Associate Principal Scientist Merck & Co, Inc - Rahway, NJ Matawan, New Jersey
Purpose: Receptor occupancy is often used to support dose selection of novel therapeutics in clinical development. Flow cytometry assays are often developed for measuring receptor occupancy in circulating blood cells in clinical trials. These assays require selective antibody reagents that directly compete with the therapeutic molecule but are not always readily available. In the absence of an available competitive antibody, we were tasked with developing a mass spectrometry assay that uses a biotinylated version of the therapeutic molecule to occupy and measure free target membrane receptors on the surface of peripheral blood mononuclear (PBMC) cells.
Methods: Whole blood is incubated with a biotinylated version of the therapeutic molecule in cell preservation tubes (CPT). The biotinylated version of the molecule binds to free target protein. CPT processed PBMC were lysed in RIPA buffer and split into two parts. In one part, streptavidin M-280 magnetic beads were used to capture free target protein. In the other part, a non-competitive monoclonal antibody specific for the target protein was conjugated to M-280 tosylated magnetic beads and used to capture the total target protein. The immunocaptured samples were washed and digested with trypsin. Anti-peptide antibodies conjugated to M-280 tosylayted beads were used to capture specific surrogate peptides to reduce sample complexity. The final 50uL eluent in 0.1% formic acid was analyzed by mass spectrometry. The assay employs a calibration curve using recombinant protein in RIPA buffer and PBMC lysate derived QC samples. An assay validation was conducted to assess sensitivity, accuracy, and precision and to support a clinical study.
Results: In proof-of-concept studies, whole blood samples were spiked with the therapeutic molecule. The IC50 values generated using this mass spectrometry assay matched favorably with IC50 values generated using a well-established pharmacodynamic assay for the same membrane receptor target. The sensitivity of the mass spectrometry assay can measure sub picomolar concentrations of free target protein in PBMC and has a dynamic range allowing for greater than 99% receptor occupancy measurements, assessed in blood from normal healthy donors. In assay validation testing, accuracy assessments were acceptable, with less than 15% bias from nominal concentration for all the calibration standards. Moreover, the precision in six replicates each of low, medium, and high derived PBMC lysate QC samples were acceptable, with less than 20% (25% for low QCs) coefficient of variance for both intra and inter day assessments.
Conclusion: We developed a receptor occupancy assay in PBMC using mass spectrometry to support a clinical program. We demonstrated that the assay was specific for the target protein in initial proof-of-concept studies. We validated the assay, ensuring the necessary sensitivity, dynamic range, accuracy and precision to support a receptor occupancy endpoint in a clinical study.
Learning Objectives:
Comprehend that receptor occupancy measurements are important biomarkers in early clinical trials, and used to inform later decision making, such as optimal dose selection in future efficacy trials.
Identify what analytical tools and platforms can be used to develop receptor occupancy assays in the clinic trial setting and see an example fit-for-purpose validation of such assay.
Describe the challenges of developing receptor occupancy assays in the clinic and learn from at least one case where selective antibodies were not available, and an alternative strategy was deployed.
