WO2019214290A1 - 一种生产嵌合抗原受体修饰的γδT细胞的方法 - Google Patents

一种生产嵌合抗原受体修饰的γδT细胞的方法 Download PDF

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WO2019214290A1
WO2019214290A1 PCT/CN2019/072285 CN2019072285W WO2019214290A1 WO 2019214290 A1 WO2019214290 A1 WO 2019214290A1 CN 2019072285 W CN2019072285 W CN 2019072285W WO 2019214290 A1 WO2019214290 A1 WO 2019214290A1
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cells
γδt
cell
fpps
tumor
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PCT/CN2019/072285
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French (fr)
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李建强
王庆龙
王琳
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河北森朗生物科技有限公司
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Priority to JP2021512982A priority Critical patent/JP7193886B2/ja
Priority to US17/054,043 priority patent/US20210154231A1/en
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Definitions

  • the present invention relates to the field of biotechnology, and in particular to a method for producing chimeric antigen receptor-modified ⁇ T cells.
  • Adoptive cellular immunotherapy is the in vitro culture, activation, and genetic modification of autologous or allogeneic immune cells to patients for antitumor activity.
  • Chimeric Antigen Receptor (CAR) modified T cell therapy technology (CAR-T technology) is to modify immune effector cells by genetic engineering technology, so that modified cells can specifically recognize and kill specific antigens. Target cells, thereby achieving the purpose of specifically clearing tumor cells.
  • CAR-T cells that specifically target the B19 lymphocyte surface marker CD19 molecule are most effective in the treatment of B lymphocyte malignancies, and can achieve complete remission in 90% of patients with relapsed and refractory B-lineage acute lymphoblastic leukemia. .
  • CAR-T cells directed against the CD19 antigen currently treat B lymphocytic tumors, they have extremely high short-term therapeutic effects. However, a significant proportion of patients will relapse after treatment has achieved complete remission. An important reason for recurrence is that the input CAR-T cells are continuously reduced in the patient's body due to the lack of effective antigenic stimulation, and thus cannot maintain long-term antitumor activity.
  • the existing CAR-T technology is designed based on the natural activity of ⁇ T cells, and the generally modified effector cells are ⁇ T cells.
  • the import of ⁇ T cells into the body can cause severe graft-versus-host response. Therefore, the current CAR-T treatment technology is mostly based on autologous cell therapy, which greatly limits the flexibility of clinical application and greatly increases the cost of treatment. While ⁇ T cells do not cause severe graft-versus-host reactions, they are a better choice for developing generic CAR-T products.
  • ⁇ T cells that recognize a particular MHC-antigen peptide complex
  • ⁇ T cells are a lower class of T lymphocyte subsets
  • most ⁇ TCRs recognize non-peptide compounds and do not require MHC presentation.
  • the diversity of the variable regions of ⁇ TCR is very limited, therefore, although the total number of ⁇ T cells is much larger than that of ⁇ T cells, the specific ability to recognize a specific compound or antigen is compared. In the case of the number of T cells, ⁇ T cells are higher than ⁇ T cells.
  • the ⁇ T cells of humans and other primates mainly include two subclasses of V ⁇ 1 and V ⁇ 9 ⁇ 2, which represent the types of surface expression of TCR, respectively.
  • V ⁇ 1 is distributed in tissues, and peripheral ⁇ T cells are mainly V ⁇ 9 ⁇ 2 subclasses.
  • the antigen recognition patterns of the two types of cells are quite different.
  • V ⁇ 1 TCR recognition antigen The type of V ⁇ 1 TCR recognition antigen is currently controversial; V ⁇ 9 ⁇ 2 TCR recognizes self or exogenous small molecule phosphorylated antigens such as HMBPP (bacterial source), IPP (human source) and BrHPP (synthetic) (Chen, ZW and NLLetvin, 2003.5(6): p.491-8; Eberl, M., et al., 2003.544(1-3): p. 4-10; Jardin, D., D. Wesch , and W. He, 2007. 67(1): p. 5-8; Sireci, G., et al., 2001. 31(5): p. 1628-35).
  • HMBPP bacterial source
  • IPP human source
  • BrHPP synthetic
  • Amino-Bisphosphonate compound drugs Zoledronate (Zol, Zetai) and Pamidornate can inhibit FPP synthase (FPPS), thereby inhibiting the metabolic pathway of HMG-CoA, leading to the in vivo or intracellular accumulation of downstream product IPP, and thus can be in vivo or Activation of V ⁇ 9 ⁇ 2T cells is efficiently stimulated in vitro (Kunzmann, V. and M. Wilhelm, 2005. 46(5): p. 671-80).
  • FPPS FPP synthase
  • ⁇ T cells Although the natural activity of ⁇ T cells makes it an excellent carrier for CAR-T cell therapy, due to the low cell content, large-scale large-scale amplification technology in vitro, and low transfection efficiency, there are no CAR-modified ⁇ T cells. Reports of successful clinical application.
  • Traditional in vitro amplification of ⁇ T cells including solid-phase anti-pan TCR ⁇ antibody amplification, or selective amplification of V ⁇ 9 ⁇ 2T cells by IPP/HMBPP/ZOL, are amplified from peripheral mononuclear cells (PBMC) due to The proportion of ⁇ T cells is low, and gene transfection is interfered by other unrelated cells, resulting in low transfection rate.
  • PBMC peripheral mononuclear cells
  • the final product amplified from PBMC has low purity and is easy to be mixed with ⁇ T cells, which is not conducive to the development of general-purpose CAR-T products.
  • Sorting and purifying ⁇ T cells can solve the above problems, but the efficiency of activation and amplification is greatly reduced without the assistance of other cells.
  • the technical problem to be solved by the present invention is how to improve the amplification efficiency of ⁇ T cells to realize large-scale production of CAR- ⁇ T cells for clinical tumor immunotherapy.
  • the present invention first provides a method for amplifying ⁇ T cells.
  • the method for amplifying ⁇ T cells comprises the step of adding a tumor cell having a reduced expression amount and/or activity of FPPS in a ⁇ T cell culture system.
  • the ratio of the number of tumor cells in which the ⁇ T cells and the FPPS expression amount and/or activity are decreased is (1-10): 1;
  • the ratio of the number of tumor cells in which the ⁇ T cells and the FPPS expression amount and/or activity are decreased is 3:1.
  • the above method further comprises the steps of: adding IL-2 to the culture system every 2-4 days when the cell concentration in the culture system reaches (1.5-2) ⁇ 10 6 /ml; when adding IL-2
  • the standard is such that the final concentration of the IL-2 in the culture system is (100-1000) IU/mL;
  • IL-2 is added to the culture system every 3 days; the standard when adding IL-2 is to make the IL
  • the final concentration of -2 in the culture system was (100-300) IU/mL.
  • IL-2 is added to the culture system every 3 days; the standard when adding IL-2 is The final concentration of IL-2 in the culture system was 200 IU/mL.
  • the present invention further provides a method of producing CAR- ⁇ T cells.
  • the method for producing CAR- ⁇ T cells comprises the steps of: co-culturing a vector expressing a chimeric antigen receptor with ⁇ T cells to obtain CAR- ⁇ T cells; wherein the culture system contains FPPS expression and/or activity decreased. Tumor cells.
  • a vector expressing a chimeric antigen receptor is added to the culture system, and cultured to obtain the CAR- ⁇ T cells.
  • the ratio of the number of tumor cells in which the ⁇ T cells and the FPPS expression amount and/or activity are decreased is (1-10): 1;
  • the ratio of the number of tumor cells in which the ⁇ T cells and the FPPS expression amount and/or activity are decreased is 3:1.
  • the culture conditions are as follows: culture at 37 ° C, 5% CO 2 for 2 days.
  • the ⁇ T cell culture system consists of TexMACS medium and human recombinant IL-2, and the concentration of the human recombinant IL-2 in the ⁇ T cell culture system is 200 U/ml.
  • the step 2) further comprises the step of adding IL-2 to the culture system every 2-4 days when the cell concentration in the culture system reaches (1.5-2) ⁇ 10 6 /ml.
  • the standard when adding IL-2 is such that the final concentration of the IL-2 in the culture system is (100-1000) IU/mL;
  • IL-2 is added to the culture system every 2 days; the standard when adding IL-2 is to make the IL
  • the final concentration of -2 in the culture system was (100-300) IU/mL.
  • IL-2 is added to the culture system every 2 days; the standard when adding IL-2 is to make the The final concentration of IL-2 in the culture system was 200 IU/mL.
  • the culture conditions are as follows: incubation at 37 ° C, 5% CO 2 for 10-14 days.
  • the vector expressing the chimeric antigen receptor is a lentiviral vector expressing a chimeric antigen receptor
  • the lentiviral vector expressing the chimeric antigen receptor further comprises a step of packaging before being added to the culture system; the method of packaging may be carried out according to the following steps: 2-1) adding 4.5 ⁇ 10 per cell culture dish 6 293FT cells and 9mL DMEM complete medium, mixed and mixed, cultured in a cell culture incubator; 2-2) On the second day of culture, each culture dish was added with the following reagent: 500 ⁇ L Buffer, 6 ⁇ g of lentiviral vector expressing chimeric antigen receptor, 3 ⁇ g psPAX2 and 1.5 ⁇ g pMD2.G, mixed evenly, then added to the system 25 ⁇ L/10 cm culture dish, mix again uniformly, and let stand for 10 min at room temperature to obtain a mixed solution; 2-3) Remove the 293FT cells used for packaging the virus from the cell culture incubator, and add the mixture to each petri dish on average.
  • DMEM complete medium containing 10% (volume fraction) of FBS was added and cultured in a cell culture incubator; 2-4) cultured for 48-72 hours and collected on culture.
  • a virus stock solution the collected virus stock solution is filtered into a centrifuge tube, centrifuged, the supernatant is discarded, and DMEM complete medium (the volume ratio of the added medium to the virus stock solution is 1:500) is added to the precipitate.
  • the virus particles are suspended to obtain lentiviral particles expressing a chimeric antigen receptor.
  • the lentiviral vector expressing the chimeric antigen receptor further comprises a step of centrifuging after being added to the culture system;
  • the conditions for the centrifugation may specifically be 35 ° C, centrifugation at 2000 rpm for 2 h.
  • the lentiviral vector expressing the chimeric antigen receptor is obtained by inserting the coding gene of the chimeric antigen receptor into a multiple cloning site of the lentiviral expression vector.
  • the lentiviral expression vector is generally used in the art.
  • the Senl_pLenti-EF1 vector is a vector obtained by adding the restriction sites PacI and SpeI to both sides of the original plasmid cloning site, and the original plasmid name is LV-pRRLEF1.WPRE (purchased from Saiye Biotechnology Co., Ltd.).
  • the chimeric antigen receptor-targeted tumor antigens include, but are not limited to, CD19, CD20, CD22, CD30, HER2, GD2, EGFR, EGFRvIII, EphA2, IL13Ra2, CD133, ROR1, IGF1R and/or L1CAM.
  • the chimeric antigen receptor targets the tumor antigen CD22;
  • the amino acid sequence of the chimeric antigen receptor targeting the tumor antigen CD22 is sequence 3 in the sequence listing;
  • the coding gene sequence is Sequence 4 in the sequence listing.
  • the present invention also provides a method of tumor immunotherapy.
  • the method for tumor immunotherapy comprises the following steps:
  • the present invention finally provides a product.
  • the active ingredient of the product provided by the invention is a tumor cell with reduced expression and/or activity of FPPS;
  • the tumor cell whose expression and/or activity of the FPPS is decreased is obtained by introducing a substance which inhibits expression of the FPPS-encoding gene into a tumor cell;
  • the substance that inhibits expression of the FPPS-encoding gene is introduced into the tumor cell through a lentivirus vector;
  • the substance that inhibits expression of the FPPS-encoding gene is a shRNA that inhibits expression of the FPPS-encoding gene.
  • the single-stranded RNA is as shown in SEQ ID NO: 2 of the Sequence Listing; the sequence of the stem I is as shown in position 1-21 of Sequence 2 of the Sequence Listing; the sequence of the loop is as in positions 22-27 of Sequence 2 of the Sequence Listing. Shown; the sequence of stem II is shown in positions 28-48 of sequence 2 of the sequence listing.
  • the coding gene sequence of the single-stranded RNA is shown in SEQ ID NO: 1 of the Sequence Listing.
  • the tumor cells may be common tumor cells, such as chronic myeloid leukemia cell lines.
  • the tumor cell is specifically a K562 cell.
  • the ⁇ T cells are V ⁇ 9 ⁇ 2T cells.
  • the ⁇ T cells are ⁇ -positive T cells sorted from PBMC.
  • Figure 1 is a structural diagram of the U6-based shRNA lentiviral plasmid.
  • Figure 2 shows Western Blot detection of protein expression levels of FPPS and housekeeping gene ⁇ -tubulin in K562-shFPPS, K562-shSRB cells and wild-type K562 cells.
  • Figure 3 shows the percentage of ⁇ T cells to CD3+ cells before and after ⁇ T cell sorting.
  • Figure 4 shows in vitro differentiation of ⁇ T cells stimulated by K562-shFPPS.
  • CFSE was labeled with K562-shFPPS, K562-shSRB and K562 cells, and mixed with ⁇ -positive T cells and ⁇ -negative T cells, respectively.
  • the ratio of ⁇ T cells to K562 cells was 3:1, in K562 group.
  • 10 ⁇ M Zoledronate was added as a positive control.
  • flow cytometry was used to detect the fluorescence intensity (abscissa) of CFSE in different groups of cells.
  • Figure 5 shows that K562-shFPPS co-culture resulted in extensive amplification of sorted ⁇ T cells.
  • K562-shFPPS, K562-shSRB and K562 cells were mixed with ⁇ -positive T cells after sorting, and the number of cells was counted at different times.
  • the ratio of ⁇ -positive T cells to K562-shFPPS or K562-shSRB or K562 cells was 5:1, and 10 ⁇ M Zoledronate was added to the K562 group as a positive control.
  • Figure 6 is a diagram showing the structure of a lentiviral plasmid carrying a CAR sequence.
  • Figure 7 shows that co-culture of K562-shFPPS with sorted ⁇ T cells can significantly improve the transfection efficiency of CAR.
  • Figure 8 shows that K+ ⁇ T cells co-cultured with K562-shFPPS specifically respond to CD22-expressing cells, secrete more IFN- ⁇ , and express more CD137 on the cell surface.
  • Figure 9 shows that CAR+ ⁇ T cells produced by K562-shFPPS co-culture can specifically lyse CD22-expressing tumor cells.
  • Example 1 a method for amplifying ⁇ T cells
  • the lentiviral recombinant plasmid was constructed using the U6-based shRNA construct of the Vectorbuilder system of Saiye Biotechnology Co., Ltd. Specific steps are as follows:
  • a specific shRNA coding sequence (shFPPS) targeting FPPS was designed and synthesized as an experimental group: CCAGCAGTGTTCTTGCAATATCTCGAGATATTGCAAGAACACTGCTGG (sequence 1); wherein the middle 6bp sequence is a stem-loop sequence, and its left side 21 bp is a sense sequence, right The 21 bp sequence was an antisense sequence; the following Scramble-shRNA coding sequence (shSRB) was synthesized as a negative control group: CCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGG; wherein the middle 6 bp sequence was a stem-loop sequence, and its left side 21 bp was a sense sequence, and the right side was 21 bp.
  • the sequence is an antisense sequence.
  • the recombinant plasmid was identified by U6 promoter universal primer, and the correct recombinant plasmid was identified by PCR for sequencing.
  • the recombinant plasmid containing the shFPPS double-stranded DNA sequence was named as the lentiviral recombinant plasmid shFPPS, and the recombinant plasmid containing the shSRB double-stranded DNA sequence was named as the lentiviral recombinant plasmid shSRB.
  • RNA sequence of shFPPS expressed by lentiviral recombinant plasmid shFPPS is as follows: CCUAAGGUUAAGUCGCCCUCGCUCGAGCGAGGGCGACUUAACCUUAGG (SEQ ID NO: 2), which is a single-stranded RNA of stem-loop structure formed by stem I, loop and stem II, and the sequence of stem I is sequence 2 of the sequence listing. The positions are shown in positions 1-21; the sequences of the loops are shown in positions 22-27 of Sequence 2 of the Sequence Listing; the sequences of Stem II are shown in positions 28-48 of Sequence 2 of the Sequence Listing.
  • the lentiviral recombinant plasmids shFPPS and shSRB were packaged to obtain lentiviral particles shFPPS and shSRB, respectively. Specific steps are as follows:
  • the 293FT cells used for packaging the virus were taken out from a 37 ° C, 5% CO 2 cell culture incubator, and the mixture was added to each Petri dish on average, gently shaken, and placed at 37 ° C, 5%. The cultivation was continued in a CO 2 incubator. After 4 hours of culture, the old medium was discarded, 5 mL of pre-warmed PBS was added to wash the cells, and 9 mL of fresh pre-warmed DMEM complete medium containing 10% (by volume fraction) FBS was added, and placed at 37 ° C, 5% CO. 2 Incubate in an incubator.
  • the culture supernatant was taken as a virus stock solution, and the collected virus stock solution was filtered through a 0.45 ⁇ m filter into a 50 mL centrifuge tube, and centrifuged at 18 ° C for 2 hours at 2 ° C for 2 hours. The supernatant was discarded, and DMEM complete medium (volume ratio of the added medium to the virus stock solution of 1:500) was resuspended to the precipitate, which was a virus concentrate.
  • the virus concentrate was dispensed at 200 ⁇ L/tube, and 10 ⁇ L was additionally taken for virus titer measurement.
  • the dispensed concentrate was stored in a -80 ° C refrigerator.
  • the lentiviral particles shFPPS and shSRB were transfected into target cells, respectively, to obtain tumor cell K562-shFPPS and control cell K562-shSRB, respectively. Specific steps are as follows:
  • K562 cells were taken out from a 37 ° C, 5% CO 2 cell culture incubator, and plated into a 24-well plate at 3 ⁇ 10 5 /500 ⁇ L medium/well, and a total of 12 wells were prepared.
  • the medium was RPMI1640 (purchased) At Gibco, catalog number 22400-089) + 10% (volume fraction) FBS (available from Excell Bio, catalog number FND500).
  • centrifuge (3) preheat the centrifuge to 35 °C in advance, and transfer the K562 cells with the virus concentrate into the centrifuge for centrifugation.
  • the centrifugation parameters are set to 2000 rpm, 20 min, 4 drops 4, 2 h, and then placed at 37 ° C after centrifugation. The cultivation was continued in a 5% CO 2 incubator.
  • the experimental group and the control group were added to G418 according to three gradients: low concentration 400 ⁇ g/mL, medium concentration 800 ⁇ g/mL, and high concentration 1200 ⁇ g/mL, and two duplicate wells were set.
  • the corresponding screening medium is changed every 3-5 days according to the color of the medium and the growth of the cells. Until the control cells all died, and the corresponding experimental group still had living cells, the living cells were expanded and cultured.
  • the expanded cells were K562 cells infected with lentiviral particles shFPPS or shSRB.
  • K562-shFPPS and K562-shSRB tumor cells and wild-type K562 cells were screened by G418. After centrifugation, 50 ⁇ L of cell lysate was added to lyse the cells, and Western Blot was used to detect the change of FPPS protein expression level.
  • the antibody for detecting the FPPS protein was a polyclonal antibody against rabbit anti-human FPPS of Abgent (animal number RB4786), and the antibody for detecting the housekeeping gene protein ⁇ -tubulin was a mouse anti-human monoclonal antibody of BD (clone number 5H1).
  • PBMC mononuclear cells
  • the CFSE-labeled ⁇ -negative T cell suspension was mixed with the tumor cell K562-shFPPS suspension at a ratio of 3:1 by volume (the ratio of ⁇ -negative T cells to tumor cell K562-shFPPS was 3:1), at 37
  • the cells in the experimental group were cultured in a °C, 5% CO 2 incubator for 72 hours;
  • the CFSE-labeled ⁇ -positive T cell suspension was mixed with the control cell K562-shSRB suspension at a ratio of 3:1 by volume (the ratio of ⁇ -positive T cells to control cells K562-shSRB was 3:1), at 37
  • the cells were cultured in a °C, 5% CO 2 incubator for 72 hours to obtain a negative control group ( ⁇ -positive T cells);
  • the CFSE-labeled ⁇ -negative T cell suspension was mixed with the control cell K562-shSRB suspension at a ratio of 3:1 by volume (the ratio of ⁇ -negative T cells to control cells K562-shSRB was 3:1), at 37
  • the cells were cultured in a °C, 5% CO 2 incubator for 72 hours to obtain a negative control group ( ⁇ -negative T cells);
  • the CFSE-labeled ⁇ -positive T cell suspension was mixed with wild type cell K562 suspension and FPPS inhibitor Zoledronate (ZOL) (Roche Pharmaceutical), and cultured in a 37 ° C, 5% CO 2 incubator for 72 hours. Positive control cells ( ⁇ -positive T cells).
  • ZOL FPPS inhibitor Zoledronate
  • Positive control cells ⁇ -positive T cells.
  • the ratio of the number of CFSE-labeled ⁇ -positive T cells to K562 cells was 3:1, and the final concentration of Zoledronate in the culture system was 5 ⁇ M;
  • the CFSE-labeled ⁇ -negative T cell suspension was mixed with wild type cell K562 suspension and FPPS inhibitor Zoledronate (ZOL) (Roche Pharmaceuticals), and cultured in a 37 ° C, 5% CO 2 incubator for 72 hours. Positive control cells ( ⁇ -negative T cells) were obtained.
  • the ratio of CFSE-labeled ⁇ -negative T cells to K562 cells was 3:1, and the final concentration of Zoledronate in the culture system was 5 ⁇ M.
  • the experimental group cells, the negative control group cells and the positive control group cells were respectively taken, and the fluorescence level of CFSE was detected by flow cytometry.
  • the specific steps are as follows: 2 ⁇ 10 5 experimental group cells, negative control group cells and positive control cells, respectively, and 1 mL Buffer (2% FBS in PBS) was added to the 3 groups of cells, and the centrifugation parameter was set to 2000 rpm, 3 min. After room temperature, after centrifugation, the supernatant was discarded, and the cells were resuspended in 200 ⁇ L Buffer (2% FBS in PBS), and the fluorescence levels of CFSE in the three groups of cells were tested on the machine.
  • Trypan Blue counting takes 10 ⁇ L of fully dispersed cell suspension, add PBS to the appropriate multiple, and add 10 ⁇ L of Trypan Blue (Gibco, Cat. No. 15250-061) to the diluted cell suspension for staining.
  • the liquid gun was evenly blown, and 10 ⁇ L of Trypan Blue-stained cell suspension was aspirated into a blood cell counting plate covered with a slide glass, and observed under a 100-fold inverted microscope, the living cells were not stained, and the dead cells were stained blue, and the count was four.
  • the invention adopts CAR structure to genetically modify ⁇ T cells, and the CAR structure is from the amino terminal to the carboxy terminal: ScFv(CD22)-Hinge(CD8)-TM(CD8)-CD137-CD3 ⁇ , which is from amino terminal to carboxyl terminal. It is: a single-chain variable region derived from a CD22 monoclonal antibody (clone number M971), a CD8a hinge region and a transmembrane region, a CD137 signal domain, and a CD3 ⁇ chain intracellular region. The amino acid sequence thereof is shown in SEQ ID NO: 3 of the Sequence Listing, and the nucleotide sequence is shown in SEQ ID NO: 4 of the Sequence Listing.
  • the DNA molecule shown in SEQ ID NO:4 was inserted into the Senl_pLenti-EF1 vector (Senl_pLenti-EF1 vector is a vector obtained by adding the cleavage sites PacI and SpeI to both sides of the original plasmid cloning site, and the original plasmid name is LV-pRRLEF1.WPRE
  • the CAR22 lentiviral vector was obtained between the cleavage sites PacI and SpeI provided by Saiye Biotechnology Co., Ltd. under contract number S1002079.
  • the CAR22 lentiviral vector was packaged according to the method of 2 in the first step of Example 1, to obtain CAR22 lentiviral particles.
  • PBMC Peripheral blood mononuclear cells
  • the ⁇ T cells sorted in the step 1 were resuspended in TexMACS medium containing 200 U/ml of human recombinant IL-2 to obtain a ⁇ T cell culture system, and the following groups were cultured according to the composition of the culture system:
  • K562-shFPPS group K562-shFPPS cells were added to the ⁇ T cell culture system, and the ratio of ⁇ T cells to K562-shFPPS cells was 3:1;
  • K562+ZOL group K562 cells and ZOL were added to the ⁇ T cell culture system, the ratio of ⁇ T cells to K562 cells was 3:1, and the final concentration of ZOL in the culture system was 10 ⁇ M;
  • PBMC+ZOL group ZOL was added to PBMC cells, and the final concentration of ZOL in the culture system was 10 ⁇ M;
  • the cells were plated in a 24-well plate at 5 ⁇ 10 5 ⁇ T cells/500 ⁇ L of the medium/well, and cultured at 37 ° C for 2 days in a 5% CO 2 cell incubator.
  • CAR22 lentiviral particles were transfected into each group of cells, respectively.
  • the specific transfection steps were as follows: 24 well plates were taken out of the incubator in advance, and 1 ⁇ Protamine sulfate (purchased from Sigama, catalog number P3369-10G) and 5 ⁇ L of CAR22 lentiviral particle solution were added to each well, and painted on the console. "8 words" and mix with a pipette. Centrifuge at 35 ° C, 2000 rpm for 2 h. After the end of the centrifugation, the 24-well plate was taken out from the centrifuge, transferred to a 37 ° C, and cultured in a 5% CO 2 cell incubator.
  • Half-liquid exchange was performed for 3 days, and 24-well plates were taken out from the incubator, half of the supernatant was aspirated per well, and half of the DMEM complete medium was added to continue the culture.
  • the cell concentration reached (1.5-2) ⁇ 10 6 /ml
  • the bottle or the bag was transferred, and 200 IU/ml of IL-2 was added every 2 days.
  • the cells were cultured until days 10-14, and 200 ⁇ L of the cell suspension was subjected to Trypan Blue counting, and subsequent cell identification, sorting, and functional tests were performed.
  • CD22-Fc protein powder 100 ⁇ g of purified CD22-Fc protein powder (purchased from Yishen Shenzhou Biotechnology Co., Ltd., Cat. No. 11958-H02H), resuspended in PBS pH 7.2 to a final concentration of (0.5-1) ⁇ g/ ⁇ L; purity >99.9% of dimethyl sulfoxide (DMSO) dissolved in a suitable amount of biotin (purchased from Suzhou Yuheng Biotechnology Co., Ltd., item number: B5026-1) formulated into a 2 mM biotin suspension; according to CD22-Fc protein: biotin Mix 1: 1 molar ratio, let stand for 1 hour at room temperature, mix once every 15 minutes; desalting with desalting column (refer to the instruction manual of desalting column for operation steps), according to the volume, PD-10 can be selected. (purchased from GE Corporation of the United States, Cat. No. 17-0851-01) or G-25 Desalting Column (purchased from
  • Transfection efficiency was determined 48-72 hours after lentivirus transfection (transfection efficiency was the percentage of ⁇ T cells positive for CD22-Fc labeling in all ⁇ T cells).
  • the specific steps are as follows: 1 (1) ⁇ 10 5 cells per tube, add 1 ⁇ L of biotin-labeled CD22-Fc, incubate at 4-8 ° C for 10 min in the dark, add 1 mL (PBS + 2% FBS), resuspend, centrifuge washing.
  • the CAR22-modified ⁇ T cells cultured on the 7th-10th day by the above method were subjected to sorting and purification to obtain purified CAR- ⁇ -positive T cells.
  • the specific steps are as follows: CAR- ⁇ T cells cultured on days 7-10 were collected, and the supernatant was centrifuged, and 80 ⁇ L of buffer (PBS + 2% FBS) and 5 ⁇ L of biotin were added per 1 ⁇ 10 7 CAR- ⁇ T cells.
  • the ratio of CD22-Fc was added to the buffer and biotin-labeled CD22-Fc, and incubated at 4-8 ° C for 15 minutes in the dark.
  • buffer and SA magnetic beads were added in a ratio of 80 ⁇ L of buffer (PBS + 2% FBS) and 10 ⁇ L of SA magnetic beads per 1 ⁇ 10 7 CAR- ⁇ T cells, and mixed, and incubated at 4-8 ° C in the dark. After the incubation period, add 1-2 mL of buffer per 1 ⁇ 10 7 CAR- ⁇ T cells, centrifuge, and add 500 ⁇ L of buffer solution per 1 ⁇ 10 8 cells to mix and mix the cell sediment layer and magnetically sort the column.
  • the CAR- ⁇ -positive T cells were sorted, and the dripped cell suspension was the CAR- ⁇ -negative T cells sorted, which was recorded as CAR- ⁇ T cells; the cells remaining in the magnetic column were CAR- ⁇ -positive. T cells are referred to as CAR+ ⁇ T cells.
  • the positive cells obtained by sorting were collected, counted, and the purity of CAR-positive T cells was detected by flow, and the remaining cells were further cultured or directly used for subsequent functional tests.
  • the full-length cDNA sequence of CD22 was cloned from healthy human PBMC (Genebank No. NM_001771.3), and the full-length cDNA sequence of CD22 was inserted into U6- carrying the Puromycine resistance gene. Based on shRNA Knockdown lentiviral plasmid (purchased from Saiye Biotechnology Co., Ltd.), a lentiviral plasmid was obtained. The lentiviral plasmid was packaged and transfected into K562 cells according to the methods of 2 and 3 of the first step of Example 1, and K562-CD22 cells with higher purity were obtained by screening.
  • the full-length cDNA sequence of CD19 was cloned from healthy human PBMC (Genebank No. NM_001770.5), and the full-length cDNA sequence of CD19 was inserted into U6- carrying the Puromycine resistance gene. Based on shRNA Knockdown lentiviral plasmid (purchased from Saiye Biotechnology Co., Ltd.), a lentiviral plasmid was obtained. The lentiviral plasmid was packaged and transfected into K562 cells according to the methods in 2 and 3 of the first step of Example 1, and K562-CD19 cells with higher purity were obtained as negative controls by screening.
  • LCL cells are immortalized cell lines obtained by Epstein-Barr virus infection of healthy adult peripheral B cells, and CD22 antigen is expressed on the cell surface.
  • the specific preparation method is as follows: B95.8 cells (purchased from the Chinese Academy of Sciences cell bank) were inoculated into a T75 cm 2 cell culture flask at a concentration of 3 ⁇ 10 5 /mL, and the medium was RPMI 1640 (purchased from gibco, catalog number is 22400-088) +10% FBS (purchased from gibco, catalog number 10099141).
  • the cells After incubating for 48 h in a 37 ° C, 5% CO 2 incubator, the cells were reseeded at a concentration of 1 ⁇ 10 6 /mL, and TPA was added at a final concentration of 20 ng / mL (purchased from cayman, catalog number 16561-29- 8) Treat cells. After 1 h, the cells were washed 3 times with RPMI 1640 complete medium to remove TPA. The obtained cells were inoculated again at a concentration of 1 ⁇ 10 6 /mL, and cultured for 96 hours at 37° C. in a 5% CO 2 incubator.
  • the culture medium was collected in a 50 mL centrifuge tube, centrifuged at 600°g for 10 min at 4° C., and the resulting supernatant was passed through 0.45 ⁇ m.
  • the filter was filtered and the filtrate was an EBV virus suspension.
  • Peripheral blood mononuclear cells (PBMC) obtained by density gradient centrifugation were sorted by CD3 (purchased from the German Meitian Biotechnology Co., Ltd., catalog number 130-050-101).
  • the resulting CD3-cell, B cells were seeded in a culture flask at a concentration of 3 ⁇ 10 5 /mL, and 1/10 volume of the EBV virus suspension was added.
  • PBMC Peripheral blood mononuclear cells
  • the target cell strain obtained in the step 1 was added to a 96-well U-bottom cell culture plate in an amount of 1 ⁇ 10 4 per well; then, the effector cells (CAR- ⁇ -positive T cells): the target cells were 10:1, respectively. 3:1, 1:1 ratio
  • the corresponding number of purified CAR- ⁇ -positive T cells were added to the corresponding wells, and effector cells and target cell blank control wells were set separately, and CAR- ⁇ -negative T cells were used as control. Effector cells, set up the same test.
  • the cell culture plate was co-cultured in a 37 ° C, 5% CO 2 incubator for 4 hours, the cell suspension was aspirated, washed twice with PBS, and resuspended in 100 ⁇ L Buffer, and 2 ⁇ L of CD3-APC-Cy7, CD4-PE were added, respectively.
  • .Cy7, CD8-VioBlue, and CD137-PE (BD, clone 4B4-1), incubated at 4-8 °C for 10 minutes in the dark, after washing, stained with BD's intracellular staining kit (Cat. No. 554714)
  • the method refers to the instructions.
  • mice anti-human IFN- ⁇ -FITC monoclonal antibody (BD, clone No. B27) was added, and the cells were washed at 4-8 ° C for 10 minutes in the dark, and washed. After washing, the flow cytometer was used. The expression levels of IFN- ⁇ and CD137 were examined.
  • the target cell lines in step 1 were subjected to CFSE labeling according to the method in step 2 of Example 1, and the cells were incubated at room temperature or at 37 ° C for 20 min in the dark, and the medium was stopped by adding 5 times volume of medium containing 10% (volume fraction) FBS. . Centrifuge at 2000 rpm for 5 min, resuspend the cells after centrifugation, incubate at room temperature or at 37 ° C for 10 min in the dark. After the incubation, the cells were washed twice and resuspended for use.
  • CAR- ⁇ -positive T cells target cells were 10:1, 3:1, 1:1, respectively.
  • the proportion of the selected number of purified CAR- ⁇ T positive cells were added to the corresponding wells, and the effector cells and the target cell blank control wells were respectively set, and the same test was established using CAR- ⁇ -negative T cells as control effect cells.
  • the cell culture plate was co-cultured in a 37 ° C, 5% CO 2 incubator for 4 hours, the cell suspension was aspirated, washed twice with PBS, and the cells were resuspended in 100 ⁇ L Buffer, and 5 ⁇ L of 7-AAD was added, and the mixture was incubated for 10 minutes in the dark. After washing and washing, the killing rate was detected by flow cytometry, and the killing rate was the percentage of the target cells (CFSE+7AAD+) killed by all target cells (CSFE+).
  • the present invention provides a novel ⁇ T cell expansion protocol, which is specifically as follows: shFPPS targeting FPP synthase (FPPS) is transfected into K562 cells by lentiviral vector.
  • FPPS FPP synthase
  • the expression of FPPS in K562 cells was down-regulated, and a K562-shFPPS cell line with reduced expression of FPPS was constructed.
  • the K562-shFPPS cell line is co-cultured with ⁇ T cells in a ⁇ T cell culture system, and it is found that the K562-shFPPS cell line can directly stimulate the differentiation and expansion of purified V ⁇ 9 ⁇ 2T cells in vitro.
  • a lentiviral vector expressing a chimeric antigen receptor is added to a ⁇ T cell culture system containing a K562-shFPPS cell line for co-culture, and it is found that the K562-shFPPS cell line can also effectively improve the transfection efficiency of the CAR gene, thereby improving Preparation efficiency of CAR- ⁇ T cells.
  • the above experimental results indicate that the K562-shFPPS cell line can effectively improve the in vitro expansion ability of ⁇ T cells and the transfection efficiency of CAR gene.
  • the solution provided by the present invention effectively solves the technical bottleneck of large-scale production of CAR- ⁇ T cells, and has good performance. Application prospects.

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Abstract

一种生产嵌合抗原受体修饰的γδT细胞的方法：将靶向于FPP合成酶的shFPPS通过慢病毒载体转染K562细胞，下调了K562细胞中FPPS的表达量，构建了FPPS表达量降低的K562-shFPPS细胞系。将K562-shFPPS细胞系加入γδT细胞培养体系中与γδT细胞进行共培养，发现K562-shFPPS细胞系能促进γδT细胞的体外分化和扩增。将表达CAR的慢病毒载体加入含有该细胞系的γδT细胞培养体系中进行共培养，发现K562-shFPPS细胞系还可以有效提高CAR基因的转染率。所提供的方案有效解决了大规模生产CAR-γδT细胞的技术瓶颈，具有良好的应用前景。

Description

一种生产嵌合抗原受体修饰的γδT细胞的方法 技术领域
本发明涉及生物技术领域，具体涉及一种生产嵌合抗原受体修饰的γδT细胞的方法。
背景技术
过继性细胞免疫治疗是将体外培养、活化、基因修饰的自体或异体免疫细胞回输给患者，用于发挥抗肿瘤活性。嵌合抗原受体(Chimeric Antigen Receptor，CAR)修饰的T细胞治疗技术(CAR-T技术)是通过基因工程技术修饰免疫效应细胞，使被修饰的细胞能特异性识别和杀伤表达特异性抗原的靶细胞，从而达到特异性清除肿瘤细胞的目的。特异靶向于B淋巴细胞表面标志物CD19分子的CAR-T细胞，在治疗B淋巴细胞恶性肿瘤中的疗效最为显著，可使90％的复发难治的B系急性淋巴细胞白血病患者获得完全缓解。虽然目前针对CD19抗原的CAR-T细胞治疗B淋巴细胞性肿瘤，具有极高的短期治疗效果。但仍有相当一部分病人在治疗达到完全缓解后会再次复发。复发的一个重要原因是输入的CAR-T细胞因为缺少有效的抗原刺激，在患者体内的数量不断减少，因此不能保持长期的抗肿瘤活性。
现有CAR-T技术是根据αβT细胞的天然活性设计的，一般修饰的效应细胞是αβT细胞。αβT细胞输入异体，会引发严重的移植物抗宿主反应，因此，目前的CAR-T治疗技术多以自体细胞治疗为主，极大的限制了临床应用的灵活性，大大提高了治疗成本。而γδT细胞不会引发严重的移植物抗宿主反应，因此是开发通用型CAR-T产品更好的选择。
αβT细胞介导的是适应性免疫应答，需要完成淋巴细胞再循环才能有效活化并发挥相应的免疫学功能。而γδT细胞介导的是天然免疫应答，能在抗原抗体发生反应的部位即时发挥免疫学效应，因此CAR-γδT细胞潜在的抗肿瘤作用会更直接、更迅速，可以通过肿瘤部位的局部注射清除肿瘤，避免脱靶效应引发的强烈不良反应。另外，αβT细胞利用多样性的αβTCR识别由主要组织相容性复合体(MHC)提呈的抗原短肽。天然情况下，识别一个特定的MHC-抗原肽复合物的αβT细胞的数量非常少，因此需要数量上的扩增才能发挥对特定抗原的免疫应答能力。而γδT细胞是一类数量较低的T淋巴细胞亚类，大多数γδTCR识别非肽类化合物，不需要MHC的提呈。不像αβTCR可变区的多样性组成，γδTCR的可变区的多样性非常有限，因此，尽管αβT细胞的总数量远多于γδT细胞，但比较对一种特定化合物或抗原具有识别能力的特定T细胞的数量的话，γδT细胞要高于αβT细胞。
人和其它灵长类动物的γδT细胞主要包括Vδ1和Vγ9δ2两个亚类，分别代表表面表达TCR的种类。Vδ1分布在组织，外周的γδT细胞主要为Vγ9δ2亚类。两类细胞的抗原识别模式截然不同，Vδ1TCR识别抗原的种类目前存在争议；Vγ9δ2TCR识别自身或外源性的小分子磷酸化抗原，诸如HMBPP(细菌来源)、IPP(人来源)和BrHPP(人工合成)(Chen,Z.W.and N.L.Letvin,2003.5(6):p.491-8；Eberl,M.,et al., 2003.544(1-3):p.4-10；Kabelitz,D.,D.Wesch,and W.He,2007.67(1):p.5-8；Sireci,G.,et al.,2001.31(5):p.1628-35)。Amino-Bisphosphonate化合物类药物Zoledronate(Zol，泽泰)和Pamidornate能抑制FPP合成酶(FPPS)，从而阻抑HMG-CoA的代谢途径，导致下游产物IPP的体内或细胞内聚集，因此能在体内或体外有效刺激活化Vγ9δ2T细胞(Kunzmann,V.and M.Wilhelm,2005.46(5):p.671-80)。
虽然γδT细胞具有的天然活性使其成为CAR-T细胞治疗的优良载体，但由于细胞含量低、体外大规模扩增技术难度大、转染率低等限制，迄今尚没有CAR修饰的γδT细胞在临床上成功应用的报道。传统的γδT细胞体外扩增技术，包括固相anti-pan TCRγδ抗体扩增，或者采用IPP/HMBPP/ZOL选择性扩增Vγ9δ2T细胞，都是从外周单个核细胞(PBMC)中进行扩增，由于γδT细胞所占比例较低，基因转染受其它无关细胞干扰导致转染率低。此外，从PBMC中扩增的终产品纯度低，容易混杂αβT细胞，不利于开发通用型CAR-T产品。分选纯化γδT细胞能解决以上问题，但是缺少其它细胞的辅助，活化和扩增效率会大大降低。
发明公开
本发明要解决的技术问题是如何提高γδT细胞的扩增效率，以实现大规模生产CAR-γδT细胞用于临床肿瘤免疫治疗。
为了解决上述技术问题，本发明首先提供了一种γδT细胞的扩增方法。
本发明提供的γδT细胞的扩增方法包括在γδT细胞培养体系中添加FPPS表达量和/或活性降低的肿瘤细胞的步骤。
上述方法中，所述γδT细胞与所述FPPS表达量和/或活性降低的肿瘤细胞的个数比为(1-10):1；
进一步的，所述γδT细胞与所述FPPS表达量和/或活性降低的肿瘤细胞的个数比为3:1。
上述方法还包括如下步骤：当培养体系中细胞浓度到达(1.5-2)×10 6个/ml时，每2-4天向所述培养体系中添加一次IL-2；添加IL-2时的标准为使所述IL-2在所述培养体系中的终浓度为(100-1000)IU/mL；
进一步的，当培养体系中细胞浓度到达(1.5-2)×10 6个/ml时，每3天向所述培养体系中添加一次IL-2；添加IL-2时的标准为使所述IL-2在所述培养体系中的终浓度为(100-300)IU/mL。
更进一步的，当培养体系中细胞浓度到达(1.5-2)×10 6个/ml时，每3天向所述培养体系中添加一次IL-2；添加IL-2时的标准为使所述IL-2在所述培养体系中的终浓度为200IU/mL。
为了解决上述技术问题，本发明又提供了一种生产CAR-γδT细胞的方法。
本发明提供的生产CAR-γδT细胞的方法包括如下步骤：将表达嵌合抗原受体的载体与γδT细胞共培养，得到CAR-γδT细胞；所述培养体系中含有FPPS表达量和/或活性降低的肿瘤细胞。
上述方法中，所述将表达嵌合抗原受体的载体与γδT细胞共培养的方法包 括如下步骤：
1)将FPPS表达量和/或活性降低的肿瘤细胞加入γδT细胞培养体系中，培养，得到培养体系；
2)将表达嵌合抗原受体的载体加入所述培养体系中，培养，得到所述CAR-γδT细胞。
上述方法中，所述1)中，所述γδT细胞与所述FPPS表达量和/或活性降低的肿瘤细胞的个数比为(1-10):1；
进一步的，所述γδT细胞与所述FPPS表达量和/或活性降低的肿瘤细胞的个数比为3:1。
上述方法中，所述1)中，所述培养的条件如下：37℃，5％CO 2培养2天。
上述方法中，所述1)中，所述γδT细胞培养体系由TexMACS培养基和人重组IL-2组成，所述人重组IL-2在γδT细胞培养体系中的浓度为200U/ml。
上述方法中，所述2)中还包括如下步骤：当培养体系中细胞浓度到达(1.5-2)×10 6个/ml时，每2-4天向所述培养体系中添加一次IL-2；添加IL-2时的标准为使所述IL-2在所述培养体系中的终浓度为(100-1000)IU/mL；
进一步的，当培养体系中细胞浓度到达(1.5-2)×10 6个/ml时，每2天向所述培养体系中添加一次IL-2；添加IL-2时的标准为使所述IL-2在所述培养体系中的终浓度为(100-300)IU/mL。
更进一步的，当培养体系中细胞浓度到达(1.5-2)×10 6个/ml时，每2天向所述培养体系中添加一次IL-2；添加IL-2时的标准为使所述IL-2在所述培养体系中的终浓度为200IU/mL。
上述方法中，所述2)中，所述培养的条件如下：37℃，5％CO 2培养10-14天。
上述方法中，所述2)中，所述表达嵌合抗原受体的载体为表达嵌合抗原受体的慢病毒载体；
所述表达嵌合抗原受体的慢病毒载体在加入所述培养体系前还包括包装的步骤；所述包装的方法具体可按照如下步骤进行：2-1)每细胞培养皿中加入4.5×10 6个293FT细胞和9mL DMEM完全培养基，混合混匀，在细胞培养箱中培养；2-2)培养第2天，每个培养皿加入如下试剂：500μL
Figure PCTCN2019072285-appb-000001
buffer、6μg表达嵌合抗原受体的慢病毒载体、3μg psPAX2和1.5μg pMD2.G，混合均匀，然后再向体系中加入
Figure PCTCN2019072285-appb-000002
25μL/10cm培养皿，再次混合均匀，室温静置10min，得到混合液；2-3)将用于包装病毒的293FT细胞从细胞培养箱中取出，将混合液平均加到每个培养皿中，混匀，放入细胞培养箱中继续培养。培养4h后，弃旧培养基，加入PBS清洗细胞，再加入含10％(体积分数)FBS的DMEM完全培养基，放入细胞培养箱中培养；2-4)培养48-72h后收取培养上清作为病毒原液，并将收集到的病毒原液过滤到离心管中，离心，弃上清液，向沉淀中加入DMEM完全培养基(加入的培养基与病毒原液的体积比为1:500)重悬病毒颗粒，得到表达嵌合抗原受体的慢病毒颗粒。
所述表达嵌合抗原受体的慢病毒载体在加入所述培养体系后还包括离心的步骤；
所述离心的条件具体可为35℃，2000rpm离心2h。
进一步的，所述表达嵌合抗原受体的慢病毒载体为将嵌合抗原受体的编码基因***慢病毒表达载体的多克隆位点间得到的。所述慢病毒表达载体为本领域常用的即可。
更进一步的，所述慢病毒表达载体为Senl_pLenti-EF1。所述表达嵌合抗原受体的慢病毒载体为将嵌合抗原受体的编码基因***慢病毒载体Senl_pLenti-EF1的PacI和SpeI酶切位点间得到的载体。
所述Senl_pLenti-EF1载体为在原始质粒克隆位点两侧增加了酶切位点PacI和SpeI后得到的载体，原始质粒名称为LV-pRRLEF1.WPRE(购自赛业生物科技有限公司)。
上述方法中，所述嵌合抗原受体靶向的肿瘤抗原包括但不限于CD19、CD20、CD22、CD30、HER2、GD2、EGFR、EGFRvIII、EphA2、IL13Ra2、CD133、ROR1、IGF1R和/或L1CAM。
在本发明的具体实施例中，所述嵌合抗原受体靶向肿瘤抗原CD22；所述靶向肿瘤抗原CD22的嵌合抗原受体的氨基酸序列为序列表中的序列3；编码基因序列为序列表中的序列4。
为了解决上述技术问题，本发明还提供了一种肿瘤免疫治疗的方法。
本发明提供的肿瘤免疫治疗的方法包括如下步骤：
(1)按照上述方法生产CAR-γδT细胞；
(2)将所述CAR-γδT细胞回输肿瘤患者体内，通过所述CAR-γδT细胞识别并杀伤所述肿瘤患者体内的肿瘤细胞，进而实现肿瘤免疫治疗的目的。
为了解决上述技术问题，本发明最后还提供了一种产品。
本发明提供的产品的活性成分为FPPS表达量和/或活性降低的肿瘤细胞；
所述产品的功能为如下B1)-B5)中任一种：
B1)促进γδT细胞扩增；
B2)促进γδT细胞分化；
B3)生产CAR-γδT细胞；
B4)提高慢病毒对γδT细胞的转染率；
B5)肿瘤免疫治疗。
上述方法或产品中，所述FPPS表达量和/或活性降低的肿瘤细胞是将抑制FPPS编码基因表达的物质导入肿瘤细胞得到的；
进一步的，所述抑制FPPS编码基因表达的物质通过慢病毒载体导入肿瘤细胞；
更进一步的，所述抑制FPPS编码基因表达的物质为抑制FPPS编码基因表达的shRNA。
在本发明的具体实施例中，所述抑制FPPS编码基因表达的shRNA为由茎I、环和茎II形成的茎环结构的单链RNA；
所述单链RNA如序列表的序列2所示；所述茎I的序列如序列表的序列2第1-21位所示；所述环的序列如序列表的序列2第22-27位所示；所述茎II的序列如序列表的序列2第28-48位所示。所述单链RNA的编码基因序列如序列表的序列1所示。
上述方法或产品中，所述肿瘤细胞可为常见的肿瘤细胞，如慢性骨髓性白血病细胞系。在本发明中，所述肿瘤细胞具体为K562细胞。
上述方法或产品中，所述γδT细胞为Vγ9δ2T细胞。所述γδT细胞为从PBMC中分选得到的γδ阳性T细胞。
附图说明
图1为U6-based shRNA慢病毒质粒结构图。
图2为Western Blot检测肿瘤细胞K562-shFPPS、K562-shSRB细胞以及野生型K562细胞中FPPS和管家基因β-tubulin的蛋白表达水平。
图3为γδT细胞分选前后γδT细胞占CD3+细胞的百分比。
图4为K562-shFPPS刺激γδT细胞的体外分化。CFSE分别标记K562-shFPPS、K562-shSRB和K562细胞，分别与分选后的γδ阳性T细胞和γδ阴性T细胞混合，γδT细胞与K562细胞的个数比均为3:1，在K562组中加入10μM的Zoledronate作为阳性对照。培养72小时后，流式细胞仪检测不同组细胞CFSE的荧光强度(横坐标)。
图5为K562-shFPPS共培养使分选的γδT细胞获得大量扩增。K562-shFPPS、K562-shSRB和K562细胞分别与分选后的γδ阳性T细胞混合培养，计数不同时间细胞数量。γδ阳性T细胞与K562-shFPPS或K562-shSRB或K562细胞的个数比均为5:1，在K562组中加入10μM的Zoledronate作为阳性对照。
图6为携带CAR序列的慢病毒质粒结构图。
图7为K562-shFPPS与分选的γδT细胞共培养可显著提高CAR的转染效率。
图8为K562-shFPPS共培养生产的CAR+γδT细胞对表达CD22的细胞发生特异性应答，分泌更多的IFN-γ，细胞表面表达更多的CD137。
图9为K562-shFPPS共培养生产的CAR+γδT细胞能特异性裂解表达CD22的肿瘤细胞。
实施发明的最佳方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、一种γδT细胞的扩增方法
一、FPPS表达量降低的K562-shFPPS肿瘤细胞系的构建
1、慢病毒重组质粒的构建
(1)重组质粒的制备
采用赛业生物科技有限公司的Vectorbuilder***的U6-based shRNA构建体系构建慢病毒重组质粒。具体步骤如下：
设计并合成特异的靶向于FPPS的shRNA编码序列(shFPPS)作为实验组：CCAGCAGTGTTCTTGCAATATCTCGAGATATTGCAAGAACACTGCTGG(序列1)；其中，中间加粗的6bp序列为茎环序列，它的左侧21bp为正义序列，右侧21bp序列为反义序列；同时合成如下Scramble-shRNA编码序列(shSRB)作为阴性对照组：CCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGG；其中，中间加粗的6bp序列为茎环序列，它的左侧21bp为正义序列，右侧21bp序列为反义序列。
分别合成以上shRNA编码序列，并分别将其克隆至U6-Based shRNA Knockdown慢病毒质粒(购于赛业生物科技有限公司，目录号LV-SGFP-0102)中的U6启动子下方，分别获得重组质粒shFPPS和shSRB。此外，在重组质粒shFPPS和shSRB中引入Neumycine抵制基因，用于阳性细胞筛选。重组质粒结构如图1所示。
(2)重组质粒的鉴定
采用U6启动子通用引物对重组质粒进行鉴定，并将PCR鉴定正确的重组质粒进行测序验证。将含有shFPPS双链DNA序列的重组质粒命名为慢病毒重组质粒shFPPS，将含有shSRB双链DNA序列的重组质粒命名为慢病毒重组质粒shSRB。慢病毒重组质粒shFPPS表达的shFPPS的RNA序列如下：CCUAAGGUUAAGUCGCCCUCGCUCGAGCGAGGGCGACUUAACCUUAGG(序列2)，其为由茎I、环和茎II形成的茎环结构的单链RNA，茎I的序列如序列表的序列2第1-21位所示；环的序列如序列表的序列2第22-27位所示；茎II的序列如序列表的序列2第28-48位所示。
2、慢病毒重组质粒的包装
分别将慢病毒重组质粒shFPPS和shSRB进行包装，分别得到慢病毒颗粒shFPPS和shSRB。具体步骤如下：
(1)将已长到80％-90％的293FT细胞培养瓶(T175)从37℃、5％CO 2的细胞培养箱中取出，消化后收集洗涤细胞，每10cm细胞培养皿中加入4.5×10 6个细胞和9mL DMEM完全培养基(购于Gibco公司，产品目录号11965-084)，轻轻摇匀，放入37℃、5％CO 2培养箱中培养。
(2)培养第2天，每个培养皿加入如下试剂：500μL
Figure PCTCN2019072285-appb-000003
buffer(购于Polyplus Transfection公司，产品目录号B161116)、6μg慢病毒重组质粒、3μg psPAX2(购于武汉淼灵生物科技有限公司，产品目录号P026)和1.5μg pMD2.G(购于广州吉赛生物科技股份有限公司，产品目录号161220L08)，混合均匀，然后再向体系中加入
Figure PCTCN2019072285-appb-000004
(购于Polyplus Transfection公司，产品目录号114-15)，25μL/10cm培养皿，再次混合均匀，室温静置10min，得到混合液。
(3)将用于包装病毒的293FT细胞从37℃、5％CO 2的细胞培养箱中取出，将混合液平均加到每个培养皿中，轻轻摇匀，放入37℃、5％CO 2培养箱中继续培养。培 养4h后，弃旧培养基，加入5mL已预热的PBS清洗细胞，再加入9mL新鲜的已预热的含10％(体积分数)FBS的DMEM完全培养基，放入37℃、5％CO 2培养箱中培养。
(4)继续培养48-72h后收取培养上清作为病毒原液，并将收集的病毒原液用0.45μm过滤器过滤到50mL离心管中，4℃、18500g高速离心2h。弃上清液，向沉淀中加入DMEM完全培养基(加入的培养基与病毒原液的体积比为1:500)重悬病毒颗粒，此即为病毒浓缩液。
(5)将病毒浓缩液按200μL/管分装，另外留取10μL进行病毒滴度测定。将分装好的浓缩液置于-80℃冰箱保存。
3、慢病毒颗粒转染靶细胞
分别将慢病毒颗粒shFPPS和shSRB转染靶细胞，分别得到肿瘤细胞K562-shFPPS和对照细胞K562-shSRB。具体步骤如下：
(1)将K562细胞从37℃、5％CO 2的细胞培养箱中取出，按3×10 5/500μL培养基/孔铺到24孔板中，共铺12孔，培养基为RPMI1640(购于Gibco公司，产品目录号22400-089)+10％(体积分数)FBS(购于Excell Bio公司，产品目录号FND500)。
(2)从12个孔中选取6孔作为实验组进行慢病毒转染，剩余6孔作为对照组。实验组，每孔加入1‰Protamine sulfate(购于Sigma公司，产品目录号P3369-10G)和5μL shFPPS病毒浓缩液，在操作台上画“8字”轻轻混匀。对照组加入5μL shSRB病毒浓缩液。
(3)提前预热离心机至35℃，将加有病毒浓缩液的K562细胞移入离心机中离心，离心参数设置为2000rpm，20min，升4降4，2h，离心结束后放入37℃、5％CO 2培养箱中继续培养。
(4)在培养48h后，实验组和对照组分别按照：低浓度400μg/mL、中浓度800μg/mL、高浓度1200μg/mL三个梯度加入G418，各设两个复孔。
(5)根据培养基的颜色和细胞生长情况，每3-5天更换一次对应的筛选培养基。直至对照孔细胞全部死亡，而对应实验组仍有活细胞时，收集活细胞扩大培养。
结果表明：K562细胞的G418最适筛选浓度为800μg/mL。扩大培养的细胞为感染慢病毒颗粒shFPPS或shSRB的K562细胞。
4、检测shFPPS感染细胞FPPS蛋白表达水平的变化
取1.5×10 6个经G418筛选的K562-shFPPS和K562-shSRB肿瘤细胞，以及野生型K562细胞，离心后加入50μL细胞裂解液裂解细胞，Western Blot检测FPPS蛋白表达水平的变化。检测FPPS蛋白的抗体为Abgent公司的兔抗人FPPS的多克隆抗体(动物号为RB4786)，检测管家基因蛋白β-tubulin的抗体为BD公司的鼠抗人单克隆抗体(克隆号为5H1)。
结果如图2所示。从图中可以看出：K562-shSRB细胞FPPS表达水平和野生型K562细胞无显著性差异，而表达shFPPS的K562-shFPPS细胞的FPPS表达水平和野生型K562细胞相比显著降低。
二、γδT细胞的体外扩增方法
1、γδT细胞的分选
取新鲜分离的来源于成人外周血或脐带血的单个核细胞(PBMC)，使用γδT细胞分选试剂盒(购于德国美天旎生物技术有限公司，产品目录号130-092-892)按照试剂盒的说明书中的操作从PBMC中分选γδT细胞，得到γδ阳性T细胞，剩余细胞为γδ阴性T细胞。分选的γδ阳性T细胞中γδT细胞的阳性百分比>90％(图3)。
2、CFSE标记γδT细胞
向装有CFSE(购于BioLegend公司，产品目录号423801)的离心管中加入36μL DMSO，使其终浓度为5mM，反复吸吹使其充分混匀，加入相应体积的PBS，使CFSE溶液终浓度为5μM，得到CFSE工作液。将1×10 7-1×10 8个γδT细胞(步骤1中的γδ阳性T细胞或γδ阴性T细胞)加入到5μL CFSE工作液中，室温或者37℃避光孵育20min，加入5倍体积含有10％(体积分数)FBS、200U/ml人重组IL-2的TexMACS培养基(购于德国美天旎生物技术有限公司，货号130-097-196)，终止染色。2000rpm离心5min，收集细胞，使用预热的TexMACS培养基重悬细胞，室温或者37℃避光孵育10min，孵育结束后，使用预热的培养基清洗细胞2次，流式检测CFSE是否染色成功，CESE成功标记的γδT细胞用于下述实验。
3、将CFSE标记γδ阳性T细胞悬液与肿瘤细胞K562-shFPPS悬液按照体积比为3：1的比例混匀(γδ阳性T细胞与肿瘤细胞K562-shFPPS个数比为3：1)，在37℃、5％CO 2培养箱中培养72小时，得到实验组细胞(γδ阳性T细胞)；
将CFSE标记γδ阴性T细胞悬液与肿瘤细胞K562-shFPPS悬液按照体积比为3：1的比例混匀(γδ阴性T细胞与肿瘤细胞K562-shFPPS个数比为3：1)，在37℃、5％CO 2培养箱中培养72小时，得到实验组细胞(γδ阴性T细胞)；
将CFSE标记γδ阳性T细胞悬液与对照细胞K562-shSRB悬液按照体积比为3：1的比例混匀(γδ阳性T细胞与对照细胞K562-shSRB个数比为3：1)，在37℃、5％CO 2培养箱中培养72小时，得到阴性对照组细胞(γδ阳性T细胞)；
将CFSE标记γδ阴性T细胞悬液与对照细胞K562-shSRB悬液按照体积比为3：1的比例混匀(γδ阴性T细胞与对照细胞K562-shSRB个数比为3：1)，在37℃、5％CO 2培养箱中培养72小时，得到阴性对照组细胞(γδ阴性T细胞)；
将CFSE标记γδ阳性T细胞悬液与野生型细胞K562悬液和FPPS的抑制剂泽泰(Zoledronate，ZOL)(罗氏制药)混匀，在37℃、5％CO2培养箱中培养72小时，得到阳性对照组细胞(γδ阳性T细胞)。其中CFSE标记γδ阳性T细胞和K562细胞的数量比为3：1，Zoledronate在培养体系中的终浓度为5μM；
将CFSE标记γδ阴性T细胞悬液与野生型细胞K562悬液和FPPS的抑制剂泽泰(Zoledronate，ZOL)(罗氏制药)混匀，在37℃、5％CO 2培养箱中培养72小时，得到阳性对照组细胞(γδ阴性T细胞)。其中CFSE标记γδ阴性T细胞和K562细胞的数量比为3：1，Zoledronate在培养体系中的终浓度为5μM。
4、分别取实验组细胞、阴性对照组细胞和阳性对照组细胞，流式检测CFSE的荧光水平。具体步骤如下：分别取2×10 5个实验组细胞、阴性对照组细胞和阳性对照 组细胞，向3组细胞中分别加入1mL Buffer(2％FBS的PBS)离心，离心参数设置为2000rpm，3min，室温，离心结束后，弃上清，用200μL Buffer(2％FBS的PBS)重悬细胞，上机测试3组细胞的CFSE的荧光水平。
结果如图4所示。从图中可以看出：K562-shFPPS细胞可以有效促进分选的γδT细胞的分化。
5、继续培养各组细胞，当细胞浓度到达(1.5-2)×10 6个/mL时，扩大培养，每3天向培养体系中补加一次IL-2，使其在培养体系中的终浓度为200IU/mL。
6、当细胞培养至第10-14天，取200μL细胞悬液进行Trypan Blue计数，并进行后续的细胞鉴定、分选以及功能测试。
Trypan Blue计数的具体步骤如下：取10μL充分打散的细胞悬液，加入PBS稀释到合适的倍数，向稀释后的细胞悬液中加入10μL Trypan Blue(Gibco，货号15250-061)染色，用移液枪吹打均匀，吸取10μL Trypan Blue染色的细胞悬液打入盖有载玻片的血球计数板上，于100倍倒立显微镜下观察，活细胞不染色，死细胞染为蓝色，计数四个大方格里的细胞总数，再除以4，乘以稀释倍数，乘以10 4，最后乘以细胞悬液总体积，即得到细胞总数。
流式细胞仪鉴定γδT细胞的具体步骤如下：分别取(1-2)×10 5个实验组细胞、阴性对照组细胞和阳性对照组细胞，分别向3组细胞悬液中加入2μL生物素标记的γδT抗体(Biolegend，331204)，4-8℃避光孵育10min，加入1mL Buffer(2％FBS的PBS)重悬，离心洗涤，再加入2μL CD3-APC(BD，555335)、αβ-PE/CY7(Biolegend，306720)、SA-PE(Biolegend，405204)和7AAD-PERCP(eBioscience，00-6993-50)，4-8℃避光孵育10min，离心洗涤，重悬于200μL的Buffer中，用MACSQuant 10流式细胞仪(购于德国美天旎生物技术有限公司)检测，FlowJo分析3组细胞的γδT比例。
结果如图5所示。从图中可以看出：扩大培养14天后，在K562-shFPPS细胞系刺激下可使分选的γδT细胞获得100倍以上的扩增。说明肿瘤细胞K562-shFPPS可促进γδT细胞的扩增，提高γδT细胞的扩增效率。
实施例2、CAR-γδT细胞的生产方法
一、CAR22慢病毒颗粒的制备
本发明应用CAR结构对γδT细胞进行基因修饰，CAR结构从氨基端到羧基端依次为：ScFv(CD22)-Hinge(CD8)-TM(CD8)-CD137-CD3ζ，即从氨基端到羧基端依次为：源于CD22单克隆抗体(克隆号为M971)的单链可变区、CD8a铰链区及跨膜区、CD137信号域和CD3ζ链胞内区。其氨基酸序列如序列表的序列3所示，核苷酸序列如序列表的序列4所示。将序列4所示的DNA分子***Senl_pLenti-EF1载体(Senl_pLenti-EF1载体为在原始质粒克隆位点两侧增加了酶切位点PacI和SpeI后得到的载体，原始质粒名称为LV-pRRLEF1.WPRE，由赛业生物科技有限公司提供，合同号为S1002079)的酶切位点PacI和SpeI之间，得到CAR22慢病毒载体。
按照实施例1步骤一的2中的方法对CAR22慢病毒载体进行包装，得到CAR22 慢病毒颗粒。
二、CAR-γδT细胞的制备
1、γδT细胞分离及分选(Day0)
无菌采集健康成人的静脉血50-100mL，密度梯度离心获取外周血单个核细胞(PBMC)。用γδT细胞分选试剂盒(购于德国美天旎生物技术有限公司，产品目录号130-092-892)从PBMC中分选γδT细胞，得到γδT细胞。
2、γδT细胞的培养
将步骤1中分选的γδT细胞用含有200U/ml人重组IL-2的TexMACS培养基重悬，得到γδT细胞培养体系，根据培养体系组成的不同分为如下各组进行培养：
K562-shFPPS组：向γδT细胞培养体系中加入K562-shFPPS细胞，γδT细胞与K562-shFPPS细胞的个数比为3:1；
K562+ZOL组：向γδT细胞培养体系中加入K562细胞和ZOL，γδT细胞与K562细胞的个数比为3:1，ZOL在培养体系中的终浓度为10μM；
PBMC+ZOL组：向PBMC细胞中加入ZOL，ZOL在培养体系中的终浓度为10μM；
按照5×10 5个γδT细胞/500μL培养基/孔铺入24孔板，置于37℃，5％CO 2细胞培养箱培养2天。
3、慢病毒转染细胞(Day2)
培养2天后，将CAR22慢病毒颗粒分别转染各组细胞。具体转染步骤如下：将24孔板提前从培养箱内取出，每孔加入1‰Protamine sulfate(购于Sigama公司，产品目录号P3369-10G)和5μL CAR22慢病毒颗粒溶液，在操作台上画“8字”，并用移液枪吹吸混匀。35℃，2000rpm离心2h。离心结束后将24孔板从离心机中取出，移入37℃，5％CO 2细胞培养箱继续培养。
4、扩大培养(Day3-14)
培养3天时进行半换液，将24孔板从培养箱取出，每孔吸出一半的上清液，并补充一半的DMEM完全培养基，继续培养。当细胞浓度到达(1.5-2)×10 6/ml的状态下，转瓶或转袋，每隔2天补加一次200IU/ml的IL-2。细胞培养至第10-14天，取200μL细胞悬液进行Trypan Blue计数，并进行后续的细胞鉴定、分选，以及功能测试。
三、流式细胞仪检测CAR-γδT细胞
1、生物素标记CD22-Fc的制备
取100μg纯化CD22-Fc蛋白粉末(购于义翘神州生物技术有限公司，货号11958-H02H)，用pH7.2的PBS重悬，使其终浓度为(0.5-1)μg/μL；用纯度>99.9％的二甲基亚砜(DMSO)溶解适量生物素(购于苏州宇恒生物科技有限公司，货号：B5026-1)配制成2mM的生物素悬液；按CD22-Fc蛋白：生物素＝1:10的摩尔比例将二者混匀，室温静置1小时，每隔15分钟混匀一次；用脱盐柱脱盐(操作步骤参考脱盐柱使用说明书)，根据体积不同，可以选择PD-10(购于美国GE公司，货 号17-0851-01)或G-25脱盐柱(购于美国GE公司，货号28-9180-04)，得到生物素标记CD22-Fc。
2、流式细胞仪检测细胞表面CAR(CD22)的表达水平
慢病毒转染后48-72小时，取各组细胞检测转染效率(转染效率为CD22-Fc标记为阳性的γδT细胞在所有γδT细胞中的百分比)。具体步骤如下：每管(1-2)×10 5个细胞，加入1μL生物素标记的CD22-Fc，4-8℃避光孵育10min，加入1mL的(PBS+2％FBS)重悬，离心洗涤。再加入SA-PE、CD3-APC、CD4-PE-Cy7、CD8-VioBlue和7AAD(购于德国美天旎生物技术有限公司)，4-8℃避光孵育10分钟，离心洗涤，200μL Buffer重悬，用MACSQuant 10流式细胞仪检测，FlowJo分析CAR(CD22)的表达水平。
检测结果如图7所示。从图中可以看出：K562-shFPPS组的转染效率显著高于K562+ZOL组和PBMC+ZOL组。说明肿瘤细胞K562-shFPPS可提高慢病毒对γδT细胞的转染效率。
四、分选纯化CAR-γδ阳性T细胞
用上述方法培养到第7-10天的CAR22修饰的γδT细胞进行分选纯化，得到纯化后的CAR-γδ阳性T细胞。具体步骤如下：收集培养到第7-10天的CAR-γδT细胞，离心弃上清，按照每1×10 7CAR-γδT细胞加入80μL缓冲液(PBS+2％FBS)和5μL的生物素标记CD22-Fc的比例加入缓冲液和生物素标记CD22-Fc，4-8℃避光孵育15分钟，孵育结束后，按照每1×10 7CAR-γδT细胞加入1-2mL缓冲液，离心洗涤，洗涤后，按照每1×10 7CAR-γδT细胞加入80μL缓冲液(PBS+2％FBS)和10μL SA磁珠的比例加入缓冲液和SA磁珠，混匀，4-8℃避光孵育30分钟，孵育结束后，按照每1×10 7CAR-γδT细胞加入1-2mL缓冲液，离心洗涤，按照每1×10 8细胞加入500μL缓冲液吹吸混匀细胞沉淀层，用磁力分选柱分选CAR-γδ阳性T细胞，滴下的细胞悬液即为分选得到的CAR-γδ阴性T细胞，将其记作CAR-γδT cells；而留在磁力柱中的细胞即为CAR-γδ阳性T细胞，将其记作CAR+γδT cells。收集分选所得阳性细胞，计数，流式检测CAR阳性T细胞的纯度，剩余细胞继续培养或直接用于后续的功能测试。
五、CAR-γδT细胞体外功能测试
1、靶细胞株的建立
(1)K562-CD22细胞
为构建表达特异性抗原CD22的肿瘤细胞株，从健康人PBMC中克隆获得CD22全长cDNA序列(Genebank号为NM_001771.3)，将CD22全长cDNA序列***到携带有Puromycine抗性基因的U6-Based shRNA Knockdown慢病毒质粒(购于赛业生物科技有限公司)中，得到慢病毒质粒。按照实施例1步骤一的2和3中的方法对慢病毒质粒进行包装并转染至K562细胞中，经筛选获得纯度较高的K562-CD22细胞。
(2)K562-CD19细胞
为构建表达特异性抗原CD19的肿瘤细胞株，从健康人PBMC中克隆获得CD19全长cDNA序列(Genebank号为NM_001770.5)，将CD19全长cDNA序列***到携带有Puromycine抗性基因的U6-Based shRNA Knockdown慢病毒质粒(购于赛业生物科技有限公司)中，得到慢病毒质粒。按照实施例1步骤一的2和3中的方法对慢病毒质粒进行包装并转染至K562细胞中，经筛选获得纯度较高的K562-CD19细胞作为阴性对照。
(3)LCL细胞
LCL细胞为EB病毒感染健康成人外周B细胞获得的永生细胞系，细胞表面表达CD22抗原。具体制备方法如下：将B95.8细胞(购于中国科学院细胞库)接种于T75cm 2细胞培养瓶中，浓度为3×10 5个/mL，培养基为RPMI1640(购于gibco，产品目录号为22400-088)+10％FBS(购于gibco，产品目录号为10099141)。37℃、5％CO 2培养箱中培养48h后，按照1×10 6个/mL浓度重新接种细胞，并加入终浓度为20ng/mL的TPA(购于cayman，产品目录号为16561-29-8)处理细胞。1h后，RPMI1640完全培养基清洗细胞3次，以去除TPA。将所得细胞按1×10 6个/mL浓度再次接种，37℃、5％CO 2培养箱中培养96h，收集培养液于50mL离心管中，4℃，600g离心10min，所得上清经0.45μm滤器过滤，滤液即为EBV病毒悬液。将密度梯度离心获取的外周血单个核细胞(PBMC)进行CD3(购于德国美天旎生物技术有限公司，产品目录号130-050-101)分选。所得CD3-细胞即B细胞按照3×10 5个/mL浓度接种于培养瓶中，加入1/10体积的EBV病毒悬液。EBV病毒感染一周后，倒置显微镜下可见细胞团簇，即为转化成功的LCL细胞。随着时间推移，细胞团簇越变越大，可根据细胞数量及培养基颜色进行扩增与冻存。
2、流式细胞仪测定细胞因子IFN-γ和表面CD137的表达水平
将步骤1获得的靶细胞株以每孔1×10 4的数量加入到96孔U型底细胞培养板中；然后按效应细胞(CAR-γδ阳性T细胞)：靶细胞分别为10:1、3:1、1:1的比例将相应数量分选纯化的CAR-γδ阳性T细胞分别加入对应各孔，分别设置效应细胞和靶细胞空白对照孔，并以CAR-γδ阴性T细胞作为对照的效应细胞，设立相同的检测。然后将细胞培养板置于37℃、5％CO 2培养箱中共培养4小时，将细胞悬液吸出，PBS洗涤两次，100μL Buffer重悬细胞，分别加入2μL CD3-APC-Cy7、CD4-PE.Cy7、CD8-VioBlue和CD137-PE(BD公司，克隆号为4B4-1)，4-8℃避光孵育10分钟，洗涤后，使用BD公司的细胞内染色试剂盒(货号554714)，染色方法参考说明书。相应试剂穿透细胞膜后，加入5μL的鼠抗人IFN-γ-FITC单克隆抗体(BD公司，克隆号为B27)，4-8℃避光孵育10分钟后进行洗涤，洗涤后流式细胞仪检测IFN-γ和CD137的表达水平。
检测结果如图8所示。从图中可以看出：K562-shFPPS细胞与γδT细胞共培养生产的CAR+γδT细胞对表达CD22的肿瘤细胞发生特异性应答，分泌更多的IFN-γ，细胞表面表达更多的CD137。
3、流式细胞仪测定CAR-γδT的特异杀伤活性
按照实施例1步骤二的2中的方法对步骤1中的各个靶细胞株进行CFSE标记，室温或者37℃避光孵育20min，加入5倍体积含有10％(体积分数)FBS的培养基终止染色。2000rpm离心5min，离心后重悬细胞，室温或者37℃避光孵育10min，孵育结束后，清洗细胞2次，重悬后待用。在96孔细胞培养板中，每孔加入1×10 4的CFSE标记的靶细胞；然后按效应细胞(CAR-γδ阳性T细胞)：靶细胞分别为10:1、3:1、1:1的比例将相应数量分选纯化的CAR-γδT阳性细胞分别加入对应各孔，分别设置效应细胞和靶细胞空白对照孔，并以CAR-γδ阴性T细胞作为对照的效应细胞，设立相同的检测。然后将细胞培养板置于37℃、5％CO 2培养箱中共培养4小时，将细胞悬液吸出，PBS洗涤两次，100μL Buffer重悬细胞，加入5μL 7-AAD，避光孵育10分钟进行洗涤，洗涤后流式细胞仪检测杀伤率，杀伤率为被杀伤的靶细胞(CFSE+7AAD+)占所有靶细胞(CSFE+)的百分比。
结果如图9所示。从图中可以看出：K562-shFPPS细胞与γδT细胞共培养生产的CAR+γδT细胞能特异性裂解表达CD22的肿瘤细胞。
工业应用
为了克服现有技术的缺陷，本发明提供了一种全新的γδT细胞扩增方案，该方案具体如下：将靶向于FPP合成酶(FPP Synthase，FPPS)的shFPPS通过慢病毒载体转染K562细胞，下调了K562细胞中FPPS的表达量，构建了FPPS表达量降低的K562-shFPPS细胞系。本发明将K562-shFPPS细胞系加入γδT细胞培养体系中与γδT细胞进行共培养，发现K562-shFPPS细胞系能直接刺激纯化的Vγ9δ2T细胞的体外分化和扩增。本发明还将表达嵌合抗原受体的慢病毒载体加入含有K562-shFPPS细胞系的γδT细胞培养体系中进行共培养，发现K562-shFPPS细胞系还可以有效提高CAR基因的转染效率，进而提高CAR-γδT细胞的制备效率。上述实验结果表明：K562-shFPPS细胞系可有效提高γδT细胞的体外扩增能力以及CAR基因的转染效率，本发明提供的方案有效解决了大规模生产CAR-γδT细胞的技术瓶颈，具有良好的应用前景。

Claims (17)

  1. 一种γδT细胞的扩增方法，包括在γδT细胞培养体系中添加FPPS表达量和/或活性降低的肿瘤细胞的步骤。
  2. 根据权利要求1所述的方法，其特征在于：所述γδT细胞与所述FPPS表达量和/或活性降低的肿瘤细胞的个数比为(1-10):1。
  3. 根据权利要求1所述的方法，其特征在于：所述方法还包括如下步骤：当培养体系中细胞浓度到达(1.5-2)×10 6个/ml时，每2-4天向所述培养体系中添加一次IL-2。
  4. 根据权利要求3所述的方法，其特征在于：添加IL-2时的标准是使所述IL-2在所述培养体系中的终浓度为(100-1000)IU/mL。
  5. 一种生产CAR-γδT细胞的方法，包括如下步骤：将表达嵌合抗原受体的载体与γδT细胞共培养，得到CAR-γδT细胞；所述培养体系中含有FPPS表达量和/或活性降低的肿瘤细胞。
  6. 根据权利要求5所述的方法，其特征在于：所述将表达嵌合抗原受体的载体与γδT细胞共培养的方法包括如下步骤：
    1)将FPPS表达量和/或活性降低的肿瘤细胞加入γδT细胞培养体系中，培养，得到培养体系；
    2)将表达嵌合抗原受体的载体加入所述培养体系中，培养，得到所述CAR-γδT细胞。
  7. 根据权利要求6所述的方法，其特征在于：所述1)中，所述γδT细胞与所述FPPS表达量和/或活性降低的肿瘤细胞的个数比为(1-10):1。
  8. 根据权利要求6所述的方法，其特征在于：所述2)中，当培养体系中细胞浓度到达(1.5-2)×10 6个/ml时，每2-4天向所述培养体系中添加一次IL-2。
  9. 根据权利要求6所述的方法，其特征在于：所述2)中，所述表达嵌合抗原受体的载体为表达嵌合抗原受体的慢病毒载体。
  10. 一种肿瘤免疫治疗的方法，包括如下步骤：
    (1)按照权利要求5-9任一所述的方法生产CAR-γδT细胞；
    (2)将所述CAR-γδT细胞回输肿瘤患者体内，通过所述CAR-γδT细胞识别并杀伤所述肿瘤患者体内的肿瘤细胞，进而实现肿瘤免疫治疗的目的。
  11. 一种产品，其活性成分为FPPS表达量和/或活性降低的肿瘤细胞；
    所述产品的功能为如下B1)-B5)中任一种：
    B1)促进γδT细胞扩增；
    B2)促进γδT细胞分化；
    B3)生产CAR-γδT细胞；
    B4)提高慢病毒对γδT细胞的转染率；
    B5)肿瘤免疫治疗。
  12. 根据权利要求1-10任一所述的方法或权利要求11所述的产品，其特征在于：所述FPPS表达量和/或活性降低的肿瘤细胞是将抑制FPPS编码基因表达的物质导入肿瘤细胞得到的。
  13. 根据权利要求12所述的方法或产品，其特征在于：所述抑制FPPS编码基因表达的物质通过慢病毒载体导入肿瘤细胞。
  14. 根据权利要求12所述的方法或产品，其特征在于：所述抑制FPPS编码基因表达的物质为抑制FPPS编码基因表达的shRNA。
  15. 根据权利要求14所述的方法或产品，其特征在于：所述抑制FPPS编码基因表达的shRNA为由茎I、环和茎II形成的茎环结构的单链RNA；
    所述茎I的序列如序列表的序列2第1-21位所示；所述环的序列如序列表的序列2第22-27位所示；所述茎II的序列如序列表的序列2第28-48位所示。
  16. 根据权利要求1-10任一所述的方法或权利要求11所述的产品，其特征在于：所述肿瘤细胞为慢性骨髓性白血病细胞系。
  17. 根据权利要求16所述的方法或产品，其特征在于：所述慢性骨髓性白血病细胞系为K562细胞。
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