Tumor antigen modified by dibenzocyclooctyne, anti-tumor vaccine and application of tumor antigen and anti-tumor vaccine

Abstract

The invention relates to a dibenzocyclooctyne modified tumor antigen, an anti-tumor vaccine and application of the dibenzocyclooctyne modified tumor antigen and the anti-tumor vaccine. The DBCO modified tumor antigen has an adjuvant effect, and the DBCO modified tumor antigen or the protein vaccine can promote DC antigen presentation and T cell activation, improve CTL killing activity and promote memory T cell formation by activating an NF-kB / iNOS signal axis, so that the tumor immunoprophylaxis and treatment effects of the tumor vaccine can be remarkably improved. Meanwhile, the DBCO modified tumor antigen or the tumor vaccine can remodel the tumor microenvironment through lung tissue targeting, significantly inhibit the progress of pulmonary metastatic tumors, and can synergistically enhance the curative effect of treating tumors by being combined with an immune checkpoint inhibitor. The invention provides a novel immune regulation strategy for the development of chemically modified vaccines, and has important significance in the field of anti-tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of vaccine and drug technology, and relates to anti-tumor vaccines, specifically to tumor antigens modified with dibenzocyclooctylene, anti-tumor vaccines, and their applications. Background Technology

[0002] The clinical translation of tumor immunotherapy has achieved significant progress from basic research to therapeutic practice, significantly changing clinical treatment strategies for malignant tumors. Currently, three main treatment strategies have emerged based on immune regulation mechanisms: 1. Immune checkpoint inhibitors (ICIs), which target immune checkpoint molecules such as PD-1 / CTLA-4 to block T cell inhibitory signaling pathways and reverse the inhibitory effect of the tumor microenvironment on T cell activity; 2. Adoptive cell therapy (ACT), which uses CAR-T or TCR-T technologies to genetically engineer the patient's T cells, enhancing their ability to specifically recognize tumor antigens; and 3. Tumor vaccines, which activate the body's specific T cell immune response through tumor-specific antigens. These three strategies function from three dimensions: relieving immunosuppression, enhancing effector cell function, and inducing immune memory.

[0003] While significant progress has been made in the clinical application of immune checkpoint inhibitors, they still face challenges related to limited treatment response rates. Clinical data shows that approximately 60% of patients develop primary resistance, and initial responders often experience disease relapse within months. Molecular mechanism studies indicate that the resistance process involves multiple pathways, including antigen presentation defects, inhibitory immune cell infiltration, and compensatory immune checkpoint activation. These issues result in an objective response rate of only 20-30% for immune checkpoint inhibitors in patients with solid tumors. Although CAR-T cell therapy has achieved breakthroughs in hematological malignancies, its efficacy in solid tumors is limited, and the treatment cost is high.

[0004] Tumor vaccines are a novel type of immunotherapy that activates the body's adaptive immune response by delivering tumor-specific antigens, thereby achieving tumor prevention or treatment. Their mechanism of action revolves around a core chain of "antigen presentation - lymphocyte activation - effector killing": The antigens carried by the vaccine are taken up by antigen-presenting cells (APCs) and processed into short peptides via the lysosomal pathway (exogenous antigens) or the cytoplasmic pathway (endogenous antigens), which are then presented to the surface of CD4+ T cells and CD8+ T cells by MHC class II or MHC class I molecules, respectively. CD4+ T cells promote the expansion and functional differentiation of cytotoxic T lymphocytes (CTLs) by secreting cytokines such as IL-2 and IFN-γ, while simultaneously enhancing the expression of APC co-stimulatory molecules (such as CD80 / 86); CTLs, in turn, directly kill tumor cells by releasing perforin and granzymes, and differentiate into memory T cells (Trms) to provide long-term immune protection. However, the efficacy of single-agent tumor vaccines remains generally limited, with low antigen cross-presentation efficiency being a key bottleneck restricting vaccine immune activation. Studies have shown that the cross-presentation efficiency of exogenous antigens via the MHC-I pathway is typically below 5%, and this inefficient antigen presentation leads to limited CTL activation.

[0005] Therefore, tumor vaccines often require optimization with adjuvants in practical applications, as unadjuvanted vaccines struggle to activate a sufficient immune response. However, currently only a small number of adjuvants have yielded promising results in clinical trials of cancer vaccines, and their effectiveness in enhancing cellular immune responses remains significant. For example, aluminum adjuvants (such as aluminum hydroxide) are the most widely used adjuvants clinically; however, they primarily promote antibody production through Th2-type immune polarization, and the induced IL-4 and IL-5 secretion is insufficient to effectively activate Th1 pathways and CD8+ T cell responses. Furthermore, the spatial mismatch between adjuvants and antigens in vivo further limits efficacy—physically mixed formulations often prevent both from being simultaneously taken up by the same APCs, reducing the synergistic activation of immunity. Even though Th1-biased adjuvants (such as the TLR4 agonist MPL) can enhance IFN-γ secretion, their driving effect on CTL amplification remains limited. These limitations highlight the necessity of developing novel adjuvant systems.

[0006] In summary, current cancer vaccine development focuses on activating cellular immune responses centered on cytotoxic T lymphocytes (CTLs), aiming to achieve durable therapeutic effects by directly killing tumor cells and forming long-term immune memory. However, due to the low presentation efficiency of exogenous antigens and the Th2-type immune polarization tendency of conventional adjuvants, the CTL activation efficacy of existing tumor vaccine systems urgently needs to be improved. Developing novel adjuvants to enhance CTL-mediated cellular immune responses is a key challenge in the field of tumor immunology. Summary of the Invention

[0007] Therefore, the purpose of this invention is to enhance CTL-mediated anti-tumor immune responses in order to improve the efficacy of tumor immunotherapy.

[0008] The technical solutions for achieving the above objectives include the following.

[0009] In one aspect, the present invention provides the application of dibenzocyclooctylene-modified tumor antigens in the preparation of antitumor vaccines;

[0010] The tumor antigen modified with dibenzocyclooctyn is obtained by reacting diphenylcyclooctyn-tetraethylene glycol-active ester with the tumor antigen.

[0011] Secondly, the present invention provides an anti-tumor vaccine obtained by incubating a tumor antigen modified with dibenzocyclooctylene as described in the present invention with dendritic cells.

[0012] Thirdly, the present invention provides the use of the dibenzocyclooctylene-modified tumor antigen described in the present invention, or the anti-tumor vaccine described in the present invention, in the preparation of drugs for treating or preventing tumors.

[0013] Fourthly, the present invention provides the use of the aforementioned dibenzocyclooctylene-modified tumor antigen in combination with an immune checkpoint inhibitor in the preparation of a drug for treating or preventing tumors; for example, the immune checkpoint inhibitor may be a PD-1 inhibitor.

[0014] Fifthly, the present invention provides a combination medicament for the prevention and / or treatment of tumors, the active ingredients of which include the dibenzocyclooctylene-modified tumor antigen and immune checkpoint inhibitor described in the present invention.

[0015] The tumor antigen and immune checkpoint inhibitor modified with dibenzocyclooctylene can be used as independent drug delivery units, or the tumor antigen and immune checkpoint inhibitor modified with dibenzocyclooctylene can be used together to form a combined drug delivery unit.

[0016] The present invention has the following beneficial effects:

[0017] This invention discovers that DBCO-modified tumor antigens possess an adjuvant effect. DBCO-modified tumor antigens or protein vaccines can significantly improve the tumor immunoprophylaxis and therapeutic efficacy of tumor vaccines by activating the NF-κB / iNOS signaling axis, promoting DC antigen presentation and T cell activation, enhancing CTL killing activity, and promoting memory T cell formation. Simultaneously, DBCO-modified tumor antigens or tumor vaccines, through lung tissue targeting, can reshape the tumor microenvironment, significantly inhibiting the progression of lung metastases. Furthermore, their combined use with immune checkpoint inhibitors can synergistically enhance the therapeutic effect on tumors. This invention provides a novel immunomodulatory strategy for the development of chemically modified vaccines, which is of great significance in the field of anti-tumor therapy. Attached Figure Description

[0018] Figure 1 This is a schematic diagram showing the formation of amide bonds between DBCO-PEG4-NHS and protein antigens via an amide coupling reaction.

[0019] Figure 2 Characterization of DBCO-OVA: A shows the modification of the DBCO group verified by Western blotting, B shows the MALDI-TOF mass spectra of DBCO-OVA and OVA, and C shows the potentials of DBCO-OVA and OVA.

[0020] Figure 3 To enhance the uptake capacity of DCs by DBCO-modified protein vaccines, A shows that DC2.4 cells showed a significant increase in DBCO-OVA uptake observed by confocal fluorescence microscopy, while B and C show that DC2.4 (B) and BMDC (C) showed increased DBCO-OVA uptake observed by flow cytometry.

[0021] Figure 4 DBCO modification promotes the antigen-presenting capacity and maturation of dendritic cells (DCs). A and B represent the increased antigen-presenting capacity of DC2.4 (A) and BMDC (B) mediated by DBCO modification as detected by flow cytometry. C represents the upregulation of surface maturation markers MHCII, CD80, CD86, and CD40 of BMDC mediated by DBCO modification as detected by flow cytometry. D represents the increase of secretory factors TNF-α, IL-1β, IL-12p60, and IL-6 of BMDC mediated by DBCO modification as detected by ELISA.

[0022] Figure 5 To detect by flow cytometry the ability of DBCO-modified protein vaccines to promote T-cell killing of tumor cells.

[0023] Figure 6DBCO-modified TCL promotes the phagocytic capacity and maturation of dendritic cells (DCs). In this study, A shows the increase in DBCO-TCL uptake by BMDCs observed by flow cytometry, and B shows the upregulation of surface maturation markers MHCII, CD80, CD86, and CD40 of BMDCs mediated by DBCO-modified TCL by flow cytometry.

[0024] Figure 7 The results show the detection of DBCO-mediated activation of the NF-κB signaling pathway. A is the immunofluorescence image of DBCO-OVA stimulating P65 nuclear translocation; B is the Western blot image of DBCO-OVA stimulating P65 nuclear translocation; C is the Western blot image of P65 and IκBα phosphorylation; and D is the flow cytometry detection of the level of antigen presentation mediated by DBCO-OVA inhibited by the NF-κB inhibitor BAY 11-7082.

[0025] Figure 8 The results of the detection of DBCO modification activating the NF-κB / iNOS signaling pathway are shown. In the figure, A is the flow cytometry detection of DBCO-OVA promoting iNOS production, B is the statistical graph of the average fluorescence intensity of iNOS, and C is the flow cytometry detection of the level of inhibition of DBCO-OVA-mediated antigen presentation by the iNOS inhibitor 1400W 2HCl.

[0026] Figure 9 To illustrate the in vivo immunogenicity of the DBCO-modified enhanced protein vaccine, the following diagrams are presented: A) Small animal imaging observation of differences in OVA-FITC / DBCO-OVA-FITC retention in various mouse organs; B) Fluorescence statistics of small animal imaging; C) Flowchart of differences in antigen uptake by lung antigen-presenting cells; D) Differences in OVA-FITC / DBCO-OVA-FITC uptake by lung macrophages; E) Differences in OVA-FITC / DBCO-OVA-FITC uptake by lung dendritic cells (DCs); F) Flowchart of differences in lung DC maturation; and G) Flow cytometry assessment of lung DC maturation (CD80). + CD86 + Differences in H. DC maturity (CD80) + CD86 + ) Statistical chart.

[0027] Figure 10The graphs show the cellular and humoral immunity of the DBCO-modified enhanced protein vaccine. In the graphs, A is a representative graph of the in vivo CTL experiment, B is a statistical graph of the spleen CTL experiment, C is a statistical graph of the lung CTL experiment, D is a graph of the OVA-specific IgG and IgG1 antibody titers in serum 7 days after OVA / DBCO-OVA immunization, E is a statistical graph of the OVA-specific IgG antibody titer, and F is a statistical graph of the OVA-specific IgG1 antibody titer.

[0028] Figure 11 To enhance the antitumor effect of DC vaccine in B16-OVA subcutaneous model by DBCO modification, A is a flowchart of DC vaccine treatment in B16-OVA subcutaneous tumor model, B is the tumor growth curve of B16-OVA tumor-bearing mice, C is the weight change of B16-OVA tumor-bearing mice, D is the anatomical diagram of B16-OVA tumor, and E is the weighing of B16-OVA tumor.

[0029] Figure 12 To enhance the antitumor effect of DC vaccine in the B16-F10 subcutaneous tumor model by DBCO modification, A is a flowchart of DC vaccine treatment in the B16-F10 subcutaneous tumor model, B is the tumor growth curve of B16-F10 tumor-bearing mice, C is the weight change of B16-F10 tumor-bearing mice, D is the anatomical diagram of B16-F10 tumor, and E is the tumor weight.

[0030] Figure 13 To promote the remodeling of the immune landscape of a B16-F10 subcutaneous tumor model by DBCO-modified protein antigen-mediated DC vaccine, A shows Ki-67 immunofluorescence staining of tumor sections, B shows CD3 immunofluorescence staining of tumor sections, and C shows the maturity of tumor DCs (CD80) as detected by flow cytometry. + CD86 + D represents MHII expression in DCs, and E represents tumor CD4. + T cell content, F represents tumor CD8 + T cell content, G represents tumor IFN-γ + T cell content, H represents tumor granzyme B. + T cell content, I represents spleen effect memory CD4 + T cells (CD44) + CD62L - CD4 + ) content, J is the spleen effect memory CD8 + T cells (CD44) + CD62L - CD4 + )content.

[0031] Figure 14To enhance the therapeutic effect of DC vaccine in the B16-F10 lung metastasis model by DBCO modification, A is a flowchart of DC vaccine treatment in the B16-F10 lung metastasis model, B is the weight change of B16-F10 lung metastasis mice during treatment, C is lung anatomical photography, and D is the statistics of lung metastasis nodules.

[0032] Figure 15 To enhance the therapeutic effect of DC vaccine in the 4T1 breast cancer orthotopic model by DBCO modification, A is a flowchart of DC vaccine treatment in the 4T1 breast cancer orthotopic model, B is the tumor growth curve of 4T1 tumor-bearing mice, C is the weight change of 4T1 tumor-bearing mice, D is the anatomical diagram of 4T1 tumor, and E is the tumor weighing.

[0033] Figure 16 The treatment of B16-OVA lung metastases with DBCO-modified protein vaccine combined with αPD-1 is shown in the following figures: A is the treatment flowchart, B is the weight change during treatment, C is the anatomical photograph of the lungs, D is the statistical graph of lung metastatic nodules, and E is the HE staining of the lungs.

[0034] Figure 17 The DBCO-modified protein vaccine was used to prevent B16-OVA lung metastases. In the figure, A is the treatment flowchart, B is the weight change during treatment, C is the lung anatomical photograph, D is the lung metastatic nodule statistics, E is the treatment survival curve, and F is the lung HE staining.

[0035] Figure 18 To enhance lung anti-tumor immunity with DBCO-modified protein vaccines, where A represents lung MHII. + CD11c + The statistical graph of DC, B represents CD3 in the blood. + T cell statistics, C represents CD4 in the blood. + T cell statistics, D represents CD8 in the blood. + T-cell statistics, E represents CD3 in the lungs + T cell statistics, F represents CD4 in the lungs + / CD8 + T cell representation diagram, G represents CD4+ in the lungs. + T cell statistics, H represents CD8+ in the lungs. + T cell statistics, I represents IFN-γ in the lungs + CD4 + T cell representation diagram, J represents IFN-γ in the lungs. + CD4 + T cell statistics, K represents IFN-γ in the lungs + CD8 + T cell representation diagram, L represents IFN-γ in the lungs. + CD8+ T-cell statistics.

[0036] Figure 19 To enhance the long-term anti-tumor immune effect of the lungs with DBCO-modified protein vaccines, A is a representative diagram of lung tissue-resident memory T cells, B is a statistical diagram of lung tissue-resident memory T cells, and C is a diagram of spleen-effect memory (CD44) cells. + CD62L - CD4 + Statistical graph of T cells, D represents spleen effect memory (CD44) + CD62L - CD8 + Statistical graph of T cells. Detailed Implementation

[0037] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0038] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual*, edited by Green and Sambrook, published in 2013, or according to the manufacturer's recommendations. All commonly used chemical reagents used in the examples are commercially available products.

[0039] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0040] Furthermore, as used herein, the term "or" is an inclusive "or" sign and is equivalent to the term "and / or" unless the context clearly specifies otherwise. The term "based on" is not exclusive and allows for basing on other factors not described unless the context clearly specifies otherwise. Additionally, throughout the specification, the meanings of "an," "a," and "the" include plural indicators. The meaning of "in" includes both "in" and "on."

[0041] Some embodiments of the present invention involve the application of dibenzocyclooctylene (DBCO) modified tumor antigens in the preparation of antitumor vaccines; the dibenzocyclooctylene modified tumor antigens are obtained by reacting diphenylcyclooctylene-tetraethylene glycol-active ester with tumor antigens.

[0042] There are no special limitations on the tumor antigens mentioned above. Any tumor antigen containing an amino group that can react with diphenylcyclooctyn-tetraethylene glycol-active ester can achieve the effects of the present invention.

[0043] For example, it can be a protein antigen, such as ovalbumin (OVA) selected as a model antigen in some embodiments of the present invention.

[0044] When the tumor antigen is a protein antigen, the molar ratio of the reaction between the diphenylcyclooctyne-tetraethylene glycol-active ester and the protein antigen is preferably 8-12:1, more preferably 9-11:1, and even more preferably 10:1.

[0045] DBCO modification in other protein antigens (such as tumor cell lysates) can also enhance the antigen uptake of dendritic cells (DCs) and the expression of co-stimulatory molecules, thereby enhancing CTL-mediated anti-tumor immune responses and improving the efficacy of tumor immunotherapy.

[0046] When the tumor antigen is a tumor cell lysis buffer (e.g., B16-F10 cell lysis buffer), the reaction mass ratio of the diphenylcyclooctyn-tetraethylene glycol-active ester to the tumor cell lysis buffer is preferably 1:4-8, more preferably 1:5-7, and even more preferably 6:1. The concentration of the tumor antigen in the tumor cell lysis buffer is 1 mg / mL-3 mg / mL, preferably 1.8 mg / mL-2.2 mg / mL.

[0047] In one embodiment of the present invention, the method for preparing the dibenzocyclooctylene-modified tumor antigen includes the following steps:

[0048] (1) Dissolve the tumor antigen in a buffer solution with a pH of 8.5-9.5, add the diphenylcyclooctyn-tetraethylene glycol-active ester, add dimethyl sulfoxide, and stir at 0℃-8℃ in the dark for 6h-10h;

[0049] (2) Add NH4Cl and stir at 0℃-8℃ in the dark for 1-3 hours to terminate the reaction. After purification, the tumor antigen modified by dibenzocyclooctylene is obtained.

[0050] In one embodiment of the present invention, the buffer solution is a 0.09 mol / L to 0.11 mol / L Na2CO3 / NaHCO3 buffer solution.

[0051] In one embodiment of the present invention, the concentration of the tumor antigen in the buffer solution is 1 mg / mL to 3 mg / mL.

[0052] In one embodiment of the present invention, the volume concentration of the dimethyl sulfoxide in the reaction system is 8%-12%.

[0053] In one embodiment of the present invention, the concentration of NH4Cl in the reaction system is 45 mmol / L-55 mmol / L.

[0054] In one embodiment of the present invention, the tumor is melanoma, breast cancer, or lung metastasis.

[0055] The dibenzocyclooctylene (DBCO) modified tumor antigen provided by this invention can be used as a tumor vaccine for the prevention and treatment of tumors, or as an adjuvant to enhance the anti-tumor immune response of existing tumor vaccines (such as DC vaccines) and improve the therapeutic effect of tumor immunity.

[0056] For example, one embodiment of the present invention also relates to an anti-tumor vaccine obtained by incubating a tumor antigen modified with dibenzocyclooctylene as described in the present invention with dendritic cells.

[0057] In one embodiment of the present invention, the ratio of the dibenzocyclooctylene-modified tumor antigen to dendritic cells is 1µg-5µg: 10,000 cells, preferably 2µg-3µg: 10,000 cells.

[0058] In one embodiment of the present invention, the incubation conditions include: a temperature of 35°C-40°C and a time of 20-28 hours.

[0059] One embodiment of the present invention also relates to the use of the aforementioned dibenzocyclooctylene-modified tumor antigen, or the aforementioned antitumor vaccine, in the preparation of drugs for treating or preventing tumors.

[0060] In one embodiment of the present invention, the application of the dibenzocyclooctylene-modified tumor antigen combined with an immune checkpoint inhibitor in the preparation of a drug for treating or preventing tumors is also involved; preferably, the immune checkpoint inhibitor is a PD-1 inhibitor, for example, αPD-1.

[0061] In one embodiment of the present invention, the tumor is melanoma, breast cancer, or lung metastasis.

[0062] The dibenzocyclooctylene-modified tumor antigen of the present invention can be used in combination with other known antitumor drugs to further enhance the antitumor effect. In combination administration, the dibenzocyclooctylene-modified tumor antigen and the known drug can be independent drug delivery units or together form a combined drug delivery unit; the dibenzocyclooctylene-modified tumor antigen can be administered simultaneously with or separately from other known antitumor drugs.

[0063] In one embodiment of the present invention, a combination drug for the prevention and / or treatment of tumors is also involved, the active ingredients of which include the dibenzocyclooctylene-modified tumor antigen and immune checkpoint inhibitor described in the present invention.

[0064] The tumor antigen and immune checkpoint inhibitor modified with dibenzocyclooctylene can be used as independent drug delivery units, or the tumor antigen and immune checkpoint inhibitor modified with dibenzocyclooctylene can be used together to form a combined drug delivery unit.

[0065] Preferably, the immune checkpoint inhibitor is a PD-1 inhibitor, such as αPD-1.

[0066] In specific embodiments of the present invention, extensive experimental data have demonstrated that the dibenzocyclooctylene-modified tumor antigen, or antitumor vaccine, or combination therapy of the present invention can significantly enhance CTL-mediated antitumor immune responses and significantly improve the efficacy of tumor immunotherapy. Specifically:

[0067] This invention successfully conjugated DBCO to the model antigen OVA via acylation. The modified DBCO-OVA significantly enhanced the antigen uptake efficiency of dendritic cells (DC2.4 and BMDC). Flow cytometry analysis showed that DBCO modification upregulated the MHC I antigen presentation efficiency on the DC surface and promoted the expression of co-stimulatory molecules (CD80, CD86, CD40) and the secretion of pro-inflammatory cytokines (TNF-α, IL-1β, IL-12p60, IL-6). In in vitro T cell killing experiments, the DBCO-modified vaccine significantly enhanced the killing ability of cytotoxic T cells against tumor cells. Generalization experiments showed that DBCO modification also enhanced antigen uptake and co-stimulatory molecule expression in DCs on other protein antigens (such as tumor cell lysates).

[0068] This invention further revealed through transcriptome sequencing that DBCO modification triggers DC transcriptional reprogramming, with differentially expressed genes significantly enriched in the NF-κB signaling pathway. Western blotting and immunofluorescence experiments showed that DBCO modification enhances p65 and IκBα phosphorylation and promotes p65 nuclear translocation, confirming that DBCO modification activates the classical NF-κB pathway. Simultaneously, a significant upregulation of iNOS gene transcription and protein expression was detected, forming an NF-κB / iNOS regulatory axis. The synergistic effect of this signaling pathway provides key molecular mechanism support for DBCO's enhancement of DC maturation and antigen presentation functions.

[0069] In tumor models such as the B16-OVA subcutaneous model, B16-F10 lung metastasis, and 4T1 in situ model, the DC vaccine mediated by the DBCO-modified protein antigen significantly inhibited tumor growth. Immune microenvironment analysis showed that the DBCO-modified group had increased infiltration of DCs and T cells within the tumor, significantly upregulated IFN-γ and Granzyme B secretion levels, and promoted the formation of spleen effector memory T cells, achieving long-term immune protection.

[0070] In vivo imaging in small animals showed that DBCO modification significantly enhanced the lung tissue targeting of OVA antigens. Flow cytometry analysis indicated that this modification promoted antigen uptake by lung macrophages and dendritic cells (DCs) and significantly upregulated the expression of DC maturation markers (CD80 / CD86). In vivo CTL assays confirmed that the modified vaccine significantly enhanced the antigen-specific T cell killing efficiency in the spleen and lungs. ELISA analysis showed significantly increased levels of OVA-specific antibody IgG and IgG1 titers, suggesting synergistic activation of cellular and humoral immunity.

[0071] DBCO-modified protein vaccines significantly inhibited metastatic nodule formation in the B16-OVA lung metastasis model. Combined with PD-1 monoclonal antibody therapy, they further synergistically reduced the number of lung nodules. Prophylactic vaccination experiments also showed significant inhibitory effects on lung metastases. Flow cytometry analysis confirmed that DBCO modification promoted CD8+ T cell infiltration and effector molecule secretion in the lungs, while simultaneously increasing the proportion of resident memory T cells in lung tissue and effector memory T cells in the spleen. This study systematically validated the anti-metastatic mechanism of DBCO modification through targeting lung APCs, activating adaptive immunity, and inducing long-term immune memory.

[0072] These experimental results all demonstrate the adjuvant effect of DBCO modification. DBCO-modified tumor antigens exhibit an adjuvant effect; DBCO-modified tumor antigens or protein vaccines can significantly improve the tumor immunoprophylaxis and therapeutic efficacy of tumor vaccines by activating the NF-κB / iNOS signaling axis, promoting DC antigen presentation and T cell activation, enhancing CTL killing activity, and promoting memory T cell formation. Simultaneously, DBCO-modified tumor antigens or tumor vaccines, through lung tissue targeting, can reshape the tumor microenvironment, significantly inhibit the progression of lung metastases, and synergistically enhance the therapeutic efficacy of tumor treatment when used in combination with immune checkpoint inhibitors. This invention provides a novel immunomodulatory strategy for the development of chemically modified vaccines, which is of great significance in the field of anti-tumor therapy.

[0073] Therefore, the dibenzocyclooctylene-modified tumor antigen or antitumor vaccine provided by the present invention can be used to prepare drugs for the prevention or treatment of tumors.

[0074] In one embodiment of the present invention, the drug can enhance the antigen presentation efficiency of antigen-presenting cells.

[0075] In one embodiment of the present invention, the drug can enhance the killing ability of cytotoxic T cells against tumor cells.

[0076] In one embodiment of the present invention, the drug can enhance cellular immunity.

[0077] In one embodiment of the present invention, the drug can enhance humoral immunity.

[0078] In one embodiment of the present invention, the drug is capable of activating the NF-κB / iNOS immune pathway.

[0079] In one embodiment of the present invention, the drug can enhance lung anti-tumor immunity.

[0080] In one embodiment of the present invention, the drug can promote the uptake of antigens by dendritic cells.

[0081] In one embodiment of the invention, the drug can enhance the ability of dendritic cells to present antigens.

[0082] In one embodiment of the present invention, the drug can promote the maturation of dendritic cells.

[0083] In one embodiment of the present invention, the drug can increase the content of cytokines in the supernatant of dendritic cells, wherein the cytokines are IL-1β, IL-6, IL-12p60 and TNF-α.

[0084] In one embodiment of the present invention, the drug can enhance the targeted delivery of antigens to secondary lymphoid organs and tumor-prone sites.

[0085] In one embodiment of the present invention, the drug can increase the titer of IgG antibodies in serum, preferably the titer of IgG1 antibodies.

[0086] In one embodiment of the present invention, the drug can increase the number of T cells within a tumor.

[0087] In one embodiment of the present invention, the drug can increase the number of mature dendritic cells within a tumor.

[0088] In one embodiment of the present invention, the drug can increase the number of T cells in lung tissue.

[0089] In one embodiment of the present invention, the drug can increase the number of mature dendritic cells in lung tissue.

[0090] In one embodiment of the present invention, the drug can upregulate CD8. + The secretion levels of IFN-γ and granzyme B by T cells.

[0091] In one embodiment of the present invention, the drug can promote the formation and expansion of spleen effector memory T cells.

[0092] In one embodiment of the present invention, the drug can enhance the enrichment of tissue-resident memory T cells in the lungs.

[0093] The present invention will be further described in detail below with reference to specific embodiments.

[0094] The experimental materials used in the examples are described below:

[0095] 1. Cell lines

[0096]

[0097] 2. Reagents and Consumables

[0098]

[0099]

[0100]

[0101] In the following examples, the cell culture method is as follows:

[0102] The mouse skin melanoma cells B16-F10, B16-F10-OVA cells (overexpressing chicken ovalbumin), and mouse dendritic cells DC2.4 were all cultured in RPMI-1640 complete medium. The mouse skin cell line 4T1 was cultured in DMEM complete medium. The cell culture procedures are as follows:

[0103] 1) Preparation of complete culture medium: According to experimental requirements, add 1% penicillin and streptomycin and 10% fetal bovine serum to DMEM or RPMI-1640 culture medium.

[0104] 2) Cell digestion is an important procedure in cell culture. When cells reach 70%-80% confluence in the culture dish, trypsin digestion is performed. First, discard the original culture medium and wash the cells twice with sterile PBS solution to remove residual culture medium. Add an appropriate amount of trypsin (0.25%), evenly coating the cell surface with trypsin, and place the dish in an incubator at 37°C for digestion. The digestion time varies depending on the cell species and culture state; generally, digestion is complete when the cells become rounded under a microscope.

[0105] 3) Neutralization: The cells digested with trypsin were neutralized using complete culture medium. Then, twice the volume of trypsin in complete culture medium was added, and the cells were gently pipetted to ensure complete dispersion. After dispersion, the cell suspension was transferred to sterile centrifuge tubes and centrifuged at 1200 rpm for 3 minutes. The supernatant was removed, and fresh cells were added as needed to resuspend the cells.

[0106] 4) Resuspension and Cell Counting: After centrifugation, discard the supernatant, add an appropriate amount of complete culture medium, and gently pipette the cell pellet to ensure complete cell dispersion and obtain a homogeneous cell suspension. At this point, the cell suspension can be seeded according to experimental needs. If subsequent experiments are required, use a Countstar cell counter to count the cells. Adjust the cell concentration according to experimental requirements and proceed with subsequent experiments.

[0107] The induction and culture methods for mouse bone marrow-derived dendritic cells (BMDC) are as follows:

[0108] BMDCs are an important model for studying immunological responses. The following examples demonstrate the induction and culture of BMDCs using standardized procedures:

[0109] 1) Removal of mouse fur: After removing the eyeballs and bleeding the mice, they were euthanized by cervical indwelling and disinfected by soaking in 75% alcohol for 10 minutes. A small incision was then made in the abdomen, the fur was torn away, and the hind legs (including the thighs and calves) were cut off. The thighs were cut off near the abdomen, and the calves were cut off at the foot. After removing the fur, the bones were disinfected by soaking in 70% ethanol for 5-10 seconds and then transferred to 15mL centrifuge tubes containing sterile PBS, which were then placed on ice.

[0110] 2) Removing the leg bone and muscle: In a biosafety cabinet, remove the hind leg and slightly dry it. After trimming away excess muscle, gently break the thigh into two parts along the joint. Place the thigh part on a piece of paper, and through the paper, use your fingers to remove the joint cover where the lower leg connects to the thigh. Continue to push the muscle to the end with the paper and cut off the tendons at the end. Wipe it clean and place it in fresh PBS on ice. Break the lower leg along the foot joint, remove the muscle, and perform the same treatment.

[0111] 3) Collecting bone marrow cells: Wash the bone marrow cells 3-5 times with 75% ethanol (inverted), then discard the ethanol. Wash 3 times with sterile 1X PBS, then add approximately 10 mL of sterile PBS and place on ice. Place the prepared leg bones in a 10 cm culture dish, hold the bone with forceps, and cut off both ends of the joint. Using a 10 mL syringe fitted with a 0.6 mm x 25-30 mm blue needle, aspirate all the PBS from the culture dish, then gently blow into the bone from one end until the bone turns white. Next, disperse the bone marrow cells with the syringe until no more red clumps are visible.

[0112] 4) Differentiation Induction: After collecting bone marrow suspension, filter through a 200-mesh nylon mesh to remove debris and muscle tissue, and centrifuge at 300 g for 5 minutes. Add 2 mL of erythrocyte lysis buffer (2 mL per mouse), lyse erythrocytes for 3 minutes, then add PBS to neutralize, and centrifuge at 300 g for 5 minutes. Prepare complete culture medium: 1640 medium + 10% inactivated FBS + 1% PS. Discard the supernatant, resuspend the cells and add complete culture medium to adjust the cell concentration to 0.5-1 x 10⁻⁶ cells / mL. 6 / mL. Add GM-CSF (20 ng / mL) and IL-4 (10 ng / mL), and incubate at 37°C in a 5% CO2 incubator; this is day 0 of culture. This process requires incubation at 37°C in a 5% CO2 incubator, with 3 / 4 of the culture medium replaced every 2 days to replenish the necessary cytokines. During culture, the concentration and conditions of cytokines play a crucial role in the culture of dendritic cells. After 7 days of culture, the dendritic cells enter the maturation stage, at which point they can be collected by centrifugation, counted, and used for subsequent immunological experiments.

[0113] Example 1: Preparation and Characterization of DBCO-OVA

[0114] 1) Dissolve OVA in 0.1 M Na2CO3 / NaHCO3 buffer solution at pH = 9 to obtain a 2 mg / mL OVA solution. Add DBCO-PEG4-NHS (diphenylcyclooctynyl tetraethylene glycol-active ester) to the OVA solution at a molar ratio of OVA:DBCO-PEG4-NHS = 1: 10. Add DMSO at a volume of 10% of the total volume of the reaction system. Stir the reaction at 4°C in the dark for 8 h.

[0115] 2) Add 5 M NH4Cl aqueous solution to the reaction system to make the final concentration of NH4Cl in the reaction system 50 mM, and stir at 4°C in the dark for 2 hours to terminate the crosslinking reaction.

[0116] 3) Use a 30 kDa ultrafiltration tube to perform ultrafiltration purification at 4°C, with a rotation speed of 8000 rpm and a time of 15 minutes. Wash the solution three times with NaHCO3 buffer to obtain the purified DBCO-OVA concentrate.

[0117] 4) Determine protein concentration using the BCA method and store at 4 ℃.

[0118] Western Blot Verification of DBCO Group Binding: To verify whether the DBCO group successfully bound to the OVA protein, Western blotting was used. An 8% separating gel and a 5% stacking gel were prepared and stored overnight at 4 °C. The protein loading was 5 μg. Electrophoresis conditions were constant voltage 100 V, and the transfer conditions were also constant voltage 100 V for 120 minutes. After transfer, the membrane was immersed in 50 μM N3-Cy5 and reacted at room temperature for 1 hour. Subsequently, the membrane was washed three times with 1X TBST for 5 minutes each time, and finally developed using a fluorescence exposure machine. The DBCO label on the OVA protein was clearly visible through fluorescence development.

[0119] Characterization of DBCO-OVA: Zeta potential was measured using a Zetasizer system: 2 mg / mL of the stock solution was prepared into a 10 μg / mL solution, and the Zeta potential of the sample was measured on the instrument; the binding of the DBCO group was analyzed by MALDI-TOF mass spectrometry.

[0120] Protein vaccines typically possess multiple lysine sites, with the ε-amino group on their side chain acting as a primary amine group. Due to its strong nucleophilicity and high reactivity, it can efficiently undergo acylation reactions with NHS esters to form stable amide bonds. In this example, OVA was selected as the model antigen. The OVA protein has a molecular weight of 42.8 kDa and consists of 386 amino acids, including 20 modifiable lysine ε-amino sites. A coupling reaction was performed between OVA and DBCO-PEG4-NHS at a molar ratio of 1:10 to successfully prepare DBCO-bonded OVA (DBCO-OVA). Figure 1 In the Western blot validation experiment, N3-Cy5 was used as the probe for detection, and DBCO-OVA could carry the Cy5 fluorescence signal via a click reaction. Figure 2 As shown in Figure A, a significant Cy5 fluorescent band was observed in the 55 kDa region, confirming the successful modification of the DBCO group. MALDI-TOF mass spectrometry analysis showed that each OVA molecule was coupled with an average of approximately 3.6 DBCO groups (…). Figure 2 (B in the text). Potential analysis shows that the average potential of the OVA is -7.39 mV, and the average potential of the DBCO-OVA is -19.47 mV. Figure 2 The C in the figure indicates that the bonding of the DBCO group enhances the negative charge on the surface of the protein vaccine.

[0121] Example 2: Preparation of DBCO-OVA-FITC

[0122] 1) Dissolve OVA in 0.1 M Na2CO3 / NaHCO3 buffer solution at pH = 9 to obtain a 2 mg / mL OVA solution. Add FITC dissolved in DMSO to the OVA solution at a molar ratio of OVA:FITC = 1: 10. Add DMSO at a volume of 10% of the total volume of the reaction system. Stir the reaction at 4 degrees Celsius in the dark for at least 8 hours.

[0123] 2) Use a 30 kDa ultrafiltration tube to perform ultrafiltration purification at 4°C, with a rotation speed of 8000 rpm and a time of 15 minutes. Wash repeatedly with NaHCO3 buffer at least 3 times until the filtrate has no visible fluorescence.

[0124] 3) The concentration of OVA-FITC was determined by the BCA method. The mother liquor was divided into two parts. One part was stored at 4 ℃ for subsequent experiments, and the other part was used to continue the synthesis of DBCO-OVA-FITC.

[0125] 4) Dilute the OVA-FITC stock solution to 2 mg / mL with 0.1 M Na2CO3 / NaHCO3 buffer at pH = 9. Add DBCO-PEG4-NHS to the OVA-FITC solution at a molar ratio of OVA:DBCO-PEG4-NHS = 1: 10. Add 10% DMSO to the reaction system and stir at 4 degrees Celsius in the dark for 8 h.

[0126] 5) Add 5 M NH4Cl to the reaction system to make the final concentration of NH4Cl in the reaction system 50 mM, and stir at 4 ℃ in the dark for 2 hours to terminate the crosslinking reaction.

[0127] 6) Use a 30 kDa ultrafiltration tube to perform ultrafiltration purification at 4°C, with a rotation speed of 8000 rpm and a time of 15 minutes. Wash the solution three times with NaHCO3 buffer to obtain the purified DBCO-OVA-FITC concentrate.

[0128] 7) Determine protein concentration using the BCA method and store at 4 ℃.

[0129] Example 3: Preparation of DBCO-bonded tumor cell lysate (DBCO-TCL)

[0130] 1) Preparation of tumor cell lysate (TCL): B16-F10 cells were seeded in 10 cm culture dishes. After the cells reached full confluence, the supernatant was discarded, the cells were washed twice with PBS, and the cells were digested with trypsin. The cell suspension was collected, centrifuged at 1500 rpm for 5 minutes, and the concentration of the cell suspension was adjusted to 2*102. 7 Cells were collected at a density of 10 cells / mL and transferred to 2 mL cryovials. The cryovials were then placed in liquid nitrogen for 15 minutes, followed by a complete thaw at 37°C for 10 minutes. This freeze-thaw cycle was repeated 6 times. After the freeze-thaw cycle, the cells were centrifuged at 12,000 rpm for 30 minutes. The supernatant was filtered through a 0.22 μm microporous membrane into sterile 2 mL EP tubes. The concentration of tumor antigen was determined to be 2 mg / mL using the BCA method. The prepared TCL solution was aliquoted and stored at -80°C.

[0131] 2) Preparation of DBCO-TCL: The TCL solution obtained in step 1) was dissolved to a concentration of 2 mg / mL using 0.1 M Na2CO3 / NaHCO3 buffer solution at pH = 9. DBCO-PEG4-NHS was added to the TCL solution at a mass ratio of 6:1 (TCL solution:DBCO-PEG4-NHS), and 10% DMSO was added to the reaction system. The mixture was stirred at 4°C in the dark for 8 h. 5 M NH4Cl was added to the reaction system to bring the final concentration of NH4Cl to 50 mM. The cross-linking reaction was terminated by stirring at 4°C in the dark for 2 h, and the mixture was stored at 4°C.

[0132] DBCO-TCL-FITC and TCL-FITC were prepared using the same method as in Example 2.

[0133] Example 4: DBCO-modified protein vaccines can effectively promote the presentation of MHC type I antigens of APCs.

[0134] 1. Detection of the difference in OVA-FITC / DBCO-OVA-FITC uptake by DC2.4 using confocal fluorescence microscopy

[0135] 1) Overnight cell culture: After digesting DC2.4 cells with trypsin, adjust the cell suspension to the required concentration, seed 100,000 cells / well into a confocal microscope culture dish, and culture overnight in a cell culture incubator at 37°C.

[0136] 2) Drug treatment: After removing the cell culture medium, the cells were washed three times with PBS. Three groups were set up: control group, OVA-FITC group, and DBCO-OVA-FITC group. The corresponding drugs were added at a concentration of 50 μg / mL for 4 hours and 12 hours, respectively.

[0137] 3) Detection on the instrument: Remove the culture medium, then rinse 3 times with PBS, add 100 μL of DMEM containing 1×Hoechst and react in the dark for 10 min. Take pictures of the corresponding fluorescence channels with a laser confocal microscope and analyze them with OlyVIA software.

[0138] 2. Flow cytometry analysis of the difference in OVA-FITC uptake by DC2.4 / BMDC and DBCO-OVA-FITC.

[0139] 1) Overnight cell seeding: DC2.4: After digesting DC2.4 cells with trypsin, adjust the cell suspension to the required concentration and seed 200,000 cells / well into a 24-well plate; BMDC: Gently pipette BMDCs with a 1 mL pipette tip until they detach, collect the cell suspension, centrifuge at 1200 rpm for 5 minutes, remove the supernatant, and wash twice with PBS. Count the cells using a Countstar cell counter and adjust the cell suspension to the required concentration. Seed 500,000 cells / well into a low-adhesion 24-well plate and incubate overnight at 37°C.

[0140] 2) Drug treatment: Three groups were set up: control group, OVA-FITC group, and DBCO-OVA-FITC group. The corresponding drugs were added at a concentration of 50 μg / mL and the treatment time was 12 hours.

[0141] 3) Cell Collection: DC2.4 cells: After washing three times with PBS, add 200 μL of trypsin to digest the cells. After complete digestion, add 200 μL of complete culture medium to stop the reaction. Collect the cells in a 1.5 mL centrifuge tube, centrifuge at 1200 rpm for 5 minutes, resuspend the cells in 200 μL of 0.5% BSA, filter through a 200-mesh nylon mesh, and keep on ice until needed. BMDC cells: Gently pipette the BMDC cells with a 1 mL pipette tip until they detach. Collect the cell suspension in a 1.5 mL centrifuge tube, centrifuge at 1200 rpm for 5 minutes, discard the culture medium, wash twice with PBS, and then resuspend the cells in 50 μL of 0.5% BSA staining system (PE anti-mouse CD11c). Stain on ice for 30 minutes. After staining, wash twice with 1 mL of 0.5% BSA, resuspend the cells in 200 μL of 0.5% BSA, filter through a 200-mesh nylon mesh, and keep on ice until needed.

[0142] 4) Flow cytometry analysis: Add 5 μg / mL of DAPI and gently vortex to mix. Perform flow cytometry analysis using a CytoflexS flow cytometer. Determine the gating settings of the flow cytometer using CytExpert software, and analyze the subsequent values ​​using Flowjo software.

[0143] The results are as follows Figure 3 As shown: Dynamic observations using laser confocal microscopy revealed that the unmodified OVA group maintained a low level of endocytosis over 12 hours, while the DBCO-OVA group exhibited significantly enhanced uptake efficiency. Figure 3 (A) Flow cytometry quantitative analysis further confirmed this phenomenon: after 12 hours of co-culture, DBCO modification significantly increased the antigen uptake rate from 13.4% to 65.7% (an increase of 15.6-fold in uptake difference). Figure 3(B in the original text). The uptake experiment was repeated using BMDC. Similarly, DBCO modification significantly increased the uptake of protein antigens by DCs, with the average uptake percentage of BMDCs increasing from 29% to 62.4%. Figure 3 The C in the figure indicates that the modification strategy has universality across cell models.

[0144] 3. Flow cytometry analysis of the antigen-presenting capacity of DBCO-modified antigens in promoting DC2.4 / BMDCs

[0145] 1) Seeding cells overnight: Seed cells as before and incubate overnight in a 37°C cell culture incubator.

[0146] 2) Drug treatment: Three groups were set up: control group, OVA group, and DBCO-OVA group. The corresponding drugs were added at a concentration of 50 μg / mL and the treatment time was 12 hours.

[0147] 3) Cell Collection: DC2.4 cells: After washing three times with PBS, 200 μL of trypsin was added to digest the cells. After complete digestion, 200 μL of complete culture medium was added to stop the reaction. The cells were collected in 1.5 mL centrifuge tubes and centrifuged at 1200 rpm for 5 minutes. The cells were then resuspended in 50 μL of 0.5% BSA staining system (PE-Cy7 anti-mouse OVAp-H-2kb) and stained on ice for 30 minutes. After staining, the cells were washed twice with 1 mL of 0.5% BSA, resuspended in 200 μL of 0.5% BSA, filtered through a 200-mesh nylon mesh, and kept on ice until needed. BMDC cells: Gently pipette the BMDCs with a 1 mL pipette tip until they detach, collect the cell suspension into a 1.5 mL centrifuge tube, centrifuge at 1200 rpm for 5 minutes, discard the culture medium, wash twice with PBS, and then resuspend the cells in 50 μL of 0.5% BSA staining system (PE anti-mouse CD11c, PE-Cy7 anti-mouse OVAp-H-2kb), and stain on ice for 30 minutes. After staining, wash twice with 1 mL of 0.5% BSA, resuspend the cells in 200 μL of 0.5% BSA, filter through a 200-mesh nylon mesh, and place on ice until needed.

[0148] 4) Flow cytometry analysis: Add 5 μg / mL of DAPI and gently vortex to mix. Perform flow cytometry analysis using a CytoflexS flow cytometer. Determine the gating settings of the flow cytometer using CytExpert software, and analyze the subsequent values ​​using Flowjo software.

[0149] 4. Flow cytometry analysis of the degree of BMDC maturation promoted by DBCO-modified antigen.

[0150] 1) Seeding cells overnight: Seed 500,000 BMDCs per well into a low-adhesion 24-well plate and incubate overnight in a 37°C cell culture incubator.

[0151] 2) Drug treatment: Three groups were set up: control group, OVA group, and DBCO-OVA group. The corresponding drugs were added at a concentration of 50 μg / mL and the treatment time was 48 hours.

[0152] 3) Cell Collection: Gently pipette the BMDCs with a 1 mL pipette tip until they detach. Collect the cell suspension into a 1.5 mL centrifuge tube, centrifuge at 1200 rpm for 5 minutes, discard the culture medium, wash twice with PBS, and then resuspend the cells in 50 μL of 0.5% BSA staining system (PE anti-mouse CD11c, AF700 anti-mouse MHC11, FITC anti-mouse CD80, BV650 anti-mouse CD86, perp / cy5.5 anti-mouse CD40). Stain on ice for 30 minutes. After staining, wash twice with 1 mL of 0.5% BSA, resuspend the cells in 200 μL of 0.5% BSA, filter through a 200-mesh nylon mesh, and store on ice until needed.

[0153] 4) Flow cytometry analysis: Add 5 μg / mL of DAPI and gently vortex to mix. Perform flow cytometry analysis using a CytoflexS flow cytometer. Determine the gating settings of the flow cytometer using CytExpert software, and analyze the subsequent values ​​using Flowjo software.

[0154] 5. ELISA method for detecting cytokine secretion in BMDC supernatant promoted by DBCO-modified antigen.

[0155] 500,000 BMDCs per well were seeded into low-adhesion 24-well plates and cultured overnight at 37°C. The experiment consisted of three groups: a control group, an OVA group, and a DBCO-OVA group. Each group received the corresponding drug at a concentration of 50 μg / mL for 48 hours. After treatment, the cell suspension was collected in 1.5 mL centrifuge tubes and centrifuged at 1200 rpm for 5 minutes. The supernatant was collected and transferred to new EP tubes. The supernatant was diluted twice and used to detect the levels of IL-1β, IL-6, IL-12p60, and TNF-α in the supernatant.

[0156] Antigen presentation capacity and maturity level are crucial foundations for APCs to exert their anti-tumor immune function. Mature DCs can efficiently present tumor antigens to T cells, thereby initiating the activation of cytotoxic T cells. Furthermore, mature DCs can upregulate the expression of co-stimulatory molecules and secrete pro-inflammatory cytokines, which further enhance T cell activation, thereby improving the intensity and effectiveness of the immune response. To investigate the effect of DBCO modification on DC antigen presentation capacity, this embodiment conducted a series of experiments. First, DCs were incubated with DBCO-OVA for 12 hours, and the effect of DBCO modification on antigen presentation efficiency was detected. Figure 4 Flow cytometry analysis showed that the mean fluorescence intensity (MFI) of H-2Kb / SIINFEKL in DC2.4 cells increased from 2092.7 to 4255.1, indicating that DBCO modification promoted the presentation of the H-2Kb / SIINFEKL complex on the surface of DC2.4 cells. Figure 4 Similarly, in BMDC, the average fluorescence intensity of H-2Kb / SIINFEKL increased from 10898.5 to 22939.5, nearly doubling the antigen-presenting capacity. This further confirms the promoting effect of DBCO modification on the antigen-presenting capacity of DCs. Figure 4 (B in the original text). Flow cytometry analysis of DC surface maturation markers revealed that DBCO modification upregulated the expression of DCMHCII, CD80, CD86, and CD40. Figure 4 The upregulation of these molecules indicates that DBCO modification promotes DC maturation. ELISA analysis of secretory factor levels in the supernatant showed that DBCO modification also mediated an increase in BMDC secretory factors TNF-α, IL-1β, IL-12p60, and IL-6. Figure 4 (D in the D). The increase of these pro-inflammatory cytokines further enhances the immune activation capacity of DCs, providing a stronger impetus for anti-tumor immune responses.

[0157] 6. Flow cytometry analysis of the ability of DBCO-modified antigen to promote the killing of OT-1 mouse T cells

[0158] 1) Magnetic bead sorting of OT-1 mouse CD8 + T cells:

[0159] OT-1 mice were euthanized by cervical dislocation. The spleens were disinfected with 75% alcohol for 10 minutes and then transferred to a biosafety cabinet for isolation. The spleens were then washed twice with PBS. The spleens were mechanically homogenized using a grinding mesh to obtain a cell suspension, which was transferred to 15 mL centrifuge tubes. The tubes were centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded. 3 mL of erythrocyte lysis buffer was added, and the cells were gently mixed by pipetting. Cell lysis was performed at room temperature for 3 minutes, and the reaction was terminated with PBS (containing 1% FBS). The cells were centrifuged again at 2000 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended in 1 mL of PBS (containing 1% FBS). After gentle pipetting, the cells were counted using a Countstar cell counter, and the cell concentration was adjusted to 3 × 10⁶ cells / mL. 8 / mL.

[0160] Transfer 100 μL of cell suspension to a sterile flow cytometry tube, add 10 μL of biotin-labeled anti-mouse CD8 antibody, mix gently, and incubate on ice for 15 minutes. Then add 10 μL of streptavidin nanobeads and incubate for another 15 minutes. Next, add 2.5 mL of MojoSort™ buffer, place the flow cytometry tube in a magnetic rack, and incubate for 5 minutes. Collect the suspension into a 50 mL centrifuge tube, wash twice, centrifuge at 2000 rpm for 5 minutes, discard the supernatant, and finally obtain sorted OT-1 CD8 cells. + T cells. After cell counting, they can be used for subsequent experiments.

[0161] 2) Antigen stimulation of BMDCs: 500,000 BMDCs per well were seeded into low-adhesion 24-well plates and cultured overnight at 37°C. Three groups were set up: control group, OVA group, and DBCO-OVA group. The corresponding drugs were added at a concentration of 50 μg / mL for 24 hours. After incubation, the cells were gently pipetted using a 1 mL pipette tip, and the cell suspension was collected into a 1.5 mL centrifuge tube. After centrifugation at 1200 rpm for 5 minutes, the culture medium was removed, the cells were washed twice with PBS, resuspended in 1 mL of 1640 medium, counted, and then placed on ice for later use.

[0162] 3) Tumor cell staining: B16-OVA tumor cells were washed three times with PBS, followed by 1 mL of trypsin digestion solution. After complete digestion, 1 mL of complete culture medium was added to stop the digestion. Cells were collected into 15 mL centrifuge tubes, centrifuged at 1200 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in 1 mL of 1640 basal medium. 5 μM CFSE staining solution was added, mixed well, and incubated at 37°C for 10 minutes. After incubation, the cells were washed twice with 1 mL of 1640 complete culture medium, counted, and stored on ice for later use.

[0163] 4) Co-culture of T cells and tumor cells: Mix T cells, DC cells and tumor cells at a ratio of 10:5:1. Seed 50,000 tumor cells / well into a 24-well plate, mix well and co-culture at 37°C for 24 hours.

[0164] 5) Flow Cytometry Detection: After trypsin digestion, dead and live cells were stained using DAPI staining, and the remaining live cells were quantified using flow cytometry. The lysis rate was obtained by calculating the number of live tumor cells in the experimental wells and the number of live cells in the tumor-only control wells. The result was normalized to the number of cells per well and expressed as the survival rate compared to the tumor-only control group. The survival rate was calculated using the formula: (Number of live cells in experimental wells ÷ Number of microbeads) ÷ (Number of live cells in tumor wells ÷ Number of microbeads) × 100%.

[0165] Activating antigen-specific cytotoxic T lymphocytes (CTLs) is crucial for enhancing the antitumor efficacy of cancer vaccines. Cancer vaccines can activate cytotoxic T lymphocytes through cellular immunity to directly recognize and kill tumor cells expressing antigens. Figure 5 As shown, the average survival rate of B16-OVA cells in the DBCO-OVA group was reduced by 15.2% compared to the OVA group. This result indicates that the DBCO-modified protein vaccine can enhance the killing ability of T cells against tumor cells.

[0166] 7. Using the DBCO-bonded tumor cell lysate prepared in Example 3, flow cytometry experiments were performed according to the method of this example to detect the difference in TCL-FITC / DBCO-TCL-FITC uptake by BMDCs and the degree to which DBCO-modified TCL promoted BMDC maturation.

[0167] In this experiment, to test the generalizability of the adjuvant effect of DBCO-modified protein vaccines, DBCO was modified onto TCL. Flow cytometry was used to assess the effect of DBCO-modified TCL on the phagocytic capacity and maturation degree of DCs. Results showed that the average uptake rate of BMDCs after DBCO modification of TCL increased from 58.1% to 73.4%, an increase of 15.3%. Figure 6 (A in the text). Correspondingly, DBCO modification also upregulated the expression of BMDC MHCII, CD80, CD86, and CD40, especially increasing the average fluorescence intensity of CD80 from 23194.3 to 39816.5 and the average fluorescence intensity of CD40 from 23286 to 38564.7, both by more than 50%. Figure 6 (B in the middle).

[0168] The above results indicate that DBCO-modified protein vaccines can significantly enhance the MHC-I antigen presentation efficiency of antigen-presenting cells.

[0169] Example 5: DBCO modification activates the NF-κB / iNOS signaling pathway

[0170] 1. Western Blot detection of NF-κB signaling pathway activation

[0171] 1) Cell treatment: BMDC cells were seeded at a density of 1 million per well in 6-well plates and cultured overnight at 37°C. Three experimental groups were set up: control group, OVA group, and DBCO-OVA group. The corresponding drug was added to each group at a concentration of 50 μg / mL for 12 hours. After treatment, the cells were gently pipetted with a 1 mL pipette tip, and the cell suspension was transferred to a 2 mL centrifuge tube. The tube was centrifuged at 1200 rpm for 5 minutes, the culture medium was discarded, the cells were washed twice with PBS, and the cells were collected by centrifugation. The supernatant was discarded, the EP tube was inverted to air dry the precipitate, and stored on ice.

[0172] 2) Cell lysis: Prepare lysis buffer (RIRA: protease inhibitor: PMSF: phosphatase inhibitor = 100:1:2:1) in 80 μL per tube. Add the lysis buffer to the EP tube, mix thoroughly with a vortex mixer, and continue lysis on ice for 30 minutes, mixing with a vortex mixer every 10 minutes. Centrifuge at 12000 rpm for 15 minutes at 4°C and collect the supernatant.

[0173] 3) BSA protein quantification: Prepare standardized samples of 2 mg / mL, setting concentration gradients of 0, 2, 4, 8, 16, and 32 mg / mL, with two replicates for each concentration. Prepare standard solutions. Prepare chromogenic working solution at a ratio of BSA sample solution A: solution B = 50:1, adding 200 μL to each well, with two replicates for each sample, and place in a 96-well plate. Add 2 μL of protein sample, incubate at 37°C for 30 minutes, and measure the absorbance at 562 nm using a microplate reader. Calculate the sample concentration based on the standard curve.

[0174] 4) Protein sample preparation: Add 5× Loading Buffer according to the actual volume of the protein sample, heat in a 95℃ metal bath for 10 minutes, dispense into new EP tubes, use one part for direct loading, and store the other part at -20℃.

[0175] 5) Electrophoresis: Clean the 1.5 mm glass plate and prepare a 10% acrylamide separating gel and a 5% stacking gel. After the gel solidifies, rinse thoroughly with water and blot dry with filter paper. Place the glass plate in the electrophoresis tank and assemble the electrophoresis module. Add an appropriate amount of electrophoresis buffer, carefully remove the comb, and rinse the sample wells with an insulin needle. Insert the pipette tip into the sample wells and slowly add 20 μg of protein sample. Fill any remaining wells with 1× Loading Buffer to the same volume. Perform electrophoresis at a constant voltage of 80 V. Once the bromophenol blue has migrated to the separating gel, increase the voltage to 100-120 V and continue electrophoresis until the gel is close to the bottom.

[0176] 6) Transfer: Cut a PVDF membrane to the appropriate size according to the number of lanes, immerse it in 100% methanol for 15 seconds, and then transfer it to transfer buffer for equilibration for 5 minutes. Quickly rinse the gel glass plate with ddH2O, pry open the glass plate, and place the gel in the transfer buffer. Stack the gel in the following order: sponge pad, filter paper, gel, PVDF membrane, filter paper, sponge pad, carefully removing air bubbles between each layer. After assembly, place the clamp into the transfer tank, fill with transfer buffer, and set the transfer time to 100 mA for 1.5 hours.

[0177] 7) Antibody incubation: Rinse the PVDF membrane with 1×TBST, block the PVDF membrane with 5% BSA at room temperature for 1 hour, then wash three times with TBST for 10 minutes each time. Dilute the primary antibody to an appropriate concentration with primary antibody dilution buffer and incubate overnight at 4°C. Wash three more times with TBST for 10 minutes each time. Incubate with HRP-labeled secondary antibody at room temperature for 1 hour, then wash three times with TBST for 10 minutes each time. Evenly drop the ECL chemiluminescence solution onto the PVDF membrane to completely cover it. Place the membrane in the dark chamber of the BIO-RAD CHEMIDOC XRS+ imaging system for chemiluminescence imaging and analyze using ImageLab software.

[0178] The extraction methods for nuclear proteins are as follows:

[0179] 1) Reagent preparation: Melt the nucleocytoplasmic separation kit at room temperature, and after dissolution, quickly place it on ice and mix thoroughly. Add 1 Mm PMSF to both cytoplasmic protein extraction reagent A and nuclear protein extraction reagent, and place them on ice for later use.

[0180] 2) Cell collection: Gently pipette the cells using a 1 mL pipette tip to transfer the cell suspension to a 2 mL centrifuge tube. Centrifuge at 1200 rpm for 5 minutes and discard the culture medium. Then wash twice with PBS, centrifuge again to collect the cells, discard the supernatant, invert the EP tube to air dry the precipitate, and store on ice.

[0181] 3) Cytoplasmic protein extraction: Resuspend the cell pellet in 200 μL of cytoplasmic protein extraction reagent A, vortex vigorously for 5 seconds, and then place in an ice bath for 15 minutes. Add 10 μL of cytoplasmic protein extraction reagent B, vortex again for 5 seconds, incubate on ice for 1 minute, vortex for another 5 seconds, and finally incubate at 12000 g for 5 minutes at 4°C. Carefully transfer the supernatant to a new EP tube to obtain the extracted cytoplasmic proteins.

[0182] 4) Extraction of nuclear proteins: After thoroughly removing the supernatant from the centrifuge tube, add 50 μL of nuclear protein lysis buffer containing the protease inhibitor PMSF, and vortex for 30 seconds until the cell pellet is fully resuspended. Incubate the sample on ice, performing high-intensity vortexing (30 seconds / time) every 2 minutes for a total processing time of 30 minutes. Then centrifuge at 12000 g for 10 minutes at 4 ℃, and transfer the supernatant to a pre-chilled centrifuge tube to obtain the nuclear protein extract. Aliquot and store at -80 ℃ for later use.

[0183] 2. Cell smear immunofluorescence

[0184] 1) Cell preparation: Add 10 μL of PBS to each well of a 24-well plate, and then carefully place the cell spreader into the well using tweezers. Seed 100,000 DC2.4 cells per well and treat with the appropriate drug for 12 hours.

[0185] 2) Cell fixation: Discard the cell culture medium and wash gently twice with PBS. Then fix the cells with 200 μL of 4% paraformaldehyde solution for 10 minutes, soak in PBS for 5 minutes, and repeat three times.

[0186] 3) Cell permeabilization: Cells were permeabilized with 0.2% Triton X-100 solution for 10 minutes, washed with PBS for 5 minutes, and repeated three times.

[0187] 4) Blocking treatment: Block with a solution containing 3% bovine serum albumin (BSA) and 1% FBS for 1 hour. After blocking, wash with PBS for 5 minutes, repeating twice.

[0188] 5) Primary antibody incubation: Add the primary antibody solution diluted with PBST solution containing 1% BSA, and then incubate overnight at 4°C.

[0189] 6) Secondary antibody incubation: Wash with PBS for 5 minutes, repeat three times. Then add secondary antibody diluted with PBS solution containing 1% BSA and incubate for 1 hour in the dark. After incubation, wash again with PBS for 5 minutes, repeat three times.

[0190] 7) Mounting: Add 100 μL of DAPI-containing anti-quenching mounting medium to the cells, cover the mounting with a coverslip, and finally fix the coverslip with nail polish.

[0191] 3. Flow cytometry analysis of the effect of BAY 11-7082 or iNOS inhibitor 1400W 2HCl on DC antigen presentation ability.

[0192] Following the method described in Example 4, BMDC cells were treated with the corresponding concentration of BAY 11-7082 or 1400W 2HCl for 1 hour, followed by treatment with 50µg DBCO-OVA. After 12 hours, the antigen-presenting capacity of the DCs was detected by flow cytometry.

[0193] Immunofluorescence staining and Western blot analysis revealed that DBCO-OVA significantly promoted p65 nuclear translocation. Figure 7 (A, B) and induce increased phosphorylation of P65 and IκBα ( Figure 7 The C in the figure indicates that DBCO modification enhances the function of DC through the NF-κB pathway.

[0194] BAY 11-7082 is a specific NF-κB signaling pathway inhibitor. Flow cytometry was used to examine the effect of BAY 11-7082 on DC antigen presentation ability. The results showed that BAY 11-7082 significantly downregulated DBCO-OVA-mediated antigen presentation. Figure 7 (D in the middle).

[0195] Furthermore, flow cytometry showed that the expression level of iNOS protein increased 1.7-fold after DBCO-OVA treatment (p<0.001). Figure 8 (A and B in the text). After treatment of DCs with the iNOS inhibitor 1400W 2HCl, the level of antigen presentation mediated by DBCO-OVA was significantly downregulated ( Figure 8 (C in the middle).

[0196] The above results indicate that DBCO can drive the MHCI-type antigen cross-presentation function of DC by activating the NF-κB / iNOS signal axis.

[0197] Example 6: In vivo lung immunization with DBCO-modified enhanced protein vaccine

[0198] 1. Small animal imaging to detect differences in the retention of DBCO-OVA-FITC / OVA-FITC in various organs of mice.

[0199] 1) Mouse grouping: Three mice were set up: Control group, OVA-FITC group and DBCO-OVA-FITC group. Each group consisted of 3 mice. Mice were injected with 500 μg of OVA-FITC / DBCO-OVA-FITC via the tail vein.

[0200] 2) Eight hours after injection of OVA / DBCO-OVA, mice were euthanized, and organs such as the heart, liver, spleen, lungs, and kidneys were collected. The blood in the organs was washed with PBS 3-4 times, the moisture was blotted with filter paper, and the FITC channel fluorescence was captured using a small animal imaging system.

[0201] 2. Flow cytometry was used to detect the difference in uptake of DBCO-OVA-FITC / OVA-FITC by lung antigen-presenting cells.

[0202] 1) Mouse grouping: Three mice were set up as Control group, OVA group and DBCO-OVA group. Mice were injected with 100ug of OVA-FITC / DBCO-OVA-FITC via the tail vein.

[0203] 2) Single-cell suspension preparation: Mice were euthanized 24 hours after injection of OVA-FITC or DBCO-OVA-FITC. Using an 18-21 gauge needle, 10 mL of cold PBS was injected into the right ventricle until the lungs were completely white to flush them. Subsequently, the thymus and heart were removed, the lungs were separated from the surrounding tissue, and placed in a centrifuge tube containing cold 0.5% BSA buffer. The lungs were transferred to a culture dish, cut into small pieces with scissors, and then placed in 5 mL EP tubes. 4 mL of digestion buffer (RPMI 1640 medium containing 2% fetal bovine serum, 1 mg / mL collagenase, 0.1 mg / mL DNase I, and 0.1 mg / mL hyaluronidase) was added. The tubes were capped, and the lung tissue was digested on a shaker at 37°C at 120 rpm for 30 minutes. The digested lung mixture was transferred to a wire mesh, and the remaining lung tissue on the mesh was ground with the rubber side of a 5 mL syringe plunger and rinsed with 0.5% BSA buffer.

[0204] 3) Cell staining: Collect cells by centrifugation at 500 g for 5 minutes. Discard the supernatant, resuspend the cells in 3 mL of 1×RBC Lysis Buffer, lyse at room temperature for 3 minutes, and then stop the reaction by adding 0.5% BSA buffer. Collect cells again by centrifugation at 500 g for 5 minutes, resuspend the cells in 1 mL of 0.5% BSA buffer, and take 300 μL of cell suspension for staining. Collect cells by centrifugation at 500 g for 5 minutes, discard the supernatant, and stain on ice for 30 minutes with flow cytometry staining solutions (FVS510, PC5.5 anti-mouse CD45, APC-A750 anti-mouse F4 / 80, BV785 anti-mouse CD11b, BV421 anti-mouse CD11c).

[0205] 4) Cell detection: After staining, wash twice with 1 mL of 0.5% BSA buffer, resuspend the cells in 200 μL of 0.5% BSA buffer, and filter through a 200-mesh nylon screen. Detect the cells using a CytoflexS flow cytometer.

[0206] 3. Flow cytometry was used to detect the differences in maturation of lung antigen-presenting cells caused by DBCO-OVA / OVA.

[0207] 1) Mouse grouping: Three mice were set up as Control group, OVA group and DBCO-OVA group. Mice were injected with 100ug OVA / DBCO-OVA via tail vein.

[0208] 2) Single-cell suspension preparation: Mice were euthanized 24 hours after injection of OVA or DBCO-OVA. Using an 18-21 gauge needle, 10 mL of cold PBS was injected into the right ventricle until the lungs were completely white to flush them. Subsequently, the thymus and heart were removed, the lungs were separated from the surrounding tissue, and placed in a centrifuge tube containing cold 0.5% BSA buffer. The lungs were transferred to a culture dish, cut into small pieces with scissors, and then placed in 5 mL EP tubes. 4 mL of digestion buffer (RPMI 1640 medium containing 2% fetal bovine serum, 1 mg / mL collagenase, 0.1 mg / mL DNase I, and 0.1 mg / mL hyaluronidase) was added. The tubes were capped, and the lung tissue was digested on a shaker at 37°C at 150 rpm for 30 minutes. The digested lung mixture was transferred to a wire mesh, and the remaining lung tissue on the mesh was ground with the rubber side of a 5 mL syringe plunger and rinsed with 0.5% BSA buffer.

[0209] 3) Cell staining: Collect cells by centrifugation at 500 g for 5 minutes. Discard the supernatant, resuspend the cells in 3 mL of 1×RBC Lysis Buffer, lyse at room temperature for 3 minutes, and then stop the reaction by adding 0.5% BSA buffer. Collect cells again by centrifugation at 500 g for 5 minutes, resuspend the cells in 1 mL of 0.5% BSA buffer, and take 300 μL of cell suspension for staining. Collect cells by centrifugation at 500 g for 5 minutes, discard the supernatant, and stain on ice for 30 minutes with flow cytometry staining solution (FVS510, FITC anti-mouse CD45, APC-A750 anti-mouse F4 / 80, BV421 anti-mouse CD11c, APC anti-mouse CD80, BV605 anti-mouse CD86).

[0210] 4) Cell detection: After staining, wash twice with 1 mL of 0.5% BSA buffer, resuspend the cells in 200 μL of 0.5% BSA buffer, and filter through a 200-mesh nylon screen. Detect the cells using a CytoflexS flow cytometer.

[0211] In vivo imaging of small animals tracked the biodistribution characteristics 8 hours after intravenous injection. Compared with unmodified OVA, the DBCO-OVA group showed a significant prolongation in systemic retention time. Notably, fluorescence statistical analysis revealed that DBCO modification could guide antigen targeting and enrichment in lung tissue, with its fluorescence signal intensity increasing by 8.9 times compared to the control group. Figure 9 (B in the figure, p<0.0001). This phenomenon suggests that the lungs may be a key site for DBCO-OVA to activate immunity—considering the high-density APC network in the lung interstitium, such as alveolar macrophages and dendritic cells, targeted enrichment may optimize antigen capture and presentation efficiency.

[0212] The differences in vaccine uptake by lung-resident APCs were quantitatively assessed using multicolor flow cytometry. Results showed that DBCO modification increased OVA endocytosis efficiency in alveolar macrophages by 5.9 times (p<0.001), while antigen uptake in the lung DC population also increased by 5.3 times (p<0.001). Figure 9 CE in the middle). Further analysis of DC maturity markers revealed ( Figure 9 FH in the group, CD80 induced by DBCO-OVA group + CD86 + The proportion of double-positive DCs reached 27.9±1%, which was significantly higher than that of the control group (15.2±3.5%) (p<0.001), indicating that DBCO modification not only enhances antigen uptake, but also amplifies the immune activation potential of DCs through maturation-promoting signals.

[0213] 4. In vivo cytotoxicity test

[0214] 1) Mouse immunization: Control group, OVA group and DBCO-OVA group were established, with 3 mice in each group. Mice were immunized by intravenous injection of 100 μg of OVA or DBCO-OVA via the tail vein.

[0215] 2) Preparation of spleen single-cell suspension: Seven days after immunization, non-immunized mice of the same strain were sacrificed, and the spleens were aseptically harvested after immersion in alcohol for 10 minutes. The spleens were washed with pre-cooled PBS, and single-cell suspensions were prepared by grinding the cells through a steel mesh using a syringe plunger method. Cells were collected by centrifugation (500 g, 5 min), resuspended in 1×RBC lysis buffer (room temperature, 3 min), and the reaction was terminated with 0.5% BSA. After a second centrifugation, the cells were resuspended in serum-containing RPMI 1640 medium and counted.

[0216] 3) Antigen peptide incubation of spleen cells: The cell suspension was divided into two groups: the experimental group was added with 1 μg / mL OVA257–263 peptide (37°C, 2 h) as target cells, and the control group was not added with peptide.

[0217] 4) Cell staining: The cell concentration in both groups was adjusted to 2.5 × 10⁻⁶. 7 Cells were collected at 1640 cells / mL, followed by differential labeling with CFSE (5 μM for target cells, 0.5 μM for control cells, 37°C, 15 min). The reaction was terminated with RPMI 1640 complete medium. Cells were collected by centrifugation at 500g for 5 min, washed twice with PBS, resuspended in RPMI 1640, and the two groups of cells were mixed at a 1:1 ratio and the concentration was adjusted to 1×10⁻⁶ cells / mL. 8 cells / mL.

[0218] 5) Inject 200 μL of the mixed cell suspension into mice via the tail vein. Sacrifice the mice 16 hours later and collect lungs and spleens to prepare single-cell suspensions (filtered through a 200-mesh screen). Add 5 μg / mL of DAPI and gently vortex to mix. Analyze using a CytoflexS flow cytometer.

[0219] 5. ELISA method for detecting IgG and IgG1

[0220] 1) Mouse immunization: Three mice were set up in the Control group, OVA group and DBCO-OVA group. Mice were immunized by intravenous injection of 100 μg of OVA or DBCO-OVA via the tail vein.

[0221] 2) Blood collection: 7 days after immunization, whole blood was collected into EP tubes, left to stand at room temperature for 2 hours, and then centrifuged at 3000 rpm / min for 15 minutes at 4°C. The supernatant was collected, aliquoted, and stored at -80°C.

[0222] 3) Coating: Prepare a 20 μg / mL OVA coating solution using 1× coating buffer, add 100 μL to each well of the ELISA plate, and incubate overnight at 4°C.

[0223] 4) Washing: Wash the ELISA plate with PBST for 5 minutes each time, repeat 3 times.

[0224] 5) Blocking: Add 3% BSA blocking solution at 200 μL / well to the well and block for 1 hour at room temperature.

[0225] 6) Washing: Wash with PBST for 5 minutes each time, repeat 3 times.

[0226] 7) Sample dilution: Dilute the sample with 1% BSA diluent at concentration gradients of 1 / 50, 1 / 100, 1 / 200, 1 / 400, 1 / 800, 1 / 1600, and 1 / 3200.

[0227] 8) Sample incubation: Add 100 μL of diluted sample to each well and incubate at 37°C for 1 hour.

[0228] 9) Washing: Wash with PBST for 5 minutes each time, repeat 3 times.

[0229] 10) Secondary antibody incubation: Dilute the secondary antibody according to the instructions, add 100 μL / well to each well, and incubate at 37°C in the dark for 1 hour.

[0230] 11) Washing: Wash with PBST for 5 minutes each time, repeat 3 times.

[0231] 12) Color development: Add 100 μL of TMB color development solution per well, incubate in the dark for about 15 minutes, then add 50 μL of stop solution, and detect the absorbance at a wavelength of 490 nm.

[0232] Cellular and humoral immunity are key indicators for evaluating the efficacy of tumor vaccines, both playing indispensable roles in the anti-tumor immune response. Humoral immunity mediates antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) through antibodies (such as IgG1), while cellular immunity relies on CTL and Th1 immune responses to directly kill tumor cells and establish immune memory. The synergistic effect of these two immune systems determines the therapeutic efficacy and long-term anti-tumor effect of tumor vaccines. Here, we systematically evaluate the regulatory role of DBCO chemical modification on the immunogenicity of OVA protein vaccines by integrating CTL response and antibody subtype analysis.

[0233] To quantitatively analyze the enhancing effect of DBCO modification on cellular immunity, this embodiment uses a CFSE-labeled SIINFEKL-sensitized spleen cell adoptive transfer model (in vivo CTL killing model) for experimental analysis. Figure 10As shown in Figure AC, the antigen-specific CTL killing efficiency of the DBCO-OVA vaccine group in spleen tissue reached 27.3±8% (n=3), which was 7.1 times higher than that of the unmodified OVA group (3.8±0.9%) (p<0.01). In the lungs, the killing efficiency of the DBCO-OVA group (28.5±5.4%) was also significantly higher than that of the OVA group (4.4±6.4%) (p<0.01). These results indicate that DBCO modification can significantly enhance the targeted delivery of antigens to secondary lymphoid organs and tumor-prone sites, thereby activating CD8+ T cell responses through the MHC-I cross-presentation pathway.

[0234] Further analysis using ELISA to detect humoral immunity levels revealed that the serum OVA-specific total IgG titer in the DBCO-OVA group reached 1048.2±119.8, a 1.9-fold increase compared to the 544.7±54.3 in the OVA group (p<0.001). Figure 10 (E in the text). Notably, the IgG1 subtype titer in the unmodified OVA group was below the detection threshold, while the IgG1 titer in the DBCO-OVA group reached 198.6±12 (p<0.0001). Figure 10 The F in the text suggests that DBCO modification can enhance the immune effect through humoral immunity.

[0235] Example 7: DBCO-modified protein antigen enhances the anti-tumor immune effect of DC vaccine

[0236] 1. Construction of a subcutaneous melanoma-bearing model in C57BL / 6 mice using B16-F10 / B16-OVA.

[0237] A subcutaneous tumor model was established using 5-6 week old C57BL / 6 mice. The hair on the back of the mice was shaved, and 50 μL of a cell suspension containing 5 × 10⁵ B16-F10 / B16-OVA was injected into the subcutaneous tissue on the right back of the mice using an insulin injector. When the tumor grew to a palpable size, its dimensions were measured using calipers. The tumor volume was calculated using the formula: Volume = Major Axis × Minor Axis. 2 ×0.5. When the tumor volume reaches approximately 50 mm. 3 (Usually around the 4th day after tumor implantation) the mice are randomly assigned to different experimental groups and the corresponding treatment regimens are implemented.

[0238] 2. DBCO-OVA-stimulated DC vaccine treatment of mouse subcutaneous tumor-bearing model

[0239] Add OVA or DBCO-OVA (50 µg OVA or DBCO-OVA per 200,000 BMDCs) to the PBS suspension of the same strain of BMDC, incubate at 37°C for 24 hours, wash thoroughly with PBS, and then prepare 10 BMDCs using PBS.7 A cell suspension of 100 μL / mL was administered to the peritumoral region of B16-OVA model mice, once every 5 days for a total of three times. Tumor volume and body weight were measured every 3-4 days. In the PBS control group, the tumor volume grew to 2000 cm³. 3 Mice were sacrificed to photograph tumors. After the photographs were taken, the tumors were divided into two halves. One half was stored at -80°C, and the other half was fixed with 4% paraformaldehyde.

[0240] like Figure 11 As shown in Figure B, the OVA group exhibited a slight tumor suppression effect, but there was no statistically significant difference compared to the PBS group; the DBCO-OVA group, on the other hand, demonstrated a significant tumor suppression effect, with a tumor volume inhibition rate of 51.8% compared to the PBS group. The body weights of the three groups were not significantly different, indicating that the DC vaccine activated by the DBCO-modified protein antigen has good biosafety. Figure 11 (C) In the PBS group, the mice had a volume close to 2000 mm. 3 At that time, mice were sacrificed, tumors were dissected, photographed, and weighed. The tumor weight in the DBCO-OVA group was significantly reduced compared to the control group, indicating that the DBCO modified protein antigen significantly enhanced the anti-tumor effect of the DC vaccine. Figure 11 (D and E in the text).

[0241] 3. Treatment of mouse subcutaneous tumor-bearing model with DBCO-modified TCL-stimulated DC vaccine

[0242] The DBCO-modified TCL-stimulated DC vaccine prepared in Example 3 was used to treat a mouse subcutaneous tumor-bearing model according to the method described in this example. The tumor-suppressive effect of the DC vaccine in the mouse B16-F10 subcutaneous model was observed. Figure 12 As shown, the DBCO-TCL group exhibited excellent tumor suppression effects. Compared to the 47.8% tumor volume inhibition rate of the TCL group, the DBCO-TCL group achieved a tumor volume inhibition rate of 71.5%, an increase of 23.7%. The weight differences among the three groups of mice were minimal, indicating the good safety profile of the DBCO-modified DC vaccine. Mice were sacrificed on day 19, and the tumors were dissected, photographed, and weighed. The tumor weight in the DBCO-TCL group was significantly lower than that in the Control and TCL groups, demonstrating the general applicability of DBCO modification in promoting the efficacy of the DC vaccine.

[0243] 4. Immunohistochemical experiments

[0244] All samples were fixed with 4% paraformaldehyde, followed by routine paraffin embedding and sectioning.

[0245] 1) Place the slices in an oven and heat at 65°C for 2 hours. After cooling slightly, place them in a xylene solution for dewaxing for 10 minutes. Repeat this step twice.

[0246] 2) Hydrate the sections using alcohol of different concentration gradients. Soak the sections in alcohol of 100%, 100%, 95%, 85%, and 75% concentrations for 3 minutes in sequence, and then rehydrate them in deionized water for 3 minutes.

[0247] 3) Add 3% hydrogen peroxide solution to the slices and incubate for 10 minutes. Then rinse three times with PBS buffer for 3 minutes each time.

[0248] 4) Heat the Tris-EDTA antigen retrieval solution (pH = 9.0) in a microwave oven until boiling. Completely immerse the slides in the retrieval solution, heat on high for 3 minutes, then on low for 15 minutes. Allow the slides to cool naturally to room temperature. Rinse the slides three times with PBS buffer for 3 minutes each time.

[0249] 5) Use an immunohistochemistry pen to draw the defined area on the slide and rinse with PBST buffer.

[0250] 6) Add 5% BSA solution and block the slides with non-specific antigens at room temperature for 30 minutes.

[0251] 7) Discard the 5% BSA solution from the slides. Dilute the primary antibody with antibody diluent according to the predetermined ratio, add it dropwise to the slides, and incubate the slides overnight at 4°C. After the primary antibody incubation is complete, allow the slides to return to room temperature. Immerse the slides in PBS solution for 5 minutes each time, for a total of 3 times.

[0252] 8) Add 30 μL of horseradish peroxidase-labeled secondary antibody to the slides and incubate at room temperature for 60 minutes. Immerse the slides in PBS solution again for 5 minutes each time, for a total of 3 times.

[0253] 9) Stain the sections with DAB chromogenic solution, observe the staining status under a microscope, and stop staining as needed. Rinse the sections with PBS solution for 5 minutes each time, for a total of 3 times.

[0254] 10) Stain the sections with hematoxylin for 1 minute. Rinse the sections under running tap water for 5 minutes.

[0255] 11) Mount the sections with neutral resin glue and let them dry for subsequent observation and analysis.

[0256] 5. Immunofluorescence chemistry experiment

[0257] 1) Refer to the immunohistochemical experiment for the primary antibody incubation procedure.

[0258] 2) Secondary antibody incubation: 30 μL of horseradish peroxidase-labeled secondary antibody was directly added to the slide and incubated at room temperature for 1 hour. Subsequently, the slide was placed in PBS solution and washed for 5 minutes each time, repeating the washing process 3 times.

[0259] 3) Fluorescence amplification: Shake off the residual washing solution on the slide, add 100 μL of dye working solution, ensuring that the sample area is completely immersed, keep it moist at room temperature and shake to incubate for 10 minutes, then wash with TBST solution for 5 minutes, repeat twice.

[0260] 4) Mounting: Add DAPI working solution and let it sit for 5 minutes, then rinse the slide with TBST solution for 5 minutes, and wash with sterile water for 5 minutes. Finally, add super quench-resistant mounting medium to completely cover the sample area, place a coverslip, and press to seal the slide.

[0261] 6. Flow cytometry to detect changes in the immune microenvironment after tumor treatment

[0262] Tumors, spleens, and lungs of mice after treatment were collected, and flow cytometry was used to analyze changes in the number and function of immune cells in the tumor microenvironment.

[0263] 1) Sample Preparation: 1. Subcutaneous Tumors: Place tumor tissue into a 5 mL round-bottom centrifuge tube, cut it into a paste-like consistency with scissors, add 4 mL of tumor digestion solution (the digestion solution ratio is: 1 mg / mL collagenase IV, 0.1 mg / mL DNase I, and 0.1 mg / mL hyaluronidase), and shake horizontally at 120 rpm for 30 minutes at 37°C for sufficient digestion. After digestion, place on ice, filter through a steel mesh, and grind the remaining tissue on the steel mesh using the rubber side of a 5 mL syringe plunger. Add sufficient 0.5% BSA solution to neutralize the digestion solution. Centrifuge at 2000 rpm for 5 minutes to collect cells, discarding the supernatant. Resuspend the cells in 6 mL of 40% Percoll solution and carefully transfer them to a 15 mL centrifuge tube. Centrifuge at 800 g for 20 minutes at 25°C with centrifugation parameters of ascending speed 6 and descending speed 2. After centrifugation, discard the supernatant, resuspend the cells in 2 mL of erythrocyte lysis buffer, and incubate on ice for 2 minutes. After incubation, add sufficient 0.5% BSA solution to neutralize, and centrifuge at 2000 rpm for 5 minutes to collect the cells. Resuspend the cells in 1 mL of 0.5% BSA to obtain a tumor single-cell suspension. 2. Spleen: Place the spleen in a 6 cm dish, grind the steel mesh with the rubber side of a 5 mL syringe plunger, add 2 mL of 0.5% BSA to rinse, collect the cell suspension into a 15 mL centrifuge tube, centrifuge at 2000 rpm for 5 minutes to collect the cells, then resuspend the cells in 3 mL of erythrocyte lysis buffer and lyse at room temperature for 3 minutes. Add sufficient 0.5% BSA to neutralize the lysis buffer. Centrifuge again at 2000 rpm for 5 minutes, collect the cells, and resuspend the cells in 1 mL of 0.5% BSA to obtain a spleen single-cell suspension.

[0264] 2) DC maturation: Transfer cells to 1.5 mL EP tubes and collect cells by centrifugation at 2000 rpm for 5 min. Add 50 μL of flow cytometry staining solution (FVS510, APC / Cy7 anti-mouse CD45, PE anti-mouse CD11c, FITC anti-mouse CD80, BV650 anti-mouse CD86, AF700 anti-mouse MHCII) and stain on ice in the dark for 30 min. Wash once with 1 mL of 0.5% BSA, collect cells by centrifugation at 2000 rpm for 5 min, resuspend in 200 μL of 0.5% BSA, pass through a 200-mesh nylon mesh, and analyze using flow cytometry.

[0265] 3) T cell count: Transfer cells to 1.5 mL EP tubes and collect cells by centrifugation at 2000 rpm for 5 min. Add 50 μL of flow cytometry staining solution (FVS510, APC / Cy7 anti-mouse CD45, BV605 anti-mouse CD3, PC5.5 anti-mouse CD4, AF700 anti-mouse CD8) and stain on ice in the dark for 30 min. Wash once with 1 mL of 0.5% BSA, collect cells by centrifugation at 2000 rpm for 5 min, resuspend in 200 μL of 0.5% BSA, pass through a 200-mesh nylon mesh, and analyze using flow cytometry.

[0266] 4) Memory T cells: Transfer cells to 1.5 mL EP tubes and collect cells by centrifugation at 2000 rpm for 5 min. Add 50 μL of flow cytometry staining solution (FVS510, APC / Cy7 anti-mouse CD45, BV605 anti-mouse CD3, PC5.5 anti-mouse CD4, AF700 anti-mouse CD8, BV650 anti-mouse CD62L, BV421 anti-mouse CD44) and stain on ice in the dark for 30 min. Wash once with 1 mL of 0.5% BSA, collect cells by centrifugation at 2000 rpm for 5 min, resuspend in 200 μL of 0.5% BSA, pass through a 200-mesh nylon mesh, and analyze using flow cytometry.

[0267] 5) T cell secretion factors: Transfer cells to 1.5 mL EP tubes and collect cells by centrifugation at 2000 rpm for 5 min. Resuspend cells in 200 μL of stimulant (1640 complete medium containing 1× cellstimulation cocktail plus protein transport inhibitor) and incubate at 37 ℃ for 2 h. After stimulation, wash away the stimulant with 1 mL of 0.5% BSA solution, collect cells by centrifugation at 2000 rpm for 5 min, discard the supernatant, add 50 μL of flow cytometry antibody staining solution (FVS510, APC / Cy7 anti-mouse CD45, BV605 anti-mouse CD3, PC5.5 anti-mouse CD4, AF700 anti-mouse CD8) and stain on ice in the dark for 30 min. After incubation, wash once with 1 mL of 0.5% BSA solution, centrifuge to collect the supernatant, add 250 μL of Fixation / Perm solution, mix well, and fix at 4 ℃ for 20 min. After fixation, cells were washed twice with 500 μL of 1× Perm / Wash™ buffer, centrifuged at 10,000 rpm for 3 minutes to collect the cells, aspirated the supernatant, and stained with 50 μL of flow cytometry antibody staining solution (APC anti-mouse Granzyme B, PE-Cy7 anti-mouse IFN-γ) on ice in the dark for 30 minutes. After labeling, cells were washed once with 500 μL of 1× Perm buffer, resuspended in 200 μL of 0.5% BSA, filtered through a 200-mesh nylon mesh, and prepared for flow cytometry analysis.

[0268] The results are as follows Figure 13 As shown.

[0269] Ki-67 immunofluorescence staining of tumor sections showed that the Ki-67 fluorescence intensity in the DBCO-TCL group was significantly lower than that in the other two groups, indicating that the DC vaccine stimulated by DBCO-TCL could effectively inhibit the proliferation of tumors in mice. Figure 13 (A) Furthermore, CD3 immunofluorescence staining showed a significant increase in the number of T cells within the tumor in the DBCO-TCL group. This may be due to the significantly improved antigen presentation capacity and maturity of the DC vaccine after DBCO-TCL stimulation, thereby enhancing the recruitment and activation of T cells. Figure 13 (B in the middle).

[0270] Tumor tissues from mice were collected 5 days after the last treatment, and their immune status was detected by flow cytometry. Results showed that mature DCs (CD80) were present in the tumors of the DBCO-TCL group.+ CD86 + The number of [something] was significantly increased compared to the control group. Figure 13 C in MHCII + The number of DCs also increased significantly. Figure 13 (D in CD4). Although CD4 + The number of T cells did not reach a statistically significant difference, but showed an upward trend. Figure 13 (E in the middle), and CD8 + The number of T cells increased significantly. Figure 13 (F in the middle).

[0271] Further detection of CD8 + T cell cytokine secretion levels were found in the DBCO-TCL group, specifically CD8. + The levels of IFN-γ and granzyme B secretion by T cells were significantly upregulated, indicating that DBCO-mediated DC vaccines not only promoted T cell proliferation but also significantly enhanced their killing ability. Figure 13 (G, H in). Similarly, spleens of mice after treatment were collected to examine changes in memory T cells, such as... Figure 13 As shown in I and J, the DBCO-TCL group significantly upregulated CD4. + and CD8 + Effector memory T cells (CD44) + CD62L - The proportion of ( ) indicates that DBCO-mediated DC vaccines can induce a specific long-term immune response against tumors.

[0272] Example 8: DBCO modification enhances the therapeutic effect of DC vaccine in a lung metastasis model.

[0273] 1. Construction of a C57BL / 6 mouse model of B16-F10 cutaneous melanoma lung metastasis

[0274] A lung metastasis model was established using 5-6 week old C57BL / 6 mice. 200 μL of a solution containing 2×10⁻⁶ ions was injected via the tail vein. 5 B16-F10 cell suspension was injected into mice. On the 4th day after tumor implantation, the mice were randomly assigned to different experimental groups and the corresponding treatment regimens were implemented. The weight of the mice was measured every 3-4 days, and the weight change curve was plotted.

[0275] 2. DC vaccine treatment of mouse lung metastasis model

[0276] Add TCL or DBCO-TCL (50µg TCL or DBCO-TCL per 200,000 BMDCs) to a PBS suspension of the same strain of BMDC, incubate at 37°C for 24 hours, wash thoroughly with PBS, and then prepare 10 BMDCs using PBS.7 A cell suspension of 100 μL / mL was administered to mice via tail vein injection, once every 5 days for a total of three times. Mice were regularly observed for their mental state and feeding status, and their body weight was measured every 3-4 days. When the control group mice showed significant weight loss, they were sacrificed. The eyeballs were removed to expel blood, and the neck was immediately dissected. After exposing the trachea, 4% paraformaldehyde was injected into the trachea using a syringe until fluid flowed from the nostrils, thus filling the lungs. The thoracic cavity was then dissected, and the heart and thymus were carefully removed. The lungs were separated from surrounding tissues, collected, washed with PBS, and photographed. After photography, the lungs were fixed with 4% paraformaldehyde.

[0277] The results are as follows Figure 14 As shown, with the extension of time, the weight gain of mice in the Control group and TCL group was relatively slow and showed a certain degree of decrease, while the weight of mice in the DBCO-TCL group was not affected by the tumor and maintained a stable increase. Figure 14 (B in the text). On day 19, the mice were sacrificed, their lungs were dissected, and photographs were taken. Figure 14 In the C group, the DBCO-TCL group significantly inhibited the growth and metastasis of lung metastases, with a lung nodule number inhibition rate of 74.2%. Figure 14 (D in the middle).

[0278] Example 9: DBCO modification enhances the therapeutic effect of DC vaccine in a mouse 4T1 breast cancer orthotopic model.

[0279] 1. Construction of Babl / c mouse 4T1 mammary gland orthotopic tumor-bearing model

[0280] An orthotopic tumor-bearing model was established using 5-6 week old Babl / c mice. Hair was shaved from the right third mammary gland region of the mice, and 50 μL of insulin containing 5 × 10⁻⁶ mg / L was injected using an insulin syringe. 5 A suspension of 4T1 cells was injected into the subcutaneous fat pad of the third pair of mammary glands on the right side of mice. When the tumor grew to a palpable size, its dimensions were measured using calipers. The tumor volume was calculated using the formula: Volume = Major Axis × Minor Axis. 2 ×0.5. When the tumor volume reaches approximately 50 mm. 3 (Usually around the 4th day after tumor implantation) the mice are randomly assigned to different experimental groups and the corresponding treatment regimens are implemented.

[0281] 2. DC vaccine treatment of mouse orthotopic tumor-bearing models

[0282] Add TCL or DBCO-TCL (50µg TCL or DBCO-TCL per 200,000 BMDCs) to a PBS suspension of the same strain of BMDC, incubate at 37°C for 24 hours, wash thoroughly with PBS, and then prepare 10 BMDCs using PBS. 7A cell suspension of 100 μL / mL was administered to mice peritumoral injections, once every 5 days for a total of three injections. Tumor volume and body weight were measured every 3-4 days. In the control group, tumor volume reached 1000 cm³. 3 Mice were sacrificed to photograph tumors. After the photographs were taken, the tumors were divided into two halves. One half was stored at -80°C, and the other half was fixed with 4% paraformaldehyde.

[0283] Triple-negative breast cancer, due to the lack of specific therapeutic targets and its highly invasive nature, high recurrence and metastasis rates, and extremely poor prognosis (with a 5-year survival rate of only about 12%-15%), poses a significant threat to patients' lives and health, necessitating new treatment methods to improve patient outcomes. This embodiment uses a mouse 4T1 breast cancer orthotopic model to test the therapeutic effect of a DBCO-modified DC vaccine on breast cancer (treatment strategy as follows...). Figure 15 As shown in A in the figure). Consistent with other tumor models, a significant tumor suppression effect was observed in the DBCO-TCL group ( Figure 15 (B in the image). The tumor anatomy diagram also shows that the tumors in the DBCO-TCL group were significantly smaller than those in other groups. Figure 15 The D and E values ​​in the text indicate the universality of DBCO modification in promoting the efficacy of DC vaccines.

[0284] Example 10: DBCO-modified protein vaccine enhances the efficacy against lung metastases.

[0285] 1. Construction of a C57BL / 6 mouse model of B16-OVA-mediated cutaneous melanoma lung metastasis

[0286] A lung metastasis model was established using 5-6 week old C57BL / 6 mice. 200 μL of a solution containing 2×10⁻⁶ ions was injected via the tail vein. 5 B16-OVA cell suspension was injected into mice. On the 4th day after tumor implantation, the mice were randomly assigned to different experimental groups and the corresponding treatment regimens were implemented. The weight of the mice was measured every 3-4 days, and the weight change curve was plotted.

[0287] 2. The therapeutic effect of DBCO-OVA combined with PD-1 monoclonal antibody on lung metastases

[0288] PD-1 monoclonal antibodies, as immune checkpoint inhibitors, restore the anti-tumor activity of T cells by blocking the PD-1 / PD-L1 signaling pathway, and have shown significant efficacy in the treatment of various tumors. However, the response rate of monotherapy is limited, which is related to insufficient infiltration of tumor antigen-specific T cells. Based on the significant effect of DBCO-OVA vaccine in enhancing antigen-specific T cell responses, this example further tests its synergistic anti-tumor effect when used in combination with PD-1 monoclonal antibodies.

[0289] Four days after lung metastasis modeling in mice, treatment began with tail vein injection of 100 μg OVA / DBCO-OVA, once every 5 days for a total of three times. One day after the first OVA / DBCO-OVA injection, tail vein injection of 1 mg / kg αPD-1 began, once every 5 days for a total of two times. Figure 16 (A) When the body weight of the control group mice decreased significantly, the lungs of the sacrificed mice were collected according to the method in Example 8.

[0290] In this experiment, the experimental groups received DBCO-OVA vaccine immunization (100 μg / dose) followed by α-PD-1 monoclonal antibody treatment (1 mg / kg) (48 hours apart). During the treatment period, the body weight of mice in each group remained stable. Figure 16 In group B), no significant toxic reactions (such as weight loss or activity inhibition) were observed in the combination therapy group, indicating that the combination therapy has good biocompatibility. After sacrifice on day 22, quantitative analysis of lung metastatic nodules showed that the number of nodules in the combination therapy group (DBCO-OVA + α-PD-1) was significantly reduced compared to the single-drug group. Figure 16 In the C and D groups, the nodule number inhibition rate reached over 72.3% (p<0.05). HE staining of lung tissue further indicated that DBCO-OVA or α-PD-1 alone had a certain inhibitory effect on lung metastases, but not as significant as the combined treatment group. The PBS control group showed significant tumor infiltration and alveolar structure destruction. Figure 16 (E in the text). The above results indicate that the combination of DBCO-OVA and PD-1 monoclonal antibody can synergistically enhance T cell infiltration and functional activation, thereby overcoming the tumor immunosuppressive microenvironment and providing a new strategy for the treatment of lung metastases.

[0291] 3. The preventive effect of DBCO-OVA vaccine on lung metastases

[0292] Mice were randomly assigned to groups and injected via tail vein with 100 μg OVA / DBCO three times, once every 5 days. Four days after the last treatment, tumor cells were injected via tail vein to construct a lung metastasis model according to the method in Example 8. Figure 17 (A) When the body weight of the control group mice decreased significantly, the lungs of the sacrificed mice were collected according to the method in Example 8.

[0293] This experiment tested the potential of DBCO-OVA vaccine as a prophylactic vaccine—by pre-inducing antigen-specific immune memory, it can block early colonization of tumor cells and the formation of metastatic lesions, thus providing a prospective protective strategy for high-risk populations. Using a C57BL / 6 mouse prophylactic immunization model, the experimental group received DBCO-OVA vaccine (100 μg / dose, intravenously, 5 days apart, for a total of 3 doses). After the last immunization, B16-OVA cells were injected intravenously to construct a lung metastasis model. Monitoring of mouse body weight changes during the experiment showed (…). Figure 17In the PBS and OVA groups, the body weight of mice gradually decreased over time, while the weight of the DBCO-OVA group remained relatively stable, suggesting that the vaccine effectively controlled the tumor. After sacrifice on day 35, quantitative analysis of lung metastatic nodules showed that the lung metastasis inhibition rate in the DBCO-OVA group reached 93.4% (n=5), an increase of 23.5% compared to the 69.8% in the OVA group (p<0.001). Figure 17 (C and D in the original text). Survival curves further confirmed that the median survival in the DBCO-OVA group was prolonged to 38 days, an improvement of 8.5 days compared to 29.5 days in the PBS group (p<0.0001). Figure 17 E in the middle). HE staining of lung tissue showed ( Figure 17 In the F group, the alveolar structure was intact, no obvious inflammatory damage was observed, and the tumor infiltration area was significantly reduced, indicating that it achieves long-term tumor monitoring through immune memory effect.

[0294] 5. Molecular mechanism by which DBCO-modified protein vaccines enhance lung anti-tumor immunity

[0295] Tumors, spleens, and lungs of mice after treatment were collected, and flow cytometry was used to analyze the changes in the number and function of immune cells in the tumor microenvironment.

[0296] Sample Preparation: Spleen: Place the spleen in a 6 cm dish, grind the steel mesh with the rubber side of a 5 mL syringe plunger, add 2 mL of 0.5% BSA to rinse, collect the cell suspension into a 15 mL centrifuge tube, centrifuge at 2000 rpm for 5 minutes to collect cells, then resuspend the cells in 3 mL of erythrocyte lysis buffer and lyse at room temperature for 3 minutes. Add sufficient 0.5% BSA to neutralize the lysis buffer. Centrifuge again at 2000 rpm for 5 minutes, collect the cells, and resuspend the cells in 1 mL of 0.5% BSA to obtain a spleen single-cell suspension. 2. Lung Metastases: The method for preparing single-cell suspensions of lung metastases is as described in Example 6, section 3.

[0297] The specific analysis and detection methods are the same as those in Example 7.

[0298] Changes in immune cell subsets in the lungs and spleen of mice after treatment were analyzed using flow cytometry. Results showed that DBCO-OVA significantly remodeled the immune landscape of the tumor microenvironment. In lung tissue, MHCII in the DBCO-OVA group... + CD11c + The proportion of DCs reached 62.52±7.11%, which was 1.6 times higher than that of the PBS group (38.22±8.91%) (p<0.05), indicating that DBCO modification significantly promoted the differentiation of DCs into the mature phenotype. Figure 18(A) Although there was no significant difference in the proportion of T cell subsets in peripheral blood among the groups (p>0.05) Figure 18 (BD in the lungs), while in the lungs, the DBCO-OVA group had CD3 + The proportion of T cells was 41.13±5.12%, significantly higher than that in the PBS group (25.50±0.96%, p<0.01) and the OVA group (29.88±2.89%, p<0.05). Figure 18 (in E), where CD4 + The proportion of T cells was 20.97±3.25%, which was 1.5-fold and 1.4-fold higher than that in the PBS group (13.79±1.01%, p<0.05) and the OVA group (15.04±0.68%, p<0.05), respectively. Figure 18 (FH in CD8) + The proportion of T cells was 20.16±2.14%, which was 1.7-fold and 1.4-fold higher than that in the PBS group (11.70±0.03%, p<0.01) and the OVA group (14.84±2.63%, p<0.05), respectively. Figure 18 The difference in T cell infiltration in the blood and lungs suggests that the immune activation effect of DBCO-OVA is tissue-specific. Next, the difference in effector T cells in the lungs was tested. The DBCO-OVA group showed increased IFN-γ in the lungs. + CD4 + The proportion of T cells reached 15.39±3.11%, which was 4.2-fold and 1.8-fold higher than that in the PBS group (3.63±1.81%, p<0.05) and the OVA group (8.75±5.18%, p<0.05), respectively. Figure 18 (IJ in the middle); IFN-γ + CD8 + The proportion of T cells was 17.74±1.67%, which was 3.4-fold and 2.0-fold higher than that in the PBS group (5.26±2.84%, p<0.01) and the OVA group (8.88±4.08%, p<0.05), respectively. Figure 18 (KL in the sample). The above results indicate that DBCO modification synergistically remodels the tumor immune microenvironment by enhancing DC maturation and T cell infiltration, providing a key cellular basis for anti-tumor effects.

[0299] The long-term antitumor immune memory effect induced by DBCO-OVA vaccine was evaluated using a multicolor flow cytometry system, with a focus on analyzing the dynamic changes of tissue-resident memory T cells (TRM) in the lungs and effector memory T cells (TEM) in the spleen. The results showed that in the lungs, DBCO-OVA vaccine significantly promoted CD8 cell growth. + CD44 + CD103 + CD69 + The enrichment of TRM cells reached 17.1±5.1%, significantly higher than that in the PBS group (4.2±0.9%, p<0.01) and the OVA group (8.2±2.2%, p<0.05), representing increases of 4.1-fold and 2.1-fold, respectively. Figure 19 (A and B in the original text). This result indicates that DBCO modification can effectively enhance the local residence of TRM cells in the lungs, thereby strengthening local immune surveillance capabilities. Simultaneously, in the spleen, the DBCO-OVA vaccine significantly promoted CD4... + and CD8 + Systematic expansion of TEM cells ( Figure 19 (C and D in the text). Among them, CD4 + The proportion of TEM cells reached 25.8±2.5%, significantly higher than that of the PBS group (17.1±2.1%, p<0.001) and the OVA group (20.2±2.5%, p<0.05), increasing by 1.5-fold and 1.3-fold, respectively; CD8 + The proportion of TEM cells significantly increased to 27.3±6.2%, significantly higher than that in the PBS group (6.8±1.2%, p<0.0001) and the OVA group (15.6±2.5%, p<0.01), representing increases of 4.0-fold and 1.7-fold, respectively. In summary, the DBCO-OVA vaccine not only enhanced the immune surveillance function of lung tissue through local enrichment of TRM cells but also established robust systemic immune memory through the systemic expansion of TEM cells, thereby effectively inducing long-term anti-tumor immune memory. These results fully demonstrate the significant advantages of the DBCO-OVA vaccine in inducing tumors with anti-immune memory.

[0300] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Application of dibenzocyclooctylene-modified tumor antigens in the preparation of antitumor vaccines; The tumor antigen modified with dibenzocyclooctyn is obtained by reacting diphenylcyclooctyn-tetraethylene glycol-active ester with the tumor antigen.

2. The application according to claim 1, characterized in that, The tumor antigen is a protein antigen.

3. The application according to claim 2, characterized in that, The molar ratio of diphenylcyclooctyne-tetraethylene glycol-active ester to the protein antigen is 8-12:1, preferably 9-11:

1.

4. The application according to claim 1, characterized in that, The tumor antigen is a tumor cell lysis buffer, preferably a B16-F10 cell lysis buffer.

5. The application according to claim 4, characterized in that, The mass ratio of the diphenylcyclooctyn-tetraethylene glycol-active ester to the tumor cell lysis buffer is 1:4-8, preferably 1:5-7.

6. The application according to any one of claims 1-5, characterized in that, The preparation method of the dibenzocyclooctylene-modified tumor antigen includes the following steps: (1) Dissolve the tumor antigen in a buffer solution with a pH of 8.5-9.5, add the diphenylcyclooctyn-tetraethylene glycol-active ester, add dimethyl sulfoxide, and stir at 0℃-8℃ in the dark for 6h-10h; (2) Add NH4Cl and stir at 0℃-8℃ in the dark for 1-3 hours to terminate the reaction. After purification, the tumor antigen modified by dibenzocyclooctylene is obtained. Preferably, the buffer solution is a 0.09 mol / L to 0.11 mol / L Na₂CO₃ / NaHCO₃ buffer solution; Preferably, the concentration of the tumor antigen in the buffer solution is 1 mg / mL to 3 mg / mL; Preferably, the volume concentration of the dimethyl sulfoxide in the reaction system is 8%-12%; Preferably, the concentration of NH4Cl in the reaction system is 45 mmol / L-55 mmol / L.

7. The application according to any one of claims 1-5, characterized in that, The tumors are melanoma, breast cancer, and lung metastases.

8. An anti-tumor vaccine, characterized in that, It is obtained by incubating the tumor antigen modified with dibenzocyclooctylene as described in any one of claims 1-7 with dendritic cells.

9. The anti-tumor vaccine according to claim 8, characterized in that, The ratio of the dibenzocyclooctylene-modified tumor antigen to dendritic cells is 1µg-5µg: 10,000 cells, preferably 2µg-3µg: 10,000 cells; And / or, the incubation conditions include: a temperature of 35°C-40°C and a time of 20-28 hours.

10. The use of the dibenzocyclooctylene-modified tumor antigen as described in any one of claims 1-7, or the antitumor vaccine as described in claim 8 or 9, in the preparation of a medicament for treating or preventing tumors.

11. The use of the dibenzocyclooctylene-modified tumor antigen combined with an immune checkpoint inhibitor as described in any one of claims 1-7 in the preparation of a medicament for treating or preventing tumors; preferably, the immune checkpoint inhibitor is a PD-1 inhibitor.

12. The application according to any one of claims 10 or 11, characterized in that, The tumors are melanoma, breast cancer, and lung metastases.

13. A combination drug for the prevention and / or treatment of tumors, characterized in that, Its active ingredients include the dibenzocyclooctylene-modified tumor antigen and immune checkpoint inhibitor as described in any one of claims 1-7; The tumor antigen and immune checkpoint inhibitor modified with dibenzocyclooctylene can be used as independent drug delivery units, or the tumor antigen and immune checkpoint inhibitor modified with dibenzocyclooctylene can be used together to form a combined drug delivery unit. Preferably, the immune checkpoint inhibitor is a PD-1 inhibitor.

14. The use of the dibenzocyclooctylene-modified tumor antigen as described in any one of claims 1-7, or the antitumor vaccine as described in claim 8 or 9, in the preparation of a medicament, wherein the medicament is capable of enhancing the antigen presentation efficiency of antigen-presenting cells; And / or, the drug can enhance the killing ability of cytotoxic T cells against tumor cells; And / or, the drug can enhance cellular immunity; And / or, the drug can enhance humoral immunity; And / or, the drug can activate the NF-κB / iNOS immune pathway; And / or, the drug can enhance lung anti-tumor immunity.

15. The use of the dibenzocyclooctylene-modified tumor antigen as described in any one of claims 1-7, or the antitumor vaccine as described in claim 8 or 9, in the preparation of a medicament, wherein the medicament is capable of promoting the uptake of the antigen by dendritic cells; And / or, the drug can enhance the ability of dendritic cells to present antigens; And / or, the drug can promote the maturation of dendritic cells; And / or, the drug can increase the cytokine content in the dendritic cell supernatant, the cytokines being IL-1β, IL-6, IL-12p60 and TNF-α; And / or, the drug can enhance the targeted delivery of antigens to secondary lymphoid organs and tumor-prone sites; And / or, the drug can increase the titer of IgG antibodies in serum, preferably the titer of IgG1 antibodies; And / or, the drug can increase the number of T cells within the tumor; And / or, the drug can increase the number of mature dendritic cells within the tumor; And / or, the drug can increase the number of T cells in lung tissue; And / or, the drug can increase the number of mature dendritic cells in lung tissue; And / or, the drug can upregulate CD8 + The secretion levels of IFN-γ and granzyme B by T cells; And / or, the drug can promote the formation and expansion of spleen effector memory T cells; And / or, the drug can increase the enrichment of tissue-resident memory T cells in the lungs.

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