Enhanced Vaccine Immunogenicity Enabled by Targeted Cytosolic Delivery of Tumor Antigens into Dendritic Cells

Abstract

Molecular vaccines comprising antigen peptides and inflammatory cues make up a class of therapeutics that promote immunity against cancer and pathogenic diseases but often exhibit limited efficacy. Here, we engineered an antigen peptide delivery system to enhance vaccine efficacy by targeting dendritic cells and mediating cytosolic delivery. The delivery system consists of the nontoxic anthrax protein, protective antigen (PA), and a single-chain variable fragment (scFv) that recognizes the XCR1 receptor on dendritic cells (DCs). Combining these proteins enabled selective delivery of the N-terminus of lethal factor (LF_N) into XCR1-positive cross-presenting DCs. Incorporating immunogenic epitope sequences into LF_N showed selective protein translocation in vitro and enhanced the priming of antigen-specific T cells in vivo. Administering DC-targeted constructs with tumor antigens (Trp1/gp100) into mice bearing aggressive B16-F10 melanomas improved mouse outcomes when compared to free antigen, including suppressed tumor growth up to 58% at 16 days post tumor induction (P < 0.0001) and increased survival (P = 0.03). These studies demonstrate that harnessing DC-targeting anthrax proteins for cytosolic antigen delivery significantly enhances the immunogenicity and antitumor efficacy of cancer vaccines.

Figure 1

Engineered anthrax proteins for targeted vaccine delivery into cross-presenting dendritic cells. (A) Designs of the engineered components: triple mutant protective antigen (mPAC), which enables side-chain bioconjugation and ablates binding to native anthrax receptors; single-chain variable fragment (scFv), which recognizes the XCR1 receptor; and a linker peptide (dotted line), which connects the scFv and mPAC. (B) Envisioned mechanism of translocation for scFv-mPAC (scFv-mPAC₈₃), which exhibits recognition of the XCR1 receptor, proteolytic cleavage (scFv-mPAC₆₃), oligomerization (scFv-mPAC)₇, and cytosolic delivery of the N-terminus of lethal factor (LF_N) with an appended antigen peptide (red).

Figure 2

Development of an anti-XCR1 scFv. (A, B) Design of the anti-XCR1 scFv, which encodes variable heavy (V_H) and light (V_L) chains from a parent anti-XCR1 IgG (MARX10). The scFv also contains a hydrophilic region (G₄S)₄ between the V_H and V_L chains and a sortase recognition tag (LPSTG₂H₆) that enables protein ligation. (C) Coomassie-visualized SDS-PAGE gel of the recombinant scFv throughout the bacterial expression and purification steps: (1) whole cell lysate; (2) Ni NTA purification; (3) treatment with SUMO protease; and (4) ion-exchange (HiTrap Q HP) chromatography. (D) Linker peptide 1, which included 1a (R = α-bromoacetyl group) and 1b (R = Alexa Fluor 647 (AF647)).

Figure 3

Engineering a DC-targeting anthrax protective antigen. (A) Schematics for the protein ligation of mPAC₈₃ and an anti-XCR1 scFv: thiol-conjugation reaction with peptide 1a, followed by sortase-mediated ligation with the scFv (scFv-LPSTG₂-H₆). (B, C) Coomassie-visualized SDS-PAGE gels of fractions obtained from size-exclusion (HiLoad 16/600 Superdex 200) and anion-exchange (HiTrap Q HP) chromatography. (D) Schematic of the sortase-mediated ligation reaction for scFv and peptide 1b.

Figure 4

ScFv-directed PA mediates selective protein translocation into XCR1-positive cells. Relative cell viability from the translocated A-chain of diphtheria toxin (DTA) into (A) XCR1⁺ and (B) XCR1^– CHO cells. Cells were incubated (72 h) with 10-fold serial dilutions of LF_N-DTA in the presence of 20 nM PA, scFv-mPAC, or scFv-mPAC[F427A]. Relative viability (% viable cells) was determined by measuring luminescence from a Cell Titer-Glo assay; the viability was normalized to untreated cells. Data represent the mean of three replicate wells ± the standard deviation (s.d.). Data are representative of two independent experiments.

Figure 5

Targeting XCR1 facilitates trafficking to lymph nodes and antigen-presenting cells. (A,C) Time-course analysis of AF647 signal in mouse organs: two mice per time point for mPAC and scFv; one mouse per time point for scFv-mPAC. Mice were treated with 1 nmol of an AF647-labeled construct: mPAC, anti-XCR1 scFv, or anti-XCR1 scFv-mPAC. The constructs were subcutaneously (sc) administered over two equal volume injections, one on each side of the tail base (n = 5 mice per group for mPAC and scFv; n = 3 mice for scFv-mPAC). (A) Representative images obtained after 2 h using an in vivo imaging system (IVIS), showing AF647 signal from resected lymph nodes (inguinal and axillary), spleen, and liver. Data represent the mean of whole-organ radiant efficiency ± s.d. (B) Quantification of the AF647 signal after 2, 24, and 48 h. (C) Flow cytometry analysis of the AF647 signal after 2 h in single-cell splenocyte populations, including CD8⁺ dendritic cells (CD11c⁺CD8⁺), medullary macrophages (CD11b⁺F4/80⁺), and T and B cells (CD3⁺B220⁺). Data represent the mean of AF647+ cells ± s.d. All data are representative of two independent experiments.

Figure 6

XCR1-targeted intracellular delivery outperforms nontargeted constructs. (A) Mice were s.c. vaccinated with LF_N-OVA (30 pmol, OVA_252–270) and c-di-GMP (25 μg), which were coadministered with mPAC, PA, or scFv-mPAC (6 pmol). (B) IFN-γ enzyme-linked immunosorbent spot (ELISpot) data are shown from 7 days after the boost (mean ± SEM; n = 10 mice per group). Statistical significance was calculated using a one-way ANOVA with the Fisher’s Least Significant Difference test. Data are compiled from two independent experiments.

Figure 7

Vaccine efficacy for cancer immunotherapy. (A) Immunization timeline. Mice (F, C57Bl/6, n = 10 per group) were s.c. inoculated with 3 × 10⁵ B16–F10 cells (day 0). On days 4, 10, 16, and 22, all mice were intraperitoneally (i.p.) injected with anti-PD1 antibody (200 μg) and were s.c. vaccinated (vacc) with c-di-GMP (25 μg) combined with either Trp1 + gp100 peptides (50 pmol each) or LF_N-Trp1-gp100 (50 pmol) + scFv-mPAC (10 pmol). (B) Mean tumor growth (mm²) per group. Data represent the mean tumor growth ± SEM. Statistical significance was calculated using two-way ANOVA with Tukey test (main effect only model) with multiple mean comparisons on the entire curves, * P < 0.05; ** P < 0.005; *** P < 0.001; and **** P < 0.0001. (C) Tumor growth plots for the individual mice over the entire experiment. (D) Body weight. Data represent the mean + SEM (E) Kaplan–Meier percent of survival curves. Statistical significance was calculated using the log-rank Mantel–Cox test. Median survival: 18 days in the naïve group, 20 days in the Trp1 + gp100 group, and 26 days in the LF_N-Trp1-gp100 + scFv-mPAC group.