Secreting type CD133 targeting CAR-T cell as well as preparation method and application of secreting type CD133 targeting CAR-T cell

Abstract

The invention discloses a secreting type CAR-T cell targeting CD133, and particularly relates to a CAR-T cell which secretes eCNTFR recombinant protein and targets CD133, and a preparation method and application of the CAR-T cell. The secreting type CAR-T cell targeting CD133 disclosed by the invention is used for expressing CD133-CAR and secreting soluble recombinant protein eCNTFR at the same time, and the CAR-T cell comprises an scFv region used for identifying a CD133 antigen. The CAR-T cell provided by the invention can express CD133-CAR targeting CD133 and secrete eCNTFR recombinant protein at the same time, can act on immune cells in a tumor inhibitory immune microenvironment, improves the activity of the immune cells, effectively overcomes the immunosuppression of the tumor microenvironment, and further enhances the killing ability of the CAR-T cell to tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a secretory CAR-T cell targeting CD133, and in particular to a CAR-T cell that secretes eCNTFR recombinant protein and targets CD133, as well as a preparation method and use of the CAR-T cell. Background Art

[0002] Primary liver cancer is one of the malignant tumors with the highest morbidity and mortality. Traditional surgery, radiotherapy and chemotherapy are still difficult to effectively treat tumors. There is an urgent need to develop new treatment methods. Immunotherapy has become a hot spot in current tumor treatment research and clinical application. Among them, CAR-T cell therapy, which modifies T cells in vitro through genetic engineering and then returns them to the patient's body, has achieved encouraging results in the treatment of hematological malignancies, but its therapeutic effect on malignant solid tumors is not good. Therefore, it is necessary to study how to improve the function of CAR-T cells so that they can achieve better therapeutic effects in malignant solid tumors.

[0003] CD133 is a transmembrane glycoprotein. Studies have reported that CD133 is highly expressed in advanced liver cancer. In addition, CD133 is also a marker of tumor stem cells and endothelial progenitor cells, and is often positively correlated with tumor metastasis and recurrence. Therefore, CAR-T cells targeting CD133 have a good effect on inhibiting the recurrence and metastasis of liver cancer in the treatment of primary liver cancer. However, in clinical trials, the remission rate of CAR-T cells targeting CD133 in patients with primary liver cancer is relatively low. The reason is that in the liver cancer microenvironment, stromal cells in contact with liver cancer cells secrete growth factors and cytokines, which can directly act by signaling to liver cancer cells or indirectly by recruiting other stromal components to resist the tumor killing function of T cells. An important aspect of this process is the expansion of tumor-associated fibroblasts (CAFs). CAFs are a variety of stromal cells with different characteristics in different tumors and tissues. CAFs support the growth of cancer cells in the body by secreting soluble factors that stimulate tumor cell growth. Cardiotrophin-like cytokine 1 (CLCF1) is a soluble factor secreted by CAFs. It binds to the CNTFR receptor on the surface of tumor cells to form the CLCF1-CNTFR signaling axis, which can induce the formation of a tumor suppressive immune microenvironment, thereby inhibiting the anti-tumor effect of T cells. Studies have reported that soluble CNTFR decoy receptor sequences (eCNTFR) can block the CLCF1-CNTFR signaling axis, hinder the formation of a tumor suppressive immune microenvironment, and thus inhibit tumor progression. Therefore, based on the CAR-T cell engineering strategy of locally blocking the CLCF1-CNTFR signaling axis, a new type of CAR-T cell that can break through the suppressive immune microenvironment mediated by the CLCF1-CNTFR signaling axis in the liver cancer tumor microenvironment has been developed, which is expected to provide a new treatment strategy for the treatment of advanced liver cancer, which is of great significance. Summary of the invention

[0004] The first purpose of the present invention is to provide a secretory CAR-T cell targeting CD133. The CAR-T cell can express CD133-CAR targeting CD133 while secreting eCNTFR recombinant protein, which can act on immune cells in the tumor suppressive immune microenvironment, improve the activity of its immune cells, effectively overcome the immunosuppression of the tumor microenvironment, and thus enhance the killing ability of CAR-T cells against tumor cells.

[0005] The secretory CD133-targeting CAR-T cells of the present invention express CD133-CAR while secreting soluble recombinant protein eCNTFR, and the CD133-CAR includes a scFv region for recognizing the CD133 antigen.

[0006] The second object of the present invention is to provide a method for preparing the secretory CAR-T cells targeting CD133. The CAR-T cells prepared by this method have both specific anti-tumor effects against tumor cells with high CD133 expression and can express soluble recombinant protein eCNTFR at high levels, thereby hindering the formation of a tumor suppressive immune microenvironment.

[0007] The method for preparing secretory CD133-targeting CAR-T cells of the present invention comprises the following steps:

[0008] A. Construct a lentiviral plasmid backbone carrying CD133-CAR and soluble recombinant protein eCNTFR gene to obtain a recombinant lentiviral plasmid;

[0009] B. transfecting the recombinant lentiviral plasmid into host cells to prepare a recombinant lentiviral vector capable of infecting T cells;

[0010] C. transducing the recombinant lentiviral vector into activated T cells to obtain CAR-T cells;

[0011] D. The CAR-T cells are cultured in vitro, expanded, and collected to obtain the secretory CD133-targeting CAR-T cells.

[0012] According to a further feature of the preparation method of the present invention, in step A, the recombinant lentiviral plasmid is a third-generation lentiviral transgenic plasmid, which is a recombinant replication-deficient lentiviral plasmid that can integrate exogenous fragments into host genes, is disposable, cannot replicate and proliferate, and is safe and reliable.

[0013] According to the further characteristics of the preparation method of the present invention, in step A, the recombinant lentiviral plasmid sequentially comprises an EF1 promoter sequence, a CD133 single-chain antibody (scFv), c-myc, a CD8 chimeric receptor transmembrane region, an intracellular signaling region, P2A, and a soluble recombinant protein eCNTFR sequence.

[0014] The intracellular signal transduction region of the CD133-CAR comprises a region encoding a co-stimulatory factor, which is selected from any combination of one or more of 4-1BB, CD28, CD27, OX40, CD30, CD40, PD-1, ICOS, LIGHT, B7-H3, a ligand that specifically binds to CD83, ICAM-1, HVEM (LIGHTR), CD160, lymphocyte function-associated antigen-1 (LFA-1), IL2Rα, CD103, CD11b, CD11c, TRANCE / RANKL, SLAMF4 (CD244, 2B4), CD69, and SLAM (SLAMF1, CD150, IPO-3).

[0015] As a preferred technical solution of the present invention, the co-stimulatory factor region is CD28; and the intracellular signal transduction region is CD28-CD3ζ.

[0016] According to a further feature of the preparation method of the present invention, in step B, the host cell is a 293T cell.

[0017] According to a further feature of the preparation method of the present invention, the step B further comprises two auxiliary plasmids PspAX and PMD2G.

[0018] The third object of the present invention is to provide the use of the secretory CD133-targeting CAR-T cells, specifically for the preparation of drugs for treating cancer.

[0019] Preferably, the secretory CD133-targeting CAR-T cells described in the present invention can be used to prepare drugs for treating liver cancer.

[0020] Preferably, the drug is a drug targeting CD133-positive tumor stem cells.

[0021] The secretory CD133-targeting CAR-T cells described in the present invention are dual-target CAR-T cells that can secrete soluble recombinant protein eCNTFR while targeting CD133. eCNTFR can improve the inhibitory immune microenvironment of liver cancer, which is beneficial to enhancing the anti-tumor ability of CD133 CAR-T cells. The intracellular signal transduction region of CD133-CAR contains a region encoding costimulatory factors. One or more combinations of costimulatory factors can cause CD133 CAR-T cells to continuously activate and proliferate, and soluble recombinant proteins are continuously secreted, improving the liver cancer tissue microenvironment, and enhancing the CD133 CAR-T cell killing of tumor cells and the memory of immune cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the pcRLenti-EF1a-MCS-WPRE backbone vector plasmid used in the present invention.

[0023] Figure 2 It is a structural diagram of the lentiviral backbone plasmid SV305 carrying CD133-CAR and soluble recombinant protein eCNTFR gene according to the present invention.

[0024] Figure 3 This is a diagram showing the results of ELISA detection of secretion of eCNTFR.

[0025] Figure 4 The figure is a graph showing the killing efficiency of target cells by the CAR-T cells expressing CD133-CAR and secreting eCNTFR under different effector-target ratios. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with examples and accompanying drawings, and the embodiments of the invention described in detail should not be construed as limiting the scope of the present invention. The disclosed content of the present invention can be improved from materials, methods and reaction conditions at the same time, and all these improvements should fall within the spirit and scope of the present invention.

[0027] The experimental methods in the following examples without detailed conditions are generally based on the conditions recommended by the manufacturer. Unless otherwise specified, percentages are calculated by weight. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0028] Although any methods and materials similar or equivalent to those described herein can be used in embodiments of the present invention, the preferred materials and methods are described herein.

[0029] In the present invention, the term CD133-CAR refers to a CD133 chimeric antigen receptor comprising a scFv sequence that recognizes CD133.

[0030] scFv: full name single-chain antibody fragment, that is, single-chain antibody fragment.

[0031] In the present invention, the term CD133-scFv refers to an anti-CD133 single-chain antibody fragment, whose nucleotide sequence is shown in SEQ ID NO.1; the nucleotide sequence is used on a lentiviral expression vector, a retroviral vector, an adenoviral expression vector, an adeno-associated viral expression vector or other types of expression vectors, preferably a lentiviral expression vector.

[0032] Example 1: Preparation of CAR-T cells that express CD133-CAR and secrete eCNTFR

[0033] The method for preparing CAR-T cells expressing CD133-CAR and secreting eCNTFR according to the present invention is as follows:

[0034] 1. Isolate PBMCs from peripheral blood.

[0035] 1. Collect 80-100 mL of human peripheral blood using an anticoagulant tube or blood collection bag (shake while collecting to fully mix the peripheral blood and anticoagulant), and separate PBMC in a sterile cell preparation workshop. Transfer the peripheral blood to a 50 ml centrifuge tube, centrifuge at 800 g for 15 minutes at room temperature, draw the upper layer of yellow autologous plasma into a new 50 ml centrifuge tube, inactivate at 56°C for 30 minutes, and place at -20°C for use; add an equal volume of PBS to the lower cell layer, and mix by inverting;

[0036] 2. Pipette 15 ml of lymphocyte separation solution (Ficoll-Paque Plus, GE) into the bottom of a new 50 ml centrifuge tube, and slowly add 30 ml of diluted blood along the tube wall to the upper layer of lymphocyte separation solution. Place the centrifuge tube in a centrifuge, 450 g, and centrifuge slowly for 25 minutes without interruption.

[0037] 3. After centrifugation, carefully aspirate the buffy coat cells above the lymphocyte separation solution, transfer to a new 50 ml centrifuge tube, add 50 ml PBS for washing, and centrifuge at 300 g for 10 min.

[0038] 4. Discard the supernatant and keep the cell pellet at the bottom of the centrifuge tube; add 50 ml of PBS again, centrifuge at 160g, and centrifuge slowly for 15 minutes;

[0039] 5. Discard the supernatant; finally add PBS, centrifuge at 300g for 10 min, and discard the supernatant to obtain PBMC cells.

[0040] 2. Activate and sort CD3-positive T cells from PBMC.

[0041] CD3 positive T cells in PBMCs were sorted (5) using the CD3 positive T cell sorting kit (EasySep™ Human T Cell Isolation Kit Catalog No.: #17951, Manufacturer: STEMCELL), as follows:

[0042] 1. Adjust the cell density to 5×10 7 cells / mL, add the cells to a 5 mL cell cryopreservation tube;

[0043] 2. Add 50ul / mL of Isolation Cocktail, mix well and let stand for 5 minutes;

[0044] 3. Vortex the RapidSheres TM reagent for 30 seconds using a vortexer to mix the particles evenly, add it to the cells at a ratio of 40ul / mL and mix well;

[0045] 4. Add PBS to 2.5 mL and pipette up and down 2-3 times, then place on the magnetic stand for 3 minutes;

[0046] 5. Tilt the magnetic rack to transfer the liquid to a new cell culture dish to obtain the sorted CD3-positive T cells.

[0047] 3. Synthesize CD133-scFv sequence.

[0048] Prepare CD133 chimeric antigen receptor (CAR), i.e. CD133-CAR, and send the scFv fragment to the company for synthesis, the sequence of which is the nucleotide sequence shown in SEQ ID NO.1.

[0049] 4. Construct a lentiviral plasmid carrying CD133-CAR and soluble recombinant protein eCNTFR gene to obtain a recombinant lentiviral plasmid.

[0050] Construction of recombinant lentiviral backbone plasmid (SV305, see Figure 2 ): The CAR structure of secreted eCNTFR was synthesized and cloned into the pcRLenti-EF1a-MCS-WPRE backbone plasmid (Heyuan Bio, H30436, see Figure 1 ), after correct sequencing, the SV305 backbone plasmid is obtained, which is connected in series with EF1a promoter-CD133scFv-c-myc-CD8TM-CD28-CD3ζ-P2A-eCNTFR, wherein the nucleotide sequence of CD133scFv is shown in SEQ ID NO.1, the nucleotide sequence of c-myc is shown in SEQ ID NO.2, the nucleotide sequence of CD8TM is shown in SEQ ID NO.3, the nucleotide sequence of CD28 is shown in SEQ ID NO.4, the nucleotide sequence of CD3ζ is shown in SEQ ID NO.5, the nucleotide sequence of P2A is shown in SEQ ID NO.6, and the nucleotide sequence of eCNTFR is shown in SEQ ID NO.7.

[0051] SEQ ID NO.1 sequence is from NCBI database (GenBank: HW350341), SEQ ID NO.2 sequence is from NCBI database human Myc gene (Gene ID: 4609), SEQ ID NO.3 sequence is from NCBI database human CD8A gene (Gene ID: 925), SEQ ID NO.4 sequence is from NCBI database human CD28 gene (Gene ID: 940), SEQ ID NO.5 sequence is from NCBI database human CD247 gene (Gene ID: 919), SEQ ID NO.6 sequence is from Porcinetes chovirus15 (UniProt Entry: A0A3B8G5R2). SEQ ID NO.7 sequence is from U.S. Patent No. 11208462 (see Table 1).

[0052] 5. Infect 293T cells with the recombinant lentiviral plasmid carrying CD133-CAR and soluble recombinant protein eCNTFR gene obtained in step 4 to prepare a lentivirus that can infect T cells.

[0053] 1. The cells after 2-3 generations of subculture after 293T cells were revived were used as working cells for lentiviral packaging, and the total number of subcultures did not exceed 10 generations.

[0054] 2. Liposome transfection (taking 10 cm culture dish as an example).

[0055] 1) One day before transfection, inoculate 293T cells into a 10 cm diameter culture dish. The number of cells inoculated should be such that the cells grow to 90% to 95% confluence on the day of transfection.

[0056] 2) On the day of transfection, remove the culture medium and add 5 ml of virus packaging culture medium;

[0057] 3) Prepare DNA-Lipofectamine3000 complex:

[0058] A. Prepare a sterile 5 mL centrifuge tube and add 1.5 mL of serum-free Opti-MEM medium and auxiliary plasmids (PspAX, PMD2G, see Figure 3 , Figure 4 ) and lentiviral expression plasmid (SV305), and mix thoroughly by pipetting;

[0059] B. Prepare another sterile 5 mL centrifuge tube, add 1.5 mL serum-free Opti-MEM culture medium and 68 μL Lipofectamine 3000, gently pipette to mix, and incubate at room temperature for 10 minutes;

[0060] C. After 30 minutes, add serum-free Opti-MEM medium containing plasmid DNA into serum-free Opti-MEM medium containing Lipofectamine3000, gently pipette to mix, and incubate at room temperature for 30 minutes;

[0061] D. Add DNA-Lipofectamine3000 complex to 293T cells drop by drop, gently shake the culture dish back and forth to mix the complex, and culture in a 37°C, 5% CO2 saturated humidity incubator.

[0062] 3. Collection and concentration of recombinant lentiviral vectors.

[0063] 1) 48 hours after transfection, collect the culture medium containing the virus. Pipette the culture medium into a sterile 50mL centrifuge tube with a lid, and then add 8mL of virus packaging culture medium to the cells that can still produce virus;

[0064] 2) Centrifuge at 4°C, 3000 rpm for 15 minutes, centrifuge the virus supernatant at low speed, remove cell debris, and recover the supernatant;

[0065] 3) 72 hours after transfection, collect the culture fluid containing the virus. Centrifuge at 4°C, 3000 rpm for 15 minutes, centrifuge the virus supernatant at low speed, remove cell debris, and recover the supernatant. Filter the virus supernatant collected at 48 hours and the virus supernatant collected at 72 hours with a 0.45 μm small filter, and collect the filtrate in a 50 mL centrifuge tube;

[0066] 4) The collected virus supernatant was concentrated using an ultracentrifuge and centrifuged at 4°C, 72,000 g / min for 120 minutes; the virus precipitate was resuspended in 500 μl of fresh culture medium, aliquoted and frozen, and a portion was taken to determine the virus titer.

[0067] 6. The recombinant lentiviral vector obtained in step 5 is used to transduce the T cells sorted in step 2 to generate CAR-T cells that express CD133-CAR and secrete soluble recombinant protein eCNTFR.

[0068] 1. T cell activation

[0069] (1) The sorted T cells were cultured at a density of 0.5 to 1 × 10 6 cells / ml were resuspended in KBM 581 medium (Cat. No. 88-581-CM, Corning) supplemented with 1% autologous plasma;

[0070] (2) Washing Dynabeads:

[0071] a) Take out the Dynabeads from the refrigerator and vortex for 30 seconds to resuspend the magnetic beads precipitated at the bottom;

[0072] b) According to the number of T cells in (1), calculate the required Dynabeads according to the ratio of Dynabeads:T = 1:1 to 1:3, use a sterile pipette to transfer the Dynabeads to a new 15ml centrifuge tube, add the same volume of DPBS, and mix by pipetting. Place the centrifuge tube on a magnetic stand, let it stand for 5 minutes, and discard the liquid with a pipette;

[0073] c) Add 2 ml of DPBS into a 15 ml centrifuge tube and repeat the washing steps in b);

[0074] d) Add 100ul of 581 culture medium to resuspend the beads, and add dropwise to the cell culture medium. Mix by pipetting and place in a cell culture incubator at 37°C, 5% CO2 for 3 days.

[0075] 2. Transduce T cells with recombinant lentiviral vector.

[0076] (1) 3 days after T cell activation, the number of T cells was counted by trypan blue staining. The required amount of virus was calculated according to MOI = 3-10: required amount of virus (ml) = (MOI * number of cells) / virus titer (Tu / ml);

[0077] (2) Take out the lentivirus from the -80°C ultra-low temperature freezer and quickly thaw it in a 37°C water bath (do not put the tube below the liquid surface);

[0078] (3) Take out the T cell culture dish, add polybrene to a final concentration of 5 ug / ml, add the required amount of virus, and mix gently with a pipette to disperse the aggregated cell clusters evenly. Seal the culture dish with a sealing film and centrifuge at 800g for 1 hour at room temperature;

[0079] (4) Remove the sealing film and place the culture dish in a 37°C, 5% CO2 incubator for 24 hours;

[0080] (5) Centrifuge at 250 g for 10 minutes at room temperature, remove the culture supernatant containing the virus, resuspend the cell pellet with fresh culture medium, transfer the cells to a new culture dish, and continue culturing.

[0081] 7. Cultivate the cells obtained in step 6 in vitro.

[0082] Gently blow the cell clusters in the culture system every day until they are dispersed, and count the number of cells using trypan blue staining to maintain the cell density at 1×10 6 Pieces / ml.

[0083] 8. Expand the cells obtained in step 7 in large quantities.

[0084] a) When the number of cells is greater than 100×10 6 At 4 pm, CAR-T cells were transferred to cell culture bags for culture. The cells were counted the next day using the trypan blue counting method to maintain the cell density at 1×10 6 Pieces / ml;

[0085] b) When CAR-T cells are cultured for 10 days, part of the cell culture fluid can be extracted for sterility testing;

[0086] c) When the total number of cells reaches 500-1000×10 6 When the CAR-T cells are collected, the magnetic beads can be removed using a magnetic pole.

[0087] d) Take part of the cell suspension and co-culture it with the target cell human liver cancer Hep3B cells (CD133 positive), and then detect the secretion of eCNTFR in the supernatant by ELISA (see Figure 4), compared with the control group, the experimental group successfully secreted eCNTFR, indicating that CAR-T cells expressing CD133-CAR and secreting soluble recombinant protein eCNTFR were successfully prepared, and the cells were expanded and cultured and frozen.

[0088] 9. Collect CAR-T cells that express CD133-CAR and secrete soluble recombinant protein eCNTFR.

[0089] Example 2: In vitro killing verification of the prepared CAR-T cells expressing CD133-CAR and secreting eCNTFR.

[0090] Studies have found that tumor cells can also autosecrete CLCF1 and bind to their own CNTFR, activating the JAK-STAT3 and Erk signaling pathways in tumor cells, enhancing the stemness of tumor cells and inducing the formation of a tumor suppressive immune microenvironment, inhibiting the anti-tumor effect of T cells. Knocking down CLCF1 in tumor cells can hinder the formation of tumor stemness, inhibit the formation of a tumor suppressive immune microenvironment, and thus inhibit tumor progression. In vitro experiments adding eCNTFR recombinant protein to tumor cells can also inhibit the proliferation of tumor cells.

[0091] Target cells (human liver cancer Hep3B cells) and effector cells (CAR-T cells expressing CD133-CAR and secreting eCNTFR) were cultured separately.

[0092] Collect 4 × 10 target cells 5 cells and effector cells (CAR-T cells), 3×10 6 cells, 300g, centrifuge for 5min, slowly rise and fall, and discard the supernatant; resuspend the target cells and effector cells separately with 1ml PBS solution, 300g, centrifuge for 5min, slowly rise and fall, and discard the supernatant; repeat once; resuspend the effector cells with 700μl culture medium (AIM-V culture medium + 10% FBS), and resuspend the target cells with 2ml culture medium (1640 culture medium + 10% FBS).

[0093] Experimental wells with effector-target ratios of 4:1, 2:1, 1:1, and 0.5:1 were set, and a control group was set, with 4 replicates in each group, and incubated in incucyte for 48 hours. Under different effector-target ratios, when the effector-target ratio was 0.5:1, the CAR-T cells expressing CD133-CAR and secreting eCNTFR had a significantly stronger killing efficiency in target cells than the CAR-T cells expressing CD133-CAR. The CAR-T cells expressing CD133-CAR and the CAR-T cells expressing CD133-CAR and secreting eCNTFR prepared by the present invention had a tumor killing ability of 69% and 80%, respectively (see Figure 4 ).

[0094] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A secretory CAR-T cell targeting CD133, characterized in that: The CAR-T cells express CD133-CAR while secreting soluble recombinant protein eCNTFR, and the CD133-CAR includes a scFv region for recognizing the CD133 antigen.

2. The method for preparing secretory CD133-targeting CAR-T cells according to claim 1, characterized in that: The steps include: A. Construct a lentiviral plasmid backbone carrying CD133-CAR and soluble recombinant protein eCNTFR gene to obtain a recombinant lentiviral plasmid; B. transfecting the recombinant lentiviral plasmid into host cells to prepare a recombinant lentiviral vector capable of infecting T cells; C. transducing the recombinant lentiviral vector into activated T cells to obtain CAR-T cells; D. The CAR-T cells are cultured in vitro, expanded, and collected to obtain the secretory CD133-targeting CAR-T cells.

3. The preparation method according to claim 2, characterized in that: In the step A, the recombinant lentiviral plasmid is a third-generation lentiviral transgenic plasmid.

4. The preparation method according to claim 2, characterized in that: In the step A, the recombinant lentiviral plasmid sequentially comprises an EF1 promoter sequence, a CD133 single-chain antibody (scFv), c-myc, a CD8 chimeric receptor transmembrane region, an intracellular signal transduction region, P2A, and a soluble recombinant protein eCNTFR sequence.

5. The preparation method according to claim 4, characterized in that: The intracellular signal transduction region is CD28-CD3ζ.

6. The preparation method according to claim 2, characterized in that: In the step B, the host cell is a 293T cell.

7. The preparation method according to claim 2, characterized in that: The step B also includes two auxiliary plasmids PspAX and PMD2G.

8. Use of the secretory CD133-targeting CAR-T cells as claimed in claim 1 for preparing a drug for treating cancer.

9. The use according to claim 8, characterized in that: The cancer is liver cancer.

10. The use according to claim 8, characterized in that: The drug is a drug targeting CD133-positive tumor stem cells.

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