Chimeric antigen receptor and use thereof

Abstract

Provided are a chimeric antigen receptor capable of simultaneously identifying two antigen ligands, a corresponding nucleic acid construct, an expression vector, a genetically modified immune cell, a pharmaceutical composition, and a use thereof. The chimeric antigen receptor comprises: an extracellular region, wherein the extracellular region comprises a first binding fragment and a second binding fragment, the first binding fragment is connected to the second binding fragment, the first binding fragment is an NKp30 extracellular domain or a fragment thereof, and the second binding fragment is an NKG2D extracellular domain or a fragment thereof; a transmembrane region, wherein the N-terminus of the transmembrane region is connected to the C-terminus of the extracellular region; and an intracellular region, wherein the N-terminus of the intracellular region is connected with the C-terminus of the transmembrane region. The chimeric antigen receptor simultaneously targets an NKp30 receptor ligand and an NKG2D receptor ligand, and expresses a transgenic immune cell of the chimeric antigen receptor, so as to effectively identify a tumor cell expressing the NKp30 receptor ligand and / or expressing the NKG2D receptor ligand, that is, for a single expression of the NKp30 receptor ligand or the NKG2D receptor ligand or simultaneous expression of both receptor ligands, effective killing effects can be exerted, and particularly, a tumor cell having one receptor ligand of NKP30 or NKG2D being lost can be effectively identified, so that tumor immune escape is prevented. Therefore, the genetically modified immune cell has high tumor cell killing activity, wide recognition spectrum, high in-vivo tumor inhibition activity, low cell infusion dosage and good curative effect.

Description

Chimeric antigen receptors and their applications Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically, to a chimeric antigen receptor and its application. More specifically, the present invention relates to a chimeric antigen receptor that can simultaneously recognize two antigen ligands, and corresponding nucleic acid constructs, expression vectors, transgenic immune cells, pharmaceutical compositions, and their uses. Background Art

[0002] Chimeric antigen receptor T (CAR-T) cells have achieved remarkable results in the treatment of hematological malignancies. The antigen recognition region of CAR molecules typically utilizes antibody-derived fragment (scFv) sequences, typically from species such as mice, rabbits, and alpacas, and possesses strong immunogenicity. Even with humanized sequences, some immunogenicity persists, generating anti-drug antibodies (ADAs) that impair the persistence of CAR-T cells in vivo. In recent years, numerous new technologies have been applied to address this issue, such as using native receptor protein sequences as receptors to reduce immunogenicity.

[0003] The NKp30 receptor is a natural NK cell-activating receptor highly expressed on NK cells. It recognizes the B7H6 molecule, thereby mediating cytotoxicity. B7H6, a member of the B7 family, is typically highly expressed on a variety of primary tumor cells, including leukemias, lymphomas, colorectal cancer, gastric cancer, and pancreatic cancer, but is rarely expressed on normal tissue cells. Currently, the function of CAR-T cells constructed based on the NKp30 extracellular segment as a receptor for B7H6 recognition has been verified in research.

[0004] The NKG2D receptor is another natural NK cell surface receptor, primarily expressed on the surfaces of NK, CD8+T, γδT, NKT, and activated macrophages. It recognizes stress proteins expressed by viral infection or cell malignant transformation, such as MHC-I class-associated proteins, MICA and MICB, and cytomegalovirus UL-16 binding proteins ULBP1-5. In patent CN109400713A, NKG2D is used as an intracellular signaling domain. CART cells constructed on this basis have significantly enhanced the ability of CAR T cells to kill tumor cells in hematologic and solid tumor killing tests, demonstrating good safety and anti-tumor activity in clinical applications.

[0005] Based on the above research and development status, further research is needed on chimeric conversion receptors with high killing activity and good efficacy to further improve the efficacy of chimeric antigen receptor immune cell therapy.

[0006] Summary of the Invention

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides a chimeric antigen receptor that can simultaneously recognize two antigen ligands and a corresponding nucleic acid construct, an expression vector, a transgenic immune cell, a pharmaceutical composition and its use. The chimeric antigen receptor of the present invention simultaneously targets NKp30 receptor ligands and NKG2D receptor ligands. The transgenic immune cell expressing the chimeric antigen receptor of the present invention can effectively identify tumor cells expressing NKp30 receptor ligands and / or expressing NKG2D receptor ligands, that is, it can effectively kill tumor cells that express a single NKp30 receptor ligand or NKG2D receptor ligand or express both receptor ligands, especially it can effectively identify tumor cells that have lost one of the NKp30 or NKG2D receptor ligands, and prevent the immune escape of the tumor. As a result, the transgenic immune cell tumor cell killing activity of the present invention is high, the recognition spectrum is wide, the in vivo tumor inhibition activity is strong, the cell infusion dose is low, and the efficacy is good.

[0008] In a first aspect of the present invention, a chimeric antigen receptor is provided. The chimeric antigen receptor comprises:

[0009] An extracellular region, wherein the extracellular region includes a first binding fragment and a second binding fragment, wherein the first binding fragment and the second binding fragment are connected, the first binding fragment is an extracellular segment of NKp30 or a fragment thereof, and the second binding fragment is an extracellular segment of NKG2D or a fragment thereof;

[0010] a transmembrane region, wherein the N-terminus of the transmembrane region is connected to the C-terminus of the extracellular region;

[0011] The intracellular region, wherein the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region.

[0012] It should be noted that the amino acid sequences involved in the present invention are all shown from N-terminus to C-terminus.

[0013] According to the chimeric antigen receptor of the embodiment of the present invention, the NKp30 receptor ligand and the NKG2D receptor ligand are simultaneously targeted. The transgenic immune cells expressing the chimeric antigen receptor of the present invention can effectively identify tumor cells expressing NKp30 receptor ligands and / or expressing NKG2D receptor ligands, that is, they can effectively kill tumor cells that express only NKp30 receptor ligands or NKG2D receptor ligands or both receptor ligands, especially tumor cells that have lost one of the NKp30 or NKG2D receptor ligands, thereby preventing the immune escape of the tumor. As a result, the transgenic immune cells expressing the chimeric antigen receptor of the present invention have high tumor cell killing activity, a wide tumor recognition spectrum, strong in vivo tumor inhibition activity, a low cell infusion dose, and good efficacy.

[0014] According to an embodiment of the present invention, the first binding fragment has the amino acid sequence shown in SEQ ID NO: 1 or a conservatively modified version thereof, and the second binding fragment has the amino acid sequence shown in SEQ ID NO: 3 or a conservatively modified version thereof. Thus, the chimeric antigen receptor of the present invention can simultaneously target both the NKp30 receptor ligand and the NKG2D receptor ligand.

[0015] According to an embodiment of the present invention, the first binding fragment has an amino acid sequence as shown in SEQ ID NO: 1, and the second binding fragment has an amino acid sequence as shown in SEQ ID NO: 3. Thus, the chimeric antigen receptor of the present invention simultaneously targets NKp30 receptor ligand and NKG2D receptor ligand.

[0016] As used herein, the term "conservatively modified amino acid sequence" refers to amino acid modifications that do not significantly affect or alter the ligand binding properties of a receptor comprising the amino acid sequence, including amino acid substitutions, additions, and deletions. Modifications can be introduced into the receptors of the present invention by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (such as threonine, valine, isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine).

[0017] According to an embodiment of the present invention, the C-terminus of the first binding fragment is connected to the N-terminus of the second binding fragment, or the N-terminus of the first binding fragment is connected to the C-terminus of the second binding fragment.

[0018] According to an embodiment of the present invention, the extracellular region further comprises a first connecting peptide, and the first binding fragment and the second binding fragment are connected by the first connecting peptide, thereby further improving the binding ability of the chimeric antigen receptor of the present invention to the NKp30 receptor ligand and the NKG2D receptor ligand.

[0019] According to an embodiment of the present invention, the C-terminus of the first binding fragment is connected to the N-terminus of the first connecting peptide, which is then connected to the N-terminus of the second binding fragment; or the C-terminus of the second binding fragment is connected to the N-terminus of the first connecting peptide, which is then connected to the N-terminus of the first binding fragment. Thus, the resulting chimeric antigen receptor of the present invention simultaneously targets both the NKp30 receptor ligand and the NKG2D receptor ligand.

[0020] According to an embodiment of the present invention, the first connecting peptide is selected from (G4S) n or at least one of the connecting peptides having an amino acid sequence as shown in SEQ ID NOs: 5, 34 to 39, wherein n is an integer other than zero.

[0021] According to an embodiment of the present invention, the first connecting peptide is (G4S) n , n is any integer between 2 and 6.

[0022] According to an embodiment of the present invention, the first connecting peptide is (G4S) 3. Thus, a chimeric antigen receptor of the present invention having better binding ability to NKp30 receptor ligand and NKG2D receptor ligand is obtained.

[0023] According to an embodiment of the present invention, the extracellular region further includes a hinge region, thereby further improving the binding ability of the chimeric antigen receptor of the present invention to NKp30 receptor ligands and NKG2D receptor ligands.

[0024] According to an embodiment of the present invention, the C-terminus of the first binding fragment is connected to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is connected to the N-terminus of the second binding fragment, and the C-terminus of the second binding fragment is connected to the N-terminus of the hinge region; or the C-terminus of the second binding fragment is connected to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is connected to the N-terminus of the first binding fragment, and the C-terminus of the first binding fragment is connected to the N-terminus of the hinge region. Thus, the chimeric antigen receptor of the present invention simultaneously targets the NKp30 receptor ligand and the NKG2D receptor ligand.

[0025] According to an embodiment of the present invention, the hinge region has an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence of a conservatively modified form thereof.

[0026] According to an embodiment of the present invention, the hinge region has an amino acid sequence as shown in SEQ ID NO:9.

[0027] According to an embodiment of the present invention, the extracellular region further includes a second connecting peptide.

[0028] According to an embodiment of the present invention, the C-terminus of the first binding fragment is connected to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is connected to the N-terminus of the second binding fragment, the C-terminus of the second binding fragment is connected to the N-terminus of the second connecting peptide, and the C-terminus of the second connecting peptide is connected to the N-terminus of the hinge region; or

[0029] The C-terminus of the second binding fragment is connected to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is connected to the N-terminus of the first binding fragment, the C-terminus of the first binding fragment is connected to the N-terminus of the second connecting peptide, and the C-terminus of the second connecting peptide is connected to the N-terminus of the hinge region.

[0030] Thus, the binding ability of the chimeric antigen receptor of the present invention to the NKp30 receptor ligand and the NKG2D receptor ligand is further improved.

[0031] According to an embodiment of the present invention, the second connecting peptide is selected from (G4S) n or at least one of the connecting peptides having an amino acid sequence as shown in SEQ ID NOs: 5, 34 to 39, wherein n is an integer other than zero.

[0032] According to an embodiment of the present invention, the amino acid sequence of the second connecting peptide is shown in SEQ ID NO: 5. Thus, a chimeric antigen receptor of the present invention having better binding ability to NKp30 receptor ligand and NKG2D receptor ligand is obtained.

[0033] According to an embodiment of the present invention, the transmembrane region includes the transmembrane segment of the CD8a molecule or a fragment thereof.

[0034] According to an embodiment of the present invention, the transmembrane segment of the CD8a molecule has an amino acid sequence as shown in SEQ ID NO: 11.

[0035] According to an embodiment of the present invention, the intracellular region includes a costimulatory domain and an intracellular signaling domain.

[0036] According to an embodiment of the present invention, the C-terminus of the costimulatory factor domain is connected to the N-terminus of the intracellular signaling domain.

[0037] According to an embodiment of the present invention, the co-stimulatory domain is selected from at least one of the intracellular segment of the CD28 molecule or a fragment thereof, the intracellular segment of the 4-1BB molecule or a fragment thereof, the intracellular segment of the 2B4 molecule or a fragment thereof, the intracellular segment of the DAP10 molecule or a fragment thereof, the intracellular segment of the DAP12 molecule or a fragment thereof, the intracellular segment of the CD27 molecule or a fragment thereof, the intracellular segment of the CD40 molecule or a fragment thereof, the intracellular segment of the OX40 molecule or a fragment thereof, and the intracellular segment of the ICOS molecule or a fragment thereof.

[0038] According to an embodiment of the present invention, the costimulatory domain is the intracellular segment of the 4-1BB molecule or a fragment thereof. Thus, the tumor killing activity of immune cells modified by the chimeric antigen receptor gene of the present invention can be further enhanced.

[0039] According to an embodiment of the present invention, the intracellular segment of the 4-1BB molecule or a fragment thereof has an amino acid sequence as shown in SEQ ID NO: 13.

[0040] According to an embodiment of the present invention, the intracellular signaling domain is the intracellular segment of the CD3ζ molecule or a fragment thereof, thereby further enhancing the tumor killing activity of immune cells modified with the chimeric antigen receptor gene of the present invention.

[0041] According to an embodiment of the present invention, the intracellular segment of the CD3ζ molecule has an amino acid sequence as shown in SEQ ID NO: 15.

[0042] According to an embodiment of the present invention, the chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO: 19 or 21. Thus, immune cells modified with the chimeric antigen receptor gene of the present invention have significantly enhanced tumor cell killing activity and in vivo tumor inhibition activity.

[0043] In a second aspect, the present invention provides a nucleic acid construct comprising: a first nucleic acid molecule encoding the aforementioned chimeric antigen receptor.

[0044] The nucleic acid construct according to an embodiment of the present invention may encode the aforementioned chimeric antigen receptor that simultaneously targets the NKp30 receptor ligand and the NKG2D receptor ligand.

[0045] According to an embodiment of the present invention, the first nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 20 or 22. Thus, the expression level of the chimeric antigen receptor of the present invention on immune cells is further improved.

[0046] According to an embodiment of the present invention, the nucleic acid construct further includes a second nucleic acid molecule, which is connected to the first nucleic acid molecule and encodes a first fusion protein or a second fusion protein, wherein the first fusion protein includes IL-15, the hinge region of the CD8a molecule, and the transmembrane region of the CD8a molecule; and the second fusion protein includes IL-15Rα and IL-15. This further promotes the activation and proliferation of immune cells expressing the aforementioned chimeric antigen receptor, maintains the number and activity of immune cells in the local tumor microenvironment, and further enhances tumor cell killing activity and in vivo tumor suppression activity.

[0047] According to an embodiment of the present invention, the C-terminus of the IL-15 of the first fusion protein is connected to the N-terminus of the hinge region of the CD8a molecule, and the C-terminus of the hinge region of the CD8a molecule is connected to the N-terminus of the transmembrane region of the CD8a molecule.

[0048] According to an embodiment of the present invention, the hinge region of the CD8a molecule has an amino acid sequence as shown in SEQ ID NO: 27. This promotes the first fusion protein to form a better conformation on the surface of immune cells.

[0049] According to an embodiment of the present invention, the transmembrane region of the CD8a molecule has an amino acid sequence as shown in SEQ ID NO: 11. Thus, the first fusion protein is expressed on the immune cell membrane.

[0050] According to an embodiment of the present invention, the first fusion protein has an amino acid sequence as shown in SEQ ID NO: 28. Thus, an immune cell that simultaneously expresses the chimeric antigen receptor and the first fusion protein is obtained.

[0051] According to an embodiment of the present invention, the second nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 29. Thus, the expression level of the chimeric antigen receptor and the first fusion protein of the present invention on immune cells is further improved.

[0052] According to an embodiment of the present invention, the C-terminus of IL-15Rα of the second fusion protein is connected to the N-terminus of IL-15, or the N-terminus of IL-15Rα is connected to the C-terminus of IL-15.

[0053] Herein, the term "second fusion protein" is equivalent to "mbIL15RF" or "super IL15", which refers to a fusion protein of IL-15Rα and IL-15.

[0054] According to an embodiment of the present invention, the second fusion protein further includes a third connecting peptide; the C-terminus of the IL-15Rα is connected to the N-terminus of the third connecting peptide, and the C-terminus of the third connecting peptide is connected to the N-terminus of the IL-15; or the C-terminus of the IL-15 is connected to the N-terminus of the third connecting peptide, and the C-terminus of the third connecting peptide is connected to the N-terminus of the IL-15Rα. In this way, a second fusion protein expressed on the cell membrane is obtained.

[0055] According to an embodiment of the present invention, the third connecting peptide is selected from (G4S)n or at least one of the connecting peptides having amino acid sequences as shown in SEQ ID NOs: 5, 34 to 39, where n is a non-zero integer.

[0056] According to an embodiment of the present invention, the third connecting peptide is selected from (G4S)n, where n is any integer between 2 and 6.

[0057] According to an embodiment of the present invention, the third connecting peptide is (G4S) 3. Thus, the second fusion protein forms a better conformation on the surface of immune cells.

[0058] According to an embodiment of the present invention, the IL-15Rα has an amino acid sequence as shown in SEQ ID NO: 25.

[0059] According to an embodiment of the present invention, the IL-15 has the amino acid sequence shown in SEQ ID NO: 26.

[0060] According to an embodiment of the present invention, the second fusion protein has an amino acid sequence as shown in SEQ ID NO: 23. Thus, an immune cell expressing the chimeric antigen receptor and the second fusion protein at the same time is obtained.

[0061] According to an embodiment of the present invention, the second nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 24. Thus, the expression level of the chimeric antigen receptor and the second fusion protein of the present invention on immune cells is further improved.

[0062] According to an embodiment of the present invention, the nucleic acid construct further comprises a third nucleic acid molecule, the third nucleic acid molecule encodes a first linker, and the first nucleic acid molecule and the second nucleic acid molecule are connected via the third nucleic acid molecule.

[0063] According to an embodiment of the present invention, the first linker is selected from at least one of P2A, T2A, E2A and F2A. Thus, immune cells can be obtained that uniformly express the chimeric antigen receptor and the first fusion protein or the second fusion protein on the cell membrane.

[0064] According to an embodiment of the present invention, the first linker is P2A, thereby further promoting the uniform expression of the chimeric antigen receptor and the first fusion protein or the second fusion protein on the cell membrane surface.

[0065] According to an embodiment of the present invention, the first linker has an amino acid sequence as shown in SEQ ID NO: 30. Thus, an immune cell is obtained in which the chimeric antigen receptor and the first fusion protein or the second fusion protein are uniformly expressed on the cell membrane.

[0066] According to an embodiment of the present invention, the third nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 31. Thus, the expression level of the chimeric antigen receptor and the first fusion protein or the second fusion protein of the present invention on immune cells is further improved.

[0067] According to an embodiment of the present invention, the nucleic acid construct has a nucleotide sequence as shown in SEQ ID NO: 32 or 33. Thus, the expression level of the chimeric antigen receptor and the first fusion protein or the second fusion protein of the present invention on immune cells is further improved.

[0068] In the third aspect of the present invention, an expression vector is provided, which carries the aforementioned nucleic acid construct.

[0069] According to an embodiment of the present invention, the expression vector is a non-pathogenic viral vector.

[0070] According to an embodiment of the present invention, the non-pathogenic virus is selected from one of retroviruses, chronic viruses and adenovirus-associated viruses.

[0071] According to an embodiment of the present invention, the non-pathogenic virus is a lentivirus. Thus, the constructed expression vector can express the chimeric antigen receptor of the present invention and / or the first fusion protein or the second fusion protein in a host cell.

[0072] When the nucleic acid molecule is linked to an expression vector, the nucleic acid molecule can be directly or indirectly linked to the control elements on the expression vector, as long as these control elements are capable of controlling the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the vector itself, or they can be exogenous, that is, not derived from the vector itself. Of course, it is sufficient that the nucleic acid molecule is operably linked to the control elements. According to an embodiment of the present invention, the expression vector is a non-pathogenic viral vector.

[0073] In this article, the term "vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and the inserted nucleic acid molecule is transferred into and / or between host cells. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for expression of the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell containing the vector.

[0074] According to an embodiment of the present invention, the nucleic acid can be obtained by operably linking the nucleic acid to a commercially available vector (such as a plasmid or viral vector). The vector in the present invention is not particularly limited, and commonly used plasmids can be used, such as pSeTag2, PEE14, pMH3, etc.

[0075] As used herein, the term "operably linked" refers to linking an exogenous gene to a vector so that control elements within the vector, such as transcriptional control sequences and translational control sequences, can function as intended to regulate the transcription and translation of the exogenous gene. Commonly used vectors include viral vectors, plasmids, bacteriophages, and the like. After the expression vectors according to some specific embodiments of the present invention are introduced into suitable immune cells, they can effectively express the aforementioned nucleic acid molecules under the mediation of a regulatory system, thereby achieving substantial expression of the protein encoded by the nucleic acid molecules on the cell membrane, thereby obtaining transgenic immune cells.

[0076] According to an embodiment of the present invention, the expression vector is a eukaryotic vector or a prokaryotic vector.

[0077] According to an embodiment of the present invention, the expression vector is a non-pathogenic viral vector.

[0078] According to an embodiment of the present invention, the non-pathogenic virus is selected from one of retrovirus, lentivirus and adenovirus-associated virus.

[0079] According to an embodiment of the present invention, the virus is a lentivirus.

[0080] In a fourth aspect, the present invention provides a transgenic immune cell that expresses the aforementioned chimeric antigen receptor or carries the aforementioned nucleic acid construct or expression vector.

[0081] The immune cells according to the embodiments of the present invention are obtained by transfecting or transforming the vector or transformant, and the cells can efficiently express the aforementioned chimeric antigen receptor or the aforementioned chimeric antigen receptor and the first fusion protein or the second fusion protein under appropriate conditions.

[0082] According to an embodiment of the present invention, the genetically modified immune cells are selected from at least one of T cells, NK cells, NKT cells, γδT cells, macrophages, peripheral blood NK cells, umbilical cord blood NK cells, NK-92 cells and iPSC-derived immune cells. The aforementioned chimeric antigen receptor, the first fusion protein or the second fusion protein of the present invention can be transduced into immune cells such as T, NK, NKT, γδT, macrophages, iPSCs through an expression vector (such as a lentiviral vector), thereby being expressed on the surface of these immune cells.

[0083] According to an embodiment of the present invention, the transgenic immune cells are NK cells.

[0084] The present invention does not impose strict restrictions on the source of NK cells, which can be peripheral blood NK cells, umbilical cord blood NK cells, iPSCs, NK-92 cells, etc.

[0085] In a fourth aspect of the present invention, a pharmaceutical composition is provided. The pharmaceutical composition comprises:

[0086] The aforementioned chimeric antigen receptor, the aforementioned nucleic acid construct, the aforementioned expression vector or the aforementioned transgenic immune cell.

[0087] According to an embodiment of the present invention, the pharmaceutical composition further comprises: a pharmaceutically acceptable excipient.

[0088] As used herein, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and thoroughly combining the active compound with a liquid carrier, a solid carrier, or both.

[0089] As used herein, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the particular intended dosage form. Except to the extent that any conventional excipient is incompatible with the chimeric antigen receptor, nucleic acid, expression vector, or transgenic immune cell of the present invention, such as by producing any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present invention.

[0090] Those skilled in the art will appreciate that the features and advantages described above for the chimeric antigen receptor, nucleic acid construct, expression vector, and transgenic immune cells are also applicable to the pharmaceutical composition and will not be described in detail here.

[0091] In the fifth aspect of the present invention, the present invention proposes the use of the aforementioned chimeric antigen receptor, the aforementioned nucleic acid construct, the aforementioned expression vector, the aforementioned transgenic immune cell or the aforementioned pharmaceutical composition in the preparation of a drug for preventing or treating tumors or cancer.

[0092] According to an embodiment of the present invention, the tumor is a solid tumor or a hematological tumor.

[0093] In some specific embodiments of the present invention, the cancer is selected from one of leukemia, lymphoma, multiple myeloma, liver cancer, bile duct cancer, esophageal cancer, lung cancer, head and neck cancer, thyroid cancer, glioma, cervical cancer, ovarian cancer, gastric cancer, gastric sarcoma, osteosarcoma, breast cancer, pancreatic cancer, melanoma, colorectal cancer, kidney cancer and prostate cancer.

[0094] In a specific embodiment of the present invention, the cancer is colorectal cancer or leukemia.

[0095] Those skilled in the art will appreciate that the features and advantages described above for the chimeric antigen receptor, nucleic acid construct, expression vector, and transgenic immune cell are also applicable to this use and will not be elaborated here.

[0096] In another aspect, the present invention also provides a method for preventing and / or treating tumors or cancers. According to an embodiment of the present invention, the method comprises: administering a pharmaceutically acceptable amount of the above-mentioned transgenic immune cells or the above-mentioned pharmaceutical composition to a subject.

[0097] According to an embodiment of the present invention, the tumor is a solid tumor or a hematological tumor.

[0098] In some specific embodiments of the present invention, the tumor or cancer is selected from at least one of leukemia, lymphoma, multiple myeloma, liver cancer, bile duct cancer, esophageal cancer, lung cancer, head and neck cancer, thyroid cancer, glioma, cervical cancer, ovarian cancer, gastric cancer, gastric sarcoma, osteosarcoma, breast cancer, pancreatic cancer, melanoma, colorectal cancer, kidney cancer and prostate cancer.

[0099] In a specific embodiment of the present invention, the cancer is colorectal cancer or leukemia.

[0100] As used herein, the term "administer" refers to the introduction of a predetermined amount of a substance into a patient via a suitable means. The transgenic immune cells or pharmaceutical compositions of the present invention may be administered via any common route, provided they reach the intended tissue. Various modes of administration are contemplated, including intraperitoneal, intravenous, intramuscular, and subcutaneous injections, but the present invention is not limited to these exemplified modes of administration. Preferably, the compositions of the present invention are administered via intravenous injection.

[0101] As used herein, the term "treatment" refers to any agent used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or transgenic immune cell to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing cells containing a chimeric antigen receptor as described herein to an individual in need.

[0102] The effective amount of the transgenic immune cells and pharmaceutical compositions described herein may vary depending on the mode of administration and the severity of the disease to be treated. Preferably, the selection of the effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, and the like. For example, depending on the urgency of the treatment, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0103] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0104] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0106] FIG1 is a schematic diagram of the structures of different chimeric antigen receptors according to Example 1 of the present invention;

[0107] FIG2 is a statistical graph showing the killing efficiency of different CAR-NK cells against colorectal cancer NCI-H716 cells according to Example 2 of the present invention;

[0108] FIG3 is a statistical diagram showing the killing efficiency of different CAR-NK cells against blood tumor K562 cells according to Example 2 of the present invention;

[0109] FIG4 is a schematic diagram of the structure of a CAR containing membrane-expressed IL15 or super IL15 according to Example 3 of the present invention, wherein (A) is a schematic diagram of the structure of a CAR containing membrane-expressed IL15; (B) is a schematic diagram of the structure of a CAR containing super IL15;

[0110] Figure 5 is a graph showing the expansion fold change of NK-92 and CAR5-NK-92 cells in Example 3 of the present invention at different IL-2 culture concentrations, wherein (A) CAR5-NK-92 membrane expresses IL15, and (B) CAR5-NK-92 membrane expresses super IL15;

[0111] Figure 6 is a graph showing the survival rate changes of NK-92 and CAR5-NK-92 cells at different IL-2 culture concentrations according to Example 3 of the present invention, wherein (A) the CAR5-NK-92 membrane expresses IL15, and (B) the CAR5-NK-92 membrane expresses super IL15;

[0112] Figure 7 shows the tumor inhibition effect of peripheral blood-derived CAR5-NK cells in Example 4 of the present invention on the human colorectal cancer NCI-H716 cell-bearing mouse model, wherein (A) the CAR5-NK-92 membrane expresses IL15, and (B) the CAR5-NK-92 membrane expresses super IL15;

[0113] FIG8 is a graph showing the statistical results of the killing efficiency of CAR4-1-NK cells against NCI-H716 cells according to Example 5 of the present invention;

[0114] FIG9 is a graph showing the statistical results of the killing efficiency of CAR5-1-NK cells against NCI-H716 cells according to Example 6 of the present invention. DETAILED DESCRIPTION

[0115] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0116] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0117] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0118] Terms and Definitions

[0119] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0120] As used herein, the terms "according to embodiments of the present invention," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0121] In this article, the term "(G4S) n " is equivalent to "(Gly4Ser)n", which means 4 glycines and 1 serine are repeated n times. It is a widely used type of linker peptide that can be located at V H C-terminal and V L N terminal, can also be located at V L C-terminal and V H Between the N-termini, (G4S)3 is commonly used. Glycine, the amino acid with the smallest molecular weight and shortest side chain, increases side chain flexibility, while serine, the most hydrophilic amino acid, increases the hydrophilicity of the linker. Therefore, (G4S)3 exhibits excellent stability and activity. Linkers of varying lengths and sequences can be designed to construct scFvs with diverse biological functions.

[0122] As used herein, the term "hinge region" refers to the heavy chain region connecting CH1 and CH2 (i.e., connecting Fab and Fc) in an antibody. It is sometimes also referred to as the "Fc hinge region" herein.

[0123] As used herein, "carbon terminus" and "C-terminus" are synonymous; "nitrogen terminus" and "N-terminus" are synonymous.

[0124] The sequences involved in the present invention are detailed in Table 1.

[0125] Table 1: Nucleotide / amino acid sequence description

[0126] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0127] It should be noted that the "plasmid" and "vector" described in the following embodiments have the same meaning and can be used interchangeably.

[0128] Experimental Example 1: Preparation of NK cells expressing different CAR structures

[0129] 1. Construction of different CAR structures

[0130] Different CAR sequences were synthesized by whole gene synthesis and cloned into the lentiviral vector pCDH-EF1-MCS-TA2-copGFP through the restriction sites XbaI and BamHI. After sequencing verification, the pCDH-EF1a-CAR1, pCDH-EF1a-CAR2, pCDH-EF1a-CAR3 and pCDH-EF1a-CAR4 plasmids were obtained.

[0131] The schematic diagram of the gene element structure of the chimeric antigen receptor of this example is shown in Figure 1, and the nucleotide / amino acid sequence is shown in Table 1. Among them, the nucleotide sequences of CAR1, CAR2, CAR3 and CAR4 are shown in SEQ ID NOs: 40-41, 20, and 22.

[0132] 2. Lentivirus packaging

[0133] 5×10 293T cells in the logarithmic growth phase were taken 6 Inoculate in a 10cm cell culture dish, add 10mL DMEM medium, and culture overnight in a 37℃, 5% CO2 incubator. When the cell density in the cell culture dish reaches 80-90%, replace with 10mL fresh DMEM medium, and continue to place the cell culture dish in the incubator for standby use. Prepare the lentiviral packaging system, add 6μg psPAX2 and 3μg pMD2.G lentiviral packaging auxiliary plasmids, and 6μg target gene vector plasmids to 250μL serum-free DMEM medium to prepare a plasmid mixture, and mix evenly. Add to 235 μL serum-free DMEM medium and mix well. Add the mixed solution to the above plasmid mixture at one time, mix well, and incubate at room temperature for 15 minutes. Add the mixed solution to the 293T cell culture dish. Change the medium after 24 hours, put the culture dish back into the 37°C, 5% CO2 incubator, collect the cell supernatant after 48 hours, centrifuge at 400×g for 5 minutes, remove cell debris, and filter the supernatant with a 0.45μm filter into a 50mL centrifuge tube. Add 5×PEG8000 solution to concentrate the virus solution, mix evenly by inverting the centrifuge tube, and place it in a 4°C refrigerator overnight. Centrifuge at 4000×g for 20 minutes at 4°C, discard the supernatant, add an appropriate amount of serum-free DMEM to resuspend the virus pellet, transfer it to an EP tube, and store it in a -80°C refrigerator.

[0134] 3. Lentiviral infection of human NK-92 cells

[0135] NK-92 cells in the logarithmic growth phase were harvested by centrifugation at 100 × g for 5 min, and appropriate amount of α-MEM medium was added to resuspend the cells to adjust the cell density to 5 × 10 5 5×10 5 NK-92 cells, 0.2 mL of virus concentrate, 0.8 mL of α-MEM medium, and protamine (final concentration 8 μg / mL) were mixed thoroughly. The cells were cultured in a 37°C, 5% CO2 incubator. After 24 hours, the cells were observed, the medium was changed, and the infected cells were transferred to an EP tube. The cells were centrifuged at 100×g for 5 minutes. A small amount of fresh α-MEM medium was added to resuspend the cells. The cells were transferred to a cell culture flask, and 10 mL of fresh α-MEM medium and IL-2 (final concentration 200 IU / mL) were added and cultured for another 48 hours. The cells were transferred to a flow cytometer, 3 mL of 1× PBS solution was added, and the cells were centrifuged at 100×g for 5 minutes. The supernatant was discarded, the cell pellet was flicked, and the cells were washed again with 1× PBS solution. GFP expression was detected using a flow cytometer. The culture was continued to expand, and the infected NK-92 cells were adjusted for amplification. The infected NK-92 cells were sorted for GFP-positive CAR-NK92 cells by flow cytometry for later experiments.

[0136] The inventors further observed through experiments that the CAR3-NK92 cells and CAR4-NK92 cells of this embodiment can effectively kill tumor cells that express only the NKp30 receptor ligand or the NKG2D receptor ligand or both receptor ligands. In particular, they can effectively identify tumor cells that have lost one of the NKp30 or NKG2D receptor ligands, thereby preventing tumor immune escape.

[0137] Experimental Example 2: Comparison of killing efficiency of different CAR structures

[0138] In this example, the inventors investigated the killing activity of different CAR-NK cells obtained in Example 1 in vitro, verified the advantages of dual-targeting over single-targeting, and selected the CAR structure with the best killing activity.

[0139] The inventors selected colorectal cancer cell line NCI-H716 cells and erythroleukemia cell line K562 cells, which highly express both NKG2D ligands MICA / B and NKp30 ligand B7-H6, as target cells, and performed in vitro killing activity tests on different CAR-NK cells.

[0140] The specific method is as follows: NCI-H716 cells or K562 cells were fluorescently labeled with CFSE and the cells were stained with 2x10 4Cells were plated into 96-well plates, and NK-92 or different CAR-NK-92 cells were then plated and incubated with NCI-H716 or K562 cells for 4 hours. Propidium Iodide (PI) was then added to distinguish live and dead cells, and the killing efficiency was measured by flow cytometry. A 2:1 ratio of effector cells to target cells was used.

[0141] The test results are shown in Figures 2 and 3.

[0142] The results of the investigation of the killing activity of different CAR structures showed that compared with single-targeted CAR1-NK-92 or CAR2-NK-92 cells, the killing efficiency of dual-targeted CAR3-NK-92 and CAR4-NK-92 cells against NCI-H716 cells and K562 cells was significantly enhanced.

[0143] Example 3: Investigation of the effect of membrane-expressed IL15 elements on the proliferation and survival of CAR-NK cells

[0144] 1. Construction of CAR sequence containing membrane-expressing IL15 element

[0145] In this example, the inventors used the CAR3 of Example 1 as an example, added a membrane-expressing IL15 element to the CAR3 sequence, and designed and constructed the CAR5 gene. The CAR5 gene was synthesized by whole gene synthesis and cloned into the lentiviral vector pCDH-EF1-MCS-TA2-copGFP through the restriction sites XbaI and BamHI. After sequencing verification, the pCDH-EF1a-CAR5 plasmid was obtained.

[0146] The schematic diagram of the gene element structure of the chimeric antigen receptor of this example is shown in Figure 4, and the nucleotide / amino acid sequence is shown in Table 1. Among them, the nucleotide sequences of CAR5A and CAR5B are shown in SEQ ID NOs: 32 and 33.

[0147] 2. Investigation of CAR5 gene modification promoting NK cell proliferation activity

[0148] The inventors further verified the effect of membrane-expressed IL15 on promoting the proliferation of NK-92 cells in vitro.

[0149] The specific method is as follows: 3×10 6 NK-92 and CAR5-NK-92 cells were plated in T25 cell culture flasks and different IL-2 concentrations (200 IU / ml and 0 IU / ml) were set. Cell counts were performed every 2 days. 3×10 6 The cells were transferred to a new T25 cell culture flask and IL-2 of corresponding concentration was added to start the next round of cell culture and counting. 6If cells are present, all cells are transferred into new T25 cell culture flasks.

[0150] The results of the NK cell proliferation fold increase on day 10 of culture are shown in Figure 5. In Figure 5 (A), the CAR5-NK-92 membrane expressed IL15, and in Figure 5 (B), the CAR5-NK-92 membrane expressed super IL15.

[0151] The results of the NK cell proliferation activity examination showed that at a concentration of 200 IU / mL IL-2, the expansion multiple of CAR5-NK-92 cells was significantly higher than that of the NK-92 cell group; at a concentration of 0 IU / mL IL-2, the expansion multiple of CAR5-NK-92 cells was also significantly higher than that of the NK-92 cell group, while the NK-92 cell group had basically no expansion.

[0152] The above results show that IL15 expressed on the membrane of CAR5-NK-92 plays an important role in promoting the expansion of NK cells, can further enhance the expansion capacity of NKR-NK cells in vivo, and can reduce the dependence of NK cell expansion on IL-2.

[0153] 3. Investigation of CAR5 gene modification to promote NK cell survival

[0154] The inventors further verified the effect of membrane-expressed IL15 on promoting the survival of NK-92 cells in vitro.

[0155] The specific method is as follows: NK-92 and CAR5-NK-92 cells of the same cell number were plated in a 24-well plate, and different IL-2 concentrations (200 IU / ml and 0 IU / ml) were set. The apoptosis rate was detected by flow cytometry every 24 hours. The flow cytometry detection of apoptosis rate was performed according to the steps in the kit instructions (Lianke Bio, product number AP101). Briefly, the cells were collected in an EP tube, centrifuged and washed once with 1xPBS solution, and the cells were resuspended. 5 μL Annexin V-FITC and 10 μL PI were added to each tube. After gentle vortex mixing, incubate at room temperature in the dark for 5 minutes, and resuspend the cells for flow cytometry. The cell viability is the proportion of double-negative Annexin V-FITC and PI staining.

[0156] The results of the NK cell survival rate test on day 8 of culture are shown in Figure 6. In Figure 6 (A), the CAR5-NK-92 membrane expressed IL15, and in Figure 6 (B), the CAR5-NK-92 membrane expressed super IL15.

[0157] The results of NK cell survival rate investigation showed that in the absence of IL-2 (IL-2 0 IU / ml), the cell survival rate of the NK-92 group decreased significantly from the 4th day, and most cells had undergone apoptosis by the 8th day; while the cells in the CAR5-NK-92 group could still maintain a high cell viability even under the condition of complete withdrawal of IL-2, and few cells underwent apoptosis.

[0158] The above results show that IL15 expressed on the membrane of CAR5-NK-92 plays an important role in promoting the survival of NK cells, which can further improve the long-term survival of NKR-NK cells in vivo and reduce the dependence of NK cell survival on IL-2.

[0159] Example 4: Investigation of the tumor inhibitory activity of primary CAR5-NK cells in vivo

[0160] The inventors established a subcutaneous tumor-bearing model in mice using human colorectal cancer NCI-H716 cells, infected primary human peripheral blood NK cells with CAR5 lentivirus to prepare peripheral blood-derived CAR-NK cells, and observed the therapeutic effect of primary CAR5-NK cells on the colorectal cancer tumor-bearing model.

[0161] The specific method is as follows: 6-week-old NCG mice were selected for subcutaneous tumor bearing in the armpits, and the tumor volume reached 50 mm. 3 NK cell therapy was initiated at 4 hr. Tumor volume was measured before treatment, and the mice were randomly divided into the untreated group, the NK cell treatment group, and the CAR5-NK cell treatment group according to tumor volume. In the NK cell treatment group, NK cells were infused through the tail vein. Mice were treated once every 2 days for a total of 3 times, with a tail vein infusion dose of 8 × 10 6 CD56 + NK cells were injected intraperitoneally every 2 days to maintain NK cell survival; in the CAR5-NK cell treatment group, CAR5-NK cells were reinfused through the tail vein for a single treatment at a dose of 4×10 6 CAR5-NK cells / cell, without IL-2 injection. Tumor volume was continuously observed and measured, and a tumor growth curve was drawn.

[0162] The experimental results are shown in Figure 7. In Figure 7 (A), the CAR5-NK-92 membrane expressed IL15, and in Figure 7 (B), the CAR5-NK-92 membrane expressed super IL15.

[0163] The results of in vivo tumor inhibition activity investigation showed that the peripheral blood-derived CAR5-NK cells prepared based on the CAR5 of the present invention had a significantly enhanced effect of inhibiting tumor growth compared with the uninfected NK cell treatment group (control group, NK cells did not carry any form of CAR or CSR), and the dose of reinfused NK cells was lower.

[0164] The above results show that the CAR5-NK cells of the present invention have a strong anti-tumor effect on colorectal cancer tumors, and the application dose is lower, which is expected to break through the bottleneck of poor treatment effect of immune cell therapy on solid tumors.

[0165] Example 5

[0166] According to the method of Example 1, the connection relationship between the first binding fragment and the second binding fragment of the extracellular region was constructed as "NKp30 extracellular segment-linker 2-NKG2D extracellular segment-Fc hinge region-CD8a transmembrane segment-4-1BB intracellular segment-CD3ζ intracellular segment" (CAR4-1) CAR structure, and NK92 cells expressing CAR4-1 (CAR4-1-NK-92) were prepared. According to the method of Example 2, the killing efficiency of CAR4-1-NK-92 and CAR4-NK-92 cells on NCI-H716 cells was investigated, and the results were statistically analyzed (Figure 8).

[0167] The results showed that the killing efficiency of CAR4-1-NK-92 cells against NCI-H716 cells was comparable to that of CAR4-NK-92 cells.

[0168] Example 6

[0169] According to the method of Example 1, a CAR structure with a connection relationship of each element of "NKG2D extracellular segment-linker 2-NKp30 extracellular segment-linker 1-Fc hinge region-CD8a transmembrane segment-4-1BB intracellular segment-CD3ζ intracellular segment" (CAR3-1) was constructed, and NK92 cells expressing CAR3-1 (CAR3-1-NK-92) were prepared. According to the method of Example 2, the killing efficiency of CAR3-1-NK-92 and CAR3-NK-92 cells on NCI-H716 cells was investigated, and the results were statistically analyzed (Figure 9).

[0170] The results showed that the killing efficiency of CAR3-1-NK-92 cells against NCI-H716 cells was comparable to that of CAR3-NK-92 cells.

[0171] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0172] Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limiting the present invention. A person skilled in the art may change, modify, replace, and modify the above embodiments within the scope of the present invention.

Claims

1. A chimeric antigen receptor, characterized in that include: An extracellular region, wherein the extracellular region includes a first binding fragment and a second binding fragment, wherein the first binding fragment and the second binding fragment are connected, wherein the first binding fragment is an extracellular segment of NKp30 or a fragment thereof, and the second binding fragment is an extracellular segment of NKG2D or a fragment thereof; A transmembrane region, wherein the N-terminus of the transmembrane region is connected to the C-terminus of the extracellular region; The intracellular region has an N-terminus connected to the C-terminus of the transmembrane region.

2. The chimeric antigen receptor according to claim 1, characterized in that The first binding fragment has an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence of a conservative modification thereof, and the second binding fragment has an amino acid sequence as shown in SEQ ID NO: 3 or an amino acid sequence of a conservative modification thereof.

3. The chimeric antigen receptor according to claim 1, characterized in that The C-terminus of the first binding fragment is connected to the N-terminus of the second binding fragment, or the N-terminus of the first binding fragment is connected to the C-terminus of the second binding fragment.

4. The chimeric antigen receptor according to claim 1, characterized in that The extracellular region further comprises a first connecting peptide, and the first binding fragment and the second binding fragment are connected by the first connecting peptide; The C-terminus of the first binding fragment is connected to the N-terminus of the first connecting peptide, and the C-terminus of the first connecting peptide is connected to the N-terminus of the second binding fragment; or The C-terminus of the second binding fragment is connected to the N-terminus of the first connecting peptide, and the C-terminus of the first connecting peptide is connected to the N-terminus of the first binding fragment.

5. The chimeric antigen receptor according to claim 4, characterized in that The first connecting peptide is selected from (G4S) n Or at least one of the connecting peptides having an amino acid sequence as shown in SEQ ID NOs: 5, 34 to 39, wherein n is an integer other than zero.

6. The chimeric antigen receptor according to claim 5, characterized in that The first connecting peptide is (G4S) n , n is any integer between 2 and 6.

7. The chimeric antigen receptor according to claim 5, characterized in that The first connecting peptide is (G4S)3.

8. The chimeric antigen receptor according to claim 1, characterized in that The extracellular region further includes a hinge region.

9. The chimeric antigen receptor according to claim 8, characterized in that The C-terminus of the first binding fragment is connected to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is connected to the N-terminus of the second binding fragment, and the C-terminus of the second binding fragment is connected to the N-terminus of the hinge region; or The C-terminus of the second binding fragment is connected to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is connected to the N-terminus of the first binding fragment, and the C-terminus of the first binding fragment is connected to the N-terminus of the hinge region.

10. The chimeric antigen receptor according to claim 8, characterized in that The hinge region has an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence of a conservatively modified form thereof.

11. The chimeric antigen receptor according to claim 8, characterized in that The extracellular region further comprises a second connecting peptide; The C-terminus of the first binding fragment is connected to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is connected to the N-terminus of the second binding fragment, the C-terminus of the second binding fragment is connected to the N-terminus of the second connecting peptide, and the C-terminus of the second connecting peptide is connected to the N-terminus of the hinge region; or The C-terminus of the second binding fragment is connected to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is connected to the N-terminus of the first binding fragment, the C-terminus of the first binding fragment is connected to the N-terminus of the second connecting peptide, and the C-terminus of the second connecting peptide is connected to the N-terminus of the hinge region.

12. The chimeric antigen receptor according to claim 11, characterized in that The second connecting peptide is selected from (G4S) n Or at least one of the connecting peptides having an amino acid sequence as shown in SEQ ID NOs: 5, 34 to 39, wherein n is an integer other than zero.

13. The chimeric antigen receptor according to claim 11, characterized in that The amino acid sequence of the second connecting peptide is shown in SEQ ID NO:

5.

14. The chimeric antigen receptor according to claim 1, characterized in that The transmembrane region includes the transmembrane segment of the CD8a molecule or a fragment thereof.

15. The chimeric antigen receptor according to claim 14, characterized in that The transmembrane segment of the CD8a molecule has an amino acid sequence as shown in SEQ ID NO:

11.

16. The chimeric antigen receptor according to claim 1, characterized in that The intracellular region includes a co-stimulatory domain and an intracellular signaling domain; The C-terminus of the co-stimulatory domain is connected to the N-terminus of the intracellular signaling domain.

17. The chimeric antigen receptor according to claim 16, characterized in that The co-stimulatory domain is selected from at least one of the intracellular segment of the CD28 molecule or a fragment thereof, the intracellular segment of the 4-1BB molecule or a fragment thereof, the intracellular segment of the 2B4 molecule or a fragment thereof, the intracellular segment of the DAP10 molecule or a fragment thereof, the intracellular segment of the DAP12 molecule or a fragment thereof, the intracellular segment of the CD27 molecule or a fragment thereof, the intracellular segment of the CD40 molecule or a fragment thereof, the intracellular segment of the OX40 molecule or a fragment thereof, and the intracellular segment of the ICOS molecule or a fragment thereof.

18. The chimeric antigen receptor according to claim 16, characterized in that The co-stimulatory domain is the intracellular segment of the 4-1BB molecule or a fragment thereof.

19. The chimeric antigen receptor according to claim 18, characterized in that The intracellular segment of the 4-1BB molecule or a fragment thereof has an amino acid sequence as shown in SEQ ID NO:

13.

20. The chimeric antigen receptor according to claim 16, characterized in that The intracellular signal transduction domain is the intracellular segment of the CD3ζ molecule or a fragment thereof.

21. The chimeric antigen receptor according to claim 20, characterized in that The intracellular segment of the CD3ζ molecule has an amino acid sequence as shown in SEQ ID NO:

15.

22. The chimeric antigen receptor according to claim 1, characterized in that The chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO: 19 or 21.

23. A nucleic acid construct, characterized in that It comprises a first nucleic acid molecule, wherein the first nucleic acid molecule encodes the chimeric antigen receptor according to any one of claims 1 to 22.

24. The nucleic acid construct according to claim 23, characterized in that The first nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 20 or 22.

25. The nucleic acid construct according to claim 23, characterized in that Further comprising a second nucleic acid molecule, the second nucleic acid molecule is connected to the first nucleic acid molecule, the second nucleic acid molecule encodes a first fusion protein or a second fusion protein, the first fusion protein comprises IL-15, a hinge region of a CD8a molecule and a transmembrane region of a CD8a molecule; the second fusion protein comprises IL-15Rα and IL-15; The C-terminus of the IL-15 of the first fusion protein is connected to the N-terminus of the hinge region of the CD8a molecule, and the C-terminus of the hinge region of the CD8a molecule is connected to the N-terminus of the transmembrane region of the CD8a molecule.

26. The nucleic acid construct according to claim 25, characterized in that The hinge region of the CD8a molecule has an amino acid sequence as shown in SEQ ID NO: 27; The transmembrane region of the CD8a molecule has an amino acid sequence as shown in SEQ ID NO:

11.

27. The nucleic acid construct according to claim 25, characterized in that The first fusion protein has the amino acid sequence shown in SEQ ID NO:

28.

28. The nucleic acid construct according to claim 25, characterized in that The second nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:

29.

29. The nucleic acid construct according to claim 25, characterized in that The C-terminus of IL-15Rα of the second fusion protein is connected to the N-terminus of IL-15, or the N-terminus of IL-15Rα is connected to the C-terminus of IL-15.

30. The nucleic acid construct according to claim 25, characterized in that The second fusion protein further comprises a third connecting peptide; the C-terminus of the IL-15Rα is connected to the N-terminus of the third connecting peptide, and the C-terminus of the third connecting peptide is connected to the N-terminus of the IL-15; or The C-terminus of the IL-15 is connected to the N-terminus of the third connecting peptide, and the C-terminus of the third connecting peptide is connected to the N-terminus of the IL-15Rα.

31. The nucleic acid construct according to claim 30, characterized in that The third connecting peptide is selected from (G4S)n or at least one of the connecting peptides having amino acid sequences as shown in SEQ ID NOs: 5, 34 to 39, wherein n is a non-zero integer.

32. The nucleic acid construct according to claim 31, characterized in that The third connecting peptide is selected from (G4S)n, where n is any integer between 2 and 6.

33. The nucleic acid construct according to claim 31, characterized in that The third connecting peptide is (G4S)3.

34. The nucleic acid construct according to claim 25, characterized in that The IL-15Rα has an amino acid sequence as shown in SEQ ID NO: 25; The IL-15 has the amino acid sequence shown in SEQ ID NO:

26.

35. The nucleic acid construct according to claim 25, characterized in that The second fusion protein has the amino acid sequence shown in SEQ ID NO:

23.

36. The nucleic acid construct according to claim 25, characterized in that The second nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:

24.

37. The nucleic acid construct according to claim 25, characterized in that It also includes a third nucleic acid molecule, which encodes a first linker, and the first nucleic acid molecule and the second nucleic acid molecule are connected through the third nucleic acid molecule.

38. The nucleic acid construct according to claim 37, characterized in that The first linker is selected from at least one of P2A, T2A, E2A and F2A.

39. The nucleic acid construct according to claim 37, characterized in that The first linker is P2A.

40. The nucleic acid construct according to claim 37, characterized in that The first linker has an amino acid sequence as shown in SEQ ID NO:

30.

41. The nucleic acid construct according to claim 37, characterized in that The third nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:

31.

42. The nucleic acid construct according to claim 37, which has a nucleotide sequence as shown in SEQ ID NO: 32 or 33.

43. An expression vector, characterized in that Carrying the nucleic acid construct according to any one of claims 23 to 42.

44. The expression vector according to claim 43, characterized in that The expression vector is a non-pathogenic viral vector.

45. The expression vector according to claim 44, characterized in that The non-pathogenic virus is selected from one of a retrovirus, a lentivirus and an adenovirus-associated virus.

46. ​​The expression vector according to claim 44, characterized in that The non-pathogenic virus is a lentivirus.

47. A transgenic immune cell, characterized in that: The transgenic immune cells include: Expressing the chimeric antigen receptor according to any one of claims 1 to 22; or Carrying the nucleic acid construct according to any one of claims 23 to 42 and the expression vector according to any one of claims 43 to 46.

48. The transgenic immune cell according to claim 47, characterized in that The transgenic immune cells are selected from at least one of T cells, NK cells, NKT cells, γδT cells, macrophages, peripheral blood NK cells, umbilical cord blood NK cells, NK-92 cells and any one of the above immune cells derived from iPSC.

49. The transgenic immune cell according to claim 47, characterized in that The transgenic immune cells are NK cells.

50. A pharmaceutical composition, characterized in that include: The chimeric antigen receptor according to any one of claims 1 to 22, the nucleic acid construct according to any one of claims 23 to 42, the expression vector according to any one of claims 43 to 46, or the transgenic immune cell according to any one of claims 47 to 49.

51. The pharmaceutical composition according to claim 50, characterized in that Further including: Pharmaceutically acceptable excipients.

52. Use of the chimeric antigen receptor of any one of claims 1 to 22, the nucleic acid construct of any one of claims 23 to 42, the expression vector of any one of claims 43 to 46, the transgenic immune cell of any one of claims 47 to 49, or the pharmaceutical composition of claim 50 or 51 in the preparation of a drug for preventing and / or treating tumors or cancer.

53. The use according to claim 52, characterized in that The tumor is a solid tumor or a blood tumor.

54. The use according to claim 52, characterized in that The tumor or cancer is selected from at least one of leukemia, lymphoma, multiple myeloma, liver cancer, bile duct cancer, esophageal cancer, lung cancer, head and neck cancer, thyroid cancer, glioma, cervical cancer, ovarian cancer, gastric cancer, gastric sarcoma, osteosarcoma, breast cancer, pancreatic cancer, melanoma, colorectal cancer, kidney cancer and prostate cancer.

55. The use according to claim 52, characterized in that The cancer is colorectal cancer or leukemia.

56. A method for treating and / or preventing tumors or cancers, characterized in that: A pharmaceutically acceptable amount of the transgenic immune cell according to any one of claims 47 to 49 or the pharmaceutical composition according to any one of claims 50 or 51 is administered to a subject.

57. The method according to claim 56, characterized in that The tumor is a solid tumor or a blood tumor.

58. The method according to claim 56, characterized in that The tumor or cancer is selected from at least one of leukemia, lymphoma, multiple myeloma, liver cancer, bile duct cancer, esophageal cancer, lung cancer, head and neck cancer, thyroid cancer, glioma, cervical cancer, ovarian cancer, gastric cancer, gastric sarcoma, osteosarcoma, breast cancer, pancreatic cancer, melanoma, colorectal cancer, kidney cancer and prostate cancer.

59. The method according to claim 56, characterized in that The cancer is colorectal cancer or leukemia.

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