Abstract 5544: Development of recombinant transcription factor proteins and antibodies for application in clinical immunoassays.
Authors: Thomas D. Pfister, Lynda J. Dieckman, Robert J. Kinders, Anne Book, Simona Colantonio, Karun Mutreja, Scott M. Lawrence, Amina Aziz, Tara Hiltke, Gordon Whiteley, Ralph E. Parchment, Joseph E. Tomaszewski, Robert A. Weinberg, James H. Doroshow
Journal: Cancer Research (2013)

Abstract EMT/stem cell transcription factors have been implicated in the differentiation of epithelial cells into mesenchymal cells (epithelial-mesenchymal transitions, EMT) during embryonic development. EMT transitions are also determinants of the progression of carcinomas, and their expression patterns are altered by chemotherapy. Methods for detection and quantitation of these transitions may have clinical importance. Specialized high quality reagents are an absolute prerequisite for formulation of stable, high-performance, clinical immunoassays. A panel of experts in the Division of Cancer Treatment and Diagnosis (DCTD, NCI) and Whitehead Institute (MIT) have identified 12 key targets in EMT/Stem cells, for which there was an unmet need for high quality reagents (SNAIL, SLUG, ZEB1, ZEB2, SOX9, LBX1, FOXC2, FOXQ1, Goosecoid, NANOG, CD133 and FOXO3). Proof of assay analytical performance and stability require both a purified, characterized antibody, and a calibrator molecule suitable for generation of an assay "standard" that may be used in several different contexts. We have developed a strategy to generate, characterize, and validate reagents for 12 EMT/Stem cell targets starting with antigen design and selection to improve the utility and minimize cross-reactivity of the antibodies produced. We have also designed and produced the corresponding recombinant protein material for these targets. For many of these we expressed one or two specific domains that were also tailored to provide maximum utility in target measurement while avoiding cross-reactivity. We have generated rabbit monoclonal antibodies to the transcription factors and tumor stem cell markers for development of pharmacodynamic assays to support both analysis of transcription factor function and for use in clinical trials for new anticancer therapies. Initial screening of rabbit bleed titers and hybridomas was performed using both the designed peptides and recombinant material. We have also tested the leading candidate hybridomas in multiple immunoassays and will present data demonstrating utility of the antibodies in these applications including ELISA, WB, immunoprecipitation and IFA in FFPE tissues. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Thomas D. Pfister, Lynda J. Dieckman, Robert J. Kinders, Anne Book, Simona Colantonio, Karun Mutreja, Scott M. Lawrence, Amina Aziz, Tara Hiltke, Gordon Whiteley, Ralph E. Parchment, Joseph E. Tomaszewski, Robert A. Weinberg, James H. Doroshow. Development of recombinant transcription factor proteins and antibodies for application in clinical immunoassays. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5544. doi:10.1158/1538-7445.AM2013-5544