Improved sample stability for clinical whole blood receptor occupancy assays with cryopreservation.

Receptor occupancy (RO) assessments are a critical component of elucidating PK/PD/safety relationships during early clinical development. As an initial measure of target engagement, RO can inform the pharmacological activity, and RO data from nonclinical studies are often translated to support human dose projection when the therapeutic target is a membrane-bound protein. Currently, across the industry, RO measurements are typically performed using freshly collected blood samples that require real-time bioanalysis, often resulting in highly variable data, missed time points, substantial operational costs, and logistical challenges. Here, we describe a novel RO method that utilizes cryopreserved whole blood, representing a substantial process improvement and enhanced robustness of RO endpoints. Instead of measuring cell surface target engagement in freshly collected whole blood, this method utilizes whole blood that is cryopreserved at the time of collection without subsequent peripheral blood mononuclear cell (PBMC) isolation. This approach extends the sample stability window from 2 to 3 days to 12 months post-collection. Internal method development demonstrated that a 1:1 dilution of whole blood with freezing media followed by cryopreservation produced RO results in T cells comparable to those obtained from fresh blood. Follow-up studies confirmed that this observation extended to multiple target binding arms of multispecific T-cell engagers, as well as RO measurements in B-cell and monocyte targets. Additional studies evaluated the feasibility of implementing this method in clinical settings by assessing assay robustness, sample freezing conditions, and shipping methods.