Abstract 2760: Novel GPCR recombinant monoclonal antibodies for cancer biology research

Abstract

Abstract Recombinant technology is becoming the preferred production mechanism for creation of antibody reagents used in academic and clinical cancer biology research. Recombinant antibodies (rAbs) are superior to standard polyclonal and hybridoma-generated monoclonal reagents as they offer reproducible performance and consistent, scalable supply. A major focus of antibody manufacturers is to leverage rAb production strategies to generate antibodies against challenging targets for which there are few, if any, reliable antibodies. G-protein coupled receptors (GPCRs) are encoded by more than 820 human genes and represent the largest membrane protein superfamily in the genome. These seven-pass transmembrane domain receptors are involved in a multitude of physiological and pathophysiological processes related to various chronic diseases, including an array of malignancies. Perhaps 30-40% of all therapeutics are directed against GPCR activity, and ~350 of the non-olfactory GPCRs are thought to be potential candidates for drug development. However, for a slew of technical reasons, the development and validation of high-quality antibodies against GPCRs has proven difficult. In an effort to facilitate further research into GPCRs, GeneTex is combining its recombinant antibody production platform with enhanced validation techniques to make specific monoclonal antibodies against GPCRs with direct relevance to cancer and other diseases. GeneTex’s rAb production protocol employs a multi-parameter fluorescence-activated single cell sorting (FACS)-based methodology to identify and select antigen-specific IgG+ memory B cells from an immunized rabbit (Starkie et al., 2016). The heavy and light chain variable region genes from single cells are amplified, cloned, and co-expressed in mammalian cells to generate a monoclonal IgG. Application-specific testing is performed in conjunction with knockout/knockdown (KO/KD), differential expression comparison in cells and tissues, cell fraction enrichment (i.e., membrane extracts versus whole cell extracts), and other methodologies. Antibodies directed against extracellular GPCR domains are validated for specificity using GPCR arrays (CDI Labs, Baltimore, MD). This workflow has resulted in the production of specific antibodies for a number of GPCRs studied by cancer biologists. These include novel recombinant monoclonal antibodies to the dopamine D2 receptor (DRD2), retinoic acid-induced protein 3 (RAI3), CXC motif chemokine receptor 2 (CXCR2), CXCR4, and CXCR7. All of these antibodies were validated for at least two applications, while the specificity of three (i.e., RAI3, CXCR4, and CXCR7) is supported by KO/KD data. GPCR array data argues for the specificity of the other two antibodies (i.e., DRD2 and CXCR2). This same production/validation approach will be applied as GeneTex targets the remaining GPCRs. Citation Format: Alexander Ball, Yung Lin Hsieh, Chun Kai Huang, Sega Huang, Shang Ru Wu, Yue Yun Chang, Jiming Liu, Po Shin Peng, Chia Yi Lin. Novel GPCR recombinant monoclonal antibodies for cancer biology research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2760.