Self-Assembling Peptide Epitopes as Novel Platform for Anticancer Vaccination

Abstract

The aim of the present study was to improve the immunogenicity of peptide epitope vaccines using novel nanocarriers based on self-assembling materials. Several studies demonstrated that peptide antigens in nanoparticulate form induce stronger immune responses than their soluble forms. However, several issues such as poor loading and risk of inducing T cell anergy due to premature release of antigenic epitopes have challenged the clinical success of such systems. In the present study, we developed two vaccine delivery systems by appending a self-assembling peptide (Ac-AAVVLLLW-COOH) or a thermosensitive polymer poly(N-isopropylacrylamide (pNIPAm) to the N-terminus of different peptide antigens (OVA_250-264, HPV-E7_43-57) to generate self-assembling peptide epitopes (SAPEs). The obtained results showed that the SAPEs were able to form nanostructures with a diameter from 20 to 200 nm. The SAPEs adjuvanted with CpG induced and expanded antigen-specific CD8⁺ T cells in mice. Furthermore, tumor-bearing mice vaccinated with SAPEs harboring the HPV E7_43-57 peptide showed a delayed tumor growth and an increased survival compared to sham-treated mice. In conclusion, self-assembling peptide based systems increase the immunogenicity of peptide epitope vaccines and therefore warrants further development toward clinical use.

Keywords: human papillomavirus (HPV); immunotherapy; nanoparticles; ovalbumin (OVA); self-assembling peptide epitopes; therapeutic cancer vaccine.

Scheme 1. Synthesis of ATRP Peptide Macroinitiator

Scheme 2. Polymerization Reaction of (a) NIPAm and (b) DMAm Using the Peptide Macroinitiator

Figure 1

AFM images of (a) OVA-SA and (b) HPV-SA peptides self-assemblies immobilized on poly-l-lysine coated mica.

Figure 2

TEM pictures of nanoparticles composed of (a) HPV-pNIPAm and (b) OVA-pNIPAm SAPEs.

Figure 3

In vivo (cross)-presentation of OVA antigen to OVA-specific CD8⁺ (OT-I) T cells. The percentage of OT-I cells relative to the total number of CD8⁺ T cells in PMBCs of the mice vaccinated with 200 μL of indicated peptide formulations (equal to 40 nmol of OVA peptide/injection) on days 2, 5, 7, and 9 postimmunization. N = 5 mice per group, representative of two experiments. The percentage of OT-I cells on day 5 postimmunization in mice treated with OVA-SA + CpG and OVA-pNIPAm + CpG vaccines is compared to that of soluble OVA peptide + CpG. Statistical significance of the compared groups is calculated by student’s t test. N = 5 mice per group, and data are presented as mean ± SE.

Figure 4

Percentage of endogenous HPV 16 E7_49–57-specific CD8⁺ T cells in peripheral blood of mice vaccinated with indicated formulations (equal to 60 nmol of HPV peptide per injection) 10 days after prime vaccination and 6 or 7 days after booster vaccination. HPV-SA + CpG was tested in a different experiment thus presented separately because of different background (untreated mice) in FACS analysis. HPV-specific CD8+T cell expansion after booster vaccination in mice treated with HPV-SA + CpG and HPV-pNIPAm + CpG vaccines is compared to untreated mice and/or soluble HPV peptide + CpG. Statistical significance of the compared groups is calculated by student’s t test. N = 5 mice per group, and data are presented as mean ± SE.

Figure 5

Survival of mice per group presented in Kaplan–Meier plot. Seven days after TC-1 tumor inoculation in wild-type C57BL/6 mice, and when tumors were palpable, mice were either left untreated or were s.c. vaccinated in the opposite flank with the indicated formulations (equal to 60 nmol of HPV peptide per injection) followed by a boost injection 14 days after the prime dose. Statistical differences between the groups were calculated using a log-rank (Mantel–Cox) test. The survival of mice vaccinated with SAPE vaccines (HPV-SA and HPV-pNIPAm) with CpG was significantly longer compared to the no treatment group (p < 0.0005 for HPV-pNIPAm + CpG and p < 0.05 for HPV-SA + CpG). However, there was no significant difference in survival between mice vaccinated with either HPV peptide + CpG or HPV-pDMAm + CpG and untreated mice. Numbers in the legends represent median survival in days. *p < 0.05, ***p < 0.001. N = 12 mice per group, pooled results from two independent experiments.