CN110129369B - Chimeric antigen receptor gene engineering vector, immune cell and application thereof - Google Patents

Chimeric antigen receptor gene engineering vector, immune cell and application thereof Download PDF

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CN110129369B
CN110129369B CN201810136172.XA CN201810136172A CN110129369B CN 110129369 B CN110129369 B CN 110129369B CN 201810136172 A CN201810136172 A CN 201810136172A CN 110129369 B CN110129369 B CN 110129369B
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chimeric antigen
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李本尚
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Shanghai Childrens Medical Center Affiliated to Shanghai Jiaotong University School of Medicine
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Abstract

The invention belongs to the field of cell therapy, and discloses a chimeric antigen receptor gene engineering vector, immune cells and application thereof, wherein the chimeric antigen receptor gene engineering vector comprises a lentiviral vector skeleton, a regulatory element connected to the lentiviral vector skeleton and a chimeric antigen receptor gene sequence, and the regulatory element comprises a WPRE element, a cPPT element and an RRE element. The chimeric antigen receptor gene sequence comprises a costimulatory signal path sequence and an effector signal path sequence which are expressed independently; alternatively, the chimeric antigen receptor gene sequence includes a fusion expressed costimulatory signaling pathway sequence and an effector signaling pathway sequence. The chimeric antigen receptor genetic engineering vector has the advantages of good safety, high transfection efficiency, rapid amplification of transfected immune cells and greatly improved killing effect. In addition, compared with the chimeric antigen receptor immune cells with single targets, the target cells are more accurately identified, and the off-target effect of the single targets is prevented.

Description

Chimeric antigen receptor gene engineering vector, immune cell and application thereof
Technical Field
The invention belongs to the field of cell therapy, and particularly relates to a chimeric antigen receptor gene engineering vector, immune cells and application thereof. The genetically engineered vector can stably transfect autologous or allogeneic immune cells after being packaged by viruses, and achieves the purpose of specifically killing target molecules in vivo through double-targeting recognition, thereby playing a clinical treatment role.
Background
Chimeric antigen receptor-Chimeric antigen receptor, abbreviated CAR. Wherein the chimeric antigen receptor cell therapy based on T cells is called CART therapy for short, the chimeric antigen receptor cell therapy based on NK cells is called CAR-NK therapy for short, and the chimeric antigen receptor cell therapy based on gamma/delta T cells is called Universal-CAR. No matter which kind of cell is adopted, the powerful killing capacity of the corresponding immune cell to the target cell is utilized to eliminate tumor and some kinds of cell.
From the first international relapse of acute lymphoblastic leukemia patients receiving CART treatment to the present, the curative effects on the relapse of refractory B-series acute lymphoblastic leukemia and lymphoma are obvious in clinical curative effects and are accepted in the industry. The CART treatment technology has been continuously updated from the earliest generation of CART with fresh clinical efficacy to the second generation of CART with significant efficacy, which is based on the generation of CART with the addition of co-stimulatory molecules such as 4-1BB or CD28. The third-generation CART is based on the second-generation CART, and two co-stimulatory molecules are added, but the third-generation CART is not considered to have advantages over the second-generation CART, so that the current mainstream is chimeric antigen receptor treatment based on the second-generation CART technology.
The second-generation CART technology mainly aims at tumor cell membrane surface antigen, light chain and heavy chain variable regions in antibody molecules capable of recognizing the antigen are connected in series through connecting peptide to form a single-chain affinity antibody structure, and then the single-chain affinity antibody structure, a hinge region/transmembrane region, a co-stimulatory molecule and an effector molecule are subjected to virus preparation and then are subjected to fusion expression in immune cells, so that the corresponding immune cells have the effect of specifically killing target cells. At present, CART vector construction based on CD19, CD20, CD30, CD33, GD2, BCMA, EGFR VIII, mesothelin, CD138, CD38 and the like adopts the same fusion expression strategy. Currently, chimeric antigen receptor cell-based therapeutic techniques have achieved very significant efficacy in CD19 positive lymphocytic leukemia and lymphoma. Combining PD-1 antibodies with CART cell therapy is a good choice and many clinical trials are currently underway.
However, CART cells are used in cancer therapy, with concomitant toxicity and risk, and even death of the patient, while exhibiting therapeutic efficacy. There are two major problems faced, first, cytokine release syndrome (cytokines release syndrome, CRS) is the most significant toxicity, a first safety risk. Cytokine release syndrome is based on activation of T cells, a response to T cell activation activity, so side effects are clinical responses positively correlated with therapeutic mechanisms of CART. Highly proliferating T cells can cause CRS, manifesting as hyperthermia and myalgia, unstable hypotension and respiratory failure. In general, the severity of CRS storm has a great correlation with tumor cell burden in patients receiving CART treatment precursors, but it is not excluded that few patients will also produce more severe CRS under low burden. Second, acute cerebral edema appears in a small number of patients, presumably due to the existence of polymorphisms (single nucleotide polymorphisms, SNPs) or mutations (single nucleotide variant, SNV) in the coding sequence of a protein transcribed and translated into neuronal cell membranes in the patient's genome, which structurally converge with the CD19 epitope, resulting in killing of CD19 positive leukemia cells by CART cells, and also in a cytotoxic effect against autologous neuronal cells (off-target effect), and we find it more appropriate to refer to the acute necrotic encephalitis associated with CART (CART related acute necrotic encephalitis, CANE). In summary, the chimeric antigen receptors of the first generation, the second generation and the third generation still have the problems of more side effects, poor specificity and the like in treatment. The present invention has been made in view of this.
Disclosure of Invention
The application aims to solve the technical problem of overcoming the defects of the prior art and providing a chimeric antigen receptor gene engineering vector, immune cells and application thereof. The chimeric antigen receptor genetic engineering vector has the advantages of good safety, high transfection efficiency, rapid amplification of transfected immune cells and greatly improved killing effect. In addition, compared with the chimeric antigen receptor immune cells with single targets, the application has more accurate recognition of target cells and prevents the off-target effect of the single targets.
In order to solve the technical problems, the application adopts the basic conception of the technical scheme that:
it is a first object of the present application to provide a chimeric antigen receptor gene engineering vector comprising a lentiviral vector backbone, regulatory elements comprising a WPRE element, a cPPT element and an RRE element, linked to the lentiviral vector backbone, and a chimeric antigen receptor gene sequence.
The application puts the slow virus elements such as Gag (coding virus structural protein), pol (coding virus required enzyme protein), env (coding virus membrane protein) and Rev (coding a protein which can act on Rev response element, namely RRE and regulate the extracellular transport of virus mRNA) into three different vectors for expression, thus forming a three-plasmid system, greatly reducing the probability of virus recombination and ensuring the safety of human body. By testing the patients in the group, the presence of lentiviral molecules was not detected in vivo for 10 months after the longest patient treatment.
The lentiviral vector backbone of the present application may be a conventional lentiviral vector, such as a viral vector derived from HIV-1. The WPRE element helps to improve the stability of the mRNA molecules of the viruses, the cPPT element helps to transfer the viruses into the nucleus, so that the genome is more easily integrated, the stable transfection efficiency is improved, the RRE element helps to transport the mRNA molecules out of the nucleus, so that the virus titer obtained by packaging is high, the virus activity is strong, and the key to improving the transfection efficiency is that. Because only cells positive to virus transfection have the effect of specifically killing tumor cells, the transfection efficiency is improved, the system for culturing the cells can be greatly reduced, and the early production cost is saved.
In a further embodiment, the chimeric antigen receptor gene sequence is inserted upstream of a mammalian constitutive promoter, preferably an EF1A promoter. The carrier of the application preferably adopts EF1A promoter to promote the expression of exogenous gene, and EF1A promoter is mammal constitutive expression promoter from human elongation factor 1 alpha source, so that exogenous gene expression is very stable and is not affected by cell types, and the carrier can be widely used for different cell types such as T cells, NK cells, macrophages and the like. Compared with promoters such as CMV, SV40 and the like, the promoter is not influenced by cell types, and more importantly, the expression of the exogenous protein is stable and not excessively high, so that the influence on the growth of cells is small, and the promoter is the basis for the high-speed amplification of the CAR positive T lymphocytes in vitro.
In a further aspect, the chimeric antigen receptor gene sequence comprises an independently expressed costimulatory signal pathway sequence and effector signal pathway sequence; alternatively, the chimeric antigen receptor gene sequence includes a fusion expressed costimulatory signaling pathway sequence and an effector signaling pathway sequence.
A self-clipping sequence is connected between the independently expressed costimulatory signal pathway sequence and the effector signal pathway sequence; the costimulatory signal pathway sequence and the effector signal pathway sequence are each independently expressed across the membrane and produce an effect when simultaneously bound to the target site and activated. The co-stimulatory signal pathway sequence and the effector signal pathway sequence on the carrier are two independent transmembrane signal pathway fusion proteins after expression, one is a co-stimulatory signal pathway and the other is an effector signal pathway; when both signal paths are activated, immune cells modified by the chimeric antigen receptor genetic engineering vector can be activated, and killing effect is generated on target cells. When only one pathway is activated, immune cells cannot be activated nor can they produce a killing effect. Thus, this approach is more accurate than the recognition of target cells by chimeric antigen receptor immune cells of a single target; meanwhile, the double-targeting combined recognition effect can also better prevent the off-target of a single target, and the specificity and the killing power of target cells are enhanced.
In the research, the serious side effects of acute cerebral edema and the like of part of patients treated by CD19-CART are found, and the analysis of the side effects is that a certain membrane protein molecule in the neuron cells of the patients is mutated, so that the structure of the membrane protein molecule is similar to that of an epitope recognized by a CD19 antibody, and off-target effect is caused. The ScFv antibody of the application aiming at different antigen epitopes (at least two antigen epitopes) of the CD19 antigen can avoid similar serious adverse events to a large extent.
The chimeric antigen receptor genetic engineering vector strategy provided by the application can also comprise chimeric antigen receptor vectors constructed by IRES and/or double-promoter vectors and/or multi-promoter vectors and the like.
According to a further scheme, the costimulatory signal path sequence comprises a first leader peptide sequence, a first antibody sequence and a costimulatory molecule sequence which are sequentially connected according to the transcription direction, and the effector signal path sequence comprises a second leader peptide sequence, a second antibody sequence and an effector molecule sequence which are sequentially connected according to the transcription direction; the self-cleaving sequence is linked between the costimulatory molecule sequence and the second leader peptide sequence, or between the effector molecule sequence and the first leader peptide sequence.
The first leader peptide sequence and the second leader peptide sequence express the same or different leader peptides and are capable of respectively guiding proteins expressed by the costimulatory signal pathway sequences and proteins expressed by the effector signal pathway sequences to cell membranes; the first antibody and the second antibody are located on the surface of the cell membrane. When molecules acting on the surface of the target cell respectively act on the two antibodies, the two antibodies of the application respectively bind to the target cell, so that the structure of the antibodies is changed, and the generated signals can activate the downstream fusion proteins of the antibodies. The signal generated by the first antibody activates the costimulatory molecule downstream thereof, and the signal generated by the second antibody activates the effector molecule downstream thereof; after both the costimulatory and effector molecules are activated, ZNP is activated, so that immune cells, i.e., CART cells, are activated and produce various cytokines that can rapidly and specifically kill target cells.
Further aspects, the first antibody sequence or the second antibody sequence include, but are not limited to: a molecular sequence that interacts with a cell membrane surface molecule of a target cell, a molecular sequence that interacts with a specific molecule that is presented inside the target cell to its cell membrane surface. Antibodies can interact with the original molecular designed sequences on the surface of the cell membrane of the target cell, including: to act on the cell membrane surface antigen of the target cell, or to act on a non-antigen protein. In addition, the target cell expressed protein through degradation process and other processes to present (including but not limited to MHC presenting pathway) to the cell membrane surface protein fragments or short peptides formed by specific chimeric antigen receptor targets, antibodies can also be activated with such targets binding. The double targeting in the application means that two ScFv antibody sequences corresponding to two identical or different molecules (antigens) aiming at the surface of target cells are simultaneously inserted into the chimeric antigen receptor genetic engineering expression vector. Preferably, the first antibody sequence or the second antibody sequence is an ScFv antibody sequence selected from the group consisting of: a molecular sequence that interacts with a cell membrane surface antigen of a target cell.
Further, the first antibody sequence or the second antibody sequence is selected from the group consisting of: at least one of CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD9, CD10, CD11a, CD11b, CD13, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD30, CD33, CD34, CD36, CD37, CD38, CD40, CD41, CD42, CD43, CD44, CD45, CD56, CD58, CD66c, CD70, CD73, CD74, CD80, CD81, CD86, CD94, CD97, CD99, CD102, CD123, CD133, CD134, CD137, CD138, CD200, GD2, EGFR VIII, GD3, NG2, CA125, a33, CEA, acam6, CS1, EGFR, ERBB2, FGF19, HER3, IL3Ra, NCAM, NKG A, BCMA, NTBA, PD-1, PDL-1, a, PSGL1, ROR1, VEGF.
Further aspects, the combination of the first antibody sequence and the second antibody sequence is selected from the group consisting of: CD19/CD19, CD19/CD20, CD19/CD123, CD19/CD66c, CD19/CD58, CD19/CD56, CD19/CD13, CD19/CD33, CD19/CD44, CD19/CD73, CD19/CD86, CD19/CD99, CD19/CD24, CD19/CD200, CD19/CD97, CD19/BDCA4, CD19/CD133, CD19/CD15, CD19/NG2, CD19/sIgM, CD20/CD20, CD20/CD123, CD20/CD66c, CD20/CD58, CD20/CD56, CD20/CD13, CD20/CD33, CD20/CD44, CD20/CD73, CD20/CD86 CD20/CD86, CD20/CD99, CD20/CD24, CD20/CD200, CD20/CD97, CD20/BDCA4, CD20/CD133, CD20/CD15, CD20/NG2, CD20/sIgM, CD22/CD22, CD123, CD22/CD66c, CD22/CD58, CD22/CD56, CD22/CD13, CD22/CD33, CD22/CD44, CD22/CD73, CD22/CD86, CD22/CD99, CD22/CD24, CD22/CD220, CD22/CD97, CD22/BDCA4, CD22/CD133, CD22/CD15, CD22/NG2, CD22/sIgM. The combinations may be used singly or in combination, thereby reducing treatment failure due to target loss. Preferably, a CD19/CD19 combination is used.
The different ScFv antibody combinations mentioned in the scheme are designed according to the unique immunophenotype characteristics of the acute leukemia cells, in the combination, CD19 and CD22 are cell membrane antigens expressed by all B-line leukemia cells, CD20 is cell membrane antigen expressed by all B-line lymphoma lymphomas, and the specific killing effect on tumor cells can be achieved by combining the same antibody sequences or other antibody sequences, and the normal B-line lymphoma cells are not influenced. We performed clinical experiments with dual targeting chimeric antigen receptor, 11 patients in the group all obtained molecular remission (MRD < 0.01%) 8-28 days after CART cell infusion, demonstrating the effectiveness of the above-described dual targeting chimeric antigen receptor-based therapies.
Alternatively, the first antibody sequence and the second antibody sequence further comprise antibody sequences designed for specific pre-nuclear antigen receptor targets formed by protein fragments or short peptides which are presented on the surface of a cell membrane by degradation and other processes of proteins expressed in target cells. In particular to a method that some tumor-derived gene mutant protein products or abnormal expression protein products in cells are degraded by proteasome or other mechanisms and presented on the surface of cell membranes by MHC molecules or other molecules, and can be used as targets for immune cell recognition. Corresponding antibody sequences are designed aiming at the presented tumor cell mutant protein products or abnormal expression protein products, and the tumor cell mutant protein products or abnormal expression protein products are assembled into chimeric antigen receptor gene engineering vectors and viruses, and specific tumor cells can be identified by transfection and expression of immune cells, so that a targeted killing effect is generated.
Preferably, mutant protein products or aberrantly expressed protein products include, but are not limited to, mutations in genes such as ABL1, ALK, ASXL1, ATM, BCOR, BCORL1, BRAF, CALR, CBL, CEBPA, CSF R, CSMD1, CUX1, DNMT3A, EP300, ETNK1, ETV6, EZH2, FLT3, GATA1, GATA2, GNAS, IDH1, IDH2, IKZF1, JAK2, JAK3, KIT, KMT2A, KMT2C, KMT2D, KRAS, MPL, NF1, NOTCH1, NPM1, NRAS, PDGFRA, PHF6, PRPF40B, PRPF, PTEN, PTPN11, RAD21, ROBO1, ROBO2, RUNX1, SETBP1, SF3A1, SH2B3, SMC1A, SMC3, SRSF2, rag 2, SUZ12, TET2, TP53, U2AF1, U2AF2, WT1, zsr 2, etc.; including but not limited to AML1-ETO, EWS-ETV1, NPM1-RARα, SCAF11-PDGFRA, AML1-MDS1/EVI1, EWS-ETV4, NUMA1-RARα, SDC4-ROS1, AML1-MTG16, EWSR1-ZNF384, NUP214-ABL1, SEC31A-ALK, ATF7IP-JAK2, EZR-ROS1, NUP98-HoxA9/11/13, SET-CAN, AXL-MBIP, FAM46C-MYC, NUP98-HoxC11 SFPQ-ABL1, BCOR-RARα, FGFR2-CIT, NUP98-HoxD13, SIL-TAL1, BCR-ABL1, FGFR2-WARS, NUP98-PMX1, SLC34A2-ROS1, BMP6-MYC, FGFR3-TACC3, OFD1-JAK2, SNX2-ABL1, CARS-ALK, FIG-ROS1, P2RY8-CRLF2, SPECC1-PDGFRB, CBFβ -MYH11, FIP1L1-PDGFRA, PAG1-ABL2, SQSTM1-ALK CCDC6-PDGFRB, FIP1L1-RARα, PAX5-ASXL1, SSBP2-CSF1R, CCDC6-ROS1, FOXJ2-MEF2D, PAX5-AUTS2, SSBP2-JAK2, CCND1-MYC, FOXO3-MYC, PAX5-CBFA2T3, SSBP2-PDGFRB, CCND3-MYC, FOXP1-ABL1, PAX5-ESRRB, STAT5b-RARα, CD74-ROS1, FUS-CHOP, PAX5-JAK2, STRN3-JAK2, CLTC-ALK FUS-FEV, PAX5-KIF3B, STRN-ALK, CREBBP-ZNF384, HBA1-CD74, PAX5-MLLT3, STRN-PDGFRA, DEK-CAN, HLXB9-ETV6, PAX5-RNF38, SYNRG-ZNF384, DGKH-ZFAND3, IQGAP2-TSLP, PAX5-SP2, TAF15-ZNF384, E2A-HLF, JAK2-SNX29, PAX5-TMEM14B, TCF-ZNF 384, E2A-PBX1, KDELR2-ROS1, PAX5-ZNF521, TERF2-JAK2, EBF1-JAK2, KIF5B-ALK, PCM1-JAK2, TFG-ALK, EBF1-PDGFRB, KIF5B-PDGFRA, PLEKHA-NTRK 3, TLS-ERG, EML4-ALK, KIF5B-RET, PLZF-RARα, TNIP1-PDGFRB, EP300-ZNF384, KLC1-ALK, PML-RARα, TPM3-ALK, ETV6-ABL, LRIG3-ROS1 PPF1BP1-JAK2, TPM3-ROS1, ETV6-ABL1, MEF2D-BCL9, PPFIBP1-ALK, TPM4-ALK, ETV6-ABL2, MEF2D-DAZAP1, PRKAR1A-RARα, TPR-JAK2, ETV6-JAK2, MEF2D-HNRNPUL1, PTK2B-KDM6A, TPR-MET, ETV6-NIPBL, MEF2D-JAK2, PTK2B-STAG2, TTL-ETV6, ETV6-NTRK3 MEF2D-SS18, PTPN3-ALK, TXNDC5-MYC, ETV6-PDGFRA, MLL rearrangement, PTPRZ1-MET, TYK2-MYB, ETV6-PDGFRB, MN1-ETV6, RANBP2-ABL1, UQCRH-EWS, ETV6-RUNX1, MN1-TEL, RB1-MYC, XBP1-MYC, ETV6-STL, MSN-ALK, RBM 15-MALK 1, ZC3HAV1-ABL2, EWS-ATF1, MYH9-ALK, RCSD1-ABL1, ZEB2-PDGFRB, EWS-CHN, MYH9-IL2RB, RCSD1-ABL2, ZMIZ1-ABL1, EWS-CHOP, NCOR1-LYN, RET-KTN1, ZM 2-FGFR1, ZALKS 1, ZSK-1, ZERS-180, REK-180, ZERF-RNF, ZERF-180, and the like; including but not limited to protein products produced by IG and TCR rearrangements, and the like.
Further, the costimulatory signal pathway sequence comprises a first leader peptide sequence, a first antibody light chain VL sequence, a first antibody connecting peptide sequence, a first antibody heavy chain VH sequence, a hinge region sequence, a transmembrane region sequence and a costimulatory molecule sequence, which are sequentially connected according to the transcription direction; the effector signal pathway sequence comprises a second leader peptide sequence, a second antibody light chain VL sequence, a second antibody connecting peptide sequence, a second antibody heavy chain VH sequence, a hinge region sequence, a transmembrane region sequence and an effector molecule sequence which are sequentially connected according to the transcription direction; and a self-shearing sequence is connected between the costimulatory molecule sequence and the second leader peptide sequence.
Further, the costimulatory molecule sequence is selected from at least one of the group consisting of 4-1BB, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS sequence; preferably, the costimulatory molecule sequence is a 4-1BB sequence; the effector molecule sequence is selected from CD3 delta; at least one of the self-cleaving sequences is selected from the group consisting of P2A, T2A, F2A, E2A, bmCPV2A, bmIFV a sequences; preferably, the self-cleaving sequence is a P2A sequence.
Further, the first antibody sequence or the second antibody sequence is selected from the nucleotide sequences shown in SEQ ID NOs 11 to 50; preferably, the sequences of SEQ ID NOs 11 to 40 are selected for acute leukemia and lymphoma; preferably, the sequences of SEQ ID NOS.41 to 50 are selected for neuroblastoma. The same sequences as described above can also be used for cellular immunotherapy of acute leukemias and lymphomas B-lineage malignancies.
Further, the first leader peptide sequence or first leader peptide sequence includes, but is not limited to: film protein guide peptide sequences of CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD9, CD10, CD11a, CD11b, CD13, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD30, CD33, CD34, CD36, CD37, CD38, CD40, CD41, CD42, CD43, CD44, CD45, CD56, CD58, CD66c, CD70, CD73, CD74, CD80, CD81, CD86, CD94, CD97, CD99, CD102, CD123, CD133, CD134, CD137, CD138, CD200, EGFR, GD3, NG2, CA125, a33, CEA, CEACAM6, CS1, EGFR, ERBB2, FGF19, HER3, IL3Ra, NCAM, NKG2A, BCMA, NTBA, PD-1, PDL-1, PSMA, PSGL1, r1, VEGF, and the like. Preferably, the leader peptide sequence is selected from SEQ ID NOS.1 to 10.
The linker peptide sequence refers to an amino acid sequence that concatenates VL and VH, and the preferred sequence is the nucleotide sequence shown as SEQ ID NO. 51. The hinge region sequence refers to the amino acid sequence that mediates the transfer of activation signals from the ScFv antibody to the transmembrane region, with the preferred sequence being the nucleotide sequence set forth in SEQ ID No. 52. The transmembrane region sequence refers to an amino acid sequence that mediates the transmembrane transfer of an activation signal from the hinge region to the intracellular region, and the preferred sequence is the nucleotide sequence set forth in SEQ ID NO. 53. Co-stimulatory molecules include, but are not limited to, the sequences of 4-1BB, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS and the like, preferably the co-stimulatory molecule has the sequence 4-1BB, preferably the nucleotide sequence set forth in SEQ ID NO. 54. Self-cleaving sequence means comprising but not limited to the P2A, T2A, F2A, E2A, bmCPV2A, bmIFV2A sequence, preferably selected from the nucleotide sequences shown in SEQ ID NOS 55-58. An effector molecule sequence is meant to include, but is not limited to, a CD3 delta sequence, preferably the nucleotide sequence shown as SEQ ID NO: 59.
The second object of the present invention is to provide a virus prepared by using the chimeric antigen receptor gene engineering vector as described above, wherein the chimeric antigen receptor gene engineering vector and the packaging plasmid are transfected into a packaging cell line to obtain the corresponding virus; preferably, the virus is selected from lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, and the like.
A third object of the present invention is to provide an immune cell transfected with the chimeric antigen receptor genetic engineering vector as described above or the virus as described above;
preferably, the immune cells include T cells, NK cells; the T cells comprise unmodified T cells, modified T cells, autologous T cells and allogeneic T cells; the NK cells comprise unmodified NK cells, modified NK cells, autologous NK cells and allogeneic NK cells; preferably, the T cells are selected from at least one of CD4 positive T lymphocytes, CD8 positive T lymphocytes, CD4 and CD8 double positive T lymphocytes;
preferably, the immune cells consist of CD4 positive T lymphocytes and CD8 positive T lymphocytes, wherein the number of the CD4 positive T lymphocytes accounts for 10% -90% of the total cell number; preferably, the number of CD 4-positive T lymphocytes is 20% to 80% of the total cell number, more preferably, the number of CD 4-positive T lymphocytes is 40% to 60% of the total cell number.
A fourth object of the present invention is to provide the use of a chimeric antigen receptor genetic engineering vector as described above, or a virus as described above, or an immune cell as described above, in the preparation of a medicament for the treatment of a neoplastic disease or an immune system disease;
preferably, the diseases for which the medicament for treating a neoplastic disease is directed include: acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphoblastic leukemia, chronic myelogenous leukemia, hairy cell leukemia, mast cell leukemia, plasma cell leukemia, myeloma, myeloproliferative diseases, juvenile myelomonocytic leukemia, mixed leukemia, various lymphomas and lymphoblastomas, hodgkin's disease, neuroblastoma, pulmonary blastoma, pancreatic blastoma, renal blastoma, primitive neuroectodermal tumors, renal cancer, bladder cancer, gonadal tumors, rhabdomyosarcoma, synovial sarcoma, bone tumors, osteosarcoma, ewing's sarcoma, soft tissue sarcoma, melanoma, various small round cell tumors, kidney, ureter, bladder, urinary tract tumors, adrenal cortex tumors, renal cell carcinoma, bladder cancer, genital gland tumors, rhabdomyosarcoma, synovial sarcoma, osteosarcoma, ewing's sarcoma, melanoma, various small round cell tumors, kidney, ureter, bladder, urinary tract tumors, adrenal cortex tumors, and renal cell tumor adrenal neuroblastoma, retinoma, astrocytoma, glioma, ependymoma, soft xanthocyst, teratoma, medulloblastoma, small and non-small cell lung cancer and bronchogenic tumors, langerhans' lymphohistiocytosis, eosinophilia syndrome, eosinophilic tumors, various types of skin cancer, various fibromas and fibrosarcomas, breast cancer, oral cancer, nasopharyngeal cancer, craniopharyngeoma, lip cancer, salivary gland tumors, esophageal cancer, gastric cancer, small intestine tumors, colorectal cancer, pancreatic cancer, liver cancer, bile duct cancer, heart tumors, vaginal tumors, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular tumors, multiple endocrine tumor syndromes, pituitary tumors, thymomas, myxomas;
Preferably, the diseases against which the medicament for treating immune system diseases is directed include: autoimmune diseases, viral infectious diseases, bacterial or fungal infectious diseases; wherein the autoimmune diseases include rheumatoid arthritis, chronic lymphothyroiditis, hyperthyroidism, insulin dependent diabetes mellitus, myasthenia gravis, chronic ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhagic nephritis syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple cerebral spinal sclerosis, acute idiopathic polyneuritis, systemic lupus erythematosus, xerophthalmia, ankylosing spondylitis, scleroderma, polyarteritis nodosa, wegener granulomatosis. Bacterial or fungal infectious diseases include staphylococci, streptococci, proteus, bacillus pyocyaneus, bacillus pertussis, actinomycetes, tetanus bacillus, clostridium perfringens, typhoid bacillus, vibrio cholerae, neisseria meningitidis, bacillus anthracis, diphtheria bacillus, pneumococci, enterococci, acinetobacter, haemophilus influenzae, escherichia coli, legionella, bacillus, anaerobic bacterial infections; various candida, aspergillus, mucor, cryptococcus, ma Nafei penicillium, sporotrichosis, and blastomycosis infections; rickettsia, spirochetes, mycoplasma, chlamydia, and various protozoa infections.
After the technical scheme is adopted, compared with the prior art, the invention has the following beneficial effects:
1. the invention improves chimeric antigen receptor gene engineering carrier, inserts regulatory element, promoter, optimizes base sequence, etc. to ensure that the carrier has good safety, high transfection efficiency, and the transfected immune cells can be rapidly amplified, thereby greatly improving killing effect.
2. The invention adopts double-targeting chimeric antigen receptor gene engineering vector as carrier for CART treatment, and the corresponding carrier can be used for carrying out transfection and expansion of autologous or allogeneic immune cells (T cells and/or NK cells and/or gamma/delta T cells) after virus packaging, so as to be used for cell immunotherapy. The double-targeting chimeric antigen receptor can be designed aiming at one or more antigens on the surface of a cell membrane, and the same antigen can be the same or different ScFv sequences. After the chimeric antigen receptor is combined with the target cell membrane surface antigen, the simultaneous and independent activation of the co-stimulatory signal molecule and the effector signal molecule can be realized through signal transduction, so that the modified immune cells are activated to play the role of resisting tumors and the like. Thus, this approach is more accurate than the recognition of target cells by chimeric antigen receptor immune cells of a single target; meanwhile, the double-targeting combined recognition effect can also better prevent the off-target of a single target, and the specificity and the killing power of target cells are enhanced. In addition, the binding effect of the similar or different molecules on the surface of the normal cells can be reduced, the probability of attacking the normal cells is reduced, and the use safety is improved.
3. In the chimeric antigen receptor sequence, a linker is not arranged between the transmembrane region sequence and the co-stimulatory signal molecule or effector signal molecule, so that a signal path can be shortened, and the signal transduction efficiency is improved, thereby improving the activation efficiency of immune cells such as T cells and the like, and being very important for improving the in vitro killing effect of CART cells.
The following describes the embodiments of the present application in further detail with reference to the accompanying drawings.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:
FIG. 1 is a schematic structural diagram of a chimeric antigen genetically engineered vector of the present application;
FIG. 2 is a core sequence of an independently expressed costimulatory molecule signaling pathway and effector molecule signaling pathway;
FIG. 3 is a block diagram of a dual targeting chimeric antigen receptor of the present application;
FIG. 4 is a comparison of transfection efficiency of vectors of the present application with Novartis lentiviral vectors; wherein a-c are transfection efficiencies of Novartis lentiviral vector packages, d-e are lentiviral transfection efficiencies of the vector packages adopting the patent, and f is an untransfected control.
FIG. 5 is an in vitro cell killing experiment of T lymphocytes of a CD19 dual targeting specific CAR of the application; wherein, fig. 5 a-5 c are the results of in vitro cell killing experiments on day 0, day 1 and day 2 of the test group, respectively; FIGS. 5 d-5 f are results of in vitro cell killing experiments on days 0, 1 and 2, respectively, of the control group;
FIG. 6 shows the results of detection of trace residual disease before and after infusion of CD19 dual-targeting specific CART for refractory acute lymphoblastic leukemia according to the application; 6A-6C are the detection results of residual disease before CD19 double-targeting specific CART cell infusion; 6D-6F is the detection result at day 8 after infusion of CD19 dual targeting specific CART cells;
FIG. 7 shows the results of detection of trace residual disease before and after double targeting specific CART infusion for secondary relapsing acute lymphoblastic leukemia CD 19;
FIG. 8 is a graph showing the results of detection of minimal residual disease before and after double targeting specific CART infusion for recurrent acute lymphoblastic leukemia CD19 after transplantation;
FIG. 9 is a comparison of in vitro proliferation experiments of CD19 double-targeting specific CART cells with other companies; wherein the number of the initial cells is 1×10 9 ；
FIG. 10 is a comparison of the killing effect of Double-CAR-CD19 of the present application with Novartis CD19-CAR (E/T=1:20);
FIG. 11 is a comparison of in vitro killing effects of transfected cells from the dual targeting vector of the present application and from Novartis lentiviral vector company;
FIG. 12 is a plot of the appropriate ratio of CD4 to CD8 versus cytotoxicity;
FIG. 13 is a Recurrence Free Survival (RFS) of the enrolled patient;
fig. 14 is the Overall Survival (OS) of the enrolled patients.
It should be noted that these drawings and the written description are not intended to limit the scope of the inventive concept in any way, but to illustrate the inventive concept to those skilled in the art by referring to the specific embodiments.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments will be clearly and completely described with reference to the accompanying drawings in the embodiments of the present invention, and the following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.
Example 1: 1) First, a chimeric antigen receptor genetic engineering vector is constructed. The corresponding sequence is synthesized by a gene synthesis method, and is verified by sequencing. The appropriate cleavage site is selected to insert the sequence into an appropriate position in an adenovirus, retrovirus or lentiviral vector, and verified by sequencing. As shown in fig. 1 and 2, the chimeric antigen receptor genetic engineering vector includes:
the RRE sequence is inserted upstream of the multiple cloning site of the vector, the WPRE sequence and the cPPT sequence are inserted downstream of the multiple cloning site, and the EF1A promoter is inserted between the RRE sequence and upstream of the multiple cloning site for initiating the chimeric antigen receptor gene inserted into the multiple cloning site. The sequence of the chimeric antigen receptor structural protein upstream is optimized, and the optimized sequence is shown as SEQ ID NO60, so that the expression of the exogenous protein is stable and continuous, and the sequence is very important for improving the in vitro killing effect of CART cells.
Leader peptide sequence construction: the membrane protein leader peptide sequence, preferably leader peptide sequence, is shown in SEQ ID NO 1-10.
The sequences of the first and second antibodies corresponding to the a or B targets on the target cells, namely ScFv antibody light chain VL sequences and heavy chain VH sequences were constructed: for acute leukemia and lymphoma, we selected the sequences in SEQ ID NOS.11-40, and can select the same or different two sequences for cellular immunotherapy of these diseases. For example, the light chain VL sequence and heavy chain VH sequence of ScFv antibody for treating CD19 positive leukemia and lymphoma by taking CD19 as target, the sequences in SEQ ID NO. 11-14 and SEQ ID NO. 19-40 can be selected, and the same CD 19-based sequences can be selected for cell immunotherapy of B-series malignant tumor. For neuroblastoma we selected the sequences in SEQ ID NOS 19-20, 41-50.
Construction of a connecting peptide sequence: refers to the amino acid sequence joining VL and VH in tandem, with the preferred sequence being as set forth in SEQ ID NO. 51. Hinge region sequence construction: refers to the amino acid sequence that mediates the transfer of activation signals from the ScFv to the transmembrane region, with the preferred sequence being as set forth in SEQ ID NO. 52. Construction of a transmembrane region sequence: refers to an amino acid sequence that mediates the transmembrane transfer of an activation signal from the hinge region to the intracellular region, with a preferred sequence being provided in SEQ ID NO. 53. Costimulatory molecule sequence construction: refers to sequences including, but not limited to, molecules such as 4-1BB, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS, and the like. Preferably, the sequence of 4-1BB is set forth in SEQ ID NO. 54. Self-cleavage sequence construction: means including but not limited to P2A, T2A, F2A, E2A, bmCPV2A, bmIFV A, etc., preferably the P2A sequence is as set forth in SEQ ID NOS.55-58. Effector molecule sequence: refers to a sequence comprising, but not limited to, the CD3 delta sequence, the corresponding sequence is provided in SEQ ID NO: 59.
Sequence assembly: in addition to the general expression vector components, the CD19 targeted chimeric antigen receptor genetically engineered vector sequence composition includes, but is not limited to, the following structures: CD8 leader-CD 19 ScFv-hinge region-CD 8 transmembrane region-4-1 BB-P2A-CD 8 leader-CD 19 ScFv-hinge region-CD 8 transmembrane region-CD 3 delta-TGA. The linker is not arranged between the transmembrane region sequence and the co-stimulatory signal molecule or the effector signal molecule, so that a signal path can be shortened, and the signal transduction efficiency is improved, thereby improving the activation efficiency of immune cells such as T cells and the like, and being very important for improving the in vitro killing effect of CART cells.
Through designing proper enzyme cutting site, the sequence is connected with the carrier adopting the same enzyme cutting after enzyme cutting, DH5 alpha or stbl3 competent coliform bacteria are transformed by the connection product, positive cloning is inoculated overnight, and enzyme cutting identification and sequencing are carried out after bacteria extract plasmid. The vector with the completely correct sequencing result is the chimeric antigen receptor gene engineering vector of the embodiment. The structure of the dual targeting chimeric antigen receptor after expression of the costimulatory signal pathway sequence and the effector signal pathway sequence, respectively, is shown in figure 3.
Next, the 293T cells were transfected by mixing the above vectors and virus-packaging plasmid in an appropriate ratio for 72 hours and 96 hours, and then the culture supernatants were collected and virus-concentrated. Again, by real-time quantitative PCR method, the determination of viral titer was performed in conjunction with different concentration gradients of virus transfected 293 cells. Again, transfection of the above viruses was performed by collecting different immune cells from human peripheral blood, ensuring transfection efficiencies above 20%, from which appropriate MOI values were found for clinical treatment. It should be noted that each batch of viruses needs to be subjected to the above operation, so that the stable and reliable curative effect of the clinical test is ensured.
Thirdly, the transfection efficiency is detected by transfecting immune cells with different peripheral blood of a human, and for CART with CD19 as a target, fab fragments aiming at ScFv are adopted as objective basis for detecting positive transfection efficiency by flow cytometry. A real-time quantitative PCR method can also be used for detecting transfection efficiency.
Thirdly, immune cells such as CART cells transfected by viruses and CD19 positive cell strains are mixed and incubated in a certain proportion, in-vitro killing detection is carried out by a flow cytometry after 0h, 24h and 48h, and the cell killing effect at different incubation times is compared with that of a control virus.
Thirdly, collecting 0.5-1 ml/Kg of peripheral blood, separating magnetic beads to obtain CD3 positive T cells or CD56 to obtain NK cells, or separating TCR gamma/delta to obtain gamma/delta T cells, adding CD3/CD28 immunomagnetic beads for stimulating activation and virus transfection, continuing to give CD3/CD28 immunomagnetic beads, interleukin7 and Interleukin15 for in vitro amplification, wherein the amplification can be carried out for more than 100 times in general 7-8 days, and the cells are washed and returned to a human body after the proper cell number is reached. Feedback personThe number of somatic CART positive cells was 1X 10 4 /Kg～5×10 7 Per Kg, and is adjusted according to the body load, preferably at 2X 10 5 /Kg～2×10 7 /Kg。
The virus transfected positive cells have the effect of specifically killing tumor cells, so that the transfection efficiency is improved, the system for culturing the cells can be greatly reduced, and the early production cost is saved. Meanwhile, cells which are negative to transfection are activated by CD3/CD28 immunomagnetic beads during culture and compete for nutrition with cells which are positive to virus transfection, and the proportion of cells which are positive to virus transfection in a transfection system is further reduced after long culture time because the cells which are negative to transfection do not generate pressure for additionally expressing foreign proteins. The double-targeting chimeric antigen receptor vector of the invention is prepared by in vitro transfection, and has the general transfection efficiency of more than 20 percent and the vast majority of 40-70 percent, and as shown in figure 4, a-c are the transfection efficiencies of other lentiviral vector packages, d-e are the lentiviral transfection efficiencies of the vector packages adopting the technology, and f is the untransfected control.
The double-targeting chimeric antigen receptor vector prepared by the invention is used for preparing the target-directed CD19 positive leukemia and lymphoma viruses, and the target-directed CD19 positive leukemia and lymphoma viruses are transfected in vitro according to CART cells: CD19 positive leukemia cell line = 1:10 to 1:20 ratio mixed and co-incubated as test group; and meanwhile, virus obtained by preparing carrier package by adopting an irrelevant sequence is used as a control group. The results of the killing of target cells by both sets of cells are shown in figure 5. Wherein, fig. 5 a-5 c are the results of in vitro cell killing experiments on day 0, day 1 and day 2 of the test group, respectively; FIGS. 5 d-5 f show the results of in vitro cell killing experiments on days 0, 1 and 2, respectively, of the control group. It can be seen that compared with the control group, the killing effect of the virus transfected CART cells prepared by the CD19 double-targeting chimeric antigen receptor vector of the scheme on target cells is greatly enhanced after 24h and 48h co-incubation.
Test example 1: one example of a recurrent patient with CD19 positive acute lymphoblastic leukemia was virally transfected CART cells prepared with this CD19 dual targeting chimeric antigen receptor vector, and the results are shown in FIG. 6. In fig. 6, 6A-6C are the results of detection of residual disease prior to CD19 dual targeting specific CART cell infusion; 6D-6F is the detection result at day 8 after infusion of CD19 double-targeting specific CART cells, with MRD of 33.1%. Indicating that on day 8 after CART treatment, bone marrow MRD achieved molecular remission (MRD < 0.01%).
Test example 2: one example of a patient with CD19 positive acute lymphoblastic leukemia that failed to re-induce after secondary recurrence was virally transfected CART cells prepared with this CD19 double-targeting chimeric antigen receptor vector, and the results are shown in FIG. 7. In FIG. 7, 7A-7C are the results of detection of residual disease before infusion of CD19 double-targeting specific CART cells, with MRD of 11.7%;7D-7F is the detection result at day 8 after infusion of CD19 double-targeting specific CART cells. Indicating that bone marrow MRD reached molecular remission (MRD < 0.01%) at day 8 after CART treatment.
Test example 3: one example of post-virus transfected CART cells from a CD19 positive lymphoblastic lymphoma/leukemia patient with relapsed bone marrow, prepared with this CD19 dual targeting chimeric antigen receptor vector, is shown in fig. 8. In the figures, as shown in fig. 8A-8C, there were a large number of tumor cells in the bone marrow before the CART cells were infused, mrd=95.6%, whereas it was only a small number of tumor cells in the bone marrow at day 8 after CART treatment (mrd=0.012%), as shown in fig. 8D-8F; on day 28 after CART treatment, bone marrow MRD achieved molecular remission (MRD < 0.01%), as shown by 8G-8I, indicating a rapid and effective course of treatment.
Test example 4: as shown in FIG. 9, in vitro amplification experiments of CART cells show that lentiviral and Novartis lentiviral vectors prepared by adopting the double-targeting chimeric antigen receptor vector of the application have different growth sizes after in vitro transfection of human T lymphocytes. The CART cells of other companies are amplified by about 14 times in 7 days after virus transfection, while the CART cells transfected by the vector of the application are amplified by 160 times in less than 7 days, and the in vitro amplification speed is greatly improved. Since these patients are at the end of the disease, shortening of the in vitro culture expansion time is important for therapeutic safety.
Test example 5: in vitro killing experiments were performed on the virus-transfected CART cells prepared according to the present application and CART cells from Novartis, and the experimental results are shown in fig. 10. The result shows that compared with the killing effect of CD19-CAR of Novartis company, the in vitro killing activity of CART cells (Double-CAR-CD 19) prepared by the Double-targeting genetic engineering vector is obviously enhanced, and the result is shown in figure 10. Wherein, fig. 10A is a comparison of effects of infusion for 0 hours, and fig. 10B is a comparison of effects of infusion for 16 hours.
Test example 6: comparing the in vitro killing effect of the application with the technology in patent CN103483452a, the application focuses on the problem of effective target ratio. The effective target ratio is effector cells (CART cells): the target cells (tumor cells) can obtain 99.9% of killing effect in the time of 1:10-1:20, and the killing effect obtained in the patent CN103483452A is 1-50:1, which does not reach the level of the application. As shown in fig. 11. Description: in vitro killing experiments were performed using different ratios of E: T, 99.9% of target cells could be killed at E: t=1:20, whereas CART of patent CN103483452a failed to achieve the above killing effect at E: t=50:1, it is noted that CART cells of patent CN103483452a were not significantly different from control due to non-specific killing of T cells to target cells at E: t=1:1 or lower.
Test example 7: in vitro studies, we found that the appropriate ratio of CD4 to CD8 cells in transfection-positive CART cells is one of the key factors for achieving better cytotoxicity, and in vitro experiments we found that when the ratio of CD4 to CD8 is between 40% and 60%, efficient killing of target cells can be achieved, whereas the ratio of CD4 to CD8 cells often deviates from the above range, and thus has a great influence on the clinical efficacy of patients receiving chemotherapeutics and the like. The corresponding detection results are shown in fig. 12. Wherein, the result of cd4:cd8=10% to 90% is similar to the result of cd4:cd8=0% to 100%; the results for cd4:cd8=90%: 10% are similar to those for cd4:cd8=100%: 0%, both of which are not shown in the figures. From the results, it can be seen that the killing effect is optimal when the amount of CD4 is 40% -60% of the total cell number. In particular CD4: cd8=60%: 40% the best killing effect.
Test example 8: the CART cells are high in transfection efficiency, high in amplification and suitable proportion are the basis for effectively killing target cells in vivo and obtaining clinical curative effects in vivo, 10 patients (9 patients with B-group acute lymphoblastic leukemia and 1 patient with B-group lymphoblastic tumor) which are difficult to treat and relapse in the group are all treated by the CART cells of the technology of the patent, morphological complete remission and molecular remission are obtained 15 days to 1 month after the treatment of the CD19-CART, and the later is detected by the flow cytometry technology to detect trace residual disease (MRD) which is less than 0.01 percent. Wherein 1 patient died early from validated central nervous system cytokine storm, 3 patients relapsed, wherein 2 patients obtained remission of morphology and molecules after secondary CART treatment, average 6 months of Relapse Free Survival (RFS) and 1 year of clinical observation, the event free survival of the group-entered patients was 52.5%. Except that 1 patient died from early central nervous system cytokine storm, 1 patient died from later chemotherapy-related infection in 3 recurrent patients, 8 patients survived, and both bone marrow morphology and MRD detection were in remission, no basis for primary disease was present, and the total survival rate reached 77.14%, and the results are shown in fig. 13 and 14.
The foregoing description is only a preferred embodiment of the present invention and is not intended to limit the invention in any way, and any person skilled in the art may make some changes or modifications to the equivalent embodiments without departing from the technical scope of the present invention, but any simple modification, equivalent changes and modifications to the above embodiments according to the technical principles of the present invention fall within the scope of the present invention.

Claims (10)

1. A chimeric antigen receptor gene engineering vector, which is characterized by comprising a lentiviral vector skeleton, a regulatory element connected to the lentiviral vector skeleton and a chimeric antigen receptor gene sequence, wherein the regulatory element comprises a WPRE element, a cPPT element and an RRE element;
an EF1A promoter is inserted into the upstream of the chimeric antigen receptor gene sequence;
the chimeric antigen receptor gene sequence comprises a costimulatory signal path sequence and an effector signal path sequence which are expressed independently;
in the independently expressed costimulatory signal path sequence and effector signal path sequence, the costimulatory signal path sequence comprises a first leader peptide sequence, a first antibody sequence and a costimulatory molecule sequence which are sequentially connected according to the transcription direction, and the effector signal path sequence comprises a second leader peptide sequence, a second antibody sequence and an effector molecule sequence which are sequentially connected according to the transcription direction; the self-shearing sequence is connected between the costimulatory molecule sequence and the second leader peptide sequence or between the effector molecule sequence and the first leader peptide sequence;
The costimulatory signal pathway sequence comprises a first leader peptide sequence, a first antibody light chain VL sequence, a first antibody connecting peptide sequence, a first antibody heavy chain VH sequence, a hinge region sequence, a transmembrane region sequence and a costimulatory molecule sequence which are sequentially connected according to the transcription direction;
the effector signal pathway sequence comprises a second leader peptide sequence, a second antibody light chain VL sequence, a second antibody connecting peptide sequence, a second antibody heavy chain VH sequence, a hinge region sequence, a transmembrane region sequence and an effector molecule sequence which are sequentially connected according to the transcription direction;
no linker is present between the transmembrane region sequence and the costimulatory signal molecule or effector signal molecule;
the first antibody sequence or the second antibody sequence is selected from nucleotide sequences shown in SEQ ID NO. 11-50; for acute leukemia and lymphoma, selecting the sequence in SEQ ID NO 11-40; aiming at neuroblastoma, selecting the sequence in SEQ ID NO. 41-50;
the sequences of the first guide peptide and the second guide peptide are selected from sequences shown in SEQ ID NO. 1-10; the first antibody connecting peptide sequence and the second antibody connecting peptide sequence are shown in SEQ ID NO. 51; the sequence of the hinge region is shown as SEQ ID NO. 52; the sequence of the transmembrane region is shown as SEQ ID NO. 53;
The costimulatory molecule sequence is a 4-1BB sequence, and the 4-1BB sequence is shown as SEQ ID NO. 54;
the self-shearing sequence is a P2A sequence, and the P2A sequence is selected from sequences shown as SEQ ID NO. 55-58;
the effector molecule sequence is a CD3 zeta sequence, and the CD3 zeta sequence is shown as SEQ ID NO: 59.
2. The chimeric antigen receptor genetic engineering vector according to claim 1, wherein the first antibody sequence or the second antibody sequence is selected from the group consisting of: a molecular sequence that interacts with a cell membrane surface molecule of a target cell, a molecular sequence that interacts with a specific molecule that is presented inside the target cell to its cell membrane surface.
3. The chimeric antigen receptor genetic engineering vector according to claim 2, wherein the combination of the first antibody sequence and the second antibody sequence is selected from the group consisting of: CD19/CD19, CD19/CD20, CD19/CD123, CD19/CD66c, CD19/CD58, CD19/CD56, CD19/CD13, CD19/CD33, CD19/CD44, CD19/CD73, CD19/CD86, CD19/CD99, CD19/CD24, CD19/CD200, CD19/CD97, CD19/BDCA4, CD19/CD133, CD19/CD15, CD19/NG2, CD19/sIgM, CD20/CD20, CD20/CD123, CD20/CD66c, CD20/CD58, CD20/CD56, CD20/CD13, CD20/CD33, CD20/CD44, CD20/CD73 CD20/CD86, CD20/CD99, CD20/CD24, CD20/CD200, CD20/CD97, CD20/BDCA4, CD20/CD133, CD20/CD15, CD20/NG2, CD20/sIgM, CD22/CD22, CD22/CD123, CD22/CD66c, CD22/CD58, CD22/CD56, CD22/CD13, CD22/CD33, CD22/CD44, CD22/CD73, CD22/CD86, CD22/CD99, CD22/CD24, CD22/CD97, CD22/BDCA4, CD22/CD133, CD22/CD15, CD22/NG2, CD22/sIgM.
4. An immune cell transfected with the chimeric antigen receptor genetically engineered vector of any one of claims 1-3.
5. The immune cell of claim 4, wherein the immune cell comprises a T cell, an NK cell; the T cells comprise unmodified T cells, modified T cells, autologous T cells and allogeneic T cells; the NK cells comprise unmodified NK cells, modified NK cells, autologous NK cells and allogeneic NK cells.
6. The immune cell of claim 5, wherein the T cell is selected from at least one of a CD4 positive T lymphocyte, a CD8 positive T lymphocyte, a CD4 and a CD8 double positive T lymphocyte.
7. The immune cell of claim 6, wherein the immune cell consists of CD 4-positive T lymphocytes and CD 8-positive T lymphocytes, wherein the number of CD 4-positive T lymphocytes is 10% to 90% of the total cell number.
8. The immune cell of claim 7, wherein the number of CD4 positive T lymphocytes is 20% to 80% of the total cell number.
9. The immune cell of claim 8, wherein the number of CD4 positive T lymphocytes is 40% to 60% of the total cell number.
10. Use of a chimeric antigen receptor genetic engineering vector according to any one of claims 1-3, or an immune cell according to any one of claims 4-9, in the manufacture of a medicament for the treatment of a neoplastic disease or an immune system disease; the tumor diseases include acute leukemia, lymphoma and neuroblastoma.
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