.

[0101] The inventors determined the improved effect of the mutant TCR affinity by studying the binding activity between the soluble-expressed MAGE-A1&HLA-A0201 complex monomer (Monomer) and the TCR expressed on the surface of JKR9. Specifically, the Monomer with an Avi-Tag was biotinylated, and then fluorescently labeled streptavidin was used to label the Monomer with fluorescence. JKR9 cells expressing the parental TCR or the mutant TCR were incubated with the fluorescently labeled Monomer at different concentrations (80 nM, 16 nM, 3.2 nM, 0.64 nM, 0.128 nM) at 4 °C for 1 h. The free Monomer was removed by washing with PBS multiple times. The fluorescence positive rate of JKR9 cells under different Monomer concentration staining conditions was detected using a flow cytometer (FACS), and the EC50 was calculated to reflect the binding activity between the TCR and the Monomer. The research results showed that JKR9 cells expressing the parental TCR hT27 could not be stained by any concentration of the Monomer. For the mutant TCR, 92% of the cells showed positive staining under the condition of staining with an 80 nM concentration of the Monomer. The positive rate of cell staining decreased with the decrease of the Monomer concentration, and the staining EC50 value was 1.137E-09. The researchers used the TCR used in the marketed drug Tebentafusp as a positive control and detected the binding activity between the TCR and the target pMHC using the same method. Under the condition of an 80 nM Monomer (gp100 &HLA-A0201) concentration, the positive rate of staining in the control group was 89.7%. The positive rate of cell staining decreased with the decrease of the Monomer concentration, and the staining EC50 value was 1.520E-09. The results proved that the TCR provided by the present invention had a significantly improved affinity compared with the parental TCR, and the binding activity between A1A2-M01-675 and its target pMHC Monomer was close to the binding activity of the TCR used in the marketed product to its target pMHC Monnomer( Figure 1 (B))。

[0102] On the other hand, the inventors determined the improvement effect of the affinity of mutant TCRs by studying the improvement of the function of TCR-mediated reporter cell recognition and activation of target cells. Specifically, T2 cells positive for HLA-A*0201 were loaded with different concentrations of the MAGE-A1 target antigen peptide (SEQ ID NO:15), and then co-incubated and activated with JKR9 reporter cells transduced with parental TCR and the TCR of the present invention, respectively. After 16 hours of activation, the reporter gene was stained for flow cytometry analysis, and the EC50 value was calculated based on the flow cytometry analysis. The research results showed that the reporter cells transduced with the parental TCR hT27 had only a weak activation response (<10%) under the condition of activation with T2 loaded with a high concentration (-5M) of the polypeptide, and the EC50 value of the mutant TCR A1A2-M01-675 was 2.36E-08. The TCR provided by the present invention has an affinity increased by at least 10,000 times compared with the parental TCR ( Figure 1 (A).

[0103] Example 2 is used to illustrate the preparation of a soluble TCR-CD3 fusion protein (CorEngager)

[0104] (1) Design of the fusion protein

[0105] A soluble TCR-CD3 fusion protein based on the high-affinity TCR obtained in Example 1 was designed and constructed. Specifically, the fusion protein includes a first polypeptide chain and a second polypeptide chain, and the soluble TCR and the anti-CD3 antibody are connected by a linker, as follows:

[0106] The first polypeptide chain contains, from the N-terminus to the C-terminus: TRAV - first linker - VH - third linker - TRAC, and the amino acid sequence is as shown in SEQ ID NO:13;

[0107] The second polypeptide chain contains, from the N-terminus to the C-terminus: VL - second linker - TRBV - TRBC, and the amino acid sequence is as shown in SEQ ID NO:14.

[0108] Among them, the amino acid sequences of each component are shown in Table 1.

[0109] Table 1

[0110]

[0111] (2) Purification and expression of the fusion protein

[0112] 1) Vector construction and plasmid extraction

[0113] The nucleic acid sequences encoding the first polypeptide chain and the second polypeptide chain as above (shown as SEQ ID NO:16 and SEQ ID NO:17 respectively) were directly cloned into the expression vector pcDNA3.4. After the obtained vector clone was confirmed by sequencing, it was amplified and cultured, and plasmid extraction was carried out.

[0114] 2) Transfection

[0115] The density of CHO cells was adjusted to 1×106 cells / ml, and the volume of cell suspension in each bottle was 40 mL. Then, the bottle mouth was tightened and placed in a shaker for continuous culture. After culturing for 2 - 4 hours at 36.5°C, 175 rpm, and 5% CO2, transfection was carried out with the plasmid. Prepare the transfection solution (1 ml): Dilute 10 μg of DNA with about 800 μL of 150 mM sterilized NaCl solution, mix well, and place it on the workbench for 5 min; add about 50 μl of transfection reagent to the DNA dilution and mix well. The final volume of the transfection solution is 1 mL. After placing it on the workbench for 10 min, add the transfection solution dropwise to the cell culture medium, shake well, tighten the bottle mouth, and put it back into the shaker (36.5°C, turn off 5% CO2, 175 rpm). After 20 - 24 h of transfection, add SMS 293-I feeding solution (0.7 mL / bottle), and then feed and culture every other day for 6 - 10 days. Then, collect the culture supernatant.

[0116] 3) Purification

[0117] The collected culture supernatant was used for nickel column affinity purification. Prepare 1 ml of packed nickel column, wash it with 10 ml of sterilized water, and equilibrate it with 10 ml of 1X Bind Buffer. Centrifuge the culture supernatant at 3000 rpm for 5 min, then filter it through a 450 nm filter membrane and load the sample. After loading the sample, wash the column with 10 ml of 1X Bind Buffer, wash the column with 10 ml of 1X Bind Buffer, wash the column with 10 ml of 1X Wash Buffer (collect 10 tubes of eluate, with a flow rate of about 0.3 - 0.4 ml / min), elute the protein with 1.2 ml of 1X Elution Buffer, and collect the eluate as the purified protein. After aliquoting, store it at -80°C.

[0118] 4) Protein purity detection

[0119] The purified protein was treated by denaturation (R) or non-denaturation (NR), and then subjected to SDS-PAGE gel electrophoresis to evaluate the protein expression purity and aggregation degree.

[0120] Based on the yield of the target protein harvested, the predicted yield of the target protein is 33 mg / L, and the purity is determined to be 85% according to the Coomassie Brilliant Blue staining result of SDS-PAGE ( Figure 2). The PAGE-SDS electrophoresis results showed that under non-reducing conditions, the CorEngager protein mainly existed in monomeric form, and a small amount of protein existed in polymeric form. The molecular weight of the monomeric protein was about 100KDa, slightly larger than the theoretical molecular weight (77KDa), which might be caused by post-translational modification of the protein; under reducing conditions, the CroEngager protein was reduced into three subunits with molecular weights between 35KDa and 70KDa. It was speculated that there was heterogeneous post-translational modification in one of the two subunits that composed CorEngager. The above results indicated that the format (CorEngager) provided by the present invention could support the expression of TCR in the form of soluble protein.

[0121] Example 3 is used to illustrate that the CorEngaer provided by the present invention can specifically mediate T cell activation

[0122] The inventors confirmed through in vitro T cell activation experiments that CorEngager could mediate effector cells to specifically recognize the target and activate effector cells. In this experiment, PBMC was used as effector cells, T2 cells expressing HLA-A0201 were used as tool cells for presenting target peptides, and MAGE-A1 (KVLEYVIKV) was used as the target peptide.

[0123] Specifically: The fusion protein of Example 2 of the present invention was serially diluted (10 -7 to 10 -11 M) with 1640 complete medium containing 10% FBS. The MAGE-A1 polypeptide was dissolved in DMSO and then diluted with water to the working concentration of 10 -4 M. T2 cells were respectively loaded with 10 -6 M of MAGE-A1 polypeptide. 1640 complete medium containing 10% FBS was added to the ELISPOT plate and blocked at room temperature for 30 min. The medium was discarded, and 5x10 5 cells / mL of PBMC (100 μL / well), 5x10 5 cells / mL of T2 cells loaded with polypeptide (100 μL / well), and different concentrations of the fusion protein (10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 ). The negative control was added with T2 cells not loaded with polypeptide and PBMC, and the concentration of the candidate polypeptide molecule was the same as that of the experimental group. After adding all the samples, the plate was covered and placed in an incubator at 37ºC and 5% CO 2 for 20 - 24 hours, and then IFN-γ secretion was determined by ELISPOT detection to evaluate the activation of immune cells induced by the target antigen peptide-MHC complex of the fusion protein of the present invention.

[0124] The experimental results show that: in the CorEngager control group using wild-type TCR hT27 ( Figure 3 B), whether under the positive reaction conditions of co-incubation of PBMC(+), T2(+), and MAGE-A1(+), or under the negative reaction conditions of co-incubation of PBMC(+), T2(+), and MAGE-A1(-), the use of any concentration of CorEngager cannot cause the activation of effector cells. In the CorEngager experimental group using mutant TCR A1A2-M01-675 ( Figure 3 A), under the co-incubation conditions of PBMC(+), T2(+), and MAGE-A1(-), due to the lack of target peptides recognizable by TCR, the use of any concentration of CorEngager cannot cause the activation of effector cells; while under the co-incubation conditions of PBMC(+), T2(+), and MAGE-A1(+), a very low concentration (-10M) of CorEngager can mediate the activation of effector cells to secrete IFN-γ, and the secretion amount of IFN-γ increases with the increase of the CorEngager concentration.

[0125] The above results indicate that the format of the present invention can mediate the specific recognition of the target pMHC complex by PBMC and activate PBMC, and shows an obvious CorEngager concentration-dependent relationship for the activation of PBMC.

[0126] Example 4 is used to illustrate the affinity of the CorEngager provided by the present invention for the target pMHC complex

[0127] The inventors detected the affinity of the CorEngager expressed by wild-type TCR hT27 and mutant TCR A1A2-M01-675 for the target pMHC complex (MAGE-A1(KVLEYVIKV) & HLA-A0201) through SPR (surface plasmon resonance) experiments. Specifically: a certain amount of MAGE-A1 was immobilized on the SA chip, the analyte was a single concentration (500 nM) of CorEngager, and zero concentration and reference channels were set. The detection results show that no binding of CorEngager hT27 to the target pMHC was detected ( Figure 4 right); CorEngager A1A2-M01-675 showed a typical binding curve of fast binding and slow dissociation with the target pMHC, with an association constant of 1.52E+05 1 / Ms, a dissociation constant of 2.08E-03 1 / s, and an equilibrium dissociation constant of 1.37E-08M (i.e., the affinity is 13.7 nM) ( Figure 4 left).

[0128] The above results indicate that the affinity of A1A2-M01-675 for the target pMHC is significantly higher than that of hT27.

[0129] Example 5 This example is used to illustrate that the CorEngager provided by the present invention can specifically mediate T cell activation and kill target cells

[0130] The inventors confirmed through an in vitro target cell killing experiment that CorEngager can specifically mediate T cell activation and kill target cells. Specifically, PBMC was used as the effector cell, and T2 cells loaded with a -7M concentration of the MAGE-A1 target peptide were used as the target cells. The effector cells and target cells were mixed at an effector-to-target ratio (E:T) of 1:1, and CorEngager A1A2-M01-675 at concentrations of (-7M, -8M, -9M, -10M, -11M) was added respectively. The cells were cultured in a 37°C CO2 incubator for 16 - 20 h. The IFN-γ ELISA kit (ExCell, Cat# EH008-96) was used to detect the secretion of IFN-γ in the supernatant. The experimental results showed that CorEngager hT27 at any concentration could not mediate PBMC to recognize target cells and produce self-activation to secrete IFN-γ; while a very low concentration (-10M) of CorEngager A1A2-M01-675 could mediate PBMC activation and secretion of IFN-γ, and as the concentration of CorEngager increased, the secretion amount of IFN-γ also increased. The IFN-γ secretion reached the maximum level (1464 pg / ml) at a CorEngager concentration of -8M, and the IFN-γ secretion EC50 value was 4.47E-10 ( Figure 5 left).

[0131] On the other hand, the T2 target cells stably express the luciferase gene. After the effector cells and target cells were incubated at an effector-to-target ratio (E:T) of 1:1 for 16 - 20 h, the luciferase substrate was added to detect the surviving target cells, and then the proportion of killed target cells was calculated. The experimental results showed that CorEngager hT27 at any concentration could not mediate PBMC to kill target cells; while a very low concentration (-11M) of CorEngager A1A2-M01-675 could mediate PBMC to kill 10% of the target cells, and as the concentration of CorEngager increased, the proportion of target cell killing also increased. The maximum killing level (60%) was reached at a CorEngager concentration of -9M, and the killing EC50 value was 1.44E-10 ( Figure 5 right).

[0132] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention. These include combinations of individual specific technical features in any suitable manner. To avoid unnecessary repetition, the present invention will not separately describe various possible combinations. However, these simple modifications and combinations should equally be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A specific binding molecule-anti-CD3 fusion protein, characterized in that: It comprises a first polypeptide chain and a second polypeptide chain, wherein the amino acid sequence of the first polypeptide chain from N-terminus to C-terminus is TRAV-first linker-VH-third linker-TRAC, and the amino acid sequence of the second polypeptide chain from N-terminus to C-terminus is: VL-second linker-TRBV-TRBC; Wherein, the specific binding molecule has the property of specifically binding to the MAGE-A1 epitope, and comprises a TCR α chain variable region (TRAV) and a TCR β chain variable region (TRBV), wherein the TCR α chain variable region comprises CDR1α as shown in SEQ ID No: 1, CDR2α as shown in SEQ ID No: 2, and CDR3α as shown in SEQ ID No: 3, and the TCR β chain variable region comprises CDR1β as shown in SEQ ID No: 4, CDR2β as shown in SEQ ID No: 5, and CDR3β as shown in SEQ ID No: 6; Wherein, the amino acid sequence of TRAV is shown in SEQ ID NO:7; The amino acid sequence of the VH is shown in SEQ ID NO:9; The amino acid sequence of TRAC is shown in SEQ ID NO: 11; The amino acid sequence of VL is shown in SEQ ID NO: 10; The amino acid sequence of TRBV is shown in SEQ ID NO:8; The amino acid sequence of TRBC is shown in SEQ ID NO: 12; The amino acid sequences of the first linker and the second linker are each independently GGGSGGGG, and the amino acid sequence of the third linker is S.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the specific binding molecule-anti-CD3 fusion protein according to claim 1.

3. An expression vector, characterized in that: The vector comprises the nucleic acid molecule of claim 2.

4. A cell, characterized in that The cell carries the nucleic acid molecule of claim 2 or the expression vector of claim 3.

5. A pharmaceutical composition comprising the specific binding molecule-anti-CD3 fusion protein of claim 1, the nucleic acid molecule of claim 2, the expression vector of claim 3, or the cell of claim 4, and optionally one or more pharmaceutically acceptable carriers or excipients.

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