Abstract 5157: Development and evaluation of a high-throughput method for rapid detection of surface antigen expression in fixed cells

Abstract

Abstract Cell surface proteins are increasingly considered in the development of novel anticancer agents, as demonstrated by recent clinical successes in the field of immuno-oncology and antibody-drug conjugates (ADCs). These conjugates specifically target antigens present on the surface of tumor cells, enabling the delivery of cytotoxic agents to specific tumor sites. During the preclinical development of such compounds, for example, when they are tested on a wide panel of cell lines, it is essential to have a rapid and reliable method for monitoring target expression and distribution. Complementing our 160 Cell line panel ProLiFiler™, we report here the development of a novel method named oncoFLOW-Profiler™ which includes steps of cell fixation, micronic storage, staining, and measurement by flow cytometry. This method is applied in a 96-well format, enabling screening of all cell lines in parallel and analysis of the expression of different targets in just one day. In this proof-of-concept study, we investigated the level of ERBB2 expression across the ProLiFiler panel of 160-cell lines in order to validate this methodology. We first compared the impact of fixation methods and sample storage on knowingly positive and negative control cell lines over a period of several months. ERBB2 measurements from the oncoFLOW-Profiler™ will be analyzed and validated by comparison with various OMICs datasets from the Cancer DataMiner platform (4HF Biotec), including RNA expression, mutational status, and protein expression levels. To determine the expression cutoff of ERBB2 amplified models, results will be analyzed according to gene copy number variation. Finally, the flow cytometry data generated will be investigated for use as a predictor of response to clinically approved ADCs targeting ERBB2: Kadcyla and Enhertu. Here we present a method that we have demonstrated is feasible for investigating cell surface receptor/antigens during preclinical phase of drug development. Our method has enabled us to successfully assess ERBB2 in biological samples and to identify models sensitive to ADCs targeting ERBB2. We will also discuss the results of ongoing experiments analyzing the applicability of this screen to intracellular targets and carbohydrate-based surface antigens, which cannot be analyzed either by transcriptomics or proteomics. Citation Format: Kaja Holtorf, Anne-Lise Peille, Daniel Feger, Vincent Vuaroqueaux, Jan Erik Ehlert, Nadine Obier. Development and evaluation of a high-throughput method for rapid detection of surface antigen expression in fixed cells [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 5157.