CN120242005A

CN120242005A - A TCR nanovesicle antibody with both T cell redirection and immunosuppression reversal, and preparation method and application thereof

Info

Publication number: CN120242005A
Application number: CN202510418508.1A
Authority: CN
Inventors: 李罗
Original assignee: Chongqing Maternal And Child Health Hospital Chongqing Obstetrics And Gynecology Hospital Chongqing Institute Of Genetics And Reproduction
Current assignee: Chongqing Maternal And Child Health Hospital Chongqing Obstetrics And Gynecology Hospital Chongqing Institute Of Genetics And Reproduction
Priority date: 2025-04-03
Filing date: 2025-04-03
Publication date: 2025-07-04
Anticipated expiration: 2045-04-03
Also published as: CN120242005B

Abstract

The present invention relates to the technical field of nano drug presentation systems, and specifically to a TCR nanovesicle antibody having both T cell redirection and immunosuppression reversal, and a preparation method and application thereof. The TCR nanovesicle antibody includes a vesicle formed by a liposome and a cell membrane; the vesicle is loaded with a TCR protein, a PD-1 antibody, and a CD3 antibody. The nanovesicle antibody has the functions of T cell redirection and immunosuppression reversal; it overcomes the difficulty of low stability of soluble TCR; it can be rapidly enriched in tumor tissues through tumor-specific TCR, and directly activate tumor-infiltrating CD8 ⁺ T cells through anti-CD3 antibodies; it can reverse the exhaustion of T cells through surface PD-1 antibodies and improve the effector function of tumor-infiltrating CD8 ⁺ T cells. This technical solution can solve the technical problems that TCR stability is not ideal and the immunosuppressive state of the tumor microenvironment is difficult to overcome, and has an ideal prospect for application and promotion.

Description

TCR nano vesicle antibody with T cell redirection and immunosuppression reversion functions, and preparation method and application thereof

Technical Field

The invention relates to the technical field of nano-drug presentation systems, in particular to a TCR nano-vesicle antibody with T cell redirection and immunosuppression reversion, and a preparation method and application thereof.

Background

T cell adapter (T-CELL ENGAGERS, TCE) is a bispecific antibody that activates T cells to achieve direct killing of tumor cells, an emerging tumor immunotherapy approach. TCE has two key binding domains, one on the T cell activation domain responsible for binding to CD3 molecules on the surface of T cells (typically CD3 single chain antibodies) and the other on the tumor targeting binding domain formed by T Cell Receptors (TCRs) or antibodies targeting tumor specific antigens or proteins expressed on the surface of tumor cells. When TCE links T cells to tumor cells, activated T cells release cytotoxic substances such as perforin and granzyme, which can directly destroy tumor cells. In addition, TCE also promotes T-cell secretion of various cytokines, further enhancing immune system response to tumors. Notably, unlike antibodies that only recognize cell membrane proteins, TCRs are capable of recognizing intracellular proteins presented by human leukocyte antigens (Human Leukocyte Antigen, HLA), which makes TCR-based TCE (TCR-TCE) a unique advantage in the treatment of solid tumors.

Despite significant advances in the field of tumor immunotherapy, only a few of these drugs are currently approved for clinical treatment. One of the major obstacles is the instability of the soluble TCR and its complex manufacturing process, resulting in high manufacturing costs. Soluble TCRs are relatively degradable due to the lack of anchoring structures to the cell membrane, which greatly limits the development process of TCR-TCE. In addition, immunosuppressive factors present in the tumor microenvironment, such as function-depleted T cells, immunosuppressive cytokines (e.g., IL-10 and TGF- β), and regulatory T cells (tregs), together constitute an environment that is detrimental to TCR-TCE effects. These factors act together to impair the effectiveness of T cell mediated anti-tumor immune responses.

Therefore, in order to drive the application of TCR-TCE in tumor therapy, a critical issue to be addressed includes improving the stability and production efficiency of soluble TCRs, while finding effective strategies to overcome the immunosuppressive state in tumor microenvironments. Only then can the potential of TCR-TCE in tumour immunotherapy be fully exploited, providing patients with a more effective treatment regimen.

Disclosure of Invention

The invention aims to provide a TCR nano vesicle antibody with T cell redirection and immunosuppression reversion, which solves the technical problems that the stability and production efficiency of a soluble TCR (T cell receptor) are not ideal and the immunosuppression state in a tumor microenvironment is difficult to overcome in the application process of TCR-TCE (T cell adapter based on T cell receptor) in tumor treatment.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

A TCR nano vesicle antibody with T cell redirection and immunosuppression reversal comprises a vesicle formed by liposome and cell membrane, wherein the vesicle is loaded with TCR protein, PD-1 antibody and CD3 antibody.

In the technical scheme, the TCR nano vesicle antibody (TPC NV) specifically recognizes a tumor through TCR on the surface of the TCR nano vesicle antibody, and activates CD8 ⁺ T cells in the tumor by utilizing the CD3 single-chain antibody so as to play a role in killing the tumor, and in addition, the TPC NV can also block a PD-1 signal path of the CD8 ⁺ T cells by utilizing the PD-1 single-chain antibody on the surface of the TPC NV, so that the depletion of the TPC NV is improved, and the effector function of the CD8 ⁺ T cells is improved.

Further, the nucleotide sequences of the TCR protein, the PD-1 antibody and the CD3 antibody are shown as SEQ ID NO.1-3 respectively.

Further, the mass ratio of liposome to cell membrane was 5:1.

Further, the raw materials of the liposome comprise SPC, DSPE-PEG2000 and cholesterol.

Further, the cell membrane is from T lymphocytes over-expressing TCR proteins, PD-1 antibodies, and CD3 antibodies.

The technical scheme also provides a preparation method of the TCR nano vesicle antibody with T cell redirection and immunosuppression reversion, which comprises the following steps in sequence:

S1, integrating nucleotide sequences of TCR protein, PD-1 antibody, CD3 antibody and CD8 protein into slow virus expression vectors respectively, and obtaining four slow viruses through virus packaging respectively; infecting T lymphocytes with four kinds of slow viruses to obtain T lymphocytes over expressing TCR protein, PD-1 antibody, CD3 antibody and CD8 protein, extracting to obtain cell membrane, dispersing the cell membrane in water to obtain cell membrane dispersion;

S2, dissolving SPC, DSPE-PEG2000 and cholesterol in chloroform, uniformly mixing, evaporating to dryness, forming a lipid film, adding water for hydration treatment, carrying out ultrasonic treatment on the hydrated lipid film, and then carrying out extrusion treatment by using a liposome extruder to obtain liposome dispersion liquid;

and S3, adding a cell membrane dispersion liquid into the liposome dispersion liquid, and carrying out ultrasonic treatment to obtain the TCR nano vesicle antibody.

By adopting the technical scheme, the nanoparticle containing the cell membrane for expressing the TCR is prepared for the first time. The technical difficulty is that the effective expression of TCR proteins requires the assistance of CD3 antibodies and CD8 proteins. The technical proposal simultaneously over-expresses CD8 protein in receptor cells so as to increase the expression of TCR protein.

Achieving high expression of OVA-TCR in Jurkat cells is one of the difficulties in this study. It was found that OVA-TCR was expressed directly in Jurkat cells and the expression level was low. However, CD8 molecules were expressed in Jurkat cells, and it was found that the expression efficiency of OVA-TCR was greatly improved after over-expression of CD 8.

Further, in S1, the nucleotide sequences of TCR protein, PD-1 antibody, CD3 antibody and CD8 protein are shown as SEQ ID NO.1-4, respectively, T lymphocyte is Jurkat cell, and lentiviral expression vector is pWPXL expression vector.

With the above scheme, since TCR protein expression is hardly achieved on non-T cells, the present technique selects Jurkat cells as T lymphocyte cell line with CD3 complex.

Further, in S2, the dosage ratio of SPC, DSPE-PEG2000, cholesterol, chloroform and water is 40-60mg:2-4 mg:2-6ml:1-4ml, and the parameters of ultrasonic treatment of the hydrated lipid membrane are 70-80W and 4-8min.

Further, in S3, the parameters of the ultrasonic treatment of the mixture of the liposome dispersion liquid and the cell membrane dispersion liquid are 40-50W for 2-5min, and the mass ratio of the liposome in the liposome dispersion liquid to the cell membrane in the cell membrane dispersion liquid is 5:1.

The technical scheme also provides application of the TCR nano vesicle antibody with T cell redirection and immunosuppression reversion in preparation of medicines for treating tumors, wherein the tumors are tumor cells expressing OVA antigens, and the TCR nano vesicle antibody is used for inhibiting tumor growth and tumor metastasis.

To sum up, the technical principle of the technical scheme is as follows:

The technical scheme develops a TCR nano vesicle 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 TCRs by anchoring the native TCR on artificial cell membranes. TCR nanovesicle antibodies can be rapidly enriched in tumor tissues by tumor-specific TCRs, and directly activate tumor-infiltrating CD8 ⁺ T cells by anti-CD 3 antibodies, thus achieving a "T cell redirection" process. In addition, TCR nanovesicle antibodies can reverse T cell depletion by surface PD-1 antibodies, improving tumor infiltrating CD8 ⁺ T cell effector function.

The beneficial effects of this technical scheme lie in:

(1) Stability and production efficiency of TCR are obviously improved:

TCR-TCE is a bispecific antibody that binds a soluble TCR to a CD3 single chain antibody, specifically recognizes a tumor antigen and activates CD8 ⁺ T cells, inducing specific killing of the tumor by CD8 ⁺ T cells. TPC NV is prepared by a cell membrane bionic nanotechnology, so that the TCR can be naturally embedded into a nanovesicle, and the specific recognition capability of the TCR on an antigen is reserved. The design overcomes the problem of easy degradation of the soluble TCR caused by lack of transmembrane anchoring, simplifies the production process and reduces the production cost. In addition, the technical scheme promotes the expression of TPC protein by over-expressing CD8 protein in T lymphocyte, and provides enough TPC protein for TPC NV to play a role in treatment.

(2) Reverse T cell depletion, enhance immune response:

The PD-1 antibody combined with the surface of TPC NV can effectively block the PD-1/PD-L1 signal path, reverse the depletion state of CD8 ⁺ T cells in the tumor microenvironment, and restore and enhance the effector functions of the CD8 ⁺ T cells. TPC NV not only restores the function of infiltrated but depleted T cells, but also expands the breadth of the anti-tumor immune response by activating new T cells, compared to PD-1 inhibitor alone.

(3) Enhancing the treatment effect on cold tumor:

For those T-cell infiltration is less (i.e. so-called "cold tumors"), traditional immune checkpoint inhibitors such as PD-1/PD-L1 antibodies tend to be of limited effect. The TPC NV can not only reverse the depletion of T cells, but also actively recruit and activate the T cells, so that the TPC NV can effectively target the tumor types which are difficult to treat, and a brand-new treatment strategy is provided.

(4) Improving local drug concentration and reducing systemic toxicity:

Free PD-1 antibodies rely on passive diffusion to the tumor site, possibly leading to systemic exposure risks. In contrast, TPC NV can form higher local drug concentrations at the tumor site by actively targeting tumor tissue, thereby reducing the risk of systemic side effects while improving the effectiveness of the treatment.

(5) Precisely controlling the amount of CD3 antibody to optimize efficacy and safety:

By accurately regulating and controlling the proportion of liposome and cell membrane, each TPC NV is ensured to contain a proper amount of CD3 antibodies (about 3000-4000), so that the T cells can be effectively activated to play an anti-tumor role, potential toxic and side effects caused by excessive activation are avoided, and the optimal balance of curative effect and safety is achieved.

(6) Wide clinical application prospect:

Given their unique structural design and mechanical advantages, TPC NV has shown great potential in the treatment of a variety of tumors expressing specific antigens. In addition, the tumor antigen can be customized and reformed according to different tumor antigens, so that the application range of the tumor antigen in personalized medicine is further widened, and more accurate and effective treatment selection is provided for cancer patients.

In conclusion, the novel TCR nano vesicle antibody not only solves the key problems of the traditional TCR-TCE, but also shows remarkable advantages in various aspects, and has important scientific value and wide application prospect.

Drawings

FIG. 1 is an experimental result of detecting TCR expression efficiency of Jurkat cells and CD 8-overexpressing Jurkat (CD 8-Jurkat) cells by flow cytometry of example 1.

Fig. 2 is a schematic diagram of the preparation process, TPC NV structure and mechanism of action of TPC NV in example 1.

Fig. 3 is a TEM micrograph of TPC NV of example 1.

FIG. 4 shows the results of protein immunoblotting of TPC cells and TPC NV in example 1.

FIG. 5 shows the in vitro functional evaluation results of TPC NV in example 2 (a: flow cytometry to detect binding efficiency of TPC NV to B16F10-OVA tumor cells; B: flow cytometry to detect binding efficiency of TPC NV to COS-7-PD-1 cells; c, d: flow cytometry to detect IFN-r and TNF-a secreted by T cells after TPC NV treatment; e: TPC NV-mediated cell killing statistical).

FIG. 6 shows tumor targeting and in vivo safety verification of TPC NV of example 3 (a: in vivo fluorescence imaging to detect TPC NV enriched in tumor sites; b: in vitro imaging to detect focused TPC NV in heart, liver, spleen, lung, kidney; c: flow cytometry to detect expression of CD8 ⁺ T cells CD69 and CD137 by tumor infiltration 12h after injection of TPC NV; d-e: ELISA to detect levels of IL-2 and IFN-r in peripheral blood of mice after injection of TPC NV; f-h: change of mice ALT, CREA, CK; i: weight curve of mice; j: HE staining to detect tissue structures of heart, liver, lung, kidney of mice).

FIG. 7 shows experimental results of TPC NV inhibition of subcutaneous tumor growth in example 4 (a: schematic in vivo experimental procedure; b: mouse tumor growth curve; c: mouse survival curve; d: immunohistochemical staining to detect tumor Ki67 expression; e-f: flow cytometry to detect the number of tumor infiltrating CD8 ⁺ T cells and CD4 ⁺ T cells, CD8 ⁺ T cell PD-1 expression; g: in vitro stimulation of tumor infiltrating CD8 ⁺ T cells; flow cytometry to detect secreted IFN-r and TNF-a levels; h: schematic antibody clearance experimental procedure; i: tumor growth curve; j: mouse survival curve).

FIG. 8 shows experimental results of TPC NV inhibition of lung metastasis tumor growth in example 5 (a: schematic in vivo experimental procedure; b: in vivo imaging of mice representative image; c: lung tumor growth curve of mice; d: in vitro imaging of mouse tissue representative image; e: fluorescence statistical image of lung tissue; f: lung tissue weight; g: survival curve of mice).

FIG. 9 is a statistical result of the number of TCR, CD3 single-chain antibody and PD-1 single-chain antibody on TPC NV of TPC NV 1-6 of comparative example 1.

FIG. 10 is an experimental result of the in vitro tumor cell recognition effect, PD-1 protein binding effect and T cell activation efficiency of TPC NV 1-6 of comparative example 1.

FIG. 11 shows the in vivo antitumor effects and in vivo safety assay results of TPC NV 1-6 of comparative example 1 (tumor growth curves, mice body weight changes, peripheral blood IFN-r, IL-2, CK, ALT and CREA changes after treatment of mice with different TPC NV).

Detailed Description

The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless otherwise indicated, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents and the like used are all commercially available.

EXAMPLE 1 TCR nanovesicle antibodies and methods of making the same

(1) Preparation of TPC cells and cell membrane acquisition

TPC cells over-express OVA specific TCR, anti-mouse CD3 single-chain antibody and anti-mouse PD-1 antibody in Jurkat cells by a genetic engineering method to obtain TPC cells. The more specific process is as follows:

(1.1) Gene sequence information

The gene sequences of OVA-specific TCRs (T cell receptors specifically recognizing the OVA ^257-264 antigen, OVA-TCRs), CD3 single chain antibodies, PD-1 single chain antibodies, CD8 were synthesized by the conventional methods of the prior art, which were assigned to biotechnology companies.

The nucleotide sequence of the OVA-TCR is as follows (SEQ ID NO.1, "GCCACC" represents a Kozak sequence for improving its expression efficiency):

GCCACCATGGACAAGATCCTGACAGCATCGTTTTTACTCCTAGGCCTTCACCTAGCTGGGGTGAATGGCCAGCAGCAGGAGAAACGTGACCAGCAGCAGGTGAGACAAAGTCCCCAATCTCTGACAGTCTGGGAAGGAGAGACCGCAATTCTGAACTGCAGTTATGAGGACAGCACTTTTAACTACTTCCCATGGTACCAGCAGTTCCCTGGGGAAGGCCCTGCACTCCTGATATCCATACGTTCAGTGTCCGATAAAAAGGAAGATGGACGATTCACAATCTTCTTCAATAAAAGGGAGAAAAAGCTCTCCTTGCACATCACAGACTCTCAGCCTGGAGACTCAGCTACCTACTTCTGTGCAGCAAGTGACAACTATCAGTTGATCTGGGGCTCTGGGACCAAGCTAATTATAAAGCCAGACATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAAAGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCACCGACTTTGACTCCCAAATCAATGTGCCGAAAACCATGGAATCTGGAACGTTCATCACTGACAAAACTGTGCTGGACATGAAAGCTATGGATTCCAAGAGCAATGGGGCCATTGCCTGGAGCAACCAGACAAGCTTCACCTGCCAAGATATCTTCAAAGAGACCAACGCCACCTACCCCAGTTCAGACGTTCCCTGTGATGCCACGTTGACTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTTATGGGACTCCGAATCCTCCTGCTGAAAGTAGCCGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTCCTCCGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCAATGTCTAACACTGTCCTCGCTGATTCTGCCTGGGGCATCACCCTGCTATCTTGGGTTACTGTCTTTCTCTTGGGAACAAGTTCAGCAGATTCTGGGGTTGTCCAGTCTCCAAGACACATAATCAAAGAAAAGGGAGGAAGGTCCGTTCTGACGTGTATTCCCATCTCTGGACATAGCAATGTGGTCTGGTACCAGCAGACTCTGGGGAAGGAATTAAAGTTCCTTATTCAGCATTATGAAAAGGTGGAGAGAGACAAAGGATTCCTACCCAGCAGATTCTCAGTCCAACAGTTTGATGACTATCACTCTGAAATGAACATGAGTGCCTTGGAACTGGAGGACTCTGCTATGTACTTCTGTGCCAGCTCTCGGGCCAATTATGAACAGTACTTCGGTCCCGGCACCAGGCTCACGGTTTTAGAGGATCTGAGAAATGTGACTCCACCCAAGGTCTCCTTGTTTGAGCCATCAAAAGCAGAGATTGCAAACAAACAAAAGGCTACCCTCGTGTGCTTGGCCAGGGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGTGAATGGCAAGGAGGTCCACAGTGGGGTCAGCACGGACCCTCAGGCCTACAAGGAGAGCAATTATAGCTACTGCCTGAGCAGCCGCCTGAGGGTCTCTGCTACCTTCTGGCACAATCCTCGAAACCACTTCCGCTGCCAAGTGCAGTTCCATGGGCTTTCAGAGGAGGACAAGTGGCCAGAGGGCTCACCCAAACCTGTCACACAGAACATCAGTGCAGAGGCCTGGGGCCGAGCAGACTGTGGAATCACTTCAGCATCCTATCATCAGGGGGTTCTGTCTGCAACCATCCTCTATGAGATCCTACTGGGGAAGGCCACCCTATATGCTGTGCTGGTCAGTGGCCTGGTGCTGATGGCCATGGTCAAGAAAAAAAATTCCTGA.

The nucleotide sequence of the CD3 single chain antibody is as follows (SEQ ID NO.2, mouse; single underlined indicates the Kozak sequence):

GCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACTATCCATATGATGTTCCAGATTATGCTGGGGCCCAGCCGGCCAGATCTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAAAGTCCCTGAAACTCTCCTGTGAGGCCTCTGGATTCACCTTCAGCGGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGGAGGGGGCTGGAGTCGGTCGCATACATTACTAGTAGTAGTATTAATATCAAATATGCTGACGCTGTGAAAGGCCGGTTCACCGTCTCCAGAGACAATGCCAAGAACTTACTGTTTCTACAAATGAACATTCTCAAGTCTGAGGACACAGCCATGTACTACTGTGCAAGATTCGACTGGGACAAAAATTACTGGGGCCAAGGAACCATGGTCACCGTCTCCTCAGGTGGCGGTGGCTCAGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGACATCCAGATGACCCAGTCTCCATCATCACTGCCTGCCTCCCTGGGAGACAGAGTCACTATCAATTGTCAGGCCAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATTATACAAATAAATTGGCAGATGGAGTCCCATCAAGGTTCAGTGGCAGTGGTTCTGGGAGAGATTCTTCTTTCACTATCAGCAGCCTGGAATCCGAAGATATTGGATCTTATTACTGTCAACAGTATTATAACTATCCGTGGACGTTCGGACCTGGCACCAAGCTGGAAATCAAAGGCAGTGGGAGTGGGAGTGGGAGTGGGAATGCTGTGGGCCAGGACACGCAGGAGGTCATCGTGGTGCCACACTCCTTGCCCTTTAAGGTGGTGGTGATCTCAGCCATCCTGGCACTGGTGGTGCTCACCATCATCTCCCTTATCATCCTCATCATGCTTTGGCAGAAGAAGCCACGTTAG.

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

the nucleotide sequence of the CD8 protein is as follows (SEQ ID No.4, mouse):

GCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGAGCCAGTTCCGGGTGTCGCCGCTGGATCGGACCTGGAACCTGGGCGAGACAGTGGAGCTGAAGTGCCAGGTGCTGCTGTCCAACCCGACGTCGGGCTGCTCGTGGCTCTTCCAGCCGCGCGGCGCCGCCGCCAGTCCCACCTTCCTCCTATACCTCTCCCAAAACAAGCCCAAGGCGGCCGAGGGGCTGGACACCCAGCGGTTCTCGGGCAAGAGGTTGGGGGACACCTTCGTCCTCACCCTGAGCGACTTCCGCCGAGAGAACGAGGGCTACTATTTCTGCTCGGCCCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACCGAAGACGTGTTTGCAAATGTCCCCGGCCTGTGGTCAAATCGGGAGACAAGCCCAGCCTTTCGGCGAGATACGTCGGCAGCGGAGCAACCAACTTTTCCCTGCTGAAGCAGGCCGGCGATGTGGAGGAGAATCCTGGACCAATGCGGCCGCGGCTGTGGCTCCTCTTGGCCGCGCAGCTGACAGTTCTCCATGGCAACTCAGTCCTCCAGCAGACCCCTGCATACATAAAGGTGCAAACCAACAAGATGGTGATGCTGTCCTGCGAGGCTAAAATCTCCCTCAGTAACATGCGCATCTACTGGCTGAGACAGCGCCAGGCACCGAGCAGTGACAGTCACCACGAGTTCCTGGCCCTCTGGGATTCCGCAAAAGGGACTATCCACGGTGAAGAGGTGGAACAGGAGAAGATAGCTGTGTTTCGGGATGCAAGCCGGTTCATTCTCAATCTCACAAGCGTGAAGCCGGAAGACAGTGGCATCTACTTCTGCATGATCGTCGGGAGCCCCGAGCTGACCTTCGGGAAGGGAACTCAGCTGAGTGTGGTTGATTTCCTTCCCACCACTGCCCAGCCCACCAAGAAGTCCACCCTCAAGAAGAGAGTGTGCCGGTTACCCAGGCCAGAGACCCAGAAGGGCCCACTTTGTAGCCCCATCACCCTTGGCCTGCTGGTGGCTGGCGTCCTGGTTCTGCTGGTTTCCCTGGGAGTGGCCATCCACCTGTGCTGCCGGCGGAGGAGAGCCCGGCTTCGTTTCATGAAACAATTTTACAAATGA.

(1.2) construction of expression vectors and lentiviruses

The expression vector for the above genes was constructed using the lentiviral transfer vector pWPXL of the prior art. That is, the above four gene fragments are respectively integrated on the multiple cloning sites of the lentiviral transfer vector pWPXL according to the conventional means of the prior art to obtain the OVA-TCR-pWPXL expression vector, the CD3 antibody-pWPXL expression vector, the PD-1 antibody-pWPXL expression vector and the CD8-pWPXL expression vector, and the construction process of the expression vectors can be completed by entrusting biotechnology companies to the conventional means of the prior art, and will not be repeated here.

The expression plasmid (one of the above expression plasmids), the packaging plasmid psPAX and the envelope plasmid pMD2.G were co-transfected into Lenti-X293T cells using Xfect transfection reagent (Takara) according to the kit protocol, and virus packaging was performed to obtain four lentiviruses, i.e., a lentivirus containing the OVA-TCR-pWPXL expression vector, a lentivirus containing the CD3 antibody-pWPXL expression vector, a lentivirus containing the PD-1 antibody-pWPXL expression vector, and a lentivirus containing the CD8-pWPXL expression vector. The method for constructing the lentivirus is completed by using a conventional means in the prior art, and can be performed by using an existing kit or entrusting biotechnology company.

(1.3) Lentiviral infection of Jurkat cells

And (3) respectively adding the prepared lentivirus into Jurkat cells for infection, and sequentially using the lentivirus containing a CD8-pWPXL expression vector, the lentivirus containing an OVA-TCR-pWPXL expression vector, the lentivirus containing a PD-1 antibody-pWPXL expression vector and the lentivirus containing a CD3 antibody-pWPXL expression vector to infect Jurkat cells so as to obtain TPC cells.

The specific operation process is as follows:

2X 10 ⁵ Jurkat cells were added with 500. Mu.l of lentivirus containing CD8-pWPXL expression vector, the functional MOI was 10, and after 8h of infection 500. Mu.l of 1640 medium of 10% FBS was supplemented, after 24h the medium was replaced with fresh 1640 medium of 10% FBS and cultured for 48h. Then, 2X 10 ⁵ cells infected in the above manner were added with 500. Mu.l of lentivirus containing OVA-TCR-pWPXL expression vector, the functional MOI was 10, and after 8 hours of infection, 500. Mu.l of 1640 medium of 10% FBS was added, and after 24 hours, the cells were cultured in fresh 1640 medium of 10% FBS. Then, 2X 10 ⁵ cells infected in the above manner were added with 500. Mu.l of lentivirus containing PD-1 antibody-pWPXL expression vector, the functional MOI was 10, and after 8 hours of infection, 500. Mu.l of 1640 medium of 10% FBS was supplemented, and after 24 hours, the cells were cultured in 1640 medium of fresh 10% FBS. Then, 2X 10 ⁵ cells infected in the above manner were added with 500. Mu.l of lentivirus containing CD3 antibody-pWPXL expression vector, the functional MOI was 10, and after 8 hours of infection, 500. Mu.l of 1640 medium of 10% FBS was supplemented, and after 24 hours, the culture was expanded by changing to fresh 1640 medium of 10% FBS. Four lentivirus infected Jurkat cells were taken and examined by flow cytometry at this time point to determine the expression of the protein of interest. The obtained cell is TPC cell, and its cell membrane will be used for subsequent preparation of nano vesicle antibody.

It should be noted that the present protocol first infects Jurkat cells with lentiviruses containing the CD8-pWPXL expression vector. This is because the inventors have first performed an operation of infecting Jurkat cells with a lentivirus containing an OVA-TCR-pWPXL expression vector (see the foregoing). That is, the infection with lentivirus containing the CD8-pWPXL expression vector was not performed in the initial step (Jurkat group). As a result, it was found that the efficiency of the overexpression of TCR protein by Jurkat cells was particularly low because the cells did not overexpress CD 8. Meanwhile, the inventors sequentially infected Jurkat cells (CD 8-Jurkat group) with lentiviruses containing CD8-pWPXL expression vector and lentiviruses containing OVA-TCR-pWPXL expression vector in the manner described above. Therefore, the plasmid for expressing the OVA-TCR is transferred into Jurkat cells, the expression plasmid is difficult to realize high-efficiency expression, in actual operation, the plasmid for expressing CD8 needs to be transfected into the cells in advance, and the CD8-pWPXL expression vector and the OVA-TCR-pWPXL expression vector exist in the Jurkat cells at the same time, so that the Jurkat cells can be ensured to express a large amount of TCR protein, and the requirement for preparing the nano vesicle antibody is met.

The experimental results are shown in detail in FIG. 1, in which the Jurkat group represents Jurkat cells infected with lentivirus containing the OVA-TCR-pWPXL expression vector (without infection with lentivirus containing the CD8-pWPXL expression vector), and in which 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. From the experimental results, if CD8 is not overexpressed in Jurkat cells, TCR protein expression efficiency is low in Jurkat cells, 93.6% of cells in the CD8-Jurkat group express TCR protein, while only 47.2% of cells in the Jurkat group express TCR protein.

(1.4) Cell membrane harvesting of TPC cells

Cell membrane separation and extraction is a conventional means of the prior art, and is described in detail by harvesting TPC cells and centrifugation at 2000rpm for 5min to collect the pellet. The collected precipitate was redispersed with PBS solution and a conventional protease inhibitor was added thereto to avoid protein degradation. The dispersed cell solution was treated with a homogenizer, and then centrifuged at 2000rpm for 5min to collect the supernatant, and the pellet was re-dispersed with PBS, and the homogenization was repeated, and centrifuged at 12000rpm for 20min to collect the pellet. The two collected cell membranes were mixed (supernatant fraction of first centrifugation + second rational pellet fraction) and protein concentration was quantified by the protein quantification kit.

(2) Preparation of TCR nanovesicle antibodies

50Mg of SPC (soybean phosphatidylcholine), 2.5mg of DSPE-PEG2000 (distearoyl phosphatidylethanolamine-polyethylene glycol 2000) and 2.5mg of cholesterol are dissolved together in 3ml of chloroform, and after being fully mixed, the mixture is evaporated under reduced pressure in a sample bottle according to a conventional means in the prior art to form a lipid film, and then 2ml of deionized water is added. Ultrasonic treatment is carried out for 5min at 80W, then a liposome extruder (polycarbonate film, aperture 100 nm) is used for 3 times of extrusion treatment, deionized water is added to fix the volume to 5mL, and liposome dispersion liquid is obtained. Adding the collected cell membrane dispersion liquid into the liposome dispersion liquid, and carrying out ultrasonic treatment for 3min at 50W to finally obtain the TCR nano vesicle antibody (TPC NV). Preferably, the mass ratio of the liposome in the liposome dispersion to the cell membrane in the cell membrane dispersion is 5:1. The mass of the liposome is measured by taking the liposome at the lower layer after solid-liquid separation is realized by the centrifugation of liposome dispersion liquid. The mass of the cell membrane is measured in the same way as the liposomes. In addition, the dosage ratio of SPC, DSPE-PEG2000, cholesterol, chloroform and water may be selected in the range of 40-60mg:2-4 mg:2-6ml:1-4ml. The parameters of ultrasonic treatment of the hydrated lipid membrane can be selected from 70-80W and 4-8min. The parameters of the ultrasonic treatment of the mixture of liposome dispersion and cell membrane dispersion may be selected in the range of 40-50W for 2-5min.

The preparation flow chart, the structure schematic diagram and the action mechanism schematic diagram of the TPC NV are shown in figure 2, the TPC NV is in a uniform spherical structure, and three proteins, namely TCR, CD3 antibody and PD-1 antibody, are connected on the surface of the TPC NV. 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 shown in figure 3. The results of western blot showed (fig. 4) that TPC NV expressed TCR, CD3 single chain antibody and PD-1 single chain antibody similar to maternal TPC cells.

Besides TPC NV, other types of nano vesicle antibodies are synthesized by the technical scheme, and the specific steps are as follows:

j NV nanovesicles prepared using the cell membrane of Jurkat cells that have not been transgenic were prepared with reference to TPC NV.

TP NV nanovesicles prepared using the cell membrane of Jurkat cells transfected with TCR and PD-1 single chain antibodies were prepared with reference to TPC NV.

TC NV nanovesicles prepared using the cell membrane of Jurkat cells transfected with CD3 single chain antibody and TCR, the preparation was performed with reference to TPC NV.

PC NV nanovesicles prepared using the cell membrane of Jurkat cells transfected with CD3 single-chain antibody and PD-1 single-chain antibody were prepared by the method described with reference to TPC NV.

Example 2 in vitro functional evaluation of TPC NV

TPC NV was studied for its ability to bind to B16F10-OVA tumor cells and the experiments were grouped as PBS group, J NV group (10. Mu.g/ml), TPC NV group (10. Mu.g/ml) and PC NV group (10. Mu.g/ml). The experimental method is that Dio marked J NV, TPC NV and PC NV are respectively incubated with 1X 10 ⁵ B16F10-OVA cells for 1h, and the percent of nano vesicle NV bound to target cells in total NV input is detected and counted after the experiment is finished.

TPC NV and EGFP-PD-1-COS-7 cell binding ability are studied, and the experimental groups are PBS group, J NV group, TPC NV group and TPC NV+aPD-1 group. The experiments were performed by incubating DiI-labeled J NV, TPC NV or PC NV (10. Mu.g/mL) with 1X 10 ⁵ COS-7-PD-1 cells for 1 hour, respectively. In addition, some cells were pre-incubated with PD-1 monoclonal antibody (aPD-1, 5. Mu.g/mL) for 1 hour, followed by the addition of DiI-TPC NV. The percent of nanovesicle NV bound to the target cells to the total nanovesicle NV input is detected and counted after the assay is completed.

TPC NV was studied for its ability to promote secretion of IFN-gamma and TNF-alpha by T cells and the experimental groups were J NV group, TPC NV group and TP NV group. The experimental procedure is as follows, 2X 10 ⁵ mouse spleen T cells were co-cultured with different concentrations of J NV, TPC NV or TP NV (1, 10, 20. Mu.g/mL) for 4 hours, followed by the addition of Brefeldin A and Monensin and incubation for a further 2 hours. Cells were collected and examined by flow cytometry for IFN-gamma and TNF-alpha secretion by T cells.

The cytotoxicity of TPC NV was studied and the experimental groups were J NV group, TPC NV group and T only group (T cells were added only in proportion). The experimental procedure was as follows, 1X 10 ³ Luciferase-B16F10-ova cells were seeded in 96-well plates and incubated overnight. Subsequently, TPC NV or J NV was added at a concentration of 10. Mu.g/mL and incubated for 2 hours. The medium was then removed and washed twice with PBS to remove free TPC NV or J NV. Then, mouse CD8 ⁺ T cells were added to the co-culture system in a ratio of T cells to Luciferase-B16F10-ova cells (2.5:1, 5:1, 10:1 or 20:1) and incubated for 24 hours. TPC NV mediated T cell cytotoxicity was detected using OneLumi ^TM Firefly Luciferase Assay Kit and Firefly Luciferase Reporter GENE ASSAY CELL LYSIS Buffer. The cytotoxicity ratio was calculated as = [1- (luminescence value of control well/luminescence value of treated well) ]. Times.100%, where wells without T cells served as control wells.

The experimental results are detailed in FIG. 5, from which it can be seen that TPC NV is able to specifically bind to B16F10 (mouse skin melanoma) tumor cells expressing OVA antigen (FIG. 5 a). In addition, TPC NV was able to bind efficiently to EGFP-PD-1-COS-7 cells and this binding could be significantly blocked by free PD-1 antibodies, demonstrating that TPC NV recognizes EGFP-PD-1-COS-7 cells by surface PD-1 antibodies (fig. 5 b). TPC NV significantly promoted secretion of IFN- γ and TNF- α by T cells compared to control NV (fig. 5 c-d). The cytotoxicity experiment results show that TPC NV is effective in inducing tumor cell death and cytotoxicity is enhanced with increasing CD8 ⁺ T cell to tumor cell ratio (fig. 5 e).

Example 3 tumor targeting and in vivo safety validation of TPC NV

Tumor targeting studies of TPC NV were performed by tail vein injection of DiO-tagged J NV or TPC NV (50 mg/kg) into C57BL/6 mice vaccinated with B16F10-ova cells. Fluorescence signals from tumor sites were measured using IVIS luminea SERIES III (PerkinElmer). After 36 hours, heart, liver, spleen, lung, kidney were collected and further IVIS imaging was performed to analyze fluorescence signals in these organs.

The in vivo safety study of TPC NV is carried out by (1) collecting tumor tissue and extracting tumor infiltrating immune cells after injecting J NV and TPC NV for 12 hours, specifically comprising the steps of removing adipose tissue and necrotic tissue by a sterile scalpel, an ophthalmic scissors and forceps, washing with 1640 culture medium containing 1% FBS and PBS containing 1% FBS for 3 times in sequence, cutting the tumor tissue into small pieces of 1-3mm, adding 10ml 1640 culture medium containing 1% FBS+1 mg/ml CollagenaseIV +10 μg/ml DNase I into the tumor tissue, blowing and mixing, transferring the heavy suspension into a 50ml conical flask, placing into a 37 ℃ and 5% CO2 suspension incubator, digesting for 1-1.5 hours, adding 1ml FBS, blowing and mixing, terminating digestion, sequentially filtering digested tissue suspension with 100 μm and 40 μm sterile filter screen, collecting filtrate, centrifuging, 350g,10min, supernatant, and bottom precipitating into tumor infiltrating lymphocytes. Flow cytometry examined CD69 and CD137 expression of tumor infiltrating CD8 ⁺ T cells.

(2) C57BL/6 tail intravenous TPC NV (50 mg/kg), peripheral blood was collected 1 day, 3 days and 10 days after injection, and ELISA kits were used to detect the levels of IL-2 and IFN-r in the peripheral blood, and at the same time, the effects of TPC NV on the biochemical indices ALT, CREA and CK of mice. Mice were continuously examined for changes in body weight following injection of TPC NV. On day 25 of TPC NV injection, heart, liver, lung and kidney tissues were collected, tissue sections were taken and HE stained, and the tissue structure was examined.

The experimental results are shown in detail in fig. 6, the tcr nanovesicle antibodies have good tumor targeting and biosafety, and the small animal fluorescence imaging shows that the tumor sites of the mice in the TPC NV group show a distinct fluorescence signal and last for at least 36 hours, while the fluorescence signal of the control NV group is weaker (fig. 6 a). The distribution of TPC NV in the main organs of the organism is further analyzed. The results showed that TPC NV accumulated most in the liver, followed by lung tissue, and quantitative analysis of fluorescence intensity showed no significant difference in distribution of TPC NV and control NV in peripheral tissue organs (fig. 6 b). Flow cytometry results showed that TPC NV was able to significantly increase the expression ratio of CD69 and CD137 in tumor-infiltrating CD8 ⁺ T cells, indicating that TPC NV was effective in enriching and activating T cells at tumor sites (fig. 6 c). ELISA experimental results show that the concentration of IL-2 in peripheral blood is significantly increased 1 day after injection of TPC NV, and is restored to the level of the control group after 3 days, and the concentration of IFN-gamma in peripheral blood is significantly higher than that in PBS group at 1 day and 3 days, and is restored to the similar level after 10 days, indicating that TPC NV can activate the peripheral immune system (FIG. 6 d-e). In terms of safety assessment, TPC NV treatment resulted in a transient elevation of ALT and CREA without significant change in CK (fig. 6 f-g), injection of TPC NV did not affect mouse body weight (fig. 6 i) and no significant damage to liver, kidney, heart, etc. organs after TPC NV treatment (fig. 6 j). The result shows that the TPC NV has good tumor targeting and biological safety.

EXAMPLE 4 Effect of TPC NV on inhibiting growth of subcutaneous tumors

TPC NV inhibition subcutaneous tumor experimental method:

(1) C57BL/6 subcutaneous injection of 2X 10 ⁵ B16F10-OVA cells, 50mg/kg J NV, TP NV, TC NV and TPC NV, PBS as negative control, respectively, were injected 7 days later.

(2) The growth of the tumor in mice was monitored, tumor volume = 0.5 x tumor long diameter x tumor short diameter ².

(3) The survival of the mice was recorded.

(4) On day 15 after tumor inoculation, tumor tissues are collected, part of the tissues are frozen and sectioned to detect Ki67 expression, the remaining tissues extract tumor-infiltrated immune cells, flow cytometry is used for detecting the numbers of tumor-infiltrated CD8 ⁺ T cells and CD4 ⁺ T cells, and meanwhile, the expression of CD8 ⁺ T cells PD-1 is analyzed.

(5) A portion of the immune cells was stimulated with PMA (250 nM) and ionomycin (250 ng/ml) for 4 hours, followed by Brefeldin A and Monensin and incubation for an additional 2 hours. Cells were collected and examined by flow cytometry for IFN-gamma and TNF-alpha secretion by T cells.

T cell clearance experimental method:

(1) C57BL/6 was subcutaneously injected with 2X 10 ⁵ B16F10-OVA cells, mice were divided into 5 groups, PBS group (negative control), TPC NV group (50 mg/kg of TPC NV was injected every three days starting from 7 days after tumor-bearing, 3 total injections), TPC NV+IgG group (100. Mu.g of IgG antibody was injected at 5, 7, 10, 13 days after tumor-bearing while TPC NV was injected), TPC NV+aCD4 group (100. Mu.g of anti-CD 4 antibody was injected at 5, 7, 10, 13 days after tumor-bearing while TPC NV was injected at 5, 7, 10, 13 days after tumor-bearing), TPC NV+aCD8 group (100. Mu.g of anti-CD 5 antibody was injected at 5, 7, 10, 13 days after tumor-bearing while TPC NV was injected at TPC group);

(3) The survival of the mice was recorded.

The experimental results are detailed in fig. 7, where tcr nanovesicle antibodies inhibit tumor growth in situ. After TPC NV treatment, tumor volume decreased (fig. 7 a-b), and survival of mice increased (fig. 7 c), ki67 expression of the tumor decreased (fig. 7 d). The TPC NV contains three proteins, and the three proteins are synergistic to play a role in inhibiting tumor growth. On day 19 in FIG. 7b, the tumor volume of TPC NV group (containing CD3 single-chain antibody, PD-1 single-chain antibody and TCR) was about 200mm ³, the tumor volume of TP NV group (containing PD-1 single-chain antibody and TCR) was about 740mm ³, and the tumor volume of TC NV group (containing CD3 single-chain antibody and TCR) was about 540mm ³. The CD3 single-chain antibody, the PD-1 single-chain antibody and the TCR are used in combination to synergistically control the tumor volume at an extremely low level, so that the tumor growth is effectively inhibited.

Flow analysis showed a significant increase in tumor-infiltrating CD8 ⁺ and CD4 ⁺ T cell numbers following TPC NV treatment (fig. 7 e). Further analysis found that the expression of PD-1 was reduced in tumor-infiltrating CD8 ⁺ T cells following TPC NV treatment and the levels of IFN- γ and TNF- α secretion were increased (fig. 7 f-g), suggesting that TPC NV treatment could improve the depletion of tumor-infiltrating CD8 ⁺ T cells and increase their effector functions. In mice injected with anti-CD 8 antibodies at the same time, the anti-tumor effect of TPC NV was almost cancelled, suggesting that the anti-tumor effect of TPC NV was dependent on CD8 ⁺ T cells (fig. 7 h-j).

EXAMPLE 5 Effect of TPC NV on inhibition of lung metastasis tumor growth

The experimental results are shown in detail in fig. 8, the tcr nanovesicle antibody inhibits tumor lung metastasis. TPC NV was found to accumulate in the lungs of mice in previous experimental studies, followed by a study of whether TPV NV inhibited lung metastatic tumor formation. Firstly, a lung metastasis model of a tumor is constructed by injecting a Luciferase-OVA-MC 38 tumor (colon cancer cells expressing the OVA) into a tail vein, wherein the mode of constructing the lung metastasis model of the tumor is a conventional mode in the prior art, and each experimental mouse adopts the same modeling mode and is not repeated here. On day 7 after tumor cell injection, mice were given different treatments of NVs in the form of tail vein injection (fig. 8 a). The experimental groups are PBS group, J NV group, TP NV group, TC NV group and TPC NV group, the drug dosage of each group is 50mg/kg, and the injection is carried out 1 time every 3 days for 3 times.

Results of in vivo imaging of the mice showed that MC38 cells metastasized to the lungs of mice 7 days after tumor injection, with progressive increase in lung fluorescence in the control group over time, while the TPC NV treated group had lower increase in lung fluorescence than the control group (fig. 8 b). Statistical analysis showed that TP NV and TC NV inhibited lung metastasis of tumors to some extent, while TPC NV significantly inhibited lung tumor growth (fig. 8 c). Subsequently, the main organs of the mice were collected and subjected to in vitro fluorescence imaging, which showed that the tumor burden of the lungs of the mice after TPC NV treatment was significantly reduced (fig. 8 d-e), along with the weight of the lung tissue also significantly lower than that of the control experimental group (fig. 8 f). Furthermore, we found that the survival of mice was significantly prolonged after TPC NV treatment (figure 8 g). The results show that TPC NV can effectively inhibit lung metastasis of tumors.

In addition, from experimental results such as a mouse lung tumor growth curve, a lung tissue fluorescence statistical image, lung tissue weight and the like, the CD3 single-chain antibody, the PD-1 single-chain antibody and the TCR are combined to form a synergistic phenomenon, so that tumor lung metastasis is inhibited.

Comparative example 1 study of the ratio of Liposome to cell membrane

The comparative example specifically investigated the manner of controlling the amounts of liposomes and cell membranes used in the preparation of nanovesicle antibodies. More specifically, this comparative example was conducted in the same manner as in example 1 except that the ratio of liposome to cell membrane was adjusted. After preparing liposomes and cell membranes as in the examples, TPC NV: 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 were further prepared as follows.

The number of TCR, CD3 single-chain antibody and PD-1 single-chain antibody on TPC NV formed by different liposome and cell membrane ratios was examined by flow cytometry, and it was found that the number of TCR, CD3 single-chain antibody and PD-1 single-chain antibody on TPC NV gradually decreased with increasing liposome to cell membrane ratio. The experimental results are shown in detail in FIG. 9.

The effect of the number of different TCR, CD3 single chain antibodies and PD-1 single chain antibodies on tumor cell recognition, PD-1 protein binding and T cell activation efficiency was subsequently assessed (see example 2 for experimental details). The experimental results found (FIG. 10) that although the numbers of TCR and PD-1 single-chain antibodies on TPC NV were different with different liposome and cell membrane ratios, they did not have a clear difference in binding efficiency to tumor cells (FIG. 10 a) and cells expressing PD-1 protein (FIG. 10 b). TPC NV-1/2/3/4/5, there was no obvious difference in T cell activation efficiency, and there was no obvious difference in IFN-r secreted by CD8 ⁺ T cells after they were treated, but the IFN-r secreted by CD8 ⁺ T cells was much lower in TPC NV-6 group than in other groups (FIG. 10 c).

To evaluate the in vivo safety line and anti-tumor effects of TPC NV in different numbers of TCR, CD3 single chain antibody and PD-1 single chain antibody, a B16F10-ova tumor-bearing mouse model was constructed and TPC NV (50 mg/kg) was injected by tail vein. Experimental methods were performed with reference to the "TPC NV inhibition subcutaneous tumor Experimental method" of example 4, C57BL/6 mice were subcutaneously injected with 2X 10 ⁵ B16F10-OVA cells, 50mg/kg of several TPC NV respectively after 7 days, PBS as a negative control, tumor size and body weight were counted 12 days after modeling, and the levels of mouse serum IFN-. Gamma.IL-2, CK, ALT and CREA were examined at 12 days. The experimental results show (FIG. 11) that TPC NV-1/2/3/4/5 can significantly inhibit tumor growth without significant differences in anti-tumor effects, however, TPC NV-6 has significantly lower anti-tumor effects than other groups, indicating poor anti-tumor ability of TPC NV-6 (FIG. 11 a). The in vivo safety of the different TPC NV was further evaluated, with no significant decrease in body weight in mice treated with TPC NV-5 and TPC NV-6, but a significant decrease in body weight in mice of the TPC NV-1/2/3/4 group (FIG. 11 b). Further, peripheral blood of mice 96h after injection of TPC NV was collected, and cytokine and biochemical indexes in the peripheral blood were analyzed, and it was found that IFN-r and IL-2 in the peripheral blood were elevated compared with PBS group after injection of TPC NV, indicating that 6 TPC NV could induce immune response in the body (FIGS. 11c, d). We further found that the CK, ALT and CREA of mice were significantly elevated after TPC NV-1/2/3/4 group treatment and far exceeded their normal reference range, indicating some impairment of heart, liver and kidney function in mice, while the indices of TPC NV-5 and TPC NV-6 groups were slightly elevated and within the normal reference range (fig. 11 e-g).

More specifically, tumor volume data on day 12 were PBS group 186+ -31.51 mm 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, and TPC NV-6 group 214+ -40.85 mm ³. It can be seen that the therapeutic effect of TPC NV-5 group was substantially consistent with TPC NV-1/2/3/4, but if the cell membrane dose was slightly reduced to the level of TPC NV-6, the tumor volume increased approximately 4 times. It can be seen that the maintenance of liposome, cell membrane is less than or equal to 5:1, has unexpected technical effect on the tumor treatment effect of the nano vesicle antibody.

The weight data of the mice on day 12 were 19.42+ -0.86 g for PBS group, 13.79+ -0.73 g for TPC NV-1 group, 13.42+ -1.51 g for TPC NV-2 group, 12.45+ -1.53 g for TPC NV-3 group, 12.42+ -1.44 g for TPC NV-4 group, 18.7+ -3.49 g for TPC NV-5 group, and 20.37 + -1.16 g for TPC NV-6 group. The effect of TPC NV-1/2/3/4 can lead to significant weight loss of mice, and compared with the PBS group, the weight loss of mice in the TPC NV-1/2/3/4 group is about 6g, and the weight loss is about 30%. It can be seen that TPC NV-1/2/3/4 produces a large side effect on mice, and that the numbers of TCR, CD3 single-chain antibodies and PD-1 single-chain antibodies on the TPC NV are too high, so that certain safety problems exist. While TPC NV-5\6 had no significant effect on mouse body weight, which was substantially consistent with the mouse body weight of the PBS group. If the cell membrane usage was slightly raised to the level of TPC NV-4, the mice body weight was reduced by almost 30%, giving a larger impact. Therefore, the adoption of the liposome for maintaining the cell membrane to be more than or equal to 5:1 has unexpected technical effects on the safety performance of the nano vesicle.

ALT level data is PBS group 47.67 + -2.52U/L, TPC NV-1 group 519+ -55.97U/L, TPC NV-2 group 491.33 + -27.75U/L, TPC NV-3 group 423.33 + -40.8U/L, TPC NV-4 group 323+ -36.17U/L, TPC NV-5 group 86.67+ -7.77U/L, and TPC NV-6 group 82.67 + -4.73U/L. Thus, TPC NV-5\6 has smaller influence on the ALT level of mice and better safety. Relative to TPC NV-5, if the cell membrane usage is slightly elevated to the level of TPC NV-4, ALT levels are elevated to about 370% of TPC NV-5.

CREA level data were PBS group 36.33 + -5.51U/L, TPC NV-1 group 292+ -27.4U/L, TPC NV-2 group 274+ -46.03U/L, TPC NV-3 group 225.33 + -39.93U/L, TPC NV-4 group 146.67 + -16.04U/L, TPC NV-5 group 49+ -2.65U/L, and TPC NV-6 group 54.33+ -6.11U/L. From this, TPC NV-5\6 has less effect on CREA levels in mice and is safer. If the cell membrane is slightly raised to the level of TPC NV-4 relative to TPC NV-5, the CREA level is raised to about 300% of TPC NV-5.

ALT levels reflect liver function status, CREA levels reflect renal function, and excessive numbers of TCR, CD3 single chain antibodies and PD-1 single chain antibodies on TPC NV can severely affect mouse liver and kidney function. When cell membrane levels are adjusted to TPC NV-5 levels, the negative effects of nanovesicle antibodies on liver and kidney function can be reduced to lower levels, within normal reference values. While a slight increase in the proportion of cell membranes has a relatively significant negative effect on liver and kidney function. Therefore, the adoption of the liposome for maintaining the cell membrane to be more than or equal to 5:1 has unexpected technical effects on the safety performance of the nano vesicle.

From these results, TPC NV-5 was the most preferred choice (liposome: cell membrane=5:1) from the viewpoints of therapeutic effect and side effects. If the cell membrane is slightly elevated, for example to 4:1 (TPC NV 4), this results in an excessive drug toxicity, and a significant increase in CK, ALT and CREA in mice. In particular, ALT levels under TPC NV4 were more than three times greater than those under TPC NV-5, and the difference in data was significant, which the inventors failed to expect prior to the experiment. In terms of therapeutic effect, the tumor growth inhibition effect of TPC NV-5 and TPC NV-1-4 is substantially consistent. If the cell membrane is slightly reduced, for example to 6:1 (TPC NV-6), the nanovesicle antibody is completely deprived of the tumor growth inhibiting effect, consistent with the effect of the normal PBS control. The maintenance of the liposome with the cell membrane not more than 5:1 can ensure the optimal tumor treatment effect (inhibiting tumor growth) of the nano vesicle antibody, and the maintenance of the liposome with the cell membrane not less than 5:1 can ensure the optimal safety performance of the nano vesicle antibody. From this, it can be seen that in the nanovesicle antibody, the maintenance of liposome: cell membrane=5:1, unexpected technical effects were obtained. If the ratio is slightly varied, the therapeutic effect is greatly deteriorated or the safety of the drug is greatly deteriorated.

The foregoing is merely exemplary embodiments of the present application, and specific structures and features that are well known in the art are not described in detail herein. It should be noted that modifications and improvements can be made by those skilled in the art without departing from the structure of the present application, and these should also be considered as the scope of the present application, which does not affect the effect of the implementation of the present application and the utility of the patent. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.

Claims

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

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

3. A TCR nanovesicle antibody having both T cell redirection and immunosuppression reversal according to claim 2, characterized in that the mass ratio of liposome to cell membrane is 5:1.

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

5. A TCR nanovesicle antibody with both T cell redirection and immunosuppression reversal according to claim 4, characterized in that the cell membrane comes from T lymphocytes overexpressing TCR protein, PD-1 antibody and CD3 antibody.

6. A method for preparing a TCR nanovesicle antibody having both T cell redirection and immunosuppression reversal according to any one of claims 1 to 5, characterized in that it comprises the following steps performed in sequence:

S1: The nucleotide sequences of TCR protein, PD-1 antibody, CD3 antibody and CD8 protein are respectively integrated into lentiviral expression vectors, and four lentiviruses are obtained by virus packaging; T lymphocytes are infected with the four lentiviruses to obtain T lymphocytes overexpressing TCR protein, PD-1 antibody, CD3 antibody and CD8 protein, and then cell membranes are extracted; the cell membranes are dispersed in water to obtain cell membrane dispersions;

S2: SPC, DSPE-PEG2000 and cholesterol are dissolved in chloroform, mixed and evaporated to dryness, and water is added to form a lipid film for hydration; the hydrated lipid film is subjected to ultrasonic treatment, and then extruded using a liposome extruder to obtain a liposome dispersion;

S3: Add cell membrane dispersion to liposome dispersion, and obtain TCR nanovesicle antibody after ultrasonic treatment.

7. A method for preparing 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 TCR protein, PD-1 antibody, CD3 antibody and CD8 protein are respectively as shown in SEQ ID NO.1-4; the T lymphocytes are Jurkat cells; and the lentiviral expression vector is a pWPXL expression vector.

8. A method for preparing 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 ultrasonic treatment of the hydrated lipid membrane are 70-80 W, 4-8 min.

9. The method for preparing a TCR nanovesicle antibody with both T cell redirection and immunosuppression reversal according to claim 8 is characterized in that: in S3, the parameters for ultrasonic treatment of the mixture formed by the liposome dispersion and the cell membrane dispersion are 40-50W, 2-5min; the mass ratio of liposomes in the liposome dispersion and cell membranes in the cell membrane dispersion is 5:1.

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