Design and Validation of a Model of a Receptor Occupancy Assay for a New Immunotherapy in Human Whole Blood Specimens
Authors: Angela Pell, Carissa Moore, Lynette Brown, and Jennifer J Stewart
Abstract: Antibody-based regimens are an important classification of therapies used to treat a variety of disease indications, including cancer. When developing therapeutic compounds with the capacity to bind to cell surface molecules, it is important to monitor drug and target engagement through receptor occupancy assays (ROA).
Flow cytometry is the primary technology used to measure receptor occupancy because it can measure receptor engagement on specific cell types within heterogeneous cell populations. Although these assays are conceptually straightforward, ROA are some of the most challenging flow cytometric methods to develop due to the requirement for very specific reagents to detect Total, Free or Bound receptors. Free receptor assays are an indirect measure of receptors that are free or not occupied by the therapeutic compound. These can be detected by using either a fluorescently labeled competitive antibody or by a fluorescently labeled therapeutic. Direct assessments of Bound receptor are designed to assess the directly bound therapeutic using a fluorescently labeled anti-drug antibody. The Free and Total receptor assay measures the total receptors available and is recognized by a noncompetitive antibody bound to a different epitope of the receptor from the one recognized by therapeutic. This design is beneficial if the goal is to determine the ratio of free sites to total sites available but is often the most limited by reagent availability. Here we describe the development of a Free and Total assay where unlabeled drug, fluorophore labeled drug and a fluorophore labeled noncompetitive antibody against human CD4 were used. For the Free receptor assay, the saturating concentration of the unlabeled drug (anti-CD4, clone SK3) was determined by titration and detection of free receptor by the fluorophore labeled drug (anti-CD4 BV421, clone SK3) showed the expected correlation of increasing fluorescence signal in response to decreasing occupied drug concentration indicating an increase in free receptors. The use of the non-compete antibody to another epitope on the CD4 receptor (anti CD4 PECy7, clone OKT4) confirmed that the change in fluorescence signal was due to differential drug occupancy and not due to changes in overall CD4+ cell number or up/down regulation or internalization of the receptor. These approaches show that ROA data can be integral for assessment target receptor engagement by the therapeutic and determining dose limit safety during a preclinical or clinical studies, where long-term receptor binding may lead to unwanted side-effects or toxicity, a hallmark of therapeutic overdosing.
