LAUSR

KRAS has been the quintessential undruggable oncogene for many years. Although there have been recently described small molecule inhibitors of KRAS, these molecules only target a specific mutant form, KRAS G12C. The KRAS G12C mutation represents just 12% of the total number of KRAS mutants across many different tumor types including CRC, NSCLC, and PDAC. We have taken a novel approach to decrease all KRAS mRNA and protein mutants using Centyrin:siRNA drug conjugates which should have applicability across a broader group of patients. Centyrins are a new class of small, stable and highly soluble proteins engineered to bind antigens with high affinity. Centyrins facilitate internalization of macromolecules, including siRNA, into cells and enable trafficking through endosomes and into the cytoplasm. Centyrins are engineered by consensus design from human tenascin C with each domain having a MW of approximately 10 kilodaltons. We have rationally designed a bispecific Centyrin conjugate which targets EpCAM and CD71 to deliver an optimized, chemically modified pan-KRAS siRNA into epithelial derived tumors. EpCAM and CD71 (the transferrin receptor) were selected due to their higher levels of expression on tumors versus normal tissue and the targeting of these receptors with a bispecific molecule enhances both potency of siRNA delivery and selectivity to tumor cells. In addition, Centyrin:siRNA conjugate candidates contain a domain to extend in vivo exposure. The designed Centyrin:siRNA drug conjugates exhibit potencies for knockdown of KRAS mRNA and protein at nM concentrations across various KRAS mutant tumor cell lines, resulting in potent inhibition of proliferation. We report activity on tumor cell lines harboring all of the most common KRAS mutants including G12D (HPAF-II), G12V (SW620), G12S (A549), G12C (H358, MIA PaCa-2), G13D (H1944), and Q61H (H460). This activity is specific for cells expressing EpCAM and CD71 and synergy of the bispecific versus the monospecific Centyrin has been demonstrated. The effect of the Centyrin:siRNA conjugate is specific for KRAS with no effect on the closely related homologs NRAS or HRAS. Pulse washout experiments suggest that in vitro treatment of cells for only 6-24 hours resulted in maximal knockdown of KRAS mRNA up to at least 72 hours. Preliminary pharmacokinetic studies have demonstrated good exposure (~100-400nM) of the siRNA in tumors in a xenograft model. Current efforts are aimed at demonstration of in vivo efficacy in multiple xenograft models with optimization of dose and scheduling. These novel Centyrin:siRNA conjugates have great potential to treat the many patients with KRAS mutations. Citation Format: Russell C. Addis, Robert Kolakowski, Swapnil Kulkarni, Joshua Gorsky, Rebecca Meyer, Yao Xin, Evana Mortezavi, Karyn T. O9Neil, Steven G. Nadler. Tumor-targeted knockdown of KRAS mutants with novel Centyrin:siRNA conjugates [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1825.