CAR-gamma delta T cell targeting CD19 and preparation method and application thereof

Abstract

The invention discloses a CAR-gamma delta T cell targeting CD19 as well as a preparation method and application thereof, and belongs to the technical field of biomedical treatment. The invention provides a CAR-gamma delta T cell targeting CD19. The CAR-gamma delta T cell can effectively recognize and kill CD19 positive tumor cells. The CAR-gamma delta T cell shows a good anti-tumor effect in vitro and in vivo, provides a novel immunotherapy strategy of non-human leukocyte antigen dependence, natural immunity enhancement and broad-spectrum tumor killing for CD19 positive tumors such as acute lymphocytic leukemia and non-Hodgkin lymphoma, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a CAR-gammadelta T cell targeting CD19 and a preparation method and application thereof. BACKGROUND

[0002] CD19 is an important marker molecule on the surface of B cells and is widely expressed in B cell-related tumors such as acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). Targeted therapy based on CD19 has become a popular direction of immunotherapy in recent years. Among them, chimeric antigen receptor T cell (CAR-T) therapy endows T cells with the ability to specifically recognize tumor antigens through genetic engineering technology, and has achieved remarkable results in clinical practice. CAR-T cell therapy has solved the limitations of traditional chemotherapy and radiotherapy to a certain extent, and has shown strong efficacy in the treatment of B cell-related malignancies.

[0003] However, traditional CAR-T cells are mainly based on alpha beta T cells, which have limitations such as difficulty in in vitro expansion and short in vivo survival time, affecting the persistence and wide applicability of the therapeutic effect. In addition, the therapeutic effect of alpha beta T cells is limited by the dependence of HLA molecules (human leukocyte antigen), which means that CAR-T cells prepared using alpha beta T cells can only be prepared using the patient's own immune cells, and the patient's own immune system is in an immunosuppressed state, affecting the universality of CAR-T cell therapy and the therapeutic effect. Therefore, although CAR-T cell therapy has achieved positive results, these limitations have affected its therapeutic effect and the diversity of treatment objects. SUMMARY

[0004] In order to solve the above-mentioned problems existing in the prior art, the purpose of the present application is to provide a CAR-gammadelta T cell targeting CD19 and a preparation method and application thereof.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0006] The present application provides a CAR-gammadelta T cell targeting CD19, wherein the CAR-gammadelta T cell is a gammadelta T cell into which a CAR gene is introduced, and the structure of the CAR gene comprises a promoter, a signal peptide, an extracellular domain recognizing a CD19 antigen, a transmembrane domain and an intracellular signal transduction domain.

[0007] Further, the promoter is EF1a, the signal peptide is SP, the extracellular domain is a single-chain antibody FMC63 Anti-CD19 scFv, the transmembrane domain is CD8a, and the intracellular signal transduction domain is 4-1BB and CD3zeta.

[0008] Further, the structure of the CAR gene is EF1a-SP-FMC63 Anti-CD19scFv-CD8a-4-1BB-CD3zeta.

[0009] The application further provides a method for preparing the CAR-gammadelta T cell, comprising the following steps:

[0010] (1) isolating peripheral blood mononuclear cells of a donor, and expanding gammadelta T cells;

[0011] (2) co-culturing the gammadelta T cells obtained in step (1) with a lentivirus loaded with a CAR gene, to obtain the CAR-gammadelta T cell.

[0012] Further, the donor in step (1) is a healthy person or a patient with CD19-positive malignant tumor;

[0013] The expansion of the gammadelta T cells comprises the following steps:

[0014] (a) culturing in a culture medium containing an activation factor;

[0015] (b) culturing in a culture medium containing an expansion factor, to obtain the gammadelta T cells;

[0016] wherein the activation factor in step (a) is at least one of interleukin and bisphosphonate, the culture medium is a serum-free culture medium, and the culturing time is 1-5 days; the expansion factor in step (b) is interleukin, the culture medium is a serum-free culture medium, and the culturing time is 1-8 days.

[0017] Further, the donor is a healthy person, the culturing time in step (a) is 3 days, and the culturing time in step (b) is 3-4 days.

[0018] Further, the interleukin in step (a) is at least one of IL-2 and IL-5, the bisphosphonate is at least one of zoledronic acid, etidronic acid, ibandronic acid, pamidronic acid, alendronic acid, risedronic acid, and milodic acid, and the serum-free culture medium is CST TM opTmizer TM culture medium; the interleukin in step (b) is at least one of IL-2 and IL-5, and the serum-free culture medium is CST TM opTmizer TM culture medium.

[0019] Further, the interleukin in step (a) is IL-2 and IL-5, and the bisphosphonate is zoledronic acid; the interleukin in step (b) is IL-2 and IL-5.

[0020] Further, the concentration of IL-2 in step (a) is 100IU / mL-800IU / mL, the concentration of IL-5 is 1ng / mL-30ng / mL, and the concentration of zoledronic acid is 1μM-10μM; the concentration of IL-2 in step (b) is 100IU / mL-800IU / mL, and the concentration of IL-5 is 1ng / mL-30ng / mL.

[0021] Further, the concentration of IL-2 in step (a) is 400IU / mL, the concentration of IL-5 is 10ng / mL, and the concentration of zoledronic acid is 4μM; the concentration of IL-2 in step (b) is 400IU / mL, and the concentration of IL-5 is 10ng / mL.

[0022] Further, the cell inoculation density of the expanded γδT cells in step (1) is (2-3)×10 6 cells / mL.

[0023] Further, the method further comprises the following steps: expanding the CAR-γδT cells obtained in step (2), and sorting and purifying the expanded CAR-γδT cells with magnetic beads containing CD19 antigens; the cell inoculation density of the expanded CAR-γδT cells is (1-2)×10 6 cells / mL.

[0024] Further, the expansion is carried out in a culture medium containing expansion factors; the expansion factors are IL-2 with a concentration of 400IU / mL and IL-5 with a concentration of 10ng / mL, and the culture medium is CST TM opTmizer TM medium; the culture time is 3-7 days.

[0025] The application also provides the use of the above-mentioned CAR-γδT cells in the preparation of a drug for treating CD19-positive malignant tumors.

[0026] Further, the CD19-positive malignant tumor is acute lymphoblastic leukemia, chronic lymphocytic leukemia or B-cell lymphoma.

[0027] The application also provides the use of the above-mentioned CAR-γδT cells in the treatment of CD19-positive malignant tumors.

[0028] Further, the CD19-positive malignant tumor is acute lymphoblastic leukemia, chronic lymphocytic leukemia or B-cell lymphoma.

[0029] The application has the following beneficial effects:

[0030] Compared with existing technologies, the CD19-targeting CAR-γδT cells of this invention have the following significant advantages and innovations:

[0031] a) Combination of innate immune function with CAR targeting: This invention combines γδT cells with innate immune function with CAR technology, which not only enhances the specific recognition and killing ability of tumor cells, but also effectively identifies immune-evading tumor cells.

[0032] b) Low immune rejection: γδT cells are not dependent on MHC molecules, have a wider range of applications, can be used in allogeneic transplantation therapy, and reduce the limitations of HLA matching;

[0033] c) High expansion and high purity: Through optimized culture and transduction conditions, CAR-γδT cells have a strong ability to expand in vitro, and high-purity CAR-γδT cells can be obtained after purification with magnetic beads.

[0034] d) Broad application prospects: The CAR-γδT cells of the present invention can effectively treat CD19-positive malignant tumors, including acute lymphoblastic leukemia and non-Hodgkin lymphoma, providing an innovative treatment option for CD19-positive patients.

[0035] In summary, this invention provides a CD19-targeting CAR-γδT cell. This CAR-γδT cell effectively recognizes and kills CD19-positive malignant tumor cells by linking a single-chain antibody (scFv) recognizing the CD19 antigen with the CD3ζ signaling chain and the T cell co-stimulatory molecule 4-1BB. Compared to traditional CAR-αβT cells, the CAR-γδT cell of this invention represents a novel immunotherapy strategy that is HLA-independent, enhances innate immunity, and exhibits broad-spectrum tumor killing. The CAR-γδT cell of this invention demonstrates good anti-tumor effects both in vitro and in vivo, and can be widely used to treat CD19-positive tumors such as acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL), showing promising application prospects.

[0036] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0037] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0038] Figure 1This is a bisphosphonate screening assay. A represents the fold increase in γδT cells with different bisphosphonates; B represents the fold increase in γδT cells with different concentrations of zoledronic acid; C represents the fold increase in γδT cells with different concentrations of IL-2; and D represents the fold increase in γδT cells with different concentrations of IL-15.

[0039] Figure 2 The images show flow cytometry plots of γδT cells. A represents the flow cytometry results from expansion to D7; B represents the flow cytometry results from expansion to D14.

[0040] Figure 3 This is a schematic diagram of the structure of the chimeric antigen receptor (CAR) gene that targets CD19.

[0041] Figure 4 Flow cytometry results for CAR-γδT cells targeting CD19. A shows the flow cytometry results after lentiviral transfection, and B shows the flow cytometry results after CD19 purification with magnetic beads.

[0042] Figure 5 The results of in vitro cytotoxicity experiments of CAR-γδT cells targeting CD19.

[0043] Figure 6 Results of cytokine release assay in CAR-γδT cells targeting CD19.

[0044] Figure 7 To assess the antitumor effect of CD19-targeted CAR-γδT cells in a mouse tumor model. Detailed Implementation

[0045] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0046] The following experiments, where no temperature is specified, are reactions conducted under normal temperature conditions, which is room temperature, or 25±5℃.

[0047] Example 1: Preparation method of CD19-targeting CAR-γδT cells

[0048] 1. Screening for high-purity γδT cell expansion methods

[0049] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor peripheral blood and obtained by density gradient centrifugation; serum-free culture medium CST was used. TM OpTmizer TM Resuspend PBMCs and seed cells at a rate of 2 × 10⁶ cells / year. 6Cells were cultured at 37°C and 5% CO2 for 3 days (defined as D0–D3). The old culture medium was removed, and new serum-free culture medium was added with the following amplification factors (concentrations refer to final concentrations): IL-2 (100 IU / mL–500 IU / mL), IL-15 (1 ng / mL–20 ng / mL), and bisphosphonates (1 μM–10 μM). Cells were then cultured in new culture containers at a density of (1–2) × 10⁶ cells / mL. 6 Cells were cultured at 100 cells / mL at 37°C and 5% CO2 until day 7. Amplification factors (concentrations below refer to final concentrations) were added: IL-2 (100 IU / mL–500 IU / mL) and IL-15 (1 ng / mL–20 ng / mL). Cells were then seeded into new culture containers for further expansion. γδT cells were harvested on day 14, and cell counts were performed. The cell count was compared with the initial cell count to calculate the fold increase. Different bisphosphonate grouping experiments were conducted according to Table 1 to screen for the optimal phosphonate for expanding and activating γδT cells. Different concentrations of zoledronic acid were conducted according to Table 2 to screen for the optimal zoledronic acid concentration for expanding γδT cells. Different concentrations of IL-2 were conducted according to Table 3 to screen for the optimal IL-2 concentration for expanding γδT cells. Different concentrations of IL-15 were conducted according to Table 4 to screen for the optimal IL-15 concentration for expanding γδT cells.

[0050] Table 1 Different bisphosphonate groups

[0051]

[0052] Table 2. Zoledronic acid usage concentration grouping

[0053]

[0054] Table 3. IL-2 grouped by concentration

[0055]

[0056] Table 4 shows IL-15 grouped by concentration.

[0057]

[0058] Results of fold expansion of γδT cells with different phosphonates are as follows Figure 1 As shown in Figure A, the amplification fold using zoledronic acid was significantly different compared to pamidronate and alendronate. Therefore, zoledronic acid was selected as the bisphosphonate for subsequent experiments.Figure 1 In group B, the γδT cells amplified using 4 μM zoledronic acid showed the highest fold increase. Figure 1 In group C, the γδT cells increased by the highest fold when IL-2 was used at a concentration of 400 IU / mL. Figure 1 In experiment D, the γδT cell amplification rate was highest when IL-15 at a concentration of 10 ng / mL. In subsequent experiments, the concentration of the γδT cell amplification factor used in this invention was consistently the aforementioned preferred concentration.

[0059] 2. Flow cytometry phenotyping analysis of γδT cells

[0060] γδT cell culture was performed according to the method described in step 1, and appropriate cell samples were taken on day 7 (D7) and day 14 (D14) of culture.

[0061] Cell samples were washed twice with PBS buffer and then incubated at room temperature in the dark for 30 minutes using human CD3 antibody (FITC-labeled) and Vδ2 antibody (PE-labeled). After staining, the cells were washed again with PBS buffer and resuspended in 500 μL of PBS for flow cytometry analysis. Results are as follows: Figure 2 The results showed that CD3+Vδ2+γδT cell populations could be detected at both D7 and D14 time points. The purity of γδT cells at D14 (95.76%) was significantly higher than that at D7 (66.32%), indicating that γδT cells continued to expand and had a stable phenotype during culture.

[0062] 3. Preparation method of CD19-targeting CAR-γδT cells

[0063] (1) γδT cell expansion

[0064] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor peripheral blood and γδT cells were expanded according to the factors and concentrations selected in step 1.

[0065] (2) Transduction of CAR gene

[0066] On day 7, γδT cells were transferred to culture vessels coated with RetroNectin (recombinant human fibronectin) and loaded with a lentivirus (LV Transm, purchased from iCarTAB, CAT#LVTran100) for expressing the anti-CD19 chimeric antigen receptor (CAR) gene. The CAR structure was as follows. Figure 3 As shown, after 24 hours of culture, the CAR gene transduction was completed. EF1α (Gene ID: 1915), SP-FMC63-CD8α sequence (i.e. Figure 3The SP-FMC63 Anti-CD9 scFv-CD8αTM sequence was obtained from (GenBank: MN702884.1); the 4-1BB-CD3ζ sequence was obtained from (GenBank: KX055828.1). All of the above sequences were obtained from NCBI.

[0067] The culture vessel coated with RetroNectin and loaded with a lentivirus for expressing the anti-CD19 chimeric antigen receptor gene was prepared by the following method:

[0068] (a) Add 10 μg / mL RetroNectin to the culture container into which γδT cells have been transferred, and coat overnight at 2℃~6℃ for 8~16 hours;

[0069] (b) Remove uncoated RetroNectin and add blocking solution (PBS solution containing 1% BSA) to seal the culture vessel;

[0070] (c) Add lentivirus loaded with the anti-CD19 chimeric antigen receptor gene to the culture vessel and centrifuge at 30℃~34℃ and 1200g~1800g for 1.5h~2.5h to remove uncoated lentivirus.

[0071] (3) Expansion and harvesting of CAR-γδT cells

[0072] After 24 hours, the medium was changed and amplification factors IL-2 and IL-15 were added (final concentrations were the preferred concentrations corresponding to those in step 1), and the cell seeding density was maintained at (1–2) × 10⁻⁶. 6 Cells / mL, continue culturing to D14, and harvest CAR-γδT cells.

[0073] (4) Purification of CAR-γδT cells

[0074] Harvested CAR-γδT cells were sorted using magnetic beads containing CD19 antigen to remove untransduced cells, ensuring high purity CAR-γδT cells. Flow cytometry was used to detect the expression rate of the CAR gene to ensure that the transduction efficiency reached more than 90%.

[0075] The following experimental examples demonstrate the beneficial effects of the present invention.

[0076] Experimental Example 1: Detection of Surface Markers in CAR-γδT Cells

[0077] CAR-γδT cells were collected before and after purification with magnetic beads (step (4) in step 3 of Example 1), and the expression of the CAR gene (CD19) on the cell surface was detected by flow cytometry to verify the phenotype and transfection success rate of CAR-γδT cells.

[0078] The results showed that the expression purity of the CAR gene (CD19) after lentiviral transfection was 51.9%. Figure 4 A) After purification with magnetic beads, the purity of CAR gene expression was 98.8%. Figure 4 B).

[0079] This invention demonstrates that the present invention yields high-purity CAR-γδT cells with surface CAR gene expression (CD19).

[0080] Experiment Example 2: Detection of CAR-γδT cell killing function in vitro

[0081] CD19-targeted CAR-γδT cells, mock CAR-γδT cells transfected with empty vectors, and CD19-positive Raji tumor cells were co-cultured for 24 hours at different effector-to-target ratios (e.g., 1:1, 5:1, 10:1). The mortality rate of tumor cells was analyzed using the lactate dehydrogenase (LDH) release assay to evaluate the in vitro killing ability of CAR-γδT cells.

[0082] The preparation method of mock CAR-γδT cells transfected with empty vector is the same as step 3 of Example 1, except that the lentivirus loaded with the gene for expressing anti-CD19 chimeric antigen receptor (CAR) is replaced with a lentivirus loaded with empty vector.

[0083] The lactate dehydrogenase (LDH) release method is described below:

[0084] https: / / www.abcam.com / en-us / knowledge-center / cell-biology / ldh-assay-kit-guide-principles-and-application

[0085] The results showed that, compared with mock CAR-γδT cells, CD19-targeted CAR-γδT cells had a significant killing effect on Raji cells, and the killing rate was directly proportional to the effector-to-target ratio. Figure 5 ).

[0086] Experiment Example 3: Detection of CAR-γδT Cytokine Release

[0087] CAR-γδT cells targeting CD19, mock CAR-γδT cells transfected with an empty vector, and CD19-positive tumor cells (Raji) were co-cultured at a 1:1 ratio, and the supernatant was collected. The levels of cytokines IFN-γ and TNF-α were detected using enzyme-linked immunosorbent assay (ELISA).

[0088] The results showed that, compared with mock CAR-γδT cells, CD19-targeting CAR-γδT cells, when co-cultured with tumor cells, significantly increased the levels of cytokines IFN-γ and TNF-α, further demonstrating their activation state and functionality. Figure 6 ).

[0089] Experiment Example 4: Antitumor Effect of CAR-γδT Cells in Animal Models

[0090] (1) Tumor model establishment

[0091] A B-cell tumor model was established by inoculating NOD / SCID mice with CD19-positive Raji cells.

[0092] (2) CAR-γδT cell therapy

[0093] Tumor model mice were randomly divided into three groups: experimental group: injected with CAR-γδT cells (5×10⁻⁶ cells / mL). 6 (5 × 10⁸ cells / cell); Negative control group: mock CAR-γδT cells injected with blank transduction vector (5 × 10⁸ cells / cell); 6 (1 cell / mouse); blank control group: injected with PBS buffer. Each group was administered to mice via tail vein infusion, and tumor volume and mouse survival were monitored.

[0094] The results showed that compared with the PBS and mock CAR-γδT groups, tumor growth in the experimental group was significantly inhibited, and the tumor volume was significantly smaller than that in the control group. Figure 7 ).

[0095] In summary, this invention provides a CD19-targeting CAR-γδT cell. This CAR-γδT cell effectively recognizes and kills CD19-positive malignant tumor cells by linking a single-chain antibody (scFv) recognizing the CD19 antigen with the CD3ζ signaling chain and the T cell co-stimulatory molecule 4-1BB. Compared to traditional CAR-αβT cells, the CAR-γδT cell of this invention represents a novel immunotherapy strategy that is HLA-independent, enhances innate immunity, and exhibits broad-spectrum tumor killing. The CAR-γδT cell of this invention demonstrates good anti-tumor effects both in vitro and in vivo, and can be widely used to treat CD19-positive tumors such as acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL), showing promising application prospects.

Claims

1. A CAR-γδT cell targeting CD19, characterized in that, The CAR-γδT cells are γδT cells infused with the CAR gene, which contains a promoter, a signal peptide, an extracellular domain that recognizes the CD19 antigen, a transmembrane domain, and an intracellular signal transduction domain.

2. The CAR-γδT cells according to claim 1, characterized in that, The promoter is EF1α, the signal peptide is SP, the extracellular domain is the single-chain antibody FMC63 Anti-CD19scFv, the transmembrane domain is CD8αTM, and the intracellular signal transduction domains are 4-1BB and CD3ζ.

3. The CAR-γδT cells according to claim 1, characterized in that, The structure of the CAR gene is EF1α—SP—FMC63 Anti-CD19 scFv—CD8αTM—4-1BB—CD3ζ.

4. A method for preparing CAR-γδT cells according to any one of claims 1 to 3, characterized in that, The method includes the following steps: (1) Isolate peripheral blood mononuclear cells from the donor and expand γδT cells; (2) Co-culture the γδT cells obtained in step (1) with lentivirus loaded with the CAR gene to obtain CAR-γδT cells.

5. The method according to claim 4, characterized in that, The donor in step (1) is a healthy person or a CD19-positive malignant tumor patient; The expansion of γδT cells includes the following steps: (a) Cultured in a medium containing activating factors; (b) Incubate in a medium containing amplification factors to obtain the product; Wherein, the activating factor in step (a) is at least one of interleukin and bisphosphonate, the culture medium is serum-free culture medium, and the culture time is 1 to 5 days; the amplification factor in step (b) is interleukin, the culture medium is serum-free culture medium, and the culture time is 1 to 8 days. Preferably, the donor is a healthy person, the culture time in step (a) is 3 days, and the culture time in step (b) is 3 to 4 days.

6. The method according to claim 5, characterized in that, In step (a), the interleukin is at least one of IL-2 and IL-5, the bisphosphonate is at least one of zoledronic acid, etidronic acid, ibandronic acid, pamidronate, alendronate, risedronate, and mirtonic acid, and the serum-free culture medium is CST. TM opTmizer TM Culture medium; the interleukin in step (b) is at least one of IL-2 and IL-5, and the serum-free culture medium is CST. TM opTmizer TM Culture medium; Preferably, the interleukins in step (a) are IL-2 and IL-5, and the bisphosphonate is zoledronic acid; the interleukins in step (b) are IL-2 and IL-5.

7. The method according to claim 6, characterized in that, In step (a), the concentration of IL-2 is 100 IU / mL to 800 IU / mL, the concentration of IL-5 is 1 ng / mL to 30 ng / mL, and the concentration of zoledronic acid is 1 μM to 10 μM; in step (b), the concentration of IL-2 is 100 IU / mL to 800 IU / mL, and the concentration of IL-5 is 1 ng / mL to 30 ng / mL. Preferably, in step (a), the concentration of IL-2 is 400 IU / mL, the concentration of IL-5 is 10 ng / mL, and the concentration of zoledronic acid is 4 μM; in step (b), the concentration of IL-2 is 400 IU / mL, and the concentration of IL-5 is 10 ng / mL.

8. The method according to claim 4, characterized in that, The cell seeding density for expanding γδT cells in step (1) is (2-3) × 10⁻⁶. 6 Cells / mL.

9. The method according to claim 4, characterized in that, The method further includes the following steps: amplifying the CAR-γδT cells obtained in step (2), and sorting and purifying the amplified CAR-γδT cells using magnetic beads containing CD19 antigen; the cell seeding density of the amplified CAR-γδT cells is (1~2)×10⁻⁶. 6 Cells / mL.

10. Use of the CAR-γδT cells of claim 1 or 2 in the preparation of a medicament for treating CD19-positive malignant tumors; preferably, the CD19-positive malignant tumor is acute lymphoblastic leukemia, chronic lymphoblastic leukemia, or B-cell lymphoma.

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