CN112159818B - Nucleic acid for treating HCC, preparation method thereof, CAR-T cell with nucleic acid and preparation method of cell - Google Patents

Abstract

The invention discloses a nucleic acid for treating HCC, a preparation method thereof, a CAR-T cell with the nucleic acid and a preparation method of the cell, wherein the sequence at least comprises a CD226 costimulatory region nucleic acid sequence as shown in SEQ ID NO.6 and a 4-1BB costimulatory region nucleic acid sequence as shown in SEQ ID NO. 7. The invention promotes the production of cytokines such as IFN-gamma and the like, thereby overcoming the inhibitory action of the liver tumor microenvironment, improving the infiltration rate, the proliferation rate and the survival rate of T cells, and leading GPC3-226BB-CART cells to survive and continuously proliferate in the tumor until the tumor is eliminated after being injected into the body.

Description

Nucleic acid for treating HCC, preparation method thereof, CAR-T cell with nucleic acid and preparation method of cell

Technical Field

The invention relates to the technical field of genes, in particular to a nucleic acid for treating HCC, a preparation method thereof, CAR-T cells with the nucleic acid and a preparation method of the cells.

Background

Hepatocellular carcinoma (HCC), abbreviated as liver cancer, is one of the most common malignant tumors worldwide at present, and the incidence rate and the mortality rate of the hepatocellular carcinoma are fifth and second globally. In 2018, in 2 months, the national cancer center issues the latest national cancer statistical data, 85.4 ten thousand new cases are issued every year in the world of primary liver cancer, and 46.6 ten thousand cases in China account for about 55 percent of the world; 81 thousands of deaths are caused by primary liver cancer every year, and the number of Chinese deaths is 42.2 thousands, which accounts for 45% -50% of the world. The current treatment means for common early liver cancer include surgery, liver transplantation and radiotherapy. However, many patients are not suitable for the above methods due to many specific situations, and in addition, these therapies are costly and present serious side effects.

Adoptive therapy of Chimeric Antigen Receptor (CAR) modified T cells is a technology emerging in recent years in the field of tumor immunotherapy. The treatment method can target T cells on tumor cells to exert the killing function of the T cells by performing gene modification on the T cells. Glypican-3 (GPC 3) is an important tumor-associated antigen of HCC. According to statistics, 72% -81% of tumor tissues of HCC patients highly express GPC3 protein. At present, the preparation of T lymphocytes (GPC 3-226 BB-CART) expressing anti-GPC 3 chimeric antigen receptor by using GPC3 as a target antigen for the treatment of liver cancer has been a research focus, but the curative effect is still unsatisfactory. This is because the EPR effect and strong immunosuppressive microenvironment of liver tumors decrease the infiltration rate, proliferation rate and survival rate of CART cells. Therefore, how to regulate and control the killing activity of GPC3-226BB-CART cells to liver cancer cells and enhance the sensitivity of liver tumor cells to the killing of GPC3-226BB-CART cells is a key problem in the current liver cancer immunotherapy research.

The CAR consists of three regions in sequence, an extracellular antigen-binding region, a transmembrane region, and an intracellular signaling region. The crucial role for the activation and proliferation of T cells is the intracellular signaling region, and in the initial studies, a single signaling region, i.e., the intracellular signaling domain immunoreceptor tyrosine-activated motif (ITAM), mostly uses CD3 ζ or fcsri γ. Activation signals from ITAMs do not provide long-term polyclonal expansion and sustained in vivo anti-tumor effects, but only cause transient T cell division and low levels of cytokine secretion. Later, CARs introduced co-stimulatory molecules, including the B7/CD28 and tumor necrosis factor/tumor necrosis factor receptor (TNF/TNFR) superfamily, commonly CD28 or 4-1BB. Studies have shown that the introduction of different or varying amounts of co-stimulatory molecules has an effect on the survival time of CAR-T cells in vivo, the number of tumor-localized CAR-T cells, and the anti-tumor capacity. Both patent cn201310164725.X and patent CN201710502507.0 disclose chimeric antigen receptor scFv (GPC 3) -CD28-4-1BB-CD3 ζ, where the killing rate of high expressing GPC3 hepatocyte cancer cell lines by introducing different or different numbers of co-stimulatory molecule CAR-T cells was compared in patent cn201310164725. X. However, the killing rate of such CAR-T cells against hepatocellular carcinoma cell lines remains to be improved, and those skilled in the art have not made much improvement.

Therefore, it is the motivation and the foundation for the present invention to develop a novel nucleic acid for treating HCC, a method for preparing the same, CAR-T cells having the same, and a method for preparing the cells, which not only have urgent research values, but also have good economic benefits and industrial application potential.

Disclosure of Invention

The present inventors have conducted intensive studies to overcome the above-identified drawbacks of the prior art, and as a result, have completed the present invention after having made a great deal of creative efforts.

Specifically, the technical problems to be solved by the invention are as follows: a nucleic acid for treating HCC, a preparation method thereof, CAR-T cells with the nucleic acid and a preparation method of the cells are provided, so that the production of cytokines such as IFN-gamma is promoted, the inhibition effect of a liver tumor microenvironment is overcome, the infiltration rate, the proliferation rate and the survival rate of the T cells are improved, and GPC3-226BB-CART cells survive and continuously proliferate in tumors after being injected into a body until the tumors are eliminated.

In order to solve the technical problem, the technical scheme of the invention is as follows:

in a first aspect, the invention provides a nucleic acid comprising at least the sequence of a nucleic acid for the treatment of HCC

The CD226 costimulatory region nucleic acid sequence of SEQ ID No.6,

and the 4-1BB costimulatory region nucleic acid sequence as shown in SEQ ID NO. 7.

In the nucleic acid of the present invention, as an improvement, the sequence thereof includes those linked sequentially

A CD8 Leader nucleic acid sequence as set forth in SEQ ID No. 2;

a GPC3scFv nucleic acid sequence as set forth in SEQ ID No. 3;

a CD8 a hinge region nucleic acid sequence as set forth in SEQ ID No. 4;

a CD8 a transmembrane region nucleic acid sequence as set forth in SEQ ID No. 5;

a CD226 costimulatory region nucleic acid sequence as set forth in SEQ ID No. 6;

a 4-1BB co-stimulatory region nucleic acid sequence as set forth in SEQ ID No. 7;

the CD3 zeta signaling region nucleic acid sequence as set forth in SEQ ID No. 8.

In the nucleic acid of the invention, as an improvement, the sequence of the nucleic acid is shown in SEQ ID NO. 3.

In a second aspect, the present invention provides a method for preparing a nucleic acid, comprising the steps of:

(1) Synthesizing the whole expression frame according to a Leader nucleic acid artificial sequence, a GPC3 single-chain antibody nucleic acid artificial sequence, a CD8 hinge region nucleic acid artificial sequence, a CD8 transmembrane region nucleic acid artificial sequence, a CD226 costimulatory region nucleic acid artificial sequence, a 4-1BB costimulatory region nucleic acid artificial sequence and a CD3 zeta signaling region nucleic acid artificial sequence respectively, and inserting the expression frame into a standard vector pUC to obtain pUC-GPC3-226BB-CAR;

(2) Performing double enzyme digestion on pUC-GPC3-226BB-CAR, cutting an agar part of a GPC3-226BB-CAR DNA fragment by using agar electrophoresis, treating by using a DNA extraction kit sol solution, passing through a DF column, discarding a filtrate, rinsing the DF column, emptying, eluting the DF column, and collecting a centrifugate to obtain the purified GPC3-226BB-CAR DNA fragment.

In a third aspect, the invention provides a CAR-T cell comprising a plasmid having a nucleic acid as described above.

In the CAR-T cell, the plasmid is a pLent-GPC3-226BB-CAR plasmid obtained by inserting a fusion gene fragment GPC3-226BB-CAR DNA into a lentivirus expression vector pLent-C-GFP.

In the CAR-T cell of the present invention, as an improvement, the plasmid is prepared by the following preparation method: the purified GPC3-226BB-CAR DNA fragment and the linearized pLent-C-GFP DNA fragment described above were ligated in a ligation system: 10 × buffer:1 mul; t4 ligase: 1 mul; GPC3-226BB-CAR DNA fragment: 4 mu l of the solution; linearized pLent-C-GFP DNA fragment: 4 μ l, ligation formation.

In a fourth aspect, the invention provides a method of making a CAR-T cell, comprising the steps of:

the pLent-GPC3-226BB-CAR plasmid described above was first lentivirally packaged and then 293T cells were infected with recombinant lentiviruses.

In a fifth aspect, the invention provides an application of the gene, which means that the gene can be applied to the preparation of a medicament for treating HCC. The pharmaceutical form includes, but is not limited to, a kit.

A kit of CAR-T cells comprising

(1) Obtaining a vector stably expressing GPC3-226BB-CART as described above;

(2) A carrier diluent.

After the technical scheme is adopted, the invention has the beneficial effects that:

the nucleic acid and CAR-T cell provided by the invention introduce two costimulatory molecules of CD226 and 4-1BB, CD226 is also called DNAX accessory molecule (DNAM-1), is an activating receptor expressed on the surfaces of various cells such as NK cells, CTL cells and platelets, belongs to tumor necrosis factor/tumor necrosis factor receptor (TNF/TNFR) superfamily costimulatory molecules, and consists of an extracellular region, a transmembrane region and a cytoplasmic region, the molecular weight is 65-67 kDa, the extracellular region has 2 IgSF V-like structural domains and potential glycosylation sites, and the cytoplasmic region has phosphorylation sites and interaction sites with signal molecules. Intracellular serine residues are phosphorylated by kinases and bind to LFA-1 molecules, the bound protein complex is recruited to lipid rafts, and tyrosine residues of the molecules are subsequently phosphorylated by one or more Src family kinases, which leads to an amplification of dependent cascade signals, leading to activation and adhesion responses of T cells. In addition, the research also shows that the CD226 cytoplasmic region recombinant protein promotes PBMC to secrete IFN-gamma.

Compared with the conventional CAR for treating HCC, the CAR disclosed by the invention can promote the production of cytokines such as IFN-gamma and the like, so that the inhibitory effect of a liver tumor microenvironment is overcome, the infiltration rate, the proliferation rate and the survival rate of T cells are improved, and GPC3-226BB-CART cells can survive and continuously proliferate in tumors after being injected into a body until the tumors are eliminated.

Drawings

FIG. 1 is a schematic design diagram of the chimeric antigen receptor Leader-GPC3scFv-CD8 α -CD226-4-1BB-CD3 ζ of the present invention.

FIG. 2 is a GPC3-226BB CAR expressing efficiency of 22.2% of GPC3-226BB CAR T cells of the invention (flow-through peak plot on right, scatter plot on left).

FIG. 3 is an ELIspot assay of the present invention. The left panel shows IFN- γ secretion from GPC3-226BB CAR T; right panel GPC3-28BB CAR T secretion of IFN- γ; the secretion capacity of GPC3-226BB-CAR T cells IFN-gamma cultured in a mixed manner with the cell strain Hep3B is remarkably higher than that of GPC3-28BB-CAR T cells.

FIG. 4 is a graph of ELISA detection of serum alpha-fetoprotein changes in tumor-bearing mice. The level of serum alpha-fetoprotein of the mice in 3 days with tumor is obviously increased.

FIG. 5 is a yellow-green fluorescence view of liver tissue. The mouse tumors contained green fluorescence throughout the GPC3-226BB-CART treated group, whereas no green fluorescence was found throughout the mouse tumors in the GPC3-28BB-CART treated group. Therefore, the GPC3-226BB CAR for treating HCC of the invention can overcome the inhibitory effect of liver tumor microenvironment, improve the infiltration rate, proliferation rate and survival rate of T cells, and allow GPC3-226BB-CART cells to survive and proliferate in tumor after being injected into body until tumor is eliminated, compared with conventional CAR for treating HCC.

Detailed Description

The invention is further illustrated by the following specific examples. The use and purpose of these exemplary embodiments are to illustrate the present invention, not to limit the actual scope of the present invention in any way, and not to limit the scope of the present invention in any way.

Example 1

A nucleic acid for treating HCC comprising in sequence at least the CD226 co-stimulatory region nucleic acid sequence as depicted in SEQ ID No.6 and the 4-1BB co-stimulatory region nucleic acid sequence as depicted in SEQ ID No. 7.

In this example, as shown in FIG. 1, the sequence of nucleic acid for treating HCC includes the sequence of the CD8 Leader nucleic acid as set forth in SEQ ID No.2, linked in series; a GPC3scFv nucleic acid sequence as set forth in SEQ ID No. 3; a CD8 a hinge region nucleic acid sequence as set forth in SEQ ID No. 4; a CD8 a transmembrane region nucleic acid sequence as set forth in SEQ ID No. 5; a CD226 costimulatory region nucleic acid sequence as set forth in SEQ ID No. 6; 4-1BB co-stimulatory region nucleic acid sequence as set forth in SEQ ID No. 7; the CD3 zeta signaling region nucleic acid sequence as set forth in SEQ ID No. 8.

Example 2

A method of preparing a nucleic acid for treating HCC, comprising the steps of:

(1) Synthesizing the whole expression frame according to a Leader nucleic acid artificial sequence, a GPC3 single-chain antibody nucleic acid artificial sequence, a CD8 hinge region nucleic acid artificial sequence, a CD8 transmembrane region nucleic acid artificial sequence, a CD226 costimulation region nucleic acid artificial sequence, a 4-1BB costimulation region nucleic acid artificial sequence and a CD3 zeta signaling region nucleic acid artificial sequence, and inserting the expression frame into a standard vector pUC to obtain pUC-GPC3-226BB-CAR;

(2) Performing double enzyme digestion on pUC-GPC3-226BB-CAR, cutting an agar part of a GPC3-226BB-CAR DNA fragment by using agar electrophoresis, treating by using a DNA extraction kit sol solution, passing through a DF column, discarding a filtrate, rinsing the DF column, emptying, eluting the DF column, and collecting a centrifugate to obtain the purified GPC3-226BB-CAR DNA fragment.

In this embodiment, the more detailed steps are:

(1) The whole expression frame was synthesized by Leader nucleic acid artificial sequence, GPC3 single-chain antibody nucleic acid artificial sequence, CD8 hinge region nucleic acid artificial sequence, CD8 transmembrane region nucleic acid artificial sequence, CD226 costimulatory region nucleic acid artificial sequence, 4-1BB costimulatory region nucleic acid artificial sequence, CD3 zeta signaling region nucleic acid artificial sequence, committee biotechnology (shanghai) ltd, and inserted into a standard vector pUC, respectively, and was named pUC-GPC3-226BB-CAR;

(2) The pUC-GPC3-226BB-CAR vector was subjected to double digestion with Fast Digest AsiSI (available from ThermoFisher) and Fast Digest NotI (available from ThermoFisher) at 37 ℃ for 20min. The 100. Mu.l enzyme system is: 10 × buffer:10 mu l of the mixture; 6 mu g of DNA; asiSI enzyme: 3 mu l of the solution; notI enzyme: 3 mu l of the solution; deionized water to make up the volume. The agar portion containing the GPC3-226BB-CAR DNA fragment was excised using agar electrophoresis and placed in two centrifuge tubes. The DNA was dissolved from the agar using a DNA extraction kit (available from ThermoFisher Co.) and concentrated by first adding 500. Mu.l DF buffer to the centrifuge tube and allowing to act at 55 ℃ for 10 minutes, shaking every 2-3 minutes until the agar was completely dissolved. The agar solution was then aspirated into the DF Column and sleeved with a Collection Tube (the filtrate was collected). Centrifuge at 8000rpm for 1 minute and pour off the filtrate. Then 500. Mu.l of Wash Buffer was added and centrifuged at 8000rpm for 1 minute, and the filtrate was decanted off. Centrifugation at 12000rpm for 2 minutes ensured that ethanol was removed. And finally transferring the DF Column to another clean micro-centrifuge tube, adding 25 mu l of Elution Buffer, standing for 2 minutes at room temperature, centrifuging for 2 minutes at 14000rpm, and obtaining the purified GPC3-226BB-CAR DNA fragment as the liquid in the micro-centrifuge tube.

Example 3

A plasmid which is a pLent-GPC3-226BB-CAR plasmid obtained by inserting a fusion gene fragment GPC3-226BB-CAR DNA into a lentiviral expression vector pLent-C-GFP. The plasmid is prepared by the following preparation method: the purified GPC3-226BB-CAR DNA fragment and the linearized pLent-C-GFP DNA fragment described above were ligated in a ligation system: 10 × buffer:1 mul; t4 ligase: 1 mul; GPC3-226BB-CAR DNA fragment: 4 mu l of the solution; linearized pLent-C-GFP DNA fragment: 4 μ l, ligation formation.

In this example, the plasmid was prepared as follows:

the whole expression cassette was synthesized by Leader nucleic acid artificial sequence, GPC3 single-chain antibody nucleic acid artificial sequence, CD8 hinge region nucleic acid artificial sequence, CD8 transmembrane region nucleic acid artificial sequence, CD226 costimulatory region nucleic acid artificial sequence, 4-1BB costimulatory region nucleic acid artificial sequence, CD3 zeta signaling region nucleic acid artificial sequence committing for Biotechnology (Shanghai) Limited and inserted into a standard vector pUC, and thus named as pUC-GPC3-226BB-CAR, while pUC-GPC3-226BB-CAR and pLent-C-GFP vectors were subjected to double enzyme digestion by Fast Digest asiSI (available from ThermoFisher) and Fast Digest NotI (available from ThermoFisher), respectively, at 37 ℃ for 20min. The 100. Mu.l enzyme system is: 10 × buffer:10 mul; 6 mu g of DNA; asiSI enzyme: 3 mul; notI enzyme: 3 mu l of the solution; deionized water to make up the volume. The agar sections containing the GPC3-226BB-CAR DNA fragment and the linearized pLent-C-GFP DNA fragment were excised by agar electrophoresis and placed in two centrifuge tubes. The DNA was dissolved out of the agar using DNA extraction kit (from ThermoFisher) and concentrated by first adding 500. Mu.l DF buffer to the centrifuge tube described above, and allowing to act at 55 ℃ for 10 minutes while shaking every 2-3 minutes until the agar was completely dissolved. The agar solution was then aspirated into the DF Column and covered with the Collection Tube (Collection of filtrate). Centrifuge at 8000rpm for 1 minute and pour off the filtrate. Then 500. Mu.l of Wash Buffer was added and centrifuged at 8000rpm for 1 minute, and the filtrate was decanted off. Centrifugation at 12000rpm for 2 minutes ensured that ethanol was removed. And finally transferring the DF Column to another clean micro-centrifuge tube, adding 25 mu l of Elution Buffer, standing for 2 minutes at room temperature, centrifuging for 2 minutes at 14000rpm, and obtaining the purified GPC3-226BB-CAR DNA fragment and the linearized pLent-C-GFP DNA fragment from the liquid in the micro-centrifuge tube.

The two DNA fragments were ligated overnight at 16 ℃ to form the pLent-GPC3-226BB-CAR plasmid. The connecting system is as follows: 10 × buffer:1 mul; t4 ligase: 1 mul; GPC3-226BB-CAR DNA:4 mu l of the solution; linearized pLent-C-GFP DNA:4 μ l.

Example 4

And (3) plasmid purification: the above-described pLent-GPC3-226BB-CAR was transformed into e. The method comprises the following specific steps: the plasmid and the competent cells are evenly mixed and incubated on ice for half an hour, then heat shock is carried out at 42 ℃ for 90 seconds, then the mixture is placed on ice for 2min, finally liquid LB culture medium is added and slowly shaken for about 1 hour, then centrifugation is carried out at 3000rpm for 5min, and 100 mul of bacterial liquid is coated on a solid plate containing ampicillin LB. The next day, single colonies were picked for overnight culture, and the pLent-GPC3-226BB-CAR plasmid was extracted using a plasmid extraction and purification kit (purchased from Qiagen, inc.), using the following specific steps: (1) centrifuging 1.5ml of bacterial solution at room temperature of 10000 Xg for 1min. (2) The supernatant was removed, 250. Mu.l of solution I (containing RNase A) was added, and the cells were shaken by a vortex shaker until they were completely suspended. (3) Adding 250 mul of solution II, and gently inverting the centrifuge tube for 4-6 times to obtain clear lysate. Preferably, the incubation is carried out at room temperature for 2min. (4) 350 ul of solution III is added and mixed by gentle inversion several times until white flocculent precipitate appears, and centrifuged 10min at room temperature at 10000 Xg. (5) The supernatant was aspirated with special care and transferred to a clean adsorption column equipped with 2ml centrifuge tubes. It is ensured that there are no aspiration deposits and cell debris. Centrifugation was carried out at room temperature at 10000 Xg for 1min until the lysate was completely passed through the column. (6) The filtrate was discarded, 500. Mu.l Buffer HBC was added, 10000 Xg was centrifuged for 1min, and the column was washed to remove residual protein to ensure the purity of DNA. (7) The filtrate was discarded, and the column was washed with 750. Mu.l of Wash Buffer diluted with 100% ethanol and centrifuged at 10000 Xg for 1min. (8) the adsorption column was washed with 750. Mu.l of Wash Buffer. (9) The column must be centrifuged at 10000 Xg for 2min to ensure that the ethanol is removed. (10) The column was placed into a clean 1.5ml centrifuge tube, 50-100. Mu.l (depending on the desired final concentration) sterile deionized water or TE buffer was added to the filter, and the plasmid DNA was collected by centrifugation at 10000 Xg for 5 min. (11) Agarose gel electrophoresis was performed with a DNA sample (Marker) of a predetermined concentration, and the result was compared to obtain pLent-GPC3-226BB-CAR plasmid having a concentration of 556 ng/. Mu.l.

The pLent-GPC3-226BB-CAR plasmid described above was sequenced by committee of Biotechnology engineering (Shanghai) Co., ltd. Sequencing is carried out correctly for later use.

Example 5

CAR-T cell comprising a plasmid having a nucleic acid as described above.

The preparation method of the CAR-T cell comprises the following steps: the pLent-GPC3-226BB-CAR plasmid described above was first lentivirally packaged and then 293T cells were infected with recombinant lentiviruses.

In this example, CAR-T cells were prepared as follows:

A. lentivirus packaging and titer detection

A Lentiviral Packaging Kit is adopted, and the specific method comprises the following steps: the lentivirus packaging cell line 293T was inoculated into a 10cm dish containing DMEM +10% FBS, cultured at 37 ℃ under 5% CO2 conditions, and prepared for transfection when the anchorage rate was 70% to 80%. A sterile 1.5ml EP tube or 15ml centrifuge tube was used to prepare the reaction system as follows: serum-free DMEM:4ml; pLent-GPC3-226BB-CAR plasmid: 10 mu g of the mixture; GM easy (TM) Lentiviral Mix:10 μ l (10 μ g); HG Transgene Reagent: 60 μ l. After mixing, standing at room temperature for 20min, adding into 293T cell culture dish, and culturing in CO2 incubator. After transfection 24, the cell culture solution was carefully aspirated off and discarded in a waste liquid cup containing a disinfectant solution, and then 15ml of fresh culture medium containing 10% serum was added to continue the culture. After 48h of liquid change, the cell supernatant was aspirated into a 50ml centrifuge tube, centrifuged at 500g for 5min at 4 ℃, filtered through a 0.45 μm filter and transferred to a new centrifuge tube. The virus particles in the supernatant can then be directly assayed for titer. The titer of 100. Mu.l of the virus solution was determined using a lentiviral vector (HIV P24) rapid test card, and the titer of the recombinant lentivirus was 1.25X 106TU/ml.

Preparation of GPC3-226BB-CAR T

Preparation of T cells

Using TBD sample density separation medium (purchased from tianjin scientific organism) from fresh peripheral blood from 75ml healthy donors, PBMCs were isolated as follows:

1) Peripheral blood 75ml was diluted with physiological saline at a ratio of 1. The diluted blood was carefully added to the same volume of lymphocyte separation medium to form distinct layers, and centrifuged horizontally at 1200rpm/min for 20min at room temperature. At the moment, 5 layers are formed in the centrifugal tube from top to bottom; serum, a white membrane layer composed of PBMCs, a lymphocyte separation liquid layer, and a lowermost erythrocyte sedimentation layer.

2) The leukocyte layer was carefully aspirated with a pipette, and the PBMC were aspirated as completely as possible. Adding 2 times of physiological saline, washing cells for 2 times, mixing uniformly each time, centrifuging at 800rpm/min for 10min. The low-speed centrifugation is favorable for removing platelet and lymphocyte separation liquid remained in the cell suspension, the supernatant is discarded after the centrifugation, and PBMC cells are collected.

The above mononuclear cells were induced to culture in a medium (purchased from CORNING corporation, 88-551-CM) containing 1000IU/ml of recombinant interferon alpha 2a (purchased from Shenyang Sansheng), and 24 hours later, 1000IU/ml of recombinant interleukin 2 (purchased from Shenyang Sansheng), 50ng/ml of OKT-3 and 5% of autologous plasma of the patients were added for further 24 hours. Every two days, the cells were inoculated at double rate, cultured to day 14, and the positive expression rate of CD3+, CD56+ in T cells was examined by flow cytometry (CD 3-FITC, CD16/CD56-PE antibody purchased from BECKMAN, A07735). The positive rate of CD3+ is more than 80%, the double positive rate of CD3+ CD56+ is more than 20%, the T induction is regarded as successful, and the T is reserved for virus infection.

Lentiviral infected T cells and expansion culture of infected T cells

T cells were infected with the recombinant lentivirus described above at MOI = 5. After the infected cells were cultured in a 37 ℃ C.5% CO2 incubator for 12 hours, the cells were collected and the supernatant was discarded, and the same amounts of virus solution and cell culture solution were added again, and the cells were cultured in a 37 ℃ C.5% CO2 incubator for further 12 hours, after which the culture supernatant was discarded by aspiration, 10ml of fresh CORNING medium was added, and the cells were further cultured for further expansion for 17 days until the cells were expanded to a sufficient amount. Chimeric antigen receptor expression was detected by FC500 flow cytometry (purchased from BECKMAN) FL1 channel (fig. 2). The positive rate of the recombinant lentivirus infected T cells is 22.2 percent by taking uninfected T lymphocytes as a negative control.

Example 6

IFN-gamma secretion detection after co-culture of GPC3-226BB-CAR T cells and target cells

The hepatocellular carcinoma target cell line Hep3B (purchased from Kaikyi Biotechnology development Co., ltd., nanjing) with high expression of GPC3 is used as a target cell, and the effector cell is GPC3-226BB-CAR T, wherein the patent CN201710502507.0 discloses the CAR T targeting GPC3 as a negative control group, and the structure of the CAR T is GPC3scFv-CD8 alpha-CD 28-4-1BB-CD3 zeta, and the GPC3-28BB-CAR T is hereinafter referred to as GPC3-28BB-CAR T.

GPC3-226BB-CAR T and GPC3-28BB-CAR T were expressed as E: t (effector cell to target cell ratio) is 1:1 was cultured in a mixed culture with a cell line Hep3B for 20 hours, and secretion of IFN-. Gamma.from GPC3-226BB-CAR T cells and GPC3-28BB-CAR T cells was detected by an ELIspot assay (purchased from eBioscience, inc.), respectively. ELIspot specific steps:

day 1: cell culture (sterile procedure) 1. Pre-coated plate activation: adding RPMI-1640 culture medium into each well, standing at room temperature for 5-10 min, and removing the culture medium by plate beating. 2. Adding a cell suspension: adding the cell suspension with the adjusted concentration into each experimental hole, wherein each experimental hole is 100 mu L; positive control wells: cell concentration 1X 105/well, adding cell culture medium and PHA; negative control wells: the cell concentration is 1 multiplied by 105 per hole, and an equal volume of cell culture medium is added; background negative control: RPMI-1640 medium containing fetal bovine serum was added. 3. And (3) incubation: culturing in a 5% CO2 incubator at 37 deg.C for 16-20 hr.

Day 2: post-culture manipulation (no longer requiring aseptic manipulation) 4 lysis of cells: pour the cells and media from the wells. The cells were hypotonic lysed by adding ice-cooled deionized water at 200. Mu.L/well and placing in a freezer at 4 ℃ for 10 minutes. And 5, washing the plate: the well contents were poured, 1 × Washing buffer,200 μ L/well, and washed 5-7 times. Standing for 30-60s each time. And finally, patting the water-absorbing paper to remove the liquid. 6. And (3) incubation of the detection antibody: biotin-labeled antibody solution was added to the wells at 100. Mu.L/well. Incubate at 37 ℃ for 1 hour. 7. Washing the plate: the well contents were poured, 1 × Washing buffer,200 μ L/well, and washed 5 times. Standing for 30-60 seconds each time. And finally, patting the water-absorbing paper to remove the liquid. 8. Incubation with enzyme-linked avidin: the diluted enzyme-labeled avidin solution was added to the plate wells at 100. Mu.L/well. Incubate at 37 ℃ for 1 hour. 9. Washing the plate: the well contents were poured, 1 × Washing buffer,200 μ L/well, and washed 5 times. Standing for 30-60 seconds each time. And finally, patting the water-absorbing paper to remove the liquid. 10. Color development: add the AEC color developing solution prepared at present into each plate well at 100. Mu.L/well. Standing at room temperature in dark place for color development for 25 minutes, and 11. Terminating the color development: the liquid in the wells was poured out, the plate base was uncovered and washed 3-5 times with deionized water to stop the development. The plate is placed in a cool place at room temperature, and the base is closed after the plate is naturally dried. 12. The ELISPOT plate spots were photographed and analyzed.

The results show that: the secretion capacity of IFN-gamma of GPC3-226BB-CAR T cells cultured in mixture with the cell line Hep3B was significantly higher than that of GPC3-28BB-CAR T cells (FIG. 3).

Example 7

GPC3-226BB-CART cell killing assay

(1) Establishment of mouse liver cancer model

Selecting a hepatocellular carcinoma target cell line Hep3B with high expression of GPC3, injecting the hepatocellular carcinoma target cell line Hep3B into a BALB/c mouse (purchased from Nanjing university model animal research institute) for abdominal cavity amplification, extracting ascites after the mouse has ascites to extract Hep3B cells, extracting the ascites, centrifuging to remove supernatant, adding a precooled PBS solution, blowing, flushing, centrifuging at 1000rpm, removing the supernatant, repeating the process for 2-3 times, adding the precooled PBS solution, uniformly blowing, centrifuging at 500rpm, leaving red blood cells in the supernatant, removing supernatant, keeping precipitated cells, adding a cell culture medium to prepare a Hep3B 1640 cell suspension, anesthetizing the BALB/c mouse with pentobarbital sodium, cutting the skin and peritoneum under the mouse protrusion to expose the left lobe of the mouse liver, extracting a 1 × 107 μ L cell suspension by using a micro-syringe, and injecting the cell suspension into the mouse liver; after the micro-syringe is pulled out, the needle hole is pressed by a 75% alcohol cotton swab until the surface of the liver does not bleed any more, and the needle hole is sealed by an adhesive. After 3 days, successful modeling was assessed by measuring AFP via mouse venous blood collection, as performed in accordance with the alpha-fetoprotein ELISA enzyme immunoassay kit (BioCheck, inc).

1) Taking blood of tumor-bearing mice and normal mice, centrifuging and taking serum;

2) Preparing a working washing liquid: concentrating the washing solution 20 ml/bottle, diluting with distilled water 1: 20;

3) Sample adding: setting blank control 1 hole, negative and positive control 2 holes, respectively adding 50 mul negative and positive control and 50 mul sample to be tested into corresponding holes;

4) Adding an enzyme: adding 1 drop (50 mu l) of enzyme marker to each hole except for a blank control hole, slightly oscillating, and sealing the hole of the plate by using a transparent adhesive tape;

5) And (3) incubation: water bath at 37 deg.C for 30min;

6) Washing: deducting liquid in the holes, filling the holes with washing liquid, standing for 20s, removing the washing liquid, repeating for 4 times, and patting on absorbent paper to dry;

7) Color development: adding 1 drop of substrate solution A and 1 drop of substrate solution B into each hole, slightly oscillating the sealing plate, and placing in a water bath at 37 ℃ for 15 minutes;

8) And (4) judging a result: 1 drop of stop solution was added to each well, and the OD value of each well (blank well zeroed) was measured with a microplate reader (wavelength 450 nm).

The result shows that the expression level of the mouse serum alpha-fetoprotein is obviously increased 3 days after tumor bearing (see figure 4), which indicates that the establishment of the mouse liver cancer model is successful.

(2) GPC3-226BB-CAR T cell in vivo killing

Tumor-bearing mice cultured for 2 weeks were randomly divided into 3 groups of 21 mice each, and an injection treatment experiment was started. The experimental groups were respectively:

a. in the control group, the tail part is injected with normal saline with the same volume;

b. one treatment group, tail vein injection of 2 × 106 cells/GPC 3-226BB-CAR T cells;

c. treatment two groups, 2X 106 cells/GPC 3-28BB-CAR T cells were injected intravenously caudally.

On the 7 th day, the 14 th day and the 21 st day after the CAR-T cell injection, each group of mice was anesthetized with 10g/L sodium pentobarbital in the abdominal cavity, and the tumor morphology was observed by dissection; a part of liver tissue was fixed with 40g/L neutral formalin, dehydrated, paraffin-embedded, sectioned, and the proliferation and persistence of CAR T in tumor were observed by green fluorescence under a fluorescence microscope.

In the GPC3-226BB-CART treatment group, the tumors of the mice gradually shrink, wherein 2 tumors of the mice completely disappear, and the tumors of the mice in the GPC3-28BB-CART treatment group are unchanged. As can be seen from the tissue section, the GPC3-226BB-CART treatment group contained GPC3-226BB-CART in the mouse tumor. In contrast, in the GPC3-28BB-CART treated group, no GPC3-28BB-CART cells were found in the mouse tumor (see FIG. 5).

Example 8

A kit of CAR-T cells comprising

(1) Obtaining a vector stably expressing GPC3-226BB-CART as described above;

(2) A carrier diluent;

(3) Instructions for use;

wherein the instructions for use comprise the method of intravenous injection described in example 7 above.

It will be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should also be understood that various alterations, modifications and/or variations can be made to the present invention by those skilled in the art after reading the technical content of the present invention, and all such equivalents fall within the protective scope defined by the claims of the present application.

Sequence listing

<110> Shandong Xingyi Biotechnology Ltd

<120> nucleic acid for treating HCC, method for preparing the same, CAR-T cell having the nucleic acid, and method for preparing the cell

<130> 2018

<160> 8

<170> SIPO Sequence Listing 1.0

<210> 1

<211> 1689

<212> DNA

<213> ethnic species (Homo sapiens)

<400> 1

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tgcatgccgc tagaccccaa gtgcagctgc agcaatctgg ggctgagctg gtgaggcctg 120

gggcttcagt gaagctgtcc tgcaaggctt cgggctacac atttaatgac tatgaaatgc 180

actgggtgaa gcagacacct gtgcatggcc tgaagtggat tggagctctt gaacctaaga 240

ctggtgatac tgcctactct cagaagttca agggcaaggc cactctgact gcagacaaat 300

cctccagcac agcctacatg gagctccgca gcctgacatc tgaggactct gccgtctatt 360

actgtacatc ttgttattat tacacttact ctggccaagg gactctggtc actgtctctg 420

caggaggtgg gggcagtggt ggcgggggat ctggaggtgg aggttccgat gttgtgatga 480

cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc tcttgcagat 540

ctagtcagag ccttgtacac agtaatggaa acacctattt acattggtac ctgcagaagc 600

caggccagtc tccaaagctc ctgatctaca aagtttccaa ccgattttct ggggtcccag 660

acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc agagtggagg 720

ctgaggatct gggagtttat ttctgctctc aaaatacaca tgttcctccc acgttcggtt 780

ctgggaccaa gctggagatt aaagctagca ccacgacgcc agcgccgcga ccaccaacac 840

cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg 900

cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatatctac atctgggcgc 960

ccttggccgg gacctgtggg gtccttctcc tgtcactggt tatcaccctt tactgcaacc 1020

acaggaacaa cagaaggaga aggagagaga gaagagatct atttacagag tcctgggata 1080

cacagaaggc acccaataac tatagaagtc ccatctctac ctctcaacct accaatcaat 1140

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aaggcggctg cgaactgaga gtgaagttca gcaggagcgc agacgccccc gcgtaccagc 1380

agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag tacgatgttt 1440

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Claims (3)

1. A nucleic acid for use in treating hepatocellular carcinoma, comprising:

the nucleic acid sequence for treating hepatocellular carcinoma is shown in SEQ ID NO. 1.

car-T cells, characterized by: a plasmid comprising the nucleic acid of claim 1.

3. Use of the CAR-T cell of claim 2 in the preparation of a medicament for the treatment of hepatocellular carcinoma.

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