Small molecule inhibitors of ARHGAP9 and their application in enhancing the anti-cancer efficacy of cells

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a small molecule inhibitor of ARHGAP9 and an inhibition method and use thereof. The present invention first provides a method for improving the anti-tumor activity of cells by in vitro treatment, wherein the cells include immune cells and immune engineering cells; the method includes knocking out the ARHGAP9 gene in the cells or culturing the cells with small molecule drugs to degrade the ARHGAP9 protein expressed by the cells; the small molecule drugs include butein.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a small molecule inhibitor of ARHGAP9 and its application in enhancing the anti-cancer efficacy of cells. Background Art

[0002] The Rho (RAS homolog) family is a class of Ras superfamily members containing a GTPase domain. Its activity is regulated by its GTPase domain, and it functions as a molecular switch by switching between the inactive Rho-GDP and active Rho-GTP forms. Rho family proteins have complex functions and are involved in cell morphology, movement, division, gene expression, and cytoskeleton regulation. They are currently known to be related to cardiovascular diseases, tumor invasion and metastasis, autoimmune diseases, Parkinson's disease, and other diseases. The Rho-GAPs subfamily is an inhibitory molecule of Rho-GTP signaling, with 47 known members. ARHGAP9 (RhoGTPase activating protein 9) is one of the members of the Rho-GAPs subfamily. However, there are contradictions in the current reports on the functions of ARHGAP9 in cancer. Some articles report that ARHGAP9 is a pro-oncogene, including that ARHGAP9 can promote the proliferation and metastasis of colon cancer cells (Sun et al. Tissuecell. 2022); ARHGAP9 is highly expressed in ovarian cancer and negatively correlated with the patient's prognosis, and can be used as a prognostic indicator for ovarian cancer (Shen et al. Transl Cancer Res. 2021;10: 4440-4453.). However, there are also contrary reports suggesting that ARHGAP9 is a tumor suppressor gene, including bioinformatics analysis that found that compared with patients with low ARHGAP9 expression, patients with breast cancer and bladder cancer with high ARHGAP9 expression had longer survival and better prognosis (Chen et al. Oncol Lett. 2019;18:6017-6025.); ARHGAP9 inhibited the proliferation and migration of liver cancer cells (Zhang et al. Cell Death Dis. 2018;9:916.); ARHGAP9 knockout can promote lung cancer metastasis (Song et al. Genomics. 2023). Therefore, the role of ARHGAP9 in the occurrence and development of cancer is still controversial. No inhibitors of ARHGAP9 have been reported so far. Currently, mainstream scientific research can only use siRNA, shRNA, and Crispr / gRNA technologies to knock down or knock out ARHGAP9, but these methods have problems such as complex operation, high cost, and poor reproducibility. Therefore, the development of small molecule inhibitors that can effectively inhibit ARHGAP9 can greatly facilitate ARHGAP9 inhibition experiments in scientific research and help subsequent research.

[0003] On the other hand, in recent years, cell therapies represented by CAR-T and CAR-NK have achieved great success in clinical cancer treatment. In 2012, after experiencing two relapses of acute lymphoblastic leukemia, American girl Emily became the first child with leukemia cured by CAR-T therapy after CAR-T treatment. At present, there are 9 CAR-T therapies on the market for clinical use, of which 6 target CD19 and 3 target BCMA, which are used for the treatment of B cell lymphoma and multiple myeloma respectively. A number of dual-target CAR-T cell products are also in clinical trials, such as simultaneously targeting CD19 / CD20, CD19 / CD22, BCMA / CD38, BCMA / CS1, etc. (Cancers. 2023 Feb 4;15(4):1003.). CAR-NK is similar to CAR-T therapy. It also uses genetic engineering methods to express CAR molecules on NK cells and inject them into the body for anti-tumor treatment. The response rate in clinical trials has also reached 73%, significantly prolonging the patient's survival. At present, CAR-NK therapy has been reported to have better safety than CAR-T therapy, will not cause fatal cytokine storm and neurotoxicity, and can be prepared in advance without HLA matching (N Engl J Med. 2020 Feb 6;382(6):545-553.). However, the current effects of CAR-T and CAR-NK therapy in solid tumors are poor, mainly due to the immunosuppressive microenvironment of solid tumors, which causes the infused CAR-T and CAR-NK to infiltrate solid tumors and inhibit their function in the solid tumor microenvironment. Existing clinical trials are being conducted to express chemokine receptors on the surface of CAR-T cells and use chemokine ligands highly expressed in solid tumors to promote the infiltration of CAR-T cells into tumor tissues (NCT03602157). Regarding the activation of CAR-T, a large number of reports have confirmed that the combined use of immune checkpoint inhibitors such as pembrolizumab, nivolumab, and ipilimumab can effectively enhance the anti-tumor effect of CAR-T therapy by blocking the PD-1 and CTLA-4 inhibitory receptors of CAR-T cells, or using gene editing strategies to knock out immune checkpoint receptors PD1 and TIGIT in CAR-T cells (MolCancer. 2022 Mar 18;21(1):78; Front Immunol. 2023 Jun 9:14:1175920.). However, there are no reports linking the inhibition of ARHGAP9 with the anti-tumor efficacy of CAR-T or CAR-NK. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a small molecule inhibitor for inhibiting ARHGAP9 in vitro and an inhibition method and its use in providing anti-cancer efficacy of immune cells and immune-engineered cells, so as to supplement the deficiencies of the prior art. The specific technical scheme is as follows.

[0005] A method for improving the anti-tumor activity of cells by in vitro treatment, wherein the cells include immune cells or immune-engineered cells; the method includes knocking out the ARHGAP9 gene in the cells or culturing the cells with small molecule drugs to degrade the ARHGAP9 protein expressed by the cells; the small molecule drugs include butenatin.

[0006] It is understandable that the “small molecule inhibitor” described in the present invention specifically refers to butea superba as a small molecule inhibitor of ARHGAP9.

[0007] Furthermore, the immune cells include T cells or NK cells; the immune engineering cells include CAR-T cells or CAR-NK cells.

[0008] Further, the operation of culturing the cells with butegrin includes adding butegrin to the cell culture medium; the addition ratio is 6 μM butegrin for treating 1×10 5 ~1×10 6 The culture time is 3-60 hours.

[0009] In one embodiment of the present invention, the components of the culture medium include RPMI1640, 10% FBS, 50 μM 2-mercaptoethanol, 1× sodium pyruvate, 1× non-essential amino acids, 1% penicillin / streptomycin, 25 mM HEPES, 50 U / mL mouse IL2, 5 ng / mL mouse IL-7 and 25 ng / mL IL-15.

[0010] It is understood that the above cell culture medium is a conventional cell culture medium, and the components of the above cell culture medium are only examples, not limitations. Other culture media that can culture T cells or NK cells can also be used.

[0011] Furthermore, the means for knocking out the ARHGAP9 gene in the cells includes using siRNA, shRNA or Crispr / gRNA.

[0012] In one embodiment of the present invention, the means for knocking out the ARHGAP9 gene in the cell is Crispr / gRNA; the sequence of the gRNA involved is shown in SEQ ID NO.2~4.

[0013] On the other hand, the present invention also provides the use of butea batatas in preparing an in vitro induced degradation agent for ARHGAP9 protein in immune cells or immune engineering cells.

[0014] Furthermore, the immune cells include T cells or NK cells; the immune engineering cells include CAR-T cells or CAR-NK cells.

[0015] On the other hand, the present invention also provides an anti-tumor agent, which includes immune-engineered cells and butenatin; the immune-engineered cells include CAR-T cells or CAR-NK cells; the butenatin is configured as an in vitro immune activator of the immune-engineered cells, used to induce in vitro degradation of ARHGAP9 protein expressed by the immune-engineered cells.

[0016] Furthermore, the butenatin and the immune engineering cells in the anti-tumor agent are independently prepared according to the unit dose, and the ratio of the butenatin and the immune engineering cells is that 6 μM butenatin is used to treat 1×10 5 ~1×10 6 Immunoengineered cells.

[0017] Furthermore, the anti-tumor agent is applicable to tumor types including solid tumors and hematological tumors.

[0018] Furthermore, the tumor targets suitable for the anti-tumor agent include CD19, BCMA, CD20, CD22, EGFRvIII, CD123, CD33, HER2, CD38, CD70, PSMA, B7-H3, GPC3, GCC, Claudin18.2, CEA, CD5, EpCAM, GD2 and / or Mesothelin, etc.

[0019] It is understandable that the above targets are common solid tumor or hematological tumor targets, and the experiments in the examples of the present invention have demonstrated that the immune cells and immune-engineered cells treated with butea bateriana are effective against a variety of solid tumors and hematological tumors.

[0020] Beneficial technical effects

[0021] The present invention provides a method for improving the anti-cancer efficacy of immune cells or immune engineering cells by knocking out the ARHGAP9 gene of immune cells or immune engineering cells or inducing the degradation of ARHGAP9 protein expressed by immune cells or immune engineering cells in vitro.

[0022] In the preliminary experiments of the present invention, after a large number of screenings, it was found that the small molecule inhibitor Butewein can effectively induce the degradation of ARHGAP9 protein of CAR-T cells or CAR-NK cells in vitro, thereby significantly improving the anti-cancer efficacy of CAR-T cells or CAR-NK cells in a variety of cancer cells. The experiment found that only a low concentration of Butewein is needed to achieve the above technical purpose, which greatly avoids the problems of complex operation, high price, low knockout efficiency, poor reproducibility, etc. of the traditional use of siRNA, shRNA, Crispr / gRNA technology to knock down or knock out ARHGAP9 of T and NK cells, and has a broader clinical application prospect.

[0023] In summary, the present invention proposes that butea superba can be used as a new small molecule inhibitor of ARHGAP9, which has both scientific research and clinical uses. In terms of scientific research, butea superba can be conveniently used to inhibit ARHGAP9 from activating NK cells and CD8 + T cells, for NK and CD8 + T cell activation experimental study. In terms of clinical use, CAR-T and CAR-NK cells can be pre-treated with butea superba before infusion, or butea superba treatment can be given after infusion of CAR-T and CAR-NK cells to enhance the anti-tumor effect of CAR-T and CAR-NK. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor.

[0025] Figure 1 In vitro co-culture experiments confirmed that knocking out ARHGAP9 can activate NK and CD8 + T cell killing cancer cell activity;

[0026] Figure 2 Conditional knockout mice confirmed that ARHGAP9 knockout can activate tumor-infiltrating NK and CD8 + T cell anticancer activity;

[0027] Figure 3 To verify that butea superba can inhibit the expression of ARHGAP9 in gradient concentration and gradient time, and activate NK and T cells to release cytokines;

[0028] Figure 4 To verify that butea superba treatment can effectively enhance the anti-tumor effects of CAR-T and CAR-NK cell therapy. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0030] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0031] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0032] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0033] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0034] In this specification, some embodiments may be disclosed in a format that is in a certain range. It should be understood that this description of "being in a certain range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0035] Specific drawings:

[0036] Figure 1AB is a Western blot detection of ARHGAP9 expression knocked out in NK92MI cells using the Crispr / gRNA method; Figure 1 C is the co-culture of control or AHRGAP9 knockout NK92MI cells with different tumor cells at different concentrations, and the cell lysis rate was detected by calcein; Figure 1 D is the expression of cytokines in NK92MI cells of the control group or AHRGAP9 knockout group detected by flow cytometry; Figure 1 E is the effect of knocking out AHRGAP9 on the mRNA expression of cytokines expressed by NK92MI detected by qPCR; Figure 1 F: Primary NK cells from WT or ARHGAP9 knockout mice co-cultured with different tumor cells; Figure 1 G is primary CD8 derived from WT or ARHGAP9 knockout OT-I mice + T cells were co-cultured with different tumor cells.

[0037] Figure 2 A is the strategy for constructing ARHGAP9 conditional knockout mice; Figure 2 B is the genotype identification of ARHGAP9 conditional knockout mice; Figure 2 CD is the effect of ARHGAP9 conditional knockout mice against leukemia E.G7 cell transplants and colon cancer MC38 transplants; Figure 2 E is the flow cytometry detection of cytokine release by tumor-infiltrating NK cells in transplanted tumors; Figure 2 F is the flow cytometry detection of cytokine release by tumor-infiltrating T cells in transplanted tumors.

[0038] Figure 3 A is a schematic diagram of the ARHGAP9 inhibitor screening strategy; Figure 3 B is the Western blot detection result of butea superba induced ARHGAP9-HA fusion protein degradation; Figure 3 CD are the Western blot results of the changes in the overall expression of ARHGAP9 protein induced by butea superba in Jurkat and NK92MI cells; Figure 3 EF is qPCR detection of the changes in the ability of butea superba to induce cytokine expression in Jurkat and NK92MI cells.

[0039] Figure 4 AB is butea superba treatment to enhance the effect of CAR-T cells against the growth of colon cancer MC38-hCD19 cell transplanted tumors; Figure 4 CD shows that treatment with butenatin enhances the anti-tumor growth effect of CAR-NK cells against human B-lymphoblastoma Daudi cells.

[0040] Example 1

[0041] In vitro knockout of ARHGAP9 verified the effect on NK cells and T cells killing tumor cells.

[0042] (1) First, ARHGAP9 was knocked out in NK cells in vitro. The control group used one Crispr / gRNA plasmid to package lentivirus, and the knockout group used three Crispr / gRNA plasmids to package lentivirus. The lentivirus was then infected with NK92MI cells (NK cells from patients with malignant non-Hodgkin's lymphoma) to construct an ARHGAP9 knockout cell line. Three monoclonal clones were selected from the control group and six monoclonal clones were selected from the treatment group. Western blot was used to verify the knockout effect. The results showed that the ARHGAP9 knockout cell line of NK92MI was successfully constructed. Monoclonal cells including three control clones (Con gRNA1-1, Con gRNA1-2, Con gRNA1-3), gARH#1-3, gARH#2-4, and gARH#3-3 were used for subsequent experiments ( Figure 1 AB). The gRNA information involved in this example is shown in Table 1.

[0043] Table 1

[0044]

[0045] (2) Calcein-AM was used to stain acute leukemia cells (THP1, U937, Molm-13, Mono-Mac-6), large B-cell lymphoma cells (DB), non-small cell lung cancer cells (NCI-H1299), prostate cancer (DU-145), ovarian cancer (OVCAR3), breast cancer cells (MCF-7), and melanoma cells (Colo-858). The above cells were co-cultured with the constructed ARHGAP9 knockout NK92MI cells. Among them, the ARHGAP9 knockout NK92MI cells are effector cells (E) and the tumor cells are target cells (T).

[0046] (3) Detecting the calcein fluorescence released into the culture medium after target cell death can reflect the death of target cells.

[0047] The results showed that ARHGAP9 knockout could significantly increase the killing effect of NK92MI cells on cancer target cells, with a good dose-effect relationship ( Figure 1 C), that is, as the effector-target ratio increases, the lysis rate of tumor cells also increases. Flow cytometry analysis further confirmed that ARHGAP9 knockout can increase the expression of NK cell activation markers (including TNFα, IFNγ, GZMB, and Perforin) ( Figure 1D). qPCR detection of mRNA expression of NK cell activation markers also confirmed that ARHGAP9 knockout could significantly increase the release of cytokines by NK92MI cells, including TNFα, IFNγ, GZMB, and Perforin ( Figure 1 E).

[0048] Furthermore, this example used NK cells isolated from the spleen of WT and ARHGAP9 knockout experimental animals (ARH- / -NK) to co-culture with various mouse tumor cell lines in vitro, including leukemia cell lines Yac-1 and C1498, and colon cancer cell line MC38. It was also confirmed that ARHGAP9 knockout can effectively increase the killing effect of NK cells on various tumor cells ( Figure 1 F). In addition, this example also isolated OT-I CD8 + T cells (which can specifically recognize target cells expressing chicken ovalbumin OVA antigen) were co-cultured with a variety of tumor cells expressing OVA at gradient concentrations, including the leukemia cell line E.G7-OVA, the melanoma cell line B16F10-OVA, and the colon cancer cell line MC38-OVA. It was also confirmed that ARHGAP9 knockout could significantly increase OT-I CD8 + T cells kill tumor cells ( Figure 1 G).

[0049] Example 2

[0050] Conditional knockout mice confirmed in vivo that ARHGAP9 knockout can activate NK cells and T cells to release cytokines, inhibit tumor growth, and prolong mouse survival.

[0051] To further verify whether ARHGAP9 has an immunosuppressive effect, this example constructed ARHGAP9 fl / fl Mice were bred with NCR1-Cre and CD4-Cre mice to obtain offspring NK cell conditional knockout ARHGAP9 mice (ARHGAP9 fl / fl NCR1-Cre and T cell conditional knockout ARHGAP9 mice (ARHGAP9 fl / fl CD4-Cre). Knockout mouse construction strategy is as follows ( Figure 2 As shown in A), the knockout mice were successfully constructed by genotyping verification ( Figure 2 B).

[0052] Then the experiment used ARHGAP9 + / + NCR1-Cre mice and ARHGAP9 + / +CD4-Cre mice were used as negative controls. Mice were subcutaneously inoculated with colon cancer cells MC38 and leukemia cells E.G7, and the volume of transplanted tumors and the survival time of mice were recorded every other day. The results showed that conditional knockout of ARHGAP9 in NK or T cells could significantly inhibit the growth of transplanted tumors and prolong the survival time of mice ( Figure 2 CD), as can be seen in the figure, as time goes by, the tumor size decreases and the survival rate of mice increases. It is worth noting that the method used in this example to record the change in tumor size of mice after inoculation of leukemia cells E.G7 is in line with the technical operation specifications in this field.

[0053] Furthermore, the infiltrating lymphocytes in the transplanted tumor tissue were separated by Percoll separation, and NK and CD8 + T cell and immune activation markers. The results showed that ARHGAP9 can significantly increase the proportion of tumor-infiltrating NK cells expressing IFNγ, TNFα, and GZMB after NK cell knockout ( Figure 2 E). At the same time, ARHGAP9 can also increase tumor infiltrating CD8 + T and CD4 + T cells express IFNγ, TNFα ( Figure 2 F).

[0054] In summary, the results of this example can confirm that ARHGAP9 is an immunosuppressive molecule, and knocking out ARHGAP9 can activate NK and T cells to release cytokines and enhance the anti-tumor immune efficacy of immune cells.

[0055] Example 3

[0056] The above examples have confirmed that knocking out ARHGAP9 in animal models in vitro and in vivo can effectively activate NK and CD8 + The killing effect of T on tumor cells suggests that inhibiting the expression of ARHGAP9 can effectively enhance the anti-tumor effect of CAR-T and CAR-NK cells. However, the use of siRNA, shRNA, and Crispr / gRNA technology to knock down or knock out ARHGAP9 in T and NK cells has problems such as complex operation, high cost, low knockout efficiency, and poor reproducibility, which will limit its clinical application. Based on this, the development of effective small molecule inhibitors can greatly solve the technical difficulties in the preparation of the above-mentioned CAR-T and CAR-NK cells and reduce costs.

[0057] The research team of the present invention targeted ARHGAP9, constructed an ARHGAP9 promoter sequence to initiate an EGFP fluorescent plasmid, and constructed an ARH coding region sequence fused HA expression plasmid, packaged them in lentivirus, infected Jurkat cells, obtained a stable expression cell line, and conducted large-scale small molecule drug screening on the FDA-approved compound library and the natural product compound library ( Figure 3 A), a variety of small molecule drugs in these compound libraries were co-incubated with infected Jurkat cells. It was found that Butein can effectively induce the degradation of ARHGAP9 fusion HA protein expressed by plasmid (the present invention constructs ARHGAP9-HA fusion expression plasmid, and uses anti-HA antibody for detection, which can avoid detecting endogenous ARHGAP9 protein in cells. Therefore, when HA expression is found to be reduced, it can be confirmed that the expressed ARHGAP9-HA fusion protein is degraded.) It has a good time-effect and dose-effect relationship ( Figure 3 B).

[0058] Jurkat and NK92MI cells were treated with different concentrations (2.5 μM, 5 μM, 10 μM, 20 μM) of Butein for 24 h, and 20 μM Butein for different time periods (3 h, 6 h, 12 h, 24 h, 48 h). Western blot analysis with anti-ARHGAP9 antibody confirmed that Butein could inhibit the expression of ARHGAP9 in a time- and dose-dependent manner. Figure 3 CD). Therefore, the experiment in this example fully confirms that butea superba, as a small molecule inhibitor, can effectively induce the degradation of ARHGAP9.

[0059] The information of Butea superba is shown in Table 2.

[0060] Table 2

[0061]

[0062] The present invention further verifies the effect of butea superba as an inhibitor of ARHGAP9 on NK and T cell activation.

[0063] Jurkat and NK92MI cells were treated with gradient concentrations of butea tinctorius (0 μM, 5 μM, 10 μM, 20 μM) in vitro. The specific method was to add gradient concentrations of butea tinctorius to the corresponding cell culture medium to culture the cells, and qPCR was used to detect the expression of cytokines after culture. The results showed that butea tinctorius could effectively promote the expression of cytokines IFNγ, TNFα, and GZMB in Jurkat and NK92MI cells, showing a good dose-effect relationship ( Figure 3EF), that is, the more Butea superba added, the higher the expression of cytokines.

[0064] In summary, the results of this example prove that butea superba can induce the degradation of ARHGAP9, thereby promoting the activation of NK and T cells.

[0065] Example 4

[0066] This example provides butea superba as a small molecule inhibitor of ARHGAP9 induced degradation to enhance the anti-tumor effect evaluation of CAR-T and CAR-NK cell therapy, and the specific steps are as follows.

[0067] (1) Day 1: 293FT cells were plated. The healing rate was about 70% on the second day. The culture medium was DMEM. + 10% FBS + 1% BSA.

[0068] (2) Day 2: Co-transfect hCD19-CAR-GFP plasmid and viral capsid plasmid. The plasmid ratio is shown in Table 3. Carefully replace the 293FT cell culture medium, add 10 mL of fresh culture medium, drop the mixture of liposomes and plasmids into the culture plate, and return it to the incubator for 48 h.

[0069] Table 3

[0070]

[0071] (3) Day 3: Add CD8 + T cells were added to each well at 1 × 10 5 Cells were cultured overnight for subsequent titer detection.

[0072] (4) Day 4: Collect the virus. Disinfect the ultracentrifuge cap, sleeve, and ultracentrifuge tube with 70% ethanol. Wash the ultracentrifuge tube twice with PBS to remove the residual ethanol, dry it, and disinfect it under UV for 30 minutes. Collect the virus supernatant with a syringe and filter it into an ultracentrifuge tube using a 0.45μm filter membrane. Add 8 ml of lentiviral culture medium to the cells and culture them for 24 hours for secondary virus collection. Pipette 2.5 mL of 10% sucrose concentrate (100 mM NaCl, 0.5 mM EDTA 2Na2H2O, 10% sucrose, 50 mM Tris-HCl (pH 7.4)) and carefully add it to the bottom of the centrifuge tube. Put the balance into the biosafety cabinet, add sterile PBS to ensure that the weight difference of the 6 tubes is less than 0.1g, insert the sleeve, and tighten the cap. Then centrifuge at 10000g and 4 °C for 4 hours. Unscrew the lid in a biosafety cabinet, take out the centrifuge tube with tweezers, pour out all the supernatant, evaporate for 3 min, resuspend the virus in 200 μL sterile PBS (without calcium and magnesium), and aliquot in 50 μL and freeze at -80℃.

[0073] (5) Leave a small amount of virus, take 10 μL and add it to 90 μL PBS, mix well, then take another 10 μL and add it to 90 μL PBS, dilute 10 times in sequence (1, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 ). The diluted virus was added to the cultured CD8 + T was placed in a 96-well plate, and 4 μL of infection enhancement reagent TransGA (Gene Gene) was added to each well and cultured overnight to determine the virus titer.

[0074] (6) Day 5: Take out the 96-well plate, count the number of GFP-positive cells under a microscope, and calculate the virus titer based on the virus dilution factor. -4 If there is one positive GFP cell in the well, the virus titer is: 1 / 10 -4 =1×10 4 TU / μL = 1×10 7 TU / ml.

[0075] (7) Infecting target cells: Isolating CD8 + T cells, the operation method is based on EasySep Mouse CD8 +T Cell Isolation Kit instructions (Stem cell# 19853). Isolated T cells were stimulated with CD3 / CD38 antibodies in vitro for 2 days. The culture medium components are: RPMI1640, 10% FBS, 50 μM 2-mercaptoethanol, 1× sodium pyruvate, 1× non-essential amino acids, 1% penicillin / streptomycin, 25 mM HEPES, 50 U / mL mouse IL2, 5 ng / mL mouse IL-7 and 25 ng / mL IL-15.

[0076] (8) Day 6: Add 50 μg / mL RetroNectin (recombinant human fibronectin, Takara, T100A) to a 6-well plate and coat the plate overnight.

[0077] (9) Day 7: Pipette out RetroNectin, add lentivirus to the plate at an MOI of 10 based on the measured viral titer, and incubate for 6 h. Discard the supernatant and wash the plate once with PBS. + T cells were collected by centrifugation at 1200 rpm for 5 min and 1×10 5 10 cells / well were added to a 6-well plate (3 mL culture system). Infection was enhanced by centrifugation at 3200 rpm, 2 h, 32°C, and then cultured for another 3 days.

[0078] (10) Day 8: 20 μM / L Butegra (6 μM: 1×10 6 The cells were treated with PBS as the control group for 2 days.

[0079] (11) Day 10: Collect cells by centrifugation, select GFP-positive cells by flow cytometry, and count 3×10 6 The dose of 10 cells / mouse was adopted by tail vein into mice that had been subcutaneously inoculated with colon cancer MC38-hCD19 cells (tumor inoculation 5 days in advance). Subsequently, the growth of transplanted tumors was monitored every other day and the survival time of mice was recorded. The transplanted tumor volume was >2000mm 3 Set as ethical death endpoint.

[0080] Experimental results: The results of CAR-T cell therapy showed that CAR-T cells treated with Butea superba had a stronger ability to kill tumor cells, significantly inhibited the growth of transplanted tumors, and prolonged the survival of mice ( Figure 4 AB).

[0081] Example 5

[0082] Similar to the CAR-T experimental scheme in Example 4, Cas9-expressing NK92MI cells were further constructed as CAR-NK source cells, and the cell culture conditions were: Alpha-MEM supplemented with 10% FBS, 0.1 mM 2-mercaptoethanol, 1% penicillin / streptomycin, 0.2 mM inositol and 0.02 mM folic acid.

[0083] (1) Using the RetroNectin-coated plate infection method, add lentivirus to the plate at an MOI of 10 and incubate for 6 h. Discard the supernatant and wash the plate once with PBS. Collect the cultured NK cells after centrifugation at 1200 rpm for 5 min and add 1×10 5 Cells / well were added to a 6-well plate (3 mL culture system). Infection was enhanced by centrifugation at 3200 rpm and 32°C for 2 h, and then cultured for another 3 days.

[0084] (2) Day 8: Add 20 μM / L Butegra (e.g. 6 μM: 1×10 6 The cells were treated with PBS as the control group for 2 days.

[0085] (3) Day 10: Collect cells by centrifugation, and select GFP-positive CAR-NK cells by flow cytometry. 6 The cells / mouse were adopted into the tail vein of tumor-bearing mice that had been subcutaneously inoculated with human B lymphoblastoma Daudi cells (tumor was inoculated 7 days in advance). CAR-NK was adopted once every 3 days, for a total of 4 times. The growth of the transplanted tumor was monitored every other day, and the survival time of the mice was recorded. The transplanted tumor volume was >2000mm 3 Set as ethical death endpoint.

[0086] Experimental results: The results of CAR-NK cell therapy showed that CAR-NK cells treated with Butea superba had a stronger ability to kill tumor cells, significantly inhibited the growth of transplanted tumors, and prolonged the survival of mice ( Figure 4 CD).

[0087] In summary, the results of Examples 5-6 show that butenatin, as a small molecule inhibitor of induced degradation of ARHGAP9, can effectively enhance the anti-tumor effect of CAR-T and CAR-NK cell therapy.

[0088] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. An anti-tumor agent, characterized in that: The anti-tumor agent includes immune-engineered cells and butenatin; the immune-engineered cells include CAR-T cells or CAR-NK cells; the butenatin is configured as an in vitro immune activator of the immune-engineered cells, and is used to induce in vitro degradation of ARHGAP9 protein expressed by the immune-engineered cells.

2. The anti-tumor agent according to claim 1, characterized in that In the anti-tumor agent, butea superba and immune engineering cells are independently prepared according to unit doses, and the ratio of butea superba to immune engineering cells is that 6 μM butea superba is used to treat 1×10 5 ~1×10 6 Immunoengineered cells.

3. The anti-tumor agent according to claim 1, characterized in that The anti-tumor agents are applicable to tumor types including solid tumors and hematological tumors.

4. The anti-tumor agent according to claim 1, wherein the tumor targets applicable to the anti-tumor agent include CD19, BCMA, CD20, CD22, EGFRvIII, CD123, CD33, HER2, CD38, CD70, PSMA, B7-H3, GPC3, GCC, Claudin18.2, CEA, CD5, EpCAM, GD2 and / or Mesothelin.

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