Tumor immunotherapy using adenovirus vaccines in combination with intratumoral doses of CpG ODN

Abstract

The combination of viral vaccination with intratumoral (IT) administration of CpG ODNs is yet to be investigated as an immunotherapeutic treatment for solid tumors. Here, we show that such a treatment regime can benefit survival of tumor-challenged mice. C57BL/6 mice bearing ovalbumin (OVA)-expressing EG.7 thymoma tumors were therapeutically vaccinated with adenovirus type 5 encoding OVA (Ad5-OVA), and the tumors subsequently injected with the immunostimulatory TLR9 agonist, CpG-B ODN 1826 (CpG), 4, 7, 10, and 13 days later. This therapeutic combination resulted in enhanced mean survival times that were more than 3.5× longer than naïve mice, and greater than 40% of mice were cured and capable of resisting subsequent tumor challenge. This suggests that an adaptive immune response was generated. Both Ad5-OVA and Ad5-OVA + CpG IT treatments led to significantly increased levels of H-2 K(b)-OVA-specific CD8+ lymphocytes in the peripheral blood and intratumorally. Lymphocyte depletion studies performed in vivo implicated both NK cells and CD8+ lymphocytes as co-contributors to the therapeutic effect. Analysis of tumor infiltrating lymphocytes (TILs) on day 12 post-tumor challenge revealed that mice treated with Ad5-OVA + CpG IT possessed a significantly reduced percentage of regulatory T lymphocytes (Tregs) within the CD4+ lymphocyte population, compared with TILs isolated from mice treated with Ad5-OVA only. In addition, the proportion of CD8+ TILs that were OVA-specific was reproducibly higher in the mice treated with Ad5-OVA + CpG IT compared with other treatment groups. These findings highlight the therapeutic potential of combining intratumoral CpG and vaccination with virus encoding tumor antigen.

Fig. 1

Comparison of the therapeutic effect of Ad5-OVA vaccination combined with no, single, or multiple IT injections of CpG on tumor volumes. Individual tumor progression (volume) over time for indicated treatments. Tumor-challenged mice were vaccinated with either Ad5-LacZ or Ad5-OVA followed by IT injections of CpG (50 μg) on days 7 (×1); days 7, 10, and 13 (×3); days 7, 10, 13, and 16 (×4) post-tumor challenge, or no CpG treatment (i.e., Ad5-OVA alone)

Fig. 2

Comparison of the therapeutic effect of Ad5-OVA vaccination combined with no, single, or multiple IT injections of CpG. a Survival curve of one experiment (log-rank (Mantel–Cox) test was used to determine statistical significance (see Table S1A in supplementary data)). b Survival curve summarizing pooled data from multiple experiments (log-rank (Mantel–Cox) test was used to determine statistical significance (see Table S1B in supplementary data). c Percentage of mice tumor-free beyond 70 days from multiple experiments. Pooled experiments (b and c) were performed using indicated number of mice (n) and indicated number of experiments (e). Naïve (n = 24, e = 6); AdLacZ (n = 12, e = 3); AdLacZ/CpG(×1) (n = 12, e = 3); AdLacZ/CpG(×3) (n = 8, e = 2); AdLacZ/CpG(×4) (n = 8, e = 2); AdOVA alone (n = 16, e = 4); AdOVA/CpG(×1) (n = 12, e = 3); AdOVA/CpG(×3) (n = 12, e = 3); and AdOVA/CpG(×4) (n = 18, e = 5)

Fig. 3

Comparative therapeutic responses to Ad5-OVA plus varied doses of IT CpG. Tumor-challenged mice were subsequently vaccinated with Ad5-OVA on day 3 (except naïve group) and then ×4 IT injections of 10, 20, 50, or 100 μg of CpG on days 7, 10, 13, and 16 post-tumor challenge. a Survival curve representing one experiment (log-rank (Mantel–Cox) test determined no significant differences between treated groups). b Mean survival graph (Day 70 was censor time point). Error bars represent standard deviation

Fig. 4

Lymphocyte depletion study. Tumor-challenged mice were subsequently vaccinated with Ad5-OVA on day 3 followed by ×4 IT injections of CpG (50 μg) (days 7, 10, 13, and 16). On days 16, 17, and 18, mice were given IP 150 μg of depleting antibodies; anti-CD4, anti-CD8, or anti-NK1.1. Depletion was maintained twice a week until day 45. a Individual tumor progression (volume) over time for indicated treatments. b Survival curve representing one experiment (log-rank (Mantel–Cox) test was used to determine statistical significance (see Table S2 for supplementary data)

Fig. 5

OVA-specific CD8+ T lymphocyte responses and Treg levels in tumor microenvironment (TME) and peripheral blood. a Percentage of CD8+ TILs that were OVA-specific. b Pooled data comparing the relative proportion of CD8+ TILs that were OVA-specific (n = 8, e = 2). c Percentage of CD4+ TILs that were Foxp3+. d Pooled data comparing the relative proportion of CD4+ TILs that were FoxP3 + (n = 8, e = 2). e Percentage of CD8 + PBLs that were OVA-specific. f Pooled data comparing the relative proportion of CD8+ PBLs that were OVA-specific (n = 11, e = 3). g Percentage of CD4+ PBLs that were Foxp3+. h Pooled data comparing the relative proportion of CD4+ PBLs that were Foxp3+ (n = 8, e = 2). Statistical significances were determined using Student’s t test as described in materials and methods (*P < 0.05, **P < 0.01, ***P < 0.001). Error bars represent standard deviation