TCR nano-vesicle antibody with functions of T cell redirection and immunosuppression reversal as well as preparation method and application of TCR nano-vesicle antibody

Abstract

The invention relates to the technical field of nano-drug presentation systems, in particular to a TCR (T cell receptor) nano-vesicle antibody with both T cell redirection and immunosuppression reversal as well as a preparation method and application of the TCR nano-vesicle antibody. The TCR nano-vesicle antibody comprises a vesicle formed by a liposome and a cell membrane; tCR protein, a PD-1 antibody and a CD3 antibody are loaded on the vesicles. The nano vesicle antibody has the functions of T cell redirection and immunosuppression reversion; the difficulty of low stability of the soluble TCR is overcome; the polypeptide can be rapidly enriched in tumor tissues through tumor specificity TCR, and CD8 + T cells infiltrated by tumors are directly activated through an anti-CD3 antibody; depletion of T cells can be reversed through the PD-1 antibody on the surface, and the effector function of tumor infiltration CD8 + T cells is improved. The technical scheme can solve the technical problems that the TCR stability is not ideal and the immunosuppression state of the tumor microenvironment is difficult to overcome, and has ideal application and popularization prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of nano drug presentation systems, and in particular to a TCR nano vesicle antibody having both T cell redirection and immunosuppression reversal, and a preparation method and application thereof. Background Art

[0002] T-cell engagers (TCE) are bispecific antibodies that can activate T cells to directly kill tumor cells and are an emerging tumor immunotherapy method. TCE has two key binding domains: one end is the T cell activation domain (usually CD3 single-chain antibody) responsible for binding to the CD3 molecule on the surface of T cells, and the other end is the tumor targeting binding domain formed by T-cell receptors (TCR) or antibodies that targets tumor-specific antigens or proteins expressed on the surface of tumor cells. When TCE connects T cells to tumor cells, the activated T cells release cytotoxic substances such as perforin and granzyme, which can directly destroy tumor cells. In addition, TCE can also induce T cells to secrete a variety of cytokines, further enhancing the immune system's response to tumors. It is worth noting that, unlike antibodies that can only recognize cell membrane proteins, TCR can recognize intracellular proteins presented by human leukocyte antigens (HLA), which makes TCR-TCE based on TCR show unique advantages in the treatment of solid tumors.

[0003] Although TCR-TCE has made significant progress in the field of tumor immunotherapy, only a few such drugs have been approved for clinical treatment. One of the main obstacles is the instability of soluble TCR and its complex production process, which leads to high production costs. Due to the lack of cell membrane anchoring structure, soluble TCR is more easily degraded, which greatly limits the research and development process of TCR-TCE. In addition, immunosuppressive factors in the tumor microenvironment, such as functionally exhausted T cells, immunosuppressive cytokines (such as IL-10 and TGF-β), and regulatory T cells (Treg), together constitute an environment that is not conducive to the effect of TCR-TCE. These factors work together to weaken the effectiveness of T cell-mediated anti-tumor immune responses.

[0004] Therefore, in order to promote the application of TCR-TCE in tumor treatment, key issues that need to be addressed include improving the stability and production efficiency of soluble TCR, and finding effective strategies to overcome the immunosuppressive state in the tumor microenvironment. Only in this way can the potential of TCR-TCE in tumor immunotherapy be fully realized and more effective treatment options be provided for patients. Summary of the invention

[0005] The object of the present invention is a TCR nanovesicle antibody with both T cell redirection and immunosuppression reversal, so as to solve the technical problems that the stability and production efficiency of soluble TCR (T cell receptor) in the application of TCR-TCE (T cell receptor-based T cell engager) in tumor treatment are not ideal and the immunosuppressive state in the tumor microenvironment is difficult to overcome.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A TCR nanovesicle antibody with both T cell redirection and immunosuppression reversal, which comprises a vesicle formed by a liposome and a cell membrane; TCR protein, PD-1 antibody and CD3 antibody are loaded on the vesicle.

[0008] In this technical solution, the TCR nanovesicle antibody (TPC NV) specifically recognizes tumors through TCR on its surface, and activates CD8 + T cells in tumors by using a CD3 single-chain antibody, thereby playing a role in killing tumors; in addition, TPC NV can also block the PD-1 signaling pathway of CD8 + T cells by using the PD-1 single-chain antibody on its surface, improve its exhaustion, and enhance the effector function of CD8 + T cells.

[0009] Furthermore, the nucleotide sequences of the TCR protein, PD-1 antibody and CD3 antibody are respectively as shown in SEQ ID NO.1-3.

[0010] Furthermore, the mass ratio of the liposome to the cell membrane is 5:1.

[0011] Furthermore, the raw materials of the liposome include SPC, DSPE-PEG2000 and cholesterol.

[0012] Furthermore, the cell membrane is from T lymphocytes overexpressing TCR protein, PD-1 antibody and CD3 antibody.

[0013] This technical solution also provides a preparation method of a TCR nanovesicle antibody with both T cell redirection and immunosuppression reversal, which includes the following steps carried out in sequence:

[0014] S1: Integrate the nucleotide sequences of the TCR protein, PD-1 antibody, CD3 antibody and CD8 protein into lentiviral expression vectors respectively, and obtain four lentiviruses through virus packaging; infect T lymphocytes with the four lentiviruses to obtain T lymphocytes overexpressing TCR protein, PD-1 antibody, CD3 antibody and CD8 protein, and then extract to obtain the cell membrane; disperse the cell membrane in water to obtain a cell membrane dispersion;

[0015] S2: Dissolve SPC, DSPE-PEG2000, and cholesterol in chloroform, mix well and evaporate to dryness. After forming a lipid film, add water for hydration treatment; the hydrated lipid film is ultrasonically treated and then extruded using a liposome extruder to obtain a liposome dispersion;

[0016] S3: Add the cell membrane dispersion to the liposome dispersion and ultrasonically treat it to obtain TCR nanovesicle antibodies.

[0017] With the above technical solution, this technical solution has prepared nanoparticles containing cell membranes overexpressing TCR for the first time. The technical difficulty lies in that the effective expression of TCR protein requires the assistance of CD3 antibody and CD8 protein. This technical solution overexpresses CD8 protein in recipient cells to increase the expression of TCR protein.

[0018] Achieving high expression of OVA-TCR in Jurkat cells is one of the difficulties in this study. It was found that when directly expressing OVA-TCR in Jurkat cells, its expression level is low. However, when CD8 molecules were expressed in Jurkat cells, it was found that after overexpressing CD8, the expression efficiency of OVA-TCR could be greatly improved.

[0019] Furthermore, in S1, the nucleotide sequences of TCR protein, PD-1 antibody, CD3 antibody, and CD8 protein are shown in SEQ ID NO.1-4 respectively; the T lymphocytes are Jurkat cells; the lentiviral expression vector is pWPXL expression vector.

[0020] With the above solution, since it is almost impossible to express TCR protein on non-T cells, this technical solution selects Jurkat cells as the T lymphocyte line, and this cell line has a CD3 complex.

[0021] Furthermore, in S2, the dosage ratio of SPC, DSPE-PEG2000, cholesterol, chloroform, and water is 40-60 mg: 2-4 mg: 2-4 mg: 2-6 ml: 1-4 ml; the parameters for ultrasonically treating the hydrated lipid film are 70-80 W, 4-8 min.

[0022] Furthermore, in S3, the parameters for ultrasonically treating the mixture formed by the liposome dispersion and the cell membrane dispersion are 40-50 W, 2-5 min; the mass ratio of liposomes in the liposome dispersion to cell membranes in the cell membrane dispersion is 5:1.

[0023] The technical solution also provides an application of a TCR nanovesicle antibody having both T cell redirection and immunosuppression reversal in the preparation of a drug for treating tumors, wherein the tumor is a tumor cell expressing an OVA antigen; the TCR nanovesicle antibody is used to inhibit tumor growth and tumor metastasis.

[0024] In summary, the technical principle of this technical solution is:

[0025] This technical solution has developed a TCR nanovesicle antibody (TPC NV), which integrates the functions of T cell redirection and immunosuppression reversal. TCR nanovesicle antibodies overcome the difficulty of low stability of soluble TCR by anchoring natural TCR on artificial cell membranes. TCR nanovesicle antibodies can be rapidly enriched in tumor tissues through tumor-specific TCRs and directly activate tumor-infiltrating CD8 + T cells, thus achieving the process of "T cell redirection". In addition, TCR nanovesicle antibodies can reverse the exhaustion of T cells through surface PD-1 antibodies and improve tumor infiltration CD8 + Effector function of T cells.

[0026] The beneficial effects of this technical solution are:

[0027] (1) Significantly improve the stability and production efficiency of TCR:

[0028] TCR-TCE is a bispecific antibody that combines soluble TCR with CD3 single-chain antibody to specifically recognize tumor antigens and activate CD8 + T cells, induced CD8 + T cells specifically kill tumors. TPC NV is prepared through cell membrane bionic nanotechnology, which allows TCR to be naturally embedded in nanovesicles, retaining its specific recognition ability for antigens. This design overcomes the problem of easy degradation of soluble TCR due to the lack of transmembrane anchoring, simplifies the production process, and reduces production costs. In addition, this technical solution promotes the expression of TPC protein by overexpressing CD8 protein in T lymphocytes, providing sufficient TPC protein for TPC NV to exert its therapeutic effect.

[0029] (2) Reverse T cell exhaustion and enhance immune response:

[0030] The PD-1 antibody bound to the surface of TPC NV can effectively block the PD-1 / PD-L1 signaling pathway and reverse the CD8 + Compared with the use of PD-1 inhibitors alone, TPCNV can not only restore the function of infiltrated but exhausted T cells, but also expand the breadth of anti-tumor immune response by activating new T cells.

[0031] (3)Enhance the therapeutic effect on "cold tumors":

[0032] For those with less T cell infiltration (so-called "cold tumors"), traditional immune checkpoint inhibitors such as PD-1 / PD-L1 antibodies often have limited effects. However, TPC NV can not only reverse T cell exhaustion but also actively recruit and activate T cells, making them more effectively target these difficult-to-treat tumor types, providing a new treatment strategy.

[0033] (4)Increase local drug concentration and reduce systemic toxicity:

[0034] Free PD-1 antibodies rely on passive diffusion to reach the tumor site, which may lead to systemic exposure risks. In contrast, TPC NV can form a higher local drug concentration at the tumor site by actively targeting tumor tissues, thus reducing the risk of systemic side effects while improving the effectiveness of treatment.

[0035] (5)Precisely control the number of CD3 antibodies to optimize efficacy and safety:

[0036] The present invention precisely regulates the ratio of liposomes to cell membranes to ensure that each TPC NV contains an appropriate amount of CD3 antibodies (about 3000 - 4000), which can not only effectively activate T cells to exert anti-tumor effects but also avoid potential toxic side effects caused by over-activation, achieving the best balance between efficacy and safety.

[0037] (6)Broad clinical application prospects:

[0038] Due to its unique structural design and mechanistic advantages, TPC NV shows great potential in treating various tumors expressing specific antigens. In addition, it can be customized according to different tumor antigens to further expand its application scope in personalized medicine, providing more precise and effective treatment options for cancer patients.

[0039] In summary, this novel TCR nanovesicle antibody not only solves the key problems of traditional TCR-TCE but also shows significant advantages in multiple aspects, having important scientific value and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Experimental results of flow cytometry for detecting TCR expression efficiency of Jurkat cells and Jurkat cells overexpressing CD8 (CD8-Jurkat) in Example 1.

[0041] Figure 2Schematic diagram of the preparation process, structure and mechanism of action of TPC NV in Example 1.

[0042] Figure 3 TEM micrograph of TPC NV in Example 1.

[0043] Figure 4 Protein immunoblotting results of TPC cells and TPC NV in Example 1.

[0044] Figure 5 In vitro functional evaluation results of TPC NV in Example 2 (a: Detection of the binding efficiency of TPC NV to B16F10-OVA tumor cells by flow cytometry; b: Detection of the binding efficiency of TPC NV to COS-7-PD-1 cells by flow cytometry; c, d: Detection of IFN-γ and TNF-α secreted by T cells after treatment with TPC NV by flow cytometry; e: Statistical chart of cell killing mediated by TPC NV).

[0045] Figure 6 Verification of tumor targeting and in vivo safety of TPC NV in Example 3 (a: Detection of TPC NV enriched in the tumor site by in vivo fluorescence imaging; b: Detection of TPC NV focused in the heart, liver, spleen, lungs and kidneys by in vitro imaging; c: Detection of the expression of CD69 and CD137 on tumor-infiltrating CD8 + T cells after injection of TPC NV for 12 h by flow cytometry; d-e: Detection of the levels of IL-2 and IFN-γ in the peripheral blood of mice by ELISA after injection of TPC NV; f-h: Changes in ALT, CREA and CK in mice; i: Body weight curve of mice; j: Detection of the tissue structure of the heart, liver, lungs and kidneys of mice by HE staining).

[0046] Figure 7 Experimental research results on the inhibition of subcutaneous tumor growth by TPC NV in Example 4 (a: Schematic diagram of the in vivo experimental procedure; b: Tumor growth curve of mice; c: Survival curve of mice; d: Detection of Ki67 expression in tumors by immunohistochemical staining; e-f: Detection of the number of tumor-infiltrating CD8 + T cells and CD4 + T cells, PD-1 expression on CD8 + T cells; g: In vitro stimulation of tumor-infiltrating CD8 + T cells, detection of the levels of IFN-γ and TNF-α secreted by flow cytometry; h: Schematic diagram of the antibody depletion experiment procedure; i: Tumor growth curve; j: Survival curve of mice).

[0047] Figure 8Experimental research results on the inhibition of lung metastatic tumor growth by TPC NV in Example 5 (a: Schematic diagram of in vivo experimental procedure; b: Representative images of in vivo imaging of mice; c: Growth curve of lung tumors in mice; d: Representative images of in vitro imaging of mouse tissues; e: Fluorescence statistical images of lung tissues; f: Lung tissue weight; g: Survival curve of mice).

[0048] Figure 9 Statistical results of the numbers of TCR, single-chain antibody against CD3, and single-chain antibody against PD-1 on TPC NV 1-6 in Comparative Example 1.

[0049] Figure 10 Experimental results of the in vitro tumor cell recognition effect, PD-1 protein binding effect, and T cell activation efficiency of TPC NV 1-6 in Comparative Example 1.

[0050] Figure 11 Experimental results of the in vivo anti-tumor effect and in vivo safety of TPC NV 1-6 in Comparative Example 1 (after treating mice with different TPC NV, tumor growth curve, changes in mouse body weight, and changes in IFN-γ, IL-2, CK, ALT, and CREA in peripheral blood). Detailed implementation manners

[0051] The present invention will be further described in detail below in conjunction with embodiments, but the implementation manners of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can all be obtained from commercial channels.

[0052] Example 1: TCR nanovesicle antibody and its preparation method

[0053] (1) Preparation of TPC cells and acquisition of cell membranes

[0054] TPC cells were obtained by overexpressing OVA-specific TCR, single-chain antibody against mouse CD3, and antibody against mouse PD-1 in Jurkat cells by genetic engineering methods. The more specific process is as follows:

[0055] (1.1) Gene sequence information

[0056] Entrust a biotechnology company to synthesize the gene sequences of OVA-specific TCR (T cell receptor that specifically recognizes OVA antigen, OVA-TCR), single-chain antibody against CD3, single-chain antibody against PD-1, and CD8 according to the conventional methods of the existing technology. 257-264 antigen, OVA-TCR), single-chain antibody against CD3, single-chain antibody against PD-1, and CD8 according to the conventional methods of the existing technology.

[0057] The nucleotide sequence of OVA-TCR is as follows (SEQ ID NO.1, "GCCACC" represents the Kozak sequence, which is used to improve its expression efficiency):

[0058] GCCACC

[0059] The nucleotide sequence of the anti-CD3 single-chain antibody is as follows (SEQ ID NO.2, mouse; the single underline indicates the Kozak sequence):

[0060] GCCACC

[0061] The nucleotide sequence of the PD-1 single-chain antibody is as follows (SEQ ID NO.3, mouse; single underline indicates the Kozak sequence; double underline indicates the EGFP sequence):

[0062]

[0063]

[0064] The nucleotide sequence of the CD8 protein is as follows (SEQ ID NO.4, mouse):

[0065] GCCACC

[0066] (1.2) Construction of expression vector and lentivirus

[0067] The lentiviral transfer vector pWPXL of the prior art is used to construct the expression vector of the above genes. That is, the above four gene fragments are respectively integrated into the multiple cloning sites of the lentiviral transfer vector pWPXL by conventional means of the prior art to obtain the OVA-TCR-pWPXL expression vector, CD3 antibody-pWPXL expression vector, PD-1 antibody-pWPXL expression vector, and CD8-pWPXL expression vector. The construction process of the expression vector can also be entrusted to a biotechnology company to be completed by conventional means of the prior art, and will not be elaborated here.

[0068] According to the operation instructions of the kit, using the Xfect transfection reagent (Takara), the expression plasmid (one of the above expression plasmids), the packaging plasmid psPAX2, and the envelope plasmid pMD2.G are co-transfected into Lenti-X 293T cells for virus packaging, and then four lentiviruses are obtained, namely lentivirus containing the OVA-TCR-pWPXL expression vector, lentivirus containing the CD3 antibody-pWPXL expression vector, lentivirus containing the PD-1 antibody-pWPXL expression vector, and lentivirus containing the CD8-pWPXL expression vector. The construction method of the above lentiviruses is completed by conventional means of the prior art. The lentiviruses can be constructed using existing kits or entrusted to a biotechnology company to complete.

[0069] (1.3) Lentivirus infection of Jurkat cells

[0070] The prepared lentiviruses are respectively added to Jurkat cells for infection. The lentivirus containing the CD8-pWPXL expression vector, the lentivirus containing the OVA-TCR-pWPXL expression vector, the lentivirus containing the PD-1 antibody-pWPXL expression vector, and the lentivirus containing the CD3 antibody-pWPXL expression vector are used to infect Jurkat cells in sequence, and then TPC cells are obtained.

[0071] The specific operation process is as follows:

[0072] 2×10 5 Jurkat cells are added to 500 μl of lentivirus containing the CD8-pWPXL expression vector, with a functional MOI of 10. After 8 h of infection, 500 μl of 1640 medium containing 10% FBS is supplemented. After 24 h, it is replaced with fresh 1640 medium containing 10% FBS and cultured for 48 h. Then, 2×10 5500 μl of lentivirus containing the OVA-TCR-pWPXL expression vector was added to the cells infected by the aforementioned method, with a functional MOI of 10. After 8 hours of infection, 500 μl of 1640 medium with 10% FBS was supplemented. After 24 hours, it was replaced with fresh 1640 medium with 10% FBS and cultured for 48 hours. Then, 2×10 5 500 μl of lentivirus containing the PD-1 antibody-pWPXL expression vector was added to the cells infected by the aforementioned method, with a functional MOI of 10. After 8 hours of infection, 500 μl of 1640 medium with 10% FBS was supplemented. After 24 hours, it was replaced with fresh 1640 medium with 10% FBS and cultured for 48 hours. Then, 2×10 5 500 μl of lentivirus containing the CD3 antibody-pWPXL expression vector was added to the cells infected by the aforementioned method, with a functional MOI of 10. After 8 hours of infection, 500 μl of 1640 medium with 10% FBS was supplemented. After 24 hours, it was replaced with fresh 1640 medium with 10% FBS for expansion culture. Jurkat cells infected with the four lentiviruses were taken, and the cells at this time point were detected by flow cytometry to determine the expression of the target protein. The obtained cells were TPC cells, and their cell membranes were used for the subsequent preparation of nanovesicle antibodies.

[0073] It should be particularly noted that in this technical solution, lentivirus containing the CD8-pWPXL expression vector was first infected into Jurkat cells. This is because the inventor first carried out the following operations: infecting Jurkat cells with lentivirus containing the OVA-TCR-pWPXL expression vector (referring to the aforementioned method). That is, the infection of lentivirus containing the CD8-pWPXL expression vector was not carried out in the initial step (Jurkat group). As a result, it was found that due to the lack of overexpression of CD8 in the cells, the efficiency of overexpression of TCR protein in Jurkat cells was extremely low. At the same time, the inventor used lentivirus containing the CD8-pWPXL expression vector and lentivirus containing the OVA-TCR-pWPXL expression vector to infect Jurkat cells in sequence (CD8-Jurkat group) referring to the aforementioned method. It can be seen that when the plasmid expressing OVA-TCR is transfected into Jurkat cells, it is difficult to achieve high-efficiency expression of this expression plasmid. In actual operation, the plasmid expressing CD8 needs to be transfected into the cells in advance. Only when both the CD8-pWPXL expression vector and the OVA-TCR-pWPXL expression vector exist in Jurkat cells can it be ensured that Jurkat cells can express a large amount of TCR protein to meet the requirements for the preparation of nanovesicle antibodies.

[0074] For the detailed experimental results, see Figure 1, the Jurkat group represents Jurkat cells infected with lentivirus containing the OVA-TCR-pWPXL expression vector (not infected with lentivirus containing the CD8-pWPXL expression vector); the CD8-Jurkat group represents Jurkat cells infected with lentivirus containing the OVA-TCR-pWPXL expression vector and lentivirus containing the CD8-pWPXL expression vector. It can be seen from the experimental results that if CD8 is not overexpressed in Jurkat cells, the expression efficiency of TCR protein in Jurkat cells is low. 93.6% of the cells in the CD8-Jurkat group express TCR protein, while only 47.2% of the cells in the Jurkat group express TCR protein.

[0075] (1.4) Obtaining the cell membrane of TPC cells

[0076] Isolating and extracting the cell membrane from cells is a conventional means of the prior art, and the specific description is as follows: Collect TPC cells and centrifuge at 2000 rpm for 5 min to collect the precipitate. Redisperse the collected precipitate with PBS solution and add a conventional protease inhibitor to avoid protein degradation. Treat the dispersed cell solution with a homogenizer, then centrifuge at 2000 rpm for 5 min to collect the supernatant, redisperse the precipitate with PBS again, repeat the homogenization treatment, and centrifuge at 12000 rpm for 20 min to collect the precipitate. Mix the cell membranes collected twice (the supernatant part of the first centrifugation + the precipitate part of the second centrifugation), and quantify the protein concentration with a protein quantification kit.

[0077] (2) Preparation of TCR nanovesicle antibody

[0078] Dissolve 50 mg of SPC (soybean phosphatidylcholine), 2.5 mg of DSPE-PEG2000 (distearoylphosphatidylethanolamine-polyethylene glycol 2000), and 2.5 mg of cholesterol in 3 ml of chloroform. After thorough mixing, evaporate under reduced pressure in a sample vial by conventional means of the prior art to form a lipid film, and then add 2 ml of deionized water. After ultrasonic treatment at 80 W for 5 min, then perform extrusion treatment 3 times using a liposome extruder (polycarbonate membrane, pore size 100 nm), and then supplement deionized water to make up the volume to 5 mL to obtain a liposome dispersion. Add the above-collected cell membrane dispersion to the above liposome dispersion. After ultrasonic treatment at 50 W for 3 min, finally obtain TCR nanovesicle antibody (TPC NV). Among them, preferably, the mass ratio of liposomes in the liposome dispersion to cell membranes in the cell membrane dispersion is 5:1. The mass of liposomes is measured by taking the lower-layer liposomes after centrifugation of the liposome dispersion to achieve solid-liquid separation. The measurement method of the mass of cell membranes is the same as that of liposomes. In addition, the optional range of the dosage ratio of SPC, DSPE-PEG2000, cholesterol, chloroform, and water is 40 - 60 mg: 2 - 4 mg: 2 - 4 mg: 2 - 6 ml: 1 - 4 ml. The optional range of the parameters for ultrasonic treatment of the hydrated lipid film is 70 - 80 W, 4 - 8 min. The optional range of the parameters for ultrasonic treatment of the mixture formed by the liposome dispersion and the cell membrane dispersion is 40 - 50 W, 2 - 5 min.

[0079] The preparation flow chart of TPC NV, the structural schematic diagram of TPC NV, and the schematic diagram of the mechanism of action are as Figure 2 shown. TPC NV forms a uniform spherical structure, and three proteins are connected to the surface of TPC NV: TCR, CD3 antibody, and PD-1 antibody. The size of TPC NV is about 181.91 ± 3.56 nm, the dispersion index is 0.21 ± 0.003, and the potential is -34.06 ± 0.27 mV. The TEM micrograph is as Figure 3 shown. The results of Western blot show ( Figure 4 ), TPC NV expresses similar TCR, CD3 single-chain antibody, and PD-1 single-chain antibody to the parental TPC cells.

[0080] In addition to TPC NV, other types of nanovesicle antibodies were also synthesized in this technical solution, specifically as follows:

[0081] J NV: Nanovesicles prepared using the cell membranes of untransfected Jurkat cells, and the preparation method refers to that of TPC NV.

[0082] TP NV: Nanovesicles prepared using the cell membranes of Jurkat cells transfected with TCR and PD-1 single-chain antibodies, and the preparation method refers to that of TPC NV.

[0083] TC NV: Nanovesicles prepared using the cell membranes of Jurkat cells transfected with single-chain antibodies against CD3 and TCR. The preparation method was referred to that of TPC NV.

[0084] PC NV: Nanovesicles prepared using the cell membranes of Jurkat cells transfected with single-chain antibodies against CD3 and single-chain antibodies against PD-1. The preparation method was referred to that of TPC NV.

[0085] Example 2: In vitro functional evaluation of TPC NV

[0086] To study the binding ability of TPC NV to B16F10-OVA tumor cells, the experimental groups were: PBS group, J NV group (10 μg / ml), TPC NV group (10 μg / ml), and PC NV group (10 μg / ml). The experimental method was as follows: Dio-labeled J NV, TPC NV, and PCNV were co-incubated with 1×10 5 B16F10-OVA cells for 1 h. After the experiment, the percentage of nanovesicles NV bound to the target cells in the total NV input was detected and counted.

[0087] To study the binding ability of TPC NV to EGFP-PD-1-COS-7 cells, the experimental groups were: PBS group, J NV group, TPCNV group, and TPC NV + aPD-1 group. The experimental method was as follows: DiI-labeled J NV, TPC NV, or PC NV (10 μg / mL) was incubated with 1×10 5 COS-7-PD-1 cells for 1 hour. In addition, some cells were pre-incubated with PD-1 monoclonal antibody (aPD-1, 5 μg / mL) for 1 hour, and then DiI-TPC NV was added. After the experiment, the percentage of nanovesicles NV bound to the target cells in the total nanovesicles NV input was detected and counted.

[0088] To study the ability of TPC NV to promote the secretion of IFN-γ and TNF-α by T cells, the experimental groups were: J NV group, TPC NV group, and TP NV group. The experimental method was as follows: 2×10 5 mouse spleen T cells were co-cultured with different concentrations of J NV, TPC NV, or TP NV (1, 10, 20 μg / mL) for 4 hours, and then Brefeldin A and Monensin were added and incubated for another 2 hours. The cells were collected, and IFN-γ and TNF-α secreted by T cells were detected by flow cytometry.

[0089] To study the cytotoxicity of TPC NV, the experimental groups were: J NV group, TPC NV group, and T only group (T cells were added only according to the ratio). The experimental method was as follows: 1×10 3 Luciferase-B16F10-ova cells were seeded into 96-well plates and incubated overnight. Subsequently, TPC NV or J NV was added at a concentration of 10 μg / mL and incubated for 2 hours. Then, the medium was removed and the cells were washed twice with PBS to remove free TPC NV or J NV. Then, mouse CD8 + T cells were added to the co-culture system according to the ratio of T cells to Luciferase-B16F10-ova cells (2.5:1, 5:1, 10:1, or 20:1) and incubated for 24 hours. The cytotoxicity of TPC NV-mediated T cells was detected using the OneLumi TM Firefly Luciferase Assay Kit and Firefly Luciferase Reporter Gene Assay Cell Lysis Buffer. The formula for calculating the cytotoxicity rate was: cytotoxicity rate = [1 - (luminescence value of the control well / luminescence value of the treated well)] × 100%, where the well without T cells was used as the control well.

[0090] The experimental results are shown in detail in Figure 5 . It can be seen from the experimental results that TPC NV can specifically bind to B16F10 (mouse skin melanoma) tumor cells expressing OVA antigen ( Figure 5 a). In addition, TPC NV can effectively bind to EGFP-PD-1-COS-7 cells, and this binding can be significantly blocked by free PD-1 antibody, demonstrating that TPC NV recognizes EGFP-PD-1-COS-7 cells through the surface PD-1 antibody ( Figure 5 b). Compared with the control group NV, TPC NV can significantly promote the secretion of IFN-γ and TNF-α by T cells ( Figure 5 c-d). The results of the cytotoxicity experiment showed that TPC NV can effectively induce tumor cell death, and the cytotoxicity increased with the increase in the ratio of CD8 + T cells to tumor cells ( Figure 5 e).

[0091] Example 3: Verification of tumor targeting and in vivo safety of TPC NV

[0092] The tumor targeting study of TPC NV was conducted as follows: DiO-labeled J NV or TPC NV (50 mg / kg) was injected via the tail vein into C57BL / 6 mice inoculated with B16F10-ova cells. The fluorescence signal at the tumor site was measured using an IVIS Lumina Series III (PerkinElmer). After 36 hours, the heart, liver, spleen, lungs, and kidneys were collected and further analyzed for fluorescence signals in these organs by IVIS imaging.

[0093] The in vivo safety study of TPC NV was conducted as follows: (1) Twelve hours after the injection of J NV and TPC NV, tumor tissues were collected and tumor-infiltrating immune cells were extracted. The specific steps were as follows: adipose tissue and necrotic tissue were removed using sterile scalpels, ophthalmic scissors, and forceps, and the tumor tissues were washed three times successively with 1640 medium containing 1% FBS and PBS containing 1% FBS. The tumor tissues were cut into small pieces 1-3 mm in size; 10 ml of 1640 medium containing 1% FBS + 1 mg / ml Collagenase IV + 10 μg / ml DNase I was added to the tumor tissues; after pipetting and mixing, the resuspended solution was transferred to a 50 ml conical flask and placed in a suspension incubator at 37°C and 5% CO2 at 120 rpm / min for digestion for 1-1.5 h. Subsequently, 1 ml of FBS was added, and the mixture was pipetted and mixed to terminate the digestion; the digested tissue suspension was filtered successively through sterile filters with pore sizes of 100 μm and 40 μm; the filtrate was collected, centrifuged at 350 g for 10 min, and the supernatant was discarded. The precipitate at the bottom was tumor-infiltrating lymphocytes. Flow cytometry was used to detect the expression of CD69 and CD137 on tumor-infiltrating CD8 + T cells.

[0094] (2) C57BL / 6 mice were injected with TPC NV (50 mg / kg) via the tail vein. Peripheral blood was collected 1 day, 3 days, and 10 days after the injection. ELISA kits were used to detect the levels of IL-2 and IFN-γ in the peripheral blood, and at the same time, the effects of TPC NV on the mouse biochemical indices ALT, CREA, and CK were detected. The body weight changes of the mice were continuously monitored after the injection of TPC NV. On the 25th day after the injection of TPC NV, the heart, liver, lungs, and kidney tissues of the mice were collected, and tissue sections were prepared and stained with HE to detect the tissue structure.

[0095] The experimental results are shown in Figure 6 , indicating that the TCR nanovesicle antibody has good tumor targeting and biological safety. Small animal fluorescence imaging showed that obvious fluorescence signals appeared at the tumor sites of the mice in the TPC NV group and lasted for at least 36 hours, while the fluorescence signals in the control NV group were weak ( Figure 6a). Further analysis was performed on the distribution of TPC NV in the main organs of the body. The results showed that TPC NV accumulated the most in the liver, followed by lung tissue. Quantitative analysis of fluorescence intensity indicated that there was no significant difference in the distribution of TPC NV and control NV in peripheral tissues and organs ( Figure 6 b). Flow cytometry results showed that TPC NV could significantly increase the expression ratios of CD69 and CD137 in CD8 + T cells infiltrating tumors, indicating that TPC NV effectively accumulated and activated T cells at the tumor site ( Figure 6 c). ELISA experiment results showed that 1 day after injection of TPC NV, the concentration of IL-2 in peripheral blood increased significantly and returned to the control group level after 3 days; at 1 day and 3 days, the concentration of IFN-γ in peripheral blood was also significantly higher than that in the PBS group and returned to a similar level after 10 days, indicating that TPC NV could activate the peripheral immune system ( Figure 6 d - e). In terms of safety assessment, treatment with TPC NV led to a transient increase in ALT and CREA while CK showed no obvious change ( Figure 6 f - g), injection of TPC NV did not affect the body weight of mice ( Figure 6 i) and no obvious damage was observed in organs such as the liver, kidney, and heart after treatment with TPC NV ( Figure 6 j). The above results indicated that TPC NV had good tumor targeting and biosafety.

[0096] Example 4: Study on the effect of TPC NV in inhibiting subcutaneous tumor growth

[0097] Experimental method for TPC NV to inhibit subcutaneous tumors:

[0098] (1) Inject 2×10 5 B16F10 - OVA cells subcutaneously into C57BL / 6. Seven days later, inject 50 mg / kg of J NV, TP NV, TC NV, and TPC NV respectively, with PBS as the negative control.

[0099] (2) Monitor the growth of tumors in mice. Tumor volume = 0.5 × tumor long diameter × tumor short diameter 2 .

[0100] (3) Record the survival period of mice.

[0101] (4) On the 15th day after tumor inoculation, collect tumor tissues. Take part of the tissues for frozen section to detect Ki67 expression; extract immune cells infiltrating tumors from the remaining tissues and detect the numbers of CD8 + T cells and CD4 + T cells by flow cytometry, and at the same time analyze the expression of PD - 1 on CD8 + T cells.

[0102] (5) Take a part of immune cells, add PMA (250 nM) and ionomycin (250 ng / ml) to stimulate for 4 hours, then add Brefeldin A and Monensin, and continue to incubate for 2 hours. Collect the cells, and detect IFN-γ and TNF-α secreted by T cells by flow cytometry.

[0103] T cell depletion experimental method:

[0104] (1) Subcutaneously inject 2×10 5 B16F10-OVA cells into C57BL / 6 mice. The mice are divided into 5 groups: PBS group (negative control), TPC NV group (starting 7 days after tumor inoculation, inject 50 mg / kg of TPC NV every three days for a total of 3 injections); TPC NV + IgG group (inject 100 μg of IgG antibody on days 5, 7, 10, and 13 after tumor inoculation, and inject TPC NV according to the TPC NV group at the same time); TPC NV + aCD4 group (inject 100 μg of anti-CD4 antibody on days 5, 7, 10, and 13 after tumor inoculation, and inject TPC NV according to the TPC NV group at the same time); TPC NV + aCD8 group (inject 100 μg of anti-CD5 antibody on days 5, 7, 10, and 13 after tumor inoculation, and inject TPC NV according to the TPC NV group at the same time);

[0105] (2) Monitor the growth of tumors in mice. Tumor volume = 0.5 × tumor long diameter × tumor short diameter 2 .

[0106] (3) Record the survival period of mice.

[0107] The experimental results are shown in Figure 7 , and TCR nanovesicle antibody inhibits the growth of orthotopic tumors. After TPC NV treatment, the tumor volume shrank ( Figure 7 a - b), and the survival period of mice was prolonged ( Figure 7 c), and the Ki67 expression of tumors decreased ( Figure 7 d). TPC NV contains three proteins, and the three proteins synergistically enhance the effect and jointly play a role in inhibiting tumor growth. On the 19th day in Figure 7 b, the tumor volume of the TPC NV group (containing single-chain CD3 antibody, single-chain PD-1 antibody, and TCR) was about 200 mm 3 , the tumor volume of the TP NV group (containing single-chain PD-1 antibody and TCR) was about 740 mm 3 , and the tumor volume of the TC NV group (containing single-chain CD3 antibody and TCR) was about 540 mm 3Around. The combined use of the CD3 single-chain antibody, PD-1 single-chain antibody, and TCR has a synergistic effect and can control the tumor volume at an extremely low level, effectively inhibiting tumor growth.

[0108] Flow cytometry analysis showed that after TPC NV treatment, the number of tumor-infiltrating CD8 + and CD4 + T cells increased significantly ( Figure 7 e). Further analysis found that the expression of PD-1 in tumor-infiltrating CD8 + T cells decreased after TPC NV treatment, and the secretion levels of IFN-γ and TNF-α increased ( Figure 7 f-g), indicating that TPC NV treatment can improve the exhaustion of tumor-infiltrating CD8 + T cells and enhance their effector functions. In mice co-injected with anti-CD8 antibody, the anti-tumor effect of TPC NV was almost completely abolished, indicating that the anti-tumor effect of TPC NV depends on CD8 + T cells ( Figure 7 h-j).

[0109] Example 5: Study on the effect of TPC NV in inhibiting the growth of lung metastatic tumors

[0110] The experimental results are shown in Figure 8 , and TCR nanovesicle antibody inhibits tumor lung metastasis. In previous experimental studies, it was found that TPC NV can accumulate in the lungs of mice. Next, it was explored whether TPV NV can inhibit the formation of lung metastatic tumors. First, a lung metastasis model of tumors was constructed by injecting Luciferase-OVA--MC38 tumors (colon cancer cells expressing OVA) via the tail vein. The above method for constructing the lung metastasis model of tumors is a conventional means of the prior art. Each experimental mouse was modeled in the same way, and details will not be elaborated here. On the 7th day after tumor cell injection, different NVs treatments were given to the mice by tail vein injection ( Figure 8 a). Experimental groups: PBS group, J NV group, TP NV group, TC NV group, TPC NV group. The drug dosage for each group was 50 mg / kg, injected once every 3 days for a total of 3 injections.

[0111] In vivo imaging results of small animals showed that 7 days after injecting the tumors, MC38 cells metastasized to the lungs of mice. As time extended, the fluorescence in the lungs of the control group gradually increased, while the fluorescence increase intensity in the lungs of the TPC NV treatment group was lower than that of the control experimental group ( Figure 8 b). Statistical analysis results showed that TP NV and TC NV could inhibit tumor lung metastasis to a certain extent, while TPCNV significantly inhibited the growth of lung tumors ( Figure 8c). Subsequently, the main organs of the mice were collected and in vitro fluorescence imaging was performed. The results showed that the tumor burden in the lungs of the mice after TPC NV treatment was significantly reduced ( Figure 8 d - e), and the weight of the lung tissue was also significantly lower than that of the control experimental group ( Figure 8 f). In addition, we also found that the survival period of the mice was significantly prolonged after TPC NV treatment ( Figure 8 g). The above results indicate that TPC NV can effectively inhibit the lung metastasis of tumors.

[0112] In addition, from the experimental results such as the tumor growth curve in the lungs of the mice, the fluorescence statistical images of the lung tissues, and the weight of the lung tissues, it can be seen that when the single-chain antibody of CD3, the single-chain antibody of PD-1, and TCR are used in combination, there is a phenomenon of synergistic enhancement, inhibiting the lung metastasis of tumors.

[0113] Comparative Example 1: Study on the ratio of liposome to cell membrane

[0114] This comparative example specifically studied the control method of the dosages of liposome and cell membrane in the preparation of nanovesicle antibodies. More specifically, this comparative example was carried out in the same way as Example 1, except that the ratio of liposome to cell membrane was adjusted. After preparing liposome and cell membrane in the way of the example, TPC NV was further prepared according to the following ratios: TPC NV-1: liposome: cell membrane = 1:1; TPC NV-2: liposome: cell membrane = 2:1; TPC NV-3: liposome: cell membrane = 3:1; TPC NV-4: liposome: cell membrane = 4:1; TPC NV-5: liposome: cell membrane = 5:1; TPC NV-6: liposome: cell membrane = 6:1.

[0115] Flow cytometry was used to detect the numbers of TCR, single-chain antibody of CD3, and single-chain antibody of PD-1 on TPC NV composed of different ratios of liposome to cell membrane, and it was found that as the ratio of liposome to cell membrane increased, the numbers of TCR, single-chain antibody of CD3, and single-chain antibody of PD-1 on TPC NV gradually decreased. The experimental results are shown in Figure 9 .

[0116] Subsequently, the effects of different numbers of TCR, single-chain antibody of CD3, and single-chain antibody of PD-1 on tumor cell recognition, PD-1 protein binding, and T cell activation efficiency were evaluated (for the experimental method, see Example 2). The experimental results showed that ( Figure 10 ), although the numbers of TCR and single-chain antibody of PD-1 on TPC NV composed of different ratios of liposome to cell membrane were different, they had similar effects on tumor cells ( Figure 10 a) and cells expressing PD-1 protein ( Figure 10There is no significant difference in the binding efficiency of (b). TPC NV-1\2\3\4\5 shows no significant difference in the activation efficiency of T cells, and after their treatment, CD8 + There is no significant difference in IFN-γ secreted by CD8 T cells in the TPC NV-6 group, but + the IFN-γ secreted by CD8 T cells in the TPC NV-6 group is much lower than that in other groups ( Figure 10 c).

[0117] To evaluate the in vivo safety and anti-tumor effect of TPC NV with different numbers of TCR, CD3 single-chain antibodies, and PD-1 single-chain antibodies, a B16F10-ova tumor-bearing mouse model was constructed, and TPC NV (50 mg / kg) was injected via the tail vein. The experimental method was carried out according to the "Experimental method for TPC NV inhibiting subcutaneous tumors" in Example 4. C57BL / 6 mice were subcutaneously injected with 2×10 5 B16F10-OVA cells. After 7 days, several TPC NVs at 50 mg / kg were injected respectively, with PBS as the negative control. The tumor size and body weight were statistically analyzed 12 days after modeling, and the levels of IFN-γ, IL-2, CK, ALT, and CREA in the mouse serum were detected on the 12th day. The experimental results showed ( Figure 11 ), TPC NV-1\2\3\4\5 could significantly inhibit tumor growth, and there was no significant difference in their anti-tumor effects. However, the anti-tumor effect of TPC NV-6 was significantly lower than that of other groups, indicating that the anti-tumor ability of TPC NV-6 was poor ( Figure 11 a). The in vivo safety of different TPC NVs was further evaluated. The body weight of mice after treatment with TPC NV-5 and TPC NV-6 did not decrease significantly, but the body weight of mice in the TPC NV-1\2\3\4 groups decreased significantly ( Figure 11 b). The peripheral blood of mice was further collected 96 h after TPC NV injection, and cytokines and biochemical indexes in the peripheral blood were analyzed. It was found that after TPC NV injection, compared with the PBS group, IFN-γ and IL-2 in the peripheral blood increased, indicating that all 6 TPC NVs could induce the immune response of the body ( Figure 11 c, d). We further found that after treatment with the TPC NV-1\2\3\4 groups, the levels of CK, ALT, and CREA in mice increased significantly, and far exceeded their normal reference value ranges, indicating that the heart, liver, and kidney functions of mice were damaged to a certain extent, while the indexes of the TPC NV-5 and TPC NV-6 groups increased slightly and were within the normal reference value ranges ( Figure 11 e-g).

[0118] More specifically, the tumor volume data on the 12th day were: 186±31.51 mm in the PBS group 3, TPC NV-1 group: 55.87 ± 6.23 mm 3 , TPC NV-2 group: 52.6 ± 5.51 mm 3 , TPC NV-3 group: 61 ± 4.07 mm 3 , TPC NV-4 group: 54.73 ± 3.1 mm 3 , TPC NV-5 group: 55.57 ± 6.63 mm 3 , TPC NV-6 group: 214 ± 40.85 mm 3 . It can be seen that the therapeutic effects of the TPC NV-5 group are basically the same as those of TPC NV-1 / 2 / 3 / 4. However, if the dosage of the cell membrane is slightly reduced to the level of TPC NV-6, the tumor volume increases to nearly 4 times the original. It can be seen that maintaining the liposome: cell membrane ≤ 5:1 produces unexpected technical effects on the treatment of tumors with nanovesicle antibodies.

[0119] The weight data of the mice on the 12th day are as follows: PBS group 19.42 ± 0.86 g, TPC NV-1 group 13.79 ± 0.73 g, TPC NV-2 group 13.42 ± 1.51 g, TPC NV-3 group 12.45 ± 1.53 g, TPC NV-4 group 12.42 ± 1.44 g, TPC NV-5 group 18.7 ± 3.49 g, TPC NV-6 group 20.37 ± 1.16 g. The effects of TPC NV-1 / 2 / 3 / 4 will cause a significant decrease in the body weight of the mice. Compared with the PBS group, the body weight of the mice in the TPC NV-1 / 2 / 3 / 4 groups decreased by about 6 g, and the body weight almost decreased by about 30%. It can be seen that TPC NV-1 / 2 / 3 / 4 have greater side effects on the mice. The number of TCR, CD3 single-chain antibodies and PD-1 single-chain antibodies on these TPC NVs is too high, and there are certain safety problems. While TPC NV-5 / 6 did not have a significant impact on the body weight of the mice, and it was basically the same as that of the mice in the PBS group. If the dosage of the cell membrane is slightly increased to the level of TPC NV-4, the body weight of the mice almost decreases by 30%, having a greater impact. It can be seen that maintaining the liposome: cell membrane ≥ 5:1 produces unexpected technical effects on the safety performance of the nanovesicles.

[0120] The data of ALT levels were as follows: 47.67±2.52 U / L in the PBS group, 519±55.97 U / L in the TPC NV-1 group, 491.33±27.75 U / L in the TPC NV-2 group, 423.33±40.8 U / L in the TPC NV-3 group, 323±36.17 U / L in the TPC NV-4 group, 86.67±7.77 U / L in the TPC NV-5 group, and 82.67±4.73 U / L in the TPC NV-6 group. It can be seen that TPC NV-5 / 6 had less impact on the ALT levels of mice and better safety. Compared with TPC NV-5, if the dosage of cell membrane was slightly increased to the level of TPC NV-4, the ALT level increased to about 370% of that in the TPC NV-5 group.

[0121] The data of CREA levels were as follows: 36.33±5.51 U / L in the PBS group, 292±27.4 U / L in the TPC NV-1 group, 274±46.03 U / L in the TPC NV-2 group, 225.33±39.93 U / L in the TPC NV-3 group, 146.67±16.04 U / L in the TPC NV-4 group, 49±2.65 U / L in the TPC NV-5 group, and 54.33±6.11 U / L in the TPC NV-6 group. It can be seen that TPC NV-5 / 6 had less impact on the CREA levels of mice and better safety. Compared with TPC NV-5, if the dosage of cell membrane was slightly increased to the level of TPC NV-4, the CREA level increased to about 300% of that in the TPC NV-5 group.

[0122] The ALT level reflects the liver function status, and the CREA level reflects the kidney function. If the quantities of TCR, CD3 single-chain antibody, and PD-1 single-chain antibody on TPC NV are too high, it will seriously affect the liver and kidney functions of mice. When the cell membrane level is adjusted to the level of TPCNV-5, the negative impact of the nanovesicle antibody on liver and kidney functions will be reduced to a lower level within the normal reference range. However, a slight increase in the cell membrane ratio will have a more significant negative impact on liver and kidney functions. It can be seen that maintaining the liposome:cell membrane ≥5:1 has an unexpected technical effect on the safety performance of the nanovesicles.

[0123] Therefore, considering various aspects such as treatment effects and side effects, TPC NV-5 is the optimal choice (liposome: cell membrane = 5:1). If the dosage of the cell membrane is slightly increased, for example, increased to 4:1 (TPC NV4), it will lead to overly strong drug toxic effects, and significant increases in CK, ALT, and CREA in mice. In particular, the ALT level under the action of TPC NV4 is more than three times that under the action of TPC NV-5, and the data difference is obvious, which was not expected by the inventors before the experiment. In terms of treatment effects, the effects of TPC NV-5 and TPC NV-1-4 in inhibiting tumor growth are basically the same. If the dosage of the cell membrane is slightly decreased, for example, increased to 6:1 (TPC NV-6), it will lead to the complete loss of the effect of the nanovesicle antibody in inhibiting tumor growth, which is the same as the effect of the ordinary PBS control. Maintaining liposome: cell membrane ≤ 5:1 can ensure the best tumor treatment effect (inhibiting tumor growth) of the nanovesicle antibody; maintaining liposome: cell membrane ≥ 5:1 can ensure the optimal safety performance of the nanovesicle antibody. Therefore, in the nanovesicle antibody, maintaining liposome: cell membrane = 5:1 achieves unexpected technical effects. If the ratio is slightly changed, it will lead to a significant deterioration in the treatment effect or a significant deterioration in drug safety.

[0124] The above are only examples of the present invention, and common knowledge such as specific structures and characteristics known in the solution is not described in detail here. It should be noted that for those skilled in the art, without departing from the structure of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope required by this application should be based on the content of its claims, and the specific implementation manners described in the specification can be used to interpret the content of the claims.

Claims

1. A TCR nanovesicle antibody with both T cell redirection and immunosuppression reversal, characterized in that: It includes vesicles formed by liposomes and cell membranes; TCR proteins, PD-1 antibodies, and CD3 antibodies are loaded on the vesicles.

2. The TCR nanovesicle antibody with both T cell redirection and immunosuppression reversal according to claim 1, wherein: The nucleotide sequences of the TCR protein, PD-1 antibody, and CD3 antibody are shown in SEQ ID NO.1-3 respectively.

3. The TCR nanovesicle antibody with both T cell redirection and immunosuppression reversal according to claim 2, wherein: The mass ratio of the liposome to the cell membrane is 5:

1.

4. A TCR nanovesicle antibody having both T cell redirection and immunosuppression reversal as described in claim 3, characterized in that: The raw materials of the liposome include SPC, DSPE-PEG2000, and cholesterol.

5. The TCR nanovesicle antibody with both T cell redirection and immunosuppression reversal according to claim 4, characterized in that: The cell membrane is from T lymphocytes overexpressing TCR proteins, PD-1 antibodies, and CD3 antibodies.

6. The preparation method of a TCR nanovesicle antibody with both T cell redirection and immunosuppression reversal according to any one of claims 1-5, characterized in that: It includes the following steps carried out in sequence: S1: The nucleotide sequences of the TCR protein, PD-1 antibody, CD3 antibody, and CD8 protein are respectively integrated into lentiviral expression vectors, and four lentiviruses are obtained through virus packaging; four lentiviruses are used to infect T lymphocytes to obtain T lymphocytes overexpressing TCR proteins, PD-1 antibodies, CD3 antibodies, and CD8 proteins, and then the cell membranes are extracted; the cell membranes are dispersed in water to obtain a cell membrane dispersion. S2: SPC, DSPE-PEG2000, and cholesterol are all dissolved in chloroform, mixed evenly and then evaporated to dryness. After forming a lipid film, water is added for hydration treatment; the hydrated lipid film is ultrasonically treated and then extruded using a liposome extruder to obtain a liposome dispersion. S3: The cell membrane dispersion is added to the liposome dispersion, and after ultrasonic treatment, TCR nanovesicle antibodies are obtained.

7. The preparation method of a TCR nanovesicle antibody with both T cell redirection and immunosuppression reversal according to claim 6, characterized in that: In S1, the nucleotide sequences of the TCR protein, PD-1 antibody, CD3 antibody, and CD8 protein are shown in SEQ ID NO.1-4 respectively; the T lymphocytes are Jurkat cells; the lentiviral expression vector is the pWPXL expression vector.

8. The preparation method of a TCR nanovesicle antibody with both T cell redirection and immunosuppression reversal according to claim 7, characterized in that: In S2, the dosage ratio of SPC, DSPE-PEG2000, cholesterol, chloroform, and water is 40-60 mg: 2-4 mg: 2-4 mg: 2-6 ml: 1-4 ml; the parameters for ultrasonically treating the hydrated lipid film are 70-80 W, 4-8 min.

9. The preparation method of a TCR nanovesicle antibody with both T cell redirection and immunosuppression reversal according to claim 8, characterized in that: In S3, the parameters for ultrasonically treating the mixture formed by the liposome dispersion and the cell membrane dispersion are 40-50 W, 2-5 min; the mass ratio of the liposomes in the liposome dispersion to the cell membranes in the cell membrane dispersion is 5:

1.

10. Use of a TCR nanovesicle antibody with both T cell redirection and immunosuppression reversal as claimed in any one of claims 1-5 in the preparation of a medicament for treating tumors, characterized in that: The tumor is tumor cells expressing OVA antigen; the TCR nanovesicle antibody is used to inhibit tumor growth and inhibit tumor metastasis.

Images