Method for rapidly generating CAR-T cells from mesenchymal stem cell-derived engineered exosome mediated mRNA

Abstract

The invention relates to a method for rapidly generating CAR-T (Chimeric Antigen Receptor-T) cells by mediating mRNA (Messenger Ribonucleic Acid) through mesenchymal stem cell-derived engineering exosome. The method for preparing the CAR-T cells comprises the following steps: (1) constructing a vector plasmid; (2) culturing and transfecting the mesenchymal stem cells; (3) separating and purifying the engineered exosome; (4) preparing initial T cells; (5) preparing CAR-T cells: co-culturing the engineered exosome and the initial T cells, centrifuging the cultured cell suspension, and culturing the precipitate by using a T cell culture medium added with IL-2 and human AB serum; and centrifuging the cell suspension, re-suspending the cell precipitate with PBS, and adjusting the density to obtain the suspension of the CAR-T cells. The method disclosed by the invention has excellent effects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a method for preparing CAR-T cells, and in particular to a method for rapidly generating CAR-T cells by using engineered exosomes derived from mesenchymal stem cells to mediate mRNA. Background Art

[0002] In recent years, as one of the leading causes of death worldwide, the incidence and mortality of cancer have shown an increasing trend year by year. According to data from the Global Cancer Observatory, there will be approximately 19.3 million new cancer cases and approximately 10 million deaths worldwide in 2020. With the aging of the population, environmental pollution and changes in lifestyle, the burden of cancer is increasing, becoming a major challenge in the field of global public health. Traditional cancer treatments mainly include surgery, radiotherapy and chemotherapy. These methods have certain efficacy in the treatment of early cancer, but due to their non-specificity and damage to normal cells, patients often face serious side effects and low quality of life. With the development of molecular biology and immunology, new cancer treatment methods continue to emerge, bringing new hope to cancer patients. In recent years, cell immunotherapy has become a research hotspot and frontier direction in the field of cancer treatment.

[0003] Chimeric Antigen Receptors (CARs) are synthetic proteins expressed on the surface of T cells, consisting of two parts, extracellular and intracellular. Such cells are usually called CAR-T cells. These receptors have the ability to recognize and bind to specific proteins on the surface of malignant cells, thereby triggering the activation and cytotoxicity of T cells to eliminate targeted malignant cells.

[0004] Chimeric antigen receptors are composed of an antigen recognition domain composed of a monoclonal antibody fragment and an intracellular signaling domain necessary to activate the effector function of CAR-T cells. The antigen recognition domain recognizes a specific protein on the surface of malignant cells, such as CD19 on B cells. The intracellular domain ensures the intracellular signaling necessary to activate the effector function of CAR-T cells.

[0005] CAR-T cells, also known as chimeric antigen receptor T cells, are an advanced personalized cellular immunotherapy, also commonly referred to as CAR-T immunotherapy. It is a cellular immunotherapy program based on chimeric antigen receptors. Generally, the gene sequence encoding the chimeric antigen receptor CAR-T is introduced into T cells through in vitro gene transduction technology to generate tumor-specific T cells that can bind to target antigens. The most common CARs include an antigen recognition region, such as the single-chain variable region (scFv) of a monoclonal antibody (mAb), and the TCRζ chain that activates the intracellular signaling domain of T cells.

[0006] There are certain differences between the first, second, and third generation CAR-T cells. CAR-T cells are divided into the first, second, and third generations according to their development stage. The first generation of CAR-T cells uses the intracellular domain in the CD3 ζ chain of the T cell receptor (TCR) to induce cytotoxicity against targeted malignant cells. However, the first generation of CAR-T cells cannot support the expansion of CAR-T cells in vivo after reinfusion. In contrast, the second and third generation CAR-T cells have additional co-stimulatory intracellular domains, such as CD28, 41BB, OX40, etc., which can enhance the ability of CAR-T cells to proliferate, expand, and persist in vivo. The third generation of CAR-T cells further enhances the ability of in vivo expansion and persistence based on the second generation.

[0007] CAR-T cells can proliferate in vivo and show a certain degree of persistence. Genetically modified CAR-T cells show strong proliferation ability and persistence in vivo. After lymphocyte clearance (usually using fludarabine and cyclophosphamide), CAR-T cells establish a good cytokine environment in the patient's body, further enhancing the proliferation ability of CAR-T cells. Once CAR-T cells are injected into the body, the number of cells can continue to increase and bind to cancer cells through engineered receptors, leading to the death of immune cancer cells. It has been reported that the persistence of CAR-T cells can be up to 3 years.

[0008] Lymphocyte clearance and engineered receptor-cancer cell binding have specific biological effects. By clearing normal lymphocytes, space is provided for the expansion of CAR-T cells; at the same time, lymphocyte clearance also clears potential immunosuppressive cells, thereby enhancing the anti-tumor effect of CAR-T cells. The binding of engineered receptors to cancer cells is the mechanism by which CAR-T cells recognize and attack cancer cells. When CAR-T cells recognize antigens on the surface of cancer cells, it triggers the activation of CAR-T cells, which in turn triggers an attack on cancer cells.

[0009] CAR-T cells have shown great potential in clinical treatment. Many CAR-T cell treatment programs have achieved remarkable results in clinical trials for different types of cancer. For example, CAR-T cell therapy for acute lymphoblastic leukemia and lymphoma has achieved a high success rate.

[0010] Mesenchymal Stem Cells (commonly abbreviated as MSCs or MSC) are a type of multipotent stem cells with multidirectional differentiation potential, which can differentiate into a variety of cell types, such as bone cells, adipocytes and chondrocytes. MSCs not only show great potential in tissue repair and regenerative medicine, but also play an important role in intercellular communication. MSCs transmit intercellular signals and exchange substances by secreting exosomes. These exosomes have a diameter of 30-150 nanometers and contain bioactive molecules such as proteins, lipids, mRNA, and miRNA.

[0011] The preparation of CAR-T cells by delivering mRNA through engineered exosomes is an emerging gene therapy technology. Its basic principle is to use exosomes derived from mesenchymal stem cells (MSCs) as carriers to deliver chimeric antigen receptor (CAR) mRNA into T cells, thereby achieving rapid preparation of CAR-T cells. In CAR-T cell therapy, the use of MSC-derived exosomes for mRNA delivery can effectively overcome the limitations of traditional viral vectors and non-viral vectors, providing a safe, efficient and rapid method for preparing CAR-T cells. This not only improves the generation efficiency of CAR-T cells, but also greatly shortens the treatment cycle, and improves the treatment effect and safety. As an emerging gene delivery vector, MSC-derived exosomes are increasingly gaining attention in modern biomedical research and show important application potential.

[0012] CAR-T cells targeting CD19 have shown amazing effects in preclinical and clinical settings. Existing studies have shown that IL-7 (interleukin-17) can prolong the survival of immune cells and stimulate the proliferation of mature T cells, so IL-7 can enhance the survival time of immune cells in the body and their anti-tumor effects. CCL-17, also known as thymic and activating regulatory chemokine (TARC), is a powerful chemokine that is usually associated with type 2 immune responses. Its encoding gene is located on chromosome 16 in humans. CCL-17 can increase the ability of endogenous immune cells to aggregate to tumor cells, further enhancing the anti-tumor effects of immune cells.

[0013] However, the prior art still expects a method for obtaining CAR-T cells, especially a method for preparing CAR-T cells by mediating mRNA through engineered exosomes derived from mesenchymal stem cells. Summary of the invention

[0014] The object of the present invention is to provide a method for obtaining CAR-T cells, in particular to provide a method for preparing CAR-T cells by mediating mRNA through engineered exosomes derived from mesenchymal stem cells. It has been unexpectedly found that the method provided by the present invention can be effectively used to prepare CAR-T cells and can obtain one or more beneficial effects, and the present invention is completed based on such findings.

[0015] To this end, the first aspect of the present invention provides a method for preparing CAR-T cells, comprising the following steps: (1) Construction of vector plasmid: The vector plasmid containing Anti-CD3-Lamp2b-MS2 element was constructed using pcDNA3.1 plasmid. Anti-CD28-Lamp2b-MS2 vector plasmid was constructed using pcDNA3.1 plasmid. The CAR-19mRNA-MS2bs vector plasmid was constructed using the pLVX-EF1α-IRES-puro plasmid; (2) Culture and transfection of mesenchymal stem cells: Primary mesenchymal stem cells are obtained by culturing Wharton's jelly stripped from the umbilical cord, and then mesenchymal stem cells of P2 to P8 are obtained by subculturing; three plasmids are transfected into mesenchymal stem cells using a plasmid transfection kit (e.g., Lipofectamine™ 3000 Reagent kit); (3) Isolation and purification of engineered exosomes: The mesenchymal stem cells transfected in the previous step were replaced with DMEM-high glucose medium without FBS, and the cells were cultured in a 37°C, 5% CO2 incubator with 2% O2 concentration (48 hours); the supernatant obtained from the cell culture was separated and purified by differential centrifugation to obtain engineered exosomes; (4) Preparation of initial T cells: The lower blood cell layer obtained by centrifugation of healthy adult peripheral blood is separated and treated with Ficoll-Hypaque lymphocyte separation solution to obtain a peripheral blood mononuclear cell (PBMC) layer; the peripheral blood mononuclear cells are then treated with magnetic beads (such as Dynabeads®) and activated in a T cell serum-free medium supplemented with IL-2 500 IU / ml and 1% autologous plasma to obtain initial T cells; (5) Preparation of CAR-T cells: The engineered exosomes were co-cultured with naive T cells, the cultured cell suspension was centrifuged, and the precipitate was cultured with T cell culture medium supplemented with IL-2 and human AB serum; The cell suspension was centrifuged, and the cell pellet was resuspended in PBS and the density was adjusted to obtain a CAR-T cell suspension.

[0016] According to the method of the first aspect of the present invention, in step (1), when the pcDNA3.1 plasmid is used to construct a vector plasmid containing the Anti-CD3-Lamp2b-MS2 element, the plasmid and gene fragments used are: pcDNA3.1 plasmid, Anti-CD3-scFv gene sequence, Lamp2b gene sequence, and MS2 Coat Protein gene sequence.

[0017] According to the method of the first aspect of the present invention, in step (1), when the vector plasmid containing the Anti-CD3-Lamp2b-MS2 element is constructed using the pcDNA3.1 plasmid, the following operations are performed: ① Design primers: designed to amplify the Anti-CD3-scFv-Lamp2b-MS2 gene sequence, ② Amplify the gene sequence: Use the PCR method to amplify the Anti-CD3-scFv-Lamp2b-MS2 sequence from the template DNA. ③ Purify PCR products: Separate PCR products by agarose gel electrophoresis, cut out the corresponding DNA bands and purify the PCR products using a gel recovery kit. ④ Double enzyme digestion: Use EcoRI and BamHI to digest the pcDNA3.1 plasmid, and digest the Anti-CD3-scFv-Lamp2b-MS2 gene fragment. ⑤ Ligation reaction: Use T4 DNA ligase to ligate the digested Anti-CD3-scFv-Lamp2b-MS2 gene fragment into the digested pcDNA3.1 plasmid. ⑥ Transformation of competent cells: The ligation reaction product was transformed into competent Stbl3 cells by heat shock method. The transformed cells were spread on LB agar plates containing antibiotics and cultured at 37°C overnight. ⑦ Screening of positive clones: Select a single colony, extract plasmid DNA using a small amount of plasmid extraction kit, and use restriction enzyme digestion and PCR methods to verify whether the plasmid contains the expected insert fragment. ⑧Sequence verification: Send samples for DNA sequencing to verify the correctness and integrity of the Anti-CD3-scFv-Lamp2b-MS2 gene sequence, to ensure that the reading frame of the inserted fragment is correct and there is no mutation. ⑨Plasmid extraction: Use a large number of plasmid extraction kits to extract plasmid DNA from positive clones, store it at low temperature and set it aside for later use.

[0018] According to the method of the first aspect of the present invention, in step (1), the operation of using pcDNA3.1 plasmid to construct a vector plasmid containing Anti-CD3-Lamp2b-MS2 elements is considered, Anti-CD3 is replaced with Anti-CD28, and the Anti-CD28-Lamp2b-MS2 vector plasmid is constructed using pcDNA3.1 plasmid.

[0019] According to the method of the first aspect of the present invention, in step (1), the sequence of CAR-19mRNA-MS2bs is composed of CD19 single-chain variable region, CD8a hinge region, CD8a transmembrane region, 4-1BB signal, CD3ζ clathrate signal, IL-7, CCL17, and MS2bs sequence.

[0020] According to the method of the first aspect of the present invention, in step (1), a suitable restriction endonuclease (for example, a restriction endonuclease with Not I (GCGGCCGC) upstream and BamH I (GGATCC) downstream) and a ligase (for example, T4 DNA ligase) are selected, and the CAR-19mRNA-MS2bs vector plasmid is constructed using the pLVX-EF1α-IRES-puro plasmid.

[0021] According to the method of the first aspect of the present invention, in step (2), primary (P0) mesenchymal stem cells are obtained from the umbilical cord Wharton's jelly using a known method, and then subcultured to obtain mesenchymal stem cells of cell generations P3 to P7, such as P3 to P6, such as P4 to P6.

[0022] According to the method of the first aspect of the present invention, in step (2), when the three plasmids are transfected into mesenchymal stem cells using a plasmid transfection kit (e.g., Lipofectamine™ 3000 Reagent kit), the DNA-Lipo3000 complex solution obtained by treatment with the kit is added dropwise to the cell culture well plate, the culture plate is gently shaken to evenly distribute the transfection complex, incubated for 4-6 hours, and then the serum-free Opti-MEM medium is replaced with DMEM high-glucose medium (containing 10% FBS), and the cells are continued to be cultured for 48-72 hours to allow plasmid expression.

[0023] According to the method of the first aspect of the present invention, in step (2), the DMEM high-glucose medium (containing 10% FBS) is a modified DMEM high-glucose medium (containing 10% FBS).

[0024] According to the method of the first aspect of the present invention, in step (2), the plasmid transfection of mesenchymal stem cells is performed using the Lipofectamine™ 3000 Reagent kit in the following manner: (a) Mesenchymal stem cells were seeded into 6-well plates and 2×10 5 Cells were cultured in a 37°C, 5% CO2 incubator until the cell density reached 50-70% confluence; (b) Dilute the three plasmid DNAs in serum-free Opti-MEM medium, and the amount of plasmid required for each well is 2.5 µg; in another centrifuge tube containing serum-free Opti-MEM medium, add Lipofectamine 3000 reagent at a ratio of 5 µL per centrifuge tube; Add 10 µL of P3000 Reagent to each plasmid DNA solution; Add Lipofectamine 3000 reagent solution dropwise to the plasmid DNA solution, mix gently, and incubate at room temperature for 15-20 minutes to form DNA-Lipo3000 complex; (c) replacing the cell culture medium in the 6-well plate with serum-free Opti-MEM medium; Add the DNA-Lipo3000 complex solution in each tube dropwise into the 6-well plate for cell culture, and gently shake the culture plate to evenly distribute the transfection complex; (d) The cells were incubated in a 37°C, 5% CO2 incubator for 4-6 hours, and then the serum-free Opti-MEM medium was replaced with modified DMEM high-glucose medium (containing 10% FBS) and the cells were cultured for another 48-72 hours to allow plasmid expression.

[0025] According to the method of the first aspect of the present invention, in step (3), the culture medium of the mesenchymal stem cells transfected in the previous step is replaced with a modified DMEM-high glucose medium (without FBS), and the cells are placed in a 37°C, 5% CO2 incubator containing 2% O2 concentration and cultured (e.g., 48 hours).

[0026] According to the method of the first aspect of the present invention, in step (3), the cell supernatant obtained by culturing mesenchymal stem cells in a modified DMEM-high glucose medium (without FBS) is placed in a centrifuge tube and subjected to the following differential centrifugation to obtain engineered exosomes: Centrifuge at 300g and 4°C for 10 min, and transfer the supernatant to another centrifuge tube; Centrifuge at 2000g and 4°C for 20 min, and transfer the supernatant to another centrifuge tube; Centrifuge at 10,000 g and 4 °C for 30 min, filter the supernatant with a 0.22 μm filter membrane and place in another centrifuge tube; Centrifuge at 100,000 g and 4 °C for 90 min and discard the supernatant; Sterile PBS was added to the centrifuge tube to resuspend the exosome precipitate, and the mixture was centrifuged at 100,000 g and 4° C. for 90 min. The supernatant was discarded, and sterile PBS was added to resuspend the exosomes to obtain an exosome suspension.

[0027] According to the method of the first aspect of the present invention, in step (2) and / or step (3), The formula of the DMEM high-glucose medium (containing 10% FBS) is: 200 mg / L anhydrous CaCl2, 0.1 mg / L nonahydrate Fe(NO3)3, 97.67 mg / L anhydrous MgSO4, 400 mg / L KCl, 3700 mg / L NaHCO3, 6400 mg / L NaCl, 109 mg / L anhydrous NaH2PO4, 434 mg / L L-alanyl-L-glutamine, 30 mg / L glycine, 84 mg / L L-arginine hydrochloride, 62.6 mg / L L-cystine dihydrochloride, 584 mg / L L-glutamine, 42 mg / L L-histidine hydrochloride monohydrate, 105 mg / L L-isoleucine, 105 mg / L L-leucine, 146 mg / L L-lysine hydrochloride, 30 mg / L / L of L-methionine, 66mg / L of L-phenylalanine, 42mg / L of L-serine, 95mg / L of L-threonine, 16mg / L of L-tryptophan, 103.79mg / L of L-tyrosine disodium salt dihydrate, 94mg / L of L-valine, 4mg / L of choline chloride, 4mg / L of D-calcium pantothenate, 4mg / L of folic acid, 4mg / L of niacinamide, 4mg / L of pyridoxine hydrochloride, 0.4mg / L of riboflavin, 4mg / L of thiamine hydrochloride, 7.2mg / L of meso-inositol, 4500mg / L of D-glucose, 110mg / L of sodium pyruvate, 15mg / L of phenol red, 100kU / L of penicillin G sodium salt, 100mg / L of streptomycin sulfate, 10% fetal bovine serum, and add appropriate amount of water to the full amount.

[0028] According to the method of the first aspect of the present invention, in step (2) and / or step (3), the formula of the DMEM high-glucose medium (without FBS) is: 200 mg / L anhydrous CaCl2, 0.1 mg / L nonahydrate Fe(NO3)3, 97.67 mg / L anhydrous MgSO4, 400 mg / L KCl, 3700 mg / L NaHCO3, 6400 mg / L NaCl, 109 mg / L anhydrous NaH2PO4, 434 mg / L L-alanyl-L-glutamine, 30 mg / L glycine, 84 mg / L L-arginine hydrochloride, 62.6 mg / L L-cystine dihydrochloride, 584 mg / L L-glutamine, 42 mg / L L-histidine hydrochloride monohydrate, 105 mg / L L-isoleucine, 105 mg / L L- Leucine, 146mg / L L-lysine hydrochloride, 30mg / L L-methionine, 66mg / L L-phenylalanine, 42mg / L L-serine, 95mg / L L-threonine, 16mg / L L-tryptophan, 103.79mg / L L-tyrosine disodium salt dihydrate, 94mg / L L-valine, 4mg / L choline chloride, 4mg / L D-calcium pantothenate , 4 mg / L folic acid, 4 mg / L niacinamide, 4 mg / L pyridoxine hydrochloride, 0.4 mg / L riboflavin, 4 mg / L thiamine hydrochloride, 7.2 mg / L meso-inositol, 4500 mg / L D-glucose, 110 mg / L sodium pyruvate, 15 mg / L phenol red, 100 kU / L penicillin G sodium salt, 100 mg / L streptomycin sulfate, add appropriate amount of water to the full amount.

[0029] According to the method of the first aspect of the present invention, in step (2) and / or step (3), the modified DMEM high-glucose medium (containing 10% The formula of FBS is: 200 mg / L anhydrous CaCl2, 0.1 mg / L nonahydrate Fe(NO3)3, 97.67 mg / L anhydrous MgSO4, 400 mg / L KCl, 3700 mg / L NaHCO3, 6400 mg / L NaCl, 109 mg / L anhydrous NaH2PO4, 124 mg / L sodium arachidonic acid, 56 mg / L zinc sulfate, 434 mg / L L-alanyl-L-glutamine, 30 mg / L glycine, 84 mg / L L-arginine hydrochloride, 62.6 mg / L L-cystine dihydrochloride, 584 mg / L L-glutamine, 42 mg / L L-histidine hydrochloride monohydrate, 105 mg / L L-isoleucine, 105 mg / L L-leucine, 146 mg / L L-lysine hydrochloride, 30mg / L L-methionine, 66mg / L L-phenylalanine, 42mg / L L-serine, 95mg / L L-threonine, 16mg / L L-tryptophan, 103.79mg / L L-tyrosine disodium salt dihydrate, 94mg / L L-valine, 4mg / L choline chloride, 4mg / L D-calcium pantothenate, 4mg / L folic acid, 4 mg / L niacinamide, 4 mg / L pyridoxine hydrochloride, 0.4 mg / L riboflavin, 4 mg / L thiamine hydrochloride, 7.2 mg / L meso-inositol, 4500 mg / L D-glucose, 110 mg / L sodium pyruvate, 15 mg / L phenol red, 100 kU / L penicillin G sodium salt, 100 mg / L streptomycin sulfate, 10% fetal bovine serum, and add appropriate amount of water to the full amount.

[0030] According to the method of the first aspect of the present invention, in step (2) and / or step (3), the formula of the modified DMEM high-glucose medium (without FBS) is: 200 mg / L anhydrous CaCl2, 0.1 mg / L nonahydrate Fe(NO3)3, 97.67 mg / L anhydrous MgSO4, 400 mg / L KCl, 3700 mg / L NaHCO3, 6400 mg / L NaCl, 109 m g / L of anhydrous NaH2PO4, 124mg / L of sodium arachidonic acid, 56mg / L of zinc sulfate, 434mg / L of L-alanyl-L-glutamine, 30mg / L of glycine, 84mg / L of L-arginine hydrochloride, 62.6mg / L of L-cystine dihydrochloride, 584mg / L of L-glutamine, 42mg / L of L-histidine hydrochloride monohydrate, 105mg / L of L-isothiazolinone. Leucine, 105mg / L L-leucine, 146mg / L L-lysine hydrochloride, 30mg / L L-methionine, 66mg / L L-phenylalanine, 42mg / L L-serine, 95mg / L L-threonine, 16mg / L L-tryptophan, 103.79mg / L L-tyrosine disodium salt dihydrate, 94mg / L L-valine, 4mg / L choline chloride, 4mg / L L of D-calcium pantothenate, 4 mg / L of folic acid, 4 mg / L of niacinamide, 4 mg / L of pyridoxine hydrochloride, 0.4 mg / L of riboflavin, 4 mg / L of thiamine hydrochloride, 7.2 mg / L of meso-inositol, 4500 mg / L of D-glucose, 110 mg / L of sodium pyruvate, 15 mg / L of phenol red, 100 kU / L of penicillin G sodium salt, 100 mg / L of streptomycin sulfate, and add appropriate amount of water to the full amount.

[0031] According to the method of the first aspect of the present invention, in step (4), initial T cells are obtained by the following operation: 100 ml of healthy adult peripheral blood containing sodium citrate anticoagulant was placed in a 250 ml centrifuge cup and centrifuged at 1500 rpm and 20°C for 15 min to collect the upper plasma; Add the lower blood cell layer to an equal volume of 0.9% sodium chloride injection and mix thoroughly. Add 20 ml of Ficoll-Hypaque lymphocyte separation solution to a 50 ml centrifuge tube, then slowly add an equal volume of blood cell suspension along the tube wall to the upper layer of lymphocyte separation solution, then centrifuge the centrifuge tube at 2000 rpm for 20 min, and adjust the centrifuge speed to 0; After discarding the supernatant of the centrifuged cell suspension, carefully aspirate the middle buffy coat layer, which is the peripheral blood mononuclear cell layer, i.e., the PBMC layer; Place the Dynabeads® magnetic beads on a vortex mixer and mix thoroughly for 30 seconds. Calculate the required number of Dynabeads® / total volume according to the ratio of Dynabeads®:PBMC=1:1. Add an equal volume of PBS to the Dynabeads® and mix thoroughly. Place Dynabeads® on the magnetic stand for 1 min, discard the supernatant, add an equal volume of PBS, and repeat this step 3 times; Remove Dynabeads® from the magnetic stand, add T cell serum-free medium, and mix thoroughly; Dynabeads® and PBMC were thoroughly mixed, vortexed for 10 seconds, and then allowed to stand at 25°C for 10 min. 6 / ml seeding density was inoculated into culture flasks, and the cells were activated in T cell serum-free culture medium supplemented with IL-2 500IU / ml and 1% autologous plasma in a 37°C, 5% CO2 incubator for 24 hours to obtain initial T cells.

[0032] According to the method of the first aspect of the present invention, in step (5), the engineered exosomes obtained in step (3) and the initial T cells obtained in step (4) are mixed at a rate of 1×10 9 Particles / mL exosomes: 1×10 6 / mL T cells were fully mixed, inoculated into culture well plates, and cultured overnight in a 37°C, 5% CO2 incubator.

[0033] According to the method of the first aspect of the present invention, in step (5), after the exosomes are co-cultured with the initial T cells overnight, the cell suspension is aspirated, centrifuged at 300g for 5 minutes, the supernatant is aspirated, T cell culture medium is added, IL-2 is added to a concentration of 500 IU / ml, and 5% human AB serum is added to the culture medium volume, and the culture medium is placed in a 37°C, 5% CO2 incubator for culture. Fresh culture medium is added every 48 hours and the cell density is adjusted to 1×10 6 / mL; According to the method of the first aspect of the present invention, in step (5), after culturing in a culture medium containing IL-2 and human AB serum for 7 days, the cell suspension is aspirated, centrifuged and the supernatant is discarded, the cell pellet is resuspended with PBS, and the density is adjusted to obtain a suspension of CAR-T cells.

[0034] The present invention can effectively obtain CAR-T cells through the above method and present the excellent effects described in the context of the present invention.

[0035] Chimeric antigen receptor (CAR) T cell therapy has become a promising technology in cancer treatment in recent years. However, the generation of most CAR-T cells currently relies on viral transduction, which can induce persistent expression of CAR but may cause serious adverse reactions. To overcome these challenges, the present invention has developed an engineered exosome delivery platform that uses the bacteriophage MS2 system to bind to the lysosomal-associated membrane protein 2B (LAMP2B) on exosomes, connects the MS2 capsid protein to the C-terminus of LAMP2B, and inserts the MS2 binding site (MS2bs) into the 3' untranslated region (UTR) of the reporter gene. LAMP2B is a marker protein of exosomes. Through the interaction between MS2 and MS2bs, the mRNA of the CAR structure is effectively loaded into the exosomes. The engineered exosomes are used to deliver CAR-19 mRNA and Anti-CD3 / CD28 single-chain variable fragments (scFvs) to T cells. The Anti-CD3 / CD28 scFvs expressed outside the exosome membrane can effectively activate primary T cells. At the same time, the exosomes deliver CAR-19 mRNA to T cells to synthesize CAR protein, and then generate CAR-T cells with cancer cell killing ability.

[0036] In response to the actual needs of current CAR-T technology, the present invention provides the construction of a CD19 CAR expression plasmid that co-expresses IL-7 and CCL17, which can not only secrete IL-7 outside T cells, but also enable CCL-17 to recruit peripheral blood immune cells to the greatest extent, further improving the tumor cell killing ability of CAR-T.

[0037] The overall technical solution of the present invention is schematically shown as follows: Figure 1 As described in detail above and below, the present invention achieves one or more beneficial effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 : Schematic diagram of the overall technical solution of the present invention.

[0039] Figure 2 : Schematic diagram of the three plasmids constructed by the present invention, which respectively show the Anti-CD3-Lamp2b-MS2 vector plasmid, the Anti-CD28-Lamp2b-MS2 vector plasmid, and the CAR-19mRNA-MS2bs vector plasmid composed of sequences such as the CD19 single-chain variable region.

[0040] Figure 3 : Typical particle size diagram of engineered exosomes.

[0041] Figure 4 : Typical electron micrographs of engineered exosomes.

[0042] Figure 5 : Typical confocal microscopy staining of T cell uptake of engineered exosomes.

[0043] Figure 6 : Typical flow cytometric plot of CAR-T cell positivity after exosome delivery of CAR-mRNA. DETAILED DESCRIPTION

[0044] The present invention can be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. The present invention provides general and / or specific descriptions of the materials and test methods used in the test. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described in as much detail as possible here.

[0045] Some of the main experimental materials used in the present invention include: Endo-Free Plasmid Maxi Kit (Omega), NaCl, peptone, EDTA, NaOH, yeast powder (Shanghai Sangon Biotechnology Co., Ltd.), Competent Stbl3 (Quanshijin Biotechnology Co., Ltd.), Lip3000 transfection reagent (containing P3000 Enhancer), Opti-MEM reduced serum medium, sodium pyruvate, DMEM-H, calcein AM (ThermoFisher), DiI cell membrane fluorescent probe (meilunbio), Millipore disposable syringe filter 0.45µm (Millipore), 96-well plate, 24-well plate, 6-well plate, 10CM, T25 culture flask, T75 culture flask, 5ml pipette, 10ml pipette, 25ml pipette (Corning-Costar), X-VIVOTM15 Medium(LONZA), Μltra-LEAF Purified anti-mouse CD28, Μltra-LEAF Purified anti-mouseCD3ε (Biolegend), IL-2 (Peprotech), BD Pharmingen™ Biotin Mouse Anti-Human CD8 (also commonly referred to in the art as CD8biotin antibody, biotin-labeled CD8 antibody, or CD8 antibody, a product of BD, catalog number 555365), PerCP-Cy™ 5.5 Mouse Anti-Human CD8 (BD), Sequence Synthesis (Universal Biology), GM-CSF ELISA kit (Abcam).

[0046] Other commonly used reagents and materials are commercially available.

[0047] Some operations of the present invention refer to the method described in CN115537389A (Chinese patent application number 202211289418.X) of this research team. If not otherwise specified, some materials used in the present invention also use the materials recorded in CN115537389A. For example, unless otherwise specified, some reagents used in the present invention are conventional in the art or can be easily purchased from commercial sources. For example, serum-free culture medium (Gibco) was purchased from ThermoFisherScientific; human platelet lysate was purchased from Precicion BioMedicals; phosphate buffer (pH6.8, PBS) was prepared by taking 250ml of 0.2mol / L potassium dihydrogen phosphate solution, adding 118ml of 0.2mol / L sodium hydroxide solution, diluting with water to 1000ml, shaking well, and sterilizing at 121°C for 15min; tissue cleaning solution (PRS-TCR-1) was purchased from Precicion. The MSC complete culture medium described herein is a serum-free culture medium containing 2% human platelet lysate.

[0048] The nucleotide sequence and amino acid sequence of Lamp2b are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively. The nucleotide sequence and amino acid sequence of MS2 are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively. The nucleotide sequence of MS2bs is shown in SEQ ID NO. 5. The nucleotide sequence and amino acid sequence of Anti-CD3 are shown in SEQ ID NO. 6 and SEQ ID NO. 7, respectively. The nucleotide sequence and amino acid sequence of Anti-CD28 are shown in SEQ ID NO. 8 and SEQ ID NO. 9, respectively. The nucleotide sequence and amino acid sequence of CAR-19 mRNA are shown in SEQ ID NO. 10 and SEQ ID NO. 11, respectively.

[0049] Example 1: Construction of vector plasmid The present invention first constructed three vector plasmids, and their plasmid maps are schematically shown as follows: Figure 2 shown.

[0050] The following describes the process of constructing the following three vector plasmids. Although the three vector plasmids designed by the present invention are novel, the construction and operation methods of these vector plasmids are conventional operation methods in the art.

[0051] 1. Construction of vector plasmid containing Anti-CD3-Lamp2b-MS2 element using pcDNA3.1 plasmid 1. Plasmids and gene fragments: pcDNA3.1 plasmid, Anti-CD3-scFv gene sequence, Lamp2b gene sequence, MS2 Coat Protein gene sequence.

[0052] 2. Restriction endonuclease: Not I (GCGGCCGC) is upstream and BamH I (GGATCC) is downstream.

[0053] 3. Other reagents: T4 DNA ligase and its buffer, Escherichia coli Stbl3 chemically competent cells (Kanglang Biotechnology), LB medium and LB agar plates (containing appropriate antibiotics), Plasmid extraction kit, PCR amplification reagents, Agarose gel electrophoresis reagents.

[0054] 4. Specific operations: ① Design primers: Design primers for amplifying Anti-CD3-scFv-Lamp2b-MS2 gene sequences. Primer design should include appropriate restriction enzyme sites for subsequent cloning.

[0055] ② Amplify gene sequence: Use PCR method to amplify Anti-CD3-scFv-Lamp2b-MS2 sequence from template DNA.

[0056] ③ Purify PCR products: Separate PCR products by agarose gel electrophoresis, cut out the corresponding DNA bands and purify the PCR products using a gel recovery kit.

[0057] ④ Double enzyme digestion: Use EcoRI and BamHI to digest the pcDNA3.1 plasmid, and then digest the Anti-CD3-scFv-Lamp2b-MS2 gene fragment.

[0058] ⑤ Ligation reaction: Use T4 DNA ligase to connect the digested Anti-CD3-scFv-Lamp2b-MS2 gene fragment to the digested pcDNA3.1 plasmid.

[0059] ⑥ Transformation of competent cells: The ligation reaction product was transformed into competent Stbl3 cells by heat shock method. The transformed cells were spread on LB agar plates containing antibiotics and cultured at 37°C overnight.

[0060] ⑦ Screening of positive clones: Select a single colony, extract plasmid DNA using a small amount of plasmid extraction kit, and use restriction enzyme digestion and PCR methods to verify whether the plasmid contains the expected insert fragment.

[0061] ⑧Sequence verification: Send samples for DNA sequencing to verify the correctness and integrity of the Anti-CD3-scFv-Lamp2b-MS2 gene sequence and ensure that the reading frame of the inserted fragment is correct and has no mutations.

[0062] ⑨Plasmid extraction: Use a large number of plasmid extraction kits to extract plasmid DNA from positive clones and store them at low temperature for later use.

[0063] 2. Construction of Anti-CD28-Lamp2b-MS2 vector plasmid using pcDNA3.1 plasmid As described above, Anti-CD3 was replaced with Anti-CD28, and the Anti-CD28-Lamp2b-MS2 vector plasmid was constructed using pcDNA3.1 plasmid.

[0064] 3. Construction of CAR-19mRNA-MS2bs vector plasmid using pLVX-EF1α-IRES-puro plasmid The CAR-19mRNA-MS2bs sequence is composed of CD19 single-chain variable region, CD8a hinge region, CD8a transmembrane region, 4-1BB signal, CD3ζ clathrate signal, IL-7, CCL17, MS2bs and other sequences. The vector used is pLVX-EF1α-IRES-PGK-puro, which is synthesized by General Biosystems (Anhui) Co., Ltd.

[0065] For the specific construction method, refer to the content section of "Construction of Anti-CD3-Lamp2b-MS2 vector plasmid using pcDNA3.1 plasmid", select appropriate restriction endonucleases and ligases, and use pLVX-EF1α-IRES-puro plasmid to construct CAR-19mRNA-MS2bs vector plasmid.

[0066] Example 2: Cultivation and transfection of mesenchymal stem cells This example prepares mesenchymal stem cells with reference to the method described in Example 1 of CN115537389A (202211289418.X) of this research team.

[0067] 1. Use tissue cleaning solution (0.9% saline containing 1% penicillin and 1% streptomycin) to clean the surface of the placenta to remove surface congestion, cut the umbilical cord and remove the internal blood vessels. This step is specifically performed as follows: Place stainless steel trays, filters, stainless steel cups, lunch boxes, tissue cleaning solution (0.9% saline containing 1% penicillin and 1% streptomycin), pipettes and other items in appropriate locations in the biosafety cabinet.

[0068] Use surgical forceps to remove the placental tissue and place it in a stainless steel tray. Use tissue cleaning solution to clean the surface of the placenta to remove the congestion on the surface of the placenta.

[0069] Use surgical scissors and forceps to cut the umbilical cord at the placenta, and cut the other end from behind the sterile umbilical cord clamp. Remove the umbilical cord and wash it three times with tissue cleaning solution to remove the congestion and blood clots on the surface. Clamp one end of the umbilical cord with forceps, and use another pair of forceps to clamp the umbilical cord and slide along the umbilical cord tissue to remove the residual umbilical cord blood in the blood vessels. First cut the umbilical cord into several sections of 2-3cm in length, then use scissors to cut it open along the longitudinal section of the umbilical cord, flatten it, and remove the 3 blood vessels inside (2 umbilical arteries and 1 umbilical vein). Transfer to a new dish.

[0070] 2. Remove the Wharton's jelly from the umbilical cord, clean it (with tissue cleaning solution), cut it into small pieces of 2~3mm^3, and transfer it to a T75 culture bottle, 1mL per bottle.

[0071] This step is specifically performed as follows: peel off the Wharton's jelly with tweezers, put it into a culture dish filled with tissue cleaning solution, wash it with the tissue cleaning solution and tear the Wharton's jelly into long strips.

[0072] After processing, transfer the Wharton's jelly to a 50 mL centrifuge tube with tweezers and cut it into 2-3 mm thick pieces with scissors. 3 Use a 3 mL sterile dropper to transfer small pieces into T75 culture bottles, 1 mL per bottle. The tip of the dropper needs to be cut off with sterile scissors before use.

[0073] 3. Slowly add 10 mL of MSC complete medium (serum-free medium containing 2% human platelet lysate) to the culture bottle, gently shake the culture bottle, so that the umbilical cord tissue block is flattened in the culture bottle, and cultured in a 37°C, 5% CO2 incubator. Replenish the fluid once every 2-3 days (add 3-4 mL of MSC complete medium each time), and after 2-3 times of repletion, remove the tissue block and replace the MSC complete medium in full to continue culturing until the cells grow to a confluency of 70%, and the obtained cells are then digested. This step is specifically performed as follows: Slowly add 10 mL of MSC complete medium (serum-free medium containing 2% human platelet lysate) to the culture bottle, gently shake the culture bottle, so that the umbilical cord tissue block is flattened in the culture bottle. Place the T75 culture bottle in a 37°C, 5% CO2 incubator for static culture. On the third day of culture, remove the culture bottle from the incubator, add 3.5 mL of MSC complete medium, and then rehydrate once every 3 days. After three times of rehydration, cells will crawl out from the bottom of the culture flask. Remove the tissue blocks and replace the MSC complete medium with complete medium to continue culturing. When the cells grow to a confluency of 70%, the obtained cells are then digested.

[0074] 4. The culture medium in the culture bottle is discarded, (20 mL) PBS is drawn to rinse the culture bottle to remove the residual culture medium, 3 mL of recombinant trypsin solution is added for digestion, the culture bottle is patted to make the cells fall off to the bottom of the culture bottle, 10 mL of MSC complete medium is added to neutralize trypsin to terminate the digestion process, the cells are blown away with a pipette, the cell suspension is collected in a centrifuge tube, centrifuged, and the supernatant is discarded to obtain a cell pellet recorded as P0 generation cells. This step is specifically operated as follows: before cell subculture, recombinant trypsin, MSC complete medium (serum-free medium containing 2% human platelet lysate), and PBS are placed at room temperature for equilibrium temperature. The culture bottle is taken out of the incubator, the culture medium in the culture bottle is discarded with a pipette, and 20 mL of PBS is drawn to rinse the culture bottle to remove the residual culture medium. Add 3mL of recombinant trypsin solution (GIBCO) to the culture flask for digestion, gently shake the culture flask to allow trypsin to fully cover the cell surface, digest for about 1min, observe the cells shrink into a circle under a microscope, gently tap the culture flask to make the cells fall off to the bottom of the culture flask in a quicksand-like state, add 10mL of MSC complete medium to neutralize trypsin to terminate the digestion process, blow the cells with a pipette, and collect the cell suspension in a 15mL centrifuge tube. Place the centrifuge tube in a centrifuge, centrifuge at 1400rpm for 5min, remove the supernatant, and the precipitated cells are recorded as P0 generation.

[0075] 5. Resuspend the P0 cell pellet in 10 mL of MSC complete medium, count the cells, and then inoculate at a density of 1×10 4 MSC cells / cm 2Inoculate into a T75 culture flask, add the above-mentioned MSC complete medium to 10 mL, and culture statically in a 37°C, 5% CO2 incubator for more than 72 hours until the cell confluence reaches 80%, discard the medium in the culture flask, rinse the culture flask with (20 mL) PBS to remove the residual medium, add 3 mL of recombinant trypsin solution for digestion, tap the culture flask to make the cells fall off to the bottom of the culture flask, add 10 mL of MSC complete medium to neutralize trypsin to terminate the digestion process, blow off the cells with a pipette, collect the cell suspension in a centrifuge tube, centrifuge, discard the supernatant, and obtain a cell precipitate as P1 cells; repeat this step 5 for MSC cell subculture until mesenchymal stem cells of cell generations P2 to P8 are obtained.

[0076] 6. Plasmid transfection of mesenchymal stem cells: In this step, Lipofectamine™ 3000 Reagent Kit (Thermo Fisher Scientific, Invitrogen) was used for transfection.

[0077] (a) P5 mesenchymal stem cells were seeded into 6-well plates and 2×10 5 Cells were cultured in a 37°C, 5% CO2 incubator until the cell density reached 50-70% confluence; (b) Dilute each plasmid DNA (plasmid 1, plasmid 2, plasmid 3) in serum-free Opti-MEM medium (CellWorld®) to a required amount of 2.5 µg per well (7.5 µg for three plasmids in total); In another centrifuge tube containing serum-free Opti-MEM medium, add 5 µL of Lipofectamine 3000 reagent to each centrifuge tube; Add 10 µL of P3000 Reagent to each plasmid DNA solution; Add Lipofectamine 3000 reagent solution dropwise to the plasmid DNA solution, mix gently (do not vortex), and incubate at room temperature for 15-20 minutes to form DNA-Lipo3000 complex; (c) Replace the cell culture medium in the 6-well plate with serum-free Opti-MEM medium (to reduce the effect of serum on transfection efficiency); Add the DNA-Lipo3000 complex solution in each tube dropwise into the 6-well plate for cell culture, and gently shake the culture plate to evenly distribute the transfection complex; (d) The cells were incubated in a 37°C, 5% CO2 incubator for 4-6 hours, and then the serum-free Opti-MEM medium was replaced with modified DMEM high-glucose medium (containing 10% FBS) and the cells were cultured for another 48-72 hours to allow plasmid expression.

[0078] Although the above-mentioned transfection is a routine operation in the art and the present invention examines the expression at the cellular level below, those skilled in the art can still use molecular level methods such as qPCR or Western Blot to detect the expression of Anti-CD3-scFv, Anti-CD28-scFv, Lamp2b, and MS2.

[0079] Example 3: Acquisition and purification of engineered exosomes 1. The mesenchymal stem cell culture medium after transfection for 48-72 days in step 6 of Example 2 was replaced with a modified DMEM-high glucose medium (without FBS), and the cells were cultured in a 37°C, 5% CO2 incubator containing 2% O2 for 48 hours (so that the MSCs cells can fully release the modified engineered exosomes).

[0080] 2. Pipette the cell supernatant (30 ml) obtained in the previous step into a (50 ml) centrifuge tube and perform the following centrifugation treatment according to the method described in Example 2 of CN115537389A: Centrifuge at 300 g and 4 °C for 10 min (to remove dead cells and larger cell debris), and transfer the supernatant to another (50 ml) centrifuge tube; Centrifuge at 2000 g and 4 °C for 20 min (to further remove cell debris and other impurities), and pipette the supernatant into another (high-speed) centrifuge tube; Centrifuge at 10,000 g and 4°C for 30 min (to further remove smaller cell debris and impurities), and filter the supernatant with a 0.22 μm filter (PES filter, millipore) and place in another (ultracentrifuge) tube; Centrifuge at 100,000 g and 4 °C for 90 min and discard the supernatant; Add 20 ml of sterile PBS to the centrifuge tube to resuspend the exosome precipitate, centrifuge at 100000g, 4°C for 90 minutes, discard the supernatant, add 1 mL of sterile PBS to resuspend the exosomes, and obtain 1 ml of exosome suspension (which can be called engineered exosomes), which can be stored at -80°C and / or packaged for performance measurement. If not specified, the centrifuge tubes and centrifuges with different centrifugal force requirements used in the present invention were purchased from Beckman Coulter.

[0081] 3. Transmission electron microscopy observation of exosomes: Referring to the operations of steps 1 to 5 of Example 4 of CN115537389A (Chinese patent application number 202211289418.X), the exosome particles obtained in this example were observed by transmission electron microscopy. The specific operation was as follows: 20µl of the exosome suspension was mixed with 20µl of 2.5% glutaraldehyde and fixed at 4°C for 45 minutes; a copper mesh was placed with the front side facing up, and 20μl of the exosome suspension was dripped in, and allowed to stand for 15 minutes; the excess solution was blotted with filter paper, 100μl of 1% phosphotungstic acid solution was added to the copper mesh and incubated for 30 minutes, the excess dye was blotted with filter paper, and dried at room temperature away from light for 30 minutes; finally, the exosomes were observed using a transmission electron microscope, and the particle size diagram of the obtained engineered exosomes was drawn. The typical particle size diagram of engineered exosomes is shown in Figure 3 , typical electron micrographs of engineered exosomes are shown in Figure 4 .

[0082] In addition, by transmission electron microscopy observation, the number of exosome particles in the exosomes obtained from step 6 (a) of Example 2, with 1×10^6 mesenchymal stem cells added, to the exosome suspension obtained in step 2 of Example 3 was calculated, and the result was 158.7×10^9 particles (per 1×10^6 mesenchymal stem cells).

[0083] 4. Detection of exosome protein content: With reference to the operation of Example 6 of CN115537389A (Chinese Patent Application No. 202211289418.X), the protein content of the exosomes (suspension) obtained in this example was detected, and the protein content in the exosomes obtained from step 6 (a) of Example 2 with 1×10^6 mesenchymal stem cells added to the exosome suspension obtained in step 2 of Example 3 was calculated, and the result was 182.4 µg (per 1×10^6 mesenchymal stem cells).

[0084] Example 3a: Using the P4 generation mesenchymal stem cells obtained in step 5 of Example 2 as the starting cells, the operations of step 6 of Example 2 and steps 1 to 2 of Example 3 were performed to obtain an engineered exosome suspension; referring to the operation of step 3 of Example 3, the engineered exosomes obtained in this example were observed by transmission electron microscopy, and the number of exosome particles in the exosomes obtained per 1×10^6 mesenchymal stem cells fed was calculated to be 146.4×10^9 particles; referring to the operation of step 4 of Example 3, the protein content of the engineered exosomes obtained in this example was detected, and the protein content in the exosomes obtained per 1×10^6 mesenchymal stem cells fed was calculated to be 188.3µg.

[0085] Example 3b: Using the P6 generation mesenchymal stem cells obtained in step 5 of Example 2 as the starting cells, the operations of step 6 of Example 2 and steps 1 to 2 of Example 3 were performed to obtain an engineered exosome suspension; referring to the operation of step 3 of Example 3, the engineered exosomes obtained in this example were observed by transmission electron microscopy, and the number of exosome particles in the exosomes obtained per 1×10^6 mesenchymal stem cells fed was calculated to be 160.2×10^9 particles; referring to the operation of step 4 of Example 3, the protein content of the engineered exosomes obtained in this example was detected, and the protein content in the exosomes obtained per 1×10^6 mesenchymal stem cells fed was calculated to be 176.8µg.

[0086] Example 3c: Referring to the methods of Examples 2-3, the only difference is that the modified DMEM high glucose medium (containing 10% FBS) used in step 6 (d) of Example 2 is replaced with conventional DMEM high glucose medium (containing 10% FBS), and the modified DMEM-high glucose medium (excluding FBS) used in step 1 of Example 3 is replaced with conventional DMEM high glucose medium (excluding FBS) to obtain an engineered exosome suspension; referring to the operation of step 3 of Example 3, the engineered exosomes obtained in this example were observed by transmission electron microscopy, and the number of exosome particles in the exosomes obtained per 1×10^6 mesenchymal stem cells fed was calculated to be 34.6×10^9 particles; referring to the operation of step 4 of Example 3, the protein content of the engineered exosomes obtained in this example was detected, and the protein content in the exosomes obtained per 1×10^6 mesenchymal stem cells fed was calculated to be 22.5µg.

[0087] Example 3d: Referring to the methods of Examples 2-3, the only difference is that sodium arachidonic acid is not added to the modified DMEM high glucose medium (containing 10% FBS) used in step 6 (d) of Example 2 and the modified DMEM-high glucose medium (not containing FBS) used in step 1 of Example 3, to obtain an engineered exosome suspension; referring to the operation of step 3 of Example 3, the engineered exosomes obtained in this example were observed by transmission electron microscopy, and the number of exosome particles in the exosomes obtained per 1×10^6 mesenchymal stem cells fed was calculated to be 43.1×10^9 particles; referring to the operation of step 4 of Example 3, the protein content of the engineered exosomes obtained in this example was detected, and the protein content in the exosomes obtained per 1×10^6 mesenchymal stem cells fed was calculated to be 29.7µg.

[0088] Example 3e: Referring to the methods of Examples 2-3, the only difference is that zinc sulfate is not added to the modified DMEM high glucose medium (containing 10% FBS) used in step 6 (d) of Example 2 and the modified DMEM-high glucose medium (not containing FBS) used in step 1 of Example 3, to obtain an engineered exosome suspension; referring to the operation of step 3 of Example 3, the engineered exosomes obtained in this example were observed by transmission electron microscopy, and the number of exosome particles in the exosomes obtained per 1×10^6 mesenchymal stem cells fed was calculated to be 38.4×10^9 particles; referring to the operation of step 4 of Example 3, the protein content of the engineered exosomes obtained in this example was detected, and the protein content in the exosomes obtained per 1×10^6 mesenchymal stem cells fed was calculated to be 24.2µg.

[0089] According to the results of Example 3 and Example 3a-3e, adding sodium arachidonic acid and zinc sulfate to the DMEM high-glucose medium can significantly increase the yield of exosome particles and the protein content in the exosomes, which is a completely unexpected finding. A higher yield of exosomes makes it possible to prepare a larger amount of CAR-T cells in the future.

[0090] In the present invention, if not otherwise specified, the conventional DMEM high glucose medium (containing 10% The formula of FBS is: 200 mg / L anhydrous CaCl2, 0.1 mg / L nonahydrate Fe(NO3)3, 97.67 mg / L anhydrous MgSO4, 400 mg / L KCl, 3700 mg / L NaHCO3, 6400 mg / L NaCl, 109 mg / L anhydrous NaH2PO4, 434 mg / L L-alanyl-L-glutamine, 30 mg / L glycine, 84 mg / L L-arginine hydrochloride, 62.6 mg / L L-cystine dihydrochloride, 584 mg / L L-glutamine, 42 mg / L L-histidine hydrochloride monohydrate, 105 mg / L L-isoleucine, 105 mg / L L-leucine, 146 mg / L L-lysine hydrochloride, 30 mg / L / L of L-methionine, 66mg / L of L-phenylalanine, 42mg / L of L-serine, 95mg / L of L-threonine, 16mg / L of L-tryptophan, 103.79mg / L of L-tyrosine disodium salt dihydrate, 94mg / L of L-valine, 4mg / L of choline chloride, 4mg / L of D-calcium pantothenate, 4mg / L of folic acid, 4mg / L of niacinamide, 4mg / L of pyridoxine hydrochloride, 0.4mg / L of riboflavin, 4mg / L of thiamine hydrochloride, 7.2mg / L of meso-inositol, 4500mg / L of D-glucose, 110mg / L of sodium pyruvate, 15mg / L of phenol red, 100kU / L of penicillin G sodium salt, 100mg / L of streptomycin sulfate, 10% fetal bovine serum, and add appropriate amount of water to the full amount.In the present invention, if not otherwise specified, the formula of the conventional DMEM high-glucose medium (without FBS) is: 200 mg / L anhydrous CaCl2, 0.1 mg / L nonahydrate Fe(NO3)3, 97.67 mg / L anhydrous MgSO4, 400 mg / L KCl, 3700 mg / L NaHCO3, 6400 mg / L NaCl, 109 mg / L anhydrous NaH2PO4, 434 mg / L L-alanyl-L-glutamine, 30 mg / L glycine, 84 mg / L L-arginine hydrochloride, 62.6 mg / L L-cystine dihydrochloride, 584 mg / L L-glutamine, 42 mg / L L-histidine hydrochloride monohydrate, 105 mg / L L-isoleucine, 105 mg / L L-leucine, 146 100mg / L L-lysine hydrochloride, 30mg / L L-methionine, 66mg / L L-phenylalanine, 42mg / L L-serine, 95mg / L L-threonine, 16mg / L L-tryptophan, 103.79mg / L L-tyrosine disodium salt dihydrate, 94mg / L L-valine, 4mg / L choline chloride, 4mg / L D-calcium pantothenate, 4mg / L g / L folic acid, 4mg / L niacinamide, 4mg / L pyridoxine hydrochloride, 0.4mg / L riboflavin, 4mg / L thiamine hydrochloride, 7.2mg / L meso-inositol, 4500mg / L D-glucose, 110mg / L sodium pyruvate, 15mg / L phenol red, 100kU / L penicillin G sodium salt, 100mg / L streptomycin sulfate, add appropriate amount of water to the full amount.In the present invention, if not otherwise specified, the modified DMEM high glucose medium (containing 10% The formula of FBS is: 200 mg / L anhydrous CaCl2, 0.1 mg / L nonahydrate Fe(NO3)3, 97.67 mg / L anhydrous MgSO4, 400 mg / L KCl, 3700 mg / L NaHCO3, 6400 mg / L NaCl, 109 mg / L anhydrous NaH2PO4, 124 mg / L sodium arachidonic acid, 56 mg / L zinc sulfate, 434 mg / L L-alanyl-L-glutamine, 30 mg / L glycine, 84 mg / L L-arginine hydrochloride, 62.6 mg / L L-cystine dihydrochloride, 584 mg / L L-glutamine, 42 mg / L L-histidine hydrochloride monohydrate, 105 mg / L L-isoleucine, 105 mg / L L-leucine, 146 mg / L L-lysine hydrochloride, 30mg / L L-methionine, 66mg / L L-phenylalanine, 42mg / L L-serine, 95mg / L L-threonine, 16mg / L L-tryptophan, 103.79mg / L L-tyrosine disodium salt dihydrate, 94mg / L L-valine, 4mg / L choline chloride, 4mg / L D-calcium pantothenate, 4mg / L folic acid, 4 mg / L niacinamide, 4 mg / L pyridoxine hydrochloride, 0.4 mg / L riboflavin, 4 mg / L thiamine hydrochloride, 7.2 mg / L meso-inositol, 4500 mg / L D-glucose, 110 mg / L sodium pyruvate, 15 mg / L phenol red, 100 kU / L penicillin G sodium salt, 100 mg / L streptomycin sulfate, 10% fetal bovine serum, and add appropriate amount of water to the full amount.In the present invention, if not otherwise specified, the formula of the modified DMEM high-glucose medium (without FBS) is: 200 mg / L anhydrous CaCl2, 0.1 mg / L nonahydrate Fe(NO3)3, 97.67 mg / L anhydrous MgSO4, 400 mg / L KCl, 3700 mg / L NaHCO3, 6400 mg / L NaCl, 109 mg / L anhydrous NaH2PO4, 124 mg / L sodium arachidonic acid, 56 mg / L zinc sulfate, 434 mg / L L-alanyl-L-glutamine, 30 mg / L glycine, 84 mg / L L-arginine hydrochloride, 62.6 mg / L L-cystine dihydrochloride, 584 mg / L L-glutamine, 42 mg / L L-histidine hydrochloride monohydrate, 105 mg / L L-isoleucine, 105 mg / L g / L L-leucine, 146mg / L L-lysine hydrochloride, 30mg / L L-methionine, 66mg / L L-phenylalanine, 42mg / L L-serine, 95mg / L L-threonine, 16mg / L L-tryptophan, 103.79mg / L L-tyrosine disodium salt dihydrate, 94mg / L L-valine, 4mg / L choline chloride, 4mg / L D- Calcium pantothenate, 4mg / L folic acid, 4mg / L nicotinamide, 4mg / L pyridoxine hydrochloride, 0.4mg / L riboflavin, 4mg / L thiamine hydrochloride, 7.2mg / L meso-inositol, 4500mg / L D-glucose, 110mg / L sodium pyruvate, 15mg / L phenol red, 100kU / L penicillin G sodium salt, 100mg / L streptomycin sulfate, water is added to the full amount. Sodium arachidonic acid and zinc sulfate used in the present invention are purchased from Sigma-Aldrich, and the purity is >98.5%. The preparation methods of the conventional DMEM high-glucose medium (containing 10% FBS), conventional DMEM high-glucose medium (excluding FBS), improved DMEM high-glucose medium (containing 10% FBS), and improved DMEM high-glucose medium (excluding FBS) described in the present invention are as follows: weigh each substance according to the specified concentration, add an appropriate amount of injection under sterile conditions to dissolve each substance, and then filter twice with a 0.45µm microporous filter membrane, and then filter twice with a 0.1µm microporous filter membrane (with filtration and sterilization capabilities). The high-glucose medium without adding sodium arachidonic acid or zinc sulfate used in Example 3d and Example 3e is only a medium prepared without adding them during preparation.

[0091] Example 4: Preparation of naive T cells 1. Place 100 ml of healthy adult peripheral blood containing sodium citrate anticoagulant in a 250 ml centrifuge cup, centrifuge at 1500 rpm, 20°C for 15 min, and collect the upper plasma.

[0092] 2. Add an equal volume of 0.9% sodium chloride injection to the lower blood cell layer and mix thoroughly. Add 20 ml of Ficoll-Hypaque lymphocyte separation solution (density 1.077, Tianjin Haoyang) to a 50 ml centrifuge tube, and then slowly add an equal volume of blood cell suspension along the tube wall to the upper layer of lymphocyte separation solution, being careful not to break the separation liquid surface. Then centrifuge the centrifuge tube at 2000 rpm for 20 minutes, and adjust the centrifuge speed to 0.

[0093] 3. After discarding the supernatant layer of the centrifuged cell suspension, carefully aspirate the middle white film layer, which is the peripheral blood mononuclear cell (PBMC) layer, and calculate the cell number and viability of the extracted mononuclear cells by trypan blue staining.

[0094] 4. Place Dynabeads® (Thermofisher) magnetic beads on a vortex mixer and mix thoroughly for 30 seconds.

[0095] 5. Calculate the required number of Dynabeads® / total volume according to the ratio of Dynabeads®:PBMC = 1:1, add an equal volume of PBS to Dynabeads® and mix thoroughly.

[0096] 6. Place Dynabeads® on the magnetic stand for 1 min, discard the supernatant, add an equal volume of PBS, and repeat this step 3 times.

[0097] 7. Remove the Dynabeads® from the magnetic stand, add T cell serum-free medium, and mix thoroughly.

[0098] 8. Mix Dynabeads® and PBMC thoroughly, vortex mixer for 10 seconds, and let stand at 25°C for 10 minutes. 6 The cells were inoculated into culture flasks at a seeding density of 1:1 / ml and activated for 24 h in a T cell serum-free medium (x-vivo-15, Lonza) supplemented with IL-2 500 IU / ml and 1% autologous plasma in a 37°C, 5% CO2 incubator to obtain initial T cells.

[0099] Example 5: Uptake of engineered exosomes by naive T cells This example demonstrates that T cells have affinity for and are able to take up engineered exosomes.

[0100] Materials: green PKH67 dye (Sigma-Aldrich), DAPI dye (Sigma-Aldrich), diluent, PBS buffer, BSA (bovine serum albumin).

[0101] 1. Dilute the extracted exosomes to 1×10 9 Particles / mL; 2. Dilute the PKH67 dye to 2× working concentration (determined according to the kit instructions); 3. Mix equal volumes of exosome suspension and PKH67 dye solution and incubate at room temperature in the dark for 5 minutes; 4. Add an equal volume of 1% BSA solution to terminate the reaction and incubate at room temperature in the dark for 1 minute; 5. Transfer the mixture to an ultrafiltration tube, ultracentrifuge (100,000 g, 70 min), and wash three times with PBS buffer to remove unbound dye; 6. Resuspend the exosome precipitate with PBS buffer and measure the exosome concentration; 7. The initial T cells obtained in step 8 of Example 4 were stained with DAPI stain, and the steps were the same as 1-7; 8. Adjust the concentration of labeled exosomes to 20 μg / μl and incubate with 1×10 primary stained T cells. 6 / mL, 1mL was incubated at room temperature for 2 hours, and after fixation, flow cytometry or confocal microscopy staining was performed with Hoechst. Typical confocal microscopy staining images of T cell uptake of engineered exosomes are shown in Figure 5 , which shows that T cells have affinity for and are able to take up engineered exosomes.

[0102] Example 6: Exosomes loaded with CAR-19 mRNA for CAR-T cell preparation and detection of CAR expression (a) Preparation of CAR-T cells: (a1) The engineered exosomes prepared in Example 3 and the initial T cells prepared in Example 4 were mixed at a rate of 1×10 9 Particles / mL exosomes: 1×10 6 / mL T cells were fully mixed, and a total of 2 mL was inoculated into a 6-well plate, and the plate was placed in a 37°C, 5% CO2 incubator for overnight culture; (a2) After overnight culture, the cell suspension was aspirated and centrifuged at 300 g for 5 minutes. The supernatant was discarded and T cell culture medium (x-vivo-15, Lonza) was added with IL-2 (to a concentration of 500 IU / ml) and 5% human AB serum (Gemini) per volume of culture medium. The cells were cultured in a 37°C, 5% CO2 incubator. Fresh culture medium was added every 48 hours and the cell density was adjusted to 1×10 6 / mL; (a3) After 7 days of culture, the cell suspension was aspirated and centrifuged at 300 g for 5 minutes. The supernatant was discarded and the cell pellet was resuspended in PBS and the density was adjusted (in this example, the density was adjusted to 1×10 7 / mL, which can also be adjusted as needed) to obtain a suspension of CAR-T cells. Unless otherwise specified, the PBS described herein refers to a phosphate buffer (pH 6.8) prepared as follows: 250 ml of 0.2 mol / L potassium dihydrogen phosphate solution, 118 ml of 0.2 mol / L sodium hydroxide solution, diluted with water to 1000 ml, shaken well, and sterilized at 121°C for 15 min to obtain a sterilized solution.

[0103] The CAR-T cell suspension obtained in the above step (a3) ​​can be subjected to the following tests.

[0104] (b) Detection of CAR expression: Protein L is an immunoglobulin binding protein derived from the bacterium Peptostreptococcus magnus that can specifically bind to the kappa light chain constant region of most mammalian antibodies. Since many CAR structures contain the Fc region of antibodies, Protein L can be used to detect CAR expression.

[0105] (b1), the density is 1×10 7 BSA solution (to a concentration of 0.1%) was added to the 100 mL CAR-T cell suspension and mixed thoroughly, then placed in a 4°C refrigerator for 15 minutes, then centrifuged at 300 g for 5 minutes, the supernatant was discarded, and the precipitate was resuspended with an appropriate amount of PBS; (b2) Centrifuge again at 300 g for 5 min, discard the supernatant, and resuspend the precipitate in an appropriate amount of PBS; (b3), add a fluorescently labeled Protein L reagent (e.g., FITC-labeled Protein L) of appropriate concentration, incubate at 4°C in the dark for 30 min, and wash the cells twice with PBS to remove unbound Protein L; (b4) Detect cell fluorescence intensity using flow cytometry.

[0106] According to the detection in this example, the typical flow cytometric graph of the CAR-T cell positive rate after exosome delivery of CAR-mRNA is shown in Figure 6 .

[0107] Example 7: Target cell killing experiment Lactate dehydrogenase (LDH) is a cytosolic enzyme present in many different cell types and is a clear and reliable indicator of cytotoxicity. Disruption of the cytoplasmic membrane releases LDH into the surrounding cell culture medium, where extracellular LDH can be quantified by coupled enzymatic reactions. LDH catalyzes the conversion of lactate to pyruvate via the NAD+ redox reaction, which is reduced to NADH. Oxidation of NADH by adenosine diphosphatase results in the reduction of tetrazolium salts (INT) to a measurable red formaldehyde product, which can be measured spectrophotometrically at 490 nm. The level of formaldehyde formation is proportional to the amount of LDH released into the culture medium and is a marker of cytotoxicity.

[0108] This example uses the CyQUANT™ LDH cytotoxicity detection kit (REF: C20301) from Thermo Fisher, and the components are shown in Table 1.

[0109] Table 1: Kit (REF: C20301) Components

[0110] 1. Cultivation of target cells: Take out the frozen target cells Raji cells (Shan En Biotech) from liquid nitrogen and quickly thaw them in a 37°C water bath. Transfer the thawed cells to a pre-prepared culture bottle containing 10 ml RPMI-1640 medium and 10% FBS, and adjust the density to 1×10 6 / mL, and culture the flask in a 37°C, 5% CO2 constant temperature incubator; 2. Prepare LDH substrate stock solution: add 11.4 mL of double distilled water to the substrate mixture and mix gently until dissolved; 3. Return the assay buffer to room temperature; 4. Prepare the reaction mixture: Mix 600 μl of assay buffer with 11.4 mL of substrate stock solution, then mix gently and protect from light until use; 5. Prepare 1×LDH positive control: Mix 1.5 μl of LDH positive control with 1 mL of PBS containing 1% BSA and store the unused portion at -20°C; 6. Determine the optimal cell number for LDH cytotoxicity assay: (1) In a 96-well plate, select two sets of parallel wells for serial cell dilution, and dilute the target cell concentration to 10,000~0 / 100 μl; one set is used to determine the maximum release of LDH, and the other set of wells is used to determine the spontaneous LDH release of cells; (2) Cultivate cells overnight in a 37°C, 5% CO2 incubator; (3) Add 10 μl of sterile water to the duplicate wells where spontaneous LDH release from cells is to be measured, and then gently tap to mix; (4) Add 10 μl of 10× cell lysis buffer to the wells where the maximum LDH release of cells is to be measured, and then gently tap to mix. Do not create bubbles during the mixing process. (5) Incubate the cells in a 37°C, 5% CO2 incubator for 45 minutes; (6) Transfer 50 μl of each sample (spontaneous LDH release and maximum LDH release groups) into a new 96-well flat-bottom plate and divide into three wells; (7) For LDH positive control assay, add 50 μl of 1× LDH positive control solution to the well plate; (8) Add 50 μl of reaction mixture to each well and tap gently to mix; (9) Incubate the plate at room temperature in the dark for 30 minutes; (10) Add 50 μl of stop solution to each well and mix gently; and measure the absorbance within 1 to 2 hours after adding the stop solution; (11) measuring the absorbance at 490 nm and 680 nm, and calculating the result by subtracting the absorbance at 680 nm from the absorbance at 490 nm; (12) The relationship between the maximum LDH release control absorbance value minus the spontaneous LDH release control absorbance value and the cell number was plotted to determine the linear range and optimal cell number of the LDH cytotoxicity assay. The optimal cell number of target cells was determined to be 1×10 4 / 100μl.

[0111] 7. Cell killing experiment (1) Based on the optimal number of target cells determined in step 6, dilute the target cells to the optimal cell number of 1×10 4 / 100 μl, placed in an incubator and cultured overnight, a positive control group, a negative control group, an experimental group, and a blank control group were set up, with 3 replicate wells in each group; (2) The effector-target ratios of CAR-T cells and Raji cells in the experimental group were set as 0.25:1, 0.5:1, 1:1, 2:1, and 4:1; (3) Each group was treated as follows: Negative control group (spontaneous LDH): add 10 μl of sterile water to three wells of this group; Positive control group (maximum LDH): add 10 μl of 10× cell lysate to three wells of this group; Experimental group: according to the effector-target ratio, the required number of effector cells / 100 μl was added to the experimental wells in sequence; Blank control group: 100 μl of basal medium was added to each well; (4) Place the 96-well plate in a 37°C, 5% CO2 incubator for 2 hours; (5) Transfer 50 μl of the cell suspension in each well to a new 96-well plate; (6) Add 50 μl of substrate reaction solution to each well, mix thoroughly, and incubate at room temperature in the dark for 30 min; (7) Add 50 μl of reaction stop solution to each well and mix thoroughly; (8) measuring the absorbance at 490 nm and 680 nm, and calculating the result by subtracting the absorbance at 680 nm from the absorbance at 490 nm; (9) The relationship between the maximum LDH release control absorbance value minus the spontaneous LDH release control absorbance value and the cell number was plotted to determine the LDH cytotoxicity and target cell killing efficiency.

[0112] Typical results of target cell killing efficiency are shown in Table 2.

[0113] In addition, in Example 6, the exosomes obtained in Example 3a were used to prepare CAR-T cells, and then the killing efficiency of the CAR-T cells on the target cells was determined according to the method of this Example 7. The results are shown in Table 2. In addition, in Example 6, the exosomes obtained in Example 3b were used to prepare CAR-T cells, and then the killing efficiency of the CAR-T cells on the target cells was determined according to the method of this Example 7. The results are shown in Table 2.

[0114] Table 2: Target cell killing efficiency results

[0115] In the table, * is the result of CAR-T cells obtained by using the exosome obtained in Example 3, ** is the result of CAR-T cells obtained by using the exosome obtained in Example 3a, and *** is the result of CAR-T cells obtained by using the exosome obtained in Example 3b. It can be seen from the results that the higher the effect-target ratio, the better the effect, and the cell death rate of the group with an effect-target ratio of 4:1 is as high as more than 80%.

[0116] In addition, in Example 6, the exosome obtained in Example 3a was used to prepare CAR-T cells, and then the killing efficiency of the CAR-T cells on target cells was determined according to the method of this Example 7. The results are shown in Table 2.

[0117] In addition, in Example 6, the exosome obtained in Example 3b was used to prepare CAR-T cells, and then the killing efficiency of the CAR-T cells on target cells was determined according to the method of this Example 7. The results are shown in Table 2.

[0118] Example 8: Cell proliferation and survival rate test 1. Preparation of CAR-mRNA vector plasmid without IL-7 and CCL17 elements Refer to Example 1, except that the nucleotide sequences of IL-7 and CCL17 are removed during the process of "constructing a CAR-19mRNA-MS2bs vector plasmid using pLVX-EF1α-IRES-puro plasmid", thereby preparing a CAR-mRNA vector plasmid without IL-7 and CCL17 elements.

[0119] 2. CAR-T cells were prepared according to the methods of Examples 2 to 6. The other functions of the CAR-T cells were the same as those of the CAR-T cells obtained in Example 6, but they did not contain the extracellular secretion function of IL-7 and CCL17. The CAR-T cells were recorded as Non-IL-7-CAR-T cells.

[0120] 3. The CAR-T cells described in Example 6 and the Non-IL-7-CAR-T cells obtained in this example were adjusted to a cell density of 1×10 6 / mL, and 100 μl of the cell suspension was inoculated into a 96-well plate and duplicate wells and blank control wells were set up. The culture plate was placed in a 37°C, 5% CO2 incubator for pre-culture for 24 hours; 4. Add 10 μl of soluble CD3 antibody and CD28 antibody at a final concentration of 10 ng / mL to the cell suspension in each well of the 96-well plate, mix thoroughly, and place the culture plate in an incubator for 4 hours; 5. Add 10 μl of CCK-8 solution to each well (be careful not to create bubbles in the wells) and place the culture plate in an incubator for 48 hours; 6. Take out the culture plate, aspirate the cell suspension, centrifuge and place the supernatant in a new 96-well plate, 50 μl per well; 7. Use an enzyme-labeled instrument to detect the absorbance of the solution at 450nm; the higher the OD value, the better the cell proliferation performance and the higher the survival rate; 8. Mix the cell pellet in step 6 with PBS, take 10 μl of the suspension for trypan blue staining, observe the cell staining under a microscope, and calculate the cell viability; 8. The OD values ​​and cell viability results of the 48-hour proliferation activity of CAR-T cells containing IL-7 and CCL17 elements and Non-IL-7-CAR-T cells are shown in Table 3 below.

[0121] Table 3:

[0122] In addition, in Example 6, the exosome obtained in Example 3a was used to prepare CAR-T cells, and then the cell proliferation and survival rate test was performed according to the method of this Example 8. The results showed that the OD value was 1.618 after 48 hours, and the cell viability was 97.6% calculated by trypan blue staining. In addition, in Example 6, the exosome obtained in Example 3b was used to prepare CAR-T cells, and then the cell proliferation and survival rate test was performed according to the method of this Example 8. The results showed that the OD value was 1.702 after 48 hours, and the cell viability was 99.2% calculated by trypan blue staining.

[0123] It can be seen from the above results that the CAR-T cells obtained in the present invention exhibit significantly better performance than Non-IL-7-CAR-T cells.

[0124] Example 9: IFN-γ interferon release experiment This example uses the Solebo human interferon gamma detection kit (Cat. No.: SEKH-0046) to conduct experiments.

[0125] 1. Gradient dilution of standard: Before opening the lid, ensure that all freeze-dried standards are at the bottom of the container, add 1 mL of SR1 standard / sample diluent to the freeze-dried standard (concentration is 1000 pg / mL), let it stand for 10 to 30 minutes until it is completely dissolved, and then gently mix it. Mix thoroughly before dilution, and dilute it 2 times according to the following concentrations: 500, 250, 125, 62.5, 31.25, 15.62, 7.81 pg / mL; 500 pg / mL is the highest point concentration of the standard curve, and SR1 standard / sample diluent is used as the 0 hole of the standard curve (0 pg / mL); calculate the amount required for the test in advance, and use SR2 detection diluent to dilute 1 100× antibody concentrate was diluted into 1× application working solution (mix thoroughly before dilution), and 100× concentrated enzyme conjugate was diluted with the diluted antibody application solution as the detection working solution, which was added to the reaction well within 30 minutes; after sealing the plate, incubated at room temperature (25±2°C) with shaking for 120 minutes, washed 4 times, and patted dry; 100μL detection working solution was added to the reaction well, and after sealing the plate, incubated at room temperature (25±2°C) with shaking for 60 minutes, washed 5 times, and patted dry; 100μL color development substrate was added to the reaction well, and after sealing the plate, color was developed at room temperature (25±2°C) in the dark for 5-30 minutes, and then 50μL stop solution was added, and the absorbance OD value was read with an enzyme reader.

[0126] 2. Draw the standard curve: Immediately use an ELISA reader for dual-wavelength detection, measure the OD values ​​at 450nm and 630nm wavelengths, and subtract the OD value at 630nm from the OD value at 450nm; calculate the average OD value of the standard: the OD value of each standard should be subtracted from the OD value of the blank control well; thus, the standard curve is drawn as y=0.0045x+0.006, R2=0.9992, where y is the OD value and x is the IFN-γ release amount (pg / mL).

[0127] 3. Prepare sample solution: The CAR-T cells prepared in Example 6 were adjusted to a density of 1×10 6 / mL, and the target cell Raji cell line expressing CD19 antigen (Shan En Biotech) was adjusted to a density of 1×10 6 / mL, inoculate the target cell suspension into a 24-well plate at 100 μl per well, inoculate CAR-T cells into each well at an effector-target ratio of 0:1 (no CAR-T cells added, also known as the negative control group), 0.5:1, 1:1, 2:1, and 4:1, and set duplicate wells, and use the culture medium x-vivo-15 to make up the reaction system to 1 mL. This group is recorded as the experimental group; At the same time, the peripheral blood mononuclear cells obtained in step 3 of Example 4 were washed by centrifugation and the density was adjusted to 1×10 6 / mL, inoculated into the target cell wells according to the same effector-target ratio, set up duplicate wells, and use the culture medium x-vivo1-5 to make up the reaction system to 1mL. This group was recorded as the control group; The 24-well plate containing the experimental group and the control group was placed in an incubator at 37°C and 5% CO2 for 4 hours, then taken out and the cell suspension in each well was pipetted into a 1.5 mL EP tube. After centrifugation at 400 g for 5 minutes, the supernatant was marked as the sample solution. 4. Calculate the IFN-γ secreted by the experimental group and the control group: According to the experimental method described in steps 1-2, the OD values ​​of the experimental group and the control group were detected, and the content of IFN-γ produced by each component was calculated according to the standard curve formula.

[0128] 5. The results of the IFN-γ release experiment are shown in Table 4.

[0129] Table 4:

[0130] In addition, in the test of the results shown in Table 4 above, the CAR-T cells prepared using the exosomes obtained in Example 3a in Example 6 were subjected to an equal test, and the results were 9.02, 16.13, 32.74, and 79.82, respectively, in terms of the effect-target ratio from low to high. In addition, in the test of the results shown in Table 4 above, the CAR-T cells prepared using the exosomes obtained in Example 3b in Example 6 were subjected to an equal test, and the results were 8.68, 15.43, 30.24, and 82.37, respectively, in terms of the effect-target ratio from low to high.

[0131] The above results show that the CAR-T cells obtained by the present invention can significantly release higher amounts of IFN-γ.

[0132] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A method for preparing CAR-T cells, comprising the following steps: (1) Construction of vector plasmid: The vector plasmid containing Anti-CD3-Lamp2b-MS2 element was constructed using pcDNA3.1 plasmid. Anti-CD28-Lamp2b-MS2 vector plasmid was constructed using pcDNA3.1 plasmid. The CAR-19mRNA-MS2bs vector plasmid was constructed using the pLVX-EF1α-IRES-puro plasmid; (2) Culture and transfection of mesenchymal stem cells: Primary mesenchymal stem cells are obtained by culturing Wharton's jelly stripped from the umbilical cord, and then mesenchymal stem cells of P2 to P8 are obtained by subculturing; three plasmids are transfected into mesenchymal stem cells using a plasmid transfection kit (e.g., Lipofectamine™ 3000 Reagent kit); (3) Isolation and purification of engineered exosomes: The mesenchymal stem cells transfected in the previous step were replaced with DMEM-high glucose medium without FBS, and the cells were cultured in a 37°C, 5% CO2 incubator with 2% O2 concentration (48 hours); the supernatant obtained from the cell culture was separated and purified by differential centrifugation to obtain engineered exosomes; (4) Preparation of initial T cells: The lower blood cell layer obtained by centrifugation of healthy adult peripheral blood is separated and treated with Ficoll-Hypaque lymphocyte separation solution to obtain a peripheral blood mononuclear cell (PBMC) layer; the peripheral blood mononuclear cells are then treated with magnetic beads (such as Dynabeads®) and activated in a T cell serum-free medium supplemented with IL-2 500 IU / ml and 1% autologous plasma to obtain initial T cells; (5) Preparation of CAR-T cells: The engineered exosomes were co-cultured with naive T cells, the cultured cell suspension was centrifuged, and the precipitate was cultured with T cell culture medium supplemented with IL-2 and human AB serum; The cell suspension was centrifuged, and the cell pellet was resuspended in PBS and the density was adjusted to obtain a CAR-T cell suspension.

2. According to the method of claim 1, in step (1): When constructing a vector plasmid containing the Anti-CD3-Lamp2b-MS2 element using the pcDNA3.1 plasmid, the plasmid and gene fragments used are: pcDNA3.1 plasmid, Anti-CD3-scFv gene sequence, Lamp2b gene sequence, MS2 Coat Protein gene sequence; or, When constructing a vector plasmid containing the Anti-CD3-Lamp2b-MS2 element using the pcDNA3.1 plasmid, proceed as follows: ① Design primers: designed to amplify the Anti-CD3-scFv-Lamp2b-MS2 gene sequence, ② Amplify the gene sequence: Use the PCR method to amplify the Anti-CD3-scFv-Lamp2b-MS2 sequence from the template DNA. ③ Purify PCR products: Separate PCR products by agarose gel electrophoresis, cut out the corresponding DNA bands and purify the PCR products using a gel recovery kit. ④ Double enzyme digestion: Use EcoRI and BamHI to digest the pcDNA3.1 plasmid, and digest the Anti-CD3-scFv-Lamp2b-MS2 gene fragment. ⑤ Ligation reaction: Use T4 DNA ligase to ligate the digested Anti-CD3-scFv-Lamp2b-MS2 gene fragment into the digested pcDNA3.1 plasmid. ⑥ Transformation of competent cells: The ligation reaction product was transformed into competent Stbl3 cells by heat shock method. The transformed cells were spread on LB agar plates containing antibiotics and cultured at 37°C overnight. ⑦ Screening of positive clones: Select a single colony, extract plasmid DNA using a small amount of plasmid extraction kit, and use restriction enzyme digestion and PCR methods to verify whether the plasmid contains the expected insert fragment. ⑧Sequence verification: Send samples for DNA sequencing to verify the correctness and integrity of the Anti-CD3-scFv-Lamp2b-MS2 gene sequence, to ensure that the reading frame of the inserted fragment is correct and there is no mutation. ⑨Plasmid extraction: Use a large number of plasmid extraction kits to extract plasmid DNA from positive clones, store it at low temperature for later use; or, Use pcDNA3.1 plasmid to construct a vector plasmid containing Anti-CD3-Lamp2b-MS2 elements, replace Anti-CD3 with Anti-CD28, and use pcDNA3.1 plasmid to construct Anti-CD28-Lamp2b-MS2 vector plasmid; or, The sequence of CAR-19mRNA-MS2bs is composed of CD19 single-chain variable region, CD8a hinge region, CD8a transmembrane region, 4-1BB signal, CD3ζ clathrate signal, IL-7, CCL17, and MS2bs sequence; or, Select appropriate restriction endonucleases and ligases, and use pLVX-EF1α-IRES-puro plasmid to construct CAR-19mRNA-MS2bs vector plasmid.

3. According to the method of claim 1, in step (2): Obtaining primary (P0) mesenchymal stem cells from the umbilical cord Wharton's jelly using a known method, and then performing subculture to obtain mesenchymal stem cells of cell generations P3 to P7, such as P3 to P6, such as P4 to P6; or, When using the plasmid transfection kit to transfect mesenchymal stem cells with three plasmids, add the DNA-Lipo3000 complex solution obtained by the kit treatment dropwise into the cell culture well plate, gently shake the culture plate to evenly distribute the transfection complex, incubate for 4-6 hours, then replace the serum-free Opti-MEM medium with DMEM high-glucose medium (containing 10% FBS), and continue to culture the cells for 48-72 hours to allow plasmid expression; or, The DMEM high-glucose medium (containing 10% FBS) is a modified DMEM high-glucose medium (containing 10% FBS); or, Plasmid transfection Mesenchymal stem cells were transfected using the Lipofectamine™ 3000 Reagent Kit as follows: (a) Mesenchymal stem cells were seeded into 6-well plates and 2×10 5 Cells were cultured in a 37°C, 5% CO2 incubator until the cell density reached 50-70% confluence; (b) Dilute the three plasmid DNAs into serum-free Opti-MEM medium, and the required amount of plasmid per well is 2.5 μg; In another centrifuge tube containing serum-free Opti-MEM medium, add 5 µL of Lipofectamine 3000 reagent to each centrifuge tube; Add 10 µL of P3000 Reagent to each plasmid DNA solution; Add Lipofectamine 3000 reagent solution dropwise to the plasmid DNA solution, mix gently, and incubate at room temperature for 15-20 minutes to form DNA-Lipo3000 complex; (c) replacing the cell culture medium in the 6-well plate with serum-free Opti-MEM medium; Add the DNA-Lipo3000 complex solution in each tube dropwise into the 6-well plate for cell culture, and gently shake the culture plate to evenly distribute the transfection complex; (d) The cells were incubated in a 37°C, 5% CO2 incubator for 4-6 hours, and then the serum-free Opti-MEM medium was replaced with modified DMEM high-glucose medium (containing 10% FBS) and the cells were cultured for another 48-72 hours to allow plasmid expression.

4. According to the method of claim 1, in step (3), the culture medium of the mesenchymal stem cells transfected in the previous step is replaced with a modified DMEM-high glucose medium (without FBS), and the cells are cultured in an incubator containing 2% O2 at 37°C and 5% CO2 (e.g., for 48 hours).

5. According to the method of claim 1, in step (3), the cell supernatant obtained by culturing mesenchymal stem cells in a modified DMEM-high glucose medium (without FBS) is placed in a centrifuge tube and subjected to the following differential centrifugation to obtain engineered exosomes: Centrifuge at 300g and 4°C for 10 min, and transfer the supernatant to another centrifuge tube; Centrifuge at 2000g and 4°C for 20 min, and transfer the supernatant to another centrifuge tube; Centrifuge at 10,000 g and 4 °C for 30 min, filter the supernatant with a 0.22 μm filter membrane and place in another centrifuge tube; Centrifuge at 100,000 g and 4 °C for 90 min and discard the supernatant; Sterile PBS was added to the centrifuge tube to resuspend the exosome precipitate, and the mixture was centrifuged at 100,000 g and 4° C. for 90 min. The supernatant was discarded, and sterile PBS was added to resuspend the exosomes to obtain an exosome suspension.

6. according to the method for claim 1, in step (2) and / or step (3), the formula of described DMEM high glucose medium (containing 10% FBS) is: 200mg / L anhydrous CaCl2, 0.1mg / L nonahydrate Fe(NO3)3, 97.67mg / L anhydrous MgSO4, 400mg / L KCl, 3700mg / L NaHCO3, 6400mg / L NaCl, 109mg / L anhydrous NaH2PO4, 434mg / L L-alanyl-L-glutamine, 30mg / L glycine, 84mg / L L-arginine hydrochloride, 62.6mg / L L-cystine dihydrochloride, 584mg / L L-glutamine, 42mg / L L-histidine hydrochloride monohydrate, 10 5mg / L L-isoleucine, 105mg / L L-leucine, 146mg / L L-lysine hydrochloride, 30mg / L L-methionine, 66mg / L L-phenylalanine, 42mg / L L-serine, 95mg / L L-threonine, 16mg / L L-tryptophan, 103.79mg / L L-tyrosine disodium salt dihydrate, 94mg / L L-valine, 4mg / L choline chloride, 4mg / L D-calcium pantothenate, 4mg / L folic acid, 4mg / L niacinamide, 4mg / L pyridoxine hydrochloride, 0.4mg / L riboflavin, 4mg / L thiamine hydrochloride, 7mg / L thiamin hydrochloride, 7 ...thiamin hydrochloride, 7mg / L choline chloride, 4mg / L D-calcium pantothenate, 4mg / L folic acid, 4mg / L niacinamide, 4mg / L pyridoxine hydrochloride, 0.4mg / L riboflavin, .2mg / L meso-inositol, 4500mg / L D-glucose, 110mg / L sodium pyruvate, 15mg / L phenol red, 100kU / L penicillin G sodium salt, 100mg / L streptomycin sulfate, 10% fetal bovine serum, and water added to the full amount; or, the formula of the DMEM high-glucose medium (without FBS) is: 200mg / L anhydrous CaCl2, 0.1mg / L nonahydrate Fe(NO3)3, 97.67mg / L anhydrous MgSO4, 400mg / L KCl, 3700mg / L NaHCO3, 6400mg / L NaCl, 109mg / L anhydrous Water NaH2PO4, 434mg / L L-alanyl-L-glutamine, 30mg / L glycine, 84mg / L L-arginine hydrochloride, 62.6mg / L L-cystine dihydrochloride, 584mg / L L-glutamine, 42mg / L L-histidine hydrochloride monohydrate, 105mg / L L-isoleucine, 105mg / L L-leucine, 146mg / L L-lysine hydrochloride, 30mg / L L-methionine, 66mg / L L-phenylalanine, 42mg / L L-serine, 95mg / L L-threonine, 16mg / L L-tryptophan, 103.79 mg / L of L-tyrosine disodium salt dihydrate, 94 mg / L of L-valine, 4 mg / L of choline chloride, 4 mg / L of D-calcium pantothenate, 4 mg / L of folic acid, 4 mg / L of niacinamide, 4 mg / L of pyridoxine hydrochloride, 0.4 mg / L of riboflavin, 4 mg / L of thiamine hydrochloride, 7.2 mg / L of meso-inositol, 4500 mg / L of D-glucose, 110 mg / L of sodium pyruvate, 15 mg / L of phenol red, 100 kU / L of penicillin G sodium salt, 100 mg / L of streptomycin sulfate, and water is added to the full amount; or, the modified DMEM high glucose medium (containing 10% The formula of FBS is: 200 mg / L anhydrous CaCl2, 0.1 mg / L nonahydrate Fe(NO3)3, 97.67 mg / L anhydrous MgSO4, 400 mg / L KCl, 3700 mg / L NaHCO3, 6400 mg / L NaCl, 109 mg / L anhydrous NaH2PO4, 124 mg / L sodium arachidonic acid, 56 mg / L zinc sulfate, 434 mg / L L-propylamine acyl-L-glutamine, 30 mg / L glycine, 84 mg / L L-arginine hydrochloride, 62.6 mg / L L-cystine dihydrochloride, 584 mg / L L-glutamine, 42 mg / L L-histidine hydrochloride monohydrate, 105 mg / L L-isoleucine, 105 mg / L L-leucine, 146 mg / L L-lysine hydrochloride, 30 mg / L L-methionine, 66 mg / L L-phenylalanine amino acid, 42mg / L L-serine, 95mg / L L-threonine, 16mg / L L-tryptophan, 103.79mg / L L-tyrosine disodium salt dihydrate, 94mg / L L-valine, 4mg / L choline chloride, 4mg / L D-calcium pantothenate, 4mg / L folic acid, 4mg / L niacinamide, 4mg / L pyridoxine hydrochloride, 0.4mg / L riboflavin, 4mg / L thiamine hydrochloride, 7. 2mg / L meso-inositol, 4500mg / L D-glucose, 110mg / L sodium pyruvate, 15mg / L phenol red, 100kU / L penicillin G sodium salt, 100mg / L streptomycin sulfate, 10% fetal bovine serum, and water added to the full amount; or, the formula of the modified DMEM high-glucose medium (without FBS) is: 200mg / L anhydrous CaCl2, 0.1mg / L Fe(NO3)3 nonahydrate, 97.67mg / L anhydrous MgSO4, 400mg / L KCl, 3700mg / L NaHCO3, 6400mg / L NaCl, 109mg / L anhydrous NaH2PO4, 124mg / L sodium arachidonic acid, 56mg / L zinc sulfate, 434mg / L L-alanyl-L-glutamine, 30mg / L glycine, 84mg / L L-arginine hydrochloride, 62.6mg / L L-cystine dihydrochloride, 584mg / L L-glutamine, 42mg / L L-histidine hydrochloride monohydrate, 105mg / L L-isoleucine, 105mg / L L-leucine, 146mg / L L-lysine hydrochloride, 30mg / L L-methionine Acid, 66mg / L L-phenylalanine, 42mg / L L-serine, 95mg / L L-threonine, 16mg / L L-tryptophan, 103.79mg / L L-tyrosine disodium salt dihydrate, 94mg / L L-valine, 4mg / L choline chloride, 4mg / L D-calcium pantothenate, 4mg / L folic acid, 4mg / L niacinamide, 4mg / L pyridoxine hydrochloride, 0.4mg / L riboflavin, 4mg / L thiamine hydrochloride, 7.2mg / L meso-inositol, 4500mg / L D-glucose, 110mg / L sodium pyruvate, 15mg / L phenol red, 100kU / L penicillin G sodium salt, 100mg / L streptomycin sulfate, add water to the full amount.

7. According to the method of claim 1, in step (4), the initial T cells are obtained by: 100 ml of healthy adult peripheral blood containing sodium citrate anticoagulant was placed in a 250 ml centrifuge cup and centrifuged at 1500 rpm and 20°C for 15 min to collect the upper plasma; Add the lower blood cell layer to an equal volume of 0.9% sodium chloride injection and mix thoroughly. Add 20 ml of Ficoll-Hypaque lymphocyte separation solution to a 50 ml centrifuge tube, then slowly add an equal volume of blood cell suspension along the tube wall to the upper layer of lymphocyte separation solution, then centrifuge the centrifuge tube at 2000 rpm for 20 min, and adjust the centrifuge speed to 0; After discarding the supernatant of the centrifuged cell suspension, carefully aspirate the middle buffy coat layer, which is the peripheral blood mononuclear cell layer, i.e., the PBMC layer; Place the Dynabeads® magnetic beads on a vortex mixer and mix thoroughly for 30 seconds. Calculate the required number of Dynabeads® / total volume according to the ratio of Dynabeads®:PBMC=1:

1. Add an equal volume of PBS to the Dynabeads® and mix thoroughly. Place Dynabeads® on the magnetic stand for 1 min, discard the supernatant, add an equal volume of PBS, and repeat this step 3 times; Remove Dynabeads® from the magnetic stand, add T cell serum-free medium, and mix thoroughly; Dynabeads® was thoroughly mixed with PBMC, suspended on a vortex mixer for 10 seconds, and then allowed to stand at 25°C for 10 minutes. 6 / ml seeding density was inoculated into culture flasks, and the cells were activated in T cell serum-free culture medium supplemented with IL-2 500IU / ml and 1% autologous plasma in a 37°C, 5% CO2 incubator for 24 hours to obtain initial T cells.

8. According to the method of claim 1, in step (5), the engineered exosomes obtained in step (3) and the initial T cells obtained in step (4) are mixed at a rate of 1×10 9 Particles / mL exosomes: 1×10 6 / mL T cells were fully mixed, inoculated into culture well plates, and cultured overnight in a 37°C, 5% CO2 incubator.

9. According to the method of claim 1, in step (5), after the exosomes are co-cultured with the initial T cells overnight, the cell suspension is aspirated, centrifuged at 300 g for 5 minutes, the supernatant is aspirated, T cell culture medium is added, IL-2 is added to a concentration of 500 IU / ml, and 5% human AB serum is added to the culture medium volume, and the culture medium is placed in a 37°C, 5% CO2 incubator for culture. Fresh culture medium is added every 48 hours and the cell density is adjusted to 1×10 6 / mL.

10. According to the method of claim 1, in step (5), after culturing in a medium containing IL-2 and human AB serum for 7 days, the cell suspension is aspirated, the supernatant is discarded by centrifugation, the cell pellet is resuspended with PBS, and the density is adjusted to obtain a suspension of CAR-T cells.

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