Chimeric antigen receptors

Abstract

The invention relates to the field of biological medicine, in particular to a chimeric antigen receptor which comprises a targeting structural domain, a spacer region structural domain, a transmembrane structural domain and a signal transduction structural domain, the targeting structural domain comprises a VL structural domain of scFv combined with MSLN, a VH structural domain of scFv combined with MSLN, a VH structural domain of scFv combined with TIGIT and a VL structural domain of scFv combined with TIGIT in any sequence, and the targeting structural domain comprises a VL structural domain of scFv combined with MSLN, a VH structural domain of scFv combined with TIGIT and a VL structural domain of scFv combined with TIGIT. The scFv combined with MSLN is positioned at an amino terminal or a carboxyl terminal of the chimeric antigen receptor, and the scFv combined with TIGIT is positioned at the other terminal of the chimeric antigen receptor. The invention also relates to an application of the chimeric antigen receptor in the aspect of treating cancers, and the chimeric antigen receptor has more efficient tumor killing activity compared with the disclosed MSLNCAR-T.

Description

Technical Field

[0001] This application relates to the field of cellular immunotherapy, and particularly to chimeric antigen receptors (CARs) and their use in the treatment of cancer. Background Art

[0002] MSLN is a glycoprotein anchored to the plasma membrane through a glycosylphosphatidylinositol (GPI) domain. It is a cell surface antigen related to tumor invasion. MSLN expression is usually limited to the surface of mesothelial cells, but it is also overexpressed in a wide range of cancers. Due to its differential expression between cancer and normal tissues and its role in tumorigenesis, MSLN can be regarded as a potential target.

[0003] Chimeric antigen receptor T cells (CAR-T cells), CAR-T cells are T cells genetically engineered to express antigen specificity, consisting of an intracellular signaling domain, a transmembrane (TM) domain, and an extracellular single-chain variable fragment (scFv) sequence. The scFv sequence is composed of the light chain V L and the heavy chain V H variable regions, which can specifically recognize and bind to tumor-associated antigens (TAAs). CAR-T cell therapy has achieved good therapeutic effects in a variety of tumors, especially hematological malignancies. However, its therapeutic effect in solid tumors is poor. The interaction between immune cell types and non-tumor cells in the TME obviously affects tumor progression, invasion, and metastasis. On this basis, it has become an urgent problem to study how to weaken the inhibitory effect of inhibitory immune checkpoints in the tumor microenvironment.

[0004] TIGIT (T cell Ig and ITIM domain) is a member of the poliovirus receptor (PVR) / Nectin family. TIGIT belongs to the immunoglobulin superfamily. TIGIT can directly inhibit the effector function of CD8+ T cells, preventing the clearance of cancer cells. CD155 (PVR / necl5 / Tage4) is a member of the nectin-like adhesion molecule family, which is highly upregulated in tumor cells of various cancer types and is associated with poor patient prognosis. Once the highly expressed CD155 on the tumor surface binds to TIGIT on the surface of NK and T cells, their killing effect on tumor cells will be inhibited. Therefore, blocking the TIGIT / CD155 signal may provide a promising supplement to current immune checkpoint-based anti-tumor immunotherapy for clinical intervention. In cancer immunotherapy, blocking the TIGIT / PVR interaction is a promising approach.

[0005] Patent (202110794199.X) discloses a chimeric antigen receptor targeting mesothelin and its application. The chimeric antigen receptor (MSLN CAR-T) includes an extracellular region, a transmembrane region, and an intracellular domain connected in sequence; the extracellular region includes an extracellular signal peptide, an antigen recognition region, and a hinge region, and the antigen recognition region is a fully human anti-mesothelin scFv; the intracellular domain includes an intracellular signal domain and an intracellular co-stimulatory signal domain. This chimeric antigen receptor T cell has strong killing activity against mesothelin-positive tumor cells. Using a fully human antibody as scfv, it has no immunogenicity and can be administered repeatedly to enhance the therapeutic effect of CAR-T cells. And it also relates to the application of the chimeric receptor targeting human Mesothelin and its immune effector cells in the preparation of drugs for preventing or treating tumors.

[0006] However, when the above MSLN CAR-T is used to inhibit tumor growth, it is not stable enough, and in some types of tumors, there may be signs of recurrence; and although there are a large number of currently disclosed CAR-Ts, the number of CAR-Ts with good therapeutic effects is actually not many, and new CAR-Ts still need to be developed and studied for the treatment of tumors. Summary of the Invention

[0007] The chimeric antigen receptor provided in this application, with the help of lentiviral transfection technology, constructs CAR-T cells targeting the mesothelin-secreting TIGIT-blocking scFv sequence. The prepared MT CAR-T cells (MLSN CAR structure with TIGIT single-chain antibody) can target MSLN and TIGIT, and have more efficient tumor killing activity compared with the disclosed MSLN CAR-T.

[0008] On the one hand, this application provides a chimeric antigen receptor, which includes a targeting domain, a spacer domain, a transmembrane domain, and a signal transduction domain. The targeting domain includes the VL domain of the scFv binding to MSLN, the VH domain of the scFv binding to MSLN, the VH domain of the scFv binding to TIGIT, and the VL domain of the scFv binding to TIGIT in any order. The scFv binding to MSLN is located at the amino terminus or carboxyl terminus of the chimeric antigen receptor, and the scFv binding to TIGIT is located at the other end.

[0009] It should be noted that for the chimeric antigen receptor provided in the present application, its targeting domain can bind to MSLN and TIGIT. Structurally, the scFv of MSLNT is located at the amino terminus or the carboxyl terminus, and the scFv of MSLN is not at the same end as the scFv of TIGIT. The "any order" herein means that for the scFv of MSLN or the scFv of TIGIT, the positions of its variable light chain (VL) and variable heavy chain (VH) can be interchanged when the antigen to which they bind remains unchanged. As known to those skilled in the art, only the VH domain and VL domain of the scFv of MSLN itself can be interchanged; only the VH domain and VL domain of the scFv of TIGIT itself can be interchanged.

[0010] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide, and the basic structure of this hybrid polypeptide includes a targeting domain (which is the antigen-binding part of an antibody), a transmembrane domain, and an intracellular signaling domain. The CAR involved in the present application adds a spacer domain. The function of the spacer domain is to provide flexibility to overcome steric hindrance and contribute to the length of the CAR, so as to allow the antigen-binding domain to access the target epitope. Importantly, the selected spacer domain seems to affect the function of the CAR, because differences in the length and composition of the spacer domain can affect flexibility, CAR expression, signal transduction, epitope recognition, the strength of activation output, and epitope recognition.

[0011] In some embodiments, such scFv is fused to a transmembrane domain and then fused to an intracellular signaling domain. Currently, with the development of technology, four generations of different CAR structures have emerged. The intracellular signaling domain of the first-generation CAR only contains a primary signaling domain, such as CD3ζ. Therefore, cells carrying the CAR (such as CAR-T cells) have poor activity and a short survival time in vivo. The second-generation CAR introduces a co-stimulatory domain, such as CD28 or 4-1BB, enabling the cells to proliferate continuously and enhancing the anti-tumor activity. The third-generation CAR contains two co-stimulatory domains (such as CD28 + 4-1BB), and the fourth-generation CAR adds a cytokine or a co-stimulatory ligand to further enhance the T cell response, or adds a suicide gene to cause the CAR cells to self-destruct when needed.

[0012] "Targeting domain" refers to any structure that can bind to an antigen or its functional variant. The targeting domain can be an antibody structure, including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and their functional fragments. For example, the targeting domain includes but is not limited to single-chain antibody (Single Chain AntibodyFragment, scFv), single-domain antibody (Single Domain Antibody, sdAb), nanobody (Nanobody, Nb), antigen-binding ligand, recombinant fibronectin domain, anticalin, and DARPIN, etc., preferably selected from scFv, sdAb, and nanobody, and more preferably selected from scFv.

[0013] "scFv" is an antibody formed by connecting the variable region of the heavy chain (VH) and the variable region of the light chain (VL) of an antibody through a linker. The optimal length and / or amino acid composition of the linker can be selected. The length of the linker significantly affects the folding and interaction of the variable regions of scFv.

[0014] In some embodiments, the scFv of MSLN and / or the scFv of TIGIT of the present application contain functional variants against the above sequences, for example, having the same CDRs as the amino acid sequence of the scFv of MSLN and / or the amino acid sequence of the scFv of TIGIT. The amino acid sequence of the scFv of MSLN includes the VL domain (SEQ ID NO: 2) of the scFv that binds to MSLN, a linker, and the VH domain (SEQ ID NO: 3) of the scFv that binds to MSLN. The amino acid sequence of the scFv of TIGIT includes the VH domain (SEQ ID NO: 4) of the scFv that binds to TIGIT, a linker, and the VL domain (SEQ ID NO: 5) of the scFv that binds to TIGIT. The amino acid sequence of the linker is: GGGGSGGGGSGGGGS.

[0015] In some embodiments, the chimeric antigen receptor sequentially includes from the amino terminus to the carboxyl terminus: the VL domain of the scFv targeting MSLN, the VH domain of the scFv targeting MSLN, a spacer domain, a transmembrane domain, a signaling domain, the VH domain of the scFv targeting TIGIT, the VL domain of the scFv targeting TIGIT, or

[0016] the VL domain of the scFv targeting MSLN, the VH domain of the scFv targeting MSLN, a spacer domain, a transmembrane domain, a signaling domain, the VL domain of the scFv targeting TIGIT, the VH domain of the scFv targeting TIGIT, or

[0017] The VH domain of the scFv targeting MSLN, the VL domain of the scFv targeting MSLN, the spacer domain, the transmembrane domain, the signaling domain, the VH domain of the scFv targeting TIGIT, the VL domain of the scFv targeting TIGIT, or

[0018] The VH domain of the scFv targeting MSLN, the VL domain of the scFv targeting MSLN, the spacer domain, the transmembrane domain, the signaling domain, the VL domain of the scFv targeting TIGIT, the VH domain of the scFv targeting TIGIT, or

[0019] The VL domain of the scFv targeting TIGIT, the VH domain of the scFv targeting TIGIT, the spacer domain, the transmembrane domain, the signaling domain, the VH domain of the scFv targeting MSLN, the VL domain of the scFv targeting MSLN, or

[0020] The VL domain of the scFv targeting TIGIT, the VH domain of the scFv targeting TIGIT, the spacer domain, the transmembrane domain, the signaling domain, the VL domain of the scFv targeting MSLN, the VH domain of the scFv targeting MSLN, or

[0021] The VH domain of the scFv targeting TIGIT, the VL domain of the scFv targeting TIGIT, the spacer domain, the transmembrane domain, the signaling domain, the VH domain of the scFv targeting MSLN, the VL domain of the scFv targeting MSLN, or

[0022] The VH domain of the scFv targeting TIGIT, the VL domain of the scFv targeting TIGIT, the spacer domain, the transmembrane domain, the signaling domain, the VL domain of the scFv targeting MSLN, the VH domain of the scFv targeting MSLN.

[0023] In some embodiments, the chimeric antigen receptor comprises, in order from the amino terminus to the carboxyl terminus: the VL domain of the scFv targeting MSLN, the VH domain of the scFv targeting MSLN, the spacer domain, the transmembrane domain, the signaling domain, the VH domain of the scFv targeting TIGIT, the VL domain of the scFv targeting TIGIT.

[0024] In some embodiments, there is also a CD8α signal peptide (CD8α Singlepeptide) at the amino terminus of the scFv of the MSLN, and the CD8α signal peptide comprises the amino acid sequence shown in SEQ ID NO:1. A signal peptide refers to the amino-terminal amino acid sequence (sometimes not necessarily at the N-terminus) in a newly synthesized polypeptide chain that guides the transmembrane transfer (localization) of a protein.

[0025] As used herein, "amino acid" means one of the 20 naturally occurring amino acids or any unnatural analog that can be present at a specific, designated position.

[0026] In some embodiments, there is also a signal peptide (SP, Singlepeptide) at the amino terminus of the scFv of the TIGIT, and the signal peptide comprises the amino acid sequence shown in SEQ ID NO:11. This signal peptide is a short (5-30 amino acids in length) peptide chain that guides the transfer of a newly synthesized protein into the secretory pathway.

[0027] In some embodiments, there is a 2A peptide between the signal peptide at the amino terminus of the scFv of the TIGIT and the carboxyl terminus of the signal transduction domain. The 2A peptide is T2A, and the T2A comprises the amino acid sequence shown in SEQ ID NO:10.

[0028] T2A is a gene sequence derived from the insect virus (Thosea asigna), encoding a short peptide with self-processing ability. It is a peptide fragment 18-22 amino acid residues in length that can induce the self-cleavage of recombinant proteins containing the 2A peptide in cells. Additionally, GSG (glycine, serine, glycine) can be added to the amino terminus of T2A (the amino acid sequence shown in SEQ ID NO:10). It should be noted that for the 2A peptides involved in this application, those skilled in the art can know that they include: P2A, T2A, E2A, and F2A. This application preferably uses T2A. Any form of replacement of P2A, E2A, and F2A falls within the protection scope of this application.

[0029] In some embodiments, there is also a tag protein (HA Tag) at the carboxyl terminus of the scFv of the TIGIT, and the tag protein comprises the amino acid sequence shown in SEQ ID NO:13.

[0030] In some embodiments, the VL domain of the scFv of the MSLN comprises the amino acid sequence shown in SEQ ID NO:2, and the VH domain of the scFv of the MSLN comprises the amino acid sequence shown in SEQ ID NO:3.

[0031] In some embodiments, the VL domain of TIGIT of MSLN comprises the amino acid sequence shown in SEQ ID NO:4, and the VH domain of the scFv of TIGIT comprises the amino acid sequence shown in SEQ ID NO:5.

[0032] Single-chain antibody (scFv) is a genetically engineered antibody in which the VH and VL domains are linked by a flexible polypeptide linker. Compared with the Fab region of the whole antibody, the single-chain antibody exhibits better tissue penetration pharmacokinetics and has complete antigen-binding specificity due to the unchanged antigen-binding surface.

[0033] In some embodiments, a linker comprising 4 - 15 amino acids is between each of the VL and VH domains.

[0034] In some embodiments, the linker comprises only G and S.

[0035] In some embodiments, the linker amino acid sequence is: GGGGSGGGGSGGGGS, denoted as (G4S)3 in the present application. Additionally, the amino acid numbering involved in the present application follows Kabat numbering. "Kabat numbering" refers to the numbering system described by Kabat et al., the content of which is recorded in the U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0036] In some embodiments, the spacer domain is selected from the group consisting of: IgG4 hinge or CD8 hinge.

[0037] In some embodiments, the spacer domain is preferably the CD8 hinge.

[0038] In some embodiments, the CD8 hinge comprises the amino acid sequence shown in SEQ ID NO:6.

[0039] The term "hinge region" generally refers to any oligopeptide or polypeptide that functions to connect the transmembrane domain to the antigen-binding region. The hinge region can contain up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.

[0040] In some embodiments, the hinge region can be derived from all or part of a natural molecule, such as the extracellular region derived from all or part of CD8, CD4, or CD28, or from all or part of an antibody constant region. Alternatively, the hinge region can be a synthetic sequence corresponding to a naturally occurring hinge sequence, or can be a fully synthetic hinge sequence. In a preferred embodiment, the hinge region comprises the hinge region portion of the CD8α chain or IgG4, more preferably the hinge of CD8, which has the amino acid sequence shown in SEQ ID NO:6.

[0041] In some embodiments, the transmembrane domain in the present application is selected from the transmembrane domains of the following proteins: CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, TCR α chain, TCR β chain, TCR γ chain, TCR δ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, or CD3δ subunit.

[0042] In some embodiments, the CD8 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:7.

[0043] The term "transmembrane domain" refers to a polypeptide structure that enables a chimeric antigen receptor to be expressed on the surface of an immune cell (such as a lymphocyte, NK cell, or NKT cell) and guides the cellular response of the immune cell against a target cell. The transmembrane domain can be natural or synthetic and can be derived from any membrane-binding protein or transmembrane protein. When the chimeric receptor polypeptide binds to the target antigen, the transmembrane domain is capable of signal transduction.

[0044] In some embodiments, the transmembrane domain can be synthetic and can mainly comprise hydrophobic residues such as leucine and valine. Preferably, the transmembrane domain is derived from the human CD8 chain, which has the amino acid sequence shown in SEQ ID NO:7.

[0045] In some embodiments, the intracellular signaling domain is selected from the signaling domains of the following proteins: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0046] In some embodiments, the CD3ζ comprises the amino acid sequence shown in SEQ ID NO:9.

[0047] The term "intracellular signaling domain" refers to the protein portion that transduces effector function signals and directs the cell to perform specified functions. The intracellular signaling domain is responsible for intracellular signal transduction after antigen binding in the antigen-binding region, thereby leading to the activation of immune cells and immune responses. In other words, the intracellular signaling domain is responsible for activating at least one of the normal effector functions of the immune cells in which the CAR is expressed. For example, the effector functions of T cells can be cytolytic activity or helper activity, including the secretion of cytokines.

[0048] In some embodiments, the intracellular signaling domain included in the chimeric antigen receptor of the present application can be the cytoplasmic sequences of the T cell receptor and co-receptors, which act together after antigen receptor binding to initiate signal transduction, as well as any derivatives or variants of these sequences and any synthetic sequences having the same or similar functions. The intracellular signaling domain contains two different types of cytoplasmic signal sequences: those that initiate antigen-dependent primary activation, and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals. The primary cytoplasmic signal sequences can contain a number of Immunoreceptor Tyrosine-based Activation Motifs (ITAMs). Non-limiting examples of the intracellular signaling domain of the present application include, but are not limited to, those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In a preferred embodiment, the signal transduction domain of the CAR of the present application can contain the CD3ζ signaling domain, which is the amino acid sequence shown in SEQ ID NO:9.

[0049] In some embodiments, the chimeric receptor further comprises one or more co-stimulatory domains.

[0050] In some embodiments, the co-stimulatory domain is a co-stimulatory signaling domain selected from the following proteins: CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134, CD137, CD150, CD152, CD223, CD270, CD272, CD273, CD274, CD276, CD278, CD357, DAP10, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, LAT, NKG2C, SLP76, LIGHT, TRIM, or ZAP70.

[0051] In some embodiments, the CD28 co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO:8.

[0052] In some embodiments, the chimeric antigen receptor of the present application further comprises one or more co-stimulatory domains. The co-stimulatory domain can be an intracellular functional signaling domain from a co-stimulatory molecule, which can comprise the entire intracellular portion of the co-stimulatory molecule, or a functional fragment thereof. A "co-stimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response (such as proliferation) of the T cell. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors. Non-limiting examples of the co-stimulatory domain of the present application include, but are not limited to, co-stimulatory signaling domains derived from the following proteins: CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134, CD137, CD150, CD152, CD223, CD270, CD272, CD273, CD274, CD276, CD278, CD357, DAP10, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, LAT, NKG2C, SLP76, LIGHT, TRIM, or ZAP70. Preferably, the co-stimulatory domain of the CAR of the present application is a CD28 fragment, more preferably the amino acid sequence shown in SEQ ID NO:8.

[0053] On the other hand, the present application provides a nucleic acid comprising a nucleic acid sequence encoding the chimeric antigen receptor.

[0054] The term "encoding", when applied to a polynucleotide, refers to a polynucleotide that is considered to "encode" a certain polypeptide, which, when in its native state or manipulated by methods well known to those skilled in the art, can be transcribed and / or translated to produce the mRNA and / or a fragment thereof of the polypeptide. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0055] It should be noted that an indefinite number of entity limitations should refer to one or more of such entities; for example, "polynucleotide" should be understood to represent one or more polynucleotides. Similarly, the terms "one or more" and "at least one", which are indefinite number limitations, can be used interchangeably herein.

[0056] In some embodiments, the chimeric antigen receptor provided by the present application comprises a targeting domain, a spacer domain, a transmembrane domain, and a signaling domain. The targeting domain comprises, in any order, the VL domain of an scFv that binds MSLN, the VH domain of an scFv that binds MSLN, the VH domain of an scFv that binds TIGIT, and the VL domain of an scFv that binds TIGIT. The scFv that binds MSLN is located at the amino terminus or carboxyl terminus of the chimeric antigen receptor, and the scFv that binds TIGIT is located at the other end. The chimeric antigen receptor sequentially comprises, from the amino terminus to the carboxyl terminus: a CD8α signal peptide, the VL domain of an scFv that targets MSLN, a (G4S)3 linker, the VH domain of an scFv that targets MSLN, a spacer domain, a CD8 transmembrane domain (CD8 TM ), a co-stimulatory domain, a signaling domain, T2A, a signal peptide, the VL domain of an scFv that targets TIGIT, a (G4S)3 linker, the VH domain of an scFv that targets TIGIT, and a tag protein. Preferably, the spacer domain is a CD8 hinge, the co-stimulatory domain is CD28, and the signaling domain is CD3ζ.

[0057] On the other hand, the present application provides a vector comprising the nucleic acid molecule described above.

[0058] In some embodiments, the vector is a lentiviral vector.

[0059] Vectors generally include targeting vectors and expression vectors. A "targeting vector" is an agent that delivers isolated nucleic acids into cells, for example, by homologous recombination or by using hybrid recombinases that recognize sequences at specific targeting sites. An "expression vector" is a vector used for transcription of heterologous nucleic acid sequences and translation of their mRNAs in appropriate immune cells. Suitable vectors for use in the present application are known in the art and many are commercially available. In some embodiments, the vectors of the present application include, but are not limited to, linear nucleic acid molecules (such as DNA or RNA), plasmids, viruses (such as retroviruses, lentiviruses, adenoviruses, vaccinia viruses, Rous sarcoma virus (RSV), polyomaviruses, and adeno-associated viruses (AAV), etc.), phages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs). A vector itself is usually a nucleotide sequence, usually a DNA sequence containing an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. Engineered vectors typically also contain an origin of replication that autonomously replicates in immune cells (if stable expression of the polynucleotide is desired), a selection marker, and restriction enzyme cleavage sites (such as a multiple cloning site, MCS). Vectors may additionally contain elements such as promoters, polyadenylation tails (polyA), 3' UTRs, enhancers, terminators, insulators, operons, selection markers, reporter genes, targeting sequences, and / or protein purification tags. In a specific embodiment, the vector is a lentiviral vector.

[0060] In another aspect, the present application provides a population of human T cells or NK cells transduced with the described vector.

[0061] Methods for introducing nucleic acids or vectors into immune cells and expressing them are known in the art. For example, nucleic acids or vectors can be introduced into immune cells by physical methods such as calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Alternatively, chemical methods can be employed, such as introducing nucleic acids or vectors through colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including water-in-oil emulsions, micelles, mixed micelles, and liposomes. In addition, biological methods can also be used to introduce nucleic acids or vectors. For example, viral vectors, especially retroviral vectors, etc., have become the most commonly used methods for inserting genes into mammals, such as human cells.

[0062] In some embodiments, a population of human T cells transduced with the described vector is provided.

[0063] In some embodiments, the population of T cells is derived from PBMCs isolated from human peripheral blood.

[0064] In another aspect, the present application provides a method for treating cancer in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of a composition comprising the described population of human T cells, thereby treating the cancer of the subject.

[0065] In some embodiments, the cancer is renal cancer, malignant mesothelioma, lung cancer, esophageal cancer, pancreatic cancer, cervical cancer, or ovarian cancer.

[0066] In some embodiments, the T cell population is autologous or allogeneic for the patient.

[0067] Autologous means any material derived from an individual that will later be reintroduced into the same individual. CAR-T cells prepared from allogeneic source T cells are off-the-shelf. Allogeneic means any material derived from a different animal or different patient of the same species as the individual into whom the material is introduced. Two or more individuals are considered allogeneic to each other when they differ genetically at one or more loci. In some cases, the genetic differences between allogeneic materials from individuals of the same species may be sufficient to cause antigenic interactions. Typically, T cells are collected from healthy donors or directly used T cells from stem cell sources, and then the genes causing immune rejection are knocked out / not knocked out through genetic engineering and CAR is loaded.

[0068] In some embodiments, the human T cell population includes cells comprising CD4+ cells and CD8+ cells.

[0069] It should be noted that the T cells may also be CD4+ / CD8+ double-positive T cells, tumor-infiltrating cells, memory T cells, naive T cells, γδ-T cells, or αβ-T cells. It is also known to those skilled in the art to introduce the vectors involved in the present application into the above-mentioned T cells.

[0070] The carrier-transduced immune cells provided by the present application can be T cells or NK cells, but are not limited thereto, and can also be macrophages, dendritic cells, monocytes. Preferably, the immune cells are T cells. The T cells can be any T cells, such as T cells cultured in vitro, such as primary T cells, or T cells from a T cell line cultured in vitro, such as Jurkat, SupT1, etc., or T cells obtained from a subject. Examples of the subject include humans, dogs, cats, mice, rats, and their transgenic species. The T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. The T cells can also be concentrated or purified. The T cells can be any type of T cells and can be at any developmental stage, including but not limited to, CD4+ / CD8+ double-positive T cells, CD4+ helper T cells (such as Th1 and Th2 cells), CD8+ T cells (such as cytotoxic T cells), tumor-infiltrating cells, memory T cells, naive T cells, γδ-T cells, αβ-T cells, etc. In a preferred embodiment, the immune cells are human T cells, more specifically derived from PBMC isolated from human peripheral blood.

[0071] On the other hand, the present application provides a pharmaceutical composition comprising the T cells and a pharmaceutically acceptable carrier, diluent or excipient.

[0072] "Pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is compatible with the subject and the active ingredient pharmacologically and / or physiologically (i.e., capable of eliciting the desired therapeutic effect without causing any undesirable local or systemic effects), and is well known to those skilled in the art. Pharmaceutically acceptable excipients include but are not limited to fillers, binders, disintegrants, coating agents, adsorbents, anti-adhesives, glidants, antioxidants, flavoring agents, coloring agents, sweetening agents, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifying agents, coating agents, isotonic agents, absorption retardants, stabilizers, and tonicity regulators. Those skilled in the art know how to select appropriate excipients to prepare the desired pharmaceutical composition of the present application.

[0073] The pharmaceutical compositions of the present application can also be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, and can particularly be in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures or fluid extracts, or in a form particularly suitable for the required administration method. The processes known in the art for producing pharmaceuticals can include, for example, conventional mixing, dissolving, granulating, sugar coating, grinding, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions containing, for example, the immune cells described herein are generally provided in solution form and preferably contain a pharmaceutically acceptable buffer. Brief Description of the Drawings

[0074] To more clearly illustrate the technical solutions of the present application, the drawings required for use in the examples will be briefly introduced below. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0075] Figure 1 The structure of MSLNCAR disclosed in the prior art and the structure of MTCAR applied to each embodiment of the present application, where the CD8α signal peptide is shown as: CD8αSignal peptide, the fusion protein linker is shown as: Linker(G4S)3, the CD8 hinge region is shown as: CD8 Hinge, the CD8 transmembrane domain is shown as: CD28 TM , the CD28 co-stimulatory domain is shown as: CD28, the 2A peptide is shown as: T2A, the signal peptide of the TIGIT single-chain antibody is shown as: SP, the tag protein is shown as: Taq, the intracellular signal domain of CD3 is shown as CD3ζ, the light chain of the MSLN single-chain antibody is shown as: V MSLNL , the heavy chain of the MSLN single-chain antibody is shown as: V MSLNH , the heavy chain of the TIGIT single-chain antibody is shown as: V TIGITL , the light chain of the TIGIT single-chain antibody is shown as: V TIGITH ;

[0076] Figure 2 It is a detection result graph of the positive rates of MSLN and CD155 antigens. Among them, a is the detection result of the positive rate of MSLN antigen. Among them, the abscissa is the fluorescence intensity of single MSLN-positive cells, and the ordinate is the number of cells with different fluorescence intensities; b is the detection result of the positive rate of CD155 antigen. The abscissa is the fluorescence intensity of single CD155-positive cells, and the ordinate is the killing rate;

[0077] Figure 3 It is the expression efficiency of MSLN scFv of T cells after lentiviral vector transfection;

[0078] Figure 4 It is a comparison chart of the TIGIT expression results in MSLN CAR-T and MT CAR-T cells for Western blot analysis. Among them, MSLN represents the TIGIT protein expression in MSLN CAR-T cells, MT represents the TIGIT expression in MT CAR-T cells, and the molecular weight of the TIGIT protein is 30 kDa;

[0079] Figure 5 It is a comparison chart of the killing effects of MSLN CAR-T and MT CAR-T effector cells on Hela CD155 target cells in a luciferase reporter experiment. Among them, the abscissa is the effector-to-target ratio, the ordinate is the cytotoxicity, MSLN represents MSLN CAR-T cells, MT represents MT CAR-T cells, and * indicates p ≤ 0.05, indicating that the results are statistically significant and the difference is significant;

[0080] Figure 6 It is a comparison chart of cytokine release in MSLN CAR-T and MT CAR-T cells detected by ELISA. T is the blank control of T cells, MSLN is MSLN CAR-T cells, MT is MT CAR-T cells, the abscissa is cytokines: IFN-γ, IL-2 and TNF-α, and the ordinate is the in vitro cytokine concentration;

[0081] Figure 7 It is a comparison chart of the tumor suppression growth experiment results of MSLN CAR-T and MT CAR-T cells. Among them, T is the blank control of T cells, MSLN is MSLN CAR-T cells, MT is MT CAR-T cells, the abscissa is time, and the ordinate is tumor volume. Detailed implementation mode

[0082] The following will describe the implementation plan of the present application in detail in combination with the examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. For those not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0083] Example 1 Preparation of lentiviral expression vector

[0084] In the example of the present application, an MLSN CAR structure with a TIGIT single-chain antibody (abbreviated as MT CAR in the example of the present application) was synthesized and cloned into the pCDH lentiviral plasmid backbone with a human CMV promoter.

[0085] See Figure 1, which is the structure of the MT CAR applied to each embodiment, includes a lentiviral vector of the MLSN CAR, including a single-chain antibody against MSLN, a CD8 hinge region, a CD8 transmembrane domain, a CD28 co-stimulatory signal molecule, and a CD3ζ signal endodomain. In the MTCAR, the T2A peptide sequence of the single-chain antibody against TIGIT was also inserted into the second-generation CAR gene, and a tag protein Tag was added for subsequent experiments.

[0086] Gene synthesis of scFV(Anti MSLN)-CD8 TM -CD28-CD3ζ-T2A-scFV(Anti TIGIT) fusion gene sequence (synthesized by GenScript), and the gene sequence is shown in SEQ ID NO: 7. It was ligated into the pLVX-EF1alpha-AcGFP1-N1 Vector vector (Clontech, catalog number: 631983) by restriction enzyme digestion and transformation. The upstream of the gene is the EP-1a promoter. The vector was transformed into the DH5α (Novizan, catalog number C502-02) Escherichia coli strain, screened with ampicillin, positive clones were obtained, plasmids were extracted, and the clones were identified by restriction enzyme digestion to obtain PLVX-scFV(Anti MSLN)-CD8 TM -CD28-CD3ζ-T2A-scFV(Anti TIGIT) lentiviral packaging vector.

[0087] Example 2 Lentivirus preparation

[0088] a. One day before transfection, 293T cells (human embryonic kidney cells, purchased from ATCC, catalog number: CL-0005) with good growth status were seeded into a T75 cell culture flask (purchased from Corning, catalog number: 430641), and the appropriate density was adjusted to make the confluence reach 80% the next day, which was suitable for lentiviral packaging.

[0089] b. Before transfection, the original culture medium was removed, and 10 mL of pre-warmed DMEM complete medium (purchased from Gibco, catalog number: 11965092) was slowly added along the wall of the culture flask.

[0090] c. Vortex and mix the Polyfect-V transfection reagent (purchased from Yingmao Shengye, catalog number: P2010-1). Prepare 2 centrifuge tubes, and prepare plasmid and transfection reagent dilutions respectively according to Table 1.

[0091] Table - 1 - Lentiviral packaging system

[0092] Centrifuge tube 1 (plasmid DNA) Centrifuge tube 2 (transfection reagent) MSLN vector 12 μg Polyfect-V transfection reagent 24 μL pH1-gag vector 9 μg Serum-free DMEM medium 476 μL pH2-VSV-G vector 3 μg Total volume 500 μL Serum-free DMEM medium X μL Total volume 500 μL

[0093] d. Mix well and incubate the transfection reagent dilution (centrifuge tube 2) at room temperature for 5 min.

[0094] e. Add the diluted transfection reagent (centrifuge tube 2) to the plasmid DNA solution (centrifuge tube 1). Note that the addition order is very important. Immediately mix well thoroughly.

[0095] f. Incubate the transfection mixture at room temperature for 15 min to form the DNA-liposome complex.

[0096] g. Add 1 mL of the transfection mixture to 9 mL of complete culture medium. Gently add it slowly along the wall of the T75 cell culture flask (note not to touch the cells). Gently shake to mix evenly and place it in an incubator at 37 °C for 24 h.

[0097] h. After 6 h of transfection, remove the culture medium containing the transfection reagent, add 20 mL of complete DMEM medium and place it in an incubator at 37 °C and 5% CO2 for culture;

[0098] i. Collect the cell culture supernatant 48 h and 72 h after transfection and store it at -80 °C.

[0099] Example 3 Preparation of MT CAR-T Cells

[0100] a. Prepare a 15 mL centrifuge tube in advance and add 7 mL of rewarmed x-vivo 15 complete culture medium (purchased from Lonza, product number: 04-418Q).

[0101] b. Take out the cryopreserved PBMC (purchased from Shanghai Miaoshun, product number: SER-PBMC-200-F) from the liquid nitrogen tank and quickly place it in a water bath at 37 °C.

[0102] c. Open the cryopreservation tube, carefully blow and mix the cells evenly, and slowly add them to the centrifuge tube. Centrifuge at 400 × g for 5 min.

[0103] d. Remove the supernatant, resuspend with PBS to make the cell concentration 5×10 7 / mL, and transfer it to a 2 mL cryopreservation tube.

[0104] e. Add 50 μL of Isolation Cocktail (Stemcell, product number: 17951) to the sample. Gently pipette and mix well and incubate at room temperature for 5 min.

[0105] f. Add 40 μL of RapidSpheres TM (Stemcell, product number: 17951) to the sample, and supplement with x-vivo15 culture medium to 2 mL. Gently pipette 2 - 3 times to mix evenly and incubate for 3 min.

[0106] g. Place the cryopreservation tube in a magnet and incubate for 3 min.

[0107] h. Pick up the magnet, pour the cell suspension into a 15 mL centrifuge tube, centrifuge at 400×g for 5 min, remove the supernatant, resuspend with 1 mL of x-vivo15 medium, and set aside for later use.

[0108] i. Add CD3 / CD28 activation beads at a ratio of bead:cell = 1:1, gently pipette to mix evenly, and incubate in a 37°C, 5% CO2 incubator for 20 min.

[0109] j. Take out the cells from the incubator, resuspend them in 5 mL of x-vivo 15 complete medium containing 100 U / mL IL-2, and transfer them to a T25 cell culture flask.

[0110] k. Viral infection can be carried out within 24 - 48 h after cell sorting activation. Seed the cells at a density of 1×10 6 into a 24-well culture plate.

[0111] l. Add 50 μL of the virus solution to the cell culture plate, change the medium after 6 - 8 h, and the CAR gene expression can be detected by flow cytometry 48 - 72 h after viral infection.

[0112] After the lentivirus packaging and preparation, the purified virus was aliquoted and stored in an -80°C refrigerator. PBMCs were activated and sorted using CD3 and 28 beads, and transfected after 24 - 48 h of in vitro culture. 3 - 4 days after transfection, flow cytometry was used to detect the MSLN scFv expression of T cells transfected with the lentiviral vector to verify the CAR transduction efficiency. The results showed that after lentiviral transfection, MT CAR-T cells with a positive rate of 48.93% were obtained (see Figure 3 ).

[0113] Example 4 Screening and Detection of Engineered Cell Lines

[0114] The MSLN CAR-T cells used in the examples of this application are publicly available CAR-Ts. For the specific structure and sequence, please refer to the patent: A Chimeric Antigen Receptor Targeting Mesothelin and Its Application (202110794199.X).

[0115] To detect the positive rates of mesothelin and CD155 in the Hela cell line, a cell line with good cell state was collected, resuspended, and incubated with anti-MSLN / anti-CD155 flow antibodies. See Figure 2 , which is the detection result graph of the positive rates of MSLN and CD155 antigens. Flow cytometry results showed that the positive rate of mesothelin in the cervical cancer cell line Hela (purchased from ATCC, catalog number: CCL-2) was approximately 99.1% (see Figure 2 a) therein, and the positive rate of CD155 was 99.4% (see Figure 2 b) therein).

[0116] Detection of TIGIT scFv Secretion in Example 5

[0117] Collect the culture supernatants of MSLN CAR-T and MT CAR-T cells, centrifuge at 1000×g for 5 min to remove cell debris. Transfer the supernatants to ultrafiltration tubes, and after centrifuging at 4000×g for 30 min, collect the enriched proteins. Refer to Figure 4 , which is a comparison chart of the TIGIT expression results in MSLN CAR-T and MT CAR-T cells analyzed by Western blot. From the Western blot analysis results, it can be seen that there is almost no secretion of TIGIT scFv in the MSLN CAR-T group of cells, while there is obvious secretion of TIGIT scFv in MT CAR-T cells.

[0118] Example 6 Cytotoxicity of MSLN CAR-T or MT CAR-T Cells against Hela CD155 Cells

[0119] Use adherent-growing tumor cells Hela CD155 (ATCC, catalog number CCL-2, stably transfected in the laboratory, the overexpression plasmid was purchased from Vigene) as target cells, count and adjust the cells to 2×10 4 per well and culture for 24 h. The next day, add MSLN CAR-T or MT CAR-T cells, set the effector-to-target ratio to 8:1, 4:1, 2:1, 1:1, and culture for about 4 h again. Determine the cytotoxicity results of effector cells by luciferase reporter assay. The results show that at different effector-to-target ratios, the group secreting TIGIT scFv shows better tumor cytotoxicity. Refer to Figure 5 , which is a comparison chart of the cytotoxicity of MSLN CAR-T and MT CAR-T effector cells against Hela CD155 target cells determined by luciferase reporter assay. The results show that at different effector-to-target ratios, the group secreting TIGIT scFv shows better tumor cytotoxicity.

[0120] Example 7 ELISA Detection of Cytokine Release from MSLN CAR-T and MT CAR-T Cells

[0121] Count the adherent-growing tumor cells Hela and distribute them into 96-well plates, with 2×10 4 cells per well. After culturing for 24 h, add T, MSLN CAR-T and MT CAR-T cells respectively, set the effector-to-target ratio to 1:1, add control antibody to the control group, and adjust the concentration to 10 4ng / mL. Incubate for about 4 h again. Collect the supernatant, centrifuge at 1000×g for 5 min to remove residual cells and debris. Use Human IFN-gamma Quantikine ELISA Kit (purchased from R&D Biotechnology Co., Ltd., catalog number: DIF50C), Human IL-2 Quantikine ELISA Kit (purchased from R&D Biotechnology Co., Ltd., catalog number: D2050), Human TNF-alpha Quantikine ELISA Kit (purchased from R&D Biotechnology Co., Ltd., catalog number: DTA00D) (R&D Systems) to detect the release of IFN-γ, IL-2 and TNF-α in the antibody group and the control group. See Figure 6 , which is a comparison chart of cytokine release detected by ELISA for MSLN CAR-T and MT CAR-T cells. The ELISA results show that more cytokines are released in the treatment group treated with MT CAR-T.

[0122] Example 8 TIGIT Antibody Inhibits Cervical Cancer Growth in Vivo

[0123] When the tumors of B-NDG mice (purchased from Beijing Biocytogen Co., Ltd.) grow to 100 mm 3 , CAR-T cell treatment is given by tail vein injection. Subcutaneously inject 5×10 6 Hela CD155 cells into the right dorsal subcutaneous area of 6-week-old B-NDG mice. When the average size reaches the standard size, count the effector cells. The tumor-bearing mice are randomly divided into three groups of four each, and 5×10 6 CAR-T cells are injected through the tail vein for treatment. During the treatment process, continuously detect the size of the tumors in the mice.

[0124] See Figure 7 , which is a comparison chart of the experimental results of tumor growth inhibition of MSLN CAR-T and MT CAR-T cells. Before treatment, there was no significant difference in the tumor size among the MT CAR-T cell treatment group, MSLN CAR-T group and T cell group. However, within 20 days after treatment, the tumor volume of the mice in the CAR-T cell treatment group continued to decrease. At the same time, the dynamic changes of the tumors in the mice were statistically analyzed. The results showed that the tumor growth rate of the mice in the CAR-T cell treatment group slowed down, while the tumors in the T cell group continued to progress. The size of the tumors in the mice was statistically analyzed. Among them, the tumor remission rate: MT CAR-T (secreting TIGIT scFv) group > MSLN CAR-T group, and signs of recurrence appeared in the MSLN CAR-T group. The experimental results show that the MT CAR-T group can significantly inhibit tumor growth.

[0125] For the similar parts between the embodiments provided in this application, reference can be made to each other. The specific embodiments provided above are only several examples under the general concept of this application and do not constitute a limitation on the protection scope of this application. For those skilled in the art, any other embodiments extended based on the solution of this application without creative efforts fall within the protection scope of this application. Sequence Listing <110> Nanjing Landun Biotechnology Co., Ltd. <120> Chimeric Antigen Receptor <160> 12 <170> SIPOSequenceListing 1.0 <210> 1 <211> 21 <212> PRT <213> Artificial Sequence <400> 1 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 2 <211> 110 <212> PRT <213> Artificial Sequence <400> 2 Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Ala Gly Ser 85 90 95 Asn Asn Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 3 <211> 123 <212> PRT <213> Artificial Sequence <400> 3 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Thr Pro Glu Lys Gly Leu Glu 35 40 45 Trp Ile Ala Tyr Ile Tyr Asn Ser Gly Thr Thr Lys Phe Asn Pro Ser 50 55 60 Leu Lys Gly Arg Val Thr Ile Ser Met Asp Ala Ser Lys Asn Gln Leu 65 70 75 80 Ser Met Lys Leu Ser Ser Val Thr Ser Ala Asp Thr Ala Val Tyr Phe 85 90 95 Cys Ala Arg Asp Gln Gly Asn Ser Pro Tyr Pro Asp Ala Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 4 <211> 109 <212> PRT <213> Artificial Sequence <400> 4 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Leu Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105 <210> 5 <211> 130 <212> PRT <213> Artificial Sequence <400> 5 Gln Val His Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly 20 25 30 Ile Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Tyr Tyr Val Ser Gly Asn Tyr Tyr Asn Val Asp Tyr 100 105 110 Tyr Phe Phe Gly Val Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val 115 120 125 Ser Ser 130 <210> 6 <211> 47 <212> PRT <213> Artificial Sequence <400> 6 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 35 40 45 <210> 7 <211> 22 <212> PRT <213> Artificial Sequence <400> 7 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 1 5 10 15 Val Ile Thr Leu Tyr Cys 20 <210> 8 <211> 42 <212> PRT <213> Artificial Sequence <400> 8 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 9 <211> 112 <212> PRT <213> Artificial Sequence <400> 9 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 10 <211> 18 <212> PRT <213> Artificial Sequence <400> 10 Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro 1 5 10 15 Gly Pro <210> 11 <211> 21 <212> PRT <213> Artificial Sequence <400> 11 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <400> 12 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5

Claims

1. A chimeric antigen receptor comprising a targeting domain, a spacer domain, a transmembrane domain, and a signaling domain, wherein the targeting domain comprises, in any order, the VL domain of an scFv that binds MSLN, the VH domain of an scFv that binds MSLN, the VH domain of an scFv that binds TIGIT, the VL domain of an scFv that binds TIGIT, and the scFv that binds MSLN is located at the amino terminus or carboxyl terminus of the chimeric antigen receptor, and the scFv that binds TIGIT is located at the other end.

2. The chimeric antigen receptor according to claim 1, which sequentially comprises, from the amino terminus to the carboxyl terminus: the VL domain of an scFv targeting MSLN, the VH domain of an scFv targeting MSLN, a spacer domain, a transmembrane domain, a signaling domain, the VH domain of an scFv targeting TIGIT, the VL domain of an scFv targeting TIGIT, or the VL domain of an scFv targeting MSLN, the VH domain of an scFv targeting MSLN, a spacer domain, a transmembrane domain, a signaling domain, the VL domain of an scFv targeting TIGIT, the VH domain of an scFv targeting TIGIT, or the VH domain of an scFv targeting MSLN, the VL domain of an scFv targeting MSLN, a spacer domain, a transmembrane domain, a signaling domain, the VH domain of an scFv targeting TIGIT, the VL domain of an scFv targeting TIGIT, or the VH domain of an scFv targeting MSLN, the VL domain of an scFv targeting MSLN, a spacer domain, a transmembrane domain, a signaling domain, the VL domain of an scFv targeting TIGIT, the VH domain of an scFv targeting TIGIT, or the VL domain of an scFv targeting TIGIT, the VH domain of an scFv targeting TIGIT, a spacer domain, a transmembrane domain, a signaling domain, the VH domain of an scFv targeting MSLN, the VL domain of an scFv targeting MSLN, or the VL domain of an scFv targeting TIGIT, the VH domain of an scFv targeting TIGIT, a spacer domain, a transmembrane domain, a signaling domain, the VL domain of an scFv targeting MSLN, the VH domain of an scFv targeting MSLN, or the VH domain of an scFv targeting TIGIT, the VL domain of an scFv targeting TIGIT, a spacer domain, a transmembrane domain, a signaling domain, the VH domain of an scFv targeting MSLN, the VL domain of an scFv targeting MSLN, or the VH domain of an scFv targeting TIGIT, the VL domain of an scFv targeting TIGIT, a spacer domain, a transmembrane domain, a signaling domain, the VL domain of an scFv targeting MSLN, the VH domain of an scFv targeting MSLN.

3. The chimeric antigen receptor according to claim 1, wherein the VL domain of the scFv of MSLN comprises the amino acid sequence shown in SEQ ID NO: 2, and the VH domain of the scFv of MSLN comprises the amino acid sequence shown in SEQ ID NO:

3.

4. The chimeric antigen receptor according to claim 1, wherein the VL domain of the TIGIT of MSLN comprises the amino acid sequence shown in SEQ ID NO: 4, and the VH domain of the scFv of TIGIT comprises the amino acid sequence shown in SEQ ID NO:

5.

5. The chimeric antigen receptor according to any one of claims 1-4, wherein a linker comprising 4-15 amino acids is between each of the VL and VH domains.

6. The chimeric antigen receptor according to claim 5, wherein the linker comprises only G and S.

7. The chimeric antigen receptor according to claim 1 or 2, wherein the spacer domain is selected from: IgG4 hinge or CD8 hinge.

8. The chimeric antigen receptor according to claim 7, wherein the CD8 hinge comprises the amino acid sequence shown in SEQ ID NO:

6.

9. The chimeric antigen receptor according to claim 1 or 2, wherein the transmembrane domain is selected from the transmembrane domains of the following proteins: CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit or CD3δ subunit.

10. The chimeric antigen receptor according to claim 9, wherein the CD8 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:

7.

11. The chimeric antigen receptor according to claim 1 or 2, wherein the intracellular signaling domain is selected from the signaling domains of the following proteins: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d.

12. The chimeric antigen receptor according to claim 11, wherein the CD3ζ comprises the amino acid sequence shown in SEQ ID NO:

9.

13. The chimeric antigen receptor according to claim 1 or 2, wherein the chimeric receptor further comprises one or more co-stimulatory domains.

14. The chimeric antigen receptor according to claim 13, wherein the co-stimulatory domain is a co-stimulatory signaling domain selected from the following proteins: CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134, CD137, CD150, CD152, CD223, CD270, CD272, CD273, CD274, CD276, CD278, CD357, DAP10, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, LAT, NKG2C, SLP76, LIGHT, TRIM or ZAP70.

15. The chimeric antigen receptor according to claim 14, wherein the CD28 co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO:

8.

16. A nucleic acid comprising a nucleic acid sequence encoding the chimeric antigen receptor according to any one of claims 1-15.

17. A vector comprising the nucleic acid molecule according to claim 16.

18. The vector according to claim 17, wherein the vector is a lentiviral vector.

19. A population of human T cells or NK cells transduced with the vector according to claim 18.

20. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising the population of human T cells according to claim 19, thereby treating the cancer of the subject.

21. The method according to claim 20, wherein the cancer is renal cancer, malignant mesothelioma, lung cancer, esophageal cancer, pancreatic cancer, cervical cancer or ovarian cancer.

22. The method according to claim 20 or 21, wherein the population of T cells is autologous or allogeneic to the patient.

23. The method according to any one of claims 20-22, wherein the population of human T cells comprises cells including CD4+ cells and CD8+ cells.

24. A method for manufacturing the population of T cells according to any one of claims 19-23, comprising the step of introducing the nucleic acid of claim 16 into T cells.

25. A pharmaceutical composition comprising the T cells according to any one of claims 19-23 and a pharmaceutically acceptable carrier, diluent or excipient.

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