Fusion proteins of binding molecules specific for MAGE-A1 and anti-CD3

Abstract

The present invention relates to the field of immunity, in particular to a fusion protein of a binding molecule and anti-CD3 having specificity for MAGE-A1. The specific binding molecule has the property of being specifically bound with the MAGE-A1 epitope, and comprises a TCR [alpha] chain variable region containing CDR1 [alpha], CDR2 [alpha] and CDR3 [alpha] and a TCR [beta] chain variable region containing CDR1 [beta], CDR2 [beta] and CDR3 [beta]. The fusion protein comprises a first polypeptide chain and a second polypeptide chain, 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. The TCR provided by the invention has the capability of high-affinity binding with target antigen peptide, and the TCE product prepared from the TCR has excellent tumor killing effect.

Description

Technical Field

[0001] The present invention relates to the field of immunity, in particular to a binding molecule with specificity for MAGE-A1 and an anti-CD3 fusion protein. Background Art

[0002] Bispecific antibody (BsAb) refers to an antibody molecule that simultaneously targets two antigens 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 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 targets of cytotoxic effector cells.

[0003] T cell-redirected bsAb (also known as T cell engager, TCE) is an important form of cytotoxic effector cell repositioning and is 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), so that bsAb can couple tumor cells and cytotoxic T cells together to activate the downstream signaling pathway of T cell TCR and kill tumor cells through T cell cytotoxicity. Blincyto (blinatumomab) is a CD19 / CD3 bispecific antibody that leads to complete remission in 69% of patients with relapsed / refractory B-precursor acute lymphoblastic leukemia (ALL). A large number of new T cell-redirected antibody forms have emerged, such as BITE, BITE-Fc, DART-Fc, TriTAC, etc.

[0004] bsAb based on antibody recognition of target cell surface molecules only recognizes tumor cell surface antigens and cannot recognize nearly 90% of intracellular proteins. TCR can recognize antigen peptides of intracellular and cell surface tumor-specific antigens that are processed and presented on the cell surface MHC molecules, and has a wider recognition range. The target cell recognition region in TCR-based T cell repositioning bispecific antibodies is replaced from traditional antibodies to TCR, so that the broad recognition characteristics 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 so far, 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 cell surface of various solid tumors such as melanoma, lung cancer, colorectal cancer, cervical cancer, and breast cancer. Therefore, the development of TCE products targeting MAGE-A1 may benefit more people with indications.

[0006] Previously, several HLA-A0201-restricted TCRs targeting MAGE-A1 have been developed, named “ht27” and “T1367” (Obenaus, M. , Leitão, C. , Leisegang, M. , Chen, X. , Gavvovidis,I. , van der Bruggen, P. , Uckert, W. et al., Identification of human T-cellreceptors with optimal affinity to cancer antigens using antigen-negativehumanized 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: 54–55). 1214–1220.). Among them, ht27 is a natural TCR sequence found in the human body, but the affinity of this TCR is low and it cannot be prepared into TCE products for use; while "T1367" is a mouse TCR sequence targeting 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 produce a strong autoimmune response when used in human experiments, resulting in ineffective treatment.

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

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

[0009] In the first aspect, the present invention provides a specific binding molecule, which 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.

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

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

[0013] In a third aspect, the present invention provides a 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.

[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 and can reach the TCE level. The TCE product prepared thereby has excellent tumor killing efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The affinity of A1A2-M01-675 provided by the present invention is shown, 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 The expression and purification results of CorEngaer provided by the present invention are shown.

[0020] Figure 3 The results show that the CorEngaer provided by the present invention has the ability to specifically mediate T cell activation.

[0021] Figure 4 The affinity of the CorEngager provided by the present invention to the target pMHC complex is shown.

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

[0023] Unless otherwise limited, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill 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 Kölbl, H., eds. (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 references 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 purpose of the present invention, the term "consisting of" is considered to be 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 of only these embodiments.

[0026] Specific binding molecules

[0027] The present invention provides a specific binding molecule, which has the property of specifically binding to a MAGE-A1 epitope and comprises a TCR α chain variable region and a TCR β chain variable region, 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.

[0028] The TCR provided by the present invention is based on ht27 as a parent, and the complementary determining region (CDR) region of the TCR is mutated by in vitro mutation to obtain a TCR clone that meets the TCE affinity level, that is, a pM level affinity. 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] The term "parent TCR" as used herein refers to the previously developed HLA-A0201-restricted human TCR ht27 targeting MAGE-A1, the specific sequence information of which can be found in the prior patent application WO 2023 / 232111 A1 of the applicant of the present invention, which can be incorporated herein by reference. In order to avoid unnecessary repetition, the present invention will not repeat it again.

[0030] The TCR domain sequences defined in this application are described with reference to the IMGT nomenclature, which is well known and available to those skilled in the art of TCR. In short, αβTCR consists of two chains connected by disulfide bonds. It is generally believed that each chain (α and β) has two domains, namely a variable region and a constant domain. The short connecting 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 generally includes a short diversity region adjacent to the connecting 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 includes three complementary determining regions (CDRs) embedded in the framework sequence (FR). CDR includes a recognition site for peptide-MHC binding. There are several genes encoding α chain variable (Vα) regions and several genes encoding β chain variable (Vβ) regions, which are distinguished by their frameworks, CDR1 and CDR2 sequences, and partially defined CDR3 sequences. Vα and Vβ genes are represented by the prefixes TRAV and TRBV, respectively, in the IMGT nomenclature. Likewise, there are several joining genes or J genes for the α and β chains, called TRAJ or TRBJ, respectively, while the diversity gene or D gene for the β chain is called TRBD. The great diversity of T cell receptor chains arises from combinatorial rearrangements between various V, J, and D genes, which include allelic variants and joining 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 (α chain variable region (Vα) and β chain variable region (Vβ)) each contain three hypervariable or complementary 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 main 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 the MHC molecules presenting the antigenic peptide.

[0032] In some embodiments, the framework sequence of the variable domain of the TCR of the present invention can be of mouse or human origin, preferably of human origin. In some preferred embodiments, the framework region is derived from the framework region of the TCR for the MAGE epitope. In some more preferred embodiments, the framework region is derived from the framework region of the TCR for MAGE-A1. In some further preferred embodiments, the framework region is derived from the framework region of the TCR for HLA-A*02 restriction of the 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 TCR α chain variable region and the TCR β chain variable region are not unique. In some preferred embodiments of the present invention, the TCR α chain variable region 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 TCR β chain variable region 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] The term "sequence identity" as used herein refers to the extent to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved and have deletions, additions or substitutions may have a lower degree of identity. Those skilled in the art will recognize that some 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] Therefore, the amino acid sequence of SEQ ID NO: 7 or 8 can be used as a "subject sequence" or "reference sequence", while the amino acid sequence of the variable region of the TCR α chain or β chain different therefrom can be used as a "query sequence".

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

[0037] The term "specific binding molecule" as used herein refers to a molecule that is 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 binding to a target antigen.

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

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

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

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

[0042] In the present invention, when the first polypeptide chain and the second polypeptide chain are combined and folded, the TRAV and TRBV are spatially close to form a binding region that specifically binds to the MAGE-A1 epitope or the complex of the epitope and the MHC molecule, and the VH and VL are spatially close 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 a heavy chain variable region VH or a light chain variable 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, and when the first binding region is VH, the second binding region is VL. Among them, the "light chain variable region (VL)" or "heavy chain variable region (VH)" consists of a "framework" region with three "complementarity determining regions" or "CDRs" interposed. The framework region is used to adjust the CDR for specific binding to antigen epitopes. CDR contains the amino acid residues in the antibody that are primarily responsible for antigen binding. From the amino terminus to the carboxyl terminus, both the VL and VH domains contain the following framework (FR) regions and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0044] 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 (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 CDRs defined by Kabat.

[0045] 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. The antibody 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.

[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 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.

[0047] As used herein, the term "antigen" refers to any substance that can induce an immune response in the body, that is, a substance that can be specifically recognized and bound by 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 or 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 following positions: position 44 of VH and position 100 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 comprises VH and VL, and cysteine ​​mutations are introduced at position 44 of VH and position 100 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 the 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 α chain constant domain TRAC and a β 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 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 α chain of the TCR molecule is "TRAC*01", and the constant domain sequence of the β chain of the TCR molecule is "TRBC1*01" or "TRBC2*01".

[0053] TRAC and TRBC can be human or mouse. TRAC and TRBC can be wild type or variants thereof. For example, variant TRAC can include one or more of T48C, N113K, PESS deletion mutation, FFPSPESS deletion mutation relative to the wild type sequence. For example, variant TRBC can include 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 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 the following positions: the 48th position of the wild type TCR α chain constant region and the 57th position of the wild type TCR β chain constant region.

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

[0055] In some embodiments, the polypeptide molecule comprises TRAC or a fragment thereof, which is not connected to TRAV by a linker. In some embodiments, the polypeptide molecule comprises TRBC or a fragment thereof, which is not connected to TRBV by a linker. In the absence of any contrary description, the term "connected by cis" refers to the connection form of the TCR constant region and the variable region in the natural state.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[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 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.

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

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

[0066] The present invention provides a specific binding molecule-anti-CD3 fusion protein, wherein it 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;

[0067] Wherein, the TRAV is the α chain variable region as described above, and the TRBV is the β chain variable region 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 N-terminus to C-terminus: TRAV-first linker-VH-third linker-TRAC, and the second polypeptide chain comprises from N-terminus to 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 an amino acid sequence as 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 an amino acid sequence as 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 an amino acid sequence as shown in SEQ ID NO: 13, and a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 14.

[0070] Nucleic Acids

[0071] The present invention provides an encoding 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] It is well known in the art that among the 20 different amino acids constituting proteins, except for Met (ATG) or Trp (TGG) which are respectively encoded by a single codon, the other 18 amino acids are respectively 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 mostly more than one codon to determine an amino acid, and the substitution of the third nucleotide in the triplet codon often does not change the composition of the amino acid, so the nucleotide sequence of the gene encoding the same protein can be different. According to the known codon table, those skilled in the art can completely deduce the nucleotide sequence of the gene that can encode them from the amino acid sequence disclosed in the present invention, and obtain the nucleotide sequence by biological methods (such as PCR method, mutation method) or chemical synthesis method, so this part of the nucleotide sequence should be included in 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 an amino acid sequence as 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 an amino acid sequence as 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] Carrier

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

[0076] The term "vector" as used herein is a nucleic acid molecule used as a medium for transferring (exogenous) genetic material into a host cell, in which the nucleic acid molecule as a vector can, for example, be replicated and / or expressed. The term "vector" encompasses, but is not limited to, plasmids, viral vectors (including retroviral vectors, lentiviral vectors, adenoviral vectors, vaccinia virus vectors, polyoma virus vectors and adenovirus-associated vectors (AAV)), bacteriophages, phagemids, cosmids and artificial chromosomes (including BACs and YACs). The vector itself is typically a nucleotide sequence, typically a DNA sequence comprising an insert (transgene) and a larger sequence as a vector "skeleton". Engineered vectors are typically included in the starting point of autonomous replication in the host cell (if stable expression of polynucleotides is required), a selection marker and a restriction enzyme cleavage site (such as a multiple cloning site, MCS). The vector may 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 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 Edition), 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 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.

[0078] cell

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

[0080] The term "cell" as used herein 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 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 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 (e.g., T cells, NK cells), monocytes (e.g., PBMCs), and stem cells. The term "stem cell" as used herein is a stem cell for expressing the TCR (particularly TCR) of the present invention. For example, a stem cell may be a lymphoid progenitor cell, an induced pluripotent stem cell (iPSC), or a hematopoietic stem cell (HSC). In some embodiments, stem cells do not include embryonic stem cells obtained by destroying human embryos, and / or do not include omnipotent stem cells for development and formation of animal individuals. Gene transfer to stem cells generally does not result in expression of TCR on the cell surface because CD3 molecules are not expressed on the stem cell surface. However, when stem cells differentiate into lymphoid precursors that migrate to the thymus, the expression of CD3 molecules will initiate expression of the introduced TCR molecules on the surface of thymocytes.

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

[0083] As used herein, the term "transfection" is a process in which nucleic acid molecules or polynucleotides (including vectors) are intentionally introduced into target cells. An example is RNA transfection, which is the process of introducing RNA (e.g., in vitro transcribed RNA, ivtRNA) into host cells. The term is mainly used for non-viral methods in eukaryotic cells. The term "transduction" is generally used to describe the transfer of viral-mediated nucleic acid molecules or polynucleotides. The transfection of animal cells generally involves opening a transient hole or "hole" in the cell membrane to allow the uptake of materials. Transfection can be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with materials to produce liposomes that fuse with the cell membrane and deposit their cargo into the interior. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate / DNA coprecipitation), microinjection, and electroporation.

[0084] In some embodiments, the method further comprises the step of expanding and / or activating the cells either 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., active agent and optional excipients) are preferably pharmaceutically acceptable, i.e., capable of inducing the desired therapeutic effect in the recipient without causing any undesirable local or systemic effects. The pharmaceutical composition of the present invention may be, for example, sterile.

[0088] The example of excipient includes but is not limited to filler, binder, disintegrant, coating agent, adsorbent, antiadhesive, glidant, preservative, antioxidant, flavoring agent, coloring agent, sweetener, solvent, cosolvent, buffer, chelating agent, viscosity imparting agent, surfactant, diluent, wetting agent, carrier, diluent, preservative, emulsifier, stabilizer and tension regulator. It is known to those skilled in the art to select suitable excipient to prepare pharmaceutical composition of the present invention. Exemplary carriers used in pharmaceutical composition of the present invention include saline, buffered saline, glucose and water. Usually, the selection of suitable excipient depends especially on the desired dosage form of the active agent used, the disease to be treated and the composition.

[0089] Depending on the active agent used, the pharmaceutical composition of the present invention can be prepared in various forms, such as solid, liquid, gaseous or lyophilized form, in particular, it can be in the form of ointment, cream, transdermal patch, gel, powder, tablet, solution, aerosol, granule, pill, suspension, emulsion, capsule, syrup, liquid, elixir, extract, tincture or fluid extract, or a form particularly suitable for the desired administration method.

[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 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.

[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 lung cancer, liver cancer, gastric cancer, esophageal cancer, melanoma and other malignant tumors.

[0093] The present invention is further described by the following specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are carried out according to the 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.

[0094] Example 1 Screening of TCRs with enhanced affinity

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

[0096] The amino acid sequences of CDR1α, CDR2α, and CDR3α are shown in SEQ ID NOs: 1-3, respectively;

[0097] The amino acid sequences of CDR1β, CDR2β, and CDR3β are shown in SEQ ID NOs: 4-6, respectively;

[0098] The amino acid sequence of the TCR α chain variable region is shown in SEQ ID NO: 7; the amino acid sequence of the TCR β chain variable region is shown in SEQ ID NO: 8; the TCR with improved affinity obtained in this way was 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 sequences provided by the present invention are cloned into TCR tandem expression lentiviral vectors respectively, the lentivirus is packaged, the reporter cell JKR9 is transduced, and then the TCR expression positive cells are sorted.

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

[0102] On the other hand, the inventors determined the effect of improving the affinity of the mutant TCR by studying the functional improvement of TCR-mediated reporter cells in recognizing target cells and activating them. Specifically, HLA-A*0201-positive T2 cells were loaded with different concentrations of MAGE-A1 target antigen peptide (SEQ ID NO: 15), and then co-incubated and activated with JKR9 reporter cells transducing the 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 results showed that the reporter cells transducing the parental TCR hT27 had only a weak activation response (<10%) under the conditions of T2 loading high concentration (-5M) polypeptide activation, and the EC50 value of the mutant TCR A1A2-M01-675 was 2.36E-08. Compared with the parental TCR, the TCR provided by the present invention has an affinity that is improved by at least 10,000 times ( Figure 1 (B)).

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

[0104] (1) Design of fusion protein

[0105] A soluble TCR-anti-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 via a linker, as follows:

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

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

[0108] The amino acid sequences of each element are shown in Table 1.

[0109] Table 1

[0110] (2) Purification and expression of fusion protein

[0111] 1) Vector construction and plasmid extraction

[0112] The nucleic acid sequences encoding the first polypeptide chain and the second polypeptide chain (shown in SEQ ID NO: 16 and SEQ ID NO: 17, respectively) were directly cloned into the expression vector pcDNA3.4, and the obtained vector clones were amplified and cultured and plasmids were extracted after being confirmed by sequencing.

[0113] 2) Transfection

[0114] Adjust the density of CHO cells to 1X106 cells / ml, and the volume of each bottle of cell solution is 40mL. Then tighten the bottle cap and place it in a shaker to continue culturing. After culturing for 2-4 hours at 36.5℃, 175rpm, and 5% CO2, use the plasmid for transfection. Prepare transfection solution (1ml): dilute 10μg DNA with about 800μL of 150 mM sterilized NaCl solution, mix well and place on the workbench for 5min; add about 50μl of transfection reagent to the DNA dilution solution and mix well. The total volume of the final transfection solution is 1mL. After placing it on the workbench for 10min, add the transfection solution dropwise to the cell culture solution, shake well, tighten the bottle cap and place it back on the shaker (36.5℃, turn off 5% CO2, 175rpm). Add SMS 293-I feed solution (0.7mL / bottle) 20-24h after transfection, and feed culture every other day for 6-10 days, and then collect the culture supernatant.

[0115] 3) Purification

[0116] Use the collected culture supernatant for nickel column affinity purification. Prepare 1ml packed nickel column, rinse with 10ml sterile water, and balance with 10ml 1XBind Buffer. Centrifuge the culture supernatant at 3000rpm for 5min, then filter with 450nm filter membrane and load the sample. After loading, wash the column with 10ml 1X Bind Buffer, wash the column with 10ml 1X Bind Buffer, wash the column with 10ml 1XWash Buffer (collect 10 tubes of eluent, flow rate is about 0.3-0.4ml / min), elute the protein with 1.2ml 1X ElutionBuffer, collect the eluent as the purified protein, and store at -80 after aliquoting.

[0117] 4) Protein purity test

[0118] The purified protein was subjected to SDS-PAGE gel electrophoresis after denaturation (R) or non-denaturation (NR) treatment to evaluate the protein expression purity and aggregation degree.

[0119] The yield of the target protein was estimated to be 33 mg / L based on the harvested target protein yield, and the purity was determined to be 85% based on the SDS-PAGE Coomassie Brilliant Blue staining results ( Figure 2). PAGE-SDS electrophoresis results showed that under non-reducing conditions, CorEngager protein mainly existed in monomeric form, with a small amount of protein existing in polymeric form. The molecular weight of the monomeric protein was about 100KDa, slightly larger than the theoretical molecular weight (77KDa), which may be caused by post-translational modification of the protein; under reducing conditions, CroEngager protein was reduced into three subunits with molecular weights between 35KDa and 70KDa. It is speculated that one of the two subunits constituting CorEngager has uneven post-translational modification of the protein. The above results show that the format (CorEngager) provided by the present invention can support the expression of TCR in the form of soluble protein.

[0120] Example 3 is used to illustrate that the CorEngaer provided by the present invention can specifically mediate T cell activation

[0121] The inventors confirmed that CorEngager can mediate effector cell-specific target recognition and activation through an in vitro T cell activation experiment. The experiment used PBMC as effector cells, T2 cells expressing HLA-A0201 as tool cells for presenting target peptides, and MAGE-A1 (KVLEYVIKV) as target peptide.

[0122] Specifically, the fusion protein of Example 2 of the present invention was diluted in a gradient manner (10 -7 Up to 10 -11 M). Dissolve the MAGE-A1 peptide in DMSO and then dilute it with water to a working concentration of 10 -4 M. T2 cells were loaded with 10 -6 M of 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 PBMCs (100 μL / well), 5x10 5 T2 cells loaded with peptides (100 μL / well) and different concentrations of fusion proteins (10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 ). T2 cells and PBMCs without peptide loading were added to the negative control, and the concentration of candidate peptide molecules was consistent with that of the experimental group. After adding all samples, the plate was covered and placed in a 37ºC, 5% CO2 incubator for 20-24 hours. ELISPOT detection was used to determine the secretion of IFN-γ to evaluate the immune cell activation induced by the fusion protein of the present invention through the target antigen peptide-MHC complex.

[0123] The experimental results showed that in the CorEngager control group using wild-type TCR hT27 ( Figure 3 B) No matter under the positive reaction condition of PBMC (+), T2 (+), MAGE-A1 (+) co-incubation or the negative reaction condition of PBMC (+), T2 (+), MAGE-A1 (-) co-incubation, the use of any concentration of CorEngager could not lead to 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, any concentration of CorEngager could not lead to the activation of effector cells; however, under the co-incubation conditions of PBMC (+), T2 (+), and MAGE-A1 (+), extremely low concentrations (-10M) of CorEngager could mediate the activation of effector cells to secrete IFN-γ, and the amount of IFN-γ secretion increased with the increase of CorEngager concentration.

[0124] The above results indicate that the format of the present invention can mediate PBMC-specific recognition of the target pMHC complex and activate PBMC, and the activation of PBMC exhibits a clear CorEngager concentration dependence.

[0125] Example 4 is used to illustrate the affinity of the CorEngager provided by the present invention for the target pMHC complex

[0126] The inventors tested the affinity of CorEngager expressed by wild-type TCR hT27 and mutant TCR A1A2-M01-675 to the target pMHC complex (MAGE-A1(KVLEYVIKV) & HLA-A0201) by SPR (surface plasmon resonance) experiment. Specifically, a certain amount of MAGE-A1 was fixed on the SA chip, and the analyte was a single concentration (500nM) of CorEngager, and the zero concentration and reference channels were set. The test results showed that CorEngager hT27 did not detect binding with the target pMHC ( Figure 4 Right); CorEngager A1A2-M01-675 exhibits a typical binding curve with fast binding and slow dissociation with the target pMHC, with a binding 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., an affinity of 13.7 nM) ( Figure 4 Left).

[0127] The above results show that the affinity of A1A2-M01-675 to target pMHC is significantly higher than that of hT27.

[0128] 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

[0129] The inventors confirmed that CorEngager can specifically mediate T cell activation and kill target cells through in vitro target cell killing experiments. Specifically, PBMCs were used as effector cells, and T2 cells loaded with -7M concentration of MAGE-A1 target peptide were used as target cells. The effector cells and target cells were mixed at an effector-target ratio (E:T) of 1:1, and CorEngager A1A2-M01-675 at concentrations of (-7M, -8M, -9M, -10M, -11M) were added respectively, and cultured in a 37°C CO2 incubator for 16-20h. The secretion of IFN-γ in the supernatant was detected using an IFN-γ ELISA kit (ExCell, Cat# EH008-96). The experimental results showed that any concentration of CorEngager hT27 could not mediate PBMC to recognize target cells and produce self-activation to secrete IFN-γ; however, an extremely low concentration (-10M) of CorEngager A1A2-M01-675 could mediate PBMC activation and secrete IFN-γ, and as the concentration of CorEngager increased, the secretion of IFN-γ also increased. At a concentration of -8M CorEngager, the secretion of IFN-γ reached the maximum level (1464 pg / ml), and the EC50 value of IFN-γ secretion was 4.47E-10 ( Figure 5 Left).

[0130] On the other hand, T2 target cells stably express the luciferase gene. After incubating the effector cells and target cells at an effector-target ratio (E:T) of 1:1 for 16-20 hours, luciferase substrate is added to detect the surviving target cells, and then the proportion of target cells killed is calculated. The experimental results show that any concentration of CorEngager hT27 cannot mediate PBMC killing of target cells; while an extremely low concentration (-11M) of CorEngager A1A2-M01-675 can mediate PBMC killing of 10% of target cells, and as the CorEngager concentration increases, the target cell killing ratio also increases, reaching the maximum killing level (60%) at a CorEngager concentration of -9M, and the killing EC50 value is 1.44E-10 ( Figure 5 right).

[0131] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications. Including the combination of various specific technical features in any suitable manner. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations. However, these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A specific binding molecule, which has the property of specifically binding to a 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.

2. The specific binding molecule according to claim 1, wherein The α chain variable region comprises the amino acid sequence shown in SEQ ID NO:7, and / or the β chain variable region comprises the amino acid sequence shown in SEQ ID NO:

8.

3. The specific binding molecule according to claim 1, wherein The specific binding molecule comprises a first polypeptide chain comprising TRAV and a first binding region of the variable region of an antibody; and a second polypeptide chain comprising TRBV and a second binding region of the variable region of an antibody, wherein the first polypeptide chain and the second polypeptide chain are combined and folded so that the specific binding molecule can simultaneously bind to an epitope of KVLEYVIKV (SEQ ID NO: 15) or a complex of the epitope with an MHC molecule and an antigen of the antibody.

4. The specific binding molecule according to claim 3, wherein The antibody is an anti-CD3 antibody, and the first binding region or the second binding region is covalently linked to the C-terminus or N-terminus of TRAV or TRBV through a linker sequence.

5. The specific binding molecule according to claim 3 or 4, wherein The specific binding molecule also includes an α chain constant domain TRAC and a β chain constant domain TRBC; The first binding region and the second binding region are each independently a heavy chain variable region VH or a light chain variable region VL of an antibody, and the amino acid sequence of the first binding region is different from the amino acid sequence of the second binding region; Wherein, the first polypeptide chain and the second polypeptide chain respectively comprise: TRAV-VH-TRAC and VL-TRBV-TRBC; TRAV-VH-TRBC and VL-TRBV-TRAC; TRAV-VL-TRAC and VH-TRBV-TRBC; TRAV-VL-TRBC and VH-TRBV-TRAC; TRBV-VH-TRAC and VL-TRAV-TRBC; TRBV-VH-TRBC and VL-TRAV-TRAC; TRBV-VL-TRAC and VH-TRAV-TRBC; or TRBV-VL-TRBC and VH-TRAV-TRAC.

6. A specific binding molecule-anti-CD3 fusion protein, characterized in that: It 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; Wherein, the TRAV is the α chain variable region of claim 2, and the TRBV is the β chain variable region of claim 2.

7. The specific binding molecule-anti-CD3 fusion protein according to claim 6, wherein: The specific binding molecule-anti-CD3 fusion protein comprises a first polypeptide chain as shown in SEQ ID NO:13 and a second polypeptide chain as shown in SEQ ID NO:

14.

8. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the specific binding molecule according to any one of claims 1 to 5, or the specific binding molecule-anti-CD3 fusion protein according to claim 6 or 7.

9. An expression vector, characterized in that The vector comprises the nucleic acid molecule of claim 8.

10. A cell, characterized in that The cell carries the nucleic acid molecule of claim 8 or the expression vector of claim 9.

11. A pharmaceutical composition comprising the specific binding molecule of any one of claims 1 to 5, the specific binding molecule-anti-CD3 fusion protein of claim 6 or 7, the nucleic acid molecule of claim 8, the expression vector of claim 9, or the cell of claim 10, and optionally one or more pharmaceutically acceptable carriers or excipients.

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