CN117467022B - Chimeric antigen receptor and uses thereof - Google Patents

Abstract

The invention provides chimeric antigen receptor capable of recognizing two antigen ligands simultaneously, corresponding nucleic acid construct, expression vector, transgenic immune cell, pharmaceutical composition and application thereof, wherein the chimeric antigen receptor comprises: an extracellular region comprising a first binding fragment and a second binding fragment, the first binding fragment and the second binding fragment being linked, the first binding fragment being an NKp30 extracellular segment or a fragment thereof, the second binding fragment being an NKG2D extracellular segment or a fragment thereof; the N end of the transmembrane region is connected with the C end of the extracellular region; and an intracellular region, wherein the N-terminal of the intracellular region is connected with the C-terminal of the transmembrane region. The chimeric antigen receptor of the invention simultaneously targets the NKp30 receptor ligand and the NKG2D receptor ligand, and the transgenic immune cells expressing the chimeric antigen receptor of the invention can effectively identify the tumor cells expressing the NKp30 receptor ligand and/or the NKG2D receptor ligand, namely can effectively kill the tumor cells which singly express the NKp30 receptor ligand or the NKG2D receptor ligand or simultaneously express the two receptor ligands, and especially can effectively identify the tumor cells losing one of the NKp30 or the NKG2D receptor ligand, thereby preventing the immune escape of tumors. Therefore, the transgenic immune cell provided by the invention has the advantages of high tumor cell killing activity, wide recognition spectrum, strong tumor inhibiting activity in vivo, low cell infusion dosage and good curative effect.

Description

Chimeric antigen receptor and its application

Technical Field

The present invention relates to the field of biotechnology, specifically, the present invention relates to a chimeric antigen receptor and its application, more specifically, the present invention relates to a chimeric antigen receptor capable of simultaneously recognizing two antigen ligands and a corresponding nucleic acid construct, an expression vector, a transgenic immune cell, a pharmaceutical composition and its use.

Background Art

Chimeric antigen receptor T (CAR-T) cells have achieved remarkable results in the treatment of hematological malignancies. The antigen recognition region of the CAR molecule usually uses the scFv sequence from the antibody, which is usually from species such as mice, rabbits, and alpacas and has strong immunogenicity. Even if the sequence is humanized, it still has a certain immunogenicity, producing anti-drug antibodies (ADA) and weakening the persistence of CAR-T cells in the body. In recent years, many new technologies have been applied to solve this problem, such as using natural receptor protein sequences as receptors to reduce immunogenicity.

The NKp30 receptor is a natural NK cell activating receptor that is highly expressed on NK cells and can recognize the B7H6 molecule, thereby mediating cytotoxicity. B7H6 is a member of the B7 family. The B7H6 molecule is usually highly expressed on a variety of primary tumor cells, including leukemia, lymphoma, colorectal cancer, gastric cancer, and pancreatic cancer, but is almost not expressed on normal tissue cells. At present, the function of CAR-T cells constructed based on the NKp30 extracellular segment as a receptor for B7H6 recognition has been verified in research.

NKG2D receptor is another natural NK cell surface receptor, mainly expressed on the surface of NK, CD8+T, γδT, NKT and activated macrophages, and recognizes stress proteins expressed by viral infection or cell malignant transformation, such as MHC-I class-related proteins, MICA and MICB, and cytomegalovirus UL-16 binding protein ULBP1-5. In the patent CN109400713A, NKG2D is used as an intracellular signal transduction domain, and the CAR T cells constructed on this basis significantly enhance the killing ability of CAR T cells against tumor cells in blood tumor and solid tumor killing tests, and show good safety and anti-tumor activity in clinical applications.

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.

Summary of the invention

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, and 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, for tumor cells that express a single NKp30 receptor ligand or NKG2D receptor ligand or express both receptor ligands, they can play an effective killing role, especially for tumor cells that lose one of the receptor ligands of NKp30 or NKG2D, and prevent the immune escape of tumors. Therefore, the transgenic immune cells of the present invention have high tumor cell killing activity, a wide recognition spectrum, strong in vivo tumor inhibition activity, a low cell infusion dose, and good efficacy.

In the first aspect of the present invention, the present invention provides a chimeric antigen receptor. The chimeric antigen receptor comprises:

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.

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

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, and the transgenic immune cells expressing the chimeric antigen receptor of the present invention can effectively identify tumor cells expressing the NKp30 receptor ligand and/or expressing the NKG2D receptor ligand, that is, they can play an effective killing role for tumor cells expressing a single NKp30 receptor ligand or a NKG2D receptor ligand or expressing both receptor ligands at the same time, especially they can effectively identify tumor cells that have lost one of the NKp30 or NKG2D receptor ligands, and prevent the immune escape of the tumor. Therefore, 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 tumor inhibition activity in vivo, a low cell infusion dose, and good efficacy.

According to an embodiment of the present invention, 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. Thus, the chimeric antigen receptor of the present invention can simultaneously target NKp30 receptor ligands and NKG2D receptor ligands.

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.

As used herein, the term "conservatively modified forms of an 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 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 by an amino acid residue with 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 non-polar 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).

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.

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 via 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.

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, 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. Thus, the chimeric antigen receptor of the present invention simultaneously targets the NKp30 receptor ligand and the NKG2D receptor ligand.

According to an embodiment of the present invention, the first connecting peptide is selected from (G ₄ S) _n or at least one of the connecting peptides having amino acid sequences as shown in SEQ ID NOs: 5, 34 to 39, and n is a non-zero integer.

According to an embodiment of the present invention, the first connecting peptide is (G ₄ S) _n , where n is any integer between 2 and 6.

According to an embodiment of the present invention, the first connecting peptide is (G ₄ S) ₃ . Thus, the chimeric antigen receptor of the present invention having better binding ability with NKp30 receptor ligand and NKG2D receptor ligand is obtained.

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 the NKp30 receptor ligand and the NKG2D receptor ligand.

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 NKp30 receptor ligands and NKG2D receptor ligands.

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.

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

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

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

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.

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.

According to an embodiment of the present invention, the second connecting peptide is selected from (G ₄ S) _n or at least one of the connecting peptides having amino acid sequences as shown in SEQ ID NOs: 5, 34 to 39, and n is a non-zero integer.

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, the chimeric antigen receptor of the present invention having better binding ability with NKp30 receptor ligand and NKG2D receptor ligand is obtained.

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

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.

According to an embodiment of the present invention, the intracellular region includes a co-stimulatory domain and an intracellular signaling domain.

According to an embodiment of the present invention, the C-terminus of the co-stimulatory factor domain is connected to the N-terminus of the intracellular signaling domain.

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.

According to an embodiment of the present invention, the co-stimulatory 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.

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.

According to an embodiment of the present invention, the intracellular signal transduction domain is the intracellular segment of the CD3ζ molecule or a fragment thereof. Thus, the tumor killing activity of the immune cells modified by the chimeric antigen receptor gene of the present invention can be further enhanced.

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.

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 by the chimeric antigen receptor gene of the present invention have significantly enhanced tumor cell killing activity and in vivo tumor inhibition activity.

In the second aspect of the present invention, the present invention provides a nucleic acid construct, which comprises: a first nucleic acid molecule, wherein the first nucleic acid molecule encodes the aforementioned chimeric antigen receptor.

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.

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.

According to an embodiment of the present invention, the nucleic acid construct further includes 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 includes IL-15, a hinge region of a CD8a molecule and a transmembrane region of a CD8a molecule; the second fusion protein includes IL-15Rα and IL-15. Thus, the activation and proliferation of immune cells expressing the aforementioned chimeric antigen receptor are further promoted, the number and activity of immune cells in the local microenvironment of the tumor are maintained, and the tumor cell killing activity and in vivo tumor inhibition activity are further improved.

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.

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. Thus, the first fusion protein is promoted to form a better configuration on the surface of immune cells.

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.

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 expressing the chimeric antigen receptor and the first fusion protein at the same time is obtained.

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.

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.

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

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. Thus, a second fusion protein expressed on the cell membrane is obtained.

According to an embodiment of the present invention, the third connecting peptide is selected from (G ₄ S) n or at least one of the connecting peptides with amino acid sequences as shown in SEQ ID NOs: 5, 34 to 39, and n is a non-zero integer.

According to an embodiment of the present invention, the third connecting peptide is selected from (G ₄ S) n, where n is any integer between 2 and 6.

According to an embodiment of the present invention, the third connecting peptide is (G ₄ S) 3. Thus, the second fusion protein forms a better configuration on the surface of immune cells.

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

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

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.

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.

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.

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.

According to an embodiment of the present invention, the first linker is P2A. Thus, the chimeric antigen receptor and the first fusion protein or the second fusion protein can be further promoted to be uniformly expressed on the cell membrane surface.

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 that uniformly expresses the chimeric antigen receptor and the first fusion protein or the second fusion protein on the cell membrane.

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.

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.

In the third aspect of the present invention, the present invention provides an expression vector, which carries the aforementioned nucleic acid construct.

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

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

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 aforementioned first fusion protein or second fusion protein in a host cell.

When the above-mentioned nucleic acid molecule is connected to an expression vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. Of course, the nucleic acid molecule can be operably connected to the control element. According to an embodiment of the present invention, the expression vector is a non-pathogenic viral vector.

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 the expression of 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 that contains the vector.

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 a 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.

In this article, the term "operably linked" refers to connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, viral vectors, plasmids, bacteriophages, etc. After the expression vectors according to some specific embodiments of the present invention are introduced into suitable immune cells, the expression of the aforementioned nucleic acid molecules can be effectively achieved under the mediation of the regulatory system, thereby achieving a large amount of expression of the protein encoded by the nucleic acid molecule on the cell membrane, thereby obtaining transgenic immune cells.

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

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

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

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

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

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.

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

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

The present invention does not impose strict restrictions on the source of NK cells, and the NK cells may be peripheral blood NK cells, umbilical cord blood NK cells, iPSCs, NK-92 cells, etc.

In a fourth aspect of the present invention, the present invention provides a pharmaceutical composition. The pharmaceutical composition comprises:

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

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

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 combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Generally, the composition is prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a solid carrier, or both.

In this article, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. Except for any conventional excipients that are incompatible with the chimeric antigen receptor, nucleic acid, expression vector or transgenic immune cell of the present invention, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of the present invention.

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 the pharmaceutical composition and will not be described in detail here.

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.

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

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.

In a specific embodiment of the present invention, the cancer is colorectal cancer.

Those skilled in the art will appreciate that the features and advantages described above for chimeric antigen receptors, nucleic acid constructs, expression vectors, and transgenic immune cells are also applicable to this use and will not be described in detail here.

In another aspect of the present invention, 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 transgenic immune cells or the above pharmaceutical composition to a subject.

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

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.

In a specific embodiment of the present invention, the cancer is colorectal cancer.

In this article, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means. The transgenic immune cells or pharmaceutical compositions of the present invention can be administered by any common route, as long as it can reach the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified modes of administration. Preferably, the composition of the present invention is administered by intravenous injection.

As used herein, the term "treatment" refers to the use of drugs to obtain the desired pharmacological and/or physiological effects. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and/or may be 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 blocking the progression of the disease; or (c) alleviating the disease, such as alleviating 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, mitigate or inhibit an 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.

The effective amount of the transgenic immune cells and pharmaceutical compositions of the present invention 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 a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, several divided doses may be administered daily, or the dose may be reduced proportionally, depending on the urgency of the treatment situation.

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.

Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

FIG1 is a schematic diagram of the structure of different chimeric antigen receptors according to Example 1 of the present invention;

FIG2 is a statistical result diagram of the killing efficiency of different CAR-NK cells on NCI-H716 cells in Example 2 of the present invention;

FIG3 is a schematic diagram of the structure of a CAR containing membrane-expressed IL15 ( FIG3-A ) or super IL15 ( FIG3-B ) according to Example 3 of the present invention;

FIG4 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 in FIG4-A , the CAR5-NK-92 membrane expresses IL15, and in FIG4-B , the CAR5-NK-92 membrane expresses super IL15;

FIG5 is a curve diagram showing the survival rate changes of NK-92 and CAR5-NK-92 cells in Example 3 of the present invention at different IL-2 culture concentrations, wherein the CAR5-NK-92 membrane in FIG5-A expresses IL15, and the CAR5-NK-92 membrane in FIG5-B expresses super IL15;

Figure 6 is a diagram showing the tumor inhibition effect of peripheral blood-derived CAR5-NK cells of Example 4 of the present invention on the human colorectal cancer NCI-H716 cell tumor-bearing mouse model, wherein the CAR5-NK-92 membrane in Figure 6-A expresses IL15, and the CAR5-NK-92 membrane in Figure 6-B expresses super IL15.

DETAILED DESCRIPTION

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.

It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

The endpoints and any values of the ranges 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 endpoint values of each range, the endpoint values of each range and the 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 as specifically disclosed in this article.

Terms and Definitions

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.

As used herein, the terms "according to an embodiment of the present invention," "optional" or "optional" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes cases where the event or circumstance occurs and cases where it does not occur.

In this article, the term "(G ₄ S) _n " is equivalent to "(Gly ₄ Ser) n ", which means that 4 glycines and 1 serine are repeated n times. It is a widely used type of connecting peptide, which can be located between the V _H C-terminus and the V _L N-terminus, or between the V _L C-terminus and the V _H N-terminus. Currently, (G ₄ S) ₃ is commonly used, in which glycine is the amino acid with the smallest molecular weight and the shortest side chain, which can increase the flexibility of the side chain, and serine is the most hydrophilic amino acid, which can increase the hydrophilicity of the connecting peptide. Therefore, (G ₄ S) ₃ has good stability and activity. Connecting peptides of different lengths and sequences can be designed to construct scFvs with different biological functions.

Herein, the term "hinge region" refers to the heavy chain region connecting _CH1 and _CH2 (i.e., connecting Fab and Fc) in an antibody. Herein, it is sometimes also referred to as the "Fc hinge region".

In this document, "carbon terminus" and "C-terminus" are synonymous; "nitrogen terminus" and "N-terminus" are synonymous.

The sequence descriptions involved in the present invention are shown in Table 1.

Table 1: Nucleotide/amino acid sequence description

The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

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

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

1. Construction of different CAR structures

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.

The schematic diagram of the gene element structure of the chimeric antigen receptor of this embodiment 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.

2. Lentivirus packaging

Take 5×10 ⁶ 293T cells in the logarithmic growth phase and inoculate them in a 10 cm cell culture dish, add 10 mL of DMEM culture medium, and culture overnight in a 37°C, 5% CO ₂ incubator. When the cell density in the cell culture dish reaches 80-90%, replace it with 10 mL of fresh DMEM culture medium, and continue to place the cell culture dish in the incubator for standby use. Prepare the lentiviral packaging system, add 6 μg psPAX2 and 3 μg pMD2.G of the lentiviral packaging auxiliary plasmids, and 6 μg of the target gene vector plasmid to 250 μL of serum-free DMEM culture medium to prepare a plasmid mixture, and mix well. Add to 235 μL serum-free DMEM medium and mix well. Add the mixture to the above plasmid mixture at one time, mix well, and incubate at room temperature for 15 minutes. Add the mixture to the 293T cell culture dish. After 24 hours, change the liquid, 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 the 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 turning the centrifuge tube upside down, 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 precipitate, transfer it to an EP tube, and store it in a -80°C refrigerator.

3. Lentivirus infection of human NK-92 cells

Pipette NK-92 cells in the logarithmic growth phase, harvest the cells by centrifugation at 100×g for 5min, add an appropriate amount of α-MEM medium to resuspend the cells, and adjust the cell density to 5×10 ⁵ /mL. In a 24-well plate, inoculate 5×10 ⁵ NK-92 cells, 0.2mL virus concentrate, 0.8mL α-MEM medium and protamine (final concentration 8μg/mL), respectively, and mix well. Place in a 37°C, 5% CO ₂ incubator for culture. After 24h, observe the cell state, change the medium, transfer the infected cells into an EP tube, centrifuge at 100×g for 5min, add a small amount of fresh α-MEM medium to resuspend the cells, transfer the cells into a cell culture flask, add 10mL fresh α-MEM medium and IL-2 (final concentration of 200IU/mL) and continue to culture for 48h. Transfer the cells into a flow tube, add 3 mL of 1×PBS solution, centrifuge at 100×g for 5 min, discard the supernatant, flick off the cell pellet, and wash again with 1×PBS solution. Use flow cytometry to detect the expression rate of GFP. Continue to expand the culture and adjust the state of the infected NK-92 cells for amplification. Use flow cytometry to sort the infected NK-92 cells for GFP-positive CAR-NK92 cells for later experiments.

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

Experimental Example 2: Comparison of killing efficiency of different CAR structures

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 preferred the CAR structure with the best killing activity.

The inventors selected colorectal cancer cell line NCI-H716 cells that highly expressed both NKG2D ligand MICA/B and NKp30 ligand B7-H6 as target cells, and performed in vitro killing activity detection on different CAR-NK cells.

The specific method is as follows: NCI-H716 cells were fluorescently labeled with CFSE, and ^2x104 cells/well were plated in a 96-well plate. NK-92 or different CAR-NK-92 cells were plated separately and incubated with NCI-H716 cells for 4 hours. PI staining was then added to distinguish between live and dead cells, and the killing efficiency was detected by flow cytometry. The ratio of effector cells to target cells was 2:1.

The test results are shown in Figure 2.

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 on NCI-H716 cells was significantly enhanced.

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

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

In this embodiment, the inventors took CAR3 of Example 1 as an example, added a membrane-expressed 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, and the pCDH-EF1a-CAR5 plasmid was obtained after sequencing verification.

The schematic diagram of the gene element structure of the chimeric antigen receptor of this embodiment is shown in Figure 3, 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 NO: 32, 33.

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

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

The specific method is as follows: 3×10 ⁶ cells of 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, and the cells were counted every 2 days. 3×10 ⁶ cells were transferred to a new T25 cell culture flask, and the corresponding concentration of IL-2 was added to start the next round of cell culture and counting. If the total number of cells is less than 3×10 ⁶ cells, all cells were transferred to a new T25 cell culture flask.

The results of the NK cell proliferation fold examination on the 10th day of culture are shown in Figure 4. In Figure 4-A, the CAR5-NK-92 membrane expresses IL15, and in Figure 4-B, the CAR5-NK-92 membrane expresses super IL15.

The results of NK cell proliferation activity investigation 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 there was basically no expansion in the NK-92 cell group.

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, which can further enhance the expansion capacity of NKR-NK cells in vivo and reduce the dependence of NK cell expansion on IL-2.

3. Investigation of CAR5 gene modification to promote NK cell survival rate

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

The specific method is as follows: the same number of NK-92 and CAR5-NK-92 cells were plated in a 24-well plate, and different IL-2 concentrations (200 IU/ml and 0 IU/ml) were set, and the apoptosis rate was detected by flow cytometry every 24 hours. The apoptosis rate was detected by flow cytometry according to the steps in the kit instructions (Lianke Bio, catalog number AP101), which is briefly as follows: collect cells in EP tubes, add 1xPBS solution, centrifuge and wash once, and resuspend the cells. Add 5 μL Annexin V-FITC and 10 μL PI 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 ratio of double negatives of Annexin V-FITC and PI staining.

The results of the NK cell survival rate test on the 8th day of culture are shown in Figure 5. In Figure 5-A, the CAR5-NK-92 membrane expresses IL15, and in Figure 5-B, the CAR5-NK-92 membrane expresses super IL15.

The results of NK cell survival rate investigation showed that in the absence of IL-2 (IL-2 0IU/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 of 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.

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.

Example 4: Investigation of the tumor-suppressing activity of primary CAR5-NK cells in vivo

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.

The specific method is as follows: 6-week-old NCG mice were selected for subcutaneous tumor bearing in the armpits, and NK cell treatment was started when the tumor volume reached 50 mm ^3. The tumor volume was measured before treatment, and the mice were randomly divided into untreated group, NK cell treatment group and CAR5-NK cell treatment group according to the tumor volume. In the NK cell treatment group, NK cells were infused back through the tail vein, and the mice were treated once every 2 days for a total of 3 times. The dose of each tail vein infusion was 8×10 ⁶ CD56 ⁺ NK cells, and IL-2 was injected intraperitoneally every 2 days to maintain NK cell survival; in the CAR5-NK cell treatment group, CAR5-NK cells were infused back through the tail vein, and the dose of a single treatment was 4×10 ⁶ CAR5-NK cells/mouse, and IL-2 was not injected. The tumor volume was continuously observed and measured, and the tumor growth curve was drawn.

The test results are shown in Figure 6. In Figure 6-A, the CAR5-NK-92 membrane expresses IL15, and in Figure 6-B, the CAR5-NK-92 membrane expresses super IL15.

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.

The above results indicate 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 therapeutic effect of immune cell therapy on solid tumors.

In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means 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 representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims (20)

1. A chimeric antigen receptor, comprising: 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 transmembrane region is a CD8a molecule transmembrane segment or a fragment thereof, and the CD8a molecule transmembrane segment has an amino acid sequence as shown in SEQ ID NO: 11; An intracellular region, wherein the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region; the intracellular region includes a costimulatory domain and an intracellular signaling domain, and the C-terminus of the costimulatory factor domain is connected to the N-terminus of the intracellular signaling domain; wherein the costimulatory domain is the intracellular segment of the 4-1BB molecule or a fragment thereof, and the intracellular signaling domain is the intracellular segment of the CD3ζ molecule or a fragment thereof, and the intracellular segment of the 4-1BB molecule or a fragment thereof has an amino acid sequence as shown in SEQ ID NO: 13, and the intracellular segment of the CD3ζ molecule has an amino acid sequence as shown in SEQ ID NO: 15; 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; The extracellular region further comprises a second connecting peptide, and the first binding fragment and the second binding fragment are connected via the second connecting peptide; the amino acid sequence of the second connecting peptide is shown in SEQ ID NO: 7; The extracellular region further comprises a hinge region, wherein the hinge region has an amino acid sequence as shown in SEQ ID NO: 9; The chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO: 21 or SEQ ID NO: 19. 2. 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 claim 1. 3 . The nucleic acid construct according to claim 2 , wherein the first nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 20 or 22. 4 . 4. The nucleic acid construct according to claim 2, further comprising a second nucleic acid molecule, wherein the second nucleic acid molecule is connected to the first nucleic acid molecule, and the second nucleic acid molecule encodes a first fusion protein or a second fusion protein, wherein the first fusion protein comprises IL-15, a hinge region of a CD8a molecule, and a transmembrane region of a CD8a molecule; and the second fusion protein comprises IL-15Rα and IL-15; Also included is a third nucleic acid molecule, the third nucleic acid molecule encodes a first linker, the first nucleic acid molecule and the second nucleic acid molecule are connected via the third nucleic acid molecule, and the first linker is P2A; The first fusion protein has an amino acid sequence as shown in SEQ ID NO: 28, and the second fusion protein has an amino acid sequence as shown in SEQ ID NO: 23. 5 . The nucleic acid construct according to claim 4 , wherein the second nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 29. 6 . The nucleic acid construct according to claim 4 , wherein the second nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 24. 7 . The nucleic acid construct according to claim 4 , wherein the third nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 31. The nucleic acid construct according to claim 4 , which has a nucleotide sequence as shown in SEQ ID NO: 32 or 33. 9. An expression vector, characterized in that it carries the nucleic acid construct according to any one of claims 2 to 8. 10. The expression vector according to claim 9, characterized in that the expression vector is a non-pathogenic viral vector. 11. The expression vector according to claim 10, characterized in that the non-pathogenic virus is selected from one of a retrovirus, a lentivirus and an adeno-associated virus. 12. The expression vector according to claim 10, characterized in that the non-pathogenic virus is a lentivirus. 13. A transgenic immune cell, characterized in that the transgenic immune cell comprises: Expressing the chimeric antigen receptor of claim 1; or Carrying the nucleic acid construct according to any one of claims 2 to 8 and the expression vector according to any one of claims 9 to 12. 14 . The genetically modified immune cell according to claim 13 , wherein the genetically modified immune cell is selected from at least one of a T cell, a NK cell, and a macrophage. 15. The genetically modified immune cell according to claim 13, characterized in that the T cell is selected from NKT cells, At least one of the cells, wherein the NK cells are selected from at least one of peripheral blood NK cells, umbilical cord blood NK cells, and NK-92 cells. 16 . The transgenic immune cell according to claim 14 or 15 , wherein the T cells, NK cells, and macrophages are derived from iPSCs. The transgenic immune cell according to claim 14 , characterized in that the transgenic immune cell is a NK cell. 18. A pharmaceutical composition, comprising: The chimeric antigen receptor according to claim 1, and the nucleic acid construct according to any one of claims 2 to 8. 19. The pharmaceutical composition according to claim 18, further comprising: a pharmaceutically acceptable excipient. 20. Use of the chimeric antigen receptor of claim 1, the nucleic acid construct of any one of claims 2 to 8, the expression vector of any one of claims 9 to 12, the transgenic immune cell of any one of claims 13 to 17, or the pharmaceutical composition of claim 18 or 19 in the preparation of a drug for preventing or treating colorectal cancer.