Construction and use of targeted exosomal chimeric antigen receptor molecule

Abstract

Provided are the construction and use of a targeted exosomal chimeric antigen receptor molecule. Provided is a fusion RNA comprising, from 5' to 3': an RNA sequence as shown in SEQ ID NO. 1 and an RNA recognition sequence of an RNA binding protein. The RNA binding protein comprises L7AE, the RNA recognition sequence of the L7AE comprises a sequence encoding a C / D box, and the sequence encoding the C / D box is as shown in SEQ ID NO. 2. There is no need to extract T cells from a patient for in vitro CAR-T construction, proliferation and reinfusion, an off-the-shelf CAR-T product is realized, and in vivo transformation of T cells into CAR-T cells is achieved, thereby achieving the applicability and timeliness of clinical treatment.

Description

Construction and application of a chimeric exosome antigen receptor molecule Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the construction and application of a targeted exosome chimeric antigen receptor molecule. Background Technology

[0002] Malignant tumors are classified into hematologic malignancies and solid organ malignancies. The World Health Organization classifies hematologic malignancies into four main categories based on cell line origin: myeloid tumors, lymphoid tumors, histiocytic tumors, and mast cell tumors. Lymphomas mainly include Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma, with B-cell lymphoma accounting for almost 95% of all lymphoma cases. Patients with B-cell lymphoma are typically characterized by enlarged lymph nodes, extranodal disease, or both, and may involve multiple organs. Research on B-cell lymphoma is crucial for improving treatment outcomes, reducing treatment side effects, and prolonging patient survival.

[0003] Current treatment options for B-cell lymphoma include surgery, chemotherapy, radiotherapy, and immunotherapy. While radiotherapy, chemotherapy, and surgery have achieved some success in controlling and treating B-cell malignancies, they still face several complex challenges, such as a lack of specific and personalized treatments, numerous adverse reactions, tumor heterogeneity, and tumor drug resistance. Immunotherapy is a relatively new cancer treatment method with the potential for high precision and personalization, and it is more effective than other types of cancer therapies. Today, immunotherapy is widely used to treat B-cell lymphoma, including antigen-specific monoclonal antibodies (e.g., anti-CD20), immune checkpoint inhibitors (ICIs), and chimeric antigen receptor T cells. CAR-T cell therapy drugs have brought new hope to lymphoma treatment.

[0004] The scFv, created from the VH and VL of the anti-CD19 antibody, is linked to the transmembrane sequence of CD28 or CD8, and then to the CD3ζ chain and the intracellular signaling domain of CD28 or CD137

[0024] . CAR simultaneously confers a binding domain from the scFv and a linking signaling domain in a single transmembrane protein, thereby activating vector-transduced T cells (CAR-T). In CAR-T cell therapy, CAR-T cells enable T cells to bind to target cell surface antigens via a single-chain variable fragment recognition domain, mediating major histocompatibility complex-restricted escape, redirecting cytotoxic T lymphocytes (CTLs) to target cells expressing the antigen, thereby killing target cell tumors and achieving precision treatment. After binding, a non-classical immune synapse is formed between the CTL and the target, mediating its anti-tumor effect through the perforin and granzyme axis, the Fas and Fas ligand axis, and cytokines. In this study, perforin and granzyme play the primary role in cell lysis and degranulation, while cytokine regulation and FasL-induced apoptosis contribute synergistically. In the granzyme-dependent pathway, perforin forms pores, allowing granzyme to enter target cells, promoting the activation of the caspase family of lethal proteases and inducing caspase-dependent apoptosis. FasL-induced cross-linking of the target cell surface death receptor Fas leads to rapid induction of the assembly of the intracellular "lethal signal complex" (DISC), which in turn activates caspase-3, resulting in cell death. Cytokines produced by activated CAR-T cells can induce the expression of interferon-γ (IFN-γ) receptors in the tumor matrix, driving antigen-independent matrix destruction-mediated tumor cell killing. Perforin, granzyme, cytokines, and FasL collectively participate in the killing of cancer cells, thus achieving therapeutic effects.

[0005] B-cell leukemia and lymphoma are the most common subtypes of hematologic malignancies, and relapse and refractory disease are the main causes of treatment failure in clinical practice. CAR-T cell therapy has revolutionized the treatment of hematologic malignancies. Currently, CARs are introduced into T cells using plasmids or viral vectors (such as adenovirus, retrovirus, or lentivirus), with lentiviruses being the most common method for transducing human T cells. The high cost, low yield, and easy loss of activity of lentiviruses directly affect the application of CAR-T products in treatment. Furthermore, severe toxicities associated with CAR-T cell therapy can also affect its efficacy and may even lead to life-threatening diseases. Therefore, the development of low-cost CAR-T cell products and the search for safe and effective alternative CAR-T cell therapies are particularly important. Summary of the Invention

[0006] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0007] The purpose of this invention is to provide a method for constructing and applying a chimeric antigen receptor molecule targeting exosomes.

[0008] On one hand, the present invention provides a fusion RNA, comprising, from 5'-3', the RNA sequence shown in SEQ ID NO.1 and the RNA recognition sequence of the RNA-binding protein.

[0009] According to a specific embodiment of the present invention, preferably, the RNA-binding protein includes L7AE.

[0010] According to a specific embodiment of the present invention, preferably, the RNA recognition sequence of the L7AE includes a sequence encoding a C / D box, the sequence encoding the C / D box being shown in SEQ ID NO.2.

[0011] On the other hand, the present invention provides a nucleic acid molecule encoding the above-mentioned fusion RNA.

[0012] In this invention, unless otherwise specified, the term "nucleic acid molecule" refers to a nucleotide sequence consisting of polynucleotides containing purine and pyrimidine bases, wherein the nucleotides represent the primary structure of a nucleic acid molecule. Here, the term "nucleic acid molecule" includes cDNA, genomic DNA, RNA, synthetic DNA, and mixed polymers containing two or more of these molecules. Furthermore, the term "nucleic acid molecule" also includes sense strands and antisense strands.

[0013] In another aspect, the present invention provides a carrier comprising the above-mentioned nucleic acid molecules.

[0014] In this invention, "vector" refers to any nucleic acid construct capable of guiding the expression of a target gene and transferring gene sequences to target cells. This term includes cloning and expression vectors, as well as integration vectors.

[0015] In another aspect, the present invention provides an RNA delivery system comprising the above-mentioned vector, a vector expressing exosomal transmembrane proteins and RNA-binding proteins, and a vector expressing a target molecule and exosomal surface membrane proteins. Preferably, the target molecule comprises a target peptide, an antibody or its antigen-binding fragment, or an affinity.

[0016] In some specific embodiments of the present invention, the RNA-binding protein includes L7AE.

[0017] In some specific embodiments of the present invention, the exosome transmembrane protein includes CD63, CD9, or CD81.

[0018] In some specific embodiments of the present invention, the RNA-binding protein is attached to the C-terminus of an exosome transmembrane protein.

[0019] In some specific embodiments of the present invention, the targeting molecule includes a targeting peptide that interacts with a cell membrane surface antigen, an antibody or its antigen-binding fragment that recognizes a specific cell surface antigen, or an affinity.

[0020] In some specific embodiments of the present invention, the exosome surface membrane protein includes Lamp2b.

[0021] In some specific embodiments of the present invention, the targeting molecule is attached to the N-terminus of an exosome surface membrane protein.

[0022] In another aspect, the present invention provides an exosome comprising the aforementioned fusion RNA, and the exosome further comprising a targeting molecule capable of recognizing a target tissue or target cell.

[0023] In some specific embodiments of the present invention, the targeting molecule is the sequence of an antibody targeting CD3, CD4, and CD8, as shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively.

[0024] In some specific embodiments of the present invention, preferably, the targeting molecule is the sequence of an antibody targeting CD8, as shown in SEQ ID NO.5.

[0025] Furthermore, the present invention provides a method for preparing the above-mentioned exosomes, the specific steps of which are as follows:

[0026] The RNA delivery system described above is introduced into cells that produce exosomes; the cells are then cultured to produce exosomes.

[0027] In another aspect, the present invention provides a cell, wherein the cell is a cell that has been introduced into the above-described RNA delivery system or that produces and secretes the above-described exosomes.

[0028] The cells or cells capable of secreting exosomes described in this invention include, but are not limited to, primary cells, cell lines, cells present in multicellular organisms, or substantially any other type of cell source. The cells of this invention include cells capable of producing exosomes in vivo.

[0029] In some specific embodiments of the present invention, the cells are 293F, 293T or 293TF cells that have been introduced into the above-described RNA delivery system or that produce and secrete the above-described exosomes.

[0030] In this invention, the methods for introducing exogenous nucleic acid molecules into exosomes or cells are known to those skilled in the art, including but not limited to lipid-mediated transfer (i.e., liposomes, including neutral and cationic lipids), electroporation, direct injection, biological projectiles, cell fusion, particle bombardment, calcium phosphate coprecipitation, DEAE-glucan-mediated transfer, and other methods such as viral vector-mediated transfer.

[0031] In another aspect, the present invention provides a method for preparing the above-mentioned cells, the method comprising the following steps: introducing the above-mentioned RNA delivery system into cells that produce exosomes.

[0032] In some specific embodiments of the present invention, the present invention constructs an exosome targeting system and an exosome mRNA targeting delivery system; specifically, it is achieved through the following steps: (1) Integrating exogenous mRNA into exosomes: L7Ae is coupled to the C-terminus of CD63, and the C / D box is inserted into the 3' untranslated region (UTR) of the CD19CAR plasmid, and L7Ae interacts with the C / D box in the 3'UTR. (1) Box interaction, packaging the mRNA encoding human CD19CAR into exosomes; (2) Using the LAMP-2B exosome targeting principle to construct a targeted exosome system: the antibody targeting CD8 (sna06) was fused into the N-terminus of LAMP-2B to construct the LAMP-2B targeting plasmid so that the targeting peptide is expressed on the surface of the exosome, and the contents of the exosome are selectively delivered to the target cells (CD8+T cells); (3) Using genetic engineering technology, the LAMP-2B targeting plasmid and the CD19CAR plasmid were co-transfected into 293F cells to obtain exosomes targeting T cells (CD19CAR targeting exosomes). The exosomes were co-cultured with PBMCs, and the exosomes targeted CD19CAR to T cells, thus constructing an exosome targeting system and improving the function of the target receptor cells of the exosomes.

[0033] In another aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned exosomes and cells.

[0034] According to a specific embodiment of the present invention, preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier or excipient.

[0035] The term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to the subject and does not eliminate the biological activity and properties of the extracellular vesicles applied in the composition. Pharmaceutically acceptable carriers can enhance or stabilize the composition, or can be used to facilitate the preparation of the composition. Pharmaceutically acceptable carriers may include physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents, etc. Carriers can be selected to minimize adverse side effects in the subject and / or minimize the degradation of one or more active ingredients. Adjuvants may also be included in any of these formulations.

[0036] The term "pharmaceuticalally acceptable excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of the active ingredient. Parenteral formulations may contain, for example, excipients such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, or hydrogenated naphthalene. Other exemplary excipients include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, ethylene-vinyl acetate copolymer particles, and surfactants (including, for example, polysorbate 20).

[0037] The pharmaceutical compositions of the present invention can be administered by a variety of methods known in the art. The route and / or manner of administration can vary depending on the desired outcome.

[0038] In another aspect, the present invention provides the use of the above-mentioned fusion RNA, nucleic acid molecule, vector, RNA delivery system, exosome or cell in the preparation of a drug for the prevention and / or treatment of CD19 expression-related tumors;

[0039] According to a specific embodiment of the present invention, preferably, the tumor is a B-cell lymphoma.

[0040] This invention provides a technique for transfecting L7Ae-containing DNA, DNA containing mRNA capable of translating CARs, and DNA containing a CD8-targeting nanobody and a LAMP-2B fusion protein into cells that secrete exosomes via plasmid transfection. Based on exosome self-assembly, CAR-exosomes can be generated in a single step. In this invention, the mRNA within the CAR-exosome CD8-targeting exosome fuses with T cells via cell receptor-mediated membrane fusion, ultimately achieving in vivo conversion of T cells into CAR-T cells.

[0041] The technical solution provided by this invention does not require the extraction of patient T cells for in vitro CAR-T construction, proliferation, and reinfusion. It can realize off-the-shelf CAR-T products and enable the in vivo transformation of one's own T cells into CAR-T, achieving universality and timeliness in clinical treatment. Attached Figure Description

[0042] Figure 1 is a schematic diagram of the mechanism of the exosome mRNA targeted delivery system.

[0043] Figure 2 shows the targeting validation of the exosome mRNA targeted delivery system.

[0044] Figure 3 shows a comparative analysis of CD19 CAR C / D box targeting exosomes to CD3+ T, CD4+ T, and CD8+ T cells.

[0045] Figure 4 shows the detection of CAR expression level in CD8+ T cells after incubation with targeted exosomes.

[0046] Figure 5 shows the detection of CAR expression levels in CD3+ T cells after incubation with targeted exosomes.

[0047] Figure 6 shows the curve of CAR expression in CD8+ T cells over time after incubation with targeted exosomes.

[0048] Figure 7 shows the assay of CAR-T positive cells prepared in vitro from CD19CAR-C / D box-CD8 targeted exosomes.

[0049] Figure 8 shows the in vitro preparation of CAR-T cells using CD19CAR-C / D box-CD8 targeted exosomes and their cytotoxicity assay.

[0050] Figure 9 shows the in vivo preparation of CAR-T cell toxicity using CD19CAR-C / D box-CD8 targeted exosomes.

[0051] Figure 10 shows the kinetics and tissue distribution of CAR-T cells prepared in vivo from CD19CAR-C / D box-CD8 targeted exosomes. Detailed Implementation

[0052] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0053] The invention can be further described through the following embodiments; however, the scope of the invention is not limited to the embodiments described below. Those skilled in the art will understand that various changes and modifications can be made to the invention without departing from its spirit and scope. The materials and methods used in the experiments are described generally and / or specifically. Although many materials and methods of operation used to achieve the objectives of the invention are well known in the art, they are still described in as much detail as possible herein. Methods not specifically described are performed according to conventional operation in the art or as recommended in the manufacturer's specification.

[0054] Example 1: Construction of an exosomal mRNA targeting delivery system specifically targeting CD3+T, CD4+T, and CD8+T cells

[0055] This embodiment provides an active packaging system for RNA entry into exosomes and an exosomal mRNA targeted delivery system that facilitates RNA delivery to target cells, the mechanism of which is shown in Figure 1. Exogenous mRNA is easily degraded in vivo, and due to the lack of a native conformation, exogenous proteins cannot perform the required functions. The archaea ribosomal protein L7Ae can bind to the C / D box structure of RNA, aiding in RNA folding and stability. L7Ae is coupled to the C-terminus of CD63, and the C / D box is inserted into the 3' untranslated region (UTR) of the CD19CAR plasmid. Through the interaction between L7Ae and the C / D box in the 3' UTR, the mRNA encoding human CD19CAR is integrated into the exosome. The targeting molecules (sequences of antibodies targeting CD3, CD4, and CD8, as shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively) were fused to the N-terminus of the lysosome-associated membrane glycoprotein Lamp2b to construct the targeting plasmids CD3-Lamp2b, CD4-Lamp2b, and CD8-Lamp2b. The exosomes functionalized with the fusion proteins were able to drive the recognized targets into the lysosomes of the recipient cells (CD8+T).

[0056] The specific construction steps are as follows:

[0057] (1) Integrating exogenous mRNA into exosomes: L7Ae is coupled to the C-terminus of CD63 and the C / D box is inserted into the 3' untranslated region (UTR) of the CD19CAR plasmid. Through the interaction between L7Ae and the C / D box in the 3'UTR, the mRNA encoding human CD19CAR is packaged into exosomes.

[0058] (2) Construction of a targeted exosome system using the LAMP-2B exosome targeting principle: The antibody sequences targeting CD3, CD4, and CD8 are fused into the N-terminus of LAMP-2B to construct a LAMP-2B targeting plasmid, so that the targeting peptides are expressed on the surface of exosomes, and the contents of exosomes are selectively delivered to target cells (CD3+T, CD4+T, CD8+T cells);

[0059] (3) Using genetic engineering technology, LAMP-2B targeting plasmid and CD19CAR plasmid were co-transfected into 293F cells to obtain exosomes targeting T cells (CD19CAR targeting exosomes). The exosomes were co-cultured with PBMCs, and the exosomes were used to target and deliver CD19CAR to T cells to construct an exosome targeting system to improve the function of the target receptor cells of exosomes.

[0060] Regarding plasmid design for the CD19CAR-C / D box: Currently, the internationally recognized CD19 monoclonal antibody scFv clones used in CD19CAR therapy are mainly FMC63 and SJ25C1. FMC63 was selected as the CD19-specific monoclonal antibody. A G4S linker exists between the VL and VH domains, sequentially tandemly linking scFv, StrepII, the CD8 hinge and transmembrane domains, ICD, and the CD3ζ intracellular domain, thus constructing the classic second-generation CAR molecule CD19CAR plasmid targeting human CD19. StrepII serves as a tag, and the CD3ζ transmembrane domain mediates the binding of the CAR dimer to endogenous TCRs, promoting T cell activation. CAR-T cells with the CD8α hinge and transmembrane domains release less IFNγ and TNFα than CAR-T cells with the CD28 domain and are less sensitive to AICD, thus linking downstream into the C / D box.

[0061] Regarding the design of the LAMP-2B targeting plasmid: Taking the BRD-PTK plasmid as an example, the specific experimental methods are described below: The nanobody sequences targeting CD3, CD4, and CD8 were synthesized by Sangon Biotech Co., Ltd. When designing primers, homologous fragments of the expression vector were introduced at both ends of the nanobody fragments. The targeting expression vector BRD-PTK-Lamp2b was purchased from Invivo. A linearized vector was obtained through double enzyme digestion. The restriction enzyme sites of the vector BRD-PTK-Lamp2b are AgeI and BsrGI. The product was subjected to 0.7% agarose gel electrophoresis, and the gel was excised and recovered into Eppendorf tubes. The corresponding fragments were recovered using a Tiangen agarose gel recovery kit, and the purity and concentration of the product were determined. The plasmids were inoculated into 5 mL of LB liquid medium and cultured at 37°C and 220 rpm for 12 hours. Plasmids were extracted using the Tiangen mini-prep kit to obtain plasmids BRD-PTK-CD3-Lamp2b, BRD-PTK-CD4-Lamp2b, and BRD-PTK-CD8-Lamp2b. These plasmids were sent to Sangon Biotech (Shanghai) Co., Ltd. for first-generation sequencing verification and were found to be correct. The plasmid construction diagram is shown in Figure 1.

[0062] In this embodiment, the sequence of the human CD19 CAR is shown in SEQ ID NO.1.

[0063] CAR sequences containing the packaging signal CD19:

[0064] The sequence of the encoded C / D box in this embodiment is shown in SEQ ID NO.2.

[0065] The sequences of the antibodies targeting CD3, CD4, and CD8 in this embodiment are shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively.

[0066] Example 2: Targeting validation of the exosome mRNA targeted delivery system

[0067] As shown in Figure 2A, the transfection rates of 293F cells in the CD19CAR-C / D box, CD19CAR-C / D box-CD3, CD19CAR-C / D box-CD4, and CD19CAR-C / D box-CD8 groups were 39.7%, 46.1%, 37%, and 40.6%, respectively, as detected by flow cytometry. The 293F group consisted of untransfected 293F cells, the CD19CAR-C / D box group was a control without the target plasmid, and the CD19CAR-C / D box-CD3, CD19CAR-C / D box-CD4, and CD19CAR-C / D box-CD8 groups were 293F cells transfected with the CD3-Lamp2b, CD4-Lamp2b, and CD8-Lamp2b target plasmids, respectively. This indicates that 293F cells can express the target protein CD19CAR-C / D box after stable transfection.

[0068] As shown in Figure 2B, flow cytometry was used to detect the percentage of CAR-T cells in CD3+T, CD4+T, and CD8+T cells. The results showed that the percentages of CAR-T cells in CD3+T cells were 3.53%, 13.3%, 7.73%, and 47.3% in the CD19CAR-C / D box, CD19CAR-C / D box-CD3, CD19CAR-C / D box-CD4, and CD19CAR-C / D box-CD8 groups, respectively. Positive CAR-T cell populations were found in the CD3-Lamp2b, CD4-Lamp2b, and CD8-Lamp2b groups.

[0069] As shown in Figure 2C, the percentages of CAR-T cells in CD4+ T cells were 2.13%, 3.85%, 12.4%, and 5.97% in the CD19CAR-C / D box, CD19CAR-C / D box-CD3, CD19CAR-C / D box-CD4, and CD19CAR-C / D box-CD8 groups, respectively. Only the CD4-Lamp2b group showed a weakly positive CAR-T cell population.

[0070] As shown in D of Figure 2, the percentages of CAR-T cells in CD8+ T cells were 2.9%, 14.7%, 4.12%, and 93.7% in the CD19CAR-C / D box, CD19CAR-C / D box-CD3, CD19CAR-C / D box-CD4, and CD19CAR-C / D box-CD8 groups, respectively. Only the CD8-Lamp2b group had a strongly positive CAR-T cell population.

[0071] In Figure 2, the PBMC group showed no exosomes. These data indicate that a positive CAR-T cell population can only be obtained in the presence of the target plasmid. CD3-Lamp2b, CD4-Lamp2b, and CD8-Lamp2b all showed targeting effects. This embodiment successfully constructed a CD19CAR-C / D box-targeting exosome-based ...

[0072] Example 3: Comparative analysis of CD19CARC / D box-targeted exosomes targeting CD3+ T, CD4+ T, and CD8+ T cells

[0073] The percentage of CAR-T cells in CD3+T, CD4+T, and CD8+T cells was statistically analyzed using GraphPadPrism8, and the results are shown in Figure 3. In the CD19CAR-C / D box-CD8 group, Strep-II expression was highest in CD3+T and CD8+T cells; the CD8-Lamp2b plasmid showed the best targeting effect in the exosome mRNA targeted delivery system.

[0074] Example 4: Detection of CAR expression level in CD8+ T cells after incubation with targeted exosomes

[0075] The percentage of CAR-T cells in CD8+ T cells on days 2, 5, 7, 9, and 12 was analyzed using FlowJO, and the results are shown in Figure 4. As shown in Figure 4A, in the CD19CAR, CD19CAR-CD8, CD19CAR-C / D box, and CD19CAR-C / D box-CD8 groups, the percentage of CAR-T cells in CD8+ T cells on day 2 was 5.57%, 66.5%, 0.22%, and 50.3%, respectively. Only when CD8-Lamp2b was present was there a positive CAR-T cell population in CD8+ T cells.

[0076] As shown in Figure 4B, in the CD19CAR, CD19CAR-CD8, CD19CAR-C / D box, and CD19CAR-C / D box-CD8 groups, the percentages of CAR-T cells in CD8+ T cells at DAY5 were 11.5%, 65.7%, 0.35%, and 84.7%, respectively. Only when CD8-Lamp2b was present were there positive CAR-T cell populations in CD8+ T cells.

[0077] As shown in Figure 4C, in the CD19CAR, CD19CAR-CD8, CD19CAR-C / D box, and CD19CAR-C / D box-CD8 groups, the percentages of CAR-T cells in CD8+ T cells at DAY7 were 0.26%, 39.7%, 0.074%, and 77.9%, respectively. Only when CD8-Lamp2b was present were there positive CAR-T cell populations in CD8+ T cells.

[0078] As shown in Figure 4D, in the CD19CAR, CD19CAR-CD8, CD19CAR-C / D box, and CD19CAR-C / D box-CD8 groups, the percentages of CAR-T cells in CD8+ T cells at DAY9 were 0.45%, 7.22%, 0.053%, and 51.8%, respectively. In the CD19CAR-CD8 and CD19CAR-C / D box-CD8 groups where CD8-Lamp2b was present, only the CD19CAR-C / D box-CD8 group had a positive CAR-T cell population.

[0079] As shown in E of Figure 4, at DAY 12, the percentages of CAR-T cells in CD8+ T cells in the CD19CAR, CD19CAR-CD8, CD19CAR-C / D box, and CD19CAR-C / D box-CD8 groups were 0.46%, 2.99%, 0.48%, and 8.14%, respectively, while the CD19CAR-C / D box-CD8 group had a weak population of positive CAR-T cells remaining.

[0080] In Figure 4, the PBMC group represents the group without exosomes. These data indicate that, in the presence of the CD8-Lamp2b targeting plasmid, both the exosome targeting system and the exosome mRNA targeting delivery system were able to obtain a positive CAR-T cell population in CD8+ T cells on Day 2. On Day 9, only the CD19CAR-C / D box-CD8 group showed a positive CAR-T cell population. The exosome mRNA targeting delivery system resulted in a longer retention time of CAR-T cells in CD8+ T cells.

[0081] Example 5: Detection of CAR expression level in CD3+ T cells after incubation with targeted exosomes

[0082] The percentage of CAR-T cells in CD3+ T cells on days 2, 5, 7, 9, and 12 was analyzed using FlowJO. As shown in Figure 5A, in the CD19CAR, CD19CAR-CD8, CD19CAR-C / D box, and CD19CAR-C / D box-CD8 groups, the percentage of CAR-T cells in CD3+ T cells on day 2 was 14.4%, 43.1%, 1.69%, and 36.2%, respectively. Only when CD8-Lamp2b was present was there a positive CAR-T cell population in CD3+ T cells.

[0083] As shown in Figure 5B, in the CD19CAR, CD19CAR-CD8, CD19CAR-C / D box, and CD19CAR-C / D box-CD8 groups, the percentages of CAR-T cells in CD3+ T cells at DAY 5 were 5.82%, 26.6%, 0.69%, and 37.4%, respectively. Only in the presence of CD8-Lamp2b were there positive CAR-T cell populations in CD3+ T cells. At DAY 7, the percentages of CAR-T cells in CD3+ T cells were 0.56%, 20.3%, 0.25%, and 35.1%, respectively. Only in the presence of CD8-Lamp2b were there positive CAR-T cell populations in CD3+ T cells, as shown in Figure 5C.

[0084] As shown in Figure 5D, in the CD19CAR, CD19CAR-CD8, CD19CAR-C / D box, and CD19CAR-C / D box-CD8 groups, the percentages of CAR-T cells in CD3+ T cells at DAY9 were 0.25%, 2.87%, 0.06%, and 17.18%, respectively. Only the CD19CAR-C / D box-CD8 group had a positive CAR-T cell population.

[0085] As shown in E of Figure 5, the percentages of CAR-T cells in CD3+ T cells at DAY 12 were 0.23%, 0.59%, 0.17%, and 1.49% in the CD19CAR, CD19CAR-CD8, CD19CAR-C / D box, and CD19CAR-C / D box-CD8 groups, respectively, and no positive CAR-T cell populations were found in any of the groups.

[0086] The PBMC group did not contain exosomes. As shown in Figure 5, these data indicate that in the presence of the CD8-Lamp2b targeting plasmid, both the exosome targeting system and the exosome mRNA targeting delivery system were able to obtain a positive CAR-T cell population in CD3+ T cells on DAY 2. On DAY 9, only the CD19CAR-C / D box-CD8 group had a positive CAR-T cell population. The exosome mRNA targeting delivery system resulted in a longer retention time of CAR-T cells in CD3+ T cells. On DAY 12, no positive CAR-T cell population was found in any group. Both the exosome targeting system and the exosome mRNA targeting delivery system were able to transiently obtain a positive CAR-T cell population in CD3+ T cells.

[0087] The percentage of CAR-T cells in CD3+ T cells and CD8+ T cells on days 2, 5, 7, 9, and 12 was statistically analyzed using GraphPadPrism8 software and plotted as a line graph, as shown in Figure 6.

[0088] As shown in Figure 6, when the CD8-Lamp2b component was present, high expression of StrepII was detected in both CD19CAR-CD8 and CD19CAR-C / D box-CD8 groups on DAY 2; the percentage of CAR-T cells began to decrease on DAY 5, and the percentage of CAR-T cells in the CD19CAR-C / D box-CD8 group was significantly higher than that on DAY 2; and the percentage of CAR-T cells decreased to about 2% on DAY 12.

[0089] The data above indicate that both the exosome targeting system and the exosome mRNA targeting delivery system can temporarily generate CAR-T cells; however, the exosome mRNA targeting delivery system is more efficient.

[0090] Example 6: In vitro preparation of CAR-T cells from CD19CAR-C / D box-CD8 targeted exosomes and its cytotoxicity assay

[0091] CD19CAR-C / D box-CD8 targeted exosomes were prepared using an exosome-Lamp2b mRNA targeted delivery system. These exosomes were co-cultured with activated PBMCs and labeled DAY1. On day 5, StrepII expression in PBMCs was detected by flow cytometry, and CAR-T cell cytotoxicity was assessed using CCK8 assay. As shown in Figure 7A, the percentage of CAR-T cells in CD3+T and CD8+T cells on day 5 was analyzed using FlowJO. In the CD19CAR-C / D box-CD8 group, the percentage of CAR-T cells in CD3+T cells was 59.5%; as shown in Figure 7B, the percentage of CAR-T cells in CD8+T cells was 94.3%. No exosomes were added to the control group. These results indicate that CD19CAR-T cells were successfully prepared using the exosome-mRNA targeted delivery system.

[0092] As shown in Figure 8, the CCK8 cytotoxicity assay showed that CD19CAR-T cells had a high killing effect on both RAJI-GL and B-LCL-GL cells in vitro when the effector-target ratio was 1:1, and CD19CAR-T cells had a better killing effect on RAJI-GL cells.

[0093] Example 7: In vivo preparation of CAR-T cytotoxicity using CD19CAR-C / D box-CD8 targeted exosomes

[0094] Further, the anti-RAJI-GL tumor activity of CAR-T cells prepared in vivo using CD19CAR-C / D box-CD8 targeted exosomes was verified using a humanized DK-NPG mouse xenograft tumor model. As shown in Figure 9, in the control exosome group, red crosses represent mice that have died on day 14; the control group, after infusion of blank exosomes, had an average luminescence intensity of 2.60E+08 Photons / s, indicating tumor cell metastasis throughout the body, and two mice died after day 14; the treatment group received 1×10⁻⁸ exosomes. 12 One exosome infusion significantly reduced the tumor burden, leaving only the tail and head remnants; the second 1×10 12 After exosome infusion, the tumor burden in the treatment group has been eliminated.

[0095] The results showed that CD19-targeting exosomes could rapidly kill tumor cells in the model. Compared with CAR-T, CAR-Exo had a stronger and faster tumor clearance ability.

[0096] Example 8: Kinetics and tissue distribution of CAR-T cells prepared in vivo from CD19CAR-C / D box-CD8 targeted exosomes

[0097] After injecting CD8+ T cell exosomes into mice via the caudal venous line, mouse tissue was collected to measure the expression of CAR molecules in total RNA, indirectly reflecting the CAR cell content. As shown in Figure 10, the results indicate that CAR-T cells are mainly distributed in the blood and peripheral lymphoid organs.

[0098] It should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A fusion RNA comprising, from 5' to 3': The RNA sequence shown in SEQ ID NO.1 and the RNA recognition sequence of the RNA-binding protein.

2. The fusion RNA according to claim 1, wherein, The RNA-binding protein includes L7AE; Preferably, the RNA recognition sequence of the L7AE includes a sequence encoding the C / D box.

3. A nucleic acid molecule encoding the fusion RNA of claim 1 or 2.

4. A carrier comprising the nucleic acid molecule of claim 3.

5. An RNA delivery system comprising the vector of claim 4, a vector expressing exosomal transmembrane proteins and RNA-binding proteins, and a vector expressing target molecules and exosomal surface membrane proteins.

6. The RNA delivery system according to claim 5, wherein, The RNA-binding protein includes L7AE; Preferably, the exosome transmembrane protein includes CD63, CD9, or CD81; Preferably, the RNA-binding protein is attached to the C-terminus of an exosome transmembrane protein; Preferably, the targeting molecule includes a targeting peptide that interacts with cell membrane surface antigens, an antibody or its antigen-binding fragment that recognizes a specific cell surface antigen, or an affinity molecule; Preferably, the exosome surface membrane protein includes Lamp2b; Preferably, the targeting molecule is attached to the N-terminus of an exosome surface membrane protein.

7. An exosome comprising the fusion RNA of claim 1 or 2, the exosome further comprising a targeting molecule capable of recognizing a target tissue or target cell.

8. The exosome according to claim 7, wherein, The target molecules include target peptides, antibodies or their antigen-binding fragments, and affinity molecules.

9. A method for preparing exosomes according to claim 7 or 8, comprising the following steps: The RNA delivery system of claim 5 or 6 is introduced into cells that produce exosomes; the cells are cultured to produce exosomes.

10. A type of cell, in which, The cells are those into which the RNA delivery system of claim 5 or 6 is introduced, or those that produce and secrete exosomes of claim 7 or 8.

11. The method for preparing cells according to claim 10, wherein, The preparation method includes the following steps: introducing the RNA delivery system of claim 5 or 6 into cells that produce exosomes.

12. A pharmaceutical composition, wherein, The pharmaceutical composition comprises the exosomes of claim 7 or 8 and the cells of claim 10.

13. The pharmaceutical composition according to claim 12, wherein, The pharmaceutical composition also includes a pharmaceutically acceptable carrier or excipient.

14. The use of the fusion RNA of claim 1 or 2, the nucleic acid molecule of claim 3, the vector of claim 4, the RNA delivery system of claim 5 or 6, the exosome of claim 7 or 8, or the cell of claim 10 in the preparation of a medicament for the prevention and / or treatment of CD19 expression-related tumors.

15. The application according to claim 14, wherein, The tumor is a B-cell lymphoma.

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