Understanding target engagement can enable direct correlations among the drug exposure at the site of action, drug efficacy and toxicity. Comparing to LBA, the hybrid LC-MS/MS assay can offer a great advantage for the total drug, total and free target, and bound target measurement to evaluate the target engagement due to its less dependence on the specificity of the capture reagents. Using IL-8 as an example, here we report the strategies for the development of the sensitive hybrid LC-MS/MS assays for target engagement evaluation in tissue samples. A Tissue Protein Extraction Reagent (T-PERTM from Thermo Fisher) was used for calibration standard and blank mouse tumor homogenate was used for quality control sample preparation. Immuno-capture was performed using an automated magnetic particle processor in a 96-well plate format (KingFisher, ThermoFisher). Several commercial antibodies were used to selectively extract and enrich free, drug-bound and total IL-8 from the homogenates. After immuno-capture, reduction, alkylation, and digestion were performed and a signature tryptic peptide was monitored for the measurement. Chromatographic separation was carried out using a Sciex M5 MicroLC system using a trap-elute mode. Selective reaction monitoring in positive ion electrospray mode was used for detection on a Sciex 5500 Triple Quadrupole mass spectrometer coupled with OptiFlow Turbo V Source. Three immuno-capture-LC-MS/MS assays were developed to selectively measure free, drug-bound and total IL-8 (both free and drug-bound IL-8). Surrogate matrix of intestinal tissue homogenates from mouse/non-human primate with exogenously added recombinant IL-8 was used during assay development to determine assay sensitivity and to test if IL-8-antibody complex can be formed ex vivo. Using this system, multiple antibodies were screened for selectivity and cross-reactivity. The assay LLOQ was optimized to the low pg/mL level to accommodate the low endogenous levels of IL-8 as well as the limited sample size. In subsequent tests with human intestinal and tumor tissue samples, the assays were used to quantify levels of unbound and total IL-8 levels and the results were comparable. In addition, the method was also optimized to avoid shifting of the equilibrium between the target and drug binding during the tissue homogenization and downstream sample processing. The blood contamination impacted on the drug concentration measurement in tissue will be discussed. The developed assays have been applied to support a phase I clinical study.
Participants will have in-depth understanding of the strategy for target engagement evaluation in tissue biopsies using immunocapture LC-MS/MS assays.
This presentation will describe the methodology, including the challenges and issues, for immunocapture LC-MS/MS assay development for total drug, total and free target, and target-drug complex measurement in tissue samples.
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