A rationally designed combined treatment with an alphavirus-based cancer vaccine, sunitinib and low-dose tumor irradiation completely blocks tumor development

Abstract

The clinical efficacy of therapeutic cancer vaccines remains limited. For effective immunotherapeutic responses in cancer patients, multimodal approaches capable of both inducing antitumor immune responses and bypassing tumor-mediated immune escape seem essential. Here, we report on a combination therapy comprising sunitinib (40 mg/kg), single low-dose (14 Gy) tumor irradiation and immunization with a therapeutic cancer vaccine based on a Semliki Forest virus vector encoding the oncoproteins E6 and E7 of human papillomavirus (SFVeE6,7). We previously demonstrated that either low-dose irradiation or sunitinib in single combination with SFVeE6,7 immunizations enhanced the intratumoral ratio of antitumor effector cells to myeloid-derived suppressor cells (MDSCs). On the basis of these results we designed a triple treatment combinatorial regimen. The trimodal sunitinib, low-dose irradiation and SFVeE6,7 immunization therapy resulted in stronger intratumoral MDSC depletion than sunitinib alone. Concomitantly, the highest levels of intratumoral E7-specific CD8+ T cells were attained after triple treatment. Approximately 75% of these cells were positive for the early activation marker CD69. The combination of sunitinib, low-dose tumor irradiation and SFVeE6,7 immunization dramatically changed the intratumoral immune compartment. Whereas control tumors contained 0.02 E7-specific CD8+ T cells per MDSC, triple treatment tumors contained more than 200 E7-specific CD8+ T cells per MDSC, a 10,000-fold increased ratio. As a result, the triple treatment strongly enhanced the immunotherapeutic antitumor effect, blocking tumor development altogether and leading to 100% tumor-free survival of tumor-bearing mice. This study demonstrates that this multimodal approach elicits superior antitumor effects and should be considered for clinical applications.

Keywords: Semliki Forest virus; cancer vaccine; low-dose local tumor irradiation; sunitinib; suppressive factors.

Figures

Figure 1.

Sunitinib combined with local tumor irradiation and therapeutic vaccination decreases intratumoral MDSCs and elevates total CD8+ and E7-specific T-cell levels and activity. (A–D) Mice were inoculated s.c. with TC-1 tumor cells. Sunitinib treatment was started day 7 after tumor inoculation and mice were injected i.p. daily for a period of 9 consecutive days. Tumors of mice were irradiated locally (14 Gy) on day 14 after tumor inoculation. Mice were then immunized with 5 × 106 SFVeE6,7 particles i.m. on day 14. On day 21 tumors (n = 3−6/group) were harvested, cells dissociated, stained with fluorophore-conjugated antibodies and analyzed by the cytofluorimetry. (A) Analysis of myeloid-derived suppressor cells (MDSCs; CD11b+Gr1+). (B) Analysis of CD8+ T cell numbers and (C) activation (CD69+CD8+) and degranulation status (CD107ab+CD8+). (D) E7-specific CD8+ T cells and their activation status. A non-parametric Kruskal-Wallis test, followed by a Dunn's multiple comparisons test was used to determine statistical differences between groups. Depicted are the statistical differences between the double- and triple-treatment groups; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2.

Intratumoral levels and ratios of E7-specific CD8+ T cells and MDSCs upon combined triple treatment. On day 7 after tumor inoculation, sunitinib treatment was started on day 7 after tumor inoculation and mice were injected daily i.p. for a period of 9 consecutive days. Mouse tumors were locally irradiated with a 14 Gy irradiation dosage at day 14 post tumor inoculation. Mice were then immunized with 5 × 106 SFVeE6,7 particles by i.m. injection on day 14. On day 21 tumors were harvested, cells dissociated, stained with fluorophore-conjugated antibodies and analyzed by the cytofluorimetry. (A) Percentages of E7-antigen specific CD8+ T cells (dark gray) and myeloid-derived suppressor cells (MDSCs; light gray) within tumors of each treatment group (n = 3−6/group). Pie chart sizes reflect the total combined numbers of E7-antigen specific CD8+ T cells and MDSCs present within tumors of each group at the time of analysis. (B) Table showing the ratios between total CD8+ T cells or HPV E7-specific cytotoxic T lymphocytes (CTLs) and MDSCs within tumors of each treatment group.

Figure 3.

Combined sunitinib, local tumor irradiation and therapeutic vaccination blocks tumor development. Mice (n = 6/group) were s.c. inoculated with TC-1 tumor cells., sunitinib treatment was started on day 7 after tumor inoculation and was administered i.p. daily for a period of 9 consecutive days. Mouse tumors were locally irradiated with 14 Gy irradiation dosage at day 14 post tumor inoculation. Mice where then immunized with 106 SFVeE6,7 particles by i.m. injections on days 14, 21 and 28. Tumor measurements were performed periodically. When the tumor size exceeded 1000 mm3 or when a tumor protruded through the skin (#) mice were sacrificed for ethical reasons.

Figure 4.

Combination of sunitinib, local tumor irradiation and therapeutic immunization leads to 100% tumor-free survival. Mice (n=6/group) were s.c. inoculated with TC-1 tumor cells., Sunitinib treatment was started on day 7 after tumor inoculation and was administered, i.p. daily for a period of 9 consecutive days. Mouse tumors were locally irradiated with 14 Gy irradiation dosage at day 14 post tumor inoculation. Mice where then immunized with 106 SFVeE6,7 particles i.m. on days 14, 21 and 28. Shown are the percentages of survival of tumor-free mice at day 60 post tumor inoculation.