Recombinant ferritin-based nanoparticles as neoantigen carri…

Mice

Female wild-type (WT) C57BL/6 and BALB/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China), and female OT-1 transgenic mice were purchased from The Jackson Laboratory. 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, 3T3, and LO2 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The B16F10-OVA cells were a gift from Prof. Song Zhang’s lab. The 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) 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 the culture of E.G7-OVA cells, and 1.5 µg/mL puromycin (Solarbio, Cat. # P8230) was used for the culture of MC-38-OVA cells to exclude cells not overexpressing OVA. The 3T3 and LO2 cells were grown in DMEM (Gibco, CA, USA) supplemented with 10% FBS and 1% penicillin-streptomycin. All cells tested negative for mycoplasma contamination according to the ATCC Universal Mycoplasma Detection Kit (ATCC, Cat. #30–1012 K) and were cultured at 37 °C in a humidified incubator with 5% CO₂.

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 with the supernatant for 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 h 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 after removal of endotoxin according to the manufacturer’s protocols (Thermo, Cat. #88272), 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 transmission electron microscopy (TEM) (QUANTA 200, FEI company, USA) operating at 100.0 KV.

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 lyse 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 containing 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% heat-inactivated fetal bovine serum (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 × 10⁵ 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 vaccines 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, 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 × 10⁶ 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 OVAT peptide

The cells and culture supernatant were harvested after 1 × 10⁶ 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, FNs, 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).

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. Then, 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, FNs 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. The attenuation of CFSE fluorescence intensity of OVA_T-specific CD8⁺ T cells in the spleen was analyzed by flow cytometry at 24, 48, and 72 h after immunization. The proliferation index, representing the total number of divisions / cells that went into division, was calculated using the Proliferation Tool in FlowJo v10.8.1 software.

In vivo target cell lysis assay

Splenocytes from C57BL/6 mice were resuspended in RPMI-1640 medium supplemented with 1% penicillin-streptomycin, and 50 µM 2-mercaptoethanol, and the cell concentration was adjusted to 1 × 10⁷ cells/mL [39]. 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, while the other was unlabeled. 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 × 10⁷ with PBS. Additional C57BL/6 mice without any treatment were immunized with PBS, OVA_T, OVA_T+Poly(I: C), OVA_T-FNs, or OVA_T-FNs + Poly(I: C). A total of 7 × 10⁶ 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 and lymph nodes were prepared, and the frequencies of splenocytes labeled with high and low concentrations of CFSE were analyzed by flow cytometry. The specific lysis percentage was calculated according to the published formula [22]:

Immunizations

A total of 6 nmol of OVA_T, M30, FNs, OVA_T-FNs, or M30-FNs 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. #3321–2 A) 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 × 10⁵ 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. #3321–2 A) for 2 h at room temperature. The plate was washed 5 times with PBS and incubated with streptavidin-ALP (MABTECH, Cat. #3321–2 A) 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 ddH₂O. 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 the peripheral blood and spleen were collected on day 21 to analyze vaccine-induced immune responses. The frequencies of OVA_T-specific CD8⁺ T cells in the peripheral blood and spleen were measured by flow cytometry. 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 secretion of IFN-γ, TNF-α, or Granzyme B by CD8⁺ T cells.

Prophylactic, therapeutic and metastasis model experiments

For prophylactic model experiments, C57BL/6 mice were immunized on days 0, 14 and 28. Fourteen days after the third immunization, 5 × 10⁵ E.G7-OVA, 1 × 10⁶ MC-38-OVA, or 3 × 10⁵ 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 × 10⁵ 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 × 10⁵ 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 × width² × 0.5. Animals were euthanized when the tumor reached 1.5 cm in diameter or surpassed 1500 mm³ in volume or when significant weight loss was observed.

ELISA

The culture medium supernatant of BMDCs cocultured with vaccines and of splenocytes restimulated 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) 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 10⁶ 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-5001–1 C) 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. 29 F.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 × 10⁶ 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 in 50 µL PBS at 4 °C overnight. The cells were washed twice with PBS and prepared for flow cytometry detection.

The cells were acquired on a BD FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA, USA) or 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.

CCK-8 assay

The 3T3 cells (5 × 10³), LO2 cells (5 × 10³), and PBMCs (1 × 10⁴) were seeded into 96-well plates and cultured for 24 h. Then, the cells were treated with different doses of FNs and OVA_T-FNs for additional 24 h. After treatment, CCK-8 solution (NCM Biotech, Cat. #C6005) was added to each well and incubated for 2.5 h. The absorbance was then measured at 450 nm and 600 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 analyses

Statistical analyses were performed using GraphPad Prism 8.0.2 software. All results were presented as the mean ± s.e.m. 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.