Application of CAR exosome drug carrier in tumor treatment

Abstract

The invention relates to an application of a CAR exosome drug carrier in tumor treatment. The application of the CAR exosome loaded medicine in tumor treatment is based on the strategy of CAR exosome coupling medicine, so that the completeness of the exosome and the controllability of the medicine loading capacity are greatly protected, the purpose of accurately delivering the medicine to tumors in a targeted manner is achieved, and a new direction is provided for tumor treatment.

Description

TECHNICAL FIELD

[0001] The application relates to the medical technology field, in particular to application of CAR exosome drug loading in tumor treatment. BACKGROUND

[0002] Immune cell therapy is a promising new type of tumor treatment method, especially CAR-T cell therapy has achieved remarkable results in treating leukemia, greatly improving the clinical remission rate of difficult-to-cure leukemia, and bringing new hope to thousands of leukemia patients around the world. However, current autologous CAR-T therapy has many problems, such as personalized customization, high cost, long preparation time, etc., and it is urgent to develop a universal vector cell and realize the "off-the-shelf" cell therapy. Therefore, it is worth further studying the new strategy of developing a universal immunotherapy, and the universal immunotherapy CAR exosome carrying drugs has great potential in precise targeted drug delivery for tumor treatment; CAR-T cell therapy for solid tumors faces many challenges, including: T cell exhaustion caused by continuous exposure to tumor antigens. The repeated stimulation of tumor antigens and the tonic signal existing in CAR-T cells make CAR-T cells over-activated and differentiated into exhausted phenotype. Off-target on-target toxicity caused by tumor antigens expressed on normal cells. Since the target of solid tumors is mostly tumor-associated antigens, these targets are also expressed in normal tissues, so CAR-T cells may damage normal tissues and cause side effects. Immunosuppressive tumor microenvironment. The microenvironment of hypoxia and nutrient deficiency is not conducive to the survival of immune cells, and the negative immune checkpoint molecules expressed by tumor cells and immunosuppressive cells such as MDSC and Treg in the tumor microenvironment and the immunosuppressive factors such as TGFβ secreted by them inhibit the immune response of CAR-T cells. And CAR-T cells are difficult to migrate and infiltrate into solid tumor lesions. Solid tumors have physical barriers of fibrous tissue, and the expression of chemotactic factors is scarce, which is not conducive to the localization and infiltration of CAR-T cells; Research is conducted to develop new CAR-T therapies for T cell malignancies, including CD5, CD7, CD4 and CD1a. Although immunotherapy as a new treatment method has developed rapidly in recent years, it faces some unique challenges when applied to the treatment of T-ALL, including CAR-T fratricide, T cell aplastic anemia, and contamination of malignant T cells produced when autologous CAR is produced. Therefore, immunotherapy for T-ALL is still mostly in the clinical trial stage, and a very effective clinical treatment method has not yet been found; Exosomes are small extracellular vesicles (EVs) produced by cells and found in all body fluids. Exosomes are considered an important pathway for the intercellular transfer of materials and signals. Different types of cells secrete exosomes containing different components, which can promote communication between cells. The recipient cells actively absorb the exosomes, and the transfer of materials and signals is achieved through material exchange or release of contents. Therefore, exosomes play a key biological role in cell interaction, affecting a variety of cellular activities in health and disease. Studies have found that exosomes produced by CAR-T cells have anti-tumor function as CAR-T cells. However, the anti-tumor effect is weak, so the development of enhanced CAR exosomes has strong application value in the treatment of tumors. SUMMARY

[0003] In view of the defects of the prior art, the application provides the application of CAR exosome drug loading in tumor treatment, which has the advantages of greatly increasing the anti-tumor effect of CAR exosomes, and solves the problems of weak anti-tumor effect of CAR exosomes.

[0004] To achieve the above-mentioned purpose, the application provides the following technical scheme: a preparation method of CAR exosome conjugated drug, comprising the following steps: adding a non-natural sugar labeled with a biological ortho group to cells to prepare exosomes containing non-natural sugars labeled with a biological ortho group on the exosome membrane of the cells, and using a biological ortho reaction to couple the exosomes with drugs, so as to achieve the effect of targeted delivery of killing.

[0005] Further, the cells are CAR cells.

[0006] Further, the CAR exosomes have anti-tumor effect.

[0007] Further, the CAR exosome conjugated drug is obtained by coupling the CAR exosome with a small molecule drug.

[0008] Further, a CAR exosome conjugated drug is prepared.

[0009] Further, the application of a CAR exosome conjugated drug in the treatment of T-cell acute lymphoblastic leukemia.

[0010] Further, the application of a CAR exosome conjugated drug in the treatment of solid tumors.

[0011] A preparation method of CAR-J-EVs-S is as follows: S1, construct plasmids containing anti-CD19-CAR, anti-CD7-CAR and anti-MSLN-CAR respectively, and co-transfect cells with the plasmids and lentivirus packaging plasmids to obtain three corresponding lentiviruses; S2, transfect Jurkat cells with the lentiviruses encoding the three target proteins respectively to obtain stable cell lines; the stably expressed cells are expanded in a culture medium containing exosomes, and after adding non-HEX 24h, all the liquid is changed to exosome-free culture medium and expanded for 24h to obtain CAR-J-EVs-S.

[0012] S3, couple CAR-J-EVs-S with DBCO-small molecule drugs at 4 DEG C for 2h, remove the uncoupled single drugs, and obtain CAR-J-EVs-S-drugs.

[0013] Here, we propose a simple method of using Jurkat cells loaded with CAR gene sequence encoding, and sugar modification engineering to obtain CAR and sugar modified exosomes, which can induce target cell death in vivo or in vitro, realize CAR exosome drug targeting delivery to tumor cells, realize efficient infiltration of tumor, efficient recognition and phagocytosis of tumor cells, so as to realize anti-tumor immune regulation and inhibition of tumor growth, and has broad application prospect.

[0014] Therefore, the application also provides the application of the CAR-J-EVs-S-drugs in anti-tumor drugs.

[0015] More preferably, the tumor is T-ALL, breast cancer and B-ALL.

[0016] Compared with the prior art, the application has the following beneficial effects: The application provides a genetically modified Jurkat cell exosome, which can target tumor cells, and is loaded with CAR genes, and the exosome contains GZMB and Perforin, which can target and kill tumor cells, and realize anti-tumor function.

[0017] The CAR-J-EVs-S prepared by the engineered CAR exosome in vitro has an azido group, which can couple small molecule drugs in need through click chemistry, thereby enhancing the anti-tumor function of the CAR exosome, realizing controllability of the drug amount of the CAR exosome drug loading and integrity of the exosome, and well playing the function of the CAR exosome, realizing high infiltration of the CAR exosome to solid tumors, and solving the problems of CAR-T self-killing, T cell aplastic anemia and malignant T cell pollution in the production of autologous CAR in the T-ALL treatment process.

[0018] The preparation process of traditional CAR-T includes the separation of PBMC, the activation of T cells, the infection of virus and the return of CAR-T, and the preparation process is extremely complex and long. In order to avoid immune rejection, the CAR-T cells need to be cultured by special personnel and special machine, so it is difficult to realize large-scale production. In order to overcome the above shortcomings, the present application discloses a method for directly producing CAR-J-EVs-S through engineered exosomes. The engineered exosomes of the present application can not only directly produce CAR-J-EVs-S in vitro, and the exosomes after sugar modification can be coupled with small molecule drugs by click chemistry, so as to greatly increase the anti-tumor effect of CAR-J-EVs-S, and realize "spot type" and "universal type" immunotherapy. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 CAR molecular structure design for generating CAR-J-EVs-S; Figure 2 Expression of CAR and sugar of CAR-J-EVs-S; Figure 3 Characterization verification diagram of CAR-J-EVs-S; Figure 4 Targeting of CAR-J-EVs-S exosomes to target cells; Figure 5 CAR-J-EVs-S-drug killing in vitro; Figure 6 Changes in tumor growth of mice after administration of CAR-J-EVs-S-drug. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0021] Example 1: Preparation of CAR exosomes (1) Construction of CAR lentivirus and stable cell line: According to the method known in the art, anti-CD19-CAR, anti-CD7-CAR and anti-MSLN-CAR gene sequences were constructed, respectively, and the complete map of the plasmid is shown in Figure 1The above each CAR plasmid was co-transfected into HEK293T cells with a lentivirus packaging plasmid system (such as psPAX2 and pMD2.G). After 48 to 72 hours of transfection, the cell supernatant containing lentivirus particles was collected, concentrated by ultracentrifugation, and three high-titer CAR lentivirus stock solutions were obtained. Jurkat cells were cultured under suitable conditions and the density was adjusted to 1 × 10 6 cells / mL. The above three lentiviruses were used to infect Jurkat cells, respectively, and puromycin (2 μg / mL) was used for pressure screening after infection, and finally a CAR-Jurkat stable cell line capable of stably expressing the corresponding CAR protein was obtained.

[0022] (2) Preparation of CAR exosomes labeled with bio-orthogonal groups: The above three stable cell lines were expanded in complete culture medium (such as RPMI-1640 supplemented with 10% exosome-depleted FBS) with exosomes removed by ultracentrifugation.

[0023] When the cells grew to the logarithmic phase, non-natural sugars containing bio-orthogonal groups (such as N3-1,6-Pr2Man) were added to the culture medium, and the cells were cultured for 24 hours. During this process, the cells metabolize the non-natural sugars and integrate them into glycoproteins, so that the exosome membranes secreted thereafter carry bio-orthogonal groups on their surfaces.

[0024] The culture medium containing non-natural sugars was removed, the cells were gently washed once with PBS, and then fresh exosome-free culture medium was added for continued culture for 24 hours to induce the cells to secrete exosomes.

[0025] The cell culture supernatant was collected, and cells and debris were removed by differential centrifugation (300 ×g 10 min, 2,000 ×g 20 min, 10,000 ×g 30 min), and finally CAR exosomes were precipitated by ultracentrifugation (100,000 ×g, 70 min). The exosome precipitate was resuspended with sterile PBS, and the protein concentration was quantified by the BCA method, and the expression of CAR and sugar on the exosomes was detected by exosome-coupled magnetic beads flow cytometry, and the results are shown in Figure 2 , that is, CAR-J exosomes loaded with bio-orthogonal group sugar modifications were obtained, and the characterization verification is shown in Figure 3 . After aliquoting, it was stored at -80°C for standby use. The CAR exosomes have targeting and anti-tumor effects due to the CAR proteins carried on their surfaces.

[0026] Example 2: Coupling of CAR exosomes with drugs Take the biorthogonal group labeled CAR exosomes prepared in Example 1 (protein concentration is about 1 mg / mL), mixed with small molecule drugs with DBCO group, such as toxin molecule MMAE. The mixed system is incubated at 4°C with gentle shaking for 2 hours. Under this condition, the biorthogonal group on the surface of the exosome reacts with the DBCO group on the drug molecule to form a stable covalent bond through high-efficiency click chemistry (Strain-Promoted Azide-Alkyne Cycloaddition, SPAAC), achieving drug conjugation.

[0027] After the reaction is completed, the reaction mixture is purified using an ultrafiltration centrifuge tube (molecular weight cutoff 100 kDa) to remove free drug molecules that are not conjugated. After washing with PBS for 3 times, the purified CAR exosome conjugated drug is obtained.

[0028] After conjugating the drug by this method, the anti-tumor function of the exosome itself can be significantly improved, achieving a synergistic effect of "targeted delivery" and "killing".

[0029] Example 3: Application and effect verification of CAR exosome conjugated drug (1) Targeted delivery effect The anti-CD19-CAR was used to verify the targeting of the CAR exosome. A human CD19-overexpressing MC38 cell line was constructed, and nude mice were selected as research objects. The prepared MC38-OE-CD19 cells were mixed, 50µL of cell suspension was extracted using a syringe, and after excluding air bubbles, it was slowly injected subcutaneously. The mice were put back into the cage for further feeding. When the tumor grew to 30mm3, CAR-J-EVs-S-Cy5 or J-EVs-S-Cy5 was injected intravenously. It was found that the exosome with CAR was more enriched in the tumor, and the results are shown in Figure 4 .

[0030] (2) Application in treating T-cell acute lymphoblastic leukemia (T-ALL): The anti-CD7-CAR exosome conjugated MMAE drug was selected, and the conjugated drug was co-cultured with human T-cell acute lymphoblastic leukemia cell lines (such as CCRF-CEM) in vitro. The cytotoxicity experiment results showed that the conjugated drug could specifically target and efficiently kill CD7-positive leukemia cells, and the half maximal inhibitory concentration (IC50) was significantly lower than that of the free drug group and the CAR exosome group without drug loading.

[0031] In vivo, a T-ALL mouse model was established, and the conjugated drug was given by tail vein injection. The results showed that the leukemia load of the treatment group mice was significantly reduced, and the survival period was significantly prolonged, proving the effectiveness and application prospect of the conjugated drug in treating T-cell acute lymphoblastic leukemia.

[0032] (3) In the treatment of solid tumors: The anti-CD19-CAR exosome conjugated MMAE drug was selected. In vivo, a CDX mouse model was established and injected with the conjugated drug. The monitoring results showed that the solid tumor growth of the treatment group mice was significantly inhibited, the tumor volume was significantly smaller than that of the control group, and no obvious toxicity was found in normal tissues, proving its effectiveness and application prospect in the treatment of solid tumors. The results are shown in Figure 6 .

[0033] It should be noted that in this paper, relational terms such as first and second are used only to distinguish one entity or operation from another, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. Moreover, the terms "include", "contain" or any other variants thereof are intended to cover non-exclusive inclusion, so that the process, method, article or equipment including a series of elements not only includes those elements, but also includes other elements not explicitly listed or inherent to such process, method, article or equipment. Without more limitations, the element defined by the statement "including a" does not exclude the presence of other identical elements in the process, method, article or equipment including the element.

[0034] Although embodiments of the present application have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a CAR-exosome conjugate drug, characterized by, The method comprises the following steps: preparing a cell exosome containing a bio-orthogonal group-labeled unnatural sugar on the cell exosome membrane by adding the bio-orthogonal group-labeled unnatural sugar to the cell, and coupling the cell exosome with a drug by using a bio-orthogonal reaction to achieve a targeted delivery killing effect.

2. The preparation method of a CAR exosome conjugated drug according to claim 1, characterized in that: The bio-orthogonal group is an azido group, and the bio-orthogonal reaction is click chemistry.

3. The preparation method of a CAR exosome conjugated drug according to claim 1, characterized in that: The cell is a CAR cell.

4. The preparation method of a CAR exosome conjugated drug according to claim 2, characterized in that: The CAR cell exosome has an anti-tumor effect.

5. The preparation method of a CAR exosome conjugated drug according to claim 3, characterized in that: The preparation of the CAR exosome comprises the following steps: S1, constructing plasmids containing a CAR molecular structure respectively, and co-transfecting the plasmids with lentivirus packaging plasmids to obtain three corresponding lentiviruses; S2, transfecting Jurkat cells with the three kinds of target proteins to obtain stable cell lines; S3, the stably expressed cells are expanded in a culture medium containing exosomes, and non-sugars are added to obtain CAR exosomes.

6. The preparation method of a CAR exosome conjugated drug according to claim 4, characterized in that: The CAR exosome can improve the anti-tumor function of the exosome after being coupled with a drug by click chemistry.

7. The preparation method of a CAR exosome conjugated drug according to claim 4, characterized in that: The CAR exosome coupled with the drug is obtained by coupling the CAR exosome with a small molecule drug.

8. A CAR exosome coupled drug prepared by the method of any one of claims 1-6.

9. The CAR exosome coupled drug of claim 7 in the treatment of T-cell acute lymphoblastic leukemia.

10. The CAR exosome coupled drug of claim 7 in the treatment of solid tumors.

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