Antigen peptide polymer vesicle tumor vaccine as well as preparation method and application thereof

Abstract

The invention discloses an antigen peptide polymer vesicle tumor vaccine as well as a preparation method and application thereof. The antigen peptide polymer vesicle tumor vaccine is obtained by assembling and cross-linking a polymer pre-grafted with antigen peptide and a block polymer and loading an immunologic adjuvant through electrostatic interaction. The vesicle system disclosed by the invention has the advantages of small size, simplicity and controllability in preparation, excellent biocompatibility, high in-vivo circulation stability, targeting of main immune organs, intracellular delivery of antigen peptides and the like. The vesicle nano vaccine displaying the antigen peptide on the surface and encapsulating the immunologic adjuvant can promote dendritic cell maturation and antigen presentation, improve the specific response of mice to tumor-associated antigens, effectively inhibit tumor growth, remarkably prolong the lifetime of tumor-bearing mice and induce a strong immune protection effect, and is high in safety and easy to popularize. A multifunctional'ready-to-use 'nano vaccine concept is embodied, a flexible platform is provided, and different peptide antigens and / or adjuvants can be used for treating different cancers.

Description

Technical Field

[0001] The present invention belongs to the technical field of polymer nanomedicines, and specifically relates to a method for preparing a polymer vesicle tumor vaccine coupled with antigen peptides and its application in blood tumors, and more particularly to a vesicle tumor vaccine loaded with an immune adjuvant and surface-grafted with tumor-associated antigen peptides. Background Art

[0002] Cancer immunotherapy, including immune checkpoint blockade (ICB) and chimeric antigen T (CAR-T) cell therapy, heralds a new era in clinical oncology and has been successful in combating a variety of malignancies. At its core is the development of cancer vaccines that use tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs) to elicit new tumor-reactive T cells and activate the immune system, thereby targeting tumor cells that escape immune surveillance through antigen-specific responses. These vaccines have great potential because they target intracellular antigens, which account for nearly 88% of total cellular proteins. The key development difficulty is that immune responses are usually transient and localized.

[0003] Subunit vaccines based on antigenic peptides have attracted extensive research in tumor therapy. However, due to the low efficiency of epitope presentation and poor stimulation of APC, peptide nanovaccines usually have moderate immunogenicity and a narrow range of immune responses, which together impair the induction of antigen-specific T cells and antibody responses. In addition, the widespread cell dissemination and innate refractory nature of AML pose a daunting challenge to the development of therapeutic vaccines. Summary of the invention

[0004] The purpose of the present invention is to disclose a method for preparing a polymer vesicle tumor vaccine coupled with an antigen peptide and its application in blood tumors, specifically a polymer vesicle tumor vaccine loaded with an immune adjuvant and surface grafted with tumor-related antigen peptides, its preparation method, and its application in the treatment of blood tumors.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical scheme: An antigen peptide polymer vesicle tumor vaccine comprises an amphiphilic block polymer vesicle grafted with an antigen peptide and an immune adjuvant.

[0006] In the present invention, the immune adjuvant includes a TLR9 agonist; and the antigen peptide includes a WT1 antigen peptide.

[0007] The present invention discloses a method for preparing the above-mentioned antigen peptide polymer vesicle tumor vaccine, comprising the following steps: using an immune adjuvant, an amphiphilic block polymer grafted with an antigen peptide, and an amphiphilic block polymer with cations as raw materials to prepare an antigen peptide polymer vesicle tumor vaccine; specifically, using an immune adjuvant, an amphiphilic block polymer grafted with an antigen peptide, and an amphiphilic block polymer with cations as raw materials to prepare an antigen peptide polymer vesicle tumor vaccine. Specifically, the polymer pre-grafted with an antigen peptide and the amphiphilic block polymer with cations are assembled and spontaneously cross-linked, and the immune adjuvant is loaded by electrostatic action to prepare the vesicle tumor vaccine.

[0008] The antigen peptide polymer vesicle tumor vaccine disclosed in the present invention is prepared from an amphiphilic block polymer, an immune adjuvant, and a tumor antigen peptide. The amphiphilic block polymer is an amphiphilic block polymer grafted with an antigen peptide and an amphiphilic block polymer with cations. As an example, the immune adjuvant is a TLR9 agonist CpG oligodeoxynucleotide; the tumor antigen peptide WT1 121-137 .

[0009] In the present invention, the amphiphilic block polymer is a hydrophilic segment-P (ester monomer unit-DTC)-cationic segment and M-hydrophilic segment-P (ester monomer unit-DTC), M is an antigenic peptide structure; the hydrophilic segment-P (ester monomer unit-DTC)-cationic segment is preferably a hydrophilic segment-P (ester monomer unit-DTC)-spe. Preferably, the hydrophilic segment is a polyethylene glycol segment (PEG); P (ester monomer unit-DTC) is a hydrophobic segment, and its molecular weight is 2 to 8 times that of the hydrophilic segment, and the molecular weight of the PDTC segment is 10% to 40% of the total molecular weight of the hydrophobic segment. The molecular weight of PEG is 2000 to 12000 Da; preferably, the molecular weight of PEG is 3000 to 8000 Da, the molecular weight of the hydrophobic segment is 2 to 6 times that of PEG, and the molecular weight of PDTC is 10 to 30% of the molecular weight of the hydrophobic segment.

[0010] The antigen peptide polymer vesicle tumor vaccine of the present invention is assembled and cross-linked by an amphiphilic block polymer grafted with an antigen peptide and an amphiphilic block polymer with a cation, and has an asymmetric membrane structure, an outer shell of polyethylene glycol (PEG), a membrane layer of reversibly cross-linked hydrophobic polycarbonate, and an inner layer of polyspermine (SPE). The spermine residues in the inner layer can be combined with immune adjuvants through electrostatic interaction, thereby achieving efficient and stable loading of adjuvants and antigens.

[0011] In the present invention, the mass ratio of the antigen peptide and the immune adjuvant is 1: (1-20), which refers to the feed mass ratio; preferably, the feed mass ratio is 1: (1-12); more preferably, the feed mass ratio is 1: (1-8), which can be selected as 1: 1, 1: 2, 1: 4, 1: 8 or any ratio within the range; forming a tumor vaccine that is co-loaded with an immune adjuvant and has tumor antigen peptides presented on the surface.

[0012] The invention discloses an anti-tumor drug, which takes antigen peptide polymer vesicle tumor vaccine as an active ingredient.

[0013] The invention discloses the application of the antigen peptide polymer vesicle tumor vaccine or anti-tumor drug in the preparation of anti-tumor drugs.

[0014] The antigen peptide polymer vesicle tumor vaccine of the present invention can be used as the sole active ingredient to prepare anti-tumor drugs, or can be used together with other active drugs (such as antibody drugs) as active ingredients to prepare anti-tumor drugs.

[0015] In the present invention, the tumor is a blood tumor or a solid tumor; preferably, the blood tumor includes leukemia.

[0016] The antigen peptide polymer vesicle tumor vaccine of the present invention can effectively promote the uptake and maturation of antigen presenting cells, especially DC cells, macrophages and B cells after intravenous administration. The biologically active immune adjuvant in the disulfide cross-linked vesicle cavity can avoid leakage during the delivery process and cause loss. After being taken up by APC cells, it can quickly escape from the inclusion body and enter the cytoplasm, effectively activating the antigen presentation of DC cells and promoting their activation. Furthermore, DC cells promote the recognition of WT1 tumor antigens by CTL cells and improve the specific killing of tumor cells.

[0017] The present invention discloses the use of the above-mentioned antigen peptide polymer vesicle tumor vaccine in the preparation of anti-tumor drugs, wherein the tumor is a blood tumor, especially in the treatment of acute myeloid leukemia. Specifically, the polymer vesicle tumor vaccine with WT1 antigen peptide grafted on the surface and an immune adjuvant inside is intravenously administered. The present invention has excellent application effect in the treatment of acute myeloid leukemia. After intravenous injection of the vaccine, the distribution of splenic B cells changes significantly, promotes the maturation of each APC cell and antigen presentation, increases the abundance of WT1-specific T cells, and has excellent anti-tumor effect in combination with anti-CTLA-4 antibodies.

[0018] Compared with the prior art, the present invention has the following advantages: 1. The present invention designs an antigen peptide polymer vesicle tumor vaccine for efficient and stable loading of immune adjuvants and tumor-related antigen peptides, and targeted enrichment of lymph nodes, which solves the problem of easy enzymatic hydrolysis of biologically active molecules and poor cellular uptake; the vesicle membrane is a reversibly cross-linked biodegradable and biocompatible PTMC, and the dithiolane ring on the side chain can provide reduction-sensitive reversible cross-linking, which can not only ensure the long circulation of the drug in the blood, but also can be rapidly degraded in the cells, while releasing immune adjuvants and tumor antigens into DC cells.

[0019] 2. The antigen peptide polymer vesicle tumor vaccine disclosed in the present invention has a significant DC cell activation effect in vivo and in vitro. The immune adjuvant and WT1 peptide are released into DC cells at the same time, which promotes the maturation of DC cells and improves the antigen presentation level, thereby increasing the proliferation of CTL cells.

[0020] 3. The antigen peptide polymer vesicle tumor vaccine disclosed in the present invention has high anti-tumor activity in vivo, can effectively inhibit the progression of blood tumors, and can completely regress tumors when used in combination with anti-CTLA-4; moreover, the cured mice have acquired long-term immune memory ability, which can significantly inhibit tumor recurrence. In addition, the present invention also has anti-tumor activity against tumors with high expression of WT1.

[0021] 4. The multifunctional antigen peptide vesicle tumor vaccine disclosed in the present invention can significantly prolong the survival of mice. Some mice can be completely cured without obvious damage to their organs. It is expected to provide an effective solution for tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Characterization diagram of antigen peptide polymers. NMR images of (A) Mal-PEG-P(TMC-DTC) and (B) S17-PEG-P(TMC-DTC) 1 H-NMR, 600 MHz, DMSO- d 6 ), (C) MicroBCA standard curve of S17 antigen peptide.

[0023] Figure 2Characterization of PASTA-NV. (A) Structural diagram of PASTA-NV and (B) particle size distribution. Endocytosis of PASTA-NV and S17 / TA-NV in BMDCs at 4 h incubation, characterized by (C) CLSM and (D) FC, (E) Endosomal escape of PASTA-NV at 4 h incubation, characterized by CLSM, (F) Expression of CD86, MHC-I and MHC-II molecules in BMDCs at 4, 12 and 24 h, (G) Uptake of PASTA-NV by spleen cells (MFI FITC), (H) Uptake (MFI FITC) and activation of spleen B cells. For CH, PASTA-NV was assembled using FITC-labeled S17; scale bars in C and E represent 20 μm, and scale bar in H represents 10 μm. DH, n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

[0024] Figure 3 The distribution of the vaccine in immune cells and the activation of immune cells after a single intravenous injection of PASTA-NV in MA9 mice. (A) Schematic diagram of AML vaccination, immunization, and analysis. Mice were vaccinated with MA9 cells on day 0 and intravenously injected with PASTA-NV (Cy5-labeled CpG) on day 3. Spleen, lymph node, bone, and serum samples were collected 6 h after injection; (B) Representative immunofluorescence (IF) images showing the colocalization of PASTA-NV in splenic DCs, macrophages, and B cells (scale bar: 100 μm); (C) Representative flow cytometry images showing the uptake of PASTA-NV by DCs, macrophages, and B cells; (D) Cy5-CpG in spleen cells, DCs, and macrophages. + (E) Expression of CD86, MHC-I, and MHC-II molecules in various immune cells in the spleen (n = 3); (F) Serum cytokines (IL-1α, IFN-γ, TNF-α, and IL-27) secreted by vaccinated mice (n = 4).

[0025] Figure 4 Representative immunofluorescence images of PASTA-NV and S17 / TA-NV (Cy5-labeled CpG) uptake by DCs, macrophages, and B cells in inguinal lymph nodes (scale bar: 100 μm).

[0026] Figure 5PASTA-NV induces potent cellular and humoral immune responses in MA9 mice. (A) Schematic diagram of AML inoculation, immunization, and analysis. PASTA-NV and S17 / TA-NV were injected intravenously, with PBS as a control; (B) Splenic DC subsets (CD103 + cDC1、CD11b + cDC2, B220 + pDCs and CD8 + DCs); (C) MFI of CD80 in cDC1 and cDC2 in spleen; (D) MFI of DC subsets (CD103 + cDC1 and CD11b + (E) MA9 cell infiltration in peripheral blood (PB) on day 32; (F) survival curve of MA9 mice; (G) NK cell activation in PB on day 23 (MFI of NKG2D and CD69); (H) IL-12p70 level in serum 6 h after the last vaccination; (I) WT1-specific expression in serum 126-134 Peptide-specific antibody titers; (J) Splenic CD4 + PD-1 in T cells + CXCR5 + The proportion of Tfhs; (K) spleen B220 + The proportion of B cells and the MFI of CD27 in B cells; (L) T cell degranulation effect (CD107a in spleen CD4 + T cells and CD8 + MFI in T cells), spleen CD4 + T cells and CD8 + (M) IFN-γ in T cells + and (N) TNF-α + The proportion of T cells; (O) Splenic CD8 + WT1 in T cells 126-134 Proportion of specific T cells; (P) titer of serum specific antibodies to the KRAS G12C mutant epitope; E, F, n = 7, analysis on day 23 (BD, GP), n = 6; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0027] Figure 6The vaccination strategy determined the antitumor efficacy and biodistribution of PASTA-NV in MA9 mice. (A) Schematic diagram of AML vaccination, immunization, and analysis; (B) Infiltration of MA9 cells in the bone marrow at day 23 (n = 6) and (C) survival curves of mice (n = 7); (D) Schematic representation of PASTA-NV (Cy5-labeled CpG) enrichment in major lymphoid organs and APCs; (E) Representative IF images of PASTA-NV colocalization with splenic B cells. Scale bars: 1 mm, 1 mm, and 200 μm (from top to bottom) of (F) Cy5-CpG in macrophages and B cells in the spleen. + Spleen cells and Cy5-CpG + DC ratio and MFI of PASTA-NV (Cy5) (n = 3); *p<0.05, **p<0.01, ***p<0.001.

[0028] Figure 7 Biodistribution of PASTA-NV was determined for vaccination strategies. (A) Semi-quantitative analysis of PASTA-NV enrichment in spleen and lymph nodes (n = 4); (B) Representative IF images of DCs and macrophages in spleen; (C) Cy5 CpG + The ratio of macrophages to B cells, and the MFI of MHC-I and MHC-II molecules in B cells (n = 4).

[0029] Figure 8 Representative IF images of PASTA-NV enrichment in DCs, macrophages, and B cells in inguinal lymph nodes; scale bar: 500 μm.

[0030] Fig. 9 To investigate the influence of vaccination strategies on the overall immune response of AML mice to PASTA-NV. (A) Schematic diagram of AML vaccination, immunization, and analysis (n = 6); (B) DCs in lymph and (C) the proportion of cDC1 and cDC2 subsets; (D) CD80 MFI values ​​of cDC1 and cDC2; (E) cDC1 and cDC2 proportions in splenic DCs; (F) B cells in spleen and (G) MFI values ​​of CD27; (H) CD107a in splenic CD4 + T cells and CD8 + MFI values ​​in T cells, after ex vivo stimulation with free S17 peptide, of splenic CD4 + T cells and CD8 + (I) Cytotoxic IFN-γ in T cells+ T cells and (J) TNF-α + The proportion of T cells. K) WT1 in spleen 126-134 CD8 + The proportion of T cells; (L) PD-1 in spleen + CD4 + and CD8 + Exhausted T cells; (M) CD206 in spleen + The number of M2 phenotype macrophages; * p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001.

[0031] Fig.10 Combination therapy of PASTA-NV (all iv) and anti-CTLA-4 (PASTA-NV+Ab) enhanced potent and durable anti-AML immune responses in MA9 mice. (A) Schematic diagram of AML inoculation, immunization, and analysis, (B) MA9 cell infiltration in PB on day 28 and (C) survival curve of mice (n = 7); (D) CD4 + and CD8 + MFI of CD69 in T cells; (E) total DC and (F) DC subsets in spleen. (G) WT1 in spleen 126-134 The number of specific T cells; (H) CD4 + The proportion of CD25+FoxP3+ Tregs in T cells; (I) On day 60, the proportion of CD8 + Central memory T cells (CD44 + CD62L + T CM ) and effector memory T cells (CD44 + CD62L - T EM ) ratio; DI, n = 6; (J) Specific killing of MA9 cells by spleen cells extracted from surviving mice (n = 3); (K) MA9 cell infiltration in PB on day 32 after re-inoculation and (L) survival curves of mice under simulated relapse conditions (previously surviving mice, n = 5, PBS, n = 7); * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

[0032] Fig.11PASTA-NV+Ab produced a general anticancer effect in mice vaccinated with PASTA-NV+Ab (n = 6). (A) Experimental flow chart; (B) Cell viability of WT1-expressing cancer cells (MC38, GL261, and LLC) co-incubated with spleen cells for 24 h was detected by CCK-8.

[0033] Fig.12 H&E staining of liver sections from surviving mice 14 months after PASTA-NV+Ab treatment. Scale bars represent 500 μm (left) and 100 μm (right).

[0034] Fig.13 Combination therapy of TA-NV and PASTA-NV (sc + iv) with anti-CTLA-4 enhanced the anti-AML effect in vivo (n = 7); (A) Schematic diagram of AML vaccination, immunization, and analysis; (B) MA9 cell infiltration in PB on day 28 and (C) survival curve of mice.

[0035] Fig.14 Figure 2. Cytokine levels in the supernatant of BMDCs and spleen cells after 24 h of incubation of nanovesicles loaded with MA9 cell lysate antigens and PASTA-NV; (A) IL-12p70 in the supernatant of BMDCs; (B) IFN-γ in the supernatant of spleen cells.

[0036] Fig.15 Figure 2 shows the tumor inhibition effect of PASTA-NV in subcutaneous colon cancer of MC38 mice. DETAILED DESCRIPTION

[0037] The present invention discloses the preparation and application of an antigen peptide polymer vesicle tumor vaccine. The polymer vesicle tumor vaccine is prepared from an amphiphilic block polymer grafted with an antigen peptide, an amphiphilic block polymer with cations, and an immune adjuvant. Preferably, the mass ratio of the amphiphilic block polymer grafted with the antigen peptide to the amphiphilic block polymer with cations is (0.1-5): (9.9-5), preferably (0.5-3): (9.5-7); preferably, the immune adjuvant includes a TLR9 agonist; the antigen peptide includes a WT1 antigen peptide; as an example, the immune adjuvant is a TLR9 agonist CpG oligodeoxynucleotide; the antigen peptide is WT1 121-137 Peptide.

[0038] In the present invention, the amphiphilic block polymer with cations is a hydrophilic segment-P (ester monomer unit-DTC)-cationic segment, such as a polymer including PEG-P (TMC-DTC)-spe. Among them, the molecular weight of the hydrophilic segment (PEG) is 2000-12000 Da; the molecular weight of the hydrophobic segment (P (TMC-DTC)) is 2-8 times that of the hydrophilic segment, and the molecular weight of PDTC is 10-40% of that of the hydrophobic segment; the cationic segment is SP.

[0039] In the present invention, in the amphiphilic block polymer grafted with antigen peptide and the amphiphilic block polymer with cation, the hydrophilic segment-P (ester monomer unit-DTC) of the amphiphilic block polymer is an existing polymer; the hydrophilic segment is a polyethylene glycol segment (PEG); the molecular weight of the hydrophobic segment is 2 to 8 times the molecular weight of the hydrophilic segment; the molecular weight of the PDTC segment is 10% to 40% of the total molecular weight of the hydrophobic segment. Preferably, the molecular weight of PEG is 3000 to 8000 Da; the molecular weight of the hydrophobic segment is 2 to 6 times the molecular weight of PEG; the molecular weight of PDTC is 10 to 30% of the molecular weight of the hydrophobic segment.

[0040] The antigen peptide polymer vesicle tumor vaccine of the present invention is a polymer vesicle with WT1 antigen peptide on the surface and loaded with immune adjuvant (such as CpG), which is obtained by self-assembly and cross-linking of a polymer pre-grafted with antigen peptide and an amphiphilic block polymer while encapsulating CpG through electrostatic action; the antigen peptide polymer vesicle vaccine of the present invention promotes the proliferation, maturation and antigen presentation of APC (such as DC, macrophage and B cells) through intravenous injection, induces cellular immune response (WT1-specific T cells) and humoral immune response (WT1-specific antibodies), can specifically identify and kill tumor cells, and after combining with anti-CTLA-4 antibodies, it achieves efficient immunotherapy for acute myeloid leukemia, and most mice can be completely cured and have long-term immune memory. In addition, the nano vaccine has the ability to induce antigen diffusion, which provides a new universal way to induce a strong and specific anti-tumor immune response, and broadens the use scenarios of peptide vaccines in tumor treatment.

[0041] In the present invention, the basic amphiphilic block polymer is PEG-P (TMC-DTC) -spe, and the polymer grafted with the WT1 antigen peptide S17 is grafted from a maleimide-functionalized polymer, represented by S17-PEG-P (TMC-DTC), and the chemical structure is shown in Figure 1. As a specific example, the present invention uses PEG- P(TMC-DTC)-spe (Mn = 5.0-(15.1-2.0)-0.2 kg / mol) amphiphilic block polymer, S17-PEG-P(TMC-DTC) amphiphilic block polymer grafted with S17 antigen peptide and immune adjuvant (CpG) as raw materials to prepare antigen peptide polymer vesicle tumor vaccine for the treatment of acute myeloid leukemia. The maleimide-functionalized polymer Mal-PEG-P (TMC-DTC) and the antigen peptide are mixed in a solvent and then dialyzed to obtain an amphiphilic block polymer grafted with the antigen peptide; wherein the solvent is an organic solvent, such as DMF, DMSO, etc., the mixing temperature is room temperature, and the mixing time is 1 to 50 hours, preferably 5 to 40 hours, and more preferably 10 to 30 hours.

[0042] Dissolved in 1 mL NN-dimethylformamide (DMF, analytical grade) and stirred at room temperature. After 24 h, the mixture was dialyzed in DMF (MWCO: 7 kDa) twice to remove unreacted free S17 peptide, and then dialyzed in water (ddH2O) twice to remove the organic solvent. After freeze-drying, the polymer S17-PEG-P (TMC-DTC) grafted with WT1 antigen peptide was obtained.

[0043] The present invention is further described below in conjunction with examples and drawings. The specific preparation methods and testing methods are conventional techniques in the art, and the statistical analysis is a conventional method.

[0044] Antigenic peptides R9 (RMFPNAPYL), S17 (SSGQARMFPNAPYLPSC), FITC-labeled S17 (FITC-SSGQARMFPNAPYLPSC), and KRAS G12C mutant peptide (KRAS 5-14 : KLVVVGACGV) is an existing product purchased from Synpeptide. CpG (TLR9 ​​agonist, sequence: 5′- TCCATGACGTTCCTGACGTT-3′) and dye-labeled CpG containing a complete phosphorothioate backbone are existing products from Sangon Biotech. Amphiphilic block copolymers poly(ethylene glycol)-b-poly(trimethylene carbonate-co-disulfane trimethylene carbonate)-b-spermine (PEG-P(TMC-DTC)-spe; Mn= 5.0-(15.0-2.0)-0.2), Mal-PEG-P(TMC-DTC) (Mn= 7.5-(15.0-2.0)) and PEG-P(TMC-DTC)-Cy5 are existing polymers. MicroBCA Protein detection kit (Thermo Scientific, 23235), mouse inflammatory factor kit (LEGENDplex, Biolegend, 76110), PE-RMFPNAPYL-tetramer (MBL, TS-M504-1), granulocyte-macrophage colony stimulating factor (GM-CSF, Peprotech, 315-03), LysoTracker (Yeasen, 40739ES50), DAPI (Beyotime), frozen section rapid antigen retrieval solution (Beyotime, P0090), endogenous peroxidase blocking buffer (Beyotime, P0100A), antifade mounting medium (Beyotime, P0126), red blood cell lysis / fixation solution (Elabscience, E-CK-A106), Brefeldin A solution (Biolegend, 420601), FoxP3 / transcription factor staining buffer kit (Multi Sciences, IC001), counting microspheres (Biolegend, 424902), Zombie-NIR live-death dye (Biolegend, 423106), 96-well plates (Corning, costar 9018) and 96-well U-bottom plates (FALCON, 353910). Roswell Park Memorial Institute (RPMI) 1640 medium (ATCC modified, A1049101), penicillin-streptomycin (PS), fetal bovine serum (FBS), and 2-mercaptoethanol were purchased from Gibco. Antibodies were existing products.

[0045] All animal experiments and operations were approved by the Experimental Animal Center of Soochow University and the Animal Care and Use Committee of Soochow University, and all protocols were in accordance with the Guide for the Care and Use of Laboratory Animals. Female C57BL / 6 mice, aged 8-12 weeks, were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. The mice were housed in a room at 20-25°C, with a 12-h light / dark cycle and a humidity of 30-70%. The AML animal model was created by injecting 5×10 5 The MA9 cell lines were established, and the mice were randomly divided into groups for in vivo experiments on the 3rd day after inoculation. The infiltration of MA9 cells in organs and the status of mice were monitored regularly thereafter.

[0046] Primary cells were extracted from C57BL / 6 mice. Bone marrow-derived dendritic cells (BMDCs) were collected from femurs and tibias and cultured in 15 mL RPMI-1640 medium supplemented with 10% FBS (Gibco), 1% penicillin / streptomycin, GM-CSF (20 ng / mL, Peprotech), and 50 μM 2-mercaptoethanol at a concentration of 5 × 10 5 / mL. These cells were cultured in untreated T75 flasks for 7 days and supplemented with 15 mL of complete medium on days 3 and 5. BMDCs were collected, counted and plated on untreated 12-well plates and, after overnight culture, were analyzed the next day.

[0047] Acquisition of spleen cells and spleen B cells: After the spleen was ground and filtered, it was centrifuged at 350 g for 5 minutes, the supernatant was discarded, and then resuspended in red blood cell lysis buffer (ACK). After 5-10 minutes, 10 times the volume of PBS was added to terminate the process, and the supernatant was discarded after centrifugation. The obtained single cell suspension was used for further experiments. MC38, GL261 and LLC cells were purchased from China Type Culture Collection Center (CCTCC). Mouse acute myeloid leukemia MLL-AF9 cells (MA9) expressing GFP are existing products.

[0048] Example 1 Grafted with tumor-associated antigen peptide WT1 121-137 Preparation of polymer (S17) 100 mg of maleimide-functionalized Mal-PEG-P (TMC DTC) (Mn = 7.5–15.0–2.0 kg / mol) and 10 mg of S17 peptide (Mn = 1826 g / mol) were dissolved in 1 mL of N,N-dimethylformamide (DMF, analytical grade) and stirred at room temperature. After 24 h, the mixture was dialyzed in DMF (MWCO: 7 kDa) twice to remove unreacted free S17 peptide, and then dialyzed in water (ddH2O) to remove the organic solvent. After lyophilization, the polymer S17-PEG-P (TMC-DTC) grafted with WT1 antigen peptide was obtained. 1 The grafting rate of peptides was determined by H-NMR (600 MHz) and MicroBCA. The results showed that the grafting rate of S17 was 100% ( Figure 1 ).

[0049] Referring to the above method, the polymer S17-PEG-P (TMC-DTC) grafted with WT1 antigen peptide can also be prepared by using DMSO solvent instead of DMF, and the S17 grafting rate is 70%.

[0050] Example 2 Preparation and Characterization of Polymersome Nanovaccine (PASTA-NV) Displaying S17 Antigen Peptide and Encapsulating TLR9 Agonist The polymer vesicle nanovaccine (PASTA-NV) with S17 antigen peptide displayed on the surface and TLR9 agonist encapsulated was produced by co-self-assembly of PEG-P(TMC-DTC)-spe and S17-PEG-P(TMC-DTC) in the presence of CpG (the nanovaccine structure is shown in Figure 2A). S17 peptide (WT1 121-137 , SSGQARMFPNAPYLPSC) is the smallest epitope R9 widely recognized by WT1 (WT1 126-134 , RMFPNAPYL) and some extended amino acids. The preparation process of the nanovaccine is as follows: S17-PEG-P (TMC-DTC) and PEG-P (TMC-DTC)-spe DMF solutions (40 mg / mL) were mixed at a mass ratio of 1:9 (at this time, the surface density of S17 was 10%), and then 225 μL of the mixed solution (containing about 75 μg S17) was added dropwise to 775 μL of phosphate buffer (2 mM, pH 6.0) containing 300 μg CpG under stirring. PASTA-NV was obtained after dialysis (MWCO: 350 kDa) twice in the same buffer and phosphate buffer (10 mM, pH7.4). The theoretical drug loading of CpG was set to 3.2wt.%, and the encapsulation efficiency (DLE) of CpG was determined by Nanodrop. The particle size, PDI and surface zeta potential of the vesicles were determined by Zetasizer Nano-ZS (Table 1). The surface density of S17 is adjustable, and the particle size is small (about 60 nm), the distribution is narrow (PDI is about 0.1), and it basically maintains electrical neutrality.

[0051] Referring to the above method, the mass ratio of S17-PEG-P (TMC-DTC) and PEG-P (TMC-DTC)-spe in DMF solution (40 mg / mL) was changed to prepare PASTA-NV with different surface densities. Specifically, 0.5:9.5 corresponds to a density of 5%, 2:8 corresponds to a density of 20%, and 3:7 corresponds to a density of 30%.

[0052] Table 1 Characterization of PASTA-NV with different surface densities, theoretical CpG drug loading of 3.2 wt.%

[0053] Referring to the above method, the nanovaccine without S17 peptide (TA-NV) was prepared by self-assembly of PEG-P(TMC-DTC)-SPE in a CpG-containing solution and had similar physical properties. The mixture of TA-NV and free S17 peptide, denoted as S17 / TA-NV (Table 2), was used as a control in subsequent studies.

[0054] Table 2 Characterization of TA-NV and S17 / TA-NV, theoretical CpG drug loading is 3.2 wt.%

[0055] Note: Group 2 (S17 / TA-NV) was characterized after physical mixing with free S17 peptide.

[0056] The preparation process of FITC-labeled antigen peptide polymer FITC-S17-PEG-P (TMC-DTC) is similar to that of the aforementioned S17-PEG-P (TMC-DTC), and the FITC-labeled S17 peptide was used directly after purchase.

[0057] The preparation process of PASTA-NV containing FITC-labeled S17 and Cy5-labeled polymers is briefly described as follows: DMF solutions (40 mg / mL) of FITC-S17-PEG-P(TMC-DTC), PEG-P(TMC-DTC)-spe, and PEG-P(TMC-DTC)-Cy5 were mixed at a volume ratio of 1:8.9:0.1, and then 225 μL of the mixed solution was added dropwise to 775 μL of phosphate buffer (2 mM, pH 6.0) containing 300 μg of CpG under stirring. PASTA-NV containing FITC-labeled S17 peptide and Cy5-labeled polymer was obtained after dialysis (MWCO: 350 kDa) twice in the same buffer and phosphate buffer (10 mM, pH 7.4).

[0058] Notably, S17 peptide displayed on the polymer surface may have advantages over free peptide and peptide encapsulated within the polymer, as reflected in better recognition by the proteasome, longer retention time in the APC cytosol, and potentially better responsiveness to eliciting the BCR.

[0059] Example 2 In vitro immune activation ability of PASTA-NV FITC-labeled S17 peptide was used to evaluate the cellular uptake, activation, and antigen presentation of PASTA-NV by BMDC. Confocal laser scanning microscopy (CLSM) images after 4 h of incubation showed that PASTA-NV effectively delivered the S17 antigen peptide into the cytoplasm of BMDCs, while the FITC fluorescence observed in the S17 / TA-NV group was negligible ( Figure 2 C). Flow cytometry confirmed that the uptake of PASTA-NV by BMDCs increased by 3.59-fold compared with S17 / TA-NV ( Figure 2 D). FITC-labeled S17 and Cy5-labeled polymer (PS) were used to study the fate of PASTA-NV in cells (S17 concentration: 0.25 μg / mL, CpG concentration: 1 μg / mL). BMDCs were seeded in 24-well plates (3×10 5 After overnight incubation, PASTA-NV and S17 / TA-NV were added and incubated for 4 h. The cells were gently washed, stained with LysoTracker and DAPI, fixed, and then imaged by CLSM. The results showed that the colocalization signals of the polymer and S17 with the inclusion body were low, indicating that PASTA-NV effectively escaped the inclusion body and released S17 into the cytosol ( Figure 2 E). Notably, PASTA-NV significantly upregulated the expression of CD86, MHC-I, and MHC-II molecules compared with S17 / TA-NV, indicating that PASTA-NV induced better activation of DCs and improved the overall antigen presentation capacity ( Figure 2 F). PASTA-NV significantly increased the maturation of DCs at 4, 12, and 24 h, and enhanced expression of MHC-I and MHC-II molecules was observed at 4 and 12 h. In addition, PASTA-NV significantly increased the mean fluorescence intensity (MFI) of FITC and activation signaling molecules (CD27 and CD69) in total splenocytes and splenic B cells ( Figure 2G, H). In order to study the uptake of PASTA-NV by splenic B cells, spleen cells were added to a 24-well plate with a glass coverslip pre-coated with anti-mouse IA / IE antibodies. After incubation at 37°C for 2 h, the suspended cells were removed, and the adherent cells were treated with PASTA-NV for 4 h. After fixation, they were blocked with 1% BSA in PBS. The cells were incubated with rat anti-mouse B220 antibody overnight at 4°C, treated with TRITC-labeled secondary antibody at 37°C for 1 h, and observed by CLSM after DAPI labeled the cell nucleus. The images show that the S17 peptide is distributed in and on B cells ( Figure 2 H). These results indicate that PASTA-NV significantly increased the uptake, activation, and overall antigen presentation capacity of APCs, including DCs and B cells.

[0060] Example 3 PASTA-NV enhances antigen cross-presentation and APC activation in vivo The orthotopic MLL-AF9 mouse AML model was routinely established. PASTA-NV and S17 / TA-NV (per mouse: S17: 5 μg, CpG: 20 μg, Cy5: 0.18 μg) were injected into the tail vein on day 3 after inoculation. Cy5-labeled CpG was used as a probe to study the effect of PASTA-NV on the uptake and activation of APC cells in vivo after a single intravenous (iv) injection (Figure 3A). Spleens and inguinal lymph nodes (LNs) were collected, fixed in 4% PFA solution, frozen and sectioned (10 μm), and blocked with 10% goat serum for 1 h. For B cell and macrophage staining, primary antibodies (B: CD45R (B220) monoclonal antibody; macrophages: F4 / 80 polyclonal antibody) were incubated overnight at 4°C. After washing three times with PBS, the tissues were incubated with secondary antibodies (B cells: rhodamine (TRITC)-conjugated goat anti-rat IgG (H+L); macrophages: CoraLite594-conjugated goat anti-rabbit IgG (H+L)) for 2 h. For staining of DCs, staining with CD11c / AF594 was performed overnight. Cell nuclei were stained with DAPI, and sections were treated with anti-fluorescence quenching mounting solution and stored at 4°C before observation using CLSM and digital pathology scanner. Figure 3B shows extensive immunofluorescence in the spleen 6 h after injection, indicating that PASTA-NV is transported to immune cells such as DCs, macrophages, and B cells. Notably, B cells and DCs that took up PASTA-NV were mainly located in the red pulp within the spleen ( Figure 3B). This is mainly due to the fact that these immune cells internalized PASTA-NV during circulation and / or migrated from the white pulp to the red pulp to interact with PASTA-NV, which indicates the importance of targeted immune regulation. At the same time, FACS analysis results showed that the total uptake of PASTA-NV by APCs increased significantly, and Cy5-CpG + Splenocytes, Cy5-CpG + DCs and Cy5-CpG + The total uptake by macrophages increased 2-3.5-fold (Fig. 3C, D). When spleen APCs were exposed to PASTA-NV, the expression of MHC I and MHC II molecules increased, indicating that spleen APCs had a stronger activation and antigen presentation capacity ( Figure 3 E). Studies on inguinal lymph nodes also found that PASTA-NV had obvious co-localization with DCs and macrophages ( Figure 4 ), which enhances immunogenicity and T cell activation in vivo. Figure 3F shows that the secretion of proinflammatory cytokines (IL-1α, IFN-γ, TNF-α, and IL-27) increased, indicating further enhancement of immune activation.

[0061] Example 4 PASTA-NV enhances anti-AML efficacy by enhancing cellular and humoral immune responses The effects of PASTA-NV after prolonged immunization in MA9 mice were evaluated by conventional methods (Figure 5A): AML mice were randomized and given four doses of PASTA-NV or S17 / TA-NV vaccine (S17: 5 μg and CpG: 20 μg per mouse) by intravenous injection on days 3, 6, 9, and 16. On day 23, peripheral blood (PB) and single cell suspensions of tissue origin (spleen, inguinal LN, and bone marrow) were harvested by a standardized process involving mechanical dissociation and filtration, resuspended in 96-well V-bottom plates, and distinguished between live and dead cells using Zombie-NIR, blocked with anti-CD16 / 32, and stained with the desired antibody cocktail (1:200 dilution) to identify immune cell subsets: CD3 + T cells, CD4 + T cells, CD8 + T cells, activated T cells (CD69, CD107a, IFN-γ, TNF-α), exhausted T cells (PD-1), follicular helper T cells (Tfh, CD4+PD-1 + CXCR5 + ), regulatory T cells (Treg, CD4 +CD25 + FoxP3 + ), B cells (B220 + )、cDC1 (CD11c + MHC-II + CD103 + )、cDC2 (CD11c + MHC-II + CD11b + )、pDC (CD11c + MHC-II + B220 + )、CD8 + DCs (CD11c + MHC II + CD8 + ), M1 macrophages (M1M, CD11b + F4 / 80 + CD86 + ), M2 macrophages (M2M, CD11b + F4 / 80 + CD206 + ) and activated NK cells (NK1.1 + NKG2D CD69). For staining of intracellular cytokines (IFN-γ, TNF-α), 50 μL of the above cell suspension was pelleted in a 96-well V-bottom plate and incubated at 37°C for 2 h in RPMI-1640 containing S17 peptide (10 μg / mL), 10% FBS, and ionomycin (1 μg / mL). Brefeldin A was added and incubated for another 4 h. The cells were then stained with Zombie-NIR, blocked with anti-CD16 / 32, and surface stained with FITC-CD3, PE-CD4, and PE / Cy7-CD8. After washing twice, the cells were fixed, permeabilized, and intracellularly stained with APC anti-mouse IFN-γ (1:400) and Per-CP / Cy5.5 anti-mouse TNF-α (1:400) according to the manual. All stained cells were resuspended in RBC lysis / fixation solution for 10 min, washed and resuspended in PBS as described above. Counting beads were added and cells were transferred to 96-well plates (FALCON, 353910), stored at 4°C in the dark, and FC measurements and analysis were performed within 1 week. 126-134For tetramer staining, splenocytes were stained with 50 μL of PE-labeled RMFPNAPYL-Tetramer plus FcR blocking reagent at a 1:50 dilution for 30 min at room temperature in the dark and then incubated with 50 μL of surface antibody cocktail (APC anti-mouse CD3 and FITC anti-mouse CD8a (clone: ​​KT15)) for 20 min on ice.

[0062] The results showed that the plasmacytoid dendritic cell (pDC) population expanded by 3.9 times, and the classical dendritic cell (cDC) population in the spleen and the activation marker (CD80) expression of cDC in LN increased significantly (Figure 5B-D), which is essential for effective T cell activation. Among the cDC subsets, type 1 (cDC1) and type 2 (cDC2) have clear division of labor during cross-presentation, which are used to activate CD8 + T cells and CD4 + These APCs expand and become activated in the spleen and inguinal lymph nodes, inducing highly efficient activation of T cells.

[0063] The results of the treatment study showed that iv injection of PASTA-NV significantly delayed the progression of acute myeloid leukemia in vivo (Figure 5E) and prolonged the median survival time (MST) of MA9 mice (MST: 55 days, 44 days in the S17 / TA-NV group and 41 days in the PBS control group, respectively) (Figure 5F). Cellular and humoral immunity stimulated by PASTA-NV were evaluated, and Figure 5G showed that NK cells in peripheral blood (PB) were significantly activated, which would control AML progression at an early stage. In addition, 6 h after the last administration, the level of IL-12p70 in the peripheral blood of the PASTA-NV group surged (Figure 5H), which is considered to be the key to stimulating T cells to continuously secrete IFN-γ. This dynamic change is crucial to promote the spread of antigens in vivo, which can drive the cancer immune cycle and achieve long-term tumor eradication.

[0064] B cells play a very important role in anti-tumor immunity. In order to detect the level of specific antibodies in mouse serum, serum WT1-specific IgG levels were measured by ELISA. Briefly, 10 μg / mL WT1126-134 peptide (R9: RMFPNAPYL) was plated on a 96-well plate (Corning, Costar 9018) and incubated overnight at 4°C. Blocked with 1% BSA for 1 h at 37°C, and then incubated with 100 μL of serially diluted serum samples (100, 200, 400, 800, 1600 and 3200) for 30 minutes. Antibodies adsorbed to serum samples were detected using HRP-conjugated IgG antibodies, and the rest of the steps were the same as those of typical ELISA. Based on the high uptake of B cells, PASTA-NV significantly enhanced the expression of specific epitopes in serum against WT1. 126-134 The specific antibodies can bind to the MHC-I epitopes on tumor cells and expose them to the immune system. Further analysis of the spleen showed that PD-1 + CXCR5 + Follicular helper T cells (Tfhs), B220 + The content of B cells was significantly upregulated, and the expression of CD27, a marker indicating memory differentiation state on B cells, was also significantly increased ( Figure 5 J, K). Tfhs are essential for B cell affinity, maturation, and differentiation into memory B cells. The results suggest that Tfh-B cell interactions are essential for initiating and maintaining humoral immune responses against AML.

[0065] In the spleen of the PASTA-NV group, the expression of CD107a (a marker related to T cell degranulation effect) and CD4 secretion of IFN-γ and TNF-α were detected. + and CD8 + T cells were significantly upregulated (Figure 5L, M, N). Notably, CD8 + T cells and CD4 + Increased secretion of IFN-γ by T cells ( Figure 3 M), which facilitates the upregulation of MHC-I molecules by tumor cells and MHC-II molecules by APCs. Upregulation of MHC-I on tumor cells and MHC-II on APCs may result in more effective antigen presentation and further elimination of tumor cells. WT1 126-134 Antigen-specific CD8 + The T cell population was significantly increased (***, Figure 5O) Further demonstrated the precision and effectiveness of PASTA-NV in inducing antigen-specific T cell responses.

[0066] In addition, the epitope spreading effect induced by PASTA-NV was detected, and the epitope against non-immune peptides and KRAS G12C mutant epitope (KRAS 5-14 This was demonstrated by the antibody response to PASTA-NV (KLVVGACGV:KLVVGACGV) (Figure 5P). In addition, a substantial increase in systemic IL-12p70, which is essential for antigen spreading, was observed (Figure 5H). This antigen spreading effect of PASTA-NV reveals the potential of this “off-the-shelf” nanovaccine. Overall, these results highlight the synergistic effect of PASTA-NV in promoting strong and durable cellular and humoral responses against AML.

[0067] Example 5: PASTA-NV vaccination strategy determines biodistribution and anti-leukemia effect The effects of vaccination strategies, i.e., all intravenous (all iv), all subcutaneous (all sc), and two subcutaneous injections followed by two intravenous injections of the same dose (sc+iv) on tumor progression and survival in tumor-bearing mice were investigated. Figure 6 A). The results showed that intravenous injection had the best inhibitory effect on MA9 cell infiltration in the bone marrow, and no AML cell infiltration was detected in 5 / 6 mice on day 23 ( Figure 6 B), and the survival rate is also the best ( Figure 6 C).

[0068] MA9 mice were injected with PASTA-NV containing Cy5-labeled CpG ( Figure 6 A, per mouse: S17: 5 μg, CpG: 20 μg, Cy5: 0.18 μg). MA9 mice were vaccinated with 3 doses of PASTA-NV or S17 / TA-NV, 3 intravenous injections (all IV), 3 subcutaneous injections (all SC), or a combination of 2 subcutaneous injections and 1 intravenous injection (sc+iv) on days 3, 6, and 9. For subcutaneous injections, the nanovaccine was injected subcutaneously on both sides close to the inguinal LN. Spleen, lymph nodes, and bone marrow were collected for analysis 6 h after the last vaccine injection ( Figure 6 D). In vivo quantitative analysis showed that the spleens of all iv group mice had obvious Cy5 signals, which exceeded those of the sc+iv group and all sc group, while the LNs of all sc group mice showed higher Cy5 intensity ( Figure 7 A). Representative IF images of spleen sections showed that in the all iv group, PASTA-NV was mainly localized in the red pulp and highly colocalized with B cells and DCs ( Figure 6 E and Figure 7 B). In stark contrast, in the all sc group, B cells were exclusively retained in the white pulp, whereas in the sc+iv group, the distribution and colocalization of B cells were intermediate between the other groups. In all three groups, macrophage localization was low, suggesting that PASTA-NV was taken up into the spleen primarily by B cells and DCs. The spleen is the gatekeeper of systemic immunity, where DCs are responsible for capturing and presenting antigens to activate T cells, and B cells are responsible for antibody responses against blood-borne antigens. Follicular B cells were observed to migrate to the marginal zone, exposed to intravenously injected PASTA-NV ( Figure 6 E), which was different from the other two groups. Detailed FACS analysis of spleens showed that the Cy5 intensity of total splenocytes in the full intravenous injection group was 2.5 and 9.7 times higher than that in the full subcutaneous and subcutaneous plus intravenous groups, and the Cy5 intensity of DC was 2.3 and 10.5 times higher than that in the other groups, while the average uptake of macrophages and B cells was less different ( Figure 6 F).

[0069] In addition, bone marrow analysis results showed that in the all iv injection group, the Cy5 signal in B cells increased significantly, and the expression of MHC-I and MHC-II also increased ( Figure 7 C). All three PASTA-NV vaccination strategies showed colocalization with DCs and macrophages within LNs ( Figure 8 ), suggesting that effective lymph node targeting is mediated by APCs migrating through blood vessels or lymphatic vessels. These findings highlight the important role of the lymphatic system in mediating local and systemic immune responses and cancer immunotherapy. Overall, PASTA-NV significantly enhanced enrichment within immune organs (especially the spleen) through all iv injections and increased access to APCs to promote T cell and humoral immune responses.

[0070] Example 6 PASTA-NV significantly targets lymph nodes and induces DC cell activation in vivo To analyze the efficacy of PASTA-NV in stimulating an anti-AML response via intravenous administration, the activation status of cDC1s and cDC2s was investigated. Fig. 9A: MA9 mice were vaccinated with 4 doses of PASTA-NV or S17 / TA-NV, 4 intravenous injections (all intravenous injections), 4 subcutaneous injections (all subcutaneous injections), or a combination of 2 subcutaneous injections and 2 intravenous injections (sc+iv) on days 3, 6, 9, and 16. For subcutaneous injections, the nanovaccine was injected subcutaneously on both sides close to the inguinal LN. The experimental procedure for the analysis of in vivo immune cell infiltration and activation was similar to that of Example 4.

[0071] A significant increase in the total number of DCs, cDC1, and cDC2 percentages was observed in the inguinal lymph nodes of the all iv group, which was significantly higher than that of the sc+iv group and the all sc group ( Fig. 9 B, C). In addition, the expression of CD80 in cDC1 and cDC2 subsets continued to increase, indicating that PASTA-NV injected all iv enhanced the activation and adaptation ability of DCs in vivo compared with the sc+iv group and the all sc group ( Fig. 9 D). It is noteworthy that subcutaneous injection of PASTA-NV led to a significant enlargement of LN, resulting in a significant decrease in the proportion of DCs compared with the PBS group. In the spleen, the cDC1 and cDC2 subsets in the all iv group also increased significantly ( Fig. 9 E), and the B cell population and CD27 expression also increased significantly ( Fig. 9 F, G). In addition, compared with the sc+iv group, the results of spleen cell detection after S17 peptide restimulation in the all iv group and all sc group showed that CD4 + and CD8 + T cell degranulation is upregulated ( Fig. 9 H), IFN-γ + and TNF-α + The proportion of T cells also increased significantly ( Fig. 9 I, J). Notably, in the spleen, more WT1-specific T cells appeared in all iv groups ( Fig. 9 K). At the same time, PD-1 + T cells ( Fig. 9 L) and CD206 + M2 phenotype macrophages ( Fig. 9In conclusion, PASTA-NV all iv injection induced a significant expansion and sustained activation of cDC1s and cDC2s, while minimizing the immunosuppressive PD-1 + T cells and M2 macrophages, thereby eliciting optimal innate and adaptive anti-AML immune responses.

[0072] Example 7 PASTA-NV and α-CTLA-4 combined therapy enhances immune response and eradicates AML cells To enhance immunotherapy for AML, the synergistic potential of PASTA-NV and ICB therapy (e.g., α-CTLA-4 antibody, InVivoMAb anti-mouse CTLA-4 (CD152), 9D9, BioXCell) was evaluated: MA9 mice were vaccinated with 4 doses of intravenous PASTA-NV on days 3, 6, 9, and 16, and tumor-bearing mice were given an additional three intravenous injections of α-CTLA-4 (Ab, 1 mg / kg) on ​​days 6, 9, and 16 to explore the effect of the combined therapy. The experimental procedure for the analysis of in vivo immune cell infiltration and activation was similar to that of Example 4 ( Fig.10 A). PASTA-NV combined with Ab intravenous injection (PASTA-NV+Ab) showed unparalleled efficacy, with almost no AML cells detected in peripheral blood on day 28 ( Fig.10 B), 6 out of 7 mice survived long term ( Fig.10 C). However, monotherapy with α-CTLA-4 antibody alone has not achieved satisfactory results.

[0073] Immunological analysis showed that compared with the PASTA-NV all iv group, the number of CD4 + T cells and CD8 + CD69 expression in T cells was significantly increased ( Fig.10 D), indicating that they play a key role in coordinating a durable and effective anti-AML response and establishing local tissue-resident memory T cells. In addition, PASTA-NV+Ab therapy significantly increased the proportion of DCs, especially the number of cells in the cDC1 and pDC subsets ( Fig.10 EF), and increased the proportion of WT1-specific CD8+ T cells ( Fig.10G). The reason for the significant increase in the number of DCs is that the chemokines secreted by activated T cells recruited more DCs and stimulated some resident monocytes to differentiate into DCs. The significant increase in the total amount of DCs is crucial for inducing cancer immunity cycles. At the same time, the CD4 + CD25 + FoxP3 + The reduction of regulatory T cells (Tregs) confirmed the further alleviation of the immunosuppressive environment ( Fig.10 H). In addition, enhanced mobility of DCs was observed, which helps improve tumor antigen cross-presentation and thus improve synergistic immune regulation. These results highlight the complex mechanism by which PASTA-NV and ICB combination therapy has excellent anti-AML efficacy and activates durable immunity in vivo.

[0074] Example 8 PASTA-NV+Ab provides long-lasting anti-tumor immunity and prevents AML recurrence To further investigate the durability of immune protection activated by PASTA-NV+Ab therapy, the memory T cells and specific killing effects of surviving mice on day 60 were studied ( Fig.10 A). On day 60, peripheral blood was collected from surviving mice that received the above vaccines, blocked, and then stained with APC-CD3, PE / Cy7-CD4, FITC-CD8, PerCP / Cy5.5-CD44, and PE-CD62L for 20 minutes on ice. The cells were resuspended in RBC lysis / fixation solution for 10 minutes, washed twice with PBS, and CD44 was detected by flow cytometry. + CD62L + Central memory T cells (T CM ) and CD44 + CD62L - Effector memory T cells (T EM ). The analysis showed that TCM and TEM increased significantly, higher than the healthy mouse control group (****, Fig.10 I). To simulate tumor relapse, the ability of memory T cells in the spleen of cured mice to be activated upon re-encountering a specific antigen (i.e., R9 or S17) was evaluated. A surviving mouse that had previously received PASTA-NV+Ab treatment was randomly selected. Splenocytes were isolated, counted, and incubated overnight (5 × 10 ) with free R9 or S17 peptides (2.74 and 5.48 μM) in 12-well plates. 6 / mL). The next day, these restimulated splenocytes were washed, counted and co-incubated with MA9 cells at a ratio of 10:1. After 24 h, all cells were collected and distinguished between live and dead cells using Zombie-NIR staining. Flow cytometry was used to detect surviving MA9 cells by GFP signal (gated on all live cells). It is generally believed that after restimulation, memory T cells in the spleen will rapidly proliferate and differentiate into effector T cells, including CTLs, which specifically kill MA9 tumor cells. Splenocytes isolated from surviving mice were restimulated with two concentrations of long peptide S17 or short peptide R9 (RMFPNAPYL), and the results showed a dose-dependent specific killing response of MA9 cells. Compared with the R9 group, splenocytes reactivated by S17 showed stronger anti-AML efficacy at low doses. This additional killing effect of S17 suggests an antigen spreading mechanism, which is CD4 + Due to the action of T cells, CD4 + This is also demonstrated by the enhanced secretion of IFN-γ by T cells.

[0075] To evaluate the role of long-term immune defense in preventing AML relapse, surviving mice were re-inoculated with MA9 cells on day 90 ( Fig.10 A). Naive mice inoculated with MA9 cells were used as controls. After 32 days, the mice were monitored for MA9 cells in the blood, body weight, and survival rate as described above. During the subsequent 100 days of continuous observation, without any therapeutic intervention, the vast majority (80%) of mice remained alive with no signs of MA9 cell infiltration in the blood or detectable abnormalities ( Fig.10 K, L). In addition, to investigate the killing of WT1-expressing tumor cell lines by spleen cells of surviving mice, spleen cells treated with S17 peptide were added to MC38, GL261, and LLC cells seeded in 96-well plates at a ratio of 10:1. After incubation for 24 h, the cells were gently washed twice with PBS to remove spleen cells and dead cancer cells, and the viability of tumor cells was determined by CCK-8 assay. The results showed that WT1-reactive spleen cells showed cytotoxicity against LLC, GL261, and MC38 cell lines ( Fig.11 ), highlighting the potential application value of this combination therapy in tumors that widely express WT1.

[0076] Furthermore, no adverse reactions were observed during the experiment and long-term monitoring. No lesions were observed in H&E liver sections of surviving mice after 14 months of PASTA-NV+Ab treatment ( Fig.12). In conclusion, PASTA-NV+Ab therapy has a profound effect on the expansion of DC subsets, amplification of T cell-mediated responses, and induction of immune memory formation. This treatment strategy can eradicate confirmed AML and enhance the host's ability to resist tumor recurrence, indicating a major advance in cancer immunotherapy.

[0077] Example 9 PASTA-NV+Ab has a better cure rate than TA-NV+Ab See also Fig.13 A: MA9 mice were vaccinated with 2 subcutaneous and 2 intravenous doses of TA-NV and PASTA-NV on days 3, 6, 9, and 16, respectively. In the ICB therapy group, tumor-bearing mice were given an additional three intravenous injections of α-CTLA-4 (Ab, 1 mg / kg) on ​​days 6, 9, and 16. Compared with the PBS group and the TA-NV (sc+iv)+Ab group, the PASTA-NV sc+iv combined therapy with Ab significantly slowed the progression of acute myeloid leukemia, and the infiltration level of MA9 cells in peripheral blood was significantly reduced ( Fig.13 B), 3 out of 7 mice were free of tumors, while in the TA-NV group without S17 antigen peptide, no cured mice were found even with combined use of Ab ( Fig.13 C).

[0078] Example 10 Proinflammatory Effects of PASTA-NV The whole tumor lysate antigen was obtained by lysing MA9 cells. Briefly described: 10 million MA9 cells were centrifuged to remove the supernatant and then added with NP-40 cell lysate containing 1mM PSMF. After incubation at 4°C for 30 min, the supernatant was centrifuged at 13,000 rpm for 10 min. The supernatant was the MA9 cell lysate antigen. The preparation process of the nanovaccine loaded with lysate antigen was as follows: 225μL of PEG-P (TMC-DTC)-spe DMF solution (40 mg / mL) was added dropwise to 775μL phosphate buffer (2 mM, pH 6.0) containing 300 μg lysate antigen and 300 μg CpG under stirring. The nanovaccine loaded with lysate antigen was obtained after dialysis (MWCO: 350 kDa) twice in the same buffer and phosphate buffer (10 mM, pH 7.4). The encapsulation efficiency (DLE) of CpG was close to 100% as determined by Nanodrop. The antigen loading was close to 100% as measured by MicroBCA. The particle size, PDI and surface zeta potential of the vesicles were measured by Zetasizer Nano-ZS, and the particle size was about 55nm, with a narrow distribution (PDI of about 0.1), and basically maintained electrical neutrality.

[0079] The ratio of antigen to CpG in the nanovesicles loaded with lysate antigen was 1:1, and the doses in the cell experiments were (Ag: 1 μg / mL, CpG: 1 μg / mL). PASTA-NV was prepared as described previously, and the doses in the cell experiments were (S17: 0.25 μg / mL, CpG: 1 μg / mL). The immunogenicity of lysate tumor antigen and WT1-based tumor-associated antigen peptide vaccines was evaluated in BMDCs and spleen cells. BMDCs and spleen cells were obtained as described previously, and 1×10 6 / mL BMDC were plated in untreated 12-well plates in advance. After standing in the incubator overnight, cell lysate antigen vesicle vaccine and PASTA-NV were added respectively. After incubation for 24 h, the culture supernatant was collected and the secretion level of IL-12p70 was tested by ELISA. In addition, 5×10 6 / mL spleen cells were plated in 48-well plates in advance. After standing in the incubator overnight, cell lysate antigen vesicle vaccine and PASTA-NV were added respectively. After 24 hours of co-incubation, the culture supernatant was collected and the secretion level of IFN-γ was tested by ELISA. The results showed that when the two types of antigen vesicles were co-incubated with BMDC, the WT1-based S17 antigen peptide vaccine could induce DC to secrete more IL-12p70 than the antigen from MA9 cell lysate, and when co-incubated with spleen cells, the WT1-based S17 antigen peptide vaccine could also induce spleen cells to secrete more IFN-γ ( Fig.14 These results suggest that PASTA-NV can induce DCs to load antigens better and promote the immune response of effector cells.

[0080] Example 11 Universality of PASTA-NV Tumor Treatment On day 0, 1×10 6 MC38 cells were injected into the right subcutaneous area of ​​mice to establish a subcutaneous colon cancer model. 1×10 5 GL261 cells were injected into the mouse brain using a stereotaxic apparatus to establish an orthotopic glioma model. PASTA-NV (S17: 5μg, CpG: 20μg) was injected into the inguinal lymph nodes and cervical lymph nodes on days 3, 6, 9, and 16, respectively. The growth of MC38 subcutaneous tumors and the survival of GL261 mice were then monitored. The results showed that PASTA-NV also had a certain therapeutic effect on colon cancer (MC38) and glioma (GL261) models: see Fig.15 Compared with the PBS group, PASTA-NV can better inhibit the growth of subcutaneous colon cancer tumors; in the glioma experiment, the median survival time of mice was extended from 20 days to 24 days; there was no significant difference between the two groups.

[0081] Antigenic peptide vaccines are a promising clinical strategy for the treatment of cancer patients. However, their anticancer immune responses remain very limited due to insufficient presentation and / or activation of antigenic peptides in antigen-presenting cells (APCs). This poses a great challenge for the development of therapeutic vaccines because acute myeloid leukemia (AML) cells spread throughout the body. In this study, we report a peptide antigen-surface TLR9 adjuvanted nanovaccine (PASTA-NV) that displays multiple Wilms' tumor 1 (WT1) peptides on its surface and encapsulates a CpG ODN adjuvant in its aqueous interior to simultaneously increase antigen presentation and APC activation / proliferation. Interestingly, systemic administration of PASTA-NV induced strong cellular and humoral anticancer immune responses in an orthotopic mouse AML, MLL-AF9 (MA9) model. When combined with anti-CTLA4 antibody, PASTA-NV achieved complete regression of AML in the mouse model and established long-lasting anti-AML immunity, effectively resisting re-attack by leukemic cells. PASTA-NV provides a new general approach to induce potent and specific anticancer immunity, which has the potential to revitalize peptide antigen-based nanovaccines for tumor therapy.

Claims

1. An antigen peptide polymer vesicle tumor vaccine, characterized in that: The invention comprises amphiphilic block polymer vesicles grafted with antigen peptides and immune adjuvants.

2. The antigen peptide polymer vesicle tumor vaccine according to claim 1, characterized in that: The immune adjuvant includes a TLR9 agonist; and the antigen peptide includes a WT1 antigen peptide.

3. The antigen peptide polymer vesicle tumor vaccine according to claim 1, characterized in that: The amphiphilic block polymer is a hydrophilic segment-P (ester monomer unit-DTC)-cationic segment and M-hydrophilic segment-P (ester monomer unit-DTC), and M is an antigen peptide structure.

4. The method for preparing the antigen peptide polymer vesicle tumor vaccine according to claim 1, characterized in that: Antigen peptide polymer vesicle tumor vaccine is prepared by using immune adjuvant, amphiphilic block polymer grafted with antigen peptide and amphiphilic block polymer with cation as raw materials.

5. The method for preparing the antigen peptide polymer vesicle tumor vaccine according to claim 4, characterized in that: The mass ratio of the antigen peptide to the immune adjuvant is 1:(1-20).

6. An anti-tumor drug, characterized in that: The active ingredient is the antigen peptide polymer vesicle tumor vaccine.

7. Use of the antigen peptide polymer vesicle tumor vaccine according to claim 1 or the anti-tumor drug according to claim 6 in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that: The tumor is a hematological tumor or a solid tumor.

9. The use according to claim 8, characterized in that: Blood cancers include leukemias.

10. Application of amphiphilic block polymer vesicles grafted with antigen peptides and immune adjuvants in the preparation of anti-tumor drugs.

Images