CN105936649A - Chimeric antigen receptor and gene and recombinant expression vector thereof, CAR133-NKT cells and preparation method and application of CAR133-NKT cells - Google Patents

Abstract

The invention belongs to the field of tumor biological products, and discloses a chimeric antigen receptor and its gene and recombinant expression vector, CAR133-NKT cells and its preparation method and application. The chimeric antigen receptor is CD133ScFv-CD8-CD137- CD3ζ, including tandem rat growth hormone signal peptide, CD133ScFv, hinge region and transmembrane region of CD8, intracellular signaling domain of CD137 and intracellular signaling domain of CD3ζ. When the CAR133-NKT cells of the present invention are co-cultured with CD133-positive epithelial tumor cells, they have good specific killing activity on CD133-positive epithelial tumor cells.

Description

Chimeric antigen receptor and its gene and recombinant expression vector, CAR133-NKT cell and preparation method thereof and application

technical field

The present invention belongs to the field of tumor biological products, and specifically relates to a chimeric antigen receptor CD133ScFv-CD8-CD137-CD3ζ in adoptive immunotherapy, its gene and recombinant expression vector, and engineered CD133-targeted NKT cells (CAR133 -NKT cell) and its preparation method and application.

Background technique

Natural killer cells (NKT) are a special type of T lymphocyte subsets with dual properties of T cells and NK cells. NKT cells can express TCR of T cells and NKR-P1 of NK cells. Under the mediation of TCR and NKR, NKT cells can produce a large amount of IL-4 and INFγ, which can kill tumor cells. NKT cells bind to the Fc region of specific antibodies through CD16 on their surface to exert antibody-dependent cell-mediated cytotoxicity (ADCC, antibody-dependent cell-mediated cytotoxicity). However, in the process of antibody-dependent cell-mediated killing, since the antibody can specifically bind to the corresponding epitope on the target cell, NKT cells can kill any target cell that has been bound to the antibody, so the antibody and the target cell Antigen binding is specific, but the killing effect of NKT cells on target cells is non-specific. In addition, under normal circumstances, the half-life of infused NKT cells in the patient's body is about 2 weeks, and the validity period is short, requiring repeated infusions. Moreover, NKT cells themselves lack specific antibodies and are insufficient to accumulate around tumors or in tumor nests, which restricts the targeted therapy of NKT cells on malignant tumors. Furthermore, studies have shown that NKT cells do not have a killing effect on all tumors, and the killing effect on some tumors is relatively weak, and the specific killing activity needs to be improved. The immune surveillance of NKT cells on tumor stem cells is still controversial.

Cancer stem cells are a special kind of tumor cells, which account for a low proportion of tumor cells, but have the potential of self-renewal and differentiation into other different tumor cells. A large number of studies have pointed out that tumor stem cells are closely related to tumor recurrence and distant metastasis, and may be the reason why tumors resist chemotherapy and radiotherapy. CD133 is a glycoprotein on the cell surface and can be used as a marker for tumor stem cells in isolated tumor tissues. In addition, CD133 is highly expressed on the surface of various tumor cells, such as colorectal cancer, liver cancer, cholangiocarcinoma, pancreatic cancer, esophageal cancer, gastric cancer, ovarian cancer, lung cancer, prostate cancer, bladder cancer, breast cancer, endometrial cancer, Brain tumors, melanoma, or glioma, etc. Studies have found that the tumor-initiating cell population contains a large number of CD133-positive cells. At the same time, the high expression of CD133 is related to the poor prognosis of tumor treatment. Due to the above characteristics, the CD133 antigen has become an ideal target for antibody-based therapy, but it is still There is no preparation with high tumor-specific killing activity.

Contents of the invention

The purpose of the present invention is to overcome the defects in the prior art that NKT cells have a relatively weak killing effect on tumors and the specific killing activity needs to be improved, make full use of the role of CD133 as an ideal target for treating tumors, and provide a chimeric antigen receptor CD133ScFv - CD8-CD137-CD3ζ and its gene and recombinant expression vector, engineered CD133-targeted NKT cells (CAR133-NKT cells) and their preparation methods and applications.

The inventors of the present invention unexpectedly found in the research that when NKT cells modified by the chimeric antigen receptor CD133ScFv-CD8-CD137-CD3ζ of the present invention are co-cultured with CD133-positive epithelial tumor cells, they have good specificity for tumor cells. lethal activity.

Therefore, in order to achieve the above object, in the first aspect, the present invention provides a chimeric antigen receptor, the chimeric antigen receptor is CD133ScFv-CD8-CD137-CD3ζ, including rat growth hormone signal peptide, CD133ScFv , the hinge region (hinge region) and transmembrane region of CD8, the intracellular signaling domain of CD137 and the intracellular signaling domain of CD3ζ.

In a second aspect, the present invention provides a gene encoding the above-mentioned chimeric antigen receptor.

In a third aspect, the present invention provides a recombinant expression vector containing the above-mentioned genes.

In a fourth aspect, the present invention provides an engineered CD133-targeted NKT cell, wherein the NKT cell is an NKT cell modified by the above chimeric antigen receptor CD133ScFv-CD8-CD137-CD3ζ.

In the fifth aspect, the present invention provides a method for preparing engineered CD133-targeted NKT cells, the method comprising: packaging a lentivirus carrying pWPXL-CD133ScFv-CD8-CD137-CD3ζ to obtain a virus concentrate; using the obtained The virus concentrate infects NKT cells, so that NKT cells express chimeric antigen receptor CD133ScFv-CD8-CD137-CD3ζ.

In a sixth aspect, the present invention provides engineered CD133-targeted NKT cells prepared by the above method.

In a seventh aspect, the present invention provides an application of the above-mentioned engineered CD133-targeted NKT cells in preparing a preparation for treating tumors.

In an eighth aspect, the present invention provides a method for treating CD133-positive epithelial tumors and/or inhibiting tumor recurrence and metastasis, the method comprising: reinfusing the engineered CD133-targeted CD133-targeting drug of the present invention into CD133-positive epithelial tumor patients. NKT cells.

When co-cultured with CD133-positive epithelial tumor cells, the NKT cells modified by the chimeric antigen receptor CD133ScFv-CD8-CD137-CD3ζ of the present invention, that is, the engineered CD133-targeted NKT cells can specifically bind to the CD133 antigen and enhance The ability of immune cells to target and recognize the CD133 antigen on the surface of cancer cells enhances the specific killing activity against CD133-positive target cells. The engineered CD133-targeted NKT cells of the present invention provide a new option for treating CD133-positive tumors and suppressing tumor metastasis and recurrence, and have good industrial application prospects.

Other features and advantages of the present invention will be described in detail in the detailed description that follows.

Description of drawings

Figure 1 is the result of flow cytometry analysis on the phenotype of isolated and cultured NKT cells.

Fig. 2 is an electrophoretic identification diagram of the restriction endonuclease MluI/NdeI double-digestion fragment of the lentiviral expression vector pWPXL-CD8-CD137-CD3ζ of the present invention.

Fig. 3 is an electrophoresis identification diagram of the restriction endonuclease BamHI/NdeI double-digestion fragment of the lentiviral expression vector pWPXL-CD133ScFv-CD8-CD137-CD3ζ of the present invention.

Fig. 4 is a schematic structural diagram of the lentiviral expression vector pWPXL-CD133ScFv-CD8-CD137-CD3ζ of the present invention, wherein the counterclockwise sequence is the forward gene fragment, and the clockwise sequence is the reverse gene fragment.

Fig. 5 shows the infection efficiency of virus concentrate containing chimeric antigen receptor CD133ScFv-CD8-CD137-CD3ζ to NKT cells detected by flow cytometry.

Fig. 6 is the result of phenotype identification of NKT cells (CAR133-NKT cells) modified by chimeric antigen receptor CD133ScFv-CD8-CD137-CD3ζ detected by flow cytometry.

Fig. 7(A)-Fig. 7(C) are diagrams of CD133 expression level analysis of epithelial tumor cells with different CD133 expression levels in Example 4 of the present invention.

Fig. 8 is an analysis diagram (4 hours) of the killing effect of CAR133-NKT cells on epithelial tumor cells with different CD133 expression levels in Example 4 of the present invention.

FIG. 9 is an analysis diagram (8 hours) of the killing effect of CAR133-NKT cells on epithelial tumor cells with different CD133 expression levels in Example 4 of the present invention.

Fig. 10 is an analysis chart of CD133 expression levels of SW620 and HT29 in Example 5 of the present invention.

Fig. 11 is a diagram of tumor tissue volume after injection of physiological saline, NKT cells, Mock cells, and CAR133-NKT cells in Example 5 of the present invention.

Fig. 12 is an analysis diagram of the effect of CAR133-NKT cells on the function of the hematopoietic system in Example 6 of the present invention.

Figure 13(A)-Figure 13(D) is an analysis diagram of the toxic and side effects of CAR133-NKT cells in the treatment of liver cancer patients in Example 7 of the present invention, showing hemoglobin (Hgb), Change trends of white blood cells (WBC), platelets (PLT) and reticulin (Ret).

Fig. 14 is a graph showing the variation of the copy number of CAR133-NKT cells and the number of CD133 positive cells in patients with liver cancer in Example 7 of the present invention.

Fig. 15 is an analysis diagram of the treatment effect of liver cancer patients in Example 7 of the present invention.

Fig. 16 is a graph showing the therapeutic effect of CAR133-NKT cells on patients with pancreatic cancer in Example 8 of the present invention.

detailed description

Specific embodiments of the present invention will be described in detail below. It should be understood that the specific embodiments described here are only used to illustrate and explain the present invention, and are not intended to limit the present invention.

The present invention provides a chimeric antigen receptor, said chimeric antigen receptor is CD133ScFv-CD8-CD137-CD3ζ, comprising rat growth hormone signal peptide in tandem, CD133 single-chain antibody CD133ScFv, hinge region of CD8 and span Membrane domain, intracellular signaling domain of CD137 and intracellular signaling domain of CD3ζ.

Preferably, the chimeric antigen receptor CD133ScFv-CD8-CD137-CD3ζ consists of rat growth hormone signal peptide, CD133ScFv, the hinge region and transmembrane region of CD8, the intracellular signaling domain of CD137 and the intracellular signaling domain of CD3ζ Composed in series. Further preferably, the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO.1, even more preferably, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.1.

The present invention provides genes encoding the above-mentioned chimeric antigen receptors. Preferably, the gene has the nucleotide sequence shown in SEQ ID NO.2, and more preferably, the nucleotide sequence of the gene encoding the chimeric antigen receptor is shown in SEQ ID NO.2.

The invention provides a recombinant expression vector containing the above gene. Preferably, the recombinant expression vector is a lentiviral expression vector. There is no particular limitation on the lentiviral expression vector, as long as it can co-transfect packaging cells such as 293T packaging cells with the helper vector to obtain NKT cells modified by virus concentrate and chimeric antigen receptor CD133ScFv-CD8-CD137-CD3ζ, preferably In some cases, the lentiviral expression vector is pWPXL-CD133ScFv-CD8-CD137-CD3ζ.

The preparation method of the lentiviral expression vector pWPXL-CD133ScFv-CD8-CD137-CD3ζ is not particularly limited, and can be various methods that can be imagined by those skilled in the art. Preferably, the lentiviral expression vector pWPXL-CD133ScFv-CD8-CD137 -The preparation method of CD3ζ comprises the following steps:

(1) The hinge region and transmembrane region of CD8, the intracellular signaling domain of CD137 and the intracellular signaling domain of CD3ζ were respectively amplified from NKT cell cDNA, and cloned into the vector pWPXL-GFP to construct pWPXL-CD8 - CD137-CD3ζ;

(2) Synthesize the nucleotide sequence encoding rat growth hormone signal peptide and CD133ScFv, and clone it into pWPXL-CD8-CD137-CD3ζ, and obtain pWPXL-CD133ScFv-CD8-CD137-CD3ζ with the correct sequence after sequencing verification.

In step (1), there is no particular limitation on the methods for respectively amplifying the hinge region and transmembrane region of CD8, the intracellular signaling domain of CD137 and the intracellular signaling domain of CD3ζ from NKT cell cDNA, which can be Various commonly used methods include, for example, RT-PCR. Among them, NKT cells can be obtained by isolating mononuclear cells in human venous blood and then culturing them.

Specifically, the method for obtaining pWPXL-CD8-CD137-CD3ζ may include: extracting the total RNA of NKT cells, reverse transcription to obtain NKT cell cDNA, using the obtained NKT cell cDNA as a template, using primers P1 (SEQ ID NO.11) and P2 (SEQID NO.12) was amplified by PCR to obtain the hinge region and transmembrane region (SEQID NO.3) of the CD8 gene; PCR amplification was performed using primers P3 (SEQID NO.13) and P4 (SEQID NO.14) to obtain CD137 Intracellular signaling domain (SEQID NO.4) of the gene; PCR amplification using primers P5 (SEQID NO.15) and P6 (SEQID NO.16) to obtain the intracellular signaling domain (SEQID NO.5) of the CD3ζ gene , the obtained PCR products were subjected to double enzyme digestion, and then ligated with the lentiviral expression vector pWPXL-GFP after MluI/NdeI double enzyme digestion.

In step (2), the method for synthesizing the nucleotide sequence encoding rat growth hormone signal peptide and CD133ScFv is not particularly limited, and can be various methods commonly used in the art, for example, it can be synthesized by total gene synthesis technology.

Specifically, the method for obtaining pWPXL-CD133ScFv-CD8-CD137-CD3ζ with the correct sequence may include: synthesizing the nucleotide sequence (SEQ ID NO. Cloned into the vector pGSI to obtain pGSI-CD133ScFv; then pGSI-CD133ScFv was subjected to BamHI/MluI double digestion and ligated with the recombinant plasmid pWPXL-CD8-CD137-CD3ζ obtained in step (1) after BamHI/MluI double digestion , and identified by sequencing, the correct sequence of pWPXL-CD133ScFv-CD8-CD137-CD3ζ was obtained. Wherein, the nucleotide sequence of rat growth hormone signal peptide is shown in SEQ ID NO.6, and the nucleotide sequence of CD133ScFv is shown in SEQ ID NO.7.

The present invention also provides an engineered CD133-targeted NKT cell, wherein the NKT cell is an NKT cell modified by the above-mentioned chimeric antigen receptor CD133ScFv-CD8-CD137-CD3ζ (ie, CAR133-NKT cell).

The present invention also provides a method for preparing engineered CD133-targeted NKT cells, the method comprising: packaging a lentivirus carrying pWPXL-CD133ScFv-CD8-CD137-CD3ζ to obtain a virus concentrate; using the obtained virus concentrate Infect NKT cells to make NKT cells express chimeric antigen receptor CD133ScFv-CD8-CD137-CD3ζ.

The method for packaging the lentivirus carrying pWPXL-CD133ScFv-CD8-CD137-CD3ζ is not particularly limited, and can be various methods commonly used by those skilled in the art. Preferably, the lentivirus expression vector pWPXL-CD133ScFv-CD8-CD137 -Co-transfect 293T packaging cells with CD3ζ and helper plasmids (such as psPAX2, pMD2.G), collect the virus supernatant at 48-72 hours after transfection, centrifuge and filter, add 5×PEG6000-NaCl to the filtrate for mixing, after centrifugation The supernatant was discarded, and the precipitate was dissolved in sterile PBS pre-cooled at 0-4°C to obtain a concentrated virus solution.

In the method of the present invention, it also includes preparing NKT cells by the following method:

(1) In the presence of CD3 monoclonal antibody, interleukin-2 and interleukin-15, the mononuclear cells are cultured in the first stage;

(2) In the presence of interleukin-2, the cells cultured in the first stage are cultured in the second stage.

Preferably, the implementation of the first stage culture includes: culturing mononuclear cells in the first NKT cell culture medium, the first NKT cell culture medium contains NKT cell culture medium, CD3 monoclonal antibody, interleukin- 2 and interleukin-15; further preferably, in the first NKT cell culture medium, the concentration of the CD3 monoclonal antibody is 30-70ng/mL, and/or the concentration of the interleukin-2 is 300-700U/mL, And/or the concentration of the interleukin-15 is 30-70ng/mL.

Preferably, the embodiment of the second-stage culture includes: culturing the cells cultured in the first stage in a second NKT cell culture fluid, and the second NKT cell culture fluid contains NKT cell culture medium and interleukin -2; Further preferably, in the second NKT cell culture medium, the concentration of interleukin-2 is 300-700U/mL.

The NKT cell culture medium is not particularly limited, and it can be various mediums commonly used in the art for culturing NKT cells, such as GT-T551 medium.

When preparing NKT cells, the conditions for the first-stage culture and the second-stage culture are not particularly limited, and can be various conditions commonly used in the art, such as 30-37° C., saturated humidity of 3-6% CO ₂ cultured in the incubator. Those skilled in the art can make adaptive adjustments to the cultivation time, which is well known to those skilled in the art and will not be repeated here.

In the NKT cells prepared by the present invention, the average ratio of CD3 ⁺ cells is >90%, the average ratio of CD3 ⁺ CD8 ⁺ cells to the total CD3 ⁺ cells is >70%; the average ratio of CD3 ⁺ CD56 ⁺ cells to the total CD3 ⁺ cells is >15% %.

The method for infecting NKT cells is not particularly limited, and can be various methods commonly used in the art. Preferably, the method includes:

(1) In the presence of virus concentrate, protamine and interleukin-2, the NKT cells are subjected to the first-stage infection culture;

(2) In the presence of CD3 monoclonal antibody, interleukin-2 and interleukin-15, the cells infected and cultured in the first stage were infected and cultured in the second stage.

Preferably, the implementation of the first-stage infection culture includes: culturing NKT cells in a third NKT cell culture solution, the third NKT cell culture solution contains NKT cell culture medium, virus concentrate, protamine and Interleukin-2; further preferably, in the third NKT cell culture medium, the concentration of the interleukin-2 is 300-700 U/mL.

Preferably, the implementation of the second-stage infection culture includes: culturing the cells of the first-stage infection culture in the first NKT cell culture medium. For the specific composition of the first NKT cell culture medium, please refer to the corresponding contents above, and details will not be repeated here.

When infecting NKT cells, the conditions for the first-stage infection culture and the second-stage infection culture are not particularly limited, and can be various conditions commonly used in the art, for example, at 30-37°C and a saturated humidity of 3-6%. cultured in a CO ₂ incubator. Those skilled in the art can make adaptive adjustments to the cultivation time, which is well known to those skilled in the art and will not be repeated here.

Specifically, the method for infecting NKT cells includes: taking 1×10 ⁷ -5×10 ⁷ NKT cells, discarding the old culture medium, adding 2-4 mL of fresh GT-T551 culture liquid, and then adding 200-400 μL of virus concentrate , 2-4μL 1× ^10-6 mg/mL protamine and IL-2 with a final concentration of 300-700U/mL, place them in a _CO2 incubator at 30-37℃ and a saturated humidity of 3-6% for infection After 12-16 hours, discard the culture medium, transfer the cells to an uncoated culture flask, add 20-50 mL of GT-T551 medium, and then add IL-2 with a final concentration of 300-700 U/mL. CD3 monoclonal antibody of 30-70ng/ml and interleukin-15 with a final concentration of 30-70ng/mL were cultured in a _CO2 incubator at 30-37°C and a saturated humidity of 3-6% for 12-18h to obtain Chimeric antigen receptor CD133ScFv-CD8-CD137-CD3ζ modified NKT cells.

Further preferably, the method of infecting NKT cells also includes:

(3) In the presence of interleukin-2, the cells infected and cultured in the second stage are induced in vitro, and when the density of the cells is 80-90%, the CD3 monoclonal antibody, interleukin-2 and interleukin-15 In the presence of , the cells are cultured for expansion.

Preferably, the implementation of the in vitro induction includes: culturing the cells cultured in the second stage of infection in the second NKT cell culture medium, and the implementation of the expansion culture includes: culturing the cells in the In the first NKT cell culture medium. For the specific composition of the first NKT cell culture solution and the second NKT cell culture solution, please refer to the corresponding content above, and will not repeat them here.

Specifically, the method for infecting NKT cells also includes: inducing in vitro the lentivirus-infected NKT cells obtained after the second-stage infection culture with GT-T551 culture medium with a final concentration of IL-2 of 300-700U/mL, and then When the cell density is 80-90%, transfer the cells into the cell culture bag, add IL-2 at a final concentration of 300-700U/mL, and CD3 monoclonal antibody at a final concentration of 30-70ng/ml every 1.5-2.5 days 1. Fresh GT-T551 culture solution with a final interleukin-15 concentration of 30-70 ng/mL is used for expansion culture and the cells are expanded to a total of 1×10 ⁹ -2×10 ⁹ cells. The chimeric antigen receptor targeting CD133 antigen is infected with NKT cells by the lentivirus of the present invention, and the infection efficiency is as high as 30%-60%, and the obtained CAR133-NKT cells account for CD3 ⁺ CD56 ⁺ cells in the total CD3 ⁺ The ratio of cells is in the range of 15%-40%.

The amino acid sequence of the chimeric antigen receptor protein expressed by the chimeric antigen receptor CD133ScFv-CD8-CD137-CD3ζ modified NKT cells is shown in SEQ ID NO.1. Among them, those skilled in the art should understand that the chimeric antigen receptor precursor protein consists of rat growth hormone signal peptide, CD133ScFv, hinge region and transmembrane region of CD8, intracellular signaling domain of CD137 and intracellular The signal domains are formed in series. After the protein is translated, the signal peptide is excised in the rough endoplasmic reticulum in the cell to become a mature chimeric antigen receptor protein, which is secreted and exported and located on the cell membrane of NKT cells. The gene coding sequence corresponding to the protein amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.2. The chimeric antigen receptor uses the structure of the hinge region and transmembrane region of the gene CD8 and the intracellular signal domains of CD137 and CD3ζ in series as the signal transduction domain, and its amino acid sequence is shown in SEQID NO.9, corresponding to The coding sequence of the gene is shown in SEQID NO.10.

The present invention also provides the engineered CD133-targeted NKT cells prepared by the above method.

The present invention also provides the application of engineered CD133-targeted NKT cells in the preparation of preparations for treating tumors. Preferably, the tumor is a CD133-positive epithelial tumor, further preferably, the epithelial tumor is colorectal cancer, liver cancer, bile duct cancer, pancreatic cancer, esophageal cancer, gastric cancer, ovarian cancer, lung cancer, prostate cancer, bladder cancer, breast cancer cancer, endometrial cancer, brain tumor, melanoma or glioma, etc.

In the application of the present invention, the composition of the preparation for treating CD133-positive tumors is not particularly limited, as long as it contains the CAR133-NKT cells described in the present invention or is prepared from CAR133-NKT cells, the specific composition of the preparation and preparation methods are well known to those skilled in the art, and will not be repeated here.

Those skilled in the art should understand that epithelial tumors are colorectal cancer, liver cancer, bile duct cancer, pancreatic cancer, esophageal cancer, gastric cancer, ovarian cancer, lung cancer, prostate cancer, bladder cancer, breast cancer, endometrial cancer, brain tumor , melanoma or glioma, etc., the CAR133-NKT cells of the present invention can recognize CD133-positive cells, including tumor initiating cells, tumor stem cells, epithelial progenitor cells and tumor cells, and exert targeted killing activity, thereby The above-mentioned different types of CD133-positive epithelial tumors all have killing effects, which can not only clear the tumor in situ, but also inhibit the metastasis of tumor cells, and may also reduce the recurrence of tumors.

The present invention also provides a method for treating CD133-positive epithelial tumors and/or inhibiting tumor recurrence and metastasis, the method comprising: reinfusing the engineered CD133-targeted NKT cells of the present invention into CD133-positive epithelial tumor patients . Preferably, the epithelial tumor is colorectal cancer, liver cancer, bile duct cancer, pancreatic cancer, esophageal cancer, gastric cancer, ovarian cancer, lung cancer, prostate cancer, bladder cancer, breast cancer, endometrial cancer, brain tumor, melanoma or Glioma.

Example

The following examples will further illustrate the present invention, but do not limit the present invention thereby.

The experimental methods in the following examples are conventional methods in the art unless otherwise specified. The experimental materials used in the following examples, unless otherwise specified, are purchased from conventional biochemical reagent stores, wherein:

NKT cell culture medium GT-T551 was purchased from TaKaRa Company.

Lymphocyte separation medium was purchased from TBD Company.

CD3 monoclonal antibody and recombinant fibronectin (retronectin) were purchased from TaKaRa Company.

Recombinant human protein interferon-γ, recombinant human interleukin 2, and recombinant human interleukin 15 were all purchased from Protech Company.

Total RNA extraction kit RNAiso Reagent, high-fidelity DNA polymerase ( HS DNA Polymerase) and T4 DNA ligase were purchased from TaKaRa Company.

RevertAid ^TM First Strand cDNA Synthesis Kit was purchased from Fermentas.

BglⅡ, EcoRI, MluI, BamHI, NdeI, EcoRⅤ were purchased from Fermentas.

Agarose gel DNA recovery kit, general DNA product purification kit, and plasmid mini-extraction kit were purchased from Tiangen Biochemical Technology Co., Ltd.

pWPXL-GFP, psPAX2, and pMD2.G were all purchased from Addgene.

pGSI was purchased from Beijing Tianyi Huiyuan Biotechnology Co., Ltd.

Trans1-T1 Phage Resistant chemically competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

Lipofectamine ^TM 2000 Transfection Reagent was purchased from Invitrogen.

293T packaging cells were purchased from ATCC, USA.

The final concentration of PEG6000 in PEG6000-NaCl was 25.5% by mass, and the final concentration of NaCl was 1.2M. Both PEG6000 and NaCl were purchased from Shanghai Suo Laibao Biotechnology Co., Ltd.

Fetal bovine serum was purchased from PAA, Germany.

CD107a-PECy5 antibody was purchased from BD Company, USA.

CD133-positive colorectal cancer cell line HT29 was purchased from ATCC, USA.

CD133-positive human liver cancer cells Hep3B were purchased from ATCC, USA.

CD133-positive human pancreatic cancer cells SW1990 were purchased from ATCC, USA.

CD133-positive human colorectal cancer cells DLD1 were purchased from ATCC, USA.

CD133-positive human colorectal cancer cells SW620 were purchased from ATCC, USA.

CD133-negative human colorectal cancer cells LOVO were purchased from ATCC, USA.

CD133-negative human liver cancer cells HepG2 were purchased from ATCC, USA.

5-Carboxyfluorescein succinimide ester was purchased from Shanghai Puzhen Biotechnology Co., Ltd.

Annexin V-RPE kit was purchased from BD Company, USA.

MethoCult ^TM H4434 classical cell culture medium was purchased from stem cell company.

All primers were synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd.

Example 1 Preparation of NKT cells

(1) Take human venous blood in a vacuum tube containing heparin. Mononuclear cells (PBMCs) were obtained by density gradient centrifugation using lymphocyte separation medium.

(2) After the PBMCs were washed three times, the final cell concentration was adjusted to 2×10 ⁶ cells/mL using NKT cell culture medium GT-T551 containing 0.6 vol% human autologous serum; mL of retronectin-coated 75cm ² cell culture flasks. Then add recombinant human interleukin 2 with a final concentration of 500U/mL, 50ng/ml CD3 monoclonal antibody and 50ng/mL recombinant human interleukin-15 to the culture medium, and incubate at 37°C and 5% saturated humidity in a _CO2 incubator cultivated in.

(3) On the 4th day of culture, transfer the cells to an uncoated culture flask, add NKT cell culture medium GT-T551 every 2 days according to the number of cell growth, control the cell concentration to ¹ ×108 cells/mL, and Add recombinant human interleukin 2 with a final concentration of 500U/ml; culture to the 12th day to obtain NKT cells, and analyze the phenotype of NKT cells by flow cytometry. The results are shown in Figure 1, where CD3 ⁺ : 95.04%; CD3 ⁺ CD8 ⁺ : 90.99%; CD3 ⁺ CD56 ⁺ : 24.12%; CD8 ⁺ CD56 ⁺ : 24.63%.

Example 2 Construction of lentiviral expression vector pWPXL-CD133ScFv-CD8-CD137-CD3ζ

(1) Preparation of NKT cell cDNA

The NKT cells cultured in Example 1 were centrifuged to precipitate, and the total RNA of the cells was extracted with the total RNA extraction kit RNAisoReagent, and stored at -80°C for future use. The extracted total RNA was reverse-transcribed with RevertAid ^TM First Strand cDNA Synthesis Kit to obtain NKT cell cDNA, which was stored at -20°C for future use.

(2) Preparation of lentiviral plasmid pWPXL-CD8-CD137-CD3ζ

The following primer sequences were designed and synthesized (wherein, the underline marks are protected bases, and the square boxes are enzyme cleavage sites):

Using the cDNA of NKT cells in step (1) as a template, PCR amplification was carried out with primers P1 and P2 to obtain the hinge region and transmembrane region of CD8 with a length of 287 bp. The nucleotide sequence is shown in SEQ ID NO.3, and the two ends respectively contain MluI and BglII restriction sites and protective bases; use primers P3 and P4 for PCR amplification to obtain a 146bp CD137 intracellular signaling domain, the nucleotide sequence is shown in SEQID NO.4, and both ends contain BglII respectively and EcoRI restriction sites and protective bases; use primers P5 and P6 to carry out PCR amplification to obtain the intracellular signaling domain of CD3ζ with a length of 359 bp, the nucleotide sequence is shown in SEQID NO.5, and both ends contain EcoRI respectively And NdeI restriction sites and protection bases. The PCR amplification reaction system of each step is the same, taking the amplification of CD137 intracellular signaling domain as an example, PCR amplification is carried out, and the PCR reaction conditions refer to Instructions for HSDNA Polymerase, the reaction system (50 μL) is as follows:

Double distilled water: 32.5 μL

5×reaction buffer: 10μL

dNTP mix (2.5 mM each): 4 μL

P3 (10mM): 1μL

P4 (10mM): 1μL

NKT cell cDNA (200ng/ul): 1μL

HS DNA Polymerase: 0.5 μL

The above PCR products were separated with 1% agarose gel, and DNA fragments were recovered with an agarose gel DNA recovery kit. After the fragments were obtained, double enzyme digestion reactions were carried out, and the enzyme digestion products were recovered with a common DNA product purification kit for future use.

The lentiviral expression vector pWPXL-GFP was double-digested with MluI/NdeI, the digested product was separated by 1% agarose gel, and the large carrier fragment was recovered with the agarose gel DNA recovery kit, and then combined with the previously recovered CD8 , CD137, and CD3ζ fragments were ligated by T4 DNA ligase, and the ligated products were transformed into Trans1-T1 Phage Resistant chemically competent cells. After culturing at 37°C for 16 hours, single clones were picked, and after culturing at 37°C and 250rpm for 12 hours, plasmids were extracted with a plasmid extraction kit. . The extracted plasmid was identified by restriction endonuclease MluI and NdeI double digestion, and the identification electropherogram is shown in Figure 2, wherein, M1: DNA molecular weight marker D15000; Lane 1: undigested fragment of plasmid pWPXL-CD8-CD137-CD3ζ; Lane 2: restriction enzyme fragment (751 bp) of plasmid pWPXL-CD8-CD137-CD3ζ; M2: DNA molecular weight marker D2000. Send the correctly identified plasmid to Beijing Tianyi Huiyuan Biotechnology Co., Ltd. to sequence the inserted fusion gene fragment. The recombinant plasmid with correct sequencing results was named pWPXL-CD8-CD137-CD3ζ, wherein the nucleotide sequence of the hinge region and transmembrane region of CD8 is shown in SEQID NO.3, and the nucleotide sequence of the intracellular signaling domain of CD137 is The acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence of the intracellular signaling domain of CD3ζ is shown in SEQ ID NO.5.

(3) Preparation of lentiviral plasmid pWPXL-CD133ScFv-CD8-CD137-CD3ζ

The whole gene synthesis codes the nucleotide sequence of rat growth hormone signal peptide and CD133ScFv fusion gene, the sequence is shown in SEQID NO.8, synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd., and its 5' end contains BamHI and kozak sequences , the 3' end contains a MluI restriction site, the aforementioned fusion gene was cloned into the plasmid pGSI, named pGSI-CD133ScFv. The plasmid was digested with BamHI/MluI double enzymes, the digested product was separated by 1% agarose gel, and the target fragment was recovered with an agarose gel DNA recovery kit for future use.

The pWPXL-CD8-CD137-CD3ζ plasmid was digested with restriction endonuclease BamHI/MluI, the digested product was separated by 1% agarose gel, and the vector fragment was recovered with an agarose gel DNA recovery kit for future use. Then it is ligated with the recovered DNA fragment containing rat growth hormone signal peptide and CD133ScFv through T4 DNA ligase, and the specific method is shown in the instructions. The ligation product was transformed into Trans1-T1 Phage Resistant chemically competent cells, cultured at 37°C for 16 hours, and single clones were picked, and after cultured at 37°C, 250rpm for 12 hours, the plasmid was extracted with a plasmid mini-extraction kit. The extracted plasmid was identified by restriction endonuclease BamHI/NdeI double digestion, and the identification results are shown in Figure 3, wherein, among them, M1: DNA molecular weight marker D15000; Lane 1: plasmid pWPXL-CD133ScFv-CD8-CD137-CD3ζnot Restriction fragment (11281p); Lane 2: restriction fragment (650bp, 956bp) of plasmid pWPXL-CD133ScFv-CD8-CD137-CD3ζ; M2: DNA molecular weight marker D2000. Send the correctly identified plasmid to Beijing Tianyi Huiyuan Biotechnology Co., Ltd. to sequence the inserted fusion gene fragment. The recombinant plasmid with the correct sequencing result was named pWPXL-CD133ScFv-CD8-CD137-CD3ζ, and its structural schematic diagram is shown in Figure 4, which includes the rat growth hormone signal peptide (nucleotide sequence shown in SEQID NO.6), Anti-CD133 single-chain antibody (nucleotide sequence shown in SEQID NO.7), the hinge region and transmembrane region of CD8, the intracellular signaling domain of CD137 and the intracellular signaling domain of CD3ζ, wherein the chimeric antigen The receptor takes the structure formed by the hinge region and transmembrane region of the gene CD8 and the intracellular signal domains of CD137 and CD3ζ in series as the signal transduction domain. Its amino acid sequence is shown in SEQID NO.9, and the corresponding gene coding sequence is shown in Shown in SEQID NO.10.

Example 3 Preparation of NKT cells modified by chimeric antigen receptor CD133ScFv-CD8-CD137-CD3ζ

(1) Packaging and concentration of lentivirus

Measure the concentration of the lentiviral expression plasmid pWPXL-CD133ScFv-CD8-CD137-CD3ζ and the helper plasmids psPAX2 and pMD2.G respectively with a spectrophotometer. The three plasmids are transfected with Lipofectamine ^TM 2000 TransfectionReagent at a mass ratio of 4:2:1 Co-transfect 293T packaging cells. Collect the virus supernatant in 50mL EP tubes at 48h and 72h of transfection respectively, centrifuge at 2000g for 10min at 4°C, transfer the supernatant obtained twice to a new EP tube, and filter the virus supernatant with a 4.5μm filter; the filtered virus Mix the supernatant with 5×PEG6000-NaCl at a volume ratio of 4:1, let stand at 4°C for 2 hours, then centrifuge at 10,000 g for 20 minutes at 4°C, discard the supernatant, and dissolve the precipitate with 1 mL of 4°C pre-cooled sterile PBS. The obtained chimeric antigen receptor virus concentrate was divided into 100 μL tubes and stored at -80°C for future use.

According to the above method, the 293T packaging cells were co-transfected with the lentiviral expression plasmid pWPXL-GFP and the helper plasmids psPAX2 and pMD2.G, and the viral supernatant was collected and concentrated to obtain the lentiviral concentrate expressing GFP green fluorescent protein.

(2) Lentivirus infection of NKT cells and expansion of infected cells

Get the ¹ × 107 NKT cells cultivated in the 75cm culture flask of Example ¹ , discard the old culture solution, add 2 mL of fresh NKT cell culture medium GT-T551, 200 μ L of the virus concentrate that step (1) obtains, 2 μL of 1×10 ^-6 mg/mL protamine and recombinant human interleukin-2 at a final concentration of 500 U/mL were placed in a CO ₂ incubator at 37°C with a saturated humidity of 5% for 12 hours, and the culture medium was discarded. At the same time, the NKT cells were simultaneously infected with the lentivirus concentrate expressing GFP green fluorescent protein (the obtained NKT cells were called CART-GFP cells), which was used to calculate the infection efficiency of the virus. Transfer the infected cells to a 75cm ² culture flask not coated with CD3 and retronectin, add 20mL of NKT cell culture medium GT-T551, and then add recombinant human interleukin 2 with a final concentration of 500U/mL and a final concentration of 50ng /ml of CD3 monoclonal antibody and recombinant human interleukin-15 at a final concentration of 50ng/mL were cultured for 18 hours at 37°C in a CO ₂ incubator with a saturated humidity of 5%, and the resulting NKT cells were called CAR133-NKT cells. Prepare CAR33-NKT cells as Mock cells according to the method disclosed in CN105384823 A. The infection efficiency of the virus was detected by flow cytometry, as shown in Figure 5, the infection efficiency of CAR133-NKT cells was 36.32%.

(3) Induction and expansion of CAR133-NKT cell population in vitro

The above-mentioned cultured NKT cells were induced in vitro with NKT cell culture medium GT-T551 with a final concentration of recombinant human interleukin 2 of 500 U/mL, and when the density of the cells was 85%, the cells were transferred into cell culture bags, and the Add fresh NKT cell medium GT-T551 with a final concentration of recombinant human interleukin 2 of 500 U/mL, a final concentration of CD3 monoclonal antibody of 50 ng/ml, and a final concentration of recombinant human interleukin 15 of 50 ng/mL on the first day for expansion and culture , after the cells expanded to a total of about 1.5×10 ⁹ cells, the infected cell population was identified by flow cytometry, and the cell phenotype generally reached the proportion of CD3-positive cells >90%; the proportion of CD3CD8 double-positive cells >70%; the proportion of CD3CD56 double-positive cells >15%, the results are shown in Figure 6, CD3 ⁺ : 94.09%; CD3 ⁺ CD4 ⁺ : 8.79%; CD3 ⁺ CD8 ⁺ : 79.95%; CD3 ⁺ CD56 ⁺ : 27.68%; CD8 ⁺ CD56 ⁺ : 24.41%.

Example 4 Analysis of the killing effect of CAR133-NKT cells on epithelial tumor cells with different CD133 expression levels

The CAR133-NKT cells prepared in Example 3, the CAR33-NKT cells and the NKT cells cultured in Example 1 were respectively inoculated in 96-well plates, and epithelial tumor cells with different CD133 expression levels (cancer cell lines expressing CD133 at a high level : Hep3B, SW1990; cancer cell lines expressing CD133 at a medium level: HT29, DLD1; and cancer cell lines LOVO, HepG2 not expressing CD133) were co-cultured at a ratio of 20:1 (killer cells: target cells), After 4 hours of co-cultivation, monensin was added at a final concentration of 2 μmol/L, incubated for 2 hours, then incubated with PE-labeled CD107a antibody for 15 minutes, washed with PBS buffer for 3 times, and analyzed by flow cytometry. The expression level.

The CAR133-NKT cells prepared in Example 3, the CAR33-NKT cells and the NKT cells cultured in Example 1 were respectively inoculated in 96-well plates, stained with 5-carboxyfluorescein succinimidyl ester (CFSE), and Epithelial tumor cells with different expression levels of CD133 (cancer cell lines with high expression of CD133: Hep3B, SW1990; cancer cell lines with medium expression of CD133: HT29, DLD1; cancer cell lines without CD133 expression LOVO, HepG2) to effectively target The ratio (killer cells: target cells) was co-cultured at a ratio of 20:1. After 8 hours of co-cultivation, the cells were stained with an Annexin V-RPE kit, and the control group was set up without immune cell killing treatment. Epithelial tumor cells with different CD133 expression levels were stained with annexin V-RPE kit. Flow cytometry was used to detect cell apoptosis, and the amount of cell apoptosis was calculated according to the following formula: apoptosis rate=(control-sample)/control×100%, and the control was the number of surviving cells without immune cell killing treatment; The sample is the survival number of immune cells treated with the addition of corresponding effect-to-target ratio (killer cells: target cells).

Among them, the analysis diagrams of CD133 expression levels of epithelial tumor cells with different CD133 expression levels are shown in Figure 7 (A)-Figure 7 (C), and the analysis diagrams of the killing effect of CAR133-NKT cells on epithelial tumor cells with different CD133 expression levels (4 hours ) is shown in Figure 8, and the analysis of the killing effect of CAR133-NKT cells on epithelial tumor cells with different CD133 expression levels (8 hours) is shown in Figure 9. It can be seen from Figures 7-9 that NKT cells modified by the chimeric antigen receptor CD133ScFv-CD8-CD137-CD3ζ have higher specific killing activity against CD133 cancer cells with high-level expression and medium-level expression, but against CD133-free cells The cell line has no killing activity, and the specific killing activity of CAR133-NKT cells is significantly better than that of NKT cells and Mock cells (CAR33-NKT cells).

Example 5 Analysis of the therapeutic effect of CAR133-NKT cells on colorectal cancer mouse model at the level of animal experiments

Human colorectal cancer cells SW620 and HT29 with positive expression of CD133 cultured to the logarithmic growth phase were used respectively (5×10 ⁶ cells each, and the expression rates of CD133 were 99.43% and 66.67% respectively, among which, SW620 and HT29 The CD133 expression level analysis chart is shown in Figure 10), diluted into 100 μl of normal saline, and injected subcutaneously into nude mice. After 15 days, a mouse model of colorectal cancer was established subcutaneously.

In the mouse model of colorectal cancer, physiological saline, NKT cells, Mock cells, and CAR133-NKT cells (the number of cells were both 2×10 ⁷ ) were administered intraperitoneally for treatment, and injected once a day for 2 consecutive days. The mice were sacrificed, the tumor tissue was removed, and the volume of the tumor tissue was measured. The results are shown in FIG. 11 . It can be seen from Figure 11 that the tumor size of the colorectal cancer mouse model injected with CAR133-NKT cells was significantly reduced, indicating that the CAR133-NKT cells of the present invention can exert the function of targeting and killing tumor cells at the animal experiment level.

Example 6 Effect of CAR133-NKT cells of the present invention on the function of the hematopoietic system

The original progenitor cells were separated from umbilical cord blood using Ficoll mononuclear separation medium, mixed with CAR133-NKT cells, NKT cells, normal saline and Mock cells (CAR33-NKT cells) at a ratio of 1:20, and inoculated in MethoCult ^TM H4434 classic Cell culture medium was cultured in an incubator with 5% CO ₂ at 37°C for 2 weeks, and then the clones were counted.

The functional impact of CAR133-NKT cells on the hematopoietic system is shown in Figure 12, which shows the clone number of hematopoietic cells, where BFU-E is a burstforming unit: early erythroid progenitor cell, CFU -GM is colony forming unit: granulocyte and macrophage, CFU-GEMM is colony forming unit: granulocyte, erythrocyte, monocyte and megakaryocyte (colony forming unit: granulocyte, erythrocyte, monocyte and megakaryocyte), CFU-E is early erythroid progenitor cell colony forming unit (colony forming unit: early erythroid progenitor cell). As shown in Figure 12, the CAR133-NKT cells of the present invention have no obvious adverse effects on the hematopoietic system. Hematopoietic stem cells also express CD133, therefore, the influence on the hematopoietic system is ruled out, ensuring the safety of CAR133-NKT cell therapy.

Example 7 The therapeutic effect of CAR133-NKT cells of the present invention on patients with CD133-positive epithelial tumors (represented by liver cancer)

CAR133-NKT cells were taken, diluted with 100ml of normal saline, and intravenously infused into CD133-positive liver cancer patients in the form of increasing doses for three consecutive days (before using the CAR133-NKT cells of the present invention for targeted immunotherapy, too much (such as radiotherapy, chemotherapy and other drug symptomatic treatment, etc.), but no obvious curative effect), in which patient 1, patient 2, and patient 3 have been infused with safe doses, and the total infusion doses for three consecutive days were respectively They were: 0.03×10 ⁷ /kg, 0.06×10 ⁷ /kg and 0.03×10 ⁷ /kg; patients 1, 2, 3 and 4 were infused with the lowest dose to exert the therapeutic effect, and the infusion was performed for three consecutive days The total injection doses were: 0.39×10 ⁷ /kg, 0.25×10 ⁷ /kg, 0.4×10 ⁷ /kg and 0.33×10 ⁷ /kg; patients 5, 6, 7 and 8 performed clinical trials. Effective dose infusion for curative effect, the total infusion doses for three consecutive days were: 0.5×10 ⁷ /kg, 0.5×10 ^{7 /kg, 0.5×10 7 /} kg and 0.79×10 ⁷ /kg; Evaluate the patient's treatment status.

The dose of infusion of CAR133-NKT cells in the treatment patients is divided into three gradients: 1) safety dose infusion, cell reinfusion volume is 0.01-0.06 (×10 ⁷ /kg), 2) the lowest dose infusion to exert therapeutic effect , the amount of cell reinfusion is 0.1-0.5 (×10 ⁷ /kg), 3) the effective dose infusion to exert clinical curative effect, the amount of cell reinfusion is 0.5-1.0 (×10 ⁷ /kg).

Figure 13(A)-Figure 13(D) are the toxic and side effects analysis diagrams of CAR133-NKT cell therapy in patients with liver cancer, showing hemoglobin (Hgb), white blood cells (WBC), and platelets (PLT) at different days after cell reinfusion and reticulin (Ret) trend. As shown in Figure 13(A)-Figure 13(D), the toxic side effects of the blood system are mainly a slight decrease in the level of hemoglobin and platelets, the side effects are relatively low, and the patients can tolerate them; other side effects such as reinfusion The associated chills, high fever, headache, dizziness, fatigue, nausea, vomiting, etc. were well tolerated by the patient and were relieved by giving antipyretic and analgesic preparations. Toxic and side effects after reinfusion, such as rash, ascites, elevated bilirubin, etc., can be relieved after giving symptomatic treatment. Other toxicity results are shown in Table 1.

Table 1

Reinfusion related fever symptoms 8/8 Grade 1 Headache 6/8 <Level 1 Dizziness 7/8 <Level 1 Hypotension 4/8 <Level 1 Acute ascites 1/8 Level 3

Figure 14 is a graph showing the change curve between the copy number of CAR133-NKT cells and the number of CD133-positive cells in patients with liver cancer, in which the copy number of CAR133-NKT cells is negatively correlated with the percentage of CD133-positive cells, indicating that CAR133-NKT can kill CD133-positive target cells .

Figure 15 shows the treatment response charts of 8 patients with liver cancer. Among them, one patient's disease progressed after the first cycle of treatment, and the disease was stable after the second cycle; the remaining seven patients had stable disease after the first cycle of treatment. It shows that CAR133-NKT cells have a certain therapeutic effect on patients with liver cancer.

Example 8 The therapeutic effect of CAR133-NKT cells of the present invention on patients with CD133-positive epithelial tumors (represented by pancreatic cancer)

CAR133-NKT cells were taken, diluted with 100ml of normal saline, and intravenously infused into CD133-positive pancreatic cancer patients in a dose-increasing manner for three consecutive days (before using the CAR133-NKT cells of the present invention for targeted immunotherapy, the Multiple treatments (such as radiotherapy, chemotherapy and other drug symptomatic treatment, etc.), but no obvious curative effect) in the body, the total amount of cells reinfused for three consecutive days was 0.8×10 ⁷ /kg, and the treatment status of the patient was evaluated after the reinfusion .

Figure 16 is a diagram of the therapeutic effect of CAR133-NKT cells on pancreatic cancer patients. It can be seen from Figure 16 that the tumor focus of pancreatic cancer patients shrank after 4 weeks of treatment, and the tumor shrinkage became more obvious after 10 weeks of treatment. It shows that CAR133-NKT cells have a certain therapeutic effect on patients with pancreatic cancer.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. These simple modifications All belong to the protection scope of the present invention.

In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way if there is no contradiction. The combination method will not be described separately.

In addition, various combinations of different embodiments of the present invention can also be combined arbitrarily, as long as they do not violate the idea of the present invention, they should also be regarded as the disclosed content of the present invention.

Claims (10)

1. A chimeric antigen receptor, characterized in that, the chimeric antigen receptor is CD133ScFv-CD8-CD137-CD3ζ, including the hinge region and transmembrane region of rat growth hormone signal peptide, CD133ScFv, CD8 in series , the intracellular signaling domain of CD137 and the intracellular signaling domain of CD3ζ; preferably, the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO.1, further preferably, the chimeric antigen receptor The amino acid sequence of the body is shown in SEQID NO.1. 2. The gene encoding the chimeric antigen receptor according to claim 1, preferably, the gene has a nucleotide sequence as shown in SEQ ID NO.2, further preferably, the nucleotide sequence of the gene As shown in SEQ ID NO.2. 3. A recombinant expression vector containing the gene of claim 2, preferably, the recombinant expression vector is a lentiviral expression vector, further preferably, the lentiviral expression vector is pWPXL-CD133ScFv-CD8-CD137-CD3ζ. 4. An engineered CD133-targeted NKT cell, characterized in that the NKT cell is an NKT cell modified by the chimeric antigen receptor according to claim 1. 5. A method for preparing engineered CD133-targeted NKT cells, characterized in that the method comprises: Pack the lentivirus carrying pWPXL-CD133ScFv-CD8-CD137-CD3ζ to obtain a virus concentrate; use the obtained virus concentrate to infect NKT cells, so that the NKT cells express chimeric antigen receptor CD133ScFv-CD8-CD137-CD3ζ. 6. The method according to claim 5, wherein the method further comprises preparing NKT cells by the following method: (1) In the presence of CD3 monoclonal antibody, interleukin-2 and interleukin-15, the mononuclear cells are cultured in the first stage; preferably, the embodiment of the first stage culture includes: culturing the mononuclear cells in the first stage In a NKT cell culture solution, the first NKT cell culture solution contains NKT cell culture medium, CD3 monoclonal antibody, interleukin-2 and interleukin-15; further preferably, in the first NKT cell culture solution, the CD3 monoclonal antibody The concentration of the cloned antibody is 30-70ng/mL, and/or the concentration of the interleukin-2 is 300-700U/mL, and/or the concentration of the interleukin-15 is 30-70ng/mL; (2) In the presence of interleukin-2, the cells cultured in the first stage are cultured in the second stage; preferably, the implementation of the second stage culture includes: culturing the cells cultured in the first stage in In the second NKT cell culture fluid, the second NKT cell culture fluid contains NKT cell culture medium and interleukin-2; further preferably, in the second NKT cell culture fluid, the concentration of the interleukin-2 is 300-700U /mL. 7. The method according to claim 6, wherein the method for infecting NKT cells comprises: (1) In the presence of virus concentrate, protamine and interleukin-2, NKT cells are subjected to the first-stage infection culture; preferably, the embodiment of the first-stage infection culture includes: NKT cells are cultivated in the third stage In the NKT cell culture fluid, the third NKT cell culture fluid contains NKT cell culture medium, virus concentrate, protamine and interleukin-2; further preferably, in the third NKT cell culture fluid, the interleukin-2 The concentration is 300-700U/mL; (2) In the presence of CD3 monoclonal antibody, interleukin-2 and interleukin-15, the cells of the first-stage infection culture are subjected to the second-stage infection culture; preferably, the embodiment of the second-stage infection culture includes : culturing the cells cultured in the first stage of infection in the first NKT cell culture medium. 8. The method according to claim 7, wherein the method for infecting NKT cells further comprises: (3) In the presence of interleukin-2, the cells infected and cultured in the second stage are induced in vitro, and when the density of the cells is 80-90%, the CD3 monoclonal antibody, interleukin-2 and interleukin-15 In the presence of the cells, the cells are expanded and cultured; preferably, the embodiment of the in vitro induction includes: the cells cultured in the second stage of infection are cultured in the second NKT cell culture medium, and the cultured cells of the expansion culture are Embodiments include: culturing cells in the first NKT cell culture medium. 9. The engineered CD133-targeted NKT cells prepared by the method according to any one of claims 5-8. 10. The application of the engineered CD133-targeted NKT cell according to claim 4 or 9 in the preparation of a preparation for treating tumors, preferably, the tumor is a CD133-positive epithelial tumor, further preferably, the The epithelial tumor is colorectal cancer, liver cancer, cholangiocarcinoma, pancreatic cancer, esophageal cancer, gastric cancer, ovarian cancer, lung cancer, prostate cancer, bladder cancer, breast cancer, endometrial cancer, brain tumor, melanoma or glioma.