Abstract 1870: A simple and quantitative immunoassay for evaluation of receptor occupancy and antibody binding capacity in fixed cells or tissue samples for immune-directing antibody development
Authors: Tatsumi Atsuro , Morichika Keisuke , Morel Caroline , Ghasemi Omid , Yokota Hiroyuki , Krueger Joseph S. 
Published: June 2022

Receptor occupancy (RO) assays are used to quantify the binding of therapeutic antibodies to their targets on the cell surface. This method can be used to in early drug discovery to determine the antibody binding capacity (ABC) of drug candidates to cells in culture to hone the desired drug characteristics during candidate screening or lead optimization. This method can also be used in xenografts or patient tissues to create pharmacodynamic (PD) data that directs the efficacy hypothesis and biomarker selection for clinical trials. The current approaches use flow cytometry on fresh specimens, which introduces many technical and logistical challenges and does not retain spatial information about the tissue. Here we introduce a new, simple method which can be performed on Formalin Fixed, Paraffin Embedded (FFPE) cells or tissues. This approach eliminates the challenges of flow cytometry methods, provides the type of quantitative data needed, and maintains the tissue contexture to study the effects of immune-directing antibodies in patient tissue samples. We describe a method for developing a standard curve to determine antibody binding capacity (ABC) using our Quanticell ™ fluorescent nanoparticle technology. We demonstrate this in a typical bispecific antibody use case, where both the immune-directing (CD3) and targeting (Her2) arms of the bispecific are modulated to create the desired efficacy characteristics. We demonstrate the establishment of a standard calibration curve for Her2 (BT474/Her2+) and CD3 (Jurkat/CD3+) in cells with titrated amounts of anti-Her2 and anti-CD3 antibodies which target the extracellular domain necessary for these studies. We also show how the same Quanticell assay can be used in clinical tissue, to evaluate the localization of the drug targets and immune cells within the tissue context of human patient samples necessary to understand how pharmacodynamic properties of the drug can modulate immune contexture changes. The Quanticell™ method is a simple immunohistochemistry (IHC) based method which allows extremely sensitive detection of therapeutic antibodies bound on FFPE fixed cells or tissue, which thus does not require the use of flow cytometry in fresh tissues, allowing simple use from early discovery through clinical research studies. Importantly, the Quanticell™ method is capable of detecting a very large and linear dynamic range of analyte concentration to allow full quantification of ABC for RO studies. Critically, the Quanticell™ method retains the tissue context, allowing for simultaneous and spatial evaluation of multiple analytes such as the target of the antibody and the immune contexture. This method allows for a consistent method for RO studies that can be applied to early discovery, translational, and clinical research to optimize drug efficacy.

Citation Format: Atsuro Tatsumi, Keisuke Morichika, Caroline Morel, Omid Ghasemi, Hiroyuki Yokota, Joseph S. Krueger. A simple and quantitative immunoassay for evaluation of receptor occupancy and antibody binding capacity in fixed cells or tissue samples for immune-directing antibody development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1870.