CN116769723B - GD2 chimeric antigen receptor modified T cell and application thereof - Google Patents
GD2 chimeric antigen receptor modified T cell and application thereof Download PDFInfo
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Abstract
The invention provides a T cell modified by a GD2 chimeric antigen receptor and application thereof, wherein the GD2 chimeric antigen receptor comprises a single-chain antibody scFv-GD2, and the nucleotide sequence of the single-chain antibody scFv-GD2 is shown as SEQ ID NO.3 in a sequence table. According to the invention, the nucleotide sequence of the single-chain antibody scFv-GD2 is optimized, the CAR-GD2 modified T cell is prepared, and after co-culture with the target cell, the release amount of IFN-gamma is obviously increased; the killing rate of the LAN-1 cells is obviously improved, and the growth of mouse neuroblastoma is obviously inhibited in vivo experiments; after the CAR-GD2-T cells and the LAN-1 cells prepared by the invention are co-cultured for 24 hours according to the effective target ratio of 10:1, the IFN-gamma release amount is 9681 pg/mL.
Description
Technical Field
The invention relates to a T cell modified by GD2 chimeric antigen receptor and application thereof, belonging to the technical field of genetic engineering.
Background
Bissialoganglioside (GD 2) is a sialic acid-containing glycosphingolipid, expressed primarily on the cell surface. GD2 is overexpressed in various embryonic carcinomas (neuroblastoma, brain tumor, retinoblastoma, ewing's sarcoma, rhabdomyosarcoma), bone tumors (osteosarcoma, ewing's sarcoma), soft tissue sarcomas (leiomyosarcoma, liposarcoma, fibrosarcoma), lung cancer, melanoma, and breast cancer. Among them, neuroblastoma is the most common extracranial solid malignancy in children, 50% of children are found with large-scale spread and metastasis of tumors, and conventional surgery, chemotherapy, radiotherapy and autologous stem cell transplantation have limited therapeutic effects on patients. Whereas GD2 is expressed in normal tissues in low amounts and in a limited manner, which makes GD 2a desirable tumor antigen for immunotherapy.
CN108728460a provides a chimeric antigen receptor targeting murine GD2, but murine scFv is easily rejected by the human immune system, resulting in CAR-T that cannot survive in vivo for a long period of time, has low cytotoxicity, and is not very killing.
Sarah A Richman et al linked the scFv fragment of murine GD2 antibody 14G2a to 4-1BB, increasing the length of the Linker between VL and VH, while allowing effective homing of the CAR-T cells to the tumor site, did not increase the anti-tumor activity of the CAR-T cells.
The prior art GD2 chimeric antigen receptor modified T cells suffer from the following disadvantages: the killing rate of target cells in vitro experiments is low, the anti-tumor activity in vivo experiments is low, and the inhibition effect on the growth of tumors is poor.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides a GD2 chimeric antigen receptor modified T cell and application thereof, and the following aims are fulfilled: the killing rate to target cells is higher, and the growth inhibition effect to mouse tumor is better.
In order to solve the technical problems, the invention adopts the following technical scheme:
a GD2 chimeric antigen receptor modified T cell comprises a single-chain antibody scFv-GD2, wherein the nucleotide sequence of the single-chain antibody scFv-GD2 is shown as SEQ ID NO.3 in a sequence table.
The GD2 chimeric antigen receptor is obtained by sequentially connecting the following modules in series: a guide, a single-chain antibody scFv-GD2, a CD8 finger region, a CD28 transmembrane region, a CD 28-4-1 BB costimulatory region, a CD3 zeta intracellular region, a self-shearing region T2A and a suicide gene RQRR 8.
The nucleotide sequence of the leader is shown in a sequence table SEQ ID NO. 2; the nucleotide sequence of the CD8 finger region is shown in a sequence table SEQ ID NO. 4; the nucleotide sequence of the CD28 transmembrane region is shown in a sequence table SEQ ID NO. 5; the nucleotide sequence of the CD 28-4-1 BB costimulatory region is shown in a sequence table SEQ ID NO. 6; the nucleotide sequence of the CD3 zeta intracellular area is shown in a sequence table SEQ ID NO. 7; the nucleotide sequence of the self-shearing region T2A is shown in a sequence table SEQ ID NO. 8; the nucleotide sequence of the suicide gene RQR8 is shown in a sequence table SEQ ID NO. 9.
The application of the GD2 chimeric antigen receptor modified T cells in preparing antitumor drugs.
Compared with the prior art, the invention has the following beneficial effects:
(1) According to the invention, the nucleotide sequence of the single-chain antibody scFv-GD2 is optimized, and the CAR-GD2 modified T cells (CAR-GD 2-T cells) are prepared, and after co-culture with target cells, the release amount of IFN-gamma is obviously increased; the killing rate of the LAN-1 cells is also obviously improved, and the growth of mouse neuroblastoma is obviously inhibited in vivo experiments.
(2) After the CAR-GD2-T cells and the LAN-1 cells prepared by the invention are co-cultured for 24 hours according to the effective target ratio of 10:1, the IFN-gamma release amount is 9681 pg/mL, and after the CAR-GD2-T cells and the LAN-1 cells are co-cultured for 24 hours according to the effective target ratio of 5:1, the IFN-gamma release amount is 7009 pg/mL.
(3) The in vitro killing rate of the CAR-GD2-T cells prepared by the invention on the LAN-1 cells is 88.7 percent (the effective target ratio is 10:1).
(4) The CAR-GD2-T cell prepared by the invention has obvious inhibition effect on the tumor growth of a C57BL6 mouse.
Drawings
FIG. 1 is a schematic structural diagram of a CAR-GD2 module;
FIG. 2 is a flow chart showing the flow cytometry detection of the expression rate of the T cell activation index CD 69;
FIG. 3 is a fluorescent image of 293T cells transfected with lentiviruses for 48 h;
FIG. 4 is a flow chart of a recombinant lentivirus containing pLent-EF1 alpha-CAR-GD 2 after infection with activated T cells;
FIG. 5 is a flow chart of a recombinant lentivirus containing pLent-EF1 alpha-CAR-GD 2-2 after infection of activated T cells;
FIG. 6 is a flow chart of infection rate of activated T cells by lentivirus containing pLent-EF 1. Alpha. -empty plasmid;
FIG. 7 is a bar graph of IFN-gamma release after 24h co-culture of CAR-GD2-T cells, CAR-GD2-2-T cells, empty T cells with target cells, respectively;
FIG. 8 is a bar graph of the in vitro killing of target cells by CAR-GD2-T cells, CAR-GD2-2-T cells, empty T cells;
FIG. 9 is a graph showing the change in body weight of mice in the in vivo toxicity test of example 5;
FIG. 10 is a graph of the inhibition efficiency of CAR-T cells on neuroblastoma growth in C57BL6 mice.
Detailed Description
EXAMPLE 1 construction of recombinant expression vector pLent-EF 1. Alpha. -CAR-GD2
The CAR-GD2 module is shown in FIG. 1 (see SEQ ID NO.1 for complete nucleic acid sequence).
The CAR-GD2 sequence is as follows:
(1) Leader (SEQ ID NO. 2)
(2) Single chain antibody scFv-GD2 (SEQ ID NO. 3)
(3) CD8 finger region (SEQ ID NO. 4)
(4) CD28 transmembrane region (SEQ ID NO. 5)
(5) CD 28-4-1 BB costimulatory region (SEQ ID NO. 6)
(6) CD3 zeta intracellular region (SEQ ID NO. 7)
(7) Self-shearing region T2A (SEQ ID NO. 8)
(8) Suicide gene RQR8 (SEQ ID NO. 9)
The whole expression cassette is synthesized by Shandong Honno biotechnology Co., ltd in the sequence from (1) to (8), the BamHI-NotI site of pLent-EF1 alpha vector (purchased from Vigene Co.) is inserted, and the plasmid is extracted by using a plasmid extraction kit from OMEGA company after the sequence is correct, so as to obtain the recombinant expression vector pLent-EF1 alpha-CAR-GD 2. In the invention, the concentration of the recombinant expression vector pLent-EF1 alpha-CAR-GD 2 is 1.1 mug/mug.
Meanwhile, the 64 th-786 th nucleotide of the Anti-GD2 single-chain antibody disclosed in CN108728460A is used as a single-chain antibody, the sequence of the invention is adopted by the other modules, a CAR structure (SEQ ID NO. 10) containing an Anti-GD2-scFv sequence disclosed in CN108728460A is constructed, a pLent-EF1 alpha vector is inserted, and a recombinant expression vector is obtained according to the operation, and is named as pLent-EF1 alpha-CAR-GD 2-2, wherein the plasmid concentration is 1.0 mug/mug.
Construction of the event-EF 1 alpha-empty plasmid: the lentivirus is directly extracted by using a kit in no-load, and the plasmid concentration is adjusted to be 1.0 mug/mug.
Example 2 preparation of pLent-EF 1. Alpha. -CAR-GD2 modified T cells
1. Preparation of activated T cells
From the peripheral blood of 75ml of the patient, peripheral blood mononuclear cells were isolated with Ficoll-Paque lymphocyte isolate. The separated cells were subjected to cell counting by separating CD8+ T cells using CD8+ sorting reagent supplied from BD company, 1X 10 6 Each cell/mL was inoculated and KBM551 cell culture medium (available from Corning under the trade designation 88-551-CM) containing IL-2 at a final concentration of 1500IU/mL was added. Then adding CD3CD28 magnetic beads with the same number as the cells for activating T cells, taking out the magnetic beads after 24 hours of activation to obtain activated T cells, and detecting the expression rate of CD69 by using a flow cytometry, wherein the expression rate of CD69 is 63.4 percent, as shown in figure 2.
2. Lentivirus package
Resuscitates 293T cells, culturesFor 3 days, passage is carried out according to the cell density, and transfection is carried out when the cell fusion degree reaches 80% after 1 passage. 6X 10 of six-hole plate 5 Cells/well were inoculated and 2ml of mem medium (available from Gibco company under the trade designation 11960-044) was added to each well in preparation for transfection the next day. Six well plates were replaced with fresh DMEM medium (available from Gibco, inc. under the trade designation 11960-044) at 2 mL/well and incubated for 1h at 37 ℃.
Preparation of transfection reagent: tube A and Tube B reagents (Tube A and Tube B) were prepared separately in 5mL centrifuge tubes;
the formulations of the A and B tubes are shown in Table 1.
TABLE 1
After being prepared, the mixture is placed for 5min, then the tube A is slowly added into the tube B, and the mixture is evenly mixed and placed for 20min at room temperature to form a liposome-DNA mixture. Adding the mixture into a culture flask, slightly mixing, standing at 37deg.C, and 5% CO 2 Culturing in an incubator.
After 48 hours, morphological changes following 293T cell transfection were observed under a microscope (FIG. 3). After 72h, the cell culture supernatant containing the virus was collected into a centrifuge tube, centrifuged at 3500rpm for 10min, cell debris was removed, filtered through a 4.5 μm filter, centrifuged at 70000g for 2h at 4℃and the pellet was resuspended in 100. Mu.L of PBS, and stored at-80℃while the virus titer was determined. The virus titer of the virus liquid containing pLent-EF1 alpha-CAR-GD 2 in the invention is 2.06X10 8 TU/mL virus solution containing pLent-EF1 alpha-CAR-GD 2-2 with a virus titer of 1.83X 10 8 TU/mL virus titer of virus solution containing pLent-EF1 alpha-empty plasmid was 2.12X10 8 TU/mL。
3. Lentivirus infects activated T cells
Extracting the above three virus solutions from-80deg.C, thawing, adding culture medium containing KBM551 with final concentration of 1500IU/mL IL-2, and diluting to virus titer of 3×10 7 TU/mL, the diluted virus solution was obtained. Resuspension 1×10 with 100 μl of diluted virus liquid 6 The number of activated T cells was adjusted to 3:1, so that virus particles and activated T cells were present in a ratio of 3:1, resulting in virus and cell suspensions. Virus and cell suspension were added to 6-well plates at 2mL per well, 37℃at 5% CO 2 Culturing in incubator for 48 hr, collecting cells, centrifuging at 400g for 5min, discarding supernatant, counting cells, and collecting the cells according to 1×10 6 Inoculating at a density of individual cells/mL, adding KBM551 culture medium containing IL-2 at a final concentration of 1500IU/mL, adding liquid at a ratio of 3 times every day, 37 ℃ and 5% CO 2 Culturing in an incubator for 13 days to expand cells to a sufficient dosage to obtain T cells after infection of the recombinant lentivirus containing pLent-EF1 alpha-CAR-GD 2, called CAR-GD2-T cells for short; t cells infected with the recombinant lentivirus containing pLent-EF1 alpha-CAR-GD 2-2, called CAR-GD2-2-T cells for short; t cells infected with the recombinant lentivirus containing pLent-EF1 alpha-empty plasmid are called empty T cells for short.
Chimeric antigen receptor expression was detected by flow cytometry. As shown in FIGS. 4-6, the infection rate of the recombinant lentivirus containing pLent-EF1 alpha-CAR-GD 2 to the activated T cells is 65.6%, the infection rate of the recombinant lentivirus containing pLent-EF1 alpha-CAR-GD 2-2 to the activated T cells is 58.7%, and the infection rate of the recombinant lentivirus containing pLent-EF1 alpha-empty plasmid to the activated T cells is 60.1%.
Example 3 in vitro IFN-gamma Release assay
LAN-1 cells (GD 2 positive neuroblastoma cell line) are used as target cells, and effector cells are CAR-GD2-T cells, CAR-GD2-2-T cells and empty T cells.
The effective target ratio is 1:1, 5:1 and 10:1 respectively, and the number of target cells is 1×10 5 And/or wells corresponding to effector cells according to different target ratios. Each group is provided with 3 compound holes, and the average value of the 3 compound holes is taken. After 200. Mu.L of DMEM medium containing 10vol% FBS was added to each well, and effector cells and target cells were co-cultured for 24 hours, cell supernatants were collected and assayed for IFN-. Gamma.content using ELISA kits.
The results are shown in Table 2 and FIG. 7, wherein LAN-1 cells are used as target cells, and compared with CAR-GD2-T cells, the CAR-GD2-T cells of the invention have significantly increased IFN-gamma release amount, stronger killing power and stronger cytotoxicity as the gradient-dependent, i.e. the higher the effective target ratio is.
TABLE 2 IFN-gamma Release (pg/mL) for in vitro killing Activity studies of CAR-GD2-T cells
。
Example 4T in vitro cell killing experiment
Killing activity assays were performed using LAN-1 cells as target cells, CAR-GD2-T cells, CAR-GD2-2-T cells and empty T cells as effector cells. According to effector cells (1X 10) 5 Well) and target cells (1X 10 4 Well) in a 10:1 ratio, the CO-cultured medium was DMEM medium containing 10vol% FBS, 200. Mu.L per well, and placed in 5% CO 2 And (3) co-culturing in a 37 ℃ incubator, adding 20 mu L of CCK-8 into each hole after 24 hours, continuously incubating for 2 hours, detecting the wavelength of 450nm by using a microplate reader, and reading an OD value.
The specific grouping is as follows:
experiment group a: co-culturing CAR-GD2-T cells and LAN-1 cells;
experimental group B: co-culturing CAR-GD2-2-T cells and LAN-1 cells;
control group C: co-culturing empty T cells and LAN-1 cells;
blank group G: LAN-1 cells.
The cell killing rate was calculated according to the following formula: killing (%) = [1- (blank OD value-effector OD value)/blank OD value ] ×100%.
The results show that the killing rate of the experimental groups A-B and the control group C is 88.7%, 43.1% and 14.9% in sequence, and the killing efficiency of the CAR-GD2-T cells is obviously higher than that of the CAR-GD2-2-T cells, and the killing efficiency of the CAR-GD2-T cells are higher than that of the control group. Therefore, the CAR-GD2-T cells prepared by the invention can enhance the killing capacity of the cells.
Example 5 in vivo toxicity experiments on CAR-T cells
C57BL6 mice (purchased from Nanjing Junker bioengineering Co., ltd.) at 6-8 weeks were divided into 5 groups of 10 each, and the CAR-T cell toxicity experiments in vivo were verified. The experimental groups were:
a. a control group, in which physiological saline of the same volume was injected into the tail vein;
b. experiment group, tail intravenous injection 2×10 7 Individual cells/activated T cells only;
c. experimental group two, tail intravenous injection 2×10 7 Individual cells/empty T cells only;
d. three groups of experiments, tail intravenous injection 2×10 7 Individual cells/CAR-GD 2-T cells only;
e. four groups of experiments, tail intravenous injection 2×10 7 Individual cells/CAR-GD 2-2-T cells only.
Mice were observed daily for performance following injection, weighed once a week, and monitored for the presence of CAR-T cells in the mice using an animal in vivo imaging system. After 45 days, the mice were dissected and pathological observations were made of the major tissues of the mice, such as brain, heart, lung, liver, colon and kidney.
During the experiment, no abnormal behavior was observed in the mice, as shown in fig. 9 and table 3, there was no significant difference in weight gain in the mice, and the CAR-T cells were detected in the blood for 35 days. Pathological observations of the major tissues after dissecting the mice, CAR-T mice did not find lesions of the tissues.
Table 3 weight gain changes in mice after 45 days of culture for each experimental group
。
EXAMPLE 6 inhibition of neuroblastoma growth by CAR-T cells on C57BL6 mice
Male C57BL6 mice (purchased from Nanjing Junko bioengineering Co., ltd.) of 6-8 weeks were kept in animal room (room temperature 23.+ -. 2 ℃ C., humidity 50%.+ -. 10%), LAN-1 cells in log phase were collected, and Phosphate Buffer (PBS) was diluted to 2X 10 6 And each mL. Under aseptic conditions, the left armpit of the mouse was inoculated with 0.2mL of LAN-1 cell suspension, and observed for two weeks until hard grain-size nodules appear in the armpit as a standard for successful modeling.
C57BL6 neuroblastoma model mouse (vernier caliper for taking subcutaneous tumor)The size of the tissue block is 90-100mm 3 ) The animals were randomized into 6 groups of 10 animals each and the injection treatment experiments were initiated. The experimental groups were:
a. a control group, in which physiological saline of the same volume was injected into the tail vein;
b. treatment group, tail intravenous injection 2×10 6 Individual cells/activated T cells only;
c. treatment two groups, tail intravenous injection 2×10 6 Individual cells/empty T cells only;
d. three groups were treated and tail vein injection was 2×10 6 Individual cells/CAR-GD 2-T cells only;
e. four groups were treated and tail vein injection was 2×10 6 Individual cells/CAR-GD 2-2-T cells only;
f. five groups were treated and tail vein injection was 2×10 6 Individual cells/CAR-GD 2-T cells, rituximab was injected 24h later, once daily.
The mice of each treatment group were immunized once every week, continuously immunized for two weeks, recorded with the day of the first injection being 0 day, the subcutaneous tumor tissue mass size of the mice of each treatment group was measured every three days by vernier calipers, tumor growth graphs were drawn by mass mean values, the mice were dissected after 5 weeks, and tumor tissues were embedded with paraffin and sectioned.
The tumor volume increase results are shown in table 4. The results show that the CAR-GD2 cells designed by the invention have the best inhibition effect on the tumor growth of the C57BL6 mice (figure 10), the CAR-T cells have no effect on the tumors after rituximab is injected, which indicates that the rituximab can start the suicide system RQR8 of the CAR-T cells in the invention, and the cell activity is controlled by the suicide gene system.
Table 4 statistics of mouse tumor volume during treatment (mm 3 ）
。
Claims (4)
1. A GD2 chimeric antigen receptor-modified T cell, characterized in that: the GD2 chimeric antigen receptor comprises a single-chain antibody scFv-GD2, and the nucleotide sequence of the single-chain antibody scFv-GD2 is shown as SEQ ID NO.3 in a sequence table.
2. A GD2 chimeric antigen receptor-modified T cell according to claim 1, wherein: the GD2 chimeric antigen receptor is obtained by sequentially connecting the following modules in series: a guide, a single-chain antibody scFv-GD2, a CD8 finger region, a CD28 transmembrane region, a CD 28-4-1 BB costimulatory region, a CD3 zeta intracellular region, a self-shearing region T2A and a suicide gene RQRR 8.
3. A GD2 chimeric antigen receptor-modified T cell according to claim 2, wherein: the nucleotide sequence of the leader is shown in a sequence table SEQ ID NO. 2; the nucleotide sequence of the CD8 finger region is shown in a sequence table SEQ ID NO. 4; the nucleotide sequence of the CD28 transmembrane region is shown in a sequence table SEQ ID NO. 5; the nucleotide sequence of the CD 28-4-1 BB costimulatory region is shown in a sequence table SEQ ID NO. 6; the nucleotide sequence of the CD3 zeta intracellular area is shown in a sequence table SEQ ID NO. 7; the nucleotide sequence of the self-shearing region T2A is shown in a sequence table SEQ ID NO. 8; the nucleotide sequence of the suicide gene RQR8 is shown in a sequence table SEQ ID NO. 9.
4. The use of GD2 chimeric antigen receptor modified T cells according to claim 1 for the preparation of a medicament for the treatment of an anti-tumor, characterized in that: the tumor is neuroblastoma.
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