Abstract 6008: Antibody validation for clinical pharmacodynamic assay reveals cross-reactivity of commercial DNMT3B clones
Authors: Benton Gabriel J. , Mohandoss Manisha , Doroshow James H. , Parchment Ralph E. , Ferry-Galow Katherine V. 
Published: April 2023

Abstract
Cooperativity between the maintenance DNA methyltransferase, DNMT1, and de novo methyltransferases, DNMT3A and 3B, is essential for proper maintenance and integrity of the methylome, and deregulation of these enzymes and associated transcriptional silencing of multiple genes has been shown in many types of cancer. Two DNA methyltransferase inhibitors are currently in clinical use with several others undergoing clinical evaluation, and the pharmacodynamic response of DNMT enzymes is a crucial aspect of understanding therapeutic effects in preclinical studies and clinical trials. Vigorous fit-for-purpose validation of research-grade reagents and the application of effective quality control testing procedures to assess lot-to-lot variability (and reject unacceptable lots) are essential to assure robust and reproducible pharmacodynamic assay performance. During the antibody validation process for the DNMT3B enzyme target, two commercial monoclonal antibody clones were found to exhibit significant cross-reactivity to another cellular protein and inappropriate subcellular localization via immunohistochemistry (IHC), including diffuse cytoplasmic staining and significant staining in a DNMT3B knock out (KO) cell line. Two other commercial monoclonal antibody clones showed the expected nuclear expression in the HCT116 parental cell line and lack of staining in the DNMT KO cells. Further evaluation via western blot revealed that the cross-reacting antibodies detected a band of the approximate expected molecular weight (MW) of DNMT3B but of a protein that was expressed equivalently in the parental and DNMT3B KO cell line pair, whereas the two other antibody clones only detected a significant band of correct MW in the parental but not the KO cell line. These results suggest that some commercial antibody clones are binding to a protein other than DNMT3B and that they are unsuitable for use with IHC and western blot to evaluate subcellular localization and expression levels of DNMT3B protein. We presume that these non-specific antibody clones were chosen for commercial development based on target screening via Western blot that did not include evaluation of a validated DNMT3B knock out cell line, as is not uncommon for research-grade antibody clones. This data highlights the critical importance of thorough fit-for-purpose validation and quality control for crucial reagents used for pharmacodynamic biomarker studies. Funded by NCI Contract No HHSN261200800001E
Citation Format: Gabriel J. Benton, Manisha Mohandoss, James H. Doroshow, Ralph E. Parchment, Katherine V. Ferry-Galow. Antibody validation for clinical pharmacodynamic assay reveals cross-reactivity of commercial DNMT3B clones. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6008.