The route of administration dictates the immunogenicity of peptide-based cancer vaccines in mice

Abstract

Vaccines consisting of synthetic peptides representing cytotoxic T-lymphocyte (CTL) epitopes have long been considered as a simple and cost-effective approach to treat cancer. However, the efficacy of these vaccines in the clinic in patients with measurable disease remains questionable. We believe that the poor performance of peptide vaccines is due to their inability to generate sufficiently large CTL responses that are required to have a positive impact against established tumors. Peptide vaccines to elicit CTLs in the clinic have routinely been administered in the same manner as vaccines designed to induce antibody responses: injected subcutaneously and in many instances using Freund's adjuvant. We report here that peptide vaccines and poly-ICLC adjuvant administered via the unconventional intravenous route of immunization generate substantially higher CTL responses as compared to conventional subcutaneous injections, resulting in more successful antitumor effects in mice. Furthermore, amphiphilic antigen constructs such as palmitoylated peptides were shown to be better immunogens than long peptide constructs, which now are in vogue in the clinic. The present findings if translated into the clinical setting could help dissipate the wide-spread skepticism of whether peptide vaccines will ever work to treat cancer.

Keywords: CD8 T cells; Melanoma; Peptide vaccines; Route of injection.

Fig. 1

Systemic vaccination is superior to subcutaneous immunization. a Congenic CD45.1 WT-B6 mice received 10,000 naïve TnTR1 CTLs followed by vaccination with pam-Trp1/TriVax administered i.v., i.m., or s.c. and numbers of TnTR1 cells (CD45.2+) in spleens were measured 7 days after the vaccination. b Similar experiment to (a) except mice was vaccinated with pam-Trp1/BiVax. c WT-B6 mice were vaccinated with pam-Trp1/TriVax administered i.v., i.m., or s.c. and numbers of endogenous Trp1 specific CTLs in spleens were measured 7 days after the vaccination. d WT-B6 mice (Thy1.2) received 10,000 naïve pmel-1 CTLs (Thy.1.1) followed by vaccination with pam-hgp100/TriVax administered i.v., i.m. or s.c. and numbers of pmel-1 cells (Thy1.1+) in spleens were measured 7 days after the vaccination. Results in (b) are presented as individual mice (each symbol) with the mean ± SD for each group. Numbers below each group represent the average CTL expansion (total numbers of CTL in spleen/number of CTLs adoptively transferred). These experiments were repeated 2–3 times with similar results

Fig. 2

Amphiphilic peptides are more immunogenic than long peptides. a Congenic CD45.1 WT-B6 mice received 10,000 naïve TnTR1 CTLs followed by vaccination with pam-Trp1/TriVax or LP-Trp1/TriVax and numbers of TnTR1 cells (CD45.2+) in spleens were measured 7 days after the vaccination. b WT mice received 10,000 naïve pmel-1 cells followed by vaccination with pam-hgp100/TriVax or LP-hgp100 TriVax and numbers of pmel-1 cells (Thy1.1+) in spleens were measured 7 days after the vaccination. c CD45.1 WT-B6 mice received 10,000 naïve TnTR1 CTLs followed by vaccination with LP-Trp1/TriVax administered i.v., i.m., or s.c. and numbers of TnTR1 cells (CD45.2+) in spleens were measured 7 days after the vaccination. d CD45.1 WT-B6 mice received 10,000 naïve TnTR1 CTLs followed pam-Trp1/BiVax or LP-Trp1/BiVax and numbers of TnTR1 cells (CD45.2+) in spleens were measured 7 days after the vaccination. Results are presented as individual mice (each symbol) with the mean ± SD for each group. Numbers below each group represent the average CTL expansion. These experiments were repeated 2–3 times with similar results

Fig. 3

Systemic vaccine induces APC-T-cell interactions at various sites. a OT-I mice were vaccinated with pam-Trp1/TriVax administered i.v., i.m., or s.c. and 24 h later, the cells from vaccinated mice were collected (inguinal, mesenteric, and axillary lymph nodes, and spleen) and cocultured with TnTR1 CTLs. The reactivity to the Trp1 epitope was examined using an IFN-γ ELISPOT assay. b CD45.1 WT-B6 mice received 10,000 naïve TnTR1 CTLs followed by pam-Trp1 peptide and poly-ICLC administered i.v. or i.m. as shown and numbers of TnTR1 cells (CD45.2+) in spleens were measured 7 days after the vaccination. c WT-B6 mice received 10,000 naïve pmel-1 cells followed by pam-gp100 peptide, poly-ICLC/antiCD40 administered i.v. or i.m. as shown and numbers of pmel-1 CTLs (Thy1.1+) in spleens were measured 7 days after the vaccination. Results are presented as individual mice (each symbol) with the mean ± SD for each group. Numbers below or above each group represent the average CTL expansion. These experiments were repeated 2–3 times with similar results

Fig. 4

Intravenous vaccination is able to recruit low numbers of antigen-specific CTL precursors. a CD45.1 WT-B6 mice received 200–2000 naïve TnTR1 CTLs followed by vaccination with pam-Trp1/TriVax administered i.v and percentages of TnTR1 cells (CD45.2) and endogenous responses (CD45.1) were assessed in the Trp1 tetramer + populations. b Number of Trp1 tetramer⁺ CTL precursors in naïve WT-B6 and Trp1-KO splenocytes. c, d OT-I mice received 20–20,000 TnTR1 naïve CTLs followed by vaccination with 2 doses of pam-Trp1/TriVax (administered 21 days apart) injected i.v. and percentages of TnTR1 cells in blood were measured 7 days after each vaccination. Results are presented as individual mice (each symbol) with the mean ± SD for each group. Numbers below the tetramer + gates represent the percentage positive CD8 T cells. These experiments were repeated 2–3 times with similar results

Fig. 5

Systemic immunization induces large diversity of polyclonal antigen-specific T cells. a Example showing the capacity of the high-resolution agarose gels to distinguish different sizes of oligonucleotides. b TCR-Vβ chain usage of CD8 spleen T cells from WT-B6 mice (note diffuse bands). c WT-B6 mice received pam-Trp1/TriVax and 7 days later Trp1 tetramer + spleen CTLs were sorted and TCR-Vβ chain usage was examined. d WT-B6 mice received pam-Trp1/TriVax and TCR-Vβ chain usage was examined 15 months later as in (c). e CDR3 and Jβ gene sequences from PCR products of gel bands excised from 2 mice immunized as described in (c). Results presented in (b–d) are from individual mice, representative of 4 mice in each treatment group

Fig. 6

Route of vaccine administration dictates the antitumor effects of peptide vaccines. CD45.1 WT-B6 mice were inoculated s.c. with 3 × 10⁵ B16F10 melanoma cells and 7 days later, when tumors were measurable the mice received 10,000 TnTR1 CTLs followed by 2 doses of pam-Trp1/TriVax administered i.v, i.m or s.c. a Mean tumor sizes in each group (n = 10 mice). b Percentages of TnTR1 CTLs in blood from (a) at day 25. c Similar experiment as in (a), but comparing the antitumor effects of pam-Trp1/TriVax and LP-Trp1/TriVax administered either i.v. or s.c. (n = 10 mice). Results in (b) are presented as individual mice (each symbol) with the mean ± SD for each group. These experiments were repeated 2–3 times with similar results