Abstract 3035: Development and characterization of CCK2R-targeted SMDCs and identification of GIST as a potential therapeutic indication
Authors: Shillingford Jonathan M. , Reddy Joseph A. , Nelson Melissa , Vetzel Marilynn , Parham Garth L. , Dorton Ryan , Guan John , Parker Nikki , Chu Haiyan , Vlahov Iontcho A. , Leamon Christopher P. 
Published: July 2016

Abstract
Cholecystokinin B receptor (CCKBR/CCK2R) is a GPCR of the cholecystokinin receptor family that is primarily expressed in the CNS and the gastrointestinal tract where it binds the ligands cholecystokinin and gastrin, respectively, and mediates their downstream actions. Since it has been reported that CCK2R is overexpressed in certain cancer types, we have explored the potential of utilizing CCK2R as a receptor system to deliver potent therapeutic warheads. To this end, we designed a series of CCK2R-targeted small-molecule drug conjugates (SMDCs) containing either a non-peptidic antagonist (Z-360) or a peptidic agonist (CCK8) as their ligand moiety and explored their activity in in vitro and in vivo models. While EC1812 (Z-360-tubulysin B) failed to elicit either competable cytotoxicity in vitro or robust therapeutic response in vivo (n = 5; 1 PR) when tested against a HEK293-CCK2R overexpressing cell line, the addition of a PEG36 linker to generate EC1977 greatly improved therapeutic efficacy in vivo (n = 5; 1 CR, 4 cures) despite having no activity in vitro. In contrast, while EC1868 (CCK8-tubulysin B) demonstrated potent, competable cytotoxicity in vitro it had only modest activity in vivo (n = 5; 5 CRs). To explore the clinical relevance of a CCK2R-targeted SMDC, we performed mRNA profiling of a human cancer array to identify indications that have high CCK2R mRNA expression. Of all the indications present on the array, those representing gastrointestinal stromal tumor (GIST) were identified as expressing the highest level of CCK2R mRNA. To further elucidate the clinical relevance of this finding, we procured human clinical samples from GIST patients and determined CCK2R-specific radioligand binding and mRNA levels. These data revealed that 50% of GIST samples express high levels of CCK2R mRNA and exhibit functional ligand binding. Interestingly, although 50% of normal stomach samples had similar mRNA levels as the high-expressing GIST samples, they failed to exhibit detectable radioligand binding. In contrast, HCC, colon cancer, and pancreatic cancer samples were all negative for both mRNA expression and ligand binding. Subsequent screening of a series of GIST patient-derived xenograft (PDX) models for functional binding and mRNA levels failed to identify an appropriate model for further in vivo testing. In conclusion, we have developed and characterized a series of CCK2R-targeted SMDCs and tested their activity in vitro and in vivo. Given the expression and functional binding of CCK2R in GIST we propose that GIST may represent an indication for CCK2R-targeted therapies.
Citation Format: Jonathan M. Shillingford, Joseph A. Reddy, Melissa Nelson, Marilynn Vetzel, Garth L. Parham, Ryan Dorton, John Guan, Nikki Parker, Haiyan Chu, Iontcho A. Vlahov, Christopher P. Leamon. Development and characterization of CCK2R-targeted SMDCs and identification of GIST as a potential therapeutic indication. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3035.