Exosomal Based micro RNA delivery for Resistant Lung Cancer

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Abstract Non-small cell lung cancers (NSCLC) have the poorest outcome of all the cancers and after a period of responsiveness, acquired resistance (e.g. T790M mutation) occurs in virtually all NSCLC tumors exposed to Tyrosine Kinase Inhibitors (TKI). Osimertinib, an irreversible EGFR inhibitor which targets EGFR- T790M mutations is the first line treatment for mutated NSCLC but resistance develops after 12-24 months. The checkpoint proteins (PD1, PDL1) and laminB2 (LMNB2) are associated with a poor prognosis in a variety of cancers. Results from our laboratory have shown that in H1975 tumors (expressing L858R/T790M-EGFR mutations), downregulation of PDL1 and LMNB2, among other proteins, could significantly reduce tumor burden in xenotransplanted mice (proteomic analysis. Further, exosomes (EVs) derived from Natural killer cells (NK92MI, NKEVs) contain various cytolytic proteins and have shown potential as anticancer agents. In our laboratory, we observed that NKEVs (using a PBS bioreactor with IL-15), showed 40 percent cell kill at concentration of 1X1010 particles when compared to EVs derived from HEK or MSC cells which showed 15-20 percent cell kill in lung PDX cells. Further, H1975 resistant (H1975R) xenotransplanted tumors when treated with NKEVs downregulated HO1, vimentin. NF-kB, P38MAPK significantly (P<0.001) as compared to control and HEK derived EVs suggesting their anticancer role via inducing apoptosis and other possible mechanisms. Also, NKEVS were found to deliver fluorescent mir3133-TYE effectively in significant amounts (as compared to control) to H1975R tumors when given intravenously showing their targeting potential. Further we explored the micro-RNA which regulate LMNB2(mir-3133) and PDL1(mir5193) and showed that they could significantly downregulate the expression of LMNB2 and PDL1 respectively in vitro and also in PDX tumors (TM00199, Jackson labs), only when delivered as EV formulations in NSG mice. Hence based on our strong preliminary data, we hypothesize that NKEVs carrying LMNB2 and PDL1 micro RNA will deliver their payload to osimertinib resistant NSCLC and will be able to overcome resistance by using in combination with carboplatin with minimal side effects. To test this hypothesis, we propose the following independent Aims: Aim 1: Formulation of NK-EVs containing PDL1 and LMNB2 micro-RNA and evaluating them in vitro in combination with carboplatin against H1975 (R and wild type) and PDX cells Aim 2: Toxicological and Pharmacodynamic evaluation of the dual micro-RNA NKEVs in H1975 resistant and PDX models. The long-term goal of this proposal is to generate enough preclinical data with bioreactor manufactured NKEVs micro RNA formulations and to understand their role in overcoming resistance so as to apply for a R01 proposal or Phase 1 clinical studies in the future.