UNIVERSAL CAR-T CELL AND PREPARATION METHOD AND USE THEREOF

Abstract

Disclosed are a universal CAR-T cell knocking out one or more of CD3 delta, CD3 gamma, CD3 epsilon and CD3 zeta, and simultaneously introducing the HSV-TK gene. Also disclosed are a method for preparing the above-mentioned CAR-T cell, a preparation comprising the CAR-T cell, and the use of the CAR-T cell.

Claims

1. A universal CAR-T cell, wherein one or more of CD3Delta, CD3Gamma, CD3 Epsilon and CD3 zeta is knocked out in said CAR-T cell.

2. The universal CAR-T cell according to claim 1, wherein an HSV-TK gene is introduced into said CAR-T cell.

3. A method of preparing said universal CAR-T cell according to claim 1, comprising the following steps: one or more of CD3Delta, CD3Gamma, CD3 Epsilon and CD3 zeta is knocked out in the CAR-T cell by a suitable gene knockout method.

4. The method of preparing said universal CAR-T cell according to claim 3, further comprising the following step: performing HSV-TK gene modification in a T cell.

5. The method of preparing said universal CAR-T cell according to claim 3, wherein said universal CAR-T cell is prepared by a gene knockout method comprising the following steps: step 1: construction of lentiviral vector and production of virus; designing an sgRNA for one or more of CD3Delta, CD3Gamma, CD3 Epsilon and CD3 zeta, cloning said sgRNA into pLenti-CrisprV2, and co-transfecting it with a lentiviral packaging plasmid; after a predetermined period of time, collecting a supernatant, filtering it and performing centrifugation to concentrate the virus, thereby obtaining a plenti-CRISPRV2-sgRNA virus; step 2: preparation of CD3-negative CAR-T cell; human PBMC is isolated and purified, and then inoculated into a culture plate with suitable stimulation conditions; after being cultured for a predetermined period of time, the cells are transfected with a CAR virus and the plenti-CRISPRV2-sgRNA virus produced in Step 1, and subjected to cell expansion with suitable stimulation conditions; and CD3-positive cells are removed from the obtained cells to get the CD3-negative CAR-T cells.

6. The method of preparing said universal CAR-T cell according to claim 5, wherein the stimulation conditions for culturing the isolated and purified human PBMC are anti-hCD3 and anti-hCD28, and the stimulation conditions for expanding the cells are stimulating with artificial antigen-presenting cells or anti-hCD3/28 every 6 days.

7. The method of preparing said universal CAR-T cell according to claim 5, wherein the lentiviral packaging plasmid in said Step 1 comprises VSV-g, pMD Gag/Pol, RSV-REV; and the centrifugation is performed using Beckman ultracentrifuge and SW28 head.

8. The method of preparing said universal CAR-T cell according to claim 5, wherein said human PBMC is mononuclear cells derived from cord blood or adult peripheral blood.

9. A formulation comprising said universal CAR-T cell according to claim 1.

10. (canceled)

11. A method of treating or preventing tumor, comprising administrating said universal CAR-T cell according to claim 1.

12. The method according to claim 11, wherein said tumor comprises solid tumor and non-solid tumor.

13. The method according to claim 11, wherein said tumor comprises lymphoma, renal tumor, neuroblastoma, germ cell tumor, osteosarcoma, chondrosarcoma, soft tissue sarcoma, liver tumor, thymoma, pulmonary blastoma, pancreatoblastoma, hemangioma.

14. The universal CAR-T cell according to claim 1, comprising an intracellular signal transduction domain, wherein said intracellular signal transduction domain further comprises at least one of CD3zeta, CD28, CD137, 4-1BB, ICOS, OX40, IL-12, 41BB, CD28, IL7R, IL2R.

15. The universal CAR-T cell according to claim 1, comprising an extracellular antigen recognition domain, wherein said extracellular antigen recognition domain is a single chain antibody or a ligand or receptor of a tumor-specific antigen.

16. The universal CAR-T cell according to claim 15, wherein said single chain antibody comprises anti-CD19 antibody, anti-CD20 antibody, EGFR antibody, HER2 antibody, EGFRVIII antibody.

17. The universal CAR-T cell according to claim 15, wherein said ligand or receptor of the tumor-specific antigen comprises NKG2D.

18. The universal CAR-T cell according to claim 1, comprising an extracellular hinge region, wherein said extracellular hinge region is a region selected from CD8a or IgG.

19. The universal CAR-T cell according to claim 1, comprising a transmembrane domain, where said transmembrane domain is one selected from CD8a, CD28, CD137 or CD3.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 is a schematic structural view of the 20BBZ CAR molecule used in an embodiment of the present invention;

[0036] FIG. 2 is a schematic view of the results of the phenotypic analysis of the CD3-negative 20BBZ CAR-T cells in an embodiment of the present invention;

[0037] FIG. 3 is a schematic view showing the regulation of ganciclovir on the survival of the CD3-negative 20BBZ CAR-T cells in an embodiment of the present invention;

[0038] FIG. 4 is a schematic view of the tumor-killing ability of the CD3-negative 20BBZ CAR-T cells and the control CAR-T cells in an embodiment of the present invention;

[0039] FIG. 5 is a schematic view of the in vivo survival ability of the CD3-negative 20BBZ CAR-T cells and the control CAR-T cell in an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0040] A universal CAR-T cell in which CD3Delta, CD3Gamma, CD3 Epsilon and CD3 zeta are knocked out, and an HSV-TK gene is introduced is provided. The present invention also relates to a method of preparing the aforesaid CAR-T cell, a formulation including the CAR-T cell and a use of the CAR-T cell.

[0041] Hereinafter the embodiments of the present invention are further described with reference to the accompanying drawings and examples. The following examples are only for more clearly illustrating the technical solutions of the present invention, but not for limiting the protective scope of the present invention.

EXAMPLE 1—PREPARATION OF CD3-NEGATIVE 20BBZ CAR-T CELLS

[0042] The preparation of the CD3-negative 20BBZ CAR-T cell of this example includes the following steps:

[0043] 1. Construction of Lentiviral Vector pLenti-CrisprV2-sgRNA and Production of Virus

[0044] Designing an sgRNA for CD3Delta, CD3Gamma, CD3 Epsilon, CD3 zeta by using crispr.mit.edu, and cloning it into pLenti-CrisprV2. Subjecting the clones sequenced correctly to a large scale endotoxin-free extraction, and co-transfecting them with a lentiviral packaging plasmid (VSV-g, pMD Gag/Pol, RSV-REV) into 293X. After 48 and 72 hours, collecting a supernatant, filtering it with a 0.45 uM filter, and performing centrifugation with Beckman ultracentrifuge and SW28 head at 25000 RPM for 2 hours to concentrate the virus to obtain plenti-CRISPRV2-sgRNA virus, which was used for the subsequent production of CAR-T cells.

[0045] 2. Preparation of CD3-Negative 20BBZ CAR-T Cells

[0046] Purifying human PBMC by a Stemcell T cell isolation kit (negative selection), and then inoculating it into a 96-well plate coated with anti-hCD3 and anti-hCD28. After 2 days, infecting the cells with 20BBZ (its structure is shown in FIG. 1, and the antibody extracellular antigen recognition domain used therein is anti-CD20 antibody) and plenti-CRISPRV2-sgRNA virus at MOI=10-20. After 1 day, continuing to culture the cells with the medium changed, and stimulating them with the artificial antigen-presenting cell or anti-hCD3/28 every 6 days. Removing the CD3-positive cells from the obtained cells by Stemcell T cell positive selection kit, thereby getting the CD3-negative 20BBZ CAR-T cells (CD3-U-CAR-T, briefly, U-CAR-T cell), which were used for the subsequent experiments and the phenotypic analysis, and the results are shown in FIG. 2. It can be seen from the figure that the obtained U-CAR-T cells are CAR-positive and CD3-negative.

EXAMPLE 2—THE SURVIVAL OF U-CAR-T CELL IS REGULATED BY GANCICLOVIR

[0047] The U-CAR-T cells obtained in Step 2 of EXAMPLE 1 and the control CAR-T cells were inoculated into 96-well plates, and ganciclovir with a concentration as shown was added. After 48 hours, the CAR-T cells were compared with the survival numbers, and the results are shown in FIG. 3. It can be seen from the figure that ganciclovir can regulate the survival of U-CAR-T, and can rapidly clear the U-CAR-T from the body in the case that the U-CAR-T causes a side effect, thereby improving the safety.

EXAMPLE 3—COMPARISON OF TUMOR-KILLING ABILITY OF U-CAR-T AND CONTROL CAR-T

[0048] The U-CAR-T cells obtained in Step 2 of EXAMPLE 1 and the control CAR-T cells were inoculated into 96-well plates, and Raji tumor cells were added at a CAR-T: tumor cells ratio of 1:1. After 24 and 48 hours, the survival rates of the tumor cells were compared, and the results are shown in FIG. 4. It can be seen from the figure that the U-CAR-T has a similar tumor killing ability to that of the control CAR-T.

EXAMPLE 4—COMPARISON OF IN VIVO SURVIVAL ABILITY OF U-CAR-T AND CONTROL CAR-T

[0049] 10.sup.6 Raji tumor cells were intravenously inoculated into B-NDG mice. After 6 days, the mice were treated with 10.sup.7 U-CAR-T and the control CAR-T, and observed for their survival rate. The results are shown in FIG. 5. It can be seen from the figure that both the U-CAR-T and the control CAR-T result in the prolonged survival time of the mice.

[0050] It can be seen from the aforesaid examples that, the universal CAR-T constructed by knockout of CD3 in the present invention exhibits a low graft-versus-host response (GVHD), and greatly enhances and expands the convenience of CAR-T cell therapy. Meanwhile, an HSV-TK is introduced, so that the U-CAR-T can be rapidly cleared from the body by the regulation of ganciclovir, thereby further improving the safety of the universal CAR-T.

[0051] Hereinbefore the specific embodiments of the present invention are described in details. However, they are only used as examples, and the present invention is not limited to the specific embodiments as described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are encompassed in the scope of the present invention. Therefore, all the equal transformations and modifications without departing from the spirit and scope of the present invention should be covered in the scope of the present invention.