CN111925990B - Anti-MUC16 CAR-T cell aiming at ovarian cancer and preparation method thereof - Google Patents
Anti-MUC16 CAR-T cell aiming at ovarian cancer and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an Anti-MUC16 CAR-T cell for ovarian cancer, which comprises a nucleic acid sequence of a single-chain antibody of Anti-MUC16, a nucleic acid sequence of a CD8 transmembrane region, a nucleic acid sequence of a 4-1BB intracellular signal region, a nucleic acid sequence of a CD3Z intracellular signal region and a nucleic acid sequence of IL-12. Also provided are methods of making the cells, (1) vector synthesis of Anti-MUC16 CAR chimeric antigen receptor; (2) virus packaging and determination of lentivirus titer; (3) preparing Anti-MUC16 CAR-T cells; (4) function test of Anti-MUC16 CAR-T cells. The invention has the advantages that the antibody of the MUC 16-resistant specific transmembrane region epitope is selected, and the current CAR-T gene means is combined to prepare the T cell capable of specifically recognizing and killing the ovarian tumor, so that a new treatment method is hopefully provided for curing the ovarian cancer.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an Anti-MUC16 CAR-T cell for ovarian cancer and a preparation method thereof.
Background
Ovarian cancer is a common malignant tumor, the mortality rate is high worldwide, and because patients have no obvious clinical symptoms in the early stage and lack a definite detection means clinically, the tumor usually worsens when the diagnosis is confirmed, so that the disease is difficult to treat and the cure rate is low. MUC16 is a high molecular weight glycoprotein, is a transmembrane protein, and at present, MUC16 is considered by scientists as an important tumor index for early diagnosis of ovarian cancer and is widely applied to clinic. Recent studies found that abnormal overexpression of MUC16 is closely related to poor prognosis and development of ovarian cancer, and that MUC16 promotes distant metastasis of ovarian cancer by binding to mesothelin and helps ovarian cancer cells achieve immune escape by inhibiting tumor cells and immune cells from forming immune synapses. Therefore, the overexpression of the MUC16 in the ovarian cancer can promote the proliferation, migration and metastasis of tumor cells and promote the occurrence and development of the ovarian cancer.
A chimeric antigen receptor T cell (CAR-T) is one of the commonly used methods in the existing immune cell therapy technology, and the T lymphocyte in a patient body is extracted and subjected to gene integration modification in vitro, so that the T lymphocyte is provided with a chimeric antibody capable of recognizing tumor cells and activating the T lymphocyte, the modified and cultured T cell is subjected to mass amplification and is returned to the patient body, the modified T cell obtains the capacity of specifically recognizing and killing the tumor cells, the tumor cells are precisely killed, and the effect of removing tumors is finally achieved. However, modified T cells may exhibit excessive activity, leading to some clinical side effects and toxicity.
How to obtain specific recognition and accurate killing and reduce side effects and toxicity as much as possible. Are the direction of consistent exploration by those skilled in the art.
Disclosure of Invention
Aiming at solving the problem of providing a treatment method for accurately killing ovarian cancer and reducing side effects and toxicity as much as possible in the prior art, the invention discloses Anti-MUC16 CAR-T cells for ovarian cancer and a preparation method thereof, and the aim of achieving the purpose is to select an antibody for resisting MUC16 specific transmembrane region epitopes, combine the current CAR-T gene means, prepare T cells capable of specifically recognizing and killing ovarian tumors, and hopefully provide a new treatment method for the cure of ovarian cancer.
In order to achieve the purpose, the technical scheme disclosed by the invention is as follows: the Anti-MUC16 CAR-T cell for ovarian cancer provided by the invention comprises a nucleic acid sequence of a single-chain antibody of Anti-MUC16, a nucleic acid sequence of a CD8 transmembrane region, a nucleic acid sequence of a 4-1BB intracellular signal region, a nucleic acid sequence of a CD3Z intracellular signal region and a nucleic acid sequence of IL-12, wherein the nucleic acid sequences are sequentially shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.
Further, the present invention also provides a method for preparing the CAR-T cell, comprising the steps of: (1) vector synthesis of Anti-MUC16-CAR chimeric antigen receptor: the single-chain antibody nucleic acid sequence of Anti-MUC16 is coded and shown in SEQ ID NO.1, the nucleic acid sequence of a CD8 transmembrane region is coded and shown in SEQ ID NO.2, the nucleic acid sequence of a 4-1BB intracellular signal region is coded and shown in SEQ ID NO.3, the nucleic acid sequence of a CD3Z intracellular signal region is coded and shown in SEQ ID NO.4, the nucleic acid sequence of IL-12 is coded and shown in SEQ ID NO.5, scFV-CD8-4-1BB-CD3Z-IL12 is introduced and synthesized on a lentivirus expression vector pCDH vector, and the construction of the chimeric antigen receptor lentivirus expression vector is completed through sequencing identification;
(2) viral packaging and determination of lentivirus titres:
(21) and (3) slow virus packaging: culturing 293T cells used for packaging lentivirus by using DMEM culture solution containing 10% FBS in mass concentration in logarithmic growth phase, digesting the 293T cells by using pancreatin, re-inoculating the cells in a 10cm cell culture dish, ensuring that the cell exchange rate is 80% -90% the next day, replacing a fresh complete culture medium (DMEM culture solution containing 10% FBS in mass concentration), taking pCDH-EF1-CAR-IL12 shuttle plasmid 30ug containing scFV-CD8-4-1BB-CD3Z-IL12 sequence, adding the pCDH-EF1-CAR-IL12 shuttle plasmid into 0.5mL serum-free DMEM, taking the packaging plasmid PG-p1-VSVG 6ug, PG-p1-RRE 7.5ug and PG-p1-REV 6ug, adding the mixture into 0.5mL serum-free DMEM, uniformly mixing, standing at room temperature for 5min, gently mixing the two-tube plasmid diluent, standing at room temperature for 20min, adding the transfection mixture into the cell culture dish dropwise, after gentle shaking, 293T cells were plated at 37 ℃ C0 2 The incubator was left to stand for 6 hours, and after 6 hours, the cells were replaced with fresh complete medium (DMEM medium containing 10% FBS by mass), and the cells were incubated at 37 ℃ under C0 2 Standing and culturing for 48 hours in an incubator, collecting virus cells at 48 hours, centrifuging for 5min at a centrifugal force of 400g, removing cells and dead cell debris, adding the supernatant into an ultrafiltration concentration tube with a cutoff amount of 100kd, concentrating the lentivirus supernatant, centrifuging for 30min at a centrifugal force of 4000g and 4 ℃, collecting virus concentrate, and subpackaging in an ultra-low temperature refrigerator at-80 ℃;
(22) determination of lentivirus titres: 10000 cells of 3T3 in each well of a 24-well plate are added with 1ml of DMEM medium containing 10% FBS, 500 microliters of virus supernatant is added in the first well after 12 hours, then three times of gradients are diluted downwards to make 5-6 gradients, and the virus titer is calculated by detecting with a flow cytometer after 3 days;
(3) preparation of Anti-MUC16 CAR-T cells:
(31) separation of peripheral blood mononuclear cells: extracting peripheral whole blood of an ovarian cancer patient to a heparin sodium vacuum blood collection tube, centrifuging anticoagulated whole blood for 2000g, centrifuging at room temperature for 10min, removing autologous plasma, adding PBS + 0.5% HSA into cell precipitates, diluting to a final volume of 20mL, fully mixing uniformly, centrifuging a human peripheral blood lymphocyte separation tube for 1min at room temperature according to 800g, sucking diluted blood by using a 25mL pipette, slowly adding the diluted blood into the human peripheral blood lymphocyte separation tube, centrifuging at 800g and 4 ℃ for 15min, separating PBMC, sucking and removing supernatant liquid by using a 10mL pipette, slowly transferring the PBMC in a new 50mL centrifugal tube, supplementing PBS + 0.5% HSA to 50mL, mixing uniformly, 400g, increasing 5 to 5, centrifuging at 4 ℃ for 10min, cleaning PBMC, discarding supernatant, sucking 20mL PBS + 0.5% HSA by mass concentration by using a 10mL pipette, blowing, mixing uniformly, counting cells, 400g, centrifuging for 10min, adding freezing medium, and freezing;
(32) taking fresh or overnight recovered PBMC cells, carrying out surface staining on the cells by using an anti-Human-CD3 antibody, carrying out on-stream detection, calculating the proportion of CD3+ T lymphocytes to obtain the total number of CD3+ T lymphocytes, and mixing CD3/CD28 immunomagnetic beads with CD3+ T lymphocytes, wherein the proportion of the numbers is that the magnetic beads: reacting CD3+ T lymphocytes at room temperature on a turnover shaker for 30min, removing supernatant suspension by using a magnet, adding 2mL of X-VIVO-15 culture solution containing 100U/mL of IL-2 cytokine, blowing and uniformly mixing, combining cells and magnetic beads, simultaneously adding the cells and the magnetic beads into a cell culture plate, and placing the cell culture plate in a 37-degree incubator for culture;
(33) the following day, the enriched CD3+ cells were counted every 10 th day 5 Adding 0.1mL of lentivirus supernatant into the cells, supplementing 400ul of X-VIVO culture medium (containing 100U/mL IL-2), adding polybrene with the final concentration of 8ug/mL into infected cell wells, carrying out centrifugal infection on the cells at 1500g and 30 ℃, centrifuging for 1.5 hours, and placing the culture plate in CO after centrifugation is finished 2 Culturing in an incubator for 5 hours, then supplementing an X-VIVO culture medium (containing 100U/mL IL-2) to 2mL, culturing in a CO2 incubator overnight, changing the culture solution the next day, and then performing cell counting and CAR-T cell function detection according to the cell growth condition;
(4) functional assay of Anti-MUC16 CAR-T cells: detection of Anti-MUC16 CAR-T cell binding ability to protein: the 293T culture supernatant can secrete and express the MUC16-Flag protein by transiently transfecting pTT5-MUC16-Flag plasmid to 293T cellsConcentrating and quantifying the clear liquid to obtain crude MUC16-Flag protein, and respectively taking 10 5 The CAR-T cell and the MOCK-T cell are incubated with 100uL of MUC16-Flag protein crude product for 30min at 4 ℃, 1mL of PBS containing 2% bovine serum is added to wash the cells, 400g of PBS is added, the cells are centrifuged for 5min at room temperature, and the supernatant is discarded; adding 2uL anti-Flag-APC flow antibody, incubating for 15min at 4 ℃, adding 1mL PBS containing 2% bovine serum to wash cells, 400g, centrifuging for 5min at room temperature, and removing supernatant; 200uL of PBS containing 2% bovine serum was added to resuspend the cells and the expression rate of positive CAR-T cells was detected on a flow machine.
In summary, the beneficial effects of the invention include: the anti-MUC16 single-chain antibody provided by the invention is a binding site of an anti-specificity transmembrane region, has strong specificity, can kill ovarian tumor cells accurately, can transfer and identify malignant tumor cells of MUC16 by chimeric antigen receptor T cells, has strong specificity, and can be added with exogenous IL-12 secretion stimulation on the basis of the existing chimeric antigen receptor to relieve an in vivo immune suppression microenvironment and enhance the anti-tumor function of the chimeric antigen receptor.
Drawings
FIG. 1 is a schematic diagram of the Anti-MUC16-CAR chimeric antigen receptor structure of the present invention;
FIG. 2 is a map of Anti-MUC16-CAR chimeric antigen receptor expression vectors of the present invention;
FIG. 3 is a diagram of a flow experiment in which Anti-MUC16-CAR T cells of the present invention do not bind to MUC16-Flag protein;
FIG. 4 is a diagram of a flow experiment of Anti-MUC16-CAR T cells binding to MUC16-Flag protein according to the present invention;
FIG. 5 is a diagram of a flow experiment of a target cell SKOV3 not overexpressing the MUC16-GFP gene according to the present invention;
FIG. 6 is a diagram of a flow experiment of a target cell SKOV3 stably overexpressing MUC16-GFP gene in accordance with the present invention;
FIG. 7 is a graph showing the killing effect of the MOCK-T cell and CAR-T-IL12 cell on target cells SKOV3 and SKOV3-MUC16-GFP of the present invention;
FIG. 8 is a graph comparing the release of interferon gamma (IFN-gamma) from the MOCK-T cells and CAR-T-IL12 cells to SKOV3 and SKOV3-MUC16-GFP cells in the killing process;
FIG. 9 is a graph showing the effect of MOCK-T cells and CAR-T-IL12 cells on killing human dendritic cell DCS according to the present invention;
FIG. 10 is a graph showing the effect of killing human ovarian normal epithelial cells IOSE80 by MOCK-T cells and CAR-T-IL12 cells according to the present invention;
FIG. 11 is a graph showing the effect of the MOCK-T cells and CAR-T-IL12 cells on killing HOMEC of human ovarian microvascular endothelial cells.
Detailed Description
The present invention is described in further detail below with reference to specific examples. The operation of the present invention is prior art without specific mention.
The first embodiment is as follows: the Anti-MUC16 CAR-T cell for ovarian cancer provided by the invention comprises a nucleic acid sequence of a single-chain antibody of Anti-MUC16, a nucleic acid sequence of a CD8 transmembrane region, a nucleic acid sequence of a 4-1BB intracellular signal region, a nucleic acid sequence of a CD3Z intracellular signal region and a nucleic acid sequence of IL-12, wherein the nucleic acid sequences are sequentially shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.
The preparation method of the cell comprises the following steps: (1) vector synthesis of Anti-MUC16-CAR chimeric antigen receptor: the nucleic acid sequence of the single-chain antibody of Anti-MUC16 is coded and shown in SEQ ID NO.1, the nucleic acid sequence of the transmembrane region of CD8 is coded and shown in SEQ ID NO.2, the nucleic acid sequence of the intracellular signal region of 4-1BB is coded and shown in SEQ ID NO.3, the nucleic acid sequence of the intracellular signal region of CD3Z is coded and shown in SEQ ID NO.4, the nucleic acid sequence of IL-12 is coded and shown in SEQ ID NO.5, scFV-CD8-4-1BB-CD3Z-IL12 is introduced and synthesized on a lentiviral expression vector pCDH, the structure of the chimeric antigen receptor is shown in figure 1 through sequencing identification, and the map of the constructed expression vector is shown in figure 2, thereby completing the construction of the lentiviral expression vector of the chimeric antigen receptor, wherein the construction method can adopt any one of the prior art, the construction of the chimeric antigen receptor lentiviral expression vector is completed by utilizing a molecular cloning technology to double-enzyme-cut the pCDH expression vector and the scFV-CD8-4-1BB-CD3Z-IL12, and then connecting the two by using T4 ligase.
(2) Viral packaging and determination of lentivirus titres:
(21) and (3) slow virus packaging: culturing 293T cells used for packaging lentivirus by using DMEM culture solution containing 10% FBS in mass concentration in logarithmic growth phase, digesting the 293T cells by using pancreatin, re-inoculating the cells in a 10cm cell culture dish, ensuring that the cell exchange rate is 80% -90% the next day, replacing a fresh complete culture medium (DMEM culture solution containing 10% FBS in mass concentration), taking pCDH-EF1-CAR-IL12 shuttle plasmid 30ug containing scFV-CD8-4-1BB-CD3Z-IL12 sequence, adding the pCDH-EF1-CAR-IL12 shuttle plasmid into 0.5mL serum-free DMEM, taking the packaging plasmid PG-p1-VSVG 6ug, PG-p1-RRE 7.5ug and PG-p1-REV 6ug, adding the mixture into 0.5mL serum-free DMEM, uniformly mixing, standing at room temperature for 5min, gently mixing the two-tube plasmid diluent, standing at room temperature for 20min, adding the transfection mixture into the cell culture dish dropwise, after gentle shaking, 293T cells were plated at 37 ℃ C0 2 The incubator was allowed to stand for 6 hours, and after 6 hours, the cells were replaced with fresh complete medium (DMEM medium containing 10% FBS by mass), and the cells were incubated at 37 ℃ and C0 2 Standing and culturing for 48 hours in an incubator, collecting virus cells at 48 hours, centrifuging for 5min at a centrifugal force of 400g, removing cells and dead cell debris, adding the supernatant into an ultrafiltration concentration tube with a cutoff amount of 100kd, concentrating the lentivirus supernatant, centrifuging for 30min at a centrifugal force of 4000g and 4 ℃, collecting virus concentrate, and subpackaging in an ultra-low temperature refrigerator at-80 ℃;
(22) determination of lentivirus titres: 10000 cells of 3T3 in each well of a 24-well plate are added with 1ml of DMEM medium containing 10% FBS, 500 microliters of virus supernatant is added in the first well after 12 hours, then three times of gradients are diluted downwards to make 5-6 gradients, and the virus titer is calculated by detecting with a flow cytometer after 3 days;
(3) preparation of Anti-MUC16 CAR-T cells:
(31) separation of peripheral blood mononuclear cells: extracting peripheral whole blood of an ovarian cancer patient to a heparin sodium vacuum blood collection tube, centrifuging anticoagulated whole blood for 2000g, centrifuging at room temperature for 10min, removing autologous plasma, adding PBS + 0.5% HSA into cell precipitates, diluting to a final volume of 20mL, fully mixing uniformly, centrifuging a human peripheral blood lymphocyte separation tube for 1min at room temperature according to 800g, sucking diluted blood by using a 25mL pipette, slowly adding the diluted blood into the human peripheral blood lymphocyte separation tube, centrifuging at 800g and 4 ℃ for 15min, separating PBMC, sucking and removing supernatant liquid by using a 10mL pipette, slowly transferring the PBMC in a new 50mL centrifugal tube, supplementing PBS + 0.5% HSA to 50mL, mixing uniformly, 400g, increasing 5 to 5, centrifuging at 4 ℃ for 10min, cleaning PBMC, discarding supernatant, sucking 20mL PBS + 0.5% HSA by mass concentration by using a 10mL pipette, blowing, mixing uniformly, counting cells, 400g, centrifuging for 10min, adding freezing medium, and freezing and storing cells;
(32) taking fresh or overnight recovered PBMC cells, performing surface staining on the cells by using an anti-Human-CD3 antibody, performing detection on a flow computer, calculating the proportion of CD3+ T lymphocytes to obtain the total number of CD3+ T lymphocytes, and mixing CD3/CD28 immunomagnetic beads with CD3+ T lymphocytes, wherein the quantity ratio is that the magnetic beads: CD3+ T lymphocytes are changed to 3:1, the mixture reacts on a turnover shaker for 30min at room temperature, supernatant suspension is removed by using a magnet, 2mL of X-VIVO-15 culture solution containing 100U/mL of IL-2 cytokine is added, after the mixture is uniformly blown, the cells and magnetic bead conjugates are added into a cell culture plate and are placed in a 37-degree incubator for culture;
(33) the following day, the enriched CD3+ cells were counted every 10 th day 5 Adding 0.1mL of lentivirus supernatant into the cells, supplementing 400ul of X-VIVO culture medium (containing 100U/mL IL-2), adding polybrene with the final concentration of 8ug/mL into infected cell wells, carrying out centrifugal infection on the cells at 1500g and 30 ℃, centrifuging for 1.5 hours, and placing the culture plate in CO after centrifugation is finished 2 The incubator was incubated for 5 hours, then X-VIVO medium (containing 100U/mL IL-2) was replenished to 2mL, CO 2 Culturing in an incubator overnight, changing the liquid for treatment the next day, and then performing cell counting and CAR-T cell function detection according to the cell growth condition;
(4) functional assay of Anti-MUC16 CAR-T cells: detection of Anti-MUC16 CAR-T cell binding ability to protein: transient transfection of pTT5-MUC16-Flag plasmid into 293T cell to make 293T culture supernatant capable of secreting and expressing MUC16-Flag protein, concentrating and quantifying culture supernatant to obtain MUC16-Flag protein crude product, respectively taking 10 5 The CAR-T cell and the MOCK-T cell are incubated with 100uL of MUC16-Flag protein crude product for 30min at 4 ℃, 1mL of PBS containing 2% bovine serum is added to wash the cells, 400g of PBS is added, the cells are centrifuged for 5min at room temperature, and the supernatant is discarded; adding 2uL anti-Flag-APC flow antibody, incubating for 15min at 4 ℃, adding 1mL PBS containing 2% bovine serum to wash cells, 400g, centrifuging for 5min at room temperature, and removing supernatant; 200uL of PBS containing 2% bovine serum was added to resuspend the cells and the expression rate of positive CAR-T cells was examined on a flow machine. Thus, whether the Anti-MUC16 CAR-T cells were successfully constructed was examined, and the results are shown in FIGS. 3 and 4, and it can be seen from FIGS. 3 and 4 that the Anti-MUC16 CAR-T cells successfully expressed the Anti-MUC16-CAR molecules, with an expression rate of about 43%.
The following is the test of Anti-MUC16 CAR-T cell Anti-tumor effect in vitro:
1.1 construction of ovarian cancer cell line SKOV3 overexpressing MUC 16: lentivirus was harvested 48 hours using pCDH-MUC16-GFP lentivirus shuttle plasmid 20ug co-transfected with packaging plasmids PG-p1-VSVG 6ug, PG-p1-RRE 7.5ug and PG-p1-REV 6ug 293T by a 10cm plate cell. SKOV3 cells were infected with a 1:1 dilution of lentiviral stock solution, harvested 2 days later, and the expression of GFP was detected on a flow-machine. The ovarian cancer cell line SKOV3 of the MUC16 with stable over-expression is obtained through monoclonal screening and detection, and simultaneously, the ovarian cancer cell line SKOV3 is provided with a GFP fluorescent label and is used for subsequent detection, the construction of the target cell SKOV3-MUC16-GFP is completed, and the results are shown in fig. 5 and fig. 6.
1.2CCK-8 cell proliferation method for detecting the killing effect of Anti-MUC16 CAR-T cells:
target cells SKOV3-MUC16-GFP are inoculated to 10000 cells/hole of the cell number in a 96-hole flat-bottom culture plate, and the cells are attached to the wall overnight. The plates were patted dry and Anti-MUC16 CAR-T cells and lentiviral-free MOCK-T cells were added at T: E1: 0, 1:1, 1:5, 1:10, 1:20, 1:50 and co-cultured at 37 ℃ for 24 hours. Spin-dry the plate, add 200ul PBS to wash the residual dead cells, discard the supernatant, wash the plate 3 times repeatedly, pat dry. 100uL/well of X-VIVO medium containing 10% CCK-8 solution was added, incubated in a CO2 incubator for 1.5 hours, and the plates were read at OD450nm of a microplate reader. The killing rate is 1- (target cell plus effector cell experimental group OD value)/(target cell control group OD value), and the Anti-MUC16 CAR-T is obtained by calculating and comparing the killing rate with the MOCK-T group, so that the Anti-MUC16 CAR-T has the capability of obviously killing ovarian cancer SKOV3-MUC16-GFP target cells, and the result is shown in figure 7.
1.3 detection of the level of cytokine interferon gamma released (IFN-. gamma.) in the killing co-culture system:
the detection of IFN-. gamma.factor in the supernatant of the killed co-culture system was carried out using the Dake human IFN-. gamma.Elisa Kit (cat # 1110002). Sample adding: 100uL/well was added to the diluted Cytokine standard to the standard well, the sample was diluted 10-fold using Dilution Buffer, 100uL/well was added to the sample well, and 100L/well was added to Dilution Buffer R (1 ×) to the blank. Adding a detection antibody: 50uL/well was added with Biotinylated antibody working solution, mixed well and covered with a sealing membrane and incubated at room temperature (18-25 ℃) for 2 hours. Washing the plate: deducting liquid in the hole, and adding 1 × Washing buffer working solution into 300 uL/well; after 1 minute of residence, the liquid in the wells was discarded. Repeat 3 times, each time on the filter paper buckle dry. Adding an enzyme: 100uL/well were added Streptavidin-HRP working solution, covered with a sealing membrane and incubated at room temperature (18-25 ℃) for 20 minutes. Repeating the plate washing step for 3 times, and developing: TMB was added at 100uL/well, incubated at room temperature (18-25 ℃) for 5-30 minutes in the absence of light, and the termination was judged by the shade of color in the wells (dark blue). Good results can be achieved by developing for 10-20 minutes. Termination reactions 100uL/well was added quickly to Stop the reaction. The plate was read at OD450nm using a microplate reader to calculate the IFN-. gamma.concentration. The higher the Anti-MUC16-CAR-T cell group effective target ratio, the higher the concentration level of IFN-gamma secreted by cell supernatant, and meanwhile, compared with the MOCK-T group, the higher the concentration level is, the Anti-MUC16-CAR-T cell has the capability of obviously killing target cells SKOV3-MUC16-GFP, and the results are shown in figure 8.
1.4 toxicity test of Anti-MUC16 CAR-T cells on human normal cells using CCK-8 method:
respectively inoculating human ovary microvascular endothelial cells HOMEC, human ovary normal epithelial cells IOSE80 and human dendritic cells DCS into a 96-hole flat-bottom culture plate according to 10000 cells/hole, and allowing the cells to adhere to the wall overnight. The plates were patted dry and Anti-MUC16 CAR-T cells and non-lentiviral MOCK-T cells were added at T: E1: 0, 1:1, 1:5, 1:10 and co-cultured for 24 hours at 37 ℃. Spin-dry the plate, add 200ul PBS to wash the remaining dead cells, discard the supernatant, wash the plate 3 times repeatedly, pat dry. 100uL/well of X-VIVO medium containing 10% CCK-8 solution was added, incubated in a CO2 incubator for 1.5 hours, and the plates read on a microplate reader OD450 nm. The killing rate is 1- (target cell plus effector cell experimental group OD value)/(target cell control group OD value), and the killing rate is calculated and compared with the MOCK-T group. The CAR-T cells have no obvious killing effect on three human normal cells, and the CAR-T cells have small toxic and side effects, and the results are shown in 9-11.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Beijing Likang Life technologies Co., Ltd
<120> Anti-MUC16 CAR-T cell aiming at ovarian cancer and preparation method thereof
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 750
<212> DNA
<213> nucleic acid sequence of Single chain antibody of Anti-MUC16 (Homo sapiens)
<400> 1
gtgaagctgc aggagtcagg gggaggcttc gtgaagcctg gagggtccct caaagtctcc 60
tgtgcagcct ctggattcac tttcagtagc tatgccatgt cctgggttcg cctgagtccg 120
gagatgaggc tggagtgggt cgcaaccatt agcagtgctg gtggttacat cttctattct 180
gacagtgtgc agggacgatt caccatttcc agagacaatg ccaagaacac cctgcacctg 240
caaatgggca gtctgaggtc tggggacacg gccatgtatt actgtgcaag gcagggattt 300
ggtaactacg gtgattacta tgctatggac tactggggcc aagggaccac ggtcaccgtc 360
tcctcaggtg gaggtggatc aggtggaggt ggatctggtg gaggtggatc tgacattgag 420
ctcacccagt ctccatcctc cctggctgtg tcagcaggag agaaggtcac tatgagctgc 480
aaatccagtc agagtctgct caacagtaga acccgaaaga accagttggc ttggtaccag 540
caaaaaccag gacagtctcc tgaactgctg atctactggg catccactag gcaatctgga 600
gtccctgatc gcttcacagg cagtggatct gggacagatt tcactctcac catcagcagt 660
gtgcaggctg aagacctggc agtttattac tgccagcaat cttataatct actcacgttc 720
ggtcctggga ccaagctgga gatcaaacgg 750
<210> 2
<211> 258
<212> DNA
<213> CD8 transmembrane region nucleic acid sequence (Homo sapiens)
<400> 2
gcggccgcat tcgtgccggt cttcctgcca gcgaagccca ccacgacgcc agcgccgcga 60
ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc 120
cggccagcgg cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatatctac 180
atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 240
tactgcaacc acaggaac 258
<210> 3
<211> 126
<212> DNA
<213> 4-1BB intracellular signal region nucleic acid sequence (Homo sapiens)
<400> 3
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 4
<211> 336
<212> DNA
<213> CD3Z intracellular Signal region nucleic acid sequence (Homo sapiens)
<400> 4
agagtgaagt tcagcaggag cgcagagccc cccgcgtacc agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgc 336
<210> 5
<211> 1623
<212> DNA
<213> IL-12 nucleic acid sequence (Homo sapiens)
<400> 5
atgtgtcacc agcagttggt catctcttgg ttttccctgg tttttctggc atctcccctc 60
gtggccatat gggaactgaa gaaagatgtt tatgtcgtag aattggattg gtatccggat 120
gcccctggag aaatggtggt cctcacctgt gacacccctg aagaagatgg tatcacctgg 180
accttggacc agagcagtga ggtcttaggc tctggcaaaa ccctgaccat ccaagtcaaa 240
gagtttggag atgctggcca gtacacctgt cacaaaggag gcgaggttct aagccattcg 300
ctcctgctgc ttcacaaaaa ggaagatgga atttggtcca ctgatatttt aaaggaccag 360
aaagaaccca aaaataagac ctttctaaga tgcgaggcca agaattattc tggacgtttc 420
acctgctggt ggctgacgac aatcagtact gatttgacat tcagtgtcaa aagcagcaga 480
ggctcttctg acccccaagg ggtgacgtgc ggagctgcta cactctctgc agagagagtc 540
agaggggaca acaaggagta tgagtactca gtggagtgcc aggaggacag tgcctgccca 600
gctgctgagg agagtctgcc cattgaggtc atggtggatg ccgttcacaa gctcaagtat 660
gaaaactaca ccagcagctt cttcatcagg gacatcatca aacctgaccc acccaagaac 720
ttgcagctga agccattaaa gaattctcgg caggtggagg tcagctggga gtaccctgac 780
acctggagta ctccacattc ctacttctcc ctgacattct gcgttcaggt ccagggcaag 840
agcaagagag aaaagaaaga tagagtcttc acggacaaga cctcagccac ggtcatctgc 900
cgcaaaaatg ccagcattag cgtgcgggcc caggaccgct actatagctc atcttggagc 960
gaatgggcat ctgtgccctg cagtggtggc ggtggaagcg gcggtggcgg aagcggcggt 1020
ggcggcagca gaaacctccc cgtggccact ccagacccag gaatgttccc atgccttcac 1080
cactcccaaa acctgctgag ggccgtcagc aacatgctcc agaaggccag acaaactcta 1140
gaattttacc cttgcacttc tgaagagatt gatcatgaag atatcacaaa agataaaacc 1200
agcacagtgg aggcctgttt accattggaa ttaaccaaga atgagagttg cctaaattcc 1260
agagagacct ctttcataac taatgggagt tgcctggcct ccagaaagac ctcttttatg 1320
atggccctgt gccttagtag tatttatgaa gacttgaaga tgtaccaggt ggagttcaag 1380
accatgaatg caaagcttct gatggatcct aagaggcaga tctttctaga tcaaaacatg 1440
ctggcagtta ttgatgagct gatgcaggcc ctgaatttca acagtgagac tgtgccacaa 1500
aaatcctccc ttgaagaacc ggatttttat aaaactaaaa tcaagctctg catacttctt 1560
catgctttca gaattcgggc agtgactatt gatagagtga tgagctatct gaatgcttcc 1620
taa 1623
Claims (1)
1. An Anti-MUC16 CAR-T cell for ovarian cancer, the cell comprising the nucleic acid sequence of a single chain antibody against Anti-MUC16, the nucleic acid sequence of a CD8 transmembrane region, the nucleic acid sequence of a 4-1BB intracellular signal region, the nucleic acid sequence of a CD3Z intracellular signal region, and the nucleic acid sequence of IL-12, wherein each of the nucleic acid sequences is represented by SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, and SEQ ID No. 5;
the preparation method of the CAR-T cell comprises the following steps:
(1) vector synthesis of Anti-MUC16-CAR chimeric antigen receptor: the nucleic acid sequence of the single-chain antibody of Anti-MUC16 is coded and shown in SEQ ID NO.1, the nucleic acid sequence of the transmembrane region of CD8 is coded and shown in SEQ ID NO.2, the nucleic acid sequence of the intracellular signal region of 4-1BB is coded and shown in SEQ ID NO.3, the nucleic acid sequence of the intracellular signal region of CD3Z is coded and shown in SEQ ID NO.4, the nucleic acid sequence of IL-12 is coded and shown in SEQ ID NO.5, scFV-CD8-4-1BB-CD3Z-IL12 is introduced and synthesized on a lentiviral expression vector pCDH vector, and the construction of the chimeric antigen receptor lentiviral expression vector is completed through sequencing identification;
(2) viral packaging and determination of lentivirus titres:
(2-1) lentivirus packaging: culturing 293T cells used for packaging lentivirus by using DMEM culture solution containing 10% FBS in a logarithmic growth phase, digesting the 293T cells by using pancreatin, re-inoculating the cells in a 10cm cell culture dish, changing a fresh complete culture medium when the cell exchange rate is 80% -90% the next day, taking 30 mu g of pCDH-EF1-CAR-IL12 shuttle plasmid containing scFV-CD8-4-1BB-CD3Z-IL12 sequence into 0.5mL serum-free DMEM, taking 6 mu g of packaging plasmid PG-p1-VSVG, 7.5 mu g of PG-p1-RRE and 6 mu g of PG-p1-REV, adding the mixture into 0.5mL serum-free DMEM, uniformly mixing, standing for 5min at room temperature, lightly mixing two-tube plasmid diluent liquid uniformly, standing for 20min at room temperature, the transfection mixture was added dropwise to the cell culture dish and after gentle shaking 293T cells were placed at 37 ℃ C0 2 The incubator is kept still for 6 hours, after 6 hours, the cells are replaced by fresh complete culture medium and DMEM containing 10% FBS by mass concentrationNourishing liquid, placing the cells at 37 deg.C, C0 2 Standing and culturing for 48 hours in an incubator, collecting virus cells at 48 hours, centrifuging for 5min at a centrifugal force of 400g, removing cells and dead cell debris, adding the supernatant into an ultrafiltration concentration tube with a cutoff amount of 100kd, concentrating the lentivirus supernatant, centrifuging for 30min at a centrifugal force of 4000g and 4 ℃, collecting virus concentrate, and subpackaging in an ultra-low temperature refrigerator at-80 ℃;
(2-2) determination of lentivirus titer: 10000 3T3 cells are distributed in each hole of a 24-hole plate, 1ml of DMEM culture medium containing 10% FBS is added, 500 microliters of virus supernatant is added in the first hole after 12 hours, then three times of gradients are diluted downwards to make 5-6 gradients, and the virus titer is calculated by detecting with a flow cytometer after 3 days;
(3) preparation of Anti-MUC16 CAR-T cells:
(3-1) isolation of peripheral blood mononuclear cells: extracting peripheral whole blood of an ovarian cancer patient to a heparin sodium vacuum blood collection tube, centrifuging anticoagulated whole blood for 2000g, centrifuging at room temperature for 10min, removing autologous plasma, adding PBS + 0.5% HSA into cell precipitates, diluting to a final volume of 20mL, fully mixing uniformly, centrifuging a human peripheral blood lymphocyte separation tube for 1min at room temperature according to 800g, sucking diluted blood by using a 25mL pipette, slowly adding the diluted blood into the human peripheral blood lymphocyte separation tube, centrifuging at 800g and 4 ℃ for 15min, separating PBMC, sucking and removing supernatant liquid by using a 10mL pipette, slowly transferring the PBMC in a new 50mL centrifugal tube, supplementing PBS + 0.5% HSA to 50mL, mixing uniformly, 400g, increasing 5 to 5, centrifuging at 4 ℃ for 10min, cleaning PBMC, discarding supernatant, sucking 20mL PBS + 0.5% HSA by mass concentration by using a 10mL pipette, blowing, mixing uniformly, counting cells, 400g, centrifuging for 10min, adding freezing medium, and freezing;
(3-2) taking fresh or overnight-recovered PBMC cells, performing surface staining on the cells by using an anti-Human-CD3 antibody, performing detection on the cells by using a flow computer, calculating the proportion of CD3+ T lymphocytes to obtain the total number of CD3+ T lymphocytes, and mixing CD3/CD28 immunomagnetic beads with CD3+ T lymphocytes in the quantity ratio of the magnetic beads: reacting CD3+ T lymphocytes at room temperature on a turnover shaker for 30min, removing supernatant suspension by using a magnet, adding 2mL of X-VIVO-15 culture solution containing 100U/mL of IL-2 cytokine, blowing and uniformly mixing, combining cells and magnetic beads, simultaneously adding the cells and the magnetic beads into a cell culture plate, and placing the cell culture plate in a 37-degree incubator for culture;
(3-3) the next day, the CD3+ cells obtained by enrichment were counted every 10 th day 5 Adding 0.1mL of lentivirus supernatant into the cell number, supplementing X-VIVO culture medium containing 100U/mL of IL-2400 mu l, adding polybrene with the final concentration of 8 mu g/mL into infected cell holes, carrying out centrifugal infection on the cells at 1500g and 30 ℃, centrifuging for 1.5 hours, and after the centrifugation is finished, placing the culture plate in CO 2 Culturing in incubator for 5 hr, supplementing X-VIVO culture medium containing IL-2 100U/mL to 2mL, and adding CO 2 Culturing in an incubator overnight, changing the culture solution for treatment the next day, and then performing cell counting and CAR-T cell function detection according to the cell growth condition;
(4) function test of Anti-MUC16 CAR-T cells: detection of Anti-MUC16 CAR-T cell binding ability to protein: transient transfection of pTT5-MUC16-Flag plasmid into 293T cell to make 293T culture supernatant capable of secreting and expressing MUC16-Flag protein, concentrating and quantifying culture supernatant to obtain MUC16-Flag protein crude product, respectively taking 10 5 The CAR-T cell and the MOCK-T cell are incubated with 100 mu L of MUC16-Flag protein crude product for 30min at 4 ℃, 1mL of PBS containing 2% bovine serum is added to wash the cells, 400g of PBS is added, the cells are centrifuged for 5min at room temperature, and the supernatant is discarded; adding 2 mu L of anti-Flag-APC flow antibody, incubating for 15min at 4 ℃, adding 1mL of PBS containing 2% bovine serum to wash cells, 400g, centrifuging for 5min at room temperature, and removing supernatant; 200. mu.L of PBS containing 2% bovine serum was added to resuspend the cells, and the expression rate of positive CAR-T cells was detected on a flow machine.
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