Anti-tumor immunotherapy despite immunity to adenovirus using a novel adenoviral vector Ad5 [E1-, E2b-]-CEA

Abstract

Adenovirus serotype 5 (Ad5) has been widely used in clinical trials because it expresses inserted transgenes robustly and augments the innate immune response. Strategies to improve Ad5 vectors that can circumvent Ad5 immunity have become a critical issue, especially for use as a cancer immunotherapeutic in which repeated immunization is required. In this study, we constructed a novel Ad5 vector with unique deletions of the viral DNA polymerase and the pre-terminal protein region (Ad5 [E1-, E2b-]). This vector contains the carcinoembryonic antigen (CEA) gene insert and is designed to induce cell-mediated immunity (CMI) against the tumor-associated target. The CEA immunogenicity and in vivo anti-tumor effects of repeated immunizations with Ad5 [E1-, E2b-]-CEA compared with those observed with current generation Ad5 [E1-]-CEA were tested in Ad5 pre-immunized mice. We report that Ad5-immune mice immunized multiple times with Ad5 [E1-, E2b-]-CEA induced CEA-specific CMI responses that were significantly increased over those detected in Ad5-immune mice immunized multiple times with a current generation Ad5 [E1-]-CEA. Ad5 immune mice bearing CEA-expressing tumors that were treated with Ad5 [E1-, E2b-]-CEA had increased anti-tumor response as compared with Ad5 [E1-]-CEA treated mice. These results demonstrate that Ad5 [E1-, E2b-]-CEA can induce CMI immune responses which result in tumor growth inhibition despite the presence of pre-existing Ad5 immunity. Multiple re-immunizations using the same vector platform are now possible with the novel Ad5 [E1-, E2b-] platform.

Fig. 1

CMI against Ad5 [E1-]-CEA and Ad5 [E1-, E2b]-CEA in Ad5 immune mice. C57Bl/6 mice (n = 5 per group) were pre-immunized two times at a 2-week interval with 10¹⁰ VP Ad5-null (containing no transgene) to induce Ad5 immunity. Separate groups were then immunized three times at 1-week intervals with 10¹⁰ VP Ad5 [E1-]-CEA or Ad5 [E1-, E2b-]-CEA. Controls received injection buffer alone. Splenocytes were collected 14 days after the final immunization and assessed for IFN-γ secreting cells (a) or IL-2 (b) by ELISpot assay. Splenocytes were also assessed for non-specific secreting cells of IFN-γ (a) or IL-2 (b) following stimulation with the non-immunizing antigens β-galactosidase (β-gal) and HIV-1 Gag. For positive controls, splenocytes were exposed to Concanavalin A (Con A) (data not shown). Data are reported as the number of spot forming cells (SPF) per 10⁶ splenocytes. The error bars depict the SEM

Fig. 2

Treatment of CEA-expressing tumors in Ad5-immune mice. C57Bl/6 mice (n = 7 per group) were immunized two times at a 2-week interval with Ad5 [E1-]-null (no transgene insert). Two weeks following the final Ad5 [E1-]-null administration mice were inoculated with 10⁶ MC38-cea2 cells subcutaneously in the left flank and administered injection buffer (tumor controls), 10¹⁰ VP of Ad5 [E1-]-CEA or 10¹⁰ VP of Ad5 [E1-, E2b-]-CEA in the right flank on days 0, 7, and 14. Tumors were measured by two opposing dimensions (a, b) and volumes were calculated according to the formula V = (a × b)²/2. Error bars represent the standard deviation of tumor volume. Asterisk indicates days that the Ad5 [E1-]-CEA treated mice had significantly smaller tumors than control mice. Hat symbol indicates days that the Ad5 [E1-, E2b-]-CEA treated mice had significantly smaller tumors than control and Ad5 [E1-]-CEA treated mice