CN114081883B

CN114081883B - Application of cloquindol in preparation of antitumor drugs

Info

Publication number: CN114081883B
Application number: CN202111301882.1A
Authority: CN
Inventors: 周翔; 代雨婷; 蔡苗苗; 黄文武; 张同存; 蒋争凡
Original assignee: Wuhan University of Science and Engineering WUSE
Current assignee: Wuhan University of Science and Engineering WUSE
Priority date: 2021-11-04
Filing date: 2021-11-04
Publication date: 2023-02-28
Anticipated expiration: 2041-11-04
Also published as: CN114081883A

Abstract

The invention discloses application of cloquindol in preparation of antitumor drugs. The invention discovers for the first time through research that antibiotic chloroquinadol can be used as an immune activator, remarkably improves the killing efficiency of CD19CAR-T on expressing CD19 antigen target cells in vitro, can also inhibit the amplification of tumors in CD19+ tumor-bearing mice, namely enhances the killing effect of the CD19CAR-T on the tumor cells, is expected to generate a better tumor treatment effect by combining the chloroquinadol with the CD-T, has no toxic or side effect on the cells, can rapidly penetrate through the cell surfaces to enter the cells as small molecules, is simple and easy to use, can induce the cells to generate a synergistic antitumor reaction by direct co-incubation, and avoids the limitations of limited activity, short half-life, blood injection administration and the like of other types of medicaments; can be suitable for various cultured cells and mouse animal models, has wide application range and is expected to be used and popularized in clinical antitumor tests.

Description

Application of cloquindol in preparation of antitumor drugs

Technical Field

The invention belongs to the technical field of immune cell therapy, and particularly relates to application of cloquindol in preparation of antitumor drugs.

Background

Acute B-lymphocytic leukemia (B-ALL) is the most common malignancy of the hematopoietic system, and it is statistically significant that adult patients have a low long-term disease-free survival (DFS) and, once relapsed, have low remission rates with conventional chemotherapy and poor prognosis. B-cell lymphoma, acute lymphoblastic leukemia and chronic lymphoblastic leukemia all originate from lymphoid B-cells. During the onset of B-lymphocyte leukemia, leukemic stem cells escape immune system monitoring by attenuating the growth of immunogenic tumor cells and exploiting the suppression of the active immune response. In recent years, a great number of new drugs and new therapies have appeared, but a therapeutic method capable of completely eradicating leukemic stem cells has not yet appeared. The clinical treatment of B lymphocyte leukemia mainly adopts radiotherapy and chemotherapy as main modes, but the relapse of patients and the refractory nature of diseases are always reasons for the ultimate failure of treatment. The development of immunotherapy offers new hopes for the radical cure of B-lymphocyte leukemia, wherein CAR-T cell therapy has good response in vitro tests and phase 1 clinical results are good. CAR-T (chimeric antigen receptor T cells) cells have significant activity against CD19 expressing B cell malignancies in humans, and these receptors fuse a single chain variable fragment of a tumor specific antibody (ScFv) with a signal molecule of an effector T cell to target tumor cells together. As one of the tumor-associated antigens, the CD19 antigen is generally expressed on normal B cells and tumor B cells, and is not expressed on other tissues and blood cells, and thus is considered to be an ideal target for CAR-T treatment of B-lymphocyte leukemia.

However, the efficacy of CAR-T cell therapy is affected by the immune status of the body, rendering part of the patients unable to benefit from CAR-T therapy. And after CAR-T cells are injected into the body, the killing efficiency will decrease with time, probably because the tumor immune regulatory cells tregs limit the killing effect of CAR-T. How to eliminate immunosuppression and activate the immune system of the body is the key point for improving the curative effect of CAR-T.

In recent years, the research on the targeting and killing performance of the CAR-T cells on tumor cells by applying natural immune activators finds that the activators can improve the response of the CAR-T cells to immunity and the transportation efficiency of the CAR-T cells reaching the tumor microenvironment by activating a STING pathway. The innate immune cGAS-STING pathway has been shown to activate the type i interferon pathway, which transforms macrophages to the lethal type M1, synergistically enhancing the tumoricidal effect, and thereby modulate downstream immune pathways. However, the activator has the defects of not obvious effect on CD19CAR-T and relatively poor tumor killing effect, so that the development of the small-molecule immune activator which can remarkably enhance the killing and inhibiting effect of the CD19CAR-T on tumors has important significance for treating malignant tumors.

Disclosure of Invention

The invention aims to provide the application of the chloroquinate in preparing the antitumor drugs, and researches show that the antibiotic chloroquinate not only has a certain effect on treating plasmodium falciparum infection, but also can be used as a novel natural immune activator, obviously improves the killing efficiency of CD19CAR-T on target cells expressing CD19 antigen in vitro, and can also inhibit the expansion of CD19+ tumor-loaded mice in vivo tumor, namely, enhances the killing effect of the CD19CAR-T on tumor cells and tumor tissues, is expected to generate a better B-ALL treatment effect by combining the chloroquinate with the CAR-T, and has a very high application prospect in clinical treatment.

In order to realize the purpose, the invention adopts the technical scheme that:

the invention provides application of chloroquinate in preparing an anti-tumor medicament.

Further, the structural formula of the chloroquinadol is as follows:

furthermore, the cloquindol achieves the anti-tumor effect by enhancing the killing effect of CD19CAR-T cells on tumor cells.

Furthermore, when the concentration of the cloquindol is 10-50 mu M, the cloquindol has the function of killing tumor cells in vitro.

Further, the tumor cell is a tumor cell expressing a CD19 surface antigen.

Further, the tumor cells comprise: human Burkitt's lymphoma cells Raji and/or human acute lymphocytic leukemia cells Nalm6.

Furthermore, when the dosage of the cloquindol is more than or equal to 5mg/Kg, the cloquindol has the in-vivo anti-tumor effect.

Furthermore, the chloroquinate is injected into the abdominal cavity, and the CD19CAR-T cells are injected into the vein, so that the in vivo synergistic anti-tumor effect is achieved.

Further, the cloquindol enhances the killing effect of CD19CAR-T cells on tumors in vivo and inhibits tumor growth.

Further, the preparation method of the CD19CAR-T cell comprises the following steps:

step one, culturing 293T cells to prepare CAR lentivirus;

collecting venous blood, removing plasma, and separating to obtain PBMC;

step three, carrying out magnetic bead sorting on the obtained PBMC to obtain T lymphocytes, and activating and culturing the T lymphocytes;

and step four, using the CAR lentivirus prepared in the step one to infect T lymphocytes, and detecting to obtain the CD19CAR-T cells.

The invention also provides application of the chloroquindol in preparation of a small-molecule immune activator.

Compared with the prior art, the invention has the beneficial effects that:

(1) The invention provides a new application of an antibiotic, namely an application in preparing an anti-tumor medicament, and researches show that the small-molecule cloquindol can remarkably enhance the killing effect of CD19CAR-T cells on tumor target cells expressing CD19 antigens.

(2) The small molecular compound of the invention, namely the cloquindol, is a safe anti-tumor reagent, and no change is found in the cell state after the cloquindol is treated in the experimental process, no matter the observation is carried out by a common microscope or a fluorescence microscope, so that the cloquindol is proved to be a safe anti-tumor reagent without obvious toxic and side effects.

(3) The small molecular immune activator cloquindol can rapidly penetrate through the cell surface to enter cells, is very simple and easy to use, and can induce the cells to generate a synergistic anti-tumor reaction by direct co-incubation. Avoids the limitations of other types of medicaments such as limited activity, short half-life, requirement of blood injection administration and the like.

(4) The small molecular compound cloquindol can be suitable for various cultured cells and mouse animal models, has wide application range, is beneficial to the use of various antitumor environments, and is expected to be used and popularized in clinical antitumor tests.

Drawings

FIG. 1 is a schematic diagram of the domain of the CD19CAR molecule in example 1 of the present invention;

FIG. 2 shows the results of flow cytometry after lentiviral transduction of T lymphocytes in example 1 of the present invention;

FIG. 3 is a graph showing the results of detecting the effect of cloquindol on CD19CAR-T killing target cells in vitro in example 2 of the present invention;

FIG. 4 is the result of testing the effect of cloquindol at different concentrations on CD19CAR-T killing target cells in vitro in example 2 of the present invention;

FIG. 5 is a graph showing in vivo imaging observations of the tumoricidal effect of cloquindol against CD19CAR-T in tumor-bearing mice in example 3 of the present invention;

FIG. 6 shows the result of detecting the cytotoxicity level of Raji and Nalm6 cell lines treated with different concentrations of chloroquinate by CCK8 in example 4 of the present invention for 24 h;

FIG. 7 is a result of observing the cytotoxic effects of chloroquinate and cisplatin on HeLa-CD19-Luc at different concentrations under a microscope in example 4 of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

Example 1 preparation of CD19CAR-T cells

The present experiment used a three-generation CAR vector to construct molecules for expressing CD19CAR, whose protein domain pattern is shown in fig. 1, where from left to right are Signal Peptide (SP), scFv, strep ii tag, CD8 hinge region (CD 8 hinger), CD28 transmembrane domain and endodomain, CD137 endodomain (41 BB) and CD3 ζ.

1. Preparation of CAR lentivirus

(1) Culturing 293T cells in a 150mm culture dish, and carrying out passage every 3-4 days at the ratio of 1:8;

(2) The day before transfection, the 293T cell is ensured to have good growth state, and the cell density is about 90%. Removing the original culture medium by using a pipettor, washing the original culture medium by using PBS (phosphate buffer solution), digesting cells by using 0.05 percent of pancreatin containing EDTA (ethylene diamine tetraacetic acid), centrifuging and collecting the cells;

(3) Cell count, density adjustment, preparation of several new 150mm dishes, addition of 293T cells 2X 10 per dish ⁷ Adding 20mL of complete culture medium into each dish, putting the dish into an incubator for overnight culture, and taking 10 dishes in total;

(4) On the next day, 1-5h before transfection, discarding the original culture medium, adding 10mL of fresh culture medium, simultaneously adding 10 μ L of 25mM chloroquine phosphate solution, and placing back to the incubator for continuous culture;

(5) Preparation of transfection system:

tube A:

serum-free DMEM medium was added according to the volume of the DNA, and the volume was filled to 500. Mu.L/dish.

And (B) tube:

(6) Respectively blowing and uniformly mixing the tube A and the tube B by using a pipettor, and standing for 5min at room temperature;

(7) Sucking the liquid in the tube B, adding the liquid in the tube A, uniformly blowing, standing at room temperature for 30min, and fully wrapping the DNA compound by the PEI;

(8) Adding 1mL of PEI-DNA mixed solution into each 150mm dish, slightly shaking the culture dish to uniformly distribute the mixed solution in the culture medium, and putting the culture dish into an incubator;

(9) Sucking out the original culture medium 6-8h after transfection, adding 25mL of fresh culture medium into each dish, and continuing to culture;

(10) After 72h, cell supernatants were carefully collected, carefully to avoid aspiration of 293T cells, centrifuged at 4 ℃,3000g,10min;

(11) Adding the centrifuged supernatant into a new 50mL centrifuge tube again, and taking care to avoid sucking the supernatant into a precipitate;

(12) Filtering the virus supernatant through 0.45 μm and 0.22 μm filters in sequence;

(13) Centrifuging the filtered virus at 4 ℃ for 30000g,3h;

(14) Discarding the supernatant, retaining the white lentivirus precipitate on the tube wall, resuspending with appropriate amount of T cell culture medium, taking 1-5 μ L for subsequent titer detection, subpackaging the rest virus solution at 100 μ L/tube, and storing in a refrigerator at-80 deg.C.

2. Isolation of PBMC

(1) Collecting peripheral blood of human by using an EDTA (ethylene diamine tetraacetic acid) anticoagulation tube, and storing at normal temperature for later use (separating within 4 h);

(2) Adding the collected venous blood into a T75 culture flask, and adding PBS or normal saline with the same volume to dilute the blood;

(3) Centrifuging with horizontal centrifuge for 300g and 10min, removing upper layer plasma, and adding PBS or physiological saline with the same volume as the lower layer;

(4) Prepare a new 50mL centrifuge tube, follow the blood cell suspension: lymphocyte separation =25, gently dripping the blood cell suspension along the vessel wall above the liquid level of the separation by using a sterile dropper or pipette;

(5) Centrifuging at 18 deg.C for 700g and 20min in a horizontal centrifuge, and regulating the lifting parameters of the centrifuge to minimum;

(6) The visible liquid level after centrifugation is divided into four layers, from top to bottom: a plasma layer, a ring-shaped milky white lymphocyte layer, a separation liquid layer and a red blood cell layer;

(7) Carefully sucking the second layer of lymphocytes by using a pipettor or a sterile pipette, and transferring the second layer of lymphocytes into a new 50mL centrifuge tube;

(8) Following the volume of lymphocyte fluid: adding a proper amount of PBS or normal saline into the normal saline/PBS, and supplementing the liquid in the tube to 45mL;

(9) Centrifuging at room temperature for 300g,10min;

(10) After the supernatant is discarded, 30mL of PBS or physiological saline is added to resuspend the cells, then the centrifugation is carried out, and the centrifugation is repeated for 1-2 times until the cell suspension is clarified;

(11) Cell counting: PBMC obtained by 100mL blood final fractionation is 3.5X 10 ⁸ Respectively, freezing the stock solution according to the ratio of 2 × 10 ⁷ Resuspending the cells at a density of one cell/mL, subpackaging and freezing at-80 ℃.

3. Human T lymphocyte magnetic bead sorting

(1) The PBMC obtained above was resuspended in the cell sorting buffer to adjust the cell density to 1X 10 ⁷ Per mL;

(2) At a rate of 5. Mu.L/1X 10 ⁶ Adding Biotinylated T cell Enrichment antibody combination (Biotinylated Human T Lymphocyte Enrichment Cocktail) into the ratio of each cell, uniformly mixing, and incubating at room temperature for 15min;

(3) Adding 10 times volume of sorting buffer, terminating incubation, centrifuging at room temperature, 300g,7min;

(4) At 5. Mu.L/1X 10 ⁶ Adding Streptavidin coupled magnetic particle solution (Streptavidin Particles Plus-DM) into the ratio of each cell, uniformly mixing, and incubating at room temperature for 30min;

(5) Adding a certain volume of sorting buffer to adjust the cell concentration to 2-8X 10 ⁷ Individual cells/mL;

(6) Transferring the cells to a sterile flow tube of 12X 75mm, the maximum volume not exceeding 1mL;

(7) Fixing the flow tube in a cell sorting magnet, standing for 6-8min, and attaching a visible brown precipitate to a magnetic pole layer;

(8) Preparing a new sterile flow tube, and sucking the liquid in the sorting tube into the new sterile flow tube;

(9) Taking out the flow tube from the magnet, adding the sorting buffer with the original volume, uniformly mixing, putting back into the magnet, and standing for 5min;

(10) Repeating the step (9) for 1-2 times;

(11) And (3) putting the flow tube with the collected liquid in the magnetic field again, standing for 8min, sucking all the liquid into a new EP tube, counting, centrifuging, and 300g and 5min to obtain the purified human T lymphocyte.

(12) Counting results are as follows: viable cells 1.9X 10 ⁸ An

4. T lymphocyte activation and culture

(1) Adding T lymphocyte activation culture medium into the sorted T lymphocyte, and adjusting cell density to 5 × 10 ⁶ Individual cells/mL;

(2) At a rate of 10. Mu.L/1X 10 ⁶ Adding T cell TransAct solution (CD 3/CD28 monoclonal antibody) into the T lymphocytes according to the proportion, and putting the T lymphocytes into an incubator for overnight culture;

(3) On the next day, the T lymphocytes are observed to aggregate into clusters with different sizes under a microscope, which indicates that the activation is successful;

(4) Adding T lymphocyte amplification culture medium, and adjusting cell density to 1-2 × 10 ⁶ Individual cells/mL;

(5) Observing and measuring cell shape, density and culture medium color every day, and changing the culture medium by half-volume liquid changing method to maintain cell density at 0.5-1 × 10 ⁶ Individual cells/mL.

5. CAR Lentiviral infection of T lymphocytes

(1) Taking the T lymphocytes 12-24h after activation, counting, centrifuging, 300g, and 5min;

(2) Take 1.5X 10 ⁸ (ii) adding CAR lentivirus at MOI =3 to each T lymphocyte, and supplementing T lymphocyte expansion medium to maintain cell density at 1X 10 ⁷ Individual cells/mL;

(3) Adding Polybrene with the final concentration of 6 mu g/mL to promote virus infection;

(4) Adding T lymphocyte amplification culture medium 6-8 hr after virus infection to maintain cell density at 1-2 × 10 ⁶ Individual cells/mL;

6. CAR lentivirus infection T lymphocyte efficiency detection

(1) The T lymphocytes 5 days after the infection of the CAR lentivirus and the T lymphocytes (NT) not infected by the virus are taken as blank control, and each sampleCollect about 5X 10 ⁵ (ii) individual cells;

(2) Centrifuging at 4 ℃ for 300g and 5min;

(3) Adding 2 μ L of 7-AAD and PE-CD19 antibody into each tube, and incubating on ice for 30min;

(4) Centrifuging at 4 ℃ for 300g and 5min;

(5) And (3) performing flow-type detection on the cells, removing dead cells through 7-AAD, then, circling out negative cell populations according to blank control, and calculating the infection efficiency of each sample.

After lentiviral transduction of T lymphocytes at MOI =3, positive expression of CD19-CAR was detected by flow cytometry on day 5, the results of which are shown in fig. 2.

Example 2 Cloquindol promotes CD19CAR-T in vitro tumoricidal assays

This example is used to verify the promoting effect of cloquindol, also known as chloroquine, promestrene (chloroquinaldol, C), on killing tumor cells in vitro by CD19CAR-T ₁₀ H ₇ Cl ₂ NO, selleck catalog No. S4192), a small molecule antibiotic of the formula:

。

1. two cancer cell lines known to express CD19 surface antigen were used in this experiment: (1) Raji: human Burkitt's lymphoma cells; (2) Nalm6: human acute lymphocytic leukemia cells, and the killing effect is detected by adopting a calcein release experiment, which comprises the following specific steps:

(1) 9X 10 cell harvest of Raji and Nalm6 cells ⁶ Cells, resuspended in 18mL of medium, added to a 12-well plate at 2 mL/well, and the plate was mixed well in a horizontal desktop cross format.

(2) After cell adherence, 800. Mu.L of fresh medium was replaced and the drug transfection system was prepared as shown in the following table:

tube A:

and a tube B:

(3) Respectively mixing the A tube and the B tube, adding the liquid of the A tube into the B tube, mixing, incubating at room temperature for 10-15min, adding into a 12-hole plate, and mixing the hole plate uniformly on a horizontal desktop in a cross way;

(4) Medicine preparation: experiments were performed with cloquindol or one of the STING-binding small molecule agonists c-di-AMP, respectively, for 24h and PBS treatment as control, and Raji and Nalm6 cells were counted in 12-well plates, 1 × 10 each ⁵ Each cell was taken into a labeled 15mL centrifuge tube.

(5) 10mL of PBS containing 5% FBS was added to each centrifuge tube, and centrifuged at 300g at room temperature for 5 minutes, and the supernatant was discarded, and then the bottom of the centrifuge tube was gently flicked with the fingertip to flick the cell mass.

(6) Add 0.1mL PBS containing 5% FBS to resuspend the cells, then add 1. Mu.L of 5mM Calcein-AM solution to the centrifuge tube cell suspension (Calcein-AM mix well before use), gently blow and mix well with a tip. The cell suspension was incubated in an electric incubator at 37 ℃ for 30min.

(7) After completion of the incubation, 10mL of PBS containing 5% FBS was added to the centrifuge tube to wash the cells, and centrifuged at room temperature and 300g for 5 minutes, and the supernatant was discarded. The washing operation was repeated twice, and after each blotting of the supernatant, the bottom of the tube was flicked with the finger to flick off the cells.

(8) Finally, 2mL of PBS containing 5% FBS was added to the centrifuge tube to resuspend the cells;

(9) Take 1.125X 10 ⁷ One CAR-T cell and 1.25X 10 ⁶ Each T lymphocyte was taken to a labeled 15mL centrifuge tube.

(10) 1mL of 5-FBS PBS was added to the centrifuge tube, and the mixture was centrifuged at room temperature and 300g for 5 minutes, and the supernatant was discarded, and then the bottom of the centrifuge tube was gently flicked with the tip of a finger to flick the cell mass. The operation was repeated once.

(11) 5% FBS-containing PBS was added to the centrifuge tube to adjust the cell density to 1.25X 10 ⁶ one/mL.

(12) Preparing a 96-pore plate with a round bottom, and paving the plate according to the following groups, wherein each group is provided with four repeats:

(13) After the plate paving is finished, uniformly mixing the pore plates in a horizontal desktop cross method, and putting the mixture back to an incubator for culturing for 2.5 hours at 37 ℃;

(14) Taking out the pore plate from the incubator, centrifuging at room temperature, 300g, and 5min;

(15) Each well was pipetted with 100. Mu.L of medium into a flat-bottomed 96-well plate and assayed using a multi-functional microplate reader.

The results of the detection of killing efficiency of CD19 CART cells against target cells after treatment of target cells Raji, nalm6 with PBS, chloroquinate (CQD, 10. Mu.M) and STING agonist c-di-AMP (8. Mu.g/mL) are shown in FIG. 3, where p is < 0.01. The results show that treatment of two tumor cell lines Nalm6 and Raji expressing CD19 surface antigen with cloquindol and one STING-binding small molecule agonist c-di-AMP, respectively, for 24 hours prior to killing with CD19CAR-T significantly enhances the killing effect of CD19CAR-T on both cell lines compared to the control group (PBS) without cloquindol; however, the effect of the STING agonist c-di-AMP on CD19CAR-T is not obvious, which indicates that the chloroquindol has the effect of remarkably enhancing the effect of killing target tumor cells by the CD19CAR-T in vitro.

2. To determine the optimal concentration of cloquindol, repeated experiments were performed using different doses of cloquindol (0, 10, 20 and 50 μ M), respectively, according to the above experimental procedure, and the results were shown in fig. 4, which were measured by the calcein release experiment. The results show that different concentrations of cloquindol can promote the killing effect of CD19CAR-T on target cells, wherein the concentration of cloquindol with the best killing effect is 10 mu M.

Example 3 Cloquindol promotes CD19CAR-T in vivo tumoricidal experiments

This example is mainly used to demonstrate that cloquindol can enhance the killing effect of CD19CAR-T on tumors expressing CD19 target molecules in tumor-bearing mice. The experiment is as follows:

1. mouse tumor model establishment and treatment

(1) 20 female NOD Scid Gamma (NOD. Cg-PrkdcsccidIL-2 rgtm1Wj1/SzJ, NSG) mice of 5 weeks of age were purchased, all of which were rigorously quarantined and housed in SPF grade housing. All animal experiments involved study protocols and objectives approved by the Wuhan scientific and university animal ethics Committee.

(2) A sufficient amount of Hela-CD19-Luc cell suspension was prepared, and each mouse was prepared for injection of 5X 10 ⁶ Preparing 1.2 times of cells;

(3) The injection part (axillary subcutaneous) was wiped with an alcohol cotton swab, and 100. Mu.L of the tumor cell suspension (5X 10) was aspirated with a 1mL sterile syringe ⁶ Individual cells) were injected subcutaneously;

(4) After injection, the injection part was pressed with a sterile dry cotton ball for 2min to prevent bleeding and leakage of tumor cell suspension.

(5) On day 3 after the mice were modeled, the mice were administered by intraperitoneal injection in groups of PBS, chloroquinate (5 mg/kg of mouse body weight) and c-di-AMP (5 mg/kg of mouse body weight) in the morning, followed by a bolus every 5 to 7 days.

(6) Sufficient numbers of CAR-T and NT cells were collected in the afternoon, and each mouse was injected with 1X 10 ⁷ 1.2 times of cells are prepared, centrifuged, 300g and 5min;

(7) Add sterile PBS to resuspend the cells to a cell density of 1X 10 ⁸ Per mL;

(8) Mice were fixed and 100 μ L of CAR-T cells or NT cells or PBS suspension was aspirated with an insulin needle for tail vein injection.

2. Small animal in vivo imaging

(1) Preparing a D-luciferin substrate with the concentration of 15mg/mL, and injecting 200 mu L of the substrate into the abdominal cavity of each mouse;

(2) The mice injected with the substrate are placed in a gas anesthesia chamber (containing sevoflurane gas) for induction of gas anesthesia, and the gas parameter is adjusted to 2;

(3) After anesthesia induction is finished, putting the mouse into an IVIS animal imaging instrument, plugging the head of the mouse into an oxygen mask, and simultaneously adjusting the gas anesthesia parameter to 1.5 for continuous anesthesia;

(4) Starting 10min after the injection of the substrate, imaging the small animal once every 30sec, and collecting biotin luminescence signals until the intensity of the luminescence signals is not increased any more, which indicates that the plateau period is reached;

(5) Collecting image and photon flux value (p/s/cm) of signal plateau ² )；

(6) After imaging, the mice were removed, placed in a 26 ℃ incubator until they were awake, and then returned to their cages.

In vivo imaging observations were made at day 3 and day 10 after injection of CD19CAR-T cells, and the results are shown in figure 5, which shows that addition of CD19CAR-T cells inhibits tumor growth, with a tendency for tumor diameter to decrease compared to mice without CAR-T cells; on the basis, the tumors in mice injected with the cloquindol in the abdominal cavity are further remarkably reduced, which shows that the cloquindol can greatly enhance the capability of CD19CAR-T cells in killing solid tumors and inhibiting tumor growth in the mice. In contrast, the STING agonist c-di-AMP had little effect on the tumoricidal effect of CAR-T cells.

Example 4 cytotoxicity assays for Cloquinaldine

In order to verify that the chloroquinate adopted by the invention is a safe anti-tumor reagent without toxic and side effects, CCK8 cytotoxicity detection is respectively carried out, and the cytotoxicity effect of chloroquinate with different concentrations on HeLa-CD19-Luc is observed under a microscope.

The CCK8 cytotoxicity detection method comprises the following steps:

(1) Collecting suspension cells, centrifuging at 300g for 5min, and adjusting cell concentration to 2 × 10 with serum-free minimal medium ⁵ Per mL;

(2) Adding the chloroquinaldol into the cell suspension according to different concentrations ((0, 10, 20 and 50 mu M)), uniformly mixing, and uniformly paving the cells in a round-bottom 96-well plate at a concentration of 100 mu L/well;

(3) The 96-well plates were incubated in a cell incubator for 24h.

(4) The 96-well plate was removed, centrifuged at 300g for 5min, 50. Mu.L of supernatant was removed, and the final medium: CCK8 reagent =9:1 CCK8 solution was prepared and added to 96 well plates and returned to the cell incubator for incubation in the dark.

(5) After incubation for 4h, the absorbance at 450nm was measured with a microplate reader and the cell viability was calculated.

The cytotoxicity level detection results of CCK8 in detection of different concentrations of cloquindol (0, 10, 20 and 50 mu M) in treatment of Raji and Nalm6 cell lines for 24h are shown in FIG. 6, and the results show that the toxic and side effects of the cloquindol with different concentrations on cells are not different from the toxic and side effects of the cloquindol with 0 mu M, namely the results prove that the cloquindol has no toxic and side effects on cells and is a safe reagent.

Then, the cytotoxicity effect of different concentrations of the chloroquinalder (0, 10, 20 and 50 mu M) and the cisplatin (5 mu g/mL) on the HeLa-CD19-Luc is observed under a microscope, the detection result is shown in figure 7, and the same microscopic observation shows that the chloroquinalder has no toxic or side effect on cells compared with the cisplatin, namely, the chloroquinalder is proved to be a safe anti-tumor agent.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

Claims

1. The application of the chloroquinate in preparing the medicine for enhancing the killing effect of CD19CAR-T cells on tumor cells expressing CD19 surface antigens, wherein the structural formula of the chloroquinate is as follows:

。

2. the use according to claim 1, wherein the cloquindol achieves an anti-tumor effect by enhancing the killing effect of CD19CAR-T cells on tumor cells.

3. The use according to claim 2, wherein the chloroquinate has an in vitro tumor cell killing effect when the concentration is 10 to 50 μ M.

4. The use of claim 3, wherein said tumor cell is a tumor cell expressing the CD19 surface antigen.

5. The use of claim 4, wherein the tumor cells comprise: human Burkitt's lymphoma cells Raji and/or human acute lymphocytic leukemia cells Nalm6.

6. The use of claim 2, wherein the cloquindol has an in vivo anti-tumor effect when the amount of the cloquindol is not less than 5 mg/Kg.

7. The use of claim 6, wherein the cloquindol is administered intraperitoneally and the CD19CAR-T cells are administered intravenously to achieve synergistic antitumor effects in vivo.

8. The use according to claim 2, wherein the process for the preparation of CD19CAR-T cells comprises:

step one, culturing 293T cells to prepare CAR lentivirus;

collecting venous blood, removing plasma, and separating to obtain PBMC;

step three, carrying out magnetic bead sorting on the PBMC to obtain T lymphocytes, and activating and culturing the T lymphocytes;