TW202330909A - Transgenic immune effector cell and use thereof - Google Patents

Abstract

The present invention relates to the field of biotechnology, and specifically relates to a transgenic immune effector cell and the use thereof. An immune checkpoint antibody in a fusion protein expressed by an immune effector cell can relieve the immunosuppression of the immune checkpoint on the immune effector cell, a cytokine can enhance the anti-tumor activity of the immune effector cell, and a chemokine receptor molecule can significantly improve the migration capability of the immune effector cell to the tumor site. Compared with the fusion protein and a cell co-expressing the above-mentioned fusion protein and a chimeric antigen receptor, the immune effector cell has a stronger killing effect on tumor cells, a low drug toxicity and high safety, and accurate treatment of CAR-T on solid tumors is effectively realized.

Description

Transgenic immune effector cells, preparation methods, lentivirus, constructs, cancer therapeutic drugs and their applications

The present invention requires the priority of the Chinese invention patent application with application number 202111055628.8, the filing date is September 9, 2021, and the invention title is a transgenic immune effector cell and its application, the entire content of which is incorporated herein by reference.

The invention belongs to the field of biotechnology. More specifically, regarding a genetically modified immune effector cell and its application.

At present, chimeric antigen receptor T cells (chimeric antigen receptor T cells; CART) have achieved very good results in the treatment of hematomas. According to public data, the efficacy of BCMA CART in multiple myeloma has reached an objective response rate ( ORR) is 100%, and the complete response rate (CR) is 70.6%, and there are already 5 CART cell products on the market around the world, all of which are targeted at hematological tumors. However, the efficacy of CART in the treatment of solid tumors is unsatisfactory. Clinical data shows that the clinical efficacy of Mesothelin CART combined with PD1 blockers for mesothelioma is 62.5% with an ORR of 62.5% and a CR of 18.75%. Therefore, the efficacy of CART for solid tumors needs to be improved.

In the process of killing solid tumors, there are three main reasons why CART cells have poor efficacy: 1) It is difficult for CART to reach and infiltrate into the tumor in large numbers for killing; 2) The activity of a small amount of CART that infiltrates into the tumor is blocked by the tumor site. Highly expressed PD1 and other immunosuppressive molecules inhibit; 3) Insufficient contact between CART cells and tumor cells significantly affects their proliferation and persistence, causing CART cells to be easily exhausted in the body.

Chemokines are small molecule cytokines that can cause cells to undergo chemotactic movement. They bind to chemokine receptors and transmit a variety of cell information. Chemokines can induce white blood cells, especially T lymphocytes, to migrate to local areas of inflammation and tumor infiltration. Compared with hematological tumors, solid tumor cells can secrete chemokines such as CXCL12 and CXCL5 to prevent CART cells from reaching tumor lesions; at the same time, solid tumors secrete very few CXCR3 and CCR5 ligands, which indicate the transport of CART cells. The two factors work together to make CART It is difficult for cells to accurately reach the site of solid tumors to exert immune effects. Therefore, how to achieve precise treatment of solid tumors is a major problem in CART cell therapy.

The present invention is based on the inventor's discovery and understanding of the following problems.

The inventor has developed a transgenic immune effector cell that co-expresses a chimeric antigen receptor, a fusion protein and a chemokine receptor molecule; among which, the chemokine receptor molecule can chemoattract more chimeric antigens receptor to the tumor site; in addition, the fusion protein can secrete immune checkpoint antibodies and cytokines, using the dual advantages of immune checkpoint antibodies and cytokines to specifically act on genetically modified immune effector cells, reducing the tumor microenvironment's effect on genetically modified immunity. While inhibiting cells, it also makes the genetically modified immune effector cells have a longer-lasting effect, and cytokines can significantly improve the killing ability of tumor cells, realizing CART's precise treatment of solid tumors.

In the first aspect of the invention, the invention proposes a transgenic immune effector cell that 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 connected in series in sequence; wherein the extracellular region specifically recognizes a tumor antigen, the transmembrane region is embedded in the cell membrane of the transgenic immune effector cell, and the intracellular region includes The intracellular segment of an immune costimulatory molecule; the fusion protein includes an immune checkpoint antibody and a cytokine; the chemokine receptor molecule is a chemokine receptor or a chemokine-binding fragment thereof.

In a second aspect of the invention, the invention proposes a lentivirus carrying the following nucleic acid: 1) Nucleic acid encoding a chimeric antigen receptor in the transgenic immune effector cell, the extracellular region of the chimeric antigen receptor specifically recognizing a tumor antigen; 2) Nucleic acid encoding a fusion protein in the transgenic immune effector cells, the fusion protein including immune checkpoint antibodies and cytokines; 3) Nucleic acid encoding a chemokine receptor molecule in the transgenic immune effector cell, where the chemokine receptor molecule is a chemokine receptor or a chemokine-binding fragment thereof.

In a third aspect of the invention, the invention proposes a construct, which includes: A first nucleic acid molecule encoding a chimeric antigen receptor in the transgenic immune effector cell, and the extracellular region of the chimeric antigen receptor specifically recognizes a tumor antigen; a second nucleic acid molecule encoding a fusion protein in the transgenic immune effector cell, the fusion protein including an immune checkpoint antibody and a cytokine; A third nucleic acid molecule encodes a chemokine receptor molecule in the transgenic immune effector cell, and the chemokine receptor molecule is a chemokine receptor or a chemokine-binding fragment thereof.

In the fourth aspect of the present invention, the present invention proposes a method for preparing the transgenic immune effector cells described in the first aspect, by introducing the lentivirus or the construct into the immune effector cells; according to the embodiment of the present invention, the The immune effector cells are selected from any one or more of T cells, NK cells, NKT cells, macrophages or CIK cells; according to embodiments of the present invention, the immune effector cells are T cells.

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

According to an embodiment of the invention, the cancer is a solid tumor.

In a sixth aspect of the present invention, the present invention provides a cancer treatment drug. According to embodiments of the present invention, the transgenic immune effector cells, the lentivirus or the construct are included.

According to an embodiment of the invention, the cancer is a solid tumor.

In a seventh aspect of the present invention, the present invention provides a method for treating cancer, which includes: administering a therapeutically effective amount of a cancer treatment drug as described above to a subject in need of treatment.

According to an embodiment of the invention, the cancer is a solid tumor.

The present invention will be further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

Unless otherwise stated, the reagents and materials used in the following examples were all commercially available.

In addition, the terms “first” and “second” are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of indicated technical features. Therefore, features defined as "first" and "second" may explicitly or implicitly include at least one of these features. In the description of the present invention, "multiple" means at least two, such as two, three, four, etc., and "multiple" and "several" mean at least two, such as two, three, etc. Four types, etc., unless otherwise clearly and specifically limited.

The invention relates to a transgenic immune effector cell, which 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 a transmembrane region in series. region and intracellular region; wherein, the extracellular region specifically recognizes a tumor antigen, 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 costimulatory molecule; The fusion protein includes an immune checkpoint antibody and a cytokine; the chemokine receptor molecule is a chemokine receptor or a chemokine-binding fragment thereof.

The above-mentioned transgenic immune effector cells according to embodiments of the present invention co-express chimeric antigen receptors, fusion proteins and chemokine receptor molecules. The fusion proteins can secrete immune checkpoint antibodies and cytokines, combining the immune checkpoint antibodies and cytokines. The dual superiority specifically acts on the transgenic immune effector cells, reducing the inhibitory effect of the tumor microenvironment on the transgenic immune effector cells and making the transgenic immune effector cells more effective. Cytokines can significantly improve the killing ability of tumor cells. Chemokine receptor molecules significantly improve the ability of immune effector cells to migrate to tumor sites. Therefore, they are comparable to fusion proteins expressing immune checkpoint antibodies or cytokines alone, and cells co-expressing the above fusion proteins and chimeric antigen receptors. Compared with the above, the above-mentioned immune effector cells have a stronger killing effect on tumor cells, and at the same time, the specificity of the combination of cytokines and immune effector cells is significantly improved, and the drug toxicity is greatly reduced.

In the present invention, the term "chemokine receptor molecule" belongs to a G protein-coupled receptor containing a seven-transmembrane structure, which mainly transduces signals through G protein, and its binding to the receptor has certain subfamily specificity. Chemokine receptor molecules are divided into CXC chemokine receptors, CC chemokine receptors, CX3C chemokine receptors and XC chemokine receptors due to different types of ligands.

In the present invention, the term "immune checkpoint" refers to a series of molecules expressed on immune cells that can regulate the degree of immune initiation. They play an important role in preventing the occurrence of autoimmunity. Immune checkpoints related to cancer include PD1, PD-L1, TIGIT, LAG3, CTLA4, BTLA, TIM3, etc.

In the present invention, the term "scFv" refers to a "single chain antibody, which is a genetically engineered antibody consisting of an antibody heavy chain variable region (VH) and a light chain variable region (VL), usually linked by 15-20 amines. Antibodies are formed by connecting short peptides (linkers) of amino acids. Single-chain antibodies can better retain their affinity activity for antigens, and have the characteristics of small molecular weight, strong penetrating power, and weak antigenicity.

In the present invention, "immune checkpoint antibodies" have anti-immune checkpoint activity and can specifically bind to immune checkpoints.

In the present invention, the term "cytokine" refers to a class of small molecule proteins with a wide range of biological activities that are synthesized and secreted by immune cells and certain non-immune cells upon stimulation. Cytokines are divided into interleukins, interferons, tumor necrosis factor superfamily, colony-stimulating factors, chemokines, growth factors, etc.

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

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.

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.

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

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.

In some embodiments, the CX3C chemokine receptor is CX3CR1.

In some embodiments, the XC chemokine receptor is XCR1.

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.

In some embodiments, the immune costimulatory molecule is selected from any one or more of 4-1BB, CD28, CD3, OX-40, CD40L, CD27, CD30, or their derivatives.

In some embodiments, the immune costimulatory molecule is 4-1BB.

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

In some embodiments, the immune checkpoint is PD1.

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

In some embodiments, the cytokine is IL-21.

In some embodiments, the fusion protein further includes a linker peptide; the immune checkpoint antibody is connected to the cytokine via the linker peptide. Short peptides containing G and S amino acids commonly used in the field can be used as the connecting peptide of the present invention.

In the present 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 be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

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

In some embodiments, the C-terminus of the PD1 antibody is connected to the N-terminus of the connecting peptide, and the N-terminus of IL-21 is connected to the C-terminus of the connecting peptide. In some embodiments, the N-terminus of the PD1 antibody is connected to the C-terminus of the connecting peptide, and the C-terminus of IL-21 is connected to the N-terminus of the connecting peptide.

The inventor found that the fusion protein obtained by the fusion of PD1 antibody and IL-21 significantly reduced the expression of PD1 on the surface of immune effector cells. The PD1 antibody specifically binds to PD1 on the surface of immune effector cells, so that IL-21 also has a specific effect. For immune effector cells, it effectively reduces the toxicity caused by the combination of IL-21 and IL-21 receptors on other cell surfaces, and the immune effector cells have a more significant killing effect on tumors.

PD1 is mainly expressed on the surface of T cells, mainly CD8+ T cells. The PD1 antibody and IL-21 of the fusion protein secreted by the transgenic immune effector cells selectively bind to the surface of T cells and CART cells. On the one hand, it blocks the PD1/PD-L1 signaling pathway, and on the other hand, it makes IL -21 specifically acts on T cells and CART cells, thereby performing its dual functions; at the same time, the fusion protein greatly increases the half-life of the drug due to its increased molecular weight.

In some embodiments, the amino acid sequence of the connecting peptide is shown in SEQ ID NO: 7. GGGGSGGGGSGGGGS (SEQ ID NO: 7).

In some embodiments, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 3 or SEQ ID NO: 9. Among them, the fusion protein PD1 scFv-IL-21 has the amino acid sequence shown in SEQ ID NO: 3, the fusion protein IL-21-PD1 scFv has the amino acid sequence shown in SEQ ID NO: 9, and PD1 scFv represents PD1 Antibody, IL-21 means that the cytokine is IL-21, the connection sequence in the fusion protein PD1 scFv-IL-21 is that the C-terminus of PD1 scFv is connected to the N-terminus of IL-21, the fusion protein IL-21-PD1 scFv The connection sequence is that the C-terminus of IL-21 is connected to the N-terminus of PD1 scFv. EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRA EDTAVYYCATNDDYWGQGTLVTVSSGGGGSGGGGSGGGGSMHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 3) HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSGGGGSMEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGS GSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS (SEQ ID NO: 9)

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 immune costimulatory molecule is 4-1BB, and the cell The inner region includes the intracellular segment of 4-1-BB and the 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 4-1- The intracellular segments of BB are connected.

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.

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

In some embodiments, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 1 or SEQ ID NO: 11. RGSATMALPVTALLLPLALLLHAARPQVQLQQSGPELEKPGASVKLSCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLA SGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGAGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGRRKP KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 1) DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:11)

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

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

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.

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.

In some embodiments, the immune effector cells are T cells.

The T cells mentioned in the present invention refer to T lymphocytes, which have the function of recognizing antigens and secreting lymphokines. When T lymphocytes receive antigens exposed to antigenic determinants, they will differentiate into effector T cells.

In some embodiments, when the chemokine receptor molecule is CCR2b, the immune costimulatory 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 When cells are constructed, the CCR2b in the chimeric antigen receptor T (CART) cells can attract more CART to the tumor site. The PD1 antibody in the fusion protein can relieve the immunosuppression of CART. At the same time, IL-21 significantly improves the response to CART. The killing ability of tumor cells.

The invention also relates to a lentivirus carrying the following nucleic acid: 1) Nucleic acid encoding the chimeric antigen receptor, the extracellular region of the chimeric antigen receptor specifically recognizes a tumor antigen; 2) Nucleic acid encoding the fusion protein, the fusion protein includes an immune checkpoint antibody and Cytokine; 3) Nucleic acid encoding the chemokine receptor molecule, which is a chemokine receptor or a chemokine-binding fragment thereof.

The above-mentioned lentivirus according to the embodiment of the present invention is introduced into recipient cells to obtain the transgenic immune effector cells, which can express and secrete fusion proteins including immune checkpoint antibodies and cytokines, and chimeric antigen receptors in the transgenic immune effector cells. and chemokine receptor molecules, reduce the expression of immune checkpoints on the surface of immune effector cells, reduce the inhibitory effect of the tumor microenvironment on immune effector cells, and chemoattract more chimeric antigen receptors to the tumor site, allowing immune effector cells to The killing effect on tumors is more significant and long-lasting, and it is highly safe.

In the present invention, the term "nucleic acid" usually refers to RNA or DNA. The nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. DNA is preferably used when ligated into a vector.

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

According to an embodiment of the present invention, the above-mentioned lentivirus further includes at least one of the following additional technical features.

In some embodiments, 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, encoding the chimeric antigen receptor. The nucleic acid has the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 12. CGCGGATCCGCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAAGTCCAGCTCCAGCAGTCGGGCCCAGAGTTGGAGAAGCCTGGGGCGAGCGTGAAGCTTTCATGCAAAGCCTCAGGCTACTCCTTTACTGGATACACGATGAATTGGGTGAAACAGTCGCATGGAAAGTCACTGGAATGGATCGGTCTGATTACGCCCTACAACGGCGCCTCCAGCTACA ACCAGAAGTTCAGGGGAAAGGCGACCCTTACTGTCGACAAGTCGTCAAGCACCGCCTACATGGACCTCCTGTCCCTGACCTCCGAAGATAGCGCGGTCTACTTTTGTGCACGCGGAGGTTACGATGGACGGGGATTCGACTACTGGGGCCAGGGAACCACTGTCACCGTGTCGAGCGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGATATCGAACTCACTCAGTCCCCAGCAATCATGTCCGCTTC ACCGGGAGAAAAGGTGACCATGACTTGCTCGGCCTCCTCGTCCGTGTCATACATGCACTGGTACCAACAAAAATCGGGGACCTCCCCTAAGAGATGGATCTACGATACCAGCAAACTGGCTTCAGGCGTGCCGGGACGCTTCTCGGGTTCGGGGAGCGGAAATTCGTATTCGTTGACCATTTCGTCCGTGGAAGCCGAGGACGACGCAACTTATTACTGCCAACAGTGGTCAAAGCACCCGCTCACTTTCGGAGCCGGCACTAAGCTG GAGATCAAGACCACGACGCCAGCGCCGCCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATTCAAACAACCATTTATGAG ACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGC AGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAATGAGAATTCCGG (SEQ ID NO: 2) GACATCCAGATGACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTT GCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGT AGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAACCACGACGCCAGCGCCCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCTGCGCCCCAGAGAGG CGTGCCGGCCAGCGGCGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAG TTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTT ACCAGGGTCCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 12)

In some embodiments, the immune checkpoint antibody is a PD1 antibody, the cytokine is IL-21, and the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 3 or SEQ ID NO: 9, encoding The nucleic acid of the fusion protein has the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 10. GAGATCGTGCTGACCCAGTCTCCAGCCACACTGAGCCTGTCTCCTGGCGAGAGAGCCACCCTGTCTTGTAGGGCCAGCCAGTCCGTGAGCTCTTACCTGGCCTGGTATCAGCAGAAGCCAGGCCAGGCCCCAAGACTGCTGATCTACGACGCCTCCAACAGAGCCACCGGCATCCCAGCCAGATTTTCTGGCTCCGGCTCTGGCACCGACTTCACACTGACCATCAGCTCTCTGGAGCCAGAGGATTTCGCCGTGT ATTACTGCCAGCAGAGCTCTAACTGGCCAAGAACATTCGGGCAGGGGACCAAGGTGGAAATCAAGAGGGGCGGCGGCGGCTCTGGCGGCGGCGGCTCCGGCGGCGGCGGCTCTCAGGTGCAGCTGGTGGAGAGCGGCGGCGGAGTGGTGCAGCCAGGCAGATCTCTGAGACTGGATTGCAAGGCCAGCGGCATCACCTTCAGCAATTCCGGCATGCACTGGGTGCGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGT CCGTGATCTGGTATGACGGCTCTAAGCGGTACTATGCCGACTCTGTGAAGGGCAGATTCACCATCTCCAGGGACAACTCCAAGAATACCCTGTTCCTGCAGATGAACAGCCTGAGGGCCGAGGATACCGCCGTGTACTATTGCGCCACCAACGACGATTACTGGGGCCAGGGCACACTGGTGACCGTGTCCAGCGGCGGCGGCGGCTCTGGCGGCGGCGGCTCCGGCGGCGGCGGCTCTATGCACAAATCAAGTCCCCAA GGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAG ACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC (SEQ ID NO: 4) CACAAATCAAGTCCCCAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAAACCACCTTCCACAAGAAATGCAGG GAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCCGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTATGGAGATCGTGCTGACCCAGTCTCCAGCCACACTGAGCCTGTCTCCTGGCGAGAGAGCCACCCTGTCTTG TAGGGCCAGCCAGTCCGTGAGCTCTTACCTGGCCTGGTATCAGCAGAAGCCAGGCCAGGCCCCAAGACTGCTGATCTACGACGCCTCCAACAGAGCCACCGGCATCCCAGCCAGATTTTCTGGCTCCGGCTGGCACCGACTTCACACTGACCATCAGCTCTCTGGAGCCAGAGGATTTCGCCGTGTATTACTGCCAGCAGAGCTCTAACTGGCCAAGAACATTCGGGCAGGGGACCAAGGGTGGAAATCAAGAGGGGCGGC GGCGGCTCTGGCGGCGGCGGCTCCGGCGGCGGCGGCTCAGGTGCAGCTGGTGGAGCGGCGGCGGAGTGGTGCAGCCAGATCTCTGAGACTGGATTGCAAGGCCAGCGGCATCACCTTCAGCAATTCCGGCATGCACTGGGTGCGGCAGGCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTATGACGGCTCTAAGCGGTACTATGCCGACTCTGTGAAGGGCAGATTCACCATCTCCAGGG ACAACTCCAAGAATACCCTGTTCCTGCAGATGAACAGCCTGAGGGCCGAGGATACCGCCGTGTACTATTGCGCCACCAACGACGATTACTGGGGCCAGGGCACACTGGTGACCGTGTCCAGC (SEQ ID NO: 10)

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

In some embodiments, the chemokine receptor molecule is CCR2.

In the present invention, the term "CCR2" is a specific receptor for monocyte chemoattractant protein-1 (CCL2), and is also a receptor for CCL7, CCL8, CCL11, CCL12, and CCL13, including two subtypes: CCR2a and CCR2b , both originate from different splicing of the same gene, only the hydroxyl end is different, but CCR2b is the main functional form.

In some embodiments, the chemokine receptor molecule is CCR2b.

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 a nucleic acid sequence as shown in SEQ ID NO: 6 nucleotide sequence. MLSTSRSRFIRNTNESGEEVTTFFDYDYGAPCHKFDVKQIGAQLLPPLYSLVFIFGFVGNMLVVLILINCKKLKCLTDIYLLNLAISDLLFLITLPLWAHSAANEWVFGNAMCKLFTGLYHIGYFGGIFFIILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWLVAVFASVPGIIFTKCQKEDSVYVCGPYFPRGWNNFHTIMRNILGLVLPLLIMVICYSG ILKTLLRCRNEKKRHRAVRVIFTIMIVYFLFWTPYNIVILLNTFQEFFGLSNCESTSQLDQATQVTETLGMTHCCINPIIYAFVGEKFRRYLSVFFRKHITKRFCKQCPVFYRETVDGVTSTNTPSTGEQEVSAGL (SEQ ID NO: 5) ATGCTGTCCCACATCTCGTTCTCGGTTTATCAGAAATACCAACGAGCGGTGAAGAAGTCACCACCTTTTTTGATTATGATTACGGTGCTCCCTGTCATAAATTTGACGTGAAGCAAATTGGGGCCCAACTCCTGCCTCCGCTCTACTCGCTGGTGTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCGTCCTCATCTTAATAAACTGCAAAAAGCTGAAGTGCTTGACTGACATTTACCTGCTCAACCTGGCCATCTCTGATC TGCTTTTTCTTATTACTCTCCCATTGTGGGCTCACTCTGCTGCAAATGAGTGGGTCTTTGGGAATGCAATGTGCAAATTATTCACAGGGCTGTATCACATCGGTTATTTTGGCGGAATCTTCTTCATCCTCCTGACAATCGATAGATACCTGGCTATTGTCCATGCTGTGTTTGCTTTAAAAGCCAGGACGGTCACCTTTGGGGTGGTGACAAGTGTGATCACCTGGTTGGTGGCTGTGTTTGCTTCTGTCCCAGGAATCATCT TTACTAAATGCCAGAAAGAAGATTCTGTTTATGTCTGTGGCCCTTATTTTCCACGAGGATGGAATAATTTCCACACAATAATGAGGAACATTTTGGGGCTGGTCCTGCCGCTGCTCATCATGGTCATCTGCTACTCGGGAATCCTGAAAACCCTGCTTCGGTGTCGAAACGAGAAGAAGAGGCATAGGGCAGTGAGAGTCATCTTCACCATCATGATTGTTTACTTTCTCTTCTGGACTCCCTATAATTGTCATTCTCCTGAACACCTT CCAGGAATTCTTCGGCCTGAGTAACTGTGAAAGCACCAGTCAACTGGACCAAGCCACGCAGGTGACAGAGACTCTTGGGATGACTCACTGCTGCATCAATCCCATCATCTATGCCTTCGTTGGGGAAGTTCAGAAGGTATCTCTCGGTGTTCTTCCGAAAGCACATCACCAAGCGCTTCTGCAAACAATGTCCAGTTTTCTACAGGGAGACAGTGGATGGAGTGACTTCAACAAACACGCCTTCCACTGGGGAGCAGGAAGTC TCGGCTGGTTTA (SEQ ID NO: 6)

In some embodiments, the lentivirus carries a nucleotide sequence containing SEQ ID NO: 4 or SEQ ID NO: 10.

The invention also relates to a construct comprising: a first nucleic acid molecule encoding the chimeric antigen receptor, and the extracellular region of the chimeric antigen receptor specifically recognizes a tumor antigen; a second nucleic acid molecule encoding the fusion protein including an immune checkpoint antibody and a cytokine; A third nucleic acid molecule encodes the chemokine receptor molecule, and the chemokine receptor molecule is a chemokine receptor or a chemokine-binding fragment thereof.

The above construct according to the embodiment of the present invention is introduced into the recipient cells to obtain the transgenic immune effector cells, which can express chimeric antigen receptors, secrete fusion proteins and chemokine receptor molecules on the surface of the transgenic immune effector cells, The killing effect on tumor cells is more significant, lasting and safe.

According to an embodiment of the present invention, the above construct further includes at least one of the following additional technical features.

In some embodiments, the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule express chimeric antigen receptors, fusion proteins and chemokine receptor molecules respectively in immune effector cells.

In some embodiments, the chimeric antigen receptor, fusion protein and chemokine receptor molecule are in unfused form.

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.

In some embodiments, the immune effector cells are T cells.

In some embodiments, the construct further includes a first promoter operably linked to the first nucleic acid molecule.

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

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

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. CGATACCGTCGACCTCGAGACCTAGAAAAACATGGAGCAATCACAAGTAGCAATACAGCAGCTACCAATGCTGATTGTGCCTGGCTAGAAGCACAAGAGGAGGAGGAGGTGGGTTTTCCAGTCACACCTCAGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAA GGCTACTTCCCTGATTGGCAGAACTACACACCAGGGCCAGGGATCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCAAGAGAAGGTAGAAGAAGCCAATGAAGGAGAGAACACCCGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCGGAGAGAGAAGTATTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCACATGGCCCGAGAGCTGCATCCGGACTCGAGATAACTTCGTATAAT GTATGCTATACGAAGTTATTCCGGACTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTAGTCAGTGTGGAAAATCTCTAGCAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGC CATCTGTTGTTTGCCCCTCCCTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAA AAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCC TAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTGTTTGCAAGCAGCAAGATT ACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCC TGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGT CACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTG CTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTT CAATATTATTGAAGCATTTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAAGGCAA GGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGCCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTT CCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGGATTTCCAAGTCTCCACCCC ATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGCGCGTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTG ACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGGATCGCGATG AAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAA GAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGGAGATATGAGGGACAATTGGAGAAGTGAATTATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAACGGTACAGGCC AGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATAACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGT GGGACAGAGAAATTAACAATTACAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTC CCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCGGCACTGCGTGCGCCAATTCTGCAGACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTTCGGGTTTACAGGGACA GCAGAGATCCAGTTTGGTTAATTAACGGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGACCCGGTGCCTAGAGAAGGTGGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTA AGTGCCGTGTGTGGTTCCCGCGGGCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTC TCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTCGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTTCCGCCCCCC TGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACTGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGT TTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGACTTAAGGCCGGCCGACGCCCTTGACGATTTTGACTTAGACATGCTCCCAGCCGATGCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTG ACGCTCTTGACGATTTTGACCTTGACATGCTCCCCGGGTAACTAAGTAAGGATCAATTCGATATCAAGCTTATCGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGT TGTCAGGCAACGTGGCGTGGTGTGCACTGTTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGA CGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCGCAT (SEQ ID NO: 8)

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.

The above-mentioned lentivirus or the above-mentioned construct according to the embodiment of the present invention is introduced into immune effector cells, and chimeric antigen receptors, fusion proteins and chemokine receptor molecules are co-expressed and secreted in the cells, thereby reducing the expression of immune checkpoints and tumor microorganisms. The inhibitory effect of the environment on immune effector cells kills tumor cells more significantly and long-term.

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.

In some embodiments, the immune effector cells are T cells.

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.

The present invention also relates to a cancer therapeutic drug, including the immune effector cell, the lentivirus or the construct.

In some embodiments, the cancer is a solid tumor.

The present invention also relates to a method of treating cancer, which includes: administering a therapeutically effective amount of a cancer treatment drug as described above to a subject in need of treatment.

In some embodiments, the cancer is a solid tumor.

The above-mentioned solid tumors include, but are not limited to, ovarian cancer and pancreatic cancer.

The present invention includes the following beneficial effects.

The inventor has developed a transgenic immune effector cell that co-expresses a chimeric antigen receptor, a fusion protein and a chemokine receptor molecule; among which, the chemokine receptor molecule can chemoattract more chimeric antigens receptor to the tumor site, significantly improving the ability of immune effector cells to migrate to the tumor site; in addition, the fusion protein can secrete immune checkpoint antibodies and cytokines, using the dual advantages of immune checkpoint antibodies and cytokines to specifically act on Genetically modified immune effector cells, immune checkpoint antibodies in the fusion protein can relieve the immune suppression of immune checkpoints on immune effector cells, and cytokines can significantly improve the killing ability of tumor cells, and the killing effect is better, long-lasting and safe, realizing CART Precision treatment of solid tumors.

Example 1 Construction of CART19 (T cells expressing chimeric antigen receptor targeting CD19) lentiviral vector expressing Anti PD1-IL-21 fusion protein and its control vector

Gene synthesize the nucleotide sequence of the structure shown in Figure 1, construct the nucleotide fragment onto the lentiviral vector according to the enzyme cutting sites of the lentiviral vector, design primers, and verify the correctness of the vector construction through sequencing results.

The amino acid sequence of CAR19 is as shown in SEQ ID NO: 11, and the nucleotide sequence is as shown in SEQ ID NO: 12.

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

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

Example 2 Packaging and concentrating lentivirus

293T was inoculated at a density of 8×10 ⁶ cells/150 mm ² culture dish. The status of the cells was observed the next day. The third generation lentiviral packaging vector was co-transfected into 293T using the PEI transfection method. After 6 hours of transfection, the vector was transferred to 293T. solution, add DMEM medium containing 10% fetal calf serum in a 15mL/150mm ² culture dish, collect the virus supernatant 48 hours and 72 hours after transfection, centrifuge at 2000rpm and 4℃ for 10min to remove cell debris, and then filter impurities with a 0.45 micron filter , the filtered virus suspension was centrifuged at 25000 rpm and 4°C for 2 hours to concentrate the lentivirus. The concentrated virus was resuspended in an appropriate amount of culture medium and stored at -80°C.

Example 3 Production of CART and control cells

20 mL of blood was drawn, PBMC were separated by Ficall gradient centrifugation, and T cells were isolated using Stemcell T cell negative selection kit (Cat. No.: 19051). The separated T cells were added with 5% human AB serum and 300 units/mL IL-2 X- Resuspend T cells in VIVO 15 medium to 1×10 ⁶ cells/mL, wash the beads with X-VIVO 15 containing 1% FBS, and add pre-washed magnetic beads (Cat#) according to the ratio of magnetic beads: T cells = 2:1. 40203D, 10ML, Life technology), 2-3 days later, resuspend the T cells in fresh culture medium to 3×10 ⁶ -5×10 ⁶ cells/mL, add lentivirus according to the MOI=10 value, and add 8µg/mL at the same time Polybrene, after 4-6 hours, add medium to dilute the cells to 1×10 ⁶ cells/mL. Replace with fresh medium the next day to maintain the cell concentration at 0.2×10 ⁶ -0.3×10 ⁶ PBMC/mL. The culture medium was replaced every 2-3 days, and the positive rate of cells was analyzed by flow cytometry 72 hours after virus infection.

The CAR19 positive rate detection results on the sixth day after T cell infection (D6) are shown in Figure 2. The results show that the 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%). It can be seen from the results that CAR19&scFv-IL21 has the highest infection efficiency.

Example 4 CART phenotypic identification

After infection, flow cytometry was used to detect the T, B, and NK cell groups of each group of cells on D6, and the CD4, CD8, and PD1 groups in T cells were also detected.

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

Table 1

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

Example 5 Construction of MSLN CART lentiviral vector expressing Anti PD1-IL-21 fusion protein and CCR2b and its control vector

The schematic structural diagram of the plasmid constructed in the embodiment of the present invention is shown in Figure 3. The nucleotide sequence of the structure shown in Figure 3 is genetically synthesized. According to the enzyme cutting site of the lentiviral vector, the nucleotide sequence is constructed into the lentiviral vector. Design primers and verify the correctness of the constructed plasmid through sequencing results.

MSLN CAR amino acid sequence: as shown in SEQ ID NO: 1, nucleotide sequence as shown in SEQ ID NO: 2.

Among them, the amino acid sequence of the CCR2b chemokine receptor molecule is as shown in SEQ ID NO: 5, and the nucleotide sequence is as shown in SEQ ID NO: 6.

The sequences of other elements are the same as in Example 1.

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.

Example 6 Packaging lentivirus

The PCDHF-85, PCDHF-86, and PCDHF-89 plasmids constructed in Example 5 were packaged with lentivirus according to the lentivirus packaging system shown in Table 2. The steps are as follows: (1) Inoculate 293T cells (cell cryopreservation density is 5 ×10 ⁶ cells/mL) into a 10cm cell culture dish, add 10 mL of DMEM medium containing 10% FBS (DMEM Gibco, 11995040-1L; FBS Gibco, 10091-148), 5% CO ₂ , CO ₂ at 37°C Cultivate in the incubator for 24 hours; (2) Pack the lentivirus according to Table 2; collect the cell supernatant 48 hours after packaging, and detect the lentivirus titer after ultracentrifugation at 25,000 rpm. The detection method is as follows: Put the gradient volume of the collected lentivirus stock solution under the same conditions. After infecting 293T cells, flow cytometry was used to detect the percentage of positive rates of MSLN antibodies and CCR2b in 293T cells 48 hours later (using FITC-labeled MSLN antigen detection and CCR2 antibody detection, ACRO, FITC-LabeLed Human MSLN (296-580) Protein, Fc Tag, Cat. No. MSN-HF253), calculate the lentivirus stock solution titer according to the following calculation formula: Lentivirus stock solution titer (TU/mL) =1.5*(10×10 ⁵ )*293T cell MSLN antibody and CCR2b positive rate percentage/lentivirus Volume of original solution μL*1000.

Table 2 Lentivirus packaging system Plasmid name Lentivirus name PCDHF-85 lentivirus PCDHF-86 lentivirus PCDHF-89 lentivirus PCDH D (PMD2.G) 36μg 36μg 36μg PCDH M (pMDLg/pRRE) 36μg 36μg 36μg PCDH N(pRSV-Rev) 36μg 36μg 36μg PCDHF-85 plasmid 72 μg 0 0 PCDHF-86 plasmid 0 72 μg 0 PCDHF-89 plasmid 0 0 72 μg PEI (PoLysciences 636951) 360 μg 360 μg 360 μg OPTI-MEMI (Gibco 31985070) 18mL 18mL 18mL

Table 3 Lentivirus titer Plasmid Lentivirus titer PCDHF-85 3.96×10 ^8TU /mL PCDHF-86 3.02×10 ^8TU /mL PCDHF-89 1.44×10 ^8TU /mL

The results of 293T cell MSLN antibody and CCR2b positive rate percentage are shown in Figure 4. The titer test results of each lentivirus are shown in Table 3. The results show that the titers of PCDHF-85, PCDHF-86, and PCDHF-89 lentiviruses are in order. are 3.96×10 ⁸ TU/mL, 3.02×10 ⁸ TU/mL, and 1.44×10 ⁸ TU/mL.

Example 7 CART cell preparation

PBMC cells were isolated from 50 mL of blood using Ficoll Lymph Separation Solution (Dakway, AS1114546), and isolated using magnetic beads coupled to CD3/CD28 antibodies (Dynabeads, CD3/CD28 CTS, Cat. No. 40203D, Lot No. A2-011710E). Obtain T cells, infect the T cells with the lentivirus prepared in Example 6 at MOI=5:1 to prepare CART cells, and set up control T cells at the same time. After the CART cells were cultured for 7 days, the MSLN antibody expression and CCR2b of the CART cells were detected. expression to determine the double-positive rate of the prepared 89CART cells; among them, PCDHF-85 lentivirus obtains 85CART cells, PCDHF-86 lentivirus obtains 86CART cells, PCDHF-89 lentivirus obtains 89CART cells, and the schematic diagram of the lentiviral vector structure of 89CART cells is shown in the figure. 5, the pCDHF empty vector nucleotide is shown in SEQ ID NO: 8.

The results of the double positive rate of 89CART cells prepared by the present invention are shown in Figure 6. The results show that the double positive rate of 89CART cells is 30.31%, indicating that the embodiment of the present invention successfully prepared 89CART cells.

Example 8 Chemotaxis of CART cells

The chemokine CCL2 can chemokine cells expressing the chemokine receptor CCR2b to migrate to areas with high concentrations of CCL2, and CCL2 is highly expressed in tumor sites. Therefore, in order to verify the migration ability of the 89CART cells prepared in Example 7 to the tumor site, human chemokine CCL2 (Genscript, product number: Z02829), X-VIVO 15 hematopoietic stem cell serum-free medium (LONZA, lot number, 8MB230), prepared to a concentration of 30ng/mL, totaling 2.4mL.

Add X-VIVO solution, 600 μL/well, to the lower layer of the 24-well plate (Corning, Cat. No. 3421, 6.5mm diameter, 5.0um pore size PC membrane) that comes with the Transwell chamber; then add 30ng/mL CCL2 solution, and set it without adding CCL2. The control group of the solution was repeated three times; slowly put it into the upper layer of the Transwell chamber, and then add the 86CART cells, 89CART cells and control T cells prepared in Example 7 to the upper layer. The cell concentration was 2×10 ⁵ cells/100 μL. Chemotaxis The experimental plan is shown in Table 4.

Incubate for 2 hours at 37°C in a 5% CO ₂ incubator. Use tweezers to carefully remove the Transwell chamber. Observe the migration of 86CART cells and 89CART cells under a microscope. Mix the lower culture medium by pipetting and transfer to a 1.5mL EP tube. Centrifuge at 2500rpm for 5 minutes and discard. Clear, resuspend in 40 μL of DPBS (Hyclone, lot number AE29431662) and count.

Table 4 Chemotaxis experimental protocol solution Chemotaxis protocol 24 well plate 600μL X-VIVO T cells 100μL T cells 100μL 86 CART cells 100μL 86 CART cells 100μL 89 CART cells 100μL 89 CART cells 100μL upper level CCL2 0 CCL2 30ng/mL CCL2 0 CCL2 30ng/mL CCL2 0 CCL2 30ng/mL lower level

The results of the migration of 89CART cells prepared in the present invention to the tumor site are shown in Figure 7. The results show that compared with the control T cells and 86CART cells, the migration ability of the 89CART cells to the tumor site is significantly improved.

Example 9 In vitro tumor cell killing performance of CART cells

Add 10,000 ovarian cancer cells (OVCAR-3 cells) (ATCC HTB161) to each well of E-Plate 16 PET (ACEA, batch number, 20190125), and the culture medium is RPMI1640 (Gibco, batch number 2215748) + 20% FBS (Gibco batch number 2152441P ), 100 μL/well, placed on the xCELLigence RTCA S16 instrument, 5% CO ₂ , cultured at 37°C for 48 hours, respectively, take T cells, 85CART cells, and 89CART cells. After counting, use T cells to dilute the 85CART cells to a positive rate of 37.79% , add each well according to the ratio of effector cells (CAR+CART cells): target cells = 10:1. The culture medium of CART cells is X-VIVO 15, 100 μL/well. CART cells and tumor cells are mixed and cultured for 25 hours, and 89CART cells are measured. The ability to kill tumor cells in vitro.

The results of the ability of 89CART cells prepared in the embodiment of the present invention to kill tumor cells in vitro are shown in Figure 8. The results show that compared with control T cells and 85CART cells, the ability of 89CART cells to kill tumor cells in vitro is significantly improved.

In addition, T cells, 85CART cells and 89CART cell culture were detected respectively (the initial cell density was 2×10 ⁵ cells/mL, the supernatant was collected after 72 hours of culture, IL-21 ELISA detection kit, Invitrogen, batch number 220657). IL-21 expression levels.

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

Example 10 Performance of CART cells in killing tumor cells in mice

Construct the ASPC-1-CCL2 cell line: Search the human CCL2 gene sequence (see SEQ ID NO: 13) from the NCBI database, construct it on the pCDHF vector, name it pCDHF-91 plasmid, and then package the lentivirus (refer to Example 6 for the packaging process) ), use packaged lentivirus to infect ASPC-1 cells (ATCC CRL1682 ^™ ). On the second day, the infected cells are plated and single-cultured. ASPC-1 cells with high CCL2 expression are selected for expansion and culture to establish the ASPC-1-CCL2 cell line.

SEQ ID NO: 13: ATGAAAGTCTCTGCCGCCCTTCTGTGCCTGCTGCTCATAGCAGCCACCTTCATTCCCCCAAGGGCTCGCTCAGCCAGATGCAATCAATGCCCCAGTCACCTGCTGTTATAACTTCACCAATAGGAAGATCTCAGTGCAGAGGCTCGCGAGCTATAGAAGAATCACCAGCAGCAAGTGTCCCAAAGAAGCTGTGATCTTCAAGACCATTGTGGCCAAGGAGATCTGTGCTGACCCCAAGCAGAAGTGGGTTCAGGATTCCATGG ACCACCTGGACAAGCAAACCCAAACTCCGAAGACTTGA.

On day 0, ASPC-1-CCL2 cells were subcutaneously inoculated into NPG mice (Biocytogram), 5.0E+06 cells/100μl+100μl Matrigel (Company BD, Cat. No. 356234)/mouse, and mice in the three groups were inoculated on day 0. T cells, 85CART cells and 89CART cells (2.0E+06 CAR+cells each) were infused back into the tail vein for 7 days, and the changes in mouse tumor volume were measured regularly (as shown in Figure 9) and peripheral blood and tumors were detected on the 20th day. The CCL2 concentration (shown in Figure 10) and the proportion of CART cells at the tumor site (shown in Figure 11).

The results show that after subcutaneous inoculation of ASPC-1-CCL2, the expression of CCL2 can be detected in both the tumor site and peripheral blood, and the CCL2 concentration in the tumor site is higher than the CCL2 concentration in the peripheral blood, which can attract more 89CART cells to the tumor site. , and can kill tumor cells in mice faster and more effectively.

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 other changes, modifications, substitutions, combinations, etc. may be made without departing from the spirit and principles of the present invention. All simplifications should be equivalent substitutions, and are all included in the protection scope of the present invention.

without

Figure 1 is a schematic structural diagram of the nucleic acid constructed in Example 1 of the present invention; "Hinge" refers to the hinge region, "Linker" refers to the connecting peptide, scFv represents PD1 scFv, and CAR19 represents the expression of a chimeric antigen receptor targeting CD19. T cell group, CAR19& scFv represents the CART19 group expressing scFv, CAR19& scFv&IL21 represents the CART19 group expressing scFv and IL-21 alone (that is, scFv and IL-21 are not fused), CAR19&scFv-IL21 represents the expression of the fusion protein scFv-IL-21 The CART19 group, CAR19&IL21-scFv represents the CART19 group expressing the fusion protein IL-21-scFv.

Figure 2 is the CAR19 positive 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 the ordinary T cell group, and CAR19 represents the chimeric antigen receptor that expresses CD19. T cell group, CAR19&scFv represents the CART19 group expressing scFv, CAR19& scFv&IL21 represents the CART19 group expressing scFv and IL-21 alone, CAR19&scFv-IL21 represents the CART19 group expressing the fusion protein scFv-IL-21, CAR19&IL21-scFv represents the fusion protein expression CART19 group of IL-21-scFv.

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

Figure 4 is a graph showing the percentage results of MSLN antibody and CCR2b positive rate of 293T cells.

Figure 5 is a schematic diagram of the vector structure of 89CART cells.

Figure 6 is a graph showing the double positive rate results of 89CART cells prepared in the present invention.

Figure 7 is a diagram showing the migration results of 89CART cells prepared in the present invention after being chemotaxis by CCL2.

Figure 8 is a graph showing the results of the ability of 89CART cells prepared in the present invention to kill tumor cells in vitro.

Figure 9 shows the detection results of tumor volume changes in mice after receiving different T cell treatments. In the figure, 85 represents 85CART cells, 89 represents 89CART cells, and T cell represents control T cells.

Figure 10 shows the detection results of cell suspension and peripheral blood CCL2 concentration in mouse tumor sites. In the figure, G3-783 represents the mouse numbered 783 in the 89CART cell therapy group, and G3-788 represents the mouse numbered 788 in the 89CART cell therapy group. mouse.

Figure 11 is the detection result of the ratio of different CART cells in cell suspension and peripheral blood of mouse tumor sites.

TW202330909A_111134036_SEQL.xml

Claims (22)

A transgenic immune effector cell, characterized in that 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 connected in series; wherein, the extracellular region specifically recognizes a tumor antigen, and the transmembrane region is embedded in the cell membrane of the transgenic immune effector cell In, the intracellular region includes an intracellular segment of an immune costimulatory molecule; The fusion protein includes immune checkpoint antibodies and cytokines; The chemokine receptor molecule is a chemokine receptor or a chemokine binding fragment thereof. The transgenic immune effector cell according to claim 1, wherein the chemokine receptor is CXC chemokine receptor, CC chemokine receptor, CX3C chemokine receptor or XC chemokine receptor. any one or more types; Preferably, the CXC chemokine receptor is any one or more of CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6 or CXCR7; Preferably, the CC chemokine receptor is any one or more of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10 or CCR11; Preferably, the CX3C chemokine receptor is CX3CR1; Preferably, the XC chemokine receptor is XCR1; Preferably, the chemokine receptor molecule is CCR2; Preferably, the amino acid sequence of the chemokine receptor molecule is shown in SEQ ID NO: 5. The transgenic immune effector cell according to claim 1 or 2, wherein the immune co-stimulatory molecule is selected from 4-1BB or its derivatives, CD28 or its derivatives, CD3 or its derivatives, OX-40 or its derivatives Any one or more of CD40L or its derivatives, CD27 or its derivatives, and CD30 or its derivatives. The transgenic immune effector cell according to any one of claims 1 to 3, wherein the immune checkpoint antibody is selected from any one or more of PD1, PD-L1, TIGIT, LAG3, CTLA4, BTLA or TIM3 . The transgenic immune effector cell according to any one of claims 1 to 4, wherein the cytokine is interleukin; Preferably, 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; Preferably, the cytokine is IL-21. The transgenic immune effector cell according to any one of claims 1 to 5, wherein the fusion protein further includes a connecting peptide; the immune checkpoint antibody is connected to the cytokine through a connecting peptide; Preferably, the connecting peptide is a short peptide containing G and S amino acids; Preferably, the amino acid sequence of the connecting peptide is as shown in SEQ ID NO: 7. The transgenic immune effector cell according to any one of claims 1 to 6, wherein the immune checkpoint antibody is a PD1 antibody and the cytokine is IL-21; Preferably, the C-terminal of the PD1 antibody is connected to the N-terminal of the connecting peptide, and the N-terminal of the IL-21 is connected to the C-terminal of the connecting peptide. The transgenic immune effector cell according to any one of claims 1 to 7, wherein the amino acid sequence of the fusion protein is shown in SEQ ID NO: 3 or SEQ ID NO: 9. The transgenic immune effector cell according to any one of claims 1 to 8, wherein the extracellular region includes an antibody that specifically recognizes a tumor antigen and a CD8 hinge region; The transmembrane region includes the transmembrane segment of CD8; The immune co-stimulatory molecule is 4-1BB, and the intracellular region includes the intracellular segment of 4-1-BB and the 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 of the intracellular region. The transgenic immune effector cell according to any one of claims 1 to 9, wherein the tumor antigen is any one or more of MSLN, GD2, GPC3, CD19, EGFR VIII, GUCY2C, HER2, MUC16 or Claudin 18.2 species. The transgenic immune effector cell according to any one of claims 1 to 10, wherein the tumor antigen is MSLN or CD19; the amino acid sequence of the chimeric antigen receptor is such as SEQ ID NO: 1 or SEQ ID NO:11 shown. The transgenic immune effector cell according to any one of claims 1 to 11, wherein the chimeric antigen receptor, the fusion protein and the chemokine receptor molecule are connected by a 2A peptide; Preferably, the 2A peptide is selected from any one or more of P2A, T2A, F2A or E2A; Preferably, the chimeric antigen receptor is connected to the fusion protein through P2A, and the fusion protein is connected to the chemokine receptor molecule through T2A. The transgenic immune effector cell according to any one of claims 1 to 12, wherein the transgenic immune effector cell is selected from any one or more of T cells, NK cells, NKT cells, macrophages or CIK cells. . A lentivirus, characterized in that the lentivirus carries the following nucleic acid: 1) Nucleic acid encoding a chimeric antigen receptor in the transgenic immune effector cell according to any one of claims 1 to 13, the extracellular region of the chimeric antigen receptor specifically recognizes a tumor antigen; 2) Nucleic acid encoding a fusion protein in the transgenic immune effector cell as described in any one of claims 1 to 13, where the fusion protein includes an immune checkpoint antibody and a cytokine; 3) Nucleic acid encoding a chemokine receptor molecule in the transgenic immune effector cell according to any one of claims 1 to 13, where the chemokine receptor molecule is a chemokine receptor or a chemokine thereof Combine fragments. The lentivirus according to claim 14, wherein the tumor antigen is MSLN or CD19, and the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO: 1 or SEQ ID NO: 11, Preferably, the nucleic acid encoding the chimeric antigen receptor has a nucleotide sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 12; Preferably, the immune checkpoint antibody is a PD1 antibody, the cytokine is IL-21, and the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 3 or SEQ ID NO: 9, preferably , the nucleic acid encoding the fusion protein has the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 10; Preferably, the chemokine receptor molecule is CCR2b, and the amino acid sequence of the chemokine receptor molecule is shown in SEQ ID NO: 5, preferably encoding the chemokine receptor molecule. The nucleic acid has the nucleotide sequence shown in SEQ ID NO: 6. A construct, characterized in that the construct includes: A first nucleic acid molecule encoding a chimeric antigen receptor in the transgenic immune effector cell according to any one of claims 1 to 13, the extracellular region of the chimeric antigen receptor is specific Identify tumor antigens; a second nucleic acid molecule encoding a fusion protein in the transgenic immune effector cell according to any one of claims 1 to 13, where the fusion protein includes an immune checkpoint antibody and a cytokine; A third nucleic acid molecule encoding a chemokine receptor molecule in the transgenic immune effector cell according to any one of claims 1 to 13, and the chemokine receptor molecule is a chemokine Receptors or chemokine-binding fragments thereof. The construct of claim 16, wherein the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule express chimeric antigen receptors, fusion proteins and chemokines respectively in immune effector cells. chemokine receptor molecules; The chimeric antigen receptor, the fusion protein and the chemokine receptor molecule are in a non-fusion form; The transgenic immune effector cells are selected from any one or more of T cells, NK cells, NKT cells, macrophages or CIK cells; Optionally, the transgenic immune effector cells are T cells. The construct of claim 16 or 17, wherein the construct further includes: a first promoter, the first promoter is operably connected to the first nucleic acid molecule; Optionally, the first promoter is selected from any one or more of U6, H1, CMV, EF-1, LTR or RSV promoters. The construct according to any one of claims 16 to 18, wherein the vector of the construct is a non-pathogenic viral vector; Optionally, the non-pathogenic viral vector is selected from any one or more of retroviral vectors, lentiviral vectors or adenovirus-associated viral vectors; Optionally, the non-pathogenic viral vector is a lentiviral vector; Optionally, the nucleic acid encoding the lentiviral vector has the nucleotide sequence shown in SEQ ID NO: 8. A method for preparing transgenic immune effector cells as described in any one of claims 1 to 13, characterized in that the lentivirus as described in claim 14 or 15, or any one of claims 16 to 19 The construct is introduced into immune effector cells; The immune effector cells are selected from any one or more of T cells, NK cells, NKT cells, macrophages or CIK cells; Optionally, the immune effector cells are T cells. A transgenic immune effector cell as described in any one of claims 1 to 13, or a lentivirus as described in claim 14 or 15, or a construct as described in any one of claims 16 to 19 is prepared Applications in cancer treatment drugs, Preferably, the cancer is a solid tumor. A cancer treatment drug, characterized by comprising the transgenic immune effector cell as described in any one of claims 1 to 13, the lentivirus as described in claim 14 or 15, or any one of claims 16 to 19 Said construct, Preferably, the cancer is a solid tumor.