A nano-operon for constructing CAR-T cells and a preparation method and application thereof

Abstract

The application discloses a kind of nano-operon for constructing CAR-T cell and its preparation method and application, belong to the field of biological medicine technology.The nano-operon for constructing CAR-T cell of the application includes nanovesicle, the nanovesicle carries CAR protein and the membrane fusion protein of target T cell, the nano-operon can utilize membrane fusion mode, so that the CAR protein is delivered to T cell by the nanovesicle carrying the membrane fusion protein of target T cell, to be able to construct CAR-T cell with tumoricidal activity directly in vivo or in vitro, reduce the waiting time of tumor treatment and reduce the required index requirements of treatment on one hand, on the other hand, effectively reduce the side effects generated in CAR-T cell therapy, realize the alleviation to patient burden.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a nano-operon for constructing CAR-T cells and a preparation method and application thereof. BACKGROUND

[0002] Chimeric antigen receptor (CAR) T cell therapy is a kind of immune cell therapy, which has shown excellent therapeutic results in the treatment of hematological tumors, and can significantly reduce the cost and time of patient treatment, and is called the next generation revolution of cell therapy.

[0003] However, as a personalized treatment scheme, the existing treatment generally needs to repeatedly separate T cells, transduce CAR genes, activate and expand, and reinfuse the patient with multiple steps for each patient, the treatment cycle is long, and the requirements for medical personnel and medical places are high, resulting in very high treatment cost; at the same time, the existing CAR-T cell therapy also causes side effects (such as cytokine release syndrome) that threaten the life and health safety of patients. SUMMARY

[0004] The purpose of the present application is to provide a nano-operon for constructing CAR-T cells and a preparation method and application thereof, aiming to solve the technical problems of high treatment cost, long cycle and obvious side effects of the existing CAR-T cell therapy.

[0005] In order to achieve the above-mentioned purpose, the technical scheme of the present application is:

[0006] The first aspect of the present application provides a nano-operon for constructing CAR-T cells, which comprises a nano-vesicle, wherein the nano-vesicle carries a CAR protein and a membrane fusion protein targeting T cells.

[0007] In a preferred implementation manner of the first aspect, the membrane fusion protein targeting T cells comprises a membrane fusion protein and a T cell surface marker protein molecule antibody sequence.

[0008] The T cell surface marker protein molecule antibody sequence is connected to the N-terminus of the membrane fusion protein.

[0009] In a preferred implementation manner of the first aspect, the membrane fusion protein comprises a p14TF protein, and a F protein and a H protein of a measles virus.

[0010] Among them, the H protein of the measles virus introduces Y481A mutation and R533A mutation.

[0011] In a preferred implementation manner of the first aspect, the H protein of the measles virus and the F protein sequence are connected through a T2A connecting peptide.

[0012] In a preferred implementation form of the first aspect, the marker protein molecule on the surface of the T cell is a CD3e molecule.

[0013] In a preferred implementation form of the first aspect, the antibody sequence of the marker protein molecule on the surface of the T cell is a scFv sequence.

[0014] In a preferred implementation form of the first aspect, the CAR protein is an aCD19-CAR protein.

[0015] The second aspect of the present application provides a preparation method of the nano-operon for constructing a CAR-T cell, and the preparation method comprises the following steps:

[0016] S101: constructing a membrane fusion protein for targeting T cells and a plasmid for expressing an aCD19-CAR, respectively;

[0017] S102: constructing an engineering cell capable of simultaneously expressing the membrane fusion protein for targeting T cells and the aCD19-CAR;

[0018] S103: using the engineering cell constructed in step S102 to produce a nano-vesicle carrying the membrane fusion protein for targeting T cells and the aCD19-CAR, thereby obtaining the nano-operon for constructing a CAR-T cell.

[0019] In a preferred implementation form of the second aspect, the method for constructing the membrane fusion protein for targeting T cells comprises:

[0020] The membrane fusion protein and antibody sequence are found from NCBI, and the membrane fusion protein and antibody sequence are sequentially subjected to mutation, connection, addition of a tag sequence, synthesis, and verification of the membrane fusion effect of the designed protein.

[0021] In a preferred implementation form of the second aspect, the method for constructing the plasmid for expressing an aCD19-CAR comprises:

[0022] After finding the 2C11 antibody light chain and heavy chain sequences, the CD8a sequence, the CD28 sequence, and the CD3ζ sequence from NCBI, the sequences are sequentially subjected to connection, addition of a tag sequence, and synthesis.

[0023] In a preferred implementation form of the second aspect, the addition of the tag sequence is the addition of a Myc tag sequence before the CD8a transmembrane sequence of the aCD19-CAR sequence.

[0024] In a preferred implementation form of the second aspect, the method for constructing the engineering cell capable of simultaneously expressing the membrane fusion protein for targeting T cells and the aCD19-CAR comprises:

[0025] The lentivirus vector plasmid is constructed, the lentivirus particles are packaged and collected, cells are infected, and drug screening is performed to obtain resistant cells.

[0026] In a preferred implementation form of the second aspect, the method for preparing the nanovesicle comprises:

[0027] The engineered cells are digested into a single cell suspension and washed with PBS;

[0028] The single cells are treated with a PBS solution containing sodium deoxycholate and PMSF, and continuously oscillated at room temperature for 20 min;

[0029] Centrifugation is performed at 6000g at 4 DEG C for 10 min, the supernatant is collected and filtered through a 0.45-micron filter membrane; centrifugation is performed at 100000g at 4 DEG C for 4 h;

[0030] The supernatant is discarded, resuspended with PBS, and the nanovesicle is obtained, which is the nanomanipulator for constructing CAR-T cells.

[0031] In a preferred implementation form of the second aspect, the concentration of the sodium deoxycholate is 0.04 w / v %.

[0032] In a preferred implementation form of the second aspect, the average particle size of the obtained nanomanipulator is 104.1-109.4 nm.

[0033] The third aspect of the present application provides an application of the nanomanipulator for constructing CAR-T cells in the first aspect in the preparation of a tumor treatment agent.

[0034] Compared with the prior art, the present application has at least the following advantages or beneficial effects:

[0035] The nanomanipulator for constructing CAR-T cells provided in the first aspect of the present application carries the CAR protein and the membrane fusion protein targeting T cells through the nanovesicle, so that the CAR protein can be delivered to T cells through the nanovesicle carrying the membrane fusion protein targeting T cells by using the membrane fusion mode, and finally the CAR-T cells having tumor killing activity can be directly constructed in vivo and in vitro. Specifically:

[0036] In the first aspect, the nanomanipulator of the present application can rapidly and simply produce CAR-T cells having tumor killing activity in vivo through intravenous injection, and no longer needs to repeatedly separate T cells, transduce CAR genes, activate and amplify, and return to patients, and the like in vitro, so that the treatment cycle is greatly reduced, the required indicators for treatment are significantly reduced, and the treatment cost is obviously reduced.

[0037] In a second aspect, the nanomanipulator of the present application carries and delivers CAR proteins through nanovesicles instead of nucleic acids, so that short-term CAR-T cells can be constructed, and side effects such as cytokine release syndrome caused by persistent existence of CAR-T cells can be effectively avoided;

[0038] In a third aspect, the nanomanipulator of the present application has high stability at a temperature of -80°C, so that it can be stored for a long time, which is beneficial to reduce the waiting time and quickly reduce the burden of patients; at the same time, the present application can be combined with immune checkpoint antibody therapy, which is beneficial to effectively inhibit the growth of B-cell lymphoma by the nanomanipulator.

[0039] Based on this, the nanomanipulator for constructing CAR-T cells provided by the present application can be used to construct CAR-T cells in vivo and in vitro, and is no longer limited to constructing CAR-T cells in vitro, thereby providing a new idea for the rapid preparation of CAR-T cells and a new method for the popularization and application of CAR-T cell therapy. BRIEF DESCRIPTION OF DRAWINGS

[0040] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments described in the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0041] Figure 1 A schematic diagram of the membrane fusion protein targeting T cells provided by the embodiments of the present application;

[0042] Figure 2 A schematic diagram of the alphaCD19-CAR protein provided by the embodiments of the present application;

[0043] Figure 3 A fluorescence micrograph of RAW264.7 cell line co-expression of the p14TF membrane fusion protein targeting T cells provided by the embodiments of the present application, wherein, Figure 3 A is the fluorescence protein expression of the RAW264.7 cell line co-expression of the p14TF membrane fusion protein targeting T cells; Figure 3 B is the bright field morphology of the RAW264.7 cell line of the p14TF membrane fusion protein targeting T cells;

[0044] Figure 4 A fluorescence micrograph of the RAW264.7 cell line of the MVTF membrane fusion protein targeting T cells provided by the embodiments of the present application, wherein, Figure 4 A is the fluorescence protein expression of the RAW264.7 cell line co-expression of the p14TF membrane fusion protein targeting T cells; Figure 4B is the bright field morphology of RAW264.7 cell line targeting T cell MVTF membrane fusion protein;

[0045] Figure 5 The detection result of fusion of HEK293T cell transiently expressing T cell membrane fusion protein and T cell membrane provided by the embodiment of the present application;

[0046] Figure 6 The detection result of fusion of RAW264.7 cell line stably expressing T cell membrane fusion protein and T cell membrane provided by the embodiment of the present application;

[0047] Figure 7 The preparation flow chart of nanomanipulator provided by the embodiment of the present application;

[0048] Figure 8 The particle size distribution chart of nanomanipulator provided by the embodiment of the present application;

[0049] Figure 9 The TEM chart of nanomanipulator provided by the embodiment of the present application;

[0050] Figure 10 The detection result of WB detecting expression of T cell membrane fusion protein carried by nanomanipulator provided by the embodiment of the present application;

[0051] Figure 11 The detection result of WB detecting expression of aCD19-CAR protein carried by nanomanipulator provided by the embodiment of the present application;

[0052] Figure 12 The detection result of membrane fusion of nanomanipulator and T cell promoted by T cell membrane fusion protein provided by the embodiment of the present application;

[0053] Figure 13 The fusion efficiency detection result of nanomanipulator and T cell prepared by the embodiment of the present application;

[0054] Figure 14 The stability detection result of nanomanipulator prepared by the embodiment of the present application under different storage temperatures;

[0055] Figure 15 The efficiency detection result of CAR-T cell constructed in vitro by nanomanipulator prepared by the embodiment of the present application;

[0056] Figure 16 The in vitro killing efficiency detection result of CAR-T cell constructed by nanomanipulator prepared by the embodiment of the present application, wherein, Figure 16 A is the cytotoxicity detection result of aCD19CAR-T cell constructed by nanomanipulator to A20 cell; Figure 16B is the detection result of cytotoxicity of aCD19 CAR-T cells constructed by nano-operon to B16-F10 cells; Figure 16 C is the detection result of cytotoxicity of aCD19 CAR-T cells constructed by nano-operon to CT26 cells;

[0057] Figure 17 The detection result of the efficiency of constructing CAR-T cells in vivo by the nano-operon prepared in the embodiment of the present application is shown in the following table: Figure 17 A is the proportion of CAR-T cells detected in blood; Figure 17 B is the proportion of CAR-T cells detected in spleen;

[0058] Figure 18 The treatment result of the nano-operon prepared in the embodiment of the present application for treating B-cell lymphoma is shown in the following table: Figure 18 A is the tumor growth in different treatment groups in the treatment experiment; Figure 18 B is the tumor growth in different treatment groups in the treatment experiment;

[0059] Figure 19 The detection result of the expression of inflammatory cytokines in vivo after treating B-cell lymphoma by the nano-operon provided in the embodiment of the present application is shown in the following table: Figure 19 A is the detection result of the expression of interleukin 1β in vivo after treating B-cell lymphoma by the nano-operon; Figure 19 B is the detection result of the expression of interleukin 6 in vivo after treating B-cell lymphoma by the nano-operon; Figure 19 C is the detection result of the expression of granulocyte-macrophage colony-stimulating factor in vivo after treating B-cell lymphoma by the nano-operon;

[0060] Figure 20 The detection result of the killing efficiency of CAR-T cells constructed by the nano-operon promoted by αOX40 provided in the embodiment of the present application is shown in the following table:

[0061] Figure 21 The experimental result of the nano-operon combined with αOX40 for treating B-cell lymphoma provided in the embodiment of the present application is shown in the following table: Figure 21 A is the tumor growth of each group of mice in the treatment experiment; Figure 21 B is the change of body weight of each group of mice in the treatment experiment. DETAILED DESCRIPTION

[0062] With reference to the drawings, the technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of the present application.

[0063] In the following description of the embodiments of the present application, the term "and / or" is used to describe the association relationship of the associated objects, which means that there can be three kinds of relationships, for example, A and / or B, which can represent the cases of A alone, B alone and A and B existing at the same time. Wherein A and B can be singular or plural. The character " / " generally represents that the front and rear associated objects are in an "or" relationship.

[0064] In the following description of the embodiments of the present application, the term "at least one" means one or more, and "multiple" means two or more. "At least one of the following (one)" or similar expressions means any combination of these items, including any combination of single (one) or multiple items. For example, "at least one of a, b or c", or "at least one of a, b and c", can represent a, b, c, a-b (i.e. a and b), a-c, b-c, or a-b-c, wherein a, b and c can be single or multiple.

[0065] Those skilled in the art should understand that in the following description of the embodiments of the present application, the order of the serial numbers does not mean the order of execution, and some or all steps can be executed in parallel or in sequence, and the execution order of each process should be determined according to its function and inherent logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0066] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a" and "the" used in the embodiments of the present application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0067] The term "nanomanipulator" used in the embodiments of the present application includes polymer nanoparticles, magnetic nanoparticles, noble metal nanoparticles, semiconductor nanoparticles, composite nanoparticles, gel nanoparticles (protein nanoparticles and nucleic acid nanoparticles) and other nanoparticles used in the field of biomedicine. Among them, the nanoparticles can be selected from naturally occurring or synthetic. The nanomanipulator of the present application specifically refers to a nanovesicle carrying a targeting T cell membrane fusion protein and aCD19-CAR.

[0068] In a first aspect, the embodiments of the present application provide a nano-operon for constructing CAR-T cells. The nano-operon for constructing CAR-T cells in the embodiments of the present application comprises a nano-vesicle, and the nano-vesicle carries a CAR protein and a membrane fusion protein for targeting T cells.

[0069] In the embodiments of the present application, the nano-operon carries the CAR protein and the membrane fusion protein for targeting T cells through the nano-vesicle contained therein, so that the CAR protein can be delivered to T cells by the nano-vesicle carrying the membrane fusion protein for targeting T cells in a membrane fusion manner, and ultimately CAR-T cells with tumor-killing activity can be directly constructed in vivo and in vitro. Specifically,

[0070] (1) The nano-operon in the embodiments of the present application can quickly and simply produce CAR-T cells with tumor-killing activity in vivo through intravenous injection, and no longer needs to repeatedly separate T cells, transduce CAR genes, activate and expand, and reinfuse into patients and other in vitro operations for each patient, so that the treatment cycle is greatly reduced, the required indicators for treatment are significantly reduced, and the treatment cost is obviously reduced;

[0071] (2) The nano-operon in the embodiments of the present application carries and delivers the CAR protein through the nano-vesicle, rather than nucleic acid, so that CAR-T cells with short-term existence can be constructed, and side effects such as cytokine release syndrome caused by the persistence of CAR-T cells can be effectively avoided;

[0072] (3) The nano-operon in the embodiments of the present application has high stability at a temperature of -80℃, so that it can be stored for a long time, which is beneficial to reduce the waiting time for tumor treatment and quickly reduce the burden on patients; at the same time, the present application can be combined with immune checkpoint antibody therapy to achieve effective inhibition of B-cell lymphoma growth by the nano-operon;

[0073] (4) The nano-operon in the embodiments of the present application is an extracellular vesicle, which is a cell-derived membrane structure particle with the advantages of low immunogenicity and good biocompatibility. Therefore, it can be used in clinical trials for treating various diseases such as tumors, diabetes, and inflammatory reactions, and has extremely high application value; and the extracellular vesicle can be used as a carrier for intercellular signal transmission to carry proteins, nucleic acids, and small molecules, and has many advantages such as stability in circulation.

[0074] In specific embodiments, the membrane fusion protein for targeting T cells is composed of a membrane fusion protein and a T cell surface marker protein molecule antibody sequence.

[0075] In the embodiments of the present application, the T cell surface marker protein molecule antibody sequence is connected to the N terminus of the membrane fusion protein.

[0076] In specific embodiments, the membrane fusion protein preferably comprises a p14TF protein, and an F protein and an H protein of a measles virus.

[0077] The H protein of the measles virus comprises a Y481A mutation and a R533A mutation.

[0078] It should be noted that the p14TF protein in the embodiments of the present application is a p14 protein targeting T cells (p14TF) formed by modifying a p14FAST protein; meanwhile, the F protein and the H protein of the measles virus in the embodiments of the present application are MVTFs (including F protein and H protein) targeting T cells formed by modifying a related fusion protein of a measles virus (MVTF). The modification of the proteins is implemented by a gene modification technique known in the art, and the embodiments of the present application do not make special limitations.

[0079] Of course, the membrane fusion protein in the embodiments of the present application can also be a membrane fusion protein of a human immunodeficiency virus of a prokaryotic cell, a membrane fusion protein of an influenza virus HA, and an EFF-1 fusion protein in a nematode cell, a Hapless2 / Germcell-specific1 (HAP2 / GCS1), a syncytin-1, a Myomaker, and the like, as long as it is a membrane fusion protein, and the embodiments of the present application do not make special limitations.

[0080] In specific embodiments, the H protein and the F protein sequence of the measles virus are preferably connected by a T2A connecting peptide.

[0081] In specific embodiments, the marker protein molecule on the surface of the T cell is preferably a CD3e molecule.

[0082] In specific embodiments, the antibody sequence of the marker protein molecule on the surface of the T cell is preferably a scFv sequence.

[0083] It should be noted that the scFv sequence can be a cell line surface specific protein molecule of human origin, mouse origin, rabbit origin, and the like, or other membrane fusion proteins known in the art, as long as it can be used for identifying cells.

[0084] In specific embodiments, the CAR protein is preferably an aCD19-CAR protein.

[0085] It should be noted that the alphaCD19-CAR protein is composed of the light chain and heavy chain variable region alphaCD19scFv of the 1D3 monoclonal antibody, the mouse CD8 alpha hinge region, the mouse CD8 transmembrane region, the intracellular CD28 costimulatory signal domain and the intracellular CD3 zeta structure in series, and a Myc tag sequence is added between the alphaCD19-scFv and the mouse CD8 alpha hinge region for subsequent detection. Among them, the CAR target can be CD22, CD20, BCMA, CD20, CD138, CD33, CD123, etc., and of course other targets known in the art can also be used.

[0086] In a second aspect, the embodiments of the present application provide a preparation method of the nano-operon for constructing CAR-T cells according to the first aspect, which specifically comprises the following steps:

[0087] S101: Constructing plasmids for expressing membrane fusion proteins targeting T cells and alphaCD19-CAR respectively;

[0088] S102: Constructing an engineering cell capable of simultaneously expressing membrane fusion proteins targeting T cells and alphaCD19-CAR;

[0089] S103: Using the engineering cell constructed in step S102 to produce nano-vesicles carrying membrane fusion proteins targeting T cells and alphaCD19-CAR, i.e. the nano-operon for constructing CAR-T cells.

[0090] In specific embodiments, the method for constructing membrane fusion proteins targeting T cells preferably comprises:

[0091] The membrane fusion protein and antibody sequences are found from NCBI, and the membrane fusion protein and antibody sequences are sequentially subjected to mutation, connection, addition of a tag sequence, synthesis and verification of the membrane fusion effect of the designed protein. Specifically:

[0092] The H protein and F protein of the measles virus and the gene sequence of the p14FAST protein are found from NCBI, and the H protein is subjected to Y481A mutation and R533A mutation (mutH protein), so as to lose the original recognition ability of CD46 and CD150. At the same time, the scFv sequence of the 2C11 clone of the CD3e molecule antibody on the surface of T cells is found from NCBI, and the anti-CD3 scFv sequence is connected to the p14 protein and the mutated H protein sequence through a linker, respectively, and the H protein and the F protein sequence are connected through a 2A connecting peptide. Finally, two membrane fusion protein sequences targeting T cells, p14TF and MVTF, are obtained.

[0093] In specific embodiments, the method for constructing alphaCD19-CAR plasmids comprises:

[0094] After finding the sequences of 2C11 antibody light chain and heavy chain, CD8a sequence, CD28 sequence and CD3ζ sequence from NCBI, the sequences are connected in turn, a tag sequence is added and synthesized. Specifically:

[0095] The light chain and heavy chain of 1D3 monoclonal antibody, the hinge region of murine CD8α, the transmembrane region of murine CD8, the intracellular CD28 costimulatory signaling domain and the intracellular CD3ζ sequence are found from NCBI, and then the light and heavy chains of 1D3 monoclonal antibody are connected by (GGGGS)3 sequence to form αCD19-scFv, which is connected with other structures to form αCD19-CAR. The Myctag sequence is added between αCD19-scFv and the hinge region of murine CD8α.

[0096] In specific embodiments, the added tag sequence is preferably Myctag sequence added before the CD8a transmembrane sequence of the αCD19-CAR sequence. The Myctag sequence can also be other tag sequences known in the art as long as it can be used for detection, and the embodiments of the present application do not make special limitations.

[0097] In specific embodiments, the method for constructing an engineered cell capable of simultaneously expressing a membrane fusion protein targeting T cells and an αCD19-CAR preferably comprises:

[0098] Constructing a lentiviral vector plasmid, packaging and collecting lentiviral particles, infecting cells, and drug screening to obtain resistant cells. Specifically:

[0099] Constructing a lentiviral vector plasmid, co-transfecting HEK293T cells with the lentiviral vector plasmid, an envelope plasmid (pMD2.0) and a packaging plasmid (psPAX2) to package lentiviral particles; then infecting RAW264.7 cells with the collected lentivirus, and further drug screening to obtain a RAW264.7 cell line capable of stably expressing a membrane fusion protein targeting T cells and an αCD19-CAR. Of course, other methods known in the art for constructing a stable expression cell line can also be used as long as a stable expression cell line can be constructed.

[0100] It should be noted that the cells used for packaging lentiviral particles can also be other cells known in the art as long as they can package lentiviral particles; the infected cells used for constructing the cell line can be conventional cells known in the art as long as they can be infected with secretory vesicles; the resistance gene for screening plasmids with resistance gene fragments in drug screening can be puromycin, bleomycin, etc., and other conventional methods for screening resistance genes in the art can also be used as long as a stable expression cell line can be constructed.

[0101] In specific embodiments, the method for preparing the nanovesicles comprises:

[0102] The engineered cells are digested into a single cell suspension and washed with PBS;

[0103] The single cells are treated with a PBS solution containing sodium deoxycholate and PMSF, shaken for 20 min at room temperature;

[0104] Centrifuged at 6000g for 10 min at 4°C, the supernatant is collected and filtered through a 0.45 μm filter membrane; centrifuged at 100000g for 4 h at 4°C;

[0105] The supernatant is discarded, resuspended with PBS, and the nanovesicles are obtained, which are the nanomanipulators for constructing CAR-T cells.

[0106] In specific embodiments, the concentration of sodium deoxycholate is preferably 0.04 w / v%.

[0107] In specific embodiments, the average particle size of the nanovesicles is preferably 104.1-109.4 nm.

[0108] In a third aspect, the embodiments of the present application provide the use of the nanomanipulators for constructing CAR-T cells in the preparation of a tumor treatment agent. Based on the characteristics of the nanomanipulators of the embodiments of the present application that can directly construct CAR-T cells with short-term existence and tumor-killing activity in vivo and in vitro, the nanomanipulators of the embodiments of the present application can not only effectively reduce the treatment cost and treatment cycle of tumors when applied to tumor treatment, but also will not cause side effects such as cytokine release syndrome; in addition, the nanomanipulators of the embodiments of the present application can be stored for a long time and can be used for tumor treatment at any time, greatly reducing the burden on patients.

[0109] The present application will be further described in detail below in combination with specific embodiments.

[0110] The raw materials used in the embodiments and their sources include:

[0111] 0.25% trypsin, cell culture medium, fetal bovine serum: American Gibco Company;

[0112] Penicillin-streptomycin mixed solution: American Introgen Company;

[0113] RIPA cell lysis solution: Shanghai Biyun Tian Biotechnology Co., Ltd.;

[0114] Bovine serum albumin (BSA): Shanghai Shenguo Biological Engineering Co., Ltd.;

[0115] Low-melting-point agarose: Guangzhou Qikexing Biological Technology Co., Ltd.;

[0116] Gelred: Guangzhou Qikexing Biological Technology Co., Ltd.;

[0117] Dimethyl sulfoxide (DMSO): Shanghai Luji Laboratory Equipment Co., Ltd.

[0118] 4% paraformaldehyde: Hefei Biosharp Co., Ltd.

[0119] DAPI: Shanghai Generay Biotech Co., Ltd.

[0120] N,N'-methylenebisacrylamide: Shanghai Generay Biotech Co., Ltd.

[0121] Tris: Sigma-Aldrich, USA

[0122] Concentrated hydrochloric acid: Shanghai Reagent Co., Ltd.

[0123] Ammonium persulfate (APS): Sigma-Aldrich, USA

[0124] Acrylamide: Shanghai Generay Biotech Co., Ltd.

[0125] TEMED (N,N,N',N'-Tetramethylethylenediamine): Sigma-Aldrich, USA; Glycerol, bromophenol blue: Shanghai Generay Biotech Co., Ltd.

[0126] Tween-20: Sigma-Aldrich, USA

[0127] Tryptone: Shanghai Generay Biotech Co., Ltd.

[0128] Yeast extract: Shanghai Generay Biotech Co., Ltd.

[0129] BCA protein concentration determination kit: Thermo, USA

[0130] ECL developing system Western Blot substrate kit: Pierce, USA

[0131] Triton X-100: Shanghai Generay Biotech Co., Ltd.

[0132] Instrument models used in examples and their sources: 12-color flow cytometer: BD, USA, FACSCelesta

[0133] Dynamic light scattering instrument: Malvern, UK, Zetasizer Nano ZSE

[0134] Microspectrophotometer: Nanodrop one, Thermo Fisher Scientific, USA;

[0135] PCR instrument: 2720 Thermal Cycler, Thermo Fisher Scientific, USA;

[0136] Ultrapure water treatment device: Millipore Milli-Q Direct, Merck, USA;

[0137] Laser confocal scanning microscope: LSM880 with Airyscan, Carl Zeiss, Germany;

[0138] Small high-speed centrifuge: Microfuge 20R, Eppendorf, Germany;

[0139] Carbon dioxide cell incubator: 3111, Thermo, USA;

[0140] Electrophoresis instrument: EPS-300, Shanghai Tian Neng Technology Co., Ltd., China;

[0141] Vertical electrophoresis tank: VE-180, Shanghai Tian Neng Technology Co., Ltd., China;

[0142] Full-automatic rotary microtome: RM2255, Leica, Germany;

[0143] Microplate reader: 800TS, BioTek, USA;

[0144] Inverted fluorescence microscope: TS2, Nikon, Japan;

[0145] pH meter: FE28-Standard, Mettler Toledo, Switzerland;

[0146] Paraffin embedding machine: Arcadia, Leica, Germany;

[0147] Fully enclosed automatic dehydration machine: ASP200S, Leica, Germany;

[0148] Digital pathology scanning system: Aperio CS2, Leica, Germany;

[0149] Super-clean workbench: SW-CJ-1FD, Suqing Antai, China;

[0150] Ultralow temperature refrigerator: 995, Thermo, USA;

[0151] -25℃ vertical low temperature storage box: DW-LY270, MIRAEUS, China;

[0152] Electronic balance: Mettler, ME204E.

[0153] Example 1

[0154] The embodiment provides a preparation method of a nano-operon for constructing a CAR-T cell, and specifically comprises the following steps:

[0155] S101: Constructing a plasmid capable of expressing a membrane fusion protein targeting T cells and a plasmid capable of expressing aCD19-CAR, respectively.

[0156] S101.1-constructing a membrane fusion protein targeting T cells, specifically comprising:

[0157] Firstly, find the p14FAST protein and the gene sequences of the H protein and the F protein of the measles virus on the NCBI, and perform Y481A and R533A mutations (mutH protein) on the H protein to cause it to lose the original recognition ability of CD46 and CD150; secondly, find the scFv sequence of the 2C11 clone of the CD3e molecular antibody on the surface of the T cell on the NCBI, and connect the anti-CD3 scFv sequence to the N-terminus of the mutated H protein and the p14 protein sequence respectively to realize the targeting membrane fusion function; thirdly, connect the H protein and the F protein sequence through the 2A self-cleavage peptide to realize the co-expression of the two proteins; finally, replace the targeting polypeptide sequence with the scFv sequence targeting the T cell to realize the targeting membrane fusion of the T cell, that is, the designed membrane fusion protein sequence targeting the T cell, including two kinds of p14TF and MVTF membrane fusion protein sequences targeting the T cell, the membrane fusion protein sequence targeting the T cell is shown in the formula (I). Figure 1 The formula (I) shows a schematic diagram of the membrane fusion protein targeting the T cell. Figure 1 The formula (I) shows a schematic diagram of the membrane fusion protein targeting the T cell.

[0158] It should be noted that we give the designed membrane fusion protein sequence targeting the T cell to Shanghaigene Biotechnology Co., Ltd. for synthesis, and express the two sequences respectively by using a CBG promoter (chicken beta-actin promoter) to obtain a p14TF protein gene sequence and a MVTF protein gene sequence;

[0159] S101.2-constructing a plasmid capable of expressing aCD19-CAR, specifically comprising:

[0160] The light chain and heavy chain of the 1D3 monoclonal antibody, the mouse CD8a hinge region, the mouse CD8 transmembrane region, the intracellular CD28 costimulatory signal domain and the intracellular CD3 zeta structure sequence were found on NCBI, and the light chain and heavy chain of the 1D3 monoclonal antibody were connected by a (GGGGS) 3 sequence to form an aCD19-scFv, which was then connected with other structures to form an aCD19-CAR. Finally, a Myctag sequence was added between the aCD19scFv and the mouse CD8a hinge region to obtain the designed aCD19-CAR protein sequence, which is shown in SEQ ID NO. 1. Figure 2 Figure 2 The aCD19-CAR protein schematic diagram is shown in FIG. 1.

[0161] It should be noted that we gave the designed aCD19-CAR protein sequence to GenScript Biotech (Shanghai) Co., Ltd. for synthesis, and used the CBG promoter to start expression.

[0162] S102: Constructing an engineering cell capable of simultaneously expressing a membrane fusion protein targeting T cells and an aCD19-CAR.

[0163] S102.1-Constructing a lentiviral vector plasmid capable of simultaneously expressing p14 protein, MVFP two T cell membrane fusion proteins and aCD19-CAR protein, specifically including:

[0164] The synthesized p14TF protein sequence was obtained by NheI / BamHI double digestion; the lentiviral vector pCDH was digested by NheI / BamHI; the linear pCDH vector and the p14TF protein sequence were connected by T4 ligase at room temperature for 30 min, and then transformed into DH5a competent cells, and after overnight culture, positive single colonies were picked and amplified and stored. The sequence was verified by sequencing, and the correct plasmid was named pCDH-EF1-p14TF;

[0165] The synthesized MVTF protein sequence was obtained by AgeI / EcoRI double digestion; the lentiviral vector pCDH-EF1-p14TF was digested by AgeI / EcoRI; the linear pCDH-EF1-p14TF vector and the MVTF protein sequence were connected by T4 ligase at room temperature for 30 min, and then transformed into DH5a competent cells, and after overnight culture, positive single colonies were picked and amplified and stored. The sequence was verified by sequencing, and the correct plasmid was named pCDH-EF1-MVTF.

[0166] S102.2-Constructing an engineering cell line capable of expressing a membrane fusion protein targeting T cells and capable of expressing an aCD19-CAR protein, specifically including: ​

[0167] The lentiviral vector plasmid, envelope plasmid (pMD2.0) and packaging plasmid (psPAX2) were co-transfected into HEK293T cells to package lentiviral particles; then the collected lentivirus was used to infect RAW264.7 cells, and after further drug screening, a RAW264.7 cell line capable of stably expressing a T cell membrane fusion protein targeting T cells was obtained. The fluorescence microscopic characterization of the RAW264.7 cell line capable of stably expressing the T cell membrane fusion protein targeting T cells is shown in Figures 3-4 , wherein Figure 3 A shows the co-expression of fluorescent proteins of the RAW264.7 cell line of the p14TF membrane fusion protein targeting T cells (the tag protein is red fluorescent protein); Figure 3 B shows the bright field morphology of the RAW264.7 cell line of the p14TF membrane fusion protein targeting T cells; Figure 4 A shows the co-expression of fluorescent proteins of the RAW264.7 cell line of the MVTF membrane fusion protein targeting T cells; Figure 4 B shows the bright field morphology of the RAW264.7 cell line of the MVTF membrane fusion protein targeting T cells. Finally, a RAW264.7 cell line stably expressing a T cell membrane fusion protein targeting T cells and a CD19-CAR was obtained by infecting with a lentivirus expressing a CD19-CAR and drug screening.

[0168] S102.3 - Detection of membrane fusion between HEK293T cells transiently expressing T cell-targeting membrane fusion proteins and T cells, specifically including:

[0169] 2x10 5 HEK293T cells were seeded in a 24-well plate and incubated overnight. Then, 500 ng of p14TF and MVTF expression plasmids were transfected into HEK293T cells using Lipofectamine 3000, and after 8 h, the culture medium was replaced and the cells were incubated for another 12 h. Mouse spleen sorted T cells were added for co-culture for 48 h. T cells were labeled with APC aCD3 antibody, and cell nuclei were labeled with DAPI, and laser confocal microscopy was used to observe the membrane fusion between HEK293T cells and T cells to form multinucleated cells, and the results are shown in Figure 5 , wherein Figure 5 The results of the detection of membrane fusion between HEK293T cells transiently expressing T cell-targeting membrane fusion proteins and T cells are shown.

[0170] According to Figure 5 It can be seen that HEK293T cells transiently expressing T cell-targeting membrane fusion proteins can fuse with T cells.

[0171] S102.4 - Detection of the fusion of the constructed RAW264.7 cell line expressing T cell membrane fusion protein with T cells, specifically including:

[0172] Take 2×10 5 RAW264.7 cells stably expressing a T-cell membrane fusion protein were seeded in 24-well plates and cultured overnight. They were then co-cultured with T cells sorted from mouse spleens for 48 hours. T cells were labeled with APCαCD3 antibody, RAW264.7 cells with FITCαCD11b antibody, and cell nuclei with DAPI. Laser confocal microscopy was used to observe the membrane fusion of the RAW264.7 cell line stably expressing the T-cell membrane fusion protein with T cells to form multicellular cells. The results were... Figure 5 As shown. Among them, Figure 6 The results show the detection of membrane fusion between RAW264.7 cells, which stably express a T cell membrane fusion protein, and T cells.

[0173] according to Figure 6 It can be seen that the RAW264.7 cell line, which can stably express the T cell membrane fusion protein, can fuse with T cells.

[0174] S103: Fabrication of FuNV nanooperons

[0175] Please see Figure 7 As shown, the specific preparation method of the nanooperon includes: discarding the original culture medium of the cells, washing the cells with pre-cooled PBS, discarding excess PBS, infiltrating the cells with 1 mL of 0.25% TE, discarding excess TE, digesting in a cell culture incubator at 37°C for 1 min, terminating digestion with DMEM medium containing 10% FBS, and resuspending the cells into a single-cell suspension. Centrifuging at 4°C at 600 rpm for 5 min; discarding the supernatant, resuspending the cells in pre-cooled PBS, counting the number of cells, and centrifuging at 4°C. Centrifuge at 600 rpm for 5 min, discard the supernatant, resuspend the cells in PBS containing 0.04% (w / v) sodium deoxycholate and 1 mM PMSF, and vortex continuously. Incubate at room temperature for 20 min, centrifuge at 6000g for 10 min at 4°C, collect the supernatant and filter through a 0.45 μm membrane. Centrifuge the collected liquid at ultra-high speed at 100000g for 4 h at 4°C, carefully discard the supernatant, and resuspend in PBS to obtain nanovesicles, which are the nanooperons for constructing CAR-T cells.

[0176] This embodiment also provides the characterization of the nanooperons used to construct CAR-T cells described above, specifically:

[0177] 1) This embodiment characterized the prepared nanooperon (FuNV), specifically including:

[0178] The nanomanipulator (FuNV) was diluted with PBS to a concentration of 1 mg / mL, and the particle size distribution was characterized by dynamic light scattering (DLS) and the particles were characterized by transmission electron microscopy (TEM), and the results are shown in Figures 8-9 The particle size distribution of the nanomanipulator is shown in the figure. Figure 8 The TEM image of the nanomanipulator is shown in the figure. Figure 9 The TEM image of the nanomanipulator is shown in the figure.

[0179] According to Figure 8 It can be seen that the particle size of the nanomanipulator is about 104.1 nm, and the particle size distribution is uniform.

[0180] According to Figure 9 It can be seen that the nanomanipulator is a spherical particle with a membrane structure, and the particle size is about 90 nm.

[0181] 2) The present embodiment detects the nanomanipulator carrying the targeting T cell membrane fusion protein and the alpha CD19 CAR protein by Western blotting (WB), which specifically includes:

[0182] After the prepared nanomanipulator (FuNV) is resuspended by RIPA lysis buffer, it is continuously shaken at room temperature for 1 min, placed on ice for 5 min, and repeated 4 times to completely lyse the nanomanipulator, obtaining a solution containing nanomanipulator proteins. For the nanomanipulator prepared by cells expressing the targeting T cell membrane fusion protein, the targeting T cell membrane fusion protein expression is detected by Protein L in WB; for the nanomanipulator prepared by cells expressing alpha CD19-CAR, the alpha CD19-CAR expression is detected by alpha Myctag antibody in WB, and the detection results are shown in Figures 10-11 In the WB detection, the internal reference protein is glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Among them, Figure 10 The detection results of the WB detection of the nanomanipulator carrying the targeting T cell membrane fusion protein expression are shown in the figure. Figure 11 The detection results of the WB detection of the nanomanipulator carrying the alpha CD19-CAR protein expression are shown in the figure.

[0183] According to Figures 10-11 It can be seen that the nanomanipulator prepared in the present embodiment can effectively carry the targeting T cell membrane fusion protein and the alpha CD19-CAR protein.

[0184] 3) Nanomanipulator (FuNV) and T cell membrane fusion efficiency detection

[0185] Take 40 μg of FuNV and NV and label with membrane dye DiI, then co-incubate with mouse spleen sorted T cells for 10 h. After washing the T cells with PBS, label the T cells with APC anti-CD3 antibody, and use laser confocal microscopy to observe the distribution of DiI on the surface of T cells, and the results are shown in Figure 10 Figure 12 The results of detecting that the membrane fusion protein targeting T cells promotes the membrane fusion of nanomanipulator and T cells are shown.

[0186] According to Figure 12 It can be seen that in the FuNV group, DiI is distributed on the surface of T cells, which can be observed by laser confocal microscopy, while no significant DiI molecules on the surface of T cells are observed in the NV group, indicating that the membrane fusion protein targeting T cells can promote the membrane fusion of nanomanipulator and T cells.

[0187] Further, the membrane fusion efficiency of FuNV and T cells is detected by flow cytometry, and the results are shown in Figure 13 Figure 13 The results of detecting the fusion efficiency of nanomanipulator and T cells are shown.

[0188] According to Figure 13 It can be seen that FuNV has significant membrane fusion compared with the NV group, about 40% and 60%.

[0189] 3) Detection of the stability of FuNV storage

[0190] The prepared FuNV is stored at 4℃, -20℃ and -80℃ respectively, and the particle size of FuNV is detected at 0h, 12h, 24h, 48h, 1 week and 4 weeks, etc. The stability of FuNV is judged by the change of particle size, and the detection results are shown in Figure 14 Figure 14 The results of detecting the stability of nanomanipulator at different storage temperatures are shown.

[0191] According to Figure 14 It can be seen that the particle size of FuNV stored at 4℃ and -20℃ changes obviously after long-term storage, while no obvious change in particle size is observed after long-term storage at -80℃. Therefore, -80℃ is the suitable storage temperature condition for FuNV.

[0192] Example 2

[0193] This embodiment provides related applications of constructing nanomanipulator for CAR-T cells, which specifically includes:

[0194] 2.1 FuNV CAR Preparation of CAR-T cells in vitro

[0195] ​​​FuNVs were produced using RAW264.7 cells co-expressing the T cell membrane targeting fusion protein and the aCD19-CAR protein CAR ) 40 pg, and incubated with mouse spleen sorted T cells for 10 h, after which the T cells were washed and cultured continuously. The FuNVs were stained with AF647 aMyc-tag antibody and detected by flow cytometry CAR The efficiency of CAR-T cell construction in vitro was detected, and the results are shown in Figure 15 . Among them, Figure 15 shows the detection results of the efficiency of the nano-operon in constructing CAR-T cells in vitro.

[0196] According to Figure 15 , it can be seen that after co-incubation and 24 h of culture, p14TF-FuNV CAR produced CAR-T cells accounted for 30.2%, while MVTF-FuNV CAR produced CAR-T cells accounted for 36.7%. With the increase of culture time, the proportion of CAR-T cells gradually decreased, and at 72 h, the proportion of CAR-T cells was less than 5%. It is proved that the CAR protein can be delivered to the surface of T cells by membrane fusion through the T cell membrane targeting fusion protein.

[0197] 2.2 FuNV CAR in vitro killing efficiency detection of CAR-T cells

[0198] Mouse spleen isolated T cells were incubated with FuNV CAR to produce aCD19 CAR-T cells, and these aCD19 CAR-T cells were co-cultured with Hoechst 33342-stained mouse B lymphoma A20 cells, mouse melanoma B16-F10 cells and mouse colorectal cancer CT26 cells (T cells and tumor cells ratio of 10:1) for 48 h. The cells were washed with PBS for 3 times, and the Hoechst 33342 fluorescence value in the tumor cells was detected to represent the killing efficiency of CAR-T cells.

[0199] Calculation formula: % cytotoxicity = [F(PBS) - F(Treat)] / F(PBS) x 100%.

[0200] The detection results are shown in Figure 16 . Among them, Figure 16 shows the in vitro killing efficiency detection results of the CAR-T cells constructed by the nano-operon, specifically: Figure 16 A shows the detection results of the cytotoxicity of aCD19 CAR-T cells constructed by the nano-operon to A20 cells; Figure 16B shows the detection results of cytotoxicity of aCD19 CAR-T cells with nano- operon construction on B16-F10 cells; Figure 16 C shows the detection results of cytotoxicity of aCD19 CAR-T cells with nano- operon construction on CT26 cells.

[0201] According to Figure 16 It can be seen that the FuNV CAR produced aCD19-CAR T cells have about 30% killing effect on A20 cells, which is significantly higher than the untreated T cells, but there is no significant killing enhancement effect on non-target cells B16-F10 and CT26. It is proved that the CAR-T cells prepared by nano-operon have killing specificity.

[0202] 2.3 FuNV CAR Efficiency of generating CAR-T cells in vivo

[0203] FuNV CAR (4.5mg / kg) was injected through the tail vein, and the mice were sacrificed after 24h, the mouse blood and spleen were collected and treated as single cell suspension, the CAR-T cell proportion in mouse blood and spleen was detected by AF647αMyctag antibody labeling CAR molecules, and the detection results were shown in the following table. Figure 15 Figure 17 The detection results of the efficiency of nano-operon in constructing CAR-T cells in vivo are shown in the following table, specifically: Figure 17 A shows the proportion of CAR-T cells detected in blood; Figure 17 B shows the proportion of CAR-T cells detected in spleen.

[0204] According to Figure 17 It can be seen that by single tail vein injection of FuNV CAR , 1.31% to 2.07% of CAR-T cells can be detected in blood and spleen. The experimental results prove that CAR-T cells can be generated in vivo by injecting nano-operon through the tail vein.

[0205] 2.4 FuNV CAR for mouse B lymphoma treatment

[0206] We constructed a mouse B cell lymphoma A20 cell subcutaneous tumor model. The tumor-bearing mice were randomly divided into 4 groups, 5 in each group, and 300μL of PBS, NV, p14TF-FuNV CAR and MVTF-FuNV CAR (nano-operon injection dose of 4.5mg / kg) were injected through the tail vein respectively. The drug was given once every two days, and a total of 5 times. During the treatment, the tumor size was measured with a vernier caliper every day, and the change of mouse body weight was detected.​

[0207] Tumor volume is calculated using the following formula: Tumor volume (mm) 3 = 0.5 × length × width 2 .

[0208] The test results are Figure 18 As shown. Among them, Figure 18 The results of using nanooperons to treat B-cell lymphoma are shown, specifically: Figure 18 A shows the tumor growth in different treatment groups during the treatment trial; Figure 18 B shows the tumor growth in different treatment groups during the treatment trial.

[0209] according to Figure 18 It can be seen that in the PBS and NV groups, tumor growth was rapid, while in the p14TF-FuNV group... CAR and MVTF-FuNV CAR Tumor growth was significantly inhibited in the experimental group. Furthermore, no significant change in body weight was observed in the treatment group mice compared to the PBS group, indicating that FuNV... CAR It has low toxicity to mice.

[0210] 2.5FuNV CAR Cytokine release syndrome detection

[0211] Cytokine release syndrome (CRS) is one of the serious side effects of CAR-T cell therapy. To detect whether CRS occurs during the treatment experiment, we used ELISA to detect the levels of CRS-related cytokines in mouse plasma after the experiment. The results were as follows: Figure 19 As shown. Among them, Figure 19 The results of in vivo detection of inflammatory cytokine expression after treatment of B-cell lymphoma with nanooperons are shown, specifically: Figure 19 A shows the results of in vivo detection of interleukin 1β expression after treatment of B-cell lymphoma with nanooperons; Figure 19 B shows the results of in vivo detection of interleukin-6 expression after treatment of B-cell lymphoma with nanooperons; Figure 19 C shows the results of detecting granulocyte-macrophage colony-stimulating factor expression in vivo after treatment of B-cell lymphoma with nanooperons.

[0212] according to Figure 19 Analysis of the levels of inflammatory cytokines IL-1β, IL-6, and GM-CSF in mouse plasma showed that the expression levels of CRS-related cytokines were not increased in the treatment group compared to the PBS group, indicating that FuNV therapy was effective. CARThe in vivo production of CAR-T cells effectively inhibited tumor growth without causing CRS with massive cytokine production, indicating that the method is highly safe.

[0213] 2.6 FuNV CAR Combination of immune checkpoint antibodies for mouse B lymphoma treatment

[0214] Compared with existing CAR-T cell therapy, the preparation method of in vivo direct production of CAR-T cells does not go through the activation process, and the tumor killing ability is limited. Therefore, FuNV CAR Combination of immune checkpoint therapy anti-OX40 antibody (aOX40) for mouse B lymphoma treatment.

[0215] In the tumor cell killing experiment at the cell level, aOX40 (10 μg / mL) was added in the co-culture experiment of aCD19-CAR-T cells prepared by nanomanipulator and A20 cells, and the killing efficiency of CAR-T cells added with aOX40 nanomanipulator was detected after co-culture for 48 h, and the results are shown in Figure 18 , wherein Figure 20 The detection results of aOX40 promoting the killing efficiency of CAR-T cells prepared by nanomanipulator are shown;

[0216] According to Figure 20 It can be seen that the killing effect of aCD19 CAR-T cells on A20 cells is significantly enhanced,

[0217] In the anti-tumor treatment experiment at the animal level, the A20B cell lymphoma tumor-bearing mice were evenly divided into 6 groups, 5 in each group, and 300 μL of PBS, p14TF-FuNV CAR , MVTF-FuNV CAR , aOX40, p14TF-FuNV CAR + aOX40 and MVTF-FuNV CAR + aOX40 (wherein the injection dose of nanomanipulator is 4.5 mg / kg, and aOX40 is intraperitoneally injected with 50 μg) were injected into the tail vein of each group. The drug was administered once every two days, and a total of 5 times. During the treatment, the tumor size was measured with a vernier caliper every day, and the body weight change of the mice was detected.

[0218] The tumor volume was calculated according to the following formula: tumor volume (mm 3 ) = 0.5 x length x width 2 .

[0219] The detection results are shown in Figure 21 , wherein Figure 21 The experimental results of nanomanipulator combined with aOX40 for treating B cell lymphoma are shown, specifically: Figure 21A shows the tumor growth of each group in the treatment experiment; Figure 21 B shows the weight change of each group of mice in the treatment experiment.

[0220] According to Figure 21 It can be seen that the use of aOX40 or nanomanipulator alone can inhibit tumor growth. Nanomanipulator combined with aOX40 can significantly enhance the inhibitory effect on tumor. During the whole treatment process, the body weight of mice in each group did not change significantly, indicating that nanomanipulator combined with aOX40 therapy did not cause serious toxicity to mice.

[0221] Each embodiment in the specification is described in a progressive manner, and the same or similar parts between each embodiment can be referred to each other, and each embodiment focuses on the difference from other embodiments.

[0222] The above examples are only used to illustrate the technical solutions of the present application, and are not limited to the present application; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can still be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the present application.

Claims

1. A nano- operon for constructing CAR-T cells, characterized in that, The nano-manipulator is a spherical particle of membrane structure, and the CAR-T cells can be generated in vivo by intravenous injection of the nano-manipulator; The nano-manipulator comprises a nanovesicle carrying a CAR protein and a T cell-targeting membrane fusion protein; The T cell-targeting membrane fusion protein comprises a membrane fusion protein and a T cell surface marker protein molecule antibody sequence; the membrane fusion protein is F protein and H protein of measles virus, the H protein is connected with the F protein sequence through a T2A connecting peptide, and the H protein is introduced with Y481A mutation and R533A mutation.

2. The nano- operon for building CAR-T cells according to claim 1, characterized in that, The T cell surface marker protein molecule antibody sequence is connected to the N-terminus of the membrane fusion protein.

3. The nano-cassette for constructing CAR-T cells according to claim 1, characterized in that, The T cell surface marker protein molecule is a CD3e molecule.

4. The nano-cassette for constructing CAR-T cells according to claim 1, characterized in that, The T cell surface marker protein molecule antibody sequence is a scFv sequence.

5. The nano-cassette for constructing CAR-T cells according to any one of claims 1-4, characterized in that, The CAR protein is an αCD19-CAR protein.

6. A method for preparing the nano-operon for constructing CAR-T cells according to any one of claims 1-5, characterized in that, The method comprises the following steps: S101: respectively constructing a plasmid capable of expressing a T cell-targeting membrane fusion protein and a plasmid capable of expressing an αCD19-CAR; S102: constructing an engineering cell capable of simultaneously expressing the T cell-targeting membrane fusion protein and the αCD19-CAR; S103: using the engineering cell constructed in step S102 to produce a nanovesicle carrying the T cell-targeting membrane fusion protein and the αCD19-CAR, thereby obtaining the nano-manipulator for constructing CAR-T cells.

7. The production method according to claim 6, characterized by, The method for constructing the T cell-targeting membrane fusion protein comprises: The membrane fusion protein and the antibody sequence are found from NCBI, and the membrane fusion protein and the antibody sequence are sequentially subjected to mutation, connection, addition of a tag sequence, synthesis, and verification of the membrane fusion effect of the designed protein.

8. The preparation method according to claim 6, characterized in that, The method for constructing the αCD19-CAR plasmid comprises: After finding the 2C11 antibody light chain and heavy chain sequences, CD8a sequences, CD28 sequences, and CD3ζ sequences from NCBI, sequentially performing connection, adding a tag sequence, and synthesis.

9. The production method according to claim 8, characterized by, The tag sequence is a Myc tag sequence added before the CD8a transmembrane sequence in the αCD19-CAR sequence.

10. The method of claim 6, wherein, The method for constructing the engineering cell capable of simultaneously expressing the T cell-targeting membrane fusion protein and the αCD19-CAR comprises: Constructing a lentiviral vector plasmid, packaging and collecting lentiviral particles, infecting cells, and drug screening to obtain resistant cells.

11. The preparation method according to claim 6, characterized in that, The preparation method of the nanovesicle comprises: The engineered cells are digested into a single cell suspension and washed with PBS; The single cells are treated with a PBS solution containing deoxycholic acid sodium and PMSF, and continuously oscillated at room temperature for 20 min; Centrifugation at 6000g at 4°C for 10 min, collection of supernatant and passage through a 0.45μm filter membrane; centrifugation at 100000g at 4°C for 4h; Discarding the supernatant, resuspending with PBS, and obtaining the nanovesicle, which is the nano-manipulator for constructing CAR-T cells.

12. The method of claim 11, wherein, The concentration of the deoxycholic acid sodium is 0.04w / v%.

13. The method of claim 11, wherein, The average particle size of the nano-manipulator is 104.1-109.4nm.

14. Use of the nano- operon for constructing CAR-T cells according to any one of claims 1-5 in the preparation of a medicament for treating a tumor.

Images