Biophysical Study of Novel Phlip-Stinga with Membrane

Immuno-oncology (IO) is a rapidly growing field that utilizes the body's own immune system to recognize and attack cancer cells. The tumor microenvironment (TME) plays a crucial role in the effectiveness of IO treatments and can be separated into two major categories: cold and hot tumors. Cold tumors contain M2 tumor-associated macrophages (M2-TAMs), cancer-associated fibroblasts (CAFs), myeloid derived suppressor cells (MDSC) and these tumors do not respond to IO treatment. On the other hand, hot tumors contain T-cells and M1 macrophages and typically respond well to IO treatments. STING agonists (STINGa) are a ligand for the STING protein, which activates the STING pathway, a fundamental pathway of innate immunity that is designed by nature to induce an inflammatory response, thus turning “cold” tumors into “hot” tumors. The non-targeted systemic use of STINGa is dangerous, since it can induce inflammation everywhere. To address these limitations, pH (low) insertion peptide (pHLIP) can be used to ensure the targeted delivery of STINGa. A biophysical study was done to assess the interaction of two novel pHLIP-STINGa constructs with membranes of liposomes, which were used to mimic the cell membrane. We monitored changes of tryptophan fluorescence and circular dichroism (CD) of pHLIP-STINGa agents in steady-state and kinetics modes to establish pK of insertion, and rates of membrane entry and exit. Obtained results indicate to a desirable pK of insertion and fast membrane insertion, which are important predictive parameters for in vivo performance of the agents.