Diabody-modified vesicle sting agonists and methods of making and using same

Abstract

This application provides a biantibody-modified vesicular STING agonist, its preparation method, and its application, relating to the field of polymer nanomedicine technology. The biantibody-modified vesicular STING agonist in this application includes vesicles, a STING agonist, and biantibodies. The vesicles are formed by cross-linking and assembling functionalized amphiphilic block polymers, and their lumens are negatively charged. The STING agonist is loaded into the vesicle lumen by electrostatic interactions. The biantibodies include anti-HER2 monoclonal antibodies and anti-PD1 monoclonal antibodies grafted onto the surface of the vesicles. The biantibody-modified vesicular STING agonist exhibits good biocompatibility and can enhance the killing effect of T cells on tumor cells, thereby killing tumor cells.

Description

Technical Field

[0001] This invention relates to the field of polymer nanomedicine technology, and in particular to a dual-antibody modified vesicle STING agonist, its preparation method, and its application. Background Technology

[0002] In recent years, T-cell-based tumor immunotherapy strategies, such as immune checkpoint blockade and T-cell connectives, have made some progress in various malignant tumors. However, their efficacy largely depends on the sufficient infiltration and sustained activation of functional T cells in the tumor microenvironment. However, for most "cold" tumors, the tumor microenvironment is usually in an immunosuppressive state, with insufficient T-cell infiltration and a high risk of T-cell exhaustion, resulting in a low overall response rate of existing immunotherapies and a tendency to develop secondary drug resistance.

[0003] Activating the Stimulator of Interferon Genes (STING) signaling pathway has potential advantages in reversing tumor immunosuppression and "heating up" the tumor microenvironment. STING agonists act on STING, a key adaptor protein in the intracellular DNA sensing pathway, activating the STING pathway and initiating a robust type I interferon response. This not only directly enhances the immunogenicity of tumor cells but also effectively bridges innate and adaptive immunity by recruiting and activating dendritic cells, thereby promoting the initiation, expansion, and invasion of tumor-specific T cells. However, STING agonists have poor cell membrane penetration due to their inherent hydrophilicity and charge properties, and systemic administration often induces systemic inflammation, causing severe toxic side effects. Although T cell connectors, immune checkpoint blockade, and STING agonists all possess certain anti-tumor effects, current technology lacks a solution that can synergistically integrate tumor-targeting antibodies, T-cell-targeting antibodies, and STING agonists. Such a solution not only possesses immune checkpoint blockade function and good biosafety but can also further enhance the overall anti-tumor effect through precise control of carrier particle size and the ratio of dual antibodies. Summary of the Invention

[0004] One objective of the first aspect of this invention is to provide a dual-antibody modified vesicle STING agonist to address the technical problem of poor anti-tumor therapeutic efficacy in the prior art.

[0005] Another objective of the first aspect of this invention is to ensure the effectiveness of anti-tumor treatment.

[0006] The second aspect of this invention aims to provide the application of a dual-antibody modified vesicle STING agonist.

[0007] The third aspect of this invention aims to provide a method for preparing a dual-antibody modified vesicle STING agonist.

[0008] According to a first aspect of the present invention, the present invention provides a dual-antibody modified vesicle STING agonist, comprising:

[0009] The vesicles are formed by cross-linking functionalized amphiphilic block polymers and amphiphilic block polymers containing negatively charged segments. The vesicles have negatively charged cavities and a particle size of 20 nm-35 nm. The functionalized amphiphilic block polymers are functional groups-second hydrophilic segments-second hydrophobic segments, wherein the functional groups are N3-, Mal-, or NHS-.

[0010] STING agonists are loaded into the lumen of the vesicles via electrostatic interactions;

[0011] The biantibody comprises an anti-HER2 monoclonal antibody and an anti-PD-1 monoclonal antibody covalently grafted onto the surface of the vesicle, wherein both the anti-HER2 monoclonal antibody and the anti-PD-1 monoclonal antibody are functional group modified antibodies, and the functional group is dibenzocyclooctylene. The ratio of the anti-HER2 monoclonal antibody and the anti-PD-1 monoclonal antibody grafted onto the surface of the vesicle is any value in the range of 1:(0.33-3). The anti-HER2 monoclonal antibody is used to target the HER2 antigen on the surface of tumor cells, and the anti-PD-1 monoclonal antibody is used to target the PD-1 antigen on the surface of T cells.

[0012] Optionally, the STING agonist is selected from any one of diABZI, STING agonist-3, SR-717, or GNE-6468.

[0013] Optionally, the amphiphilic block polymer is a first hydrophilic segment-first hydrophobic segment-Ac-KD. z In this structure, the first hydrophilic segment is polyethylene glycol, the first hydrophobic segment is a random copolymer of trimethylene carbonate monomer and disulfide five-membered ring carbonate monomer, Ac is an acetyl group, and KD... z The negatively charged linker is represented by K, which is lysine, D is aspartic acid, and z represents a repeating unit.

[0014] Optionally, the second hydrophilic segment is polyethylene glycol, and the second hydrophobic segment is a random copolymer of trimethylene carbonate monomer and disulfide pentaneous ring carbonate monomer.

[0015] Optionally, the mass of the STING agonist accounts for 1%-11.2% of the total mass of the vesicle and the STING agonist.

[0016] In accordance with a second aspect of the present invention, the present invention also provides the application of the above-described dual-antibody modified vesicle STING agonist in antitumor immunotherapy.

[0017] According to a third aspect of the present invention, the present invention also provides a method for preparing the above-mentioned dual-antibody modified vesicle STING agonist, comprising the following steps:

[0018] Amphiphilic block polymers and functionalized amphiphilic block polymers are provided;

[0019] An organic solution containing the amphiphilic block polymer and the functionalized amphiphilic block polymer, and an acidic buffer solution containing the STING agonist were prepared. The organic solution and the acidic buffer solution were mixed and then subjected to dialysis and ultrafiltration to obtain the vesicular STING agonist.

[0020] Functional group-modified anti-HER2 monoclonal antibodies and functional group-modified anti-PD-1 monoclonal antibodies were prepared.

[0021] The vesicle STING agonist, the anti-HER2 monoclonal antibody, and the anti-PD-1 monoclonal antibody are mixed at a preset molar ratio to ensure that the anti-HER2 monoclonal antibody and the anti-PD-1 monoclonal antibody are covalently grafted onto the surface of the vesicle STING agonist, thereby preparing the dual-antibody modified vesicle STING agonist.

[0022] Optionally, in the step of preparing the dual-antibody modified vesicular STING agonist by mixing the vesicular STING agonist, the anti-HER2 monoclonal antibody, and the anti-PD-1 monoclonal antibody at a preset molar ratio so that the anti-HER2 monoclonal antibody and the anti-PD-1 monoclonal antibody are covalently grafted onto the surface of the vesicular STING agonist, the preset molar ratio of the functional group of the vesicular STING agonist, the anti-HER2 monoclonal antibody, and the anti-PD-1 monoclonal antibody is any value in the range of 1:(0.17-0.5):(0.17-0.5).

[0023] Optionally, in the step of preparing an organic solution containing the amphiphilic block polymer, the functionalized amphiphilic block polymer, and an acidic buffer solution containing a STING agonist, and then mixing the organic solution and the acidic buffer solution to obtain the vesicle STING agonist by dialysis and ultrafiltration, the STING agonist is encapsulated in the vesicle lumen while the functionalized amphiphilic block polymer and the amphiphilic block polymer are assembled and crosslinked to form vesicles.

[0024] In this invention, a dual-antibody modified vesicle STING agonist is introduced onto the surface of nanovesicles with synergistic anti-HER2 and anti-PD-1 monoclonal antibodies, achieving dual targeting and physical bridging of tumor cells and T cells. On one hand, it precisely targets tumor cells expressing HER2 antigen; on the other hand, it recruits and activates T cells, while simultaneously blocking the PD-1 / PD-L1 immunosuppressive pathway and promoting the efficient formation of functional immune synapses. Furthermore, the vesicle-delivered STING agonist is efficiently released and activates the STING pathway within tumor cells, synergistically upregulating MHC-I expression and inducing the secretion of immunostimulatory cytokines, thereby enhancing antigen presentation and dendritic cell-mediated adaptive immune responses, achieving a transformation from local innate immunity to systemic immune responses. Simultaneously, by limiting the vesicle size and the ratio of the two antibodies, intercellular spacing is regulated and the target-activation kinetics are balanced, improving the T cell recognition and killing efficiency of tumor cells. Moreover, the vesicle system exhibits good biocompatibility, degradability, and tumor-targeting delivery capabilities, reducing the non-specific release of the STING agonist into normal tissues, thus improving both anti-tumor therapeutic efficacy and biosafety.

[0025] Furthermore, the mass of STING agonist is set to account for 1%-11.2% of the total mass of vesicles and STING agonist to ensure that the STING agonist content in the vesicles can balance the anti-tumor therapeutic effect with safety and avoid overtreatment.

[0026] The above and other objects, advantages and features of the present invention will become more apparent to those skilled in the art from the following detailed description of specific embodiments of the invention in conjunction with the accompanying drawings. Attached Figure Description

[0027] The following sections will describe some specific embodiments of the invention in detail by way of example and not limitation, with reference to the accompanying drawings. The same reference numerals in the drawings denote the same or similar parts or portions. Those skilled in the art should understand that these drawings are not necessarily drawn to scale. In the drawings:

[0028] Figure 1 This is a schematic flowchart of a method for preparing a dual-antibody modified vesicle STING agonist according to an embodiment of the present invention;

[0029] Figure 2 This is a process flow diagram of a method for preparing a biantibody-modified vesicle STING agonist according to an embodiment of the present invention;

[0030] Figure 3 The particle size distribution diagram is based on TCE-nSTING from Example 1;

[0031] Figure 4The TCE-nSTING, PBS, TCE-n, diABZI, IP-nSTING, and IT-nSTING in Example 1 and Comparative Examples 1 to 5 are the effects of these substances on B16F10. HER2+ STING pathway-related genes (A) interferon in tumor cells - β 、 (B) Effect of CXC motif chemokine ligand 10 and (C) interleukin-6 expression on the graph;

[0032] Figure 5 In Example 1 and Comparative Examples 1 to 5, TCE-nSTING, PBS, TCE-n, diABZI, IP-nSTING, and IT-nSTING (A) upregulated B16F10. HER2+ Graph showing the expression of MHC-I on the surface of tumor cells and (B) its promotion of naive T cell activation;

[0033] Figure 6 This is the TCE-nSTING of Example 1 for B16F10 HER2+ Graph showing the toxic effects on tumor cells;

[0034] Figure 7 This is a statistical graph showing the apoptosis rate of T cells killing tumor cells mediated by TCE-nSTING, PBS, TCE-n, diABZI, IP-nSTING and IT-nSTING in Example 1 and Comparative Examples 1 to 5.

[0035] Figure 8 The results of the detection of TCE-nSTING, PBS, TCE-n, diABZI, IP-nSTING and IT-nSTING enhancing T cell killing function and promoting cytokine secretion in Example 1 and Comparative Examples 1 to 5 are as follows: (A) Expression of lysosome-associated membrane protein-1, (B) Expression of perforin, (C) Expression of granzyme B, (D) Secretion of interferon-γ, and (E) Secretion of tumor necrosis factor-α.

[0036] Figure 9 B16F10 treated with TCE-nSTING, PBS, TCE-n, diABZI, IP-nSTING, and IT-nSTING in Example 1 and Comparative Examples 1 to 5 HER2+The indirect activation of dendritic cells and promotion of T cell activation by tumor cell culture supernatant; (A) representative flow cytometry and (B) statistical analysis of maturation of mouse bone marrow-derived dendritic cells; (C) MHC-I expression level on the surface of dendritic cells; (D) interleukin-12, (E) interleukin-6 and (F) tumor necrosis factor-α; (G) activation of dendritic cells with naïve CD8+. + The effects of T cells;

[0037] Figure 10 It is subcutaneous B16F10 HER2+ After melanoma mice were injected via the tail vein with fluorescently labeled TCE-nSTING, diABZI, IP-nSTING, IT-nSTING and nSTING of Examples 1 and Comparative Examples 3 to 6, respectively, for 8 hours, (A) in vitro fluorescence imaging images of each major tissue and (B) drug distribution of diABZI in each tissue.

[0038] Figure 11 Examples 1, 2, 1, 2, 4, and 5 are subcutaneous B16F10. HER2+ The in vivo antitumor efficacy in mouse models includes (A) a single treatment flowchart, (B) the average tumor growth curve of mice in different treatment groups, (C) the tumor growth curve of each mouse, (D) the mouse survival curve, and (E) the change in body weight.

[0039] Figure 12 The effects of TCE-nSTING, PBS, and TCE-n on (A) the tumor immune microenvironment and (B) the immune cell phenotype in the spleen, as shown in Examples 1, 1, and 2, respectively.

[0040] Figure 13 It is subcutaneous B16F10 HER2+ After mice were treated with TCE-nSTING, PBS, and TCE-n as described in Examples 1, 1, and 2, the following changes were observed in the draining lymph nodes: (A) maturation of dendritic cells, (B) expression of MHC-I on the surface of dendritic cells, and (C) CD4 expression. + T and (D)CD8 + T cell activation status;

[0041] Figure 14 It is subcutaneous B16F10 HER2+ Mice treated with TCE-nSTING, PBS, and TCE-n as described in Examples 1, 1, and 2, respectively, had (A)CD8 levels in their peripheral blood. + The number of T cells and the levels of (B) interferon-β, (C) interferon-γ and (D) interleukin-6 cytokines in the serum;

[0042] Figure 15 Mice cured by TCE-nSTING (A) in Example 2 were re-inoculated with B16F10. HER2+ A schematic diagram of tumor cells and (B) mice cured by treatment in Example 2, re-inoculated with B16F10. HER2+ Tumor cells and healthy mice inoculated with B16F10 HER2+ Tumor growth curves of tumor cells, CD8+ in mouse peripheral blood + Quantitative analysis of the proportions of (C) effector memory T cells, (D) central memory T cells, and (E) representative flow cytometry of memory T cells, CD8+ in mouse spleen. + Quantitative analysis of the proportions of (F) effector memory T cells and (G) central memory T cells in T cells, and representative flow cytometry plots of (H) memory T cells;

[0043] Figure 16 TCE-nSTING, PBS, and TCE-n in Example 1, Comparative Example 1, and Comparative Example 2 were used in CT-26 HER2+ - The antitumor efficacy of Luc colorectal cancer in a mouse model of metastatic colorectal cancer, including (A) bioluminescence images and semi-quantitative analysis, (B) survival curves and (C) changes in body weight of different groups of mice;

[0044] Figure 17 The particle size distribution (A), STING agonist loading (B), and STING agonist release (C) of nSTING in Comparative Example 6 are shown.

[0045] Figure 18 It is PEG-P(TMC-DTC)-Ac-KD in Example 1 10 (A) structural formula and (B) proton NMR spectrum and (C) proton NMR spectrum of Ps in Comparative Example 8.

[0046] Figure 19 These are the potential diagrams of TCE-n in Comparative Example 2 and Ps in Comparative Example 8.

[0047] Figure 20 This is the particle size distribution diagram of TCE-n in Comparative Example 2;

[0048] Figure 21 TCE-n-mediated T cell killing of B16F10 in Comparative Examples 2 and 7 HER2+ The condition of tumor cells;

[0049] Figure 22 It is (A)B16F10 HER2+Flow cytometry analysis of tumor cells and (B)T cells after incubation for 4 h with fluorescently labeled PBS, TCE-n, Ps, IP-n, and IT-n of comparative examples 1, 2, 8 to 10, respectively; (C)B16F10 HER2+ Confocal laser scanning microscopy images of tumor cells and (D)T cells after incubation with fluorescently labeled TCE-n (component 2) and Ps (component 8) for 4 h. The fluorescently labeled TCE-n (component 2) is in (E)B16F10. HER2+ Internalization and surface binding in tumor cells and (F)T cells;

[0050] Figure 23 The fluorescently labeled TCE-n, Ps, IP-n, and IT-n of Comparative Examples 2, 8, to 10 are physically bridged with B16F10. HER2+ Laser confocal scanning microscopy image of tumor cells and T cells;

[0051] Figure 24 This is a diagram illustrating the mechanism of TCE-nSTING in treating tumor cells in Example 1. Detailed Implementation

[0052] Unless otherwise specified, all terms (including technical and scientific terms) used in the description of this embodiment have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0053] The biantibody-modified vesicle STING agonist provided by this invention comprises vesicles, a STING agonist, and a biantibody. The vesicles are formed by cross-linking a functionalized amphiphilic block polymer with and without a negatively charged segment, resulting in a negatively charged lumen. The vesicles have a particle size of 20 nm-35 nm. The functionalized amphiphilic block polymer is a functional group-second hydrophilic segment-second hydrophobic segment polymer, wherein the functional group is N3-, Mal-, or NHS-. The STING agonist is loaded into the lumen of the vesicle through electrostatic interactions. The biantibody consists of an anti-HER2 monoclonal antibody and an anti-PD-1 monoclonal antibody covalently grafted onto the surface of vesicles, respectively. Both the anti-HER2 monoclonal antibody and the anti-PD-1 monoclonal antibody are functional group modified antibodies, and the functional group is dibenzocyclooctylene. The ratio of anti-HER2 monoclonal antibody to anti-PD-1 monoclonal antibody grafted onto the vesicle surface is any value in the range of 1:(0.33-3). The anti-HER2 monoclonal antibody is used to target the HER2 antigen on the surface of tumor cells, and the anti-PD-1 monoclonal antibody is used to target the PD-1 antigen on the surface of T cells.

[0054] In this embodiment, the dual-antibody modified vesicle STING agonist achieves dual targeting and physical bridging of tumor cells and T cells by synergistically introducing anti-HER2 and anti-PD-1 monoclonal antibodies onto the surface of nanovesicles. On the one hand, it precisely targets tumor cells expressing HER2 antigen; on the other hand, it recruits and activates T cells, while simultaneously blocking the PD-1 / PD-L1 immunosuppressive pathway and promoting the efficient formation of functional immune synapses. Furthermore, the vesicle-delivered STING agonist efficiently releases and activates the STING pathway within tumor cells, synergistically upregulating MHC-I expression and inducing the secretion of immunostimulatory cytokines, thereby enhancing antigen presentation and dendritic cell-mediated adaptive immune responses, realizing the transformation from local innate immunity to systemic immune responses. Simultaneously, by limiting the vesicle size and the ratio of the two antibodies, intercellular spacing regulation and target-activation kinetics balance are achieved, improving the T cell recognition and killing efficiency of tumor cells. In addition, the vesicle system exhibits good biocompatibility, degradability, and tumor-targeting delivery capabilities, reducing the non-specific release of the STING agonist in normal tissues, thus improving both anti-tumor therapeutic efficacy and biosafety.

[0055] In this embodiment, the STING agonist is released into tumor cells. Activating the STING pathway upregulates the expression level of MHC-I on the tumor cell surface. MHC-I molecules are loaded with tumor-specific antigen peptides that T cell receptors can specifically recognize and bind to, enhancing the T cell's sensitivity to tumor cells. Simultaneously, activation of the STING pathway prompts tumor cells to secrete immunostimulatory cytokines. These cytokines act on dendritic cells via the paracrine pathway, promoting dendritic cell maturation and antigen cross-presentation, transforming the innate immune response to the tumor into systemic adaptive immunity. The synergistic effect of MHC-I upregulation and immunostimulatory cytokine secretion enhances the T cell's killing efficacy against tumor cells.

[0056] In this embodiment, the particle size of the vesicles is set to 20nm-35nm, which can effectively shorten the spatial distance between the T cells and tumor cells physically bridged on the vesicle surface, further promote the efficient formation of immune synapses, thereby enhancing the recognition and binding of T cell receptors to specific antigen peptides on the surface of tumor cells, and achieving excellent anti-tumor therapeutic effects.

[0057] In this embodiment, the STING agonist can be delivered into tumor cells and specifically accumulate in tumor tissue, avoiding premature release of the STING agonist in the bloodstream and normal tissues. Furthermore, vesicles exhibit good biocompatibility and degradability, and their metabolites are non-toxic and harmless to the human body, thus enabling the dual-antibody modified vesicle STING agonist to possess good biosafety.

[0058] In this embodiment, compared to loading a negatively charged STING agonist within the vesicle, a positively charged STING agonist within the vesicle lumen, after being released into tumor cells, exhibits better affinity for the negatively charged endoplasmic reticulum membrane within the tumor cells. This is because the STING protein is located at the endoplasmic reticulum membrane, allowing the STING agonist to accumulate more efficiently in the microenvironment surrounding the STING protein, thereby effectively activating the STING pathway and enhancing the killing effect on tumor cells.

[0059] In this embodiment, the ratio of anti-HER2 monoclonal antibody and anti-PD-1 monoclonal antibody grafted onto the vesicle surface is set to 1:(0.33-3) to achieve a dynamic balance between precise targeting of tumor cells and efficient activation of T cells. This ratio ensures both sufficient anti-HER2 monoclonal antibody density to mediate efficient targeting of vesicles to HER2 antigen on tumors and appropriate anti-PD-1 monoclonal antibody density to effectively recruit T cells and block the interaction between PD-1 antigen and PD-L1, thereby synergistically promoting the stable formation of immune synapses and achieving precise and efficient killing of tumors by the dual-antibody modified vesicle STING agonist.

[0060] In this embodiment, the particle size of the vesicles can be, for example, 20 nm, 25 nm, 30 nm, or 35 nm, or any other value between 20 nm and 35 nm. The ratio of anti-HER2 monoclonal antibody and anti-PD-1 monoclonal antibody grafted onto the vesicle surface can be, for example, 1:0.33, 1:1, 1:2, or 1:3, or any other value between 1:(0.33-3). Anti-HER2 monoclonal antibody is understood as a monoclonal antibody against HER2. In this embodiment, the anti-HER2 monoclonal antibody is preferably trastuzumab (Tra), and the anti-PD-1 monoclonal antibody is understood as a monoclonal antibody against PD-1. In other embodiments, the anti-HER2 monoclonal antibody in the dual-antibody modified vesicle STING agonist can be an antibody targeting tumor-associated antigens.

[0061] In this embodiment, two antibodies are covalently grafted onto the vesicle surface using a chemical method, forming a stable connection between the antibodies and the vesicles, thereby improving the binding strength of the antibodies on the vesicle surface. Compared to physical adsorption or simple coating methods, this method effectively avoids the problem of antibodies detaching from the vesicle surface in the complex physiological environment of the human body, reduces instability during use, and helps improve the stability and functional consistency of the dual-antibody modified vesicle STING agonist in vivo, thus enhancing the anti-tumor therapeutic effect.

[0062] In this embodiment, through endocytosis by tumor cells, most of the vesicles in the dual-antibody modified vesicle STING agonist are endocytosed into the tumor cells, while the remaining vesicles remain localized on the surface of the tumor cells, serving as a bridge between tumor cells and T cells. This achieves a synergistic effect, mediating spatial bridging between T cells and tumor cells and promoting contact between immune cells, while simultaneously delivering and releasing the STING agonist into the tumor cells, thereby activating the STING signaling pathway.

[0063] In this embodiment, the synthesis of the amphiphilic block polymer and the functionalized amphiphilic block polymer can be found in the inventor's published patents or articles.

[0064] In a further embodiment, the STING agonist is selected as any one of diABZI, STING agonist-3, SR-717 or GNE-6468. diABZI, STING agonist-3, SR-717 or GNE-6468 can effectively activate the STING signaling pathway and have shown excellent anti-tumor effects in a variety of preclinical models.

[0065] In a further embodiment, the amphiphilic block polymer is a first hydrophilic segment-first hydrophobic segment-Ac-KD. z The first hydrophilic segment is polyethylene glycol, and the first hydrophobic segment is a random copolymer of trimethylene carbonate monomer and disulfide five-membered ring carbonate monomer. Ac is an acetyl group, and KD... z This is a negatively charged chain segment, where K is lysine, D is aspartic acid, and z represents a repeating unit. Specifically, D is aspartic acid, making KD z It is negatively charged. z can be any value from 5 to 15. The higher the value of z in this range, the longer the aspartic acid chain segment, which is more conducive to the formation of smaller vesicles, thereby shortening the spatial distance between anti-HER2 monoclonal antibodies and anti-PD-1 monoclonal antibodies on the vesicle surface and improving the therapeutic effect of anti-tumor treatment.

[0066] In a further embodiment, the second hydrophilic segment is polyethylene glycol, and the second hydrophobic segment is a random copolymer of a trimethylene carbonate monomer and a disulfide pentacyclic carbonate monomer. The formed vesicle surface has functional groups N3-, Mal-, or NHS-, which are used for grafting anti-HER2 monoclonal antibodies and anti-PD-1 monoclonal antibodies.

[0067] The first and second hydrophobic segments can specifically be polyethylene glycol-poly(trimethylene carbonate-) co -Disulfide pentacyclic carbonate), polyethylene glycol-poly(lactic acid- co -Disulfide pentacyclic carbonate) or polyethylene glycol-poly(caprolactone- co - Disulfide pentacyclic carbonate). Polyethylene glycol-poly(trimethylene carbonate- co - Disulfide pentacyclic carbonate) is abbreviated as PEG-P (TMC-DTC), polyethylene glycol-poly(lactic acid-) co - Disulfide pentacyclic carbonate) is abbreviated as PEG-P (LA-DTC), polyethylene glycol-poly(caprolactone- co - Disulfide pentacyclic carbonate (PEG-P) is abbreviated as PEG-P (CL-DTC).

[0068] In a further embodiment, the mass of the STING agonist accounts for 1%-11.2% of the total mass of the vesicles and the STING agonist, for example, it can be 1%, 4%, 8%, or 11.2%, or any value within the range of 1%-11.2%. This range ensures, on the one hand, that the vesicles have high drug loading efficiency, enabling effective enrichment of the STING agonist at the tumor site and full activation of downstream immune signaling pathways. On the other hand, it ensures that the release level of the STING agonist within the tumor is at a safe level, avoiding excessive release that could lead to potential toxicity or adverse reactions.

[0069] In a further embodiment, the dual antibody-modified vesicle STING agonist can be used in antitumor immunotherapy, where the tumor can be a malignant solid tumor, such as melanoma or colorectal cancer.

[0070] Figure 1 This is a schematic flowchart of a method for preparing a biantibody-modified vesicle STING agonist according to an embodiment of the present invention. Figure 2 This is a process flow diagram of a method for preparing a biantibody-modified vesicle STING agonist according to an embodiment of the present invention.

[0071] In a further embodiment, such as Figure 1 and Figure 2 As shown, the preparation method of the biantibody-modified vesicle STING agonist includes the following steps:

[0072] Step S100: Provide an amphiphilic block polymer and a functionalized amphiphilic block polymer;

[0073] Step S200: Prepare an organic solution containing an amphiphilic block polymer and a functionalized amphiphilic block polymer, and an acidic buffer solution containing a STING agonist. Mix the organic solution and the acidic buffer solution and then obtain the vesicular STING agonist by dialysis and ultrafiltration.

[0074] Step S300: Prepare functional group-modified anti-HER2 monoclonal antibody and functional group-modified anti-PD-1 monoclonal antibody;

[0075] In step S400, the vesicle STING agonist, anti-HER2 monoclonal antibody, and anti-PD-1 monoclonal antibody are mixed in a preset molar ratio to ensure that both anti-HER2 monoclonal antibody and anti-PD-1 monoclonal antibody are covalently grafted onto the surface of the vesicle STING agonist, thereby preparing a dual-antibody modified vesicle STING agonist.

[0076] In step S200, the STING agonist itself is not positively charged, but the basic group of the STING agonist absorbs protons in the acidic solution, making the STING agonist in the acidic buffer solution positively charged. Furthermore, while the functionalized amphiphilic block polymers assemble and crosslink to form vesicles, the STING agonist is encapsulated within the vesicle lumen. It is important to note that the vesicle STING agonist should be understood as the STING agonist encapsulated within the vesicle lumen.

[0077] In step S300, the functional groups modified on the anti-PD-1 monoclonal antibody and the anti-HER2 monoclonal antibody can be dibenzocyclooctyne (DBCO).

[0078] In step S400, through click chemistry, the DBCO groups on the anti-PD-1 monoclonal antibody and the anti-HER2 monoclonal antibody react with the N3- functional group on the surface of the vesicular STING agonist, thereby grafting the anti-PD-1 monoclonal antibody and the anti-HER2 monoclonal antibody onto the surface of the vesicular STING agonist. The preset molar ratio of the functional group (N3-, Mal-, or NHS-) of the vesicular STING agonist, the anti-HER2 monoclonal antibody, and the anti-PD-1 monoclonal antibody is set to 1:(0.17-0.5):(0.17-0.5), which can precisely control the grafting density of the two antibodies on the vesicle surface, ensuring that the dual-antibody modified vesicular STING agonist simultaneously possesses sufficient tumor targeting ability and T cell recruitment ability. The functional groups of the vesicle STING agonist and the preset molar ratio of the anti-HER2 monoclonal antibody to the anti-PD-1 monoclonal antibody can be, for example, 1:0.17:0.17, 1:0.33:0.33, 1:0.5:0.17 or 1:0.5:0.5, or any other value among 1:(0.17-0.5):(0.17-0.5).

[0079] The following detailed description uses specific embodiments and comparative examples.

[0080] Example 1

[0081] Example 1 provides a method for preparing a dual-antibody modified vesicle STING agonist, specifically including the following steps:

[0082] Step S111, providing PEG-P(TMC-DTC)-Ac-KD 10As an amphiphilic block polymer, and providing N3-PEG-P(TMC-DTC) as a functionalized amphiphilic block polymer;

[0083] Step S121, PEG-P(TMC-DTC)-Ac-KD 10 The N3-PEG-P(TMC-DTC) polymer was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 40 mg / mL to obtain PEG-P(TMC-DTC)-Ac-KD. 10 Solution and N3-PEG-P(TMC-DTC) solution, followed by PEG-P(TMC-DTC)-Ac-KD 10 The organic solution was prepared by mixing the solution and N3-PEG-P(TMC-DTC) solution at a mass ratio of 96:4. Then, 10 mg of the STING agonist diABZI powder was dissolved in 0.01 M hydrochloric acid to obtain a diABZI solution with a concentration of 1 mg / mL. Next, 900 μL of 5 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer (pH 6.8) and 300 μL of diABZI solution were mixed thoroughly to obtain an acidic buffer solution. Then, 100 μL of the organic solution was slowly added dropwise to the acidic buffer solution under stirring at 37 °C and 300 rpm. After the addition was complete, stirring was continued for 5 min, followed by incubation overnight at 37 °C. Then, dialysis was performed using a 5 mM HEPES buffer (pH 7.4) and a dialysis membrane with a molecular weight cutoff of 3.5 kDa to remove DMSO, yielding the azide-functionalized vesicle STING agonist solution to be ultrafiltered. The vesicle STING agonist was then concentrated by ultrafiltration until the concentration was approximately 20 mg / mL, thus obtaining the vesicle STING agonist (nSTING for short).

[0084] In step S131, 200 μL of Tra stock solution with a concentration of 20 mg / mL was added to 200 μL of phosphate buffer with a concentration of 10 mM and a pH of 8.5 for dilution. Then, 8.7 μL of DMSO solution (10 mg / mL) containing N-hydroxysuccinimide-tetraethylene glycol-dibenzocyclooctylene (NHS-PEG4-DBCO) was added to the solution. After thorough mixing, the solution was reacted overnight at 25°C and 120 rpm in the dark. After the reaction, the sample was washed three times using an ultrafiltration tube with a molecular weight cutoff of 10 kDa, with phosphate-buffered saline (PBS) as the medium, to remove unreacted small molecules NHS-PEG4-DBCO and DMSO, yielding functionalized Tra (tra-DBCO). The functionalized anti-PD-1 monoclonal antibody was prepared in the same way as Tra-DBCO, i.e., by reacting NHS-PEG4-DBCO and anti-PD-1 monoclonal antibody at a molar ratio of 5:1, thus preparing the functionalized anti-PD-1 monoclonal antibody (aPD-1-DBCO).

[0085] Step S141: Mix 50 μL of nSTING (20 mg / mL), 7.4 μL of Tra-DBCO (10.8 mg / mL), and 7.1 μL of aPD-1-DBCO (11.3 mg / mL) thoroughly. After the reaction is complete, dilute to 1 mL with 5 mM HEPES buffer (pH 7.4). Remove unreacted monoclonal antibody by ultracentrifugation at 4°C and 58,000 rpm for 1 h to obtain the biantibody-modified vesicle STING. The agonist (TCE-nSTING) contains vesicle surface Tra and anti-PD-1 monoclonal antibody in a 1:1 ratio, meaning that TCE-nSTING contains an average of 4 Tra and 4 anti-PD-1 monoclonal antibody. The molar ratio of Tra-DBCO, aPD-1-DBCO and the functional group N3 of the vesicle STING agonist is 0.33:0.33:1. The mass of the STING agonist diABZI accounts for 5.6% of the total mass of nSTING.

[0086] Example 2

[0087] The difference between Example 2 and Example 1 is that the mass of the STING agonist accounts for 11.2% of the total mass of the vesicles and the STING agonist.

[0088] Comparative Example 1:

[0089] The difference between Comparative Example 1 and Example 1 is that it does not include TCE-nSTING, but only PBS buffer solution.

[0090] Comparative Example 2:

[0091] The difference between Comparative Example 2 and Example 1 is that the STING agonist diABZI is not present in the vesicle lumen, so as to obtain a dual antibody modified vesicle (TCE-n for short). The ratio of Tra on the vesicle surface to anti-PD-1 monoclonal antibody in TCE-n is 4:4.

[0092] Comparative Example 3:

[0093] The difference between Comparative Example 3 and Example 1 is that it does not include vesicles, anti-PD-1 monoclonal antibody and Tra, but only the free STING agonist diABZI.

[0094] Comparative Example 4:

[0095] The difference between Comparative Example 4 and Example 1 is that the Tra modified on the vesicle surface was replaced with non-specific immunoglobulin G (IgG) to obtain the first monoantibody modified vesicle STING agonist (IP-nSTING for short).

[0096] Comparative Example 5:

[0097] The difference between Comparative Example 5 and Example 1 is that the anti-PD-1 monoclonal antibody modified on the vesicle surface was replaced with IgG to obtain a second monoclonal antibody-modified vesicle STING agonist (IT-nSTING for short).

[0098] Comparative Example 6:

[0099] Comparative Example 6 provides a vesicle STING agonist (nSTING for short), and differs from Example 1 in that it does not include steps S131 and S141 of Example 1.

[0100] Comparative Example 7:

[0101] The difference between Comparative Example 7 and Comparative Example 2 is that the average particle size of TCE-n is 250 nm.

[0102] Comparative Example 8:

[0103] Comparative Example 8 provides a vesicle (Ps) that differs from Example 1 in that it does not include Tra, the anti-PD-1 monoclonal antibody, and the STING agonist diABZI.

[0104] Comparative Example 9:

[0105] The difference between Comparative Example 9 and Comparative Example 2 is that the Tra on the vesicle surface was replaced with IgG to obtain the first monoantibody-modified vesicle (IP-n).

[0106] Comparative Example 10:

[0107] The difference between Comparative Example 10 and Comparative Example 2 is that the anti-PD-1 monoclonal antibody modified on the vesicle surface was replaced with IgG to obtain a second monoclonal antibody modified vesicle (IT-n for short).

[0108] TCE-nSTING, TCE-n, IP-nSTING, IT-nSTING, nSTING, Ps, IP-n, and IT-n are all formulations. The raw materials involved in this invention are all conventional or commercially available products, and the specific preparation operations and testing methods are conventional methods in the field.

[0109] Figure 3 The particle size distribution diagram is based on TCE-nSTING from Example 1.

[0110] Depend on Figure 3 As shown, the average particle size of TCE-nSTING is 44 nm. This smaller particle size facilitates the penetration and accumulation of TCE-nSTING in vivo and in tumor tissues, indicating that the vesicles also have a smaller particle size. This helps to shorten the spatial distance between the two antibodies on the vesicle surface, thereby bringing the targeting of tumor cells and T cells closer together, promoting the formation of immune synapses, and ultimately achieving highly efficient killing of tumor cells. Furthermore, the polydispersity index (PDI) of TCE-nSTING is less than 0.2, indicating that TCE-nSTING has a relatively uniform distribution and good dispersibility and reproducibility.

[0111] Figure 4 The TCE-nSTING, PBS, TCE-n, diABZI, IP-nSTING, and IT-nSTING in Example 1 and Comparative Examples 1 to 5 are the effects of these substances on B16F10. HER2+ STING pathway-related genes (A) interferon-β in tumor cells 、 (B) Effects of CXC motif chemokine ligand 10 and (C) interleukin-6 expression.

[0112] The study of B16F10 using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) HER2+ Changes in STING pathway-related mRNAs and B16F10 in tumor cells after treatment with different agents. HER2+ This refers to tumor cells constructed by introducing and expressing the human HER2 receptor into B16F10 mouse melanoma cells. Specifically, B16F10... HER2+ Tumor cells were distributed at 1 × 10⁻⁶ per well. 5The samples were seeded at a density of [number] units in a 12-well plate, and the corresponding formulations from Example 1 and Comparative Examples 1 to 5 were then placed in the 12-well plate. The concentrations of Tra, anti-PD-1 monoclonal antibody, and IgG in the wells of the 12-well plate were all 10 μg / mL, and the concentration of diABZI in the wells was 1 μg / mL. Finally, after incubation for 24 h, B16F10 was extracted. HER2+ Total RNA from tumor cells was analyzed by reverse transcription-real-time RT-qPCR.

[0113] like Figure 4 As shown, compared to Comparative Examples 1 to 5, the TCE-nSTING and B16F10 of Example 1... HER2+ After tumor cells are incubated, downstream genes of the STING pathway, such as interferon, are affected. - β 、 CXC motif chemokine ligand 10 (abbreviated as CXC motif chemokine ligand 10) Cxcl10 The relative mRNA expression levels of both α and β-interleukin-6 were increased, indicating that TCE-nSTING can efficiently activate the STING signaling pathway. Furthermore, Tra in IT-nSTING targets B16F10. HER2+ After tumor cells, B16F10 HER2+ Interferon-β in tumor cells Cxcl10 The relative mRNA expression level of Tra was the smallest compared to that of interleukin-6, indicating that Tra targets B16F10. HER2+ Tumor cells enhanced intracellular delivery of STING agonists and activation of the STING pathway.

[0114] Figure 5 In Example 1 and Comparative Examples 1 to 5, TCE-nSTING, PBS, TCE-n, diABZI, IP-nSTING, and IT-nSTING (A) upregulated B16F10. HER2+ A diagram showing the expression of MHC-I on the surface of tumor cells and (B) its promotion of naive T cell activation.

[0115] B16F10 was detected by flow cytometry. HER2+ Tumor cells were treated with different formulations in Example 1 and Comparative Examples 1 to 5, B16F10 HER2+ Expression levels of MHC-I molecules on the surface of tumor cells. (B16F10) HER2+ Tumor cells were distributed at 1 × 10⁻⁶ per well. 5 The B16F10 molecules were seeded at a density of 1000 μg / mL in a 12-well plate, and the formulations corresponding to Examples 1 and 5 were then placed in the 12-well plate. The total concentration of Tra and anti-PD-1 monoclonal antibody in the wells of the 12-well plate was 20 μg / mL, and the concentration of diABZI in the wells was 1 μg / mL. Finally, after incubation for 24 h, B16F10 was collected. HER2+Tumor cells were stained and tested using an anti-H-2Kb antibody labeled with phycoerythrin. The anti-H-2Kb antibody is a type of MHC-I molecule. Results are as follows... Figure 5 As shown in A, after TCE-nSTING or IT-nSTING processing, B16F10 HER2+ The expression level of MHC-I molecules on the surface of tumor cells was significantly increased, and higher than that of comparative examples 1 to 4, indicating that TCE-nSTING can precisely deliver STING agonists into tumor cells through the specific recognition of Tra, thereby enhancing the antigen presentation ability of tumor cells.

[0116] Since TCE-nSTING can activate the STING pathway in tumor cells and upregulate MHC-I expression, further research was conducted on B16F10 cells treated with different formulations. HER2+ The effect of tumor cells on promoting the activation of naïve T cells. B16F10 cells pretreated with the formulations corresponding to Examples 1 and Comparative Examples 1 to 5 for 24 h were compared. HER2+ Tumor cells, at 10 per well 5 The cells were co-cultured with unactivated T cells extracted from the spleen at a density of [number] cells for 24 hours, followed by antibody staining, and the expression of CD69 protein, an early activation marker on the T cell surface, was detected by flow cytometry. Figure 5 As shown in B, the B16F10 processed by TCE-nSTING HER2+ Tumor cells can efficiently promote T cell activation, CD8-positive cytotoxic T lymphocytes (CD8+). + Cytotoxic T Lymphocytes, abbreviated as CD8 + CD69 in T cells + The proportion was 31.6%, higher than that of comparative examples 1 to 5. Overall, this indicates that TCE-nSTING can activate the STING pathway in tumor cells and upregulate MHC-I expression on the surface of tumor cells, promoting their antigen-presenting ability and thus effectively activating T cells. Additionally, CD8... + T cells are the body's main cytotoxic immune cells, capable of specifically recognizing and eliminating tumor cells. CD69 is a type C lectin receptor, belonging to the membrane protein family. + A positive result indicates that T cells are in an early stage of activation.

[0117] Figure 6 This is the TCE-nSTING of Example 1 for B16F10 HER2+ A diagram illustrating the toxic effects on tumor cells.

[0118] The effect of TCE-nSTING on B16F10 was determined using the Cell Counting Kit-8 (CCK-8) method. HER2+Tumor cell toxicity. Specifically, B16F10... HER2+ Tumor cells were distributed at 3 × 10⁻⁶ cells per well. 3 B16F10 was seeded at a density of [number] cells per well in a 96-well plate, and TCE-nSTING from Example 1 was added to the wells. The concentration of diABZI in the 96-well plate was diluted to a range of 0.01 µg / mL to 100 µg / mL. The plates were then incubated for 24 hours, and B16F10 was detected using the CCK-8 assay. HER2+ Tumor cell survival rate.

[0119] Depend on Figure 6 As shown, TCE-nSTING exhibited a concentration-dependent inhibitory effect on tumor cell proliferation. When diABZI was at a concentration of 1 µg / mL in a 96-well plate, B16F10... HER2+ The tumor cell survival rate remained at around 85%, indicating that at the working concentration of TCE-nSTING in this experiment, diABZI itself had an effect on B16F10. HER2+ The direct anti-proliferative effect of tumor cells is limited.

[0120] Figure 7 This is a statistical graph showing the apoptosis rate of T cells killing tumor cells mediated by TCE-nSTING, PBS, TCE-n, diABZI, IP-nSTING and IT-nSTING in Example 1 and Comparative Examples 1 to 5.

[0121] Based on Figure 6 The conclusion was that the TCE-nSTING-mediated T cell killing of B16F10 cells was assessed by double staining with Annexin V and 7-aminoactinomycin D (Annexin V / 7-AAD). HER2+ The effects on tumor cells were assessed using PBS, TCE-n, diABZI, IP-nSTING, and IT-nSTING as controls. T cells derived from the spleen were extracted and resuspended in complete T cell culture medium (containing 10% FBS, 1% penicillin / streptomycin, 0.05 mM β-mercaptoethanol, 10 ng / mL interleukin-2, 10 ng / mL interleukin-7, and 0.5 μg / mL anti-CD28 antibody in RPMI 1640) at a concentration of 5 × 10⁻⁶ cells / mL. 6 T cells were seeded at a density of 1 / mL in 6-well plates. The 6-well plates were pre-coated with anti-CD3 antibody (2 μg / mL) and anti-CD28 antibody (5 μg / mL) and incubated overnight at 4°C. The cells were then cultured for 48 hours to complete activation, yielding activated T cells. Specifically, B16F10 cells labeled with carboxyfluorescein succinimidyl ester (CFSE) fluorescent dye were used. HER2+Tumor cells were distributed at 1 × 10⁻⁶ per well. 5 The B16F10 particles were densely packed in a 12-well plate, and then... HER2+ After the tumor adheres to the inner wall of the well, add 1×10 to each well. 6 Activated T cells were co-cultured, and then PBS, TCE-n, diABZI, IP-nSTING, IT-nSTING, and TCE-nSTING were added to different wells, respectively. The concentrations of Tra, anti-PD-1 monoclonal antibody, and diABZI in each well were 10 µg / mL and 1 µg / mL, respectively. After incubation for 24 h, all cells in each well were collected, stained with Annexin V / 7-AAD labeled with phycocyanin, and CFSE-positive cell populations were screened by flow cytometry and defined as B16F10. HER2+ The tumor cell population was then analyzed for Annexin V / 7-AAD staining to assess B16F10. HER2+ The level of apoptosis in tumor cells. For example... Figure 7 As shown, TCE-nSTING in Example 1 can induce 56.4% of B16F10 HER2+ Tumor cells underwent apoptosis, which was superior to that in comparative examples 1 to 5, indicating that TCE-nSTING can induce apoptosis in B16F10 cells. HER2+ Establishing an effective functional connection between tumor cells and T cells can promote the killing of tumor cells by T cells.

[0122] Figure 8 The results show the detection results of TCE-nSTING, PBS, TCE-n, diABZI, IP-nSTING and IT-nSTING enhancing T cell killing function and promoting cytokine secretion in Example 1 and Comparative Examples 1 to 5. (A) Expression of lysosome-associated membrane protein-1, (B) Expression of perforin, (C) Expression of granzyme B, (D) Secretion of interferon-γ, and (E) Secretion of tumor necrosis factor-α.

[0123] To further elucidate the underlying immunological mechanisms, flow cytometry was used to analyze the execution of B16F10 by T cells. HER2+ The expression levels of key functional markers of tumor cell killing, lysosomal-associated membrane protein-1 (LAMP-1, also known as CD107a), granzyme B, and perforin, were studied. These levels were observed in B16F10. HER2+In the tumor cell and T cell co-culture system, the formulations corresponding to Example 1 and Comparative Examples 1 to 5 were added and incubated for 24 hours. When detecting CD107a, anti-CD107a antibody was added to each group at the beginning of co-culture to label CD107a exposed on the T cell surface during degranulation during the killing process. For the detection of granzyme B and perforin, after incubation, all cells in each group, including B16F10 cells, were collected. HER2+ Tumor cells and T cells were selected, and all cells in each system were surface-stained using anti-T cell differentiation antigen CD3 and anti-T cell differentiation antigen CD8 antibodies. Afterwards, all cells in each system were fixed and permeabilized, and intracellular staining was performed using anti-granzyme B and anti-perforin antibodies. The anti-T cell differentiation antigen CD3 antibody was used to identify T cells, and the anti-T cell differentiation antigen CD8 antibody was used to differentiate CD8 cells. + T cell subsets, anti-granzyme B antibody was used to detect the expression level of granzyme B in T cells and T cell subsets, and anti-perforin antibody was used to detect the expression level of perforin in T cells and T cell subsets. Figure 8 As can be seen from A in the figure, compared with Comparative Examples 1 to 5, CD8 in Example 1 is... + CD107a in T cells + The increased proportion indicates that TCE-nSTING treatment enhances CD8. + The degranulation activity and cytotoxic killing efficacy of T cells. Figure 8 As can be seen from B in the diagram, compared to Comparative Examples 1 to 5, the perforin positivity in Example 1 was observed on CD8. + The increased proportion of T cells indicates that TCE-nSTING treatment enhances CD8. + The ability of T cells to form pores in target cell membranes. Figure 8 As shown in C, compared to Comparative Examples 1 to 5, the granzyme B positivity in Example 1 was observed in CD8. + The increased proportion of T cells indicates that TCE-nSTING treatment enhances CD8. + T cell-induced B16F10 HER2+ The ability of tumor cells to undergo apoptosis. In summary, TCE-nSTING treatment of B16F10... HER2+ Tumor cells enhanced CD8 + The killing power of T cells.

[0124] like Figure 8 As indicated by D and E in the data, enzyme-linked immunosorbent assay (ELISA) was used to detect B16F10. HER2+The supernatant of a tumor cell-T cell co-culture system was analyzed. The concentrations of interferon-γ and tumor necrosis factor-α in the supernatant treated with TCE-nSTING were both increased, and superior to comparative examples 1 to 5, indicating that TCE-nSTING enhances the anti-tumor effector function of T cells. Specifically, increased concentrations of interferon-γ and tumor necrosis factor-α generally indicate a higher activation state of T cells. Therefore, TCE-nSTING can physically bridge B16F10... HER2+ Simultaneous targeted delivery of diABZI to B16F10 tumor cells and T cells. HER2+ Within tumor cells, it promotes the activation of the STING pathway and antigen presentation, thereby enhancing the killing effect of T cells on B16F10. HER2+ Tumor cells.

[0125] Figure 9 B16F10 treated with TCE-nSTING, PBS, TCE-n, diABZI, IP-nSTING, and IT-nSTING in Example 1 and Comparative Examples 1 to 5 HER2+ The indirect activation of dendritic cells and promotion of T cell activation by tumor cell culture supernatant; (A) representative flow cytometry and (B) statistical analysis of maturation of mouse bone marrow-derived dendritic cells; (C) MHC-I expression level on the surface of dendritic cells; (D) interleukin-12, (E) interleukin-6 and (F) tumor necrosis factor-α; (G) activation of dendritic cells with naïve CD8+. + The effect of T cells.

[0126] Given that TCE-nSTING can promote T cell killing of B16F10 by physically bridging and activating the tumor STING pathway. HER2+ Tumor cells were further investigated to determine the mechanism of TCE-nSTING through B16F10. HER2+ The ability of tumor cells to indirectly activate dendritic cells (DCs) through paracrine effects was investigated. First, bone marrow-derived dendritic cells (BMDCs) were extracted from mouse bone marrow. Specifically, leg bones of C57BL / 6J mice were aseptically collected, and the bone marrow cavity was washed with PBS to obtain a single-cell suspension. Cells were collected by centrifugation, and erythrocytes were lysed using erythrocyte lysis buffer. The resulting cells were then resuspended in BMDC induction medium and cultured for 7 days. This medium consisted of RPMI 1640 basal medium supplemented with 10% fetal bovine serum, 1% penicillin / streptomycin, 0.05 mM β-mercaptoethanol, and 20 ng / mL recombinant mouse granulocyte-macrophage colony-stimulating factor. B16F10... HER2+Tumor cells were distributed at 1 × 10⁻⁶ per well. 5 The antibodies were seeded at a density of 10 μg / mL in 12-well plates, and the corresponding formulations were added. The plates were incubated for 24 h. The concentrations of Tra, anti-PD-1 monoclonal antibody, and IgG in each well were 10 μg / mL, and the concentration of diABZI in each well was 1 μg / mL. Finally, the supernatant from each well was collected and mixed with BMDCs at a ratio of 1 × 10⁻⁶ per well. 6 The cells were incubated for 24 hours, and then the maturation status and antigen presentation capacity of the DCs were detected by flow cytometry.

[0127] Depend on Figure 9 From A to C, we can see that CD80 in BMDCs + (Differentiation antigen 80 positive) CD86 + The proportion of (differentiation antigen 86 positive) increased from 14.3% in Comparative Example 1 to 29.9% in Example 1, and the expression of MHC-I molecules on the surface of BMDCs in Example 1 was upregulated by 2.0-fold compared to Comparative Example 1, indicating that TCE-nSTING treated B16F10 HER2+ The supernatant from tumor cells effectively promoted the maturation of BMDCs. Figure 9 As shown in D to F, compared with comparative examples 1 to 5, the levels of interleukin-12, interleukin-6, and tumor necrosis factor-α secreted by BMDCs were all increased in the TCE-nSTING treatment group. Figure 9 As can be seen from G in the figure, compared with Comparative Examples 1 to 5, the CD8 treated in Example 1... + CD69 in T cells + The increased proportion indicates that TCE-nSTING can efficiently activate T cells through mature DCs. In summary, by Figure 9 It is known that TCE-nSTING can effectively stimulate B16F10 HER2+ Tumor cells release immune-active factors and efficiently drive DC maturation through the paracrine pathway, activating T cells and thereby enhancing the synergistic anti-tumor effect between innate and adaptive immunity.

[0128] Figure 10 It is subcutaneous B16F10 HER2+ After melanoma mice were injected via the tail vein with fluorescently labeled TCE-nSTING, diABZI, IP-nSTING, IT-nSTING, and nSTING (Examples 1 and 3 to 6), respectively, for 8 hours, (A) in vitro fluorescence imaging images of major tissues and (B) drug distribution of diABZI in each tissue.

[0129] To evaluate the in vivo targeting and biodistribution of TCE-nSTING, a subcutaneous B16F10 assay was established. HER2+Mouse melanoma model. To establish the model, 1×10 6 B16F10 HER2+ Mouse melanoma cells were resuspended in 50 µL of PBS buffer containing 50% Matrigel and subcutaneously inoculated above the left hind leg of C57BL / 6J mice. Examples 1 and Comparative Examples 4 to 6 were labeled with Cyanine 5 (Cy5), respectively. Cy5 was mixed with diABZI from Comparative Example 3 to obtain TCE-nCy5 / STING, IP-nCy5 / STING, IT-nCy5 / STING, nCy5 / STING, and Cy5 / diABZI, respectively, until the tumor volume reached approximately 100 mm². 3 -150mm 3 The experiment began with tail vein injections of 200 μL each of TCE-nCy5 / STING, nCy5 / STING, IP-nCy5 / STING, IT-nCy5 / STING, and Cy5 / diABZI. The Cy5 dose was 0.6 μg / mouse, the Tra, anti-PD-1 monoclonal antibody, and IgG doses were all 1 mg / kg, and the diABZI dose was 3 mg / kg. Eight hours after injection, mice were sacrificed, and their hearts, livers, spleens, lungs, kidneys, tumors, and lymph nodes were collected for in vitro imaging using near-infrared fluorescence imaging. Cy5 appeared red in the near-infrared fluorescence imaging. Figure 10 As shown in Figure A, TCE-nCy5 / STING showed the highest enrichment level at the tumor site, with a fluorescence signal significantly higher than that of comparative examples 3 to 6, demonstrating a higher enrichment capacity at the tumor site and achieving precise delivery to the tumor microenvironment.

[0130] To further investigate the distribution of diABZI in mice, after imaging, each organ of each mouse was weighed and homogenized in PBS solution containing 20 mM dithiothreitol. The homogenate was then incubated overnight at 37°C and 200 rpm with shaking to release the diABZI drug, yielding a homogenate. The homogenate was then acidified with 0.1% hydrochloric acid and extracted overnight with ethyl acetate. The supernatant organic phase was collected by centrifugation and vacuum dried. Subsequently, it was reconstituted with the mobile phase for high-performance liquid chromatography (HPLC), filtered, and subjected to quantitative analysis. Figure 10 As shown in B, the content of the STING agonist diABZI in the tumor tissue of the TCE-nCy5 / STING group was higher than that of the free Cy5 / diABZI group, the nCy5 / STING group, and the IP-nCy5 / STING group, with an increase of more than 5 times, indicating that TCE-nCy5 / STING can efficiently target and deliver the STING agonist to the tumor site.

[0131] Figure 11Examples 1, 2, 1, 2, 4, and 5 are subcutaneous B16F10. HER2+ In vivo antitumor efficacy in mouse models: (A) Individual treatment flowchart, (B) average tumor growth curve of mice in different treatment groups, (C) tumor growth curve of each mouse, (D) mouse survival curve and (E) weight change.

[0132] Subcutaneous B16F10 HER2+ A mouse melanoma model was used to evaluate the in vivo antitumor efficacy of TCE-nSTING, and the model was established using the same method as the model described above. The tumor volume in mice reached 50 mm². 3 -100mm 3 Day 0 was recorded as the start date, and mice were randomly divided into 6 groups, each containing five mice. On days 0, 3, 6, and 9, each group of mice was injected via the tail vein with the formulations corresponding to Examples 1, 2, 1, 2, 4, and 5, respectively. The doses of Tra, anti-PD-1 monoclonal antibody, and IgG were all 1 mg / kg. Results are as follows: Figure 11 As shown in Figures A to D, tumors in the PBS group mice grew rapidly, with a median survival of only 13 days. The TCE-n group, the single-target IP-nSTING group, and the IT-nSTING group could, to some extent, delay tumor growth and prolong the survival of mice, indicating that both TCE-n-mediated physical bridging and single-target delivery of STING agonists can produce good anti-tumor effects. The TCE-nSTING group exhibited a more efficient tumor suppression effect, with superior survival benefits compared to the TCE-n group, IP-nSTING group, and IT-nSTING group; one mouse even experienced complete tumor regression (e.g., ...). Figure 11 C in the middle), long-term survival (such as C) Figure 11 (D in the original text). Furthermore, compared to Example 1, Example 2 showed a further enhanced tumor suppression effect, with a median survival of 53.5 days in mice and 50% of mice achieving complete remission; no recurrence was observed during the 90-day observation period. (As shown in the original text). Figure 11 As shown in E, the body weight of mice in each group remained relatively stable during the administration of the drug formulation, and no deaths due to toxicity occurred, indicating that IP-nSTING, IT-nSTING, TCE-n, and TCE-nSTING all have biocompatibility.

[0133] Figure 12 The effects of TCE-nSTING, PBS, and TCE-n on (A) the tumor immune microenvironment and (B) the immune cell phenotype in the spleen, corresponding to Examples 1, 1, and 2, are described. Figure 13 It is subcutaneous B16F10 HER2+After mice were treated with TCE-nSTING, PBS, and TCE-n as described in Examples 1, 1, and 2, the following changes were observed in the draining lymph nodes: (A) maturation of dendritic cells, (B) expression of MHC-I on the surface of dendritic cells, and (C) CD4 expression. + T and (D)CD8 + T cell activation status Figure 14 It is subcutaneous B16F10 HER2+ Mice treated with TCE-nSTING, PBS, and TCE-n as described in Examples 1, 1, and 2, respectively, had (A)CD8 levels in their peripheral blood. + The number of T cells and the levels of (B) interferon-β, (C) interferon-γ and (D) interleukin-6 cytokines in the serum.

[0134] Subcutaneous B16F10 HER2+ The effect of TCE-nSTING on regulating anti-tumor immunity in mice was further evaluated using a mouse melanoma model. Mice were inoculated with B16F10... HER2+ After melanoma was induced in mice, the tumor volume reached approximately 100 mm in the mice around day 9. 3 -150mm 3 Mice were randomly assigned to three groups to begin the treatment experiment. They were injected via tail vein with TCE-nSTING, PBS, and TCE-n (Examples 2, 1, and 2), one injection every three days for a total of three injections. The doses of Tra, anti-PD-1 monoclonal antibody, and IgG were all 1 mg / kg, and the diABZI dose was 1.5 mg / kg. Twenty-four hours after the last administration, mice were sacrificed, and peripheral blood, tumors, spleen, and lymph nodes were collected and prepared into single-cell suspensions for staining and flow cytometry analysis. Peripheral blood was centrifuged to collect serum, and the concentrations of interferon-β, interferon-γ, and interleukin-6 in the serum were then detected using an enzyme-linked immunosorbent assay (ELISA) kit.

[0135] like Figure 12 As shown in A, compared to Comparative Example 1, the TCE-nSTING treatment group in Example 1 showed reduced tumor cell proliferation activity and Ki-67 + The proportion of mature dendritic cells (DCs) decreased significantly, while the expression of MHC-I molecules on their surface was upregulated by 3.0-fold, indicating stronger immunogenicity and superior efficacy compared to the TCE-n group. Furthermore, compared to Comparative Example 1, the TCE-nSTING treatment group in Example 1 showed a 3.0-fold increase in the number of mature DCs within the tumor tissue and a 2.8-fold upregulation of MHC-I, providing antigen presentation support for the sustained amplification of T cell responses. After TCE-nSTING treatment, CD8+ expression in the tumor site increased. + The percentage of T cells was 5.7 times higher in the PBS group and superior to the control group (Rule 2). CD8 + PD-1 in T cells+ Ki67 + The proportion of T cells increased from 12.4% in the PBS group to 23.9% in the TCE-nSTING treatment group, indicating that T cells not only increased in number at the tumor site but were also in an active proliferative state. To assess the functional status of infiltrating T cells, their exhaustion phenotype was further analyzed. T cell exhaustion is a dynamic differentiation process that can produce a precursor exhaustion subset (PD-1) that can still respond to treatment. + TIM-3 - ) and the dysfunctional, treatment-resistant end-exhaustion subgroup (PD-1) + TIM-3 + CD8 + Ki67 in T cells + The proportion increased from 24.8% in the PBS group to 43% in the TCE-nSTING treatment group, indicating that CD8+ in the tumor sites of mice treated with TCE-nSTING increased. + T cells not only proliferate actively, but their differentiation pathways are also effectively directed towards a functional precursor depletion state. PD-1 + TIM-3 - The subgroup proportion increased from 7.4% in the PBS group to 12.4% in the TCE-nSTING treatment group, and PD-1 + TIM-3 + The final exhaustion rate decreased to 20.6%.

[0136] like Figure 12 As shown in B, in the spleen, the CD8+ in the TCE-nSTING treatment group + T cell activation (CD69), proliferation (Ki67), and killing ability (granulase B, perforin) all reached their highest levels. Simultaneously, CD4... + Within T cell subsets, TCE-nSTING suppressed the proportion of regulatory T cells with immunosuppressive functions and upregulated activation levels (CD69). TCE-nSTING also induced high activation of splenic T cells (PD-1). + At the same time, it effectively improved PD-1 + TIM-3 - The subpopulation ratio was adjusted, and the occurrence of terminal exhaustion was effectively suppressed.

[0137] In draining lymph nodes, TCE-nSTING promotes the maturation of dendritic cells (DCs) and enhances their antigen-presenting capacity. Figure 13 (A and B in the text), and efficiently initiated the initial activation of T cells ( Figure 13 (C and D in the text). For example... Figure 14 As shown in A, CD8+ levels in peripheral blood of the TCE-nSTING group +The number of T cells increased 2.0 times compared to the PBS group, indicating that TCE-nSTING can significantly enhance the systemic expansion and mobilization of cytotoxic T cells, which is beneficial for improving the body's ability to recognize and kill tumor cells. Figure 14 As shown in B to D, compared with the PBS group, the serum levels of key cytokines interferon-β, interferon-γ, and interleukin-6 in the TCE-nSTING group were increased by 6.4 times, 10.5 times, and 8.9 times, respectively, indicating that both the body's innate and adaptive immunity were efficiently activated.

[0138] Figures 12 to 14 A "+" sign indicates that the corresponding cells express the marker molecule, i.e., positive, while a "-" sign indicates that the corresponding cells do not express the marker molecule, i.e., negative. For example, CD80 + CD86 + PD-1 is commonly used to characterize mature dendritic cells. + Ki-67 + This suggests that T cells are in a state of proliferation and activation, while PD-1 + Tim-3 - With PD-1 + Tim-3 + These correspond to precursor depletion and terminal depletion T cell subsets, respectively.

[0139] Figure 12 In this study, CD11c serves as a marker for dendritic cells, CD80 and CD86 are co-stimulatory molecules that stimulate T cell activation, CD69 is an activation marker reflecting early T cell activation, Tim-3 is an immune checkpoint molecule related to T cell exhaustion, and Ki-67 is a cell proliferation marker.

[0140] Figure 13 In this study, CD11c serves as a marker for dendritic cells, CD80 and CD86 are co-stimulatory molecules that stimulate T cell activation, and CD69 is an activation marker reflecting early T cell activation.

[0141] Figure 14 In China, CD45 is a common leukocyte antigen. + Cells typically represent the total white blood cell population; CD3 is a marker for T cells, CD8 is a marker for cytotoxic T cells, and CD3... + CD8 + Cells are generally defined as CD8 cells + T cells.

[0142] Figure 15 Mice cured by TCE-nSTING (A) in Example 2 were re-inoculated with B16F10. HER2+ A schematic diagram of tumor cells and (B) mice cured by treatment in Example 2, re-inoculated with B16F10.HER2+ Tumor cells and healthy mice inoculated with B16F10 HER2+ Tumor growth curves of tumor cells, CD8+ in mouse peripheral blood + Quantitative analysis of the proportions of (C) effector memory T cells, (D) central memory T cells, and (E) representative flow cytometry of memory T cells, CD8+ in mouse spleen. + Quantitative analysis of the proportions of (F) effector memory T cells, (G) central memory T cells, and (H) representative flow cytometry of memory T cells.

[0143] like Figure 15 As shown in A, to further evaluate the antitumor immune memory effect in mice, B16F10 was used. HER2+ Ninety days after the onset of treatment, tumor-bearing mice were subcutaneously injected again with B16F10 above the right hind leg of mice cured by TCE-nSTING as described in Example 2. HER2+ Tumor cells (1×10) 6 (each tumor), observe and record its growth. For example... Figure 15 As shown in B, healthy mice were inoculated with the same number of B16F10. HER2+ Tumor cells served as the control group. Compared with the control group, cured mice were re-inoculated with B16F10. HER2+ The tumor cells were completely protected afterward, and no signs of tumor regeneration or recurrence were observed.

[0144] B16F10 revaccination HER2+ On day 20 after tumor cell inoculation, all mice were sacrificed and spleens and peripheral blood were collected. Changes in memory T cell subsets were analyzed by flow cytometry. Figure 15 In this context, E represents peripheral blood CD8. + T cell memory T cell populations Figure 15 The H in the text represents the spleen's CD8+. + T cell memory T cell populations Figure 15 The values ​​shown in the upper right quadrant of E and H represent the central memory T cells in CD8. + The percentage of T cells, with the values ​​shown in the lower right quadrant representing effector memory T cells in CD8... + The percentage of T cells. For example... Figure 15 As shown in C to H, compared with the control group mice, the peripheral blood and spleen CD8 levels of the TCE-nSTING group mice were significantly higher. + The proportions of effector memory T cells and central memory T cells were significantly increased, with the increase in effector memory T cells being more pronounced, reaching 35.7% and 20.8% in peripheral blood and spleen, respectively. Overall, this indicates that TCE-nSTING can not only promote CD8... +T cells proliferate, activate, and exert their killing function, and can also inhibit their terminal depletion, expand the effector memory T cell population, and induce a potent anti-tumor immune memory effect.

[0145] Figure 15 CD44 is a surface marker molecule associated with T cell activation and memory differentiation, while L-selectin is an adhesion molecule that mediates lymphocyte homing to lymph nodes.

[0146] Figure 16 TCE-nSTING, PBS, and TCE-n in Example 1, Comparative Example 1, and Comparative Example 2 were used in CT-26 HER2+ -The antitumor efficacy of Luc colorectal cancer in a mouse model of metastatic colorectal cancer, including (A) bioluminescence images and semi-quantitative analysis, (B) survival curves and (C) changes in body weight of different groups of mice.

[0147] To further verify the antitumor efficacy of TCE-nSTING, CT-26 was injected via the tail vein. HER2+ -Luc cell suspension (4 × 10⁻⁶ per mouse) 5 A colon cancer lung metastasis mouse model was constructed in BALB / c mice using [number of cells] for evaluation. The day of inoculation was designated as day 0. On days 3, 6, 9, and 12, mice were injected via the tail vein with TCE-nSTING, PBS, and TCE-n (from Examples 1, 1, and 2, respectively). The doses of Tra and anti-PD-1 monoclonal antibody were 1 mg / kg, and the dose of diABZI was 1.5 mg / kg. From day 5 onwards, tumor growth was monitored every 10 days using bioluminescence imaging, and mouse body weight was recorded every 3 days. Figure 16 As shown in Figure A, the bioluminescent signal originating from tumor cells in the PBS group mice increased rapidly, with a median survival of 39.5 days. TCE-n treatment significantly inhibited tumor cell proliferation, with the bioluminescent signal in 50% of the mice returning to near background levels. The TCE-nSTING group showed excellent tumor suppression, with the bioluminescent signal gradually weakening after administration; by day 55, no significant bioluminescent signal was observed in 5 out of 6 mice. Figure 16 As shown in B, TCE-nSTING treatment prolonged the survival time of mice, improved the cure rate, with 83% of mice achieving complete cure and remaining healthy at 112 days. Figure 16 As shown in C, the body weight of mice in each group remained relatively stable during the treatment period, and no deaths due to toxicity occurred.

[0148] Figure 17 The diagram shows the particle size distribution (A), STING agonist loading (B), and STING agonist release (C) of nSTING in Comparative Example 6.

[0149] The particle size and particle size distribution of nSTING were determined by DLS. Figure 17 As shown in section A, the nSTING particle size exhibits a unimodal distribution with an average particle size of 35 nm and a PDI less than 0.1, indicating a narrow particle size distribution and good uniformity of the vesicles. A portion of nSTING was taken, and 20 mM dithiothreitol was added to fully disrupt the disulfide cross-linking structure of the vesicles in nSTING. The diABZI content in nSTING was determined by high-performance liquid chromatography (HPLC). Encapsulation efficiency refers to the percentage of STING agonist actually encapsulated inside the vesicles compared to the initial total STING agonist feed. Figure 17 As shown in section B, when the theoretical drug loading of diABZI is between 2 wt.% and 7 wt.%, the actual drug loading increases linearly. Although there is a small amount of diABZI loss during the preparation process, the encapsulation efficiency is above 80%, indicating that the vesicles have excellent loading capacity and process stability. To further verify the stability and responsive release behavior of nSTING under different physiological environments, a normal physiological environment without glutathione (GSH) and a simulated tumor environment containing 10 mM GSH were simulated, and the cumulative release rate of diABZI in nSTING was monitored. Figure 17 As shown in C, nSTING exhibits excellent stability in a normal physiological environment, with drug leakage of less than 20% within 24 hours. However, in a simulated tumor cell environment, the disulfide bonds in the vesicle structure undergo reducing breakage, leading to the disintegration of the vesicle skeleton and triggering the rapid release of the loaded drug diABZI. Drug leakage reaches more than 85% within 12 hours, indicating that nSTING can achieve precise trigger-based release of the drug diABZI at the lesion site, indirectly demonstrating that TCE-nSTING also has the same efficacy.

[0150] Figure 18 It is PEG-P(TMC-DTC)-Ac-KD in Example 1 10 (A) Structural formula and (B) 1H NMR spectrum and (C) 1H NMR spectrum of Ps in Comparative Example 8.

[0151] Depend on Figure 18 From A, we can know that KD 10 It contains aspartic acid and lysine. The aspartic acid side chain contains a carboxyl group, which ionizes to -COO under physiological conditions. - Lysine carries a negative charge, and its side chain contains an ε-amino group, giving it a positive charge under physiological conditions. However, aspartic acid is more abundant than lysine, which can lead to KD... 10 It becomes a negative charge link. (By...) Figure 18 As shown in B, PEG-P(TMC-DTC)-Ac-KD can be observed in the hydrogen nuclear magnetic resonance spectrum. 10 The characteristic peaks corresponding to each chain segment, but Figure 18 As indicated by C in the diagram, the characteristic peak labeled b belongs to the PEG chain segment. The proton NMR spectrum primarily detects the characteristic signal of the outer PEG layer of Ps, but does not detect KD. 10 The characteristic peaks indicate that KD 10 The chain segment is located within the cavity of Ps, thus making the cavity of Ps negatively charged. Since the TCE-nSTING of Examples 1 and 2 includes Ps, it indicates that the vesicle cavities in the TCE-nSTING obtained in Examples 1 and 2 are negatively charged.

[0152] Figure 19 These are the potential diagrams of TCE-n (Comparative Example 2) and Ps (Comparative Example 8).

[0153] Depend on Figure 19 It can be seen that the zeta potentials of TCE-n in Comparative Example 2 and Ps in Comparative Example 8 are both negative, indicating that TCE-n and Ps are negatively charged as a whole. Ps and TCE-n are part of the structure of TCE-nSTING in Example 1, reflecting that the vesicle lumen of TCE-nSTING is negatively charged and can load a positively charged STING agonist in the vesicle lumen through electrostatic interaction.

[0154] Figure 20 This is the particle size distribution diagram of TCE-n in Comparative Example 2.

[0155] Depend on Figure 20 The average particle size of TCE-n is approximately 32 nm, indicating that TCE-n remains within the nanoscale range, with a small size and no significant aggregation or excessive growth. The PDI of TCE-n is less than 0.1, indicating that the size distribution of TCE-n is highly uniform and the system has good dispersibility.

[0156] Figure 21 TCE-n-mediated T cell killing of B16F10 in Comparative Examples 2 and 7 HER2+ The condition of tumor cells.

[0157] The effects of TCE-n particles of different sizes on T cell-mediated tumor cell killing were investigated using Annexin V / 7-AAD double staining and flow cytometry. CFSE-prelabeled B16F10... HER2+ Cells were co-cultured with activated T cells at a ratio of 1:10, and incubated with the corresponding TCE-n for Comparative Examples 2 and 7 for 24 h, respectively, with the total antibody concentration kept constant at 20 µg / mL. After incubation, the cells were washed, stained with Annexin V / 7-AAD, and analyzed by flow cytometry.

[0158] like Figure 21As shown, TCE-n with different particle sizes in Comparative Example 2 and Comparative Example 7 were compared. Compared with Comparative Example 7, TCE-n in Comparative Example 2 induced tumor cell apoptosis more effectively. This indicates that the smaller particle size of the vesicles shortened the spatial distance between the two antibodies in Comparative Example 2, which is conducive to the formation and stability of immune synapses, thereby enhancing the killing effect on tumor cells. This shows that the smaller particle size vesicles used in TCE-nSTING help to enhance the killing effect on tumor cells.

[0159] Figure 22 It is (A)B16F10 HER2+ Flow cytometry analysis of tumor cells and (B)T cells after incubation for 4 h with fluorescently labeled PBS, TCE-n, Ps, IP-n, and IT-n of comparative examples 1, 2, 8 to 10, respectively; (C)B16F10 HER2+ Confocal laser scanning microscopy images of tumor cells and (D)T cells after incubation with fluorescently labeled TCE-n (component 2) and Ps (component 8) for 4 h. The fluorescently labeled TCE-n (component 2) is in (E)B16F10. HER2+ Internalization and surface binding in tumor cells and (F)T cells.

[0160] To investigate the ability of TCE-nSTING to target and bind to T cells and tumor cells, Cy5 was used to fluorescently label TCE-n (Comparative Example 2), Ps (Comparative Example 8), IP-n (Comparative Example 9), and IT-n (Comparative Example 10) to obtain TCE-nCy5, nCy5, IP-nCy5, and IT-nCy5, respectively. PBS from Comparative Example 1 was mixed with Cy5, and the results were analyzed by flow cytometry and confocal laser scanning microscopy (CLSM). Specifically, B16F10... HER2+ Cells or T cells at 1×10 5 Cells were seeded at a density of [number] cells / well in 12-well plates and co-incubated for 4 hours with nCy5, IP-nCy5, IT-nCy5, and TCE-nCy5 (Cy5: 50 nM), respectively, with PBS as a blank control. After washing, the mean fluorescence intensity (MFI) of the cells was quantitatively analyzed by flow cytometry. Figure 22 As shown in A, compared to nCy5, TCE-nCy5 and B16F10 HER2+ The binding amount to tumor cells increased 3.2 times. For example... Figure 22 As shown in Figure B, TCE-nCy5 binds to T cells 3.6 times more than nCy5. In contrast, when IgG is used instead of anti-PD-1 monoclonal antibodies or Tra, IP-nCy5 and IT-nCy5 can only target one type of cell. For example, compared with TCE-nCy5, IT-nCy5 modified with Tra and IgG binds to B16F10 cells more effectively.HER2+ The binding amount to tumor cells was comparable, but the binding amount to T cells was significantly reduced, and comparable to that of nCy5.

[0161] Further investigation was conducted using CLSM to examine the relationship between TCE-nCy5 and T cells and B16F10. HER2+ Tumor cell binding was assessed. After 4 hours of incubation, cells were fixed with paraformaldehyde and labeled with WGA-AF488 (0.2 µM) and DAPI (15 µM), respectively, for T cells or B16F10 cells. HER2+ The cell membrane and nucleus of tumor cells. (Example) Figure 22 As shown in C and D, compared to nCy5, B16F10 treated with the TCE-nCy5 group... HER2+ Tumor cells ( Figure 22 C in the middle) and T cells ( Figure 22 Both D) showed effectively enhanced and dense red fluorescence signals, indicating that TCE-n can precisely target T cells and B16F10. HER2+ Tumor cells. Specifically, Cy5 is a far-red fluorescent dye that emits red fluorescence upon excitation, used to trace the distribution and localization of vesicles. As seen in the overlay image, the red fluorescent signal exhibits extremely high spatial co-localization with the green tumor cell membrane boundary, indicating that TCE-nCy5 can specifically anchor to B16F10 cells. HER2+ On the surface of tumor cells and T cells, it crosses the cell membrane barrier and enters B16F10 via a highly efficient endocytic pathway. HER2+ Intracellular tumor cells. Furthermore, the fluorescence intensity of Cy5 in tumor cells incubated with TCE-nCy5 was significantly higher than that in tumor cells incubated with nCy5, which is highly consistent with the aforementioned quantitative analysis results from flow cytometry, indicating that TCE-n can accurately target T cells and B16F10. HER2+ Tumor cells.

[0162] Then, the TCE-n in B16F10 was quantitatively analyzed by trypan blue fluorescence quenching experiment. HER2+ Internalization and surface binding in cells and T cells. Trypan Blue (TB), as a non-membrane permeability quencher, can selectively eliminate B16F10. HER2+ The Dil / FITC signal bound to the surface of tumor cells effectively distinguishes extracellular and intracellular vesicle signals.

[0163] Specifically, B16F10 HER2+ Tumor cells or T cells at 1×10 5Cells were seeded at a density of [number] cells / well in 12-well plates and incubated for 4 hours with either Dil-labeled TCE-n or FITC-labeled TCE-n. After washing, an equal volume of 0.4% trypan blue solution was added to the resuspended cells, and the cells were immediately tested. Figure 22 As shown in E, B16F10 HER2+ After TB treatment, the Dil fluorescence signal of tumor cells decreased significantly. Calculations showed that approximately 80% of TCE-n was internalized, indicating that TCE-n can efficiently recognize and bind to tumor cells, and that most of it can enter the cell via endocytosis. Figure 22 As shown in F, for T cells, the fluorescence signal was significantly reduced after trypan blue treatment, but there was no significant difference compared with untreated blank T cells, indicating that TCE-n mainly binds to the T cell membrane surface and does not exhibit significant internalization behavior. TCE-n in B16F10 HER2+ Tumor cells and T cells exhibit different internalization kinetics, B16F10 HER2+ The efficient endocytosis of TCE-n by tumor cells and the partial retention of TCE-n on its surface enable TCE-n to bridge T cells and B16F10. HER2+ Tumor cells can also ensure the precise delivery of the STING agonist diABZI and the activation of the cGAS-STING pathway, thereby effectively reshaping tumor immunogenicity. Furthermore, the surface binding of T cells can effectively bridge B16F10... HER2+ It exerts PD-1 blocking efficacy on tumor cells and T cells, and on the other hand, it can reduce the effect of STING agonists on T cells. This mechanism also indirectly demonstrates that TCE-nSTING has the same working principle, that is, TCE-nSTING can also precisely bridge to B16F10. HER2+ It enters the surface of tumor cells and T cells, and crosses the tumor cell membrane barrier via a highly efficient endocytic pathway to reach B16F10. HER2+ Inside tumor cells.

[0164] Figure 22 C and D in the formula represent DAPI, a blue fluorescent nuclear dye used to label the cell nucleus, and WGA-AF488, a green fluorescent cell membrane dye used to label cell membrane structures.

[0165] Figure 22 In the E, Dil is a lipophilic fluorescent dye used to track the binding and internalization of TCE-n in tumor cells. "+" indicates that the corresponding TCE-nDil or 0.4% trypan blue was added in the experiment, and "-" indicates that the corresponding TCE-nDil or 0.4% trypan blue was not added in the experiment. Figure 22In F, FITC stands for fluorescein isothiocyanate, used to detect the binding of TCE-n on the surface of T cells and for quantitative fluorescence analysis. "+" indicates that the corresponding TCE-nFITC or 0.4% trypan blue was added in the experiment, and "-" indicates that the corresponding TCE-nFITC or 0.4% trypan blue was not added in the experiment.

[0166] Figure 23 The fluorescently labeled TCE-n, Ps, IP-n, and IT-n of Comparative Examples 2, 8, to 10 are physically bridged with B16F10. HER2+ Laser confocal scanning microscopy images of tumor cells and T cells.

[0167] Cy5 was used to fluorescently label TCE-n (Comparative Example 2), Ps (Comparative Example 8), IP-n (Comparative Example 9), and IT-n (Comparative Example 10) to obtain TCE-nCy5, nCy5, IP-nCy5, and IT-nCy5, respectively. The bridging of B16F10 by TCE-nCy5 was evaluated using CLSM. HER2+ Effects on tumor cells and T cells. B16F10 HER2+ Tumor cells were labeled with 1 µM Dil fluorescent dye and cultured adherently at room temperature, while CD8+ was added. + T cells were labeled with 5 µM DiO fluorescent dye, which turned green upon excitation at room temperature. The two cell lines were then co-cultured at a 1:10 ratio, incubated with TCE-nCy5, nCy5, IP-nCy5, and IT-nCy5 for 4 h, respectively. The final Cy5 concentration was 3 μM. nCy5, IP-nCy5, and IT-nCy5 served as controls. Figure 23 It can be seen that in B16F10 HER2+ In a co-culture system of tumor cells and T cells, the addition of TCE-nCy5 can alter the pseudo-blue color of B16F10. HER2+ Tumor cells adhered tightly to green T cells, and a distinct red vesicle signal was observed at the interface. However, in the single-target (IP-nCy5, IT-nCy5) or non-targeted nCy5 control groups, almost no T cells were observed. This indicates that TCE-n can not only target and bind to B16F10... HER2+ Tumor cells and T cells can also be effectively bridged, which indirectly proves that TCE-nSTING has the same working principle.

[0168] Figure 24 This is a diagram illustrating the mechanism of TCE-nSTING in treating tumor cells in Example 1.

[0169] In short, by Figure 24It can be seen that TCE-nSTING induces tumor cell apoptosis in three ways. Firstly, after TCE-nSTING bridges tumor cells and T cells via Tra and anti-PD-1 monoclonal antibodies, it relieves PD-1-mediated immunosuppression and promotes CD8 cell growth. + T cell activation upregulates the expression levels of granzyme B, perforin, interferon-γ, and tumor necrosis factor-α, enhancing the killing effect of T cells on tumor cells. Secondly, TCE-nSTING releases STING agonists into tumor cells. The free STING agonist specifically binds to STING protein located on the endoplasmic reticulum, activating the STING signaling pathway. Activated STING further recruits and activates the TBK1-IRF3 signaling axis. Subsequently, phosphorylated transcription factor IRF3 translocates into the nucleus, upregulating MHC-I to activate naive T cells, enhance tumor antigen presentation, and promote CD8+ expression. + T cells recognize and eliminate tumor cells. Thirdly, tumor antigens released during tumor cell apoptosis are taken up by dendritic cells, promoting their maturation and subsequently upregulating the expression levels of interleukin-6, interleukin-12, and tumor necrosis factor-α. Mature dendritic cells activate T cells, further amplifying the immune response and forming a sustained anti-tumor immune cycle. Through these three synergistic effects, TCE-nSTING can efficiently kill tumor cells.

[0170] Therefore, those skilled in the art should recognize that although numerous exemplary embodiments of the present invention have been shown and described in detail herein, many other variations or modifications conforming to the principles of the present invention can be directly determined or derived from the disclosure of the present invention without departing from the spirit and scope of the invention. Thus, the scope of the present invention should be understood and construed as covering all such other variations or modifications.

Claims

1. A biantibody-modified vesicle STING agonist, characterized in that, include: The vesicles are formed by cross-linking functionalized amphiphilic block polymers and amphiphilic block polymers containing negatively charged segments. The vesicles have negatively charged cavities and a particle size of 20 nm-35 nm. The functionalized amphiphilic block polymers are functional groups-second hydrophilic segments-second hydrophobic segments, wherein the functional groups are N3-, Mal-, or NHS-. STING agonists are loaded into the lumen of the vesicles via electrostatic interactions; The biantibody comprises an anti-HER2 monoclonal antibody and an anti-PD-1 monoclonal antibody covalently grafted onto the surface of the vesicle, wherein both the anti-HER2 monoclonal antibody and the anti-PD-1 monoclonal antibody are functional group modified antibodies, and the functional group is dibenzocyclooctylene. The ratio of the anti-HER2 monoclonal antibody and the anti-PD-1 monoclonal antibody grafted onto the surface of the vesicle is any value in the range of 1:(0.33-3). The anti-HER2 monoclonal antibody is used to target the HER2 antigen on the surface of tumor cells, and the anti-PD-1 monoclonal antibody is used to target the PD-1 antigen on the surface of T cells. The mass of the STING agonist accounts for 1%-11.2% of the total mass of the vesicles and the STING agonist.

2. The dual-antibody modified vesicle STING agonist according to claim 1, characterized in that, The STING agonist is selected from any one of diABZI, STING agonist-3, SR-717, or GNE-6468.

3. The dual-antibody modified vesicle STING agonist according to claim 2, characterized in that, The amphiphilic block polymer is a first hydrophilic segment - a first hydrophobic segment - Ac-KD. z ;in, The first hydrophilic segment is polyethylene glycol, and the first hydrophobic segment is a random copolymer of trimethylene carbonate monomer and disulfide five-membered ring carbonate monomer. Ac is an acetyl group, and KD... z The negatively charged linker is represented by K, which is lysine, D is aspartic acid, and z represents a repeating unit.

4. The dual-antibody modified vesicle STING agonist according to claim 3, characterized in that, The second hydrophilic segment is polyethylene glycol, and the second hydrophobic segment is a random copolymer of trimethylene carbonate monomer and disulfide five-membered ring carbonate monomer.

5. The use of the biantibody-modified vesicle STING agonist according to any one of claims 1-4 in the preparation of antitumor immunotherapy drugs.

6. A method for preparing a biantibody-modified vesicle STING agonist as described in any one of claims 1-4, characterized in that, Includes the following steps: Amphiphilic block polymers and functionalized amphiphilic block polymers are provided; An organic solution containing the amphiphilic block polymer and the functionalized amphiphilic block polymer, and an acidic buffer solution containing the STING agonist were prepared. The organic solution and the acidic buffer solution were mixed and then subjected to dialysis and ultrafiltration to obtain the vesicular STING agonist. Functional group-modified anti-HER2 monoclonal antibodies and functional group-modified anti-PD-1 monoclonal antibodies were prepared. The vesicle STING agonist, the anti-HER2 monoclonal antibody, and the anti-PD-1 monoclonal antibody are mixed at a preset molar ratio to ensure that the anti-HER2 monoclonal antibody and the anti-PD-1 monoclonal antibody are covalently grafted onto the surface of the vesicle STING agonist, thereby preparing the dual-antibody modified vesicle STING agonist.

7. The preparation method according to claim 6, characterized in that, In the step of preparing the biantibody-modified vesicular STING agonist by mixing the vesicular STING agonist, the anti-HER2 monoclonal antibody, and the anti-PD-1 monoclonal antibody at a preset molar ratio to covalently graft the anti-HER2 monoclonal antibody and the anti-PD-1 monoclonal antibody onto the surface of the vesicular STING agonist, the preset molar ratio of the functional group of the vesicular STING agonist and the anti-HER2 monoclonal antibody to the anti-PD-1 monoclonal antibody is any value in the range of 1:(0.17-0.5):(0.17-0.5).

8. The preparation method according to claim 6, characterized in that, In the step of preparing an organic solution containing the amphiphilic block polymer, the functionalized amphiphilic block polymer, and an acidic buffer solution containing a STING agonist, and then mixing the organic solution and the acidic buffer solution to obtain a vesicle STING agonist by dialysis and ultrafiltration, the STING agonist is encapsulated in the vesicle lumen while the functionalized amphiphilic block polymer and the amphiphilic block polymer are assembled and crosslinked to form vesicles.

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