Transgenic immune effector cells and uses thereof

Abstract

The application relates to the field of biotechnology, in particular to a transgenic immune effector cell and application thereof. The immune checkpoint antibody in the fusion protein expressed by the immune effector cell can relieve the immune suppression of the immune checkpoint on the immune effector cell, the cytokine can enhance the antitumor activity of the immune effector cell, and the chemokine receptor molecule can significantly improve the migration ability of the immune effector cell to the tumor site; compared with the fusion protein and the cell co-expressing the fusion protein and a chimeric antigen receptor, the immune effector cell has stronger killing effect on tumor cells, has low drug toxicity, high safety, and effectively realizes the precise treatment of a solid tumor by CART.

Description

[0001] Priority Statement

[0002] This application claims priority to Chinese Patent Application No. 202111055628.8, filed on September 9, 2021, entitled "A Transgenic Immune Effector Cell and Its Application", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of biotechnology. More specifically, it relates to a transgenic immune effector cell and its application. Background Technology

[0004] Currently, chimeric antigen receptor T cell (CART) has achieved very good results in the treatment of hematologic malignancies. According to publicly available data, BCMACART for multiple myeloma has achieved an objective response rate (ORR) of 100% and a complete response rate (CR) of 70.6%, and five CART cell products are already on the market globally, all targeting hematologic malignancies. However, the efficacy of CART in treating solid tumors is less satisfactory. Clinical data shows that the ORR of Mesothelin CART combined with a PD-1 inhibitor for mesothelioma is 62.5%, and the CR is 18.75%. Therefore, the efficacy of CART in treating solid tumors urgently needs improvement.

[0005] The main reasons for the poor efficacy of CAR-T cells in killing solid tumors are as follows: 1) CAR-T cells have difficulty reaching and infiltrating the tumor in large quantities to kill tumor cells; 2) The activity of a small number of CAR-T cells that infiltrate the tumor is inhibited by immunosuppressive molecules such as PD1, which are highly expressed in the tumor site; 3) Insufficient contact between CAR-T cells and tumor cells significantly affects their proliferation and persistence, leading to easy depletion of CAR-T cells in vivo.

[0006] Chemokines are small cytokines that induce chemotaxis in cells. After binding to chemokine receptors, they transmit various cellular signals, inducing leukocytes, especially T lymphocytes, to migrate to inflammatory sites and tumor infiltration sites. Compared to hematologic malignancies, solid tumor cells can secrete chemokines such as CXCL12 and CXCL5 to prevent CAR-T cells from reaching the tumor lesion. Simultaneously, solid tumors secrete very little CXCR3 and CCR5 ligands, which aid in CAR-T cell transport. These two factors combined make it difficult for CAR-T cells to precisely reach the solid tumor site and exert their immune effect. Therefore, achieving precise treatment of solid tumors is a major challenge in CAR-T cell therapy. Summary of the Invention

[0007] This invention is based on the inventor's discoveries and understanding of the following problems:

[0008] The inventors have developed a transgenic immune effector cell that co-expresses a chimeric antigen receptor, a fusion protein, and a chemokine receptor molecule. The chemokine receptor molecule can attract more chimeric antigen receptors to the tumor site. Furthermore, the fusion protein can secrete immune checkpoint antibodies and cytokines, leveraging the dual advantages of both to specifically target the transgenic immune effector cell. This reduces the inhibitory effect of the tumor microenvironment on the transgenic immune effector cell while making its effects more long-lasting. Additionally, the cytokines significantly enhance the killing ability against tumor cells, enabling precise CAR-T therapy for solid tumors.

[0009] In a first aspect, the present invention provides a transgenic immune effector cell, wherein the transgenic immune effector cell co-expresses a chimeric antigen receptor, a fusion protein, and a chemokine receptor molecule; the chimeric antibody receptor includes an extracellular region, a transmembrane region, and an intracellular region sequentially connected in series; wherein the extracellular region specifically recognizes tumor antigens, the transmembrane region is embedded in the cell membrane of the transgenic immune effector cell, and the intracellular region includes an intracellular segment of an immune co-stimulatory molecule; the fusion protein includes an immune checkpoint antibody and a cytokine; and the chemokine receptor molecule is a chemokine receptor or a chemokine-binding fragment thereof.

[0010] In a second aspect, the present invention provides a lentivirus carrying the following nucleic acid:

[0011] 1) The nucleic acid encoding the chimeric antigen receptor in the transgenic immune effector cells, wherein the extracellular region of the chimeric antigen receptor specifically recognizes tumor antigens;

[0012] 2) Nucleic acid encoding a fusion protein in the transgenic immune effector cells, the fusion protein comprising immune checkpoint antibodies and cytokines;

[0013] 3) The nucleic acid encoding the chemokine receptor molecule in the transgenic immune effector cells, wherein the chemokine receptor molecule is a chemokine receptor or its chemokine-binding fragment.

[0014] In a third aspect, the present invention provides a construct comprising:

[0015] A first nucleic acid molecule, which encodes a chimeric antigen receptor in the transgenic immune effector cells, wherein the extracellular region of the chimeric antigen receptor specifically recognizes tumor antigens;

[0016] A second nucleic acid molecule, the second nucleic acid molecule encoding a fusion protein in the transgenic immune effector cells, the fusion protein comprising immune checkpoint antibodies and cytokines;

[0017] A third nucleic acid molecule, wherein the third nucleic acid molecule encodes a chemokine receptor molecule in the transgenic immune effector cell, wherein the chemokine receptor molecule is a chemokine receptor or a chemokine-binding fragment thereof.

[0018] In a fourth aspect of the invention, the invention provides a method for preparing the transgenic immune effector cells described in the first aspect, wherein the lentivirus or the construct is introduced into the immune effector cells; according to an embodiment of the invention, the immune effector cells are selected from any one or more of T cells, NK cells, NKT cells, macrophages or CIK cells; according to an embodiment of the invention, the immune effector cells are T cells.

[0019] In a fifth aspect, the present invention proposes the use of the transgenic immune effector cells, the lentivirus, or the construct in the preparation of cancer therapeutic drugs.

[0020] According to an embodiment of the present invention, the cancer is a solid tumor.

[0021] In a sixth aspect, the present invention provides a cancer therapeutic agent. According to embodiments of the invention, it includes the transgenic immune effector cells, the lentivirus, or the construct.

[0022] According to an embodiment of the present invention, the cancer is a solid tumor.

[0023] In a seventh aspect, the present invention provides a method for treating cancer, comprising: administering a therapeutically effective amount of the cancer treatment drug as described above to a subject requiring treatment.

[0024] According to an embodiment of the present invention, the cancer is a solid tumor. Attached Figure Description

[0025] Figure 1This is a schematic diagram of the structure of the nucleic acid constructed in Example 1 of the present invention; wherein, "Hinge" refers to the hinge region, "Linker" refers to the linker peptide, scFv represents PD1 scFv, CAR19 represents the T cell group expressing the chimeric antigen receptor targeting CD19, CAR19&scFv represents the CART19 group expressing scFv, CAR19&scFv&IL21 represents the CART19 group expressing scFv and IL-21 alone (i.e., scFv and IL-21 are not fused), CAR19&scFv-IL21 represents the CART19 group expressing the fusion protein scFv-IL-21, and CAR19&IL21-scFv represents the CART19 group expressing the fusion protein IL-21-scFv.

[0026] Figure 2 This is the CAR19 positivity rate detection result of D6 after T cell infection in Example 3 of the present invention; wherein, scFv represents PD1 scFv, T cell group represents ordinary T cell group, CAR19 represents T cell group expressing chimeric antigen receptor targeting CD19, CAR19&scFv represents CART19 group expressing scFv, CAR19&scFv&IL21 represents CART19 group expressing scFv and IL-21 alone, CAR19&scFv-IL21 represents CART19 group expressing fusion protein scFv-IL-21, and CAR19&IL21-scFv represents CART19 group expressing fusion protein IL-21-scFv.

[0027] Figure 3 This is a schematic diagram of the structure of the plasmid constructed in Example 5 of the present invention; wherein, Hinge refers to the hinge region, Linker refers to the linker peptide, SS1 refers to the MSLN antibody (tumor antigen is MSLN); Anti PD1scFv(VH+VL) refers to the PD1 antibody; Anti PD1-IL-21 refers to the fusion protein composed of the PD1 antibody and IL-21.

[0028] Figure 4 This is a graph showing the percentage results of MSLN antibody and CCR2b positivity rates in 293T cells.

[0029] Figure 5 This is a schematic diagram of the carrier structure of 89CART cells.

[0030] Figure 6 This is a graph showing the double seroconversion rate of 89CART cells prepared according to the present invention.

[0031] Figure 7 This is a graph showing the migration of 89CART cells prepared in this invention after being chemotactically activated by CCL2.

[0032] Figure 8 This is a graph showing the in vitro tumor cell killing ability of the 89CART cells prepared according to the present invention.

[0033] Figure 9 The results show the changes in tumor volume in mice after receiving different T cell therapies. In the figure, 85 represents 85CART cells, 89 represents 89CART cells, and T cell represents control T cells.

[0034] Figure 10 The results show the concentration of CCL2 in cell suspensions and peripheral blood from mouse tumor sites. In the figure, G3-783 represents mouse number 783 in the 89CART cell therapy group, and G3-788 represents mouse number 788 in the 89CART cell therapy group.

[0035] Figure 11 The results show the proportions of different CAR-T cells in cell suspensions and peripheral blood from mouse tumor sites. Detailed Implementation

[0036] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0037] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0038] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, four, etc., and "multiple" or "several" means at least two, such as two, three, four, etc., unless otherwise explicitly specified.

[0039] This invention relates to a transgenic immune effector cell, wherein the transgenic immune effector cell co-expresses a chimeric antigen receptor, a fusion protein, and a chemokine receptor molecule; the chimeric antigen receptor includes an extracellular region, a transmembrane region, and an intracellular region sequentially connected in series; wherein the extracellular region specifically recognizes tumor antigens, the transmembrane region is embedded in the cell membrane of the immune effector cell, and the intracellular region includes an intracellular segment of an immune co-stimulatory molecule; the fusion protein includes an immune checkpoint antibody and a cytokine; and the chemokine receptor molecule is a chemokine receptor or a chemokine-binding fragment thereof.

[0040] According to embodiments of the present invention, the transgenic immune effector cells co-express chimeric antigen receptors, fusion proteins, and chemokine receptor molecules. The fusion protein can secrete immune checkpoint antibodies and cytokines, thus specifically targeting the transgenic immune effector cells with the dual advantages of immune checkpoint antibodies and cytokines. This reduces the inhibitory effect of the tumor microenvironment on transgenic immune effector cells while making the transgenic immune effector cells more effective over a longer period. Cytokines can significantly enhance the killing ability against tumor cells, and chemokine receptor molecules significantly enhance the migration ability of immune effector cells to the tumor site. Therefore, compared with fusion proteins that express immune checkpoint antibodies or cytokines alone, and cells that co-express the above-mentioned fusion proteins and chimeric antigen receptors, the above-mentioned immune effector cells have a stronger killing effect on tumor cells, while significantly improving the specificity of cytokine binding to immune effector cells and greatly reducing drug toxicity.

[0041] In this invention, the term "chemokine receptor molecule" refers to G protein-coupled receptors containing a seven-transmembrane structure. These receptors primarily transduce signals via G proteins, and their binding to receptors exhibits certain subfamily specificity. Chemokine receptor molecules are classified into CXC chemokine receptors, CC chemokine receptors, CX3C chemokine receptors, and XC chemokine receptors based on the type of ligand they contain.

[0042] In this invention, the term "immune checkpoint" refers to a series of molecules expressed on immune cells that regulate the level of immune activation and play an important role in preventing autoimmune reactions. Cancer-related immune checkpoints include PD1, PD-L1, TIGIT, LAG3, CTLA4, BTLA, and TIM3.

[0043] In this invention, the term "scFv" refers to "single-chain antibody, a genetically engineered antibody composed of a heavy chain variable region (VH) and a light chain variable region (VL) typically linked by a short peptide (linker) of 15-20 amino acids. Single-chain antibodies can better retain their affinity activity for antigens and have characteristics such as small molecular weight, strong penetrability, and weak antigenicity."

[0044] In this invention, the "immune checkpoint antibody" has anti-immune checkpoint activity and can specifically bind to immune checkpoints.

[0045] In this invention, the term "cytokine" refers to a class of small molecule proteins with broad biological activity synthesized and secreted by immune cells and certain non-immune cells upon stimulation. Cytokines are classified into interleukins, interferons, the tumor necrosis factor superfamily, colony-stimulating factors, chemokines, growth factors, etc.

[0046] In this invention, the term "chimeric antigen receptor" is abbreviated as CAR, which is the core component of CAR-T therapy. It includes an extracellular region that can specifically recognize tumor antigens, a transmembrane region embedded in the cell membrane, and an intracellular region that includes an intracellular segment of immune co-stimulatory molecules. Applying CAR engineering technology to CAR-T therapy composed of T cells can be widely used in CAR-T cell therapy.

[0047] According to embodiments of the present invention, the above-mentioned transgenic immune effector cells further include at least one of the following additional technical features:

[0048] In some embodiments, the chemokine receptor is any one or more of CXC chemokine receptor, CC chemokine receptor, CX3C chemokine receptor or XC chemokine receptor.

[0049] In some embodiments, the CXC chemokine receptor is any one or more of CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6 or CXCR7.

[0050] In some embodiments, the CC chemokine receptor is any one or more of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10 or CCR11.

[0051] In some embodiments, the CX3C chemokine receptor is CX3CR1.

[0052] In some embodiments, the XC chemokine receptor is XCR1.

[0053] In some embodiments, the chemokine receptor molecule is CCR2b, and the amino acid sequence of the chemokine receptor molecule is shown in SEQ ID NO: 5.

[0054] In some embodiments, the immune co-stimulatory molecule is selected from any one or more of 4-1BB, CD28, CD3, OX-40, CD40L, CD27, CD30, or derivatives thereof.

[0055] In some embodiments, the immune co-stimulatory molecule is 4-1BB.

[0056] In some embodiments, the immune checkpoint is selected from any one or more of PD1, PD-L1, TIGIT, LAG3, CTLA4, BTLA, or TIM3.

[0057] In some implementations, the immune checkpoint is PD1.

[0058] In some embodiments, the cytokine is an interleukin; the cytokine is selected from any one or more of IL-21, IL-23, IL-2, IL-7, IL-9, IL-12, IL-15 or IL-18.

[0059] In some embodiments, the cytokine is IL-21.

[0060] In some embodiments, the fusion protein further includes a linker peptide; the immune checkpoint antibody is linked to the cytokine via the linker peptide. Short peptides containing G and S amino acids commonly used in the art can be used as the linker peptides of this invention.

[0061] In this invention, the term "short peptide" refers to a peptide with no more than 50 amino acid residues, for example, it can be 4-50, for example, it can also be 4-30, for example, it can also be 4-20, or it can also be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0062] In some embodiments, the immune checkpoint antibody is a PD1 antibody, and the cytokine is IL-21.

[0063] In some embodiments, the C-terminus of the PD1 antibody is linked to the N-terminus of the linker peptide, and the N-terminus of the IL-21 is linked to the C-terminus of the linker peptide.

[0064] The inventors discovered that the fusion protein obtained by fusing PD1 antibody with IL-21 significantly reduces the expression level of PD1 on the surface of immune effector cells. The PD1 antibody specifically binds to PD1 on the surface of immune effector cells, thereby enabling IL-21 to also specifically act on immune effector cells, effectively reducing the toxicity caused by IL-21 binding to other IL-21 receptors on the surface of cells, and making the tumor-killing effect of immune effector cells more significant.

[0065] PD1 is mainly expressed on the surface of T cells, primarily CD8+ T cells. The fusion protein secreted by the transgenic immune effector cells selectively binds to the surface of T cells and CAR-T cells with its PD1 antibody and IL-21. On the one hand, it blocks the PD1 / PD-L1 signaling pathway; on the other hand, it enables IL-21 to specifically act on T cells and CAR-T cells, thus performing its dual function. At the same time, the increased molecular weight of the fusion protein significantly improves the drug's half-life.

[0066] In some embodiments, the amino acid sequence of the linker peptide is shown in SEQ ID NO: 7.

[0067] GGGGSGGGGSGGGGS (SEQ ID NO: 7).

[0068] In some embodiments, the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 3 or SEQ ID NO: 9. Specifically, the fusion protein PD1 scFv-IL-21 has the amino acid sequence shown in SEQ ID NO: 3, and the fusion protein IL-21-PD1 scFv has the amino acid sequence shown in SEQ ID NO: 9. PD1 scFv represents a PD1 antibody, IL-21 represents the cytokine IL-21, and the linking order in the fusion protein PD1 scFv-IL-21 is that the C-terminus of PD1 scFv is linked to the N-terminus of IL-21, and the linking order in the fusion protein IL-21-PD1 scFv is that the C-terminus of IL-21 is linked to the N-terminus of PD1 scFv.

[0069] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDN SKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSGGGGSGGGGSGGGGSMHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO: 3)

[0070] HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSGGGGSMEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQA PRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS(SEQ ID NO:9)

[0071] In some embodiments, the extracellular region includes an antibody that specifically recognizes a tumor antigen and a CD8 hinge region; the transmembrane region includes a transmembrane segment of CD8; the immunostimulatory molecule is 4-1BB; the intracellular region includes an intracellular segment of 4-1-BB and a CD3 zeta chain; the CD8 hinge region of the extracellular region is connected to the transmembrane region, and the transmembrane region is connected to the intracellular segment of 4-1-BB in the intracellular region.

[0072] In some embodiments, the tumor antigen is any one or more of MSLN, GD2, GPC3, CD19, EGFR VIII, GUCY2C, HER2, MUC16, or Claudin 18.2.

[0073] In some embodiments, the tumor antigen is MSLN or CD19.

[0074] In some embodiments, the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO: 1 or SEQ ID NO: 11.

[0075] RGSATMALPVTALLLPLALLLHAARPQVQLQQSGPELEKPGASVKLSCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGAGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:1)

[0076] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGS GGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQ GTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC ELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ IDNO:11)

[0077] In some embodiments, the chimeric antigen receptor, fusion protein, and chemokine receptor molecule are linked by a 2A peptide.

[0078] In some embodiments, the 2A peptide is selected from any one or more of P2A, T2A, F2A, or E2A.

[0079] In some embodiments, the chimeric antigen receptor is linked to the fusion protein via P2A, and the fusion protein is linked to the chemokine receptor molecule via T2A.

[0080] In some embodiments, the immune effector cells are selected from any one or more of T cells, NK cells, NKT cells, macrophages, or CIK cells.

[0081] In some embodiments, the immune effector cells are T cells.

[0082] The T cells mentioned in this invention refer to T lymphocytes, which have the function of recognizing antigens and secreting lymphokines. When T lymphocytes receive antigens exposed by antigen determinants, they will differentiate into effector T cells.

[0083] In some embodiments, when the chemokine receptor molecule is CCR2b, the immune co-stimulatory molecule is 4-1BB, the immune checkpoint is PD1, the tumor antigen is MSLN, the cytokine is IL-21, and the immune effector cell is T cell, the CCR2b in the constructed chimeric antigen receptor T (CART) cells can chemotact more CART to the tumor site, the PD1 antibody in the fusion protein can relieve the immunosuppression of CART, and IL-21 significantly enhances the killing ability of tumor cells.

[0084] The present invention also relates to a lentivirus carrying the following nucleic acids:

[0085] 1) A nucleic acid encoding the chimeric antigen receptor, wherein the extracellular region of the chimeric antigen receptor specifically recognizes tumor antigens; 2) A nucleic acid encoding the fusion protein, wherein the fusion protein includes immune checkpoint antibodies and cytokines; 3) A nucleic acid encoding the chemokine receptor molecule, wherein the chemokine receptor molecule is a chemokine receptor or its chemokine-binding fragment.

[0086] The lentivirus described above according to an embodiment of the present invention is introduced into recipient cells to obtain the transgenic immune effector cells. The transgenic immune effector cells can express and secrete fusion proteins including immune checkpoint antibodies and cytokines, chimeric antigen receptors, and chemokine receptor molecules, thereby reducing the expression of immune checkpoints on the surface of immune effector cells, reducing the inhibitory effect of the tumor microenvironment on immune effector cells, and chemotactically attracting more chimeric antigen receptors to the tumor site, making the killing effect of immune effector cells on tumors more significant and long-lasting, with high safety.

[0087] In this invention, the term "nucleic acid" generally refers to RNA or DNA. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence. DNA is preferably used when it is ligated into a vector.

[0088] In this invention, the term "lentivirus" refers to a viral vector that can effectively integrate exogenous genes or exogenous shRNA into the host chromosome, thereby achieving the effect of persistent expression of the target sequence.

[0089] According to embodiments of the present invention, the lentivirus further includes at least one of the following additional technical features:

[0090] In some embodiments, the tumor antigen is MSLN or CD19, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 1 or SEQ ID NO: 11, and the nucleic acid encoding the chimeric antigen receptor has a nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 12.

[0091]

[0092]

[0093] In some embodiments, the immune checkpoint antibody is a PD1 antibody, the cytokine is IL-21, the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 3 or SEQ ID NO: 9, and the nucleic acid encoding the fusion protein has a nucleotide sequence as shown in SEQ ID NO: 4 or SEQ ID NO: 10.

[0094]

[0095]

[0096] In some embodiments, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 3, and the nucleic acid encoding the fusion protein has the nucleotide sequence shown in SEQ ID NO: 4.

[0097] In some embodiments, the chemokine receptor molecule is CCR2.

[0098] In this invention, the term "CCR2" refers to the specific receptor for monocyte chemoattractant protein-1 (CCL2), and also the receptor for CCL7, CCL8, CCL11, CCL12, and CCL13. It includes two subtypes, CCR2a and CCR2b, which are derived from different splices of the same gene and differ only in their hydroxyl ends, but CCR2b is the main functional form.

[0099] In some embodiments, the chemokine receptor molecule is CCR2b.

[0100] In some embodiments, the amino acid sequence of the chemokine receptor molecule is as shown in SEQ ID NO: 5, and the nucleic acid encoding the chemokine receptor molecule has the nucleotide sequence shown in SEQ ID NO: 6.

[0101] MLSTSRSRFIRNTNESGEEVTTFFDYDYGAPCHKFDVKQIGAQLLPPLYSLVFIFGFVGNMLVVLILINCKKLKCLTDIYLLNLAISDLLFLITLPLWAHSAANEWVFGNAMCKLFTGLYHIGYFGGIFFIILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWLVAVFASVPGIIFTKCQ KEDSVYVCGPYFPRGWNNFHTIMRNILGLVLPLLIMVICYSGILKTLLRCRNEKKRHRAVRVIFTIMIVYFLFWTPYNIVILLNTFQEFFGLSNCESTSQLDQATQVTETLGMTHCCINPIIYAFVGEKFRRYLSVFFRKHITKRFCKQCPVFYRETVDGVTSTNTPSTGEQEVSAGL(SEQ ID NO:5)

[0102]

[0103] In some embodiments, the lentivirus carries a nucleotide sequence containing the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 10.

[0104] The present invention also relates to a construct comprising:

[0105] A first nucleic acid molecule, which encodes the chimeric antigen receptor, wherein the extracellular region of the chimeric antigen receptor specifically recognizes tumor antigens;

[0106] A second nucleic acid molecule, the second nucleic acid molecule encoding the fusion protein, the fusion protein comprising immune checkpoint antibodies and cytokines;

[0107] A third nucleic acid molecule, wherein the third nucleic acid molecule encodes the chemokine receptor molecule, wherein the chemokine receptor molecule is a chemokine receptor or a chemokine-binding fragment thereof.

[0108] The above-described construct according to embodiments of the present invention is introduced into recipient cells to obtain the transgenic immune effector cells. The transgenic immune effector cells can express chimeric antigen receptors on their surface and secrete fusion proteins and chemokine receptor molecules, resulting in a more significant, sustained, and safe killing effect on tumor cells.

[0109] According to embodiments of the present invention, the above-described construct further includes at least one of the following additional technical features:

[0110] In some embodiments, the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule sequentially express a chimeric antigen receptor, a fusion protein, and a chemokine receptor molecule in immune effector cells, respectively.

[0111] In some embodiments, the chimeric antigen receptor, fusion protein, and chemokine receptor molecule are in a non-fusion form.

[0112] In some embodiments, the immune effector cells are selected from any one or more of T cells, NK cells, NKT cells, macrophages, or CIK cells.

[0113] In some embodiments, the immune effector cells are T cells.

[0114] In some embodiments, the construct further includes a first promoter operatively linked to the first nucleic acid molecule.

[0115] In some implementations, the first promoter is selected from any one or more of the U6, H1, CMV, EF-1, LTR, or RSV promoters.

[0116] In some embodiments, the vector for the construct is a non-pathogenic viral vector. In some embodiments, the non-pathogenic viral vector is selected from one or more of retroviral vectors, lentiviral vectors, or adenovirus-associated viral vectors.

[0117] In some embodiments, the non-pathogenic viral vector is a lentiviral vector; the nucleic acid encoding the lentiviral vector has the nucleotide sequence shown in SEQ ID NO: 8.

[0118]

[0119] The present invention also relates to a method for preparing the immune effector cells by introducing the lentivirus or the construct into the immune effector cells.

[0120] When the lentivirus or construct described above according to embodiments of the present invention is introduced into immune effector cells, chimeric antigen receptors, fusion proteins and chemokine receptor molecules are co-expressed and secreted in the cells, reducing the expression of immune checkpoints and the inhibitory effect of the tumor microenvironment on immune effector cells, resulting in more significant and long-lasting killing of tumor cells.

[0121] In some embodiments, the immune effector cells are selected from any one or more of T cells, NK cells, NKT cells, macrophages, or CIK cells.

[0122] In some embodiments, the immune effector cells are T cells.

[0123] The present invention also relates to the use of the immune effector cells, the lentivirus, or the construct in the preparation of cancer therapeutic drugs.

[0124] The present invention also relates to a cancer therapeutic agent comprising the immune effector cells, the lentivirus, or the construct.

[0125] In some implementations, the cancer is a solid tumor.

[0126] The present invention also relates to a method for treating cancer, comprising: administering a therapeutically effective amount of the cancer treatment drug as described above to a subject requiring treatment.

[0127] In some implementations, the cancer is a solid tumor.

[0128] The aforementioned solid tumors include, but are not limited to, ovarian cancer and pancreatic cancer.

[0129] The present invention has the following beneficial effects:

[0130] The inventors have developed a transgenic immune effector cell that co-expresses a chimeric antigen receptor, a fusion protein, and a chemokine receptor molecule. The chemokine receptor molecule attracts more chimeric antigen receptors to the tumor site, significantly enhancing the migration ability of the immune effector cell to the tumor. Furthermore, the fusion protein secretes immune checkpoint antibodies and cytokines, specifically targeting the transgenic immune effector cell with the dual advantages of both. The immune checkpoint antibodies in the fusion protein relieve the immunosuppression of immune effector cells by immune checkpoints, while the cytokines significantly enhance the killing ability against tumor cells, resulting in a better, longer-lasting, and safer killing effect, thus achieving precise CAR-T therapy for solid tumors.

[0131] Example 1: Construction of a CART19 (a T cell expressing a chimeric antigen receptor targeting CD19) lentiviral vector expressing the AntiPD1-IL-21 fusion protein and its control vector.

[0132] Gene synthesis Figure 1 Based on the nucleotide sequence of the structure shown, the nucleotide fragments were constructed into a lentiviral vector according to the lentiviral vector restriction enzyme sites. Primers were designed, and the correctness of the vector construction was verified by sequencing results.

[0133] CAR19 amino acid sequence: as shown in SEQ ID NO: 11, nucleotide sequence: as shown in SEQ ID NO: 12;

[0134] The amino acid sequence of the fusion protein PD1 scFv-IL-21 is shown in SEQ ID NO:3; the nucleotide sequence is shown in SEQ ID NO:4.

[0135] The amino acid sequence of the fusion protein IL-21-PD1 scFv is shown in SEQ ID NO:9; the nucleotide sequence is shown in SEQ ID NO:10.

[0136] Example 2: Packaging and Concentrating Lentiviral Viruses

[0137] 293T will be processed according to 8×10 6 Cells / 150mm 2 The cells were seeded at the appropriate density in culture dishes, and their condition was observed the following day. The lentiviral packaging vector (generation 3) was co-transfected into 293T cells using PEI transfection. The medium was changed 6 hours after transfection, at a rate of 15 mL / 150 mL. 2 DMEM medium containing 10% fetal bovine serum was added to the culture dish. Viral supernatant was collected at 48 and 72 hours after transfection. The virus was centrifuged at 2000 rpm and 4℃ for 10 min to remove cell debris. Then, impurities were filtered through a 0.45 μm filter. The filtered virus suspension was centrifuged at 25000 rpm and 4℃ for 2 hours to concentrate the lentivirus. The concentrated virus was resuspended in an appropriate amount of medium and stored at -80℃.

[0138] Example 3 Production of CART and Control Cells

[0139] 20 mL of blood was drawn, and PBMCs were separated by Ficall gradient centrifugation. T cells were then isolated using the Stemcell T cell negative selection kit (catalog number: 19051). The isolated T cells were resuspended in medium supplemented with 5% human AB serum and 300 units / mL IL-2X-VIVO 15 to a final volume of 1×10⁻⁶. 6T cells / mL were collected and the beads were washed with X-VIVO 15 containing 1% FBS. Pre-washed magnetic beads (Cat#40203D, 10 mL, Life Technology) were added at a magnetic bead:T cell ratio of 2:1. After 2-3 days, the T cells were resuspended in fresh culture medium to a concentration of 3 × 10⁶ cells / mL. 6 -5×10 6 lentivirus was added at a concentration of 10 cells / mL, with an MOI of 10, along with 8 μg / mL of polybrene. After 4-6 hours, culture medium was added to dilute the cells to 1 × 10⁻⁶. 6 Cells / mL, replace with fresh culture medium the next day to maintain a cell concentration of 0.2 × 10⁻⁶ cells / mL. 6 -0.3×10 6 PBMC / mL, and the culture medium was changed every 2-3 days. The cell positivity rate was analyzed by flow cytometry 72 hours after the virus infection was completed.

[0140] The CAR19 positivity rate was measured on day 6 (D6) after T-cell infection. Figure 2 As shown, the results indicate that the infection rates were as follows: CAR19 group (66.97%), CAR19&scFv group (65.07%), CAR19&scFv&IL21 group (56.31%), CAR19&scFv-IL21 group (78.1%), and CAR19&IL21-scFv group (57.78%). The results show that CAR19&scFv-IL21 had the highest infection efficiency.

[0141] Example 4: CART phenotypic identification

[0142] After infection, the T, B, and NK cell populations of each D6 group were detected by flow cytometry, and the CD4, CD8, and PD1 populations of T cells were also detected.

[0143] The results are shown in Table 1. It can be seen that only a small proportion (2.87%) of T cells in the CAR19&scFv-IL21 group expressed PD1 molecules, suggesting that CAR19&scFv-IL21 may regulate the expression of PD1 molecules on the surface of T cells, greatly reducing the proportion of PD1 expression on the surface of T cells.

[0144] Table 1

[0145]

[0146] Note: In Table 1, scFv represents PD1 scFv, T cell group represents ordinary T cell group, CAR19 represents T cell group expressing chimeric antigen receptor targeting CD19, CAR19&scFv represents CART19 group expressing scFv, CAR19&scFv&IL21 represents CART19 group expressing scFv and IL-21 alone, CAR19&scFv-IL21 represents CART19 group expressing fusion protein scFv-IL-21, and CAR19&IL21-scFv represents CART19 group expressing fusion protein IL-21-scFv.

[0147] Example 5: Construction of an MSLN CART lentiviral vector expressing AntiPD1-IL-21 fusion protein and CCR2b, and its control vector.

[0148] A schematic diagram of the structure of the plasmid constructed in this embodiment of the invention is shown below. Figure 3 As shown, gene synthesis Figure 3 The nucleotide sequence of the structure shown was constructed into a lentiviral vector according to the lentiviral vector restriction enzyme sites. Primers were designed, and the correctness of the constructed plasmid was verified by sequencing results.

[0149] MSLN CAR amino acid sequence: as shown in SEQ ID NO: 1, nucleotide sequence as shown in SEQ ID NO: 2;

[0150] The amino acid sequence of the CCR2b chemokine receptor molecule is shown in SEQ ID NO: 5, and the nucleotide sequence is shown in SEQ ID NO: 6.

[0151] The sequence of other components is the same as in Example 1.

[0152] The three constructed plasmids MSLN CAR, MSLN CAR+Anti PD1-IL-21, and MSLN CAR+Anti PD1-IL-21+CCR2b were named PCDHF-85, PCDHF-86, and PCDHF-89, respectively; among them, Anti PD1-IL-21 represents a fusion protein composed of PD1 antibody and IL-21.

[0153] Example 6: Packaging Lentiviral Virus

[0154] The PCDHF-85, PCDHF-86, and PCDHF-89 plasmids obtained in Example 5 were packaged into lentiviruses according to the lentivirus packaging system shown in Table 2. The steps are as follows:

[0155] (1) Seed 293T cells (cell cryopreservation density was 5×10⁻⁶) 6(cells / mL) were added to a 10cm cell culture dish, along with 10mL of DMEM medium containing 10% FBS (DMEM Gibco, 11995040-1L; FBS Gibco, 10091-148), and cultured in a CO2 incubator at 37℃ for 24h with 5% CO2.

[0156] (2) Lentiviral packaging was performed according to Table 2; cell supernatant was collected 48 h after packaging, and the lentivirus titer was detected after ultracentrifugation at 25,000 rpm. The detection method is as follows:

[0157] The collected lentiviral stock solution was used to infect 293T cells under the same conditions at gradient volumes. After 48 hours, the percentage of MSLN antibody and CCR2b positivity in 293T cells was detected by flow cytometry (using MSLN antigen labeled with FITC and CCR2 antibody detection, ACRO, FITC-Labeled Human MSLN (296-580) Protein, Fc Tag, Cat. No. MSN-HF253). The lentiviral stock solution titer was calculated according to the following formula:

[0158] Lentiviral stock solution titer (TU / mL) = 1.5 * (10 × 10⁻⁶) 5 *293T cells MSLN antibody and CCR2b positivity rate percentage / lentivirus stock solution volume μL*1000.

[0159] Table 2 Lentiviral Packaging Systems

[0160]

[0161] Table 3 Lentiviral titers

[0162] plasmid Lentiviral titer PCDHF-85 <![CDATA[3.96×10 8 TU / mL]]> PCDHF-86 <![CDATA[3.02×10 8 TU / mL]]> PCDHF-89 <![CDATA[1.44×10 8 TU / mL]]>

[0163] The percentage results of MSLN antibody and CCR2b positivity rates in 293T cells are as follows: Figure 4 As shown in Table 3, the titers of the lentiviruses PCDHF-85, PCDHF-86, and PCDHF-89 were 3.96 × 10⁻⁶, respectively. 8 TU / mL, 3.02×10 8 TU / mL, 1.44×10 8 TU / mL.

[0164] Example 7 CART cell preparation

[0165] PBMCs were isolated from 50 mL of blood using Ficoll lymphocyte separation medium (Dacco, AS1114546). T cells were obtained by positive selection using magnetic beads conjugated with CD3 / CD28 antibodies (Dynabeads, CD3 / CD28 CTS, catalog number 40203D, batch number A2-011710E). The lentivirus prepared in Example 6 was used to infect the T cells at an MOI of 5:1 to prepare CAR-T cells. Control T cells were also included. After 7 days of CAR-T cell culture, the double positivity rate of the prepared 89CART cells was determined by detecting the expression of MSLN antibody and CCR2b in the CAR-T cells. Specifically, 85CART cells were obtained from PCDHF-85 lentivirus, 86CART cells from PCDHF-86 lentivirus, and 89CART cells from PCDHF-89 lentivirus. A schematic diagram of the lentiviral vector structure for 89CART cells is shown below. Figure 5 As shown, the pCDHF empty vector nucleotide is shown in SEQ ID NO: 8.

[0166] The results of the double seroconversion rate of 89CART cells prepared in this invention are as follows: Figure 6 As shown, the results indicate that the double positivity rate of 89CART cells was 30.31%, demonstrating that the 89CART cells were successfully prepared in this embodiment of the invention.

[0167] Example 8: Chemotaxis of CART cells

[0168] Chemokine CCL2 can attract cells expressing the chemokine receptor CCR2b to migrate to sites with high CCL2 concentrations, and tumor sites highly express CCL2. Therefore, to verify the migration ability of the 89CART cells prepared in Example 7 to tumor sites, human chemokine CCL2 (Genscript, product number: Z02829) and X-VIVO 15 serum-free hematopoietic stem cell culture medium (LONZA, batch number: 8MB230) were purchased and prepared to a concentration of 30 ng / mL, totaling 2.4 mL.

[0169] X-VIVO solution was added to the lower layer of a 24-well plate (Corning, catalog number 3421, 6.5 mm diameter, 5.0 μm pore size PC membrane) provided with the Transwell chamber, 600 μL / well; then 30 ng / mL CCL2 solution was added, with a control group without CCL2 solution included, and this was repeated 3 times; the upper layer of the Transwell chamber was slowly placed in the plate, and then 86CART cells, 89CART cells, and control T cells prepared in Example 7 were added to the upper layer, with a cell concentration of 2 × 10⁻⁶ cells. 5 The chemotaxis assay protocol is shown in Table 4, with cells / 100 μL.

[0170] Incubate at 37°C in a 5% CO2 incubator for 2 hours. Carefully remove the Transwell chamber with tweezers and observe the migration of 86CART and 89CART cells under a microscope. Mix the lower culture medium by pipetting and transfer it to a 1.5 mL EP tube. Centrifuge at 2500 rpm for 5 min, discard the supernatant, resuspend in 40 μL of DPBS (Hyclone, batch number AE29431662), and count the cells.

[0171] Table 4 Chemotaxis Experimental Scheme

[0172]

[0173] The migration results of the 89CART cells prepared in this invention to the tumor site are as follows: Figure 7 As shown, the results indicate that 89CART cells have a significantly enhanced ability to migrate to the tumor site compared with control T cells and 86CART cells.

[0174] Example 9: In vitro tumor cell killing performance of CART cells

[0175] 10,000 ovarian cancer cells (OVCAR-3 cells) (ATCC HTB161) were added to each well of an E-Plate 16 PET (ACEA, batch number 20190125) plate. The culture medium was RPMI 1640 (Gibco, batch number 2215748) + 20% FBS (Gibco, batch number 2152441P), 100 μL / well. The plate was placed on an xCELLigence RTCAS16 instrument and cultured at 37°C for 48 h with 5% CO2. T cells, 85CART cells, and 89CART cells were collected and counted. The 85CART cells were diluted with T cells to a positive rate of 37.79%. The effector cells (CAR+CART cells): target cells were added to each well at a ratio of 10:1. The culture medium for CART cells was X-VIVO 15, 100 μL / well. The CART cells and tumor cells were co-cultured for 25 h. The ability of 89CART cells to kill tumor cells in vitro was then measured.

[0176] The results of the ability of 89CART cells prepared in this embodiment to kill tumor cells in vitro are as follows: Figure 8 As shown, the results indicate that 89CART cells significantly enhanced the ability to kill tumor cells in vitro compared with control T cells and 85CART cells.

[0177] In addition, the cultures of T cells, 85CART cells, and 89CART cells were tested separately (all starting at a cell density of 2×10⁶). 5Cells / mL, supernatant collected after 72h culture, IL-21 expression level in supernatant (IL-21 ELISA kit, Invitrogen, batch number 220657).

[0178] The results showed that the 89CART cells prepared in this invention secreted IL-21 and PD1 antibodies normally.

[0179] Example 10: Performance of CART cells in killing tumor cells in mice

[0180] Construction of the ASPC-1-CCL2 cell line: The human CCL2 gene sequence (see SEQ ID NO: 13) was retrieved from the NCBI database and constructed into the pCDHF vector, named pCDHF-91 plasmid, and then packaged with lentivirus (packaging process referred to Example 6). ASPC-1 cells (ATCC CRL1682) were then infected with the packaged lentivirus. TM On the second day, the infected cells were seeded into single clones, and ASPC-1 cells with high CCL2 expression were selected for expanded culture to establish the ASPC-1-CCL2 cell line.

[0181] SEQ ID NO: 13:

[0182] ATGAAAGTCTCTGCCGCCCTTCTGTGCCTGCTGCTCATAGCAGCCACCTTCATTCCCCAAGGGCTCGCTCAGCCAGATGCAATCAATGCCCCAGTCACCTGCTGTTATAACTTCACCAATAGGAAGATCTCAGTGCAGAGGCTCGCGAGC TATAGAAGAATCACCAGCAGCAAGTGTCCCAAAGAAGCTGTGATCTTCAAGACCATTGTGGCCAAGGAGATCTGTGCTGACCCCAAGCAGAAGTGGGTTCAGGATTCCATGGACCACCTGGACAAGCAAACCCAAACTCCGAAGACTTGA.

[0183] On day 0, ASPC-1-CCL2 cells were subcutaneously inoculated into NPG mice (Biocytogen), 5.0E+06 cells / 100μl + 100μl of matrix gel (BD, catalog number 356234) / mouse. On day 7, mice in the three groups received tail vein infusions of T cells, 85CAR cells, and 89CAR cells (2.0E+06CAR+ cells each). Tumor volume changes in the mice were measured periodically (e.g., ...). Figure 9 (as shown) and on day 20, the concentration of CCL2 in peripheral blood and tumor was measured (e.g. Figure 10(as shown) and the proportion of CART cells in the tumor site (e.g.) Figure 11 (As shown).

[0184] The results showed that CCL2 expression could be detected in both the tumor site and peripheral blood after subcutaneous inoculation with ASPC-1-CCL2. The concentration of CCL2 in the tumor site was higher than that in the peripheral blood. This can chemotize more 89CART cells to the tumor site and kill tumor cells in mice more quickly and effectively.

[0185] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A transgenic immune effector cell, characterized in that, The transgenic immune effector cells co-expressed chimeric antigen receptors, fusion proteins, and chemokine receptor molecules; The chimeric antigen receptor comprises an extracellular region, a transmembrane region, and an intracellular region connected in series; wherein the extracellular region specifically recognizes tumor antigens, the transmembrane region is embedded in the cell membrane of the transgenic immune effector cell, and the intracellular region includes an intracellular segment of an immune co-stimulatory molecule; The fusion protein includes immune checkpoint antibodies and cytokines; The chemokine receptor molecule is a chemokine receptor or its chemokine-binding fragment. The immune checkpoint antibody is a PD1 antibody, and the cytokine is IL-21; The fusion protein also includes a linker peptide, through which the immune checkpoint antibody is linked to the cytokine. The linker peptide is a short peptide composed of G and S amino acids. The C-terminus of the PD1 antibody is linked to the N-terminus of the linker peptide, and the N-terminus of the IL-21 is linked to the C-terminus of the linker peptide. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 3 or SEQ ID NO:

9.

2. The transgenic immune effector cells according to claim 1, characterized in that, A) The chemokine receptor is any one or more of CXC chemokine receptor, CC chemokine receptor, CX3C chemokine receptor or XC chemokine receptor. B) The immune co-stimulatory molecules are selected from any one or more of 4-1BB, CD28, CD3, OX-40, CD40L, CD27, CD30 or their derivatives.

3. The transgenic immune effector cells according to claim 2, characterized in that, The CXC chemokine receptor is any one or more of CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6 or CXCR7.

4. The transgenic immune effector cells according to claim 2, characterized in that, The CC chemokine receptor is any one or more of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10 or CCR11.

5. The transgenic immune effector cells according to claim 2, characterized in that, The CX3C chemokine receptor is CX3CR1.

6. The transgenic immune effector cells according to claim 2, characterized in that, The XC chemokine receptor is XCR1.

7. The transgenic immune effector cells according to claim 2, characterized in that, The chemokine receptor molecule is CCR2.

8. The transgenic immune effector cells according to claim 2, characterized in that, The amino acid sequence of the chemokine receptor molecule is shown in SEQ ID NO:

5.

9. The transgenic immune effector cells according to claim 1, characterized in that, The amino acid sequence of the linker peptide is shown in SEQ ID NO:

7.

10. The transgenic immune effector cells according to claim 1, characterized in that, The PD1 antibody is a single-chain antibody.

11. The transgenic immune effector cells according to claim 10, characterized in that, The heavy chain variable region of the PD1 single-chain antibody includes the three complementarity-determining regions of the heavy chain variable region in SEQ ID NO: 3, and the light chain variable region of the PD1 single-chain antibody includes the three complementarity-determining regions of the light chain variable region in SEQ ID NO:

3.

12. The transgenic immune effector cells according to claim 10, characterized in that, The heavy chain variable region of the PD1 single-chain antibody includes amino acid residues 124 to 236 of SEQ ID NO: 3, and the light chain variable region of the PD1 single-chain antibody includes amino acid residues 1 to 108 of SEQ ID NO:

3.

13. The transgenic immune effector cells according to claim 10, characterized in that, The PD1 single-chain antibody is scFv as specified in SEQ ID NO:

3.

14. The transgenic immune effector cells according to claim 10, characterized in that, The amino acid sequence of the PD1 single-chain antibody is the sequence of amino acid residues from position 1 to position 236 of SEQ ID NO:

3.

15. The transgenic immune effector cells according to claim 1, characterized in that, The cytokine is the IL-21 portion of SEQ ID NO:

3.

16. The transgenic immune effector cells according to claim 1, characterized in that, The amino acid sequence of the cytokine IL-21 is the sequence of amino acid residues from position 252 to position 390 of SEQ ID NO:

3.

17. The transgenic immune effector cells according to any one of claims 1-16, characterized in that, A) The extracellular region includes antibodies that specifically recognize tumor antigens and the CD8 hinge region; B) The transmembrane region includes the transmembrane segment of CD8; and / or C) The immune co-stimulatory molecule is 4-1BB, and the intracellular region includes an intracellular segment of 4-1-BB and a CD3 Zeta chain; The CD8 hinge region of the extracellular region is connected to the transmembrane region, and the transmembrane region is connected to the intracellular segment of 4-1-BB in the intracellular region.

18. The transgenic immune effector cells according to claim 17, characterized in that, The tumor antigen is MSLN or CD19.

19. The transgenic immune effector cells according to claim 17, characterized in that, The antibody that specifically recognizes tumor antigens is an anti-MSLN single-chain antibody.

20. The transgenic immune effector cells according to claim 19, characterized in that, The heavy chain variable region of the MSLN single-chain antibody includes the three complementary determinant regions of the heavy chain variable region in SEQ ID NO: 1, and the light chain variable region of the MSLN single-chain antibody includes the three complementary determinant regions of the light chain variable region in SEQ ID NO:

1.

21. The transgenic immune effector cells according to claim 19, characterized in that, The heavy chain variable region of the MSLN single-chain antibody includes amino acid residues 1 to 145 of SEQ ID NO: 1, and the light chain variable region of the MSLN single-chain antibody includes amino acid residues 161 to 266 of SEQ ID NO:

1.

22. The transgenic immune effector cells according to claim 19, characterized in that, The MSLN single-chain antibody is scFv as specified in SEQ ID NO:

1.

23. The transgenic immune effector cells according to claim 19, characterized in that, The amino acid sequence of the MSLN single-chain antibody is the sequence of amino acid residues from position 1 to position 266 of SEQ ID NO:

1.

24. The transgenic immune effector cells according to claim 17, characterized in that, The antibody that specifically recognizes tumor antigens is an anti-CD19 single-chain antibody.

25. The transgenic immune effector cells according to claim 24, characterized in that, The heavy chain variable region of the CD19 single-chain antibody includes the three complementary determinant regions of the heavy chain variable region in SEQ ID NO: 11, and the light chain variable region of the CD19 single-chain antibody includes the three complementary determinant regions of the light chain variable region in SEQ ID NO:

11.

26. The transgenic immune effector cells according to claim 24, characterized in that, The heavy chain variable region of the CD19 single-chain antibody includes amino acid residues 123 to 242 of SEQ ID NO: 11, and the light chain variable region of the CD19 single-chain antibody includes amino acid residues 1 to 107 of SEQ ID NO:

11.

27. The transgenic immune effector cells according to claim 24, characterized in that, The CD19 single-chain antibody is scFv as specified in SEQ ID NO:

11.

28. The transgenic immune effector cells according to claim 24, characterized in that, The amino acid sequence of the CD19 single-chain antibody is the sequence of amino acid residues from position 1 to position 242 of SEQ ID NO:

11.

29. The transgenic immune effector cells according to claim 24, characterized in that, The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 1 or SEQ ID NO:

11.

30. The transgenic immune effector cells according to claim 1, characterized in that, The chimeric antigen receptor, fusion protein, and chemokine receptor molecules are linked by a 2A peptide.

31. The transgenic immune effector cells according to claim 30, characterized in that, The 2A peptide is selected from any one or more of P2A, T2A, F2A or E2A.

32. The transgenic immune effector cells according to claim 24, characterized in that, The chimeric antigen receptor is linked to the fusion protein via P2A, and the fusion protein is linked to the chemokine receptor molecule via T2A.

33. The transgenic immune effector cells according to claim 1, characterized in that, The transgenic immune effector cells are selected from any one or more of T cells, NK cells, NKT cells, macrophages, or CIK cells.

34. A lentivirus, characterized in that, The lentivirus carries the following nucleic acid: 1) The nucleic acid encoding the chimeric antigen receptor in the transgenic immune effector cells of any one of claims 1-33, wherein the extracellular region of the chimeric antigen receptor specifically recognizes tumor antigens; 2) A nucleic acid encoding a fusion protein in a transgenic immune effector cell according to any one of claims 1-33, wherein the fusion protein comprises an immune checkpoint antibody and a cytokine; and 3) The nucleic acid encoding the chemokine receptor molecule in the transgenic immune effector cells of any one of claims 1-33, wherein the chemokine receptor molecule is a chemokine receptor or a chemokine-binding fragment thereof.

35. The lentivirus according to claim 34, characterized in that, A) The tumor antigen is MSLN or CD19, and the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 1 or SEQ ID NO: 11; B) The immune checkpoint antibody is a PD1 antibody, the cytokine is IL-21, the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 3 or SEQ ID NO: 9, and the nucleic acid encoding the fusion protein has the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 10; and / or C) The chemokine receptor molecule is CCR2, the amino acid sequence of the chemokine receptor molecule is shown in SEQ ID NO: 5, and the nucleic acid encoding the chemokine receptor molecule has the nucleotide sequence shown in SEQ ID NO:

6.

36. The lentivirus according to claim 35, characterized in that, The nucleic acid encoding the chimeric antigen receptor has a nucleotide sequence as shown in SEQ ID NO: 2 or SEQ ID NO:

12.

37. A construct, characterized in that, The construct comprises: a first nucleic acid molecule encoding a chimeric antigen receptor in a transgenic immune effector cell according to any one of claims 1-33, wherein the extracellular region of the chimeric antigen receptor specifically recognizes a tumor antigen; a second nucleic acid molecule encoding a fusion protein in a transgenic immune effector cell according to any one of claims 1-33, wherein the fusion protein comprises an immune checkpoint antibody and a cytokine; and a third nucleic acid molecule encoding a chemokine receptor molecule in a transgenic immune effector cell according to any one of claims 1-33, wherein the chemokine receptor molecule is a chemokine receptor or a chemokine-binding fragment thereof.

38. The construct according to claim 37, characterized in that, A) The first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule sequentially express a chimeric antigen receptor, a fusion protein, and a chemokine receptor molecule in immune effector cells, respectively. B) The chimeric antigen receptor, fusion protein, and chemokine receptor molecules are in non-fusion form; C) The transgenic immune effector cells are selected from any one or more of T cells, NK cells, NKT cells, macrophages, or CIK cells; D) The construct further includes: a first promoter, which is operatively linked to the first nucleic acid molecule; and / or E) The vector of the construct is a non-pathogenic viral vector.

39. The construct according to claim 38, characterized in that, The transgenic immune effector cells are T cells.

40. The construct according to claim 38, characterized in that, The first promoter is selected from any one or more of the U6, H1, CMV, EF-1, LTR or RSV promoters.

41. The construct according to claim 38, characterized in that, The non-pathogenic viral vector is selected from any one or more of retroviral vectors, lentiviral vectors, or adenovirus-associated viral vectors.

42. The construct according to claim 38, characterized in that, The non-pathogenic viral vector is a lentiviral vector.

43. The construct according to claim 42, characterized in that, The nucleic acid encoding the lentiviral vector has the nucleotide sequence shown in SEQ ID NO:

8.

44. A method for preparing transgenic immune effector cells according to any one of claims 1-33, characterized in that, The lentivirus of any one of claims 34-36, or the construct of any one of claims 37-43, is introduced into immune effector cells.

45. The method according to claim 44, characterized in that, The immune effector cells are selected from any one or more of T cells, NK cells, NKT cells, macrophages, or CIK cells.

46. ​​The method according to claim 45, characterized in that, The immune effector cells are T cells.

47. The use of the transgenic immune effector cells according to any one of claims 1-33, the lentivirus according to any one of claims 34-36, or the construct according to any one of claims 37-43 in the preparation of cancer therapeutic drugs; The cancer in question is a solid tumor.

48. A cancer treatment drug, characterized in that, Includes the transgenic immune effector cells according to any one of claims 1-33, the lentivirus according to any one of claims 34-36, or the construct according to any one of claims 37-43; The cancer in question is a solid tumor.

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