Chimeric antigen receptor targeting HCC (Hepatitis C Cell) as well as application of chimeric antigen receptor

Abstract

The invention belongs to the biomedical technology, and particularly relates to a chimeric antigen receptor targeting HCC cells and application thereof, the chimeric antigen receptor comprises an extracellular domain, a transmembrane domain (TM) and an intracellular signal domain, and is characterized in that the extracellular domain comprises a GPC3 antigen binding domain, and the intracellular signal domain comprises a cytokine IL-7 and a chemotactic factor CCL19. Tumor antigen phosphatidylinositol proteoglycan 3 (GPC3) highly expressed on the surface of an HCC cell is selected as a main target spot of hepatocellular carcinoma CAR-T treatment, a second-generation GPC3-CAR-T cell is constructed, a transgenic cell factor IL-7 and a chemotactic factor CCL19 are increased to enter the CAR-T cell, and the cell factor IL-7 and the chemotactic factor have the effects of starting cell system immunity, promoting cell apoptosis, promoting cell apoptosis and the like. NK cells and cytotoxic T cells are gathered to a solid tumor area, and the problem that CAR-T cells are immersed into solid tumors is solved.

Description

Technical Field

[0001] The present invention belongs to the field of biological medicine technology, and particularly relates to a chimeric antigen receptor targeting HCC cells and its applications. Background Art

[0002] Hepatocellular carcinoma (HCC) is a highly malignant tumor. Currently, surgical resection, liver transplantation, and transcatheter arterial chemoembolization are the main strategies for treating early HCC patients. However, most HCC patients are diagnosed at an advanced stage, and these treatments do not produce significant effects. Chimeric antigen receptor (CAR)-modified T cell (CAR-T) therapy refers to using a patient's own T cells, which are genetically engineered in vitro and then intravenously infused back into the patient. After that, they target and kill tumor cells in the body. CAR-T therapy has achieved remarkable results in hematological malignancies, but it still faces great challenges in the treatment of solid malignancies.

[0003] Glypican-3 (GPC3), whose Chinese name is phosphatidylinositol proteoglycan 3, belongs to a GPI-anchored membrane protein of the heparan sulfate proteoglycan family. Studies have found that GPC3 is overexpressed in more than 70% of HCCs, and its expression level increases with the increase in the malignancy of HCC. Therefore, GPC3 is considered a very promising new target for the treatment of HCC. Existing technologies use fusion proteins to modify T cells, enabling the fusion proteins to bind to the natural T cell receptor / CD3 complex to form engineered T cells. However, research has shown that the treatment effect still needs to be improved. Summary of the Invention

[0004] In CAR-T therapy, the design and expression of chimeric antigen receptors are crucial and related to the effectiveness of treatment. The present invention selects the tumor antigen phosphatidylinositol proteoglycan 3 (GPC3) highly expressed on the surface of HCC cells as the attack target of CAR-T cells. Using this target as the main target for CAR-T treatment of hepatocellular carcinoma, a second-generation GPC3-CAR-T cell is constructed. The transgenic cytokines IL-7 and chemokine CCL19 are introduced into the CAR-T cells. The cytokines IL-7 and chemokines can initiate the systemic immunity of cells, aggregate NK cells and cytotoxic T cells to the solid tumor region, and solve the problem of CAR-T cell infiltration into solid tumors.

[0005] The present invention adopts the following technical solutions: A chimeric antigen receptor targeting HCC cells, comprising an extracellular domain, a transmembrane domain (TM), and an intracellular signaling domain. The extracellular domain includes a GPC3 antigen-binding domain, and the intracellular signaling domain includes the cytokine IL-7 and the chemokine CCL19.

[0006] In the present invention, the extracellular domain further includes a promoter and a leader peptide. Preferably, the promoter includes a GAG promoter.

[0007] In the present invention, a hinge domain is provided between the extracellular domain and the transmembrane domain. Preferably, the hinge domain includes a CD8 hinge domain.

[0008] In the present invention, the transmembrane domain includes one or more of CD28, CD5, CD16, CD22, and CD33.

[0009] In the present invention, the intracellular signaling domain includes a co-stimulatory domain and an intracellular signal transduction domain. Preferably, the co-stimulatory domain includes a 4-1BB co-stimulatory domain.

[0010] Preferably, the chimeric antigen receptor targeting HCC cells in the present invention includes a GAG promoter, a leader peptide, GPC3 antigen binding, a CD8α hinge region, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, IL-7, and CCL19.

[0011] The present invention discloses a nucleic acid molecule encoding the chimeric antigen receptor targeting HCC cells as described above, or a vector containing the nucleic acid molecule; specific vectors and construction methods are conventional techniques, for example, the vector is selected from DNA vectors, RNA vectors, plasmids, lentiviral vectors, adenoviral vectors, and retroviral vectors.

[0012] An immune effector cell includes the chimeric antigen receptor targeting HCC cells as described above, or a nucleic acid molecule encoding the chimeric antigen receptor targeting HCC cells, or a vector containing the nucleic acid molecule. Preferably, the immune cell is selected from T lymphocytes, natural killer (NK) cells, and macrophage (M) cells.

[0013] A drug includes a chimeric antigen receptor targeting HCC cells or an immune effector cell, and further includes one or more pharmaceutically acceptable excipients, which is a conventional technique.

[0014] Preferably, the present invention discloses a CAR T cell, whose chimeric antigen receptor is the chimeric antigen receptor targeting HCC cells as described above; it is an immune cell drug, specifically a chimeric antigen receptor T cell targeting the GPC3 antigen, for the treatment of liver cancer.

[0015] The present invention discloses the use of the above-mentioned chimeric antigen receptor targeting HCC cells in the preparation of CAR immune cells, especially CAR T cells, or in the preparation of immunotherapeutic drugs; the present invention also discloses the use of the above-mentioned CAR immune cells in the preparation of immunotherapeutic drugs. Specifically, the present invention discloses the use of the above-mentioned CAR, nucleic acid molecule, vector, immune effector cell or drug for the treatment of diseases or cancers related to the expression of CPG3. Preferably, the immunotherapeutic drug is a drug for the treatment of liver cancer.

[0016] The present invention incorporates IL-7 and CCL19 into the CAR structure, which improves the tumor immune microenvironment and simultaneously attracts the aggregation of DC cells, NK cells and cytotoxic T cells to solid tumors, activates local tumor immunity, and thus creates a microenvironment for the activation of immune cells. CAR-T cells are activated and migrate into solid tumors.

[0017] CAR (chimeric antigen receptor) technology is a perfect integration of genetic engineering and immunotherapy, and is one of the most promising research directions in the current field of tumor treatment. Among them, CAR is the key to treatment. The present invention designs and constructs second-generation CARs, namely GPC3-CAR and GPC3-IL-7-CCL19-CAR. The co-stimulatory molecule is selected as 4-1BB. The above two kinds of CAR lentiviruses and the empty virus of the control group have been prepared. Using sorted CD4 + , CD8 + T cells, the above-mentioned CAR viruses are introduced into the cells, and the preparation of two kinds of CAR-T cells and the control group is completed. It has been confirmed that CAR-modified T cells have specific killing effects on liver cancer cells. After stimulation with GPC3-IL-7-CCL19-CAR cells, the secretion of IL-7 and CCL19 increases significantly, and the ability of CAR-T cells to kill tumor cells is significantly better than that of the other two groups of CAR-T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic structural diagram of the chimeric antigen receptor targeting HCC cells of the present invention.

[0019] Figure 2 It shows that the CAR T cells of the present invention successfully secrete IL-7 and CCL19 and show proliferation, chemotaxis and subtype advantages in vitro.

[0020] Figure 3 It shows that the secretion of IL-7 and CCL19 is detected in the culture supernatant.

[0021] Figure 4 It shows that the co-expression of IL-7 and CCL19 enhances the in vivo expansion, persistence and anti-tumor activity of GPC3-CAR-T cells. DETAILED DESCRIPTION OF THE INVENTION

[0022] CAR (Chimeric Antigen Receptor) technology is a perfect integration of genetic engineering and immunotherapy, and is one of the most promising research directions in the current field of cancer treatment. Among them, CAR is the key to treatment. The present invention discloses a chimeric antigen receptor targeting HCC cells, which includes an extracellular domain, a transmembrane domain (TM), and an intracellular signaling domain. The extracellular domain includes a GPC3 antigen-binding domain, and the intracellular signaling domain includes cytokines IL-7 and chemokine CCL19.

[0023] Specifically, see Figure 1 , the CAR structure is composed of scFv, hinge region (CD8α), transmembrane region (CD28), and intracellular signaling domain (4-1BB-CD3ζ, IL-7, CCL19) connected in sequence. The scFv sequence is constructed and regulated by innovative genetic technology to make it have the best and most suitable affinity in order to reduce the off-target effect. The present invention has successfully constructed the CAR structure of GPC3-IL-7-CCL19-CAR independently. The above CD8α, CD28, linker, 4-1BB, and CD3ζ sequences are obtained by analyzing protein structure bioinformatics data and through structural analysis of protein characteristics such as protein secondary structure, solvent accessibility, protein flexibility, disulfide bridges, and binding sites.

[0024] Innovation in the in vivo activation of CAR-T. After CAR-T cells enter solid tumors, in the tumor microenvironment, they are quickly affected by immunosuppressive factors and inhibitory cell subsets, and CAR-T cells quickly enter a senescent and exhausted state, losing the activity of killing tumor cells. In the present invention, on the genetic engineering fragment of the viral vector, the co-expressed IL-7 gene is added. In the tumor microenvironment, with the large expression of IL-7, the number of the naive subset of CAR-T cells is greatly increased, and the duration of maintaining young CAR-T cells in the tumor is extended several times.

[0025] The following illustrates the technological progress of the present invention through experiments. The specific reagents are conventional products of existing CAR technology, and the specific preparation operations and performance tests are conventional technologies. Nucleic acid molecules encoding chimeric antigen receptors targeting HCC cells are industrially synthesized according to conventional technologies. Conventional statistical methods are used to reflect the differences. Example 1

[0026] (1) Transfer 20 μl of bacterial liquid into 200 ml of LB medium (containing 50 μg / mL Ampicilin), and place it in a shaker for overnight incubation (37 °C, 200 rpm); (2) Add 2.5 mL of equilibration buffer BL to adsorption column CP6 (the adsorption column is placed in a 50 mL collection tube), centrifuge at 8000 rpm for 2 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube; (3) Add 200 mL of the overnight cultured bacterial solution to a centrifuge tube, centrifuge at 8000 rpm for 3 min at room temperature, discard the supernatant and collect the bacteria; (4) Add 8 mL of solution PI (RNase A has been added) to the centrifuge tube with the bacterial cell pellet, and use a pipette to thoroughly suspend the bacterial cell pellet; (5) Add 8 mL of solution P2 to the centrifuge tube, immediately gently invert it up and down 8 times, and let it stand at room temperature for 5 min; (6) Add 8 mL of solution P4 to the centrifuge tube, immediately gently invert it up and down 8 times, mix well until the solution shows a white dispersed flocculent precipitate. Then let it stand at room temperature for about 10 min. Centrifuge at 8000 rpm for 10 min, carefully pour all the solution into the filter CSI, slowly push the push handle to filter, and collect the filtrate in a clean 50 mL centrifuge tube; (7) Add isopropanol with a volume 0.3 times that of the filtrate to the filtrate, invert it up and down to mix well and then transfer it to the adsorption column CP6; (8) Centrifuge at 8000 rpm for 2 min at room temperature, pour out the waste liquid in the collection tube, and put the adsorption column CP6 back into the collection tube; (9) Add 10 mL of washing solution PW (anhydrous ethanol has been added) to the adsorption column CP6, centrifuge at 8000 rpm for 2 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube. Repeat once; (10) Put the adsorption column CP6 back into the collection tube, centrifuge at 8000 rpm for 5 min, open the lid, and place it at room temperature for 10 min; (11) Place the adsorption column CP6 in a clean 50 mL collection tube, suspend and drop 50 μL of elution buffer TB in the middle of the adsorption membrane, let it stand at room temperature for 5 min, and then centrifuge at 8000 rpm for 2 min at room temperature. Transfer all the eluate in the 50 mL centrifuge tube to a clean 1.5 mL EP tube and store it at -20 °C. Example 2

[0027] Isolation and culture of peripheral human blood T lymphocytes (1) Transfer 10 mL of heparinized whole blood into a 50 mL centrifuge tube, add 10 mL of PBS solution, and mix gently; (2) Take two 15 mL centrifuge tubes, first add 5 mL of Ficoll solution. Then gently add the diluted blood to the upper layer of Ficoll in the two centrifuge tubes, avoiding mixing the two solutions. Add 10 mL of diluted blood to each centrifuge tube; (3) Centrifuge at 2500 rpm for 20 min at room temperature (acceleration setting is 9 and deceleration setting is 0 during centrifugation); (4) Carefully aspirate the middle cloudy layer (i.e., mononuclear cell PBMC) with a sharp pipette into another 15 mL centrifuge tube, add 10 mL of PBS solution, centrifuge at 1500 rpm for 10 min, discard the supernatant, resuspend with 1 mL of PBS, and perform cell counting; (5) Sorting: Use Pan T Cell Isolation Kit human. After cell counting in step (4), centrifuge again and resuspend with PBS (40 μl / 1×10 7 cells). Add Pan T Cell Biotin Antibody Cocktail (10 μl / 1×10 7 cells), mix well, and incubate at 4°C for 5 min. Add PBS (30 μl / 1×10 7 cells), add Pan T Cell Microbead Cocktail (20 μl / 1×10 7 cells), mix well, and incubate at 4°C for 10 min. Place the MS Column in the MACS magnetic field, install the column, place it on the test tube rack, and add 3 mL of PBS to rinse the column. After the column is rinsed, add the cell suspension. After the column is emptied, add 3 mL of PBS-Ab to rinse each time, for a total of three times; the effluent is T cells. Centrifuge at 500 g for 5 min, resuspend with 1 mL of AIM-V containing 10% FBS. After cell counting, add Human T-Activator CD3 / CD28 beads (25 μl / 1×10 7 cells) and IL-2 (300 U / mL), and culture in a 37°C, 5% CO2 incubator; (6) Culture: The sorted T lymphocytes are half-changed every other day (i.e., after collecting the cells, centrifuge at 300 g for 5 min, discard half of the supernatant, resuspend the remaining cell solution with cells, and add new complete medium at a final cell concentration of 5×10 5 / mL), and add IL-2 (300 U / mL) each time the medium is changed. Example 3

[0028] Preparation and packaging of chronic viral vectors

[0029] (1) Prepare a 10 cm culture dish 18 h in advance. Inoculate 4×10 6 293FT cells. After overnight incubation, when the cell confluence reaches about 80%, lentivirus can be packaged. Replace the medium with 10 mL of DMEM medium containing 10% FBS 2 h before packaging; (2) Prepare the solution; (3) Take a 1.5 LEP tube, allocate and configure tubes A and B, then gently mix and let stand for 5 min. Add A to B, gently mix well, and incubate at room temperature for 30 min. Add the DNA-liposome complex to the 293FT medium, mix gently by shaking, observe fluorescence after 6 h, and change the medium; (4) Collect the virus supernatants at 24 h and 48 h respectively into 50 mL centrifuge tubes, store at 4 °C, and replace with 13 mL of fresh DMEM medium containing 10% FBS; (5) After 72 h, collect the virus supernatant into the centrifuge tube in step (4) and mix well; (6) Centrifuge at 2000 g for 1 h at 4 °C, collect the supernatant into a clean 50 mL centrifuge tube, and filter the virus supernatant using a 20 mL syringe and a 0.22 µm filter.

[0030] Tube A Tube B Example 4

[0031] (1) Take 6 Ultra-clear SW28 centrifuge tubes, disinfect them with 70% ethanol, and place them in a laminar flow hood. Sterilize with ultraviolet light for 30 min; (2) Add approximately 30 mL of the pre-treated virus supernatant and then 10 mL of PBS to each Ultra-clear SW28 centrifuge tube; (3) Take a 10 mL pipette and aspirate 5 mL of 20% sugar solution. Insert the pipette all the way to the bottom of the centrifuge tube and slowly dispense the sucrose solution; (4) Adjust the weight of each tube with PBS so that the weight difference between the corresponding centrifuge tubes does not exceed 0.1 g; (5) Place all 6 centrifuge tubes in the Beckman SW28 ultracentrifuge rotor in sequence; (6) Centrifuge at 30000 rpm for 2 h at 4 °C; (7) Carefully remove the tubes from the rotor. Pour out the supernatant, invert the centrifuge tubes on a paper towel and let stand for 10 min to allow the remaining supernatant to drain. Aspirate the remaining droplets. There should be visible precipitate at the bottom of the tube; (8) Add 200 μL of serum-free AIM-V medium to each tube to wash down the precipitate. Aliquot the virus suspension into 50 μL per 1.5 mL EPP tube and store at -80 °C. Example 5

[0032] Infection of T lymphocytes with GPC3-CAR lentiviral vector

[0033] (38) Seed T lymphocytes at 5×10 5 / well 24 h in advance, and the steps are the same as before.

[0034] (1) Infection: The steps are the same as before. First, add polybrene (final concentration 10 μg / mL) to each well. Then, infect sorted T lymphocytes 24 h later with GccD, G7×19ccD, and Mock lentiviruses at a level of 20 MOI respectively. Gently mix and culture in an incubator at 37°C with 5% CO2. After 18 h, centrifuge the cells at room temperature at 300 g for 4 min, remove the old supernatant, and replace it with fresh AIM-V medium containing 10% FBS and IL-2 (300 U / L), and continue the culture; (2) Cell culture: Change the medium by half every other day (i.e., centrifuge the cells at 300 g for 5 min after collecting the cells, discard half of the supernatant, resuspend the cell solution with the remaining cell solution, and add fresh complete medium according to the final cell concentration of 5×10 5 / mL), and add IL-2 (300 U / mL) each time the medium is changed. Count the cells at 24 h, 72 h, 96 h, 7 d, and 14 d respectively.

[0035] See Figure 2 and Figure 3 , CAR-T successfully secretes IL-7 and CCL19 and shows proliferation, chemotaxis, and subtype advantages in vitro; the secretion of IL-7 and CCL19 is detected in the culture supernatant. Starting from the 7th day, the number of GPC3-ccD-7×19 CAR-T cells is significantly more than that of GPC3-ccD-CAR-T cells. The cells grow in clusters, and the density is greater than that of GPC3-ccD CAR-T and Non-CAR-T cells; in the wells with the culture supernatant of GPC3-ccD-7×19 CAR-T cells, the number of non-CAR-T cells migrating to the lower chamber is more than that in the other two groups; in GPC3-ccD-7×19 CAR-T cells, the proportion of naive T cells (CAR+CD62L+CD45RA+CD45RO–) and T memory stem cells (CAR+CD62L+CD45RA+CD45RO+) is higher than that of GPC3-ccD CAR-T. Example 6

[0036] Hepatocarcinoma cell culture

[0037] Select two hepatocarcinoma cell lines, HepG2 with high GPC3 expression and SK-Hep-1 without GPC3 expression.

[0038] Turn on the water bath in advance and preheat the complete medium and trypsin at 37°C. After the medium is fully preheated, take out the cryopreservation tube containing the cells from liquid nitrogen and quickly place it in a 37°C constant temperature water bath, shaking continuously. The whole process should not exceed 1 minute. After thawing, quickly transfer the cells in the cryopreservation tube to a centrifuge tube containing 4 ml of preheated medium with a pipette gun, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add 1 ml of preheated medium to the centrifuge tube, and gently pipette the cells up and down several times with a pipette gun. Then transfer the cell suspension to a culture dish containing 3 ml of preheated medium and culture it under the culture conditions of saturated humidity, 37°C and 5% CO2. Observe the cells under the microscope. If the coverage rate reaches 70% to 80%, passage is carried out.

[0039] Construction of mouse hepatoma tumor-bearing model

[0040] NOD / SCID mice, 6 - 8 weeks old, a total of 45 mice, weighing 18 - 22 g. Constant temperature (22 - 24°C), constant humidity (relative humidity 40 - 60%), cages, bedding, drinking water and feed are all sterilized.

[0041] Tumor cell inoculation

[0042] One day before inoculating hepatoma tumor cells, weigh the mice routinely and record, make marks with mouse ear tags and shave the hair for skin preparation. All injection supplies are disinfected by autoclaving. When the number of hepatoma cells meets the requirements, take the logarithmic growth phase of hepatoma cells, rinse twice with 1×PBS, terminate after trypsin digestion, collect into a 50 ml centrifuge tube, centrifuge at 1000 rpm for 5 minutes to remove the supernatant, and wash twice with PBS again. Take 5 μl and dilute it 20 times for resuspension and counting. Calculate the number of cells in the whole cell suspension, resuspend the cells with a certain volume of PBS to contain 5×10 cells in 200 μl, and bring the cells to the animal experiment center with an ice box.

[0043] Dip a cotton ball in alcohol to routinely disinfect the subcutaneous inoculation site on the back of the mouse's rib. Use a needle to aspirate the hepatoma cell suspension in the ice box, which has been diluted to 5×10 6 / 0.2 ml, mix with 50% Matrigel before injection, shake well and slowly inject subcutaneously into the mouse thigh. Gently press the injection site to prevent leakage of the cell suspension and subcutaneous bleeding. After the mice are successfully injected and tumor formation occurs in about 7 - 10 days, measure the size of the mouse tumor with a vernier caliper and record. After successfully establishing the subcutaneous tumor model of hepatoma tumor-bearing, randomly divide the mice with high expression of GPC3 in HepG2 with established tumors into cages, 5 mice in each cage, a total of 4 cages. The operation for the mice with established tumors of SK-Hep-1 without GPC3 expression is the same as above.

[0044] See Figure 4 , A is a schematic diagram of the in vivo evaluation protocol for the persistence of GPC3-CAR T (GHccD) cells. NSG mice were injected with 2×106 HepG2 cells, injected with 2×10 6 Ffluc + CAR T cells after 2 weeks. B shows the monitoring of bioluminescent GPC3-CAR T cells at the specified time points after injection. C shows the bioluminescence counts of GPC3-CAR T cells (mean ± SEM) over the course of the experiment. D shows the ratio of CD4+ to CD8+ measured by flow cytometry, with GPC3-CART cells relative to mouse CD45-expressing cells in the spleen tissue on day 18. The GHccD T cells effectively expanded for 8 days, and then the cell population contracted and completely disappeared 15 days after injection. Compared with the GHccD T cells, the peak expansion timeline of the 7×19.GHccD T cells was longer (day 12, P < 0.001). The co-expression of IL7 and CCL19 induced the highest amplification and persistence of GPC3-CAR-T cells in vivo.

[0045] The present invention constructs a liver tumor-bearing model in SCID mice (HepG2 and SK-HEP-1) and conducts research on the inhibition of transplanted tumors; the technical results obtained from the project are as follows: (1) Using a humanized GPC3 single-chain antibody as the extracellular antigen-binding region, a second-generation CAR and a control group: empty virus were designed. The CAR co-stimulatory molecule 4-1BB was selected, and the above-mentioned CAR lentiviral expression vector was constructed; (2) The use of sorted CD4 + , CD8 + T cells in human peripheral blood lymphocytes, the CAR lentivirus was introduced, the preparation of CAR-T cells was completed, and the preparation standard of CAR-T cells was optimized; (3) It has been confirmed that CAR-modified T cells have a specific killing effect on hepatocellular carcinoma cells, and the effect of shrinking the tumor mass is obvious. The empty virus CAR-T in the control group does not have the effect of killing hepatocellular carcinoma cells. The following indicators have been completed: the purity of transfected cells has reached 90%; the viability of transfected cells has reached more than 85%; the tumor killing ability, the killing rate of co-culture with in vitro tumor cell lines exceeds 85%; the number of T cells has reached 10 E10 cells / ml or more.

[0046] The present invention selects GPC3 as the CAR-T treatment target for hepatocellular carcinoma, adds IL-7 and CCL19, so that GPC3-CAR-T cells containing IL-7 and CCL19 secrete a large amount of IL-7 and CCL19 near solid tumors, change the local immune environment, attract DC cells, NK cells and cytotoxic T cells to aggregate towards the tumor mass in large numbers, activate CAR-T cells, change the local environment of the tumor, increase the infiltration of immune cells into the tumor mass, and target and attack the tumor mass. At the same time, 4-1-BB is selected as the co-stimulatory factor.

[0047] The chimeric antigen receptor constructed by the present invention can be more stably expressed on the surface of T cells. The CAR-T cells exhibit the maximum cell proliferation ability and effective target-binding characteristics, and have a better ability to eliminate tumor cells. The cells in the experimental group have no toxic and side effects on antigen-negative cells and can be used for targeted tumor therapy.

Claims

1. A chimeric antigen receptor targeting HCC cells, comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain, characterized in that, The extracellular domain includes the GPC3 antigen-binding domain, and the intracellular signaling domain includes the cytokine IL-7 and the chemokine CCL19.

2. The chimeric antigen receptor targeting HCC cells according to claim 1, wherein The extracellular domain further includes a promoter and a leader peptide, and the promoter includes the GAG promoter.

3. The chimeric antigen receptor targeting HCC cells according to claim 1, wherein A hinge region domain is provided between the extracellular domain and the transmembrane domain.

4. The chimeric antigen receptor targeting HCC cells according to claim 3, wherein The hinge region domain includes the CD8 hinge region domain; the transmembrane domain includes one or more of CD28, CD5, CD16, CD22, and CD33.

5. The chimeric antigen receptor targeting HCC cells according to claim 1, characterized in that, The intracellular signaling domain includes a co-stimulatory domain and an intracellular signal transduction domain.

6. The chimeric antigen receptor targeting HCC cells according to claim 5, wherein The co-stimulatory domain includes the 4-1BB co-stimulatory domain.

7. A nucleic acid molecule encoding the chimeric antigen receptor targeting HCC cells according to claim 1, or a vector comprising the nucleic acid molecule.

8. An immune effector cell comprising the chimeric antigen receptor targeting HCC cells according to claim 1, or the nucleic acid molecule encoding the chimeric antigen receptor targeting HCC cells according to claim 7, or a vector comprising the nucleic acid molecule.

9. A drug comprising the chimeric antigen receptor targeting HCC cells according to claim 1 or the immune effector cell according to claim 8.

10. Use of the chimeric antigen receptor targeting HCC cells according to claim 1 in the preparation of CAR immune cells, or in the preparation of immunotherapeutic drugs; use of the CAR immune cells according to claim 8 in the preparation of immunotherapeutic drugs.

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