Tailoring the bioactivities of IL-12 for cancer immunotherapy with mRNA-based technology: from immunocytokines to armored T cell transfer

Resumen

IL-12 is a potent cytokine for cancer immunotherapy. However, its systemic delivery as a recombinant protein has shown unacceptable toxicity in early clinical trials, mostly related to the secondary increase in IFN- levels in circulation. In the last two decades, a wide plethora of methodologies has been employed to harness the desired anti-tumor features of IL-12 whilst limiting its side effects. Pro-cytokines and immunocytokines are some of the examples used to favor IL-12 bioactivity selectively in the TME. Also, the intra-tumoral delivery of IL-12-encoding mRNA or DNA is being evaluated in clinical trials. The first chapter of the thesis provides evidence of a viable platform to generate mRNAs encoding for chimeric constructs designed to customize the desired properties of the therapeutic cytokines. Upon intra-tumor injection of naked mRNA in murine models, our results demonstrated that the in vivo translated and secreted molecules encompassing single-chain IL-12 fused to diabodies targeting CSF1R, PD-L1 or both tether this potent cytokine onto the surface of tumor-infiltrating myeloid cells, preventing its systemic leakage, when compared to non-chimerized IL-12. To our surprise, the IL-12-diabody targeting PD-L1 is sufficient to achieve high tethering capacity, refuting our prediction that the double targeting would be more effective than the single ones. Furthermore, the anti-tumor efficacy of the IL-12-diabodies was comparable to that achieved by non-targeted IL-12, demonstrating that the fusion of the diabody to the cytokine does not hinder its potent bioactivity in vivo. The transient expression of IL-12 in the tumor niche, as well as the prevented leakage to the periphery mediated by the mRNA-encoded immunocytokines, holds promise for expanding the methodologies aimed at making the most of IL-12-anti-tumor properties whilst limiting its associated side effects. In the second chapter of the thesis we evaluated the effectiveness of tumor-specific CD8+ T cells, engineered with IL-12-encoding mRNA, against murine models of peritoneal metastasis. In order to recapitulate the human disease where the omentum represents a preferential location of metastasis, we employed intraperitoneal injection of tumor cells that preferentially localized to the omentum. We compared the intravenous and locoregional routes of administration of engineered OT-I T cell therapy in terms of anti-tumoral efficacy and reprogramming of the tumor microenvironment. Our findings clearly demonstrated the superiority of intracavitary immunotherapy: local delivery rapidly localized the armored T cell therapy to the omentum, where early encounter with tumor cell antigens and localized exposure of the tumor niche to secreted IL-12 resulted in a significantly higher anti-tumor response and in a more favorable transcriptional reprogramming of the omentum. Transient IL-12 expression was responsible for higher activation status of the immune cells in the TME, longer persistence of transferred T lymphocytes in vivo, and the development of immunity to endogenous antigens following primary tumor eradication. The anti-tumor efficacy of T cells with lower affinity TCRs was also improved by IL-12-mRNA-engineering, but to a lesser extent than T cells bearing higher affinity TCRs. Our data point to an alternative and safer route for the intracavitary delivery of adoptive T-cell therapies engineered to express IL-12 transiently.