CAR molecules, cells or exosomes containing the same, and uses thereof

Abstract

Disclosed herein are CAR molecules, cells or exosomes containing the same, and uses thereof. The CAR molecules are CD19-targeting CAR molecules and / or CD22-targeting CAR molecules. Also disclosed are nucleic acids encoding the CAR molecules, recombinant expression vectors containing the nucleic acids, and pharmaceutical compositions containing the CAR molecules, cells, or exosomes containing the same. CAR-T cells containing the CAR molecules have higher therapeutic efficacy and therapeutic safety in clinical treatments, and the exosomes provided have better targeting and tumor penetration, potentially opening up a new direction in tumor treatment using CAR-T cell therapy.

Description

[Technical Field]

[0001] The present invention belongs to the field of biotechnology, and specifically relates to CAR molecules, cells or exosomes containing the same, and uses thereof. [Background technology]

[0002] This application claims priority from Chinese Patent Application No. 2023100571558, filed on January 19, 2023. This application cites the above Chinese patent application in its entirety.

[0003] Cancer is one of the most lethal diseases in the world. The main treatments for tumors include surgical resection, chemotherapy, radiation therapy, and immunotherapy. Balancing the two issues of drug toxicity to tumors and precise drug delivery to the tumor site can improve drug efficacy while avoiding toxic side effects of drugs on normal cells and tissues.

[0004] The rationale for tumor immunotherapy is the ability of the immune system to recognize tumor-associated antigens and modulate the body's ability to attack tumor cells (highly specific cytolysis). This biological process is highly complex and is still under investigation. In the 1990s, several research groups discovered tumor antigens, and T lymphocytes can recognize these tumor antigens in a major histocompatibility complex (MHC)-dependent manner.

[0005] Tumor immunotherapy is generally divided into two categories: nonspecific immunotherapy and specific immunotherapy. Nonspecific immunotherapy mainly includes cytokines and toxins such as interleukin-2 (IL-2), interferon α (IFN-α), tumor necrosis factor (TNF-α), BCG vaccine, and adoptive cellular immunotherapy. Specific immunotherapy mainly involves tumor vaccines.

[0006] Chimeric antigen receptors (CARs) are composed of a fusion of an extracellular antigen target-binding domain, a transmembrane domain, and an intracellular T cell activation signaling domain. They are introduced into T cells via exogenous genes, and after effector T cells express the CAR molecule, they confer specific antigen recognition capabilities to the cells. CAR-T cell therapy involves expanding these cells ex vivo and then returning them to the patient for treatment. Currently, CAR-T therapy has shown promising results in the treatment of B-cell-derived tumors and leukemia.

[0007] Compared with conventional adoptive cellular immunotherapy, CAR-T therapy has two advantages: First, CAR-T cells directly bind to antigen epitopes in a major histocompatibility complex (MHC)-independent manner, endowing immune cells with the ability to specifically recognize and kill tumors. Second, receptor molecules that specifically recognize natural antigens can be highly expressed on the cell membrane without relying on antigen-presenting cells to process natural antigens. Target antigens can be proteins, carbohydrates, or lipids.

[0008] CARs consist of three modules: an extracellular target-binding module (ligand-binding domain), a transmembrane domain that anchors the cell membrane, and an intracellular signaling module (signaling and cosignaling domains) that transmits activation signals. The target-binding module is typically based on the variable region of a single-chain antibody (single-chain variable fragment, scFv), linking the VH and VL sequences. The intracellular signaling module typically consists of intracellular segments, such as the signaling molecule CD3ζ chain and the costimulatory molecule CD28, involved in T cell activation, thereby transmitting TCR (T cell receptor) activation signals to the interior of the cell. Such CAR-transduced T cells possess the antigen specificity and cytotoxicity of effector T cells. When tumor-associated antigens themselves function as CAR-T cell receptors, the extracellular domain of CAR-T cells is primarily their ligand or peptide, thereby conferring tumor-associated antigen specificity to CAR-T cells. When CARs recognize specific tumor-associated antigens, the combined action of intracellular tandem T cell activation signal motifs (CD3ζ) and costimulatory signal motifs leads to specific activation of CAR-T cells, which manifests as massive proliferation, secretion of functional cytokines, and CAR-T cell-dependent killing.

[0009] Currently, CAR-T technology has reached the fourth generation and is primarily classified by the number of signaling motifs tandemly connected in the intracellular domain of the CAR molecule. First-generation CARs are defined as those with a single intracellular signaling motif. Because the intracellular signaling domain of first-generation CAR molecules contains only one CD3ζ, cell activation signals are primarily transmitted through its tyrosine kinase activation motif (ITAM), activating the associated signaling pathway. Clinical studies of early-stage tumor treatment have shown that CAR-T cell therapy has limited therapeutic efficacy in most clinical cases due to the inability of reinfused cells to persist in the body for long periods.

[0010] Because the half-life of adoptively transferred CAR-T cells in clinical cases is closely related to their potential therapeutic efficacy, researchers typically consider adding signaling motifs when designing CAR structures to further amplify the CAR-T cell activation signal and extend their half-life in vivo.

[0011] However, existing CAR-T cell therapies have certain limitations, such as antigen escape, on-target off-tumor effects, CAR-T cell migration and tumor infiltration, and an immunosuppressive microenvironment. Compared to hematological malignancies, solid tumor CAR-T cell therapies are limited by the ability of CAR-T cells to migrate and infiltrate within solid tumors, as their penetration and migration are restricted by the immunosuppressive tumor microenvironment and physical tumor barriers, such as the tumor stroma. In the tumor microenvironment, many cell types that cause immunosuppression, such as myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and regulatory T cells (Tregs), can infiltrate solid tumors. These tumor-infiltrating cells promote the production of tumor-promoting cytokines, chemokines, and growth factors. Furthermore, immune checkpoint pathways, such as PD-1 or CTLA-4, can be used to suppress antitumor immunity. One of the main reasons for the lack of response or poor response to CAR-T cell therapy is insufficient T cell proliferation and short T cell persistence.

[0012] Exosomes (Exo) are small vesicles with a lipid bilayer structure and particle size ranging from 30 to 150 nm. First, the plasma membrane invaginates to form early endosomes, which then develop into late endosomes or multivesicular bodies (MVBs). Finally, the MVBs fuse with the plasma membrane to release exosomes. The exosome formation process is closely related to the association of lipids, proteins, and other molecules with the plasma membrane. The exosome membrane expresses several marker proteins, including those of the tetraspanin superfamily (CD63, CD9, CD81, and CD82), heat shock protein 70 (HSP70), and tumor susceptibility gene 101 (T101) protein, in addition to related plasma membrane proteins. Almost all cells can secrete exosomes, and these exosomes exhibit distinct membrane proteins, contents, and functions. For example, mesenchymal stem cell-derived exosomes can inhibit tumor initiation, growth, and invasion through the miRNAs they contain, while tumor cell-derived exosomes can promote tumor migration or aid in immune evasion by creating a tumor microenvironment. Exosomes with different target categories and functions can be customized by genetically engineering exosome-derived cells or by directly modifying exosomes, demonstrating the broad potential of exosomes in the field of targeted tumor therapy.

[0013] Exosomes are characterized by their high biocompatibility, low immunogenicity, and ease of modification, leading to rapid development of research into their use as drug delivery carriers. Targeting determines whether exosomes can accurately and efficiently deliver drugs to diseased cells and tissues. Therefore, to improve exosome specificity for tumor cells, exosomes with specific natural targeting properties can be selected as drug delivery carriers, or exosomes can be modified by other means to enhance their targeting function, thereby further improving the potential and value of exosomes for clinical application. Summary of the Invention

[0014] To solve the technical problem of insufficient therapeutic efficacy and safety in clinical treatment using CAR-T cells targeting CD19 or CD22 in conventional technologies, the present invention uses gene editing technology to replace the variable region of T cells with a single-chain antibody, thereby expressing chimeric antigen receptors (CARs) on the surface of T cells. The single-chain variable fragment (scFv) or ligand of the CAR provides antibody-like specificity to T cells, enabling them to bind to target cells expressing the corresponding antigen. The resulting T cells are chimeric antigen receptor T cells (CAR-T cells), which can be used to treat tumors.

[0015] Furthermore, the present invention solves the problems of the prior art, namely, that the matrix of solid tumors affects the penetration of CAR-T cells, making it difficult for CAR-T to infiltrate solid tumors, that the immunosuppressive tumor microenvironment inhibits the immune response induced by CAR-T cell therapy, and that clinical application is often accompanied by toxic effects such as cytokine release syndrome and CAR-T cell-associated encephalopathy syndrome, limiting the therapeutic efficacy of CAR-T cell therapy in solid tumors. To address these problems, the present invention utilizes exosomes containing CAR molecules to target and treat tumors, based on the property that tumor cells actively take up exosomes.

[0016] In order to solve the above technical problems, the present invention provides a technical solution, which is a CAR molecule that targets CD19 and / or a CAR molecule that targets CD22, wherein the amino acid sequence of the CAR molecule that targets CD19 is shown in SEQ ID NO: 1, and the amino acid sequence of the CAR molecule that targets CD22 is shown in SEQ ID NO: 2.

[0017] In the present invention, a CAR molecule (chimeric antigen receptor) is an artificially designed transmembrane protein that combines the specificity of an antigen-specific antibody with the function of a T cell receptor. Generally, a CAR molecule is obtained by fusing an extracellular antigen target binding domain, a transmembrane domain, and an intracellular T cell activation signaling domain.

[0018] In order to solve the above technical problems, the present invention provides a technical solution, which is an isolated nucleic acid encoding a CAR molecule according to the present invention.

[0019] In a preferred embodiment of the present invention, the nucleotide sequence encoding the CD19-targeting CAR molecule is set forth in SEQ ID NO: 3, and / or the nucleotide sequence encoding the CD22-targeting CAR molecule is set forth in SEQ ID NO: 4.

[0020] As is well known in the art, "nucleic acid" as used herein refers to a chain of nucleotides of any length, including DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase.

[0021] In order to solve the above technical problems, the present invention provides a technical solution, which is a recombinant expression vector comprising the nucleic acid according to the present invention.

[0022] In a preferred embodiment of the invention, the recombinant expression vector is a lentiviral vector. In a more preferred embodiment of the present invention, the backbone of the lentiviral vector is pLVX-EF1α-IRES-Puro.

[0023] The term "recombinant expression vector" refers to a genetically engineered oligonucleotide or polynucleotide construct that allows a host cell to express mRNA, protein, or peptide when the construct contains a nucleotide sequence encoding mRNA, protein, or peptide and the vector is contacted with a cell under conditions sufficient for expression of the mRNA, protein, or peptide in the cell. Vectors of the invention are typically not naturally occurring, although portions of the vector may be naturally occurring. Recombinant expression vectors of the invention include, but are not limited to, DNA and RNA, which may contain any type of nucleotide, be single-stranded or double-stranded, be synthetic, or be derived in part from natural sources, and may contain natural, non-natural, or modified nucleotides. Recombinant expression vectors may contain naturally occurring or non-naturally occurring internucleotide linkages, or a combination of the two. In exemplary aspects, modified nucleotides or non-naturally occurring internucleotide linkages do not inhibit transcription or replication of the vector.

[0024] The recombinant expression vector of the present invention may be any suitable recombinant expression vector that can be used for transformation or transfection to deliver one or more target genes or sequences into any suitable host cell, and preferably to express said genes or sequences in the host cell. Suitable vectors include vectors designed for propagation and amplification, or expression, or both, and examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, cationic coagulant-associated DNA or RNA expression vectors, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

[0025] In order to solve the above technical problems, the present invention provides a technical solution, which is a transformant expressing the CAR molecule described in the present invention.

[0026] In a preferred embodiment of the present invention, the transformant is a host cell into which the recombinant expression vector according to the present invention has been introduced. In a more preferred embodiment of the invention, said host cell is a mammalian cell that is a T cell, a 293 cell, or a cell line derived therefrom, such as, for example, a 293T cell or a 293F cell.

[0027] As used herein, the term "host cell" refers to any type of cell that may contain a nucleic acid or vector described herein. In exemplary embodiments, the host cell is derived from or obtained from a mammal. A particularly preferred mammal is a human. As used herein, the term "host cell" may include cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" or "transformed cells," which include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. The invention includes mutant progeny that have the same function or biological activity as the cells screened or selected for in the primary transformed cell.

[0028] For purposes herein, T cells may be any T cell, such as cultured T cells (e.g., primary T cells), T cells from a cultured T cell line (e.g., Jurkat, SupT1, etc.), or T cells obtained from a mammal. If obtained from a mammal, T cells may be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells may be enriched or purified. T cells may be obtained by maturing hematopoietic stem cells into T cells in vitro or in vivo. In exemplary aspects, the T cells are human T cells. In exemplary aspects, the T cells are T cells isolated from a human. The T cells may be any type of T cell, preferably, the T cells are CD3-positive T cells.

[0029] In the present invention, 293T cells are a cell line derived from 293 cells (human fetal kidney cells) by genetic engineering, into which the adenovirus E1A gene has been introduced, capable of expressing the SV40 large T antigen, and containing the SV40 replication origin and promoter region. 293F cells are a type of wild-type 293 cell line that can express high levels of proteins under serum-free conditions and can grow in suspension in culture medium.

[0030] According to the transformant of the present invention, the transformant further expresses granzyme and / or perforin, the amino acid sequence of the granzyme being shown in SEQ ID NO: 10, and the amino acid sequence of the perforin being shown in SEQ ID NO: 8.

[0031] In a preferred embodiment of the present invention, the granzyme and / or perforin is fused to CD63 (LAMP-3, lysosome-associated membrane protein-3), the amino acid sequence of which is shown in SEQ ID NO:6.

[0032] In the present invention, the fusion refers to linking the nucleotide sequence of granzyme and / or perforin with that of CD63 using a linker commonly used in the art to form a fusion protein after expression.

[0033] In a more preferred embodiment of the present invention, the transformant further overexpresses a protein that promotes exosome secretion, and the protein that promotes exosome secretion is CX43, KIBRA, or Rab27a. The NCBI reference sequence number for the amino acid sequence of CX43 is NP_000156.1, and preferably contains an S to A substitution at position 368. The NCBI reference sequence number for the amino acid sequence of KIBRA is NP_056053.1. The NCBI reference sequence number for the amino acid sequence of Rab27a is NP_899058.1.

[0034] In a further preferred embodiment of the present invention, the nucleotide sequence encoding the granzyme is set forth in SEQ ID NO: 9. The nucleotide sequence encoding the perforin is set forth in SEQ ID NO: 7. The nucleotide sequence encoding the CD63 is set forth in SEQ ID NO: 5. The NCBI reference SEQ ID NO: of the nucleotide sequence encoding the CX43 is NM_000165.5, preferably containing an AGC to GCC substitution at positions 1317 to 1319. The NCBI reference SEQ ID NO: of the nucleotide sequence encoding the KIBRA is XM_005265853.3. And / or the NCBI reference SEQ ID NO: of the nucleotide sequence encoding the Rab27a is NM_183236.3.

[0035] In the present invention, the term "NCBI Reference Sequence Number" refers to the nucleotide sequence or amino acid sequence of a gene obtained by searching the biological database National Center for Biotechnology Information (https: / / www.ncbi.nlm.nih.gov / ) using the NCBI Reference Sequence Number.

[0036] In the transformant according to the present invention, the genes encoding the granzyme, perforin, CD63, KIBRA and / or Rab27a are placed in an expression plasmid.

[0037] In a preferred embodiment of the present invention, the backbone of the expression plasmid is PcDNA3.1 or pCMV. PcDNA3.1 or pCMV according to the present invention are both plasmid vectors well known in the art.

[0038] In order to solve the above technical problems, the present invention provides a technical solution, which is a method for producing exosomes, comprising the steps of culturing the transformant described in the present invention and isolating and purifying exosomes from the culture medium of the transformant. The method for culturing the transformant is a method conventionally used in the technical field.

[0039] In order to solve the above technical problems, the present invention provides a technical solution, which is an exosome comprising a CAR molecule according to the present invention. In a preferred embodiment of the present invention, the exosomes are produced by the method for producing exosomes described in the present invention.

[0040] In order to solve the above technical problems, the present invention provides a technical solution, which is a pharmaceutical composition comprising one or more components selected from the CAR molecule described in the present invention, the transformant described in the present invention, and the exosome described in the present invention. In a preferred embodiment of the invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0041] The pharmaceutical compositions of the present invention contain suitable pharmaceutically acceptable carriers, e.g., carriers, pharmaceutical excipients well known in the art, including buffering agents. As used herein, "pharmaceutically acceptable carrier" or "pharmaceutical carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic and absorption delaying agents.

[0042] In order to solve the above technical problems, the present invention provides use of a CAR molecule described in the present invention, a transformant described in the present invention, an exosome described in the present invention, or a pharmaceutical composition described in the present invention in a medicament for preventing and / or treating a tumor associated with CD19 and / or CD22 expression.

[0043] In a preferred embodiment of the invention, the tumor is an acute B-lymphocytic tumor. In a more preferred embodiment of the present invention, said acute B-lymphocytic neoplasm is B-ALL leukemia or aggressive B-cell lymphoma. In order to solve the above technical problems, the present invention provides a method for diagnosing, treating, and / or preventing tumors associated with CD19 and / or CD22 expression, comprising the step of administering to a patient in need thereof a therapeutically effective amount of a CAR molecule described in the present invention, a transformant described in the present invention, an exosome described in the present invention, or a pharmaceutical composition described in the present invention.

[0044] In a preferred embodiment of the invention, the tumor is an acute B-lymphocytic tumor. In a more preferred embodiment of the present invention, said acute B-lymphocytic neoplasm is B-ALL leukemia or aggressive B-cell lymphoma.

[0045] As used herein, the term "effective amount" refers to an amount of a drug or agent that elicits the biological or pharmacological response in a tissue, system, animal, or human that is desired, for example, by a researcher or clinician. The term "therapeutically effective amount" also refers to an amount that improves the treatment, cure, prevention, or alleviation of a disease, condition, or side effect, or reduces the rate of progression of a disease or condition, compared to a corresponding subject not receiving the amount. The term also encompasses within its scope an amount effective to enhance normal physiological function.

[0046] In order to solve the above technical problems, the present invention provides a technical solution, which is a combination therapy comprising a step of administering to a patient in need thereof a CAR molecule described in the present invention, a transformant described in the present invention, an exosome described in the present invention, or a pharmaceutical composition described in the present invention, and a second therapeutic agent.

[0047] In a preferred embodiment of the present invention, the second therapeutic agent comprises another anti-tumor antibody, or the other anti-tumor pharmaceutical composition, and / or another anti-tumor drug.

[0048] In order to solve the above technical problems, the present invention provides a technical solution, which is a CAR molecule described in the present invention, a transformant described in the present invention, an exosome described in the present invention, or a pharmaceutical composition described in the present invention, used for the diagnosis, prevention, and / or treatment of tumors associated with CD19 and / or CD22 expression.

[0049] In a preferred embodiment of the invention, the tumor is an acute B-lymphocytic tumor. In a more preferred embodiment of the present invention, said acute B-lymphocytic neoplasm is B-ALL leukemia or aggressive B-cell lymphoma.

[0050] The above-mentioned preferred conditions can be arbitrarily combined within the scope of common knowledge in the art to obtain each preferred embodiment of the present invention. The reagents and raw materials used in the present invention are commercially available.

[0051] The inventive effects of the present invention are as follows: The CAR-T cells CAR19T and CAR22T produced by the present invention have higher therapeutic efficacy and safety in clinical treatment compared to similar CAR-T cells produced by conventional technologies, specifically manifested by a higher complete remission rate, a higher overall response rate, and a lower rate of severe CRS reactions and neurotoxicity in patients.

[0052] Compared with CAR-T cells chimerized with CD19 monoclonal antibody scFv (CAR19T) or CD22 monoclonal antibody scFv (CAR22T), exosomes Exo-CAR-CD19 or Exo-CAR-CD22 produced by 293F cells not only effectively lysed Raji-luc cells, which are CD19- and CD22-positive tumor cells, but also reduced the off-target side effects of CAR-T cells. This indicates that exosomes Exo-CAR-CD19 or Exo-CAR-CD22 have excellent targeting ability and high tumor penetration. Therefore, Exo-CAR exosomes are expected to become a new direction for CAR-T cell therapy in tumor treatment. [Brief explanation of the drawings]

[0053] [Figure 1] FIG. 1 is a schematic diagram of the partial structures of CAR19 and CAR22. [Figure 2] FIG. 1 is a schematic diagram of the structure of the recombinant lentiviral vector pCAR19. [Figure 3] FIG. 1 is a schematic diagram of the structure of the recombinant lentiviral vector pCAR22. [Figure 4] This shows the concentration detection results of the plasmid pCAR19. [Figure 5] The results of detecting the concentration of the plasmid pCAR22 are shown below. [Figure 6] 1 shows the results of enzyme digestion detection of plasmid pCAR19 and plasmid pCAR22. [Figure 7] These are the results of an introduction efficiency test after introduction of CAR19T cells and CAR22T cells. [Figure 8] Results of tumor killing detection by CAR19 T cells and CAR22 T cells. [Figure 9] Experimental results of the effects of CAR19T and CAR22T on tumor growth in mice. [Figure 10] This is the result of promoting secretion of exosome secretion-promoting genes. [Figure 11-1] Electron microscopy, particle size and molecular phenotype flow cytometry analysis of Exo-CAR-CD19 and Exo-CAR-CD22. [Figure 11-2] Electron microscopy, particle size and molecular phenotype flow cytometry analysis of Exo-CAR-CD19 and Exo-CAR-CD22. [Figure 12] In vivo antitumor effects of Exo-CAR-CD19 and Exo-CAR-CD22. [Figure 13] Measurement of granzyme and perforin content in exosomes modified with Exo-CAR-CD19 and Exo-CAR-CD22, where part A represents measurement of perforin content and part B represents measurement of granzyme content. [Figure 14] Figure 1 shows the effect of tumor killing efficiency of Exo-CAR-CD19 and Exo-CAR-CD22 exosomes, where part A represents the killing efficiency of Exo-CAR-CD22 and part B represents the killing efficiency of Exo-CAR-CD19. [Figure 15] Killing results of Exo-CAR-CD19 and Exo-CAR-CD22 exosome concentration gradients. [Figure 16] Standard curves for perforin or granzymes. [Figure 17]This is the total protein amount of exosome secretion-promoting CX43(S368A). Here, -CX43 means that the CX43(S368A) exosome secretion-promoting gene was not introduced, and + means that the CX43(S368A) exosome secretion-promoting gene was introduced. DETAILED DESCRIPTION OF THE INVENTION

[0054] The present invention will be further described below with reference to examples, but the present invention is not limited to these examples. In the following examples, experimental methods for which specific conditions are not described are carried out according to conventional methods and conditions or selected according to product specifications.

[0055] Sources of biological and reagent materials of the present invention: pLVX-EF1α-IRES-Puro: purchased from Clontech. DH5alpha competent cells: purchased from Takara. EndoFree plasmid mega kit: Purchased from Qiagen. Includes QIAfilter Cartridge, Buffers P1, P2, P3, Buffer FW, Buffer ER, Buffer QBT, Buffer QC, Buffer QN, endotoxin-free water, and Buffer TE. Gag plasmid and vsvg plasmid: purchased from Addgene. 293T cells: purchased from Takara.

[0056] Definitions of abbreviations and keywords of the present invention: CAR-T: chimeric antigen receptor modified T, chimeric antigen receptor modified T cells. Exo-CAR: Exosome-chimeric antigen receptor, exosome carrying CAR molecules.

[0057] Example 1 Construction of recombinant lentiviral vectors pCAR19 and pCAR22 The amino acid sequence of CAR19 is shown in SEQ ID NO: 1: MDFQVQIFSFLLISASVIMSRMADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGST SGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTV

[0058] The amino acid sequence of CAR22 is shown in SEQ ID NO:2: MALPVTALLLPLALLLHAAIPDTEVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKRLEWVAYISSGGGTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHSGYGTHWGVLFA YWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPWTFGGGTKLEIK ATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQ TTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0059] Gene synthesis was carried out at Nanjing GenScript Biotech Corp. according to sequence 1 (CAR19, SEQ ID NO: 3) and sequence 2 (CAR22, SEQ ID NO: 4), and the synthesized sequence was cloned into a T vector.

[0060] CAR19 nucleotide sequence (SEQ ID NO:3): ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAATGTCTAGAATGGCCGACATCCAGATGACCCAGACCACCTCCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCCGACGGCACCG TCAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTTAGCGGCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCCACCTACTTTTGCCAGCAGGGCAACACACTGCCCTACACCTTTGGCGGCGGAACAAAGCTGGAAATCACCGGCAGCACC TCCGGCAGCGGCAAGCCTGGCAGCGGCGAGGGCAGCACCAAGGGCGAGGTGAAGCTGCAGGAAAGCGGCCCTGGCCTGGTGGCCCCCAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCCGGCAGCCCCCAGGAAGGGGCCTGGAATGGCTGGGCGTGATCT GGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTG

[0061] CAR22 nucleotide sequence (SEQ ID NO:4):

[0062] The structures of the CAR19 and CAR22 moieties are shown in Figure 1. Both CD19 and CD22 are third-generation CARs that use CD28 and 4-1BB as costimulatory signals. Sequence comparison of CAR19 and CAR22 showed that the sequences are completely identical.

[0063] The CAR19 sequence fragment was 798 bp long and equipped with EcoRI and MluI restriction enzyme sites at both ends. It was then cloned into the multiple cloning site of the lentiviral backbone plasmid pLVX-EF1α-IRES-Puro to complete the vector construction. The complete construction map is shown in Figure 2, where WPRE stands for woodchuck hepatitis virus posttranscriptional regulatory element, RRE stands for Rev response element, and cPPT / CTS stands for central polypurine tract / CTS (central termination sequence).

[0064] The CAR22 fragment, 1608 bp in length, was terminated at both ends with EcoRI and MluI and cloned into the multiple cloning site of the lentiviral backbone plasmid pLVX-EF1α-IRES-Puro to complete the vector construction. The complete construction map is shown in Figure 3.

[0065] The lentiviral backbone plasmid pLVX-EF1α-IRES-Puro and the synthetic sequence were double-digested with EcoR I-HF and Mlu I restriction endonucleases, and the product was subjected to 1.5% agarose gel electrophoresis. The gel was excised and collected into Eppendorf tubes. The corresponding fragments were recovered using a QIAGEN agarose gel recovery kit, and the purity and concentration of the product were determined.

[0066] The fragments were added to an Eppendorf tube at a 1:1 molar ratio, and T4 DNA ligase (NEB) and T4 DNA ligase buffer were added. The mixture was incubated at 22°C for 2 hours. Eight microliters of the ligation solution was added to 100 μL of DH5alpha competent cells, which were then immersed in ice water for 30 minutes and then heat-shocked at 42°C for 90 seconds. After this, 500 μL of soc medium was added and the cells were incubated at 37°C and 220 rpm for 2 hours. After 2 hours, the Eppendorf tube was centrifuged at 4000 g for 1 minute to remove the remaining liquid (400 μL). The remaining liquid was spread on an LB plate and incubated at 37°C for 12 hours. A single colony was picked from the plate and inoculated into 5 mL of LB liquid medium, which was then incubated at 37°C and 220 rpm for 12 hours.

[0067] Plasmids were extracted using a QIAGEN Mini Kit to obtain pCAR19 and pCAR22 plasmids, and the first-generation sequences were verified by Nanjing GenScript Biotech Corp. to confirm their accuracy. DH5alpha strains containing the pCAR19 or pCAR22 plasmids were then stocked.

[0068] Example 2: Preparation of pCAR19 and pCAR22 plasmids The DH5alpha strain carrying the pCAR19 or pCAR22 plasmid was inoculated into 250 mL of LB medium containing 100 μg / mL ampicillin and cultured overnight at 37°C and 220 rpm. The culture was centrifuged at 6000 g for 20 minutes at 4°C, and the supernatant was discarded.

[0069] Buffer P1 was removed from the EndoFree plasmid mega kit (Qiagen), and 120 mL of pre-chilled buffer P1 was added to the E. coli precipitate obtained by centrifugation. The centrifuge bottle was then capped and vigorously shaken to completely disperse the E. coli precipitate in buffer P1.

[0070] 120 mL of buffer solution P2 was added to the centrifuge bottle, the bottle cap was closed, and the bottle was placed on a roller mixer. The rotation speed was slowly increased to 50 rpm, and after thorough mixing, the mixture was left at room temperature for 5 minutes.

[0071] Add 120 mL of Buffer P3 to the centrifuge bottle, cap it, place it on a roller mixer, slowly increase the rotation speed to a maximum of 70 rpm, and mix thoroughly until a white, fluffy mixture is obtained. Centrifuge at 9000 g for 15 minutes at 4°C.

[0072] 50 mL of Buffer FW was poured into the QIAfilter cartridge, and the supernatant was added to the QIAfilter cartridge and gently mixed. The mixture was then filtered into a correspondingly labeled glass bottle.

[0073] 20 mL of buffer solution ER was added to each vial, mixed evenly by inverting six times, and incubated at -20°C for 30 minutes. The marked mega columns were placed in the corresponding racks and 35 mL of Buffer QBT was added to each mega column to balance it and allowed to drain under gravity.

[0074] The liquid in the glass bottles was poured into the corresponding marked mega columns in a single batch. After the liquid in the columns had drained, 200 mL of Buffer QC was added to each mega column in a small batch to wash it. After the liquid in the columns had drained, the waste liquid in the waste collection tray was poured into a clean 50 mL centrifuge tube.

[0075] 40 mL of Buffer QN was added to each mega column, and the flow-through was collected in a clean 50 mL centrifuge tube, inverted six times to mix evenly, and 20 mL was dispensed into another clean, labeled 50 mL centrifuge tube.

[0076] 14 mL of isopropanol (room temperature) was added to each 50 mL centrifuge tube, and the tubes were inverted six times to mix evenly. The tubes were then centrifuged at 4°C and 15,000 g for 50 minutes.

[0077] The supernatant was aspirated in a clean bench, and 3.5 mL of endotoxin-free water was added to each tube to rinse the tubes without scattering any sediment at the bottom. The tubes were then centrifuged at 15,000 g for 30 minutes at 4°C. Buffer TE included in the EndoFree Plasmid Mega Kit was preheated in an oven.

[0078] After centrifugation in a clean bench, the supernatant was removed by suction and the mixture was dried by blowing air in the clean bench (to evaporate the remaining absolute ethanol, for about 10 minutes). The TE buffer solution was removed from the oven, and 1 mL of TE buffer solution was added to each tube in a clean bench. The tubes were pipetted 10 times with a pipette, then placed in a 65°C oven. The tube walls were continuously tapped during this time to completely dissolve the precipitate. The tubes were then centrifuged at 4°C and 4000 g for 1 minute, and the liquid on the tube walls was shaken off to the bottom of the tube and mixed uniformly by pipetting.

[0079] All liquids were transferred to corresponding labeled EP tubes that were endotoxin-, pyrogen-, and nuclease-free in a clean bench. 2 μL of each aliquot was aspirated, and the plasmid concentration was measured using a microspectrophotometer. The corresponding EP tube was marked to obtain the pCAR19 or pCAR22 plasmid.

[0080] Plasmid detection 1. Plasmid Concentration Detection After receiving the sample, 1 μL was taken to measure the concentration, and then the nucleic acid measurement module was entered using an ultra-low-volume UV spectrophotometer (Nanodrop). The parameters were set, and after blank correction, the sample for detection was loaded and detected. The results are shown in Figures 4 and 5, and Table 1 below.

[0081] Table 1 [Table 1]

[0082] 2. Plasmid DNA (enzyme digestion) detection Principle: Agarose gel electrophoresis is a standard method for separating, identifying, and purifying DNA fragments. Agarose is a polysaccharide extracted from agar. It is hydrophilic but uncharged, making it an excellent electrophoretic carrier. DNA is negatively charged under alkaline conditions (pH 8.0 buffer) and migrates through the gel medium toward the positive electrode under an electric field. Different DNA molecule fragments migrate at different speeds in the electric field due to their different molecular structure. Ethidium bromide (EB) can be embedded between the base pairs of DNA molecules to form fluorescent complexes, which separate into distinct zones after ultraviolet irradiation, achieving the purposes of separation, molecular weight identification, and recombinant screening.

[0083] The results of enzyme digestion identification can be used to preliminarily determine the content of supercoiled plasmid in the stock solution. The higher the supercoiled content, the higher the purity of the plasmid and the higher the efficiency of subsequent viral packaging.

[0084] Methods: 200ng of each sample was digested with EcoRI, KpnI, and MluI, followed by EcoRI single digestion, and then detected by 0.7% agarose gel electrophoresis. Sample numbers were marked above the gel wells. M1: 10,000 kb DNA marker (10,000, 8,000, 6,000, 5,000, 4,000, 3,500, 3,000, 2,000, 1,500, 1,200, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100), M2: D15,000 kb DNA marker (5,000, 3,000, 2,000, 1,000, 750, 500, 250, 100). The results of sample agarose gel electrophoresis (sample load: approximately 100 ng) are shown in Figure 6.

[0085] 3. Sequencing of Target Genes 20 μL (500 ng) of plasmid DNA was taken and sent for sequencing to confirm whether the target gene of the product produced by the plasmid had changed based on the original seed sequence. Under a stable process, the target gene remained unchanged during the fermentation and amplification process of the working seed, and could be used for the next production step and correct protein expression.

[0086] Example 3 Production of recombinant lentiviruses LVCAR19 and LVCAR22 Multilayer cell culture flask (Hyperflask) (Corning) was used. 130.0–140.0 × 10 6 293T cells (Takara) were inoculated into a total of 560 mL of DMEM complete medium (50 mL fetal bovine serum, 5 mL antibiotic-antimycotic (100x)) and cultured for 24 hours in an incubator at 37°C with 5% CO2. 320 μg of plasmid (pCAR19 or pCAR22:gag plasmid:vsvg plasmid = 6:3:2) was added to the 960 μg PEI tube, vortexed, and then equilibrated at room temperature for 10 minutes. 35 mL of the PEI / plasmid mixture was homogenously mixed with 525 mL of DMEM complete medium and transferred to the multi-layer cell culture flask. The multi-layer cell culture flask was cultured in an incubator at 37°C with 5% CO2 for 3 days, after which the cell culture supernatant was collected.

[0087] The supernatant was centrifuged at 4,000 rpm (or 3,000 g) for 30 minutes, after which cryonase (Takara) was added to the supernatant and placed at 4°C. After 6 hours, the lentiviral supernatant was filtered through a 0.22 μm filter, filtered by suction, and centrifuged at 30,000 g for 2.5 hours at 4°C. The supernatant was removed, and 1 mL of T cell medium was added to resuspend the precipitate. After resuspension, 20 μL was left over for titer activity detection, and the remaining lentiviral concentrate was aliquoted, labeled LVCAR19 or LVCAR22, and stored at -80°C for later use.

[0088] Lentivirus activity titer detection: Principle: Protein-L is labeled with fluorescein, which can specifically bind to the Kappa region of the single-chain antibody light chain in CAR. The fluorescent signal detected by flow cytometry indirectly reflects the expression of CAR in 293T cells.

[0089] Methods: 293T cells were cultured in 6-well plates at 5.0 × 10 5 293T cells were inoculated at 1000 cells / well, and 0.1 μL, 0.5 μL, or 1 μL of lentivirus concentrate was added to each well to set up one negative control. The cells were cultured at 37°C in a 5% CO2 incubator. After 3 days, 293T cells were harvested with Versene solution (Gibco) and subjected to flow cytometry to detect the percentage of CAR-positive 293T cells, which was then converted to the activity titer of the LVCAR19 or LVCAR22 lentivirus concentrate.

[0090] The current activity titer of the lentivirus stock solution is in the range of 1 to 10E+05, and the activity titer of the concentrated solution is in the range of 1 to 10E+08. The detection analysis results are as shown in Tables 2 and 3.

[0091] Table 2 [Table 2]

[0092] Table 3 [Table 3]

[0093] Example 4 Production of CAR19T and CAR22T 100 mL of peripheral blood was collected from healthy individuals, and mononuclear cells were isolated using Ficoll lymphocyte separation solution. After counting, CD3-positive cells were selected using an appropriate amount of CD3 MicroBeads, human (Miltenyi Biotec), and 1.0–2.0 × 10 6 The cells were cultured at a density of 1000 cells / mL in complete T cell culture medium (OpTmizer® CTS® T-Cell Expansion Basal Medium, OpTmizer® CTS T-Cell Expansion Supplement (Invitrogen), 500 units of IL-2 (Beijing SL Pharmaceutical Co., Ltd.)) and simultaneously cultured in 25 μL / 10 6T cells were activated by adding Dynabeads Human T-Activator CD3 / CD28 (Invitrogen) to each cell.

[0094] After 24 hours, LVCAR19 or LVCAR22 virus was added at an MOI of 3 for transfection, mixed uniformly, and placed in a CO2 incubator. After 4 hours, an appropriate amount of complete T cell medium was added and cultured.

[0095] Twenty-four hours after lentiviral transduction, transduced CAR19 or CAR22 T cells were replaced with fresh complete T cell medium to achieve a viable cell density of 1.0 × 10 6 The culture density was adjusted to 1.0 × 10 / mL, and the cells were cultured and grown for 10 to 20 days. The cells were observed and counted daily, and the medium was replenished depending on the counted cell number. The cell culture density was always 1.0 × 10 / mL. 6 The blood pressure was maintained at 1 / mL.

[0096] CAR-T cell manufacturing: CAR19 or CAR22 T cells were collected according to the expected cell dose, resuspended in 100 mL of saline containing 2% human albumin, transferred to a cell reinfusion bag, and heat-sealed to produce the CAR19 or CAR22 T cell formulation product.

[0097] Detection of CAR19T and CAR22T transduction efficiency: 1.0×10 6 Transduced T cells were cultured with 1 μg / ml FITC-Protein-L at room temperature for 30 minutes, washed twice with saline, and then the FITC fluorescent signal was detected by flow cytometry to determine the FITC-positive cell rate, which reflects the percentage of CAR cells in the total cell count. The test results are shown in Figure 7 and Table 4, demonstrating that CAR19 T cells and CAR22 T cells were successfully generated.

[0098] Table 4 [Table 4]

[0099] Example 5 In vitro functional detection of CAR19T and CAR22T Calcein-AM was used to detect the in vitro tumor-killing function of CAR19T and CAR22T.

[0100] Take an appropriate amount of K562 or RAJI target cells and incubate them at 1 × 10 6 Calcein-AM was added to a final concentration of 25 μM to a 1.5 × 10 cells / mL cell suspension (PBS, 5% fetal bovine serum) and cultured in an incubator for 30 minutes. After washing twice at room temperature, 1.5 × 10 cells were cultured. 5 The cells were resuspended at 100 μg / mL. CAR19T or CAR22T cells were added at different effect-target ratios, centrifuged at 200 g for 30 seconds, and incubated at 37°C for 2-3 hours. After incubation, the supernatant was collected and the calcein fluorescence intensity was measured. The target cell lysis rate was calculated based on the spontaneous release control and maximum release control.

[0101] 1. Tumor killing experiment data Before re-injection, different batches of virus were subjected to functional detection such as tumor killing in cell lines using calcein test method. The formula is as follows:

number

[0102] F CTL detection = mean fluorescence value of triplicate wells containing target cells and a given concentration of effector T cells F spontaneous release = mean fluorescence value of three replicate wells containing target cells and 1xPBS Fmax emission = mean fluorescence value of three replicate wells containing target cells and lysate NOTE: Simultaneously, the fluorescence value of the cell pellet lysate in the 96-well plate was measured. The total fluorescence value of each well was equal to the sum of the fluorescence values ​​of the supernatant and cell pellet, and the difference in the total fluorescence value was kept less than 5%.

[0103] The results can be seen in Figure 8 and Table 6 below. Anti-CD19-CAR-T are CAR-T cells that target CD19, anti-CD22-CAR-T are CAR-T cells that target CD22, positive cells were RAJI cells, and negative cells were K562 cells.

[0104] Table 5 Positive target cells: [Table 5]

[0105] Table 6 Negative target cells: [Table 6]

[0106] The results showed that the killing rates of anti-CD19-CAR molecule-loaded T cells at multiple effector / target ratios of 50:1, 10:1, and 2:1 were 85%, 46%, and 15%, respectively, and that of anti-CD22-CAR molecule-loaded T cells at multiple effector / target ratios of 50:1, 10:1, and 2:1 were 94%, 47%, and 11%, respectively, indicating that tumor cell killing was efficient and dose-dependent.

[0107] Example 6 Sequential injection of CART19T and CAR22T in a mouse tumor-bearing model To detect the in vivo antitumor effects of CAR-T cells, immunodeficient (NOD-SCID) mice were selected to construct tumor models with Raji-luc cells. After successful modeling, CAR-T cells were injected and the experimental results were monitored using an IVIS small animal in vivo imaging system (Xenogen, Hopkinton, USA). The experimental steps are as follows:

[0108] 1. Modeling: 5 x 10 cells / mL were injected into 5-week-old NOD-SCID mice. 6 Raji-luc cells / mouse were injected subcutaneously.

[0109] After 2.7 days, the animals were photographed, and mice were anesthetized by intraperitoneal injection of D-luciferin (Molecular Imaging Product, Bend, USA) at a dose of 150 mg / kg and intraperitoneal injection of 75 mg / kg sodium pentobarbital, and 10 min later, optical signals were collected using an IVIS small animal in vivo imaging system (Xenogen, Hopkinton, USA).

[0110] 3. After successful modeling, CAR-T cells were injected on the same day, and the groups were as follows: (1) CD19 group, 5 × 10 CAR19 T cells 6 injected at 1000x / mouse. (2) CD22 group, CAR22 T cells were injected at 5 × 106 cells / mouse. (3) CD19+CD22 group: 5 × 10 CAR19 T cells 6 CAR22 T cells were injected at 5 x 10 cells / mouse, and 3 days later 6 injected at 1000x / mouse. (4) NT group, 5 × 10 normal T cells 6 injected at 1000x / mouse. (5) GFP group: 5 × 10 T cells transduced with GFP lentivirus 6 T cells / mouse.

[0111] 4. 21 days after CAR-T cell injection, imaging was performed and the experimental results were analyzed. See Figure 9, where NT: conventional T cell injection group, CD22: CAR22T injection group, GFP: GFP lentivirus-transduced T cell injection group, CD19+CD22: CAR19T and CAR22T injection group, CD19: CAR19T injection group.

[0112] The results showed that compared with the conventional T cell-injected group and the GFP-T-injected group, the tumor burden in both the CAR19T-injected group and the CAR22T-injected group was significantly reduced, and the tumors in the CD19+CD22-injected group completely disappeared, demonstrating that the efficacy of sequential injection of dual-targeted CAR T cells was superior to that of single-targeted injection and non-injected groups.

[0113] Example 7 Clinical Use of CAR19T and CAR22T Sequential infusion of anti-CD19 CAR-T and anti-CD22 CAR-T was used to treat 59 patients with relapsed / refractory / high-risk acute B-cell lymphoma, including 42 patients with B-ALL and 17 patients with aggressive B-cell lymphoma.

[0114] Clinical background of enrolled patients: Regarding B-ALL leukemia patients, the median age was 28 years, with a male-to-female ratio of approximately 3:2. Nearly half of the patients had relapsed more than twice or had primary refractory disease. Prior to enrollment in this study, seven patients underwent transplantation, and six received CAR19 therapy, but all relapsed. The percentage of dysregulated early B cells collected from bone marrow ranged from 0 to 92%. Six patients had dysregulated early B cells in their cerebrospinal fluid. Most patients had high-risk genetic abnormalities, including BCR / ABL translocations, T315I mutations, 17p deletions, or TP53 mutations. MLL rearrangements were also detected, primarily MLL / AF4 and hypodiploidy.

[0115] Regarding patients with aggressive B-cell lymphoma: The median age of lymphoma patients was 38 years, and the male-to-female ratio was approximately 1:1. All pathological subtypes were high-grade B-cell lymphoma, including one case of dual lymphoma. Approximately 65% ​​of patients had relapsed more than once or had primary refractory disease. Three patients underwent transplantation but relapsed. Nearly half of the patients had Myc translocations, 17p deletions, or TP53 mutations.

[0116] CAR-T cell therapy effects: For leukemia patients, 40 patients received pretreatment with fludarabine and cyclophosphamide before reinfusion of CAR19T and CAR22T. The mean doses of CAR19T and CAR22T were 2.44 ± 1.11 × 10 6 / kg and 2.19±1.22×10 6 / kg.

[0117] Thirty-eight of 42 (90.5%) patients achieved complete remission (CR or CRi), slightly higher than the 83% complete remission rate achieved by Kymriah, a CD19 CART drug marketed by Novartis. Thirty-three of 42 (78.6%) patients achieved molecular remission and were MRD-negative as detected by flow cytometry.

[0118] Overall survival and event-free survival at 6 months were 80.0% and 60.8%, respectively. At 12 months, overall survival and event-free survival approached approximately 60% and 40%, respectively.

[0119] In a follow-up study, 23 patients ultimately relapsed, although antigen escape of CD19 and CD22 had not previously been detected in relapsed cases after hybrid CAR-T cell therapy.

[0120] For patients with aggressive lymphoma, the majority achieved complete remission (CR). The overall response rate was over 80%, which is higher than the 72% achieved by Yescarta, a CD19 CART drug developed by FosunKite. Overall survival and early survival rates at 6 months were approximately 90% and 60%, respectively, higher than the 79% and 55% achieved by Yescarta.

[0121] Safety of CAR-T cell therapy: Among leukemia patients, the majority (88%) developed CRS, but only 20% had severe CRS (≥3 grade), a significantly lower response rate compared with 75% (47 of 63) for Kymriah, a CD19 CART drug marketed by Novartis.

[0122] Aggressive lymphoma patients overall experienced CRS, with only 10% experiencing severe but reversible CRS, slightly lower than the 13% with Yescarta, while only 17.6% experienced neurotoxicity, significantly lower than the 87% with Yescarta.

[0123] Example 8 Screening of exosome secretion-promoting genes The eukaryotic expression vector selected for this experiment was PCDNA3.1 (purchased from Invitrogen). Double enzyme digestion was performed in the laboratory on the original plasmid, with BamhI and NotI digestion yielding a linear vector with sticky ends. Double enzyme digestion prevents the vector from self-circularizing. The digestion product was amplified by PCR, and the size of the vector fragment was preliminarily verified by agarose gel electrophoresis. The gel was then recovered and the vector concentration was measured. This study investigated the effects of four genes that promote exosome secretion (including Booster, CX43, KIBRA, and Rab27a) on increasing exosome production. Here, Booster is composed of three genes (SDC4, STEAP3, and Ndab), of which the NCBI reference sequence number for the nucleotide sequence of SDC4 is NM_002999.4, the NCBI reference sequence number for the nucleotide sequence of STEAP3 is NM_001008410.2, and the nucleotide sequence of Ndab is shown in SEQ ID NO: 11 (the NCBI reference sequence number for the amino acid sequence of SDC4 is NP_002990.2, the NCBI reference sequence number for the amino acid sequence of STEAP3 is NP_878919.2, and the amino acid sequence of Ndab is shown in SEQ ID NO: 12). The NCBI reference sequence number for the nucleotide sequence of CX43 is NM_000165.5 (NCBI reference sequence number for amino acid sequence: NP_000156.1), the NCBI reference sequence number for the nucleotide sequence of KIBRA is XM_005265853.3 (NCBI reference sequence number for amino acid sequence: NP_056053.1), and the NCBI reference sequence number for the nucleotide sequence of Rab27a is NM_183236.3 (NCBI reference sequence number for amino acid sequence: NP_899058.1).

[0124] Nucleotide sequence of Ndab (SEQ ID NO: 11):

[0125] Amino acid sequence of Ndab (SEQ ID NO: 12): MNTLPEHSCDVLIIGSGAAGLSLALRLADQHQVIVLSKGPVTEGSTFYAQGGIAAVFDETDSIDSHVEDTLIAGAGICDRHAVEFVASNARSCVQWLIDQGVLFDTHIQPNGEESYHLTREGGHSHRRILHAADA TGREVETTLVSKALNHPNIRVLERSNAVDLIVSDKIGLPGTRRVVGAWVWNRNKETVETCHAKAVVLATGGASKVYQYTTNPDISSGDGIAMAWRAGCRVANLEFNQFHPTALYHPQARNFLLTEALRGEGAYLK RPDGTRFMPDFDERGELAPRDIVARAIDHEMKRLGADCMFLDISHKPADFIRQHFPMIYEKLLGLGIDLTQEPVPIVPAAHYTCGGVMVDDHGRTDVEGLYAIGEVSYTGLHGANRMASNSLLECLVYGWSAAED ITRRMPYAHDISTLPPWDESRVENPDERVVIQHNWHELRLFMWDYVGIVRTTKRLERALRRITMLQQEIDEYYAHFRVSNNLLELRNLVQVAELIVRCAMMRKESRGLHFTLDYPELLTHSGPSILSPGNHYINR

[0126] Common cell transfection methods include artificial liposomes, viral transfection, and PEI transfection. However, artificial liposomes are relatively expensive, and viral transfection may cause some damage to the HEK293T or HEK293F cells used in this experiment. Therefore, we adopted the PEI transfection method in this study. DNA can be transfected into host cells using polyethyleneimine (PEI), a stable cationic polymer. PEI condenses DNA into positively charged particles that bind to the anionic cell surface. Therefore, the DNA:PEI complex is taken up by cells and releases the DNA into the cytoplasm. Considering experimental costs, the use of PEI is preferable to other cell transfection reagents. PEI transfection requires high requirements for cell viability, number, and viability. After 24 hours of culture, HEK293T or HEK293F cells are harvested and observed under a microscope. When cell fusion rates reach 60%-70%, the cells have entered logarithmic growth phase, indicating optimal cell transfection. Cell viability is above 90%. Typically, 1 μg of DNA is used per mL of culture for transfection. The PEI and DNA should be diluted to 1 / 20 of the total culture volume before mixing. 30 μL of pre-prepared PEI (1 μg / μL) was added to 100 μL of pure DMEM medium, placed in a 1.5 mL centrifuge tube, and vortexed to mix. 275 ng of CD63-nanoluc plasmid (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) and 725 ng of the exosome secretion plasmid constructed above (mass ratio 1:3) were added to a new 1.5 mL centrifuge tube containing 100 μL of pure DMEM medium, vortexed to mix, and left to stand for 5 minutes.The incubated PEI was added to the centrifuge tube containing the plasmid mixture. The recommended mass ratio of PEI to plasmid is 1:3. The mixture was allowed to stand for 30 minutes to allow the PEI to fully encapsulate the double plasmid, forming a PEI-DNA complex. CD63-nanoluc was used to quantify exosomes. This ensured equal amounts of each plasmid, promoting exosome secretion. A blank control was also set up, containing the same concentrations of CD63-nanoluc and empty vector (pcDNA3.1). Finally, the PEI-DNA mixture was added dropwise to the cell culture medium of the monolayer cells. The plate was gently shaken to mix, taking care not to disrupt the adherent cells, and then cultured in a cell incubator at 37°C and 5% CO2. After 24 hours of culture, 10 mL of DMEM complete medium was added to replenish the liquid and maintain a favorable culture environment for the cells. The cells were then cultured in a cell incubator at 37°C and 5% CO2.

[0127] As shown in Figure 10, the quantitative results of exosome purification showed that KIBRA and Rab27a can significantly increase exosome production.

[0128] Example 9 Collection of exosomes Exo-CAR-CD19 and Exo-CAR-CD22 (I) Construction of HEK-293T or HEK-293F cells transfected with recombinant lentivirus LVCAR19 or LVCAR22, an exosome secretion-promoting plasmid, and granzyme and perforin overexpression plasmids.

[0129] <1> Plasmids used in transfection in the examples and their construction methods (1) The recombinant lentivirus LVCAR19 or LVCAR22 was constructed in accordance with Examples 1 to 3 of the present invention. (2) The construction method of the exosome secretion-promoting plasmid can be seen in Example 8 of the present invention. The exosome secretion-promoting gene of the exosome-promoting plasmid used in this example was KIBRA. The exosome secretion-promoting gene KIBRA was purchased from Wuhan Miaoling Biotechnology Co., Ltd. or obtained by RT-PCR in cells. The vector was digested with NheI and XhoI pcDNA3.1, and homologous recombination was performed. The vector was then verified by first-generation sequencing by Sangon Biotech. (3) Construction of the CD63-fused perforin overexpression plasmid, PcDNA3.1-CD63-PRF1, and the CD63-fused granzyme overexpression plasmid, PcDNA3.1-CD63-GZMB: Plasmid CD63-nanoluc was digested with endonucleases EcoR I and Age I at 37°C to obtain the CD63 sequence (nucleotide sequence shown in SEQ ID NO:5, amino acid sequence shown in SEQ ID NO:6). The nucleotide sequences of perforin PRF1 and granzyme GZMB are shown in SEQ ID NO:7 and SEQ ID NO:9, respectively, and their amino acid sequences are shown in SEQ ID NO:8 and SEQ ID NO:10, respectively. The vector, pcDNA3.1, was digested with Nhe I and Xho I, and homologous recombination was performed. The results were verified by first-generation sequencing by Sangon Biotech.

[0130] Nucleotide sequence of CD63 (SEQ ID NO:5): Atggcggtggaaggaggaatgaaatgtgtgaagttcttgctctacgtcctcctgctggccttttgcgcctgtgcagtgggactgattgccgtgggtgtcggggcacagcttgtcctgagtcagaccataatccaggggctacccctggctctctgttgccagtggtcatcatcgcag tgggtgtcttcctcttcctggtggcttttgtgggctgctgcggggcctgcaaggagaactattgtcttatgatcacgtttgccatctttctgtctcttatcatgttggtggaggtggccgcagccattgctggctatgtgtttagagataaggtgatgtcagagtttaataacaacttc cggcagcagatggagaattacccgaaaaataaacccacactgcttcgatcctggacaggatgcaggcagattttaagtgctgtggggctctaactacacagattgggaagaaaatcccttccatgtcgaagaaccgagtccccgactcctgctgcattaatgttactgtgggctgtggga ttaatttcaacgagaaggcgatccataaggagggctgtgtggagaagattggggggctggctgaggaaaaaatgtgctggtggtagctgcagcagcccttggaattgcttttgtcgaggttttgggaattgtctttgcctgctgcctcgtgaagagtatcagagtggctacgaggtgat

[0131] CD63's amino acid sequence(sequence number 6): MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNNNF RQQMENYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVM

[0132] Nucleotide sequence of perforin PRF1 (SEQ ID NO:7):

[0133] Amino acid sequence of perforin PRF1 (SEQ ID NO:8): MAARLLLLGILLLLLPLPVPAPCHTAARSECKRSHKFVPGAWLAGEGVDVTSLRRSGSFPVDTQRFLRPDGCTLCENALQEGTLQRLPLALTNWRAQGSGCQRHVTRAKVSSTEAVARDAARSIRNDWKVGLDVTPK PTSNVHVSVAGHSQAANFAAQKTHQDQYSFSTDTVECRFYSFHVVHTPPLHPDFKRALGDLPHHFNASTQPAYLRLISNYGTHFIRAVELGGRISALTALRTCELALEGLTDNEVEDCLTVEAQVNIGIHGSISEAAK ACEEKKKKHKMTASFHQTYRERHSEVVGGHHTSINDLLFGIQAGPEQYSAWVNSLPGSPGLVDYTLEPLHVLLDSQDPRREALRRALSQYLTDRARWRDCSRPCPPGRQKSPRDPCQCVCHGSAVTTQDCCPRQRGLAQ LEVTFIQAWGLWGDWFTATDAYVKLFFGGQELRTSTVWDNNNPIWSVRLDFGDVLLATGGPLRLQVWDQDSGRDDDLLGTCDQAPKSGSHEVRCNLNHGHLKFRYHARCLPHLGGGTCLDYVPQMLLGEPPGNRSGAVW

[0134] Nucleotide sequence of granzyme GZMB (SEQ ID NO:9): Atgcaaccaatcctgcttctgctggccttctctcctgctgcccagggcagatgcaggggagatcatcgggggacatgaggccaagccccactcccgcccctacatggcttatcttatgatctgggatcagaagtctctgaagaggtgcggtggcttcctgatacaagacgacttcgtgctgacagct gctcactgttgggggaagctccataaatgtcaccttggggcccacaatatcaaagaacaggagccgacccagcagtttatccctgtgaaaagacccatcccccatccagcctataatcctaagaacttctccaacgacatcatgctactgcagctggagagaaaggccaagcggaccagagctgtg cagcccctcaggctacctagcaacaaggcccaggtgaagccagggcagacatgcagtgtggccggctggggggcagacggcccccctgggaaaacactcacacacactacaagaggtgaagatgacagtgcaggagaatcgaaagtgcgaatctgacttacgccattattacgacagtaccattgag ttgtgcgtggggacccagagattaaaaagacttcctttaaggggactctggaggccctcttgtgtgtaacaaggtggcccagggcattgtctcctatggacgaaacaatggcatgcctccacgagcctgcaccaaagtctcaagctttgtacactggataaagaaaaccatgaaacgccactaa

[0135] グランザイムGZMBのamino acid sequence(sequence number 10): MQPILLLLAFLLLPRADAGEIIGGHEAKPHSRPYMAYLMIWDQKSLKRCGGFLIQDDFVLTAAHCWGSSINVTLGAHNIKEQEPTQQFIPVKRPIPHPAYNPKNFSNDIMLLQLERKAKRTRA VQPLRLPSNKAQVKPGQTCSVAGWGQTAPLGKHSHTLQEVKMTVQEDRKCESDLRHYYDSTIELCVGDPEIKKTSFKGDSGGPLVCNKVAQGIVSYGRNNGMPPRACTKVSSFVHWIKKTMKRH

[0136] <2> Cell culture and transfection HEK-293T cells (DSMZ:ACC-635) were cultured in DMEM (Gibco) supplemented with 10% (v / v) fetal bovine serum (FBS, VIVA) and 1% (v / v) penicillin / streptomycin solution (Gibco). HEK-293F cells were cultured in S-F medium, which was prepared by mixing equal volumes of SMM 293-TII medium and OPM-293 CD05 Medium, in a humidified atmosphere containing 5% CO. 0.05% trypsin-EDTA (Gibco) was used for serial passage of these cells.

[0137] Transfection (HEK-293T, HEK-293F) and 5 × 10 24 h before transfection. 6 Seed the cells into a 10 cm cell culture dish and add 30 µL of PEI (PEI, 20000MW, Polysciences, stock solution 1 mg / mL in dHO) to 10 µg of total DNA ( <1> recombinant lentivirus LVCAR19 or LVCAR22, <2> KIBRA exosome secretion promoting plasmid, <3> PcDNA 3.1-CD63-PRF1, <4> HEK-293T or HEK-293F cells were transfected into 10 cm cell culture dishes by culturing them with pcDNA3.1-CD63-GZMB. 16 hours after transfection, the medium was replaced with fresh DMEM.

[0138] (II) Collecting exosomes from the cells constructed in step (I) of this example HEK-293F cells transfected with the recombinant lentivirus LVCAR19 or LVCAR22 obtained in step (I), KIBRA exosome secretion-promoting plasmid, and granzyme and perforin overexpression plasmids were cultured in CM-conditioned medium, and exosomes were collected using the following steps.

[0139] 1. Pre-cleared CM (conditioned medium) (starting from step 2) by differential centrifugation and filtration as follows: (1) Centrifuge at 500 × g for 5 minutes at 4 °C, transfer the supernatant to a new tube, and discard the pellet. Repeat once more, collecting the supernatant and discarding the pellet. (2) The supernatant from step (1) was centrifuged at 2000 × g for 15 minutes at 4 °C, and the pellet was discarded. The collected supernatant was filtered once through a 0.22 μm filter.

[0140] 2. For pre-cleaning, 1.9 g (±0.001 g) of sucrose was accurately weighed into a universal test tube and topped up with deuterium until the weight reached 7.6 g (±0.001 g) to prepare a 25% (w / w) sucrose deuterium solution.

[0141] 3. 22.5 ml of pre-cleared CM was added to an ultracentrifuge tube. If the volume was less than 22.5 ml, the CM was topped up to 22.5 ml with 0.22 μm-filtered PBS.

[0142] 4. Place a glass pipette into the centrifuge tube and add 3 ml of sucrose solution so that the sucrose solution forms a separate layer below the CM.

[0143] 5. Carefully place the centrifuge tube containing the layered CM / sucrose solution into the bucket of the spin-out rotor and secure the bucket to the rotor.

[0144] 6. The rotor was placed in an ultracentrifuge and centrifuged at 100,000 xg at 4°C for 1.5 hours.

[0145] 7. 2 ml of the sucrose layer was collected and added to an ultracentrifuge tube containing 20 ml of filtered PBS to wash.

[0146] 8. The tube was placed in a fixed-angle rotor and centrifuged at 100,000 xg for 1.5 hours at 4°C.

[0147] 9. The supernatant was carefully removed using a 10 ml pipette and the pellet was resuspended in 400 μL of filtered PBS.

[0148] 10. Exosomes were stored short-term at 4°C and long-term at -80°C for use in subsequent experiments.

[0149] Example 10 Identification and phenotypic analysis of exosomes Exo-CAR-CD19 and Exo-CAR-CD22 Electron microscopy, particle size and flow cytometry analyses were performed on Exo-CAR-CD19 and Exo-CAR-CD22 collected in Example 9, and the results are shown in Figure 11.

[0150] 10.1 Exosome TEM Detection / Transmission Electron Microscopy Process 1. Exosomes were fixed with glutaraldehyde. 2. 5-10 μL of exosome solution was added to the copper mesh and allowed to adsorb for approximately 10 minutes at room temperature. Excess liquid was then carefully removed by suction using filter paper. Next, 10 μL of 2% phosphotungstic acid solution (pH 6.5) was added dropwise to the copper mesh and stained for 2 minutes at room temperature. Excess staining solution was then carefully removed by suction using filter paper, and the copper mesh was allowed to dry at room temperature. 3. The observation was carried out by machine, and the observation voltage was 120kV.

[0151] 10.2 Nanoflow cytometry Exosomes were stained with monoclonal antibodies (MABs) that recognize exosome protein markers (CD9, CD63, and CD81) using phycoerythrin (PE, purchased from Biolegend) and detected by nanoflow cytometry. Exosomes were counted one by one at the single particle level, and single or two protein markers present on the exosome membrane surface were quantitatively analyzed individually and in combination. The specific steps are as follows: 1. A wash buffer containing an enhancer (purchased from Wako, code number: 297-79701) was prepared. (1) 10x washing buffer was diluted to 1x with ultrapure water. (2) 100x binding enhancer was added to 1x wash buffer at a ratio of 1:100 to obtain wash buffer containing enhancer.

[0152] 2. Magnetic Bead Pretreatment (1) The required amount of magnetic bead suspension was added at 10 μL per tube, and 100 μL of wash buffer containing an enhancer was added for every 10 μL of magnetic beads (purchased from Wako, code number: 297-79701). The mixture was vortexed for 5 seconds, quickly centrifuged, and then allowed to stand on a magnetic rack for 1 minute, and the supernatant was carefully removed. (2) The above steps were repeated once.

[0153] 3. Exosome binding (1) 100 μL of concentrated exosome suspension was taken. (2) 100 μL of the exosome suspension was added to the EP tube containing the magnetic beads and vortexed for 5 seconds. (3) 1 μL of exosome-binding enhancer (100×) was added to the above EP tube and vortexed for 5 seconds. (4) The EP tube was left standing at room temperature for 1 hour and vortexed every 20 minutes to ensure complete capture of exosomes. (5) The EP tube was quickly centrifuged, placed on a magnetic stand for 1 minute, and the supernatant was carefully removed. 500 μL of washing buffer containing a binding promoter was added to the EP tube and mixed by vortexing for 5 seconds. The EP tube was quickly centrifuged, placed on a magnetic stand for 1 minute, and the supernatant was removed. (6) The above steps were repeated once. (7) 100 μL of washing buffer containing a binding enhancer was added to the EP tube, and the tube was vortexed for 3 seconds to obtain a suspension of magnetic beads bound to exosomes.

[0154] 4. Exosome Immunostaining (1) 100 μL of the exosome-bound magnetic bead suspension was added to each EP tube. (2) The labeled anti-exosome surface marker protein antibody and the labeled isotype control antibody were added to the above EP tube and vortexed for 5 seconds. (3) The EP tube was left at room temperature for 1 hour and vortexed every 20 minutes to allow the antibody to bind. (4) The cultured EP tube was quickly centrifuged and placed on a magnetic stand for 1 minute to separate the magnetic beads from the supernatant, after which the supernatant was removed. (5) 300 μL of washing buffer (containing enhancer) was added to each EP tube, vortexed for 5 seconds, quickly centrifuged, and placed on a magnetic stand for 1 minute to separate the magnetic beads from the supernatant, after which the supernatant was removed. (6) The above steps were repeated once. (7) 300 μL of washing buffer (containing an enhancer) was added to each EP tube, which was then vortexed for 5 seconds and used for flow cytometry detection. Exosome phenotyping by flow cytometry showed that 94.2% of the exosomes contained the CAR molecular structure, and all exosomes expressed the exosome-specific proteins CD81, CD63, and CD9.

[0155] 10.3 NTA (for exosome samples, to detect particle size distribution and concentration): <1> The sample pool was washed with deionized water. <2> The instrument was calibrated with polystyrene microspheres (100 nm). <3> The sample pool was washed with 1× PBS buffer (Biological Industries, Israel). <4> The sample was diluted with 1x PBS buffer (BI, Israel) and injected for detection. The results showed that the diameter distribution of exosomes was mainly concentrated around 150 nm.

[0156] Example 11 Detection of granzymes and perforin in 293F recombinant exosomes 11.1 The culture supernatant from the Exo-CAR cell killing experiment was centrifuged at 1000 × g for 10 minutes at 4 °C, and 200 μL was dispensed into each EP tube and frozen at -80 °C to avoid repeated freezing and thawing. 11.2 The experiments were performed referring to the instructions for the two ELISA kits (Human Perforin ELISA Kit, Solarbio, Catalog Number: SEKH-0295, Human Granzyme ELISA Kit, Solarbio, Catalog Number: SEKH-0193). Because the steps and reagents are almost the same, the Human Perforin ELISA Kit was used as an example.

[0157] 11.2.1 Warming of reagents: The reagent kit and test sample were left at room temperature 30 minutes before the start of the experiment. If crystallization occurred, the kit was placed in a 37°C water bath to completely dissolve the crystals. 11.2.2 Preparation of cleaning solution: The 20x concentrated cleaning solution was diluted with deionized water to a 1x working solution, and the working solution volume was 300ml (30ml increase). The cleaning solution was standard, and when preparing two kits at the same time, 560ml (20ml increase) was prepared. 11.2.3 Gradient dilution of standard solution (the two kits are different): Standard solution (SR1) (0.5 mL of perforin, 1 mL of granzyme) was added to the lyophilized standard solution and left for 15 minutes to dissolve completely, then gently mixed and gradient diluted according to the instructions. The reconstituted standard solution was stored at -20°C or -80°C. 11.2.4 Preparation of biotinylated antibody working solution: 100x antibody concentration was diluted to 1x with detection diluent (SR2) (mix thoroughly before dilution) and added to the reaction well within 30 minutes. 6500μL of working solution (65μL + 6435μL) was prepared. 11.2.5 Preparation of Enzyme-Conjugated Working Solution (the two kits are different): Dilute to a 1x working solution (40x for Granzyme and 100x for Perforin) with Enzyme-Conjugated Diluent (SR3) (centrifuge before dilution) and add to the reaction wells within 30 minutes. For Perforin, prepare 6500 µL of working solution (65 µL + 6435 µL), and for Granzyme B, prepare 6000 µL of working solution (150 µL + 5850 µL). 11.2.6 Automatic plate washing settings: Shake off the liquid in the wells of the ELISA plate, tap them dry on absorbent paper, inject 300 μL of washing solution, and wash the plate 5 times with a 30-second interval between injection and aspiration.

[0158] 11.2.7 Discovery Step: Detection step: Granzyme B 1. 30 minutes before the experiment, remove the human granzyme ELISA kit and allow it to warm to room temperature. Before adding the standards / samples, wash the plate three times, shake it off, and dry it. 2. 100 μL of the standard solution and test sample were added to the reaction wells, the plate was sealed, and the plate was incubated in a 37°C incubator for 90 minutes. The tapping and washing was repeated four times. 3. 10 μL of biotinylated antibody working solution was added to the reaction wells, and the plate was sealed and incubated in a 37°C incubator for 60 minutes. The tapping and washing was repeated four times. 4. 100 μL of the enzyme conjugate working solution was added to the reaction well, and the plate was sealed and incubated in a 37°C incubator for 30 minutes. The beating and washing was repeated five times. 5. 100 μL of colorimetric substrate was added to the reaction well, the plate was sealed, and the plate was incubated at 37°C in the dark for 15 minutes. 6. 50 μL of stop solution was added, and the OD at 450 nm was immediately measured using an ELISA reader (within 5 minutes).

[0159] Detection step: Perforin 1. 30 minutes before the experiment, remove the human perforin ELISA kit and allow it to warm to room temperature. Wash the plate three times and shake dry before adding the standards / samples. 2. 100 μL of the standard solution and the detection sample were added to the reaction wells, the plate was sealed, and the plate was incubated at room temperature (25 + 2°C) for 120 minutes. The tapping and washing was repeated four times. 3. 10 μL of biotinylated antibody working solution was added to the reaction wells, and the plate was sealed and incubated at room temperature (25 + 2°C) for 60 minutes. The tapping and washing was repeated four times. 4. 100 μL of the enzyme conjugate working solution was added to the reaction well, the plate was sealed and incubated at room temperature (25 + 2°C) for 60 minutes. The beating and washing was repeated five times. 5. 100 μL of colorimetric substrate was added to the reaction well, the plate was sealed, and the plate was incubated at room temperature (25 + 2°C) in the dark for 10 to 30 minutes. 6. 50 μL of stop solution was added, and the optical density (OD) at 450 nm was immediately measured using an ELISA reader (within 5 minutes). NOTE: The speed of the microoscillator was 300–400 rpm (preferably to prevent the liquid in the microwells from splashing out).

[0160] Result judgment: 1. The OD value at 450 nm was measured using a microplate reader. Dual wavelength detection was selected, with 630 nm as the reference wavelength. If dual wavelength detection is not possible, the OD value at 630 nm can be subtracted from the OD value at 450 nm. 2. Calculate the average OD values ​​of the standard solutions and samples: The OD value of the zero well was subtracted from the OD value of each standard solution and sample. 3. A standard curve was created using software with the concentration of the standard solution on the horizontal axis and the absorbance OD value on the vertical axis (see Figure 16), and the granzyme B content of the sample was converted to the corresponding concentration using the corresponding OD value from the standard curve. 4. If the OD value of the sample was higher than the upper limit of the standard curve, it was diluted appropriately and retested, and the dilution factor was multiplied when calculating the concentration.

[0161] The detection results are shown in Figure 13. The following exosome groups were collected for quantification of granzymes and perforin: PB-PRF1 is a perforin co-expression plasmid (backbone: PcDNA 3.1), PcDNA 3.1-CD63-PRF1 is a perforin-fused to CD63 expression plasmid, PB-GZMB is a granzyme-expression plasmid (backbone: PcDNA 3.1), and PcDNA 3.1-CD63-GZMB is a granzyme-fused to CD63 expression plasmid. Cell, exo, and cell supernatant represent the granzyme and perforin contents in transfected cells, exosomes, and cell culture supernatant, respectively. The results show that granzymes and perforin were enriched in exosomes, reaching 4700 pg / ml and 25,000 pg / ml, respectively, significantly higher than their intracellular expression levels. When the cells were not transfected with a plasmid containing a granzyme or perforin gene, the granzyme and perforin contents in the cells, exosomes, and cell culture supernatant were 0 pg / ml.

[0162] The granzyme and perforin contents of the modified exosomes of the present invention compared to exosomes isolated from CAR19T and CAR22T are shown in Table 7 below.

[0163] Table 7 [Table 7]

[0164] As can be seen from Table 7, the perforin and granzyme contents of the CD22-modified exosomes of the present invention are significantly higher than the granzyme and perforin contents of the exosomes isolated from CAR22T, while the perforin and granzyme contents of the CD19-modified exosomes of the present invention can reach substantially the same level as the granzyme and perforin contents of the exosomes isolated from CAR19T.

[0165] Example 12 In vivo antitumor effect of exosomes Exo-CAR-CD19 and Exo-CAR-CD22 To verify the in vivo antitumor effect of Exo-CAR, immunodeficient (NOD-SCID) mice and Raji-luc cells were used to construct a tumor model. After successful model construction, Exo-CAR was injected and the experimental results were detected using an IVIS small animal in vivo imaging system (Xenogen, Hopkinton, USA). The experimental steps are as follows:

[0166] 1. Modeling: 5 x 10 cells / mL were injected into 5-week-old NOD-SCID mice. 6 Raji-luc cells / mouse were injected subcutaneously. After 2.7 days, animals were imaged by intraperitoneal injection of 150 mg / kg D-luciferin (Molecular Imaging Products, Bend, USA) and 75 mg / kg sodium pentobarbital to anesthetize the mice. After 10 minutes, optical signals were collected using an IVIS small animal in vivo imaging system (Xenogen, Hopkinton, USA). 3. After successful modeling, Exo-CAR was injected on the same day, and the groups were as follows: (1) CD19 group, injected with Exo-CAR-CD19, 5 × 10 12 pieces / fish. (2) CD22 group, injected with Exo-CAR-CD22, 5 × 10 12 pieces / fish. (3) CD19 + CD22 group: First, Exo-CAR-CD19 was added to 5 × 10 12 5 × 10 cells / mouse, and 3 days later, Exo-CAR-CD22 was administered at 5 × 10 cells / mouse. 12 Inject one per animal. (4) CD22 + CD19 group: First, Exo-CAR-CD22 was administered at 5 × 10 12 5 × 10 cells / mouse, and 3 days later, Exo-CAR-CD19 was administered at 5 × 10 cells / mouse. 12 Inject one per animal. (5) NT group, negative control exosomes. (6) EGFP group, non-targeted control exosomes. 4. Imaging was performed 21 days after Exo-CAR injection, and the experimental results were analyzed. The results are shown in Figure 12. Compared with the NT group and EGFP group, the tumor-bearing tumors in the CAR19 group, CAR22 group, CD19+CD22 group, and CD22+CD19 group were significantly reduced, and eventually (on day 21) the tumors completely disappeared, indicating that Exo-CAR has an antitumor effect in vivo.

[0167] Example 13 Killing effect of genetically engineered exosomes on tumor cells Detection of cell death by double staining with fluorescent dyes Fluorescent dye double-staining flow cytometry is a method in which target cells are labeled with two different fluorescent dyes and then killed after co-culture with effector cells. Dead cells were determined by detecting the decrease in double-fluorescent cells on the flow cytometer, and the killing efficiency was calculated.

[0168] Specific steps: (1) EGFP-expressing cells were used as target cells and resuspended in medium containing NK-EVs. (2) 2 × 10 cells 4 The cells were seeded into a 96-well plate at a density of 100 cells / well. (3) 400 μg of exosomes were taken from the experimental group, and the final volume of each well was 100 μL. (4) No exosomes were added to the positive control group. (5) After mixing all the wells, the mixture was cultured at 37°C in a CO2 incubator for approximately 12 or 24 hours. (6) After the culture was completed, the cells could be directly detected by flow cytometry. The results are shown in Figure 14. The cell killing was divided into the PBS control group, the 293F cell exosome group, the EGFP CAR exosome control group, the anti-CD19 CAR exosome group, and the anti-CD22 CAR exosome group. The target cells were RAJI-EGFP-LUC, and the negative control cells were K562-EGFP-LUC. The killing results showed that the killing rates of the anti-CD19 CAR exosome group and the anti-CD22 CAR exosome group against the positive target cells were 63% and 98%, respectively.

[0169] To detect cell killing, we used the same fluorescent dye double-staining flow cytometry method, but in step (3), we set the exosome concentration gradient from 0 μg to 200 μg. The results are shown in Figure 15. The killing effect of anti-CD19 CAR exosomes and anti-CD22 CAR exosomes is dose-dependent, and at sufficiently high doses (>200 μg), the killing effect of CAR-T can be achieved or even exceeded.

[0170] Example 14 Exosome secretion-promoting gene CX43 (S368A) Using the same method as in Example 9, HEK-293T cells or HEK-293F cells were constructed by introducing recombinant lentivirus LVCAR19 or LVCAR22, an exosome secretion-promoting plasmid, and granzyme and perforin overexpression plasmids. The difference is that the exosome secretion-promoting gene of the exosome secretion-promoting plasmid used in this example is CX43 (S368A), whose amino acid sequence is NCBI reference sequence number: NP_000156.1, with S at position 368 substituted with A (the nucleotide sequence encoding this is NCBI reference sequence number NM_000165.5, with AGC substituted with GCC at positions 1317-1319). The total amount of protein secreted by the constructed cells was measured, and the results are shown in Figure 17. Through preliminary optimization of the plasmid ratio, including co-transfection of the secretion-promoting plasmid CX43(S368A), the secretion amount of artificial exosomes specifically loaded with granzyme and perforin CAR-CD19 and CAR-CD22 could be increased by 3-4 times, although there is still room for improvement in large-scale exosome production.

[0171] The above embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Any equivalent replacement or modification made by those skilled in the art based on the present invention falls within the scope of protection of the present invention. The scope of protection of the present invention is subject to the claims.

Claims

1. A CAR molecule targeting CD19 and / or a CAR molecule targeting CD22, wherein the amino acid sequence of the CAR molecule targeting CD19 is set forth in SEQ ID NO: 1 and the amino acid sequence of the CAR molecule targeting CD22 is set forth in SEQ ID NO:

2.

2. Encoding the CAR molecule of claim 1, Preferably, the nucleotide sequence encoding the CD19-targeting CAR molecule is set forth in SEQ ID NO: 3, and / or the nucleotide sequence encoding the CD22-targeting CAR molecule is set forth in SEQ ID NO:

4.

3. The nucleic acid of claim 2, Preferably, the recombinant expression vector is a lentiviral vector; More preferably, the recombinant expression vector is characterized in that the backbone of the lentiviral vector is pLVX-EF1α-IRES-Puro.

4. Expressing the CAR molecule of claim 1, Preferably, the recombinant expression vector according to claim 3 is introduced into a host cell, More preferably, the host cell is a mammalian cell, for example, a T cell, a 293 cell, or a cell line derived from a 293 cell, such as a 293T cell or a 293F cell.

5. expressing granzyme and / or perforin, the amino acid sequence of said granzyme being set forth in SEQ ID NO: 10, and the amino acid sequence of said perforin being set forth in SEQ ID NO: 8; Preferably, the granzyme and / or perforin is fused to CD63, the amino acid sequence of which is set forth in SEQ ID NO: 6; More preferably, a protein that promotes exosome secretion is further overexpressed, and the protein that promotes exosome secretion is CX43, KIBRA, or Rab27a, and the NCBI reference sequence number of the amino acid sequence of CX43 is NP_000156.1, preferably, S is substituted with A at position 368, and the NCBI reference sequence number of the amino acid sequence of KIBRA is NP_056053.1, and the NCBI reference sequence of the amino acid sequence of Rab27a is NP_899058.1; More preferably, the nucleotide sequence encoding the granzyme is set forth in SEQ ID NO: 9, the nucleotide sequence encoding the perforin is set forth in SEQ ID NO: 7, the nucleotide sequence encoding the CD63 is set forth in SEQ ID NO: 5, the NCBI reference sequence of the nucleotide sequence encoding the CX43 is NM_000165.5, preferably, AGC is substituted with GCC at positions 1317 to 1319, the NCBI reference sequence of the nucleotide sequence encoding the KIBRA is XM_005265853.3, and / or the NCBI reference sequence of the nucleotide sequence encoding the Rab27a is NM_183236.

3.

6. the genes encoding the granzyme, perforin, CD63, KIBRA and / or Rab27a are placed in an expression plasmid; The transformant according to claim 5, wherein the backbone of the expression plasmid is preferably PcDNA3.1 or pCMV.

7. A method for producing exosomes, comprising the steps of culturing the transformant according to any one of claims 4 to 6, and isolating and purifying exosomes from a culture medium of the transformant.

8. comprising the CAR molecule of claim 1, Preferably, the exosome is produced by the method according to claim 7.

9. The present invention relates to a method for producing a CAR molecule comprising administering to a subject an effective amount of the CAR molecule of claim 1, a method for producing a CAR molecule of claim 2, a method for producing a CAR molecule of claim 3, a method for producing a CAR molecule of claim 4, a method for producing a CAR molecule of claim 5, a method for producing a CAR molecule of claim 6, a method for producing a CAR molecule of claim 7, a method for producing a CAR molecule of claim 8, a method for producing Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

10. Use of the CAR molecule according to claim 1, the transformant according to any one of claims 4 to 6, the exosome according to claim 8, or the pharmaceutical composition according to claim 9 in the manufacture of a medicament for preventing and / or treating a tumor associated with CD19 and / or CD22 expression, Preferably, the tumor is an acute B-lymphocytic tumor; More preferably, said acute B-lymphoid tumor is B-ALL leukemia or aggressive B-cell lymphoma.

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