CN119529098A

CN119529098A - Fusion protein of binding molecule specific for MAGE-A1 and anti-CD3

Info

Publication number: CN119529098A
Application number: CN202510101832.0A
Authority: CN
Inventors: 王雪艳; 毕晶磊; 翟佳慧; 闫宝琪
Original assignee: Beijing Kerui Biotechnology Co ltd
Current assignee: Beijing Kerui Biotechnology Co ltd
Priority date: 2025-01-22
Filing date: 2025-01-22
Publication date: 2025-02-28
Anticipated expiration: 2045-01-22
Also published as: CN119529098B

Abstract

The present invention relates to the field of immunity, in particular to a binding molecule specific for MAGE-A1 and an anti-CD3 fusion protein. The specific binding molecule has the property of specifically binding to the MAGE-A1 epitope, including a TCR alpha chain variable region containing CDR1α, CDR2α and CDR3α and a TCR beta chain variable region containing CDR1β, CDR2β and CDR3β. The fusion protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising from the N-terminus to the C-terminus: TRAV-linker-VH-optional linker-TRAC, and the second polypeptide chain comprising 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 the target antigen peptide with high affinity, and the characteristics of the TCE product prepared thereby have excellent tumor killing efficacy.

Description

Binding molecules specific for MAGE-A1 and anti-CD 3 fusion proteins

Technical Field

The present invention relates to the field of immunology, in particular to fusion proteins of a binding molecule specific for MAGE-A1 and anti-CD 3.

Background

Bispecific antibodies (bispecific antibody, bsAb, abbreviated as "diabodies") refer to an antibody molecule that targets two antigens simultaneously or two different epitopes of one antigen. Compared with the common antibody, the bispecific antibody has stronger specificity, can more accurately target tumor cells and reduce off-target toxicity. With the development of recombinant protein expression techniques and antibody engineering techniques, many different antibody formats have been produced. Multispecific antibodies are used for a variety of purposes, including (1) receptor activation (2) blocking (3) internalization (4) aggregation, (5) binding of membrane-associated proteins, or (6) targeting of cytotoxic effector cells.

T cell relocated bsAb (also known as T CELL ENGAGER, TCE) is an important form of cytotoxic effector cell relocation, a central mainstay of current cancer immunotherapy. Such bsAb recognizes a target on the surface of a tumor cell, and at the same time recognizes a molecule on the surface of a T cell (which in most cases is CD 3), so that bsAb can couple the tumor cell to a cytotoxic T cell to cause activation of a signal pathway downstream of the T cell TCR, and thereby kill the tumor cell through cytotoxicity of the T cell. Blincyto (blinatumomab) is a CD19/CD3 bispecific antibody that results in complete remission in 69% of relapsed/refractory B-precursor Acute Lymphoblastic Leukemia (ALL) patients. A number of new T cell repositioning antibody formats have emerged, such as BITE, BITE-Fc, DART-Fc, triTAC, and the like.

Based on the recognition of bsAb by antibodies to target cell surface molecules, only tumor cell surface antigens are recognized and not nearly 90% of intracellular proteins. The TCR can recognize antigen peptides of tumor specific antigens in cells and on cell surfaces, which are presented on MHC molecules on the cell surfaces after processing, and the recognition range is wider. The T cell repositioning of the TCR-based bispecific antibody changes the target cell recognition region from a traditional antibody to a TCR, which can take advantage of the broad recognition properties of TCRs to increase target selectivity.

Melanoma-associated antigen Gene family MAGE is the most well studied cancer testosterone antigen to date, and MAGE-A1 is the first identified cancer testosterone antigen gene. Numerous 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, breast cancer, etc., and therefore the development of TCE products for MAGE-A1 would likely benefit more of the indicated populations from this.

Several HLA-A 0201-restricted TCRs targeting MAGE-A1 have been developed previously, designated "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-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.)., respectively, wherein ht27 is a native TCR sequence found in humans, but has a low affinity for TCR production and cannot be used as TCE products, whereas "T1367" is a murine TCR sequence targeting the human MAGE-A1 antigen found in transgenic mice, which has a high affinity for efficient killing of tumor cells in vitro experiments, but may give rise to a strong autoimmune response in human experiments, resulting in ineffective therapy.

Thus, there is a need to develop TCR products that specifically target MAGE-A1 with high affinity.

Disclosure of Invention

The invention takes a humanized TCR ht27 sequence as a template, and uses an in vitro mutation mode to mutate a Complementarity Determining Region (CDR) region of ht27, so as to obtain a mutant TCR sequence with high affinity, and further develops a TCE product targeting MAGE-A1, wherein the TCE product has excellent tumor cell killing activity.

In a first aspect, the invention provides a specific binding molecule having the property of specifically binding to a MAGE-A1 epitope and comprising a TCR alpha chain variable region (TRAV) comprising a CDR1 alpha as set forth in SEQ ID No. 1, a CDR2 alpha as set forth in SEQ ID No. 2 and a CDR3 alpha as set forth in SEQ ID No. 3 and a TCR beta chain variable region (TRBV) comprising a CDR1 beta as set forth in SEQ ID No. 4, a CDR2 beta as set forth in SEQ ID No. 5 and a CDR3 beta as set forth in SEQ ID No. 6.

In some preferred embodiments, the alpha chain variable region comprises the amino acid sequence shown in SEQ ID NO. 7 and/or the beta chain variable region comprises the amino acid sequence shown in SEQ ID NO. 8.

In a second aspect, the invention provides a specific binding molecule-anti-CD 3 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, a TRAV-linker-VH-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus, a VL-linker-TRBV-optional linker-TRBC;

wherein the TRAV is an alpha chain variable region as described above and the TRBV is a beta chain variable region as described above.

In a third aspect, the invention provides a nucleic acid molecule, wherein the nucleic acid molecule encodes a specific binding molecule as described above, or a specific binding molecule-anti-CD 3 fusion protein as described above.

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

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

In a sixth aspect, the invention provides a pharmaceutical composition comprising a specific binding molecule as described above, a specific binding molecule-anti-CD 3 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.

The TCR provided by the invention has the capability of binding target antigen peptide with high affinity, and can reach the TCE grade, so that the prepared TCE product has excellent tumor killing effect.

Drawings

FIG. 1 shows the affinity of A1A2-M01-675 provided by the invention, wherein FIG. 1 (A) shows the binding activity of A1A2-M01-675 to its target pMHC Monomer, and FIG. 1 (B) shows the activation of JKR9 reporter cells by A1A 2-M01-675.

FIG. 2 shows the results of expression and purification of CorEngaer provided by the present invention.

FIG. 3 shows the ability of CorEngaer provided by the present invention to specifically mediate T cell activation.

Fig. 4 shows the affinity of CorEngager provided by the present invention for a target pMHC complex.

FIG. 5 shows the ability of CorEngager provided by the present invention to specifically mediate T cell activation and kill target cells.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, terms such as those described in Janeway CA Jr, travers P, walport M et al (Immunobiology), fifth edition, new York: GARLANDSCIENCE (2001) and "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and K, lbl, H.edit (1995), HELVETICA CHIMICA ACTA, CH-4010 Basel, switzerland, are used herein.

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" are used interchangeably. Similarly, the terms "comprising," "including," and "having" are used interchangeably.

The term "comprising" when used herein and in the appended claims does not exclude other elements. For the purposes 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 to include or contain at least a certain number of embodiments, it should also be understood that a group consisting of preferably only these embodiments is disclosed.

Specific binding molecules

The present invention provides a specific binding molecule having the property of specifically binding to a MAGE-A1 epitope and comprising a TCR alpha chain variable region comprising a CDR1 alpha as shown in SEQ ID No. 1, a CDR2 alpha as shown in SEQ ID No. 2 and a CDR3 alpha as shown in SEQ ID No. 3 and a TCR beta chain variable region comprising a CDR1 beta as shown in SEQ ID No. 4, a CDR2 beta as shown in SEQ ID No. 5 and a CDR3 beta as shown in SEQ ID No. 6.

The TCR provided by the invention takes ht27 as a parent, and adopts an in vitro mutation mode to mutate a Complementarity Determining Region (CDR) region of the TCR to obtain TCR clones meeting the affinity level of TCE, namely, pM-grade affinity, then prepares TCE based on the obtained TCR with high affinity to verify the efficacy, and finally determines the TCR clones with high affinity.

The term "parent TCR" as used herein refers to a previously developed human TCR ht27 targeting the HLA-A0201 restriction of MAGE-A1, the specific sequence information of which may be found in the applicant's prior patent application WO 2023/232111 A1 of the present invention, which may be incorporated herein by reference, again without further elaboration in order to avoid unnecessary repetition.

The TCR domain sequences defined in the present application are described with reference to IMGT nomenclature, which is well known and available to those skilled in the TCR art. Briefly, αβ TCRs consist of two disulfide-linked chains. Each chain (α and β) is generally considered to have two domains, a variable region and a constant domain. The short linking region connects the variable and constant domains and is generally considered to be part of the alpha variable region. In addition, the β chain typically comprises a short diversity region adjacent to the linking region, which is also typically considered part of the β variable region. The variable region of each chain is located at the N-terminus and comprises three Complementarity Determining Regions (CDRs) embedded in a framework sequence (FR). CDRs contain the recognition sites for peptide-MHC binding. There are several genes encoding the alpha chain variable (vα) region and several genes encoding the beta chain variable (vβ) region, differing in their framework, CDR1 and CDR2 sequences, as well as in the partially defined CDR3 sequences. The vα and vβ genes are denoted by the prefixes TRAV and TRBV, respectively, in IMGT nomenclature. Similarly, the α and β chains have several linked genes or J genes, known as TRAJ or TRBJ, respectively, while the β chain diversity gene or D gene is known as TRBD. The vast diversity of T cell receptor chains arises from the combined rearrangements between the various V, J and D genes, which include allelic variants and linkage diversity. The constant regions or C-domains of the TCR α and β chains are referred to as TRAC and TRBC, respectively.

Antigen specificity is conferred by the variable regions of the alpha and beta chains. Both variable regions of the TCR alpha and beta chains (alpha chain variable region (vα) and beta chain variable region (vβ)) comprise three hypervariable or complementarity determining regions (CDR 1 alpha/β, CDR2 alpha/β and CDR3 alpha/β) surrounded by 4 Framework (FR) regions (FR 1 alpha/β, FR2 alpha/β, FR3 alpha/β and FR4 alpha/β). CDR3 is the primary determinant of antigen recognition and specificity (i.e., the ability to recognize and interact with a specific antigen), while CDR1 and CDR2 interact primarily with MHC molecules presenting antigenic peptides.

In some embodiments, the framework sequences of the TCR variable domains of the invention may be murine or human, preferably human. In some preferred embodiments, the framework region is derived from the framework region of a TCR for a MAGE epitope. In some more preferred embodiments, the framework regions are derived from framework regions of TCRs directed against MAGE-A1. In some further preferred embodiments, the framework regions are derived from framework regions of HLA-A.times.02 restricted TCRs for mutated KRAS G12V. In a most preferred embodiment, the framework region is derived from the framework region of ht 27.

Thus, after the 6 CDR domains of the TCRs of the present invention are determined, the tcra chain variable region and the tcra chain variable region are not unique. In some preferred embodiments of the invention, the TCR a 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 to SEQ ID NO 7. In some preferred embodiments of the 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 to SEQ ID No. 8.

The term "sequence identity" as used herein refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at identical 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 of the identical sequence have 100% identity, but are less highly conserved and sequences with deletions, additions or substitutions may have a lower degree of identity. Those skilled in the art will recognize that some algorithms may 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.

Thus, the amino acid sequence of SEQ ID NO. 7 or 8 may be used, for example, as a "subject sequence" or "reference sequence", whereas the TCR alpha-chain or beta-chain variable region amino acid sequence, which differs therefrom, may be used as a "query sequence".

In some embodiments, the TCR alpha chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 7. In some embodiments, the TCR.beta.chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 8. In some embodiments, the TCR alpha chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 7 and the TCR beta chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 8.

The term "specific binding molecule" as used herein refers to a molecule capable of binding a target antigen. These molecules may take several different forms as discussed herein, for example, may be fragments of specific binding molecules of the invention. Fragment refers to the portion of a specific binding molecule that remains bound to a target antigen.

In some embodiments of the invention, the TCRs provided herein are 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.

The term "epitope" generally refers to a site on an antigen, typically a (poly) peptide, that is recognized by a binding domain. The term "binding domain" refers in its broadest sense 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 may comprise a single epitope, but typically comprises at least two epitopes, and may comprise any number of epitopes, depending on the size, conformation and type of antigen. The term "epitope" generally includes both linear epitopes and conformational epitopes. A linear epitope is a contiguous epitope comprised in the primary sequence of amino acids, and generally comprises at least 2 amino acids or more. Conformational epitopes are formed by the side-by-side non-contiguous amino acids by folding of the target antigen, and in particular the target (poly) peptide.

In some embodiments, the MHC molecule is of type HLA-A 02, e.g., type 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, or type HLA-A 02:11. In preferred embodiments, the MHC molecule is of type HLA-A 02:01 or type HLA-A 02:05.

The TCR molecules provided by the invention have pM affinity and, therefore, can be used to prepare TCE. In some embodiments of the invention, the specific binding molecule comprises a first polypeptide chain comprising the alpha chain variable region TRAV and a first binding region of the variable region of an antibody, and a second polypeptide chain comprising the beta chain variable region TRBV and a second binding region of the variable region of an antibody, wherein the first and second polypeptide chains are combined and folded such that the specific binding molecule is capable of simultaneously binding 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.

In the present invention, when the first and second polypeptide chains are combined and folded, the TRAV and TRBV are spatially close together to form a binding region that specifically binds to a MAGE-A1 epitope or a complex of the epitope and an MHC molecule, and the VH and VL are spatially close together to form a binding region that specifically binds to the antigen.

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. Wherein a "light chain variable region (VL)" or a "heavy chain variable region (VH)" consists of a "framework" region interspersed with three "complementarity determining regions" or "CDRs". The framework regions are used to modulate the CDRs for specific binding to the epitope. CDRs comprise amino acid residues in antibodies that are primarily responsible for antigen binding. From amino-terminus to carboxy-terminus, the VL and VH domains each comprise the Framework (FR) and CDR regions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

Amino acid assignments for each VL and VH domain are according to any conventional definition of CDR. Conventional definitions include Kabat definitions (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), chothia definitions (Chothia & Lesk, J. Mol. Biol. 196:901-917, 1987; chothia et al, nature 342:878-883, 1989), chothia Kabat CDR, where CDR-H1 is a complex of Chothia and Kabat CDRs, abM definitions used by antibody modeling software for Oxford Molecular, and Martin et al, CONTACT definitions (world Wide Web bifo. 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 Kabat-defined CDRs.

The term "antibody" as used herein is to 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) that comprise 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 comprising an epitope of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site, so long as the antibodies exhibit the desired biological activity.

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 full length antibodies.

The term "antigen" as used herein refers to any substance capable of inducing an immune response in the body. I.e., a substance that is specifically recognized and bound by antigen receptors (TCR/BCR) on the surface of T/B lymphocytes, activates T/B cells, proliferates and differentiates them, generates immune response products (sensitized lymphocytes or antibodies), and specifically binds to the corresponding products in vivo and in vitro.

In some embodiments, VH and VL comprise 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 FR2 in the case of VH. In a preferred embodiment, the cysteine mutation is selected from the group consisting of position 44 of VH and position 100 of VL.

In some embodiments, the antigen is selected from the group consisting of CD3, CD28, and 4-1BB (CD 137). 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.

In some preferred embodiments, the antigen is UCHT-1, which comprises a VH and a VL, and a cysteine mutation is introduced at position 44 of the VH and position 100 of the VL. In a more preferred embodiment, the antigen has a VH as shown in SEQ ID NO.9 and a VL as shown in SEQ ID NO.10.

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

In some embodiments, the specific binding molecule further comprises an alpha chain constant domain TRAC and a beta chain constant domain TRBC, in preferred embodiments, 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 published databases of the international immunogenetic information system (IMGT), for example the constant domain sequence of the α chain of a TCR molecule is "TRAC x 01" and the constant domain sequence of the β chain of a TCR molecule is "TRBC1 x 01" or "TRBC2 x 01".

TRAC and TRBC may be of human or murine origin. TRAC and TRBC may be wild-type or variants thereof. For example, variant TRAC may comprise one or more of a T48C, N113K, PESS deletion mutation, a FFPSPESS deletion mutation, relative to the wild-type sequence. For example, the variant TRBC may comprise one or more of the S57C, C187A, N loop deletion mutations, D, FG, relative to the wild-type sequence. In a preferred embodiment, the TRAC and/or TRBC comprises at least one cysteine mutation relative to the wild-type sequence to form a disulfide bond between the TRAC and TRBC, more preferably at position 48 of the wild-type TCR alpha chain constant region and 57 of the wild-type TCR beta chain constant region.

In a preferred embodiment, the TRAC comprises a cysteine mutation at position 48, a N113K and FFPSPESS deletion mutation, having the amino acid sequence shown in SEQ ID NO. 11. In some embodiments, the TRBC comprises a cysteine mutation at position 57, a C187A, N D mutation, having the amino acid sequence shown in SEQ ID NO. 12.

In some embodiments, the polypeptide molecule comprises TRAC or a fragment thereof, which is not homeotropically linked to TRAV via a linker. In some embodiments, the polypeptide molecule comprises TRBC or fragment thereof that is not homeotropically linked to TRBV via a linker. Without being bound by a contrary statement, the term "homeotropically linked" refers to the linkage of the constant region to the variable region of the TCR in its native state.

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

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

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

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

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 a TRBV-linker-VH-optional linker-TRAC and the second polypeptide chain comprises from N-terminus to C-terminus a VL-linker-TRAV-optional linker-TRBC.

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 a TRBV-linker-VH-optional linker-TRBC and the second polypeptide chain comprises from N-terminus to C-terminus a VL-linker-TRAV-optional linker-TRAC.

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 a TRBV-linker-VL-optional linker-TRAC and the second polypeptide chain comprises from N-terminus to C-terminus a VH-linker-TRAV-optional linker-TRBC.

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 a TRBV-linker-VL-optional linker-TRBC and the second polypeptide chain comprises from N-terminus to C-terminus a VH-linker-TRAV-optional linker-TRAC.

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.

Specific binding molecule-anti-CD 3 fusion proteins

The present invention provides a specific binding molecule-anti-CD 3 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, a TRAV-linker-VH-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus, a VL-linker-TRBV-optional linker-TRBC;

In a preferred embodiment, the specific binding molecule-anti-CD 3 fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises TRAV-first linker-VH-third linker-TRAC from the N-terminus to the C-terminus and the second polypeptide chain comprises VL-second linker-TRBV-TRBC from the N-terminus to the C-terminus, 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.

In some preferred embodiments, the fusion protein comprises a first polypeptide chain having an amino acid sequence as set forth 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 to SEQ ID NO. 13, and a second polypeptide chain having an amino acid sequence as set forth 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 to 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.

Nucleic acid

The present invention provides a coding nucleic acid molecule, wherein the nucleic acid molecule encodes a specific binding molecule as described above, or a specific binding molecule-anti-CD 3 fusion protein as described above.

It is well known in the art that of the 20 different amino acids that make up a protein, other than Met (ATG) or Trp (TGG) are each encoded by a single codon, the 18 other amino acids are each encoded by 2-6 codons (Sambrook et al, molecular cloning, cold spring harbor laboratory Press, new York, U.S. second edition, 1989, see page 950 appendix D). That is, due to the degeneracy of the genetic code, the nucleotide sequence of the gene encoding the same protein may differ, since the substitution of the third nucleotide in the triplet codon, which determines most of the codons of one amino acid, does not change the composition of the amino acid. The nucleotide sequences of the genes encoding them can be deduced entirely from the amino acid sequences disclosed in the present invention according to known codon tables by those skilled in the art, and they can be obtained by biological methods (e.g., PCR methods, mutation methods) or chemical synthesis methods, and thus, all such partial nucleotide sequences are intended to be included in the scope of the present invention.

In some preferred embodiments, the nucleic acid molecule encoding the fusion protein comprises a first polypeptide chain having an amino acid sequence as set forth 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 to SEQ ID NO. 16, and a second polypeptide chain having an amino acid sequence as set forth 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 to SEQ ID NO. 17.

Carrier body

The invention provides vectors comprising the nucleic acids provided herein.

The term "vector" as used herein is a nucleic acid molecule that serves as a vehicle for the transfer of (foreign) genetic material into a host cell in which said 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 viral vectors, polyomaviral vectors, and adeno-associated vectors (AAV)), phages, 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 that acts as the "backbone" of the vector. The engineered vector typically comprises an origin of autonomous replication in the host cell (if stable expression of the polynucleotide is desired), a selectable marker, and a restriction enzyme cleavage site (e.g., a multiple cloning site, MCS). The vector may additionally comprise promoters, genetic markers, reporter genes, targeting sequences and/or protein purification tags. As known to those skilled in the art, a number of suitable carriers 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 (4 th edition), cold Spring HarborLaboratory, cold Spring Harbor Laboratory Press, new York (2012), which is incorporated herein by reference in its entirety.

In some embodiments, the vector is preferably selected from the group consisting of lentiviral vectors, retroviral vectors, plasmids, DNA vectors, mRNA vectors, transposon-based vectors, and artificial chromosomes.

Cells

The invention provides a cell comprising a TCR, nucleic acid, or vector provided according to the invention.

The term "cell" as used herein refers to any type of cell capable of expressing a TCR of the invention. The cells may be eukaryotic cells, e.g., plants (not having the potential to develop into plants), animals, fungi, or algae, or may be prokaryotic cells, e.g., bacteria or protozoa. The cells may be cultured cells or primary cells, i.e. isolated directly from an organism, such as a human. The cells may be adherent cells or suspension cells, i.e. cells grown in suspension. Suitable host cells are known in the art and include, for example, DH 5. Alpha. E.coli cells, chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For the purpose of producing the TCR of the invention, the cell is preferably a mammalian cell. Most preferably, the host cell is a human cell.

In some embodiments, the cells are 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 a TCR of the invention (particularly a TCR). For example, the stem cells may be lymphoprogenitor cells, induced pluripotent stem cells (ipscs), or Hematopoietic Stem Cells (HSCs). In some embodiments, the stem cells do not include embryonic stem cells obtained by disrupting a human embryo, and/or totipotent stem cells for development and formation of an animal individual. Transfer of genes to stem cells typically does not result in expression of TCR on the cell surface, as the stem cell surface does not express CD3 molecules. However, when stem cells differentiate into lymphoid precursors that migrate to the thymus (lymphoid precursor), expression of the CD3 molecule will initiate expression of the introduced TCR molecule on the surface of the thymocytes.

In some embodiments, the methods of making the cells of the invention comprise the step of transducing or transfecting the cells with the vectors of the invention.

The term "transfection" as used herein is the process of deliberately introducing a nucleic acid molecule or polynucleotide (including vectors) into a target cell. One example is RNA transfection, a process whereby RNA (e.g., in vitro transcribed RNA, ivtRNA) is introduced into a host cell. The term is mainly used for non-viral methods in eukaryotic cells. The term "transduction" is generally used to describe viral-mediated transfer of a nucleic acid molecule or polynucleotide. Transfection of animal cells typically involves opening a transient pore or "hole" in the cell membrane to allow uptake of the material. Transfection may be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with materials to create 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 co-precipitation), microinjection, and electroporation.

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

Pharmaceutical composition

In another aspect, the invention provides a pharmaceutical composition comprising a specific binding molecule as described above, a specific binding molecule-anti-CD 3 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.

The pharmaceutical composition of the 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 optional excipients) are preferably pharmaceutically acceptable, i.e., capable of eliciting a desired therapeutic effect in a recipient without causing any undesired local or systemic effects. The pharmaceutical composition of the invention may be, for example, sterile.

Examples of excipients include, but are not limited to, fillers, binders, disintegrants, coating agents, adsorbents, anti-adherent agents, glidants, preservatives, antioxidants, flavoring agents, colorants, sweeteners, solvents, co-solvents, buffers, chelating agents, viscosity-imparting agents, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifiers, stabilizers, and tonicity adjusting agents. The selection of suitable excipients to prepare the pharmaceutical compositions of the present invention is known to those skilled in the art. Exemplary carriers for use in the pharmaceutical compositions of the invention include saline, buffered saline, dextrose, and water. In general, the choice of suitable excipients depends inter alia on the active agent used, the disease to be treated and the desired dosage form of the composition.

Depending on the active agent employed, the pharmaceutical compositions of the present invention may be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, and may be in particular 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 extract, or in a form particularly suitable for the desired method of administration.

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

Preferred examples of the second therapeutic agent include known anticancer drugs such as cisplatin, maytansine derivatives, rapamycin (rachelmycin), calicheamicin (calicheamicin), docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium porphyrin II (sorfimer sodiumphotofrin II), temozolomide, topotecan, glucuronic acid Qu Meisha t (TRIMETREATE GLUCURONATE), oriostatin E (auristatin E), vincristine, and doxorubicin, and peptide cytotoxins such as ricin, diphtheria toxin, pseudomonas exotoxin A, DNA enzyme, and rnase, radionuclides such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210, and 213, actinium 225, and astatin 213, prodrugs such as antibody-directed enzyme prodrugs, immunostimulants such as IL-2, chemotactic factors such as IL-8, platelet factor 4, antibodies or fragments thereof such as anti-CD 3 antibodies or fragments thereof, complement activators, viral/bacterial domains, and bacterial peptide domains.

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

The invention is further illustrated by the following specific examples. It should be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address specific conditions in the examples below, is carried out by molecular cloning according to conditions conventional in the art, for example, sambrook and Russeii et al, a laboratory Manual (third edition) (2001), conditions described in CSHL publishing, or conditions suggested by the manufacturer. Unless otherwise indicated, the experimental materials and reagents used in the following examples are all commercially available.

Example 1 is used to demonstrate the screening of affinity-enhanced TCRs

The inventors replaced the Complementarity Determining Region (CDR) region of ht27 with the humanized TCR ht27 sequence as a template using in vitro substitution, and obtained high affinity substitution clones by in vitro positive screening of the substituted TCR. By this method, the selected preferred TCRs were subjected to in vitro positive selection to obtain high affinity surrogate clones. By this method, the sequence information of the preferred TCR sequences screened is as follows:

The amino acid sequences of CDR1 alpha, CDR2 alpha and CDR3 alpha are shown in SEQ ID NO 1-3 respectively;

The amino acid sequences of CDR1 beta, CDR2 beta and CDR3 beta are respectively shown in SEQ ID NO 4-6;

The amino acid sequence of the TCR alpha chain variable region is shown as SEQ ID NO. 7, the amino acid sequence of the TCR beta chain variable region is shown as SEQ ID NO. 8, and the affinity-enhanced TCR thus obtained is designated A1A2-M01-675.

The affinity of the TCR provided by the present invention is determined by:

The parent TCR and the TCR sequence provided by the invention are cloned into TCR tandem expression lentiviral vectors respectively, lentivirus is packaged, report cell JKR9 is transduced, and then TCR expression positive cells are sorted.

The inventors judged the effect of increasing mutant TCR affinity by studying the binding activity of a soluble expressed MAGE-A1& HLA-A0201 complex Monomer (Monomer) to a TCR expressed on the surface of JKR 9. Specifically, the Monomers carrying Avi-Tag were biotinylated and then fluorescent labeled with fluorescent labeled streptavidin as Monomers, the Monomers labeled with different concentrations (80 nM, 16nM, 3.2nM, 0.64nM, 0.128 nM) were incubated with JKR9 cells expressing the parent TCR or mutant TCR, respectively, at 4℃for 1h, the free Monomers were removed by multiple rinsing with PBS, and the fluorescent positive rate of the JKR9 cells at different Monomer concentrations was detected using a flow cytometer (FACS), and the EC50 was calculated to reflect the binding activity of the TCR to the Monomers. The study results showed that JKR9 cells expressing parental TCR hT27 could not be stained with either concentration of Monomer, that 92% of cells showed positive staining with a mutant TCR at 80nM concentration of Monomer, that the cell staining positive rate decreased with decreasing Monomer concentration, that the staining EC50 value was 1.137E-09, that the study was examined for TCR binding activity to the target pMHC using the same method as the positive control using TCR used by marketed drug Tebentafusp, that the control staining positive rate was 89.7% at 80nM Monomer (gp 100 & HLA-A 0201) concentration, that the cell staining positive rate decreased with decreasing Monomer concentration, and that the staining EC50 value was 1.520E-09. The results demonstrate that the affinity of the TCR provided by the invention is significantly improved compared with the parent TCR, and the binding activity of A1A2-M01-675 to its target pMHC Monomer is close to that of the TCR used by the marketed product to its target pMHC Monnomer (figure 1 (a)).

On the other hand, the inventors judged the enhancement effect of mutant TCR affinity by studying the enhancement of TCR-mediated reporter cell function that recognizes and activates target cells. Specifically, HLA-A 0201 positive T2 cells were loaded with varying concentrations of MAGE-A1 target antigen peptide (SEQ ID NO: 15) and then co-incubated with the transduced parent TCR, JKR9 reporter cells of the TCR of the invention, respectively, for activation. After 16h activation, the stained reporter gene was subjected to flow analysis and EC50 values were calculated based on the flow analysis. The results of the study showed that reporter cells transduced with the parental TCR hT27 had a weak activation response (< 10%) only under T2 loading conditions with high (-5M) polypeptide activation, with an EC50 value of 2.36E-08 for the mutant TCR A1A 2-M01-675. The TCRs provided by the present invention have at least 10000 fold improved affinity compared to the parent TCR (fig. 1 (B)).

Example 2 is used to demonstrate the preparation of soluble TCR-anti-CD 3 fusion protein (CorEngager)

(1) Design of fusion proteins

The soluble TCR-anti-CD 3 fusion protein based on the high affinity TCR obtained in example 1 was designed and constructed, in particular, comprising a first polypeptide chain and a second polypeptide chain, the soluble TCR and the anti-CD 3 antibody being linked by a linker, in particular as follows:

The first polypeptide chain comprises TRAV-first connector-VH-third connector-TRAC from N end to C end, and the amino acid sequence is shown as SEQ ID NO. 13;

The second polypeptide chain comprises VL-second linker-TRBV-TRBC from the N end to the C end, and the amino acid sequence is shown as SEQ ID NO. 14.

Wherein the amino acid sequences of the elements are shown in Table 1.

TABLE 1

(2) Purification and expression of fusion proteins

1) Vector construction and plasmid extraction

The nucleic acid sequences (shown as SEQ ID NO:16 and SEQ ID NO: 17) encoding the first polypeptide chain and the second polypeptide chain are directly cloned into an expression vector pcDNA3.4, and the obtained vector clone is amplified, cultured and extracted by plasmid after sequencing and confirmation.

2) Transfection

The CHO cell density was adjusted to 1X106 cells/mL, the cell fluid volume per bottle was 40mL, then the flask was screwed down and placed into a shaker to continue culturing, and after culturing for 2-4 hours at 36.5℃175rpm,5% CO2, transfection with plasmid was performed. The transfection solution (1 mL) was prepared by diluting 10. Mu.g of DNA with about 800. Mu.L of 150 mM sterile NaCl solution, mixing the solution and placing the solution on a bench for 5min, adding about 50. Mu.L of transfection reagent to the DNA dilution solution, mixing the solution and finally obtaining a total volume of 1mL of the transfection solution. After 10min of standing on the bench, the transfection solution was added dropwise to the cell culture solution, after shaking, the flask was screwed back to the shaker (36.5 ℃ C., 5% CO2 was closed, 175 rpm). SMS 293-I stock solution (0.7 mL/bottle) was added 20-24h after transfection, followed by culture at intervals of 6-10 days, and then culture supernatant was collected.

3) Purification

Nickel column affinity purification was performed with the collected culture supernatant. A1 ml packed nickel column was prepared, rinsed with 10ml sterilized water, and equilibrated with 10ml 1XBind Buffer. The culture supernatant was centrifuged at 3000rpm for 5min, and then filtered through a 450nm filter, and loaded. After the sample loading is completed, the column is washed by 10ml 1X Bind Buffer, 10ml 1X Bind Buffer and 10ml 1X Wash Buffer (10 tubes of eluent are collected and the flow rate is about 0.3-0.4 ml/min), the protein is eluted by 1.2ml 1X Elution Buffer, the eluent is collected to be the purified protein, and the purified protein is sub-packaged and stored at-80.

4) Protein purity detection

Purified proteins were subjected to SDS-PAGE gel electrophoresis after denaturation (R) or non-denaturation (NR) treatment to evaluate the purity of protein expression and the degree of aggregation.

The yield of the target protein was predicted to be 33mg/L based on the yield of the target protein obtained, and the purity was determined to be 85% based on SDS-PAGE Coomassie Brilliant blue staining (FIG. 2). The result of PAGE-SDS electrophoresis shows that CorEngager proteins exist mainly in a monomer form under non-reducing condition, a small amount of proteins exist in a polymer form, the molecular weight of the monomer proteins is about 100kDa and slightly larger than the theoretical molecular weight (77 kDa), which is possibly caused by post-translational modification of the proteins, and CroEngager proteins are reduced into three subunits with molecular weights of 35kDa to 70kDa under reducing condition, and it is presumed that heterogeneous post-translational modification of the proteins exists in one of two subunits constituting CorEngager. The above results indicate that format (CorEngager) provided by the present invention can support TCR expression as a soluble protein.

Example 3 for the purpose of demonstrating CorEngaer provided by the present invention can specifically mediate T cell activation

The inventors confirm through in vitro T cell activation experiments that CorEngager can mediate effector cell specific recognition of targets and activate effector cells. The experiment uses PBMC as effector cells, T2 cells expressing HLA-A0201 as tool cells for presenting target peptides, and MAGE-A1 (KVLEYVIKV) as target peptides.

Specifically, the fusion protein of example 2 of the present invention was diluted in gradient (10 ^-7 to 10 ^-11 M) with 1640 complete medium containing 10% FBS. MAGE-A1 polypeptide was dissolved in DMSO and then diluted with water to a use concentration of 10 ^-4 M. T2 cells were used to load 10 ^-6 M MAGE-A1 polypeptide, respectively. To the ELISPOT plate was added 1640 complete medium with 10% FBS and blocked at room temperature for 30min. Media was discarded, 5x10 ⁵ cells/mL PBMC (100 μl/well), 5x10 ⁵ cells/mL polypeptide-loaded T2 cells (100 μl/well), and different concentrations of fusion protein (10 ^-7、10^-8、10^-9、10^-10、10^-11) were added to the plates. Negative controls were added to T2 cells and PBMCs that did not carry polypeptide, and the concentration of candidate polypeptide molecules was consistent with the experimental group. After all samples were added, the plate cover was covered and secretion of IFN-gamma was determined by ELISPOT assay after incubation in a 37oC, 5% CO ₂ incubator for 20-24 hours to assess immune cell activation induced by the fusion proteins of the invention by the target antigen peptide-MHC complex.

The experimental results show that no activation of effector cells could be induced by any concentration of CorEngager in CorEngager control group using wild-type TCR hT27 (FIG. 3B), either under positive reaction conditions for co-incubation with PBMC (+), T2 (+), MAGE-A1 (+), or under negative reaction conditions for co-incubation with PBMC (+), T2 (+), MAGE-A1 (-). In CorEngager experiments with mutant TCR A1A2-M01-675 (FIG. 3A), IFN-gamma secretion was mediated by very low concentrations (-10M) of CorEngager under the conditions of PBMC (+), T2 (+), MAGE-A1 (+), and IFN-gamma secretion was increased with increasing CorEngager concentration, since no concentration of CorEngager resulted in activation of effector cells due to lack of TCR recognizable target peptide.

The results show that the format of the invention can mediate the specific recognition of the target pMHC complex by PBMC and activate the PBMC, and the activation of the PBMC shows obvious CorEngager concentration dependence.

Example 4 is used to demonstrate the affinity of CorEngager provided by the present invention for the target pMHC complex

The inventors examined the affinity of CorEngager expressed by wild-type TCR hT27, mutant TCR A1A2-M01-675, with the target pMHC complex (MAGE-A1 (KVLEYVIKV) & HLA-A 0201) by SPR (surface plasmon resonance) experiments. Specifically, a certain amount of MAGE-A1 was immobilized with SA chip, and CorEngager of single concentration (500 nM) was used as the analyte, and zero concentration and reference channel were set. The detection results showed that CorEngager hT did not detect binding to the target pMHC (FIG. 4 right), corEngager A A2-M01-675 exhibited a typical binding profile 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., affinity of 13.7 nM) (FIG. 4 left).

The above results indicate that A1A2-M01-675 has significantly higher affinity for the target pMHC than hT27.

Example 5 this example demonstrates that CorEngager provided by the present invention can specifically mediate T cell activation and kill target cells

The inventors confirm through in vitro target cell killing experiments that CorEngager can specifically mediate T cell activation and kill target cells. Specifically, PBMC was used as effector cells, MAGE-A1 target peptide at a concentration of-7M was used as target cells, effector cells and target cells were mixed at an effective target ratio (E: T) of 1:1, corEngager A A2-M01-675 at a concentration of (-7M, -8M, -9M, -10M, -11M) were added, respectively, and cultured in a 37℃CO2 incubator for 16-20 hours. IFN-. Gamma.secretion was detected in the supernatant using IFN-. Gamma.ELISA kit (Excell, cat#EH008-96). The experimental results showed that CorEngager hT at any concentration could not mediate the production of self-activating IFN-gamma secretion by target cells, while CorEngager A A2-M01-675 at very low concentrations (-10M) mediated the activation and secretion of IFN-gamma by PBMC, and that the IFN-gamma secretion increased with increasing CorEngager concentration, and the IFN-gamma secretion reached the maximum level at-8M concentration CorEngager (1464 pg/ml) with an EC50 value of 4.47E-10 (FIG. 5 left).

On the other hand, T2 target cells stably express Luciferase (Luciferase) gene, and after effector cells and target cells are incubated for 16-20h according to an effective target ratio (E: T) of 1:1, luciferase substrate is added to detect surviving target cells, and the proportion of target cells killed is calculated. The experimental results showed that CorEngager hT at any concentration failed to mediate PBMC killing of target cells, while CorEngager A A2-M01-675 at very low (-11M) concentrations mediated PBMC killing of 10% of target cells, and as CorEngager concentration increased, the target cell killing rate increased, reaching a maximum killing level (60%) at-9M CorEngager concentration, with a killing EC50 value of 1.44E-10 (FIG. 5 right).

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. The technical solution of the invention can be subjected to a plurality of simple variants within the scope of the technical idea of the invention. Including the various specific features being combined in any suitable manner. The various possible combinations of the invention are not described in detail in order to avoid unnecessary repetition. Such simple variations and combinations are likewise to be regarded as being within the scope of the present disclosure.

Claims

1. A specific binding molecule having the property of specifically binding to a MAGE-A1 epitope and comprising a TCR a chain variable region (TRAV) comprising CDR1 a as shown in SEQ ID No. 1, CDR 2a as shown in SEQ ID No. 2 and CDR 3a as shown in SEQ ID No. 3, and a TCR β chain variable region (TRBV) comprising CDR1 β as shown in SEQ ID No. 4, CDR2 β as shown in SEQ ID No. 5 and CDR3 β as shown in SEQ ID No. 6.

2. The specific binding molecule according to claim 1, wherein the alpha chain variable region comprises the amino acid sequence shown as SEQ ID No. 7 and/or the beta chain variable region comprises the amino acid sequence shown as 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 the antibody, and a second polypeptide chain comprising TRBV and a second binding region of the variable region of the antibody, wherein the first and second polypeptide chains are combined and folded such that the specific binding molecule is capable of simultaneously binding 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.

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

5. The specific binding molecule of claim 3 or 4, wherein the specific binding molecule further comprises an alpha chain constant domain TRAC and a beta 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 and the amino acid sequence of the second binding region are different;

wherein the first polypeptide chain and the second polypeptide chain each 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-CD 3 fusion protein comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises TRAV-linker-VH-optional linker-TRAC from N-terminus to C-terminus and the second polypeptide chain comprises VL-linker-TRBV-optional linker-TRBC from N-terminus to C-terminus;

Wherein the TRAV is the alpha chain variable region of claim 2 and the TRBV is the beta chain variable region of claim 2.

7. The specific binding molecule-anti-CD 3 fusion protein of claim 6, wherein the specific binding molecule-anti-CD 3 fusion protein comprises a first polypeptide chain as set forth in SEQ ID No. 13 and a second polypeptide chain as set forth in SEQ ID No. 14.

8. A nucleic acid molecule encoding the specific binding molecule of any one of claims 1-5, or the specific binding molecule-anti-CD 3 fusion protein of claim 6 or 7.

9. An expression vector comprising the nucleic acid molecule of claim 8.

10. A cell carrying 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 1-5, the specific binding molecule-anti-CD 3 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.