Abstract 3228: Development and characterization of in vitro assays to detect and quantitate tubulysin B hydrazide in biological samples
Authors: Parker Nikki L. , Shillingford Jonathan M. , Nelson Melissa , Reddy Joseph A. , Leamon Christopher P. 
Published: July 2017

Abstract
EC1456 is a folate-targeted small molecule drug conjugate (SMDC) of tubulysin B hydrazide (TubBH) that is currently in Phase I clinical studies for treatment of patients with folate receptor (FR)-positive tumors. The ability to detect, quantitate, and localize drug in tissues and biological fluids is particularly important for the characterization of its biodistribution and pharmacodynamic properties. To aid in the biological characterization of our targeted TubBH conjugates, our lab had a rabbit polyclonal antibody produced against TubBH for use in various assays. An IgG-purified fraction of antiserum was used to develop competitive ELISA, immunohistochemical, and flow cytometry methods for analysis of biological samples, such as xenograft tumors from animals dosed with drug. Before assay development, a direct ELISA was performed to determine antibody titer and specificity. The TubBH antibody showed excellent binding to immobilized TubBH, whereas a rabbit IgG negative control antibody had minimal binding. Next, a competitive ELISA was developed in which soluble TubBH competed for antibody binding to immobilized antigen. Multiple experimental parameters were optimized, including coating antigen concentration, antibody dilution, and sample matrix extraction method. Using the optimized conditions, the resulting standard curves for TubBH in acetonitrile-extracted KB tumor sample matrix showed good sensitivity and dose-response. It was determined that, in addition to untargeted TubBH, the antibody could also be used to quantitate intact EC1456, as well as a metabolite of the drug lacking the hydrazide. The utility of the antibody was further demonstrated utilizing a PSMA-targeted version of TubBH. Once optimized, the ELISA method was used to analyze FR-positive KB and FR-negative A549 xenograft tumor homogenates from animals dosed with either EC1456 or untargeted TubBH. The levels of TubBH were higher 4h-post EC1456 dose in the FR-positive KB tumors compared to the FR-negative A549 tumors; however, levels were similar after 24h. Moreover, untargeted TubBH resulted in lower tumor concentrations of the drug compared to EC1456 in KB tumors. An immunohistochemical method developed using the antibody showed strong, focal, cytoplasmic staining in FFPE tumor sections of KB tumors from animals dosed with EC1456, while no staining was observed in tumors from undosed animals. Finally, the antibody was successfully used to detect cell surface FR-bound EC1456 on fixed and unfixed KB cells in vitro by flow cytometry. This experiment confirmed that the antibody is able to recognize the conjugate even when it is situated in the FR binding pocket. Collectively, these results show that this antibody against TubBH can be used as a powerful tool for analysis of tumor drug uptake for folate-targeted TubBH in xenograft models and could hold great potential for use in patient sample analysis as well.
Citation Format: Nikki L. Parker, Jonathan M. Shillingford, Melissa Nelson, Joseph A. Reddy, Christopher P. Leamon. Development and characterization of in vitro assays to detect and quantitate tubulysin B hydrazide in biological samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3228. doi:10.1158/1538-7445.AM2017-3228