WO2019076149A1 - 通用型car-t细胞及其制备方法和应用 - Google Patents

通用型car-t细胞及其制备方法和应用 Download PDF

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WO2019076149A1
WO2019076149A1 PCT/CN2018/103714 CN2018103714W WO2019076149A1 WO 2019076149 A1 WO2019076149 A1 WO 2019076149A1 CN 2018103714 W CN2018103714 W CN 2018103714W WO 2019076149 A1 WO2019076149 A1 WO 2019076149A1
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cells
car
cell
hla
universal
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French (fr)
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张巍
李戊玲
张茜真
单娟娟
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重庆精准生物技术有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/26—Universal/off- the- shelf cellular immunotherapy; Allogenic cells or means to avoid rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
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    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464493—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; Prostatic acid phosphatase [PAP]; Prostate-specific G-protein-coupled receptor [PSGR]
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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Definitions

  • the invention belongs to the field of immunotherapy, relates to general-purpose CAR-T cells and preparation methods and applications thereof, and particularly relates to a general-purpose T cell and a general-purpose CAR-T cell, a preparation method thereof and application in medicine.
  • Adoptive cell therapy is a kind of biological therapy technology. It is considered that the autoimmune cells (mainly T cells) are expanded in vitro and then returned to tumor patients for therapeutic purposes. It is the fourth treatment method after surgery, radiotherapy and chemotherapy, and is widely used in clinical treatment.
  • the chimeric antigen receptor also known as CAR, is an artificial receptor that mimics the function of TCR.
  • CAR The chimeric antigen receptor
  • the antigen (receptor) on the surface of the tumor cell binds to the antibody (ligand) of the chimeric antigen receptor, the signal is transmitted to the intracellular region through the hinge region and the transmembrane region, and the intracellular signal domain converts the signal into an activation signal.
  • the effector cells are activated, and the effector cells kill tumor cells by secreting perforin or producing cytokines, and the effector cells themselves are also expanded to further expand the immune killing effect.
  • CAR chimeric antigen receptor
  • Allogeneic cells refer to cells belonging to the same species but genetically different; although more T lymphocytes can be isolated from healthy human peripheral blood, the positive rate of CAR infection is high and the viability and function are superior to the patient-derived T lymphocytes.
  • the advantages of cells are treated with allogeneic cells, but allogeneic cell therapy still faces many obstacles: due to immunogenetic differences between receptors, on the one hand, immune-active donor T lymphocytes enter the receptor. After the patient has proliferated to a certain extent, the normal cells or tissues of the recipient patient are mistaken for the target to attack to produce a graft-versus-host response (GVHD).
  • GVHD graft-versus-host response
  • HVGR host anti-graft response
  • immunosuppressive drugs are mainly used to inhibit receptor immune system activity before treatment, but immunosuppressive drugs may affect the efficacy of returning cells in adoptive cell therapy; for GVHD medical treatment mainly through HLA matching
  • HLA matching the success probability of HLA matching is low, time consuming and costly.
  • an object of the present invention is to provide a universal type CAR-T cell, which can target a specific tumor-associated marker and inactivate cell surface TCR and MHC, and can reduce the same species. Immune rejection caused by allogeneic cell therapy and safe and effective removal of tumor cells in patients.
  • TCR T cell antigen receptor
  • MHC I, MHC II major histocompatibility complex
  • the CAR-T treatment of autologous cells requires the preparation of blood-separated patients' own T lymphocytes.
  • the factors affecting the production process of CAR-T are not standardized and the production safety is affected.
  • some The patient's autologous T lymphocytes have insufficient activity and quantity after chemotherapy, or are affected by the tumor environment, resulting in limited T lymphocyte activity and proliferative capacity. Such cells are often difficult to prepare for CAR-T, and the safety and efficacy of treatment are high.
  • the prepared cells Being affected; or if there is a sudden situation in the preparation of CAR-T cells, the prepared cells can not be returned to the patient in time, which will affect the therapeutic effect, and even affected by the state of autologous T lymphocytes, some tumor patients cannot accept autologous CAR- T cell adoptive therapy; the development of universal CAR-T or universal T lymphocyte therapy for allogeneic therapy is imminent.
  • knockout of the universal CAR-T cells includes the TRAC, B2M and CIITA genes while also expressing HLA-E or HLA-F.
  • HVGR and GVHR-related genes include TCR, MHC class I and II molecules (main histocompatibility complex MHC I, MHC II) related genes, and a single TRAC gene is a gene encoding a TCRa chain and two genes encoding TCR ⁇ .
  • the TRBC gene forms a complete functional TCR complex.
  • Knock out TRAC is the optimal solution for inducing TCR inactivation.
  • B2M and CIITA are MHC I and MHC II related genes, respectively.
  • T lymphocytes are knocked out by the above three genes. Cells do not cause graft-versus-host disease (GVHD) when they are returned to the same allogeneic patient.
  • the inventors found that the universal CAR-T cells were quickly cleared by allogeneic NK cells and had a short survival time. Probably because B2M interference will cause complete inactivation of all genes of the MHC class I family. Although this can completely eliminate GVHD, the expression of MHC class I molecules is a key molecule for NK cells to recognize "autologous cells", and MHC class I molecules are completely inactivated. It will cause NK cells to treat the returned cells as "allogene" to clear the returned cells, affecting the effectiveness of universal CAR-T cells against tumors.
  • Perfecting is used to achieve the goal of attenuating GVDH without causing NK cell attack.
  • the universal type CAR-T cells knock out the TRAC, B2M and CIITA genes while also expressing HLA-E or HLA-F.
  • the universal type CAR-T cells knock out the TRAC, B2M and CIITA genes while also expressing HLA-E.
  • HLA is a highly polymorphic allogeneic antigen controlled by the human major histocompatibility complex (MHC) gene cluster, HLA It is equivalent to the above MHC molecules; it is found that HLA-A is mainly concentrated in 8 types of samples, and 8 types are A*02, A*11, A*24, A*30, A*33 respectively.
  • MHC human major histocompatibility complex
  • HLA-A knockout can cause HLA inactivation but does not affect HLA-E, HLA-F function in humans (human conserved HLA molecules are consistent in all humans), select HLA-A gene replacement
  • the B2M gene knocks out TRAC, HLA-A, and CIITA to achieve universal CAR-T cells that do not cause GVHD and do not cause NK-specific recognition.
  • the universal type CAR-T cells are knocked out of the TRAC, HLA-A or B2M, CIITA genes.
  • a second object of the present invention is to provide a method for preparing the universal CAR-T cell, comprising the following steps:
  • step 2) Transfecting the T cells of step 1) with a lentiviral vector encoding a CAR or CAR co-expressing different tumor-associated molecules with HLA-E or HLA-F to obtain CAR-T cells targeting different tumor-associated molecules ;
  • the CAR-T cells obtained in step 2) are subjected to multi-gene knockout including TRAC, HLA-A and CIITA or TRAC, B2M and CIITA by TALEN or zinc finger method or CRISPR/Cas9 system method to obtain universal CAR- T cells.
  • the universal CAR-T cells are capable of targeting specific tumor-associated markers and inactivating TCR and MHC functions on the cell surface, and the preparation method needs to overcome two technical obstacles.
  • One is to simultaneously achieve multi-gene knockout in T lymphocytes.
  • the feasibility of multi-gene expression; the second is whether the obtained universal CAR-T or T lymphocytes can safely and effectively target tumor cells with specific targets.
  • Multi-knockout studies are basically done on T lymphocytes.
  • Multi-gene knockout on CAR-T requires both gene knockout and CAR-T expression to be technically difficult to achieve;
  • the gene knockout method is a CRISPR/Cas9 vector and a CAR vector in which a sgRNA having a gene of interest to be knocked out is repeatedly infected with a viral vector, and the cell activity is decreased due to multiple virus infections, and the multi-gene knockout causes the vector to be too large.
  • the transfection efficiency of virus or transfection reagent is extremely low, and only the electrotransfer can be used to achieve the purpose of transfecting the vector.
  • Value-added survival even in the case of stimulation with an activating reagent, the survival time in vitro is generally up to 2-3 weeks. After the electrospinning, the infection efficiency of the CAR is low and the cell activity is poor;
  • the applicant optimized the infection conditions to prepare CAR-T cells by CAR infection after 24 hours of T cell activation, and then electroporated after 24 hours of culture, transfecting the CRISPR/Cas9 vector carrying the sgRNA of the gene of interest to be knocked out, Construction of universal CAR-T cells.
  • the knockout efficiency of the method is high, the universal CAR-T cell obtained by the universal allogeneic return is safe, and the positive rate of the infected CAR is stable and has good killing activity and specificity.
  • CAR-T or T lymphocytes (T cells) used for adoptive transfer or allogeneic reinfusion are prepared from peripheral blood-separated T cells, but T cells isolated from peripheral blood are already mature T
  • the cells maintain proliferative and effective function time in vitro, so the genetically modified universal cells need to be rapidly expanded in vitro to the patient for the required number of treatments and then returned to the patient.
  • the inventors explored through a large number of experiments, combined with viral infection and electroporation. In this way, optimal methods for constructing universal CAR-T or T cells were obtained: 1) multi-gene expression using viral infection; 2) multi-gene interference using CRISPR/Cas9 technology.
  • the commonly used virus-infected expression genes are relatively mature for single-gene infection technology.
  • Multi-gene infections are often inefficient, and involve multiple gene knockouts.
  • CRISPR/Cas9 technology can achieve stable knockout of target genes, Multi-gene interference has a low efficiency in knockout by means of viral infection, and the use of electroporation has a great influence on cell activity and the optimal electroporation conditions require a large number of exploration experiments.
  • step 2) CAR-T cells are first obtained by infecting T lymphocytes containing a virus encoding a specific tumor target and a virus co-expressing HLA-E or HLA-F, and further multi-gene knocking is performed.
  • the target molecules recognized by the CAR in the step 2) include CD19, PSCA, CD123, CD20, CEA (carcinoembryonic antigen), FAP, CD133, EGFR, EGFRVIII, BCMA, PSMA, Her2, CA125, EphA2, C-met , L1CAM, VEGFR, CS1, ROR1, EC, NY-ESO-1, MUC1, MUC16, mesothelin, LewisY, GPC3, GD2, EPG, DLL3, CD99, 5T4, CD22, CD30, CD33, CD138, CD171 .
  • the method of multi-gene knockout in step 3) is a method of the CRISPR/Cas9 system.
  • the CRISPR/Cas9 system consists of gRNA and Cas9 nuclease or endonuclease.
  • the multi-gene knockout is preferably performed by the CRISPR/Cas9 system, and the multi-gene gRNA to be knocked out is constructed on the same vector, and transfected by electroporation to perform gene knockout.
  • gRNA sequences targeting the B2M, HLA-A, CIITA and TRAC genes are set forth in SEQ ID NOS: 5-19.
  • gRNA sequences targeting TRAC, B2M and CIITA or HLA-A, CIITA and TRAC genes were constructed on vector PX330A.
  • the expressed HLA-E gene sequence is SEQ ID NO: 33, the protein sequence is SEQ ID NO: 34; and the expressed HLA-F gene sequence is SEQ ID NO: 32.
  • 293T cells are transfected with GFP.
  • the electroporation conditions are PBMC or T cells activated for 48 hours, the voltage is 1000V, the pulse width is 35ms, the number of electric shocks is twice, and the total number of cells is 2 ⁇ 10 5 .
  • a third object of the present invention is to provide a CRISPR/Cas9 gene knockout expression vector obtained by the above production method, which comprises the gene sequence shown in SEQ ID NOs: 26-31.
  • universal CAR-T cells which are targeted to specific tumors and simultaneously inactivated cell surface TCR and MHC class I and class II molecules, or cell surface TCR and MHC one can be obtained.
  • Universal T lymphocytes which are simultaneously inactivated by two types of molecules, can be used for tumor treatment to improve the safety and effectiveness of CAR-T cells in clearing tumors.
  • the cell or tissue of the malignant tumor or infectious disease is capable of expressing, including CD19, PSCA, CD123, CD20, CEA (carcinoembryonic antigen), FAP, CD133, EGFR, EGFRVIII, BCMA, PSMA, Her2, CA125, EphA2, C-met, L1CAM, VEGFR, CS1, ROR1, EC, NY-ESO-1, MUC1, MUC16, mesothelin, LewisY, GPC3, GD2, EPG, DLL3, CD99, 5T4, CD22, CD30, CD33, CD138, CD171
  • the universal type CAR-T comprises UCAR-19 and UCAR-PSCA.
  • the malignant tumor or infectious disease includes: hematological tumor, solid tumor, immune rejection caused by allogeneic transplantation, and autoimmune diseases such as allergic reaction or systemic lupus erythematosus.
  • TCR T cell antigen receptor
  • MHC I, MHC II major histocompatibility complex
  • the gene encoding the TCR includes TRAC and/or TRBC; the genes encoding the major histocompatibility complex include HLA, B2MH and CIITA.
  • the universal type CAR-T cells simultaneously knock out the TRAC, HLA-A and CIITA genes or simultaneously knock out TRAC, and the B2M and CIITA genes also express HLA-E or HLA-F.
  • This universal T cell can be used for allogeneic treatment.
  • the malignant tumor or infectious disease includes: hematological tumor, solid tumor, immune rejection caused by allogeneic transplantation, and autoimmune diseases such as allergic reaction or systemic lupus erythematosus.
  • Allogeneic universal T cells obtained a wider application space for allogeneic cell therapy to promote the standardized production of cell therapy; Applicants further use the universal T lymphocytes to successfully carry out CAR-T transformation to obtain the same species Allogeneic universal CAR-T cells further enhance the safety and efficacy of CAR-T treatment.
  • the universal T cells and universal CAR-T cells of the present invention can be applied to the treatment of malignant tumors or infectious diseases by means of allogeneic reinfusion, and the rejection is low in the patient for a long period of time.
  • the safety of survival is high, which promotes the treatment of most patients, especially tumor patients, and the radiotherapy treatment leads to fewer autologous T cells or even the treatment effect and safety of cell therapy due to the attenuation or loss of T cell function.
  • the promotion and development of T cell therapy provides assistance.
  • the method for preparing universal CAR-T cells can obtain universal CAR-T cells in which TCR and MHC are simultaneously inactivated with high CAR expression rate, and the universal CAR-T cells are applied to malignant tumors or infectivity.
  • the treatment of the disease has low rejection and is highly effective in the patient's body.
  • the general-purpose CAR-T cells provided by the present invention are applied to the treatment of allogeneic adoptive cells without being affected by the patient's own condition or treatment mode, and the general-purpose CAR-T cells can be prepared at any time. Good time to treat patients to ensure the effectiveness of treatment.
  • the universal T cells provided by the present invention can be applied to immunotherapy T cells of allogeneic reinfusion, and the universal T cells are convenient for small-scale rejection after programmed allogeneic return.
  • Figure 1 shows the results of HLA-A typing frequency detection in Chinese population.
  • Figure 2 shows the design of knockout TCR and MHC class I and II molecular-related gene vectors using the CRISPR/Cas9 system.
  • Figure 3 shows the designed gRNA targeting site detection.
  • Figure 4 shows the expression of B2M, CIITA and TRAC on CAR-T cell surface after knockout of CRISPR/Cas9 system.
  • Figure 5 shows the CAR expression assay.
  • Figure 6 shows the simultaneous inactivation efficiency of TCR and MHC class I and II molecules in CAR-T cells after knockout of CRISPR/Cas9 system and the three-negative CAR-T phenotype after sorting.
  • Figure 7 shows the CRISPR/Cas9 off-target detection.
  • Figure 8 shows the detection of allogeneic NK cell reactivity by triple negative CAR-T cells.
  • Figure 9 is a general-purpose CAR-T HLA-E expression assay.
  • Figure 10 shows the ability of universal CAR-T killing tumor cells.
  • Figure 11 shows the detection of universal CAR-T cell allogeneic NK reactivity.
  • This method belongs to the HLA high resolution typing technique.
  • the EDTA anticoagulation tube (blood routine tube) was used to take 4 ml of venous blood from the subject, and was temporarily stored at 4° refrigerated and stored at -20° for a long time. Then sent to Huada for HLA typing.
  • HLA high-resolution typing (MHC-I and MHC-II, 4 positions) of the sample and the corresponding expression values and p-values were calculated directly from the raw data of RNA-Seq sequencing (fastq format) using seq2HLA software.
  • B2M, CIITA, and TRAC genes are closely related to TCR and MHC class I and class II related gene functions to confirm knockout of B2M, CIITA, and TRAC genes.
  • the B2M, CIITA, and TRAC gene sequences were obtained at NCBI to obtain the CDS sequence, B2M sequence number AH002619.2, CIITA at NCBI gene ID: 4261, and TRAC at NCBI gene ID: 28755, as shown in SEQ ID NO: 1-3.
  • gRNAs were designed for the targeted SEQ ID NO: 1-3 regions of the CDS region, respectively, as shown in Table 1.
  • the following primers were designed and synthesized by Nanjing Kingsray Biotech Co., Ltd.
  • the specific primers are as follows:
  • Primer sg-B2M-3-up 5'-CACCGGCCGAGATGTCTCGCTCCG-3'; SEQ ID NO: 20;
  • Primer sg-B2M-3-down 5'-AAACCGGAGCGAGACATCTCGGCC-3'; SEQ ID NO: 21;
  • Primer sg-CIITA2-up 5'-CACCGATATTGGCATAAGCCTCCC-3'; SEQ ID NO: 22;
  • Primer sg-CIITA2-down 5'-AAACGGGAGGCTTATGCCAATATC-3'; SEQ ID NO: 23;
  • Primer sg-TRAC-1-up 5'-CACCAGAGTCTCTCAGCTGGTACA-3'; SEQ ID NO: 24;
  • Primer sg-TRAC-1-down 5'-AAACTGTACCAGCTGAGAGACTCT-3'; SEQ ID NO: 25.
  • the two upstream and downstream primers were annealed into three double-stranded gRNA sequences, and the reaction system was loaded according to the specification of Annealing Buffer for DNA Oligos (5X) (purchased from Biyuntian).
  • PX330A-1X3, PX330S-2, and PX330S-3 (all purchased from Addgene Plasmid) were digested with restriction endonuclease BbsI (purchased from NEB), and the digestion reaction was carried out according to the instructions.
  • the digested product was separated by agarose gel electrophoresis, and the DNA fragment was recovered.
  • the corresponding annealed primer fragment and the purified vector fragment were respectively ligated by T4 ligase (purchased from Promega) to obtain expression with only one gRNA. Carrier.
  • T4 ligase purchased from Promega
  • the expression vector containing only one gRNA constructed above was digested with restriction endonuclease BsaI (purchased from NEB) according to the Golden gate cloning technique.
  • Quick ligase using Quick Ligation TM Kit (available from NEB) ligation reaction was performed, in accordance with instructions loaded.
  • Six-white screening was performed using X-gal and IPTG (both purchased from Biotech Biotech Co., Ltd.) to obtain six combinatorial vectors containing three different gRNA expression cassettes:
  • step 3 The vector obtained in step 3 was transfected into competent cells, and the sequence correctness was verified by PCR.
  • the specific steps are shown in the molecular cloning experiment guide (third edition, J. Sambrook et al.).
  • the experimental results are shown in Fig. 2. Three bands and sequencing results were correct.
  • Example 3 general-purpose CAR-T cell construction and phenotypic detection
  • PBMC transfection was further performed after better electroporation conditions.
  • the experimental results are shown in Table 2, Table 3.
  • Preferred electroporation conditions were PBMC or T cell activation. After 48 hours, the voltage was 1000V, the pulse width was 35ms, and the number of electric shocks was twice. The total amount of cells was 2 ⁇ 10 5 , and after electroporation, it was cultured in a medium containing fresh IL-2.
  • Collect 60 ml of peripheral blood with a blood collection tube supplemented with anticoagulant dispense 30 ml each in a 50 ml centrifuge tube, dilute with 7.5 ml of hydroxyethyl starch; allow natural sedimentation at room temperature (18-25 ° C) for about 30 min, collect the upper plasma, centrifuge 15 min; then resuspended the precipitate with physiological saline, added to the lymphocyte separation solution at a volume ratio of 1:1, centrifuged by centrifugation, and centrifuged for 20 min; after centrifugation, a second layer of white lymphocytes was taken and washed twice with physiological saline.
  • the cells were resuspended in physiological saline, and cultured in RPMI 1640 complete medium containing 10% FBS to obtain human peripheral blood mononuclear cells.
  • the newly prepared mononuclear cell PBMC was cultured in RPMI 1640 complete medium containing 10% FBS, and the anti-CD3 monoclonal antibody was activated for lentivirus infection; the CAR gene containing the targeted CD19 or PSCA and/or the coding HLA-E were added respectively.
  • the lentiviral vector expressing the gene, uninfected peripheral blood lymphocytes (PBMC) was used as a blank control; 24 hours later, the cell count was collected to prepare universal CAR-T cells, and the 24-well plate was plated with 2 ⁇ 10 5 cells per well. 1000V, electric pulse 35ms, electric shock twice to complete the electric rotation. Specific steps See Transfection System for details. Transfection System operating instructions.
  • the preferred electroporation conditions were used: electroporation was performed twice with a voltage of 1000 V and a pulse width of 35 ms, and the CRISPR/Cas9 expression vector containing the gRNA targeting the B2M, CIITA and TRAC genes was electrotransferred ( 2) CAR-T cells targeting CD19 or PSCA obtained universal pUCAR-19 and pUCAR-PSCA cells, and were infected with CAR virus for 10 days and knocked out TCR and MHC by electroporation using CRISPR/Cas9 system.
  • T cells of the second type of molecular related genes 300g/min, centrifuged for 5min, discard the supernatant to collect the cells; adjust the density of the cells to 1 ⁇ 10 6 /ml; separately collect the collected cells by flow cytometry
  • the positive rate of Protein-L was detected to detect the positive rate of CAR expression in general-purpose CAR-T cells.
  • the PBS was washed twice to wash away excess unbound Protein-L antibody and labeled with HLA-DR, B2M, CIITA, HLA-E and CD3 antibodies to detect TCR. And the expression of MHC class I and class II molecules.
  • the modified T lymphocytes had a surface B2M ratio of about 30%, and CIITA and TRAC molecules were not expressed.
  • 43.6% of CAR was expressed, and the method used in the invention was simultaneously Knocking out 3 genes is highly efficient and does not affect CAR expression.
  • the PUCAR-CD19 or pUCAR-PSCA cells which were triple-negative and CAR-positively expressed by B2M, CIITA and TRAC, were sorted, as shown in Figure 6, for the obtained pUCAR-CD19 cells.
  • Sorting B2M, CIITA and TRC molecules were negative and CAR and HLA-E positively expressed pUCAR-CD19 cells, triple negative and co-expressing CAR and HLA-E cells were improved universal CAR-T cells with pUCAR-019 HLA -E+ indicates that the expression of HLA-E is shown in Fig. 7.
  • the universal CAR-T cell obtained in the step 3 was further purchased from the Qiagen (Cat. No. 511004) genomic extraction kit for extraction of the genome using the QIAamp DNA Blood Mini Kit. According to the kit instructions, the designed gRNA-targeted region was verified by RT-PCR and sequencing. The results are shown in Figure 3. The bold or missing region of the font is the region targeted by the gRNA designed by the developer. The results show that the researchers The designed gRNA can target the target regions of B2M, CIITA and TRAC genes to inactivate B2M, CIITA and TRAC genes, wherein - is a deletion; + is an insertion; M is a mutation.
  • Example 4 CRISPR/cas9 Knockout B2M, CIITA, and TRAC off-target detection
  • the off-target test results are shown in Figure 8.
  • the off-target-analysis results were 0/6 or 0/5, indicating that the gRNAs designed for B2M, CIITA, and TRAC did not show off-target after repeated experiments.
  • the gRNA sequence has high specificity and stability, and is safer in vivo.
  • the corresponding CAR-T cells were plated according to the pre-designed effective target ratio, and every 15 minutes after the CAR-T cells were plated. Record the resistance index and judge the proliferation or death of the adherent target cells by the resistance index. See Figure 9.
  • the cell base number is 1. When the Y-axis number is greater than 1, it is proved that the tumor cells proliferate. When the Y-axis number is less than 1, the tumor is indicated. Cells were killed, X-axis TBC, BTC, TCB were expressed in PSCA-CAR-T cells transfected with PX330A-1X3-TRAC-1-B2M-3-CIITA2, PX330-A-1X3-B2M-3-TRAC-1, respectively.
  • PSCA group is auto-infected CAR-T cells infected with CAR without untransfected knockout system
  • Medium is normal culture Tumor cells
  • PBMC group is T lymph without uninfected CAR or transfected knockout vector
  • the killing results are shown in Figure 9.
  • the universal CAR-T exhibited significant killing at 24 hours, especially in the BTC (PX330-A-1X3-B2M-3-TRAC-1-CIITA2) group. Killing activity and specificity.
  • CD19-positive Raji cells (abbreviated as Raji-luc) stably expressing firefly luciferase were used as target cells, and UCAR-19 was used as effector cells to evaluate the killing results of pUCAR-019.
  • 1 indicates that CAR-019 cells are infected with pUCAR-019-1 cells of PX330-A-1X3-B2M-3-TRAC-1-CIITA2, and 2 indicates that CAR-019 cells are infected with PX330A-1X3-TRAC-1-CIITA2-B2M- 3 pUCAR-019-2 cells; used
  • the killing effect is measured by the standard method provided by the Luciferase Assay System (Promega Cat. #E2520) kit.
  • the kill rate is calculated by the following formula:
  • Cell killing rate (1-effect cell target cell co-culture well fluorescence intensity / single target cell well fluorescence intensity) ⁇ 100%
  • the killing results are shown in Fig. 10, and the cells of the pUCAR-019-1 group exhibited high killing activity.
  • NK-92 cells were mixed with PBMC or pUCAR-19 according to the ratio of the effective target ratio to 1:1, and the culture was continued as usual.
  • the cell culture plate was taken out from the cell culture incubator 1 hour before the co-culture, and the LDH release reagent provided by the kit was added to the "sample maximum enzyme activity control well" in an amount of 10% of the volume of the original culture solution. After adding the LDH release reagent, mix repeatedly by pipetting several times and continue to incubate in the cell culture incubator.
  • the cell culture plates were centrifuged for 5 min in a multiwell plate centrifuge at 400 g. 120 ⁇ l of the supernatant of each well was taken and added to the corresponding well of a new 96-well plate, and the sample was measured.
  • pUCAR-019 represents a triple-negative CAR cell that knocks out B2M, CIITA, and TRAC and targets CD19
  • pUCAR-019 HLA-E+ indicates that CAR-T cells knocked out by B2M, CIITA, and TRAC co-express HLA-E.

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Abstract

提供了一种通用型CAR-T细胞及其制备方法和应用。所述通用型CAR-T细胞中经过多基因敲除同时抑制了T细胞抗原受体(TCR)和主要组织相容性复合体(MHCⅠ,MHCⅡ)在T细胞中的功能。编码所述TCR的基因包括TRAC和/或TRBC。编码所述主要组织相容性复合体的基因包括HLA-A、B2MH和CIITA。所述通用型CAR-T细胞能够靶向特异的肿瘤相关标志并且细胞表面TCR和MHC功能失活，可以降低同种异体细胞治疗引起的免疫排斥反应并且清除病人体内的肿瘤细胞，且在使用中不受病人自身病情或治疗方式影响。

Description

通用型CAR-T细胞及其制备方法和应用 技术领域
本发明属于免疫治疗领域，涉及通用型CAR-T细胞及其制备方法和应用，具体涉及一种通用型T细胞和通用型CAR-T细胞及其制备方法和在药物中的应用。
背景技术
过继细胞治疗(adoptive cell therapy，ACT)是生物治疗技术的一种，对自体免疫细胞(主要是T细胞)进行体外扩增，然后将其回输给肿瘤患者以达到治疗目的的方法，被认为是继手术、放、化疗后的第4种治疗方式，在临床治疗中受到广泛应用。
嵌合抗原受体又称CAR，是模拟TCR功能的人工受体。肿瘤细胞表面的抗原(受体)与嵌合抗原受体的抗体(配体)结合时，通过铰链区和跨膜区将信号传递至胞内，胞内信号域再将信号转化为活化信号，激活效应细胞，效应细胞通过分泌穿孔素或者产生细胞因子杀伤肿瘤细胞，同时效应细胞本身也发生扩增，进一步扩大免疫杀伤作用。
近年来随着嵌合抗原受体(CAR)修饰的T细胞在血液病肿瘤治疗上的显著效果，过继细胞治疗的需求不断升高。但是目前过继细胞治疗还是基于自体细胞的回输治疗方式，这种方式在步骤上需要抽取病人一定量的外周血，之后再进行外周血单个核细胞分离，T淋巴细胞分离，进一步对分离的T淋巴细胞进行修饰改造以及体外增殖，修饰的T细胞增殖到一定数目再回输回病人体内才能起到一定的治疗效果。但是大部分病人尤其是肿瘤病人在进行CAR-T细胞过继治疗前均进行过其他方式的治疗，例如：化疗，放射性治疗，这就导致获得的T淋巴细胞较少，甚至由于药物的作用很多病人的T细胞在体外扩增困难并且病人自身来源的T细胞还可能出现功能衰减或缺失，并且对于T细胞基因改造的难度加大并且治疗效果和安全性不易控制，这些障碍均影响着CAT-T细胞治疗和肿瘤临床治疗的应用推广和发展。
同种异体细胞是指属于相同物种但在遗传上有所差异个体的细胞；虽然能够利用健康人外周血分离的T淋巴细胞较多，CAR感染阳性率高并且活力和功能优于病人来源T淋巴细胞的优势进行同种异体细胞治疗，但是同种异体细胞治疗仍然面临诸多障碍：由于供受体之间存在免疫遗传学差异，一方面，具有免疫活性的供体的T淋巴细胞在进入受体病人体内并增殖到一定程度后，将受体病人的正常细胞或组织误认为靶标进行攻击从而产生移植物抗宿主反应(GVHD)，另一方面，作为异体细胞受体体内的正常免疫系统也可能会对其进行清 除产生宿主抗移植物反应(HVGR)从而影响治疗效果。针对HVGR临床上主要在治疗前应用免疫抑制类药物抑制受体免疫系统活性，但是在过继细胞治疗时免疫抑制类药物会影响回输细胞的疗效；针对GVHD医学上主要通过HLA配型来进行预防，但是HLA配型成功概率低耗时长而且费用高昂。
因此需要开发一种可以降低同种异体细胞治疗引起的免疫排斥反应并安全有效的通用型CAR-T细胞。
发明内容
有鉴于此，本发明的目的在于提供一种通用型CAR-T细胞，本发明的通用型CAR-T细胞能够靶向特异的肿瘤相关标志并且细胞表面TCR和MHC功能失活，可以降低同种异体细胞治疗引起的免疫排斥反应并且安全有效的清除病人体内的肿瘤细胞。
为实现上述目的，本发明的技术方案为：
通用型CAR-T细胞，所述通用型CAR-T细胞中经过多基因敲除同时抑制了T细胞抗原受体(TCR)和主要组织相容性复合体(MHC Ⅰ,MHC Ⅱ)在T细胞中的功能；编码所述TCR的基因包括TRAC和/或TRBC；编码所述主要组织相容性复合体的基因包括HLA-A，B2M和CIITA。
自体细胞的CAR-T治疗需要抽取血液分离病人自身T淋巴细胞制备，一方面由于病人自身病情以及T淋巴细胞状态不同CAR-T生产过程影响因素较多不能标准化生产影响安全性，另一方面有些病人自体T淋巴细胞经过化疗后活性和数量不足，或受到肿瘤环境影响导致T淋巴细胞活性和增殖能力受限，这样的细胞在制备CAR-T时往往难度较大，治疗的安全性和有效性受到影响；或者在CAR-T细胞制备过程中若出现突发状况已准备细胞不能及时回输给病人也会影响治疗效果，甚至于受到自体T淋巴细胞状态影响有些肿瘤患者不能接受自体的CAR-T细胞过继治疗；可用于同种异体治疗的通用型CAR-T或通用型T淋巴细胞治疗的研发技术就显得迫在眉睫。
我们同时敲除基因TRAC，B2M和CIITA或TRAC、HLA-A和CIITA基因，TCR和MHC一、二类分子(主要组织相容性复合体)功能全部失活，获得的通用型CAR-T安全性高，另一方面考虑到体内NK细胞通过MHCI来识别“自体和非自体”并对识别的“非自体”进行攻击，导致病人的NK细胞会识别通用型CAR-T并对其清除，这样该通用型CAR-T在病人体内存活时间短效果差，所以我们进行了改进：同时表达HLA-E或HLA-F可以与NK细胞表面的分子识别，让NK细胞认为该细胞为“自体”细胞不进行攻击。
进一步，所述通用型CAR-T细胞中敲除包括TRAC，B2M和CIITA基因的同时还表达 HLA-E或HLA-F。
宿主抗移植物反应(host versus graft reaction,HVGR)与移植物抗宿主反应(graft versus host reaction,GVHR)是除细胞因子释放综合征(Cytokine release syndrome,CRS)等以外CAR-T治疗存在的最大难题之一，HVGR与GVHR相关基因包含TCR、MHC一、二类分子(主要组织相容性复合体MHC Ⅰ、MHC Ⅱ)相关基因，单个TRAC基因是编码TCRa链的基因与编码TCRβ的两个TRBC基因形成完整的有功能的TCR复合物，敲除TRAC是可以达到致使TCR失活的最优方案，B2M和CIITA分别为MHC Ⅰ、MHC Ⅱ相关基因，以上三个基因同时敲除的T淋巴细胞在回输入同种异体病人时不会引起移植物抗宿主病(GVHD)。
但是利用上述构建的通用型CAR-T细胞进行试验时发明人发现：该通用型CAR-T细胞会很快被异体的NK细胞清除，存活时间短。可能是因为B2M干扰后会引起MHCI类家族所有基因的完全失活，虽然这样能够更完全消除GVHD但是MHCI类分子的表达又是NK细胞识别“自体细胞”的关键分子，MHCI类分子完全失活会导致NK细胞将回输细胞认为“异体”对回输细胞进行清除，影响通用型CAR-T细胞针对肿瘤的有效性。
在感染CAR的同时共表达MHCI类亚型中在人类基因组中保守的基因如HLA-E或HLA-F，再利用CRISPR/Cas9敲除TRAC、B2M和CIITA基因，让通用型CAR-T细胞重新“贴上自体标签”，以达到在减弱GVDH的同时又不引起NK细胞的攻击的目的。
作为一种优选，所述通用型CAR-T细胞中敲除TRAC，B2M和CIITA基因的同时还表达HLA-E或HLA-F。
作为一种优选，所述通用型CAR-T细胞中敲除TRAC，B2M和CIITA基因的同时还表达HLA-E。
申请人团队之前进行过不同健康供者的血液的HLA分型检测，HLA是具有高度多态性的同种异体抗原，受控于人类主要组织相容性复合体(MHC)的基因簇，HLA等同于前述MHC分子；发现所检测样本中HLA-A主要集中在8种分型中，8种分型分别为分别为A*02，A*11，A*24，A*30，A*33，A*03，A*01和A*26，对这8种分型进行PCR扩增获得PCR片段进行gRNA设计时又发现其中7种分型可在同一区域设计gRNA，进一步发明人团队利用生物信息技术通过大数据分析不同HLA-A转录本比例发现该8种基因分型在包含90％以上中国人中频率最高。8种频率最高的分型gRNA设计区域基因序列如SEQ ID NO:4，gRNA序列如SEQ ID NO:8-12。
HLA-A基因敲除可以引起HLA功能失活但是对于人类保守的HLA-E，HLA-F功能并不影响(人类保守的HLA分子在所有人类中表型均一致)，选择HLA-A基因替换B2M基因，敲除 TRAC、HLA-A和CIITA从而达到既不引起GVHD又不会引起NK特异识别的通用型CAR-T细胞。
进一步，所述通用型CAR-T细胞中敲除TRAC、HLA-A或B2M、CIITA基因。
本发明的目的之二在于提供一种所述通用型CAR-T细胞的制备方法，包括以下步骤：
1)得到活化的T细胞；
2)用编码靶向不同肿瘤相关分子的CAR或CAR与HLA-E或HLA-F共表达的慢病毒载体来转染步骤1)的T细胞，获得靶向不同肿瘤相关分子的CAR-T细胞；
3)将步骤2)获得的CAR-T细胞通过TALEN或锌指方法或CRISPR/Cas9系统的方法进行包括TRAC、HLA-A和CIITA或者TRAC、B2M和CIITA的多基因敲除获得通用型CAR-T细胞。
所述的通用型CAR-T细胞能够靶向特异的肿瘤相关标志并且细胞表面TCR和MHC功能失活，制备方法需要逾越两个技术障碍，一是在T淋巴细胞内同时实现多基因敲除与多基因表达的可行性；二是获得的通用型CAR-T或T淋巴细胞是否能够安全有效的针对特异靶点的肿瘤细胞。
多基因敲除的研究，基本是在T淋巴细胞上完成的，在CAR-T上进行多基因敲除既要完成基因敲除又要实现CAR-T的表达在技术上实现较为困难；一般的基因敲除方法是，利用病毒载体多次感染带有要敲除的目的基因的sgRNA的CRISPR/Cas9载体和CAR载体，并且因多次病毒感染细胞活性降低，而多基因敲除导致载体过大利用病毒或者转染试剂转染效率极低，只能用电转才能实现转染载体的目的，但是申请人发现经过电转的细胞状态较差需要经过一定的恢复期而T淋巴细胞不能在体外长期增值存活(即便使用活化试剂刺激在体外生存时间一般也是最多在2-3周)电转之后再进行CAR的感染效率低并且细胞活性差；
因此，申请人优化感染条件在T细胞活化24小时后先进行CAR感染制备CAR-T细胞，24小时培养后再进行电转，转染带有要敲除的目的基因的sgRNA的CRISPR/Cas9载体，构建通用型CAR-T细胞。该方法敲除效率高，获得的通用型CAR-T细胞异体回输安全性高，感染CAR阳性率稳定具有很好的杀伤活性和特异性。
为了达到治疗的目的，过继转输或者同种异体回输所用的CAR-T或T淋巴细胞(T细胞)均为外周血分离的T细胞制备，但是外周血分离的T细胞是已经成熟的T细胞在体外保持增殖和有效功能时间有限，所以经过基因修饰的通用型细胞需要在体外快速扩增到病人治疗所需数目后回输给病人，发明人通过大量实验摸索，结合病毒感染和电转的方式获得了最优的构建通用型CAR-T或T细胞的方法：1)利用病毒感染的方式进行多基因表达；2)利用CRISPR/Cas9技术进行多基因干扰。但是目前普遍采用的病毒感染表达基因对于单基因感染技术较为成熟，多基因感染往往感染效率不高，并且同时涉及到多基因敲除， CRISPR/Cas9技术虽然可以实现目标基因的稳定敲除，但是多基因干扰采用病毒感染的方式敲除效率低，利用电转的方式对细胞活性影响大并且最佳的电转条件需要大量摸索实验。
步骤2)中先利用含有编码针对特异性肿瘤靶点的CAR和共表达HLA-E或HLA-F的病毒感染T淋巴细胞获得CAR-T细胞，再进一步进行多基因敲除。
进一步，步骤2)中所述CAR识别的靶标分子包括CD19，PSCA，CD123，CD20，CEA(癌胚抗原)，FAP，CD133，EGFR，EGFRVIII，BCMA，PSMA，Her2，CA125，EphA2，C-met，L1CAM，VEGFR，CS1，ROR1，EC，NY-ESO-1，MUC1，MUC16，mesothelin，LewisY，GPC3，GD2，EPG，DLL3，CD99，5T4，CD22，CD30，CD33，CD138，CD171中的一种。
进一步，步骤3)中多基因敲除的方法为CRISPR/Cas9系统的方法。
进一步，所述CRISPR/Cas9系统由gRNA和Cas9核酸酶或内切酶组成。
进一步，通过CRISPR/Cas9系统进行多基因敲除优选电转转染方式，将要敲除的多基因的gRNA构建在同一载体，利用电转的方式转染进行基因敲除。
进一步，靶向B2M，HLA-A，CIITA和TRAC基因的gRNA序列如SEQ ID NO:5-19所示。
进一步，靶向TRAC、B2M和CIITA或者HLA-A，CIITA和TRAC基因的gRNA序列构建在载体PX330A。
表达的HLA-E基因序列为SEQ ID NO:33，蛋白序列为SEQ ID NO:34；表达的HLA-F基因序列为SEQ ID NO:32。
如何选择敲除方式达到转染效率高细胞存活率高这是一个难题。申请人通过多次试验发现多次转染以达到多基因敲除的方法会严重影响细胞活性，申请人设计靶向B2M，CIITA和TRAC或者HLA-A，CIITA和TRAC三种基因的gRNA构建在同一载体，利用电转的方式转染进行基因敲除。在未有更有效的提示技术下，申请人通过大量实验摸索电转条件的优化：在进行电转时所用电转Buffer、电压、脉冲大小以及电击次数的不同都会影响着细胞存活率和电转效率。
作为一种优选，293T细胞进行GFP的转染，电转条件为PBMC或T细胞活化48小时后电压1000V，脉宽为35ms电击次数两次进行电转，细胞总量2×10 5，电转后用新鲜含IL-2的培养基培养。
本发明的目的之三在于提供一种上述制备方法中所得的CRISPR/Cas9基因敲除的表达载体，所述表达载体包含如SEQ ID NO:26-31所示的基因序列。
本发明的目的还在于提供一种所述通用型CAR-T细胞在异体治疗的药物中的应用。
在所述的方法下得到所述的通用型细胞，可以获得靶向特异性肿瘤并且细胞表面TCR和 MHC一、二类分子同时失活的通用型CAR-T细胞，或者细胞表面TCR和MHC一、二类分子同时失活的通用型T淋巴细胞，应用于肿瘤治疗可以提高CAR-T细胞清除肿瘤的安全性和有效性。
本发明的目的还在于提供一种所述通用型CAR-T细胞在用于制备针对恶性肿瘤或感染性疾病的药物中的应用。
进一步，所述恶性肿瘤或感染性疾病的细胞或组织能够表达包括CD19，PSCA，CD123，CD20，CEA(癌胚抗原)，FAP，CD133，EGFR，EGFRVIII，BCMA，PSMA，Her2，CA125，EphA2，C-met，L1CAM，VEGFR，CS1，ROR1，EC，NY-ESO-1，MUC1，MUC16，mesothelin，LewisY，GPC3，GD2，EPG，DLL3，CD99，5T4，CD22，CD30，CD33，CD138，CD171中的一种。所述通用型CAR-T包含UCAR-19和UCAR-PSCA。
进一步，所述恶性肿瘤或感染性疾病包括：血液系统肿瘤、实体瘤、异体移植所引起的免疫排斥反应以及自身免疫性疾病如过敏反应或者系统性红斑狼疮。
本发明的目的还在于提供一种用于同种异体治疗药物中的通用型T细胞，所述通用型T细胞中同时抑制了T细胞抗原受体(TCR)和主要组织相容性复合体(MHC Ⅰ,MHC Ⅱ)在T细胞中的功能并且表达识别恶性肿瘤或者感染细胞的嵌合抗原受体。
进一步，编码所述TCR的基因包括TRAC和/或TRBC；编码所述主要组织相容性复合体的基因包括HLA，B2MH和CIITA。
进一步，所述通用型CAR-T细胞中同时敲除了TRAC、HLA-A和CIITA基因或同时敲除TRAC，B2M和CIITA基因还表达HLA-E或HLA-F。
该通用型T细胞可以用于同种异体治疗。
本发明的目的还在于提供一种上述的通用型T细胞在用于制备针对恶性肿瘤或感染性疾病的药物中的应用。
进一步，所述恶性肿瘤或感染性疾病包括：血液系统肿瘤、实体瘤、异体移植所引起的免疫排斥反应以及自身免疫性疾病如过敏反应或者系统性红斑狼疮。
申请人在与HVGR和GVHR相关的基因中，通过基因编辑技术设计不同的研究方案对健康供着的T淋巴细胞进行基因编辑改造，最终获得排斥反应低在病人体内长期存活并且安全性高的同种异体通用T细胞，通用型T细胞的获得使得同种异体细胞治疗获得更广泛的应用空间促进细胞治疗的标准化生产；申请人进一步利用通用型的T淋巴细胞成功进行CAR-T改造获得同种异体通用型CAR-T细胞，让CAR-T治疗的安全性和有效性进一步提升。
总的来说，本发明所述的通用型T细胞和通用型CAR-T细胞可以通过同种异体回输的方式应用于恶性肿瘤或感染性疾病的治疗，并且排斥反应低在病人体内可以长期存活安全性 高，促进了大部分病人尤其是肿瘤病人经过化疗，放射性治疗导致自体T细胞较少甚至由于病情T胞功能衰减或缺失进而影响细胞治疗的治疗效果和安全性问题的解决，为CAR-T细胞治疗的推广和发展提供帮助。
本发明的有益效果在于：
1)本发明提供的制备通用型CAR-T细胞的方法能够获得CAR表达率高TCR和MHC同时失活的通用型CAR-T细胞，所述通用型CAR-T细胞应用于恶性肿瘤或感染性疾病的治疗排斥反应低并且在病人体内有效性安全性高。
2)本发明提供的通用型CAR-T细胞应用于异体的过继细胞治疗不受病人自身病情或治疗方式影响通用型CAR-T细胞的制备，可以随时制备所需通用型CAR-T，在最佳的时机给予病人进行治疗，确保治疗的有效性。
3)本发明提供的通用型T细胞能够应用于同种异体回输的免疫治疗的T细胞，所述通用型T细胞便于程序化制备异体回输后排斥反应小。
附图说明
图1为中国人群HLA-A分型频率检测结果。
图2为利用CRISPR/Cas9系统敲除TCR和MHC一、二类分子相关基因载体设计。
图3为设计的gRNA靶向位点检测。
图4为CRISPR/Cas9系统敲除后CAR-T细胞表面B2M，CIITA和TRAC表达检测。
图5为CAR表达检测。
图6为CRISPR/Cas9系统敲除后CAR-T细胞TCR和MHC一、二类分子同时失活效率以及分选后获得三阴性CAR-T表型检测。
图7为CRISPR/Cas9脱靶检测。
图8为三阴性CAR-T细胞对异体NK细胞反应性检测。
图9为通用型CAR-T HLA-E表达检测。
图10为通用型CAR-T杀伤肿瘤细胞能力检测。
图11为通用型CAR-T细胞异体NK反应性检测。
具体实施方式
以下将参照附图，对本发明的优选实施例进行详细描述。优选实施例中未注明具体条件的实验方法，通常按照常规条件，例如分子克隆实验指南(第三版，J.萨姆布鲁克等著)中所 述的条件，或按照制造厂商所建议的条件。所举实施例是为了更好地对本发明的内容进行说明，但并不是本发明的内容仅限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整，仍属于本发明的保护范围。
实施例1、中国人群HLA-A分型频率检测
1.SBT(Sequenced Based Typing)检测：
该方法属于HLA高分辨率分型技术。
EDTA抗凝管(血常规管)抽取受检者静脉血4ml，4°冷藏暂存，-20°长期冻存。然后送华大进行HLA分型检测。
2.生物信息学方法：参考Boegel,Sebastian,Valesca et al.(2013)
利用seq2HLA软件，直接从RNA-Seq测序的原始数据(fastq格式)中计算该样本的HLA高分辨率分型(MHC-I和MHC-II，4位)及相应的表达值和p值。
实验结果如图1所示获得8种中国人频率最高的8种HLA-A分型，分别为A*02，A*11A*24，A*30，A*33，A*03，A*01和A*26，分别针对这8种亚型设计gRNA分子，gRNA序列如SEQ ID NO:8-12。
实施例2、敲除TCR和MHC一、二类分子相关基因载体构建
1.sgRNA分子设计：
确认与TCR和MHC一、二类分子相关的基因：B2M、CIITA以及TRAC基因与TCR和MHC一、二类分子相关的基因功能密切相关确认敲除B2M、CIITA以及TRAC基因。在NCBI分别获得B2M、CIITA以及TRAC基因序列号获得CDS序列，B2M序列号AH002619.2，CIITA在NCBI基因ID：4261，TRAC在NCBI基因ID：28755，如SEQ ID NO：1-3所示。分别针对CDS区的确定靶向的SEQ ID NO：1-3区域设计gRNA，gRNA序列如表1。
表1：gRNA序列
Figure PCTCN2018103714-appb-000001
Figure PCTCN2018103714-appb-000002
2.构建CRISPR/Cas9基因敲除的表达载体
设计如下引物，并将其由南京金斯瑞生物科技公司合成，具体引物如下：
引物sg-B2M-3-up：5’-CACCGGCCGAGATGTCTCGCTCCG-3’；SEQ ID NO:20；
引物sg-B2M-3-down：5’-AAACCGGAGCGAGACATCTCGGCC-3’；SEQ ID NO:21；
引物sg-CIITA2-up：5’-CACCGATATTGGCATAAGCCTCCC-3’；SEQ ID NO:22；
引物sg-CIITA2-down：5’-AAACGGGAGGCTTATGCCAATATC-3’；SEQ ID NO:23；
引物sg-TRAC-1-up：5’-CACCAGAGTCTCTCAGCTGGTACA-3’；SEQ ID NO:24；
引物sg-TRAC-1-down：5’-AAACTGTACCAGCTGAGAGACTCT-3’；SEQ ID NO:25。
然后以上述所示序列为引物，将两条上下游引物退火成三条双链的gRNA序列，反应体系按Annealing Buffer for DNA Oligos(5X)(购自碧云天公司)说明书加样。
用限制性内切酶BbsI(购自NEB公司)分别酶切PX330A-1X3,PX330S-2，PX330S-3(均购自Addgene Plasmid)，酶切反应按说明书进行。酶切产物经琼脂糖凝胶电泳分离后进行DNA片段回收，然后分别将对应退火后的引物片段和纯化后载体片段通过T4连接酶(购自Promega公司)进行连接，获得只带一个gRNA的表达载体。大肠杆菌表达后用质粒抽提试剂盒(Invitrogen公司)抽提质粒，具体方法见说明书。
3.参照Golden gate cloning技术，用限制性内切酶BsaI(购自NEB公司)分别酶切上述构建的只带一个gRNA的表达载体。利用快速连接酶Quick Ligation TMKit(购自NEB)进行连接反应，按照说明书加样。用X-gal和IPTG(均购自生工生物工程股份有限公司)进行蓝白斑筛选得到六个包含三个不同gRNA表达框的组合载体：
PX330A-1X3-B2M-3-CIITA2-TRAC-1，PX330-A-1X3-B2M-3-TRAC-1-CIITA2，
PX330A-1X3-CIITA2-B2M-3-TRAC-1，PX330A-1X3-CIITA2-TRAC-1-B2M-3，
PX330A-1X3-TRAC-1-B2M-3-CIITA2，PX330A-1X3-TRAC-1-CIITA2-B2M-3；大肠杆菌表达后用质粒抽提试剂盒(Invitrogen公司)抽提质粒，具体方法见说明书。载体图谱及PCR结果如图2所示。
4.检测构建的载体是否正确
将步骤3中获得的载体分别转染感受态利用小抽质粒PCR验证序列正确性，具体步骤 见分子克隆实验指南(第三版，J.萨姆布鲁克等著)，实验结果见图2可见有三条带并且进行测序结果正确。
实施例3、通用型CAR-T细胞构建以及表型检测
1.电转条件确认
在不同的条件下利用293T细胞进行GFP的转染进行电转条件的优化确认较好的电转条件后进一步进行PBMC转染，实验结果见表2，表3，优选的电转条件为PBMC或T细胞活化48小时后电压1000V，脉宽为35ms电击次数两次进行电转，细胞总量2×10 5，电转后用新鲜含IL-2的培养基培养。
表2：不同电转条件下转染293T细胞转染效率以及细胞活性比较
序号 效率％ 存活率％ 电压/V 脉宽/ms 电击次数
1 0 100 0 1 1
2 83.1 35.7 1400 20 1
3 89.8 27.9 1500 20 1
4 91.8 21.2 1600 20 1
5 85.4 11.8 1700 20 1
6 60.6 23.2 1100 30 1
7 80.3 27.8 1200 30 1
8 83.9 23.9 1300 30 1
9 90 14.7 1400 30 1
10 48.2 34.8 1000 40 1
11 73.7 22.4 1100 40 1
12 87.4 15.6 1200 40 1
13 65.3 28.6 1100 20 2
14 83.6 22.8 1200 20 2
15 82.6 20.8 1300 20 2
16 94 12.8 1400 20 2
17 20.3 42.1 850 30 2
18 47.3 42.8 950 30 2
19 77.3 32.7 1050 30 2
20 90.6 23.6 1000 30 2
21 78.2 26.3 1300 10 3
22 87.8 23.8 1400 10 3
23 92.3 14.6 1500 10 3
24 89.7 8.9 1600 10 3
表3：PBMC和T淋巴细胞电转条件优化结果
Figure PCTCN2018103714-appb-000003
Figure PCTCN2018103714-appb-000004
2.包含CAR载体的慢病毒感染T淋巴细胞进行CAR-T细胞制备
1)人外周血单核细胞的分离
用加有抗凝剂的采血管采集外周血约60ml，分装于50ml离心管各30ml，加入7.5ml羟乙基淀粉稀释；室温(18～25℃)自然沉降约30min，收集上层血浆，离心15min；然后用生理盐水重悬沉淀，按体积比为1:1加到淋巴细胞分离液上，梯度离心，离心20min；离心后，取第二层白色淋巴细胞层，并用生理盐水洗涤2次，生理盐水重悬细胞，加入含有10％FBS的RPMI 1640完全培养基培养，得人外周血单核细胞。
2)慢病毒载体感染T淋巴细胞
用含10％FBS的RPMI 1640完全培养基培养新制备的单个核细胞PBMC，抗CD3单克隆抗体活化后进行慢病毒感染；分别加入含有靶向CD19或PSCA的CAR基因和/或编码HLA-E表达基因的慢病毒载体，未感染的外周血淋巴细胞(PBMC)作为空白对照；24小时后收集细胞计数制备通用型CAR-T细胞，24孔板铺板每孔2×10 5细胞，电转条件电压1000V，电脉冲35ms，电击两次完成电转。具体步骤采用
Figure PCTCN2018103714-appb-000005
Transfection System详见
Figure PCTCN2018103714-appb-000006
Transfection System操作说明书。
3.CAR-T细胞表型检测：
以靶向CD19和PSCA为例，利用优选的电转条件：电压1000V，脉宽为35ms电击次数两次进行电转，将含有靶向B2M、CIITA以及TRAC基因的gRNA的CRISPR/Cas9表达载体电转进(2)中靶向CD19或PSCA的CAR-T细胞获得通用型pUCAR-19和pUCAR-PSCA细胞，对培养至10天的已感染包含CAR病毒并通过电转利用CRISPR/Cas9系统敲除TCR和MHC一、二类分子相关基因的T细胞，300g/min，离心5min，弃尽上清以收集细胞；将细胞调整密度为1×10 6个/ml；将收集的细胞分别分装利用流式细胞术检测Protein-L阳性率以检测通用型CAR-T细胞CAR表达阳性率，PBS清洗2次洗去多余未结合Protein-L抗体后标记HLA-DR，B2M，CIITA，HLA-E以及CD3抗体检测TCR和MHC一、二类分子的表达。检测结果如图4所示，经过修饰的T淋巴细胞存在有30％左右比例的表面B2M，CIITA 以及TRAC分子均不表达，如图5所示有43.6％的CAR表达，发明所采用的方法同时敲除3个基因效率高并且不影响CAR表达。
分选B2M，CIITA以及TRAC分子三阴性并且CAR阳性表达的pUCAR-CD19或pUCAR-PSCA细胞，如图6所示为获得的pUCAR-CD19细胞。
分选B2M，CIITA以及TRAC分子三阴性并且CAR和HLA-E阳性表达的pUCAR-CD19细胞，三阴性并共表达CAR和HLA-E的细胞为改进的通用型CAR-T细胞用pUCAR-019 HLA-E+表示，如图7所示为HLA-E的表达。
4.gRNA靶向性检测
进一步将步骤3中获得的通用型CAR-T细胞细胞用QIAamp DNA Blood Mini Kit购于Qiagen公司(货号511004)基因组抽提试剂盒抽提基因组。按试剂盒说明书操作，RT-PCR并测序方式验证设计的gRNA靶向的区域，结果见图3，字体加粗或者缺码的区域即为研发人员所设计gRNA所靶向区域，结果显示研究人员所设计的gRNA可以对应的靶向B2M、CIITA以及TRAC基因的目标区域致使B2M、CIITA以及TRAC基因失活，其中—为缺失；+为插入；M为突变。
实施例4、CRISPR/cas9敲除B2M、CIITA以及TRAC脱靶检测
1.将gRNA在设计网站比对，找出预测人全基因组上的脱靶位点，具体基因位点如表4。(http://crispr.mit.edu)
2.在NCBI网站找出脱靶位点所在基因位置。(http://blast.ncbi.nlm.nih.gov)
3.设计相应基因位点PCR引物，进行PCR，连接T载体并测序。
4.根据测序结果分析是否发生脱靶。
表4：预测人全基因组上的脱靶位点
Figure PCTCN2018103714-appb-000007
Figure PCTCN2018103714-appb-000008
脱靶检测结果如图8所示，off-target-analysis结果均为0/6或0/5，表明分别针对B2M、CIITA以及TRAC设计的gRNA经过多次重复试验均未出现脱靶现象，申请所提及的gRNA序列特异性高稳定性好，在体内更安全。
实施例5、通用型CAR-T细胞抗肿瘤效果验证
1.以PSCA高表达Hela细胞和PSCA不表达的T24细胞作为靶细胞检测构建的通用型CAR-T细胞针对PSCA靶点的有效性和特异性，pUCAR-PSCA作为效应细胞按照1:1效靶比铺板效应细胞，验证pUCAR-PSCA的杀伤效果。杀伤实验通过ACEA xCELLigence RTCA MP仪器完成，实验步骤依据仪器说明书进行；第一天将靶细胞(表达PSCA的肿瘤细胞)以2-5*10^4每孔铺板于仪器配备的96孔板中，附着于孔底的肿瘤细胞以电阻指数为数据每15分钟记录一次，24小时后依据预先设计的效靶比每孔铺入相应的CAR-T细胞，CAR-T细胞铺入后每隔15分钟记录一次电阻指数，通过电阻指数判断贴壁的靶细胞的增殖或者死亡情况，见图9，细胞基数为1当Y轴数字大于1是证明肿瘤细胞增殖了，当Y轴数字小于1时表明肿瘤细胞被杀伤，X轴T-B-C、B-T-C、T-C-B分别表示在PSCA-CAR-T细胞分别转染PX330A-1X3-TRAC-1-B2M-3-CIITA2、PX330-A-1X3-B2M-3-TRAC-1-CIITA2、以及PX330A-1X3-TRAC-1-CIITA2-B2M-3载体的通用型pUCAR-PSCA细胞，PSCA组为未转染敲除系统只感染了CAR的自体CAR-T细胞，Medium为正常培养的肿瘤细胞，PBMC组为未感染CAR也未转染敲除载体的T淋巴细胞，杀伤结果如图9所示：在24小时时通用型CAR-T展现了显著的杀伤作用，尤其是B-T-C(PX330-A-1X3-B2M-3-TRAC-1-CIITA2)组展现了高的杀伤活性和特异性。
2.以稳定表达萤火虫荧光素酶的CD19阳性Raji细胞(简称为Raji-luc)作为靶细胞，UCAR-19作为效应细胞按照1:1效靶比铺效应细胞，验证pUCAR-019的杀伤结果。其中1表示CAR-019细胞感染PX330-A-1X3-B2M-3-TRAC-1-CIITA2的pUCAR-019-1细胞，2表示CAR-019细胞感染PX330A-1X3-TRAC-1-CIITA2-B2M-3的pUCAR-019-2细胞；使用
Figure PCTCN2018103714-appb-000009
Luciferase Assay System(Promega Cat.#E2520)试剂盒提供的标准方法检测杀伤效果，杀伤率用下列公式计算：
细胞杀伤率＝(1-效应细胞靶细胞共培养孔荧光强度/单独靶细胞孔荧光强度)×100％
杀伤结果如图10所示，1即pUCAR-019-1组的细胞展现了高的杀伤活性。
实施例6、通用型CAR-T细胞对异体NK细胞反应性检测
1.根据细胞的大小和生长速度将适量同种异体T淋巴细胞或者pUCAR-19细胞接种到96孔细胞培养板中，使待检测时细胞密度不超过80-90％满。
2.吸去培养液，用PBS液洗涤一次。换新鲜培养液。将NK-92细胞依据效靶比1:1比例分别与PBMC或pUCAR-19混合继续按常规培养。共培养24小时前1小时从细胞培养箱里取出细胞培养板，在“样品最大酶活性对照孔”中加入试剂盒提供的LDH释放试剂，加入量为原有培养液体积的10％。加入LDH释放试剂后，反复吹打数次混匀，然后继续在细胞培养箱中孵育。
3.共培养24小时后，将细胞培养板用多孔板离心机400g离心5min。分别取各孔的上清液120μl，加入到一新的96孔板相应孔中，随即进行样品测定。
其中pUCAR-019表示敲除B2M、CIITA和TRAC并靶向CD19的三阴性CAR细胞，pUCAR-019 HLA-E+表示敲除B2M、CIITA和TRAC的CAR-T细胞共表达HLA-E。
具体测定方式参考：碧云天的乳酸脱氢酶细胞毒性检测试剂盒(LDH Cytotoxicity Assay Kit)，产品编号C0017。
实验结果如图11所示：LDH细胞毒性检测中B2M被敲除后NK细胞对其有明显杀伤，敲除B2M(HLA-I类分子通用亚基)后，保守型HLA-I类分子无法表达，即无法保护其免受NK细胞介导的细胞毒性。未敲除组(PBMC)与表达HLA-E的通用型CAR-T细胞组杀伤基本一致，HLA-E的表达可以弥补保守型HLA-I类分子的功能，抑制NK细胞活化降低对通用型CAR-T细胞的杀伤。表5中列出了细胞毒性检测各细胞的裂解率。
表5：NK细胞介导的细胞毒性裂解异体免疫细胞裂解率
细胞类型 细胞裂解率24h(％) 细胞裂解率48h(％) 重复次数
PBMC 38.95873525 63.3938706 2
pUCAR-019 HLA-E+ 39.08128334 57.8858351 2
pUCAR-019 51.00717304 81.0123622 2
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。

Claims (20)

  1. 通用型CAR-T细胞，其特征在于，所述通用型CAR-T细胞中经过多基因敲除同时抑制了T细胞抗原受体(TCR)和主要组织相容性复合体(MHCⅠ,MHCⅡ)在T细胞中的功能；编码所述TCR的基因包括TRAC和/或TRBC；编码所述主要组织相容性复合体的基因包括HLA-A，B2MH和CIITA。
  2. 根据权利要求1所述的通用型CAR-T细胞，其特征在于，所述通用型CAR-T细胞中敲除包括TRAC，B2M和CIITA基因的同时还表达HLA-E或HLA-F。
  3. 根据权利要求1所述的通用型CAR-T细胞，其特征在于，所述通用型CAR-T细胞中敲除包括TRAC、HLA-A或B2M、CIITA基因。
  4. 权利要求1-3任一项所述通用型CAR-T细胞的制备方法，其特征在于，包括以下步骤：
    1)得到活化的T细胞；
    2)用编码靶向不同肿瘤相关分子的CAR或CAR与HLA-E或HLA-F共表达的慢病毒载体来转染步骤1)的T细胞，获得靶向不同肿瘤相关分子的CAR-T细胞；
    3)将步骤2)获得的CAR-T细胞通过TALEN或锌指方法或CRISPR/Cas9系统的方法进行包括TRAC、HLA-A和CIITA或者TRAC、B2M和CIITA的多基因敲除获得通用型CAR-T细胞。
  5. 根据权利要求4所述的制备方法，其特征在于，步骤2)中所述CAR识别的靶标分子包括CD19，PSCA，CD123，CD20，CEA(癌胚抗原)，FAP，CD133，EGFR，EGFRVIII，BCMA，PSMA，Her2，CA125，EphA2，C-met，L1CAM，VEGFR，CS1，ROR1，EC，NY-ESO-1，MUC1，MUC16，mesothelin，LewisY，GPC3，GD2，EPG，DLL3，CD99，5T4，CD22，CD30，CD33，CD138，CD171中的一种。
  6. 根据权利要求4所述的制备方法，其特征在于，步骤3)中多基因敲除的方法为CRISPR/Cas9系统的方法。
  7. 根据权利要求6所述的制备方法，其特征在于，所述CRISPR/Cas9系统由gRNA和Cas9核酸酶或内切酶组成。
  8. 根据权利要求7所述的制备方法，其特征在于，通过CRISPR/Cas9系统进行多基因敲除优选电转转染方式，将要敲除的多基因的gRNA构建在同一载体，利用电转的方式转染进行基因敲除。
  9. 根据权利要求8所述的制备方法，其特征在于，靶向B2M，HLA-A，CIITA和TRAC基因的gRNA序列如SEQ ID NO:5-19所示。
  10. 根据权利要求9所述的制备方法，其特征在于，靶向TRAC、B2M和CIITA或者HLA-A， CIITA和TRAC基因的gRNA序列构建在载体PX330A。
  11. 权利要求4的制备方法中所得的CRISPR/Cas9基因敲除的表达载体，其特征在于，所述表达载体包含如SEQ ID NO:26-31所示基因序列。
  12. 所述通用型CAR-T细胞在异体治疗的药物中的应用。
  13. 所述通用型CAR-T细胞在用于制备针对恶性肿瘤或感染性疾病的药物中的应用。
  14. 根据权利要求13所述的应用，其特征在于，所述恶性肿瘤或感染性疾病的细胞或组织能够表达包括CD19，PSCA，CD123，CD20，CEA(癌胚抗原)，FAP，CD133，EGFR，EGFRVIII，BCMA，PSMA，Her2，CA125，EphA2，C-met，L1CAM，VEGFR，CS1，ROR1，EC，NY-ESO-1，MUC1，MUC16，mesothelin，LewisY，GPC3，GD2，EPG，DLL3，CD99，5T4，CD22，CD30，CD33，CD138，CD171中的一种。
  15. 根据权利要求13所述的应用，其特征在于，所述恶性肿瘤或感染性疾病包括：血液系统肿瘤、实体瘤、异体移植所引起的免疫排斥反应以及自身免疫性疾病如过敏反应或者系统性红斑狼疮。
  16. 用于同种异体治疗药物中的通用型T细胞，其特征在于，所述通用型T细胞中同时抑制了T细胞抗原受体(TCR)和主要组织相容性复合体(MHCⅠ,MHCⅡ)在T细胞中的功能。
  17. 根据权利要求16所述的通用型T细胞，其特征在于，编码所述TCR的基因包括TRAC和/或TRBC；编码所述主要组织相容性复合体的基因包括HLA，B2MH和CIITA。
  18. 根据权利要求16所述的通用型T细胞，其特征在于，所述通用型T细胞中同时敲除了TRAC、HLA-A和CIITA基因或TRAC，B2M和CIITA基因的同时还表达HLA-E或HLA-F。
  19. 权利要求16-18任一项所述的通用型T细胞在用于制备针对恶性肿瘤或感染性疾病的药物中的应用。
  20. 根据权利要求19所述的应用，其特征在于，所述恶性肿瘤或感染性疾病包括：血液系统肿瘤、实体瘤、异体移植所引起的免疫排斥反应以及自身免疫性疾病如过敏反应或者系统性红斑狼疮。
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