Tcrs recognizing mage-a3 and uses thereof

Abstract

The present application discloses TCRs recognizing MAGE-A3 and uses thereof, the TCRs comprising alpha and / or beta chains, the alpha or beta chains comprising CDR amino acid sequences or variable region amino acid sequences according to any one of SEQ ID NO. 1-48. The present application also discloses bispecific TCRs, recombinant TCRs, nucleic acid molecules, vectors, engineered cells and compositions.

Description

Technical Field

[0001] This invention belongs to the fields of cellular immunology and genetic engineering, and relates to TCRs that recognize MAGE-A3 and their applications. Background Technology

[0002] Melanoma-associated antigen A3 (MAGE-A3) is a tumor-associated testis antigen that is expressed in various 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] Meanwhile, the MAGE-A3 gene exhibits a strict expression pattern, not being expressed in normal tissues outside the testes and placenta. MAGE-A3 possesses innate immunogenicity, presenting itself to specific T cells via human leukocyte antigen (HLA) molecules on the cell surface, thus eliciting an immune response. Testicular cells, lacking HLA class I molecule expression, avoid autoimmune responses against these tissues. The tumor specificity of MAGE-A3 makes it a potentially safe and valuable target for immunotherapy. The presentation of MAGE-A3-derived epitopes on HLA class I molecules suggests they are suitable as promising targets for TCR-mediated cancer immunotherapy.

[0003] T-cell adoptive immunotherapy based on T-cell receptor (TCR) engineered cells holds great promise for the treatment of solid tumors. The TCR recognizes antigens by recognizing the major histocompatibility complex (MHC)-epitope peptide complex, triggering a signaling cascade that activates T cells, enabling them to perform anti-tumor immune surveillance. However, the anti-tumor immune process of T cells in cancer patients is often suppressed by tumor cells. TCR therapy involves screening and identifying TCR sequences that specifically bind to target antigens, modifying T cells, and reinfusing them into the patient to specifically kill tumor cells. In fact, two early clinical trials of MAGE-A3-targeted specific TCR immunotherapy were terminated due to severe side effects, attributed to TCR recognition of homologous peptides. A clinical trial of an HLA-A*01:01-restricted MAGE-A3 epitope (EVDPIGHLY)-specific TCR showed severe cardiovascular toxicity. The study revealed that the main reason was that the TCR recognized an epitope with a similar sequence to titin (ESDPIVAQY), leading to T-cell infiltration in cardiac tissue and causing myocardial damage. [2-3]In a clinical trial of HLA-A*02:01-restricted MAGE-A3 epitope (KVAELVHFL)-specific TCR, 5 / 9 patients achieved clinical cancer regression, but 2 patients experienced neurotoxicity and ultimately died. The neurotoxicity is thought to be due to low expression of the similar sequence (KMVKLVHFL) MAGE-A12 in brain tissue. [4] However, later studies using RNA probe technology to identify MAGE-A12 in brain tissue did not observe its expression exceeding the detection limit determined by the negative control probe, indicating that its expression was extremely limited. In contrast, the EPS8L2 protein, which possesses a homologous epitope (SAAELVHFL), showed higher expression levels in brain tissue. Furthermore, researchers validated in vitro experiments the endogenous presentation of the corresponding EPS8L2 peptide and the targeted killing of EPS8L2-expressing tumor cells by TCR. [5] Clinical cases highlighting the need for continued exploration of MAGE-A3-specific TCRs applicable to cancer immunotherapy, particularly those exhibiting no cross-reactivity or extremely low cross-reactivity with healthy tissues. Therefore, screening for highly specific TCRs is crucial for reducing side effects and enhancing tumor-killing efficacy.

[0004] Cited References

[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] This invention induces novel specific TCRs by selectively using the HLA-A*02:01-restricted MAGE-A3 epitope FLWGPRALV (SEQ ID NO: 49), thereby screening for T cell receptors or their antigen-binding fragments with high affinity and cytotoxic effects. The specific protocol is as follows:

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

[0012] A 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 its antigen-binding portion described in the first aspect of the present invention.

[0013] A third aspect of the present invention provides a recombinant TCR comprising the TCR or its antigen-binding portion as described in the first aspect of the present invention, and a co-stimulatory region.

[0014] A 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 as described in the first aspect of the present invention, the multispecific antibody as described in the second aspect of the present invention, or the recombinant TCR as described in the third aspect of the present invention.

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

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

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

[0018] The eighth aspect of the present invention provides a kit comprising the TCR or its antigen-binding portion as described in the first aspect of the present invention, the multispecific antibody as described in the second aspect of the present invention, the recombinant TCR as described in the third aspect of the present invention, the vector as described in the fifth aspect of the present invention, the engineered cells as described in the sixth aspect of the present invention, or the composition as 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 this invention provides the use of the TCR or its antigen-binding portion described in the first aspect of this invention, the multispecific antibody described in the second aspect of this invention, the recombinant TCR described in the third aspect of this invention, the nucleic acid molecule described in the fourth aspect of this invention, the vector described in the fifth aspect of this invention, the engineered cell described in the sixth aspect of this invention, or the composition described in the seventh aspect of this invention in 1) the preparation of a medicament for treating and / or preventing diseases, 2) the preparation of a product for detecting diseases, 3) the preparation of an adoptive cell transfer therapy product, 4) the preparation of a targeted product, and 5) the preparation of an immune-enhancing product.

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

[0023] The beneficial effects of this invention are:

[0024] This invention discovers a TCR specific to MAGE-A3, which exhibits high affinity and antigen sensitivity. Furthermore, the application of this TCR in cancer treatment demonstrates high therapeutic efficacy. Attached Figure Description

[0025] Figure 1 This image shows the results of inducing MAGE-A3-specific T cells after co-incubating DC cells loaded with MAGE-A3 antigen peptide (FLWGPRALV) from different donors with T cells.

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

[0027] Figure 3 This is a structural diagram of an HLA expression vector.

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

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

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

[0031] Figure 7 This is a graph showing the relative expression of the T cell activation marker 4-1BB and EC50 values ​​after T cells with different TCRs from the same donor peripheral blood were co-incubated overnight with T2 cells loaded with different concentration gradient antigen peptides.

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

[0033] Figure 9This is the identification motif diagram of the ZZ07 sequence.

[0034] Figure 10 This is a graph showing the killing effect of TCR-T cells stably expressing ZZ07 on target cells expressing natural antigens.

[0035] Figure 11 This is an in vivo anti-tumor effect diagram of TCR-T cells that stably express ZZ07. Detailed Implementation

[0036] This invention relates to TCRs or their antigen-binding moieties that specifically bind to epitopes on MAGE-A3 or MAGEA3, multispecific antibodies, recombinant TCRs, encoding nucleic acid molecules, vectors, and engineered cells. Some aspects of this invention relate to methods for treating cancer in subjects of need.

[0037] In this invention, the term "T cell receptor" (TCR) refers to a heterogeneous cell surface receptor capable of specifically interacting with a target antigen. It contains variable α and β chains (also referred to as TCRα and TCRβ, respectively) or variable γ and δ chains (also referred to as TCRγ and TCRδ, respectively) or their antigen-binding moieties, and is capable of specifically binding to an antigen (e.g., an antigen or peptide epitope bound to an MHC molecule). In some embodiments, the TCR is in the αβ form. Typically, TCRs present in αβ and γδ forms are generally structurally similar, but T cells expressing them may have different anatomical locations or functions. TCRs may be present on the cell surface or in a soluble form. Generally, TCRs are found on the surface of T cells (or T lymphocytes) that are typically responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.

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

[0039] A TCR may comprise two chains, an α-chain and a β-chain (or less commonly, a γ-chain and a δ-chain), interconnected by disulfide bonds. Each chain contains variable domains (α-chain variable domain and β-chain variable domain) and constant regions (α-chain constant region and β-chain constant region). The variable domain is located distal to the cell membrane and interacts with the antigen. The constant region is located proximal to the cell membrane. A TCR may also include a transmembrane region and a short cytoplasmic tail. As used herein, the term “constant region” encompasses both the transmembrane region and the cytoplasmic tail (where present) as well as the conventional “constant region.” Each chain of the 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-terminus. In some embodiments, the TCR, for example via the cytoplasmic tail, associates with a constant protein of the CD3 complex involved in mediating signal transduction. In some cases, this structure allows the TCR to associate with other molecules, such as CD3 or its subunits. For example, TCRs containing both constant and transmembrane domains can anchor proteins in the cell membrane and associate with invariant subunits of CD3 signaling transducers or complexes. The intracellular tail regions of CD3 signaling subunits (e.g., CD3γ, CD3δ, CD3ε, and CD3ζ chains) contain one or more activating motifs or ITAMs based on immunoreceptor tyrosine residues and are typically involved in the signal transduction capabilities of the TCR complex.

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

[0041] In this invention, "antigen-binding molecule," "part of TCR," or "TCR fragment" refers to any portion of the TCR smaller than the whole. The antigen-binding molecule may include the antigen complementarity-determining region (CDR).

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

[0043] In some embodiments, the TCR or its antigen-binding portion comprises an α-chain CDR3 having any of the amino acid sequences shown in SEQ ID NO: 9-14 and / or a β-chain CDR3 having any of the amino acid sequences shown in SEQ ID NO: 35-41.

[0044] In some embodiments, the TCR or its antigen-binding portion further comprises an α-chain CDR1 having any amino acid sequence of SEQ ID NO: 1-4 and a CDR2 having any amino acid sequence of SEQ ID NO: 5-8; and / or a β-chain CDR1 having any amino acid sequence of SEQ ID NO: 21-27 and a β-chain CDR2 having any amino acid sequence of SEQ ID NO: 28-34.

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

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

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

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

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

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

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

[0052] In some embodiments, the TCR or its antigen-binding portion comprises an α-chain variable region having at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 99% identity with any amino acid sequence shown in SEQ ID NO:15-20 and / or a β-chain variable region having at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 99% identity with any amino acid sequence shown in SEQ ID NO:42-48.

[0053] In some embodiments, the TCR or its antigen-binding portion comprises an α-chain variable region represented by any amino acid sequence of SEQ ID NO:15-20 and / or a β-chain variable region represented by any amino acid sequence of SEQ ID NO:42-48.

[0054] In some embodiments, the TCR or its antigen-binding portion includes the α-chain variable region shown in SEQ ID NO:15 and the β-chain variable region shown in SEQ ID NO:42.

[0055] In some embodiments, the TCR or its antigen-binding portion includes the α-chain variable region shown in SEQ ID NO:16 and the β-chain variable region shown in SEQ ID NO:43.

[0056] In some embodiments, the TCR or its antigen-binding portion includes the α-chain variable region shown in SEQ ID NO:17 and the β-chain variable region shown in SEQ ID NO:44.

[0057] In some embodiments, the TCR or its antigen-binding portion includes the α-chain variable region shown in SEQ ID NO:18 and the β-chain variable region shown in SEQ ID NO:45.

[0058] In some embodiments, the TCR or its antigen-binding portion includes the α-chain variable region shown in SEQ ID NO:16 and the β-chain variable region shown in SEQ ID NO:46.

[0059] In some embodiments, the TCR or its antigen-binding portion includes the α-chain variable region shown in SEQ ID NO:19 and the β-chain variable region shown in SEQ ID NO:47.

[0060] In some embodiments, the TCR or its antigen-binding portion comprises the α-chain variable region shown in SEQ ID NO:20 and the β-chain variable region shown in SEQ ID NO:48.

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

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

[0063] In some implementations, the TCR or its antigen-binding portion further includes a detectable marker.

[0064] In some implementations, the detectable markers include enzymes, radionuclides, fluorescent dyes, luminescent substances, and biotin.

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

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

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

[0068] In some implementations, Cα and Cβ are mouse homeostatic regions.

[0069] In some implementations, Cα and Cβ are human constant regions.

[0070] In some implementations, the provided TCR is fully human. The provided TCR 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, are unaffected or substantially unaffected by the presence of an endogenous human TCR. When formatted with a human constant region, it exhibits substantial activity in primary human T cells containing an endogenous TCR.

[0071] In some embodiments, the TCR or its antigen-binding fragment comprises variants of the α chain and / or β chain. In some embodiments, the variant comprises the amino acid sequence of any of the TCRs described herein, containing one, two, three, or four or more amino acid substitutions in the constant region of the α chain or β chain. In some embodiments, a TCR (or its functional portion) comprising the substituted amino acid sequence advantageously provides one or more of the following: reduced mispairing with the endogenous TCR chain, increased expression in host cells, and increased antitumor activity compared to a parental TCR comprising the unsubstituted amino acid sequence.

[0072] In some embodiments, the Cα region and / or the Cβ region contains one or more cysteine ​​residues capable of forming one or more non-natural disulfide bridging bonds between the α and β chains. The constant domain of the TCR may contain short linker sequences in which cysteine ​​residues form disulfide bonds, thereby connecting the two chains of the TCR. In some embodiments, the TCR may have additional cysteine ​​residues in each of the α and β chains, such that the TCR contains two disulfide bonds in the constant domain. In some embodiments, both the constant and variable domains each contain disulfide bonds formed by cysteine ​​residues.

[0073] In some embodiments, the α chain and 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 α and β chains capable of binding to MHC-peptide complexes. Typically, scTCRs can be generated using methods known to those skilled in the art.

[0075] In some embodiments, the scTCR comprises a first segment consisting of an amino acid sequence corresponding to the sequence of the provided TCRα chain variable region; a second segment consisting of an amino acid sequence corresponding to the sequence of the provided TCRβ chain variable region, the sequence being fused to the N-terminus of an amino acid sequence corresponding to the extracellular sequence of the TCRβ 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 includes a first segment consisting of an α-chain variable region sequence fused to the N-terminus of an α-chain extracellular constant domain sequence; a second segment consisting of a β-chain variable region sequence fused to the N-terminus of a β-chain extracellular constant and transmembrane sequence; 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 includes a first segment consisting of an α-chain variable region sequence fused to the N-terminus of an α-chain extracellular constant domain sequence; a second segment consisting of a β-chain variable region sequence fused to the N-terminus of a β-chain extracellular constant and transmembrane sequence; 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 comprises a first segment consisting of a TCR β-chain variable region sequence fused to the N-terminus of the β-chain extracellular constant domain sequence; a second segment consisting of an α-chain variable region sequence fused to the N-terminus of the α-chain extracellular constant and transmembrane sequence; 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, for the scTCR to bind to the MHC-peptide complex, the α and β chains must be paired such that their variable region sequences are oriented for such binding. Various methods for facilitating α and β pairing in the scTCR are well known in the art. In some embodiments, a linker sequence is included, which connects the α and β chains to form a single polypeptide chain. In some embodiments, the linker should be of 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 so long as to block or reduce 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-stranded (dTCR). In some embodiments, the dTCR contains a first polypeptide wherein a sequence corresponding to the provided TCR α chain variable region sequence is fused to the N-terminus of the extracellular sequence corresponding to the TCR α chain constant region; and a second polypeptide wherein a sequence corresponding to the provided TCR β chain variable region sequence is fused to the N-terminus of the TCR β chain constant region extracellular sequence, the first polypeptide and the second polypeptide being linked by a disulfide bond. In some embodiments, this bond may correspond to a natural interchain disulfide bond present in a naturally occurring dimerized αβTCR. In some embodiments, interchain disulfide bonds are not present in a natural TCR. For example, in some embodiments, one or more cysteine ​​residues may be incorporated into the extracellular sequence of the constant region of the dTCR polypeptide pair. In some cases, both natural and non-natural disulfide bonds are desirable. 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 some embodiments, the TCR or its antigen-binding portion includes one or more amino acid variations, 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, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the T-cell receptor or antigen-binding fragment. Any combination of deletions, insertions, and substitutions can be fabricated to obtain a final construct, subject to the limitation that the final construct has the desired characteristics, such as antigen binding.

[0082] In some embodiments, the TCR or its antigen-binding portion includes one or more amino acid substitutions, for example, compared to the sequence of a binding molecule (e.g., a TCR) described herein and / or compared to the sequence of a natural library, such as a human library. Substitution mutations induce sites of interest including CDRs, FRs, and / or constant regions, especially FRs or constant regions. Amino acid substitutions can be introduced into the binding molecule of interest and products can be screened for desired activities, such as retained / improved antigen affinity or cohesion, 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, the TCR or its antigen-binding portion may contain one or more modifications in the α chain and / or β chain such that when the TCR or its antigen-binding fragment is expressed in cells, the frequency of mispairing between the TCR α and β chains and endogenous TCR α and β chains is reduced, the expression of the TCR α and β chains is increased, and / or the stability of the TCR α and β chains is increased.

[0084] A 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 its antigen-binding portion 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. Epitopes can be located on the same antigen or on different antigens. Multispecific antibodies can be, for example, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, etc. In some embodiments, multispecific antibodies bind to two, three, four, five, six, or more different epitopes.

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

[0087] In some implementations, the first antigen-binding domain comprises a single-stranded variable fragment (“scFv”).

[0088] In some implementations, the multispecific antibody further includes a second antigen-binding domain.

[0089] In some implementations, the second antigen-binding domain specifically binds to proteins expressed on the surface of immune cells.

[0090] In some implementations, the immune cells are T cells or natural killer cells.

[0091] In some implementations, the T cells are CD8 cells. + 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 implementations, the second antigen-binding domain includes scFv.

[0095] In some implementations, the first antigen-binding domain and the second antigen-binding domain are connected or associated by covalent bonds.

[0096] In some implementations, the first antigen-binding domain and the second antigen-binding domain are linked by peptide bonds.

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

[0098] A third aspect of the present invention provides a recombinant TCR comprising the TCR or its antigen-binding portion as 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 CD28 peptide, 4-1BB peptide, OX40 peptide, ICOS peptide, DAP-10 peptide, and any combination thereof.

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

[0101] A 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 as described in the first aspect of the present invention, the multispecific antibody as described in the second aspect of the present invention, or the recombinant TCR as described in the third aspect of the present invention.

[0102] In this invention, the terms "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 understand that, due to the degeneracy of the genetic code, many different polynucleotides can encode the same polypeptide. Furthermore, it should be understood that those skilled in the art can use conventional techniques to perform nucleotide substitutions, additions, or deletions of the polypeptide sequence encoded by the polynucleotides of the present invention to reflect the codon selection of any particular host organism to express the polypeptides 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 present invention.

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

[0106] In some implementations, the nucleic acid molecule contains a nucleic acid sequence encoding the α chain and / or a nucleotide sequence encoding the β chain.

[0107] In some implementations, the nucleotide sequences encoding the α-chain and / or the nucleotide sequences encoding the β-chain are codon-optimized. Typically, codon optimization involves balancing the percentage of codons selected from a large pool of publicly available human transfer RNAs, ensuring that none are overloaded or restrictive. Different cells exhibit variations in their specific codon selections. This codon bias corresponds to the bias in the relative abundance of a particular tRNA in a cell type. By altering codons in the sequence to match the relative abundance of the corresponding tRNA, expression can be increased. Similarly, expression can be reduced by intentionally selecting codons known to be rare in a particular cell type for the corresponding tRNA. Thus, an additional degree of translational control is obtained. Generally, for codon optimization, codons are selected to balance those with human usage frequencies. Typically, codon redundancy for amino acids ensures that different codons encode a single amino acid. In some implementations, when selecting codons for substitution, it is desirable that the resulting mutation be a silent mutation, such that the codon change does not affect the amino acid sequence. Typically, the last nucleotide of the codon can remain unchanged without affecting the amino acid sequence. In some cases, the nucleic acid sequence encoding binding molecules, such as TCRs or their antigen-binding fragments, is modified to remove hidden splicing sites.

[0108] In some embodiments, the nucleotide sequence encoding the α chain and the nucleotide sequence encoding the β chain are separated by a peptide sequence that causes ribosome jumping.

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

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

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

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

[0113] In some implementations, 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, adenovirus vectors, lentiviruses, Sendai virus vectors, baculovirus vectors, Epstein-Barr virus vectors, multivaccinia virus 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 biocidal resistance, such as resistance to antibiotics, heavy metals, etc., and the provision of prototrophic complementation in auxotrophic host cells. Suitable marker genes for the vectors of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidine resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0117] In some embodiments, the recombinant vector may be prepared using standard recombinant DNA techniques. In some embodiments, the vector may contain regulatory sequences, such as transcription and translation start and stop codons, and, where appropriate and considering whether the vector is DNA-based or RNA-based, specific to the type of host (e.g., bacteria, fungi, plants, or animals) to which the vector is to be introduced. In some embodiments, the vector may contain a non-natural promoter operatively linked to a nucleotide sequence encoding a binding molecule, such as a TCR, antibody, or an antigen-binding fragment thereof. In some embodiments, the promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and promoters present in long terminal repeat sequences of mouse stem cell viruses. Other promoters known to those skilled in the art are also covered.

[0118] A sixth aspect of this invention provides an engineered cell comprising the TCR or its antigen-binding portion as described in the first aspect of this invention, the multispecific antibody as described in the second aspect of this invention, the recombinant TCR as described in the third aspect of this invention, the nucleic acid molecule as described in the fourth aspect of this invention, or the vector as described in the fifth aspect of this invention. The cell can be of any type. It can be a eukaryotic cell, such as a plant, animal, fungus, or algae, or a prokaryotic cell, such as a bacterium or protozoan. The cell can be a cultured cell or a primary cell, i.e., a cell directly isolated from an organism (e.g., a human). The cell can be an adherent cell or a suspension cell, i.e., a cell growing 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 the recombinant expression vector, the cell is preferably a prokaryotic cell, such as DH5α cells. For the purpose of producing a recombinant TCR, polypeptide, or protein, the cell is preferably a mammalian cell.

[0119] In some embodiments, the cells are derived from blood, bone marrow, lymph nodes, or lymphoid organs, and are immune system cells, such as innate or adaptive immune cells, like 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 the subject and / or cells isolated from and frozen from the subject. In some embodiments, the cells include one or more subgroups of T cells or other cell types, such as intact T cell populations, CD4+, etc. + Cells, CD8 + Cells and their subpopulations, such as those defined by: function, activation state, maturity, differentiation potential, expansion, recycling, localization and / or persistence, antigen specificity, antigen receptor type, presence in a specific organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. When referring to the subject being treated, cells may be allogeneic cells and / or autologous cells. These methods include off-the-shelf methods. In some aspects, such as in off-the-shelf techniques, cells are pluripotent and / or multipotent cells, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the method includes isolating cells from the subject as described herein, preparing, processing, culturing and / or engineering them, and reintroducing them into the same patient before or after cryopreservation.

[0120] In some embodiments, the engineered cells are primary cells obtained from the subject. Samples include tissues, body fluids, and other samples obtained directly from the subject, as well as samples generated 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 can be samples obtained directly from biological sources 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; and tissue and organ samples, including processed samples obtained therefrom.

[0121] In some implementations, the sample from which the cells are derived or from which the cells are isolated is a blood sample or a blood-derived sample, or is derived from apheresis or leukocyte removal products. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumors, leukemia, lymphoma, lymph nodes, intestinal-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissues, liver, lungs, stomach, intestines, colon, kidneys, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils, or other organs, and / or cells obtained therefrom. In the case of cell therapy, such as adoptive cell therapy, the samples include autologous and allogeneic samples.

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

[0123] In some implementations, the cells are selected from T cells, natural killer cells, natural killer T cells, or ILC cells.

[0124] In some embodiments, the cells are T cells. This includes, but is not limited to, subtypes and subsets of T cells and / or CD4+ T cells and / or CD8+ T cells, specifically naive T cells (T...). N ) cells, effector T cells (T cells) EFF ), memory T cells and their subtypes, such as stem cell memory T (T12) SCM ), central memory T(T) CM ), effect memory T (T EM These include late-stage differentiation effector memory T cells, tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-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 implementations, the TCR or its antigen-binding portion is heterologous to the cells.

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

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

[0128] A seventh aspect of the present invention provides a composition comprising a TCR or its antigen-binding portion as described in the first aspect of the present invention, a multispecific antibody as described in the second aspect of the present invention, a recombinant TCR as described in the third aspect of the present invention, a nucleic acid molecule as described in the fourth aspect of the present invention, a vector as described in the fifth aspect of the present invention, or an engineered cell as described in 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 the prevention / treatment of diseases, and / or for detection, diagnosis, and prognosis.

[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 formulation in which the biological activity of the active ingredient contained therein is permitted and which does not contain any additional components that would have unacceptable toxicity to the subject to whom the formulation will be administered.

[0131] "Pharmaceutically acceptable carriers" refer to components in a drug formulation that are non-toxic to the subjects, excluding the active ingredient. 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 the specific cells or binding molecules and / or by the method of administration. Therefore, a variety of suitable formulations exist. 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. The preservative or mixture thereof is typically present in an amount from about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed. 1980). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations used, 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 octadecyl dimethyl benzyl ammonium chloride; hexahydroxy quaternary ammonium chloride; benzyl chloride; benzyl chloride; phenolic alcohols, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); and low molecular weight carriers (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 dextrin; 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, the composition includes a buffer. 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 mixture thereof is typically present in an amount from about 0.001% to about 4% by weight of the total composition. Methods for preparing administerable pharmaceutical compositions are known.

[0134] Pharmaceutical formulations may include lyophilized formulations and aqueous solutions. Formulations or compositions may also contain more than one active ingredient suitable for a specific indication, disease, or condition that can be treated with a TCR or cell therapy, preferably having activity complementary to that TCR or cell, wherein the individual activities do not adversely affect each other. Such active ingredients are suitable to be present in a combination of amounts that can effectively achieve the intended purpose. Therefore, in some embodiments, the pharmaceutical composition further includes other pharmaceutical active agents or drugs, such as chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In some implementations, the TCR or its antigen-binding fragment is administered, for example, in a pharmaceutically acceptable salt form. Suitable pharmaceutically acceptable acid addition salts include salts derived from inorganic 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 encapsulated in microcapsules, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or crude emulsions. In some 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 respects, the pharmaceutical composition may employ time-release, delayed-release, and sustained-release delivery systems, such that delivery of the composition occurs prior to sensitization of the treatment site and the delivery time is sufficient to induce sensitization at the treatment site. A variety of release delivery systems are available and known. Such systems avoid repeated administration of the composition, thereby improving convenience for both the subject and the physician.

[0137] In some embodiments, the pharmaceutical composition contains a binding amount of molecules and / or cells that can effectively treat or prevent the disease or symptom, such as a therapeutically effective amount or a preventatively effective amount. In some embodiments, therapeutic or preventative efficacy is monitored by periodically evaluating the treated subject. For repeated administration over several days or longer, depending on the symptom, treatment is repeated until the desired suppression of disease symptoms is achieved. However, other dosing regimens may be applicable and can be determined. The desired dose may be delivered by a single bolus injection, by multiple bolus injections, or by continuous infusion.

[0138] Formulations include those intended for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, percutaneous, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, cell populations are administered parenterally. As used herein, the term "parenterally" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, intracranial, intrathoracic, and intraperitoneal administration. In some embodiments, cell populations are administered to subjects via peripheral systemic delivery, via intravenous, intraperitoneal, or subcutaneous injection.

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

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

[0141] In some implementations, the kit may be used for therapeutic purposes and / or diagnostic purposes.

[0142] In some implementations, the kit may include one or more other elements, including: instructions for use; other reagents, such as labels, therapeutic agents, or pharmaceutical agents or radiation protection compositions that can be used to chelate or otherwise conjugate antibodies with labels or therapeutic agents; devices or other materials for preparing the antibody for administration; pharmaceutically acceptable carriers; and devices 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 implementations, the cells are derived from cells collected from subjects who require cell therapy;

[0145] In some implementations, the cells are T cells or natural killer cells.

[0146] In some implementations, the introduction is carried out using a transduction method.

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

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

[0149] In some implementations, the MAGE-A3 + The disease is cancer. The cancer can be any type of cancer, including sarcomas (e.g., synovial sarcoma, osteoblastic sarcoma, uterine leiomyosarcoma, and alveolar rhabdomyosarcoma), lymphomas (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), hepatocellular carcinoma, gliomas, head cancers (e.g., squamous cell carcinoma), neck cancers (e.g., squamous cell carcinoma), acute lymphoblastic cancer, leukemias (e.g., acute myeloid leukemia and chronic lymphocytic leukemia), bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer or rectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer, vulvar cancer, chronic myeloid cancer, and colon cancer (e.g., colon cancer). The cancers include any one of the following: 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 carcinoma, 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 bowel cancer, soft tissue cancer, gastric cancer, testicular cancer, thyroid cancer, and urothelial carcinoma (e.g., ureteral cancer and bladder cancer).

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

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

[0152] In some implementations, the adoptive T cell transfer is an allogeneic adoptive T cell transfer, an autologous adoptive T cell transfer, or a universal allogeneic reactive adoptive T cell transfer.

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

[0154] In this invention, the terms "treatment" and "prevention," and those derived therefrom, do not necessarily imply 100% or complete treatment or prevention. Rather, there exist varying degrees of treatment or prevention in which those skilled in the art consider to have potential benefit or therapeutic effect. In this respect, the methods of the present invention can provide any amount of cancer treatment or prevention at any level in mammals. Furthermore, the treatment or prevention provided by the methods of the present invention may include treatment of one or more symptoms or conditions of a disease (e.g., 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 a TCR or its antigen-binding portion as described in the first aspect of the present invention, a multispecific antibody as described in the second aspect of the present invention, a recombinant TCR as described in the third aspect of the present invention, a nucleic acid molecule as described in the fourth aspect of the present invention, a vector as described in the fifth aspect of the present invention, engineered cells as described in the sixth aspect of the present invention, or a composition as described in the seventh aspect of the present invention to form a complex, and detecting the complex, wherein the detection of the complex indicates the presence of cancer in the mammal.

[0156] For the method of detecting cancer in mammals of the present invention, the cancer cell sample may be a sample containing whole cells, their lysates, or fractions of whole cell lysates (e.g., nucleus or cytoplasm fractions, all protein fractions, or nucleic acid fractions).

[0157] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0158] Example 1: Expanding MAGE-A3-specific T cells

[0159] 1. Sorting CD14 from PBMC + Monocytes and their induced differentiation

[0160] HLA-A*02:01 type cryopreserved PBMCs were purchased from Miaoshun Biotechnology. After thawing, the PBMCs were resuspended in RPMI 1640 medium, and cell counts were performed and cell viability recorded. The cells were then centrifuged at 350g for 5 minutes, and the supernatant was discarded. Based on the counting results, the cells were sorted using magnetic bead separation buffer at a concentration of 1×10⁻⁶. 8 Resuspend cells at / mL, then at 1×10 8 Add cells to EasySep at a ratio of 100 μL. TM Human CD14 Positive Selection Cocktail II, mix thoroughly by blowing and let stand at room temperature for 15 minutes; add magnetic beads (EasySep) TM Dextran RapidSpheres TM 50100) Vortex oscillation to ensure thorough mixing, according to every 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 thoroughly, transfer to sorting tubes, place on a sorting rack, and let stand for 15 minutes to complete sorting. The sorted CD14 cells are then ready. + Monocytes were resuspended in 1640 medium containing 10% FBS and supplemented with 20 ng / mL IL-4 and 100 ng / mL GM-CSF. Cells were cultured at a rate of 1 × 10⁻⁶ cells / mL. 6 Cells were seeded at a density of 1 / mL in six-well plates and incubated in a 37°C CO2 incubator. After 3 days of culture, the culture medium was adjusted, and 50 ng / mL TNF-α was added for 16 hours of further culture. Mature dendritic cells or cells loaded with the antigenic peptide FLWGPRALV (SEQ ID NO: 49) were then cultured.

[0161] 2. From CD14 - T cells sorted in PBMC

[0162] From frozen CD14 - T cells were enriched in PBMCs by negative selection. 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 ready to be co-cultured with DC cells.

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

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

[0165] Figure 1 MAGE-A3-specific T cells induced from different donors were demonstrated. The proportions of specific T cells induced by co-culturing DCs loaded with antigenic peptides with T cells were 0.28%, 0.33%, and 0.8%, 0.11%, 0.42%, and 0.13%, respectively.

[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 the TCR were fused with the α and β constant regions of the mouse, respectively. The TCRα and TCRβ chains were linked by the P2A sequence, and the structural diagram is shown below. Figure 2 As shown. 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 TCRα / β gene, after codon optimization, was synthesized and digested with BamHI and SacI restriction endonucleases. It was then cloned into an in vitro transcription (IVT) plasmid vector between the BamHI and SacI restriction sites. A Kozak (GCCACC) sequence was added downstream of the BamHI site, and a double-stop codon (TGATAA) sequence was added upstream of the SacI site. After constructing the IVT plasmid vector, mRNA was synthesized and temporarily stored at -80°C for in vitro functional verification. The exemplary codon-optimized α-strand variable region and β-strand variable region nucleic acid sequences of 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 overexpressing HLA-A*02:01

[0173] Plasmids for constructing HLA expression genes were created using 293FT tool cells. Figure 3 The product is packaged as a lentivirus, and HLA-A*02:01 is integrated into the genome of K562 cells via lentiviral infection, achieving stable inheritance and expression. K562-HLA-A*02:01 OE Target cells used to validate specific TCR activation.

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

[0175] The activation status of the TCR pathway in genetically engineered Jurkat-NFAT-luc cells can be verified by detecting Luciferase fluorescence values. TCR mRNA was transduced into Jurkat-NFAT-luc cells via electroporation to induce specific TCR expression. The detailed steps are as follows: Jurkat-NFAT-luc cells were collected, centrifuged at 350g for 5 minutes, washed once with DPBS (Cytiva), and then resuspended in R solution (Thermo Fisher Scientific) at a density of 2×10⁶ cells / mL. 7 / mL, add 100μL of cell suspension to a 1.5mL EP tube, along with 5μg CD8αβ mRNA and 5μg TCR mRNA, and mix thoroughly. Add 3-5mL of E2 electroporation buffer (Thermo Fisher Scientific) to the electroporation cuvette, place it in the thermopneumatic electroporator (Thermo Fisher Scientific), and carefully aspirate the cell suspension containing mRNA using a 100μL electroporator tip, avoiding air bubbles. Insert the electroporator into the E2 buffer of the electroporator, set the electroporation conditions to 1400V, 20ms, 2 pulses, and start electroporation. Transfect TCR mRNA into Jurkat-NFAT-luc cells to obtain TCR-expressing cells. 24 hours after electroporation, perform MAGE-A3 tetramer staining on Jurkat-NFAT-luc cells, and analyze the transduction results using flow cytometry. Figure 4 Experimental results showed that Jurkat cells transduced from ZZ01, ZZ04, ZZ05, ZZ06, ZZ07, and ZZ09 all contained Tetramer-positive cells, with positive rates of 93.66%, 79.95%, 21.41%, 60.44%, 86.30%, and 62.04%, respectively. Jurkat cells transduced from ZZ08 did not bind to Tetramer.

[0176] 3. Detection results of TCR pathway activation status

[0177] Jurkat-NFAT-luc cells electroporated for 24 hours have been shown to express TCR, and when combined with K562-HLA-A*02:01 cells loaded with an overnight antigen peptide (10 μM)... OE The samples were co-incubated at a 20:1 target-effect ratio in 96-well plates at 37°C for 4 hours in a CO2 incubator. Simultaneously, the Bio-Lite assay reagent (Nanjing Novizan Biotechnology Co., Ltd.), stored at -20°C, was thawed in the dark until it reached room temperature before use. After incubation, the samples were centrifuged and washed once with DPBS to remove the supernatant. 100 μL of Bio-Lite assay reagent was added to each well, and the samples were incubated in the dark for 3–5 minutes before being analyzed using a microplate reader.

[0178] Figure 5 The fluorescence values ​​of Jurkat-NFAT-luc cells transduced with different TCRs are shown, with untransduced cells serving as the control group. Activation results varied among the different TCRs. ZZ08 cells were not activated, while Jurkat-NFAT-luc cells transduced with ZZ07 showed the highest fluorescence value. Except for ZZ08, all other TCRs underwent further validation.

[0179] Example 4: Peptide Sensitivity Detection of MAGE-A3-Specific TCR

[0180] 1. Preparation of T cells expressing TCR

[0181] Cryopreserved peripheral blood mononuclear cells (PBMCs) from healthy donors (Miaoshun Biotechnology) were thawed and resuspended in X-VIVO15 medium. T cells were purified and isolated using magnetic beads, and activated with CD3 / CD28 magnetic beads. The cells were then resuspended in X-VIVO15 medium containing 2.5% human serum and 30 IU / mL IL-2 and cultured for three days. On the third day, the magnetic beads were removed, and the cells were cultured for another three days. On the fourth day, the cells were collected and centrifuged at 350g for 5 minutes. After washing once with DPBS, the cells were resuspended in R solution to a concentration of 2 × 10⁶ cells / mL. 7 / mL, add 100μL of cell suspension to a 1.5mL EP tube, and simultaneously add 5μg of TCR mRNA, mix thoroughly; add 3-5mL of E2 electroporation buffer (Thermo Fisher Scientific) to the electroporation cuvette, place it in the electroporator cuvette, carefully aspirate the cell suspension mixed with mRNA using a 100μL electroporator tip, avoiding air bubbles, insert the electroporator into the E2 solution of the electroporator, set the electroporation conditions to 1400V, 10ms, 3 pulses, start electroporation, and transduce TCR mRNA into activated T cells to obtain TCR-expressing T cells. 24 hours after electroporation, perform MAGE-A3 tetramer staining on the T cells, and analyze the transduction results by flow cytometry. Figure 6Experimental results demonstrate that ZZ01, ZZ04, ZZ05, ZZ06, ZZ07, and ZZ09 were all successfully transduced into activated T cells, and CD4+ were also activated. + and CD8 + Tetramer-positive cells were detected in all T cells, and Tetramer-positive cells were present in CD3+ cells. + The proportions of 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, thus failing to transduce endogenous peptides to MHC loading sites. Furthermore, their HLA type is HLA-A*02:01, making them suitable target cells for TCR peptide sensitivity validation. Cultured T2 cells were collected, washed once, and resuspended in RPMI 1640 medium containing 10% FBS. Cells were then cultured at a density of 1×10⁻⁶ cells / cells. 6 The antigen peptide was seeded at a density of 10 / mL in 24-well plates, and different concentrations of antigen peptide were added in a concentration gradient of 10. -12 M~10 -5 M, a total of 8 groups, with an antigen peptide loading time of 4 hours.

[0184] 3. Results of peptide sensitivity testing

[0185] Activated T cells expressing TCR were mixed with T2 cells loaded with different concentrations of antigenic peptides at a ratio of 1×10⁻⁶. 5 The cells were seeded in a mixture of samples from each well into 96-well plates and incubated overnight at 37°C using a CO2 incubator. The following day, cells from each group were collected and stained with flow cytometry antibodies CD3-Violet786, CD8-BV510, CD4-APC-Cy7, and 4-1BB-BV421, and then analyzed. The CD8+ levels in each sample were... + The expression level of the T cell activation marker 4-1BB can reflect the activation effect of target cells loaded with different concentrations of antigenic peptides on T cells. Using the highest 4-1BB expression level group as 100%, each TCR was analyzed, and the EC50 value was calculated. Figure 7 The EC50 value reflects the peptide sensitivity of TCR. Experimental results show that the EC50 value of ZZ01 is 4.865 × 10⁻⁶. -9 The EC50 value of M,ZZ04 is 2.487×10⁻⁶. -9 The EC50 value of M,ZZ05 is 9.935×10 -9 The EC50 value of M,ZZ06 is 3.076×10⁻⁶. -9 The EC50 value of M,ZZ07 is 6.174×10⁻⁶.-10 The EC50 value of M,ZZ09 is 1.449×10⁻⁶. -8 M, among which ZZ07 has the smallest EC50 value, indicating that it has the best antigen sensitivity.

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

[0187] ZZ07 was selected to verify the cytotoxic ability of TCR-transduced T cells against target cells loaded with antigenic peptides. TCR-transduced T cells were co-incubated overnight with T2 cells loaded with antigenic peptides, and the cytotoxic effect was detected the next day using a microplate reader. Figure 8 The experimental results showed that, compared with the control group, the killing efficiency of ZZ07 increased with the increase of the effector-to-target ratio, at 35.59%, 46.36%, 58.29%, and 72.01%, respectively. This indicates 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 standard method for identifying specific amino acid sites closely related to TCR function, stability, and conformation. Replacing amino acids at various positions in a peptide with alanine removes the active group on the side chain and replaces it with a small methyl group without other functional groups, thus having minimal impact on protein structure and being able to distinguish the effect of a specific amino acid on TCR recognition. In this experiment, an alanine scanning peptide library was constructed. Since the 7th position is itself alanine, it was mutated to glycine for verification.

[0190] First, the TCR mRNA to be validated was transduced into T cells via electroporation. Four hours later, it was co-incubated with T2 cells loaded with various mutant peptides (the mutant peptides need to be added one day in advance and loaded overnight). The secretion of IFN-γ in each group was detected by enzyme-linked immunospot (ELISPOT) assay. The ELISPOT procedure was as follows: After washing both effector cells and target cells once, they were resuspended in serum-free ELISPOT medium and the cell density was adjusted to 5 × 10⁶ cells / year. 5 Effector cells and target cells were seeded at a 1:1 ratio into pre-washed ELISPOT-IFN-γ detection plates at a density of / mL. After overnight incubation, color development was performed, and the plates were then placed in a cool, dark place at room temperature to air dry. The immunospots were then imaged and read using an ELISPOT analyzer (CTL S6). The ELISPOT detection results and statistical results of ZZ07 transduced T cells were presented in [the table / data missing]. Figure 9The results show that the activated T sample, without resting treatment, also showed a certain number of spots in the control group. In the statistical results, the background values ​​of the corresponding control group were removed. Therefore, the recognition motif of ZZ07 can be determined to be FLWG-R--V. The ZZ07 recognition sequence is relatively conserved and has strong specificity.

[0191] Example 7: Preparation and Functional Verification of Bispecific Antibodies

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

[0193] TCR-CD3 bispecific antibodies (composed of a high-affinity TCR and a low-affinity CD3 antibody) were prepared. The binding affinity of ZZ07 in the bispecific antibody to the peptide-HLA-A*02:01 molecule, as well as the binding affinity of the CD3 antibody used in the bispecific antibody to the CD3 molecule, were detected by surface plasmon resonance (SPR) technique.

[0194] 2. The killing function of dual antibodies against target cells expressing naturally occurring antigens.

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

[0196] The results showed that the dual antibodies had a good killing effect 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 various cell subsets

[0198] PBMCs were combined with target cells NCI-H1299-LUC (MAGE-A3). + HLA-A*02:01 OE Cells were co-incubated at a 5:1 ratio with 1 nM of prepared penicillin antibody. After 48 hours of co-incubation, cells were stained with flow cytometry antibodies against CD3, CD4, CD8, CD107a, CD27, and CD45RO, and analyzed to determine the presence of CD45RO in activated T cells expressing CD107a. + CD27 +Central memory T cells (Tcm) and CD45RO + CD27 - The proportion of effector memory T cells (Tem). Detection of CD8 levels after the addition of bispecific antibody. + T cells and CD4 + T cell killing efficiency and CD8 + Subgroups and CD4 + Subgroups including Tcm, Temra, Tem, and The cell killing efficiency.

[0199] The results showed that the dual antibodies affected T cell immune responses by influencing the composition of memory T cells; the Tem and Temra subsets showed the best killing effect on tumor cells.

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

[0201] Day 0 will use human malignant melanoma cells A375 (MAGE-A3) + HLA-A*02:01 + After mixing human PBMCs, the bispecific antibody was subcutaneously transplanted into severely immunodeficient mice. From Day 1 to Day 5, mice were injected with different doses of the bispecific antibody daily via tail vein injection. Tumor volume was measured and recorded twice a week to observe the inhibitory effect of the bispecific antibody 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 had 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 against target cells with naturally expressed antigens

[0205] The Maestro Z cell non-destructive real-time monitoring system was used to evaluate the effects of ZZ07 stably expressing T cells on multiple naturally expressed antigen target cells, including NCI H1299 and HLA-A. * 02:01 * KYSE410 (HLA-A) * 02:01 OE It is also represented as KYSE410 HLA-A * 02:01 * The killing functions of A375 and U251 were assessed. Target cells were first digested with trypsin, then resuspended at a concentration of 1×10⁻⁶. 5 At a concentration of [number] cells / mL, seed 100 μL per well in a 96-well impedance plate. After the target cells have adhered and grown for 24 hours, add 4 × 10⁴ cells / mL to each well.4 ZZ07 TCR-T cells were used, with untransduced T cells serving as control cells. Impedance analysis was used to monitor the killing effect of effector cells on target cells in real time.

[0206] The results are as follows Figure 10 As shown, ZZ07 TCR-T can effectively kill target cells NCI H1299 (HLA-A) that naturally express antigens. * 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) were selected. - / - To verify the in vivo antitumor effect of ZZ07 TCR-T cells, each mouse was injected subcutaneously into the axilla with 4 × 10⁴ cells. 6 NCI H1703-MAGEA3 OE Cells were used to establish a tumor model. On day 7, the experimental animals were randomly assigned to three groups based on tumor volume, and the effector cell group and control group were injected. Each mouse received 1×10⁻⁶ cells via the tail vein. 7 The number of cells was 40.5% (Tetramer-positive cell rate in the experimental group). At the time of injection, the average tumor volume in each group was smaller.

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

[0210] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope 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 an epitope of MAGEA3, the sequence of which is set forth in SEQ ID NO: 49; the TCR or antigen-binding portion thereof comprises an alpha chain CDR1, CDR2, and CDR3 of the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10, respectively; and a beta chain CDR1, CDR2, and CDR3 of the amino acid sequence set forth in SEQ ID NO: 25, SEQ ID NO: 32, and SEQ ID NO: 39, respectively.

2. The TCR or antigen-binding portion thereof of claim 1, wherein, the TCR or antigen-binding portion thereof comprises an alpha chain variable region that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 16 and a beta chain variable region that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:

46.

3. The TCR or antigen-binding portion thereof of claim 2, wherein, the TCR or antigen-binding portion thereof comprises an alpha chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 16 and a beta chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:

46.

4. The TCR or antigen-binding portion thereof of claim 3, wherein, the TCR or antigen-binding portion thereof comprises an alpha chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 16 and a beta chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:

46.

5. The TCR or antigen-binding portion thereof of claim 4, wherein, the TCR or antigen-binding portion thereof comprises an alpha chain variable region that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 16 and / or a beta chain variable region that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO:

46.

6. The TCR or antigen-binding portion thereof of claim 5, wherein, the TCR or antigen-binding portion thereof comprises an alpha chain variable region set forth in SEQ ID NO: 16 and / or a beta chain variable region set forth in SEQ ID NO:

46.

7. The TCR or antigen-binding portion thereof of any of claims 1-6, wherein, the TCR or antigen-binding portion thereof is a soluble TCR that lacks a transmembrane domain.

8. The TCR or antigen-binding portion thereof of any of claims 1-6, wherein, the TCR or antigen-binding portion thereof binds to an MHC I and / or MHC II peptide complex.

9. The TCR or antigen-binding portion thereof of any of claims 1-6, wherein, the TCR or antigen-binding portion thereof further comprises a detectable label.

10. The TCR or antigen-binding portion thereof of claim 9, wherein, the detectable label comprises an enzyme, a radionuclide, a fluorescent dye, a luminescent substance, biotin.

11. The TCR or antigen-binding portion thereof of any of claims 1-6, wherein, the TCR or antigen-binding portion thereof further comprises a therapeutic agent.

12. The TCR or antigen-binding portion thereof of any of claims 1-6, wherein, the alpha chain further comprises an alpha constant region Ca and / or the beta chain further comprises a beta constant region Cb.

13. The TCR or antigen-binding portion thereof of claim 12, wherein, the Ca and Cb are mouse constant regions.

14. The TCR or antigen-binding portion thereof of claim 12, wherein, the Ca and Cb are human constant regions.

15. The TCR or antigen-binding portion thereof of claim 12, wherein, the Ca region and / or the Cb region comprises the introduction of one or more cysteines capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain.

16. The TCR or antigen-binding portion thereof of any of claims 1-6, wherein, the alpha chain and the beta chain further comprise a signal peptide.

17. The TCR or antigen-binding portion thereof of any of claims 1-6, wherein, the TCR or antigen-binding portion thereof is a single chain.

18. The TCR or antigen-binding portion thereof of any of claims 1-6, wherein, the TCR or antigen-binding portion thereof is a double chain.

19. A multispecific antibody, characterized in that comprises a first antigen binding domain, the first antigen binding domain comprising the TCR or antigen-binding portion thereof of any one of claims 1-18.

20. The multispecific antibody of claim 19, wherein the first antigen binding domain comprises a single chain variable fragment.

21. The multispecific antibody of claim 19, wherein The multispecific antibody further comprises a second antigen binding domain that specifically binds to a protein CD3 expressed on the surface of an immune cell.

22. The multispecific antibody of claim 21, wherein The immune cell is a T cell or a natural killer cell.

23. The multispecific antibody of claim 22, wherein The T cells are CD8 + T cells.

24. The multispecific antibody of any one of claims 21-23, wherein, The second antigen binding domain comprises an scFv.

25. The multispecific antibody of any one of claims 21-23, wherein, The first antigen binding domain and the second antigen binding domain are linked or associated by a covalent bond.

26. The multispecific antibody of any one of claims 21-23, wherein, The first antigen binding domain and the second antigen binding domain are linked by a peptide bond.

27. A recombinant TCR, characterized in that, The recombinant TCR comprises the TCR or antigen binding portion thereof of any one of claims 1-18, and a costimulatory region.

28. The recombinant TCR of claim 27, wherein, The costimulatory region comprises a costimulatory molecule selected from the group consisting of a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, and any combination thereof.

29. The recombinant TCR of claim 28, wherein, The costimulatory region comprises a CD28 polypeptide.

30. A nucleic acid molecule, wherein, The nucleic acid molecule encodes the TCR or antigen binding portion thereof of any one of claims 1-18, the multispecific antibody of any one of claims 19-26, or the recombinant TCR of any one of claims 27-29.

31. The nucleic acid molecule of claim 30, wherein The nucleotide sequence of the nucleic acid molecule is codon optimized.

32. The nucleic acid molecule of claim 30 or 31, wherein The nucleotide sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain are separated by a peptide sequence that causes ribosomal skipping.

33. The nucleic acid molecule of claim 30 or 31, wherein The nucleic acid molecule is synthetic.

34. The nucleic acid molecule of claim 30 or 31, wherein The nucleic acid molecule is a cDNA.

35. A vector comprising the nucleic acid of claim 34. The vector comprises the nucleic acid molecule of any one of claims 30-34.

36. The carrier of claim 35, wherein, The vector is a viral vector, a mammalian vector, or a bacterial vector.

37. The vector of claim 36, wherein The vector is a viral vector.

38. The vector of claim 37, wherein, The viral vector is a retroviral vector.

39. The vector of claim 38, wherein, The retroviral vector is selected from the group consisting of an adenoviral vector, a lentivirus, a Sendai virus vector, a baculovirus vector, an Epstein-Barr virus vector, a papovaviral vector, a vaccinia virus vector, a herpes simplex virus vector, a hybrid vector, and an adeno-associated virus (AAV) vector.

40. The vector of claim 39, wherein, The retroviral vector is a lentivirus vector.

41. An engineered cell, comprising: The engineered cell comprises the TCR or antigen binding portion thereof of any one of claims 1-18, the multispecific antibody of any one of claims 19-26, the recombinant TCR of any one of claims 27-29, the nucleic acid molecule of any one of claims 30-34, or the vector of any one of claims 35-40.

42. The engineered cell of claim 41, wherein, The engineered cell expresses CD3.

43. The engineered cell of claim 41 or 42, wherein, The cell is selected from the group consisting of a T cell, a natural killer cell, or an ILC cell.

44. The engineered cell of claim 41 or 42, wherein, The cell is a natural killer T cell.

45. The engineered cell of claim 41 or 42, wherein, The TCR or antigen binding portion thereof is heterologous to the cell.

46. The engineered cell of claim 41 or 42, wherein, The engineered cell is a primary cell obtained from a subject.

47. The engineered cell of claim 46, wherein, The subject is a mammalian subject.

48. The engineered cell of claim 46, wherein, The subject is a human.

49. A composition comprising, comprises the TCR or antigen binding portion thereof of any one of claims 1-18, the multispecific antibody of any one of claims 19-26, the recombinant TCR of any one of claims 27-29, the nucleic acid molecule of any one of claims 30-34, the vector of any one of claims 35-40, or the engineered cell of any one of claims 41-48.

50. The composition of claim 49, wherein, The composition further comprises a pharmaceutically acceptable carrier.

51. A kit comprising, The kit comprises a TCR or antigen-binding portion thereof according to any one of claims 1-18, a multispecific antibody according to any one of claims 19-26, a recombinant TCR according to any one of claims 27-29, a vector according to any one of claims 35-40, an engineered cell according to any one of claims 41-48, or a composition according to any one of claims 49-50.

52. A method of engineering a cell targeting an antigen, the method comprising introducing a cell with a nucleic acid molecule according to any one of claims 30-34 or a vector according to any one of claims 35-40.

53. The method of claim 52, wherein, The cell is derived from a cell collected from a subject in need of a cell therapy.

54. The method of claim 52, wherein, The cell is a T cell or a natural killer cell.

55. The method of claim 52, wherein, The introducing is by a method of transduction.

56. Use of a TCR or antigen-binding portion thereof according to any one of claims 1-18, a multispecific antibody according to any one of claims 19-26, a recombinant TCR according to any one of claims 27-29, a nucleic acid molecule according to any one of claims 30-34, a vector according to any one of claims 35-40, an engineered cell according to any one of claims 41-48, or a composition according to any one of claims 49-50, for the manufacture of a medicament for the treatment and / or prevention of a disease, the disease being lung cancer, esophageal cancer, melanoma, glioma, liver cancer, bone cancer, multiple myeloma, colon cancer, pancreatic cancer, gastric cancer, cervical cancer, renal cancer, breast cancer, urothelial cancer, bladder cancer, synovial sarcoma.

57. Use of the TCR or antigen-binding portion thereof of any one of claims 1-18, the multispecific antibody of any one of claims 19-26, the recombinant TCR of any one of claims 27-29, the nucleic acid molecule of any one of claims 30-34, the vector of any one of claims 35-40, the engineered cell of any one of claims 41-48, or the composition of any one of claims 49-50 in the manufacture of a product for detecting whether a disease is MAGE A3 + .

58. The use according to claim 57, wherein The disease is cancer.

59. The use according to claim 58, wherein The cancer comprises melanoma, liver cancer, breast cancer, lung cancer, prostate cancer, synovial cell sarcoma, head and neck cancer, esophageal cancer, or ovarian cancer.

60. Use of a TCR or antigen-binding portion thereof according to any one of claims 1-18, a multispecific antibody according to any one of claims 19-26, a recombinant TCR according to any one of claims 27-29, a nucleic acid molecule according to any one of claims 30-34, a vector according to any one of claims 35-40, an engineered cell according to any one of claims 41-48, or a composition according to any one of claims 49-50, for the manufacture of a product for adoptive cell transfer therapy for a disease, the disease being selected from lung cancer, esophageal cancer, melanoma, glioma, liver cancer, bone cancer, multiple myeloma, colon cancer, pancreatic cancer, gastric cancer, cervical cancer, renal cancer, breast cancer, urothelial cancer, bladder cancer, synovial sarcoma.

61. The use of claim 60, wherein, The adoptive cell transfer is adoptive T cell transfer.

62. The use of claim 61, wherein, The adoptive T cell transfer is allogeneic adoptive T cell transfer, autologous adoptive T cell transfer, or universal non-allogeneic reactive adoptive T cell transfer.

63. Use of the TCR or antigen-binding portion thereof of any one of claims 1-18, the multispecific antibody of any one of claims 19-26, the recombinant TCR of any one of claims 27-29, the nucleic acid molecule of any one of claims 30-34, the vector of any one of claims 35-40, the engineered cell of any one of claims 41-48, or the composition of any one of claims 49-50 in the manufacture of a MAGE A3 targeting product that directs a therapeutic molecule to a tumor site, the tumor selected from the group consisting of lung cancer, esophageal cancer, melanoma, glioma, liver cancer, bone cancer, multiple myeloma, colon cancer, pancreatic cancer, gastric cancer, cervical cancer, renal cancer, breast cancer, urothelial cancer, bladder cancer, synovial sarcoma.

64. Use of the TCR or antigen-binding portion thereof of any one of claims 1-18, the multispecific antibody of any one of claims 19-26, the recombinant TCR of any one of claims 27-29, the nucleic acid molecule of any one of claims 30-34, the vector of any one of claims 35-40, the engineered cell of any one of claims 41-48, or the composition of any one of claims 49-50 in the manufacture of a product that enhances the immune response of a subject to a cancer, the cancer comprising lung cancer, esophageal cancer, melanoma, glioma, liver cancer, bone cancer, multiple myeloma, colon cancer, pancreatic cancer, gastric cancer, cervical cancer, renal cancer, breast cancer, urothelial cancer, bladder cancer, synovial sarcoma.

Images