Cell-penetrating peptides enhance peptide vaccine accumulation and persistence in lymph nodes to drive immunogenicity

Abstract

Peptide-based cancer vaccines are widely investigated in the clinic but exhibit modest immunogenicity. One approach that has been explored to enhance peptide vaccine potency is covalent conjugation of antigens with cell-penetrating peptides (CPPs), linear cationic and amphiphilic peptide sequences designed to promote intracellular delivery of associated cargos. Antigen-CPPs have been reported to exhibit enhanced immunogenicity compared to free peptides, but their mechanisms of action in vivo are poorly understood. We tested eight previously described CPPs conjugated to antigens from multiple syngeneic murine tumor models and found that linkage to CPPs enhanced peptide vaccine potency in vivo by as much as 25-fold. Linkage of antigens to CPPs did not impact dendritic cell activation but did promote uptake of linked antigens by dendritic cells both in vitro and in vivo. However, T cell priming in vivo required Batf3-dependent dendritic cells, suggesting that antigens delivered by CPP peptides were predominantly presented via the process of cross-presentation and not through CPP-mediated cytosolic delivery of peptide to the classical MHC class I antigen processing pathway. Unexpectedly, we observed that many CPPs significantly enhanced antigen accumulation in draining lymph nodes. This effect was associated with the ability of CPPs to bind to lymph-trafficking lipoproteins and protection of CPP-antigens from proteolytic degradation in serum. These two effects resulted in prolonged presentation of CPP-peptides in draining lymph nodes, leading to robust T cell priming and expansion. Thus, CPPs can act through multiple unappreciated mechanisms to enhance T cell priming that can be exploited for cancer vaccines with enhanced potency.

Keywords: cancer immunotherapy; cell penetrating peptides; peptide vaccines.

Fig. 1.

Conjugating peptide antigens to CPPs increases antigen-specific T cell responses in vitro and in vivo. (A) Schematic structure of antigen-CPP conjugates prepared using azide/alkyne click chemistry and sequences of eight tested CPPs. (B and C) Splenocytes from C57BL/6 mice were pulsed for 1 h with the indicated gp100 peptide or CPP conjugate then cocultured with naïve CFSE-labeled pmel-1 CD8⁺ T cells. Pmel-1 T cell activation was assessed by flow cytometry analysis of CD69 up-regulation at 24 h (B) and proliferation (CFSE dilution) at 72 h (C). Significance relative to the gp100 long peptide was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test: ****P < 0.0001. (D–H) C57BL/6 mice (n = 4 to 5 animals per group) were immunized twice with 5 nmol of the indicated antigen and 25 µg cyclic-di-GMP, followed by restimulation of peripheral blood mononuclear cells (PBMCs) at day 21 with peptide to detect cytokine-producing antigen-specific T cells by flow cytometry. (D) Timeline of immunization experiments. (E) Representative flow cytometry plots (Left) and quantification (Right) of gp100-specific IFN-γ⁺/TNF-α⁺ CD8⁺ T cells. (F) Representative flow cytometry plots (Left) and quantification (Right) of Adpgk-specific IFN-γ⁺/TNF-α⁺ CD8⁺ T cells. (G) Linker placement for the antigen gp100 to pAntp and MPG. The original, a click linkage between the antigen C terminus and the CPP N terminus, is denoted “c-n”; “c-c” denotes a click linker between the antigen C terminus and the CPP C terminus; “–“ denotes a variant with a peptide bond linking the antigen and the CPP (i.e., synthesizing the antigen-CPP as a single long peptide). (H) Percentage of IFN-γ⁺ of CD8⁺ T cells after a prime and boost with the CPP conjugates shown in G. Significance relative to the gp100 long peptide (or Adpgk) was determined by two-way ANOVA followed by Dunnett’s multiple comparisons test: ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05.

Fig. 2.

Conjugation of peptide antigens to pAntp enhances vaccine potency without skewing T cell phenotypes, leading to robust expansion of therapeutically effective T cells. (A) Timeline of immunization experiments. (B) Percentage of IFN-γ⁺ of CD8⁺ T cells (or CD4⁺ cells for M30 and M48) after a prime and boost using the indicated long antigen peptide or antigen-pAntp, along with c-di-GMP adjuvant, as compared to the respective antigen. (C and D) C57BL C57BL/6 mice (n = 5 animals per group) were immunized with 5 nmol Adpgk or Adpgk-pAntp peptide combined with 25 µg cyclic-di-GMP on days 0 and 14, and then on day 21 PBMCs were restimulated with optimal Adpgk peptide ex vivo and stained for flow cytometry analysis. Shown are representative flow cytometry plots, percentages, and quantification of: (C) CD8⁺/IFN-γ⁺/IL-7R^hi/KLRG1^lo MPECs, CD8⁺/IFN-γ⁺/IL-7R^lo/KLRG1^hi SLECs, and CD8⁺/IFN-γ⁺/IL-7R^lo/KLRG1^lo early effectors (EE); (D) PD-1⁺/TCF-1⁺ expression. Statistical analyses for C and D were performed using a two-way ANOVA with Šídák’s multiple comparisons test. (E) Timeline for therapeutic efficacy study using the MC-38 tumor model. (F) Corresponding tumor growth curves with a two-way ANOVA statistical comparison followed by Tukey posttest of untreated control to Adgpk-pAntp. (G) overall survival curves, analyzed with the Mantel–Cox log-rank test. For all sections ****P < 0.0001; ***P < 0.001; **P < 0.01, *P < 0.05; n.s., not significant.

Fig. 3.

CPPs enhance peptide antigen uptake in vitro but are dependent on cross-presenting cells to promote T cell priming in vivo. (A) Representative histograms and median fluorescence intensities of cy5-gp100 and cy5-gp100-CPP in DC2.4 cells after 1-h incubation as determined by flow cytometry. ****P < 0.0001, **P < 0.01 vs. cy5-gp100 determined by one-way ANOVA using Bonferroni’s multiple comparisons test. (B) Confocal microscopy of DC2.4 cells stained with a membrane dye (CellBright Steady Membrane 550), Hoechst, and anti-FITC-AF647 to discern membrane-bound and internalized FITC-gp100-pAntp after 1-h incubation with the peptide. (C) Percentage of antigen-FITC internalized or associated with the surface of DC2.4 cells after 4 h of incubation with 2.5 µM peptide. Surface-localized antigen was detected using anti–FITC-cy5. ****P < 0.0001, ***P < 0.001 vs. nonfluorescent gp100 control determined by two-way ANOVA with Šídák’s multiple comparisons test. (D) C57BL/6 (wild-type, WT) or CD11c-DTR mice (n = 3 animals per group) were treated with 550 ng diphtheria toxin followed 24 h later by immunization with 5 nmol gp100-pAntp and c-di-GMP adjuvant. Six days after the priming dose, T cell responses were measured by ELISpot of splenocytes. (Upper) Representative images. (Lower) Quantification of IFN-γ⁺ spot-forming units (SFUs) *P < 0.05 by Student’s t test vs. CD11c-DTR. (E) Batf3^−/− mice (n = 5 animals per group) were immunized twice with 5 nmol gp100-pAntp and c-di-GMP adjuvant and the percentage of antigen-specific IFN-γ⁺/CD8⁺ T cells was analyzed as in Fig. 2A. n.s., not significant by one-way ANOVA followed by Bonferroni posttest vs. adjuvant-only control.

Fig. 4.

pAntp CPP conjugates exhibit enhanced LN accumulation and uptake by APCs in dLNs relative to free peptide antigen. Groups of C57BL/6 mice (n = 3 per group) were immunized subcutaneously with 25 nmol cy5-labeled gp100 or cy5-gp100-pAntp with 25 µg c-di-GMP, and then at selected times inguinal dLNs were isolated for histology or flow cytometry analysis. (A and B) Immunofluorescence of dLNs 48 h after immunization with cy5-gp100 (A) or cy5-gp100-pAntp (B). (Scale bars, 100 µm for main images and 50 µm for Insets.) (C–F) Representative dot plots from day 2 and quantification of percentage of peptide⁺ cells at 2- or 6-d postvaccination with cy5-gp100 and cy5-gp100-pAntp for (C) CD19⁺ cells; (D) F4/80⁺ macrophages; (E) cDC1 cells; and (F) cDC2 cells. **P < 0.01, *P < 0.05, by unpaired Student’s t test. (G) Representative flow cytometry plots showing up-regulation of MHCII and CD86 on CD11c⁺ DCs in response to the vaccination with the gp100 peptides with or without addition of c-di-GMP adjuvant. (H) Quantification (n = 3 animals per group) of percentage of MHCII^hi/CD86^hi DCs in each condition. n.s., not significant by two-way ANOVA with a Bonferroni posttest.

Fig. 5.

Antigen-CPP conjugates exhibit increased trafficking to dLNs and associate with serum proteins. (A) Whole-tissue fluorescence imaging of inguinal LNs 48 h after immunization with 25 nmol-labeled gp100 or gp100-CPPs with 25 µg of cyclic-di-GMP (n = 4 LNs per group, image shown at 1.5X)). (B) Quantification of total peptide fluorescence from each dLN, compared to control. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; n.s., not significant by one-way ANOVA with Dunnett’s posttest. (C) Schematic of antigen pull-down experiment. (D) Native-PAGE analysis of proteins pulled down by gp100 or gp100-CPPs following incubation with mouse serum. Several bands identified by LC-MS/MS are indicated.

Fig. 6.

CPPs protect linked antigens from degradation in serum and prolong the duration of antigen presentation in dLNs. (A) Timeline of serum exposure T cell activation experiments. (B) Representative concentration dependence plots for CD69 up-regulation by pmel-1 T cells cocultured with splenocytes pulsed with the indicated concentration of gp100 or gp100-pAntp peptide with or without preincubation with serum. (C) Log fold-change in EC₅₀ values for fresh vs. serum-treated peptides for each antigen construct. (D) Percentage of intact peptide remaining after incubation in 10% fresh mouse serum as analyzed by LC-QTOF-MS. (E) Timeline for experiments assessing the duration of antigen presentation following a single injection of gp100 or gp100-pAntp in C57BL/6 mice. AT, adoptive transfer. (F) Levels of available antigen as a function of time postimmunization in dLNs read out by pmel-1 T cell CD69 up-regulation (n = 5 animals per group). A two-way ANOVA with a Tukey posttest was used to compare gp100 and gp100-pAntp for each point, with: ****P < 0.0001, **P < 0.01, and *P < 0.05.