LAUSR

Background: Treatment of immunologically cold tumors is a major challenge. IL-12 can activates both innate and adaptive immunity to elicit strong antitumor immunity. Nevertheless, the poor pharmacokinetic and severe side effects of IL-12 limit the application. We have recently developed pH-sensitive polymeric micelles loading IL-12 (IL-12/m) that can selective activate in tumor tissues to invigorate immunotherapy and enhance efficacy. Herein, we elucidated the mechanisms behind the enhanced efficacy of IL-12/m and their ability to synergize with immune checkpoint inhibitors (ICIs). Methods: To determine the contribution of the immune cells on the efficacy on IL-12/m, mice bearing orthotopic 4T1 tumor models were treated with anti-CD4 (for depleting CD4+ cells), anti-CD8 (for depleting CD8+ cells), anti-asialo GM1 (for depleting NK cells). The tumor volume was tracked after treatment with IL-12/m. The immune status of the tumors was studied by histology, flow cytometry and RNA-seq after treatment with free IL-12, IL-12/m and their combination with ICIs. The efficacy of the treatments was assessed by following the tumor growth and mice survival. Results: IL-12 can promote the growth and function of T cells to initiate the enhanced anti-tumoral immune reactions. Flow cytometry analysis IL-12/m significantly enhanced tumor infiltration of CD8+ T cells compared to free IL-12. The immune cell depletion study revealed that the depletion of CD8+ cytotoxic T cells, CD4+ T cells and NK cells significantly lessen efficacy of IL-12/m, which is consistent with the biological function of IL-12. The depletion experiment suggests CD8+ cytotoxic T cells are the most important fraction for IL-12/m efficacy. Moreover, immunohistochemistry analysis of 4T1 revealed IL-12/m promoted high infiltration of CD8+ cells and the upregulation of Granzyme B in CD8 + Cells. IL-12/m also increased PD-L1 levels in tumors. The effects of IL-12/m on the TME was further investigated as a monotherapy and in combination with anti-PD-1 antibodies. The combination of IL-12/m with anti-PD-1antibodies presented high CD45+ cells in tumors. Lymphoid cells and T cells were upregulated by IL-12 and the anti-PD-1 antibody combination. The general CD4+ T cells also showed upregulation in the IL-12/m treated groups. While anti-PD-1 monotherapy did not show any suppression on Tregs in the tumor model, the combination of IL-12/m plus anti-PD-1 depleted Tregs. Moreover, IL-12/m increase the presence of Th cells. We then investigated the activation by transcriptome analysis. The heatmap of global gene expression indicated differentiated gene profiles. GOBP and KEGG analysis showed that IL-12/m upregulated the stimulation of proliferation, differentiation and activation of various effector immune cells, differentiation of T cells into Th cells, and T cell activation and T cell receptor signaling pathway. The enhanced the intratumoral immunity of IL-12/m plus ICIs allowed eradicating 4T1 tumors. Conclusions: Systemically injected IL-12/m ensured strong stimulation of antitumor immunity leading to high safety and strong therapeutic outcomes in cold tumor models. The enhanced immune response of IL-12 synergize with checkpoint blockade to eradicate cold tumors. The approach has potential for developing safer and more effective immunotherapies. Citation Format: Horacio Cabral, Pengwen Chen, Koji Nagaoka, Takuya Miyazaki, Kazunori Kataoka, Kazuhiro Kakimi. pH-sensitive polymeric micelles loading IL-12 profoundly inflame the tumor microenvironment to eradicate cold tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB336.