Comparison Of Receptor Occupancy Methods Of Anti-PD-L1 Monoclonal Antibody In Whole Blood And Pbmc Of Cynomolgus Monkey

Flow cytometry-based receptor occupancy is a detection method commonly used to quantify the interaction between monoclonal antibody drugs and receptors,and plays an important role in vitro,preclinical and early clinical research stages of drug development.Many factors need to be considered during the establishment of the RO experimental method in clinical trials,including clinical centers,sample types,testing centers,analysts,etc.The choice of sample types in method establishment is related to practical issues such as science,cost,cold chain logistics and the feasibility of multicenter clinical trials.Samples of receptor occupancy usually include whole blood and PBMC.In this paper,the free receptor detection mode was used to establish the receptor occupancy methods of anti-PD-L1 antibody RC98(humanized IgG1 antibody)in cynomolgus monkey whole blood and PBMC,and were fully verified respectively.Compare the characteristics and advantages and disadvantages of the two sample types for the receptor occupancy experiment.We coupled fluorescein AF647 to RC98 to obtain a detection antibody for measuring free receptors.We explored and determined the best concentration of the detection antibody(RC98-AF647)and the drug concentration point of high and low receptor occupancy in cynomolgus monkey whole blood and PBMC.Establish and verify anti-PD-L1 antibody RC98 receptor occupancy method in the whole blood and PBMC.Whole blood verification parameters include: intra-and inter-run precision(P),stability after sample collection and fixation,specificity,selectivity and sensitivity.PBMC verification parameters include: intra-and inter-run precision(P),stability after sample collection and fixation,long-term stability,specificity,selectivity,and sensitivity.The intra-and inter-run precision(% CV)of the whole blood was 2.4% to 24.4%and 11.4%～12.9%.The whole blood was stable within 24 hours after collection and the sample was stable within 24 hours after fixation.The intra-and inter-run precision(%CV)of the PBMC was 1.3% to 11.5% and 4.8% to 8.3%.It was stable within 24 hours after whole blood collection and stable within 24 hours after fixation,and PBMC stable within 3 months after freezing.The selectivity of the two samples is somewhat different,but there is no significant difference in specificity and sensitivity.Cynomolgus monkey PBMC can replace whole blood as the receptor occupancy sample in multi-center clinical trials to ensure the timely processing of samples and reduce logistics costs.