Background We previously developed a saline-formulated mixture of 4 synthetic messenger ribonucleic acids (mRNAs) encoding the cytokines IL-12, IFN-α, GM-CSF, and IL-15 sushi (IL-15-IL-15Rα receptor fusion) that promoted systemic immunity and tumor eradication upon intratumoral administration in multiple mouse tumor models.

Here, we explored whether stabilization of the mRNAs through a lipoplex (LPX) formulation could confer increased expression of the encoded cytokines to result in improved antitumor efficacy. To this end, we developed a pH-responsive amphoteric LPX by mixing preformed liposomes with mRNA in aqueous/aqueous solutions. The resulting complex was designed to destabilize under neutral pH in circulation, leading to a limited exposure in non-target organs following intratumoral administration.

Methods Biological activity of LPX-formulated mRNA-encoded cytokines or reporter genes were compared to that of naked mRNA in vivo using C57BL/6 and BALB/c mice harboring TC-1, B16F10 and CT26 tumors, respectively. Following intratumoral administration, cytokines were analyzed in serum and organs by ELISA and luciferase expression was followed by bioluminescent imaging. Anti-tumor efficacy was assessed by tumor growth inhibition and survival of CT26-tumor bearing mice and expansion of tumor-reactive T cells were analyzed by flow cytometry. Abscopal effects were assessed in C57BL/6 mice bearing B16F10 subcutaneous tumors on both flanks.

Results In vivo studies demonstrated a favorable tumor-to-liver translation ratio of luciferase reporter mRNA formulated with the LPX, supporting the hypothesis that the LPX would destabilize in circulation. The LPX formulation further increased target expression of each of the four encoded cytokines in tumors ranging from 7 to 47-fold compared to saline solution in B16F10-tumor bearing animals. Intratumoral administration of LPX-mRNA to mice with subcutaneous CT26 colorectal tumors significantly inhibited tumor growth, with stronger tumor growth inhibition when compared to treatment with saline-formulated mRNA. This increased antitumor activity was correlated with stronger pharmacodynamic responses, including expansion of T cells reactive against the tumor antigen gp70. C57BL/6 mice bearing B16F10 subcutaneous tumors on both flanks received an intratumoral injection of LPX-mRNA on one side. Treatment resulted in significant inhibition of tumor growth and prolonged survival. Antitumor efficacy was further enhanced when combined with anti-PD-1 antibody, resulting in complete tumor regression in a subset of mice, consistent with an abscopal response.

Conclusions Together, these results demonstrate the potent anticancer activity of a novel LPX-formulated mRNA mixture.

Ethics Approval Animal experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) and in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).