Chimeric receptors targeting mesothelin and their uses

Abstract

The present disclosure relates to a chimeric receptor targeting mesothelin and its uses. The chimeric antigen receptor comprises a first fusion peptide and a second fusion peptide, wherein: the first fusion peptide comprises a mesothelin antigen-binding domain and a transmembrane domain; the second fusion peptide comprises a transmembrane domain, a cytoplasmic domain and a co-stimulatory domain. The chimeric antigen receptor of the present disclosure has good killing activity against mesothelin-expressing positive solid tumor cells.

Description

Technical Field

[0001] The present disclosure relates to the field of tumor cell therapy, and more particularly, to chimeric receptors targeting mesothelin that express a signal domain containing DAP12 and a costimulatory signaling molecule, immune effector cells thereof, compositions containing the same, methods for preparing the same, and uses thereof. Background Art

[0002] Mesothelin (MSLN) is a glycoprotein linked to the cell membrane through a glycosylphosphatidylinositol region (GPI domain). It was initially identified as a tumor-associated antigen due to its limited expression in normal tissues and overexpression in tumors. The mesothelin gene encodes a 71-kDa precursor protein. After the N-terminus is cleaved by furin protease, the 40-kDa C-terminus (mesothelin) remains on the cell membrane, and the soluble 31-kDa N-terminal fragment MPF (megakaryotic potentiating factor) is released.

[0003] Mesothelin is overexpressed in the vast majority of primary pancreatic adenomas, while rare and weak expression is seen in benign pancreatic tissues. Epithelial malignant pleural mesothelioma (MPM) universally expresses mesothelin, while sarcomatoid MPM does not. Most serous epithelial ovarian tumors and related primary peritoneal tumors express mesothelin. In ovarian cancer, mesothelin is a target of the innate immune response and has been proposed as a target for cancer immunotherapy. In patients with pancreatic cancer, the presence of mesothelin-specific CTLs is associated with overall survival. Soluble antibody fragments of anti-mesothelin antibodies conjugated with immunotoxins have been used to treat cancer patients with mesothelin-positive tumors. This method has demonstrated sufficient safety and some clinical activity in pancreatic cancer. In ovarian cancer, according to the RECIST criteria, this treatment strategy produced a minor response and stable disease in a second patient who had completely resolved their ascites.

[0004] In recent years, although conventional tumor treatment methods such as radiotherapy, chemotherapy, and surgical treatment can achieve certain therapeutic effects, problems such as tumor metastasis, recurrence, and low patient survival rates have not been properly solved. With the development of tumor immunology theory and technology, the role of immune cells in tumor treatment has been increasingly emphasized. Among them, chimeric antigen receptor T cell (CAR-T) technology is a rapidly developing cell therapy technology in recent years.

[0005] At present, the CAR structure designs at home and abroad mainly include the traditional second-generation and third-generation CARs. The second-generation CAR is scFv-41BB-CD3ζ or scFv-CD28-CD3ζ, and the third-generation CAR is scFv-41BB-CD28-CD3ζ or scFv-CD28-41BB-CD3ζ. Currently, the most widely used in clinical applications is the traditional second-generation CAR, and it has significant efficacy in hematological tumors.

[0006] CN107580628A discloses targeted cytotoxic cells with chimeric receptors for adoptive immunotherapy, in which DAP12 / KIRS2 is used as the intracellular signaling domain of the CAR structure. However, due to the lack of co-stimulatory signal molecules, it has the technical problem of poor sustainability. When co-cultured with tumor cells in experiments, the IL-2 secretion of DAP12 / KIRS2 CAR-T is very low, and it leads to ineffective treatment in clinical therapy.

[0007] Therefore, new strategies are needed to enhance clinical efficacy to maximize the potential of CAR-T cell therapy. Summary of the Invention

[0008] Aiming at the problems existing in the prior art, the present disclosure introduces the co-stimulatory signal molecule 4-1BB and a single-chain antibody against mesothelin into DAP12 / KIRS2 to form a CAR structure SS1-KIRS2 / DAP12-4-1BB against mesothelin (hereinafter referred to as SS1) antigen, and verifies the in vitro pharmacodynamics (killing, cytokine secretion, proliferation, etc.) based on this CAR structure, demonstrating that this CAR structure has an effective killing effect on solid tumor cells with positive mesothelin expression.

[0009] Therefore, on the one hand, the present disclosure provides a chimeric antigen receptor targeting mesothelin.

[0010] On the one hand, the present disclosure provides a nucleic acid encoding the chimeric antigen receptor as described above.

[0011] On the one hand, the present disclosure provides a vector containing the nucleic acid as described above.

[0012] On the one hand, the present disclosure provides a cell containing the vector as described above.

[0013] On the one hand, the present disclosure provides a pharmaceutical composition containing the chimeric antigen receptor, its nucleic acid and / or cell as described above, and a pharmaceutically acceptable carrier.

[0014] On the one hand, the present disclosure provides a method for preparing cells by introducing the nucleic acid or vector as described above into immune effector cells.

[0015] In one aspect, the present disclosure provides the use of the aforementioned chimeric antigen receptor, nucleic acid, vector, cell, and / or pharmaceutical composition in the preparation of a medicament for treating and / or preventing a disease or disorder.

[0016] In one aspect, the present disclosure provides a method for providing anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of a composition comprising the aforementioned chimeric antigen receptor, nucleic acid, vector, cell, and / or pharmaceutical composition.

[0017] In one aspect, the present disclosure provides a method for treating a mammal suffering from a disease or disorder, comprising administering to the mammal an effective amount of the aforementioned chimeric antigen receptor, nucleic acid, vector, cell, and / or pharmaceutical composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Shows the mesothelin-DAP12 / KIRS2 CAR construct design.

[0019] Figure 2 Shows the expansion and volume changes of two CAR-T cells.

[0020] Figure 3 Shows the positive rates of two CAR-T cells and NTD cells.

[0021] Figure 4 Shows the detection results of the differentiation subtypes of two CAR-T cells and NTD cells.

[0022] Figure 5 Shows the target cell lysis rates of two CAR-T cells and NTD cells.

[0023] Figure 6 Shows the detection results of IL-2 and IFN-γ of two CAR-T cells and NTD cells.

[0024] Figure 7 Shows the flow cytometry detection results of the proliferation of two CAR-T cells and NTD cells.

[0025] Figure 8 Shows the tumor change trends in mice in different CAR-T administration groups.

[0026] Figure 9 Shows the flow chart of the clinical treatment plan.

[0027] Figure 10 Shows the statistical analysis of the survival of solid tumors treated with SS1-CAR-T.

[0028] Figure 11 Shows the statistical analysis of the progression-free survival of solid tumors treated with SS1-CAR-T. DETAILED DESCRIPTION

[0029] I. Definitions

[0030] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures used herein are terms and conventional procedures widely used in the respective fields. At the same time, for a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0031] As used herein and unless otherwise stated, the term "about" or "approximately" means within plus or minus 10% of a given value or range. In cases where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0032] "Chimeric antigen receptor" or "CAR", as the term is used herein, refers to a chimeric polypeptide that shares structural and functional characteristics with a cellular immune function receptor or adaptor molecule from, for example, a T cell or NK cell. In embodiments, the CAR comprises an antigen-binding domain that binds a cognate antigen (such as a tumor antigen described herein). After binding the cognate antigen, the CAR can activate or inactivate the cytotoxic cell in which it is located, or modulate the anti-tumor activity of the cell or modulate the immune response of the cell.

[0033] The term "antibody" as used herein refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to a target antigen. An antibody can be a complete immunoglobulin derived from a natural source or from a recombinant source, and can be an immunoreactive portion of a complete immunoglobulin. An antibody can be polyclonal or monoclonal, multi-chain or single-chain or a complete immunoglobulin, and can be from a natural source or from a recombinant source. Antibodies are typically tetramers of immunoglobulin molecules. The antibody molecules described herein can exist in a variety of forms, in which the antigen-binding portion of the antibody is expressed as part of a continuous polypeptide chain, including, for example, single-domain antibody fragments (sdAb), single-chain antibodies (scFv), and humanized or human antibodies, as described herein, for example.

[0034] With respect to an antibody chain polypeptide sequence, the phrase "substantially identical" can be understood to mean an antibody chain that exhibits at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polypeptide sequence. With respect to a nucleic acid sequence, the term can be understood to mean a nucleotide sequence that exhibits at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to a reference nucleic acid sequence.

[0035] The term "sequence 'identity'" or "identity" has its recognized meaning in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using publicly available techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule (see, e.g., Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Although there are many methods for measuring identity between two polynucleotides or polypeptides, the term "identity" is well known to those of skill in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0036] A "substitution" variant is a variant in which at least one amino acid residue in the native sequence has been removed and a different amino acid inserted in its place. The substitution can be single, where only one amino acid in the molecule is replaced; or it can be multiple, where two or more amino acids in the same molecule are replaced. Multiple substitutions can be at contiguous sites. Also, one amino acid can be replaced by multiple residues, where such variants include both substitutions and insertions. An "insertion" variant is a variant in which one or more amino acids are inserted adjacent to an amino acid at a particular position in a native sequence. Adjacent amino acid means adjacent to the α-carboxyl or α-amino functional group of the amino acid. A "deletion" variant is a variant in which one or more amino acids have been removed from the native amino acid sequence. Typically, deletion variants have one or two amino acids deleted in a particular region of their molecule.

[0037] As used in reference to the variable domains of antibodies, the term "variable" refers to certain portions of the molecule where there is extensive sequence variation between antibodies, and which are used for the specific recognition and binding of a particular antibody to its specific target. However, the variability is not evenly distributed throughout the entire variable domain of an antibody. The variability is concentrated in three segments called complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) or hypervariable regions, which are located within the variable domains of both the light and heavy chains. The more conserved portions within the variable domain are called framework (FR) regions or framework sequences. Each variable domain of a native heavy chain and light chain includes four FR regions, which predominantly adopt a β-sheet conformation and are connected by three CDRs, which form loops that connect the β-sheet structures and in some cases form part of the β-sheet structure. The CDRs of each chain are typically connected in proximity by FR regions and, with the CDRs from other chains, contribute to the formation of the antibody target binding site (epitope or determinant) (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD (1987)). As used herein, the numbering of immunoglobulin amino acid residues is according to the Kabat et al. immunoglobulin amino acid residue numbering system, unless otherwise specified. A CDR can have the ability to specifically bind an associated epitope.

[0038] The term "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full-length but that at least includes the portion of the variable region of the antibody that binds an antigen (e.g., one or more CDRs and / or one or more antibody binding sites) and thus retains the binding specificity and at least a portion of the specific binding ability of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that includes an antigen-binding portion that binds the same antigen as the antibody from which the antibody fragment is derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of a full-length antibody, as well as synthetically produced derivatives, such as recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2, single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragments may include multiple chains joined together, e.g., by disulfide bonds and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by replacing the corresponding region), acquires the ability to bind immunologically specifically (i.e., exhibits a Ka of at least or at least about 10 7 -10 8 M-1) to an antigen. A "functional fragment" is a fragment or analogue that can prevent or substantially reduce the ability of the receptor to bind a ligand or initiate signal transduction. As used herein, a functional fragment generally has the same meaning as "antibody fragment" and, with respect to an antibody, can refer to a fragment that can prevent or substantially reduce the ability of the receptor to bind a ligand or initiate signal transduction, e.g., Fv, Fab, F(ab') 2 , etc. An "Fv" fragment is a dimer (V H -V L dimer) formed by non-covalent association of the variable domain of a heavy chain and the variable domain of a light chain. In this configuration, the three CDRs of each variable domain interact to define the target-binding site on the surface of the V H -V L dimer, as in the case of a complete antibody. The six CDRs together confer the target-binding specificity of the complete antibody. However, even a single variable domain (or half of an Fv that includes only 3 target-specific CDRs) can still have the ability to recognize and bind a target.

[0039] The term "monoclonal antibody" refers to a population of identical antibodies, meaning that each individual antibody molecule in the monoclonal antibody population is identical to the other antibody molecules. This property is contrary to that of a polyclonal population of antibodies, which contains antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by many well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912 - 917; and Nelson et al., J Clin Pathol (2000), 53, 111 - 117). For example, monoclonal antibodies can be prepared by immortalizing B cells, such as by fusing them with myeloma cells to produce a hybridoma cell line or by infecting B cells with a virus such as EBV. Recombinant techniques can also be used to prepare antibodies from a cloned population of host cells in vitro by transforming the host cells with a plasmid carrying an artificial sequence encoding the antibody.

[0040] The term full-length antibody is an antibody having two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region, such as an antibody naturally produced by an antibody-secreting B cell and a synthetically produced antibody having the same domains.

[0041] The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a murine antibody and the constant region sequence is derived from a human antibody.

[0042] A "humanized" antibody is a non-human (e.g., murine) antibody form that is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of an antibody), containing minimal sequences derived from a non-human immunoglobulin. Preferably, a humanized antibody is a human immunoglobulin (recipient antibody) in which the residues of the complementarity-determining regions (CDRs) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) such as a mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

[0043] In addition, in humanization, amino acid residues within the CDR1, CDR2, and / or CDR3 regions of VH and / or VL may also be mutated, thereby improving one or more binding properties (e.g., affinity) of the antibody. Mutations can be introduced, for example, by PCR-mediated mutagenesis, and the effects of such mutations on antibody binding or other functional properties can be evaluated using the in vitro or in vivo assays described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. Additionally, mutations within the CDRs generally do not exceed one or two. Accordingly, the humanized antibodies described herein also encompass antibodies that contain one or two amino acid mutations within the CDRs.

[0044] The term "CDR" refers to the complementarity-determining region, and it is known that each heavy and light chain of an antibody molecule has three CDRs. CDRs are also referred to as hypervariable regions and are present in the variable regions of each heavy and light chain of an antibody, having sites of very high variability in the primary structure of the CDRs. In this specification, the CDRs of the heavy chain are represented by CDR1, CDR2, CDR3 from the amino-terminal of the amino-terminal sequence of the heavy chain, and the CDRs of the light chain are represented by CDR1, CDR2, CDR3 from the amino-terminal of the amino-terminal sequence of the light chain. These sites are adjacent to each other in the tertiary structure and determine the specificity of the antigen to which the antibody binds.

[0045] The term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitope determinants generally comprise the chemically active surface moieties of a molecule, such as amino acids or sugar side chains, and generally have specific three-dimensional structural features as well as specific charge characteristics.

[0046] The terms "specifically binds" or "immunologically specifically binds" with respect to an antibody or an antigen-binding fragment thereof are used interchangeably herein and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with a cognate antigen through non-covalent interactions between the antibody-binding site of the antibody and the antigen. The antigen can be an isolated antigen or present on a tumor cell. Typically, an antibody that immunologically specifically binds (or specifically binds) an antigen has an affinity constant Ka of about or 1×10 7 M -1 or 1x10 8 M -1 or greater (or 1x10 -7 M or 1×10 -8bind to the antigen with an affinity constant (Kd) of M or lower. The affinity constant can be determined by standard kinetic methods of antibody reactions, such as immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art (see, for example, Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); see also U.S. Patent No. 7,229,619, which describes exemplary SPR and ITC methods for calculating the binding affinity of antibodies). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (see, BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare LifeSciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335).

[0047] The terms "polynucleotide" and "nucleic acid molecule" refer to oligomers or polymers containing at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which are typically linked together by phosphodiester bonds. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded, and can be cDNA.

[0048] As used herein, an isolated nucleic acid molecule is a nucleic acid molecule that has been separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or medium when prepared by recombinant techniques, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding the provided antibodies or antigen-binding fragments.

[0049] As used herein, "operably linked" with respect to a nucleic acid sequence, region, element, or domain means that the nucleic acid regions are functionally related to each other. For example, a promoter can be operably linked to a nucleic acid encoding a polypeptide such that the promoter regulates or mediates transcription of the nucleic acid.

[0050] "Conservative sequence modifications" of the sequences described in the sequence listing herein are also provided, i.e., nucleotide and amino acid sequence modifications that do not abolish the binding of an antibody encoded by or containing an amino acid sequence to an antigen. These conservative sequence modifications include conservative nucleotide and amino acid substitutions, as well as nucleotide and amino acid additions and deletions. For example, modifications can be introduced into the sequence listing described herein by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative sequence modifications include conservative amino acid substitutions, where an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are well defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids having β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Methods for identifying nucleotide and amino acid conservative substitutions that do not abolish antigen binding are well known in the art (e.g., see Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0051] The term "expression" refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. The expression level of a polypeptide can be evaluated using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from a host cell. Such methods can include, but are not limited to, quantifying the polypeptide in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.

[0052] The term "host cell" is a cell that is used to receive, maintain, replicate, and amplify a vector. A host cell can also be used to express a polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. A host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.

[0053] The term "vector" refers to a replicable nucleic acid into which one or more heterologous proteins can be expressed when the vector is transformed into a suitable host cell. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can typically be introduced by restriction enzyme digestion and ligation. Vectors also include those that contain nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for amplifying the nucleic acids or for expressing / displaying the polypeptides encoded by the nucleic acids. Vectors generally remain free, but can be designed to integrate a gene or part thereof into the chromosomes of the genome. Vectors of artificial chromosomes are also contemplated, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vectors are well known to those skilled in the art.

[0054] As used herein, the term "vector" also includes "viral vector" or "vector of a virus". A vector of a virus is an engineered virus that is operably linked to a foreign gene to transfer (as a vehicle or shuttle) the foreign gene into a cell.

[0055] The term "expression vector" includes a vector capable of expressing DNA that is operably linked to regulatory sequences capable of affecting the expression of such DNA fragments, such as a promoter region. Such additional fragments can include promoter and terminator sequences and optionally can include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors generally are derived from plasmid or viral DNA or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, that, when introduced into a suitable host cell, results in the expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that can replicate in eukaryotic cells and / or prokaryotic cells and expression vectors that remain free or integrate into the genome of the host cell.

[0056] The term "stimulation" refers to the initial response that mediates a signal transduction event by binding of a stimulatory molecule (e.g., the TCR / CD3 complex) to its cognate ligand, such as, but not limited to, signal transduction via the TCR / CD3 complex. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-β, and / or reorganization of the cytoskeletal structure, etc.

[0057] The term "stimulatory molecule" refers to a molecule expressed by a T cell that provides a primary cytoplasmic signaling sequence that regulates, in a stimulatory manner, the primary activation of the TCR complex for at least some aspects of the T cell signaling pathway. In one aspect, the primary signal is initiated, for example, by the binding of the TCR / CD3 complex to a peptide-loaded MHC molecule, and this results in mediating T cell responses, including, but not limited to, proliferation, activation, differentiation, etc. The primary cytoplasmic signaling sequence (also referred to as "primary signaling domain") that functions in a stimulatory manner can contain a signaling motif called an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAM-containing primary cytoplasmic signaling sequences particularly useful in the present invention include, but are not limited to, those derived from: TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS"), FcεRI, CD66d, DAP10, and DAP12. In the specific CARs of the present invention, the intracellular signaling domain in any one or more of the CARs of the present invention includes an intracellular signaling sequence.

[0058] The term "antigen-presenting cell" or "APC" refers to an immune system cell that presents on its surface a foreign antigen complexed with a major histocompatibility complex (MHC), such as an accessory cell (e.g., B-cells, dendritic cells, etc.). T-cells can recognize these complexes using their T-cell receptor (TCR). The APC processes the antigen and presents it to the T-cells.

[0059] The term "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain can generate signals that promote the immune effector functions of a CAR-containing cell (e.g., a CAR-T cell or an NK cell expressing a CAR). Examples of immune effector functions (e.g., in a CAR-T cell or an NK cell expressing a CAR) include cytolytic activity and accessory activity, including the secretion of cytokines. In an embodiment, the intracellular signal domain transduces effector function signals and directs the cell to perform specialized functions. While the entire intracellular signaling domain can be used, in many cases, it is not necessary to use the entire chain. In terms of using a truncated portion of the intracellular signaling domain, such truncated portions can be used in place of the full chain as long as they transduce effector function signals. The term intracellular signaling domain is thus intended to include any truncated portion of the intracellular signaling domain that is sufficient to transduce effector function signals.

[0060] In one embodiment, the intracellular signaling domain can include a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules responsible for primary stimulation or antigen-dependent stimulation. In one embodiment, the intracellular signaling domain can include a co-stimulatory intracellular domain. Exemplary co-stimulatory intracellular signaling domains include those derived from molecules responsible for co-stimulatory signals or antigen-independent stimulation. For example, in the case of immune effector cells expressing a CAR, such as CAR-T cells or NK cells expressing a CAR, the primary intracellular signaling domain can comprise the cytoplasmic sequence of the T cell receptor, and the co-stimulatory intracellular domain can comprise the cytoplasmic sequence from a co-receptor or co-stimulatory molecule.

[0061] The primary intracellular signaling domain can include a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of primary cytoplasmic signaling sequences containing an ITAM include, but are not limited to, those derived from: CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (“ICOS”), FcεRI, CD66d, DAP10, and DAP12.

[0062] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand to mediate a costimulatory response of the T cell, such as but not limited to proliferation. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand required for an effective immune response. Costimulatory molecules include but are not limited to MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptors, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a and a ligand that specifically binds to CD83.

[0063] A costimulatory intracellular signaling domain refers to the intracellular portion of a costimulatory molecule. The intracellular signaling domain can include the entire intracellular portion of the molecule or the entire native intracellular signaling domain, or a functional fragment thereof.

[0064] The term "4-1BB" refers to a member of the TNFR superfamily having the amino acid sequence provided in GenBank Acc. No. AAA62478.2, or equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc.; and "4-1BB co-stimulatory domain" is defined as amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc. In one aspect, the "4-1BB co-stimulatory domain" is the sequence provided in SEQ ID NO:5 or equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc.

[0065] The term "immune effector cell" refers to a cell that participates in an immune response, e.g., a cell that promotes an immune effector response. Examples of immune effector cells include T cells, e.g., α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

[0066] The term "immune effector function or immune effector response" refers to the function or response of immune effector cells, e.g., a function or response that enhances or promotes the immune attack on a target cell. For example, immune effector function or response refers to the property of T cells or NK cells that promotes the killing of a target cell or inhibits growth or proliferation. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector function or response.

[0067] In some embodiments, a cell population (e.g., a harvested cell population) comprises, e.g., T cells or a population of T cells at different stages of differentiation. The stages of T cell differentiation from least to most differentiated include naive T cells, stem central memory T cells, central memory T cells, effector memory T cells, and terminal effector T cells. After antigen exposure, naive T cells proliferate and differentiate into memory T cells, e.g., stem central memory T cells and central memory T cells, and then differentiate into effector memory T cells. Memory T cells further differentiate into terminal effector T cells upon receipt of appropriate T cell receptor, co-stimulation, and inflammatory signals.

[0068] Naive T cells (TN) are characterized by the expression pattern of the following cell surface markers: CCR7+, CD62L+, CD45RO-, CD95-. Stem cell central memory T cells (TSCM) are characterized by the expression pattern of the following cell surface markers: CCR7+, CD62L+, CD45RO-, CD95+. Central memory T cells (TCM) are characterized by the following expression pattern of cell surface markers: CCR7+, CD62L+, CD45RO+, CD95+. Effector memory T cells (TEM) are characterized by the expression pattern of the following cell surface markers: CCR7-, CD62L-, CD45RO+, CD95+. Terminally differentiated effector T cells (TEff) are characterized by the following expression pattern of cell surface markers: CCR7-, CD62L-, CD45RO-, CD95+.

[0069] Fresh T cells from healthy donors are usually classified into four subsets based on CD45RA and CD62L expression: 1) naive T cells (CD45RA+CD62L+, called Tn), 2) central memory T cells (CD45RA-CD62L+, termed Tcm), 3) effector memory T cells (CD45RA-CD62L-, called Tem) and 4) CD45RA+ effector T cells (CD45RA+CD62L-, abbreviated as Temra). The expression of CCR7, CD27, CD28 and CD95 is then further evaluated in each subset. CD95 expression is significantly upregulated after lentiviral transduction. The latter three T cell subsets are positive for CD95, while only a small fraction of Tn express CD95 (3.6 ± 1.4% in CD4+ and 3.7 ± 1.3% in CD8+ T cells). This small population also co-expresses CD27, CD28 and CCR7 and is considered to be stem cell memory (Tscm).

[0070] The term "effector function" refers to the specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including the secretion of cytokines.

[0071] The term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with the administration of a drug. Suitable pharmaceutically acceptable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, which is a standard reference in the art and is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous carriers, such as fixed oils, can also be used. The use of such media and reagents with pharmaceutically active substances is well known in the art. Except for any conventional media or reagents that are incompatible with the chimeric antigen receptor, their use in the compositions is contemplated.

[0072] The term "treating" an individual having a disease or medical condition means that the symptoms of the individual are partially or completely alleviated, or remain unchanged after treatment. Thus, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing a potential disease and / or preventing the worsening of symptoms or the development of a disease. Treatment also includes any pharmaceutical use of any chimeric antigen receptor and any composition provided herein.

[0073] The term "efficacy" means the effect resulting from the treatment of an individual that modifies, usually improves or ameliorates the symptoms of a disease or medical condition, or cures the disease or medical condition.

[0074] The term "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, is at least sufficient to produce an efficacy. Thus, it is the amount necessary to prevent, cure, improve, arrest, or partially arrest the symptoms of a disease or disorder.

[0075] The term "prophylactically effective amount" or "prophylactically effective dose" refers to the amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the desired prophylactic effect, e.g., preventing or delaying the onset or recurrence of a disease or symptoms, reducing the likelihood of the occurrence or recurrence of a disease or symptoms. A full prophylactically effective dose need not occur by administering a single dose and may occur only after administering a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.

[0076] The term "patient" refers to a mammal, such as a human.

[0077] II. SPECIFIC EMBODIMENTS

[0078] In one aspect, the present disclosure provides a chimeric antigen receptor targeting mesothelin (MSLN), which comprises a first fusion peptide and a second fusion peptide, wherein:

[0079] The first fusion peptide comprises a mesothelin antigen-binding domain and a transmembrane domain;

[0080] The second fusion peptide comprises a transmembrane domain, a cytoplasmic domain, and a co-stimulatory domain.

[0081] In some embodiments, the transmembrane domain of the second fusion peptide interacts with the transmembrane domain of the first fusion peptide through charge interactions, or the second fusion peptide interacts with a signaling molecule through a phosphorylated ITAM sequence within the cytoplasmic domain.

[0082] For the chimeric antigen receptor according to the previous aspect, the transmembrane domain of the second fusion peptide interacts with the transmembrane domain of the first fusion peptide through charge interactions, or the second fusion peptide interacts with a signaling molecule through a phosphorylated ITAM sequence within the cytoplasmic domain.

[0083] In some embodiments, the transmembrane domain of the first fusion peptide is a KIR transmembrane domain; preferably, the KIR transmembrane domain is selected from KIR2DS2, KIR2DL3, KIR2DL1, KIR2DL2, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DS1, KIR3DL2, KIR3DL3, KIR2DP1, and KIR3DP1; more preferably, the KIR transmembrane domain is KIRS2 or KIR2DS2; preferably, the KIRS2 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO:2, preferably having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably having 98% or 99% or more identity; more preferably, the amino acid sequence of the KIRS2 is as shown in SEQ ID NO:2; preferably, the KIR2DS2 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO:3, preferably having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably having 98% or 99% or more identity; more preferably, the amino acid sequence of the KIR2DS2 is as shown in SEQ ID NO:3.

[0084] In some embodiments, the first fusion peptide comprises a signal peptide, and the signal peptide is a CD8α signal peptide; preferably, the CD8α signal peptide comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, preferably having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably having 98% or 99% or more identity; more preferably, the amino acid sequence of the CD8α signal peptide is as shown in SEQ ID NO: 1.

[0085] In some embodiments, the transmembrane domain of the second fusion peptide is a DAP12 transmembrane domain; preferably, the transmembrane domain of DAP12 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 5, preferably having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably having 98% or 99% or more identity; more preferably, the amino acid sequence of the transmembrane domain of DAP12 is as shown in SEQ ID NO: 5.

[0086] In some embodiments, the cytoplasmic domain is a DAP12 cytoplasmic domain or a KIR cytoplasmic domain; preferably, the cytoplasmic domain is the cytoplasmic domain of DAP12; preferably, the cytoplasmic domain of DAP12 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 6, preferably having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably having 98% or 99% or more identity; more preferably, the amino acid sequence of the cytoplasmic domain of DAP12 is as shown in SEQ ID NO: 6.

[0087] In some embodiments, the co-stimulatory domain is 4-1BB; preferably, 4-1BB comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 9, preferably having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably having 98% or 99% or more identity; more preferably, the amino acid sequence of 4-1BB is as shown in SEQ ID NO: 9.

[0088] In some embodiments, the second fusion peptide comprises a DAP12 transmembrane domain, a DAP12 cytoplasmic domain, and a co-stimulatory domain 4-1BB, or comprises a truncated DAP12 transmembrane domain, a DAP12 cytoplasmic domain, and a co-stimulatory domain 4-1BB; preferably, the DAP12 transmembrane domain and the DAP12 cytoplasmic domain comprise an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO:7, preferably having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably having 98% or 99% or more identity; more preferably, the amino acid sequences of the DAP12 transmembrane domain and the DAP12 cytoplasmic domain are as shown in SEQ ID NO:7; preferably, the truncated DAP12 transmembrane domain and the DAP12 cytoplasmic domain comprise an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO:8, preferably having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably having 98% or 99% or more identity; more preferably, the amino acid sequences of the truncated DAP12 transmembrane domain and the DAP12 cytoplasmic domain are as shown in SEQ ID NO:8.

[0089] In some embodiments, the second fusion peptide comprises a signal peptide, and the signal peptide is a DAP12 signal peptide or a CD8α signal peptide; preferably, the DAP12 signal peptide comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO:4, preferably having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably having 98% or 99% or more identity; more preferably, the amino acid sequence of the DAP12 signal peptide is as shown in SEQ ID NO:4; preferably, the CD8α signal peptide comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO:1, preferably having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably having 98% or 99% or more identity; more preferably, the amino acid sequence of the CD8α signal peptide is as shown in SEQ ID NO:1.

[0090] In some embodiments, the mesothelin antigen-binding domain comprises a mesothelin antibody or an antigen-binding fragment thereof; preferably, the mesothelin antigen-binding domain comprises the heavy-chain variable region and the light-chain variable region of a mesothelin antibody; preferably, the mesothelin antigen-binding domain comprises Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabody, Fd, and Fd' fragments.

[0091] In some embodiments, the antibody or antigen-binding fragment thereof comprises SS1 scFv1 shown in SEQ ID NO:11 and SS2 scFv2 shown in SEQ ID NO:12.

[0092] In some embodiments, the first fusion peptide comprises SS1 scFv and KIRS2, or comprises SS1 scFv and KIR2DS2.

[0093] In some embodiments, the chimeric antigen receptor is an SS1 scFv1-KIRS2 / Dap12-BB chimeric antigen receptor, and the SS1 scFv1-KIRS2 / Dap12-BB chimeric antigen receptor is formed by cleavage of a fusion protein by a T2A peptide. The fusion protein comprises a DAP12 signal peptide, DAP12 (including a transmembrane domain and a cytoplasmic domain), 4-1BB, a T2A cleavage site, a CD8α signal peptide, SS1 scFv1, and KIRS2; preferably, the fusion protein comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO:13, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity; more preferably, the amino acid sequence of the fusion protein is as shown in SEQ ID NO:13.

[0094] In some embodiments, the chimeric antigen receptor is the SS1 scFv2-KIRS2 / Dap12-BB chimeric antigen receptor, and the SS1 scFv2-KIRS2 / Dap12-BB chimeric antigen receptor is formed by cleavage of a fusion protein by the T2A peptide. The fusion protein comprises a CD8α signal peptide, truncated DAP12 (tDap-12, including a truncated transmembrane domain and cytoplasmic domain), 4-1BB, a T2A cleavage site, a CD8α signal peptide, SS1 scFv2, and KIRS2; preferably, the fusion protein comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO:15, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity; more preferably, the amino acid sequence of the fusion protein is as shown in SEQ ID NO:15.

[0095] In some embodiments, the T2A cleavage site comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO:10, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity; more preferably, the amino acid sequence of the T2A cleavage site is as shown in SEQ ID NO:10.

[0096] In one aspect, the present disclosure provides a nucleic acid encoding the foregoing chimeric antigen receptor; preferably, the nucleic acid comprises a nucleotide sequence having 80% or more identity with the nucleotide sequence shown in SEQ ID NO:14 or SEQ ID NO:16, preferably a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence having 98% or 99% or more identity; preferably, the nucleic acid is selected from the nucleic acids shown in SEQ ID NO:14 or SEQ ID NO:16; more preferably, the encoding nucleic acid is the nucleic acid shown in SEQ ID NO:14.

[0097] In one aspect, the present disclosure provides a vector comprising the foregoing nucleic acid.

[0098] In one aspect, the present disclosure provides a cell comprising the foregoing nucleic acid or vector.

[0099] In one aspect, the present disclosure provides a composition comprising the foregoing chimeric antigen receptor, nucleic acid, vector, and / or cell and a pharmaceutically acceptable carrier.

[0100] In one aspect, the present disclosure provides a method for preparing cells, the method comprising introducing the foregoing nucleic acid and vector into immune effector cells.

[0101] In one aspect, the present disclosure provides the use of the foregoing chimeric antigen receptor, nucleic acid, vector, cell, and / or composition in the preparation of a medicament for treating and / or preventing a disease or disorder.

[0102] In some embodiments, the chimeric antigen receptor can be used as a therapeutic agent. Such agents will generally be used to treat, alleviate, and / or prevent diseases or pathologies in a subject associated with abnormal mesothelin (MSLN) expression, activity, and / or signaling. A treatment regimen can be implemented by identifying a subject, such as a human patient suffering from (or at risk of or developing) a disease or disorder associated with abnormal MSLN expression, activity, and / or signaling, such as cancer or other neoplastic disorders, using standard methods. An antibody preparation, preferably one with high specificity and high affinity for its target antigen, is administered to the subject and will generally produce an effect due to its binding to the target. The administered chimeric antigen receptor can eliminate, inhibit, or interfere with the expression, activity, and / or signaling function of the target (such as MSLN). The administered chimeric antigen receptor can eliminate, inhibit, or interfere with the binding of the target (such as MSLN) to its endogenous ligand to which it naturally binds. For example, the chimeric antigen receptor binds to the target and modulates, blocks, inhibits, reduces, antagonizes, neutralizes, or otherwise interferes with MSLN expression, activity, and / or signaling. In some embodiments, a chimeric antigen receptor having heavy and light chain CDRs can be administered to a subject for treating a disease or disorder associated with abnormal MSLN expression. In one embodiment, the disease or disorder associated with abnormal MSLN expression can be cancer.

[0103] As a non-limiting example, diseases or disorders associated with abnormal MSLN expression, activity, and / or signaling include solid tumors. Preferably, the mesothelin expression-related diseases are selected from mesothelioma, epithelioid malignant pleural mesothelioma, ovarian cancer, lung cancer, esophageal cancer, pancreatic cancer, gastric cancer, biliary tract cancer, endometrial cancer, thymic cancer, colon cancer, breast cancer.

[0104] Symptoms associated with cancer and other neoplastic disorders include, for example, inflammation, fever, general malaise, fever, pain, frequent local inflammation, loss of appetite, weight loss, edema, headache, fatigue, rash, anemia, muscle weakness, muscle fatigue, and abdominal symptoms such as abdominal pain, dysentery, or constipation.

[0105] In one aspect, the present disclosure provides a method for providing anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of a composition comprising the foregoing chimeric antigen receptor, nucleic acid, vector, and / or cell.

[0106] In one aspect, the present disclosure provides a method of treating a mammal suffering from a disease or disorder, comprising administering to the mammal an effective amount of the aforementioned chimeric antigen receptor, nucleic acid, vector, and / or cell.

[0107] The pharmaceutical compositions of the embodiments are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: a sterile diluent for injection, such as water, saline solution, fixed oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetate, citrate, or phosphate, and agents for regulating osmotic pressure, such as sodium chloride or dextrose. The pH may be adjusted with an acid or a base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0108] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (which are water-soluble here) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable pharmaceutically acceptable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy injection is possible. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. For example, by using coatings such as lecithin, maintaining the desired particle size in the case of dispersions, and using surfactants, suitable fluidity can be maintained. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include in the composition isotonic agents, such as sugars, polyols (such as mannitol, sorbitol), sodium chloride. Prolonged absorption of injectable compositions can be achieved by including in the composition agents that delay absorption, such as aluminum monostearate and gelatin.

[0109] Sterile injectable solutions can be prepared by incorporating the chimeric antigen receptor in the required amount into a suitable solvent having one or a combination (as required) of the ingredients listed above, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the chimeric antigen receptor into a sterile vehicle containing a basic dispersion medium and the required other ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the method of preparation is vacuum drying and freeze drying of the powder, which contains the active ingredient and any additional desired ingredients from a sterile filtered solution of these ingredients described above.

[0110] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser or nebulizer containing a suitable propellant such as a gas like carbon dioxide.

[0111] Systemic administration can also be effected by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants suitable for the permeation barrier are used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, one or more of the chimeric antigen receptors can be formulated as an ointment, plaster, gel, or cream as is generally known in the art.

[0112] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter or other glycerides) or retention enemas for rectal delivery.

[0113] In one embodiment, the chimeric antigen receptor can be prepared with a carrier that prevents its rapid elimination from the body, such as a sustained-release / controlled-release formulation, including implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The methods for preparing such formulations will be apparent to those skilled in the art.

[0114] It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, a dosage unit form refers to physically discrete units suitable as unit doses for the subject to be treated; each unit contains a predetermined quantity of one or more of the chimeric antigen receptors calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification of the dosage unit forms of this embodiment is dictated by and directly depends on: the unique characteristics of the chimeric antigen receptor and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such chimeric antigen receptors for the treatment of individuals.

[0115] The pharmaceutical composition can be placed in a container, package, or dispenser together with instructions for administration.

[0116] The formulations described herein may also contain more than one of said chimeric antigen receptors, preferably those having complementary activities and no negative impact on each other, depending on the particular condition to be treated. Alternatively or in addition, the composition may contain, for example, reagents that enhance its function, such as cytotoxin reagents, cytokines, chemotherapeutic agents, or growth inhibitors. Such molecules are present in appropriate combination in an amount effective for the intended purpose. For example, they may be present in combination in a kit or in use.

[0117] In one embodiment, the production of the cytokine IL-6 is reduced or inhibited. In another embodiment, the production of the cytokine IL-10 is reduced or inhibited.

[0118] In one embodiment, cytokine release syndrome is graded. In another embodiment, grade 1 describes a cytokine release syndrome in which the symptoms are not life-threatening and only require symptomatic treatment, such as fever, nausea, fatigue, headache, myalgia, malaise. In another embodiment, the symptoms of grade 2 require and respond to moderate intervention, such as oxygen supply, fluids, or vasopressors for hypotension. In another embodiment, the symptoms of grade 3 require and respond to aggressive intervention. In another embodiment, the symptoms of grade 4 are life-threatening symptoms that require a ventilator and the patient exhibits organ toxicity.

[0119] In one embodiment, one or more of said chimeric antigen receptors may be administered in combination therapy, i.e., in combination with other reagents such as therapeutic agents (which may be used to treat pathological conditions or disorders such as various forms of cancer, autoimmune disorders, and inflammatory diseases). The term "in combination" as used herein means that the reagents are administered substantially synchronously, simultaneously, or sequentially. If administered sequentially, the first of the two compounds is still preferably detected at an effective concentration at the treatment site when the second compound is started. In one case, "in combination" may also mean that the chimeric antigen receptors of the present disclosure and other therapeutic agents are included simultaneously in a kit.

[0120] For example, combination therapy may comprise co-formulating and / or co-administering one or more of the chimeric antigen receptors described herein with one or more additional therapeutic agents (such as one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and / or cytotoxins or cell growth inhibitors, as described in more detail below). Such combination therapy may advantageously utilize lower doses of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with various single therapies.

[0121] The present disclosure adds a costimulatory signal molecule element by linking DAP12 in series with 4-1BB, links different single-chain variable fragment (scFV) antibodies against mesothelin, designs a chimeric antigen receptor for solid tumors positive for mesothelin expression, and forms a chimeric antigen receptor structure in which a fusion peptide containing an antigen-binding domain and a transmembrane domain and another fusion peptide containing a transmembrane domain, a cytoplasmic domain, and a costimulatory domain are connected in parallel. The chimeric antigen receptor has good killing activity against mesothelin-positive solid tumor cells and can promote the secretion of cytokines (including IL-2 and INFγ). The CAR structure of the SS1-KIRS2 / DAP12-4-1BB molecular combination of the present disclosure is expected to significantly improve the clinical treatment effect of CAR-T cells for MSLN-positive solid tumors in response to the low effectiveness problem of CAR-T clinical treatment of solid tumors.

[0122] For the purposes of clarity and concise description, features are described herein as part of the same or separate embodiments. However, it will be understood that the scope of the present invention may include embodiments having combinations of all or some of the described features.

[0123] Examples

[0124] Example 1: Structural Design of Mesothelin-Targeted Chimeric Antigen Receptor

[0125] In this example, the following two mesothelin-targeted chimeric antigen receptors (MSLN CAR) were designed ( Figure 1 ):

[0126] (1) MSLN SS1 scFv1-KIRS2 / Dap12-BB chimeric antigen receptor

[0127] As shown in Figure 1 (A), the MSLN SS1 scFv1-KIRS2 / Dap12-BB chimeric antigen receptor comprises a first fusion peptide MSLN SS1 scFv1-KIRS2 and a second fusion peptide Dap12-BB, wherein:

[0128] The first fusion peptide MSLN SS1 scFv1-KIRS2 contains an antigen-binding domain and a transmembrane domain. The antigen-binding domain is MSLN SS1 scFv1, and the transmembrane domain is the KIRS2 transmembrane domain;

[0129] The second fusion peptide Dap12-BB contains a DAP12 transmembrane domain, a DAP12 cytoplasmic domain, and a costimulatory domain 4-1BB.

[0130] The MSLN SS1 scFv1-KIRS2 / Dap12-BB chimeric antigen receptor is formed by cleavage of the pKT032 fusion protein by the T2A peptide. The pKT032 fusion protein contains the DAP12 signal peptide, DAP12 (including the transmembrane domain and cytoplasmic domain), 4-1BB, the T2A cleavage site, the CD8α signal peptide, MSLN SS1 scFv1, and KIRS2. The amino acid sequence of the pKT032 fusion protein is shown in SEQ ID NO:13, and its encoding nucleic acid is shown in SEQ ID NO:14.

[0131] pKT032: DAP12 signal peptide + DAP12 + 4-1BB + T2A + CD8α signal peptide + SS1scFv1 + KIRS2

[0132] (2) MSLN SS1 scFv2-KIRS2 / Dap12-BB chimeric antigen receptor

[0133] As Figure 1 (B) shows, the MSLN SS1 scFv2-KIRS2 / Dap12-BB chimeric antigen receptor comprises a first fusion peptide MSLN SS1 scFv2-KIRS2 and a second fusion peptide Dap12-BB, wherein:

[0134] The first fusion peptide MSLN SS1 scFv2-KIRS2 contains an antigen-binding domain and a transmembrane domain. The antigen-binding domain is MSLN SS1 scFv2, and the transmembrane domain is the KIRS2 transmembrane domain;

[0135] The second fusion peptide Dap12-BB contains the DAP12 transmembrane domain, the DAP12 cytoplasmic domain, and the co-stimulatory domain 4-1BB.

[0136] The MSLN SS1 scFv2-KIRS2 / Dap12-BB chimeric antigen receptor is formed by cleavage of the pKT108 fusion protein by the T2A peptide. The pKT0108 fusion protein contains the DAP12 signal peptide, DAP12 (including the transmembrane domain and cytoplasmic domain), 4-1BB, the T2A cleavage site, the CD8α signal peptide, MSLN SS1 scFv2, and KIRS2. The amino acid sequence of the pKT108 fusion protein is shown in SEQ ID NO:15, and its encoding nucleic acid is shown in SEQ ID NO:16.

[0137] pKT108: DAP12 signal peptide + DAP12 + 41BB + T2A + CD8α signal peptide + SS1scFv2 + KIRS2

[0138] Wherein:

[0139] The amino acid sequence of the CD8α signal peptide is as follows:

[0140] MALPVTALLLPLALLLHAARP (SEQ ID NO 1)

[0141] The amino acid sequence of KIRS2 is as follows:

[0142] SKTGNPRHLHVLIGTSVVKIPFTILLFFLLHRWCSNKKNAAVMDQEPAGNRTVNSEDSDEQDHQEVSYA (SEQ ID NO 2)

[0143] The amino acid sequence of KIR2DS2 is as follows:

[0144] HEGVHRKPSLLAHPGPLVKSEETVILQCWSDVRFEHFLLHREGKYKDTLHLIGEHHDGVSKANFSIGPMMQDLAGTYRCYGSVTHSPYQLSAPSDPLDIVITGLYEKPSLSAQPGPTVLAGESVTLSCSSRSSYDMYHLSREGEAHERRFSAGPKVNGTFQADFPLGPATHGGTYRCFGSFRDSPYEWSNSSDPLLVSVTGNPSNSWPSPTEPSSKTGNPRHLHVLIGTSVVKIPFTILLFFLLHRWCSNKKNAAVMDQEPAGNRTVNSEDSDEQDHQEVSYA (SEQ ID NO 3)

[0145] The amino acid sequence of the DAP12 signal peptide is as follows:

[0146] MGGLEPCSRFLLLPLLLAVSG (SEQ ID NO 4)

[0147] The amino acid sequence of the DAP12 transmembrane domain is as follows:

[0148] GVLAGIVMGDLVLTVLIALAV (SEQ ID NO 5)

[0149] The amino acid sequence of the DAP12 cytoplasmic domain is as follows:

[0150] YFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK (SEQ ID NO 6)

[0151] The amino acid sequence of DAP12 contains the DAP12 transmembrane domain and the DAP12 cytoplasmic domain sequence, and the specific sequences are as follows:

[0152] LRPVQVQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK(SEQ ID NO 7)

[0153] The truncated amino acid sequence of DAP12 contains the truncated DAP12 transmembrane domain and the DAP12 cytoplasmic domain, and the specific sequences are as follows:

[0154] CSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK(SEQ ID NO 8)

[0155] The amino acid sequence of 4-1BB is as follows:

[0156] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO 9)

[0157] The amino acid sequence of the T2A cleavage site is as follows:

[0158] EGRGSLLTCGDVEENPG(SEQ ID NO 10)

[0159] The amino acid sequence of SS1 scFv1 is as follows:

[0160] QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGGGGSGGGGSSGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTYGAGTKLEIK(SEQ ID NO 11)

[0161] The amino acid sequence of SS1 scFv2 is as follows:

[0162] QVQLQQSGPELEKPGASVKISCHASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGGGGSGGGGSSGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSHLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCKQWSKHPLTYGAGTKLEIK(SEQ ID NO 12)

[0163] The amino acid sequence of pKT032 is as follows:

[0164] MGGLEPCSRFLLLPLLLAVSGLRPVQVQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKDIKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSRVEGGGEGRGSLLTCGDVEENPGPRMALPVTALLLPLALLLHAARPGSQVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGGGGSGGGGSSGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTYGAGTKLEIKASGGGGSGGGGSSPTEPSSKTGNPRHLHVLIGTSVVKIPFTILLFFLLHRWCSNKKNAAVMDQEPAGNRTVNSEDSDEQDHQEVSYA(SEQ ID NO 13)

[0165] The nucleotide sequence of pKT032 is as follows:

[0166]

[0167] The amino acid sequence of pKT108 is as follows:

[0168] MGGLEPCSRFLLLPLLLAVSGLRPVQVQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKDIKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSRVEGGGEGRGSLLTCGDVEENPGPRMALPVTALLLPLALLLHAARPGSQVQLQQSGPELEKPGASVKISCHASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGGGGSGGGGSSGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSHLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCKQWSKHPLTYGAGTKLEIKASGGGGSGGGGSSPTEPSSKTGNPRHLHVLIGTSVVKIPFTILLFFLLHRWCSNKKNAAVMDQEPAGNRTVNSEDSDEQDHQEVSYA (SEQ ID NO 15)

[0169] The nucleotide sequence of pKT108 is as follows:

[0170]

[0171] Example 2: Preparation of Lentivirus

[0172] (1) Passage 293T cells every other day

[0173] Seed 5×10 6 cells into each T150 cell flask. After 48 hours, the cell number should reach 20 - 25 million per flask.

[0174] (2) Seeding 293T cells in flasks

[0175] a) Taking one T150 cell culture flask as an example, gently wash the cells twice with about 15 ml of 1×PBS.

[0176] b) Add 3 ml of 0.25% trypsin - 2.21 mM EDTA

[0177] c) Wait until the cells detach, then add 12 ml of DMEM medium (purchased from corning) containing 10% (wt) FBS (purchased from Gibico) to the detached cells.

[0178] d) Collect and transfer the cells to a sterile centrifuge tube, centrifuge at 1000 rpm for 10 minutes.

[0179] e) Aspirate the supernatant, and resuspend the pellet in 10 ml of DMEM culture medium containing 10% (wt) FBS.

[0180] f) Count the cells and calculate the volume required for 12×10 6 cells according to the cell concentration.

[0181] g) Combine the cells with 25 ml of DMEM culture medium containing 10% (wt) FBS, place them in a T150 cell flask, gently shake to evenly distribute the cells on the bottom of the flask, and culture overnight in an incubator at 37°C and 5% CO 2 .

[0182] (3) Cell transfection

[0183] Observe the cells. When the cell density reaches approximately 80% - 90%, start the transfection.

[0184] a) Gently aspirate the culture medium 30 - 60 minutes before transfection.

[0185] b) Mix the plasmid DNA and calcium chloride solution. Taking one T150 flask as an example, 28 μg of pRSV.rev (purchased from Invitrogen), 28 μg of pGAG-Pol (purchased from Invitrogen), 11 μg of pVSVG (purchased from Invitrogen), and 23 μg of lentiviral expression plasmid (plasmids pKT032 and pKT108, synthesized by Sangon Biotech, Shanghai) are required. Add the lentiviral expression plasmids into 1.5 ml of calcium chloride solution respectively and mix well.

[0186] c) Add 1.5 ml of borate buffer solution into a 15 ml sterile centrifuge tube. Use a 1 ml pipette tip to mix the DNA-calcium chloride solution well and then drop it into the borate buffer solution. Mix quickly for 15 - 20 times and incubate at room temperature for 25 - 30 minutes.

[0187] d) Use a 5 ml pipette to add the DNA-calcium chloride-borate buffer mixture (purchased from Beyotime Biotechnology, Shanghai) drop by drop evenly into the T150 flask. Culture it in a 37 °C cell culture incubator containing 5% carbon dioxide and change the medium after 6 h.

[0188] e) Change the medium after 6 h. Gently shake the culture plate several times to fully suspend some calcium phosphate precipitates, aspirate the culture medium containing calcium phosphate precipitates, and add 20 ml of fresh DMEM medium containing 5% (wt) FBS, and continue the culture.

[0189] (4) Collect the virus supernatant for the first time

[0190] a) Collect the culture supernatant of the 293T cells transfected the previous day into a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, label it, and temporarily store it in a 4 °C refrigerator.

[0191] b) Add 20 ml of pre-warmed DMEM medium containing 5% (wt) FBS into the cell flask and continue to culture it overnight in a 37 °C cell culture incubator.

[0192] (5) Collect the virus supernatant for the second time (48 h / the fourth day).

[0193] (6) Filter the supernatant

[0194] Pool the supernatants collected twice and filter them through a 0.45 μm filter membrane to remove cell debris.

[0195] (7) Virus concentration

[0196] Centrifuge at 12000 - 24000 rpm overnight at 4 °C.

[0197] (8) Virus storage

[0198] After centrifugation, pour out all the supernatant, resuspend with fresh DMEM medium containing 5% (wt) FBS, aliquot the virus (denoted as lentivirus pKT032 and pKT108), and quickly store it in a -80°C refrigerator for later use.

[0199] Example 3: In vitro functional test of MSLN CAR-T cells

[0200] (1) Cell preparation

[0201] Separate plasma from fresh blood samples (inactivate for later use), separate PBMC from the blood cell suspension using lymphocyte separation medium, and perform T cell sorting on the obtained PBMC using a T cell sorting kit. Activate T cells by adding Dynabeads CD3 / CD28 (added at a ratio of cell:Dynabeads = 1:1), and use activation medium (X-VIVO15, 5% plasma, 300 IU / ml IL-2) to adjust the final cell concentration to 1×10 6 / ml, and culture at 37°C in 5% CO 2 . After 24 h, transfect T cells with the CAR lentivirus prepared in Example 2 (virus pKT032 and pKT108), and remove the lentivirus 48 h after infection. Start observing cell rehydration culture every 1 - 2 days from the 4th day (D4), and maintain the cell density at 0.8×10 6 cells / mL. Use activation medium on the 4th - 5th days (D4 - D5), and use amplification medium (X-VIVO15, 300 IU / ml IL-2) after the 5th day (D5). Continuously culture until the 11th day (D11) to obtain two types of mesothelin-targeted CAR-T cells (CAR-T cells pKT032 and pKT108).

[0202] There was no significant difference in the activation and amplification of T cells with two different CAR structures ( Figure 2 A); both types of CAR-T cells were in an amplified state during the culture process, with the volume gradually increasing in the first 4 days and then gradually decreasing ( Figure 2 B).

[0203] (2) Detection of CAR-T cell positive rate

[0204] Take the pKT032 and pKT108 CAR-T cells on the 8th day (D8), add anti-mouse Fab antibody (PE) primary antibody and streptavidin-PE secondary antibody for incubation, and incubate untransduced T cells in the same way as a control. Detect the positive rate of CAR-T cells by flow cytometry. Detect the positive rate of two types of SS1 CAR-T cells, and there was little difference in the positive rate expression of the two types of CAR-T on the 8th day ( Figure 3 ).

[0205] (3) Detection of T cell differentiation subsets

[0206] CAR-T cell subset detection antibodies: anti-human CD3 (APC-Cy7), anti-human CCR7 antibody (BV421), anti-human CD45RO antibody (APC), CD4-BB515, CD8-BV510, CD45RA (PE-Cy7), CD62L-PE.

[0207] On the 12th day of culturing two types of CAR-T cells, namely pKT032 and pKT108, samples were taken and CAR-T cell subset detection antibodies were added for flow cytometry detection. CAR-T cell typing was detected, including the proportions of Tn, Teff, Tcm, and Tem in CD4+ and CD8+ T cells. The T cell differentiation subtypes of the two types of CAR-T (pKT032, pKT108) showed that compared with untransduced T cells (NTD) (the proportion of Tcm was 29.28%), pKT032 (73.67%) and pKT108 (74.21%) had more memory group cells ( Figure 4 ). The present invention can achieve a lower differentiation state of CAR-T, which will be more persistent and have memory activity in vivo.

[0208] (4) Target cell culture

[0209] SK-OV-3 cells were cultured using Myco5A medium (Myco5A + 10% FBS + 1% Penicillin / Streptomycine); OVCAR-3 cells were cultured using 1640 medium (1640 + 10% FBS + 1% Penicillin / Streptomycine).

[0210] (5) CAR-T specific killing

[0211] a. Digest and count the target cells, and adjust them with the culture medium (Myco5A or 1640 + 10% FBS) to a target cell suspension with a density of 2×10 5 cells / ml;

[0212] b. Add 50 μl of cell culture medium to the Eplate plate, place the Eplate in the DP monitoring slot, and prepare to measure the baseline.

[0213] c. After the baseline measurement is completed, take out the Eplate and add 1×10 4 (50 μl) target cells, and place them at room temperature for 30 min.

[0214] d. Put the E-plate plate with the cell suspension added back into the corresponding monitoring slot and start monitoring.

[0215] e. After 18 h, count the CAR-T and NTD cells, and adjust the effector cells to the corresponding density according to the effector-to-target ratio (E:T) in the following table.

[0216] E:T Number of target cells Cell density and volume added per well 0:1 0 50 μl culture medium 1:1 <![CDATA[1×10 4 > <![CDATA[2×10 5 cells / ml, 50 μl]]> 5:1 <![CDATA[5×10 4 > <![CDATA[1×10 6 cells / ml, 50 μl]]> 10:1 <![CDATA[1×10 5 > <![CDATA[2×10 6 cells / ml, 50 μl]]>

[0217] f. Pause the data acquisition of all detection wells.

[0218] g. Take out the E-plate that has been placed overnight, and add 50 μl of the corresponding effector cells to each well.

[0219] h. Put the E-plate back into the corresponding detection well, and continue the data acquisition of the real-time cell analysis (RTCA) system.

[0220] i. After adding the effector cells, monitor for no more than 48 h.

[0221] Detect the killing effects of two kinds of SS1 CAR-T cells, pKT032 and pKT108, and untransduced T cells (NTD) on tumor cells SK-OV-3 and OVCAR-3, and compare the killing abilities of CAR-T cells containing different anti-mesothelin scFvs ( Figure 5 ). The results show that NTD has no killing effect on SK-OV-3 and OVCAR-3 cells; at the effector-to-target ratios (E:T) of 0:1, 1:1, 5:1, and 10:1, all three CAR-T cells show obvious killing effects on SK-OV-3 and OVCAR-3 at the effector-to-target ratios of 5:1 and 10:1. There is no significant difference in the lysis rates of the target cells by the two CAR-T cells, pKT032 and pKT108.

[0222] (6) Detection of cytokine secretion

[0223] Co-culture pKT032 and pKT108 CAR-T with SK-OV-3 and OVCAR-3 cells respectively at E:T = 2:1 for 24 h; collect the cell supernatants and detect IL-2 and IFN-γ by ELISA.

[0224] Analyze the supernatants taken after co-culturing two kinds of CAR-T cells and NTD with SK-OV-3 and OVCAR-3 cells for 24 hours, and detect cytokines by ELISA. After co-culturing the two CAR-T cells with the positively selected target cells, both secrete IFN-γ and IL-2, and the secretion levels of IFN-γ (2997.99 pg / ml and 4622.92 pg / ml respectively) and IL-2 (1214.24 pg / ml and 4769.07 pg / ml respectively) of the pKT032 CAR-T cells are significantly higher than those of the pKT108 CAR-T group ( Figure 6) Overall, under the stimulation of tumor antigens, pKT032 CAR-T cells secrete the optimal amounts of IFN-γ and IL-2.

[0225] (7) Proliferation upon antigen stimulation

[0226] a. On the 8th day (D8) of CAR-T cell culture, remove the magnetic beads and continue culturing for 2 days using a medium without IL-2.

[0227] b. On the 10th day (D10), count the effector cells (CAR-T) and NTD.

[0228] c. Take the required CAR-T cells (5×10 6 / ml), wash twice with PBS, and finally resuspend the cells in 500 μl of PBS to a concentration of 1×10 7 / ml.

[0229] d. Add the fluorescent dye CFSE (purchased from BD Biosciences, original concentration 5 mM) to a final concentration of 5 μM, and place in an incubator for 10 min.

[0230] e. Add 9 volumes of PBS equal to the incubation volume and centrifuge.

[0231] f. After discarding the supernatant, resuspend in 10 ml of medium containing 10% FBS, centrifuge, and discard the supernatant.

[0232] g. Resuspend in 5 ml of medium (10% FBS, 300 UI / ml IL-2) to 1×10 6 / ml for later use.

[0233] h. Count the target cells (SK-OV-3 and OVCAR-3), and adjust the density to 1×10 6 / ml with the culture medium (Myco5A or 1640 + 10% FBS + 300 UI / ml IL-2) for standby.

[0234] i. Add 1 ml of CAR-T cells per well according to the corresponding effector-to-target ratio (E:T), place in a 37°C, 5% CO 2 incubator for culture, and centrifuge and change the medium after 1 - 2 days.

[0235] j. Perform flow cytometry on the fifth day (D5).

[0236] Flow cytometry results of the CFSE fluorescence intensity of two types of CAR-T cells (pKT032, pKT108) showed that Figure 7) After stimulation with mesothelin-positive target cells, compared with the NTD control group, the fluorescence intensity of CFSE in pKT032 CAR-T shifted to the left and weakened, confirming that the positive target cells could stimulate the proliferation of pKT032 CAR-T; while the fluorescence intensity of CFSE in pKT108 CAR-T shifted to the left very little, confirming that the proliferation of pKT108 CAR-T under the stimulation of positive target cells was weak and not as good as that of pKT032 CAR-T.

[0237] Example 4: In vivo function test of MSLN CAR-T cells

[0238] Tumor cells: SKOV-3 (5×10 6 cells)

[0239] Tumor formation method: subcutaneous tumor formation

[0240] Drug administration groups: NTD, pKT032, pKT108

[0241] Drug administration dose: 5×10 6 CAR-T

[0242] In vivo drug efficacy:

[0243] The anti-tumor activities of three kinds of CAR-T cells (NTD, pKT032 and pKT108) in animals were further verified ( Figure 8 ). The results showed that the tumor volume of mice in the NTD group increased rapidly and reached the experimental endpoint soon; the clearance efficiencies of pKT032 and pKT108 on mouse tumors were similar, and pKT032 made the tumor regress more rapidly and could bring more durable efficacy.

[0244] Example 5: Clinical study of MSLN CAR-T cells

[0245] (1) Cell preparation

[0246] Separate plasma from fresh blood samples (inactivated for standby), separate PBMC from the blood cell suspension with lymphocyte separation medium, and perform T cell sorting on the obtained PBMC with a T cell sorting kit. Activate T cells by adding Dynabeads CD3 / CD28 (added according to cell:Dynabeads = 1:1), and use activation medium (X-VIVO15, 5% plasma, 300 IU / ml IL-2) to adjust the final cell concentration to 1×10 6 / ml, and culture at 37°C and 5% CO 2 . After 24 h, transfect T cells with pKT032 lentivirus, and remove the lentivirus 48 h after infection. Start observing cell fluid replacement culture every 1-2 days from the 4th day (D4) to maintain the cell density at 0.8×10 6cells / mL. On the 4th - 5th day (D4 - D5), activation medium was used, and after the 5th day (D5), expansion medium (X - VIVO15, 300 IU / ml IL - 2) was used. Culturing was continued until the 7th - 12th day (D7 - D12).

[0247] The CAR clinical - grade vector was manufactured by Nanjing Aide Immunotherapy Research Institute Co., Ltd. At the end of CAR - T cell culture, the cells were refrigerated in an injectable cryoprotectant. CAR - T cells conforming to the administration dose were administered. Each bag contained an aliquot of cryopreservation medium (volume determined by the dose), and the cryopreservation solution was CS5. Bags containing mesothelin CAR - T cells (10 - 100 ml) were stored in a - 135°C liquid nitrogen tank for testing. The cryopreserved bags were stored in the freezer until needed. For increased safety, the first dose was given in divided doses on the 0th and 1st days, approximately 30% of the cells on the 0th day and 70% of the cells on the 1st day.

[0248] (2) Cell thawing

[0249] The frozen cells were transported to the laboratory or the patient in dry ice. Using a water bath maintained at 37°C, the cells were thawed and gently massaged until just thawed. There should be no frozen chunks left in the container. If the mesothelin CAR - T cell product shows a damaged or leaking bag, it should not be transfused back.

[0250] (3) Premedication

[0251] After the mesothelin CAR - T preparation is warmed, it should not be left at room temperature for too long. Therefore, the warming time needs to be determined after all the preparations for the subject's treatment are completed. Necessary first - aid equipment and drugs should be prepared before transfusion. 30 - 60 minutes before transfusion, the subject was pre - medicated with acetaminophen, Depakine, and diphenhydramine or other H1 antihistamine drugs. Since mesothelin CAR - T is an autologous T - cell therapy and the packaging bag is patient - specific, the subject information on the bag should be confirmed before transfusion. Before transfusion, check whether the packaging is damaged. If it is damaged, do not transfuse.

[0252] (4) Transfusion

[0253] Re - confirm the subject information on the packaging bag; rinse the injection pipeline with normal saline; flush the pipeline with 50 ml of normal saline before and after CAR - T cell infusion; complete intravenous transfusion within 30 minutes.

[0254] (5) Inclusion and exclusion criteria

[0255] (1) Recurrent and refractory ovarian and pancreatic cancers positive for mesothelin; (2) Aged 18 - 70 years, regardless of gender; (3) Recurrence after receiving second-line or above treatments such as chemotherapy or targeted drugs; (4) The immunohistochemical results of tumor tissues show positive expression of MSLN antigen, and the antigen expression rate ≥ 15%; (5) According to the RECIST 1.1 standard, the patient has at least 1 evaluable tumor lesion and can be accurately measured at baseline; (6) ECOG score of 0 - 2, with an expected survival period of more than 12 weeks; (7) Having a venous access for intravenous blood sampling or apheresis; (8) The patient voluntarily participates and signs an informed consent form in writing.

[0256] (6) Exclusion criteria

[0257] Patients who meet any of the following criteria cannot be included in this study:

[0258] (1) Pregnant or lactating women; (2) Those who have used chemotherapy or radiotherapy within 3 days before the blood sampling period; (3) Those who have used systemic steroid drugs in combination within 5 days before the blood sampling period (except those who are currently or recently using inhaled steroids); (4) Those who have used drugs that stimulate the production of bone marrow hematopoietic cells within 5 days before the blood sampling period; (5) Those who have used any gene or cell therapy products; (6) Those with a history of epilepsy or other central nervous system diseases; (7) Subjects with active hepatitis B or hepatitis C (defined as: positive for hepatitis B surface antigen HBsAg or hepatitis B core antibody HBcAb and the peripheral blood HBV DNA titer is higher than the upper limit of detection); those positive for hepatitis C virus HCV antibody and positive for peripheral blood HCV RNA; those infected with human immunodeficiency virus or syphilis; (8) Those who have suffered from other tumors in the past 5 years; (9) Patients with severe pleural or ascites; (10) Those with active or uncontrollable infections that require systemic treatment within 14 days before enrollment; (11) Those who have received other anti-tumor treatments (except pre-treatment chemotherapy) within two weeks before the start of the study; (12) Those who the investigator assesses as unable or unwilling to comply with the requirements of the study protocol.

[0259] (7) Treatment plan

[0260] Figure 9 The flow chart of the clinical treatment plan is shown.

[0261] (8) Pre-treatment plan

[0262] On the 3rd day before infusion: Intravenous drip of fludarabine, with a dose of 75 - 100 mg / m 2 / day;

[0263] On the 3rd day before infusion: Intravenous drip of cyclophosphamide, with a dose of 750 mg / m 2 / day.

[0264] (9) Administration regimen

[0265] (1) Infusion dose: Based on the existing peer doses internationally

[0266] For the test drug pKT032, the test administration is carried out according to the clinical situation, 3×10 6 CAR-T cells / kg are intravenously infused once. The above administration regimen is taken as one course of treatment.

[0267] (10) Treatment results

[0268]

[0269] Note: ① + represents as of the statistical date; ② caused by pretreatment; PR, partial remission; SD, stable disease.

[0270] All solid tumor patients treated with pKT032 CAR-T cells are recurrent and refractory types, and have undergone more than 4 previous treatment methods, and the condition cannot be controlled. For example, patient 2A was enrolled after 40 chemotherapy sessions, with chemotherapy and targeted drug resistance, and then received CAR-T treatment. According to the literature report, the median survival period of recurrent and refractory ovarian cancer patients is 4.8 months, and the median survival period of solid tumor patients treated with pKT032 CAR-T cells is 11.6 months( Figure 10 ). The median progression-free survival period of solid tumors treated with pKT032 CAR-T cells is 7 months( Figure 11 ), showing a significant benefit compared with the data of the median survival period of 2.1 months in the first phase of CAR-T cell treatment at the University of Pennsylvania (Mol Ther. 2019 Nov 6; 27(11):1919-1929). SEQUENCE LISTING <110> Nanjing Kati Medical Technology Co., Ltd. <120> Chimeric receptor targeting mesothelin and its use <130> MTI21129 <160> 16 <170> PatentIn version 3.5 <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> 69 <212> PRT <213> artificial sequence <400> 2 Ser Lys Thr Gly Asn Pro Arg His Leu His Val Leu Ile Gly Thr Ser 1 5 10 15 Val Val Lys Ile Pro Phe Thr Ile Leu Leu Phe Phe Leu Leu His Arg 20 25 30 Trp Cys Ser Asn Lys Lys Asn Ala Ala Val Met Asp Gln Glu Pro Ala 35 40 45 Gly Asn Arg Thr Val Asn Ser Glu Asp Ser Asp Glu Gln Asp His Gln 50 55 60 Glu Val Ser Tyr Ala 65 <210> 3 <211> 283 <212> PRT <213> artificial sequence <400> 3 His Glu Gly Val His Arg Lys Pro Ser Leu Leu Ala His Pro Gly Pro 1 5 10 15 Leu Val Lys Ser Glu Glu Thr Val Ile Leu Gln Cys Trp Ser Asp Val 20 25 30 Arg Phe Glu His Phe Leu Leu His Arg Glu Gly Lys Tyr Lys Asp Thr 35 40 45 Leu His Leu Ile Gly Glu His His Asp Gly Val Ser Lys Ala Asn Phe 50 55 60 Ser Ile Gly Pro Met Met Gln Asp Leu Ala Gly Thr Tyr Arg Cys Tyr 65 70 75 80 Gly Ser Val Thr His Ser Pro Tyr Gln Leu Ser Ala Pro Ser Asp Pro 85 90 95 Leu Asp Ile Val Ile Thr Gly Leu Tyr Glu Lys Pro Ser Leu Ser Ala 100 105 110 Gln Pro Gly Pro Thr Val Leu Ala Gly Glu Ser Val Thr Leu Ser Cys 115 120 125 Ser Ser Arg Ser Ser Tyr Asp Met Tyr His Leu Ser Arg Glu Gly Glu 130 135 140 Ala His Glu Arg Arg Phe Ser Ala Gly Pro Lys Val Asn Gly Thr Phe 145 150 155 160 Gln Ala Asp Phe Pro Leu Gly Pro Ala Thr His Gly Gly Thr Tyr Arg 165 170 175 Cys Phe Gly Ser Phe Arg Asp Ser Pro Tyr Glu Trp Ser Asn Ser Ser 180 185 190 Asp Pro Leu Leu Val Ser Val Thr Gly Asn Pro Ser Asn Ser Trp Pro 195 200 205 Ser Pro Thr Glu Pro Ser Ser Lys Thr Gly Asn Pro Arg His Leu His 210 215 220 Val Leu Ile Gly Thr Ser Val Val Lys Ile Pro Phe Thr Ile Leu Leu 225 230 235 240 Phe Phe Leu Leu His Arg Trp Cys Ser Asn Lys Lys Asn Ala Ala Val 245 250 255 Met Asp Gln Glu Pro Ala Gly Asn Arg Thr Val Asn Ser Glu Asp Ser 260 265 270 Asp Glu Gln Asp His Gln Glu Val Ser Tyr Ala 275 280 <210> 4 <211> 21 <212> PRT <213> artificial sequence <400> 4 Met Gly Gly Leu Glu Pro Cys Ser Arg Phe Leu Leu Leu Pro Leu Leu 1 5 10 15 Leu Ala Val Ser Gly 20 <210> 5 <211> 21 <212> PRT <213> artificial sequence <400> 5 Gly Val Leu Ala Gly Ile Val Met Gly Asp Leu Val Leu Thr Val Leu 1 5 10 15 Ile Ala Leu Ala Val 20 <210> 6 <211> 52 <212> PRT <213> Artificial sequence <400> 6 Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala 1 5 10 15 Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu 20 25 30 Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg 35 40 45 Pro Tyr Tyr Lys 50 <210> 7 <211> 92 <212> PRT <213> Artificial sequence <400> 7 Leu Arg Pro Val Gln Val Gln Ala Gln Ser Asp Cys Ser Cys Ser Thr 1 5 10 15 Val Ser Pro Gly Val Leu Ala Gly Ile Val Met Gly Asp Leu Val Leu 20 25 30 Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu Gly Arg Leu Val Pro 35 40 45 Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg Lys Gln Arg Ile Thr 50 55 60 Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly Gln Arg Ser Asp Val 65 70 75 80 Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr Lys 85 90 <210> 8 <211> 79 <212> PRT <213> artificial sequence <400> 8 Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met Gly Asp 1 5 10 15 Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu Gly Arg 20 25 30 Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg Lys Gln 35 40 45 Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly Gln Arg 50 55 60 Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr Lys 65 70 75 <210> 9 <211> 42 <212> PRT <213> Artificial Sequence <400> 9 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> 10 <211> 17 <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 <210> 11 <211> 240 <212> PRT <213> Artificial Sequence <400> 11 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Ser Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile 130 135 140 Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser 145 150 155 160 Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser 165 170 175 Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro 180 185 190 Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile 195 200 205 Ser Ser Val Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys Gln Gln Trp 210 215 220 Ser Lys His Pro Leu Thr Tyr Gly Ala Gly Thr Lys Leu Glu Ile Lys 225 230 235 240 <210> 12 <211> 240 <212> PRT <213> artificial sequence <400> 12 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys His Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Ser Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile 130 135 140 Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser 145 150 155 160 Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser 165 170 175 Pro Lys Arg Trp Ile Tyr Asp Thr Ser His Leu Ala Ser Gly Val Pro 180 185 190 Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile 195 200 205 Ser Ser Val Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys Lys Gln Trp 210 215 220 Ser Lys His Pro Leu Thr Tyr Gly Ala Gly Thr Lys Leu Glu Ile Lys 225 230 235 240 <210> 13 <211> 533 <212> PRT <213> artificial sequence <400> 13 Met Gly Gly Leu Glu Pro Cys Ser Arg Phe Leu Leu Leu Pro Leu Leu 1 5 10 15 Leu Ala Val Ser Gly Leu Arg Pro Val Gln Val Gln Ala Gln Ser Asp 20 25 30 Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met 35 40 45 Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu 50 55 60 Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg 65 70 75 80 Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly 85 90 95 Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr 100 105 110 Lys Asp Ile Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 115 120 125 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 130 135 140 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Ser Arg Val 145 150 155 160 Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val 165 170 175 Glu Glu Asn Pro Gly Pro Arg Met Ala Leu Pro Val Thr Ala Leu Leu 180 185 190 Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Gly Ser Gln Val 195 200 205 Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala Ser Val 210 215 220 Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met 225 230 235 240 Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly Leu 245 250 255 Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe Arg Gly 260 265 270 Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Asp 275 280 285 Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg 290 295 300 Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr 305 310 315 320 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser 325 330 335 Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile Met Ser 340 345 350 Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser 355 360 365 Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys 370 375 380 Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly Arg 385 390 395 400 Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser 405 410 415 Val Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Lys 420 425 430 His Pro Leu Thr Tyr Gly Ala Gly Thr Lys Leu Glu Ile Lys Ala Ser 435 440 445 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Pro Thr Glu Pro Ser 450 455 460 Ser Lys Thr Gly Asn Pro Arg His Leu His Val Leu Ile Gly Thr Ser 465 470 475 480 Val Val Lys Ile Pro Phe Thr Ile Leu Leu Phe Phe Leu Leu His Arg 485 490 495 Trp Cys Ser Asn Lys Lys Asn Ala Ala Val Met Asp Gln Glu Pro Ala 500 505 510 Gly Asn Arg Thr Val Asn Ser Glu Asp Ser Asp Glu Gln Asp His Gln 515 520 525 Glu Val Ser Tyr Ala 530 <210> 14 <211> 1599 <212> DNA <213> artificial sequence <400> 14 atggggggac ttgaaccctg cagcaggttc ctgctcctgc ctctcctgct ggctgtaagt 60 ggtctccgtc ctgtccaggt ccaggcccag agcgattgca gttgctctac ggtgagcccg 120 ggcgtgctgg cagggatcgt gatgggagac ctggtgctga cagtgctcat tgccctggcc 180 gtgtacttcc tgggccggct ggtccctcgg gggcgagggg ctgcggaggc agcgacccgg 240 aaacagcgta tcactgagac cgagtcgcct tatcaggagc tccagggtca gaggtcggat 300 gtctacagcg acctcaacac acagaggccg tattacaaag atatcaaacg gggcagaaag 360 aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 420 gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact gtctagagtc 480 gagggcggcg gagagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc 540 ggccctagga tggccttacc agtgaccgcc ttgctcctgc cgctggcctt gctgctccac 600 gccgccaggc cgggatccca ggtacaactg cagcagtctg ggcctgagct ggagaagcct 660 ggcgcttcag tgaagatatc ctgcaaggct tctggttact cattcactgg ctacaccatg 720 aactgggtga agcagagcca tggaaagagc cttgagtgga ttggacttat tactccttac 780 aatggtgctt ctagctacaa ccagaagttc aggggcaagg ccacattaac tgtagacaag 840 tcatccagca cagcctacat ggacctcctc agtctgacat ctgaagactc tgcagtctat 900 ttctgtgcaa gggggggtta cgacgggagg ggttttgact actggggcca agggaccacg 960 gtcaccgtct cctcaggtgg aggcggttca ggcggcggtg gctctagcgg tggtggatcg 1020 gacatcgagc tcactcagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 1080 atgacctgca gtgccagctc aagtgtaagt tacatgcact ggtaccagca gaagtcaggc 1140 acctccccca aaagatggat ttatgacaca tccaaactgg cttctggagt cccaggtcgc 1200 ttcagtggca gtgggtctgg aaactcttac tctctcacaa tcagcagcgt ggaggctgaa 1260 gatgatgcaa cttattactg ccagcagtgg agtaagcacc ctctcacgta cggtgctggg 1320 acaaagttgg aaatcaaagc tagcggtggc ggaggttctg gaggtggggg ttcctcaccc 1380 actgaaccaa gctccaaaac cggtaacccc agacacctgc atgttctgat tgggacctca 1440 gtggtcaaaa tccctttcac catcctcctc ttctttctcc ttcatcgctg gtgctccaac 1500 aaaaaaaatg ctgctgtaat ggaccaagag cctgcaggga acagaacagt gaacagcgag 1560 gattctgatg aacaagacca tcaggaggtg tcatacgca 1599 <210> 15 <211> 533 <212> PRT <213> Artificial Sequence <400> 15 Met Gly Gly Leu Glu Pro Cys Ser Arg Phe Leu Leu Leu Pro Leu Leu 1 5 10 15 Leu Ala Val Ser Gly Leu Arg Pro Val Gln Val Gln Ala Gln Ser Asp 20 25 30 Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met 35 40 45 Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu 50 55 60 Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg 65 70 75 80 Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly 85 90 95 Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr 100 105 110 Lys Asp Ile Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 115 120 125 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 130 135 140 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Ser Arg Val 145 150 155 160 Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val 165 170 175 Glu Glu Asn Pro Gly Pro Arg Met Ala Leu Pro Val Thr Ala Leu Leu 180 185 190 Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Gly Ser Gln Val 195 200 205 Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala Ser Val 210 215 220 Lys Ile Ser Cys His Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met 225 230 235 240 Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly Leu 245 250 255 Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe Arg Gly 260 265 270 Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Asp 275 280 285 Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg 290 295 300 Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr 305 310 315 320 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser 325 330 335 Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile Met Ser 340 345 350 Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser 355 360 365 Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys 370 375 380 Arg Trp Ile Tyr Asp Thr Ser His Leu Ala Ser Gly Val Pro Gly Arg 385 390 395 400 Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser 405 410 415 Val Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys Lys Gln Trp Ser Lys 420 425 430 His Pro Leu Thr Tyr Gly Ala Gly Thr Lys Leu Glu Ile Lys Ala Ser 435 440 445 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Pro Thr Glu Pro Ser 450 455 460 Ser Lys Thr Gly Asn Pro Arg His Leu His Val Leu Ile Gly Thr Ser 465 470 475 480 Val Val Lys Ile Pro Phe Thr Ile Leu Leu Phe Phe Leu Leu His Arg 485 490 495 Trp Cys Ser Asn Lys Lys Asn Ala Ala Val Met Asp Gln Glu Pro Ala 500 505 510 Gly Asn Arg Thr Val Asn Ser Glu Asp Ser Asp Glu Gln Asp His Gln 515 520 525 Glu Val Ser Tyr Ala 530 <210> 16 <211> 1599 <212> DNA <213> artificial sequence <400> 16 atggggggac ttgaaccctg cagcaggttc ctgctcctgc ctctcctgct ggctgtaagt 60 ggtctccgtc ctgtccaggt ccaggcccag agcgattgca gttgctctac ggtgagcccg 120 ggcgtgctgg cagggatcgt gatgggagac ctggtgctga cagtgctcat tgccctggcc 180 gtgtacttcc tgggccggct ggtccctcgg gggcgagggg ctgcggaggc agcgacccgg 240 aaacagcgta tcactgagac cgagtcgcct tatcaggagc tccagggtca gaggtcggat 300 gtctacagcg acctcaacac acagaggccg tattacaaag atatcaaacg gggcagaaag 360 aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 420 gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact gtctagagtc 480 gagggcggcg gagagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc 540 ggccctagga tggccttacc agtgaccgcc ttgctcctgc cgctggcctt gctgctccac 600 gccgccaggc cgggatccca ggtacaactg cagcagtctg ggcctgagct ggagaagcct 660 ggcgcttcag tgaagatatc ctgccacgct tctggttact cattcactgg ctacaccatg 720 aactgggtga agcagagcca tggaaagagc cttgagtgga ttggacttat tactccttac 780 aatggtgctt ctagctacaa ccagaagttc aggggcaagg ccacattaac tgtagacaag 840 tcatccagca cagcctacat ggacctcctc agtctgacat ctgaagactc tgcagtctat 900 ttctgtgcaa gggggggtta cgacgggagg ggttttgact actggggcca agggaccacg 960 gtcaccgtct cctcaggtgg aggcggttca ggcggcggtg gctctagcgg tggtggatcg 1020 gacatcgagc tcactcagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 1080 atgacctgca gtgccagctc aagtgtaagt tacatgcact ggtaccagca gaagtcaggc 1140 acctccccca aaagatggat ttatgacaca tcccacctgg cttctggagt cccaggtcgc 1200 ttcagtggca gtgggtctgg aaactcttac tctctcacaa tcagcagcgt ggaggctgaa 1260 gatgatgcaa cttattactg caagcagtgg agtaagcacc ctctcacgta cggtgctggg 1320 acaaagttgg aaatcaaagc tagcggtggc ggaggttctg gaggtggggg ttcctcaccc 1380 actgaaccaa gctccaaaac cggtaacccc agacacctgc atgttctgat tgggacctca 1440 gtggtcaaaa tccctttcac catcctcctc ttctttctcc ttcatcgctg gtgctccaac 1500 aaaaaaaatg ctgctgtaat ggaccaagag cctgcaggga acagaacagt gaacagcgag 1560 gattctgatg aacaagacca tcaggaggtg tcatacgca 1599

Claims

1. A chimeric antigen receptor targeting mesothelin (MSLN), which comprises a first fusion peptide and a second fusion peptide, wherein: the first fusion peptide comprises a mesothelin antigen-binding domain and a transmembrane domain; the second fusion peptide comprises a transmembrane domain, a cytoplasmic domain and a co-stimulatory domain; the transmembrane domain of the second fusion peptide interacts with the transmembrane domain of the first fusion peptide through charge interaction, or the second fusion peptide interacts with a signaling molecule through a phosphorylated ITAM sequence within the cytoplasmic domain; the chimeric antigen receptor is selected from any one of the following: the chimeric antigen receptor is an SS1 scFv1-KIRS2 / Dap12-BB chimeric antigen receptor, and the SS1 scFv1-KIRS2 / Dap12-BB chimeric antigen receptor is formed by cleavage of a fusion protein by a T2A peptide. The fusion protein comprises a DAP12 signal peptide, DAP12 comprising a DAP12 transmembrane domain and a DAP12 cytoplasmic domain, 4-1BB, a T2A cleavage site, a CD8α signal peptide, SS1 scFv1 and KIRS2; or the chimeric antigen receptor is an SS1 scFv2-KIRS2 / Dap12-BB chimeric antigen receptor, and the SS1 scFv2-KIRS2 / Dap12-BB chimeric antigen receptor is formed by cleavage of a fusion protein by a T2A peptide. The fusion protein comprises a DAP12 signal peptide, DAP12 comprising a DAP12 transmembrane domain and a DAP12 cytoplasmic domain, 4-1BB, a T2A cleavage site, a CD8α signal peptide, SS1 scFv2 and KIRS2; the amino acid sequence of the DAP12 signal peptide is as shown in SEQ ID NO:4, the amino acid sequence of the DAP12 transmembrane domain is as shown in SEQ ID NO:5, the amino acid sequence of the DAP12 cytoplasmic domain is as shown in SEQ ID NO:6, the amino acid sequence of 4-1BB is as shown in SEQ ID NO:9, the amino acid sequence of the T2A cleavage site is as shown in SEQ ID NO:10, the amino acid sequence of the CD8α signal peptide is as shown in SEQ ID NO:1, the amino acid sequence of KIRS2 is as shown in SEQ ID NO:2, the amino acid sequence of SS1 scFv1 is as shown in SEQ ID NO:11 or the amino acid sequence of SS1 scFv2 is as shown in SEQ ID NO:

12.

2. The chimeric antigen receptor according to claim 1, wherein, the amino acid sequence of the DAP12 is as shown in SEQ ID NO:

7.

3. The chimeric antigen receptor according to claim 1 or 2, wherein, the amino acid sequence of the SS1 scFv1-KIRS2 / Dap12-BB chimeric antigen receptor fusion protein is as shown in SEQ ID NO:13; or the amino acid sequence of the SS1 scFv2-KIRS2 / Dap12-BB chimeric antigen receptor fusion protein is as shown in SEQ ID NO:

15.

4. A nucleic acid encoding the chimeric antigen receptor according to any one of claims 1-3.

5. The nucleic acid according to claim 4, wherein the nucleic acid is selected from the nucleic acids shown in SEQ ID NO:14 and SEQ ID NO:

16.

6. The nucleic acid according to claim 4 or 5, wherein the encoding nucleic acid is the nucleic acid shown in SEQ ID NO:

14.

7. A vector comprising the nucleic acid according to any one of claims 4-6.

8. A cell comprising the nucleic acid according to any one of claims 4-6 or the vector according to claim 7.

9. A composition comprising the chimeric antigen receptor according to any one of claims 1-3, the nucleic acid according to any one of claims 4-6, the vector according to claim 7, and / or the cell according to claim 8, and a pharmaceutically acceptable carrier.

10. A method for preparing a cell, the method comprising introducing the nucleic acid according to any one of claims 4-6 and the vector according to claim 7 into immune effector cells.

11. Use of the chimeric antigen receptor according to any one of claims 1-3, the nucleic acid according to any one of claims 4-6, the vector according to claim 7, the cell according to claim 8, and / or the composition according to claim 9 in the preparation of a medicament for treating ovarian cancer or pancreatic cancer.

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