Background Interleukin-12 (IL-12) is a potent immunotherapeutic cytokine in mouse models which application as a systemic agent in the clinical setting is hampered by IFNγ-dependent toxicity.¹ Engineering T cells with IL-12 is highly efficacious in mouse models² but has resulted in serious adverse events in clinical settings.³ On the other hand, IL-18 is a myeloid-derived cytokine that elicits IFN-γ expression on T and NK lymphocytes.⁴

IL-12 and IL-18 are known to synergize in terms of eliciting massive IFNγ production.⁵ For cancer immunotherapy, IL-18 has the caveat of being down-regulated in its function by a decoy receptor termed IL-18BP,⁶ which is reportedly abundant in tumor tissues.^7,8 Recently, a mutant sequence of mouse IL-18 termed DRIL18 which preserves its bioactivity but lacks binding to IL-18BP has been reported to exert T-cell-dependent antitumor activity upon systemic delivery.⁷

We previously reported that transient engineering of tumor-specific CD8 T cells with IL-12 mRNA enhanced their systemic therapeutic efficacy when delivered intratumorally.⁹ In this study, we sought to improve the therapeutic strategy of intratumoral delivery of T cells transiently engineered to express IL-12 with IL-18 mRNA electroporation.

Methods We mixed CD8⁺ T cells (TCR transgenic, TILs and CAR-T) engineered with mRNAs to transiently express either single-chain IL-12 (scIL-12) or an IL-18 decoy-resistant variant (DRIL18) that is not functionally hampered by IL-18BP. CD8⁺ T cells were injected repeatedly into mouse tumors for antitumor efficacy experiments. RNA-seq was performed to assess the functional changes induced after mRNA electroporation. Additionally, T-cell metabolic modifications and glycosylation profile functional changes were analyzed using seahorse and cell adhesion assays.

Results Pmel-1 TCR-transgenic T cells electroporated with scIL-12 or DRIL18 mRNAs exerted powerful therapeutic effects in local and distant melanoma lesions. These effects were associated with T-cell metabolic fitness, enhanced miR-155 control of immunosuppressive target genes, enhanced expression of various cytokines and unique changes in the glycosylation profile of surface proteins, enabling enhanced adhesiveness to E-selectin. Efficacy of this intratumoral immunotherapeutic strategy was recapitulated using other clinically relevant adoptive T cell therapies as tumor-infiltrating lymphocytes (TILs) and CAR T cells upon IL-12 and DRIL18 mRNA electroporation.

Conclusions We report on a substantial improvement of adoptive T-cell therapies strategy based on mRNA transient gene-transfer and repeated intratumoral delivery. The synergistic immunobiology of IL-12 and IL-18, best represented in the form of DRIL18, holds promise for efficacious outcomes in the treatment of metastatic cancer patients.

Acknowledgements This project has been supported by MINECO SAF2017-83267-C2-1-R and PID2020-112892RB-100 (AEI/FEDER, UE), by Instituto de Salud Carlos III (PI19/01128), by the I-ON network supported by Bristol Myers Squibb to I.M. and cofinanced by Fondos FEDER ”A way to make Europe”. This project has received funding from the European Union’s Horizon 2020 research and innovation program (grant agreement n° 945393- T2EVOLVE) and (Marie-Sklodowska-Curie grant agreement n° 765394), Fundación de la Asociación Española Contra el Cáncer (AECC) GCB15152947MELE, Fundación La Caixa and Fundación BBVA, Gobierno de Navarra Salud, Gobierno de Navarra Proyecto LINTERNA Ref: 0011–1411-2020-000075, Mark Foundation, Fundación BBVA and Fundación Olga Torres

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