BackgroundBO-112 is a nanoplexed form of polyinosinic:polycytidylic acid that acting on toll-like receptor 3 (TLR3), melanoma differentiation-associated protein 5 (MDA5) and protein kinase RNA-activated (PKR) elicits rejection of directly injected transplanted tumors, but has only modest efficacy against distant untreated tumors. Its clinical activity has also been documented in early phase clinical trials. The 5,6-dimethylxanthenone-4-acetic acid (DMXAA) stimulator of interferon genes (STING) agonist shows a comparable pattern of efficacy when used via intratumoral injections.MethodsMice subcutaneously engrafted with bilateral MC38 and B16.OVA-derived tumors were treated with proinflammatory immunotherapy agents known to be active when intratumorally delivered. The combination of BO-112 and DMXAA was chosen given its excellent efficacy and the requirements for antitumor effects were studied on selective depletion of immune cell types and in gene-modified mouse strains lacking basic leucine zipper ATF-like transcription factor 3 (BATF3), interferon-α/β receptor (IFNAR) or STING. Spatial requirements for the injections were studied in mice bearing three tumor lesions.ResultsBO-112 and DMXAA when co-injected in one of the lesions of mice bearing concomitant bilateral tumors frequently achieved complete local and distant antitumor efficacy. Synergistic effects were contingent on CD8 T cell lymphocytes and dependent on conventional type 1 dendritic cells, responsiveness to type I interferon (IFN) and STING function in the tumor-bearing host. Efficacy was preserved even if BO-112 and DMXAA were injected in separate lesions in a manner able to control another untreated third-party tumor. Efficacy could be further enhanced on concurrent PD-1 blockade.ConclusionClinically feasible co-injections of BO-112 and a STING agonist attain synergistic efficacy able to eradicate distant untreated tumor lesions.

Interleukin-12-based local gene therapy of cancer constitutes an active area of clinical research using plasmids, mRNAs and viral vectors. To improve antitumor effects, we have experimentally tested the combination of mRNA constructs encoding interleukin-12 (IL-12) and interleukin-18 (IL-18). Moreover, we have used a form of IL-18 (DR-18) which has preserved bioactivity but does not bind to the IL-18BP decoy receptor. Both cytokines dramatically synergize to induce IFNγ release from mouse splenocytes, and, if systemically co-transferred to the liver, they mediate lethal toxicity. However, if given intratumorally to B16OVA tumor-bearing mice, the combination attains efficacy against the directly treated tumor and moderate tumor-delaying activity on distant uninjected lesions. Co-treatment was conducive to the presence of more activated CD8+ T cells in the treated and non-injected tumors. In keeping with these findings, the efficacy of treatment was contingent on the integrity of CD8+ T cells and cDC1 dendritic cells in the treated mice. Furthermore, efficacy of IL-12 plus DR-18 local mRNA co-injection against distant concomitant tumors could be enhanced upon combination with anti-PD-1 mAb systemic treatment, thus defining a feasible synergistic immunotherapy strategy.

SpHPP HPP GuidelinesMissing Proteins Validation EXPERIMENTAL DATABASES TRANSCRIPTOMICSSpHPP cell lines dataset (shotgun and microarrays). GEO microarrays (normal tissues, cell lines, cancer samples).RNA-Seq (HBM, ENCODE and CCLE datasets).

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