Protein degradation-inducing chimeric molecular tumor vaccine

Abstract

This specification discloses compounds comprising an active agent covalently linked to a tumor antigen. It also discloses nanoparticles comprising a tumor antigen covalently linked to an active agent and a plurality of lipids or lipidoids.

Description

Cross-reference of related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 538,122, filed on 13 September 2023; the contents of the application are incorporated herein by reference in their entirety. [Background technology]

[0002] Cytotoxic T cells initiate a robust adaptive immune response by recognizing peptide-major histocompatibility complex (MHC) class I complexes on the surface of antigen-presenting cells (APCs). In vaccine-based cancer immunotherapy, tumor antigen processing and MHC-I cross-presentation are crucial steps. Specifically, antigens in cancer vaccines must be 1) taken up by endocytosis by APCs, 2) transported from endolysosomes into the cytoplasm, and further 3) broken down into short peptides (approximately 8-13 amino acids) via the ubiquitin-proteasome pathway (UPP). These peptide fragments are then loaded into MHC-I binding grooves and positioned on the APC surface for cross-priming. Strategies using cell-permeable peptides, "proton sponge" polymers, and pore-forming molecules have been developed to promote endolysosomal escape of tumor antigens and enhance their immunogenicity. However, due to the limited efficiency of cytosolic transport, the development of highly effective antitumor vaccines remains a challenge. Improving endolysosome extrusion can increase the amount of tumor antigen in the cytosol, thereby increasing MHC-I pathway-mediated cross-presentation. This also increases the risk of leakage of digestive enzymes that cause cell necrosis from endolysosomes. Therefore, maximizing the conversion of extruded tumor antigens to antigenic peptides is expected to enhance antitumor activity, but this has been little studied.

[0003] In UPP-mediated proteolysis, the rate-limiting step is the recruitment of E3 ubiquitin ligase to the protein of interest (POI). However, because there is no mechanism for the escaped tumor antigen to bind to the E3 ubiquitin ligase, UPP-mediated degradation of tumor antigens into antigenic peptides is not an efficient process. Furthermore, the macromolecular crowding effect within living cells further reduces the probability of collision between tumor antigens and E3 ubiquitin ligase. [Overview of the Initiative] [Means for solving the problem]

[0004] Activation of cytotoxic T cells typically requires the presentation of tumor antigen-derived epitopes on MHC class I cells expressed by antigen-presenting cells (APCs). Ubiquitin-proteasome pathway (UPP)-mediated proteolysis (degradation), i.e., antigen processing, of tumor antigens is a crucial step. Directing tumor antigens to the UPP can be an alternative pathway to enhance antigen processing and cross-presentation. This specification discloses a targeted antigen degradation-based tumor vaccine (TAgD-TVac) using tumor antigens pre-fused with proteolysis-targeting chimeras (PROTACs). When delivered using lymph node-targeted lipid nanoparticles, the PROTAC conjugation targets tumor antigens to the UPP in APCs under the induction of E3 ligase ligands, thereby increasing antigen cross-presentation and initiating robust cytotoxic T cell activation. TAgD-TVac, prepared using related tumor lysates, has been demonstrated to induce potent antigen-specific adaptive immunity and immunological memory against a variety of solid tumors, including malignant melanoma, triple-negative breast cancer, Lewis lung cancer, and (clinically inoperable) ovarian cancer, indicating its potential for clinical application.

[0005] In one embodiment, the present disclosure provides a compound having a structure represented by formula I, or a pharmaceutically acceptable salt thereof: [ka] [In the formula, A is a tumor antigen; E is the active agent (e.g., a drug); Y is a linker that covalently connects A to E, and includes alkylene, alkenylene, alkynylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene, or a combination thereof, and the linker contains 1 to 100 carbon atoms, preferably 10 to 50 carbon atoms.

[0006] In another embodiment, the disclosure provides nanoparticles comprising a tumor antigen covalently linked to an active agent, and a plurality of lipids or lipidoids.

[0007] In another embodiment, the present disclosure provides a pharmaceutical composition comprising nanoparticles described herein and pharmaceutically acceptable excipients.

[0008] In another aspect, the Disclosure provides a method for treating cancer in a subject requiring such treatment, comprising administering a therapeutically effective amount of a compound or nanoparticle described herein.

[0009] In another aspect, the Disclosure provides a method for treating an infectious disease in a subject requiring such treatment, comprising administering a therapeutically effective amount of a compound or nanoparticle described herein. [Brief explanation of the drawing]

[0010] [Figure 1]Figure 1 shows a schematic diagram illustrating that a targeted antigen degradation-based tumor vaccine (TAgD-TVac) enhances antigen cross-presentation and induces a robust anti-tumor immune response. TAgD-TVac is prepared by loading pre-fused PROTAC tumor antigen onto adjuvant lipid nanoparticles (LNPs) with lymph node (LN) targeting capability. The pre-fused PROTAC tumor antigen is obtained by covalently modifying the tumor antigen with (S,R,S)-AHPC. After being taken up by antigen-presenting cells (APCs), the pre-fused PROTAC tumor antigen is released from the LNP and binds to E3 ubiquitin ligase, where it undergoes ubiquitination and proteasome-mediated antigen degradation. The resulting tumor antigen peptide binds to major histocompatibility complex class I (MHC-I) and induces robust cytotoxic T cell cross-priming. TAgD-TVac shows potent antitumor therapeutic outcomes in (subcutaneous) breast cancer, (orthotopic) Lewis lung cancer, and (unresectable) ovarian cancer. [Figure 2A] Figures 2A-2K show the preparation of TAgD-TVac and the enhancement of cross-presentation in DCs by improved proteasome-mediated antigen degradation. Figure 2A shows the preparation of pre-fused PROTAC tumor antigens using 2-aminothiophene (Trout's reagent) and sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) as a linker. The antigen is modified with Trout's reagent and then reacted with a maleimidized analog of (S,R,S)-AHPC. [Figure 2B] Figure 2B shows fluorescence images of a mixture of pre-fused AHPC-GFP-Cre (green) and von Hippel-Lindau protein (pVHL, red), where pVHL was pre-labeled with RhB. The pre-fused AHPC-GFP-Cre and pVHL were mixed in a molar ratio of 1.5:1 and incubated at 37°C for 2 hours. Scale bar, 5 μm. [Figure 2C]Figure 2C shows that pVHL was immobilized on the surface of PLGA NPs to evaluate the interaction between pre-fused AHPC-GFP-Cre and pVHL. [Figure 2D] Figure 2D shows the agarose gel electrophoresis results of pre-fused AHPC-GFP-Cre after incubation with pVHL-modified PLGA NPs. A mixture of GFP-Cre, AHPC, and pVHL-modified PLGA NPs is used as a control. [Figure 2E] Figure 2E shows the chemical structure of the adjuvant (113-O12B) lipidoid. [Figure 2F] Figure 2F shows typical transmission electron microscope (TEM) images of adjuvant LNPs and TAgD-TVac. Scale bar: 200 nm. [Figure 2G] Figure 2G shows representative immunofluorescence images of DC2.4 cells after 12 hours of incubation with GFP-Cre+AHPC LNPs and pre-fused AHPC-GFP-Cre LNPs. LNP / protein = 10 / 1 (w / w). Pre-fused AHPC-GFP-Cre fluoresces green. pVHL is stained with anti-VHL antibody, followed by staining with a fluorescent secondary antibody. Scale bar, 10 μm. [Figure 2H] Figure 2H shows the degradation kinetics of GFP-Cre in DC2.4 cells after incubation with different LNP formulations for 3 hours. GPF-Cre fluorescence was monitored for 24 hours (h). [Figure 2I] Figure 2I shows Western blotting analysis of cellular OVA levels in bone marrow-derived dendritic cells (BMDCs) after incubation with different LNP formulations for 3 hours. Cellular OVA levels are assessed after a further 24-hour incubation. LNP / protein = 10 / 1 (w / w). [Figure 2J] Figure 2J shows representative flow cytometry analyses of H2kb-SIINFEKL expression in BMDCs after different in vitro treatments. [Figure 2K]Figure 2K shows representative flow cytometry and statistical analyses of H2kb-SIINFEKL expression in BMDCs after different in vitro treatments. Data are presented as mean ± standard deviation (sd) from n independent experiments (n=3). Statistical significance was analyzed by one-way ANOVA with Tukey's multiple comparison test (***p<0.001, ****p<0.0001). [Figure 3A] Figures 3A-3R demonstrate that TAgD-TVacOVA targets lymph nodes (LNs) and induces robust antitumor immunity in vivo. Figure 3A shows representative IVIS images of fluorescence distribution in BALB / c mice 6 hours after subcutaneous (SC) injection of Cy5.5-labeled ALC-0315OVA and TAgD-TVacOVA. Pre-fused AHPC-OVA is pre-labeled with Cy5.5. [Figure 3B] Figure 3B shows representative ex vivo fluorescence images of major organs. [Figure 3C] Figure 3C shows the ratio of radiance in the inguinal lymph nodes (LN) and the liver. [Figure 3D] Figure 3D shows that TAgD-TVacOVA reaches the lymphatic nephrite (LN) via the afferent lymphatic vessel. [Figure 3E] Figure 3E shows Western blotting analysis of OVA levels in LN after administration of different LNP formulations. A mixture of OVA and LN tissue lysates is used as a positive control (PC). [Figure 3F] Figure 3F shows Western blotting analysis of OVA levels in LN after administration of different LNP formulations. A mixture of OVA and LN tissue lysates is used as a positive control (PC). [Figure 3G]Figure 3G illustrates the mechanism of subcellular analysis of Cre-mediated gene recombination in Ai14 mice. The gene encoding tdTomato is blocked by a stop gene between two loxP segments. The loxP gene is cleaved when Cre recombinase is delivered to the cytoplasm. Subsequently, the tdTomato gene is activated, and the cell expresses tdTomato, which exhibits red fluorescence. [Figure 3H] Figure 3H shows the percentage of tdTomato-positive (tdTomato+) cells in various types of immune cells after SC injection of GFP-Cre-loaded adjuvant LNPs. Unprotected GFP-Cre is used as a control. [Figure 3I] Figure 3I shows macrophage depletion in mice by injection of Clodrosome®. The mice were treated with subcutaneous administration of Clodrosome® 18 hours (-18h) prior to administration of Cy5.5-labeled TAgD-TVacOVA. Clodrosome® is a well-characterized liposomal drug used to deplete macrophages in either local tissue or peripheral blood, depending on the injection route. [Figure 3J] Figure 3J shows the ex vivo fluorescence image of LN isolated 6 hours (6h) after injection of Cy5.5-labeled TAgD-TVacOVA. [Figure 3K] Figure 3K shows a schematic timeline of B16F10-OVA tumor inoculation and vaccination in the C57BL / 6 mouse model. B16F10-OVA cells (1 × 10⁶ per mouse) were subcutaneously injected into the left flank of C57BL / 6 mice. [Figure 3L] Figure 3L shows individual tumor growth curves after administration of different LNP formulations. [Figure 3M] Figure 3M shows the average tumor growth kinetics after administration of different LNP formulations. [Figure 3N] Figure 3N shows the survival rate of B16F10-OVA tumor-bearing mice. [Figure 3O] Figure 3O shows flow cytometry analysis of H2kb-SIINFEKL expression in DCs (gated with CD11C+ cells) within the LN after different treatments. [Figure 3P] Figure 3P shows flow cytometry analysis of the CD8+ T cell population (gated with CD45+CD3+ cells) in tumor tissue. [Figure 3Q] Figure 3Q shows flow cytometry analysis of GzmBhigh expression in CD8+ T cells (gated with CD45+CD3+ cells) in tumor tissue. [Figure 3R] Figure 3R shows flow cytometry analysis of CD62LowCD44High T cells (gated with CD3+CD8+ cells) in the spleen. Data are shown as mean ± standard deviation (sd) from n independent experiments (n=3 for c, f, h, o, p, q, and r; n=4 for j; n=6 for m and n). Statistical significance was analyzed by one-way ANOVA with Tukey's multiple comparison test for c, f, o, p, q, and r; two-way ANOVA with Tukey's test for h and m; Log-rank (Mantel-Cox) test for n; and t-test for c and j (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 4A] Figures 4A-4L show that TAgD-TVac4T1 induces robust antitumor immunity and suppresses lung metastasis in a breast cancer model. Figure 4A shows a schematic timeline of 4T1-Luc tumor inoculation and vaccination in a BALB / c mouse model. 4T1-Luc tumor cells (1 × 10⁶ per mouse) were subcutaneously injected into the left flank of the mice. [Figure 4B] Figure 4B shows in vivo bioluminescence imaging of 4T1-Luc tumor growth after administration of different LNP formulations. [Figure 4C]Figure 4C shows the individual tumor growth curves. CR: complete response. [Figure 4D] Figure 4D shows the mean tumor growth dynamics. [Figure 4E] Figure 4E shows the survival rate of 4T1-Luc tumor-bearing mice. [Figure 4F] Figure 4F shows flow cytometry analysis of CD80 and CD86 expression in DCs (gated with CD11C+ cells) within the LN after different treatments. [Figure 4G] Figure 4G shows flow cytometry analysis of the CD8+ T cell population (gated with CD45+CD3+ cells) in tumor tissue. [Figure 4H] Figure 4H shows flow cytometry analysis of GzmBhigh expression in CD8+ T cells (gated with CD45+CD3+ cells) in tumor tissue. [Figure 4I] Figure 4I shows flow cytometry analysis of CD62LowCD44High T cells (gated with CD3+CD8+ cells) in the spleen. [Figure 4J] Figure 4J shows a schematic diagram of the tumor rechallenge experiment design. Mice, treated with different methods, are rechallenged with 4T1-Luc cells (1 × 10⁶ per mouse) via IV injection on day 18. [Figure 4K] Figure 4K shows bioluminescence images of mice after re-challenge with 4T1-Luc tumor cells. [Figure 4L] Figure 4L shows bioluminescence images of the lung after rechallenge with 4T1-Luc tumor cells. Data are shown as mean ± standard deviation (sd) from n independent experiments (n=6 for d and e; n=3 for f, g, h, and i). Statistical significance was analyzed by one-way ANOVA with Tukey's multiple comparison test for f, g, h, and i; two-way ANOVA with Tukey's test for d; and log-rank (Mantel-Cox) test for e (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 5A] Figures 5A-5M show the results of TAgD-TVac LLC in combination with ICB therapy for orthotopic Lewis lung cancer. Figure 5A shows a schematic timeline of LLC-Luc tumor inoculation (0.5 × 10⁶ per mouse) and treatment in a C57BL / 6 mouse model. [Figure 5B] Figure 5B shows in vivo bioluminescence imaging of LLC-Luc tumor growth after administration of different formulations. [Figure 5C] Figure 5C shows ex vivo bioluminescence imaging and photographs of lungs collected from different treatment groups (taken with an iPhone 14 Pro Max). [Figure 5D] Figure 5D shows ex vivo bioluminescence imaging and photographs of lungs collected from different treatment groups (taken with an iPhone 14 Pro Max). [Figure 5E] Figure 5E shows the body weight of LLC-Luc tumor-bearing mice at different time points. Mice were euthanized if they showed a weight loss of more than 15% from baseline (reaching the human endpoint). [Figure 5F] Figure 5F shows the survival rate of LLC-Luc tumor-bearing mice. [Figure 5G] Figure 5G shows flow cytometry analysis of CD80 and CD86 expression in DCs (gated with CD11C+ cells) within the LN after different treatments. [Figure 5H] Figure 5H shows the IL-6 levels in the blood. [Figure 5I] Figure 5I shows flow cytometry analysis of CD62LowCD44High T cells (gated with CD3+CD8+ cells) in the spleen. [Figure 5J] Figure 5J shows a schematic diagram of the design for a tumor rechallenge experiment. [Figure 5K] Figure 5K shows that LLC-Luc cells (1 × 10⁶ per mouse) were subcutaneously injected into the left flank of C57BL / 6 mice, and B16F10 tumor cells (1 × 10⁶ per mouse) were inoculated into the flank opposite to the LLC-Luc tumor. [Figure 5L]Figure 5L shows photographs of LLC-Luc tumors and B16F10 tumors. [Figure 5M] Figure 5M shows the mean tumor growth dynamics. TFS: tumor-free survival (percentage of animals showing complete regression at the end of the study). Data are shown as mean ± standard deviation (sd) from n independent experiments (n=6 for e and f; n=3 for g, h, and i; n=5 for m). Statistical significance was analyzed by one-way ANOVA using Tukey's multiple comparison test for g, h, and i; two-way ANOVA using Tukey's test for e and m; and the Log-rank (Mantel-Cox) test for f (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 6A] Figures 6A-6O show the results of TAgD-TVacID8 in combination with ICB therapy for inoperable ovarian cancer. Figure 6A shows a schematic timeline of ID8-Luc tumor inoculation and treatment in a C57BL / 6 mouse model. To establish an inoperable ovarian cancer model, ID8-Luc (1 × 10⁷ per mouse) tumor cells were intraperitoneally injected into female C57BL / 6 mice. [Figure 6B] Figure 6B shows in vivo bioluminescence imaging of ID8-Luc tumor growth after administration of different formulations. [Figure 6C] Figure 6C shows ex vivo bioluminescence imaging of the abdominal cavity and major organs collected from different treatment groups. [Figure 6D] Figure 6D shows hematoxylin and eosin (H&E) staining of ovarian sections in different treatment groups. [Figure 6E] Figure 6E shows peritoneal images from different treatment groups (taken with an iPhone 14 Pro Max). [Figure 6F] Figure 6F shows the normalized intensity data analysis of bioluminescence signals in mice after different treatments. [Figure 6G]Figure 6G shows the survival rate of ID8-Luc tumor-bearing mice. Mice were euthanized if they developed ascites and gained more than 30% body weight. [Figure 6H] Figure 6H shows the volume measurement of the collected ascites fluid. [Figure 6I] Figure 6I shows flow cytometry analysis of CD80 and CD86 expression in DCs (gated with CD11C+ cells) within the LN after different treatments. [Figure 6J] Figure 6J shows the levels of pro-inflammatory cytokines (M-CSF) in the blood. M-CSF: Macrophage colony-stimulating factor. [Figure 6K] Figure 6K shows the levels of pro-inflammatory cytokines (IFN-γ) in the blood. IFN-γ: Interferon-γ. [Figure 6L] 6L indicates the level of pro-inflammatory cytokine (IL-6) in the blood. IL-6: Interleukin 6. [Figure 6M] Figure 6M shows the levels of pro-inflammatory chemokine (MCP-1). MCP-1: Monocyte chemoattractant protein-1. [Figure 6N] Figure 6N shows the levels of the pro-inflammatory chemokine (MIP-1α). MIP-1α: Macrophage inflammatory protein-1α. [Figure 6O]Figure 6O shows the levels of the pro-inflammatory chemokine (MIP-1β). MIP-1β: Macrophage inflammatory protein-1β. Data are shown as mean ± sd from n independent experiments (n=6 for f and g; n=3 for h, i, j, k, l, m, n, and o). Statistical significance was analyzed by one-way ANOVA using Tukey's multiple comparison test for h, i, j, k, l, m, n, and o; two-way ANOVA using Tukey's test for f; and log-rank (Mantel-Cox) test for g (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 7A] Figures 7A-7C show the characterization of adjuvant LNPs and their lymph node (LN) targeting ability. Figure 7A shows the particle size of the adjuvant LNPs. [Figure 7B] Figure 7B shows a representative IVIS image of the fluorescence distribution in BALB / c mice 6 hours after SC injection of DiR-labeled adjuvant LNP into the tail base. [Figure 7C] Figure 7C shows the quantitative analysis of bioluminescence signals in LN. Data are presented as mean ± standard deviation (sd) from n independent experiments (n=3). [Figure 8A] Figures 8A–8F show TAgD-TVacOVA-mediated proteasomal degradation of OVA in BMDCs. Figure 8A shows flow cytometry analysis of the internal distribution of different formulations by DC2.4 cells by analyzing GFP fluorescence. [Figure 8B] Figure 8B shows a quantitative analysis of the colocalization of GFP and pVHL signals. [Figure 8C] Figure 8C shows a quantitative analysis of the colocalization of GFP and pVHL signals. The coefficient is close to 1 when they are highly colocalized. [Figure 8D] Figure 8D shows the SDS polyacrylamide gel electrophoresis of OVA and pre-fused AHPC-OVA. [Figure 8E] Figure 8E shows the fluorescence spectra of a mixture of pre-fused AHPC-OVA and pVHL after 1 hour incubation. For comparison, a mixture of OVA, AHPC, and pVHL was used. OVA and pVHL were pre-labeled with FITC and RITC, respectively. [Figure 8F] Figure 8F shows Western blotting analysis of OVA levels in the cytoplasm of BMDCs after incubation for 3 hours with different LNP formulations. OVA levels are assessed after a further 24-hour incubation. LNP / protein = 10 / 1 (w / w). Data are presented as mean ± standard deviation (sd) from n independent experiments (n=3). Statistical significance was analyzed by t-test (*p<0.05). [Figure 9A] Figures 9A-9C show TAgD-TVacOVA-mediated antigen cross-presentation in BMDCs. Figure 9A shows a fluorescence image of H2kb-SIINFEKL complex expression in BMDCs (stained with anti-CD11c antibody). [Figure 9B] Figure 9B shows flow cytometry analysis of the CD80+CD86+BMDC population after various treatments. [Figure 9C] Figure 9C shows flow cytometry analysis of the CD80+CD86+BMDC population after various treatments. Data are presented as mean ± standard deviation (sd) from n independent experiments (n=3). Statistical significance was analyzed by one-way ANOVA with Tukey's multiple comparison test (**p<0.01). [Figure 10A] Figures 10A-10C show the LN targeting ability of Cy5.5-labeled TAgD-TVacOVA in C57BL / 6 mice. Figure 10A shows a representative IVIS image of the fluorescence distribution in C57BL / 6 mice 6 hours after SC injection of Cy5.5-labeled TAgD-TVacOVA. Pre-fused AHPC-OVA is pre-labeled with Cy5.5. [Figure 10B] Figure 10B shows that TAgD-TVacOVA reaches the lymphatic nucleus (LN) via the afferent lymphatic vessel. [Figure 10C]Figure 10C shows ex vivo fluorescence images of major organs. [Figure 11] Figure 11 shows the delivery of GFP-Cre to the linden (LN) in Ai14 mice using adjuvant LNPs. The percentage of tdTomato-positive (tdTomato+) cells in various types of immune cells 48 hours after SC injection of GFP-Cre-loaded adjuvant LNPs into the tail root. Naked GFP-Cre was used as a control. [Figure 12A] Figures 12A and 12B show the antitumor effect of TAgD-TVacOVA in a melanoma mouse model. Figure 12A is a photograph of B16F10-OVA tumors in different treatment groups. [Figure 12B] Figure 12B shows hematoxylin and eosin (H&E) staining of tumor tissue. [Figure 13A] Figure 13A shows a gating strategy for identifying CD11C+ cells within lymph nodes. [Figure 13B] Figure 13B shows a gating strategy for identifying CD3+ T cells within tumor tissue. [Figure 13C] Figure 13C shows a gating strategy for identifying CD3+CD8+ T cells in the spleen. [Figure 14A] Figures 14A–14H show antitumor immunity induced by TAgD-TVacOVA in a melanoma mouse model. Figure 14A shows flow cytometry analysis of H2kb-SIINFEKL expression in DCs (gated with CD11C+ cells) within the LN after different treatments. [Figure 14B] Figure 4B shows flow cytometry analysis of CD80 and CD86 expression in DCs (gated with CD11C+ cells) within the LN. [Figure 14C] Figure 14C shows flow cytometry analysis of the CD8+ T cell population (gated with CD45+CD3+ cells) in tumor tissue. [Figure 14D]Figure 14D shows flow cytometry analysis of GzmBhigh expression in CD8+ T cells (gated with CD45+CD3+ cells) in tumor tissue. [Figure 14E] Figure 14E shows flow cytometry analysis of CD62LlowCD44high and CD62LhighCD44high T cells (gated with CD3+CD8+ cells) in the spleen. [Figure 14F] Figure 14F shows flow cytometry analysis of CD80 and CD86 expression in DCs (gated with CD11C+ cells) within the LN. [Figure 14G] Figure 14G shows flow cytometry analysis of CD62LlowCD44high and CD62LhighCD44high T cells (gated with CD3+CD8+ cells) in the spleen. [Figure 14H] Figure 14H shows the mean tumor growth dynamics in CD8+ T cell depleted mice. Data are shown as mean ± standard deviation (sd) from n independent experiments (n=3 for f and g; n=5 for h). Statistical significance was analyzed by one-way ANOVA with Tukey's multiple comparison test for f and g, and by two-way ANOVA with Tukey's test for h (***p<0.001, ****p<0.0001). [Figure 15A] Figures 15A and 15B show pro-inflammatory cytokines and chemokines in the blood of a melanoma mouse model. Figure 15A shows the levels of pro-inflammatory cytokines (including M-CSF, IFN-γ, and IL-6) after different treatments. M-CSF: macrophage colony-stimulating factor; IFN-γ: interferon-γ; IL-6: interleukin-6. [Figure 15B]Figure 15B shows the levels of pro-inflammatory chemokines (including MCP-1, MIP-1α, and MIP-1β). MCP-1: monocyte chemotactic protein-1; MIP-1α: macrophage inflammatory protein-1α; MIP-1β: macrophage inflammatory protein-1β. Data are presented as mean ± standard deviation (sd) from n independent experiments (n=3). Statistical significance was analyzed by one-way ANOVA with Tukey's multiple comparison test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 16] This study demonstrates the antitumor effect of TAgD-TVac4T1 in a mouse model of breast cancer. H&E staining of tumor tissue and lung tissue is shown. [Figure 17A] Figures 17A–17E show antitumor immunity induced by TAgD-TVac4T1 in a mouse model of breast cancer. Figure 17A shows flow cytometry analysis of CD80 and CD86 expression in DCs (gated with CD11C+ cells) within the LN. [Figure 17B] Figure 17B shows flow cytometry analysis of the CD8+ T cell population (gated with CD45+CD3+ cells) in tumor tissue. [Figure 17C] Figure 17C shows flow cytometry analysis of GzmBhigh expression in CD8+ T cells (gated with CD45+CD3+ cells) in tumor tissue. [Figure 17D] Figure 17D shows flow cytometry analysis of CD62LlowCD44high and CD62LhighCD44high T cells (gated with CD3+CD8+ cells) in the spleen. [Figure 17E] Figure 17E shows flow cytometry analysis of CD62LlowCD44high and CD62LhighCD44high T cells (gated with CD3+CD8+ cells) in the spleen. Data are presented as mean ± standard deviation (sd) from n independent experiments (n=3). Statistical significance was analyzed by one-way ANOVA with Tukey's multiple comparison test (*p<0.05, ***p<0.001). [Figure 18] This shows the antitumor effect of TAgD-TVacLLC in an orthotopic lung cancer mouse model. H&E staining of the lungs. [Figure 19A] Figures 19A-19E show antitumor immunity induced by TAgD-TVacLLC in an orthotopic lung cancer mouse model. Figure 19A shows flow cytometry analysis of CD80 and CD86 expression in DCs (gated with CD11C+ cells) within the LN. [Figure 19B] Figure 19B shows flow cytometry analysis of CD62LlowCD44high and CD62LhighCD44high T cells (gated with CD3+CD8+ cells) in the spleen. [Figure 19C] Figure 19C shows flow cytometry analysis of CD62LlowCD44high and CD62LhighCD44high T cells (gated with CD3+CD8+ cells) in the spleen. [Figure 19D] Figure 19D shows flow cytometry analysis of the CD8+ T cell population (gated with CD45+CD3+ cells) in LLC-Luc tumor tissue. [Figure 19E] Figure 19E shows flow cytometry analysis of the CD8+ T cell population (gated with CD45+CD3+ cells) in LLC-Luc tumor tissue. Data are presented as mean ± standard deviation (sd) from n independent experiments (n=3). Statistical significance was analyzed by one-way ANOVA with Tukey's multiple comparison test (**p<0.01, ***p<0.001, ****p<0.0001). [Figure 20A] Figures 20A-20C show the antitumor effect of TAgD-TVacID-8 in an inoperable ovarian cancer model. Figure 20A shows H&E staining of the ovary. T: tumor; F: follicle; CL: corpus lutea. [Figure 20B] Figure 20B shows H&E staining of the spleen. [Figure 20C]Figure 20C shows flow cytometry analysis of CD80 and CD86 expression in DCs (gated with CD11C+ cells) within the LN. [Modes for carrying out the invention]

[0011] Developing an effective tumor vaccine requires inducing robust antigen-specific T cell-based cellular immunity capable of targeting and eliminating tumor cells. Cytotoxic T cells initiate the immune response by recognizing peptide-major histocompatibility complex class I (MHC-I) complexes on the surface of antigen-presenting cells (APCs). Tumor antigen processing and MHC-I cross-presentation are crucial steps. Specifically, the antigen in the tumor vaccine must be 1) endocytotic by APCs, 2) transported from endolysosomes into the cytoplasm, and further 3) degraded into short peptides (approximately 8-13 amino acids) via the ubiquitin-proteasome pathway (UPP). These peptide fragments are then loaded into MHC-I binding grooves and positioned on the APC surface for cross-priming. Cell-permeable peptides are used to facilitate endolysosome extrusion of tumor antigens and enhance their immunogenicity. 4 Strategies using "proton sponge" polymers and pore-forming molecules have been developed. However, due to limited antigen presentation efficiency, the development of highly effective antitumor vaccines remains a challenge. Improving endolysosome escape can increase the amount of tumor antigen in the cytosol, thereby increasing MHC-I-dependent cross-presentation. This also increases the risk of digestive enzymes that cause cell necrosis leaking from the endolysosome. Therefore, maximizing the conversion of escaped tumor antigens to antigenic peptides is expected to improve antitumor activity, but this has been little studied.

[0012] In UPP-mediated proteolysis, the rate-limiting step is the recruitment of E3 ubiquitin ligase to the target protein (POI). However, because there is no mechanism for the escaped tumor antigen to bind to the E3 ubiquitin ligase, UPP-mediated degradation of tumor antigens to antigenic peptides is not actually an efficient process. Furthermore, the macromolecular crowding effect within living cells further reduces the probability of collision between tumor antigens and E3 ubiquitin ligase.

[0013] Recent advances in chemical biology have developed a powerful tool called proteolysis-targeting chimeras (PROTACs), which can target specific proteins for ubiquitination and proteasomal degradation by recruiting E3 ubiquitin ligases to point of interest (POIs). Inspired by these findings, we hereby demonstrate a targeted antigen degradation-based tumor vaccine (TAgD-TVac) using a pre-fused tumor antigen with a PROTAC that targets tumor antigens to UPPs for increased cross-presentation and robust cytotoxic T cell activation. TAgD-TVac was constructed by loading a pre-fused PROTAC tumor antigen onto adjuvant lipid nanoparticles (LNPs) with excellent lymph node (LN) targeting ability. The pre-fused PROTAC tumor antigen was obtained by coupling an E3 ubiquitin ligase ligand with the tumor antigen. Lymph nodes (LNs) are major organs responsible for immune surveillance and activation, and contain various lymphocytes, including T cells, B cells, and APCs. TAgD-TVac efficiently accumulated in LNs after subcutaneous (SC) administration (Figure 1). Mechanistic studies showed that adjuvant LNPs can reach LNs via lymphocyte homing, initially taken up by APCs (which include dendritic cells (DCs) and macrophages). In APCs, pre-fused PROTAC tumor antigens were released from LNPs and bound to E3 ubiquitin ligase under the induction of E3 ligase ligands, thereby undergoing ubiquitination and proteasome-mediated antigenic degradation. The resulting antigen peptides efficiently bound to MHC-I, inducing robust cytotoxic T cell cross-priming (Figure 1). The increased cross-presentation was confirmed by formulating TAgD-TVac using ovalbumin (OVA, as a model antigen). The inventors found that APC treated with TAgD-TVac showed stronger cross-presentation of OVA-derived peptides to MHC-I on its surface.As a result, TAgD-TVac induced enhanced cross-priming of cytotoxic T cells and potent antitumor immunity in an OVA-expressing melanoma mouse model.

[0014] To evaluate the generality and clinical translatability of TAgD-TVac, the inventors utilized TAgD-TVac to combat multiple types of solid tumors, including triple-negative breast cancer (subcutaneous tumor model), Lewis lung cancer (orthotopic tumor model), and ovarian cancer (clinically inoperable tumor model) (Figure 1). Tumor lysate protein (TLP) isolated from tumor cells was used to prepare personalized TAgD-TVac. The inventors found that TAgD-TVac significantly suppressed tumor growth, metastasis, and recurrence. Furthermore, TAgD-TVac can synergistically act with immune checkpoint blockade (ICB) therapy to further improve treatment outcomes, offering a promising method for increasing clinically objective response rates.

[0015] PROTVAC delivers pre-fused PROTAC tumor antigens to APCs, and then to UPPs via E3 ligase ligand induction, thereby increasing MHC-1 pathway-mediated antigen cross-presentation and initiating activation of cytotoxic T cells within the LN. Compared to conventional approaches to enhance tumor antigen cytosolic transport, our PROTVAC strategy does not require intentional disruption of endolysosomes, making it an attractive solution for increasing the immunogenicity of tumor antigens and inducing adaptive anti-tumor immunity.

[0016] Disclosed herein are exemplary individualized PROTVAC tumor vaccines using relevant tumor lysates, and their ability to induce potent antigen-specific adaptive immunity and immune memory against multiple solid tumors has been successfully demonstrated. Combining PROTVAC with ICB therapy further suppressed tumor growth, reduced the risk of tumor metastasis and recurrence, and retained the potential for clinical application. These efforts provide a new pathway for initiating adaptive immune responses via simple chemistry and nanotechnology, informing the design of effective cancer immunotherapies. Success in PROTAC-based vaccine technology may represent a significant breakthrough in bridging fundamental research of the ubiquitin-proteasome protein degradation system and vaccine development.

[0017] In one aspect, the present disclosure provides a compound having a structure represented by Formula I, or a pharmaceutically acceptable salt thereof: [Chemical Formula] [In the formula, A is a tumor antigen; E is an active agent (e.g., a drug); Y is a linker that covalently links A to E, comprising alkylene, alkenylene, alkynylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene, or a combination thereof, and the linker contains 1 to 100 carbon atoms, preferably 10 to 50 carbon atoms].

[0018] In certain embodiments, the linker is -CH2-, -C(=O)-, -C(=O)-NR 1 -, -NR 1 -C(=O)-, -C(=O)-O-, -O-C(=O)-, -NR 1 -C(=O)-NR 2 -, -O-C(=O)-NR 1 -, -NR 1 -C(=O)-O-, -O-, and -S-, and is a heteroalkylene comprising 5 to 50 moieties selected therefrom; each R 1 and R 2Each of these is independently selected from H and alkyl. In certain embodiments, the linker is a heteroalkylene comprising 5 to 50 moieties selected from -CH2- and -O-.

[0019] In certain preferred embodiments, the linker comprises ethylene glycol. In certain further preferred embodiments, the linker comprises 1 to 10 ethylene glycol moieties (e.g., 2, 4, 6, or 8 ethylene glycol moieties).

[0020] In certain embodiments, the linker is an alkylene containing 5 to 50 methylene moieties.

[0021] In certain embodiments, the linker is -C(=O)-, -C(=O)-NR 2 -, -NR 2 -C(=O)-, -C(=O)-O-, -OC(=O)-, -NR 2 -C(=O)-NR 3 -, -OC(=O)-NR 2 -, -NR 2 -C(=O)-O-, -O-, and -S- further include a portion selected from -C(=O)-O-, -O-, and -S-, where each R 2 and R 3 Each of these is independently selected from H and alkyl.

[0022] In certain embodiments, the linker has 1 to 5 -C(=O), -C(=O)-NR 2 -, or -NR 2 -C(=O)- parts (for example, one, two, or three parts), where each R 2 and R 3 Each is independently selected from H and alkyl. In certain preferred embodiments, at least one -C(=O)- moiety is adjacent to E.

[0023] In certain embodiments, the linker is [ka] This further includes: In certain embodiments, the linker is [ka] This further includes: In certain embodiments, the linker is [ka] It also includes.

[0024] In certain embodiments, the compound has a structure represented by formula Ia, or a pharmaceutically acceptable salt thereof: [ka] [In the formula, n1 and n2 are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. n3 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0025] In certain embodiments, the compound has a structure represented by formula Ib or a pharmaceutically acceptable salt thereof: [ka]

[0026] In certain embodiments, E is a ligand for a ligase (e.g., E3 ligase). In certain embodiments, E is a ligand for von Hippel-Lindau (VHL), a cellular inhibitor of apoptosis (cIAP), cereblon (CRBN), or murine double minute 2 (MDM2). In certain embodiments, E is [ka] [ka] [ka] [In the formula, each X 1 and X 2 O, NR are independent of each other. 4 Selected from S; Each R 3 Each is independently selected from halos (e.g., fluoro or bromo); Each R 4 [These are independently H, alkyl, or aralkyl] That is the case.

[0027] In certain embodiments, E is [ka] In another embodiment, E is [ka] In yet another embodiment, E is [ka] That is the case.

[0028] In certain embodiments, the compound has a structure represented by formula Ic, or a pharmaceutically acceptable salt thereof: [ka]

[0029] In certain embodiments, the compound has a structure represented by formula Id, or a pharmaceutically acceptable salt thereof: [ka]

[0030] In some embodiments, a tumor antigen is an antigenic substance produced in tumor cells that elicits an immune response in the host. In certain embodiments, A is a T antigen. In certain embodiments, the antigen is derived from a tumor (e.g., tumor cells). In certain embodiments, the antigen is derived from an infection pathogen (e.g., bacteria or viruses). In certain embodiments, the antigen is derived from prostate cancer tumor cells, liver tumor cancer cells, colorectal tumor cancer cells, ovarian tumor cancer cells, pancreatic tumor cancer cells, breast tumor cancer cells, melanoma tumor cancer cells, or bladder tumor cancer cells. In certain embodiments, the antigen is prostate-specific antigen (PSA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen 125 (CA-125), mucin-1 (MUC-1), melanoma-associated antigen (MAGE), or New York esophageal squamous cell carcinoma 1 (NY-ESO-1).

[0031] In another embodiment, the disclosure provides nanoparticles comprising a tumor antigen covalently linked to an active agent, and a plurality of lipids or lipidoids.

[0032] In certain embodiments, the tumor antigen covalently linked to the active agent has a structure represented by formula I: [ka] [In the formula, A is a tumor antigen; E is the active agent (e.g., a drug); Y is a linker (L) that covalently connects A to E, and includes alkylene, alkenylene, alkynylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene, or a combination thereof, and L contains 1 to 100 carbon atoms, preferably 10 to 50 carbon atoms.

[0033] In certain embodiments, the tumor antigen is [ka] It is covalently linked to the active drug via [a specific mechanism].

[0034] In certain embodiments, the active agent is a compound described herein.

[0035] In certain embodiments, the nanoparticles comprise a plurality of lipids. In certain embodiments, the nanoparticles comprise a plurality of lipids. In certain preferred embodiments, the nanoparticles comprise a plurality of lipids and lipidoids. In certain embodiments, the lipids and lipidoids are 113-O12B, cholesterol (Chol), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG). 2k Selected from the group consisting of ).

[0036] In another embodiment, the present disclosure provides a pharmaceutical composition comprising nanoparticles described herein and pharmaceutically acceptable excipients.

[0037] In another embodiment, the Disclosure provides a method for treating cancer in a subject requiring such treatment, comprising administering a therapeutically effective amount of a compound or nanoparticle described herein. In certain embodiments, the cancer is malignant melanoma, breast cancer, ovarian cancer, splenic cancer, or lung cancer. In certain embodiments, the method further comprises administering an immune checkpoint inhibitor.

[0038] In another embodiment, the Disclosure provides a method for treating an infection in a subject requiring such treatment, comprising administering a therapeutically effective amount of a compound or nanoparticle described herein. In a particular embodiment, the infection is a bacterial infection. In a particular embodiment, the infection is a viral infection.

[0039] Pharmaceutical composition The compositions and methods of the present invention can be used to treat individuals that require them. In certain embodiments, the individuals are mammals such as humans, or non-human mammals. When administered to animals such as humans, the compositions or compounds are preferably administered as a pharmaceutical composition comprising, for example, the compounds of the present invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions (e.g., water or physiologically buffered saline), or other solvents or vehicles (e.g., glycols, glycerols, oils such as olive oil, or injectable organic esters). In preferred embodiments, when such a pharmaceutical composition is for human administration, particularly for invasive administration routes (i.e., routes such as injection or transplantation that avoid transport or diffusion across the epithelial barrier), the aqueous solution is pyrogen-free or substantially pyrogen-free. Excipients can be selected, for example, to result in delayed release of the drug, or to selectively target one or more cells, tissues, or organs. Pharmaceutical compositions may be in the form of dosage unit forms such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, lyophilized products for reconstitution, powders, solutions, syrups, suppositories, and injections. Compositions may also be present in transdermal delivery systems (e.g., skin patches). Compositions may also be present in solutions suitable for topical administration, such as lotions, creams, or ointments.

[0040] A pharmaceutically acceptable carrier may contain, for example, a physiologically acceptable agent that acts to stabilize, increase the solubility of, or enhance the absorption of compounds such as the compounds of the present invention. Such physiologically acceptable agents include, for example, carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; or other stabilizers or excipients. The selection of a pharmaceutically acceptable carrier, including physiologically acceptable agents, depends, for example, on the route of administration of the composition. The formulation or pharmaceutical composition may be a selfemulsifying drug delivery system or a selfmicroemulsifying drug delivery system. The pharmaceutical composition (formulation) may be, for example, a liposome or other polymer matrix in which the compounds of the present invention can be incorporated therein. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively easy to produce and administer.

[0041] Pharmaceutical compositions (formulations) can be administered to a subject by any of a number of routes of administration, such as, for example, oral (e.g., aqueous or non-aqueous solutions or suspensions such as drenches, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingual); subcutaneous; transdermal (e.g., as a patch applied to the skin); and topical (e.g., as a cream, ointment, or spray applied to the skin). The compound may also be formulated for inhalation. In certain embodiments, the compound may simply be dissolved or suspended in sterile water. Details of suitable administration routes and compositions can be found, for example, in U.S. Patents 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970, and 4,172,896, and the patents cited therein.

[0042] The formulation may be conveniently provided in unit dosage forms and can be prepared by any method well known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to prepare a single dosage form varies depending on the host being treated and the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to prepare a single dosage form is usually the amount of the compound that produces the therapeutic effect. Generally, out of 100%, this amount is in the range of about 1% to about 99% of the active ingredient, preferably in the range of about 5% to about 70%, and most preferably in the range of about 10% to about 30%.

[0043] Methods for preparing these formulations or compositions include the step of associating an active compound, such as the compound of the present invention, with a carrier and optionally one or more accessory ingredients. Generally, formulations are prepared by homogeneously and closely associating the compound of the present invention with a liquid carrier, a fine solid carrier, or both, and then, if necessary, shaping the product.

[0044] Formulations of the present invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (flavored basis, usually using sucrose and acacia or tragacanth), lyophilized products, powders, granules, or as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or as lozenges (pastilles) (using an inert base such as gelatin and glycerin, or sucrose and acacia), and / or as mouthwash, each containing a predetermined amount of the compound of the present invention as an active ingredient. The composition or compound may also be administered as a bolus, lick, or paste.

[0045] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, sugar-coated tablets, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or bulking agents, e.g., starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, e.g., carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, e.g., glycerol; (4) disintegrants, e.g., agar, charcoal. (1) Calcium stearate, potato starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate, etc.; (5) Dissolution retarders, e.g., paraffin; (6) Absorption enhancers, e.g., quaternary ammonium compounds; (7) Wetting agents, e.g., cetyl alcohol and glycerol monostearate; (8) Absorbents, e.g., kaolin and bentonite clay; (9) Lubricants, e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; (10) Complexing agents, e.g., modified cyclodextrin and unmodified cyclodextrin; and (11) Coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical composition may also include buffering agents. Similar types of solid compositions may also be used as fillers in soft and hard-filled gelatin capsules with excipients such as lactose or milk sugar and high molecular weight polyethylene glycol.

[0046] Tablets can be prepared by compression or molding, with one or more optional auxiliary components. Compressed tablets can be prepared using a binder (e.g., gelatin or hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked carboxymethylcellulose sodium), a surfactant, or a dispersant. Molded tablets can be prepared by molding a mixture of powder compounds moistened with an inert liquid diluent using appropriate machinery.

[0047] Tablets, as well as other solid dosage forms of pharmaceutical compositions such as sugar-coated tablets, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored, or prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulation field. These may also be formulated to provide sustained or controlled release of the active ingredient therein, for example, by using varying proportions of hydroxypropyl methylcellulose, other polymer matrices, liposomes and / or microspheres to provide a desired release profile. They may be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating a sterilizer in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injection medium immediately before use. These compositions may also optionally contain an opaque agent, and may optionally release the active ingredient in a delayed manner, either only or preferentially, in specific parts of the gastrointestinal tract. Examples of embedding compositions that may be used include polymer substances and waxes. The active ingredient may also be in a microencapsulated form together with one or more of the excipients, where appropriate.

[0048] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophilized products for reconstitution, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms may contain, for example, inert diluents commonly used in the art, such as water or other solvents, cyclodextrins and their derivatives, solubilizers, and emulsifiers (e.g., ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, as well as mixtures thereof).

[0049] In addition to inert diluents, oral compositions may also contain adjuvants such as humectants, emulsifiers and suspending agents, sweeteners, flavorings, colorants, fragrances and preservatives.

[0050] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, hydroxyaluminum oxide (aluminum metahydroxide), bentonite, agar, and tragacanth, as well as mixtures thereof.

[0051] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants as needed.

[0052] Ointments, pastes, creams, and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof, in addition to the active compound.

[0053] Powders and sprays may contain excipients in addition to the active compound, such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures thereof. Sprays may further contain conventional propellants, such as chlorofluorocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

[0054] Transdermal patches offer the additional advantage of providing controlled delivery of the compounds of the present invention to the body. Such dosage forms can be prepared by dissolving or dispersing the active compound in a suitable medium. Absorption enhancers may also be used to increase the flux of the compound through the skin. The rate of such flux can be controlled by providing a rate-controlled membrane or by dispersing the compound in a polymer matrix or gel.

[0055] Pharmaceutical compositions suitable for parenteral administration include one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injection solutions or dispersions immediately before use, and these may contain antioxidants, buffers, bacteriostatic agents, solutes that make the blood and preparation of the target recipient isotonic, or suspending agents or thickeners.

[0056] Suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, and polyethylene glycol), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0057] These compositions may contain adjuvants such as preservatives, humectants, emulsifiers, and dispersants. The inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, and phenolsorbic acid, can ensure prevention of microbial action. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. Furthermore, the inclusion of absorption-delaying agents such as aluminum monostearate and gelatin can lead to longer absorption of injectable pharmaceutical formulations.

[0058] In some cases, it is desirable to delay the absorption of a drug from subcutaneous or intramuscular injection in order to prolong its effects. This can be achieved by using a liquid suspension of a crystalline or amorphous substance with low water solubility. In this case, the rate of drug absorption depends on the rate of dissolution, which in turn may depend on the crystal size and crystal form. Alternatively, delayed absorption of a parenterally administered drug form can be achieved by dissolving or suspending the drug in an oily vehicle.

[0059] Depot formulations for injection are prepared by forming a microencapsulation matrix of the target compound in a biodegradable polymer such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the ratio of drug to polymer and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoester) and poly(anhydride). Depot formulations for injection are also prepared by encapsulating the drug in liposomes or microemulsions that are compatible with body tissues.

[0060] For use in the method of the present invention, the active compound may be provided by itself or, for example, in combination with a pharmaceutically acceptable carrier, as a pharmaceutical composition containing 0.1 to 99.5% (more preferably 0.5 to 90%) of the active ingredient.

[0061] The delivery method may also be provided by a refillable or biodegradable device. In recent years, various sustained-release polymer devices have been developed and tested in vivo for the controlled delivery of drugs, including protein-based biopharmaceuticals. Various biocompatible polymers (including hydrogels), including both biodegradable and non-biodegradable polymers, can be used to form implants for the sustained release of compounds at specific target sites.

[0062] The actual dose levels of the active ingredient in a pharmaceutical composition may be varied to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration without causing toxicity to the patient.

[0063] The selected dosage level depends on a variety of factors, including the activity of the specific compound or combination of compounds used, or its ester, salt, or amide; the route of administration; the time of administration; the rate of excretion of the specific compound used; the duration of treatment; other drugs, compounds, and / or substances used in combination with the specific compound used; the age, sex, weight, condition, overall health, and medical history of the patient being treated; and similar factors well known in the medical field.

[0064] A physician or veterinarian with ordinary skill in the art can easily determine and prescribe the therapeutically effective dose of the required pharmaceutical composition. For example, a physician or veterinarian can start administration of the pharmaceutical composition or compound at a level lower than the level required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. "Therapeutic dose" means a concentration of the compound sufficient to produce the desired therapeutic effect. It is generally understood that the effective dose of a compound varies depending on the subject's weight, sex, age, and medical history. Other factors that influence the effective dose include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent administered with the compound of the present invention. A larger total dose may be delivered by multiple administrations of the drug. Methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, incorporated herein by reference).

[0065] Generally, the appropriate daily dose of the active compound used in the compositions and methods of the present invention is the amount of the compound that is the minimum effective dose to produce a therapeutic effect. Such an effective dose usually depends on the factors mentioned above.

[0066] If desired, an effective daily dose of the active compound may be administered separately at appropriate intervals throughout the day as one, two, three, four, five, six, or more subdoses, or optionally in unit dosage form. In certain embodiments of the present invention, the active compound may be administered two or three times a day. In preferred embodiments, the active compound is administered once a day.

[0067] Patients receiving this treatment include any animal in need, including primates, especially humans; as well as other mammals such as horses, cattle, pigs, sheep, cats and dogs; poultry; and pets in general.

[0068] In certain embodiments, the compounds of the present invention may be used alone or conjointly administered with other types of therapeutic agents.

[0069] This disclosure includes the use of pharmaceutically acceptable salts of the compounds of the present invention in compositions and methods of the present invention. In certain embodiments, the salts intended for the present invention include, but are not limited to, alkyl, dialkyl, trialkyl, or tetraalkylammonium salts. In certain embodiments, the salts intended for the present invention include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydravamin, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, the salts intended for the present invention include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts.In certain embodiments, the salts intended for use in the present invention include 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, l-ascorbic acid, l-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carboxylic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, and d-glucoic acid. This includes, but is not limited to, heptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphate, glycolic acid, hypolic acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, l-malic acid, malonic acid, mandelic acid, methanesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, l-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, l-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenate.

[0070] Pharmacopoecially acceptable acid addition salts can also exist as various solvates with water, methanol, ethanol, dimethylformamide, and others. Mixtures of such solvates can also be prepared. The source of such solvates may be derived from the crystallization solvent, inherent to the preparation or crystallization solvent, or associated with such solvent.

[0071] Wetting agents, emulsifiers, and lubricants (e.g., sodium lauryl sulfate and magnesium stearate), as well as colorants, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the composition.

[0072] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bicarbonate, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0073] definition Unless otherwise defined herein, scientific and technical terms used herein shall have the meanings commonly understood by those skilled in the art. Generally, the nomenclature and techniques used in relation to chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry described herein are commonly used and well known in the art.

[0074] The methods and techniques described herein are, unless otherwise indicated, carried out in accordance with conventional methods commonly known in the art and described in various general and more specific references cited and discussed herein. See, for example, “Principles of Neural Science”, McGraw-Hill Medical, New York, NY (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4th ed.”, WH Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed.”, WH Freeman & Co., NY (1999); and Gilbert et al., “Developmental Biology, 6th ed.”, Sinauer Associates, Inc., Sunderland, MA (2000).

[0075] Unless otherwise defined herein, chemical terms used herein are used in accordance with their prior art usage, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, CA (1985).

[0076] All of the above and any other publications, patents, and published patent applications referenced in this application are incorporated herein by reference. In case of any conflict, this specification shall prevail, including its specific definitions.

[0077] The term "agent" is used herein to refer to compounds (e.g., organic or inorganic compounds, mixtures of compounds), biomacromolecules (e.g., nucleic acids, antibodies (including parts thereof, as well as humanized, chimeric and human antibodies, and monoclonal antibodies), proteins or parts thereof, such as peptides, lipids, carbohydrates, etc.), or extracts made from biological materials such as bacterial, plant, fungal, or animal (especially mammalian) cells or tissues. Agents include, for example, agents with known structures and agents with unknown structures.

[0078] The terms "patient," "subject," or "individual" are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, domestic animals (including cattle and pigs), companion animals (e.g., dogs and cats), and rodents (e.g., mice and rats).

[0079] To “treat” a condition or patient means to take steps to obtain a beneficial or desired outcome, including clinical outcomes. Beneficial or desired clinical outcomes may include, but are not limited to, the alleviation or improvement of one or more symptoms or conditions, whether detectable or undetectable; reduction of disease severity; stabilization (i.e., non-exacerbation) of the disease; prevention of disease spread; delay or slowing of disease progression; improvement or alleviation of the disease state; and remission (whether partial or total), whether detectable or undetectable. “Treatment” may also mean extending survival compared to the survival expected without treatment.

[0080] The term “preventing” is recognized and well understood in the art when used in relation to conditions such as local recurrence (e.g., pain), diseases such as cancer, complex syndromes such as heart failure, or any other medical condition, and includes the administration of a composition that reduces the frequency of symptoms of a medical condition or delays its onset in a subject compared to a subject not administered the composition. Therefore, cancer prevention includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving prophylactic treatment by a statistically and / or clinically significant amount compared to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population compared to an untreated control population.

[0081] The "administration" of a substance, compound, or drug to a subject, or the "administration" thereof, can be carried out using one of the various methods known to those skilled in the art. For example, compounds or drugs can be administered intravenously, intra-arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, intraocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., via vascular ducts). Compounds or drugs can also be appropriately introduced by refillable or biodegradable polymer devices or other devices (e.g., patches and pumps) or formulations that provide sustained release, sustained release, or controlled release of the compound or drug. Administration can be, for example, one time, multiple times, and / or over one or more long periods.

[0082] The appropriate method for administering a substance, compound, or drug to a subject depends, for example, on the subject's age and / or physical condition, as well as the chemical and biological properties of the compound or drug (e.g., solubility, digestibility, bioavailability, stability, and toxicity). In some embodiments, the compound or drug is administered orally to the subject, for example, by ingestion. In some embodiments, the orally administered compound or drug is in the form of a sustained-release or extended-release formulation, or is administered using a device for such sustained-release or extended-release.

[0083] As used herein, the term “conjoint administration” refers to any form of administration of two or more different therapeutic agents such that a second agent is administered while a previously administered therapeutic agent is still effective in the body (for example, the two agents are effective simultaneously in the patient, which may include a synergistic effect between the two agents). For example, different therapeutic compounds may be administered simultaneously or sequentially, either in the same formulation or in separate formulations. Thus, an individual receiving such treatment may benefit from the combined effects of different therapeutic agents.

[0084] The "therapeutic effective dose" of a drug or medication is the amount of the drug or medication that, when administered to a subject, produces the intended therapeutic effect. The complete therapeutic effect may not necessarily occur with a single dose, but may only occur after a series of doses. Therefore, the therapeutic effective dose may be administered in one or more doses. The exact effective dose required for a subject depends, for example, on the subject's size, health, and age, as well as the nature and severity of the condition being treated. Those skilled in the art can readily determine the effective dose for a given situation through routine experiments.

[0085] As used herein, the terms “optional” or “optionally” mean that the events or circumstances described below may or may not occur, and that the description includes both cases in which the events or circumstances occur and cases in which they do not occur. For example, “optionally substituted alkyl” means that the alkyl may be substituted, but also includes cases in which the alkyl is not substituted.

[0086] It is understood that the substituents and substitution patterns of the compounds of the present invention can be selected by those skilled in the art to yield chemically stable compounds that can be readily synthesized from readily available starting materials by known techniques in the art and by the methods described below. If the substituent itself is substituted with multiple groups, it is understood that these multiple groups may be on the same carbon or different carbons, as long as a stable structure is obtained.

[0087] As used herein, the term “optionally substituted” means that 1 to 6 hydrogen radicals in a given structure are replaced by radicals of certain substituents, including but not limited to: hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH2-O-alkyl, -OP(O)(O-alkyl)2, or -CH2-OP(O)(O-alkyl)2. Preferably, “optionally substituted” means that 1 to 4 hydrogen radicals in a given structure are replaced by the substituents described above. More preferably, 1 to 3 hydrogen radicals are replaced by the substituents described above. It should be understood that substituents may be further substituted.

[0088] As used herein, the term "alkyl" refers to a saturated aliphatic group, C1-C 10 Linear alkyl groups or C1-C 10Branched alkyl groups are included, but are not limited to, these. Preferably, the "alkyl" group refers to a C1-C6 linear alkyl group or a C1-C6 branched alkyl group. Most preferably, the "alkyl" group refers to a C1-C4 linear alkyl group or a C1-C4 branched alkyl group. Examples of "alkyl" include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, neopentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3-octyl, or 4-octyl. The "alkyl" group may be optionally substituted.

[0089] The term "acyl" is recognized in the relevant art and refers to a group represented by the general formula hydrocarbyl C(O)-, preferably alkyl C(O)-.

[0090] The term "acylamino" is recognized in the relevant technical field and refers to an amino group substituted with an acyl group, which can be represented, for example, by the formula hydrocarbyl C(O)NH-.

[0091] The term "acyloxy" is recognized in the relevant technical field and refers to a group represented by the general formula hydrocarbyl C(O)O-, preferably alkyl C(O)O-.

[0092] The term "alkoxy" refers to an alkyl group that has an oxygen atom bonded to it. Representative alkoxy groups include methoxy, ethoxy, propoxy, and tert-butoxy.

[0093] The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group, and can be represented by the general formula alkyl-O-alkyl.

[0094] The term "alkyl" refers to saturated aliphatic groups, including linear alkyl groups, branched alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, linear or branched alkyl groups have 30 or fewer carbon atoms in their skeleton (for example, C for linear groups). 1-30 Regarding branched chains, C 3-30 ), more preferably having 20 or fewer carbon atoms.

[0095] Furthermore, when the term "alkyl" is used herein, in the examples and claims, it is intended to include both unsubstituted alkyl groups and substituted alkyl groups, the latter of which refers to an alkyl moiety having substituents that replace hydrogens on one or more carbons of a hydrocarbon skeleton, and includes haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl.

[0096] "C x-y " or "C x ~C y The term "alkyl" means a group containing x to y carbon atoms in a chain, when used with chemical parts such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy. C0 alkyl indicates hydrogen if the group is at a terminal position, and a bond if it is in the middle. For example, C 1-6 Alkyl groups contain 1 to 6 carbon atoms in their chain.

[0097] As used herein, the term "alkylamino" refers to an amino group substituted with at least one alkyl group.

[0098] The term "alkylthio," as used herein, refers to a thiol group substituted with an alkyl group, and may be represented by the general formula: alkylS-.

[0099] As used herein, the term "amide" refers to the following base: [ka] [In the formula, R9 and R 10 each independently represent hydrogen or a hydrocarbyl group, or R 9 and R 10 together with the N atom to which they are attached, complete a heterocycle having 4 to 8 atoms in the ring structure].

[0100] The terms "amine" and "amino" are recognized in the art and refer to both unsubstituted amines and substituted amines, as well as salts thereof, for example, [Chemical Formula] [wherein R 9 , R 10 , and R 10 '] each independently represent hydrogen or a hydrocarbyl group, or R 9 and R 10 together with the N atom to which they are attached, complete a heterocycle having 4 to 8 atoms in the ring structure] refers to the moiety represented by

[0101] The term "aminoalkyl" as used herein refers to an alkyl group substituted with an amino group.

[0102] The term "aralkyl" as used herein refers to an alkyl group substituted with an aryl group.

[0103] The term "aryl" as used herein includes substituted or unsubstituted monocyclic aromatic groups in which each atom of the ring is carbon. Preferably, the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. The term "aryl" also encompasses polycyclic ring systems having two or more cyclic rings where two or more carbons are common to two adjacent rings, and at least one of the rings is aromatic, for example, the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0104] The term "carbamate" is recognized in the relevant technical field and refers to the following bases: [ka] [In the formula, R 9 and R 10 [This independently represents a hydrogen atom or a hydrocarbyl group.]

[0105] As used herein, the term "carbocykrylalkyl" refers to an alkyl group substituted with a carbocyclic group.

[0106] The term "carbocyclic ring" includes monocyclic rings with 5 to 7 members and bicyclic rings with 8 to 12 members. Each ring in a bicyclic carbocyclic ring may be selected from saturated, unsaturated, and aromatic rings. A carbocyclic ring includes a bicyclic molecule in which one, two, or three or more atoms are shared between two rings. The term "condensed carbocyclic ring" refers to a bicyclic carbocyclic ring in which each ring shares two adjacent atoms with the other ring. Each ring in a condensed carbocyclic ring may be selected from saturated, unsaturated, and aromatic rings. In exemplary embodiments, an aromatic ring, such as phenyl, may be condensed with a saturated or unsaturated ring, such as cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated, and aromatic bicyclic rings is included in the definition of a carbocyclic ring, as long as the valence allows. Exemplary “carbocyclic” compounds include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octa-3-ene, naphthalene, and adamantane. Exemplary condensed carbocyclic compounds include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene, and bicyclo[4.1.0]hepta-3-ene. The “carbocyclic” compounds can be substituted at any one or more positions where hydrogen atoms may be present.

[0107] As used herein, the term "carbocykrylalkyl" refers to an alkyl group substituted with a carbocyclic group.

[0108] The term "carbonate" is recognized in the relevant technical field and refers to the group -OCO2-.

[0109] As used herein, the term "carboxy" refers to the group represented by the formula -CO2H.

[0110] The term "cycloalkyl" includes substituted or unsubstituted non-aromatic monocyclic structures, preferably 4- to 8-membered rings, more preferably 4- to 6-membered rings. The term "cycloalkyl" also includes polycyclic ring systems having two or more cyclic rings, where two or more carbons are common to two adjacent rings, and at least one of the rings is cycloalkyl, with substituents (e.g., R) 100 The group is bonded to a cycloalkyl ring, and for example, the other cyclic ring may be a cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Examples of heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, benzozodioxane, and tetrahydroquinoline.

[0111] The term "ester" as used herein refers to the group -C(O)OR 9 It refers to R 9 This represents a hydrocarbyl group.

[0112] As used herein, the term “ether” refers to a hydrocarbyl group bonded to another hydrocarbyl group via oxygen. Therefore, the ether substituent of a hydrocarbyl group can be hydrocarbyl-O-. Ethers may be symmetric or asymmetric. Examples of ethers include, but are not limited to, heterocyclic-O-heterocyclic and aryl-O-heterocyclic groups. Ethers also include “alkoxyalkyl” groups, which can be represented by the general formula alkyl-O-alkyl.

[0113] As used herein, the terms "halo" and "halogen" mean halogens, including chloro, fluoro, bromo, and iodine.

[0114] The terms "hetaralkyl" and "heteroaralkyl," as used herein, refer to alkyl groups substituted with heteroaryl groups.

[0115] The terms "hetaliarl" and "hetaliarl" include substituted or unsubstituted aromatic monocyclic structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, wherein the ring structure contains at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "hetaliarl" and "hetaliarl" also include polycyclic ring systems having two or more cyclic rings, wherein two or more carbons are common to two adjacent rings, and at least one of the rings is heteroaromatic, for example, the other cyclic ring may be a cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Examples of heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0116] As used herein, the term "heteroatom" means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0117] As used herein, the term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclic group.

[0118] The terms "heterocyclyl", "heterocycle" and "heterocyclic" refer to a substituted or unsubstituted non-aromatic ring structure, preferably a 3- to 10-membered ring, more preferably a 3- to 7-membered ring, and the ring structure contains at least 1 heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heterocyclyl" and "heterocyclic" also include polycyclic ring systems having two or more cyclic rings, where two or more carbon atoms are common to two adjacent rings, and at least one of the rings is heterocyclic, for example, the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Examples of the heterocyclyl group include piperidine, piperazine, pyrrolidine, morpholine, lactone, lactam and the like.

[0119] The term "hydrocarbyl", as used herein, refers to a group bonded via a carbon atom that has no =O or =S substituents, typically has at least one carbon-hydrogen bond and a main carbon skeleton, but may optionally contain heteroatoms. Accordingly, groups such as methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered hydrocarbyl for the purposes of this application, while substituents such as acetyl (which has a =O substituent on the linking carbon) and ethoxy (which is linked via oxygen rather than carbon) are not. Hydrocarbyl groups include, but are not limited to, aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

[0120] The term "hydroxyalkyl", as used herein, refers to an alkyl group substituted with a hydroxy group.

[0121] The term “lower” means, when used in conjunction with chemical parts such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, a group having 10 or fewer atoms, preferably 6 or fewer, in the substituent. “Lower alkyl” refers to an alkyl group having, for example, 10 or fewer, preferably 6 or fewer carbon atoms. In certain embodiments, the acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents as defined herein are lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, respectively, whether they appear alone or in combination with other substituents, such as in the descriptions of hydroxyalkyl and aralkyl (in this case, for example, when counting carbon atoms in the alkyl substituent, atoms in the aryl group are not counted).

[0122] The terms “polycyclyl,” “polycyclic,” and “polycyclic formula” refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl) in which two or more atoms are common to two adjacent rings, for example, the rings are “fused rings.” Each of the rings in a polycyclic compound may or may not be substituted. In certain embodiments, each ring in a polycyclic compound contains 3 to 10, preferably 5 to 7, atoms.

[0123] The term "sulfate" is recognized in the relevant technical field and refers to the group -OSO3H or its pharmaceutically acceptable salts.

[0124] The term "sulfonamide" is recognized in the relevant technical field and refers to a group represented by the following general formula: [ka] [In the formula, R 9 and R 10 [This independently represents hydrogen or hydrocarbyl.]

[0125] The term "sulfoxide" is recognized in this technical field and refers to the group -S(O)-.

[0126] The term "sulfonate" is recognized in the art and refers to the group SO3H or its pharmaceutically acceptable salts.

[0127] The term "sulfone" is recognized in the relevant technical field and refers to the group -S(O)2-.

[0128] The term “substituted” refers to a portion of a skeleton having substituents that replace hydrogens on one or more carbon atoms. “Substituted” or “substituted with” should be understood to imply that such substitutions, according to the permissible valencies of the substituted atom and substituent, result in a stable compound that does not spontaneously undergo transformation, such as rearrangement, cyclization, or elimination. As used herein, the term “substituted” is intended to include all permissible substituents of an organic compound. In a broader embodiment, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of an organic compound. There may be one or more permissible substituents for a given organic compound, and they may be the same or different. For the purposes of the present invention, heteroatoms such as nitrogen may have any permissible substituents of the organic compounds described herein that satisfy the valencies of the hydrogen substituent and / or the heteroatom. Substituents may include any substituents described herein, e.g., halogens, hydroxyls, carbonyls (e.g., carboxyls, alkoxycarbonyls, formyls, or acyls), thiocarbonyls (e.g., thioesters, thioacetic acids, or thioformates), alkoxyls, phosphoryls, phosphates, phosphonates, phosphinates, aminos, amides, amidines, imines, cyanos, nitros, azides, sulfhydryls, alkylthios, sulfates, sulfonates, sulfamoyls, sulfonamides, sulfonyls, heterocyclyls, aralkyls, or aromatic or heteroaromatic moieties. It will be understood by those skilled in the art that substituted portions on a hydrocarbon chain may, where appropriate, be substituted themselves.

[0129] As used herein, the term "thioalkyl" refers to an alkyl group substituted with a thiol group.

[0130] The term "thioester," as used herein, refers to the group -C(O)SR 9 or -SC(O)R 9 This refers to R 9 This represents hydrocarbyl.

[0131] As used herein, the term "thioether" refers to an equivalent of an ether in which oxygen is replaced by sulfur.

[0132] The term "urea" is recognized in the relevant technical field and can be represented by the following general formula: [ka] [In the formula, R 9 and R 10 [represents hydrogen or hydrocarbyl independently.]

[0133] As used herein, the term “modulate” includes inhibition or suppression of a function or activity (such as cell proliferation), as well as enhancement of a function or activity.

[0134] The phrase "pharmaceutically acceptable" is recognized in the art. In certain embodiments, this term includes compositions, excipients, adjuvants, polymers, and other materials and / or dosage forms that are suitable for use in contact with human and animal tissues within reasonable benefit / risk ratios, within the bounds of sound medical judgment, without causing excessive toxicity, irritation, allergic reactions, or other problems or complications.

[0135] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be compatible with the other components of the formulation and “acceptable” in the sense that it is not harmful to the patient. Some examples of substances that may serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil. (10) Oils such as propylene glycol; (11) Glycols such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) Esters such as ethyl oleate and ethyl laurate; (13) Agar; (14) Buffers such as magnesium hydroxide and aluminum hydroxide; (15) Alginic acid; (16) Pyrogen-free water (water that does not contain pyrogens); (17) Isotonic saline; (18) Ringer's solution; (19) Ethyl alcohol; (20) Phosphate buffer; and (21) Other non-toxic suitable substances used in pharmaceutical formulations.

[0136] "Pharmacologically acceptable salt" or "salt" is used herein to mean an acid addition salt or base addition salt that is suitable for or adapted to the treatment of a patient.

[0137] As used herein, the term “pharmaceutically acceptable acid addition salt” means any non-toxic organic or inorganic salt of any basic compound represented by Formula I. Exemplary inorganic acids that form suitable salts include hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, as well as metal salts such as sodium monohydrogen orthophosphate and potassium bisulfate. Exemplary organic acids that form suitable salts include mono-, di-, and tri-carboxylic acids such as glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, benzoic acid, phenylacetic acid, cinnamic acid, and salicylic acid, as well as sulfonic acids such as p-toluenesulfonic acid and methanesulfonic acid. Either mono- or di-salts can be formed, and such salts may exist in hydrated, solvated, or substantially anhydrous forms. Generally, acid addition salts of compounds of Formula I are more soluble in water and various hydrophilic organic solvents and generally exhibit higher melting points compared to their free base forms. The selection of appropriate salts is known to those skilled in the art. Other pharmaceutically unacceptable salts, such as oxalates, may also be used, for example, in the isolation of compounds of formula I for laboratory use or for subsequent conversion to pharmaceutically acceptable acid addition salts.

[0138] As used herein, the term “pharmaceutically acceptable base addition salt” means any non-toxic organic or inorganic base addition salt of any acidic compound represented by formula I or any intermediate thereof. Exemplary inorganic bases that form suitable salts include lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or barium hydroxide. Exemplary organic bases that form suitable salts include aliphatic, alicyclic, or aromatic organic amines, such as methylamine, trimethylamine, and picoline, or ammonia. The selection of suitable salts is known to those skilled in the art.

[0139] Many of the compounds useful in the methods and compositions of this disclosure have at least one stereocenter in their structure. This stereocenter may be in an R or S configuration, and the R and S notations are used in accordance with the rules set out in Pure Appl. Chem. (1976), 45, 11-30. This disclosure intends all stereoisomer forms (including all possible mixtures of stereoisomers), such as enantiomers and diastereomers, of compounds, salts, prodrugs, or mixtures thereof. See, for example, International Publication No. 01 / 062726.

[0140] Furthermore, certain compounds containing alkenyl groups may exist as Z (tuzamen) or E (entgegen) isomers. In each case, the disclosure includes both mixtures and separate individual isomers.

[0141] A “prodrug” or “pharmaceutically acceptable prodrug” refers to a compound that, after administration, is metabolized in a host, for example, by hydrolysis or oxidation, to form a compound of the Disclosure (e.g., a compound of formula I). ​​Typical examples of prodrugs include compounds having a biologically unstable or cleavable (protecting) group on the functional group of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce an active compound. Examples of prodrugs using esters or phosphoramidates as biologically unstable or cleavable (protecting) groups are disclosed in U.S. Patents 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference. The prodrugs of the Disclosure are metabolized to produce compounds of formula I. The Disclosure, to its extent, includes prodrugs of the compounds described herein. Conventional procedures for the selection and preparation of appropriate prodrugs are described, for example, in “Design of Prodrugs” Ed. H. Bundgaard, Elsevier, 1985.

[0142] As used herein, the terms “logarithm of solubility,” “LogS,” or “LogS” are used in the art to quantify the water solubility of a compound. The water solubility of a compound significantly affects its absorption and distribution characteristics. Low solubility is often associated with poor absorption. The LogS value is the unitless logarithm (base 10) of solubility measured in moles (mol) / liter.

[0143] As used herein, the terms “parenteral administration” and “administered parenterally” usually refer to, but are not limited to, modes of administration other than enteral and local administration by injection, including, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions. [Examples]

[0144] While the present invention has been described in general terms, it will be more readily understood by referring to the following examples. These examples are included solely for illustrative purposes of specific aspects and embodiments of the present invention and are not intended to limit the invention.

[0145] Example 1: Synthesis and activity of exemplary PROTVAC Characterization of pre-fusion PROTAC tumor antigens and PROTVAC Affinity between pre-fusion PROTAC tumor antigen and pVHL TAgD-TVac consists of a pre-fused PROTAC tumor antigen and an adjuvant LNP with LN targeting ability. As proof of concept, (S,R,S)-AHPC, a ligand that recruits von Hippel-Lindau protein (pVHL, E3 ubiquitin ligase), was used. The pre-fused PROTAC tumor antigen was synthesized by covalently modifying the tumor antigen with (S,R,S)-AHPC (Figure 2A). Because it is easy to visualize under excitation light, green fluorescent protein (GFP)-Cre (obtained by fusing with Cre recombinase) was first used as a substitute for the tumor antigen to prepare a pre-fused formulation (denoted as pre-fused AHPC-GFP-Cre). The integration of AHPC with GFP-Cre was confirmed by evaluating the affinity of pre-fused AHPC-GFP-Cre to pVHL using confocal laser scanning microscopy (CLSM). pVHL was pre-labeled with rhodamine-B-isothiocyanate (RITC) and then incubated with pre-fused AHPC-GFP-Cre. A mixture of GFP-Cre, AHPC, and pVHL was used as a control. As shown in Figure 2B, co-localization of pre-fused AHPC-GFP-Cre and pVHL was observed, suggesting binding of pre-fused AHPC-GFP-Cre to pVHL and the formation of a pre-fused AHPC-GFP-Cre / pVHL complex. In contrast, little co-localization was observed when a mixture of GFP-Cre, AHPC, and pVHL was incubated. The affinity between pre-fused AHPC-GFP-Cre and pVHL was further confirmed by agarose gel electrophoresis. pVHL was immobilized on the surface of poly(lactic acid-coglycolic acid) (PLGA) nanoparticles (NPs) and incubated with pre-fused GFP-AHPC in the presence of polyethylene glycol (PEG) (Figure 2C). PEG was used to mimic the crowding state within cells. Subsequently, the samples were loaded into gel wells and electrophoresis was performed.When pVHL-modified PLGA nanoparticles were incubated with pre-fused AHPC-GFP-Cre, almost all GFP signals were observed near the gel wells (Figure 2D). In contrast, the affinity between GFP-Cre and pVHL-modified PLGA nanoparticles was negligibly low, causing all GFP-Cre to flow out of the wells. These results confirm that AHPC successfully integrated with GFP-Cre and demonstrate that pre-fused AHPC-GFP-Cre has the ability to target pVHL.

[0146] Ability of pre-fused PROTAC tumor antigens to target intracellular pVHL Next, we investigated the ability to target intracellular pVHL in mouse DCs (DC2.4). For intracellular delivery of pre-fused AHPC-GFP-Cre, we constructed an adjuvant LNP with lymph node (LN) target specificity. The adjuvant tripidoid (113-O12B, Figure 2E, and Schemes 1 and 2), cholesterol (Chol), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG). 2k Adjuvant LNPs were prepared using a molar ratio of 48.5:38:10:3.5. Transmission electron microscopy images of the adjuvant LNPs showed a closed vesicle morphology with a diameter of approximately 120 nm (Figures 2F and 7A). These adjuvant LNPs could specifically accumulate in lymph nodes (LNs) after subcutaneous administration to the ridge, which was demonstrated using a DiR-labeled formulation and observed with the IVIS imaging system (PerkinElmer, Figures 7B and 7C).

[0147] Next, we evaluated the ability of pre-fused AHPC-GFP-Cre to recruit intracellular pVHL under the induction of AHPC ligand. If pre-fused AHPC-GFP-Cre cannot recruit cytosolic pVHL, GFP will be detected in the nucleus due to the intrinsic nuclear targeting ability of Cre recombinase. Using adjuvant LNPs, the pre-fused formulation (hereinafter, pre-fused AHPC-GFP-Cre LNP) and the co-loaded control formulation (hereinafter, GFP-Cre+AHPC LNP) were administered. AHPC inclusion was confirmed using high-performance liquid chromatography (HPLC). These formulations were first incubated with DC2.4 cells for 3 hours, which was sufficient time for intracellular uptake (Figure 8A). After intracellular uptake, incubation for a further 9 hours revealed that pre-fused AHPC-GFP-Cre co-localized with pVHL stained with anti-VHL antibody, and as shown in Figure 2G, there was little accumulation in the nucleus. In contrast, co-localization of GFP-Cre and pVHL was weak in the control GFP-Cre and AHPC, and significant accumulation of GFP-Cre in the nucleus was observed. These results suggest that pre-fused AHPC-GFP-Cre preferentially bound to intracellular pVHL. Co-localization based on CLSM images was quantified by calculating the Pearson's co-localization coefficient (PCC) between GFP fluorescence (green) and pVHL (red). PCC values ​​for the pre-fused AHPC-GFP-Cre LNP group (PCC 事前融合AHPC-GFP-Cre LNP =0.89) is the GFP-Cre+AHPC LNP group (PCC GFP-Cre+AHPC LNP The result was significantly higher than (=0.58) (Figures 8B and 8C). These results support the ability of pre-fused AHPC-GFP-Cre to recruit intracellular pVHL.

[0148] PROTVAC promotes antigen cross-presentation. It was demonstrated that GFP-Cre fused with AHPC ligand recruits pVHL in both cell-free systems and at the cellular level. Next, ubiquitination and proteasome-mediated degradation of GFP-Cre in DC2.4 cells were evaluated by monitoring the fluorescence of GFP-Cre using flow cytometry. Different formulations were incubated with DC2.4 cells for 3 hours, then the culture medium was changed to fresh medium and incubated further. As shown in Figure 2H, during the 24-hour observation period, pre-fused AHPC-GFP-Cre showed faster degradation of GFP-Cre (reduced to 8.3%) compared to GFP-Cre alone (24.7%) and GFP-Cre + AHPC (31.3%). These results suggest that modification of GFP-Cre with AHPC significantly accelerates GFP-Cre degradation.

[0149] Next, pre-fused PROTAC tumor antigens (denoted as pre-fused AHPC-OVA) were constructed using OVA and characterized by SDS-polyacrylamide gel electrophoresis (Figure 8D). Förster resonance energy transfer (FRET) analysis showed the formation of a pre-fused AHPC-OVA / pVHL binary complex. OVA and pVHL were pre-labeled with fluorescein isothiocyanate (FITC) and RITC, respectively. FRET analysis showed effective energy transfer after co-incubating pre-fused AHPC-OVA and pVHL for 2 hours, suggesting that the pre-fused AHPC-OVA and pVHL molecules were in close proximity to each other. When OVA, AHPC, and pVHL were mixed, negligible energy transfer was observed (Figure 8E). These results supported the ability of pre-fused AHPC-OVA to recruit pVHL. Next, proteasome-mediated OVA degradation in mouse bone marrow-derived dendritic cells (BMDCs) was tested by Western blotting. Semi-quantitative analysis revealed intracellular delivery of pre-fused AHPC-OVA via LNP (TAgD-TVac OVAFigure 2I shows that TAgD-TVac, compared to OVA delivery (denoted as OVA LNP) and co-delivery of OVA and AHPC (denoted as OVA+AHPC LNP), resulted in the highest OVA degradation. To further confirm proteasome degradation, cytoplasmic OVA levels were evaluated. OVA A significantly reduced OVA level was observed in the group, confirming enhanced OVA degradation (Figure 8F). Cross-presentation of OVA fragments in BMDC was evaluated by flow cytometry and confocal laser scanning microscopy (CLSM). As shown in Figures 2J and 2K, TAgD-TVac OVA BMDC treated with TAgD-TVac for 12 hours showed the most efficient cross-presentation of OVA fragments in MHC-I, as demonstrated by staining for the H2kb-SIINFEKL complex. Extending the incubation time to 24 hours further increased cross-presentation. These results were confirmed by immunofluorescence analysis. OVA The highest H2kb-SIINFEKL complex signal (green) was observed in the group (Figure 9A). The inventors also found that TAgD-TVac OVA In BMDCs treated with TAgD-TVac, we found that the expression of the co-stimulatory markers CD80 and CD86 was significantly upregulated, suggesting enhanced DC maturation (Figures 9B and 9C). These results suggest that TAgD-TVac OVA This study shows that UPP can improve the cross-presentation of tumor antigens in DCs, which is important for cross-priming of cytotoxic T cells.

[0150] TAgD-TVac induces robust cytotoxic T cell activation by improving the cross-presentation of tumor antigens. Lymph nodes (LNs) are important sites for antigen presentation and cytotoxic T cell stimulation. To achieve robust T cell cross-priming, pre-fused AHPC-OVA needs to be delivered to LNs. To demonstrate this, TAgD-TVac OVADuring preparation, pre-fused AHPC-OVA was pre-labeled with cyanine 5.5NHS ester (Cy5.5). Cy5.5-labeled TAgD-TVac in BALB / c mice. OVA The in vivo distribution of was visualized using the IVIS imaging system. As shown in Figures 3A and 3B, LNP (ALC-0315) was prepared using ALC-0315, an important component in Pfizer-BioNTech COVID-19 Vaccine Community (registered trademark). OVA (as indicated) Compared to TAgD-TVac OVA This showed significantly reduced accumulation in the liver and higher accumulation in the liver (LN). ALC-0315 OVA After treatment with TAgD-TVac, the fluorescence radiance in the liver was approximately 2.2 times higher than in LN. OVA In the group, the fluorescence intensity in the liver decreased to 36.1% of the intensity in the LN (lymph node), confirming excellent LN targeting ability (Figure 3C). Since fluorescence signals were observed in lymphatic vessels connected to the LN (Figure 3C), TAgD-TVac OVA It is possible that TAgD-TVac enters the LN via lymphatic vessels after SC administration. Similar results were observed in C57BL / 6 mice (Figure S7). Next, the degradation efficiency of OVA in the LN was evaluated by Western blotting. Compared with the OVA LNP group and the OVA+AHPC LNP group, TAgD-TVac OVA A significant decrease in OVA levels was observed in the group, suggesting enhanced OVA degradation (Figures 3E and 3F).

[0151] For efficient antigen cross-presentation, OVAs need to undergo proteasomal degradation in APCs. Therefore, we investigated which cell types within the LN are targets for TAgD-TVac. We used genetically engineered Ai14 mice, which result in activatable and stable expression of the red fluorescent protein tdTomato after cytosolic delivery of Cre recombinase (Figure 3G). Fluorescent cells can be detected by flow cytometry. GFP-Cre was used as an activator and loaded onto adjuvant LNPs. As shown in Figures 3H and 11, administration of GFP-Cre-loaded adjuvant LNPs resulted in positive expression of tdTomato in 23.6% of DCs and 25.4% of macrophages. These results indicate that the adjuvant LNPs effectively delivered GFP-Cre to the cytoplasm of APCs. A potential mechanism is that the adjuvant LNPs were taken up by APCs after injection and then reached the LNs via lymphocyte homing through the afferent lymphatic vessels. To confirm this hypothesis, Cy5.5-labeled TAgD-TVac OVA The drug was injected into a macrophage-depleted mouse model. As shown in Figure 3I, compared to untreated mice, the fluorescence signal at the LN was significantly reduced in macrophage-depleted mice. Quantitative results showed that the fluorescence intensity was reduced to 34.5% of the original level (Figure 3J). Although the molecular-level mechanisms of the in vivo distribution of TAgD-TVac in mice remain unclear, our results support the important role of APC in the LN targeting ability of TAgD-TVac.

[0152] TAgD-TVac for increased cross-presentation and induction of antitumor T cell response OVA The effects were tested in a mouse model of OVA-expressing melanoma (B16F10-OVA). Mice were given phosphate-buffered saline (PBS), OVA LNP, OVA+AHPC LNP, or TAgD-TVac. OVA Tumor growth was monitored after vaccination with TAgD-TVac (protein, 20 μg per dose, Figure 3K) via SC. As shown in Figures 3L, 3M, and 12A, TAgD-TVacOVA Treatment with TAgD-TVac effectively suppressed the growth of B16F10-OVA tumors. Consistent with this, during the 40-day observation period, TAgD-TVac showed superior growth compared to the other treatment group (0%, Figure 3N). OVA A significant improvement in survival rate was observed in treated mice (survival rate 16.7%). Subsequently, tumors were excised for hematoxylin and eosin (H&E) staining (Figure 12B). TAgD-TVac OVA Tumors collected from the group showed a dramatic decrease in nuclear number and significant nuclear enrichment (karyopyknosis). In contrast, other treatment groups showed little to no apoptosis. TAgD-TVac OVA The enhanced antitumor effect is thought to be due to increased cross-presentation and robust cytotoxic T cell activation.

[0153] For verification, TAgD-TVac OVA Antitumor immunity induced by TAgD-TVac was evaluated by flow cytometry. OVA The procedure involves cross-presentation of OVA fragments in MHC-1 on the surface of DC in LN (H2Kb-SIINFEKL high This significantly improved the rate (19.2%). In contrast, moderate cross-presentation efficiency was observed in the other treatment groups (OVA LNP, 10.1%; OVA+AHPC LNP, 9.76%) (Figures 3O, 13A, and 14A). Furthermore, TAgD-TVac OVA DCs derived from treated mice showed significantly upregulated expression of CD80 and CD86, and enhanced DC maturation was confirmed (Figures 14A and 14F). Improved OVA cross-presentation and DC maturation were associated with TAgD-TVac OVA This is thought to be due to its superior LN targeting ability and more efficient degradation of pre-fused AHPC-OVA in DCs. Activation of innate immunity was confirmed by evaluating the levels of cytokines and chemokines in the blood. As shown in Figure 15, TAgD-TVac OVAThe treatment elevated the levels of pro-inflammatory cytokines (such as M-CSF, IFN-γ, and IL-6) and pro-inflammatory chemokines (such as MCP-1, MIP-1α, and MIP-1β). Among them, IL-6 plays a critical role in the proliferation and differentiation of cytotoxic T cells for adaptive immunity. Therefore, intratumoral cytotoxic CD8, which are the major effector for tumor suppression + T cell activation was analyzed. The highest CD8 OVA T cell infiltration was observed in the TAgD-TVac group + (CD8, 31.1%), which is considered to be attributed to enhanced antigen cross-presentation in LNs (Figures 3P and S14C). The CD8 + T cells in the TAgD-TVac group showed a significant increase in the expression of Granzyme B (GzmB, which mediates apoptosis, 27.8%), further supporting T cell activation (Figures 3Q and 11D). Efficient antigen cross-presentation also induces strong immune memory, which was confirmed by evaluating the proportions of effector memory T (T OVA ) cells and central memory T (T + ) cells in the spleen. Treatment with TAgD-TVac resulted in a significant increase in the proportions of CD62L high CD44 EM T cells (32.6%) and CD62L CM CD44 OVA T cells (24.8%) in the spleen compared with other treatment groups (Figures 3R, 14E, and 14G). The critical role of T cells was further confirmed using B16F10-OVA tumor-bearing mice with CD8 low T cell depletion. As shown in Figure 14H, the anti-tumor effect of TAgD-TVac was found to be significantly reduced. high EM high high CM + OVA

[0154] Anti-tumor effect of TAgD-TVac in a subcutaneous breast cancer model ​​​​​​​Breast cancer is the second most common cancer in women. Therefore, we investigated the antitumor activity of TAgD-TVac in a 4T1 triple-negative breast cancer model. To mimic antigens derived from patient tumor tissue, we isolated tumor lysate proteins (TLP) from 4T1 (4T1-Luc) tumor cells transfected with luciferase. 4T1 Using ), TAgD-TVac(TAgD-TVac 4T1 (Notation) was prepared. TLP 4T1 And adjuvant LNPs with co-loaded AHPCs were used as a control (TLP 4T1 (Denoted as +AHPC LNP). Tumor-bearing mice were given TAgD-TVac on day 0, day 5, and day 10. 4T1 Prime and boost doses of TAgD-TVac (protein, 20 μg per dose) were administered (Figure 4A). 4T1 The treatment effectively suppressed the growth of 4T1-Luc tumors, as demonstrated by measuring the bioluminescence and volume of 4T1-Luc tumors in vivo (Figures 4B, 4C, and 4D). These results were confirmed by histological analysis of H&E-stained tumor sections (Figure 15). As a result, the other treatment groups (PBS: 0%, TLP) 4T1 Compared to +AHPC LNP: 16.7%, (Figure 4E), TAgD-TVac over a 40-day observation period showed a higher percentage. 4T1 A significant improvement in survival rate was observed in mice treated with (survival rate, 33.3%). Importantly, one TAgD-TVac 4T1 The treated mice were found to have no detectable tumors at the end of this experiment (Figure 4C). This may be due to the abundance of tumor antigens in the tumor cell lysates, which may have induced a more efficient anti-tumor immune response.

[0155] Next, TAgD-TVac 4T1 We investigated the antitumor immunity induced by TAgD-TVac. First, we studied the maturation of DCs in the LN by measuring CD80 and CD86, which are co-stimulatory markers essential for cross-priming. As shown in Figures 4F and 11A, TAgD-TVac 4T1The treatment significantly upregulated the expression of CD80 and CD86 in DCs (CD80 high CD86 high , 20.4%). In contrast, TLP 4T1 In the +AHPC LNP group, moderate expression of CD80 and CD86 was observed (9.75%). Based on these results, TAgD-TVac 4T1 Induced DC maturation was confirmed, which is important for efficient cross-priming of cytotoxic T cells. Subsequently, intratumoral cytotoxic CD8 + T cell activation was analyzed compared to other treatment groups. 4T1 The highest CD8 in the group + T cell infiltration (CD8 + 32.1% was observed (Figures 4G and 11B). TAgD-TVac OVA The procedure is CD8 + The expression of granzyme B by T cells was also significantly increased (GzmB high , 19.1%) (Figures 4H and 17C). These results are from TAgD-TVac 4T1 This study confirmed cytotoxic T cell-mediated antitumor immunity after vaccination using SC administration.

[0156] TAgD-TVac 4T1 Vaccination with TAgD-TVac also induced strong immunological memory. 4T1 The procedure there is TLP 4T1 +AHPC LNP(T EM Cells: 23.0%, T CM Compared to cells: 26.9%, CD62L in the spleen low CD44 high T EM Cells (31.6%) and CD62L high CD44 high T CM This resulted in an increase in the percentage of cells (34.4%) (Figures 4I, 17D, and 17E). Considering the important role of memory T cells in the prevention of tumor metastasis, we used the IVIS imaging system to study the effects of TAgD-TVac on the suppression of tumor metastasis. 4T1 The effects of TLP were evaluated. 4T1+AHPC LNP group and TAgD-TVac 4T1 4T1-Luc tumor-carrying mice were re-challenged with 4T1-Luc cells via IV injection. The control group (normal mice) were also treated with 4T1-Luc cells via IV injection. As shown in Figures 4K and 4L, TAgD-TVac 4T1 Treatment with TAgD-TVac effectively suppressed tumor metastasis. In contrast, all mice treated with other methods showed significant lung metastasis. This result was supported by histological analysis of H&E-stained lung sections. 4T1 The treatment resulted in a significant reduction in 4T1-Luc tumor nodules in lung tissue compared to other treatment groups (Figure 16). These results are related to TAgD-TVac 4T1 However, it was shown that this can induce efficient T cell-dependent antitumor immunity for the suppression of primary and metastatic tumor growth.

[0157] Antitumor effect of TAgD-TVac in an orthotopic lung cancer model Lung cancer (carcinoma) is the third most common cancer in the United States. The potential of TAgD-TVac to suppress the development of orthotopic lung cancer in mice was investigated, and its ability to improve treatment outcomes with ICB therapy was demonstrated. Lewis lung cancer (LLC-Luc) cells, which stably express luciferase, were intercostal inoculated into the right lung of C57BL / 6 mice. Using tumor lysate proteins isolated from LLC-Luc cells, TAgD-TVac (TAgD-TVac LLC A preparation (denoted as) was made. TAgD-TVac was administered to tumor-bearing mice on days 0, 4, and 8. LLC (Protein, 20 μg per dose) was administered three times. Anti-PD-1 antibody (aPD-1, 200 μg per dose) was administered by intraperitoneal (IP) injection on days 1, 5, and 9 (Figure 5A). TAgD-TVac LLC The antitumor effect of monotherapy with TAgD-TVac was also evaluated. Monotherapy with a PD-1 inhibitor was used for comparison. Since LLC-Luc tumors are expected to grow in the lungs and externally visible tumor masses are not expected, the inventors used body weight as the basis for evaluating tumor progression. As shown in Figure 5B, TAgD-TVac LLCTreatment with TAgD-TVac in combination with aPD-1 significantly suppressed the growth of LLC-Luc tumors. LLC Treatment (aPD-1 + TAgD-TVac LLC (indicated as) further suppressed tumor growth. Next, lung tissue was collected. As shown in Figures 5C and 5D, aPD-1+TAgD-TVac LLC Following treatment, a significant reduction in LLC-Luc tumor nodules in the lungs was observed. This result was supported by histological analysis of H&E-stained lung sections (Figure 18). As a result, TAgD-TVac in combination with aPD-1 LLC The treatment effectively reduced weight loss in tumor-bearing mice and extended survival over a 40-day observation period (survival rate 50.0%, Figures 5E and 5F). ICB therapy augmentation was found to enhance TAgD-TVac in LN. LLC This is thought to be due to the activation of DCs induced by [the phenomenon].

[0158] For verification, the activation of DC in the LN was investigated by measuring CD80 and CD86. aPD-1(CD80CD86 high , 6.17%) or TAgD-TVac LLC Compared to monotherapy (7.24%), TAgD-TVac LLC Treatment significantly upregulated CD80 and CD86 expression (12.9%) and enhanced DC maturation was observed (Figure 5G and S19A). Due to its important role in T cell-mediated adaptive antitumor immunity, serum IL-6 levels were assessed. aPD-1 and TAgD-TVac LLC Both were able to raise the level of IL-6. aPD-1 and TAgD-TVac LLC The combination therapy increased IL-6 levels 2.2-fold compared to aPD-1 monotherapy, and robust T cell activation was confirmed (Figure 5H). Next, the establishment of immunological memory was evaluated. As shown in Figures 5I, 19A, and 19C, TAgD-TVac LLC The treatment involves CD62L in the spleen. low CD44 high T EM Cells (23.6%) and CD62L high CD44high T CM The percentage of cells (27.1%) was improved. Furthermore, when combined with aPD-1, CD62L in the spleen improved. low CD44 high T EM Cells and CD62L high CD44 high T CM The percentages of cells increased to 37.1% and 33.2%, respectively, indicating stronger immunological memory.

[0159] The T cell-dependent adaptive immunity formed by APC is antigen-specific. To demonstrate this, aPD-1+TAgD-TVac LLC LLC-Luc tumor-bearing mice that received combination therapy were re-challenged on day 40 with LLC-Luc (left flank) and non-cognate B16F10 tumor cells (right flank) via SC injection to mimic tumor recurrence (Figures 5J and 5K). The control group (normal mice) were also treated with LLC-Luc and B16F10 tumor cells via SC injection. As shown in Figures 5L and 5M, aPD-1+TAgD-TVac LLC In two out of five mice in the group, recurrence of LLC-Luc tumors was completely suppressed. In contrast, only slight suppression of B16F10 tumor growth was observed. Selective suppression of LLC-Luc tumor growth was associated with intratumoral CD8 + T cell infiltration (CD8 + As evidenced by the detection of 20.9% (control, 5.37%), the results are thought to be due to T cell activation. These results are consistent with TAgD-TVac in combination with ICB therapy. LLC However, it effectively induced a protective cognate anti-tumor immune response, confirming robust T-cell-dependent adaptive immunity and strong immunological memory.

[0160] Antitumor effects of TAgD-TVac in an inoperable ovarian cancer model Ovarian cancer is a highly malignant and serious tumor. Because it lacks typical clinical symptoms in its early stages, many patients are diagnosed with metastasis to intra-abdominal organs, making surgical treatment impossible. In light of this clinical challenge, we established an advanced ovarian cancer model to test the therapeutic outcomes of the TAgD-TVac technology developed by our inventors. In this study, TAgD-TVac was prepared using tumor lysate protein isolated from ovarian (ID8-Luc) tumor cells that stably express luciferase. ID8 (as indicated). Tumor-bearing mice were given TAgD-TVac on day 0, day 4, and day 8. ID8 (Protein, 20 μg per dose) was administered three times, and aPD-1 (200 μg per dose) was administered three more times on days 1, 5, and 9 (Figure 6A). As shown in Figures 6B and 6F, TAgD-TVac ID8 The treatment effectively suppressed the growth of intraperitoneal tumors, and its effect was slightly superior to ICB monotherapy. TAgD-TVac ID8 and combination therapy with aPD-1 (aPD-1 + TAgD-TVac ID8 (as indicated) aPD-1 or TAgD-TVac ID8 It showed significantly higher inhibition of tumor growth compared to administration of aPD-1+TAgD-TVac alone. Next, bioluminescence signals in the abdominal cavity and each major organ were analyzed. As shown in Figure 6C, mice showed significantly higher inhibition of tumor growth when aPD-1+TAgD-TVac was administered. ID8 Following the procedure, a significant reduction in ID8-Luc tumor nodules was observed in the abdominal cavity (and peritoneum), ovaries, and spleen. This result was confirmed by histological analysis and photographs (Figures 6D, 6E, 20A, and 20B). In consistency with this, TAgD-TVac ID8 During the 80-day observation period, this extended the survival time of tumor-bearing mice, which was longer than in the PBS group (Figure 6G). Combination therapy with aPD-1 further improved survival, with 66.7% of mice surviving. aPD-1 + TAgD-TVac ID8In treated mice, ascites volume also decreased significantly (Figure 6H). These results demonstrate that the combination of ICB therapy and the TAgD-TVac strategy can synergistically enhance the antitumor effect. ID8 We also evaluated the antitumor immunity induced by combination therapy. As shown in Figures 6I and 20C, TAgD-TVac ID8 (CD80 high CD86 high , 11.3%, and combination therapy (15.0%) effectively enhanced DC activation in the LN. Furthermore, the inventors have found that TAgD-TVac ID8 The treatment also increased levels of pro-inflammatory cytokines (including M-CSF, IFN-γ, and IL-6, Figures 6J-6L) and pro-inflammatory chemokines (including MCP-1, MIP-1α, and MIP-1β, Figures 6M-6O) in the blood, confirming the activation of innate immunity. Combination therapy with aPD-1 further enhanced the activation of innate immunity. These cytokines and chemokines induce T cell proliferation and differentiation for robust adaptive anti-tumor immunity.

[0161] summary We developed TAgD-TVac by loading pre-fused PROTAC tumor antigens onto adjuvant LNPs with LN-targeting capabilities. TAgD-TVac delivered the pre-fused PROTAC tumor antigen to APCs, and then to UPPs under the induction of E3 ligase ligands, thereby increasing MHC-I-dependent antigen cross-presentation and initiating activation of cytotoxic T cells within the LN. Four individualized TAgD-TVacs were prepared using relevant tumor lysates, and their ability to induce potent antigen-specific adaptive immunity and immunological memory against a variety of solid tumors was successfully demonstrated. Combining TAgD-TVac with ICB therapy further suppressed tumor growth and reduced the risk of tumor metastasis and recurrence, preserving its potential for clinical application. Current efforts provide a novel pathway for initiating adaptive immune responses via simple chemistry and nanotechnology, signaling the design of effective cancer immunotherapies. Success in PROTAC-based vaccine technology could represent a significant breakthrough in bridging the gap between fundamental research on ubiquitin-proteasome protein degradation systems and vaccine development.

[0162] Materials and methods Unless otherwise stated, reagents and solvents were used as received from commercial suppliers without further purification. All reagents and solvents for 113O-12B lipid synthesis were purchased from Sigma-Aldrich or Fisher Scientific. 2-Aminotifen (Trout's Reagent), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), and tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP·HCl) were purchased from Fisher Scientific. Protamine sulfate (salmon-derived) was purchased from Sigma-Aldrich. Ovalbumin was purchased from Abnova. (S,R,S)-AHPC-PEG4-amine was purchased from Broadpharm. Von Hippel-Lindau recombinant protein was purchased from Novus Biologicals®. Fluorescein isothiocyanate (FITC) and rhodamine B isothiocyanate (RhB) were purchased from Sigma-Aldrich. 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine iodide (DiR) was purchased from Invitrogen. Cholesterol (Chol), D-luciferin potassium salt, and Cell Counting Kit-8 (CCK-8) were purchased from Thermo Scientific®. 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG) were purchased from Avanti Polar Lipids. A 5-6 nm thick carbon-supported film formed on a 200 square-mesh copper grid was purchased from Electron Microscopy Sciences. Disodium bicinconate hydrate (BCA) was purchased from Solarbio.Dulbecco's Modified Eagle Medium (DMEM) growth medium, fetal bovine serum (FBS), and Dulbecco's phosphate-buffered saline (D-PBS, 10 mM, pH 7.2) were purchased from Gibco Corporation. Trypsin-EDTA (0.25%) and LysoTracker Red were purchased from Invitrogen. B16F10, 4T1-Luc, LLC-Luc, ID8-Luc tumor cell lines, and DC2.4 cell line were purchased from the American Type Culture Collection (ATCC). The Lysosome Enrichment Kit for Tissues and Cultured Cells was purchased from Thermo Scientific®. Antibodies for Western blotting analysis, flow cytometry, and confocal laser scanning microscopy (CLSM) were purchased from Invitrogen (Thermo Scientific), Cell Signaling Technology (USA), and Biolegend (USA).

[0163] Device all 1 H nuclear magnetic resonance ( 1¹H NMR spectra were recorded using a Bruker AVIII 500MHz NMR spectrometer operating in Fourier transform mode. ESI-MS spectra were collected by a Finningan LTQ. Hydrodynamic size and polydispersity index of nanoparticles were measured using a Zeta-PALS particle size analyzer (Brookhaven Instruments). Transmission electron microscopy (TEM) measurements were performed using an FEI Technai Spirit transmission electron microscope. Cells were observed using a confocal laser scanning microscope (Eclipse Ti2, Nikon, Japan). Flow cytometry analysis was performed using a BD Accuri C6 flow cytometer or LSR-II flow cytometer (BD, USA). In vivo imaging of mice was performed using a Perkin Elmer® IVIS® system (Bioluminescence Imaging, Boston, USA).

[0164] Preparation of pre-fused AHPC-GFP-Cre and pre-fused AHPC-OVA [ka]

[0165] Preparation of pre-fused AHPC-GFP-Cre GFP-Cre (0.5 mg) was dissolved in 0.5 mg of phosphate-buffered saline (PBS, 10 mM, pH 7.4), and then Trout's reagent (5 equivalents) and tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP·HCl, 10 equivalents) were added. The solution was stirred at room temperature for 3 hours, and then dialyzed against PBS at 4°C to obtain thiol-modified GFP-Cre.

[0166] (S,R,S)-AHPC-PEG4-amine (20 mg, 0.03 mmol) was dissolved in 0.4 ml of dimethylformamide (DMF) containing triethylamine (TEA, 0.06 mmol, 2 equivalents). Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (28 mg, 0.06 mmol) dissolved in 0.2 mL of DMF was added dropwise to the stirred mixture and the mixture was reacted at room temperature for 24 hours. The solvent was then evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane / methanol, 10 / 1, v / v) to obtain 22 mg of SMCC-modified AHPC (0.02 mmol, yield 77.9%) as a white solid. TLC: Rf = 0.51 (dichloromethane / methanol, 6 / 1, v / v). ESI-MS: m / z[M+Na + ]C 57 H 111 N3O8S8 calculated value: 905.42; measured value: 905.64.

[0167] Thiol-modified GFP-Cre and SMCC-modified AHPC (15 / 1, w / w) were mixed in PBS, then TCEP (thiol-modified GFP-Cre / TCEP, 1 / 10, n / n) was added, and the mixture was stirred at room temperature for 8 hours. The mixture was dialyzed in PBS to obtain purified pre-fused AHPC-GFP-Cre, which was stored at 4°C for further use.

[0168] Preparation of pre-fused AHPC-OVA Pre-fused AHPC-OVAs were prepared using the same method and evaluated by SDS-polyacrylamide gel electrophoresis.

[0169] Synthesis of 113-O12B lipids [ka]

[0170] Synthesis of Compound 3 Compound 1 is mentioned in previous literature. 1The compound was synthesized according to the following procedure. A solution of compound 1 (0.6 g, 2 mmol) in 10 mL of acetonitrile (ACN), cesium carbonate (2.0 g, 6 mmol), and iodomethane (0.15 mL, 2.4 mmol) was added under an argon atmosphere and stirred overnight at room temperature. The solvent was removed under reduced pressure. Next, crude compound 2 was dissolved in a mixture of ethyl acetate (10 mL) and water (10 mL). The aqueous layer was extracted twice with ethyl acetate (10 mL). The combined organic layers were dried over anhydrous magnesium sulfate (MgSO4), filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol, 95 / 5~90 / 10, v / v) to obtain 0.5 g of compound 2 (1.6 mmol, yield 78.1%). TLC: Rf = 0.68 (dichloromethane / methanol, 6 / 1, v / v). ESI-MS: m / z[M+H + ]C 15 H 31 N3O4 calculated value: 318.23; measured value: 318.55.

[0171] To a solution of compound 2 (1.0 g, 3 mmol) in 10 mL of ethyl acetate, 4N HCl in dioxane (4.5 mL, 18 mmol) was added dropwise under an argon atmosphere at 4°C. The reaction mixture was stirred at room temperature for 12 hours, and the solvent was removed under reduced pressure. The residue was triturated three times with diethyl ether and with a small amount of ethyl acetate. The resulting solid was dissolved in 10N NaOH and extracted with dichloromethane (6 × 20 mL). The combined organic layers were dried over anhydrous MgSO4, filtered, and the solvent was removed to obtain 0.3 mg of compound 3 (0.026 mmol, yield 85.4%). ESI-MS: m / z[M+H] + ]C5H 15 N3 calculated value: 118.13; measured value: 118.28.

[0172] Synthesis of 113-O12B lipids Compound 3 (headamine) and compound 6 (bioreducible acrylate-modified alkyl chain) were mixed in a molar ratio of 1:4.8 in a Teflon®-lined glass screw-top vial and heated for 48 hours (70°C). The crude product was purified by silica gel column chromatography to obtain 113-O12B lipid. 1 H NMR(CDCl3,500MHz,ppm):4.35(t,J=5.0Hz,8H),2.92(t,J=5.0Hz,8H),2.82(t,J=5.0Hz,8H),δ2.72(t,J=5.0Hz,8H),2.58(t,J=5 .0Hz,4H),2.51-2.45(m,12H),2.26(s,3H),1.69(t,J=5.0Hz,8H),1.40(t,J=5.0Hz,8H),1.34-1.27(m,32H),0.93-0.89(m,12H). 13 C NMR(CDCl3,ppm):δ172.3,62.6,56.2,51.9,49.6,43.1,39.3,37.1,32.6,31.8,29.7,29.2,29.1,28.5,22.6,14.1.ESI-MS:m / z[M+H + ]C 57 H 111 For N3O8S8, the calculated value is 1221.61; the measured value is 1222.23.

[0173] Preparation of Adjuvant LNPs Preparation of Adjuvant LNPs Adjuvant LNP contains adjuvant tripidoid (113-O12B), cholesterol (Chol), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG). 2k The formulation was prepared using ethanol dilution with the ingredients in a molar ratio of 48.5:38:10:3.5.

[0174] For the preparation of DiR-labeled adjuvant LNPs, a mixture of total lipids and DiR in a weight ratio of 20 / 1 was dissolved in ethanol. Then, 100 μL of the above solution was added dropwise to 300 μL of sodium acetate buffer (25 mM, pH 5.2) and vigorously vortexed. Next, ethanol was removed from the formulation by dialysis against PBS for 12 hours (molecular weight cutoff, 35 kDa). The resulting DiR-labeled adjuvant LNPs were stored at 4°C for further use (protected from light exposure).

[0175] Preparation of pre-fused AHPC-GFP-Cre LNPs, GFP-Cre-containing adjuvant LNPs, and TAgD-TVac To prepare LNP formulations loaded with different proteins, different protein payloads were mixed with adjuvant LNP at a weight ratio of 1 / 10 and vigorously vortexed. The solutions were then incubated at room temperature for 5 minutes. The resulting formulations were stored at 4°C for further use.

[0176] Characterization of adjuvant LNPs and TAgD-TVac TEM measurement To prepare the TEM sample, 10 μL of the sample solution was dropped onto a carbon-coated copper grid and allowed to stand for 10–15 minutes. Excess liquid was then absorbed with filter paper. Next, the sample was negatively stained with phosphotungstic acid (EPTA) (5–10 μL) for 1–2 minutes, absorbed again, air-dried, and then analyzed by TEM.

[0177] DLS measurement The hydrodynamic size of different formulations was evaluated in PBS at 37°C.

[0178] Lymph node (LN) targeting ability of adjuvant LNPs All animal experiments were conducted in accordance with and approved by the Institutional Animal Care and Use Committee at the Tufts University's Animal Protocol # M2023-33. The lymph node targeting ability of adjuvant LNPs was evaluated in BALB / c and C57BL / 6 mice using a DiR-labeled formulation. 50 μg of DiR-labeled adjuvant LNPs were subcutaneously administered to the tail ridge of mice. Six hours later, the in vivo distribution of DiR-labeled adjuvant LNPs was visualized using the IVIS imaging system (PerkinElmer, Boston, Massachusetts).

[0179] Ability of pre-fused AHPC-GFP-Cre to target intracellular pVHL Adjuvant LNPs were used to deliver pre-fused AHPC-GFP-Cre to DC2.4 cells (this formulation is referred to as pre-fused AHPC-GFP-Cre LNP). Flow cytometry and confocal laser scanning microscopy (CLSM) observations were used to study the intracellular uptake of pre-fused AHPC-GFP-Cre LNPs and the ability of pre-fused AHPC-GFP-Cre to target intracellular pVHL.

[0180] For flow cytometry assays, DC2.4 cells (5 × 10⁶ cells per well) 4 Cells were seeded in 12-well plates and incubated overnight for cell adhesion. Different formulations containing pre-fused AHPC-GFP-Cre LNP and GFP-Cre+AHPC LNP (GFP-Cre, 2 μg / mL; adjuvant LNP, 20 μg / mL) were incubated with the cells for 3 hours, then the cells were washed three times with PBS and subsequently digested with 0.25% trypsin for flow cytometry analysis.

[0181] For confocal microscopy (CLSM) observation, DC2.4 cells were seeded in a confocal dish and grown to approximately 80% confluence. Various formulations, including pre-fused AHPC-GFP-Cre LNP and GFP-Cre+AHPC LNP (GFP-Cre, 2 μg / mL; adjuvant LNP, 20 μg / mL), were incubated with the cells for 3 hours, then the culture medium was changed to fresh medium and incubated for a further 9 hours. The cells were washed three times with PBS and fixed with 4% paraformaldehyde. Intracellular pVHL was stained with anti-VHL antibody and fluorescent secondary antibody (red). The nuclei were stained with DAPI (blue).

[0182] OVA TAgD-TVac OVA Mediated proteasome degradation and antigen cross-presentation Bone marrow-derived dendritic cells (BMDCs) were generated from the bone marrow of C57BL / 6 mice.

[0183] Western blotting was performed to evaluate the OVA levels of cells in BMDCs. For Western blotting, BMDCs (1 × 10⁶ per well) were used. 5Cells were seeded in a 6-well plate and incubated overnight for cell adhesion. Different formulations (OVA, 20 μg / mL; adjuvant LNP, 200 μg / mL) were incubated with the cells for 3 hours, then replaced with fresh culture medium and incubated for a further 24 hours. The cells were then washed three times with PBS and lysed in 0.5 mL of lysis buffer (PBS containing 1% Triton X-100 and 1 mM PMSF) at 4°C for 30 minutes. Equal volumes of protein loading were separated by SDS-PAGE and transferred to a PVDF membrane by electrophoresis. Nonspecific binding sites were blocked at room temperature for 2 hours using 5% skim milk in PBS (PBST) containing 0.05% Tween20. The membrane was incubated overnight at 4°C for 24 hours in a primary antibody of appropriate concentration, followed by three rinses in PBST buffer for 15 minutes each. The membrane was then incubated at room temperature for 1 hour in a secondary antibody (1:10000). Membranes were detected and analyzed using a computer-controlled chemiluminescence imaging system. Cytosolic fractions were prepared using a lysosome enrichment kit to evaluate the level of OVA in the cytoplasm. Antibody list: Anti-chicken ovalbumin antibody (Invitrogen, catalog number PA1196, clone number polyclonal), anti-mouse GAPDH antibody (Novus Biologicals, catalog number NB300326, clone number polyclonal).

[0184] BMDCs were incubated with different formulations (OVA, 20 μg / mL; adjuvant LNP, 200 μg / mL) for 12 or 24 hours. The BMDCs were then harvested and stained with fluorescently conjugated antibodies for flow cytometry analysis. Antibody list: PE-anti-mouse CD11C antibody (BioLegend, catalog number 117308, clone number N418), APC-anti-mouse H2kb-SIINFEKL antibody (eBioscience, catalog number 17-5743-82, clone number eBio25-D1.16), FITC-anti-mouse CD80 antibody (BD Biosciences, catalog number 561954, clone number 16-10A1), and APC-anti-mouse CD86 antibody (BioLegend, catalog number 105012, clone number GL-1).

[0185] TAgD-TVac OVA LN targeting capability TAgD-TVac OVA For the preparation of the compound, pre-fused AHPC-OVA was pre-labeled with cyanine 5.5NHS ester (Cy5.5). Cy5.5-labeled TAgD-TVac OVA The LN targeting ability was evaluated in BALB / c and C57BL / 6 mice. Cy5.5-labeled TAgD-TVac OVA (OVA, 10 μg; adjuvant LNP, 100 μg per dose) was subcutaneously injected into the tail ridge of mice. Six hours later, Cy5.5-labeled TAgD-TVac OVA The in vivo distribution of [the substance] was visualized using the IVIS imaging system.

[0186] Delivery of GFP-Cre to lymph nodes (LNs) in Ai14 mice using adjuvant LNPs Ai14 mice were subcutaneously injected twice into the tail root with GFP-Cre-loaded adjuvant LNP (20 μg GFP-Cre; 200 μg adjuvant LNP per injection) at 0 and 24 hours. Forty-eight hours after the last injection, the mice were euthanized and inguinal LN was collected. A cell suspension was prepared by grinding and filtering through a 70 μm strainer. Then, 2 × 10⁶ cells were extracted. 6Each cell was incubated on ice for 1 hour in 100 μL of flow cytometry staining buffer containing the recommended concentration of fluorescent conjugate antibody. The cells were then washed twice with PBS and kept at 4°C for flow cytometry analysis. Antibody list: APC / Cy7-anti-mouse CD49b antibody (BioLegend, catalog number 108920, clone number DX5), APC / Cy7-anti-mouse CD3 antibody (BioLegend, catalog number 100222, clone number 17A2), Brilliant Violet 650-anti-mouse B220 antibody (BioLegend, catalog number 103241, clone number RA3-6B2), PE / Cy7-anti-mouse CD11C antibody (BioLegend, catalog number 117318, clone number N418), and PerCP / Cy5.5-anti-mouse F4 / 80 antibody (BioLegend, catalog number 123128, clone number BM8).

[0187] To establish a macrophage depletion mouse model, macrophages were depleted by injecting mice with Clodrosome® (10 mL / kg). Clodrosome® is a well-characterized liposomal drug used to deplete macrophages in either local tissue or peripheral blood, depending on the injection route. Briefly, it is a Cy5.5-labeled TAgD-TVac OVA Cldrosome® was administered subcutaneously to mice 18 hours prior to administration of [substance name]. Six hours later, the mice were euthanized, and inguinal lymph nodes (LNs) were collected. The Cy5.5 signal in the LNs was visualized using the IVIS imaging system.

[0188] TAgD-TVac for inducing antitumor T cell responses in vivo OVA The effect Female C57BL / 6 mice (Charles River) aged 6-8 weeks and B16F10 tumor cells expressing ovalbumin (B16F10-OVA) were used in this study. B16F10-OVA tumor cells (1 × 10⁶ per mouse) were placed in the left flank of the C57BL / 6 mice. 6Tumor-bearing mice were established by subcutaneous (SC) injection of ). Then, 6 days after tumor inoculation, the mice were randomly divided into four groups (n=6). The mice were given phosphate-buffered saline (PBS), OVA LNP, OVA+AHPC LNP, and TAgD-TVac. OVA Tumor growth was monitored after each individual was vaccinated at the base of the tail using SC (protein, 20 μg per dose; LNP, 200 μg per dose; 3 doses). Tumor volume (V) was calculated as V = L × W 2 The calculation was performed according to / 2 (wherein L and W are the maximum and minimum diameters of the tumor (mm), respectively). If signs of health impairment are present, or if the tumor volume is 1.5 cm 3 If the threshold was exceeded, the mice were euthanized.

[0189] For the flow cytometry assay, freshly collected tumor tissue was digested with collagenase IV and prepared as a single-cell suspension according to the manufacturer's instructions. The tumor cell suspension was then prepared by grinding and filtering through a 70 μm strainer. Subsequently, for flow cytometry analysis, 2 × 10⁶ cells were collected. 6 The cells were incubated on ice for 1 hour in 100 μL of flow cytometry staining buffer containing the recommended concentration of fluorescent conjugate antibody. Antibody list: APC / Cy7-anti-mouse CD45 antibody (BioLegend, catalog number 103116, clone number 30-F11), APC-anti-mouse CD3 antibody (BD Biosciences, catalog number 565643, clone number 17A2), PE-anti-mouse CD8 antibody (BD Biosciences, catalog number 553032, clone number 53-6.7), FITC-anti-mouse CD4 antibody (BD Biosciences, catalog number 553730, clone number GK1.5), and FITC-anti-mouse GzmB antibody (BioLegend, catalog number 515403, clone number GB11).

[0190] The same procedure was used to analyze immune activation in lymph nodes and spleen using fluorescently conjugated antibodies. Antibody list: PE-anti-mouse CD11C antibody (BioLegend, catalog number 117308, clone number N418), APC-anti-mouse H2kb-SIINFEKL antibody (eBioscience, catalog number 17-5743-82, clone number eBio25-D1.16), FITC-anti-mouse CD80 antibody (BD Biosciences, catalog number 561954, clone number 16-10A1), and APC-anti-mouse CD86 antibody (BioLegend, catalog number 105012, clone number GL-1), APC-anti-mouse CD3 antibody (BD Biosciences, catalog number 565643, clone number 17A2), PE-anti-mouse CD8 antibody (BD Biosciences, catalog number 553032, clone number 53-6.7), APC / Cy7-anti-mouse CD62L antibody (BioLegend, catalog number 104428, clone number MEL-14), and FITC-anti-mouse CD44 antibody (BioLegend, catalog number 163606, clone number QA19A43).

[0191] Levels of pro-inflammatory cytokines (including M-CSF, IFN-γ, and IL-6) and pro-inflammatory chemokines (including MCP-1, MIP-1α, and MIP-1β) in the blood were measured using an ELISA kit according to the manufacturer's instructions.

[0192] CD8 in vivo + To deplete T cells, an anti-CD8a antibody (aCD8, 300 μg / mouse, BioXcell, catalog number BE0061, clone number 2.43) was administered by intraperitoneal injection.

[0193] TAgD-TVac in a mouse model of breast cancer 4T1 antitumor effect Tumor lysate protein solutions were prepared by freeze-thaw method. Cultured 4T1-Luc tumor cells were rapidly frozen on dry ice for 5 minutes and immediately thawed at 37°C. The solutions were then vortexed and thoroughly mixed. This procedure was repeated twice. Protamine sulfate (derived from salmon) was added to the resulting solutions to bind and remove nucleic acids. The final solutions were centrifuged at 13,000 rpm for 10 minutes. The supernatant was collected, and the concentration of the lysate was measured by bicinchoninic acid (BCA) assay.

[0194] Six-to-eight-week-old female BALB / c mice (Charles River) and luciferase-transfected 4T1 (4T1-Luc) tumor cells were used in this study. 4T1-Luc tumor cells (1 × 10⁶ per mouse) were placed in the left flank of the BALB / c mice. 6 Tumor-carrying mice were established by SC injection of ) . Then, 6 days after tumor inoculation, the mice were randomly divided into three groups (n=6). These groups were PBS and TLP, respectively. 4T1 +AHPC LNP and TAgD-TVac 4T1 (Protein, 20 μg per dose; LNP, 200 μg per dose, total of 3 doses) were subcutaneously inoculated into the tail ridge of mice, and tumor growth was monitored. Tumor growth was monitored by measuring in vivo bioluminescence using the IVIS imaging system (every 5 days). Tumor volume (V) was given by the formula: V = L × W 2 Calculated according to / 2. If signs of health impairment are present, or if the tumor volume is 1.5 cm², 3 If the threshold was exceeded, the mice were euthanized.

[0195] For the flow cytometry assay, freshly collected tumor tissue was digested with collagenase IV and prepared as a single-cell suspension according to the manufacturer's instructions. The tumor cell suspension was then prepared by grinding and filtering through a 70 μm strainer. Subsequently, for flow cytometry analysis, 2 × 10⁶ cells were collected. 6Cells were incubated on ice for 1 hour in 100 μL of flow cytometry staining buffer containing the recommended concentration of fluorescent conjugate antibodies (including APC-Cy7-anti-mouse CD45 antibody, APC-anti-mouse CD3 antibody, PE-anti-mouse CD8 antibody, FITC-anti-mouse CD4 antibody, and FITC-anti-mouse GzmB antibody).

[0196] By performing a similar procedure, immunoactivation in lymph nodes and the spleen was analyzed using fluorescently conjugated antibodies, including PE-anti-mouse CD11C antibody, FITC-anti-mouse CD80 antibody, APC-anti-mouse CD86 antibody, APC-anti-mouse CD3 antibody, PE-anti-mouse CD8 antibody, APC-Cy7-anti-mouse CD62L antibody, and FITC-anti-mouse CD44 antibody.

[0197] TLP without obvious tumors 4T1 +AHPC LNP and TAGD-TVac 4T1 On day 18, the mice in the group were injected intravenously (IV) with 4T1-Luc tumor cells (1 × 10⁶ cells per mouse). 6 The cells were challenged again. The PBS control group also received IV injections of 4T1-Luc tumor cells (1 × 10⁶ per mouse). 6 The mice were treated with (cells). On day 32, all mice were euthanized, their lungs were collected, and observed using the IVIS imaging system.

[0198] TAGD-TVac in an orthotopic lung cancer mouse model LLC antitumor effect Six-to-eight-week-old female C57BL / 6 mice (Charles River) and luciferase-stably expressed Lewis lung carcinoma (LLC-Luc) cells were used in this study. The C57BL / 6 mice were anesthetized with isoflurane in an anesthesia box and then maintained with the same flow rate of isoflurane using an anesthesia mask on a heated (37°C) heating pad. The rib layer was exposed by removing hair from below the left scapula, and the left rib region of the mice was disinfected with 70% ethanol. The prepared LLC-Luc cell suspension (0.5 × 10⁶) was then prepared.6 50 μL of insulin was drawn into a mouse syringe. The needle was inserted through the chest wall between the 3rd and 4th ribs and carefully punctured into the left lung. While keeping the syringe upright, the cells were slowly injected to a depth of approximately 3-5 mm. Six days later, the establishment of an orthotopic lung cancer mouse model was verified using the IVIS imaging system. The mice were then randomly divided into four groups (n=6). TAgD-TVac was administered to tumor-bearing mice. LLC (Protein, 20 μg per dose; LNP, 200 μg per dose; administered via SC injection into the ridge) was given in three doses. Anti-PD-1 antibody (aPD-1, 200 μg per dose; administered in three doses) was administered by intraperitoneal (IP) injection. TAgD-TVac LLC The inhibitory effect of monotherapy was also evaluated. Monotherapy with aPD-1 was used as a comparison. Tumor growth was monitored by measuring in vivo bioluminescence using the IVIS imaging system (every 3 days). Since LLC-Luc tumors are expected to grow in the lungs and externally observable tumor masses are not expected, body weight was used to assess tumor progression. Mice were euthanized if they showed a body weight loss of more than 15%.

[0199] Lymph nodes and spleens were collected to prepare single-cell suspensions. Immune activation in the lymph nodes and spleens was analyzed by staining cells with fluorescent conjugate antibodies, including PE-anti-mouse CD11C antibody, FITC-anti-mouse CD80 antibody, APC-anti-mouse CD86 antibody, APC-anti-mouse CD3 antibody, PE-anti-mouse CD8 antibody, APC-Cy7-anti-mouse CD62L antibody, and FITC-anti-mouse CD44 antibody.

[0200] aPD-1+TAgD-TVac LLC LLC-Luc tumor-carrying mice that received combination therapy were re-challenged on day 40 by SC injection with LLC-Luc (left flank) and Cognate B16F10 tumor cells (right flank) to mimic tumor recurrence (n=5). LLC-Luc cells (1 × 10⁶ per mouse) 6 ) was subcutaneously injected into the left flank of C57BL / 6 mice, and B16F10 tumor cells (1 × 10 per mouse) were administered.6 The solution was inoculated into the flank opposite the LLC-Luc tumor. LLC-Luc and B16F10 tumor cells were also injected via SC into the control group (normal mice, n=5). Tumor volume (V) was calculated using the formula: V = L × W 2 The calculation was performed according to / 2. All mice were euthanized on day 55, and the tumors were collected and photographed.

[0201] TAgD-TVac in a mouse model of inoperable ovarian cancer ID8 antitumor effect Female C57BL / 6 mice (Charles River) aged 6-8 weeks and luciferase-stable expressing ovarian tumor cells (ID8-Luc) were used in this study. ID8-Luc (1 × 10⁶ cells per mouse) 7 Tumor cells were intraperitoneally injected into C57BL / 6 mice to establish an inoperable ovarian cancer model. After 7 days, the establishment of the inoperable ovarian cancer mouse model was verified using the IVIS imaging system. The mice were then randomly divided into four groups (n=6). Tumor-carrying mice were given TAgD-TVac ID8 (Protein, 20 μg per dose; LNP, 200 μg per dose; administered via SC to the ridge) was given in three doses. Anti-PD-1 antibody (aPD-1, 200 μg per dose; administered in three doses) was administered by intraperitoneal (IP) injection. aPD-1 or TAgD-TVac ID8 The antitumor effect of monotherapy was also evaluated. Tumor growth was monitored by measuring in vivo bioluminescence using the IVIS imaging system (every 5 days). In accordance with institutional guidelines, mice were euthanized if they developed ascites or gained more than 30% body weight.

[0202] Blood samples were collected from the mice on day 10. Blood levels of pro-inflammatory cytokines (including M-CSF, IFN-γ, and IL-6) and pro-inflammatory chemokines (including MCP-1, MIP-1α, and MIP-1β) were measured using an ELISA kit according to the manufacturer's instructions. When any of the tumor-bearing mice in the PBS group reached the human endpoint criteria (weight gain >30%), all mice were euthanized and ascites volume was assessed.

[0203] statistical analysis Data were expressed as mean ± standard deviation (SD). All data were analyzed using Graphpad Prism (8.0) software.

[0204] Embedding by reference All publications and patents referenced herein are incorporated herein by reference in whole, as if each individual publication or patent were explicitly incorporated by specific and individual reference. In case of any conflict, this application shall prevail, including the definitions herein.

[0205] Equal portions While specific embodiments of the present invention have been described, the above specification is illustrative and not limiting. Many variations of the present invention will become apparent to those skilled in the art upon examination of the specification and the following claims. The full scope of the present invention should be determined by reference to the full scope of the claims and their equivalents, as well as the specification and such variations.

Claims

1. Compounds having the structure represented by formula I, or pharmaceutically acceptable salts thereof: 【Chemistry 1】 [In the formula, A is a tumor antigen; E is the active agent (e.g., a drug); Y is a linker (L) that covalently connects A to E, and includes alkylene, alkenylene, alkynylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene, or a combination thereof, and L contains 1 to 100 carbon atoms, preferably 10 to 50 carbon atoms.

2. The aforementioned linker is -CH ２ -, -C(=O)-, -C(=O)-NR １ -, -NR １ -C(=O)-, -C(=O)-O-, -O-C(=O)-, -NR １ -C(=O)-NR ２ -, -O-C(=O)-NR １ -, -NR １ -C(=O)-O-, -O-, and -S-, a heteroalkylene comprising 5 to 50 moieties selected therefrom, wherein each R １ and R ２ are each independently selected from H and alkyl, the compound according to claim 1.

3. The linker is -CH ２ The compound according to claim 1 or 2, which is a heteroalkylene comprising 5 to 50 moieties selected from - and -O-.

4. The linker is a compound according to any one of claims 1 to 3, comprising ethylene glycol.

5. The compound according to claim 4, wherein the linker comprises 1 to 10 ethylene glycol moieties (for example, 2, 4, 6, or 8 ethylene glycol moieties).

6. The compound according to claim 1, wherein the linker is an alkylene containing 5 to 50 methylene moieties.

7. The linker is -C(=O)-, -C(=O)-NR ２ -, -NR ２ -C(=O)-, -C(=O)-O-, -OC(=O)-, -NR ２ -C(=O)-NR ３ -, -OC(=O)-NR ２ -, -NR ２ It further includes a portion selected from -C(=O)-O-, -O-, and -S-, where each R ２ and R ３ The compound according to claim 6, wherein each is independently selected from H and alkyl.

8. The linker has 1 to 5 -C(=O), -C(=O)-NR ２ -, or -NR ２ -C (=O)- part (for example, one, two, or three parts), where each R ２ and R ３ The compound according to any one of claims 2 to 7, wherein each is independently selected from H and alkyl.

9. The compound according to any one of claims 2 to 8, wherein at least one -C(=O)- moiety is adjacent to E.

10. The aforementioned linker is 【Chemistry 2】 A compound according to any one of claims 2 to 9, further comprising:

11. The aforementioned linker is 【Transformation 3】 A compound according to any one of claims 2 to 9, further comprising:

12. The linker is 【Chemistry 4】 A compound according to any one of claims 2 to 9, further comprising:

13. Structure represented by formula Ia: 【Transformation 5】 [In the formula, n1 and n2 are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. n3 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The compound according to any one of claims 1 to 12, having or a pharmaceutically acceptable salt thereof.

14. Structure represented by formula Ib: 【Transformation 6】 The compound according to any one of claims 1 to 13, having or a pharmaceutically acceptable salt thereof.

15. The compound according to any one of claims 1 to 14, wherein E is a ligand for a ligase (e.g., E3 ligase).

16. The compound according to any one of claims 1 to 14, wherein E is a ligand for von Hippel-Lindau (VHL), cell apoptosis inhibitor protein (cIAP), cereblon (CRBN), or mouse double microchromosome 2 (MDM2).

17. E is, 【Transformation 7】 【Transformation 8】 【Chemistry 9】 [In the formula, Each X １ and X ２ O, NR ４ Selected from S; Each R ３ Each is independently selected from halos (e.g., fluoro or bromo); Each R ４ [These are independently H, alkyl, or aralkyl.] The compound according to any one of claims 1 to 16.

18. E is, 【Chemistry 10】 The compound according to claim 17.

19. E is, 【Chemistry 11】 The compound according to claim 17.

20. E is, 【Chemistry 12】 The compound according to claim 17.

21. E is, 【Chemistry 13】 The compound according to claim 17.

22. Structure represented by formula Ic: 【Chemistry 14】 The compound according to claim 1, having or a pharmaceutically acceptable salt thereof.

23. Structure represented by formula Id: 【Chemistry 15】 The compound according to claim 1, having or a pharmaceutically acceptable salt thereof.

24. A is a T antigen, the compound according to any one of claims 1 to 23.

25. The antigen is derived from a tumor (for example, from tumor cells), and is a compound according to any one of claims 1 to 24.

26. The antigen is a compound according to any one of claims 1 to 24, derived from an infectious pathogen (e.g., bacteria or a virus).

27. The compound according to any one of claims 1 to 24, wherein the antigen is derived from prostate cancer cells, liver cancer cells, colorectal cancer cells, ovarian cancer cells, pancreatic cancer cells, ovarian cancer cells, breast cancer cells, malignant melanoma cancer cells, or bladder cancer cells.

28. The compound according to any one of claims 1 to 24, wherein the antigen is prostate-specific antigen (PSA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen 125 (CA-125), mucin-1 (MUC-1), malignant melanoma-associated antigen (MAGE), or New York esophageal squamous cell carcinoma 1 (NY-ESO-1) antigen.

29. A is the Simian Vacuolating Virus 40 T antigen (SV40 large T antigen), a compound according to any one of claims 1 to 24.

30. Nanoparticles comprising a tumor antigen covalently linked to an active drug, and multiple lipids or lipidoids.

31. The tumor antigen covalently linked to the active agent has a structure represented by formula I: 【Chemistry 16】 [In the formula, A is a tumor antigen; E is the active agent (e.g., a drug); Y is a linker (L) that covalently connects A to E, and includes alkylene, alkenylene, alkynylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene, or a combination thereof, and L contains 1 to 100 carbon atoms, preferably 10 to 50 carbon atoms. The nanoparticles according to claim 30, having the properties of the nanoparticles described in claim 30.

32. The aforementioned tumor antigen is 【Chemistry 17】 The nanoparticles according to claim 30 or 41, which are covalently linked to the active agent via a .

33. The nanoparticle according to claim 30, wherein the active agent is a compound according to any one of claims 1 to 29.

34. Nanoparticles according to any one of claims 30 to 33, comprising a plurality of lipidoids.

35. Nanoparticles according to any one of claims 30 to 33, comprising a plurality of lipids.

36. Nanoparticles according to any one of claims 30 to 33, comprising a plurality of lipids and lipidoids.

37. The aforementioned lipids and lipidoids are 113-O12B, cholesterol (Chol), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG). ２ｋ ), MC-3, SM-102, ALC0315, L88, and 93-O17S, or any combination thereof, nanoparticles according to any one of claims 30 to 36.

38. The aforementioned lipids and lipidoids are 113-O12B, cholesterol (Chol), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG). ２ｋ A nanoparticle according to any one of claims 27 to 33, selected from the group consisting of ).

39. A pharmaceutical composition comprising nanoparticles according to any one of claims 30 to 38, and a pharmaceutically acceptable excipient.

40. A method for treating cancer, comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound according to any one of claims 1 to 29, or a pharmaceutically acceptable salt thereof, or nanoparticles according to any one of claims 30 to 38.

41. The method according to claim 40, wherein the cancer is malignant melanoma, breast cancer, ovarian cancer, splenic cancer, or lung cancer.

42. The method according to claim 40 or 41, further comprising administering an immune checkpoint inhibitor (e.g., a PD-1 inhibitor).

43. A method for treating an infectious disease, comprising administering to a subject in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 29, or a pharmaceutically acceptable salt thereof, or nanoparticles according to any one of claims 30 to 38.

44. The method according to claim 43, wherein the pathogen of the infectious disease is a bacterium.

45. The method according to claim 43, wherein the pathogen of the infectious disease is a virus.

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