CN112574953A

CN112574953A - Mesothelin chimeric antigen receptor exosome, and preparation method and application thereof

Info

Publication number: CN112574953A
Application number: CN202011460904.4A
Authority: CN
Inventors: 杨鹏翔; 接晶; 杨宇民
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2020-12-11
Filing date: 2020-12-11
Publication date: 2021-03-30

Abstract

The invention discloses an exosome of a targeting mesothelin chimeric antigen receptor immune cell, a preparation method and an application thereof, belonging to the technical field of tumor immunity. After the immune cells are activated by specific mesothelin antigen, the generated exosome is further analyzed, separated, purified and enriched to finally obtain the immune cell exosome carrying the CAR. The exosome can be used for treating various cancers, such as breast cancer, pancreatic cancer, ovarian cancer and the like, has the advantages of overcoming adverse reactions such as immune inflammation storm and the like of CAR cell therapy, enhancing the tissue infiltration capacity of CAR, being convenient to store and transport, and providing a new strategy for treating related diseases.

Description

Mesothelin chimeric antigen receptor exosome, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of tumor immunology, and particularly relates to an exosome derived from a Chimeric Antigen Receptor (CAR) immune cell of targeted mesothelin, a preparation method of the exosome and application of the exosome in preparation of a tumor immunotherapy medicament.

Background

The traditional treatment methods of tumors mainly comprise surgical treatment, chemotherapy and radiotherapy, and immunotherapy is particularly concerned as a novel treatment method. Immunotherapy includes immune checkpoint antibody therapy, monoclonal antibody therapy, tumor vaccines, chimeric antigen receptor cell therapy, and the like. Chimeric antigen receptor-modified immune cells have rapidly moved from preclinical models of tumor immunity to commercial applications. It combines monoclonal antibody technology with cell signal transduction technology, and recognizes tumor surface specific antigen through extracellular single-chain antibody variable region segment, and attacks tumor cells directly by bypassing the limitation of antigen presentation and MHC. The U.S. FDA has now approved Kymrian (CTL019), Yescata therapy for The treatment of acute leukemias and lymphomas that targets The treatment differentiation Antigen 19 (cluster of differentiation 19, CD 19) positive B-cell leukemia and lymphoma with a total remission rate of greater than 80% (June CH, O' Connor RS, Kawalekar OU, Ghassemi S, Millone MC: CAR T cell immunology for human cancer 2018, 359(6382): 1361-.

Mesothelin is a glycoprotein anchored on cell membranes and regulates the growth and metastasis of tumors by activating signal pathways such as NF-kappa B, MAPK and PI 3K. Studies have shown that Mesothelin is abnormally expressed in a variety of tumor tissues, including breast, pancreatic, ovarian, etc. (Morello A, Sadelain M, Adusumil PS: Mesothelin-Targeted CARs: Driving T Cells to Solid tumors 2016, 6(2): 133. sup. 146; Rafiq S, Yeku OO, Jackson HJ, Purdon TJ, van Leuwen DG, Drakes DJ, Song M, Miele MM, Li Z, Wang P et al: Targeted Cancer of a PD-1-blocking by CAR-T Cells enhanced anti-tissue in vivo. biological technology 2018, 36(9): 847. step R, Saattache molecular R, Sapotein et al: sample polypeptide, Schedulcorant et al: heat Cancer, and molecular tissue culture of Schedulcoration T cell culture J, 669 and 697, and is associated with poor prognosis. Therefore, mesothelin is a very promising therapeutic target.

Cells, including immune cells, secrete large numbers of exosomes (exosomes), and it has been found that CAR-T cells can secrete exosomes expressing CAR molecules that are targeted to kill specific target-positive tumor cells directly by encapsulated perforin, granzyme, lysosomal enzyme, etc. (Fu W, Lei C, Liu S, Cui Y, Wang C, Qian K, Li T, Shen Y, Fan X, Lin F et al: CAR exosomes derived from effector CAR-T cells have positive antigen effects and low toxicity. Nature immunity 2019, 10(1): 4355). However, no mesothelin-targeted CAR immune cell-derived exosomes and no reports of their efficacy in treating diseases have been found at present.

Disclosure of Invention

The invention aims to provide a Chimeric Antigen Receptor (CAR) immune cell-derived exosome (hereinafter referred to as a ' Chimeric Antigen Receptor or ' mesothelin CAR exosome ') targeting mesothelin, a preparation method thereof and application thereof in preparing a medicament for treating tumors.

The inventor finds that the CAR-T cell targeting mesothelin is used to activate a specific antigen, and then the secreted exosomes are purified and enriched to prepare specific exosomes carrying CAR proteins. The exosome has very strong anti-tumor effect, and in addition, due to the membrane structure of the exosome, the exosome can be further engineered, such as coated with drugs, antibodies and the like. The exosome can realize the treatment of diseases such as tumors and has important significance for a new tumor treatment method. In addition, the exosome has the advantages of overcoming adverse reactions such as immune inflammation storm and the like in CAR cell therapy, enhancing the tissue soaking capacity of CAR, having the advantages of convenient storage and transportation, and providing a new strategy for the treatment of related diseases.

In a first aspect of the invention, a mesothelin CAR exosome is provided, wherein the exosome comprises a mesothelin-targeting moiety having the amino acid sequence of SEQ ID number 1.

In a second aspect of the present invention, there is provided a method of preparing the mesothelin CAR exosomes described above, wherein the method comprises:

(1) separating and culturing to obtain immune cells;

(2) adopting an activator containing or expressing mesothelin to perform antigen-specific activation on the immune cells and culturing to obtain CAR immune cells;

(3) collecting a precipitate comprising CAR immune cell exosomes from the culture supernatant of the CAR immune cells;

(4) and purifying and enriching the precipitate to obtain the purified mesothelin CAR exosomes.

In a third aspect of the invention, there is provided a pharmaceutical composition or antibody conjugate comprising the mesothelin CAR exosomes described above.

In a fourth aspect of the invention, there is provided an application of the mesothelin CAR exosome, a pharmaceutical composition or an antibody conjugate comprising the mesothelin CAR exosome in preparation of anti-tumor drugs.

Drawings

Figure 1 is an electron microscopy of recombinant mesothelin protein activated CAR-T derived CAR exosome morphology.

Figure 2 is the particle size of recombinant mesothelin protein-activated CAR-T derived CAR exosomes under electron microscopy.

Fig. 3 is the result of ELISA detection of exosome surface CAR binding to antigen.

FIG. 4 is a test result of significant killing of mesothelin-overexpressing MDA-231-MSLN cells by CAR-T derived CAR exosomes.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. See, e.g., Singleton et al, Dictionary of Microbiology and Molecular Biology 2nd ed., J.Wiley & Sons (New York, NY 1994); sambrook et al, Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989).

Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. In fact, the present invention is not limited to the methods and materials described herein, but various conventional modifications and adaptations can be made based on the spirit of the present invention, and the modified or adapted solution still falls within the scope of the present invention.

As used herein, the terms "a" and "an" and the like encompass a plurality of the subject matter unless the context clearly dictates otherwise.

In this document, an object modified by the term "about" encompasses approximations within the error range due to measurement errors and the like.

In one embodiment, a mesothelin CAR exosome is provided, wherein the exosome comprises a mesothelin-targeting moiety having the amino acid sequence of SEQ ID number 1.

1 Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr

21 Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly

41 Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Arg Val Ala Ser Gly Val Pro Gly Arg

61 Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu

81 Asp Asp Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Lys His Pro Leu Thr Phe Gly Cys Gly

101 Thr Lys Leu Glu Ile Lys Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gln Val

121 Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala Ser Val Lys Ile Ser Cys

141 Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly

161 Lys Cys Leu Glu Trp Ile Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Gln Asn

181 Lys Phe Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Asp

201 Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Gly Gly Tyr Asp

221 Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser（SEQ ID NO. 1）。

In some preferred embodiments, the disclosure relates to a mesothelin CAR exosome, wherein the exosome has an amino acid sequence represented by SEQ ID number 2. In the amino acid sequence shown in SEQ ID number 2, amino acids 1-22 are leader peptide Ig kappa leader sequence, amino acids 23-259 are the sequence shown in SEQ ID number 1, and amino acids 260-269 are MYC tag sequence.

1 Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser Val Ile Met Ser

21 Arg Gly Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys

41 Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys

61 Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Arg Val Ala Ser Gly Val Pro

81 Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu

101 Ala Glu Asp Asp Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Lys His Pro Leu Thr Phe Gly

121 Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

141 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala Ser Val Lys Ile

161 Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser

181 His Gly Lys Cys Leu Glu Trp Ile Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr

201 Gln Asn Lys Phe Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

221 Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Gly Gly

241 Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Glu

261 Gln Lys Leu Ile Ser Glu Glu Asp Leu（SEQ ID NO. 2）。

In one embodiment, there is provided a method of making the mesothelin CAR exosomes described above, wherein the method comprises:

(1) separating and culturing to obtain immune cells;

Herein, the immune cells can be isolated using conventional methods known in the art.

In some preferred embodiments, the immune cell may be a T cell, NK cell, or the like. More preferably, the immune cells are derived from blood of healthy volunteers.

In one embodiment of the present invention, the immune cell is a T cell, and the immune cell is prepared according to the following steps:

a) isolating PBMCs (peripheral blood mononuclear cells) from peripheral blood of the subject, isolating and activating T cells in the presence of magnetic beads;

b) the immune cells are obtained by culturing and expanding the T cells in a minimal medium supplemented with interleukins (e.g., IL-2, IL-15, etc.).

Herein, after obtaining a large amount of CAR immune cells, antigen-specific activation of the CAR immune cells is required.

In some preferred embodiments, the antigen-specific activation can be performed by: adding the antigen or the immobilized antigen or the virus particles expressing the antigen into a culture system of the immune cells, co-culturing the immune cells and the inactivated engineering cells expressing the antigen, co-culturing the immune cells and the inactivated tumor cells expressing the antigen, and the like.

In some preferred embodiments, the activator can be a recombinant mesothelin protein, a recombinant mesothelin protein crosslinked by magnetic beads, mesothelin-expressing MDA-MB-231 cells, mesothelin-expressing viral particles, and the like.

In one embodiment of the invention, the activator is a recombinant mesothelin protein. More preferably, the antigen-specific activation is performed by: the nucleic acid sequence encoding the recombinant mesothelin protein is inserted into a lentiviral vector, and then transfected into a host cell (e.g., HEK-293T cells) for amplification, and the resulting viral particle expressing the recombinant mesothelin protein is transfected into T cells.

In a specific embodiment of the invention, the CAR immune cell is a CAR-T cell. The scFv of the exosome secreted by the cell targets mesothelin.

Herein, the precipitate comprising CAR immune cell exosomes may be collected from the culture supernatant of CAR immune cells according to conventional means known in the art.

In a specific embodiment of the invention, the CAR immune cell exosome-containing precipitate is prepared by the following method: centrifuging the culture supernatant at 200-500g for 3-8 min to remove dead cells and large debris to obtain a primary centrifuged supernatant; transferring the supernatant fluid after the primary centrifugation into a new centrifuge tube, and centrifuging for 20-40 minutes at 8000-12000g to remove organelles and small particles to obtain supernatant fluid after secondary centrifugation; transferring the supernatant of the secondary centrifugation to a new centrifuge tube, centrifuging for 50-80 minutes at 120000g of 80000-.

Herein, the specific binding capacity of CAR exosomes to antigen is exploited to purify and enrich CAR exosomes.

In some preferred embodiments, the pellet is resuspended in PBS and then the antigen coated magnetic body (i.e., CAR capture magnet) is added thereto; and performing primary separation by magnetic force to obtain a separated magnetic body, adding a PBS (phosphate buffer solution) into the magnetic body for resuspension, and performing separation by elution of the buffer solution to obtain the purified mesothelin CAR exosomes.

In a specific embodiment of the present invention, the antigen-coated magnetic body is Dynabeads coated with recombinant mesothelin protein. At this time, the magnetic beads are added into the heavy suspension of the exosome precipitate, the test tube is placed in a magnetic field, and the exosomes specifically bound with the magnetic beads are adsorbed by the magnetic field; after the magnetic beads are adsorbed, the supernatant is aspirated, and the test tube is moved out of the magnetic field; adding PBS buffer solution for resuspension, adding into a sorting column, discarding the unbound components flowing out, washing the sorting column with buffer solution, and washing the exosome vesicles without antigen binding capacity. And (4) removing the sorting column from the magnetic field, quickly eluting the exosomes retained on the sorting column by using a buffer solution, and balancing to physiological pH, thereby obtaining the CAR exosomes. Total protein concentration was measured using Bradford kit and stored in aliquots at-80 ℃.

The prepared mesothelin CAR exosome is subjected to biological activity detection, the exosome carries CAR protein, the average diameter of the exosome is about 30-150nm, and the morphology observed under a transmission electron microscope meets the characteristics of the exosome. Further research shows that the mesothelin CAR exosome can well target cells and tissues expressed by target targets, and can inhibit tumor cell proliferation and in-vivo tumor growth.

In one embodiment, a pharmaceutical composition or antibody conjugate comprising the mesothelin CAR exosomes described above is provided.

In some preferred embodiments, the pharmaceutical composition or antibody conjugate may further comprise: cytotoxic drugs such as cisplatin, carboplatin, and platinic oxalate; drugs that affect nucleic acid synthesis, such as methotrexate, fluorouracil, and the like; drugs acting on nucleic acid transcription, such as actinomycin D, daunorubicin, doxorubicin, epirubicin, and the like; drugs mainly acting on tubulin synthesis, such as paclitaxel, vinblastine, vinorelbine, etc.; hormonal antiestrogens such as droloxifene, exemestane, etc.; aromatase inhibitors, agonists/antagonists such as letrozole, renningde, etanerone, and the like; biological response modifier: tumor interferon is mainly inhibited through the immune function of the organism; interleukin-2; thymosin peptide drugs; or monoclonal antibodies such as rituximab, herceptin, Avastin, and the like.

In one embodiment, there is provided a use of the mesothelin CAR exosome, the pharmaceutical composition or the antibody conjugate comprising the same as described above in preparation of an anti-tumor drug.

In preferred embodiments, the tumor includes, but is not limited to, triple negative breast cancer, pancreatic tumor, or ovarian tumor; more preferably, the tumor is a mesothelin-positive tumor.

In one embodiment of the invention, the anti-tumor agent inhibits the cell viability and tumor growth rate of mesothelin-overexpressing MDA-MB-231 and BT-549.

Exemplary aspects of the present invention may be illustrated by the following numbered paragraphs, but the scope of the present invention is not limited thereto:

1. a mesothelin CAR exosome, wherein the exosome comprises a mesothelin-targeting moiety having the amino acid sequence of SEQ ID number 1.

2. The exosome according to paragraph 1, characterized in that it has the amino acid sequence shown in SEQ ID number 2.

3. A method of making the mesothelin CAR exosomes of paragraph 1 or 2, wherein the method comprises:

(1) separating and culturing to obtain immune cells;

4. The method of paragraph 3, wherein the immune cell is a T cell or an NK cell.

5. The method of paragraph 4, wherein the immune cells are derived from blood of healthy volunteers.

6. The method of any one of paragraphs 3-5, wherein the immune cell is a T cell and the immune cell is prepared by:

a) isolating PBMCs from the peripheral blood of the subject, and isolating and activating T cells in the presence of magnetic beads;

b) and culturing and expanding the T cells in a minimal medium supplemented with interleukin to obtain the immune cells.

7. The method of any one of paragraphs 3-6, wherein the antigen-specific activation is performed by: adding the antigen or the immobilized antigen or the virus particles expressing the antigen into a culture system of the immune cells, or co-culturing the immune cells and the inactivated engineering cells expressing the antigen, or co-culturing the immune cells and the inactivated tumor cells expressing the antigen.

8. The method of any one of paragraphs 3-7, wherein the activator is a recombinant mesothelin protein, a recombinant mesothelin protein cross-linked with magnetic beads, MDA-MB-231 cells expressing mesothelin, or viral particles expressing mesothelin.

9. The method of paragraph 8, wherein the activator is a recombinant mesothelin protein.

10. The method of paragraph 9, wherein the antigen-specific activation is performed by: inserting a nucleic acid sequence coding the recombinant mesothelin protein into a lentiviral vector, then transfecting host cells for amplification, and transfecting the T cells with the obtained viral particles expressing the recombinant mesothelin protein.

11. The method of any of paragraphs 3-10, wherein the CAR immune cell is a CAR-T cell.

12. The method of any of paragraphs 3-11, wherein the precipitate comprising CAR immune cell exosomes is prepared by: centrifuging the culture supernatant at 200-500g for 3-8 min to remove dead cells and large debris to obtain a primary centrifuged supernatant; transferring the supernatant fluid after the primary centrifugation into a new centrifuge tube, and centrifuging for 20-40 minutes at 8000-12000g to remove organelles and small particles to obtain supernatant fluid after secondary centrifugation; transferring the supernatant of the secondary centrifugation to a new centrifuge tube, centrifuging for 50-80 minutes at 120000g of 80000-.

13. The method according to any one of paragraphs 3 to 12, wherein the pellet is resuspended in PBS and the antigen-coated magnetic body is then added thereto; and performing primary separation by magnetic force to obtain a separated magnetic body, adding a PBS (phosphate buffer solution) into the magnetic body for resuspension, and performing separation by elution of the buffer solution to obtain the purified mesothelin CAR exosomes.

14. The method according to paragraph 13, wherein the antigen-coated magnetic body is Dynabeads coated with a recombinant mesothelin protein.

15. A pharmaceutical composition or antibody conjugate comprising the mesothelin CAR exosomes of paragraph 1 or 2.

16. The pharmaceutical composition or antibody conjugate of paragraph 15, further comprising: cytotoxic drugs; agents that affect nucleic acid synthesis; an agent that acts on nucleic acid transcription; drugs that act primarily on tubulin synthesis; hormonal antiestrogens; aromatase inhibitors, agonists/antagonists; a biological response modifier; interleukin-2; thymosin peptide drugs; or a monoclonal antibody.

17. The pharmaceutical composition or antibody conjugate of paragraph 16, wherein the cytotoxic drug is selected from the group consisting of cisplatin, carboplatin, and platinic oxalate; the drug affecting nucleic acid synthesis is selected from methotrexate and fluorouracil drugs; the drug acting on nucleic acid transcription is selected from actinomycin D, daunorubicin, adriamycin and epirubicin; the drug mainly acting on the synthesis of tubulin is selected from paclitaxel, vinblastine and vinorelbine; the hormone antiestrogen is selected from droloxifene and exemestane; the aromatase inhibitor, agonist/antagonist is selected from letrozole, renningde, etanerone; the biological response regulator is selected from tumor interferon which is mainly inhibited through the immune function of the organism; and the monoclonal antibody is selected from the group consisting of rituximab, herceptin, and Avastin.

18. Use of the mesothelin CAR exosome of paragraph 1 or 2, a pharmaceutical composition or antibody conjugate comprising the same in the preparation of an anti-tumor medicament.

19. The use of paragraph 18, wherein the tumor comprises a triple negative breast cancer, a pancreatic tumor, or an ovarian tumor.

20. The use of paragraph 18 or 19, wherein the tumour is mesothelin-positive tumour.

21. The use of any one of paragraphs 18-20, wherein the anti-neoplastic agent inhibits the cell viability and tumor growth rate of mesothelin-overexpressing MDA-MB-231 and BT-549.

The following examples are for illustrative purposes only and are not intended to limit the scope of the present application. Unless otherwise indicated, all reagents, materials and equipment used in the following examples are commercially available or can be formulated or obtained according to the prior art well known in the art. Unless otherwise stated, specific experimental means mentioned in the following examples are conventional in the art (for example, molecular cloning experimental guidelines (4 th edition), written by J. SammBruk et al, Proc. congress, scientific Press, 2017; medical immunology (7 th edition), edited by Cao Xuan Tao, public health Press, 2018).

Example 1

Preparing mesothelin-targeted MSLN-CAR-T cell-derived exosomes, comprising the steps of:

step 1, T cell isolation culture: PBMCs were isolated from human peripheral blood collected and purified using CD3/CD28Dynabeads (Miltenyi Biotec) at a rate of 3: 1, and cultured in 1640 medium, to which IL-2 (20 IU/mL) and IL-15 (290U/mL) were added every other day for passage every 3 to 4 days.

Step 2, preparing CAR-T cells: the nucleic acid sequence encoding the CAR sequence comprising the MSLN single chain antibody (entrusted sumau biosciences limited; i.e., "mesothelin recombinant protein") was synthesized genetically. The structure of the CAR sequence comprises: anti-MSLN single chain antibody scFv, the CD8 hinge domain, the Transmembrane (TM) region of the 4-1BB molecule, and the intracellular segment of the CD3 signaling molecule. For ease of detection, a Myc tag was inserted between the scFv and the hinge region of the single chain antibody. The above nucleic acid sequence is inserted into a lentiviral vector. Vectors were transfected into HEK-293T cells using a lentiviral expression system. The supernatant was collected, filtered to remove cell debris, and then centrifuged at 12000rpm for 1min to collect viral particle pellets. Transfecting the activated T cells obtained in the step 1 by using the viral particle sediment, culturing for 48 hours to obtain MSLN-targeted CAR-T cells, culturing the CAR-T cells in a culture medium containing puromycin at a proper concentration for 7 days, and screening to obtain the MSLN-CAR-T cells successfully transfected by the virus.

Step 3, preparation of an exosome precipitate: exosomes were harvested from the culture supernatant by centrifugation. First, centrifuge at 300 Xg for 5 minutes, and then at 10,000 Xg for 30 minutes to remove cells and debris. The supernatant was then centrifuged at 100,000 Xg for 60 minutes to pellet the exosomes. Finally, PBS was added to the pellet, washed and centrifuged at 100,000 × g for 1 hour to obtain an exosome pellet. All centrifugation should be performed at 4 ℃.

Step 4, purifying exosomes: magnetic beads Dynabeads coated by mesothelin recombinant protein are adopted. Adding the magnetic beads into the PBS solution of the exosome precipitate, placing the test tube in a magnetic field, and adsorbing the exosomes specifically bound with the magnetic beads by the magnetic field; after the magnetic beads are adsorbed, the supernatant is aspirated, and the test tube is moved out of the magnetic field; adding PBS buffer solution for resuspension, adding into a sorting column, discarding the unbound components flowing out, washing the sorting column with buffer solution, and washing the exosome vesicles without antigen binding capacity. The sorting column was removed from the magnetic field and the exosomes retained on the sorting column were rapidly eluted with running buffer and equilibrated to physiological pH, at which time purified mesothelin CAR exosomes were obtained.

The prepared exosomes were subjected to morphological characterization and performance measurement as follows.

1. Exosome identification

First, exosome structures were detected by transmission electron microscopy. And (3) taking exosomes, diluting with PBS in an equal time, dripping the exosomes on a copper net, standing for 1 minute, and wiping redundant liquid. And (3) carrying out negative dyeing on 3% sodium phosphotungstate for 5 minutes, airing at room temperature, and observing the morphological structure of the sodium phosphotungstate under a transmission electron microscope. As shown in FIG. 1 and FIG. 2, the prepared exosomes are uniform vesicles with diameters of 30-150 nm.

2. Expression of exosome surface CAR

For ease of detection, a Myc tag is inserted between the scFv and the hinge region, so Myc expression can be detected by flow. Since the exosomes were too small to be detected by direct flow, exosomes were mixed with 4 μm magnetic beads (Interfacial Dynamics Corporation, Portland, OR, USA) and incubated overnight. And incubating with MYC antibody for 40 minutes the next day, washing twice, and detecting on a machine. CAR expression was detected to be over 90%.

3. Detection of exosome surface CAR binding capacity: to demonstrate that exosomes can efficiently bind MSLN antigen, an antigen-based double antibody sandwich ELISA was used.

The method comprises the following steps: recombinant MSLN was coated in 96-well plates overnight at 4 ℃. Blocking buffer was added to the plate for 2 hours at room temperature. The purified exosomes prepared in the above examples were serially diluted and added to each well overnight at 4 ℃. anti-Myc antibody was added to each well and incubated for 1 hour at room temperature. A substrate solution to be reacted with HRP was added to each well. Addition of 0.5N H₂SO₄The reaction was terminated and the absorbance was measured at 450 nm. As shown in figure 3, the OD values were significantly higher than the control with increasing CAR exosome concentration, suggesting that CAR-T cell exosomes can efficiently bind MSLN antigen.

4. CAR-T exosomes kill MSLN-positive tumor cells.

The stably overexpressed MDA231-MSLN cell line was used as target cell. Will be 1 × 10⁴Individual cells/well target cells were plated in U-bottom 96-well plates. Expressing 100. mu.L of the above-mentioned plasmidEXAMPLES preparation of Mesothelin CAR exosomes effector CAR-T cells were added to reaction wells at different concentrations (exosomes amounts 50, 100 and 300 μ g) and at 5% CO₂Incubate at 37 ℃ for 4 hours. 50 μ L of supernatant was collected from each well and transferred to a new 96 well plate. The Promega LDH lactate dehydrogenase kit was used, the substrate mixture was added and incubated at room temperature for 30 minutes. Finally, a stop solution was added, and absorbance at 490 nm was measured using a microplate reader. Three duplicate wells were designed for each experimental group, and medium was added to the other 3 wells as controls.

The kill calculation is as follows: specific killing rate (%) = (effector cell and target cell mixing well A value-effector cell natural release A value-target cell natural release A value)/(target cell maximum release A value-target cell natural release A value) × 100%

As shown in FIG. 4, with exosomes secreted by untransfected T-cells as controls, exosomes of MSLN-targeted CAR-T cells killed significantly more efficiently than control exosomes against the MDA231-MSLN cell line at 50, 100 and 300 μ g doses: (P<0.05). However, no significant difference was observed for the MDA231 cell line, which had lower MSLN expression than the MDA231-MSLN cell line, suggesting that exosomes have targeted killing characteristics.

Sequence listing

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His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

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Asp Thr Ser Arg Val Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser

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145 150 155 160

Lys Cys Leu Glu Trp Ile Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser

165 170 175

Ser Tyr Gln Asn Lys Phe Arg Gly Lys Ala Thr Leu Thr Val Asp Lys

180 185 190

Ser Ser Ser Thr Ala Tyr Met Asp Leu Leu Ser Leu Thr Ser Glu Asp

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Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

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Claims

1. A mesothelin CAR exosome, wherein the exosome comprises a mesothelin-targeting moiety having the amino acid sequence of SEQ ID number 1;

preferably, the exosome has an amino acid sequence shown as SEQ ID number 2.

2. A method of making the mesothelin CAR exosome of claim 1, wherein the method comprises:

(1) separating and culturing to obtain immune cells;

3. The method of claim 2, wherein the immune cell is a T cell or NK cell;

preferably, the immune cells are derived from blood of healthy volunteers;

preferably, the immune cell is a T cell, and the immune cell is prepared according to the following steps:

4. The method according to claim 2 or 3, characterized in that the antigen-specific activation is carried out by: adding the antigen or the immobilized antigen or virus particles expressing the antigen into a culture system of the immune cells, or co-culturing the immune cells and inactivated engineering cells expressing the antigen, or co-culturing the immune cells and inactivated tumor cells expressing the antigen;

preferably, the activator is mesothelin recombinant protein, mesothelin recombinant protein crosslinked by magnetic beads, MDA-MB-231 cells expressing mesothelin, and virus particles expressing mesothelin; more preferably, the activator is a recombinant mesothelin protein;

preferably, the antigen-specific activation is performed by: inserting a nucleic acid sequence encoding the recombinant mesothelin protein into a lentiviral vector, then transfecting host cells for amplification, and transfecting the obtained viral particles expressing the recombinant mesothelin protein into T cells;

preferably, the CAR immune cell is a CAR-T cell.

5. The method according to any one of claims 2-4, wherein the precipitate comprising CAR immune cell exosomes is prepared by: centrifuging the culture supernatant at 200-500g for 3-8 min to remove dead cells and large debris to obtain a primary centrifuged supernatant; transferring the supernatant fluid after the primary centrifugation into a new centrifuge tube, and centrifuging for 20-40 minutes at 8000-12000g to remove organelles and small particles to obtain supernatant fluid after secondary centrifugation; transferring the supernatant obtained by the secondary centrifugation into a new centrifuge tube, centrifuging for 50-80 minutes at 120000g of 80000-;

preferably, the pellet is resuspended with PBS, and then the antigen-coated magnetic body is added thereto; performing primary separation by magnetic force to obtain a separated magnetic body, adding a PBS buffer solution for resuspension, and performing sorting by buffer solution elution to obtain a purified mesothelin CAR exosome;

preferably, the antigen-coated magnetic body is Dynabeads coated with mesothelin recombinant protein.

6. A pharmaceutical composition or antibody conjugate comprising the mesothelin CAR exosome of claim 1.

7. The pharmaceutical composition or antibody conjugate of claim 6, further comprising: cytotoxic drugs; agents that affect nucleic acid synthesis; an agent that acts on nucleic acid transcription; drugs that act primarily on tubulin synthesis; hormonal antiestrogens; aromatase inhibitors, agonists/antagonists; a biological response modifier; interleukin-2; thymosin peptide drugs; or a monoclonal antibody;

preferably, the cytotoxic drug is selected from cisplatin, carboplatin, and platinic oxalate; the drug affecting nucleic acid synthesis is selected from methotrexate and fluorouracil drugs; the drug acting on nucleic acid transcription is selected from actinomycin D, daunorubicin, adriamycin and epirubicin; the drug mainly acting on the synthesis of tubulin is selected from paclitaxel, vinblastine and vinorelbine; the hormone antiestrogen is selected from droloxifene and exemestane; the aromatase inhibitor, agonist/antagonist is selected from letrozole, renningde, etanerone; the biological response regulator is selected from tumor interferon which is mainly inhibited through the immune function of the organism; and the monoclonal antibody is selected from the group consisting of rituximab, herceptin, and Avastin.

8. Use of the mesothelin CAR exosome of claim 1, a pharmaceutical composition or antibody conjugate comprising the same in the preparation of an anti-tumor drug.

9. The use of claim 8, wherein the tumor comprises a triple negative breast cancer, pancreatic tumor, or ovarian tumor;

preferably, the tumor is a mesothelin-positive tumor.

10. The use of claim 8 or 9, wherein the anti-tumor agent inhibits the cell viability and tumor growth rate of mesothelin-overexpressing MDA-MB-231 and BT-549.