Abstract 1544: Cell penetrating single domain antibody (sdAb) SBT-100 binds KRAS and inhibits growth of human cancers with KRAS activating mutations

Background: Despite nearly fourth years of research, scientists have failed to develop a clinically viable therapy against KRAS, one of the deadliest families of cancer-causing proteins. Mutations in KRAS are prevalent amongst the top three most deadly cancer types in the United States: pancreatic (95%), colorectal (45%), and lung (35%). KRAS has been thought to be undruggable due to: 1) its intracellular location and lack of binding pockets for small molecules; 2) the high (pM) affinity of RAS for GTP precludes direct targeting of the nucleotide binding pocket; 3) high intracellular concentrations of GTP (uM) inhibits competition for the nucleotide-binding pocket by small molecules; and 4) possible toxicity. Mutations of KRAS result in it being perpetually turned on to propagate signal down the MAPK pathway. This results in constant production of P-ERK and plays an important role in malignant development. To overcome these challenges, Singh Biotechnology has developed SBT-100 a first in class & best in class novel sdAb that penetrates the cell membrane to bind KRAS to inhibit its GTPase activity. Methods: Human cancer cell lines were purchased from ATCC. BIAcore affinity studies were conducted by Precision Antibody. KRAS GTPase assay was purchased from Promega. Levels of P-ERK were determined using western blots. In vitro cell growth suppression was tested with MTT assay. Athymic nude mice for xenograft studies were purchased from Envigo. Results: SBT-100 binds KRAS with KD=10-9 and KRAS(G12D) with KD=10-7 as demonstrated by BIAcore affinity assay. Both SBT-100 and SBT-102 significantly inhibit KRAS GTPase activity in vitro and inhibition is comparable to polyclonal antibody to KRAS. Growth of MDA-MB-231 cells with KRAS(G13D) mutation and PANC-1 cells with KRAS(G12D) mutation are significantly decreased in the MTT assay when incubated with SBT-100. Additionally, same cell lines have significantly decreased P-ERK expression when cultured with SBT-100. Xenograft studies demonstrate significant growth suppression of MDA-MB-231 and PANC-1 when treated with SBT-100 in vivo. Conclusion: SBT-100, crosses the cell membrane, binds to KRAS intracellularly and its most common mutant with nanomolar affinity, inhibits KRAS GTPase activity, downregulates P-ERK signaling, and suppresses the growth of cancers cells in vitro and in vivo without showing any toxic effects. Citation Format: Sunanda Singh, Genoveva Murillo, Avani Singh, Samara Singh, Meenakshi Parihar, Anjali Singh, Rajendra Mehta, Ashutosh Parihar. Cell penetrating single domain antibody (sdAb) SBT-100 binds KRAS and inhibits growth of human cancers with KRAS activating mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1544.