TCR for identifying MAGE-A3 and application thereof

Abstract

The invention discloses a TCR (T cell receptor) for identifying MAGE-A3 and application thereof, the TCR comprises an alpha chain and / or a beta chain, and the alpha chain or the beta chain comprises a CDR amino acid sequence or a variable region amino acid sequence described in any one of SEQ ID NO.1-48. The invention also discloses a bispecific TCR, a recombinant TCR, a nucleic acid molecule, a vector, an engineered cell and a composition.

Description

Technical Field

[0001] The invention belongs to the field of cellular immunology and genetic engineering, and relates to TCR for recognizing MAGE-A3 and application thereof. Background Art

[0002] Melanoma associated antigen A3 (MAGE-A3) is a tumor testis antigen that is expressed in many tumor types, including melanoma (up to 76%), non-small cell lung cancer (35%-50%), bladder cancer (30%-58%), and hepatocellular carcinoma (24%-78%), and affects tumor proliferation, metastasis, and pathogenicity. [1] . At the same time, the MAGE-A3 gene has a strict expression pattern and is not expressed in normal tissues other than the testis and placenta. MAGE-A3 has natural immunogenicity and can be presented to specific T cells through human leukocyte antigen molecules (HLA) on the cell surface to stimulate an immune response. Testicular cells lack the expression of HLA class I molecules, avoiding autoimmune responses against these tissues. The tumor specificity of MAGE-A3 makes it a potentially safe and valuable target for immunotherapy. MAGE-A3-derived epitopes are presented on HLA class I molecules, indicating that they are suitable as promising targets for TCR-mediated cancer immunotherapy.

[0003] T cell adoptive immunotherapy based on T cell receptor (TCR) engineering has great prospects in the treatment of solid tumors. The role of TCR is to recognize antigens by recognizing major histocompatibility complex (MHC)-epitope peptide complexes and triggering signal cascade reactions, thereby activating T cells and playing an anti-tumor immune surveillance role. The anti-tumor immune process of T cells in tumor patients is often inhibited by tumor cells. TCR therapy transforms T cells and returns them to patients by screening and identifying TCR sequences that specifically bind to target antigens, enabling them to specifically kill tumor cells. In fact, two early clinical trials of specific TCR immunotherapy targeting MAGE-A3 were terminated due to serious side effects, which were attributed to TCR recognition of homologous peptides. Severe cardiovascular toxicity occurred in clinical trials of HLA-A*01:01-restricted MAGE-A3 epitope (EVDPIGHLY)-specific TCRs. Studies have revealed that the main reason is that TCR recognizes an epitope (ESDPIVAQY) with a similar sequence to titin, resulting in T cell infiltration in the heart tissue and myocardial damage. [2-3]In a clinical trial of a TCR specific for the HLA-A*02:01 restricted MAGE-A3 epitope (KVAELVHFL), 5 / 9 patients achieved clinical regression of cancer, but 2 patients developed neurological toxicity and eventually died. The neurological toxicity is believed to be caused by the small amount of MAGE-A12 with a similar sequence (KMVKLVHFL) expressed in brain tissue. [4] However, in later studies, RNA probe technology was used to identify MAGE-A12 in brain tissue, and its expression was not observed to exceed the detection limit determined by the negative control probe, indicating that its expression was extremely limited. The EPS8L2 protein with a homologous epitope (SAAELVHFL) was expressed at a high level in brain tissue, and the researchers verified the endogenous presentation of the corresponding peptide of EPS8L2 and the targeted killing of tumor cells expressing EPS8L2 by TCR in in vitro experiments. [5] . Clinical cases of specific TCR targeting MAGE-A3 warn that there is still a need to continue to explore MAGE-A3-specific TCRs that can be used in cancer immunotherapy, and such TCRs are required to have no cross-reaction to healthy tissues or have an acceptable very low cross-reaction. Therefore, screening TCRs with high specificity is of great significance for reducing side effects and improving tumor killing effects.

[0004] Cited Literature

[0005] [1]Kruit WH et.al.Selection of immunostimulant AS15 for activeimmunization with MAGE-A3 protein:results of a randomized phase II study of the European Organization for Research and Treatment of Cancer Melanoma Groupin Metastatic Melanoma.J Clin Oncol.2013 Jul 1;31(19):2413-20.

[0006] [2]Cameron BJ et.al.Identification of a Titin-derived HLA-A1-presentedpeptide as a cross-reactive target for engineered MAGE A3-directed Tcells.Sci Transl Med.2013Aug 7;5(197):197ra103.

[0007] [3]Linette GP et al. Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced T cells in myeloma and melanoma. Blood. 2013Aug 8; 122(6):863-71.

[0008] [4]Morgan RA et al.Cancer regression and neurological toxicity following anti-MAGE-A3 TCR gene therapy.J Immunother.2013 Feb;36(2):133-51.

[0009] [5]MartinAD et al.Re-examination of MAGE-A3 as a T-cell TherapeuticTarget.J Immunother.2021 Apr 1;44(3):95-105. Summary of the invention

[0010] The present invention selects and uses the HLA-A*02:01 restricted MAGE-A3 epitope FLWGPRALV (SEQ ID NO: 49) to induce a new specific TCR, thereby screening out a T cell receptor or an antigen binding fragment thereof with high affinity and killing effect. The specific scheme is as follows:

[0011] In a first aspect, the present invention provides a TCR or an antigen binding portion thereof, characterized in that the TCR or the antigen binding portion thereof specifically binds to an epitope of MAGE-A3, and the epitope sequence is shown in SEQ ID NO:49.

[0012] The second aspect of the present invention provides a multispecific antibody comprising a first antigen binding domain, wherein the first antigen binding domain comprises the TCR or antigen binding portion thereof as described in the first aspect of the present invention.

[0013] The third aspect of the present invention provides a recombinant TCR, which comprises the TCR or antigen binding portion thereof described in the first aspect of the present invention, and a co-stimulatory region.

[0014] The fourth aspect of the present invention provides a nucleic acid molecule, characterized in that the nucleic acid molecule encodes the TCR or its antigen binding portion described in the first aspect of the present invention, the multispecific antibody described in the second aspect of the present invention, or the recombinant TCR described in the third aspect of the present invention.

[0015] The fifth aspect of the present invention provides a vector, characterized in that the vector comprises the nucleic acid molecule according to the fourth aspect of the present invention.

[0016] The sixth aspect of the present invention provides an engineered cell, which comprises the TCR or its antigen binding portion described in the first aspect of the present invention, the multispecific antibody described in the second aspect of the present invention, the recombinant TCR described in the third aspect of the present invention, the nucleic acid molecule described in the fourth aspect of the present invention, or the vector described in the fifth aspect of the present invention.

[0017] The seventh aspect of the present invention provides a composition comprising the TCR or its antigen binding portion described in the first aspect of the present invention, the multispecific antibody described in the second aspect of the present invention, the recombinant TCR described in the third aspect of the present invention, the nucleic acid molecule described in the fourth aspect of the present invention, the vector described in the fifth aspect of the present invention, or the engineered cell described in the sixth aspect of the present invention.

[0018] The eighth aspect of the present invention provides a kit, which comprises the TCR or its antigen binding portion described in the first aspect of the present invention, the multispecific antibody described in the second aspect of the present invention, the recombinant TCR described in the third aspect of the present invention, the vector described in the fifth aspect of the present invention, the engineered cell described in the sixth aspect of the present invention, or the composition described in the seventh aspect of the present invention.

[0019] The ninth aspect of the present invention provides a method for engineering cells targeting antigens, the method comprising introducing the nucleic acid molecule described in the fourth aspect of the present invention or the vector described in the fifth aspect of the present invention into the cells.

[0020] The tenth aspect of the present invention provides the use of the TCR or its antigen binding portion described in the first aspect of the present invention, the multispecific antibody described in the second aspect of the present invention, the recombinant TCR described in the third aspect of the present invention, the nucleic acid molecule described in the fourth aspect of the present invention, the vector described in the fifth aspect of the present invention, the engineered cell described in the sixth aspect of the present invention or the composition described in the seventh aspect of the present invention in 1) preparing a drug for treating and / or preventing a disease, 2) preparing a product for detecting a disease, 3) preparing an adoptive cell transfer therapy product, 4) preparing a targeted product, and 5) preparing a product for enhancing immunity.

[0021] The eleventh aspect of the present invention provides a method for preventing or treating a disease, comprising administering to a subject in need thereof the TCR or antigen-binding portion thereof described in the first aspect of the present invention, the multispecific antibody described in the second aspect of the present invention, the recombinant TCR described in the third aspect of the present invention, the nucleic acid molecule described in the fourth aspect of the present invention, the vector described in the fifth aspect of the present invention, the engineered cell described in the sixth aspect of the present invention, or the composition described in the seventh aspect of the present invention.

[0022] The twelfth aspect of the present invention provides a method for detecting a disease, comprising contacting a sample with the TCR or its antigen-binding portion described in the first aspect of the present invention, the multispecific antibody described in the second aspect of the present invention, the recombinant TCR described in the third aspect of the present invention, the nucleic acid molecule described in the fourth aspect of the present invention, the vector described in the fifth aspect of the present invention, the engineered cell described in the sixth aspect of the present invention, or the composition described in the seventh aspect of the present invention to form a complex and perform detection.

[0023] Beneficial effects of the present invention:

[0024] The present invention discovers a TCR specific to MAGE-A3, which has high affinity and antigen sensitivity. At the same time, the TCR is applied to the treatment of cancer and has a high therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a graph showing the results of inducing MAGE-A3-specific T cells after DC cells from different donors were loaded with MAGE-A3 antigen peptide (FLWGPRALV) and co-incubated with T cells.

[0026] Figure 2 This is a diagram of the structure of TCR.

[0027] Figure 3 This is a diagram of the structure of the HLA expression vector.

[0028] Figure 4 This is a flow cytometry result of pMHC tetramer staining of Jurkat-NFAT-luc cells transduced with different TCRs by electroporation.

[0029] Figure 5 This is a graph showing the detection results of the Jurkat-NFAT-luc reporter system expressing different TCRs.

[0030] Figure 6 This is a flow cytometry result of pMHC tetramer staining of peripheral blood activated T cells transduced with different TCRs by electroporation.

[0031] Figure 7 The graph shows the relative expression detection results and EC50 value of the T cell activation marker 4-1BB after the same donor peripheral blood activated T cells transduced with different TCRs by electroporation were co-incubated with T2 cells loaded with different concentration gradient antigen peptides overnight.

[0032] Figure 8 This is a graph showing the killing test results of activated T cells transduced with ZZ07 by electroporation against T2 cells loaded with antigen peptides.

[0033] Fig. 9This is the recognition motif diagram of the ZZ07 sequence.

[0034] Fig.10 This is a graph showing the killing test results of TCR-T cells that stably express ZZ07 against naturally expressing antigen target cells.

[0035] Fig.11 This is a diagram of the in vivo anti-tumor effect of TCR-T cells stably expressing ZZ07. DETAILED DESCRIPTION

[0036] The present invention relates to TCRs or antigen binding portions thereof, multispecific antibodies, recombinant TCRs, encoding nucleic acid molecules, vectors, engineered cells that specifically bind to MAGE-A3 or an epitope on MAGEA3. Some aspects of the present invention relate to methods of treating cancer in a subject in need thereof.

[0037] In the present invention, the term "T cell receptor" (TCR) refers to a heterogeneous cell surface receptor that can specifically interact with a target antigen. It contains variable α chain and β chain (also referred to as TCRα and TCRβ, respectively) or variable γ chain and δ chain (also referred to as TCRγ and TCRδ, respectively) or its antigen binding portion, and can be specifically bound to an antigen (for example, an antigen or peptide epitope bound to an MHC molecule). In some embodiments, TCR is in the form of αβ. Generally, TCRs in the form of αβ and γδ are generally similar in structure, but the T cells expressing them may have different anatomical locations or functions. TCR may be present on the cell surface or in a soluble form. In general, TCR is found on the surface of T cells (or T lymphocytes) that are generally responsible for identifying antigens bound to major histocompatibility complex (MHC) molecules.

[0038] Unless otherwise indicated, the term "TCR" should be understood to encompass a full TCR and its antigen binding portion or antigen binding fragment. In some embodiments, the TCR is a complete or full-length TCR, such as a TCR containing an alpha chain and a beta chain. In some embodiments, the TCR is an antigen binding portion that is smaller than the full-length TCR but binds to a specific peptide bound in an MHC molecule (such as binding to an MHC-peptide complex). In some cases, the antigen binding portion or fragment of a TCR may contain only a portion of the domain of the full-length or complete TCR, but is still able to bind to a peptide epitope bound by the full TCR, such as an MHC-peptide complex. In some cases, the antigen binding portion contains the variable domains of the TCR, such as the variable alpha (V) domain of the TCR. α ) chain and variable β(V β ) chain or an antigen-binding fragment thereof sufficient to form a binding site for binding to a specific MHC-peptide complex.

[0039] TCR may include two chains, α chain and β chain (or less commonly γ chain and δ chain) interconnected by a disulfide bond. Each chain includes a variable domain (α chain variable domain and β chain variable domain) and a constant region (α chain constant region and β chain constant region). The variable domain is located at the distal end of the cell membrane, and the variable domain interacts with the antigen. The constant region is located at the proximal end of the cell membrane. TCR may also include a transmembrane region and a short cytoplasmic tail. As used herein, the term "constant region" encompasses transmembrane regions and cytoplasmic tails (when present) as well as traditional "constant regions". Each chain of TCR (e.g., α or β) may have an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end. In some embodiments, TCR, for example, via a cytoplasmic tail, associates with an invariant protein of a CD3 complex involved in mediating signal transduction. In some cases, this structure allows TCR to associate with other molecules (such as CD3) or their subunits. For example, a TCR containing a constant domain and a transmembrane region can anchor the protein in the cell membrane and associate with the invariant subunits of the CD3 signaling transducer or complex. The intracellular tail of the CD3 signaling subunits (e.g., CD3γ, CD3δ, CD3ε, and CD3ζ chains) contains one or more immunoreceptor tyrosine-based activation motifs or ITAMs and is generally involved in the signaling ability of the TCR complex.

[0040] The variable domains can be further subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each α chain variable domain and β chain variable domain contains three CDRs and four FRs: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Each variable domain contains a binding domain that interacts with an antigen. Although all three CDRs on each chain participate in antigen binding, CDR3 is the primary antigen binding region. CDR1 also interacts with antigens, while CDR2 primarily recognizes HLA complexes.

[0041] In the present invention, "antigen binding molecule", "portion of TCR" or "TCR fragment" refers to any part of TCR that is smaller than the whole. Antigen binding molecules may include antigen complementarity determining regions (CDRs).

[0042] In a first aspect, the present invention provides a T cell receptor or an antigen binding molecule thereof, wherein the TCR or the antigen binding portion thereof specifically binds to an epitope of MAGE-A3, and the epitope sequence is shown in SEQ ID NO:50.

[0043] In some embodiments, the TCR or antigen binding portion thereof comprises an alpha chain CDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 9-14 and / or a beta chain CDR3 having the amino acid sequence set forth in any one of SEQ ID NOs: 35-41.

[0044] In some embodiments, the TCR or its antigen-binding portion further comprises an α chain CDR1 shown in any one of the amino acid sequences of SEQ ID NOs: 1-4 and a CDR2 shown in any one of the amino acid sequences of SEQ ID NOs: 5-8; and / or a β chain CDR1 shown in any one of the amino acid sequences of SEQ ID NOs: 21-27 and a β chain CDR2 shown in any one of the amino acid sequences of SEQ ID NOs: 28-34.

[0045] In some embodiments, the TCR or its antigen binding portion comprises CDR1, CDR2, CDR3 of the α chain variable region shown in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, and CDR1, CDR2, CDR3 of the β chain variable region shown in SEQ ID NO: 21, SEQ ID NO: 28, SEQ ID NO: 35.

[0046] In some embodiments, the TCR or its antigen binding portion comprises CDR1, CDR2, CDR3 of the α chain variable region shown in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, and CDR1, CDR2, CDR3 of the β chain variable region shown in SEQ ID NO:22, SEQ ID NO:29, SEQ ID NO:36.

[0047] In some embodiments, the TCR or its antigen binding portion comprises CDR1, CDR2, CDR3 of the α chain variable region shown in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:11, and CDR1, CDR2, CDR3 of the β chain variable region shown in SEQ ID NO:23, SEQ ID NO:30, SEQ ID NO:37.

[0048] In some embodiments, the TCR or its antigen binding portion comprises CDR1, CDR2, CDR3 of the α chain variable region shown in SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:12, and CDR1, CDR2, CDR3 of the β chain variable region shown in SEQ ID NO:24, SEQ ID NO:31, SEQ ID NO:38.

[0049] In some embodiments, the TCR or its antigen binding portion comprises CDR1, CDR2, CDR3 of the α chain variable region shown in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, and CDR1, CDR2, CDR3 of the β chain variable region shown in SEQ ID NO:25, SEQ ID NO:32, SEQ ID NO:39.

[0050] In some embodiments, the TCR or its antigen binding portion comprises CDR1, CDR2, CDR3 of the α chain variable region shown in SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:13, and CDR1, CDR2, CDR3 of the β chain variable region shown in SEQ ID NO:26, SEQ ID NO:33, SEQ ID NO:40.

[0051] In some embodiments, the TCR or its antigen binding portion comprises CDR1, CDR2, CDR3 of the α chain variable region shown in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:14, and CDR1, CDR2, CDR3 of the β chain variable region shown in SEQ ID NO:27, SEQ ID NO:34, SEQ ID NO:41.

[0052] In some embodiments, the TCR or its antigen binding portion comprises an α chain variable region that is at least 80%, preferably at least 90%, preferably at least 95%, and preferably at least 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 15-20 and / or a β chain variable region that is at least 80%, preferably at least 90%, preferably at least 95%, and preferably at least 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 42-48.

[0053] In some embodiments, the TCR or antigen binding portion thereof comprises an α chain variable region set forth in any one of the amino acid sequences of SEQ ID NOs: 15-20 and / or a β chain variable region set forth in any one of the amino acid sequences of SEQ ID NOs: 42-48.

[0054] In some embodiments, the TCR or antigen binding portion thereof comprises an α chain variable region set forth in SEQ ID NO:15 and a β chain variable region set forth in SEQ ID NO:42.

[0055] In some embodiments, the TCR or antigen binding portion thereof comprises an α chain variable region set forth in SEQ ID NO:16 and a β chain variable region set forth in SEQ ID NO:43.

[0056] In some embodiments, the TCR or antigen binding portion thereof comprises an α chain variable region set forth in SEQ ID NO:17 and a β chain variable region set forth in SEQ ID NO:44.

[0057] In some embodiments, the TCR or antigen binding portion thereof comprises an α chain variable region set forth in SEQ ID NO:18 and a β chain variable region set forth in SEQ ID NO:45.

[0058] In some embodiments, the TCR or antigen binding portion thereof comprises an α chain variable region set forth in SEQ ID NO:16 and a β chain variable region set forth in SEQ ID NO:46.

[0059] In some embodiments, the TCR or antigen binding portion thereof comprises an α chain variable region set forth in SEQ ID NO:19 and a β chain variable region set forth in SEQ ID NO:47.

[0060] In some embodiments, the TCR or antigen binding portion thereof comprises an α chain variable region set forth in SEQ ID NO:20 and a β chain variable region set forth in SEQ ID NO:48.

[0061] In some embodiments, the TCR, or antigen binding portion thereof, is a soluble TCR lacking a transmembrane domain.

[0062] In some embodiments, the TCR, or antigen binding portion thereof, binds to MHC I and / or MHC II peptide complexes.

[0063] In some embodiments, the TCR, or antigen binding portion thereof, further comprises a detectable label.

[0064] In some embodiments, the detectable label comprises an enzyme, a radionuclide, a fluorescent dye, a luminescent substance, or biotin.

[0065] In some embodiments, the TCR, or antigen binding portion thereof, further comprises a therapeutic agent.

[0066] In some embodiments, the α chain further comprises an α constant region, Ca, and / or the β chain further comprises a β constant region, Cβ.

[0067] In some embodiments, the α chain constant domain (Cα) and the β chain constant domain (Cβ) are individually mammalian.

[0068] In some embodiments, the Ca and Cβ are mouse constant regions.

[0069] In some embodiments, the Ca and Cβ are human constant regions.

[0070] In some embodiments, the TCR provided is fully human. The TCR provided is a TCR containing a human constant region, such as a fully human TCR, whose expression and / or activity, such as when expressed in human cells, such as human T cells, such as primary human T cells, is not affected or substantially not affected by the presence of endogenous human TCRs. When formatted with a human constant region, it exhibits substantial activity in primary human T cells containing endogenous TCRs.

[0071] In some embodiments, TCR or its antigen binding fragment comprises variants of alpha chain and / or beta chain. In some embodiments, variant comprises the amino acid sequence of any one of TCR described herein, which contains one, two, three or four or more amino acid substitutions in the constant region of alpha chain or beta chain. In some embodiments, TCR (or its functional part) comprising substituted amino acid sequence advantageously provides one or more of the following: compared with parent TCR comprising unsubstituted amino acid sequence, mispairing with endogenous TCR chain is reduced, expression of host cells is increased, and anti-tumor activity is increased.

[0072] In some embodiments, the Cα region and / or the Cβ region include the introduction of one or more cysteines capable of forming one or more non-natural disulfide bridges between the α chain and the β chain. The constant domain of the TCR may contain a short connection sequence in which the cysteine ​​residues form a disulfide bond, thereby connecting the two chains of the TCR. In some embodiments, the TCR may have additional cysteine ​​residues in each of the α chain and the β chain so that the TCR contains two disulfide bonds in the constant domain. In some embodiments, the constant domain and the variable domain each contain a disulfide bond formed by a cysteine ​​residue.

[0073] In some embodiments, the α chain and the β chain further comprise a signal peptide.

[0074] In some embodiments, the TCR or its antigen binding portion is a single chain (scTCR). It contains an alpha chain and a beta chain that can bind to an MHC-peptide complex. Generally, scTCRs can be produced using methods known to those skilled in the art.

[0075] In some embodiments, the scTCR contains a first segment composed of an amino acid sequence corresponding to the sequence of a provided TCR α chain variable region; a second segment composed of an amino acid sequence corresponding to the sequence of a provided TCR β chain variable region, which is fused to the N-terminus of an amino acid sequence corresponding to the extracellular sequence of a TCR β chain constant domain; and a linker sequence that connects the C-terminus of the first segment to the N-terminus of the second segment.

[0076] In some embodiments, the scTCR contains a first segment consisting of a provided α chain variable region sequence fused to the N-terminus of the α chain extracellular constant domain sequence; and a second segment consisting of a provided β chain variable region sequence fused to the N-terminus of the β chain extracellular constant and transmembrane sequences; and optionally, a linker sequence that connects the C-terminus of the first segment to the N-terminus of the second segment.

[0077] In some embodiments, the scTCR contains a first segment consisting of a provided α chain variable region sequence fused to the N-terminus of the α chain extracellular constant domain sequence; and a second segment consisting of a provided β chain variable region sequence fused to the N-terminus of the β chain extracellular constant and transmembrane sequences; and optionally, a linker sequence that connects the C-terminus of the first segment to the N-terminus of the second segment.

[0078] In some embodiments, the scTCR contains a first segment consisting of a provided TCR β chain variable region sequence fused to the N-terminus of the β chain extracellular constant domain sequence; and a second segment consisting of a provided α chain variable region sequence fused to the N-terminus of the α chain extracellular constant and transmembrane sequences; and optionally, a linker sequence that connects the C-terminus of the first segment to the N-terminus of the second segment.

[0079] In some embodiments, in order for the scTCR to bind to the MHC-peptide complex, the α chain and the β chain must be paired so that their variable region sequences are oriented for such binding. Various methods for promoting the pairing of α and β in the scTCR are well known in the art. In some embodiments, a linker sequence is included that connects the α chain and the β chain to form a single polypeptide chain. In some embodiments, the linker should have a sufficient length to cover the distance between the C-terminus of the α chain and the N-terminus of the β chain or vice versa, while also ensuring that the linker length is not too long so that it blocks or reduces the binding of the scTCR to the target peptide-MHC complex.

[0080] In some embodiments, the TCR or its antigen binding portion is a double chain (dTCR). In some embodiments, the dTCR contains a first polypeptide, wherein the sequence corresponding to the TCR α chain variable region sequence provided is fused to the N-terminus corresponding to the extracellular sequence of the TCR α chain constant region; and a second polypeptide, wherein the sequence corresponding to the TCR β chain variable region sequence provided is fused to the N-terminus corresponding to the extracellular sequence of the TCR β chain constant region, and the first polypeptide and the second polypeptide are connected by a disulfide bond. In some embodiments, the bond may correspond to the natural interchain disulfide bond present in the natural dimeric α β TCR. In some embodiments, there is no interchain disulfide bond in the natural TCR. For example, in some embodiments, one or more cysteines can be incorporated into the constant region extracellular sequence of the dTCR polypeptide. In some cases, both natural and non-natural disulfide bonds are desired. In some embodiments, the TCR contains a transmembrane sequence to anchor to the membrane.

[0081] In some embodiments, the T cell receptor or its antigen binding portion is modified. In certain embodiments, the TCR or its antigen binding portion, includes one or more amino acid changes, such as substitution, deletion, insertion and / or mutation. Exemplary variants include variants designed to improve the binding affinity and / or other biological properties of the binding molecule. Amino acid sequence variants can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the T cell receptor or antigen binding fragment or by peptide synthesis. Such modifications include, for example, the deletion and / or insertion and / or substitution of residues in the amino acid sequence of the T cell receptor or antigen binding fragment. Any combination of deletion, insertion and substitution can be made to obtain the final construct, with the limiting condition that the final construct has the desired characteristics, such as antigen binding.

[0082] In certain embodiments, TCR or its antigen binding portion thereof, including one or more amino acid substitutions, for example, compared with binding molecules (e.g., TCR) sequences described herein and / or compared with natural spectral libraries, for example, sequences of human spectral libraries. Substitution mutations induce sites of interest including CDR, FR and / or constant regions, especially FR or constant regions. Amino acid substitutions can be introduced into binding molecules of interest and the product can be screened for desired activity, such as retained / improved antigen affinity or avidity, reduced immunogenicity, improved half-life, CD8 independent binding or activity, surface expression, promotion of TCR chain pairing and / or other improved properties or functions.

[0083] In some embodiments, a TCR or antigen binding portion thereof may contain one or more modifications in the α chain and / or β chain such that when the TCR or antigen binding fragment thereof is expressed in a cell, the frequency of mispairing between the TCR α chain and β chain and endogenous TCR α chain and β chain is reduced, the expression of the TCR α chain and β chain is increased, and / or the stability of the TCR α chain and β chain is increased.

[0084] The second aspect of the present invention provides a multispecific antibody comprising a first antigen binding domain, wherein the first antigen binding domain comprises the TCR or antigen binding portion thereof as described in the first aspect of the present invention.

[0085] The term "multispecific antibody" refers to an antibody that binds to two or more different epitopes. The epitopes can be located on the same antigen or on different antigens. The multispecific antibody can be, for example, a bispecific antibody, a trispecific antibody, a tetraspecific antibody, etc. In some embodiments, the multispecific antibody binds to two, three, four, five, six or more different epitopes.

[0086] In some embodiments, the first antigen binding domain comprises a scFv, (scFv)2, Fv, Fab, F(ab')2, Fd, dAb, or VHH.

[0087] In some embodiments, the first antigen binding domain comprises a single chain variable fragment ("scFv").

[0088] In some embodiments, the multispecific antibody further comprises a second antigen binding domain.

[0089] In some embodiments, the second antigen binding domain specifically binds to a protein expressed on the surface of an immune cell.

[0090] In some embodiments, the immune cell is a T cell or a natural killer cell.

[0091] In some embodiments, the T cells are CD8 + T cells.

[0092] In some embodiments, the proteins expressed on the surface of the immune cells include CD3, CD2, CD5, CD6, CD8, CD11a (LFA-1α), CD43, CD45 and CD53, KLR4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, NKG2C, PD-L1, CD80, CD86, LAG3, CTLA4, TIM3, CD40, CD28 or 4-1BB.

[0093] In a preferred embodiment, the protein is CD3.

[0094] In some embodiments, the second antigen binding domain comprises a scFv.

[0095] In some embodiments, the first antigen binding domain and the second antigen binding domain are linked or associated by a covalent bond.

[0096] In some embodiments, the first antigen binding domain and the second antigen binding domain are linked by a peptide bond.

[0097] In a preferred embodiment, the multispecific antibody is a bispecific antibody.

[0098] The third aspect of the present invention provides a recombinant TCR, which comprises the TCR or antigen binding portion thereof described in the first aspect of the present invention, and a co-stimulatory region.

[0099] In some embodiments, the co-stimulatory region comprises a co-stimulatory molecule selected from a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, and any combination thereof.

[0100] In a preferred embodiment, the co-stimulatory region comprises a CD28 polypeptide.

[0101] The fourth aspect of the present invention provides a nucleic acid molecule, characterized in that the nucleic acid molecule encodes the TCR or its antigen binding portion described in the first aspect of the present invention, the multispecific antibody described in the second aspect of the present invention, or the recombinant TCR described in the third aspect of the present invention.

[0102] In the present invention, "nucleic acid molecule", "nucleic acid" and "polynucleotide" are used interchangeably and refer to nucleotide polymers. Such nucleotide polymers may contain natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA and PNA. "Nucleic acid sequence" refers to the linear sequence of nucleotides that constitute a nucleic acid molecule or polynucleotide.

[0103] Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, many different polynucleotides can encode the same polypeptide. In addition, it should be understood that the skilled person can use routine techniques to replace, add or delete nucleotides that do not affect the polypeptide sequence encoded by the polynucleotide of the present invention to reflect the codon selection of any specific host organism to express the polypeptide of the present invention.

[0104] The nucleotides described herein can be modified by any method available in the art. Such modifications can be made to enhance the in vivo activity or lifespan of the polynucleotides of the invention.

[0105] Polynucleotides such as DNA polynucleotides can be produced recombinantly, synthetically, or by any means available to those skilled in the art. They can also be cloned by standard techniques.

[0106] In some embodiments, the nucleic acid molecule contains a nucleic acid sequence encoding an alpha chain and / or a nucleotide sequence encoding a beta chain.

[0107] In some embodiments, the nucleotide sequence encoding the alpha chain and / or the nucleotide sequence encoding the beta chain are codon optimized. Generally, codon optimization involves balancing the percentage of codons selected by a large number of disclosed human transfer RNAs so that none of them is overloaded or restrictive. Different cells differ in their specific codon selection. The codon bias corresponds to the bias of the relative abundance of a specific tRNA in a cell type. By changing the codons in the sequence so that it is adapted to match the relative abundance of the corresponding tRNA, expression can be increased. Similarly, expression can be reduced by intentionally selecting codons that are known to be rare in a specific cell type. Therefore, an additional degree of translation control can be obtained. In general, for codon optimization, codons are selected to select those codons that are balanced with human usage frequency. Generally, the codon redundancy of an amino acid encodes an amino acid in different codons. In some embodiments, when a codon is selected for replacement, it is expected that the resulting mutation is a silent mutation so that the codon change does not affect the amino acid sequence. Generally, the last nucleotide of a codon can remain unchanged without affecting the amino acid sequence. In some cases, the nucleic acid sequence encoding a binding molecule, such as a TCR or antigen-binding fragment thereof, is modified such that a cryptic splice site is removed.

[0108] In some embodiments, the nucleotide sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain are separated by a peptide sequence that causes ribosome skipping.

[0109] In some embodiments, the nucleic acid molecule is synthetic.

[0110] In some embodiments, the nucleic acid molecule is cDNA.

[0111] The fifth aspect of the present invention provides a vector, characterized in that the vector comprises the nucleic acid molecule according to the fourth aspect of the present invention. In the present invention, "vector", "recombinant vector" and "recombinant expression vector" are used interchangeably. In some embodiments, one or more nucleic acids encoding one or both chains of a binding molecule (e.g., TCR) are cloned into one or more suitable expression vectors. The expression vector may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host. Suitable vectors include vectors designed for proliferation and amplification or for expression or for both, such as plasmids and viruses.

[0112] In some embodiments, the vector is a viral vector, a mammalian vector, or a bacterial vector.

[0113] In some embodiments, the vector is a viral vector.

[0114] In some embodiments, the viral vector is a retroviral vector. The retroviral vector includes, but is not limited to, adenoviral vectors, lentivirus, Sendai virus vectors, baculovirus vectors, Epstein-Barr virus vectors, papovavirus vectors, vaccinia virus vectors, herpes simplex virus vectors, hybrid vectors, and adeno-associated virus (AAV) vectors.

[0115] In some embodiments, the vector is a bacterial vector, including but not limited to vectors of the pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, La Jolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), or pEX series (Clontech, Palo Alto, Calif.).

[0116] The recombinant vector may include one or more marker genes that enable selection of transformed or transfected cells. Marker genes include biocide resistance, for example, resistance to antibiotics, heavy metals, etc., providing prototrophic complementation in auxotrophic host cells, etc. Suitable marker genes for carriers of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0117] In some embodiments, recombinant vectors can be prepared using standard recombinant DNA techniques. In some embodiments, the vector may contain regulatory sequences, such as transcription and translation initiation and termination codons, and when appropriate and considering that the vector is based on DNA or RNA, it has specificity for the type of host (e.g., bacteria, fungi, plants or animals) to be introduced into the vector. In some embodiments, the vector may contain a non-natural promoter operably connected to a nucleotide sequence encoding a binding molecule, such as a TCR, an antibody or its antigen-binding fragment. In some embodiments, the promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, a SV40 promoter, an RSV promoter and a promoter present in the long terminal repeat sequence of a mouse stem cell virus. Other promoters known to those skilled in the art are also contemplated.

[0118] The sixth aspect of the present invention provides an engineered cell, the engineered cell comprising the TCR or its antigen binding portion described in the first aspect of the present invention, the multispecific antibody described in the second aspect of the present invention, the recombinant TCR described in the third aspect of the present invention, the nucleic acid molecule described in the fourth aspect of the present invention, or the vector described in the fifth aspect of the present invention. The cell can be any type of cell. It can be a eukaryotic cell, for example, a plant, an animal, a fungus or an algae, or it can be a prokaryotic cell, for example, a bacterium or a protozoan. The cell can be a cultured cell or a primary cell, that is, a cell directly isolated from an organism (for example, a human). The cell can be an adherent cell or a suspension cell, that is, a cell grown in suspension. Suitable cells are known in the art and include, for example, DH5α Escherichia coli cells, Chinese hamster ovary cells, VERO cells, COS cells, HEK293 cells, etc. For the purpose of amplifying or replicating a recombinant expression vector, the cell is preferably a prokaryotic cell, for example, a DH5α cell. For the purpose of producing a recombinant TCR, a polypeptide or a protein, the cell is preferably a mammalian cell.

[0119] In some embodiments, the cells are derived from blood, bone marrow, lymph or lymphoid organs, and are immune system cells, such as innate or adaptive immune cells, for example myeloid or lymphoid cells, including lymphocytes, typically T cells and / or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells are typically primary cells, such as cells isolated directly from a subject and / or isolated and frozen from a subject. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as a complete T cell population, a CD4 ... + Cells, CD8 + Cells and their subpopulations, such as those defined below: function, activation state, maturity, differentiation potential, amplification, recirculation, location and / or persistence, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion profile and / or degree of differentiation. When referring to the treated subject, the cell can be an allogeneic cell and / or an autologous cell. These methods include existing methods. In some aspects, such as in existing technologies, the cell is a multipotent and / or multipotent cell, such as a stem cell, such as an induced pluripotent stem cell (iPSC). In some embodiments, the method includes isolating a cell from a subject, preparing it, processing it, culturing it and / or engineering it, and reintroducing it into the same patient before or after cryopreservation.

[0120] In some embodiments, the engineered cells are primary cells obtained from a subject. Samples include tissues, body fluids, and other samples obtained directly from a subject, as well as samples produced by one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with a viral vector), washing, and / or incubation. Biological samples may be samples directly obtained from a biological source or processed samples. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat; tissue and organ samples, including processed samples obtained therefrom.

[0121] In some embodiments, the sample from which the cell comes or the sample from which the cell is separated is a blood sample or a blood-derived sample or is or is derived from apheresis or leukocyte removal product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMC), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, intestinal associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil or other organs, and / or cells obtained therefrom. In cell therapy, such as adoptive cell therapy, samples include samples of autologous and allogeneic origin.

[0122] In some embodiments, the engineered cells express CD3.

[0123] In some embodiments, the cell is selected from a T cell, a natural killer cell, a natural killer T cell, or an ILC cell.

[0124] In some embodiments, the cell is a T cell. Including but not limited to subtypes and subpopulations of T cells and / or CD4+T cells and / or CD8+T cells are naive T (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes, such as stem cell memory T (T SCM ), central memory T(T CM ), effect memory T(T EM ) or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells and δ / γ T cells.

[0125] In some embodiments, the TCR or antigen binding portion thereof is heterologous to the cell.

[0126] In some embodiments, the subject is a mammal.

[0127] In some embodiments, the subject is a human.

[0128] The seventh aspect of the present invention provides a composition, comprising the claim comprising the TCR or antigen binding portion thereof according to the first aspect of the present invention, the multispecific antibody according to the second aspect of the present invention, the recombinant TCR according to the third aspect of the present invention, the nucleic acid molecule according to the fourth aspect of the present invention, the vector according to the fifth aspect of the present invention, or the engineered cell according to the sixth aspect of the present invention. The composition includes pharmaceutical compositions and formulations, and methods and uses of these molecules and compositions, such as for preventing / treating diseases, and / or detection, diagnosis and prognosis methods.

[0129] Pharmaceutical compositions and formulations typically include one or more optional pharmaceutically acceptable carriers or excipients.In some embodiments, the composition includes at least one additional therapeutic agent.

[0130] The term "pharmaceutical formulation" refers to a preparation that is in such form as to permit the biological activity of the active ingredient contained therein to be effective, and contains no additional components that would be unacceptably toxic to a subject to which the formulation would be administered.

[0131] "Pharmaceutically acceptable carrier" refers to ingredients in a pharmaceutical formulation other than the active ingredient that are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0132] In some aspects, the choice of carrier is determined in part by specific cells or binding molecules and / or by the method of administration. Therefore, there are a variety of suitable formulations. For example, the pharmaceutical composition may contain a preservative. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. Preservatives or their mixtures are usually present in an amount of about 0.0001% to about 2% of the total composition weight. Carriers are described, for example, by Remington's Pharmaceutical Sciences 16th edition, Osol, A. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexahydroxyquaternium chloride; benzalkonium chloride; benzethonium chloride; phenolic alcohol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other sugars including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG).

[0133] In some aspects, a buffer is included in the composition. Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and a variety of other acids and salts. In some aspects, a mixture of two or more buffers is used. The buffer or a mixture thereof is typically present in an amount of about 0.001% to about 4% of the total composition weight. Methods for preparing administrable pharmaceutical compositions are known.

[0134] The pharmaceutical formulation may include a lyophilized formulation and an aqueous solution. The formulation or composition may also contain more than one specific indication, disease or condition applicable to available TCR or cell therapy, preferably those indications, diseases or conditions with activity complementary to the TCR or cell, wherein each activity has no adverse effect on each other. Such active ingredients are suitable for being present in an amount combination that can effectively achieve the intended purpose. Therefore, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In some embodiments, the TCR or its antigen binding fragment is administered, for example, in the form of a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include salts derived from inorganic acids and organic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid and sulfuric acid, and organic acids such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid and arylsulfonic acids, such as p-toluenesulfonic acid.

[0135] The active ingredient can be embedded in a microcapsule, a colloidal drug delivery system (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or a macroemulsion. In certain embodiments, the pharmaceutical composition is formulated as an inclusion complex, such as a cyclodextrin inclusion complex; or as a liposome. Liposomes can be used to target host cells (e.g., T cells or NK cells) to specific tissues.

[0136] In some aspects, the pharmaceutical composition can be delivered in a timed release, delayed release, and sustained release delivery system, so that the delivery of the composition occurs before the sensitization of the treated area and the delivery time is sufficient to cause the sensitization of the treated area. Various types of release delivery systems are available and known. Such systems can avoid repeated administration of the composition, thereby improving the convenience of the subject and the physician.

[0137] In some embodiments, the pharmaceutical composition contains binding molecules and / or cells in an amount that can effectively treat or prevent a disease or condition, such as a therapeutically effective amount or a preventive effective amount. In some embodiments, the therapeutic or preventive efficacy is monitored by regularly assessing the treated subject. For repeated administration over several days or longer, depending on the condition, repeated treatment is performed until the desired inhibition of disease symptoms occurs. However, other dosing regimens may be applicable and may be determined. The desired dose may be delivered by a single bolus administration of the composition, by multiple bolus administration of the composition, or by continuous infusion administration of the composition.

[0138] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual or suppository administration. In some embodiments, the cell colony is administered parenterally. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, intracranial, intrathoracic and intraperitoneal administration. In some embodiments, the cell colony is administered to a subject by intravenous, intraperitoneal or subcutaneous injection using peripheral systemic delivery.

[0139] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0140] The eighth aspect of the present invention provides a kit, which comprises the TCR or its antigen binding portion described in the first aspect of the present invention, the multispecific antibody described in the second aspect of the present invention, the recombinant TCR described in the third aspect of the present invention, the vector described in the fifth aspect of the present invention, the engineered cell described in the sixth aspect of the present invention, or the composition described in the seventh aspect of the present invention.

[0141] In some embodiments, the kits can be used for therapeutic uses, and / or diagnostic uses.

[0142] In some embodiments, the kit may contain one or more other elements, including: instructions for use; other reagents, such as labels, therapeutic agents, or agents that can be used to chelate or otherwise couple the antibody to the label or therapeutic agent, or a radiation protecting composition; a device or other materials for preparing the antibody for administration; a pharmaceutically acceptable carrier; and a device or other materials for administration to a subject.

[0143] The ninth aspect of the present invention provides a method for engineering cells targeting antigens, the method comprising introducing the nucleic acid molecule described in the third aspect of the present invention or the vector described in the fifth aspect of the present invention into the cells.

[0144] In some embodiments, the cells are derived from cells collected from a subject in need of cell therapy;

[0145] In some embodiments, the cell is a T cell or a natural killer cell.

[0146] In some embodiments, the introducing is by transduction.

[0147] The tenth aspect of the present invention provides the use of the TCR or its antigen binding portion described in the first aspect of the present invention, the multispecific antibody described in the second aspect of the present invention, the recombinant TCR described in the third aspect of the present invention, the nucleic acid molecule described in the fourth aspect of the present invention, the vector described in the fifth aspect of the present invention, the engineered cell described in the sixth aspect of the present invention or the composition described in the seventh aspect of the present invention in 1) preparing a drug for treating and / or preventing a disease, 2) preparing a product for detecting a disease, 3) preparing an adoptive cell transfer therapy product, 4) preparing a targeted product, and 5) preparing a product for enhancing immunity.

[0148] In some embodiments, the disease is MAGE-A3 + disease.

[0149] In some embodiments, the MAGE-A3 + The disease is cancer. The cancer can be any cancer, including sarcoma (e.g., synovial sarcoma, osteogenic sarcoma, uterine leiomyosarcoma, and alveolar rhabdomyosarcoma), lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), hepatocellular carcinoma, glioma, head cancer (e.g., squamous cell carcinoma), neck cancer (e.g., squamous cell carcinoma), acute lymphocytic cancer, leukemia (e.g., acute myeloid leukemia and chronic lymphocytic leukemia), bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer, vulvar cancer, chronic myeloid cancer, colon cancer (e.g., colon cancer). any one of esophageal cancer, cervical cancer, gastric cancer, gastrointestinal carcinoid tumors, hypopharyngeal cancer, laryngeal cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer), malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer (e.g., renal cell carcinoma), small intestine cancer, soft tissue cancer, gastric cancer, testicular cancer, thyroid cancer and urothelial cancer (e.g., ureteral cancer and bladder cancer).

[0150] In some embodiments, the cancer comprises melanoma, liver cancer, breast cancer, lung cancer, prostate cancer, synovial cell sarcoma, head and neck cancer, esophageal cancer, or ovarian cancer.

[0151] In some embodiments, the adoptive cell transfer is adoptive T cell transfer.

[0152] In some embodiments, the adoptive T cell transfer is an allogeneic adoptive T cell transfer, an autologous adoptive T cell transfer, or a universal non-alloreactive adoptive T cell transfer.

[0153] The eleventh aspect provides a method for preventing or treating a disease, comprising administering to a subject in need thereof the TCR or antigen-binding portion thereof described in the first aspect of the invention, the multispecific antibody described in the second aspect of the invention, the recombinant TCR described in the third aspect of the invention, the nucleic acid molecule described in the fourth aspect of the invention, the vector described in the fifth aspect of the invention, the engineered cell described in the sixth aspect of the invention, or the composition described in the seventh aspect of the invention.

[0154] In the present invention, "treatment" and "prevention" and words derived therefrom do not necessarily mean 100% or complete treatment or prevention. On the contrary, there are different degrees of treatment or prevention where one of ordinary skill in the art considers that there is a potential benefit or therapeutic effect. In this regard, the methods of the present invention can provide any level of cancer treatment or prevention in any amount in a mammal. In addition, the treatment or prevention provided by the methods of the present invention can include the treatment of one or more conditions or symptoms of a disease (e.g., a cancer to be treated or prevented).

[0155] The twelfth aspect of the present invention provides a method for detecting a disease, comprising contacting a sample with the TCR or its antigen-binding portion described in the first aspect of the present invention, the multispecific antibody described in the second aspect of the present invention, the recombinant TCR described in the third aspect of the present invention, the nucleic acid molecule described in the fourth aspect of the present invention, the vector described in the fifth aspect of the present invention, the engineered cell described in the sixth aspect of the present invention, or the composition described in the seventh aspect of the present invention to form a complex, and detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0156] For the method of detecting cancer in a mammal of the present invention, the cancer cell sample may be a sample comprising whole cells, lysates thereof, or fractions of whole cell lysates (eg, nuclear or cytoplasmic fractions, total protein fractions, or nucleic acid fractions).

[0157] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0158] Example 1 Expansion of MAGE-A3-specific T cells

[0159] 1. Isolation of CD14 from PBMC + Monocytes and differentiation induction

[0160] HLA-A*02:01 type cryopreserved PBMCs were purchased from Miaoshun Biotechnology. The resuscitated PBMCs were resuspended in RPMI 1640 medium and the cell count was completed and the cell viability was recorded. Then, they were centrifuged at 350g for 5 minutes, the supernatant was fully discarded, and the magnetic bead sorting buffer was used according to the counting results at 1×10 8 / mL resuspend the cells, and then 8 Add 100 μL of EasySep TM Human CD14 Positive Selection Cocktail Ⅱ, pipette to mix well and let stand at room temperature for 15 minutes; TM Dextran RapidSpheres TM 50100) vortex to ensure sufficient mixing, and then add 1×10 8 Add 100 μL of magnetic beads to the cell suspension, mix well by pipetting, and let stand at room temperature for 6 minutes. Add an appropriate volume of sorting buffer, mix well, transfer to a sorting tube, and place it on a sorting rack. Let stand for 15 minutes to complete the sorting. The sorted CD14 + Monocytes were resuspended in 1640 medium containing 10% FBS and supplemented with 20 ng / mL IL-4 and 100 ng / mL GM-CSF. 6 / mL density was seeded in a six-well plate and placed in a 37°C CO2 incubator for culture. After 3 days of cell culture, the culture medium was adjusted and supplemented with 50ng / mL TNF-α for 16 hours. Mature DC cells or loaded with antigen peptide FLWGPRALV (SEQ ID NO: 49).

[0161] 2. From CD14 - Isolation of T cells from PBMC

[0162] From frozen CD14 - T cells were enriched in PBMC by negative selection, and the sorted T cells were resuspended in X-VIVO 15 medium containing 5% human serum and supplemented with 5 ng / mL IL-7. After being cultured in a 37°C carbon dioxide incubator for 1 day, they were prepared for co-culture with DC cells.

[0163] 3. Co-culture of DCs and T cells

[0164] DC cells and T cells were co-cultured for two rounds at a ratio of 1:2. The co-culture medium was X-VIVO15 medium containing 5% human serum and supplemented with 10ng / mL IL-7, 10ng / mL IL-15, and 20ng / mL IL-2. After 14 days of culture, the specific T cells induced by each group of samples were detected by flow cytometry.

[0165] Figure 1 The results show that MAGE-A3-specific T cells induced from different donors were 0.28%, 0.33%, 0.8%, 0.11%, 0.42% and 0.13% respectively when DC cells loaded with antigen peptides were co-cultured with T cells.

[0166] Example 2 Construction of in vitro transcription plasmid vector expressing MAGE-A3 specific TCR and preparation of mRNA

[0167] The α and β variable region sequences of the MAGE-A3 specific TCR obtained by sequencing are as follows:

[0168]

[0169] The α and β variable region sequences of TCR were fused with the α constant region and β constant region of the mouse, respectively. The TCRα and TCRβ chains were connected by the P2A sequence. The structure is shown in the figure Figure 2 The sequence of the α constant region is shown in SEQ ID NO: 50; the sequence of the β constant region is shown in SEQ ID NO: 51; and the exemplary complete ZZ07 sequence is shown in SEQ ID NO: 52.

[0170] The codon-optimized exogenous TCR gene of TCRα / β was synthesized and digested with BamHI and SacI restriction endonucleases, and cloned into the BamHI and SacI restriction sites of the in vitro transcription (IVT) plasmid vector. A Kozak (GCCACC) sequence was added downstream of the BamHI restriction site, and a double stop codon (TGATAA) sequence was added upstream of the SacI restriction site. After the in vitro transcription plasmid vector was constructed, mRNA synthesis was performed, and the synthesized mRNA was temporarily stored at -80°C for in vitro functional verification. The nucleic acid sequences of the α chain variable region and the β chain variable region of the exemplary codon-optimized ZZ07 are shown in SEQ ID NO: 53 and SEQ ID NO: 54, respectively.

[0171] Example 3 Activation verification of MAGE-A3 specific TCR

[0172] 1. Preparation of K562 cells overexpressing HLA-A*02:01

[0173] The plasmid constructed by HLA expression gene was constructed using 293FT tool cells ( Figure 3 ) was packaged into lentivirus, and HLA-A*02:01 was integrated into the K562 cell genome by lentivirus infection to obtain stable inheritance and expression. K562-HLA-A*02:01 OE Serves as target cells for validation of specific TCR activation.

[0174] 2. Preparation of Jurkat-NFAT-luc cells expressing TCR

[0175] The activation state of the TCR pathway can be verified by detecting the fluorescence value of Luciferase in genetically engineered Jurkat-NFAT-luc cells. TCR mRNA was transduced into Jurkat-NFAT-luc cells by electroporation to express specific TCR. The detailed steps are as follows: Jurkat-NFAT-luc cells were collected and centrifuged at 350g for 5 minutes. After washing once with DPBS (Cytiva), the cells were resuspended in R solution (Thermo Fisher Scientific) at 2×10 7 / mL, take 100μL of cell suspension and add it to a 1.5mL EP tube, add 5μg CD8αβmRNA and 5μg TCR mRNA at the same time, and mix thoroughly; add 3-5mL E2 electrotransfer buffer (Thermo Fisher Scientific) to the electrotransfer cup, place it in the cup slot of the electrotransfer instrument (ThermoFisher Scientific), carefully absorb the cell suspension mixed with mRNA with a 100μL electrotransfer gun tip to avoid bubbles, insert the electrotransfer gun into the E2 buffer of the electrotransfer instrument, set the electrotransfer conditions to 1400V, 20ms, 2pulse, start electrotransfer, transduce TCR mRNA into Jurkat-NFAT-luc cells, and obtain cells expressing TCR. 24 hours after the electrotransfer is completed, the Jurkat-NFAT-luc cells are stained with MAGE-A3 tetramers, and the results of transduction are analyzed on a flow cytometer ( Figure 4 ). The experimental results showed that Jurkat cells transduced with ZZ01, ZZ04, ZZ05, ZZ06, ZZ07, and ZZ09 all had Tetramer-positive cells, with positive rates of 93.66%, 79.95%, 21.41%, 60.44%, 86.30%, and 62.04%, respectively. Jurkat cells transduced with ZZ08 did not bind to Tetramer.

[0176] 3. TCR pathway activation status detection results

[0177] Jurkat-NFAT-luc cells electroporated for 24 hours have been shown to express TCR and were incubated with K562-HLA-A*02:01 loaded with antigen peptide (10 μM) overnight. OE The samples were co-incubated in a 96-well plate at a 20:1 effector-target ratio and incubated in a 37°C carbon dioxide incubator for 4 hours. At the same time, the Bio-Lite detection reagent (Nanjing Novozyme Biotech Co., Ltd.) stored at -20°C was placed in a dark environment to thaw and allowed to return to room temperature before use. After incubation, the samples were centrifuged and washed once with DPBS, the supernatant was removed, 100 μL Bio-Lite detection reagent was added to each well, and the samples were kept away from light for 3 to 5 minutes before detection on an ELISA instrument.

[0178] Figure 5 The fluorescence values ​​of Jurkat-NFAT-luc cells transduced with different TCRs are shown, and the control group is non-transduced cells. There are differences in the activation results of different TCRs. ZZ08 was not activated, and the fluorescence value of Jurkat-NFAT-luc cells transduced with ZZ07 was the highest. Except for ZZ08, the remaining TCRs were subsequently verified.

[0179] Example 4 Peptide-sensitive detection of MAGE-A3-specific TCR

[0180] 1. Preparation of T cells expressing TCR

[0181] Cryopreserved PBMC (Miaoshun Biotechnology) from the peripheral blood of healthy donors were revived and resuspended in X-VIVO15 medium. T cells were purified and separated by magnetic beads, activated with CD3 / CD28 magnetic beads, and resuspended in X-VIVO15 medium containing 2.5% human serum and 30 IU / mL IL-2 for three days. The magnetic beads were removed on the third day and cultured again. On the fourth day, the cells were collected and centrifuged at 350g for 5 minutes. After washing with DPBS, the cells were resuspended in R solution to 2×10 7 / mL, take 100μL of cell suspension and add it to a 1.5mL EP tube, add 5μg TCR mRNA at the same time, and mix thoroughly; add 3-5mL E2 electrotransfer buffer (Thermo Fisher Scientific) to the electrotransfer cup, place it in the electrotransfer cup slot, carefully absorb the cell suspension mixed with mRNA with a 100μL electrotransfer gun tip to avoid bubbles, insert the electrotransfer gun into the E2 solution of the electrotransfer instrument, set the electrotransfer conditions to 1400V, 10ms, 3pulse, start electrotransfer, transduce TCR mRNA into activated T cells, and obtain TCR-expressing T cells. 24 hours after electrotransfer, the T cells were stained with MAGE-A3 tetramers, and the results of transduction were analyzed on a flow cytometer ( Figure 6). The experimental results show that ZZ01, ZZ04, ZZ05, ZZ06, ZZ07, and ZZ09 were successfully transduced into activated T cells, and CD4 + and CD8 + Tetramer-positive cells can be detected by T cells. Tetramer-positive cells are in the CD3 + The proportions in T cells were 77.59%, 55%, 32.42%, 27.29%, 77.54% and 12.88% respectively.

[0182] 2. Prepare T2 cells

[0183] T2 cells lack peptide transporters (TAPs) involved in antigen processing and are unable to transfer endogenous peptides to the MHC loading site. Their HLA type is HLA-A*02:01, and they can be used as target cells for TCR peptide sensitivity verification. The cultured T2 cells were collected, washed once, and resuspended in RPMI 1640 medium containing 10% FBS. The cells were cultured at 1×10 6 / mL were inoculated in a 24-well plate, and different concentrations of antigen peptides were added, with a concentration gradient of 10 -12 M~10 -5 M, 8 groups in total, antigen peptide loading time was 4 hours.

[0184] 3. Peptide sensitivity test results

[0185] Activated T cells expressing TCR were incubated with T2 cells loaded with different concentrations of antigen peptides at a rate of 1×10 5 The number of cells per well was mixed and inoculated in a 96-well plate and placed in a 37°C carbon dioxide incubator for overnight culture. The cells of each group were collected on the second day, stained with flow cytometry antibodies CD3-Violet786, CD8-BV510, CD4-APC-Cy7, and 4-1BB-BV421, and tested on the machine. + The expression level of the T cell activation marker 4-1BB can reflect the activation effect of target cells loaded with different concentrations of antigen peptides on T cells. Taking the group with the highest expression level of 4-1BB as 100%, each TCR was analyzed and the EC50 value was calculated ( Figure 7 ), the EC50 value can reflect the peptide sensitivity of TCR. The experimental results showed that the EC50 value of ZZ01 was 4.865×10 -9 The EC50 value of M and ZZ04 was 2.487×10 -9 The EC50 value of M and ZZ05 was 9.935×10 -9 The EC50 value of M and ZZ06 was 3.076×10 -9 M, the EC50 value of ZZ07 was 6.174×10-10 M, the EC50 value of ZZ09 was 1.449×10 -8 M, among which ZZ07 had the smallest EC50 value, indicating that it had the best antigen sensitivity.

[0186] Example 5 Detection of the killing function of MAGE-A3 specific TCR on target cells loaded with antigen peptides

[0187] ZZ07 was selected to verify the killing ability of TCR-transduced T cells on target cells loaded with antigen peptides. TCR-transduced T cells were co-incubated with T2 cells loaded with antigen peptides overnight, and the killing effect was detected by microplate reader the next day ( Figure 8 ). The experimental results showed that compared with the control group, the killing efficiency of ZZ07 increased with the increase of the effect-target ratio, which was 35.59%, 46.36%, 58.29%, and 72.01% in sequence. This shows that ZZ07 has a good ability to kill target cells.

[0188] Example 6 Recognition motif of MAGE-A3 specific TCR

[0189] Alanine scanning is a conventional method used to identify specific amino acid sites that are closely related to TCR function, stability, and conformation. The amino acids at various positions of the peptide are replaced with alanine, removing the active groups on the side chain and replacing them with methyl groups that are small in size and have no other functional groups. This has little effect on the protein structure and can distinguish the effect of a specific amino acid on TCR recognition. In this experiment, an alanine scanning peptide library was constructed, and since the 7th position itself was alanine, it was chosen to be mutated to glycine for verification.

[0190] First, the TCR mRNA to be verified was transduced into T cells by electroporation, and then co-incubated with T2 cells loaded with each mutant peptide 4 hours later (mutant peptides must be added one day in advance and loaded overnight), and the secretion of IFN-γ in each group was detected by enzyme-linked immunosorbent spot (ELISPOT). The ELISPOT operation steps are as follows: After both effector cells and target cells are washed once, they are resuspended in serum-free ELISPOT medium and the cell density is adjusted to 5×10 5 / mL, the effector cells and target cells were inoculated in a pre-washed ELISPOT-IFN-γ test plate at a ratio of 1:1. After overnight incubation, the color was developed and then placed in a cool dark place at room temperature. After drying naturally, the immune spots were imaged and read using an ELISPOT analyzer (CTL S6). The ELISPOT test results and statistical results of ZZ07-transduced T cells are shown in Fig. 9The activated T samples were not treated with resting cells, and a certain number of spots were detected in the control group. In the statistical results, the statistical values ​​removed the background values ​​of the corresponding control group. Therefore, it can be determined that the recognition motif of ZZ07 is FLWG-R--V. The recognition sequence of ZZ07 is relatively conservative and has strong specificity.

[0191] Example 7 Preparation of bispecific antibodies and functional verification

[0192] 1. Preparation of bispecific antibodies and their affinity testing

[0193] TCR-CD3 bispecific antibodies (composed of higher affinity TCR and lower affinity CD3 antibody) were prepared, and the binding affinity between ZZ07 and peptide-HLA-A*02:01 molecules in the bispecific antibodies, as well as the binding affinity between the CD3 antibody used in the bispecific antibodies and the CD3 molecules were detected by surface plasmon resonance technology (SPR).

[0194] 2. The killing function of bispecific antibodies on naturally expressed antigen target cells

[0195] PBMCs were mixed with target cells NCI-H1299-LUC (MAGE-A3 + ; HLA-A*02:01 OE )、A375-LUC(MAGE-A3 + ; HLA-A*02:01 + ) were co-incubated in a 5:1 ratio, and gradiently diluted dual antibodies were added respectively. The target cell group alone was set as the negative control. After 48 hours of incubation, the fluorescence value of the unkilled cells was detected by an ELISA instrument, and the killing effect on the target cells was calculated. The Luminex multifactor detection platform was used to detect the secretion levels of cytokines such as IFN-γ, TNF-α, IL-2, IL-6, and MIP-1β in the co-culture supernatant.

[0196] The results showed that the bispecific antibody had a good killing function on target cells that naturally expressed antigens.

[0197] 3. Effects of dual antibodies on the composition of memory T cells and the killing efficiency of each cell subset

[0198] PBMCs were mixed with target cells NCI-H1299-LUC (MAGE-A3 + ; HLA-A*02:01 OE ) were co-incubated at a ratio of 5:1, and 1 nM of the prepared double antibody was added. After 48 hours of co-incubation, the cells were stained with CD3, CD4, CD8, CD107a, CD27, and CD45RO flow cytometry antibodies and tested on the machine to analyze the CD45RO in activated T cells expressing CD107a. + CD27 +Central memory T cells (Tcm) and CD45RO + CD27 - The proportion of effector memory T cells (Tem). + T cells and CD4 + T cell killing efficiency and CD8 + Subpopulations and CD4 + Tcm, Temra, Tem and The cell killing efficiency.

[0199] The results showed that the dual antibody affects the T cell immune response by affecting the composition of memory T cells; the Tem and Temra subgroups have the best killing effect on tumor cells.

[0200] 4. In vivo detection of the effect of dual antibodies on melanoma

[0201] Day 0 Human malignant melanoma cells A375 (MAGE-A3 + ; HLA-A*02:01 + ) were mixed with human PBMC and then subcutaneously transplanted into severely immunodeficient mice. From Day 1 to Day 5, different doses of dual antibodies were injected into the mice via tail vein injection every day. The tumor volume was then measured and recorded twice a week to observe the inhibitory effect of the dual antibody drug on the tumor.

[0202] The results showed that compared with the control group, the tumor volume in the dual antibody group was significantly reduced in a dose-dependent manner, indicating that the dual antibody has a good inhibitory effect on melanoma.

[0203] Example 8 TCR-T functional verification

[0204] 1. Detection of the killing function of TCR-T cells stably expressing ZZ07 on naturally expressed antigen target cells

[0205] Maestro Z cell non-destructive real-time monitoring system was used to evaluate the effect of ZZ07 stably expressed T cells on a variety of naturally expressed antigen target cells NCI H1299 HLA-A * 02:01 * KYSE410(HLA-A * 02:01 OE ) is also denoted as KYSE410 HLA-A * 02:01 * , A375, and U251. The target cells were first digested with trypsin and then resuspended at 1×10 5 The target cells were grown for 24 hours and 4×10 cells / mL were added to each well.4 ZZ07 TCR-T cells were used as ZZ07 TCR-T cells, and T cells not transduced with ZZ07 were used as control cells. The killing of target cells by effector cells was monitored in real time using an impedance meter.

[0206] The results are as follows Fig.10 As shown, ZZ07 TCR-T can effectively kill target cells NCI H1299 (HLA-A * 02:01 OE ), KYSE410(HLA-A * 02:01 OE ), A375, U251.

[0207] 2. In vivo detection of the effect of TCR-T cells stably expressing ZZ07 on lung cancer

[0208] Ten immunodeficient NOG mice (NOD-scidIl2rg - / - ) to verify the anti-tumor effect of ZZ07 TCR-T cells in vivo. Each mouse was injected subcutaneously in the armpit with 4×10 6 NCI H1703-MAGEA3 OE On the 7th day, the experimental animals were randomly divided into 3 groups according to the tumor volume, and the effector cell group and the control group were injected. Each mouse was injected with 1×10 7 cells (Tetramer positive cell rate in the experimental group was 40.5%). At the time of injection, the average tumor volume of each group was smaller.

[0209] The results are as follows Fig.11 As shown, the tumors of mice in the ZZ07 group were completely eliminated, indicating that ZZ07 has a good anti-tumor effect in vivo.

[0210] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A TCR or an antigen-binding portion thereof, characterized in that The TCR or antigen binding portion thereof specifically binds to an epitope of MAGEA3, the epitope sequence of which is shown in SEQ ID NO: 49; Preferably, the TCR or antigen-binding portion thereof comprises an α chain CDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 9-14 and / or a β chain CDR3 having an amino acid sequence as shown in any one of SEQ ID NOs: 35-41; Preferably, the TCR or its antigen-binding portion further comprises an α chain CDR1 having an amino acid sequence as shown in any one of SEQ ID NOs: 1-4 and a CDR2 having an amino acid sequence as shown in any one of SEQ ID NOs: 5-8; and / or a β chain CDR1 having an amino acid sequence as shown in any one of SEQ ID NOs: 21-27 and a β chain CDR2 having an amino acid sequence as shown in any one of SEQ ID NOs: 28-34; Preferably, the TCR or antigen-binding portion thereof comprises an α chain variable region having at least 80% identity to the amino acid sequence shown in any one of SEQ ID NOs: 15-20 and / or a β chain variable region having at least 80% identity to the amino acid sequence shown in any one of SEQ ID NOs: 42-48; Preferably, the TCR or antigen-binding portion thereof comprises an α chain variable region having at least 90% identity to the amino acid sequence shown in any one of SEQ ID NOs: 15-20 and / or a β chain variable region having at least 90% identity to the amino acid sequence shown in any one of SEQ ID NOs: 42-48; Preferably, the TCR or antigen-binding portion thereof comprises an α chain variable region having at least 95% identity to the amino acid sequence shown in any one of SEQ ID NOs: 15-20 and / or a β chain variable region having at least 95% identity to the amino acid sequence shown in any one of SEQ ID NOs: 42-48; Preferably, the TCR or antigen-binding portion thereof comprises an α chain variable region having at least 99% identity to the amino acid sequence shown in any one of SEQ ID NOs: 15-20 and / or a β chain variable region having at least 99% identity to the amino acid sequence shown in any one of SEQ ID NOs: 42-48; Preferably, the TCR or antigen-binding portion thereof comprises an α chain variable region shown in any one of the amino acid sequences of SEQ ID NOs: 15-20 and / or a β chain variable region shown in any one of the amino acid sequences of SEQ ID NOs: 42-48; Preferably, the TCR or antigen-binding portion thereof is a soluble TCR lacking a transmembrane domain; Preferably, the TCR or antigen binding portion thereof binds to MHC I and / or MHC II peptide complexes; Preferably, the TCR or antigen binding portion thereof further comprises a detectable label; Preferably, the detectable label comprises an enzyme, a radionuclide, a fluorescent dye, a luminescent substance, or biotin; Preferably, the TCR or antigen binding portion thereof further comprises a therapeutic agent; Preferably, the α chain further comprises an α constant region Cα and / or the β chain further comprises a β constant region Cβ; Preferably, the Cα and Cβ are mouse constant regions; Preferably, the Cα and Cβ are human constant regions; Preferably, the Cα region and / or the Cβ region comprises the introduction of one or more cysteines capable of forming one or more non-native disulfide bridges between the α chain and the β chain; Preferably, the α chain and the β chain further comprise a signal peptide; Preferably, the TCR or antigen-binding portion thereof is a single chain; Preferably, the TCR or antigen binding portion thereof is double-chain.

2. A multispecific antibody, characterized in that: comprising a first antigen binding domain, wherein the first antigen binding domain comprises the TCR or antigen binding portion thereof of claim 1; Preferably, the first antigen binding domain comprises a single chain variable fragment; Preferably, the multispecific antibody further comprises a second antigen binding domain; Preferably, the second antigen binding domain specifically binds to a protein expressed on the surface of an immune cell; Preferably, the immune cells are T cells or natural killer cells; Preferably, the T cells are CD8 + T cells; Preferably, the proteins expressed on the surface of the immune cells include CD3, CD2, CD5, CD6, CD8, CD11a (LFA-1α), CD43, CD45 and CD53, KLR4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, NKG2C, PD-L1, CD80, CD86, LAG3, CTLA4, TIM3, CD40, CD28 or 4-1BB; Preferably, the protein is CD3; Preferably, the second antigen binding domain comprises a scFv; Preferably, the first antigen-binding domain and the second antigen-binding domain are linked or associated via a covalent bond; Preferably, the first antigen binding domain and the second antigen binding domain are linked by a peptide bond.

3. A recombinant TCR, characterized in that The recombinant TCR comprises the TCR or antigen binding portion thereof of claim 1, and a co-stimulatory region; Preferably, the co-stimulatory region comprises a co-stimulatory molecule selected from the group consisting of CD28 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, DAP-10 polypeptide, and any combination thereof; Preferably, the co-stimulatory region comprises a CD28 polypeptide.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the TCR or antigen-binding portion thereof of claim 1, the multispecific antibody of claim 2, or the recombinant TCR of claim 3; Preferably, the nucleotide sequence of the nucleic acid molecule is codon optimized; Preferably, said nucleotide sequence encoding said α chain and said nucleotide sequence encoding said β chain are separated by a peptide sequence that causes ribosome skipping; Preferably, the nucleic acid molecule is synthetic; Preferably, the nucleic acid molecule is cDNA.

5. A carrier, characterized in that The vector comprises the nucleic acid molecule according to claim 4; Preferably, the vector is a viral vector, a mammalian vector or a bacterial vector; Preferably, the vector is a viral vector; Preferably, the viral vector is a retroviral vector; Preferably, the retroviral vector is selected from adenoviral vectors, lentivirus, Sendai virus vectors, baculovirus vectors, Epstein-Barr virus vectors, papovavirus vectors, vaccinia virus vectors, herpes simplex virus vectors, hybrid vectors and adeno-associated virus (AAV) vectors; Preferably, the retroviral vector is a lentiviral vector.

6. An engineered cell, characterized in that: The engineered cell comprises the TCR or antigen binding portion thereof of claim 1, the multispecific antibody of claim 2, the recombinant TCR of claim 3, the nucleic acid molecule of claim 4, or the vector of claim 5; Preferably, the engineered cells express CD3; Preferably, the cell is selected from T cells, natural killer cells, natural killer T cells or ILC cells; Preferably, the cell is a T cell; Preferably, said TCR or antigen binding portion thereof is heterologous to said cell; Preferably, the engineered cells are primary cells obtained from a subject; Preferably, the subject is a mammalian subject; Preferably, the subject is a human.

7. A composition, characterized in that Comprising the TCR or antigen-binding portion thereof according to claim 1, the multispecific antibody according to claim 2, the recombinant TCR according to claim 3, the nucleic acid molecule according to claim 4, the vector according to claim 5 or the engineered cell according to claim 6; Preferably, the composition further comprises a pharmaceutically acceptable carrier.

8. A kit, characterized in that: The kit comprises the TCR or antigen binding portion thereof of claim 1, the multispecific antibody of claim 2, the recombinant TCR of claim 3, the vector of claim 5, the engineered cell of claim 6 or the composition of claim 7.

9. A method for engineering a cell targeting an antigen, the method comprising introducing the nucleic acid molecule of claim 3 or the vector of claim 5 into the cell; Preferably, the cells are derived from cells collected from a subject in need of cell therapy; Preferably, the cell is a T cell or a natural killer cell; Preferably, the introduction is by transduction.

10. Applications as described in any of the following: (1) Use of the TCR or antigen-binding portion thereof according to claim 1, the multispecific antibody according to claim 2, the recombinant TCR according to claim 3, the nucleic acid molecule according to claim 4, the vector according to claim 5, the engineered cell according to claim 6 or the composition according to claim 7 in the preparation of a drug for treating and / or preventing a disease; Preferably, the disease is MAGEA3 + disease; Preferably, the MAGEA3 + The disease is cancer; Preferably, the cancer comprises melanoma, liver cancer, breast cancer, lung cancer, prostate cancer, synovial cell sarcoma, head and neck cancer, esophageal cancer or ovarian cancer; (2) Use of the TCR or antigen-binding portion thereof according to claim 1, the multispecific antibody according to claim 2, the recombinant TCR according to claim 3, the nucleic acid molecule according to claim 4, the vector according to claim 5, the engineered cell according to claim 6, or the composition according to claim 7 in the preparation of a product for detecting a disease; Preferably, the disease is MAGEA3 + disease; Preferably, the MAGEA3 + The disease is cancer; Preferably, the cancer comprises melanoma, liver cancer, breast cancer, lung cancer, prostate cancer, synovial cell sarcoma, head and neck cancer, esophageal cancer or ovarian cancer; (3) Use of the TCR or antigen-binding portion thereof according to claim 1, the multispecific antibody according to claim 2, the recombinant TCR according to claim 3, the nucleic acid molecule according to claim 4, the vector according to claim 5, the engineered cell according to claim 6 or the composition according to claim 7 in the preparation of an adoptive cell transfer therapy product; Preferably, the adoptive cell transfer is adoptive T cell transfer; Preferably, the adoptive T cell transfer is an allogeneic adoptive T cell transfer, an autologous adoptive T cell transfer, or a universal non-alloreactive adoptive T cell transfer; (4) Use of the TCR or antigen-binding portion thereof of claim 1, the multispecific antibody of claim 2, the recombinant TCR of claim 3, the nucleic acid molecule of claim 4, the vector of claim 5, the engineered cell of claim 6 or the composition of claim 7 in the preparation of a targeted product that guides therapeutic molecules to the tumor site; (5) Use of the TCR or antigen-binding portion thereof according to claim 1, the multispecific antibody according to claim 2, the recombinant TCR according to claim 3, the nucleic acid molecule according to claim 4, the vector according to claim 5, the engineered cell according to claim 6, or the composition according to claim 7 in the preparation of a product that enhances immunity.

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