Abstract 1804: Durable eradication of tumors by single injections of a pHLIP-STING agonist

Abstract

Abstract Purpose: pHLIPs (pH-Low Insertion Peptides) target cell surface acidity resulting from the metabolism of cells in a solid tumor. The acidity causes pHLIPs to insert as transmembrane helices, and attached cargoes can be delivered into targeted cells. STING agonists can trigger an immune response when they are inside cells. pHLIP targeted delivery of a STING agonist (STINGa) effectively triggers cytokine secretion to mobilize the immune system to eradicate tumors while avoiding side effects. Experimental Procedures: Constructs consisting of a pHLIP peptide (Var3) linked to a STINGa (diABZI) via a self-immolating linker were synthesized and characterized. Activation of the STING pathway by the agent was confirmed in cells. Small (100 mm3) and large (400-700 mm3) tumors were grown in female Balb/c mice following flank injections of CT26 colorectal cancer cells. Randomized groups of mice were injected i.p. or i.v. with pHLIP-STINGa made with L- or D- amino acid pHLIP peptides, STINGa alone, pHLIP alone or vehicle. Tumor size and body weight were then measured 3 times per week. The durability of the response to the agent was tested by a second injection of CT26 cells after 60 days. In separate experiments, the identities of targeted immune and stromal cells, the production of cytokines, the PK, tumor targeting and biodistribution were assessed. Results and Conclusions: pHLIP extends the lifetime of a STINGa in the blood 6-fold and delivers STINGa to acidic cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), myeloid derived suppressor cells (mMDSCs), and dendritic cells (DCs). The resulting activation of cytokines within the tumor microenvironment (TME) triggers the eradication of small and large CT26 tumors in mice after a single dose of pHLIP-STINGa. The tumor stroma was destroyed, intratumoral hemorrhage developed, and the TME pH increased, which is important for the efficient cytotoxic activity of immune cells. Further, no tumors developed in 20 out of 25 tumor-free mice re-challenged by an additional injection of cancer cells. The therapeutic effect on CT26 tumors was insignificant in nude mice lacking T-cells. Inhibition of MHC-I negative B16F10 melanoma tumor growth was also observed in Balb/c mice. Thus, targeted delivery of the STINGa to the tumor stroma and TAMs activates signaling, potentially resulting in the recruitment and infiltration of both T-cells and NK-cells, which gain access to the tumor core. The cytotoxic activity of immune cells is enhanced, and immune memory is developed. Citation Format: Donald M. Engelman, Yana K. Reshetnyak, Oleg A. Andreev. Durable eradication of tumors by single injections of a pHLIP-STING agonist [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1804.