Anti CD70 CAR-T Cell, and Preparation Method Therefor and Use Thereof

Abstract

Provided are a CAR-T cell specifically recognizing CD70, and a preparation method therefor. The method comprises expressing a SCFV(Anti CD70)-CD8-4-1BB-CD3ζ molecule in a T cell. The CAR-T cell has tumor killing activity and may be used for preparing a medication in the treatment of a tumor with the high expression level of CD70 molecules.

Claims

1. An Anti CD70 CAR-T cell, wherein the Anti CD70 CAR-T cell targets the CD70 domain and expresses SCFV-CD8-4-1BB-CD3ζ fusion protein in T lymphocytes; wherein the amino acid sequence of the SCFV-CD8-4-1BB-CD3ζ fusion protein is as shown in SEQ ID NO. 7.

2. A method for preparing an Anti CD70 CAR-T cell the following steps: (1) Synthesis and amplification of SCFV-CD8-4-1BB-CD3ζ fusion protein gene, and cloning the SCFV-CD8-4-1BB-CD3ζ fusion protein gene into lentivirual expression vector; (2) Infecting 293T cells with lentivirual packaging plasmid and lentivirual expression vector plasmid obtained in step (1), packaging and preparing lentivirus; (3) Separating T lymphocytes, culturing and amplifying T lymphocytes, infecting T lymphocytes with lentivirus obtained in step (2), and making the T lymphocytes express SCFV-CD8-4-1BB-CD3ζ fusion protein to obtain the Anti CD70 CAR-T cells; wherein the CAR-T cell targets the CD70 domain and expresses SCFV-CD8-4-1BB-CD3ζ fusion protein in T lymphocytes; and wherein the amino acid sequence of the SCFV-CD8-4-1BB-CD3ζ fusion protein is as shown in SEQ ID NO. 7.

3. The Anti CD70 CAR-T cell of claim 1, wherein the Anti CD70 CAR-T cell is used to treat a subject in need of treatment.

4. The method of claim 3, wherein the tumor is renal cell carcinoma or brain glioma.

5. The Anti CD70 CAR-T cell of claim 1, wherein the T lymphocyte is derived from PBMC isolated from human peripheral blood.

6. The Anti CD70 CAR-T cell of claim 1, wherein in the SCFV-CD8-4-1BB-CD3ζ fusion protein, an amino acid sequence of a CD8 Leader is as shown in SEQ ID NO. 1 and an amino acid sequence of CD70 SCFV (containing (G.sub.4S).sub.3) is as shown in SEQ ID NO. 2.

7. The Anti CD70 CAR-T cell of claim 1, wherein in the SCFV-CD8-4-1BB-CD3ζ fusion protein, an amino acid sequence of a CD8 Hinge is as shown in SEQ ID NO. 3 and an amino acid sequence of CD8 is as shown in SEQ ID NO. 4.

8. The Anti CD70 CAR-T cell of claim 1, wherein in the SCFV-CD8-4-1BB-CD3ζ fusion protein, an amino acid sequence of 4-1BB is shown as SEQ ID NO. 5 and an amino acid sequence of CD3ζ is as shown in SEQ ID NO. 6.

9. The Anti CD70 CAR-T cell of claim 1, wherein a molecule of an Anti CD70 SCFV sequence is expressed on the surface of the Anti CD70 CAR-T cell, and a T cell activation signal is transmitted intracellular of the Anti CD70 CAR-T cell by a 4-1BB-CD3ζ molecule.

10. The Anti CD70 CAR-T cell of claim 1, wherein the Anti CD70 CAR-T cell is in a formulation comprising one of a carrier, diluent, or excipient.

11. An Anti CD70 CAR-T cell, wherein the CAR-T cell targets the CD70 domain and expresses SCFV-CD8TM-4-1BB-CD3ζ fusion protein in T lymphocytes, wherein the T lymphocyte is derived from PBMC isolated from human peripheral blood and the amino acid sequence of the SCFV-CD8TM4-1BB-CD3ζ fusion protein is as shown in SEQ ID NO. 7.

Description

DESCRIPTION OF THE FIGURES

[0021] FIG. 1 is a structure diagram of the lentivirual plasmid vector PLV-SCFV (anti CD70)-CD8-4-1BB -CD3ζ of the present invention;

[0022] FIG. 2 is a diagram showing the flow detection results of renal cells (786-0) with high expression of CD70 antigen of the present invention;

[0023] FIG. 3 is a flow assay result diagram of the constructed Anti CD70 CAR-T cells of the present invention;

[0024] FIG. 4 is an in vitro killing effect diagram of the CAR-T of the present invention (786-0 is the experimental group and MCF-7 is the negative control group);

[0025] FIG. 5 is an in vitro killing effect diagram of CAR-T cells under different effector-target ratios of the present invention (measured by RTCA);

[0026] FIG. 6 is a graph showing the results of survival rate change of mice that obtained by animal level experiments of the present invention;

[0027] FIG. 7 is a graph showing the results of volume change of subcutaneous transplanted tumor in mice that obtained by animal level experiments of the present invention;

[0028] FIG. 8 is a graph showing the results of changes in body weight of mice during the experimental period that obtained by animal level experiments of the present invention.

SEQUENCE LISTING

[0029] The sequence listing file entitled USPTOSequenceList004.txt, created on Mar. 10, 2022, having a size of 8.84 KB, and the sequence listing file entitled WO2021047208A1sequencelisting.txt, created on Mar. 4, 2022, having a size of 8.81 KB, are both submitted to the USPTO with this application, the disclosures of each of which are hereby entirely incorporated by reference herein.

DETAILED DESCRIPTION

[0030] The technical solution in the present invention will be clearly and completely described combined with specific embodiments below.

EXAMPLE 1

preparation of lentivirual expression vector

[0031] SCFV (anti CD70)-CD8-4-1BB-CD3ζ fusion gene sequence was gene synthesised. The gene sequence is shown in SEQ ID NO. 7, which is connected to the PLV vector by restriction digestion, and the upstream of the gene is the EP-1a promoter. The vector was transformed into DH5α Escherichia coli strain and the screening was performed by ampicillin to obtain the positive clone. The plasmid was extracted and the clone was digested to identify, then the PLV-SCFV (anti CD70)-CD8-4-1BB-CD3ζ lentivirual packaging vector was obtained, see FIG. 1.

EXAMPLE 2

preparation of lentivirus

[0032] 24 hours before transfection, 293T cells were inoculated into T75 culture flasks at a rate of approximately 8×10.sup.6 per flask. Ensure that lentivirus packing is performed when the cells are evenly distributed in the culture flask with a fusion degree of about 80%.

[0033] Prepare plasmids and transfection reagent diluent

[0034] 1. Mix PEI 40K transfection reagent evenly by vortex oscillation.

[0035] 2. Prepare two centrifuge tubes, and prepare plasmids and transfection reagent diluent in the following order.

TABLE-US-00002 Centrifuge tube 1 Centrifuge tube 2 (plasmid DNA) (transfection reagent) Lentivirual vector 10 μg PEI 40K transfection  40 μl VSVG vector 7.5 μg reagent G-Puro vector 2.5 μg DMEM serum-free 460 μl medium DMEM serum-free X μl Total volume 500 μl medium Total volume 500 μl

[0036] 3. Mix well.

[0037] 4. Add the diluent of transfection reagent (centrifuge tube 2) to the plasmid DNA solution (centrifuge tube 1), and immediately mix well. It is important to note the order of addition.

[0038] 5. Incubate the transfection mixture at room temperature for 15-20 minutes.

[0039] 6. Add each 1 ml of transfection mixture to the 293T cell culture flask, and gently pipette the medium to mix.

[0040] 7. Incubate at 37° C. for 6 hours.

[0041] 8. Remove the culture medium containing transfection reagent, and change the medium with 20 ml virus culture medium.

[0042] 9. Collect cell culture supernatant 48 hours after transfection. 500 g centrifuge for 10 min to remove cell debris. The supernatant can be directly used for lentivirus infection, and can also be used for virus titer determination or virus concentration. For long time storage, it can be cryopreserved at −80° C.

EXAMPLE 3

Preparation of Anti CD70 CAR-T cells

[0043] 0.5 ml blood was taken for rapid detection of pathogenic microorganisms, and microbial infections such as HBV, HCV, HDV, HEV, HIV-1/2, treponema pallidum and parasites were excluded. Peripheral blood mononuclear cells (PBMC) were collected by apheresis machine. Complete growth medium was prepared, 5% autologous AB or FBS was added to PBS, with IL-2 at the concentration of 20 ng/ml. The separated PBMC was diluted with medium to 2×10/ml, and 50u1 was taken to detect the purity of T cells in PBMC by flow cytometry. Day 0, buffer 1 was configured, and 1% FBS was added to PBS. Beads were oscillated for 30 s or shaken up and down for 5 min manually. CD3/CD8 beads were taken out in 1.5 ml EP tube according to the ratio of beads to T cells of 3:1. 1 ml Buffer 1 was added to wash beads, then beads1min was sucked out of EP tube with magnet, and washing solution was discarded. Repeat twice. Then beads were suspended to original volume with culture medium. Cells and beads were mixed and added to the appropriate culture flask at 2×10.sup.5 PBMC/ml. The cell density was adjusted to 2×10.sup.5/ml the next day, and virus vectors were added according to the ratio of virus vector: cell=1: 5. 4 ug/ml of polybrene and 20 ng/ml IL-2 were added at the same time. After 4 hours, the cell density was adjusted to 5×10.sup.4/ml by adding fresh and complete culture medium. All the cells were centrifuged, and fresh culture medium was added to continue culture. The cell density was maintained at 0.5−1×10%/ml by changing the medium half every 2-3 days. After 10-12 days, the number of cells reached a level of 10.sup.6. The immune cells were obtained by centrifuged at 400 g for 5min, and then washed twice with precooled PBS (400 g, 5 min). The cell population and CAR-T cell ratio were detected by blood cell counting plate and flow cytometry, as shown in FIG. 3. According to the illustration of FIG. 3, the positive rate of CAR-T cells prepared by the method of this example is over 97%. The color change of the culture medium, cell density and cell morphology were observed every day and recorded accordingly. In the process of gradually expanding culture, IL-2 needed for the total volume was added.

EXAMPLE 4

Screening and detection of engineering cell lines

[0044] (1) Screening cancer cell lines with high expression of CD70 for culture;

[0045] (2) Each 20,000 different cells were taken, centrifuged at 400 g, 5 min, and washed twice with precooled PBS. Then 2 μ1 of CD70 antibody (BD) was added respectively to incubate for 20 min in the dark. After centrifugation, the cells were washed with precooled PBS for one time, and the cells were re-suspended with 200 μ1 PBS. The expression of CD70 was detected by flow cytometry (see FIG. 2). The experimental results verified that renal cell line 786-0 highly expresses CD70 (closed to 100%), and glioma cell line MO59K also has a high expression rate, which can be used as a target cell for subsequent killing experiment. Breast cancer cell line MCF-7 almost does not express CD70 and can be used as a negative control.

EXAMPLE 5

[0046] The killing effect of 786-0 and M059K mediated by CD70 CAR-T cell was monitored by RTCA in real time (MCF-7 was used as a negative control)

[0047] 1. Before inoculation of target cells, 100 μ1 1640 medium was added to each well and the plate was put into RTCA. The value at this time was recorded as “0”;

[0048] 2. Each well was added with 1×10.sup.5 target cells, and they were incubated at 37° C. with 5% CO.sub.2 for 24 hours until the cell confluence reached over 90%;

[0049] 3. The 96-well plate was taken out and 200 μ1 CD70 CAR-T cells were added to each well according to E/T ratio of 1: 1, 2: 1, 4: 1 and 8: 1.

[0050] 4. The electrode plate was put back into RTCA and fasten, and continued to culture;

[0051] 5. After 48 hours, the experiment was finished, and the cell killing results were observed under microscope, as shown in FIG. 4. According to the illustration, the killing effect of CD70 CAR-T on renal cell carcinoma cells (786-0) and glioma (M059K) increases with the increase of the effector-target ratio, while no killing effect is found on cells that do not express CD70 (MCF-7) in different effector-target ratios.

[0052] 6. Analyzing the data, the results are as shown in FIG. 5. According to the illustration, it can be seen that the time required for CD70 CAR-T to achieve the same killing effect on renal cell cells (786-0) and glioma cells (M059K) is shortened with the increase of the effector-target ratio, and the cancer cells can be almost eliminated by different effector-target ratios, while no killing effect is found on breast cancer cells (MCF-7) without CD70 expression in different effector-target ratios.

EXAMPLE 6

[0053] NCG mice (purchased from Biocytogen Co., Ltd.) were subcutaneously inoculated with human renal cell carcinoma cell 786-0 with high expression of CD70 on the left and right flanks of their backs to establish the subcutaneous transplanted tumor model. After 20 and 25 days after tumor injection, mice were divided into groups (5 mice in each group) and treated with Anti CD70 CAR-T cells, mock T cells and PBS via tail vein infusion. The survival of mice was observed and recorded every three days. The results are as shown in FIG. 6. According to the illustration of FIG. 6, the remission rate of mice injected with CD70 CAR-T group was 94%, that of mice injected with mock T group was 25%, and all mice injected with PBS group died on the 22nd day. The tumor size was measured every five days. The results are shown in FIG. 7. According to the illustration, the subcutaneous transplanted tumors of mice injected with three different doses of CD70 CAR-T cells disappeared 20 days after injection of CD70 CAR-T cells, and the subcutaneous transplanted tumors of mice injected with mock T group reached an average of 510 mm.sup.2, while the subcutaneous transplanted tumor of mice injected with PBS reached an average of 700 mm.sup.2. The weight changes of mice were recorded. The results are shown in FIG. 8. According to the illustration, the weight of mice injected with CD70 CAR-T and mock T increased, while the weight of mice injected with PBS decreased from 21 g to 19 g on average. All the above results show that CD70 CAR-T has a good effect on tumor inhibition, and the mortality and survival status of transplanted tumor mice are well improved.