Background: IL-12 is a pro-inflammatory cytokine with antitumor potential that activates both innate and adaptive immunity. However, IL-12 shows poor pharmacokinetics and intolerable toxicities due to unspecific distribution. Herein, we present pH-sensitive polymeric micelles loading IL-12 (IL-12/m) that release fully active IL-12 after sensing intratumoral pH to safely potentiate immunotherapy. Methods: IL-12/m with 40 nm in diameter were prepared by self-assembly in phosphate buffer. The IL-12 release was tested at different pH. The stimulation of IFN-γ release from IL-12 and IL-12/m was studied in mouse splenocytes. Pharmacokinetic, pharmacodynamic, and antitumor activity were studied in immunosuppressed (B16F10 and 4T1) mouse tumor models (staged ≥ 200 mm3) and in a lung metastasis model (4T1). The safety of IL-12 was evaluated by tracing levels of cytokines and blood markers of toxicity. Results: IL-12/m selectively activated at intratumoral pH 6.5, releasing more than 80% of IL-12 in 24 h. While IL-12/m did not induce IFN-γ production in splenocytes, the released IL-12 showed similar activity to native IL-12. In mice, IL-12/m prolonged blood circulation with minimal leakage, yielding a 3-fold longer half-life than IL-12. Moreover, the accumulation in tumor for IL-12/m (7.5%ID/g of tumor) was 3-fold higher compared that of free IL-12, with more than 90% of IL-12/m being activated. The tumors treated with IL-12/m showed 2-3-fold higher levels of IFN-γ, IL-6 and TNF-α than those treated with IL-12, whereas anti-inflammatory IL-10 was 2-fold lower for IL-12/m. In blood and organs, IL-12/m lowered the cytokine levels compared to IL-12. Also, IL-12/m avoided the tachyphylaxis of IL-12 without peak effects of IFN-γ, and the blood markers of toxicity, i.e., BUN, ALT and AST, remained close to control levels, supporting the tumor selective activation of IL-12/m. IL-12/m was efficacious in the B16F10 tumor model (10 µg iv 3 injections every 4 days) as monotherapy, and in combination with anti-PD-1 (10 mg/kg IP 3 injections every 4 days), which led to 6 of 10 complete regressions (CR). IL-12/m was also effective at 1 µg (5 injections every 2 days) against 4T1 tumors. IL-12/m increase the number and activation of CD8+ T cells and NK cells in tumors. The combination of IL-12/m (10 µg iv 3 injections every 4 days) with anti-PD1 and anti-CTLA-4 (both 10 mg/kg IP 3 injections every 4 days) achieve complete CR in a spontaneous model of lung metastasis. Responders showed immunological memory after being rechallenged with fresh tumor cells. Conclusions: IL-12/m, a pH-activated polymeric micelle loading IL-12, masked the activity of IL-12 in healthy tissues, while it unleashed full potency of IL-12 in tumors, improving tolerability and efficacy. IL-12/m presented greater antitumor activity than IL-12 as monotherapy and in combination with immune checkpoint inhibitors. Citation Format: Horacio Cabral, Pengwen Chen, Kazuhiko Kakimi, Kazunori Kataoka, Takuya Miyazaki, Koji Nagaoka. pH-activatable IL-12-loaded polymeric micelles safely enhance antitumor efficacy as monotherapy and in combination with immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2072.
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.
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