Recombinant ferritin-based nanoparticles as neoantigen carriers significantly inhibit tumor growth and metastasis

Recombinant ferritin-based nanoparticles as neoantigen carriers significantly inhibit tumor growth and metastasis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Recombinant ferritin-based nanoparticles as neoantigen carriers significantly inhibit tumor growth and metastasis Wei Zheng, Shixiong Li, Zhongliang Shi, Kailing Su, Yu Ding, Luyue Zhang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4654130/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Sep, 2024 Read the published version in Journal of Nanobiotechnology → Version 1 posted 11 You are reading this latest preprint version Abstract Background Tumor neoantigen peptide-based vaccines, systemic immunotherapies that enhance antitumor immunity by activating and expanding antigen-specific T cells, have achieved remarkable results in the treatment of a variety of solid tumors. However, how to effectively deliver neoantigens to induce robust antitumor immune responses remains a major obstacle. Results Here, we developed a safe and effective neoantigen peptide delivery system (neoantigen-ferritin nanoparticles, neoantigen-FNs) that successfully achieved effective lymph node targeting and induced robust antitumor immune responses. Genetically engineered self-assembled particles with a size of 12 nm were obtained by fusing a neoantigen with optimized ferritin, which rapidly migrates to and continuously accumulates in lymph nodes. The neoantigen-FNs vaccine induced a greater quantity and quality of antigen-specific CD8 + T cells and resulted in significant growth control of multiple tumors, dramatic inhibition of melanoma metastasis and regression of established tumors. In addition, no obvious toxic side effects were detected in the various models, indicating the high safety of optimized ferritin as a vaccine carrier. Conclusions Homogeneous and safe neoantigen-FNs could be a very promising system for neoantigen peptide delivery because of their ability to efficiently migrate to lymph nodes and induce efficient antitumor immune responses. Tumor neoantigen peptide-based vaccine vaccine platform lymph node-targeting ferritin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Therapeutic tumor vaccines are personalized immunotherapies that recognize and eliminate malignant cells by stimulating the body to generate tumor antigen-specific CD8 + T cells[ 1 ]. Several clinical trials have demonstrated that personalized therapeutic vaccines can significantly prolong the survival of patients with solid tumors[ 2 , 3 ]. In 2010, the U.S. Food and Drug Administration (FDA) approved Provenge (sipuleucel-T) for the therapy of advanced prostate cancer, suggesting that therapeutic tumor vaccines have promising prospects for the treatment of solid tumors[ 4 , 5 ]. Advances in rapid genome sequencing technology have driven the development of neoantigen therapeutic vaccines[ 6 – 8 ]. Neoantigens are tumor-specific antigens derived from nonsynonymous mutations and have become ideal targets for therapeutic tumor vaccines because they do not elicit autoimmune responses or central tolerance. Neoantigen peptides have been widely applied in clinical research due to their favorable biosafety, abundant modifiable sites and mature synthetic routes[ 9 , 10 ]. However, neoantigen peptides usually fail to elicit sufficient antigen-specific cytotoxic T lymphocyte (CTL) responses in vivo because of their lower immunogenicity and poor lymph node-targeted delivery[ 11 , 12 ]. Therefore, it is imperative to develop an ideal neoantigen peptide delivery system that not only enhances the immunogenicity of neoantigens, but also enables targeted delivery to lymph nodes to stimulate effective antitumor immune responses. To address these issues, a variety of neoantigen peptide delivery platforms, including polymeric nanoparticles[ 13 ], inorganic nanoparticles[ 14 ], and liposomes[ 15 ], which are able to induce robust CTL responses and significantly inhibit tumor growth in mice, have been developed and validated in preclinical studies. Unfortunately, poor biocompatibility, particle heterogeneity, or inconsistent manufacturing processes have limited the application of these systems in clinical trials[ 16 ]. However, protein-caged nanoparticles such as virus-like particles (VLPs) and ferritin family proteins have attracted much attention in drug delivery due to their good biodegradability, highly ordered structures, and simple and repeatable preparation methods[ 17 , 18 ]. These proteins tend to self-assemble into nanoscale core-shell structures, which would facilitate effective targeting to lymph nodes and persistence in vivo[ 19 ]. Tumor peptide vaccines based on VLPs and heat shock proteins have shown strong efficacy in triggering protective immunity against different tumors[ 20 – 22 ]. Lymph node targeting is often considered critical for the effective in vivo delivery of tumor vaccines and the induction of robust antitumor immune responses[ 23 ]. A study comparing the lymph node-targeting ability of four different protein-caged nanoparticles demonstrated that engineered human ferritin heavy chain (hFTN) nanoparticles have greater potential for direct lymph node targeting and tumor immunotherapy[ 24 ]. Ferritin is a 12 nm spherical particle self-assembled from 24 monomers with the advantages of nanoscale size[ 25 ], multiple peptide modification sites, good biocompatibility, and thermal stability[ 26 ]. In addition, ferritin has been extensively investigated for the delivery of viral antigens such as influenza and SARS-CoV-2 antigens[ 27 – 29 ], and several clinical studies have shown that ferritin has excellent biosafety as an antigen delivery vehicle[ 30 , 31 ]. However, studies on ferritin as a delivery platform for neoantigen peptides in tumor vaccines have been very limited[ 32 – 34 ]. Therefore, we expect to develop a ferritin-based tumor vaccine platform for the delivery of neoantigen peptides to enhance antitumor immune responses. In this study, Helicobacter pylori -derived ferritin[ 25 ], which is distantly related to human evolution, was chosen as a delivery platform for tumor neoantigen peptides due to its advantage to prevent the generation of autoimmune responses or immune tolerance[ 35 ]. The results showed that OVA T -ferritin nanoparticles (OVA T -FNs) are homogeneous and stable 12 nm particles and have significantly greater immunogenicity than OVA T free peptides. Moreover, OVA T -FNs can rapidly migrate to lymph nodes and reside for a long time to enhance uptake by antigen-presenting cells (APCs) in lymph nodes. Effective antigen-specific immune responses were induced by OVA T -FNs vaccine in vivo, resulting in significant inhibition of tumor growth and metastasis. In addition, OVA T -FNs possessed excellent biosafety, which will facilitate subsequent clinical translation. Methods Mice Female wild-type (WT) C57BL/6 and BALB/c mice and female OT-1 transgenic mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All mice used in this study were between 6 and 8 weeks old and were maintained under specific pathogen-free conditions in the animal facility at Nankai University. All mice were housed in groups of 5 under conditions of a 12-hour light-dark cycle (8:00–20:00, light; 20:00–8:00, dark), constant room temperature (21°C), suitable humidity (40%~60%), and free access to food and water. The animal procedures were performed with ethical compliance and approval from the Institutional Animal Care and Use Committee at Nankai University. Cell lines The E.G7-OVA, MC-38-OVA, B16F10-OVA, B16F10, and DC2.4 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). E.G7-OVA, MC-38-OVA, B16F10-OVA, and B16F10 cells were grown in RPMI 1640 (Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS, Biological Industries, Kibbutz, Israel) and 1% penicillin-streptomycin (NCM Biotech, Cat. #C100C5). In addition, 50 µM 2-mercaptoethanol (Aladdin, Cat. #M301574) and 0.4 mg/mL G-418 (Solarbio, Cat. #IG0010) were used for culture of E.G7-OVA, and 1.5 µg/mL puromycin (Solarbio, Cat. # P8230) was used for culture of MC-38-OVA and B16F10-OVA to exclude cells not overexpressing OVA. DC2.4 cells were grown in DMEM (Gibco, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS), 1% penicillin-streptomycin and 50 µM 2-mercaptoethanol. All cells tested negative for mycoplasma contamination according to the ATCC Universal Mycoplasma Detection Kit (ATCC, Cat. #30-1012K) and were cultured at 37°C in a humidified incubator with 5% CO 2 . Protein biosynthesis and purification The gene encoding Helicobacter pylori ferritin (residues 5-167) was codon-optimized to adapt to the Escherichia coli ( E. coli ) expression system, and a point mutation (Asn19Gly) was designed to remove a potential N-linked glycosylation site. The OVA T peptide (SIINFEKL) followed by a (GGS) 3 linker and the M30 peptide (PSKPSFQEFVDWENVSPELNSTDQPFL) followed by a SASGG linker were fused to the N-terminus of ferritin (residues 5-167) to generate OVA T -FNs and M30-FNs, respectively[ 7 ]. Moreover, FNs without peptides at the N-terminus of ferritin (residues 5-167) were prepared as a control. The constructs were cloned and inserted into the pET-28a (+) expression vector (Novagen, Madison, WI, USA) using the NcoI and XhoI restriction sites. The recombinant plasmids were transformed into E. coli BL21(DE3) competent cells (ZOMANBIO, Cat. #ZK201), and the bacterial cells were grown in LB media supplemented with 50 µg/mL kanamycin (Solarbio, Cat. #K8020) at 37°C until an absorbance of 0.6 was reached at 600 nm. The proteins were induced to be overexpressed by 0.7 mM isopropyl β -D-1-thiogalactopyranoside (IPTG) (Solarbio, Cat. #II0130) for 16 h at 16°C. The bacterial cells were harvested and resuspended in lysis buffer (50 mM Tris, 500 mM NaCl, 5% glycerol, pH 8.0) and then homogenized at a pressure of 700 bar. The supernatant containing the recombinant proteins was obtained by centrifugation at 18,000 rpm for 40 min at 4°C, and the proteins were isolated by Ni-nitrilotriacetic acid affinity chromatography. Briefly, 2 mL of Ni-NTA resin (TransGen, Cat. #DP101) was used to purify proteins, which were then equilibrated with PBS (pH 7.2 ~ 7.4) and incubated 2 times. Nontarget proteins were removed by washing with 10 mM, 50 mM, or 100 mM imidazole, and target proteins were eluted with 300 mM imidazole. The size and purity of the harvested proteins were determined by SDS-PAGE. The eluted proteins were buffer exchanged into PBS (pH 7.2 ~ 7.4) containing 1 mM EDTA (Solarbio, Cat. #E1170) and 5% glycerol and concentrated to less than 5 mL using Amicon-Ultra15 centrifugal filters (EMD Millipore) with a 100-kDa molecular weight cutoff (MWCO). The concentrated proteins were further purified by size exclusion chromatography using a HiPrep 16/60 Sephacryl S-500 HR size-exclusion column in the above buffer at a flow rate of 0.8 mL/min. The purified proteins were concentrated to 2.5 mg/mL using Amicon-Ultra15 centrifugal filters with a 100-kDa MWCO, and then 5% glycerol was added, followed by storage at -80°C. Negative-stain electron microscopy For the negative-staining study, 4 µL of 100 µg/mL protein sample was applied to a carbon film-coated 300-mesh Cu grid (Beijing Zhongjingkeyi Technology Co., Ltd., Beijing, China) for 1 min. After air-drying, the sample was negatively stained with 1% (w/v) uranyl acetate for 1 min and then observed by scanning electron microscopy (HITACHI HT7700 Exalens). Dynamic light scattering (DLS) and zeta potential analysis Particle size and zeta potential analysis of FNs and OVA T -FNs at a concentration of approximately 2 mg/mL were performed using a Zetasizer Nano ZS (Malvern Instruments Ltd., UK). Blood and tissue processing Approximately 150 µL of peripheral blood was collected into a 1.5 mL EP tube containing 30 µL of 0.5 M EDTA, and 1.5 mL of ACK buffer (Solarbio, Cat. #R1010) was added to the lysed red blood cells twice for 5 min each. Lymph nodes or spleen placed between two 40 µm filters were mechanically prepared into single cells in a 12-well plate containing 1 mL of MACS buffer (1×PBS containing 0.5% BSA and 2 mM EDTA). In addition, the splenocytes were lysed at room temperature for 5 min with 2 mL of ACK buffer to remove red blood cells. The obtained single-cell suspension was filtered through a 40 µm nylon mesh filter for subsequent flow cytometry detection or transfer experiments. Preparation of bone marrow-derived dendritic cells (BMDCs) Bone marrow was flushed from the femurs and tibias of C57BL/6 mice with ice-cold RPMI 1640 medium supplemented with 10% HI-FBS and 2% penicillin-streptomycin. Red blood cells were lysed, and the remaining cells were seeded in a 10 cm tissue culture dish containing 12 mL of BMDC medium consisting of RPMI 1640 medium supplemented with 10% HI-FBS, 1% penicillin-streptomycin, 50 µM 2-mercaptoethanol, 20 ng/mL IL-4 (PeproTech, Cat. #214 − 14) and 40 ng/mL GM-CSF (PeproTech, Cat. #315-03). On day 2, nonadherent and loosely adherent cells were collected and transferred to a new culture dish, after which 4 mL of fresh BMDC medium was added. On day 4, half of the medium was gently removed, and an equal volume of fresh medium was added. On day 6, BMDCs were identified by flow cytometry and used for subsequent experiments. In vitro cellular uptake assay First, suitable cells cover glasses (NEST, Cat. #801010) were placed in a 24-well plate and 5×10 5 immature BMDCs obtained as described above were added and cultured for 24 h. BMDCs were then incubated with 2 nmol Cy5-labeled OVA T , FNs or OVA T -FNs for 4 h. After the unphagocytosed antigens were washed away with PBS, the BMDCs were stained with CD11c-FITC antibody at 4°C for 30 min, followed by fixation with 200 µL of 1% paraformaldehyde (Solarbio, Cat. #P1111) at 4°C for 20 min. The cover glasses were harvested and stained with DAPI for 5 min, after which the localization of Cy5-labeled antigen in FITC-positive cells was detected by confocal laser scanning microscopy (CLSM; TCS SP5, Leica, Germany). Evaluation of endosomal escape ability A total of 1×10 6 immature BMDCs were grown in 35 mm confocal dishes (NEST, Cat. #801001) for 24 h. Then, 2 nmol of FITC-labeled OVA T , FNs or OVA T -FNs were added and cocultured for 4 h. After being washed 5 times with PBS, the BMDCs were stained with LysoTracker (Invitrogen, Waltham, MA, USA, Cat. #L7528) for 1 h, followed by Hoechst (US Everbright, Cat. #H4078) for 30 min. The colocalization of FITC and LysoTracker was detected by confocal laser scanning microscopy (CLSM; TCS SP5, Leica, Germany) to analyze the endosomal escape ability of FNs and OVA T -FNs. Activation and maturation of BMDCs and cross-presentation of OVA T peptide Cells and culture supernatants were harvested after 1×10 6 BMDCs were cocultured with 2 nmol of OVA T , FNs or OVA T -FNs for 24 h. BMDCs were then stained with CD80, CD86, MHC-I, MHC-II, CD40, and SIINFEKL-H2Kb antibodies for 30 min at 4°C, and the frequencies of activation markers were analyzed with a BD FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA, USA). The concentrations of IL-12 (P70), IFN-α1, IL-6, IFN-γ and TNF-α in the culture supernatant were determined by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocols. In vivo imaging and lymph node targeting BALB/c or C57BL/6 mice were immunized with PBS, 6 nmol Cy5-labeled OVA T or OVA T -FNs in the groin. The fluorescence intensities at the injection site, isolated lymph nodes and various organs were analyzed 24, 48, and 72 h after immunization using an IVIS Lumina imaging system (IVIS Lumina II, Xenogen, USA). Moreover, the sizes of the isolated lymph nodes in each group were recorded 24 h after immunization. Antigen uptake and activation of APCs in lymph nodes C57BL/6 mice were immunized in the groin with Cy5-labeled OVA T , FNs or OVA T -FNs at a dose of 6 nmol per side. Twenty-four hours after immunization, the lymph nodes were harvested and prepared into single-cell suspensions, and the frequencies of Cy5 in APCs, such as DCs (CD11c + ), macrophages (CD11b + F4/80 + ), and B cells (B220 + ), as well as the frequency of activated DCs (CD80 + CD86 + CD11c + ), were analyzed by flow cytometry. In vivo proliferation assay Splenocytes from OT-1 mice were labeled with 5 µM CFSE (Invitrogen, Cat. #65–0850) for 1 h at 37°C and then transferred intravenously to C57BL/6 mice, which were then immunized subcutaneously in the groin with 6 nmol of OVA T and OVA T -FNs in the presence or absence of 50 µg of Poly(I:C) (InvivoGen, Cat. #tlrl-pic-5) 24 h after transfer. Three days after immunization, the attenuation of CFSE fluorescence intensity in CD8 + tetramer + cells in the spleen were analyzed by flow cytometry. In vivo target cell lysis assay Splenocytes from C57BL/6 mice were resuspended in RPMI-160 medium supplemented with 10% FBS, 1% penicillin-streptomycin, and 50 µM 2-mercaptoethanol, and the cell concentration was adjusted to 1×10 7 cells/mL[ 36 ]. The above cells were divided into two aliquots, one of which was labeled with OVA T peptide at a final concentration of 25 µM for 2 h at 37°C in the dark, while the other was unlabeled. After washing once with PBS to remove unbound OVA T peptide, the cells were resuspended in PBS to a final concentration of 1×10 7 cells/mL. Splenocytes loaded with OVA T peptide and unloaded with OVA T peptide were labeled with 5 µM and 0.5 µM CFSE, respectively, for 1 h at 37°C. The cells were washed twice with PBS to remove unbound CFSE dye and subsequently mixed at a ratio of 1:1 after adjusting the cell concentration to 5×10 7 with PBS. Additional C57BL/6 mice without any treatment were immunized with PBS, OVA T , OVA T vaccine, OVA T -FNs, or OVA T -FNs vaccine. A total of 7×10 6 mixed cells were injected intravenously into mice immunized for 8 days at a dose of 200 µL per mouse. Eighteen hours after injection, single-cell suspensions of the spleens were prepared, and the frequencies of splenocytes labeled with high and low concentrations of CFSE, were analyzed by flow cytometry. Immunizations A total of 6 nmol of OVA T , M30 peptides, or OVA T -FNs, M30-FNs nanoparticles alone, or prepared by mixing with 50 µg of Poly(I:C) were formulated in 100 µL of PBS and immunized subcutaneously via the groin. Enzyme-linked immunospot (ELISpot) assay ELISpot plates (EMD Millipore, MSIPS4510) were pretreated with 50 µL of 35% ethanol for 30 s and washed 5 times with sterile water before being coated with 100 µL of 15 µg/mL anti-IFN-γ (MABTECH, Cat. #3321-2A) overnight at 4°C. After coating, the antibody was discarded, and the plate was blocked for 1 h with complete medium (RPMI-1640 supplemented with 10% FBS and 1% penicillin-streptomycin). A total of 2×10 5 splenocytes were added to each well and restimulated for 48 h at 37°C with complete medium containing OVA T or M30 peptide at a final concentration of 10 µg/mL. The cells were washed away, and the plates were incubated with 100 µL of 1 µg/mL biotinylated detection antibody (MABTECH, Cat. #3321-2A) for 2 h at room temperature. The plate was washed 5 times with PBS and incubated with streptavidin-ALP (MABTECH, Cat. #3321-2A) diluted at a ratio of 1:1000 for 1 h at room temperature. After washing with PBS 5 times, 100 µL of the substrate NBT&BCIP (Sangon Biotech, Cat. #C510032) prepared with 1× color development buffer was added to each well until obvious spots appeared, after which the reaction was stopped with 200 µL of ddH 2 O. After dying, the spots were counted with an ELISpot reader (AID iSpot, AID-Autoimmune Diagnostika GmbH, Strassberg, Germany). Experiments to detect immune responses induced by vaccines C57BL/6 mice were immunized on days 0 and 14, and peripheral blood and spleen were collected on day 21 to analyze vaccine-induced immune responses. The frequencies of tetramer + CD8 + T cells in the peripheral blood and spleen were measured by flow cytometry to assess the ability of OVA T -FNs vaccine to induce antigen-specific CD8 + T cells. The phenotypes of CD8 + T cells in peripheral blood were evaluated by flow cytometry analysis of the proportions of PD-1 + TIM-3 − cells, effector memory T (Tem) cells, and central memory T (Tcm) cells. Vaccine-induced CTL responses were measured by the frequency of IFN-γ, TNF-α, or Granzyme B in CD8 + T cells. Prophylactic, therapeutic and metastasis vaccine experiments For prophylactic model experiments, C57BL/6 mice were immunized on days 0, 14 and 28. Fourteen days after the third immunization, 5×10 5 E.G7-OVA, 1×10 6 MC-38-OVA, or 3×10 5 B16F10-OVA cells in 100 µL of PBS were subcutaneously implanted into the right flank of each mouse. For metastasis model experiments, C57BL/6 mice were immunized on days 0, 14 and 28, 1×10 5 B16F10 cells in 200 µl of PBS were injected through the tail vein on day 35, and the lungs of the mice were removed on day 57 to count the number of metastatic foci. For therapeutic model experiments, C57BL/6 mice were subcutaneously implanted in the right flank with 1×10 5 B16F10 cells in 100 µL of PBS on day 0 and then immunized three times on days 5, 8, and 12. The tumor volume was estimated by the following formula: tumor volume = length × width 2 × 0.5. Animals were euthanized when the tumor reached 1.5 cm in diameter or surpassed 1500 mm 3 in volume or when significant weight loss was observed. ELISA The culture medium supernatants of BMDCs after 24 h of coculture and of splenocytes after 48 h of restimulation with OVA T peptide were collected, and the concentrations of cytokines were detected by ELISA kits (BioLegend, San Diego, CA, USA). The concentrations of IL-12 (p70) (Cat. #433604), IFN-α1 (Cat. #447904), TNF-α (Cat. #430904), IFN-γ (Cat. #430804), and IL-6 (Cat. #431304) in the medium supernatant of BMDCs and of IFN-γ (Cat. #430804) in the medium supernatant of splenocytes were detected according to the manufacturer’s protocols. Flow cytometry analysis For the APCs uptake analysis, cells were preincubated with 0.25 µg of TruStain FcX™ PLUS anti-mouse CD16/32 blocking antibody (BioLegend, clone. S17011E) per 10 6 cells in a volume of 100 µl for 10 min on ice to reduce nonspecific binding. After FcR blocking, the cells were then stained for 30 min at 4°C with surface antibodies diluted at 1:300 in 100 µL of FACS buffer (fluorochrome-conjugated antibodies purchased from BioLegend unless otherwise indicated): CD45 (Cat. #103133. clone. 30-F11), CD11b (Cat. #101292. clone. M1/70), CD11c (Cat. #117306 and 117307. clone. N418), CD11c (BD Biosciences, Cat. #612797, clone. HL3), CD80 (Cat. #104705. clone. 16-10A1), CD86 (Cat. #105025. clone. GL-1), CD40 (Cat. #157506. clone. FGK45), MHC-I (Cat. #114612. clone. 28-8-6), MHC-II (Cat. #107625. clone. M5/114.15.2), B220 (Cat. #103205. clone. RA3-6B2), F4/80 (Cat. #123110. clone. BM8), and SIINFEKL-H2Kb (Cat. #141605. clone. 25-D1.16). After staining, the cells were washed once with FACS buffer and resuspended in 300 µL of PBS for flow cytometry detection. For T-cell tetramer analysis, single cells derived from the peripheral blood or spleen were first incubated with 100 µL of PBS containing 50 nM dasatinib (MACKLIN, Cat. #D828602) for 30 min at room temperature. The samples were washed once and treated with an anti-CD16/32 antibody for 10 min on ice. The cells were then stained with tetramer antibody (MBL, Cat. #TS-5001-1C) diluted 1:20 in 50 µL of FACS buffer containing 50 nM dasatinib for 45 min at 4°C under light protection. After washing once with FACS buffer, the cells were stained with surface antibodies diluted at 1:300 in 100 µL of FACS buffer (fluorochrome-conjugated antibodies purchased from BioLegend unless otherwise indicated) for 30 min at 4°C: CD8 (GeneTex, Cat. #GTX76348. clone. KT15), PD-1 (Cat. #135209. clone. 29F.1A12), TIM-3 (Cat. #119718. clone. RMT3-23), CD44 (Cat. #103043. clone. IM7), and CD62L (BD Biosciences, Cat. #564109, clone. MEL-14). For intracellular cytokine analysis, 1×10 6 splenocytes were first stimulated with 10 µg/mL OVA T peptide and Golgi plug (BD Biosciences, Cat. #555029) at 37°C for 6 h. The cells were then treated with an anti-CD16/32 blocking antibody at 4°C for 10 min and subsequently stained with CD3 (BD Biosciences, Cat. #564379, clone. 145-2C11), CD4 (Cat. #100491. clone. GK1.5) and CD8 (Cat. #100706. clone. 53 − 6.7) diluted at 1:300 for 30 min at 4°C. The cells were washed once with FACS buffer and fixed and permeabilized at 4°C for 1 h using the FoxP3/Transcription Factor Staining Buffer Set (Invitrogen, Cat. #00-5523-00). The cells were washed once with 1× wash buffer and then stained with the following intracellular antibodies: IFN-γ (Cat. #505808. clone. XMG1.2), TNF-α (Cat. #506313. clone. MP6-XT22), and Granzyme B (Cat. #372216. clone. QA16A02) diluted at 1:300 at 4°C overnight. The cells were washed twice with wash buffer and prepared for flow cytometry detection. The cells were acquired on a BD FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA, USA) and a BD LSRFortessa X-20 (BD Biosciences) using BD FACSDiva Software v8.0.3 (BD Biosciences). All collected data were analyzed with FlowJo version V10.8.1. MTT assay DC2.4 cells (1×10 5 ) were incubated with different doses of FNs and OVA T -FNs in 96-well plates for 24 h. After discarding the supernatant, 0.5 mg/mL MTT (Solarbio, Cat. #IM0280) was added, and the cells were incubated for 4 h. After incubation, 100 µL of DMSO was added to each well, and the absorbance was subsequently measured at 490 nm using a Cell Imaging Multi-Mode Reader (Cytation 5, BioTek, Winooski, VT, USA). Hematoxylin and eosin (H&E) staining and determination of various enzymes in serum Fourteen days after the third immunization, 200 µL of peripheral blood was collected and placed at 4°C overnight, followed by centrifugation at 1,000 rpm for 20 min to harvest the serum. The heart, liver, spleen, lung and kidney were removed and fixed in 15 mL of 4% paraformaldehyde (Solarbio, Cat. #P1110) for 4 days. Organ damage and the serum concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine (CRE) were determined by Tianjin JingNuo Pathological Diagnostic Co. Statistical analysis Statistical analyses were performed using GraphPad Prism 8.0.2 software. All results are presented as the mean ± s.e.m., except where otherwise specified. An unpaired Student’s t test, one-way ANOVA with Tukey’s multiple-comparison test, two-way ANOVA with Tukey’s multiple-comparison test or log rank test was used for comparisons between the groups. P values less than 0.05 were considered to indicate statistical significance. No sample in any representative experiment was excluded from the analysis. Results 1. Engineering and characterization of OVA T -FNs tumor vaccines The homogeneity, biocompatibility, and size of tumor vaccines are critical to their effectiveness in suppressing tumors[ 37 ]. We expect to develop a more effective and safer peptide delivery platform for tumor neoantigens relying on Helicobacter pylori -derived ferritin nanoparticles. SIINFEKL (OVA T ) was fused to optimized ferritin proteins to prepare OVA T -FNs, in which the first four amino acids at the N-terminus were omitted because the fifth amino acid is better displayed on the particle surface, and an N19Q mutation was introduced to abolish a potential N-linked glycosylation site (Fig. 1 a)[ 27 ]. To test whether the OVA T -FNs protein subunits could be synthesized in an E. coli expression system and subsequently self-assembled into nanoparticles, we investigated the purity, structural integrity, and size of the obtained OVA T -FNs. SDS-PAGE of purified FNs and OVA T -FNs revealed a single protein band corresponding to the protein subunit (Fig. 1 b). The peak shape of OVA T -FNs analyzed by molecular exclusion chromatography was similar to that of FNs, suggesting that the fusion of OVA T peptide at the N-terminus of FNs did not affect the self-assembly of the optimized ferritin into nanoparticles (Fig. 1 c). OVA T -FNs were homogeneous 12 nm spherical particles similar to the native structure, as revealed by transmission electron microscopy (TEM) and DLS (Fig. 1 d, e). Moreover, the particle size of OVA T -FNs was not affected after treatment at 4°C for 7 days or 65°C for 10 min, suggesting that OVA T -FNs are able to tolerate high temperatures and have good stability, which is important for the clinical translation of these vaccines (Fig. S1 a, b). In addition, the absolute zeta potentials of FNs and OVA T -FNs were approximately 10 mV and 8 mV, respectively, indicating that the individual particles were well dispersed and stable (Fig. 1 f). Together, these data suggested that OVA T -FNs prepared by fusion expression have superior purity, a uniform particle size, and good thermal stability. 2. Activation and cross-presentation of BMDCs after the uptake of OVA T -FNs To demonstrate that optimized ferritin can be used as a tumor vaccine vector, we first investigated whether OVA T -FNs could be taken up by BMDCs in vitro and efficiently cross-present OVA T peptides on the cell surface. Fluorescence confocal microscopy images showed that the uptake of OVA T -FNs by CD11c + BMDCs was dramatically greater than that of free OVA T after coincubation with BMDCs for 4 h (Fig. 2 a). Moreover, the uptake of OVA T -FNs by BMDCs significantly increased with prolonged coculture time (Fig. S2). In addition, OVA T -FNs did not fully colocalize with lysosomes but were predominantly localized in the cytoplasm (Fig. 2 b), suggesting that OVA T -FNs can escape from lysosomes into the cytoplasm and be degraded by the proteasome into peptide fragments. The maturation of BMDCs plays an essential role in triggering tumor-specific CTL responses[ 38 ]. The expression of the surface activation markers CD80, CD86, MHC-I, MHC-II, and CD40 was significantly upregulated in BMDCs after coculture with OVA T -FNs, indicating that OVA T -FNs induced the activation of BMDCs (Fig. 2 c-g). The frequency of SIINFEKL-H2Kb + cells on the surface of BMDCs was analyzed by flow cytometry to verify whether BMDCs cross-present OVA T antigen via MHC-I molecules, and the results showed that 10% of BMDCs cocultured with OVA T -FNs were able to cross-present SIINFEKL to the cell surface through MHC-I (Fig. 2 h and Fig. S3a, b). In addition, cytokines secreted by mature BMDCs are also critical for downstream immune activation[ 39 ]. IL-12 (p70) acts as a polarizing factor that promotes the differentiation of naive CD4 + T cells to T helper 1 (Th1) cells and mediates an effective adaptive immune response[ 40 ]. The concentrations of cytokines in the culture supernatant of BMDCs cocultured with OVA T -FNs for 24 h were determined by ELISA, and the results showed that the concentrations of IL-12 (p70), IFN-α1, IL-6, IFN-γ and TNF-α were significantly increased (Fig. 2 i, j and Fig. S4a-c). Together, these results confirmed that OVA T -FNs can be taken up by APCs, induce APCs activation and maturation, and enhance antigen presentation via MHC-I. In vivo targeting and activation of APCs in lymph nodes by OVA T -FNs. The effective delivery of tumor vaccines to lymph nodes and the maturation and cross-presentation of APCs in vivo are critical for triggering powerful antitumor responses[ 41 , 42 ]. Therefore, we investigated whether OVA T -FNs can migrate to lymph nodes and be taken up by APCs. In vivo fluorescence images revealed highly distinct Cy5 fluorescence in the groin of mice immunized with OVA T -FNs at 24, 48, and 72 h after immunization, suggesting that compared with free OVA T , OVA T -FNs could persist at the injection site (Fig. 3 a, b). Next, we isolated the lymph nodes and various organs of immunized mice to test whether OVA T -FNs could selectively localize to the lymph nodes. Consistent with the results shown in Fig. 3 a, b, remarkable localization of OVA T -FNs was detected in the lymph nodes at 24 h and even 72 h after immunization, suggesting that OVA T -FNs can effectively target the lymph nodes (Fig. 3 c, d). Among the remaining organs, Cy5 fluorescence could be detected only in the liver and kidney at 24 h post injection, which might be related to the metabolic function of the liver and kidney as normal metabolic organs (Fig. S5a, b). In addition, the isolated lymph nodes were significantly swollen 24 h after immunization with OVA T -FNs compared to 24 h after immunization with OVA T (Fig. S5c). The above results suggest that OVA T -FNs can be retained in vivo for a long time and can rapidly migrate to lymph nodes. Prolonged retention of OVA T -FNs in lymph nodes would facilitate their uptake by APCs. To verify whether OVA T -FNs can be taken up by APCs in lymph nodes. C57BL/6 mice were injected subcutaneously with Cy5-labeled OVA T -FNs, and the lymph nodes were collected 24 h later. The frequencies of Cy5 + in CD11c + DCs, macrophages, and B cells were significantly increased in OVA T -FNs-treated group, but these cells largely did not take up free OVA T peptide (Fig. 3 e-g). Moreover, immunization with OVA T -FNs resulted in a significant increase in the frequency of the activation marker CD80 + CD86 + in CD11c + DCs compared to that in cells immunized with OVA T free peptides (Fig. 3 h). These results suggest that OVA T -FNs can migrate to lymph nodes, reside there for long periods of time. This may provide an opportunity for APCs to continuously take up OVA T -FNs and come into full contact with T cells. 4. Triggering and expansion of antigen-specific T cells induced by OVA T -FNs The ability to induce the production and activation of antigen-specific CD8 + T cells is an important criterion for evaluating the efficacy of tumor vaccines. First, we investigated whether OVA T -FNs could induce the proliferation of antigen-specific CD8 + T cells. In vitro, OT-1 CD8 + T cells cocultured with OVA T -FNs-treated BMDCs showed more pronounced attenuation of CFSE fluorescence and a greater proliferative index, with 70% of OT-1 CD8 + T cells dividing for 1–3 generations. In contrast, no significant attenuation of CFSE fluorescence intensity was detected in OT-1 CD8 + T cells cocultured with OVA T -loaded BMDCs (Fig. S6a-d). Since almost all peptide vaccines currently in clinical trials require adjuvants to enhance their induced immune responses[ 10 , 43 ], consistent with previous studies, we also included Poly(I:C) as an adjuvant in all of our in vivo experiments (OVA T + Poly(I:C), referred to as OVA T vaccine; OVA T -FNs + Poly(I:C), referred to as OVA T -FNs vaccine). Similar to the in vitro results, compared with OVA T vaccine, OVA T -FNs vaccine induced a more significant decrease in CFSE fluorescence intensity and an increase in the proliferation index in vivo (Fig. 4 a-c). Approximately 80% of CD8 + T cells divided for 7–9 generations after immunization with OVA T -FNs vaccine (Fig. S6e). OVA-specific CD8 + T-cell immune responses induced by OVA T -FNs vaccine were investigated 7 days after the second immunization (Fig. 4 d). A 6-fold higher proportion of antigen-specific CD8 + T cells could be detected in the peripheral blood after immunization with OVA T -FNs vaccine than after immunization with OVA T vaccine (Fig. 4 e, f and Fig. S7). The proportion of tetramer + CD8 + T cells in the spleen of mice immunized with OVA T -FNs vaccine also doubled compared with that in the other groups (Fig. 4 g). These results indicated that OVA T -FNs vaccine prepared by fusing OVA T peptide with optimized ferritin to efficiently deliver the antigenic peptide significantly enhanced CD8 + T-cell immune responses. Furthermore, the quality of triggered CD8 + T-cell response is also an important determinant of the therapeutic efficacy of tumor vaccines[ 44 , 45 ]. To investigate the phenotype of OVA-specific CD8 + T cells induced by OVA T -FNs vaccine, we evaluated the levels of cell surface markers indicating activation, exhaustion, and dysfunction. Four percent of CD8 + T cells in the peripheral blood of mice immunized with OVA T -FNs vaccine expressed PD-1 but not TIM-3 (Fig. 4 h), and a very limited number of cells expressed both PD-1 and TIM-3 (Fig. S8a, b)[ 46 ]. Based on the expression levels of CD44 and CD62L, half of the activated CD8 + T cells in OVA T -FNs vaccine-treated group were effector memory T (Tem) cells and central memory T (Tcm) cells, which could continue to differentiate into effector cells (Fig. 4 i and Fig. S8c). In conclusion, OVA T -FNs vaccine induced a large number of antigen-specific CD8 + T cells in vivo that maintained favorable early differentiation characteristics. Enhancement of CTL responses by OVA-FNs vaccine Antigen-specific CD8 + T cells generate strong CTL responses after a second exposure to the same antigen. To explore the level of CTL responses induced by OVA T -FNs vaccine, we measured the levels of key effectors secreted by CD8 + T cells after OVA T peptide restimulation. As shown in Fig. 5 a, b, after restimulation for 48 h, approximately 180 IFN-γ-secreting CD8 + T cells were detected among 2×10 5 splenocytes from mice immunized with OVA T -FNs vaccine. Similarly, the concentration of IFN-γ secreted by splenocytes was much higher in mice immunized with OVA T -FNs vaccine (Fig. 5 c). After restimulation with OVA T peptide and Golgi plug for 6 h, the frequency of IFN-γ- secreting CD8 + T cells was significantly elevated in the mice immunized with OVA T -FNs vaccine than in the other groups (Fig. 5 d, e and Fig. S9). As mentioned above, comparable amounts of IFN-γ were detected in mice immunized with OVA T vaccine or OVA T -FNs vaccine, as shown by ELISpot and ELISA. However, the flow cytometry results revealed that the frequency of IFN-γ secretion by CD8 + T cells was significantly higher in the mice immunized with the OVA T -FNs vaccine than in the other groups. The possible reason might be that prolonged restimulation of splenocytes in ELISpot and ELISA may lead to a sustained accumulation of secreted IFN-γ around the cells. The level of Granzyme B, another important effector molecule, was also dramatically increased in mice immunized with OVA T -FNs vaccine (Fig. 5 f). To directly verify the cytotoxic function of antigen-specific CD8 + T cells induced by OVA T -FNs vaccine, we performed a target cell lysis assay based on labeling with different concentrations of CFSE. Eighty percent of splenocytes labeled with high concentrations of CFSE were lysed in the spleen and lymph nodes of mice immunized with OVA T -FNs vaccine, while only 60% were lysed in mice immunized with OVA T vaccine (Fig. 5 h, i and Fig. S10a, b). Overall, the increased secretion of multiple effectors by antigen-specific CD8 + T cells suggested that OVA T -FNs vaccine can induce robust CTL responses in vivo and can specifically kill target cells. 6. Growth inhibition of multiple primary tumors induced by OVA T -FNs vaccine To further evaluate the antitumor immune responses induced by OVA T -FNs vaccine in vivo, we employed a variety of prophylactic tumor models to investigate the inhibition of tumor growth. C57BL/6 mice were immunized every 14 days and challenged with E.G7-OVA lymphoma cells 2 weeks after the third immunization. As demonstrated by the average growth curves and the individual tumor growth curves, compared with immunization with other control vaccines, immunization with OVA T -FNs vaccine significantly inhibited tumor growth. In addition, the tumors were isolated 16 days after implantation, and the tumor size was markedly smaller in the mice vaccinated with OVA T -FNs vaccine than in the other groups (Fig. 6 a-d). Next, the immune protection triggered by OVA T -FNs vaccine was explored in a highly immunogenic MC-38-OVA colon cancer model, and immunization with OVA T -FNs vaccine significantly inhibited tumor growth and prolonged survival. In addition, on day 60 after tumor implantation, a slightly palpable tumor was observed in one mouse in the OVA T -FNs vaccine group, while the other four tumors remained undetectable (Fig. 6 e-g and Fig. S11a). Moreover, in the poorly immunogenic B16F10-OVA melanoma model, tumor growth was significantly controlled in mice immunized with OVA T -FNs vaccine, whereas there were no significant differences between mice vaccinated with other vaccines or PBS (Fig. S11b, c). In all three different prophylactic tumor models, there was no weight loss in any of the immunized mice, suggesting that OVA T -FNs vaccine had no obvious side effects in vivo (Fig. S11d-f). The safety of tumor vaccines is an important factor that must be evaluated in preclinical studies, so we assessed the side effects of OVA T -FNs on cells or mice. In vitro, neither FNs nor OVA T -FNs affected the viability of DC2.4 cells even at a concentration of 1 mg /mL (Fig. 6 h). In vivo, H&E staining data showed that mice immunized three times with OVA T -FNs vaccine did not exhibit damage to various tissues or organs (Fig. 6 i). The expression of molecules associated with liver and kidney impairment was also not abnormally elevated in the serum (Fig. S12). The above results indicated that OVA T -FNs had satisfactory biosafety and did not cause hepatotoxicity, nephrotoxicity or pathological damage in vivo. Taken together, these results demonstrated that vaccination with OVA T -FNs vaccine not only significantly inhibited the growth of a wide range of tumors but also had a favorable safety profile. 7. M30-FNs vaccine showed significant efficacy in both metastatic and primary tumor-bearing models We demonstrated that delivery of the model antigen peptide OVA T using optimized ferritin as a vaccine carrier significantly inhibited tumor growth. Next, we explored whether the fusion of neoantigens with optimized ferritin could also induce robust immune responses and inhibit tumor growth. M30-FNs were prepared by fusing M30, a B16F10 melanoma neoantigen, to the N-terminus of optimized ferritin[ 7 ]. SDS-PAGE image demonstrated that high-purity M30-FNs were obtained (Fig. S13). TEM image showed that M30-FNs self-assembled into homogeneous particles with a particle size of 12 nm (Fig. 7 a). Splenocytes from mice immunized with M30-FNs vaccine 3 times were restimulated with the M30 peptide, and IFN-γ secretion was detected by ELISpot. More IFN-γ was secreted by 2×10 5 splenocytes from mice immunized with M30-FNs vaccine than those immunized with M30 vaccine (Fig. 7 b, c). The highly metastatic property of melanoma is one of the major causes of death in melanoma patients. The 5-year survival rate of primary melanoma patients is 99%, but that of metastatic melanoma patients is only 27%[ 47 ]. We therefore explored whether the neoantigen-FNs delivery system could function similarly in a metastatic tumor model. B16F10 cells were injected intravenously 7 days after the last immunization, and the lungs were removed 22 days after the injection to count the number of lung metastases (Fig. 7 d). The numbers of metastatic foci were significantly reduced after immunization with M30-FNs vaccine compared to approximately 15 metastatic foci that could be detected in each of the other groups (Fig. 7 e, f). In addition, the area of lung tumor invasion was significantly reduced in mice immunized with M30-FNs vaccine (Fig. S14). These results showed that T-cell immune responses induced by a neoantigen-FNs vaccine corresponding to B16F10 melanoma significantly inhibited tumor metastasis to the lungs. Currently, personalized therapeutic tumor vaccines are being investigated extensively in the clinic[ 48 ]. We next explored the ability of M30-FNs vaccine to induce regression of established tumors. C57BL/6 mice were subcutaneously implanted with B16F10 cells and then immunized 3 times on days 5, 8, and 12 (Fig. 7 g). M30-FNs vaccine significantly controlled tumor growth and had no effect on body weight compared to that in mice vaccinated with various other controls (Fig. 7 h, i). On day 16, only 1 of the 5 mice showed visible tumors (Fig. 7 j). These data suggested that neoantigen-FNs vaccine induced robust immune responses, significantly inhibited tumor metastasis, and promoted regression of established tumors. Discussion Although tumor neoantigen peptide vaccines have achieved remarkable results in the treatment of solid tumors, it is clear that low immunogenicity, poor lymph node targeting, and insufficient immune responses remain major obstacles[ 11 , 49 , 50 ]. In this study, we developed a tumor neoantigen peptide delivery system based on ferritin nanoparticles (neoantigen-FNs), which significantly enhanced the proportion and function of antigen-specific CD8 + T cells in vivo compared to those of free peptides. The neoantigen-FNs vaccine remarkably inhibited tumor growth and metastasis in a variety of prophylactic, B16F10 metastatic, and B16F10 therapeutic models and did not cause in vivo toxicity in mice with a favorable safety profile. The homogeneity, stability and biosafety of therapeutic tumor vaccines are crucial for their clinical translation[ 37 ]. In this study, OVA T -FNs self-assembled into well-homogenized spherical particles with particle sizes of approximately 12 nm, similar to the natural structure of ferritin nanoparticles. In addition, the structural properties of OVA T -FNs did not change when they were heated at 4°C for 7 days or even at 65°C for 10 min. A high thermal stability will be beneficial for maximizing the application of neoantigen-FNs. In addition, OVA T -FNs had no effect on the viability of DC2.4 cells in vitro. In vivo, OVA T -FNs also did not cause organ damage or serotoxicity, and there was no weight loss in the immunized mice, which suggested that the neoantigen-FNs peptide delivery system has good biosafety and has the potential to be applied in the clinic. The antitumor effects of tumor vaccines depend on the uptake of antigens by APCs and their presentation to CD8 + T cells in lymph nodes. Therefore, effective delivery of tumor vaccines to lymph nodes to increase the probability of contact with APCs would facilitate the activation of antitumor immune responses[ 51 ]. A well-established strategy to promote direct lymph node targeting is to design vaccines in particle form, and the size of the particles has a significant impact on selective lymph node targeting[ 42 ]. OVA T -FNs are spherical particles with a particle size of 12 nm, which strongly facilitates their localization in lymph nodes. The results of ex vivo lymph node imaging showed that OVA T -FNs could directly migrate to and reside in lymph nodes, where they were then taken up by APCs, inducing the activation of APCs. In clinical trials, failure to elicit effective antigen-specific T-cell responses in patients is a major obstacle for tumor neoantigen peptide vaccines[ 11 ]. Fusion of OVA T peptides with FNs to enhance the immunogenicity and lymph node targeting of OVA T peptides significantly increased the frequency of OVA T tetramer + CD8 + T cells in the peripheral blood and spleen. Moreover, not only the quantity but also the quality of antigen-specific CD8 + T cells improved after OVA T -FNs vaccine treatment. We examined the expression levels of CD8 + T-cell surface markers in the peripheral blood and showed that OVA T -FNs vaccine induced functional PD-1 + TIM-3 − rather than exhausted CD8 + T cells. Half of the activated cells retained their memory properties and could continue to differentiate into effector cells when re-exposed to the same antigens, which is also consistent with the more significant inhibition of tumor growth after OVA T -FNs vaccine immunization. Various experiments have confirmed that these CD8 + T cells can secrete large amounts of effectors, such as IFN-γ and Granzyme B, thereby significantly inducing lysis of target cells. In various prophylactic tumor models, OVA T -FNs vaccine prepared based on the model antigen significantly inhibited tumor growth, suggesting that the use of FNs as an antigen delivery vehicle can dramatically enhance the antitumor efficacy of peptide vaccines. Moreover, melanoma is a highly malignant and extremely metastatic tumor, and the antitumor immune responses induced by M30-FNs vaccine, prepared by fusion of the neoantigen, also significantly inhibited melanoma metastasis to the lungs. Similarly, for established melanoma, M30-FNs vaccine likewise induced regression. The above results suggested that the use of FNs as delivery vectors for mutation-derived neoantigens, which are less effective in the clinic, could also significantly enhance tumor suppression. Conclusions We have developed an excellent platform for tumor neoantigen delivery, FNs, which are not only homogeneous in terms of particle size and structural stability, but also highly biocompatible. Fusion with FNs significantly enhanced the immunogenicity of antigen peptides and induced the activation and maturation of APCs. Notably, neoantigen-FNs selectively targeted lymph nodes, activated a large number of early-differentiated antigen-specific CD8 + T cells in vivo, and exhibited a striking ability to kill target cells. Both activated model antigen- and neoantigen-specific immune responses significantly inhibit tumor growth. In conclusion, FNs can be used as ideal neoantigen delivery platforms to further improve the effectiveness of neoantigen peptide vaccines, which not only broadens the road of neoantigen peptide vaccine delivery vehicles but also has great potential for clinical application. Abbreviations Neoantigen-ferritin nanoparticles (neoantigen-FNs) Food and Drug Administration (FDA) Cytotoxic T lymphocyte (CTL) Virus-like particles (VLPs) Human ferritin heavy chain (hFTN) OVA T -ferritin nanoparticles (OVA T -FNs) Antigen presenting cells (APCs) Wild-type (WT) American Type Culture Collection (ATCC) Fetal bovine serum (FBS) Heat-inactivated fetal bovine serum (HI-FBS) Escherichia coli ( E. coli ) Isopropyl β -D-1-thiogalactopyranoside (IPTG) Molecular weight cutoff (MWCO) Dynamic light scattering (DLS) Bone marrow-derived dendritic cells (BMDCs) Enzyme-Linked Immunosorbent Assay (ELISA) Enzyme-Linked Immunospot (ELISpot) Hematoxylin and eosin (H&E) Lymph node (LN) T helper 1 (Th1) Effector memory T (Tem) Central memory T (Tcm) Transmission electron microscopy (TEM) Aspartate aminotransferase (AST) Alanine aminotransferase (ALT) Blood urea nitrogen (BUN) Creatinine (CRE) Declarations Ethics approval and consent to participate All of the animal experimental manipulations included in this study were approved by the policies and guidelines of the Animal Ethics Committee of Nankai University (2022-SYDWLL-000639). Consent for publication Not applicable. Availability of data and materials The data and materials used and analyzed during this research are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This research was supported by the National Key R&D Program of China (No. 2022YFC2304202) and the National Natural Science Foundation of China (No.82073341). Authors' contributions Authors and affiliations WZ, SL, ZS, KS, YD, LZ, QT, JH, and HZ: State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Protein Sciences, Cancer Biology Center, College of Life Sciences, Nankai University, Tianjin 300071, PR China FW: School of Medicine, Nankai University, Tianjin 300071, PR China People’s Hospital of Tianjin, Tianjin 300180, PR China Contributions WZ, SL, KS, and FW conceived and designed the experiments; WZ, SL, ZS, HZ, KS, YD, LZ, QT, and JH performed all the experiments; WZ, SL, ZS, LZ, and HZ analyzed the data obtained; WZ, SL, and FW wrote the manuscript. Corresponding authors HZ, ZH State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Protein Sciences, Cancer Biology Center, College of Life Sciences, Nankai University, Tianjin 300071, PR China Nankai International Advanced Research Institute (SHENZHEN FUTIAN), Shenzhen 518045, PR China Acknowledgements The authors acknowledge support from the National Key R&D Program of China (No. 2022YFC2304202) and the National Natural Science Foundation of China (No.82073341). We sincerely appreciate the help and guidance of teachers Yajuan Wan, Rui Wang, Ruming Liu, Li Jiao, Di An and Ying Zhou from the Instrumentation Platform of Nankai University, as well as Xiaomin Su and Yanfang Chen from the Animal Experiment Center of Nankai University. References Saxena M, van der Burg SH, Melief CJM, Bhardwaj N. Therapeutic cancer vaccines. Nat Rev Cancer. 2021;21(6):360-78. Weber JS, Carlino MS, Khattak A, Meniawy T, Ansstas G, Taylor MH, et al. 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Personal neoantigen vaccines induce persistent memory T cell responses and epitope spreading in patients with melanoma. Nat Med. 2021;27(3):515-25. Oliveira G, Stromhaug K, Klaeger S, Kula T, Frederick DT, Le PM, et al. Phenotype, specificity and avidity of antitumour CD8(+) T cells in melanoma. Nature. 2021;596(7870):119-25. Philip M, Schietinger A. CD8(+) T cell differentiation and dysfunction in cancer. Nat Rev Immunol. 2022;22(4):209-23. Baharom F, Ramirez-Valdez RA, Tobin KKS, Yamane H, Dutertre CA, Khalilnezhad A, et al. Intravenous nanoparticle vaccination generates stem-like TCF1(+) neoantigen-specific CD8(+) T cells. Nat Immunol. 2021;22(1):41-52. Eddy K, Shah R, Chen S. Decoding Melanoma Development and Progression: Identification of Therapeutic Vulnerabilities. Front Oncol. 2020;10:626129. Lin MJ, Svensson-Arvelund J, Lubitz GS, Marabelle A, Melero I, Brown BD, et al. Cancer vaccines: the next immunotherapy frontier. Nat Cancer. 2022;3(8):911-26. Blass E, Ott PA. Advances in the development of personalized neoantigen-based therapeutic cancer vaccines. Nat Rev Clin Oncol. 2021;18(4):215-29. Sahin U, Tureci O. Personalized vaccines for cancer immunotherapy. Science. 2018;359(6382):1355-60. Liu H, Moynihan KD, Zheng Y, Szeto GL, Li AV, Huang B, et al. Structure-based programming of lymph-node targeting in molecular vaccines. Nature. 2014;507(7493):519-22. Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstract.png Additionalfile.docx Cite Share Download PDF Status: Published Journal Publication published 14 Sep, 2024 Read the published version in Journal of Nanobiotechnology → Version 1 posted Editorial decision: Revision requested 24 Jul, 2024 Reviews received at journal 14 Jul, 2024 Reviews received at journal 12 Jul, 2024 Reviews received at journal 05 Jul, 2024 Reviewers agreed at journal 04 Jul, 2024 Reviewers agreed at journal 02 Jul, 2024 Reviewers agreed at journal 02 Jul, 2024 Reviewers invited by journal 02 Jul, 2024 Editor assigned by journal 01 Jul, 2024 Submission checks completed at journal 01 Jul, 2024 First submitted to journal 28 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4654130","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":326878893,"identity":"20057564-c6ad-485c-8ae7-0796607f27a9","order_by":0,"name":"Wei Zheng","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Zheng","suffix":""},{"id":326878894,"identity":"5277e571-d559-4f1c-9f9e-66a70cbed01b","order_by":1,"name":"Shixiong Li","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"Shixiong","middleName":"","lastName":"Li","suffix":""},{"id":326878895,"identity":"af98ba51-6f33-49c7-95db-a03430abf1d5","order_by":2,"name":"Zhongliang Shi","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"Zhongliang","middleName":"","lastName":"Shi","suffix":""},{"id":326878896,"identity":"9e49164c-7990-4bfe-91f9-878e6450de53","order_by":3,"name":"Kailing Su","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"Kailing","middleName":"","lastName":"Su","suffix":""},{"id":326878897,"identity":"8b5a26cf-bc22-42df-8b3d-7a1d0cb69ef4","order_by":4,"name":"Yu Ding","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Ding","suffix":""},{"id":326878898,"identity":"8b44149d-ee6c-4182-9a13-1772ea261d8b","order_by":5,"name":"Luyue Zhang","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"Luyue","middleName":"","lastName":"Zhang","suffix":""},{"id":326878899,"identity":"c9549e9b-fb96-42ba-ac3a-a09604da96f8","order_by":6,"name":"Qian Tang","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Tang","suffix":""},{"id":326878900,"identity":"ee9f5f53-c886-4493-9d8b-a150be6a0431","order_by":7,"name":"Jiani Han","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"Jiani","middleName":"","lastName":"Han","suffix":""},{"id":326878901,"identity":"bf482e2b-46b1-4414-a7bd-6e706e582851","order_by":8,"name":"Han Zhao","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"Han","middleName":"","lastName":"Zhao","suffix":""},{"id":326878902,"identity":"a00108e4-635b-4608-904f-6d7997d2b0a3","order_by":9,"name":"Fengwei Wang","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"Fengwei","middleName":"","lastName":"Wang","suffix":""},{"id":326878903,"identity":"0e3dbc56-6f27-48fe-88ae-0e7a4c29da95","order_by":10,"name":"Hongru Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYBACAwYGNhAtB8TMYC7DASK1GJOuJbEBrIWBCC3m0sefPfi5ozZ9O/vZwwY/Chjk+G4kMH4uwKPFsi8h3bD3zPHcnT15yYk9BgzGkjcSmKVn4HPYGYZjErxtx3I3HMgxPsBjwJC44UYCGzMPXi2MbZJ/246lG5x/Y3zwjwFDPRFamNmkedtqEgxu5BgnA20BMghqYWOTlm07YLjhxhtjYxkDCcOZZx42S+PXwv5M8m1bnbzB+RxjyTd/bOT5jicf/IxPCxQchjEkgJixgbAGBoY6YhSNglEwCkbBSAUAxFlK249+TU0AAAAASUVORK5CYII=","orcid":"","institution":"Nankai University","correspondingAuthor":true,"prefix":"","firstName":"Hongru","middleName":"","lastName":"Zhang","suffix":""},{"id":326878904,"identity":"5897b79d-e305-45cf-82f1-88320fd85753","order_by":11,"name":"Zhangyong Hong","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"Zhangyong","middleName":"","lastName":"Hong","suffix":""}],"badges":[],"createdAt":"2024-06-28 10:37:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4654130/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4654130/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12951-024-02837-2","type":"published","date":"2024-09-14T15:58:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60935936,"identity":"257b84e6-fec1-4829-a8c5-f8f970afcc3c","added_by":"auto","created_at":"2024-07-23 19:01:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":388811,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDesign and characterization of neoantigen-FNs.\u003c/strong\u003e \u003cstrong\u003ea,\u003c/strong\u003e Schematic diagram of the design of neoantigen-FNs. \u003cstrong\u003eb,\u003c/strong\u003e SDS-PAGE analysis of purified FNs (monomer ~19.94 kDa) and OVA\u003csub\u003eT\u003c/sub\u003e-FNs (monomer ~21.49 kDa). \u003cstrong\u003ec,\u003c/strong\u003e Size exclusion chromatography analysis of purified FNs and OVA\u003csub\u003eT\u003c/sub\u003e-FNs. The proteins formed ~478.56 kDa (FNs) and ~515.76 kDa (OVA\u003csub\u003eT\u003c/sub\u003e-FNs) nanoparticles. \u003cstrong\u003ed,\u003c/strong\u003e TEM images of FNs (top) and OVA\u003csub\u003eT\u003c/sub\u003e-FNs (bottom) stained with 1% uranyl acetate. Scale bar, 100 nm. \u003cstrong\u003ee,\u003c/strong\u003e DLS measurements of FNs (left) and OVA\u003csub\u003eT\u003c/sub\u003e-FNs (right)(n=3). \u003cstrong\u003ef,\u003c/strong\u003e Zeta potential measurements of FNs and OVA\u003csub\u003eT\u003c/sub\u003e-FNs. The mean zeta potentials were -10 mV and -8 mV for FNs and OVA\u003csub\u003eT\u003c/sub\u003e-FNs, respectively(n=3). \u003cstrong\u003ee, f,\u003c/strong\u003e Data are presented as the mean ± s.e.m. \u003cstrong\u003ef,\u003c/strong\u003e The statistical significance of differences between the groups was assessed using two-tailed unpaired Student’s \u003cem\u003et\u003c/em\u003e test. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4654130/v1/31f56d5a1ca460c0f7892cd4.png"},{"id":60935939,"identity":"c1ab7dd3-eb2d-422e-823b-8f48a6a4ecb0","added_by":"auto","created_at":"2024-07-23 19:01:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":415594,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe uptake of OVA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-FNs by BMDCs induced their activation and maturation in vitro.\u003c/strong\u003e \u003cstrong\u003ea, b,\u003c/strong\u003e OVA\u003csub\u003eT\u003c/sub\u003e-FNs were taken up by BMDCs and escaped from lysosomes into the cytoplasm. \u003cstrong\u003ea,\u003c/strong\u003e Confocal fluorescence images of BMDCs treated with OVA\u003csub\u003eT\u003c/sub\u003e-Cy5 (red), FNs-Cy5 (red), or OVA\u003csub\u003eT\u003c/sub\u003e-FNs-Cy5 (red) for 4 h. The nuclei and surface markers of BMDCs were stained with DAPI (blue) and a FITC-conjugated CD11c antibody (green), respectively. Scale bar, 25 μm. \u003cstrong\u003eb,\u003c/strong\u003e Confocal fluorescence images of BMDCs treated with OVA\u003csub\u003eT\u003c/sub\u003e-FITC (green), FNs-FITC (green), or OVA\u003csub\u003eT\u003c/sub\u003e-FNs-FITC (green) for 4 h. The nuclei and lysosomes of BMDCs were stained with Hoechst (blue) and LysoTracker (red), respectively. Scale bar, 10 μm. \u003cstrong\u003ec-g,\u003c/strong\u003e Activation of BMDCs by OVA\u003csub\u003eT\u003c/sub\u003e-FNs. Flow cytometry analysis of the frequencies of CD80\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003ec\u003c/strong\u003e), CD86\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003ed\u003c/strong\u003e), MHC-I\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003ee\u003c/strong\u003e), MHC-II\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003ef\u003c/strong\u003e), and CD40\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003eg\u003c/strong\u003e) CD11c\u003csup\u003e+\u003c/sup\u003e cells in BMDCs treated with OVA\u003csub\u003eT\u003c/sub\u003e, FNs, or OVA\u003csub\u003eT\u003c/sub\u003e-FNs for 24 h (n=3). \u003cstrong\u003eh,\u003c/strong\u003e Cross-presentation efficiency of BMDCs was characterized by flow cytometry analysis of the frequency of SIINFEKL-H2Kb\u003csup\u003e+\u003c/sup\u003e cells among CD11c\u003csup\u003e+\u003c/sup\u003e BMDCs (n=3). \u003cstrong\u003ei, j,\u003c/strong\u003e ELISA analysis of IL-12(P70) (\u003cstrong\u003ei\u003c/strong\u003e) and IFN-α1 (\u003cstrong\u003ej\u003c/strong\u003e) in the culture supernatant of BMDCs after treatment with OVA\u003csub\u003eT\u003c/sub\u003e, FNs, or OVA\u003csub\u003eT\u003c/sub\u003e-FNs for 24 h (n=3). \u003cstrong\u003ec-j,\u003c/strong\u003e Data are presented as the mean ± s.e.m. Statistical significance between the groups was assessed using two-tailed unpaired Student’s \u003cem\u003et\u003c/em\u003e test. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4654130/v1/c1329b59d17912a5938284c9.png"},{"id":60935937,"identity":"d96ab033-b016-44a4-a6ee-cdf0d5140cb3","added_by":"auto","created_at":"2024-07-23 19:01:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":426733,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOVA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-FNs for lymph node targeting and were taken up by APCs in the lymph nodes.\u003c/strong\u003e \u003cstrong\u003ea,\u003c/strong\u003e \u003cstrong\u003eb,\u003c/strong\u003e Live imaging of the location of OVA\u003csub\u003eT\u003c/sub\u003e-FNs in vivo. BALB/c mice were subcutaneously administered in the left groin with 6 nmol Cy5-labeled vaccines. Fluorescence images (\u003cstrong\u003ea\u003c/strong\u003e) and total efficiency (\u003cstrong\u003eb\u003c/strong\u003e) at 24 h, 48 h, and 72 h after administration are shown (n=3). \u003cstrong\u003ec, d,\u003c/strong\u003e Representative fluorescence images (\u003cstrong\u003ec\u003c/strong\u003e) and total efficiency (\u003cstrong\u003ed\u003c/strong\u003e) of proximal lymph nodes (left) and distal lymph nodes (right). C57BL/6 mice were subcutaneously administered in the left groin with PBS or 6 nmol Cy5-labeled vaccine, and the lymph nodes were removed at 24 h, 48 h, and 72 h after administration (n=3). \u003cstrong\u003ee-h,\u003c/strong\u003e Flow cytometry analysis of the frequencies of vaccine\u003csup\u003e+\u003c/sup\u003e cells among CD11c\u003csup\u003e+\u003c/sup\u003e DCs (\u003cstrong\u003ee\u003c/strong\u003e), macrophages (\u003cstrong\u003ef\u003c/strong\u003e) and B cells (\u003cstrong\u003eg\u003c/strong\u003e) and the frequency of CD80\u003csup\u003e+\u003c/sup\u003e CD86\u003csup\u003e+\u003c/sup\u003e CD11c\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003eh\u003c/strong\u003e) cells in the lymph nodes (n=3). \u003cstrong\u003eb,\u003c/strong\u003e \u003cstrong\u003ed-h,\u003c/strong\u003e Data are presented as the mean ± s.e.m. \u003cstrong\u003eb, d,\u003c/strong\u003e Statistical significance between the groups was assessed using one-way ANOVA. \u003cstrong\u003ee-f,\u003c/strong\u003e Statistical significance between the groups was assessed using two-tailed unpaired Student’s \u003cem\u003et\u003c/em\u003e test. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4654130/v1/65a8b872e1c9c4986e9d83f6.png"},{"id":60936252,"identity":"df2dd644-ed8f-4eb4-a0ff-d1a82d4982b5","added_by":"auto","created_at":"2024-07-23 19:09:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":420640,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of OVA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-FNs on the proliferation and production of specific CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cells.\u003c/strong\u003e \u003cstrong\u003ea-c,\u003c/strong\u003e Proliferation of antigen-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells induced by OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine in vivo. Schematic of the experimental design (\u003cstrong\u003ea\u003c/strong\u003e), flow cytometry analysis data (\u003cstrong\u003eb\u003c/strong\u003e) and proliferation index (\u003cstrong\u003ec\u003c/strong\u003e) for the proliferation of CFSE-labeled OT-1 CD8\u003csup\u003e+\u003c/sup\u003e T cells (n=3). \u003cstrong\u003ed,\u003c/strong\u003e Schematic diagram for evaluating immune responses induced by OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine. \u003cstrong\u003ee-i,\u003c/strong\u003e C57BL/6 mice (n=5) were vaccinated subcutaneously with OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine or controls. Whole blood (\u003cstrong\u003ee, f, h, i\u003c/strong\u003e) and the spleen (\u003cstrong\u003eg\u003c/strong\u003e) were collected on day 21. \u003cstrong\u003ee-g,\u003c/strong\u003e Flow cytometry analysis of the frequency of OVA\u003csub\u003eT\u003c/sub\u003e tetramer\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cells in the blood (\u003cstrong\u003ee, f\u003c/strong\u003e) and spleen (\u003cstrong\u003eg\u003c/strong\u003e). \u003cstrong\u003eh,\u003c/strong\u003e Flow cytometry analysis of the frequency of PD-1\u003csup\u003e+\u003c/sup\u003e TIM-3\u003csup\u003e-\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cells in the blood. \u003cstrong\u003ei,\u003c/strong\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cells were subdivided into CD62L\u003csup\u003e+\u003c/sup\u003e CD44\u003csup\u003e-\u003c/sup\u003e (gray, naïve T), CD62L\u003csup\u003e+\u003c/sup\u003e CD44\u003csup\u003e+\u003c/sup\u003e (yellow, Tcm), CD62L\u003csup\u003e-\u003c/sup\u003e CD44\u003csup\u003ehi\u003c/sup\u003e (purple, Tem), and CD62L\u003csup\u003e-\u003c/sup\u003e CD44\u003csup\u003elo\u003c/sup\u003e (blue, Teff) subsets based on CD62L and CD44 expression. \u003cstrong\u003ec, f-i,\u003c/strong\u003e Data are presented as the mean ± s.e.m. Statistical significance between the groups was assessed using two-tailed unpaired Student’s \u003cem\u003et\u003c/em\u003e test. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4654130/v1/328780bb4934972e429df01a.png"},{"id":60936438,"identity":"5f74653d-f541-48d0-a6f6-fb90d286ef86","added_by":"auto","created_at":"2024-07-23 19:17:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":513731,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOVA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-FNs for the elicitation of CTL responses.\u003c/strong\u003e \u003cstrong\u003ea, b,\u003c/strong\u003e ELISpot of IFN-γ spot-forming cells in 2×10\u003csup\u003e5\u003c/sup\u003e splenocytes restimulated with OVA\u003csub\u003eT\u003c/sub\u003e peptide for 48 h on day 21 (n=5). \u003cstrong\u003ec,\u003c/strong\u003e IFN-γ production in the supernatant of 2×10\u003csup\u003e5\u003c/sup\u003e splenocytes restimulated with OVA\u003csub\u003eT\u003c/sub\u003e peptide for 48 h, as measured by ELISA (n=5). \u003cstrong\u003ed-f, \u003c/strong\u003eFlow cytometry data showing the frequencies of IFN-γ\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003ed, e\u003c/strong\u003e) and Granzyme B\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003ef\u003c/strong\u003e) CD8\u003csup\u003e+\u003c/sup\u003e T cells in the spleen on day 21. A total of 1×10\u003csup\u003e6\u003c/sup\u003e splenocytes were restimulated with OVA\u003csub\u003eT\u003c/sub\u003e peptide and a Golgi plug for 6 h (n=5). \u003cstrong\u003eg-i,\u003c/strong\u003e CTL assay of specific CD8\u003csup\u003e+\u003c/sup\u003e T cells induced by OVA\u003csub\u003eT\u003c/sub\u003e-FNs in vivo (n=3). Flow cytometry analysis of the lysis of OVA\u003csub\u003eT\u003c/sub\u003e-pulsed splenocytes in the spleen (\u003cstrong\u003eh\u003c/strong\u003e) and lymph nodes (LN) (\u003cstrong\u003ei\u003c/strong\u003e). \u003cstrong\u003eb, c, h, i,\u003c/strong\u003e Representative data are shown as the mean ± s.e.m. The statistical significance between the groups was assessed using two-tailed unpaired Student’s \u003cem\u003et\u003c/em\u003e test. \u003cstrong\u003ee, f,\u003c/strong\u003e Statistical significance of differences between the groups was assessed using one-way ANOVA. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4654130/v1/459e6240444e1f201d621d91.png"},{"id":60935940,"identity":"b3fa25e6-9498-4f44-bdb5-6043d6f08417","added_by":"auto","created_at":"2024-07-23 19:01:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1034794,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOVA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-FNs vaccine inhibited tumor growth and were nontoxic.\u003c/strong\u003e \u003cstrong\u003ea-d,\u003c/strong\u003e The prophylactic effects of OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine against E.G7-OVA tumors (n=5). Schematic illustration of the experimental design (\u003cstrong\u003ea\u003c/strong\u003e), average tumor growth curves (\u003cstrong\u003eb\u003c/strong\u003e) and tumor size on day 16 (\u003cstrong\u003ec\u003c/strong\u003e) are presented. \u003cstrong\u003ed, \u003c/strong\u003eIndividual tumor growth curves of mice vaccinated with PBS (black), OVA\u003csub\u003eT\u003c/sub\u003e (green), OVA\u003csub\u003eT\u003c/sub\u003e vaccine (blue), OVA\u003csub\u003eT\u003c/sub\u003e-FNs (purple) and OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine (red) are shown. \u003cstrong\u003ee-g,\u003c/strong\u003e The prophylactic effects of OVA\u003csub\u003eT\u003c/sub\u003e-FNs against MC-38-OVA colon tumors (n=5). Schematic illustration of the experimental design (\u003cstrong\u003ee\u003c/strong\u003e), average tumor growth curves (\u003cstrong\u003ef\u003c/strong\u003e) and survival curves (\u003cstrong\u003eg\u003c/strong\u003e) are shown. \u003cstrong\u003eh, i, \u003c/strong\u003eBiosafety assessment of OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine in vitro (\u003cstrong\u003eh\u003c/strong\u003e) and in vivo (\u003cstrong\u003ei\u003c/strong\u003e). \u003cstrong\u003eh, \u003c/strong\u003eEvaluation of the cytotoxicity of FNs and OVA\u003csub\u003eT\u003c/sub\u003e-FNs to the DC2.4 cell line by MTT assay (n=3). \u003cstrong\u003ei, \u003c/strong\u003eRepresentative H\u0026amp;E-stained sections of the heart, liver, spleen, lung and kidney.\u003cstrong\u003e \u003c/strong\u003eScale bar, 100 μm. \u003cstrong\u003eb, f, h, \u003c/strong\u003eData are presented as the mean ± s.e.m. \u003cstrong\u003eb, f,\u003c/strong\u003e The statistical significance of differences between the groups was assessed using two-way ANOVA. \u003cstrong\u003eg,\u003c/strong\u003e Statistics were assessed by log rank test. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4654130/v1/72dd27c5a1896dfe44a871ae.png"},{"id":60936254,"identity":"8f442019-3602-4aca-a547-b4873bf74435","added_by":"auto","created_at":"2024-07-23 19:09:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":464195,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeoantigen-based M30-FNs vaccine controlled the metastasis and growth of melanoma.\u003c/strong\u003e \u003cstrong\u003ea,\u003c/strong\u003e TEM image of M30-FNs stained with 1% uranyl acetate. Scale bar, 100 nm. \u003cstrong\u003eb, c,\u003c/strong\u003e ELISpot of IFN-γ spot-forming cells in 2×10\u003csup\u003e5\u003c/sup\u003e splenocytes restimulated with M30 peptide (n=5). C57BL/6 mice were immunized with M30-FNs vaccine on days 0 and 14, and the spleens were removed on day 21. \u003cstrong\u003ed-f, \u003c/strong\u003eThe antitumor capacity of M30-FNs in a prophylactic metastatic B16F10 melanoma model (n=4). A schematic of the experimental design (\u003cstrong\u003ed\u003c/strong\u003e), pictures of the lungs (\u003cstrong\u003ee\u003c/strong\u003e) and the number of metastatic lung nodules (\u003cstrong\u003ef\u003c/strong\u003e) are shown. \u003cstrong\u003eg-i,\u003c/strong\u003e Antitumor therapeutic effects of M30-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells induced by M30-FNs vaccine against B16F10 tumors (n=5). Schematic illustration of the experimental design (\u003cstrong\u003eg\u003c/strong\u003e), average tumor growth curves (\u003cstrong\u003eh\u003c/strong\u003e), body weights (\u003cstrong\u003ei\u003c/strong\u003e) and individual tumor growth curves (\u003cstrong\u003ej\u003c/strong\u003e) are shown. \u003cstrong\u003ec, f, h, i,\u003c/strong\u003e Data are presented as the mean ± s.e.m. \u003cstrong\u003ec, f,\u003c/strong\u003e Statistical significance between the groups was assessed using a two-tailed unpaired Student’s \u003cem\u003et\u003c/em\u003e test. \u003cstrong\u003eh, i,\u003c/strong\u003e Statistical significance between the groups was assessed using two-way ANOVA. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4654130/v1/56cf069a69dafe8739ac2586.png"},{"id":64619345,"identity":"033afef3-4091-4db4-a20e-43ddeb6b87aa","added_by":"auto","created_at":"2024-09-16 16:14:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4865778,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4654130/v1/cd2e7f00-2ce3-4a6b-a8d0-05c6cc4931d6.pdf"},{"id":60935943,"identity":"0ecf9dae-e38e-446c-bde3-78dcb83ba644","added_by":"auto","created_at":"2024-07-23 19:01:31","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3200276,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-4654130/v1/28dbb4d5f4ec2a25a5325cb0.png"},{"id":60935941,"identity":"64b2c42c-3065-4774-81da-204ec37f16af","added_by":"auto","created_at":"2024-07-23 19:01:31","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3820523,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-4654130/v1/56f5a48f447ce01bd9ca88bc.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Recombinant ferritin-based nanoparticles as neoantigen carriers significantly inhibit tumor growth and metastasis","fulltext":[{"header":"Background","content":"\u003cp\u003eTherapeutic tumor vaccines are personalized immunotherapies that recognize and eliminate malignant cells by stimulating the body to generate tumor antigen-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Several clinical trials have demonstrated that personalized therapeutic vaccines can significantly prolong the survival of patients with solid tumors[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In 2010, the U.S. Food and Drug Administration (FDA) approved Provenge (sipuleucel-T) for the therapy of advanced prostate cancer, suggesting that therapeutic tumor vaccines have promising prospects for the treatment of solid tumors[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdvances in rapid genome sequencing technology have driven the development of neoantigen therapeutic vaccines[\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Neoantigens are tumor-specific antigens derived from nonsynonymous mutations and have become ideal targets for therapeutic tumor vaccines because they do not elicit autoimmune responses or central tolerance. Neoantigen peptides have been widely applied in clinical research due to their favorable biosafety, abundant modifiable sites and mature synthetic routes[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, neoantigen peptides usually fail to elicit sufficient antigen-specific cytotoxic T lymphocyte (CTL) responses in vivo because of their lower immunogenicity and poor lymph node-targeted delivery[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, it is imperative to develop an ideal neoantigen peptide delivery system that not only enhances the immunogenicity of neoantigens, but also enables targeted delivery to lymph nodes to stimulate effective antitumor immune responses.\u003c/p\u003e \u003cp\u003eTo address these issues, a variety of neoantigen peptide delivery platforms, including polymeric nanoparticles[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], inorganic nanoparticles[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and liposomes[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], which are able to induce robust CTL responses and significantly inhibit tumor growth in mice, have been developed and validated in preclinical studies. Unfortunately, poor biocompatibility, particle heterogeneity, or inconsistent manufacturing processes have limited the application of these systems in clinical trials[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, protein-caged nanoparticles such as virus-like particles (VLPs) and ferritin family proteins have attracted much attention in drug delivery due to their good biodegradability, highly ordered structures, and simple and repeatable preparation methods[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These proteins tend to self-assemble into nanoscale core-shell structures, which would facilitate effective targeting to lymph nodes and persistence in vivo[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Tumor peptide vaccines based on VLPs and heat shock proteins have shown strong efficacy in triggering protective immunity against different tumors[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLymph node targeting is often considered critical for the effective in vivo delivery of tumor vaccines and the induction of robust antitumor immune responses[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. A study comparing the lymph node-targeting ability of four different protein-caged nanoparticles demonstrated that engineered human ferritin heavy chain (hFTN) nanoparticles have greater potential for direct lymph node targeting and tumor immunotherapy[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Ferritin is a 12 nm spherical particle self-assembled from 24 monomers with the advantages of nanoscale size[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], multiple peptide modification sites, good biocompatibility, and thermal stability[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In addition, ferritin has been extensively investigated for the delivery of viral antigens such as influenza and SARS-CoV-2 antigens[\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and several clinical studies have shown that ferritin has excellent biosafety as an antigen delivery vehicle[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. However, studies on ferritin as a delivery platform for neoantigen peptides in tumor vaccines have been very limited[\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Therefore, we expect to develop a ferritin-based tumor vaccine platform for the delivery of neoantigen peptides to enhance antitumor immune responses.\u003c/p\u003e \u003cp\u003eIn this study, \u003cem\u003eHelicobacter pylori\u003c/em\u003e-derived ferritin[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], which is distantly related to human evolution, was chosen as a delivery platform for tumor neoantigen peptides due to its advantage to prevent the generation of autoimmune responses or immune tolerance[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The results showed that OVA\u003csub\u003eT\u003c/sub\u003e-ferritin nanoparticles (OVA\u003csub\u003eT\u003c/sub\u003e-FNs) are homogeneous and stable 12 nm particles and have significantly greater immunogenicity than OVA\u003csub\u003eT\u003c/sub\u003e free peptides. Moreover, OVA\u003csub\u003eT\u003c/sub\u003e-FNs can rapidly migrate to lymph nodes and reside for a long time to enhance uptake by antigen-presenting cells (APCs) in lymph nodes. Effective antigen-specific immune responses were induced by OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine in vivo, resulting in significant inhibition of tumor growth and metastasis. In addition, OVA\u003csub\u003eT\u003c/sub\u003e-FNs possessed excellent biosafety, which will facilitate subsequent clinical translation.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003eFemale wild-type (WT) C57BL/6 and BALB/c mice and female OT-1 transgenic mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All mice used in this study were between 6 and 8 weeks old and were maintained under specific pathogen-free conditions in the animal facility at Nankai University. All mice were housed in groups of 5 under conditions of a 12-hour light-dark cycle (8:00\u0026ndash;20:00, light; 20:00\u0026ndash;8:00, dark), constant room temperature (21\u0026deg;C), suitable humidity (40%~60%), and free access to food and water. The animal procedures were performed with ethical compliance and approval from the Institutional Animal Care and Use Committee at Nankai University.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell lines\u003c/h3\u003e\n\u003cp\u003eThe E.G7-OVA, MC-38-OVA, B16F10-OVA, B16F10, and DC2.4 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). E.G7-OVA, MC-38-OVA, B16F10-OVA, and B16F10 cells were grown in RPMI 1640 (Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS, Biological Industries, Kibbutz, Israel) and 1% penicillin-streptomycin (NCM Biotech, Cat. #C100C5). In addition, 50 \u0026micro;M 2-mercaptoethanol (Aladdin, Cat. #M301574) and 0.4 mg/mL G-418 (Solarbio, Cat. #IG0010) were used for culture of E.G7-OVA, and 1.5 \u0026micro;g/mL puromycin (Solarbio, Cat. # P8230) was used for culture of MC-38-OVA and B16F10-OVA to exclude cells not overexpressing OVA. DC2.4 cells were grown in DMEM (Gibco, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS), 1% penicillin-streptomycin and 50 \u0026micro;M 2-mercaptoethanol. All cells tested negative for mycoplasma contamination according to the ATCC Universal Mycoplasma Detection Kit (ATCC, Cat. #30-1012K) and were cultured at 37\u0026deg;C in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eProtein biosynthesis and purification\u003c/h2\u003e \u003cp\u003eThe gene encoding \u003cem\u003eHelicobacter pylori\u003c/em\u003e ferritin (residues 5-167) was codon-optimized to adapt to the \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) expression system, and a point mutation (Asn19Gly) was designed to remove a potential N-linked glycosylation site. The OVA\u003csub\u003eT\u003c/sub\u003e peptide (SIINFEKL) followed by a (GGS)\u003csub\u003e3\u003c/sub\u003e linker and the M30 peptide (PSKPSFQEFVDWENVSPELNSTDQPFL) followed by a SASGG linker were fused to the N-terminus of ferritin (residues 5-167) to generate OVA\u003csub\u003eT\u003c/sub\u003e-FNs and M30-FNs, respectively[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Moreover, FNs without peptides at the N-terminus of ferritin (residues 5-167) were prepared as a control. The constructs were cloned and inserted into the pET-28a (+) expression vector (Novagen, Madison, WI, USA) using the NcoI and XhoI restriction sites. The recombinant plasmids were transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) competent cells (ZOMANBIO, Cat. #ZK201), and the bacterial cells were grown in LB media supplemented with 50 \u0026micro;g/mL kanamycin (Solarbio, Cat. #K8020) at 37\u0026deg;C until an absorbance of 0.6 was reached at 600 nm. The proteins were induced to be overexpressed by 0.7 mM isopropyl \u003cem\u003eβ\u003c/em\u003e-D-1-thiogalactopyranoside (IPTG) (Solarbio, Cat. #II0130) for 16 h at 16\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe bacterial cells were harvested and resuspended in lysis buffer (50 mM Tris, 500 mM NaCl, 5% glycerol, pH 8.0) and then homogenized at a pressure of 700 bar. The supernatant containing the recombinant proteins was obtained by centrifugation at 18,000 rpm for 40 min at 4\u0026deg;C, and the proteins were isolated by Ni-nitrilotriacetic acid affinity chromatography. Briefly, 2 mL of Ni-NTA resin (TransGen, Cat. #DP101) was used to purify proteins, which were then equilibrated with PBS (pH 7.2\u0026thinsp;~\u0026thinsp;7.4) and incubated 2 times. Nontarget proteins were removed by washing with 10 mM, 50 mM, or 100 mM imidazole, and target proteins were eluted with 300 mM imidazole. The size and purity of the harvested proteins were determined by SDS-PAGE. The eluted proteins were buffer exchanged into PBS (pH 7.2\u0026thinsp;~\u0026thinsp;7.4) containing 1 mM EDTA (Solarbio, Cat. #E1170) and 5% glycerol and concentrated to less than 5 mL using Amicon-Ultra15 centrifugal filters (EMD Millipore) with a 100-kDa molecular weight cutoff (MWCO). The concentrated proteins were further purified by size exclusion chromatography using a HiPrep 16/60 Sephacryl S-500 HR size-exclusion column in the above buffer at a flow rate of 0.8 mL/min. The purified proteins were concentrated to 2.5 mg/mL using Amicon-Ultra15 centrifugal filters with a 100-kDa MWCO, and then 5% glycerol was added, followed by storage at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eNegative-stain electron microscopy\u003c/h2\u003e \u003cp\u003eFor the negative-staining study, 4 \u0026micro;L of 100 \u0026micro;g/mL protein sample was applied to a carbon film-coated 300-mesh Cu grid (Beijing Zhongjingkeyi Technology Co., Ltd., Beijing, China) for 1 min. After air-drying, the sample was negatively stained with 1% (w/v) uranyl acetate for 1 min and then observed by scanning electron microscopy (HITACHI HT7700 Exalens).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDynamic light scattering (DLS) and zeta potential analysis\u003c/h2\u003e \u003cp\u003eParticle size and zeta potential analysis of FNs and OVA\u003csub\u003eT\u003c/sub\u003e-FNs at a concentration of approximately 2 mg/mL were performed using a Zetasizer Nano ZS (Malvern Instruments Ltd., UK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBlood and tissue processing\u003c/h2\u003e \u003cp\u003eApproximately 150 \u0026micro;L of peripheral blood was collected into a 1.5 mL EP tube containing 30 \u0026micro;L of 0.5 M EDTA, and 1.5 mL of ACK buffer (Solarbio, Cat. #R1010) was added to the lysed red blood cells twice for 5 min each. Lymph nodes or spleen placed between two 40 \u0026micro;m filters were mechanically prepared into single cells in a 12-well plate containing 1 mL of MACS buffer (1\u0026times;PBS containing 0.5% BSA and 2 mM EDTA). In addition, the splenocytes were lysed at room temperature for 5 min with 2 mL of ACK buffer to remove red blood cells. The obtained single-cell suspension was filtered through a 40 \u0026micro;m nylon mesh filter for subsequent flow cytometry detection or transfer experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of bone marrow-derived dendritic cells (BMDCs)\u003c/h2\u003e \u003cp\u003eBone marrow was flushed from the femurs and tibias of C57BL/6 mice with ice-cold RPMI 1640 medium supplemented with 10% HI-FBS and 2% penicillin-streptomycin. Red blood cells were lysed, and the remaining cells were seeded in a 10 cm tissue culture dish containing 12 mL of BMDC medium consisting of RPMI 1640 medium supplemented with 10% HI-FBS, 1% penicillin-streptomycin, 50 \u0026micro;M 2-mercaptoethanol, 20 ng/mL IL-4 (PeproTech, Cat. #214\u0026thinsp;\u0026minus;\u0026thinsp;14) and 40 ng/mL GM-CSF (PeproTech, Cat. #315-03). On day 2, nonadherent and loosely adherent cells were collected and transferred to a new culture dish, after which 4 mL of fresh BMDC medium was added. On day 4, half of the medium was gently removed, and an equal volume of fresh medium was added. On day 6, BMDCs were identified by flow cytometry and used for subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro cellular uptake assay\u003c/h2\u003e \u003cp\u003eFirst, suitable cells cover glasses (NEST, Cat. #801010) were placed in a 24-well plate and 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e immature BMDCs obtained as described above were added and cultured for 24 h. BMDCs were then incubated with 2 nmol Cy5-labeled OVA\u003csub\u003eT\u003c/sub\u003e, FNs or OVA\u003csub\u003eT\u003c/sub\u003e-FNs for 4 h. After the unphagocytosed antigens were washed away with PBS, the BMDCs were stained with CD11c-FITC antibody at 4\u0026deg;C for 30 min, followed by fixation with 200 \u0026micro;L of 1% paraformaldehyde (Solarbio, Cat. #P1111) at 4\u0026deg;C for 20 min. The cover glasses were harvested and stained with DAPI for 5 min, after which the localization of Cy5-labeled antigen in FITC-positive cells was detected by confocal laser scanning microscopy (CLSM; TCS SP5, Leica, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of endosomal escape ability\u003c/h2\u003e \u003cp\u003eA total of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e immature BMDCs were grown in 35 mm confocal dishes (NEST, Cat. #801001) for 24 h. Then, 2 nmol of FITC-labeled OVA\u003csub\u003eT\u003c/sub\u003e, FNs or OVA\u003csub\u003eT\u003c/sub\u003e-FNs were added and cocultured for 4 h. After being washed 5 times with PBS, the BMDCs were stained with LysoTracker (Invitrogen, Waltham, MA, USA, Cat. #L7528) for 1 h, followed by Hoechst (US Everbright, Cat. #H4078) for 30 min. The colocalization of FITC and LysoTracker was detected by confocal laser scanning microscopy (CLSM; TCS SP5, Leica, Germany) to analyze the endosomal escape ability of FNs and OVA\u003csub\u003eT\u003c/sub\u003e-FNs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eActivation and maturation of BMDCs and cross-presentation of OVA\u003csub\u003eT\u003c/sub\u003e peptide\u003c/h2\u003e \u003cp\u003eCells and culture supernatants were harvested after 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e BMDCs were cocultured with 2 nmol of OVA\u003csub\u003eT\u003c/sub\u003e, FNs or OVA\u003csub\u003eT\u003c/sub\u003e-FNs for 24 h. BMDCs were then stained with CD80, CD86, MHC-I, MHC-II, CD40, and SIINFEKL-H2Kb antibodies for 30 min at 4\u0026deg;C, and the frequencies of activation markers were analyzed with a BD FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA, USA). The concentrations of IL-12 (P70), IFN-α1, IL-6, IFN-γ and TNF-α in the culture supernatant were determined by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer\u0026rsquo;s protocols.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo imaging and lymph node targeting\u003c/h2\u003e \u003cp\u003eBALB/c or C57BL/6 mice were immunized with PBS, 6 nmol Cy5-labeled OVA\u003csub\u003eT\u003c/sub\u003e or OVA\u003csub\u003eT\u003c/sub\u003e-FNs in the groin. The fluorescence intensities at the injection site, isolated lymph nodes and various organs were analyzed 24, 48, and 72 h after immunization using an IVIS Lumina imaging system (IVIS Lumina II, Xenogen, USA). Moreover, the sizes of the isolated lymph nodes in each group were recorded 24 h after immunization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAntigen uptake and activation of APCs in lymph nodes\u003c/h2\u003e \u003cp\u003eC57BL/6 mice were immunized in the groin with Cy5-labeled OVA\u003csub\u003eT\u003c/sub\u003e, FNs or OVA\u003csub\u003eT\u003c/sub\u003e-FNs at a dose of 6 nmol per side. Twenty-four hours after immunization, the lymph nodes were harvested and prepared into single-cell suspensions, and the frequencies of Cy5 in APCs, such as DCs (CD11c\u003csup\u003e+\u003c/sup\u003e), macrophages (CD11b\u003csup\u003e+\u003c/sup\u003e F4/80\u003csup\u003e+\u003c/sup\u003e), and B cells (B220\u003csup\u003e+\u003c/sup\u003e), as well as the frequency of activated DCs (CD80\u003csup\u003e+\u003c/sup\u003e CD86\u003csup\u003e+\u003c/sup\u003e CD11c\u003csup\u003e+\u003c/sup\u003e), were analyzed by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo proliferation assay\u003c/h2\u003e \u003cp\u003eSplenocytes from OT-1 mice were labeled with 5 \u0026micro;M CFSE (Invitrogen, Cat. #65\u0026ndash;0850) for 1 h at 37\u0026deg;C and then transferred intravenously to C57BL/6 mice, which were then immunized subcutaneously in the groin with 6 nmol of OVA\u003csub\u003eT\u003c/sub\u003e and OVA\u003csub\u003eT\u003c/sub\u003e-FNs in the presence or absence of 50 \u0026micro;g of Poly(I:C) (InvivoGen, Cat. #tlrl-pic-5) 24 h after transfer. Three days after immunization, the attenuation of CFSE fluorescence intensity in CD8\u003csup\u003e+\u003c/sup\u003e tetramer\u003csup\u003e+\u003c/sup\u003e cells in the spleen were analyzed by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo target cell lysis assay\u003c/h2\u003e \u003cp\u003eSplenocytes from C57BL/6 mice were resuspended in RPMI-160 medium supplemented with 10% FBS, 1% penicillin-streptomycin, and 50 \u0026micro;M 2-mercaptoethanol, and the cell concentration was adjusted to 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e cells/mL[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The above cells were divided into two aliquots, one of which was labeled with OVA\u003csub\u003eT\u003c/sub\u003e peptide at a final concentration of 25 \u0026micro;M for 2 h at 37\u0026deg;C in the dark, while the other was unlabeled. After washing once with PBS to remove unbound OVA\u003csub\u003eT\u003c/sub\u003e peptide, the cells were resuspended in PBS to a final concentration of 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e cells/mL. Splenocytes loaded with OVA\u003csub\u003eT\u003c/sub\u003e peptide and unloaded with OVA\u003csub\u003eT\u003c/sub\u003e peptide were labeled with 5 \u0026micro;M and 0.5 \u0026micro;M CFSE, respectively, for 1 h at 37\u0026deg;C. The cells were washed twice with PBS to remove unbound CFSE dye and subsequently mixed at a ratio of 1:1 after adjusting the cell concentration to 5\u0026times;10\u003csup\u003e7\u003c/sup\u003e with PBS. Additional C57BL/6 mice without any treatment were immunized with PBS, OVA\u003csub\u003eT\u003c/sub\u003e, OVA\u003csub\u003eT\u003c/sub\u003e vaccine, OVA\u003csub\u003eT\u003c/sub\u003e-FNs, or OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine. A total of 7\u0026times;10\u003csup\u003e6\u003c/sup\u003e mixed cells were injected intravenously into mice immunized for 8 days at a dose of 200 \u0026micro;L per mouse. Eighteen hours after injection, single-cell suspensions of the spleens were prepared, and the frequencies of splenocytes labeled with high and low concentrations of CFSE, were analyzed by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eImmunizations\u003c/h2\u003e \u003cp\u003eA total of 6 nmol of OVA\u003csub\u003eT\u003c/sub\u003e, M30 peptides, or OVA\u003csub\u003eT\u003c/sub\u003e-FNs, M30-FNs nanoparticles alone, or prepared by mixing with 50 \u0026micro;g of Poly(I:C) were formulated in 100 \u0026micro;L of PBS and immunized subcutaneously via the groin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunospot (ELISpot) assay\u003c/h2\u003e \u003cp\u003eELISpot plates (EMD Millipore, MSIPS4510) were pretreated with 50 \u0026micro;L of 35% ethanol for 30 s and washed 5 times with sterile water before being coated with 100 \u0026micro;L of 15 \u0026micro;g/mL anti-IFN-γ (MABTECH, Cat. #3321-2A) overnight at 4\u0026deg;C. After coating, the antibody was discarded, and the plate was blocked for 1 h with complete medium (RPMI-1640 supplemented with 10% FBS and 1% penicillin-streptomycin). A total of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e splenocytes were added to each well and restimulated for 48 h at 37\u0026deg;C with complete medium containing OVA\u003csub\u003eT\u003c/sub\u003e or M30 peptide at a final concentration of 10 \u0026micro;g/mL. The cells were washed away, and the plates were incubated with 100 \u0026micro;L of 1 \u0026micro;g/mL biotinylated detection antibody (MABTECH, Cat. #3321-2A) for 2 h at room temperature. The plate was washed 5 times with PBS and incubated with streptavidin-ALP (MABTECH, Cat. #3321-2A) diluted at a ratio of 1:1000 for 1 h at room temperature. After washing with PBS 5 times, 100 \u0026micro;L of the substrate NBT\u0026amp;BCIP (Sangon Biotech, Cat. #C510032) prepared with 1\u0026times; color development buffer was added to each well until obvious spots appeared, after which the reaction was stopped with 200 \u0026micro;L of ddH\u003csub\u003e2\u003c/sub\u003eO. After dying, the spots were counted with an ELISpot reader (AID iSpot, AID-Autoimmune Diagnostika GmbH, Strassberg, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eExperiments to detect immune responses induced by vaccines\u003c/h2\u003e \u003cp\u003eC57BL/6 mice were immunized on days 0 and 14, and peripheral blood and spleen were collected on day 21 to analyze vaccine-induced immune responses. The frequencies of tetramer\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cells in the peripheral blood and spleen were measured by flow cytometry to assess the ability of OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine to induce antigen-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells. The phenotypes of CD8\u003csup\u003e+\u003c/sup\u003e T cells in peripheral blood were evaluated by flow cytometry analysis of the proportions of PD-1\u003csup\u003e+\u003c/sup\u003e TIM-3\u003csup\u003e\u0026minus;\u003c/sup\u003e cells, effector memory T (Tem) cells, and central memory T (Tcm) cells. Vaccine-induced CTL responses were measured by the frequency of IFN-γ, TNF-α, or Granzyme B in CD8\u003csup\u003e+\u003c/sup\u003e T cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eProphylactic, therapeutic and metastasis vaccine experiments\u003c/h2\u003e \u003cp\u003eFor prophylactic model experiments, C57BL/6 mice were immunized on days 0, 14 and 28. Fourteen days after the third immunization, 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e E.G7-OVA, 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e MC-38-OVA, or 3\u0026times;10\u003csup\u003e5\u003c/sup\u003e B16F10-OVA cells in 100 \u0026micro;L of PBS were subcutaneously implanted into the right flank of each mouse.\u003c/p\u003e \u003cp\u003eFor metastasis model experiments, C57BL/6 mice were immunized on days 0, 14 and 28, 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e B16F10 cells in 200 \u0026micro;l of PBS were injected through the tail vein on day 35, and the lungs of the mice were removed on day 57 to count the number of metastatic foci.\u003c/p\u003e \u003cp\u003eFor therapeutic model experiments, C57BL/6 mice were subcutaneously implanted in the right flank with 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e B16F10 cells in 100 \u0026micro;L of PBS on day 0 and then immunized three times on days 5, 8, and 12.\u003c/p\u003e \u003cp\u003eThe tumor volume was estimated by the following formula: tumor volume\u0026thinsp;=\u0026thinsp;length \u0026times; width\u003csup\u003e2\u003c/sup\u003e \u0026times; 0.5. Animals were euthanized when the tumor reached 1.5 cm in diameter or surpassed 1500 mm\u003csup\u003e3\u003c/sup\u003e in volume or when significant weight loss was observed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eThe culture medium supernatants of BMDCs after 24 h of coculture and of splenocytes after 48 h of restimulation with OVA\u003csub\u003eT\u003c/sub\u003e peptide were collected, and the concentrations of cytokines were detected by ELISA kits (BioLegend, San Diego, CA, USA). The concentrations of IL-12 (p70) (Cat. #433604), IFN-α1 (Cat. #447904), TNF-α (Cat. #430904), IFN-γ (Cat. #430804), and IL-6 (Cat. #431304) in the medium supernatant of BMDCs and of IFN-γ (Cat. #430804) in the medium supernatant of splenocytes were detected according to the manufacturer\u0026rsquo;s protocols.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry analysis\u003c/h2\u003e \u003cp\u003eFor the APCs uptake analysis, cells were preincubated with 0.25 \u0026micro;g of TruStain FcX\u0026trade; PLUS anti-mouse CD16/32 blocking antibody (BioLegend, clone. S17011E) per 10\u003csup\u003e6\u003c/sup\u003e cells in a volume of 100 \u0026micro;l for 10 min on ice to reduce nonspecific binding. After FcR blocking, the cells were then stained for 30 min at 4\u0026deg;C with surface antibodies diluted at 1:300 in 100 \u0026micro;L of FACS buffer (fluorochrome-conjugated antibodies purchased from BioLegend unless otherwise indicated): CD45 (Cat. #103133. clone. 30-F11), CD11b (Cat. #101292. clone. M1/70), CD11c (Cat. #117306 and 117307. clone. N418), CD11c (BD Biosciences, Cat. #612797, clone. HL3), CD80 (Cat. #104705. clone. 16-10A1), CD86 (Cat. #105025. clone. GL-1), CD40 (Cat. #157506. clone. FGK45), MHC-I (Cat. #114612. clone. 28-8-6), MHC-II (Cat. #107625. clone. M5/114.15.2), B220 (Cat. #103205. clone. RA3-6B2), F4/80 (Cat. #123110. clone. BM8), and SIINFEKL-H2Kb (Cat. #141605. clone. 25-D1.16). After staining, the cells were washed once with FACS buffer and resuspended in 300 \u0026micro;L of PBS for flow cytometry detection.\u003c/p\u003e \u003cp\u003eFor T-cell tetramer analysis, single cells derived from the peripheral blood or spleen were first incubated with 100 \u0026micro;L of PBS containing 50 nM dasatinib (MACKLIN, Cat. #D828602) for 30 min at room temperature. The samples were washed once and treated with an anti-CD16/32 antibody for 10 min on ice. The cells were then stained with tetramer antibody (MBL, Cat. #TS-5001-1C) diluted 1:20 in 50 \u0026micro;L of FACS buffer containing 50 nM dasatinib for 45 min at 4\u0026deg;C under light protection. After washing once with FACS buffer, the cells were stained with surface antibodies diluted at 1:300 in 100 \u0026micro;L of FACS buffer (fluorochrome-conjugated antibodies purchased from BioLegend unless otherwise indicated) for 30 min at 4\u0026deg;C: CD8 (GeneTex, Cat. #GTX76348. clone. KT15), PD-1 (Cat. #135209. clone. 29F.1A12), TIM-3 (Cat. #119718. clone. RMT3-23), CD44 (Cat. #103043. clone. IM7), and CD62L (BD Biosciences, Cat. #564109, clone. MEL-14).\u003c/p\u003e \u003cp\u003eFor intracellular cytokine analysis, 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e splenocytes were first stimulated with 10 \u0026micro;g/mL OVA\u003csub\u003eT\u003c/sub\u003e peptide and Golgi plug (BD Biosciences, Cat. #555029) at 37\u0026deg;C for 6 h. The cells were then treated with an anti-CD16/32 blocking antibody at 4\u0026deg;C for 10 min and subsequently stained with CD3 (BD Biosciences, Cat. #564379, clone. 145-2C11), CD4 (Cat. #100491. clone. GK1.5) and CD8 (Cat. #100706. clone. 53\u0026thinsp;\u0026minus;\u0026thinsp;6.7) diluted at 1:300 for 30 min at 4\u0026deg;C. The cells were washed once with FACS buffer and fixed and permeabilized at 4\u0026deg;C for 1 h using the FoxP3/Transcription Factor Staining Buffer Set (Invitrogen, Cat. #00-5523-00). The cells were washed once with 1\u0026times; wash buffer and then stained with the following intracellular antibodies: IFN-γ (Cat. #505808. clone. XMG1.2), TNF-α (Cat. #506313. clone. MP6-XT22), and Granzyme B (Cat. #372216. clone. QA16A02) diluted at 1:300 at 4\u0026deg;C overnight. The cells were washed twice with wash buffer and prepared for flow cytometry detection.\u003c/p\u003e \u003cp\u003eThe cells were acquired on a BD FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA, USA) and a BD LSRFortessa X-20 (BD Biosciences) using BD FACSDiva Software v8.0.3 (BD Biosciences). All collected data were analyzed with FlowJo version V10.8.1.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eMTT assay\u003c/h2\u003e \u003cp\u003eDC2.4 cells (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e) were incubated with different doses of FNs and OVA\u003csub\u003eT\u003c/sub\u003e-FNs in 96-well plates for 24 h. After discarding the supernatant, 0.5 mg/mL MTT (Solarbio, Cat. #IM0280) was added, and the cells were incubated for 4 h. After incubation, 100 \u0026micro;L of DMSO was added to each well, and the absorbance was subsequently measured at 490 nm using a Cell Imaging Multi-Mode Reader (Cytation 5, BioTek, Winooski, VT, USA).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin and eosin (H\u0026amp;E) staining and determination of various enzymes in serum\u003c/h2\u003e \u003cp\u003eFourteen days after the third immunization, 200 \u0026micro;L of peripheral blood was collected and placed at 4\u0026deg;C overnight, followed by centrifugation at 1,000 rpm for 20 min to harvest the serum. The heart, liver, spleen, lung and kidney were removed and fixed in 15 mL of 4% paraformaldehyde (Solarbio, Cat. #P1110) for 4 days. Organ damage and the serum concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine (CRE) were determined by Tianjin JingNuo Pathological Diagnostic Co.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism 8.0.2 software. All results are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;s.e.m., except where otherwise specified. An unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test, one-way ANOVA with Tukey\u0026rsquo;s multiple-comparison test, two-way ANOVA with Tukey\u0026rsquo;s multiple-comparison test or log rank test was used for comparisons between the groups. \u003cem\u003eP\u003c/em\u003e values less than 0.05 were considered to indicate statistical significance. No sample in any representative experiment was excluded from the analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e1. Engineering and characterization of OVA\u003csub\u003eT\u003c/sub\u003e-FNs tumor vaccines\u003c/h2\u003e \u003cp\u003eThe homogeneity, biocompatibility, and size of tumor vaccines are critical to their effectiveness in suppressing tumors[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. We expect to develop a more effective and safer peptide delivery platform for tumor neoantigens relying on \u003cem\u003eHelicobacter pylori\u003c/em\u003e-derived ferritin nanoparticles. SIINFEKL (OVA\u003csub\u003eT\u003c/sub\u003e) was fused to optimized ferritin proteins to prepare OVA\u003csub\u003eT\u003c/sub\u003e-FNs, in which the first four amino acids at the N-terminus were omitted because the fifth amino acid is better displayed on the particle surface, and an N19Q mutation was introduced to abolish a potential N-linked glycosylation site (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea)[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo test whether the OVA\u003csub\u003eT\u003c/sub\u003e-FNs protein subunits could be synthesized in an \u003cem\u003eE. coli\u003c/em\u003e expression system and subsequently self-assembled into nanoparticles, we investigated the purity, structural integrity, and size of the obtained OVA\u003csub\u003eT\u003c/sub\u003e-FNs. SDS-PAGE of purified FNs and OVA\u003csub\u003eT\u003c/sub\u003e-FNs revealed a single protein band corresponding to the protein subunit (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The peak shape of OVA\u003csub\u003eT\u003c/sub\u003e-FNs analyzed by molecular exclusion chromatography was similar to that of FNs, suggesting that the fusion of OVA\u003csub\u003eT\u003c/sub\u003e peptide at the N-terminus of FNs did not affect the self-assembly of the optimized ferritin into nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). OVA\u003csub\u003eT\u003c/sub\u003e-FNs were homogeneous 12 nm spherical particles similar to the native structure, as revealed by transmission electron microscopy (TEM) and DLS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, e). Moreover, the particle size of OVA\u003csub\u003eT\u003c/sub\u003e-FNs was not affected after treatment at 4\u0026deg;C for 7 days or 65\u0026deg;C for 10 min, suggesting that OVA\u003csub\u003eT\u003c/sub\u003e-FNs are able to tolerate high temperatures and have good stability, which is important for the clinical translation of these vaccines (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea, b). In addition, the absolute zeta potentials of FNs and OVA\u003csub\u003eT\u003c/sub\u003e-FNs were approximately 10 mV and 8 mV, respectively, indicating that the individual particles were well dispersed and stable (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Together, these data suggested that OVA\u003csub\u003eT\u003c/sub\u003e-FNs prepared by fusion expression have superior purity, a uniform particle size, and good thermal stability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e2. Activation and cross-presentation of BMDCs after the uptake of OVA\u003csub\u003eT\u003c/sub\u003e-FNs\u003c/h2\u003e \u003cp\u003eTo demonstrate that optimized ferritin can be used as a tumor vaccine vector, we first investigated whether OVA\u003csub\u003eT\u003c/sub\u003e-FNs could be taken up by BMDCs in vitro and efficiently cross-present OVA\u003csub\u003eT\u003c/sub\u003e peptides on the cell surface. Fluorescence confocal microscopy images showed that the uptake of OVA\u003csub\u003eT\u003c/sub\u003e-FNs by CD11c\u003csup\u003e+\u003c/sup\u003e BMDCs was dramatically greater than that of free OVA\u003csub\u003eT\u003c/sub\u003e after coincubation with BMDCs for 4 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Moreover, the uptake of OVA\u003csub\u003eT\u003c/sub\u003e-FNs by BMDCs significantly increased with prolonged coculture time (Fig. S2). In addition, OVA\u003csub\u003eT\u003c/sub\u003e-FNs did not fully colocalize with lysosomes but were predominantly localized in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), suggesting that OVA\u003csub\u003eT\u003c/sub\u003e-FNs can escape from lysosomes into the cytoplasm and be degraded by the proteasome into peptide fragments. The maturation of BMDCs plays an essential role in triggering tumor-specific CTL responses[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The expression of the surface activation markers CD80, CD86, MHC-I, MHC-II, and CD40 was significantly upregulated in BMDCs after coculture with OVA\u003csub\u003eT\u003c/sub\u003e-FNs, indicating that OVA\u003csub\u003eT\u003c/sub\u003e-FNs induced the activation of BMDCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-g). The frequency of SIINFEKL-H2Kb\u003csup\u003e+\u003c/sup\u003e cells on the surface of BMDCs was analyzed by flow cytometry to verify whether BMDCs cross-present OVA\u003csub\u003eT\u003c/sub\u003e antigen via MHC-I molecules, and the results showed that 10% of BMDCs cocultured with OVA\u003csub\u003eT\u003c/sub\u003e-FNs were able to cross-present SIINFEKL to the cell surface through MHC-I (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh and Fig. S3a, b).\u003c/p\u003e \u003cp\u003eIn addition, cytokines secreted by mature BMDCs are also critical for downstream immune activation[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. IL-12 (p70) acts as a polarizing factor that promotes the differentiation of naive CD4\u003csup\u003e+\u003c/sup\u003e T cells to T helper 1 (Th1) cells and mediates an effective adaptive immune response[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The concentrations of cytokines in the culture supernatant of BMDCs cocultured with OVA\u003csub\u003eT\u003c/sub\u003e-FNs for 24 h were determined by ELISA, and the results showed that the concentrations of IL-12 (p70), IFN-α1, IL-6, IFN-γ and TNF-α were significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei, j and Fig. S4a-c). Together, these results confirmed that OVA\u003csub\u003eT\u003c/sub\u003e-FNs can be taken up by APCs, induce APCs activation and maturation, and enhance antigen presentation via MHC-I.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eIn vivo targeting and activation of APCs in lymph nodes by OVA\u003c/b\u003e \u003csub\u003e \u003cb\u003eT\u003c/b\u003e \u003c/sub\u003e \u003cb\u003e-FNs.\u003c/b\u003e \u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eThe effective delivery of tumor vaccines to lymph nodes and the maturation and cross-presentation of APCs in vivo are critical for triggering powerful antitumor responses[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Therefore, we investigated whether OVA\u003csub\u003eT\u003c/sub\u003e-FNs can migrate to lymph nodes and be taken up by APCs. In vivo fluorescence images revealed highly distinct Cy5 fluorescence in the groin of mice immunized with OVA\u003csub\u003eT\u003c/sub\u003e-FNs at 24, 48, and 72 h after immunization, suggesting that compared with free OVA\u003csub\u003eT\u003c/sub\u003e, OVA\u003csub\u003eT\u003c/sub\u003e-FNs could persist at the injection site (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b).\u003c/p\u003e \u003cp\u003eNext, we isolated the lymph nodes and various organs of immunized mice to test whether OVA\u003csub\u003eT\u003c/sub\u003e-FNs could selectively localize to the lymph nodes. Consistent with the results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b, remarkable localization of OVA\u003csub\u003eT\u003c/sub\u003e-FNs was detected in the lymph nodes at 24 h and even 72 h after immunization, suggesting that OVA\u003csub\u003eT\u003c/sub\u003e-FNs can effectively target the lymph nodes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d). Among the remaining organs, Cy5 fluorescence could be detected only in the liver and kidney at 24 h post injection, which might be related to the metabolic function of the liver and kidney as normal metabolic organs (Fig. S5a, b). In addition, the isolated lymph nodes were significantly swollen 24 h after immunization with OVA\u003csub\u003eT\u003c/sub\u003e-FNs compared to 24 h after immunization with OVA\u003csub\u003eT\u003c/sub\u003e (Fig. S5c). The above results suggest that OVA\u003csub\u003eT\u003c/sub\u003e-FNs can be retained in vivo for a long time and can rapidly migrate to lymph nodes. Prolonged retention of OVA\u003csub\u003eT\u003c/sub\u003e-FNs in lymph nodes would facilitate their uptake by APCs.\u003c/p\u003e \u003cp\u003eTo verify whether OVA\u003csub\u003eT\u003c/sub\u003e-FNs can be taken up by APCs in lymph nodes. C57BL/6 mice were injected subcutaneously with Cy5-labeled OVA\u003csub\u003eT\u003c/sub\u003e-FNs, and the lymph nodes were collected 24 h later. The frequencies of Cy5\u003csup\u003e+\u003c/sup\u003e in CD11c\u003csup\u003e+\u003c/sup\u003e DCs, macrophages, and B cells were significantly increased in OVA\u003csub\u003eT\u003c/sub\u003e-FNs-treated group, but these cells largely did not take up free OVA\u003csub\u003eT\u003c/sub\u003e peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-g). Moreover, immunization with OVA\u003csub\u003eT\u003c/sub\u003e-FNs resulted in a significant increase in the frequency of the activation marker CD80\u003csup\u003e+\u003c/sup\u003e CD86\u003csup\u003e+\u003c/sup\u003e in CD11c\u003csup\u003e+\u003c/sup\u003e DCs compared to that in cells immunized with OVA\u003csub\u003eT\u003c/sub\u003e free peptides (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). These results suggest that OVA\u003csub\u003eT\u003c/sub\u003e-FNs can migrate to lymph nodes, reside there for long periods of time. This may provide an opportunity for APCs to continuously take up OVA\u003csub\u003eT\u003c/sub\u003e-FNs and come into full contact with T cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e4. Triggering and expansion of antigen-specific T cells induced by OVA\u003csub\u003eT\u003c/sub\u003e-FNs\u003c/h2\u003e \u003cp\u003eThe ability to induce the production and activation of antigen-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells is an important criterion for evaluating the efficacy of tumor vaccines. First, we investigated whether OVA\u003csub\u003eT\u003c/sub\u003e-FNs could induce the proliferation of antigen-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells. In vitro, OT-1 CD8\u003csup\u003e+\u003c/sup\u003e T cells cocultured with OVA\u003csub\u003eT\u003c/sub\u003e-FNs-treated BMDCs showed more pronounced attenuation of CFSE fluorescence and a greater proliferative index, with 70% of OT-1 CD8\u003csup\u003e+\u003c/sup\u003e T cells dividing for 1\u0026ndash;3 generations. In contrast, no significant attenuation of CFSE fluorescence intensity was detected in OT-1 CD8\u003csup\u003e+\u003c/sup\u003e T cells cocultured with OVA\u003csub\u003eT\u003c/sub\u003e-loaded BMDCs (Fig. S6a-d). Since almost all peptide vaccines currently in clinical trials require adjuvants to enhance their induced immune responses[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], consistent with previous studies, we also included Poly(I:C) as an adjuvant in all of our in vivo experiments (OVA\u003csub\u003eT\u003c/sub\u003e + Poly(I:C), referred to as OVA\u003csub\u003eT\u003c/sub\u003e vaccine; OVA\u003csub\u003eT\u003c/sub\u003e-FNs\u0026thinsp;+\u0026thinsp;Poly(I:C), referred to as OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine). Similar to the in vitro results, compared with OVA\u003csub\u003eT\u003c/sub\u003e vaccine, OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine induced a more significant decrease in CFSE fluorescence intensity and an increase in the proliferation index in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-c). Approximately 80% of CD8\u003csup\u003e+\u003c/sup\u003e T cells divided for 7\u0026ndash;9 generations after immunization with OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine (Fig. S6e).\u003c/p\u003e \u003cp\u003eOVA-specific CD8\u003csup\u003e+\u003c/sup\u003e T-cell immune responses induced by OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine were investigated 7 days after the second immunization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). A 6-fold higher proportion of antigen-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells could be detected in the peripheral blood after immunization with OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine than after immunization with OVA\u003csub\u003eT\u003c/sub\u003e vaccine (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, f and Fig. S7). The proportion of tetramer\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cells in the spleen of mice immunized with OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine also doubled compared with that in the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). These results indicated that OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine prepared by fusing OVA\u003csub\u003eT\u003c/sub\u003e peptide with optimized ferritin to efficiently deliver the antigenic peptide significantly enhanced CD8\u003csup\u003e+\u003c/sup\u003e T-cell immune responses.\u003c/p\u003e \u003cp\u003eFurthermore, the quality of triggered CD8\u003csup\u003e+\u003c/sup\u003e T-cell response is also an important determinant of the therapeutic efficacy of tumor vaccines[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. To investigate the phenotype of OVA-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells induced by OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine, we evaluated the levels of cell surface markers indicating activation, exhaustion, and dysfunction. Four percent of CD8\u003csup\u003e+\u003c/sup\u003e T cells in the peripheral blood of mice immunized with OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine expressed PD-1 but not TIM-3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh), and a very limited number of cells expressed both PD-1 and TIM-3 (Fig. S8a, b)[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Based on the expression levels of CD44 and CD62L, half of the activated CD8\u003csup\u003e+\u003c/sup\u003e T cells in OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine-treated group were effector memory T (Tem) cells and central memory T (Tcm) cells, which could continue to differentiate into effector cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei and Fig. S8c). In conclusion, OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine induced a large number of antigen-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells in vivo that maintained favorable early differentiation characteristics.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEnhancement of CTL responses by OVA-FNs vaccine\u003c/h3\u003e\n\u003cp\u003eAntigen-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells generate strong CTL responses after a second exposure to the same antigen. To explore the level of CTL responses induced by OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine, we measured the levels of key effectors secreted by CD8\u003csup\u003e+\u003c/sup\u003e T cells after OVA\u003csub\u003eT\u003c/sub\u003e peptide restimulation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b, after restimulation for 48 h, approximately 180 IFN-γ-secreting CD8\u003csup\u003e+\u003c/sup\u003e T cells were detected among 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e splenocytes from mice immunized with OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine. Similarly, the concentration of IFN-γ secreted by splenocytes was much higher in mice immunized with OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). After restimulation with OVA\u003csub\u003eT\u003c/sub\u003e peptide and Golgi plug for 6 h, the frequency of IFN-γ- secreting CD8\u003csup\u003e+\u003c/sup\u003e T cells was significantly elevated in the mice immunized with OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine than in the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, e and Fig. S9). As mentioned above, comparable amounts of IFN-γ were detected in mice immunized with OVA\u003csub\u003eT\u003c/sub\u003e vaccine or OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine, as shown by ELISpot and ELISA. However, the flow cytometry results revealed that the frequency of IFN-γ secretion by CD8\u003csup\u003e+\u003c/sup\u003e T cells was significantly higher in the mice immunized with the OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine than in the other groups. The possible reason might be that prolonged restimulation of splenocytes in ELISpot and ELISA may lead to a sustained accumulation of secreted IFN-γ around the cells. The level of Granzyme B, another important effector molecule, was also dramatically increased in mice immunized with OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eTo directly verify the cytotoxic function of antigen-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells induced by OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine, we performed a target cell lysis assay based on labeling with different concentrations of CFSE. Eighty percent of splenocytes labeled with high concentrations of CFSE were lysed in the spleen and lymph nodes of mice immunized with OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine, while only 60% were lysed in mice immunized with OVA\u003csub\u003eT\u003c/sub\u003e vaccine (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh, i and Fig. S10a, b). Overall, the increased secretion of multiple effectors by antigen-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells suggested that OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine can induce robust CTL responses in vivo and can specifically kill target cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e6. Growth inhibition of multiple primary tumors induced by OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine\u003c/h2\u003e \u003cp\u003eTo further evaluate the antitumor immune responses induced by OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine in vivo, we employed a variety of prophylactic tumor models to investigate the inhibition of tumor growth. C57BL/6 mice were immunized every 14 days and challenged with E.G7-OVA lymphoma cells 2 weeks after the third immunization. As demonstrated by the average growth curves and the individual tumor growth curves, compared with immunization with other control vaccines, immunization with OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine significantly inhibited tumor growth. In addition, the tumors were isolated 16 days after implantation, and the tumor size was markedly smaller in the mice vaccinated with OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine than in the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-d).\u003c/p\u003e \u003cp\u003eNext, the immune protection triggered by OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine was explored in a highly immunogenic MC-38-OVA colon cancer model, and immunization with OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine significantly inhibited tumor growth and prolonged survival. In addition, on day 60 after tumor implantation, a slightly palpable tumor was observed in one mouse in the OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine group, while the other four tumors remained undetectable (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee-g and Fig. S11a). Moreover, in the poorly immunogenic B16F10-OVA melanoma model, tumor growth was significantly controlled in mice immunized with OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine, whereas there were no significant differences between mice vaccinated with other vaccines or PBS (Fig. S11b, c). In all three different prophylactic tumor models, there was no weight loss in any of the immunized mice, suggesting that OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine had no obvious side effects in vivo (Fig. S11d-f).\u003c/p\u003e \u003cp\u003eThe safety of tumor vaccines is an important factor that must be evaluated in preclinical studies, so we assessed the side effects of OVA\u003csub\u003eT\u003c/sub\u003e-FNs on cells or mice. In vitro, neither FNs nor OVA\u003csub\u003eT\u003c/sub\u003e-FNs affected the viability of DC2.4 cells even at a concentration of 1 mg /mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh). In vivo, H\u0026amp;E staining data showed that mice immunized three times with OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine did not exhibit damage to various tissues or organs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei). The expression of molecules associated with liver and kidney impairment was also not abnormally elevated in the serum (Fig. S12). The above results indicated that OVA\u003csub\u003eT\u003c/sub\u003e-FNs had satisfactory biosafety and did not cause hepatotoxicity, nephrotoxicity or pathological damage in vivo. Taken together, these results demonstrated that vaccination with OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine not only significantly inhibited the growth of a wide range of tumors but also had a favorable safety profile.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003e7. M30-FNs vaccine showed significant efficacy in both metastatic and primary tumor-bearing models\u003c/h2\u003e \u003cp\u003eWe demonstrated that delivery of the model antigen peptide OVA\u003csub\u003eT\u003c/sub\u003e using optimized ferritin as a vaccine carrier significantly inhibited tumor growth. Next, we explored whether the fusion of neoantigens with optimized ferritin could also induce robust immune responses and inhibit tumor growth. M30-FNs were prepared by fusing M30, a B16F10 melanoma neoantigen, to the N-terminus of optimized ferritin[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. SDS-PAGE image demonstrated that high-purity M30-FNs were obtained (Fig. S13). TEM image showed that M30-FNs self-assembled into homogeneous particles with a particle size of 12 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Splenocytes from mice immunized with M30-FNs vaccine 3 times were restimulated with the M30 peptide, and IFN-γ secretion was detected by ELISpot. More IFN-γ was secreted by 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e splenocytes from mice immunized with M30-FNs vaccine than those immunized with M30 vaccine (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, c).\u003c/p\u003e \u003cp\u003eThe highly metastatic property of melanoma is one of the major causes of death in melanoma patients. The 5-year survival rate of primary melanoma patients is 99%, but that of metastatic melanoma patients is only 27%[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. We therefore explored whether the neoantigen-FNs delivery system could function similarly in a metastatic tumor model. B16F10 cells were injected intravenously 7 days after the last immunization, and the lungs were removed 22 days after the injection to count the number of lung metastases (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). The numbers of metastatic foci were significantly reduced after immunization with M30-FNs vaccine compared to approximately 15 metastatic foci that could be detected in each of the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee, f). In addition, the area of lung tumor invasion was significantly reduced in mice immunized with M30-FNs vaccine (Fig. S14). These results showed that T-cell immune responses induced by a neoantigen-FNs vaccine corresponding to B16F10 melanoma significantly inhibited tumor metastasis to the lungs.\u003c/p\u003e \u003cp\u003eCurrently, personalized therapeutic tumor vaccines are being investigated extensively in the clinic[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. We next explored the ability of M30-FNs vaccine to induce regression of established tumors. C57BL/6 mice were subcutaneously implanted with B16F10 cells and then immunized 3 times on days 5, 8, and 12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg). M30-FNs vaccine significantly controlled tumor growth and had no effect on body weight compared to that in mice vaccinated with various other controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eh, i). On day 16, only 1 of the 5 mice showed visible tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ej). These data suggested that neoantigen-FNs vaccine induced robust immune responses, significantly inhibited tumor metastasis, and promoted regression of established tumors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough tumor neoantigen peptide vaccines have achieved remarkable results in the treatment of solid tumors, it is clear that low immunogenicity, poor lymph node targeting, and insufficient immune responses remain major obstacles[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In this study, we developed a tumor neoantigen peptide delivery system based on ferritin nanoparticles (neoantigen-FNs), which significantly enhanced the proportion and function of antigen-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells in vivo compared to those of free peptides. The neoantigen-FNs vaccine remarkably inhibited tumor growth and metastasis in a variety of prophylactic, B16F10 metastatic, and B16F10 therapeutic models and did not cause in vivo toxicity in mice with a favorable safety profile.\u003c/p\u003e \u003cp\u003eThe homogeneity, stability and biosafety of therapeutic tumor vaccines are crucial for their clinical translation[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In this study, OVA\u003csub\u003eT\u003c/sub\u003e-FNs self-assembled into well-homogenized spherical particles with particle sizes of approximately 12 nm, similar to the natural structure of ferritin nanoparticles. In addition, the structural properties of OVA\u003csub\u003eT\u003c/sub\u003e-FNs did not change when they were heated at 4\u0026deg;C for 7 days or even at 65\u0026deg;C for 10 min. A high thermal stability will be beneficial for maximizing the application of neoantigen-FNs. In addition, OVA\u003csub\u003eT\u003c/sub\u003e-FNs had no effect on the viability of DC2.4 cells in vitro. In vivo, OVA\u003csub\u003eT\u003c/sub\u003e-FNs also did not cause organ damage or serotoxicity, and there was no weight loss in the immunized mice, which suggested that the neoantigen-FNs peptide delivery system has good biosafety and has the potential to be applied in the clinic.\u003c/p\u003e \u003cp\u003eThe antitumor effects of tumor vaccines depend on the uptake of antigens by APCs and their presentation to CD8\u003csup\u003e+\u003c/sup\u003e T cells in lymph nodes. Therefore, effective delivery of tumor vaccines to lymph nodes to increase the probability of contact with APCs would facilitate the activation of antitumor immune responses[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. A well-established strategy to promote direct lymph node targeting is to design vaccines in particle form, and the size of the particles has a significant impact on selective lymph node targeting[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. OVA\u003csub\u003eT\u003c/sub\u003e-FNs are spherical particles with a particle size of 12 nm, which strongly facilitates their localization in lymph nodes. The results of ex vivo lymph node imaging showed that OVA\u003csub\u003eT\u003c/sub\u003e-FNs could directly migrate to and reside in lymph nodes, where they were then taken up by APCs, inducing the activation of APCs.\u003c/p\u003e \u003cp\u003eIn clinical trials, failure to elicit effective antigen-specific T-cell responses in patients is a major obstacle for tumor neoantigen peptide vaccines[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Fusion of OVA\u003csub\u003eT\u003c/sub\u003e peptides with FNs to enhance the immunogenicity and lymph node targeting of OVA\u003csub\u003eT\u003c/sub\u003e peptides significantly increased the frequency of OVA\u003csub\u003eT\u003c/sub\u003e tetramer\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cells in the peripheral blood and spleen. Moreover, not only the quantity but also the quality of antigen-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells improved after OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine treatment. We examined the expression levels of CD8\u003csup\u003e+\u003c/sup\u003e T-cell surface markers in the peripheral blood and showed that OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine induced functional PD-1\u003csup\u003e+\u003c/sup\u003e TIM-3\u003csup\u003e\u0026minus;\u003c/sup\u003e rather than exhausted CD8\u003csup\u003e+\u003c/sup\u003e T cells. Half of the activated cells retained their memory properties and could continue to differentiate into effector cells when re-exposed to the same antigens, which is also consistent with the more significant inhibition of tumor growth after OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine immunization. Various experiments have confirmed that these CD8\u003csup\u003e+\u003c/sup\u003e T cells can secrete large amounts of effectors, such as IFN-γ and Granzyme B, thereby significantly inducing lysis of target cells.\u003c/p\u003e \u003cp\u003eIn various prophylactic tumor models, OVA\u003csub\u003eT\u003c/sub\u003e-FNs vaccine prepared based on the model antigen significantly inhibited tumor growth, suggesting that the use of FNs as an antigen delivery vehicle can dramatically enhance the antitumor efficacy of peptide vaccines. Moreover, melanoma is a highly malignant and extremely metastatic tumor, and the antitumor immune responses induced by M30-FNs vaccine, prepared by fusion of the neoantigen, also significantly inhibited melanoma metastasis to the lungs. Similarly, for established melanoma, M30-FNs vaccine likewise induced regression. The above results suggested that the use of FNs as delivery vectors for mutation-derived neoantigens, which are less effective in the clinic, could also significantly enhance tumor suppression.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe have developed an excellent platform for tumor neoantigen delivery, FNs, which are not only homogeneous in terms of particle size and structural stability, but also highly biocompatible. Fusion with FNs significantly enhanced the immunogenicity of antigen peptides and induced the activation and maturation of APCs. Notably, neoantigen-FNs selectively targeted lymph nodes, activated a large number of early-differentiated antigen-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells in vivo, and exhibited a striking ability to kill target cells. Both activated model antigen- and neoantigen-specific immune responses significantly inhibit tumor growth. In conclusion, FNs can be used as ideal neoantigen delivery platforms to further improve the effectiveness of neoantigen peptide vaccines, which not only broadens the road of neoantigen peptide vaccine delivery vehicles but also has great potential for clinical application.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eNeoantigen-ferritin nanoparticles (neoantigen-FNs)\u003c/p\u003e\n\u003cp\u003eFood and Drug Administration (FDA)\u003c/p\u003e\n\u003cp\u003eCytotoxic T lymphocyte (CTL)\u003c/p\u003e\n\u003cp\u003eVirus-like particles (VLPs)\u003c/p\u003e\n\u003cp\u003eHuman ferritin heavy chain (hFTN)\u003c/p\u003e\n\u003cp\u003eOVA\u003csub\u003eT\u003c/sub\u003e-ferritin nanoparticles (OVA\u003csub\u003eT\u003c/sub\u003e-FNs)\u003c/p\u003e\n\u003cp\u003eAntigen presenting cells (APCs)\u003c/p\u003e\n\u003cp\u003eWild-type (WT)\u003c/p\u003e\n\u003cp\u003eAmerican Type Culture Collection (ATCC)\u003c/p\u003e\n\u003cp\u003eFetal bovine serum (FBS)\u003c/p\u003e\n\u003cp\u003eHeat-inactivated fetal bovine serum (HI-FBS)\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e)\u003c/p\u003e\n\u003cp\u003eIsopropyl \u003cem\u003e\u0026beta;\u003c/em\u003e-D-1-thiogalactopyranoside (IPTG)\u003c/p\u003e\n\u003cp\u003eMolecular weight cutoff (MWCO)\u003c/p\u003e\n\u003cp\u003eDynamic light scattering (DLS)\u003c/p\u003e\n\u003cp\u003eBone marrow-derived dendritic cells (BMDCs)\u003c/p\u003e\n\u003cp\u003eEnzyme-Linked Immunosorbent Assay (ELISA)\u003c/p\u003e\n\u003cp\u003eEnzyme-Linked Immunospot (ELISpot)\u003c/p\u003e\n\u003cp\u003eHematoxylin and eosin (H\u0026amp;E)\u003c/p\u003e\n\u003cp\u003eLymph node (LN)\u003c/p\u003e\n\u003cp\u003eT helper 1 (Th1)\u003c/p\u003e\n\u003cp\u003eEffector memory T (Tem)\u003c/p\u003e\n\u003cp\u003eCentral memory T (Tcm)\u003c/p\u003e\n\u003cp\u003eTransmission electron microscopy (TEM)\u003c/p\u003e\n\u003cp\u003eAspartate aminotransferase (AST)\u003c/p\u003e\n\u003cp\u003eAlanine aminotransferase (ALT)\u003c/p\u003e\n\u003cp\u003eBlood urea nitrogen (BUN)\u003c/p\u003e\n\u003cp\u003eCreatinine (CRE)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eAll of the animal experimental manipulations included in this study were approved by the policies and guidelines of the Animal Ethics Committee of Nankai University (2022-SYDWLL-000639).\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe data and materials used and analyzed during this research are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThis research was supported by the National Key R\u0026amp;D Program of China (No. 2022YFC2304202) and the National Natural Science Foundation of China (No.82073341).\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/h3\u003e\n\u003ch4\u003eAuthors and affiliations\u003c/h4\u003e\n\u003cp\u003eWZ, SL, ZS, KS, YD, LZ, QT, JH, and HZ:\u003c/p\u003e\n\u003cp\u003eState Key Laboratory of Medicinal Chemical\u0026nbsp;Biology, Tianjin Key Laboratory of Protein\u0026nbsp;Sciences, Cancer Biology Center, College of\u0026nbsp;Life Sciences, Nankai University, Tianjin 300071, PR China\u003c/p\u003e\n\u003cp\u003eFW:\u003c/p\u003e\n\u003cp\u003eSchool of Medicine, Nankai University, Tianjin 300071, PR China\u003c/p\u003e\n\u003cp\u003ePeople\u0026rsquo;s Hospital of Tianjin, Tianjin 300180, PR China\u003c/p\u003e\n\u003ch4\u003eContributions\u003c/h4\u003e\n\u003cp\u003eWZ, SL, KS, and FW conceived and designed the experiments; WZ, SL, ZS, HZ, KS, YD, LZ, QT, and JH performed all the experiments; WZ, SL, ZS, LZ, and HZ analyzed the data obtained; WZ, SL, and FW wrote the manuscript.\u003c/p\u003e\n\u003ch4\u003eCorresponding authors\u003c/h4\u003e\n\u003cp\u003eHZ, ZH\u003c/p\u003e\n\u003cp\u003eState Key Laboratory of Medicinal Chemical\u0026nbsp;Biology, Tianjin Key Laboratory of Protein\u0026nbsp;Sciences, Cancer Biology Center, College of\u0026nbsp;Life Sciences, Nankai University, Tianjin 300071, PR China\u003c/p\u003e\n\u003cp\u003eNankai International Advanced Research Institute (SHENZHEN FUTIAN), Shenzhen 518045, PR China\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe authors acknowledge support from the National Key R\u0026amp;D Program of China (No. 2022YFC2304202) and the National Natural Science Foundation of China (No.82073341). We sincerely appreciate the help and guidance of teachers Yajuan Wan, Rui Wang, Ruming Liu, Li Jiao, Di An and Ying Zhou from the Instrumentation Platform of Nankai University, as well as Xiaomin Su and Yanfang Chen from the Animal Experiment Center of Nankai University.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSaxena M, van der Burg SH, Melief CJM, Bhardwaj N. Therapeutic cancer vaccines. Nat Rev Cancer. 2021;21(6):360-78.\u003c/li\u003e\n\u003cli\u003eWeber JS, Carlino MS, Khattak A, Meniawy T, Ansstas G, Taylor MH, et al. Individualised neoantigen therapy mRNA-4157 (V940) plus pembrolizumab versus pembrolizumab monotherapy in resected melanoma (KEYNOTE-942): a randomised, phase 2b study. Lancet. 2024;403(10427):632-44.\u003c/li\u003e\n\u003cli\u003ePant S, Wainberg ZA, Weekes CD, Furqan M, Kasi PM, Devoe CE, et al. Lymph-node-targeted, mKRAS-specific amphiphile vaccine in pancreatic and colorectal cancer: the phase 1 AMPLIFY-201 trial. 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Nature. 2014;507(7493):519-22.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Tumor neoantigen, peptide-based vaccine, vaccine platform, lymph node-targeting, ferritin","lastPublishedDoi":"10.21203/rs.3.rs-4654130/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4654130/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTumor neoantigen peptide-based vaccines, systemic immunotherapies that enhance antitumor immunity by activating and expanding antigen-specific T cells, have achieved remarkable results in the treatment of a variety of solid tumors. However, how to effectively deliver neoantigens to induce robust antitumor immune responses remains a major obstacle.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHere, we developed a safe and effective neoantigen peptide delivery system (neoantigen-ferritin nanoparticles, neoantigen-FNs) that successfully achieved effective lymph node targeting and induced robust antitumor immune responses. Genetically engineered self-assembled particles with a size of 12 nm were obtained by fusing a neoantigen with optimized ferritin, which rapidly migrates to and continuously accumulates in lymph nodes. The neoantigen-FNs vaccine induced a greater quantity and quality of antigen-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells and resulted in significant growth control of multiple tumors, dramatic inhibition of melanoma metastasis and regression of established tumors. In addition, no obvious toxic side effects were detected in the various models, indicating the high safety of optimized ferritin as a vaccine carrier.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHomogeneous and safe neoantigen-FNs could be a very promising system for neoantigen peptide delivery because of their ability to efficiently migrate to lymph nodes and induce efficient antitumor immune responses.\u003c/p\u003e","manuscriptTitle":"Recombinant ferritin-based nanoparticles as neoantigen carriers significantly inhibit tumor growth and metastasis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-23 19:01:26","doi":"10.21203/rs.3.rs-4654130/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-24T16:24:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-15T03:00:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-12T06:25:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-05T23:19:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"155787421174112962627704445412605823804","date":"2024-07-05T00:40:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"130360519821339271520643467539602167512","date":"2024-07-02T15:34:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"323073078132778122197459331639582620482","date":"2024-07-02T13:38:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-02T13:14:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-01T12:01:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-01T11:59:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2024-06-28T10:36:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f819b22f-483b-4ce4-9026-021deffb0721","owner":[],"postedDate":"July 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-16T16:05:54+00:00","versionOfRecord":{"articleIdentity":"rs-4654130","link":"https://doi.org/10.1186/s12951-024-02837-2","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2024-09-14 15:58:19","publishedOnDateReadable":"September 14th, 2024"},"versionCreatedAt":"2024-07-23 19:01:26","video":"","vorDoi":"10.1186/s12951-024-02837-2","vorDoiUrl":"https://doi.org/10.1186/s12951-024-02837-2","workflowStages":[]},"version":"v1","identity":"rs-4654130","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4654130","identity":"rs-4654130","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}