The antitumor effects of adenoviral-mediated, intratumoral delivery of interleukin 23 require endogenous IL-12

Abstract

Interleukin (IL)-23 is a member of the IL-12 family of heterodimeric cytokines, comprised of p19 and p40 subunits, which exhibits immunostimulatory properties similar to IL-12. We have demonstrated previously that adenoviral-mediated, intratumoral delivery of IL-23 (Ad.IL-23) was able to induce systemic antitumor immunity. Here we demonstrate that Ad.IL-23 requires endogenous IL-12 for conferring an antitumor effect after adenoviral-mediated, intratumoral delivery. In contrast, Ad.IL-12 does not require IL-23 for its antitumor effects although endogenous IL-23 appears important for induction of systemic antitumor immunity by IL-12. However, despite the requirement for endogenous IL-12, co-delivery of IL-23 and IL-12 does not provide even an additive local or systemic antitumor effect, regardless of the dose. We further demonstrate that although the use of a single-chain IL-23 (scIL-23) results in higher level of expression and a more pronounced IL-23-mediated antitumor effect, there is still no synergy with IL-12. These results demonstrate that although significant antitumor effects are achieved by intratumoral injection of adenovirus expressing either scIL-23 or IL-12 alone and that IL-23 requires endogenous IL-12 for maximum antitumor benefit, the combined use of these cytokines provides no additive or synergistic effect.

Figure 1. Ad.IL-23 anti-tumor activity is dependent on endogenous IL-12, but Ad.IL-12 is not

To determine the roles of endogenous IL-12 and IL-23 in the anti-tumor activities of Ad.IL-12 and Ad.IL-23, MCA205-tumor bearing C57BL/6 mice deficient in IL-12 and IL-23 p40 (a and b) or IL-23 p19 (c and d) were treated on days 7, 9 and 11-post tumor inoculation with 5×10¹⁰ particles of Ad.IL-12, Ad.IL-23 or saline and survival and tumor volume monitored. Data is represented both as percent animals with tumors and as animal survival using Kaplan-Meier survival curves.

Figure 2. Ad.IL-12 and Ad.IL-23 co-treatment does not synergistically enhance anti-tumor effects

To determine if Ad.IL-12 and Ad.IL-23 co-treatment results in synergistic anti-tumor activity either locally or systemically, C57BL/6 mice bearing two MCA205 tumors were treated in one on day 7 with 5×10⁸ particles of Ad.IL-12 or 2.5×10¹⁰ particles of Ad.IL-23 as follows: Ad.IL-12 on day 7, followed by Ad.IL-23 on day 11; Ad.IL-23 on day 7 followed by Ad.IL-12 on day 11; Ad.IL-12 on days 7 and 11; Ad.IL-23 on days 7 and 11; or Ad.Psi5 on days 7 and 11 (a and b). Alternatively, mice were treated simultaneously with 1×10⁹ particles of Ad.IL-12 and 5×10¹⁰ particles of Ad.IL-23 or either virus alone (c and d). Data is represented both as percent animals with tumors and as animal survival using Kaplan-Meier survival curves.

Figure 3. Generation and characterization of adenovirus expressing single chain IL-23

Single-chain IL-23 (Ad.scIL-23) was designed to link the p40 and p19 subunits using a 15-amino acid (Gly₄Ser)₃ spacer (a). To ensure cytokine production, MCA205 cells were infected with an MOI 500 of Ad.scIL-23 and supernatants analyzed for IL-23 by ELISA 72 hours post-infection (b). To assess scIL-23 biological activity, Ad.IL-23-infected MCA205 supernatants were incubated with splenocytes for 48 hours and analyzed for IL-17 expression by ELISA (c). To assess the anti-tumor effects of Ad.scIL-23, C57BL/6 mice bearing MCA205 tumors were treated on days 7, 9 and 11 post-tumor inoculation with 5×10¹⁰ particles of Ad.scIL-23 and tumor volume and survival monitored. Data is represented both as tumor volume (d) and animal survival using Kaplan-Meier survival curves (e).

Figure 4. Ad.IL-12 and Ad.scIL-23 co-treatment does not synergistically enhance anti-tumor effects

To determine if co-delivery of Ad.IL-12 and Ad.scIL-23 results in synergistically enhanced anti-tumor activity, mice bearing two day 7 MCA205 tumors were treated once intratumorally with 1×10⁸ particles of Ad.IL-12 and 1×10⁹ particles of Ad.scIL-23, either virus alone or Ad.Psi5. Data is represented as percent animals with tumors for both injected (a) and contralateral tumors (b).

Figure 5. Ad.IL-12 and Ad.IL-23 co-treatment does not alter tumor CD8+ T-cell infiltration or angiogenesis

To determine if use of single chain IL-23 in combination with Ad.IL-12 altered patterns of CD8+ T-cell infiltration and angiogenesis compared to use of its double chain counterpart, day 15 injected tumors from mice co-treated with Ad.IL-12 and Ad.scIL-23, or either virus alone, were harvested and analyzed for CD8 (a) or CD31 (b) expression. Images were quantified using MetaMorph software. Statistically significant differences were determined using the T-test.