T cell-specific membrane-fusogenic virus-like particles, preparation method therefor and use thereof

Abstract

Disclosed are T cell-specific membrane-fusogenic virus-like particles, a preparation method therefor and a use thereof. The T cell-specific membrane-fusogenic virus-like particles are prepared by transiently transfecting 293T cells with expression plasmids for human T cell-specific membrane-fusogenic protein, expression plasmids for mRNA-loaded protein, and expression plasmids for CAR mRNA; the molar ratio of the expression plasmids for the mRNA-loaded protein to the expression plasmids for the CAR mRNA is 1:2-4. In the present invention, by using a viral budding mechanism to produce T cell-specific membrane-fusogenic virus-like particles, a loaded CAR mRNA drug can be specifically delivered into cytoplasms of T cells, thereby generating CAR-T cells.

Description

T cell specific membrane fusion type viro-mimetic particles, and preparation method and application thereof

[0001] The present application claims priority to the Chinese patent application No. 2024112862949, filed on September 13, 2024, and entitled "T cell specific membrane fusion type viro-mimetic particles, and preparation method and application thereof", the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application belongs to the technical field of biological medicine, and more specifically, the present application relates to a T cell specific membrane fusion type viro-mimetic particle for generating human CAR-T cells in vivo, and a preparation method and application thereof. BACKGROUND

[0003] Chimeric antigen receptor (CAR) T cell therapy is a precise targeted therapy for tumors, and has achieved remarkable clinical efficacy. Among them, the chimeric antigen receptor is artificially constructed, mainly composed of three domains: extracellular region targeting binding domain, transmembrane region and one or more intracellular signal transduction domains. In recent years, the number of clinical trials related to CAR-T cell therapy has rapidly increased. Since the first CAR-T cell drug was approved by the US Food and Drug Administration (FDA) in 2017, fifteen CAR-T cell drugs have been approved for marketing worldwide, two of which are in China.

[0004] So far, all the marketed CAR-T cell therapy drugs need to prepare T cells in vitro, and the steps include isolating T cells from the patient's body, transducing CAR genes into T cells through viral vectors, then activating and expanding these T cells, and finally returning them to the patient's body. CAR-T cell therapy is a highly personalized treatment method, each patient is a separate batch, which cannot be mass-produced, and has strict requirements for operators and production environment. In addition, its preparation process takes a long time, which may delay the condition of tumor patients and limit the application of CAR-T cell therapy. Moreover, the side effects of CAR-T cell therapy cannot be ignored, especially cytokine release syndrome (CRS), which has clinical manifestations including fever, tachycardia, hypoxia and other organ dysfunction, and can be fatal in severe cases. In order to alleviate CRS, clinicians can only use corticosteroid hormones to temporarily suppress part of the inflammatory response in the body, and cannot eliminate inflammation from the root. In addition, there is a risk of viral vector insertion into the host cell genome in clinical CAR-T cell therapy, leading to genetic toxicity problems such as insertion mutation, activation of proto-oncogenes, ectopic gene expression, etc.

[0005] To solve the problem of complex CAR-T cell preparation process, some researchers have begun to try to prepare CAR-T cells in vivo. Waqas Nawaz et al. used adeno-associated virus to deliver CD4-targeted CAR gene, which can produce CAR-T cells with targeted killing activity, and has significant therapeutic effect on T cell lymphoma. However, this preparation method still produces continuously activated CAR-T cells in vivo, which can cause cytokine storm and other side effects. Some studies use transient transfection of CAR mRNA into T cells to prepare CAR-T cells with short-term existence and controllable expression. Researchers in M.T. Stephan's group use lipid nanoparticles such as LNP (Lipid Nanoparticles) to encapsulate CAR mRNA, and use transient transfection to generate CAR-T cells in vivo for a short period of time, hoping to achieve safer therapeutic effect. LNP can effectively protect mRNA in physiological environment, and can be endocytosed into cells, and with the help of ionizable lipids, it can destroy endosomes in acidified environment, thereby delivering mRNA to the cytoplasm. However, LNP administered systemically is mainly enriched in the liver, and it is difficult to target other tissue cells. LNP mainly relies on passive uptake mechanism to enter cells, and the proportion of endosomes that can successfully escape endosomes is low, so that most of the mRNA is degraded and cannot be released into the cytoplasm to exert efficacy.

[0006] HIV efficiently delivers its genomic RNA into the cytoplasm of T cells through the mechanism of "active recognition triggering membrane fusion". HIV uses the gp120 subunit of the envelope human membrane fusion protein gp160 on the envelope to recognize the CD4 molecule of T cells, and generates a fusion pore on the cell membrane through the gp41 subunit, so that the viral envelope and the T cell membrane are fused with each other, thereby directly delivering the genomic RNA into the cytoplasm for efficient infection. This mechanism provides a way to design an efficient mRNA drug delivery vector.

[0007] Studies have shown that RNA virus-mimicking virus-like particles can be used to deliver mRNA drugs. For example, some studies use endogenous retroviral capsid proteins Peg10 or Gag-MS2 to encapsulate mRNA, and induce endosome membrane fusion through membrane fusion protein VSV-G to deliver mRNA into the cytoplasm. However, the existing virus-like particles lack the ability of "active recognition triggering membrane fusion", resulting in insufficient cell specificity and delivery efficiency, making it difficult to target mRNA delivery to T cells.

[0008] Therefore, there is still a need to develop a virus-like particle that can accurately and efficiently deliver mRNA drugs directly to T cells to produce human CAR-T cells in vivo. SUMMARY

[0009] Based on this, the purpose of the present application is to provide a T cell specific membrane fusion type of virus-like particles and its preparation method and application, the virus-like particles of the present application can directly and accurately and efficiently deliver CAR mRNA drugs to T cells, and effectively and rapidly prepare transient human CAR-T cells in vivo.

[0010] In the first aspect of the present application, a T cell specific membrane fusion type of virus-like particles is disclosed, which is prepared by transiently transfecting a human T cell specific membrane fusion protein expression plasmid, an mRNA loading protein expression plasmid and a CAR mRNA expression plasmid into 293T cells; the molar ratio of the human T cell specific membrane fusion protein expression plasmid to the mRNA loading protein expression plasmid is 1:1.8-2.2; and the molar ratio of the mRNA loading protein expression plasmid to the CAR mRNA expression plasmid is 1:2-4.

[0011] In the second aspect of the present application, a preparation method of the above-mentioned T cell specific membrane fusion type of virus-like particles is disclosed, which comprises the following steps:

[0012] (1) constructing a human T cell specific membrane fusion protein expression plasmid, an mRNA loading protein expression plasmid and a UTR-anti-human CD19 CAR expression plasmid;

[0013] (2) co-transfecting the three expression plasmids constructed in step (1) into 293T cells for incubation for 45-55 hours, followed by ultracentrifugation and purification, to obtain the virus-like particles.

[0014] In the third aspect of the present application, the application of any of the above-mentioned T cell specific membrane fusion type of virus-like particles in the preparation of a drug for treating tumors is disclosed.

[0015] The T cell specific membrane fusion type virus-like particle of the present application is prepared by co-transfecting the expression plasmid of human T cell specific membrane fusion protein, the expression plasmid of mRNA loading protein and the expression plasmid of CAR mRNA into 293T cells according to a certain molar ratio by using the virus budding mechanism, which can be fused with the T cell membrane by the human T cell specific membrane fusion protein, thereby the loaded CAR mRNA drug can be specifically delivered into the cytoplasm of T cells (bypassing the endosome, reducing the low drug delivery efficiency and liver toxicity problems when delivering mRNA drugs, effectively improving the stability and targeting of CAR mRNA acting on T cells) to prepare CAR-T cells, and the CAR-T cells are transiently produced, which cannot continuously activate the CAR-T cells (can realize the rapid and efficient induction of CAR protein expression while avoiding genomic integration and providing rapid response, improving the safety and controllability of treatment), the T cell specific membrane fusion type virus-like particle prepared by the present application can specifically deliver the CAR mRNA drug (such as anti-human CD19 CAR mRNA) to T cells in vivo (also in vitro) through intravenous injection, realize the specific expression of CAR protein in T cells, and modify the T cells into CAR-T cells in situ in vivo, greatly simplify the preparation process of CAR-T cell product, shorten the treatment cycle, and can accurately target and kill tumor cells, significantly enhance the activity of T cells and the anti-tumor effect, and is expected to reduce the production of cytokine storm in vivo treatment and improve the safety. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a schematic diagram of the plasmid required for constructing and preparing the T cell specific membrane fusion type virus-like particle (T-FVLP mCAR ) in the embodiment 1 of the present application.

[0017] Figure 2 is a preparation and characterization result diagram of the T cell specific membrane fusion type virus-like particle (T-FVLP mCAR ) in the embodiment 2 of the present application; A is the preparation method; B is the particle size of the virus-like particles in different groups; C is the yield of the virus-like particles in different groups; D is the protein map of the virus-like particles in different groups.

[0018] Figure 3 is a loading efficiency optimization result diagram of the T cell specific membrane fusion type virus-like particle (T-FVLP mCAR ) in the embodiment 3 of the present application; wherein A is the CAR mRNA loading efficiency result under different molar ratios of pCMV-Peg10 expression plasmid and pCMV-UTR-anti-human CD19 CAR expression plasmid; B is the result of the proportion of CAR mRNA encapsulated particles.

[0019] Figure 4 is a graph of T cell-specific membrane fusion and its efficiency detection results of T cell-specific membrane fusion type of the virus-like particles (T-FVLP mCAR ) in Example 4 of the present application; wherein A is a fluorescence signal graph; B is a fusion efficiency result.

[0020] Figure 5 is a graph of mRNA delivery efficiency and the proportion of CAR-T cells produced of T cell-specific membrane fusion type of the virus-like particles (T-FVLP mCAR ) in Example 5 of the present application; wherein A is a red fluorescence protein expression result; B is a result of Western blot detection of CAR protein expression; C is the proportion of T cells expressing CAR protein.

[0021] Figure 6 is a graph of expression duration detection results of CAR-T cells produced by T cell-specific membrane fusion type of the virus-like particles (T-FVLP mCAR ) in Example 6 of the present application.

[0022] Figure 7 is a graph of tumor cell killing ability detection results of CAR-T cells produced by T cell-specific membrane fusion type of the virus-like particles (T-FVLP mCAR ) in Example 7 of the present application.

[0023] Figure 8 is a graph of Raji-Luc B cell lymphoma growth detection results of humanized mice after treatment with CAR-T cells produced by T cell-specific membrane fusion type of the virus-like particles (T-FVLP mCAR ) in Example 8 of the present application; wherein A is a bioluminescence signal graph; B is a statistical graph of fluorescein intensity.

[0024] Figure 9 is a graph of in vivo safety evaluation detection results of CAR-T cells produced by T cell-specific membrane fusion type of the virus-like particles (T-FVLP mCAR ) in Example 9 of the present application; wherein A is the level of main inflammatory cytokines IL-1β, IL-6 and GM-CSF; B is a H&E staining tissue image of the forehead and hippocampus. DETAILED DESCRIPTION

[0025] In order to facilitate the understanding of the present application, the present application will be described more fully below. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0027] Unless otherwise indicated, all experiments were carried out in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning: A Laboratory Manual (2001), or as suggested by the manufacturer's instructions.

[0028] All starting materials and / or reagents were purchased on the market or prepared according to conventional methods well known to those skilled in the art.

[0029] In some embodiments of the application, a T cell specific membrane fusion type virosome is disclosed, which is prepared by transient transfection of 293T cells with an expression plasmid of human T cell specific membrane fusion protein, an expression plasmid of mRNA loading protein and an expression plasmid of CAR mRNA; the molar ratio of the expression plasmid of human T cell specific membrane fusion protein to the expression plasmid of mRNA loading protein is 1:1.8-2.2; the molar ratio of the expression plasmid of mRNA loading protein to the expression plasmid of CAR mRNA is 1:2-4.

[0030] In some of the embodiments, the molar ratio of the expression plasmid of mRNA loading protein to the expression plasmid of CAR mRNA is 1:3-4, more preferably 1:3.

[0031] In some embodiments, the human T cell specific membrane fusion protein is obtained by removing the original receptor recognition ability (e.g., introducing amino acid mutations into multiple sites of the recognition subunit gp120 of gp160, introducing Y481A and R533A mutations into the H protein of MV-H / MV-F, or introducing K47A and R354A mutations into the VSVG protein) in the gene sequence of the target subunit of HIV virus gp160, measles virus MV-H / MV-F, or vesicular stomatitis virus VSV-G, and then connecting anti-CD3, anti-CD4, anti-CD8, etc. scFv to the extracellular end of the corresponding target subunit using synthetic biology methods. The anti-CD3, anti-CD4, anti-CD8, etc. scFv sequences are mainly obtained by cutting the heavy chain variable region and light chain variable region sequences of the antibody from the sequences of the anti-human CD3, CD4, and CD8 monoclonal antibodies disclosed in the IMGT database, and connecting them through a flexible short peptide.

[0032] In some embodiments, the human T cell specific membrane fusion protein is obtained by connecting the anti-CD3 scFv sequence as shown in SEQ ID NO: 2 to the extracellular end of the mutated gp160 gene sequence as shown in SEQ ID NO: 1.

[0033] In some embodiments, the mRNA loading protein includes mRNA loading protein Peg10 and mRNA loading protein Gag-MS2 or mRNA loading protein Gag-Tat.

[0034] In some embodiments, the nucleotide sequence of the protein Peg10 is as shown in SEQ ID NO: 3. Peg10 is derived from the capsid protein of an endogenous retrovirus, which recognizes and encapsulates CAR mRNA carrying the corresponding loading signal UTR sequence. In addition, the capsid protein Gag of HIV can be connected to bacteriophage capsid protein MS2 or trans-activating protein Tat, etc. through synthetic biology methods to obtain new capsid proteins such as Gag-MS2 and Gag-Tat. Similarly, Gag-MS2 and Gag-Tat can also recognize and encapsulate MS2 loop-CAR mRNA and TAR-CAR mRNA carrying their corresponding loading signals, respectively.

[0035] In some embodiments, the CAR mRNA has a UTR signal and carries the anti-CD19 or anti-EGFRvIII targeting human tumor cell target, and the CAR protein expressed therefrom is an anti-CD19 protein or an anti-EGFRvIII CAR protein.

[0036] In some embodiments, the CAR mRNA has a UTR signal and carries a target anti-CD19 targeting human tumor cells, and the sequence is shown as SEQ ID NO: 4.

[0037] In some embodiments, the preparation method of the T cell specific membrane fusion type viro-mimic particles is disclosed, comprising the following steps:

[0038] (1) Constructing an expression plasmid of human T cell specific membrane fusion protein, an expression plasmid of mRNA loading protein, and an expression plasmid of CAR mRNA;

[0039] (2) After co-transfecting the three expression plasmids constructed in step (1) into 293T cells for 45-55 hours, ultracentrifugation and purification are performed, and the T cell specific membrane fusion type viro-mimic particles are obtained.

[0040] In some embodiments, the preparation method of the T cell specific membrane fusion type viro-mimic particles is disclosed, comprising the following steps:

[0041] (1) Constructing an expression plasmid of human T cell specific membrane fusion protein, an expression plasmid of mRNA loading protein, and an expression plasmid of CAR mRNA;

[0042] (2) After co-transfecting the three expression plasmids constructed in step (1) into 293T cells for 45-55 hours, ultracentrifugation and purification are performed, and the T cell specific membrane fusion type viro-mimic particles are obtained.

[0043] In some embodiments of the present invention, gene fragments of the human T cell-specific membrane fusion protein anti-CD3 scFv-mut gp160, the mRNA loading protein Peg10, and the UTR-anti-human CD19 CAR mRNA are first synthesized using biological methods, introduced into expression plasmids, and sequenced for verification. Then, the three expression plasmids are transiently transfected together to obtain engineered cells capable of simultaneously expressing the human T cell-specific membrane fusion protein anti-CD3 scFv-mut gp160, the mRNA loading protein Peg10, and the UTR-anti-human CD19 CAR mRNA. The three components assemble and bud in the engineered cells, and after separation and purification by ultracentrifugation, a highly efficient CAR-loaded protein can be obtained. The mRNA-based T-cell-specific membrane-fusion viral-like particles (CAR molecules are mainly composed of three parts: an extracellular segment, a transmembrane segment, and an intracellular segment of the human sequence. The extracellular segment is the single-chain variable region (scFv) of an antibody targeting human targets, such as hematologic malignancies, solid tumors, immune cells, and viruses, and is mainly responsible for recognizing and binding specific antigens on the tumor surface; when CAR-T cells bind to tumor cells, the intracellular segment is responsible for signal transduction and completing T-cell activation and effector functions). The average particle size of the viral-like particles is approximately 90 nm.

[0044] In other embodiments of the present invention, the use of the above-mentioned T cell-specific membrane fusion-type viral-like particles in the preparation of medicaments for treating tumors is disclosed.

[0045] In the following examples, the raw materials used are: Tryptone, Yeast extract, BSA purchased from Shanghai Shengong Bioengineering Co., Ltd.; DMEM cell culture medium, fetal bovine serum, 0.25% trypsin purchased from American Gibco company; PEI purchased from Shanghai Liji Biological Technology Co., Ltd.; RIPA cell lysis buffer purchased from Shanghai Biyun Tian Biological Technology Co., Ltd.; BCA protein concentration determination kit, SYTO dye purchased from American Thermo company; Tween-20 purchased from American Sigma-Aldrich company; Peg10 antibody purchased from American Proteintech Group company; ECL developing system Western Blot substrate kit purchased from American Pierce company; DiI dye purchased from Dalian Melon Biotechnology Co., Ltd.; PE anti-human CD3, PerCP / Cy5.5 anti-human CD3, AF488 anti-human CD11b, BV421 anti-human CD19, AF647 anti-Myc tag flow cytometry antibody, anti-human CD3, anti-human CD28 antibody, Anti-human GAPDH, Anti-gp160, Anti-Peg10, Anti-Myc purchased from American Biolegend company; anti-Myc anti-human GAPDH antibody purchased from Beijing Yiqiao God State Technology Co., Ltd.; anti-Myc tag antibody purchased from American CST company; NCG mice purchased from Guangdong Yaoke Biological Technology Co., Ltd.; PBMC purchased from Shanghai Aoneng Biological Technology Co., Ltd.; CD3 sorting magnetic beads purchased from Germany Miltenyi Biotec company; potassium salt of fluorescein purchased from Dalian Melon Biotechnology Co., Ltd.

[0046] In the following examples, the instruments used are from the following sources: Electronic balance: Model ME204E, Mettler Toledo Group, Switzerland. Clean bench: Model SW-CJ-1FD, Suzhou Antai Air Technology Co., Ltd.; Inverted fluorescence microscope: Model TS2, Nikon Corporation, Japan; Ultra-low temperature freezer: Model 995, Thermo Fisher Scientific, USA; -25℃ vertical cryogenic storage box: Model DW-LY270, Zhongke Meiling Cryogenic Technology Co., Ltd.; Carbon dioxide cell culture incubator: Model 3111, Thermo Fisher Scientific, USA; Mini high-speed centrifuge: Model Microfuge 20R, Eppendorf GmbH, Germany; Floor-standing intelligent refrigerated ultracentrifuge: Model Optima XPN-100, Beckman Coulter Ltd., USA; Ultrapure water treatment system: Model Millipore Milli-Q Direct, Merck, USA; Nanoflow cytometer: Model NanoFCM U30, Guangzhou Lianbo Biotechnology Co., Ltd.; Flow cytometer: Model FACSCelesta, BD Biosciences, USA; Laser confocal scanning microscope: Model LSM 880 with Airyscan (Carl Zeiss, Germany); Microplate reader: Model 800TS (BioTek, USA); In vivo imaging system: Model IVIS Lumina III (PerkinElmer, USA); Digital Pathology microscope: Model Aperio CS2 (Leica, USA).

[0047] In this specification and accompanying drawings, mCAR and CAR mRNA have the same meaning.

[0048] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0049] Example 1: Construction and preparation of T cell-specific membrane fusion-type virus-like particles (T-FVLP) mCAR ) Required plasmid

[0050] Please refer to Figure 1. Using conventional biological methods, three T-cell-specific membrane fusion-type viral-like particles (hereinafter named T-FVLP) were constructed. mCAR The required plasmid.

[0051] 1. The anti-CD3 scFv gene sequence (SEQ ID NO:2) was linked to the mutated gp160 gene sequence (SEQ ID NO:1) at the 5' end, and the expression plasmid pCMV-T-FP of human T cell specific membrane fusion protein (T-FP) was constructed using CMV as the promoter.

[0052] SEQ ID NO: 1 (mutant gp160 gene sequence 5' end to 3' end)

[0053] SEQ ID NO: 2 (anti-human CD3 scFv gene sequence, 5' end to 3' end)

[0054] 2. Construct the expression plasmid pCMV-Peg10 of mRNA loading protein Peg10 (the gene sequence is shown in SEQ ID NO: 3).

[0055] SEQ ID NO: 3 (gene sequence of Peg10, 5' end to 3' end)

[0056] 3. Design the mRNA loading signal UTR and anti-human-CD19 CAR gene sequence (SEQ ID NO: 4), construct the expression plasmid pCMV-UTR-anti-human CD19 CAR of UTR-anti-human CD19 CAR.

[0057] SEQ ID NO: 4 (UTR-anti-human CD19 CAR gene sequence, 5' end to 3' end)

[0058] Example 2 Preparation and characterization of T cell specific membrane fusion type viro-mimetic particles (T-FVLP mCAR )

[0059] The three expression plasmids pCMV-T-FP, pCMV-Peg10, and pCMV-UTR-anti-human CD19 CAR constructed in Example 1 were co-transiently transfected into 293T cells using PEI transfection reagent, and the cell supernatant was collected after 48 h, centrifuged at 1000 g for 10 min, then filtered with a 0.45 μm sterile filter membrane, and then ultracentrifuged at 150 000 g for 100 min at 4°C. The centrifugation precipitate was resuspended with sterile 1xPBS, and after standing at 4°C for 1 h, it was centrifuged at 3000 rpm for 5 min, and the obtained supernatant was the T cell specific membrane fusion type viro-mimetic particles T-FVLP mCAR .

[0060] The viro-mimetic particles VLP (the plasmid pCMV-Peg10 was transiently transfected into 293T cells, and the other preparation steps were the same as T-FVLP mCAR ) and VLP mCAR(The plasmids pCMV-Peg10 and pCMV-UTR-anti-human CD19 CAR were simultaneously and transiently transfected into 293T cells. Other preparation steps were the same as those for T-FVLP.) mCAR (As a comparison)

[0061] T-FVLP mCAR The preparation method is shown in Figure 2A, and the characterization results of each group are shown in Figures 2B to 2D, respectively. Nanoflow cytometry analysis revealed that T-FVLP… mCAR The particle size is approximately 90 nm, and the yield is 1.5 × 10⁻⁶. 8 10 7 Cells, with VLP group and VLP mCAR There were no significant differences between the groups (Figures 2B and 2C). Western blot results confirmed the T-FVLP... mCAR Simultaneous expression of T-FP and Peg10 proteins indicates that the prepared T-FVLP mCAR It contains two components: anti-CD3 scFv-gp160 and Peg10, while VLP and VLP mCAR It contains only the Peg10 component, without the anti-CD3 scFv-gp160 component, and its surface is modified with human T cell-specific membrane fusion protein (Fig. 2D).

[0062] Example 3: T-cell specific membrane fusion-type viral-like particle (T-FVLP) mCAR Optimization of loading efficiency

[0063] This embodiment explored T-FVLP. mCAR The effect of the transfection molar ratio of pCMV-Peg10 to pCMV-UTR-anti-human CD19 CAR on the loading efficiency of CAR mRNA during the preparation process.

[0064] Using PEI transfection reagent, to 1×10 7 4 μg pCMV-T-FP, 6 μg pCMV-Peg10, and different masses of pCMV-UTR-anti-human CD19 CAR plasmid were transiently and simultaneously transfected into 293T cells. Cell supernatant was collected after 48 h to obtain T-FVLP. mCAR The CAR mRNA was lysed (operation method as in Example 2), and the loading amount of CAR mRNA was detected by real-time PCR.

[0065] The experimental results are shown in Figure 3. When the molar ratio of the two plasmids transfected was 1:3, T-FVLP mCAR The highest loading efficiency for CAR mRNA was achieved at 1.5 × 10⁻⁶. 8 T-FVLPmCAR i.e. 5 ng of CAR mRNA can be loaded, which is significantly higher than the loading efficiency when the molar ratio of the two plasmids is 1:1 and 1:2 (Figure 3A). The T-FVLPs are detected by nano-flow cytometry to be close to 100% T-FVLPs mCAR All of them effectively encapsulate CAR mRNA; VLPs mCAR Since the CAR mRNA therein contains a UTR sequence, almost complete effective encapsulation of CAR mRNA is also achieved; and a part of the VLPs may encapsulate their own mRNA at random (Figure 3B).

[0066] Example 4 T-cell specific membrane fusion of T-cell specific membrane fusion type imitated virus-like particles (T-FVLPs mCAR ) and efficiency thereof

[0067] The cryopreserved human peripheral blood mononuclear cells PBMCs (Shanghai Aobio Biotech Co., Ltd.) are thawed in a water bath and left to stand in an incubator overnight, and anti-human CD3 and anti-human CD28 antibodies are added to stimulate activation. 5 x 10 5 activated PBMCs are inoculated in each well of a 96-well plate, and DiD-labeled VLPs mCAR , T-FVLPs mCAR are added, respectively, and the cells are collected after 12 h of co-incubation; antibody blocking and staining (PEaCD3, AF488aCD11b, BV421aCD19) are performed. After staining, the cells are resuspended in 1x PBS and transferred to a confocal dish, and the confocal microscope is used to observe the T-cell specific membrane fusion specificity of VLPs (all imitated virus-like particles, including VLPs mCAR and T-FVLPs mCAR ) with PBMCs.

[0068] The experimental results are shown in Figure 4. The envelope DiI fluorescence of T-FVLPs mCAR is obviously co-localized with the T-cell membrane, and no DiI fluorescence signal is observed on the B-cell and monocyte membranes, which indicates that T-FVLPs mCAR specifically fuse with the T-cell membrane, and no DiI fluorescence signal is observed on the T-cells, B-cells and monocytes of VLPs mCAR (Figure 4A). Through flow cytometry detection, T-FVLPs mCAR fuse with about 30% of the T-cells, and VLPs mCAR do not fuse with the T-cells (Figure 4B).

[0069] The results of this example show that the T-FVLPs mCAR prepared by the application can efficiently fuse with the T-cell membrane by means of the human T-cell specific membrane fusion protein T-FP.

[0070] Example 5 mRNA delivery efficiency of T cell specific membrane fusion type viro-mimetic particles (T-FVLP mCAR ) and the proportion of CAR-T cells produced

[0071] Preparation of T-FVLP using UTR-mRFP encapsulating RFP expressing cells mRFP (preparation method same as Example 2). Human CD3 + T cells were obtained by magnetic bead sorting, and T-FVLP mRFP were co-incubated with the activated human CD3 + T cells for 24 h.

[0072] In addition, activated human CD3 + T cells were seeded in 96-well plates at 5x10 5 cells per well, and VLP or VLP mCAR or T-FVLP mCAR was added respectively, and after co-incubation for 24 h, T cells were collected by centrifugation at 450g at 4°C for 5 min; then RIPA lysis was used to obtain protein samples, which were quantified using a BCA protein quantification kit, and the concentration was adjusted to 0.5 μg / μL.

[0073] The experimental results are shown in Figure 5. Flow cytometry detection showed that 21.6% of T cells expressed red fluorescent protein RFP after T-FVLP mRFP transfection, while T cells in the VLP and VLP mRFP groups had no significant RFP expression (Figure 5A). In addition, Western blot experiments showed that after T-FVLP mCAR transfection of human T cells, the T cells had obvious CAR protein expression, while the T cells in the VLP and VLP mCAR groups did not express CAR protein (Figure 5B). Flow cytometry detection results also showed that 19.7% of T cells became CAR-T cells after T-FVLP mCAR transfection, while VLP and VLP mCAR could not convert T cells into CAR-T cells (Figure 5C).

[0074] The results of this example show that the T cell specific membrane fusion type viro-mimetic particles prepared by the present application can deliver mRNA to T cells for CAR-T cell preparation.

[0075] Example 6 Expression duration of CAR-T cells produced by T cell specific membrane fusion type viro-mimetic particles (T-FVLP mCAR )

[0076] Magnetic bead sorting of activated human peripheral mononuclear cells PBMCs obtained CD3+ T cells. 5 x 10 5 CD3 + T cells were seeded in each well of 96-well plate, and VLP or VLP mCAR or T-FVLP mCAR were added respectively. After 24h, 48h and 72h incubation, human CD3 + T cells were collected for flow cytometry detection. Cells were blocked and stained (PerCP / Cy5.5 aCD3, AF647 anti-Myc tag), and the proportion of CAR-T cells generated after different incubation times was detected by flow cytometry.

[0077] The results are shown in Figure 6. No CAR-T cells were generated after T cells were incubated with VLP; a very low proportion of CAR-T cells were generated after 24h incubation of T cells with VLP mCAR , and the proportion decreased rapidly; a large number of transient CAR-T cells were generated after T cells were incubated with T-FVLP mCAR , and the number of cells decreased from 24h to 72h, being 18.4% at 24h, 9.8% at 48h, and 3.6% at 72h.

[0078] The results of this example demonstrate that the T cell-specific membrane fusion type virosome (T-FVLP mCAR ) prepared in the application can generate transient CAR-T cells, and does not continuously activate CAR-T cells, so as to reduce the occurrence of cytokine storm in in vivo treatment and improve safety.

[0079] Example 7 Tumor cell killing ability of CAR-T cells produced by T cell-specific membrane fusion type virosome (T-FVLP mCAR )

[0080] Raji-Luc cells (Shanghai Fuheng Biotechnology Co., Ltd.) are a kind of CD19-positive B cell lymphoma cells. 1 x 10 4 Raji-Luc cells were seeded in each well of 96-well plate. Human peripheral mononuclear cells PBMC were sorted by magnetic beads to obtain CD3 + T cells, which were incubated with VLP or VLP mCAR or T-FVLP mCAR for 24h. 5 x 10 4 CD3 + T cells were seeded into the wells containing Raji-Luc cells. After 24h incubation, the Raji-Luc cells were removed, fresh culture medium containing 2 x luciferase substrate was added, and after sufficient reaction, the bioluminescence signal was read by a microplate reader.

[0081] The results are shown in Figure 7. As shown in Figure 7, the T-FVLP mCAR group had the strongest killing ability on tumor cells, and the viability of Raji-Luc cells was reduced by 35.8%, which was higher than that of the VLP or VLP + group. The CD3 mCAR group had a significant difference.

[0082] Example 8 T cell-specific membrane fusion type of virus-like particles (T-FVLP mCAR ) in vivo generated CAR-T cells for treating humanized mouse Raji-Luc B lymphoma

[0083] On the day before treatment, 1 x 10 7 activated human peripheral mononuclear cells PBMC were injected into immunodeficient NCG mice, which had been previously xenotransplanted with Raji-Luc B cell lymphoma. Subsequently, the mice were intravenously injected with PBS, VLP, VLP mCAR and T-FVLP mCAR at 0, 3, 6, 9, 12 days, respectively, with a dose of 2 x 10 10 VLP per mouse each time, for a total of 5 doses. The growth of Raji-Luc B cell lymphoma expressing luciferase in the mice was continuously monitored by bioluminescence imaging.

[0084] The experimental results are shown in Figure 8. Compared with the negative control group (PBS, VLP or VLP mCAR ), T-FVLP mCAR treatment can significantly inhibit the growth of Raji-Luc B cell lymphoma, and the tumor growth inhibition rate reaches 83.5%. By the 6th day, the tumor growth of the T-FVLP mCAR group was significantly slower than that of the other groups, and this effect became more pronounced on the 9th, 12th and 15th days. By the 15th day, the bioluminescence signal of the T-FVLP mCAR group mice was relatively weak and localized to individual tissues, while the bioluminescence signal of the mice in the other groups was significantly stronger and more widely distributed in multiple tissues (Figure 8A and Figure 8B).

[0085] The experimental results of this example show that the T cell-specific membrane fusion type of virus-like particles (T-FVLP mCAR ) prepared by the present application can effectively target and kill CD19-positive B cell lymphoma cells in vivo generated αCD19 CAR-T cells.

[0086] Example 9 T cell-specific membrane fusion type of virus-like particles (T-FVLP mCAR ) generated CAR-T cells for evaluating the safety in vivo

[0087] At the end of the treatment in Example 8, the NCG mice were sacrificed, and the peripheral blood and other major organs (including brain, liver, kidney, lung, heart) were collected. The levels of major inflammatory cytokines IL-1β, IL-6 and GM-CSF in serum were detected by ELISA. Histopathological analysis of each organ was performed using hematoxylin and eosin (H&E) staining. H&E-stained tissue images were observed using a Digital Pathology Aperio CS2 microscope.

[0088] The results are shown in Figure 9. In the T-FVLP mCAR After treatment, the concentrations of key inflammatory cytokines such as IL-1β, IL-6 and GM-CSF did not increase, similar to the levels observed in the PBS and other control groups (Figure 9A). No obvious lesions were observed in the tissues of each organ in the experimental group (Figure 9B). In addition, no neurological symptoms such as seizures, limb disorders or paralysis were observed during the treatment. Histopathological examination of brain tissue showed no signs of damage or toxicity. This is most likely due to the transient nature of the aCD19 CAR-T cells produced by the T-FVLP mCAR mediated production, which did not result in long-term activation of CAR-T cells, thus reducing excessive cytokine release and greatly reducing the associated side effects.

[0089] The experimental results of this embodiment show that the T cell-specific membrane fusion type virus-like particles (T-FVLP mCAR ) prepared by the present application have good biocompatibility and safety.

[0090] Each technical feature of the above-described embodiments can be combined arbitrarily. To make the description concise, not all possible combinations of each technical feature in the above-described embodiments are described, however, as long as the combination of these technical features does not exist contradictory, it should be considered as within the scope of the present disclosure.

[0091] The above-described embodiments only express several embodiments of the present application, which are described in more detail and in more detail, but should not be construed as limiting the scope of the patent. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. Therefore, the scope of protection of the present patent should be subject to the appended claims.

Claims

1. A T cell specific membrane fusogenic viro-mimetic particle, characterized in that, It is prepared by transiently transfecting 293T cells with an expression plasmid of a human T cell specific membrane fusion protein, an expression plasmid of an mRNA loading protein and an expression plasmid of a CAR mRNA; the molar ratio of the expression plasmid of the human T cell specific membrane fusion protein to the expression plasmid of the mRNA loading protein is 1:1.8-2.2; and the molar ratio of the expression plasmid of the mRNA loading protein to the expression plasmid of the CAR mRNA is 1:2-4.

2. The T cell specific membrane fusion type viroparticle according to claim 1, characterized in that, The molar ratio of the expression plasmid of the mRNA loading protein to the expression plasmid of the CAR mRNA is 1:3-4.

3. The T cell specific membrane fusion type viroparticle according to claim 2, characterized in that, The molar ratio of the expression plasmid of the mRNA loading protein to the expression plasmid of the CAR mRNA is 1:

3.

4. The T cell specific membrane fusion type viroparticle according to any one of claims 1 to 3, characterized in that, The human T cell specific membrane fusion protein is obtained by introducing mutations into the gene sequence of the gp160 of HIV virus, the MV-H / MV-F of measles virus or the VSV-G targeting subunit of vesicular stomatitis virus to remove the original receptor recognition ability, and then connecting anti-CD3 scFv, anti-CD4 scFv or anti-CD8 scFv to the extracellular end of the corresponding targeting subunit.

5. The T cell specific membrane fusion type viroparticle according to claim 4, characterized in that, The human T cell specific membrane fusion protein is obtained by connecting anti-CD3 scFv with the sequence shown in SEQ ID NO:2 to the extracellular end of the mutated gp160 gene with the sequence shown in SEQ ID NO:

1.

6. The T cell specific membrane fusion pseudovirion of any one of claims 1-3, wherein, The mRNA loading protein includes mRNA loading protein Peg10 and mRNA loading protein Gag-MS2 or mRNA loading protein Gag-Tat.

7. The T cell specific membrane fusion type viroparticle according to claim 6, characterized in that, The nucleotide sequence of the protein Peg10 is shown in SEQ ID NO:

3.

8. The T cell specific membrane fusion pseudovirion of any one of claims 1-3, wherein, The CAR mRNA has a UTR signal and loads the anti-CD19 or anti-EGFRvIII targeting human tumor cell target point.

9. The T cell specific membrane fusion type viroparticle of claim 8, wherein, The CAR mRNA is anti-CD19 targeting human tumor cell target point with a UTR signal, and its sequence is shown in SEQ ID NO:

4.

10. A method for the preparation of a T cell specific membrane fusogenic virosome according to any one of claims 1 to 9, characterized in that, The method comprises the following steps: (1) constructing an expression plasmid of a human T cell specific membrane fusion protein, an expression plasmid of an mRNA loading protein and an expression plasmid of a CAR mRNA; (2) after co-transfecting the three expression plasmids constructed in step (1) into 293T cells for 45-55 hours, ultracentrifugation and purification are performed, and the product is obtained.

11. Use of the T cell specific membrane fusion type viro-mimetic particle according to any one of claims 1-9 in the preparation of a medicament for treating tumors.

Images