A methodology was developed for the isolation of cell surface, intracellular and extracellular PD-1 with cell surface labeling technique using mouse T cell line 3A9 transfected with human PD-1. By using Pierce Cell Surface Protein Isolation Kit from Thermo Scientific, cell surface PD-1, intracellular PD-1 and extracellular PD-1 were successfully isolated and quantified using immunocapture LC-MS/MS analysis. To improve the measurement accuracy, an in-sample calibration curve using multiple isotopologue reactive monitoring technique was used to quantify the PD-1 levels in different isolated samples. The measurement of RO of an anti-PD-1 drug’s binding on PD-1 in tumor tissue samples is critical to establishing the PK/PD relationship. Traditionally, free and total target, total drug and target/drug complex could be measured with immuno-capture LC-MS/MS assays using tumor tissue homogenates. However, as only cell surface PD-1 is active, the data generated with the traditional approach may not reflect the actual RO. Therefore, it is highly desired to develop a procedure capable of differentiating surface PD-1 from extracellular and intracellular PD-1 proteins. By using a cell surface protein isolation kit from ThermoFisher, extracellular, intracellular and cell surface PD-1 were successfully isolated for mouse T cell line 3A9 transfected with human PD-1. Extracellular PD-1 was harvested in the supernatant after low speed centrifugation (sample 1). After cells were washed with PBS buffer twice (sample 2 and 3), the harvested cells were labeled with cell impermeant cell surface labeling reagent, Sulfo-NHS-SS-Biotin, followed by quench with glycine (sample 4). The labeled samples were washed twice (sample 5 and 6) before cell lysis. The labeled cell surface proteins were then isolated using streptavidin magnetic beads (sample 7), while intracellular non-labeled proteins were collected in the supernatant (sample 8). All samples were further enriched using an anti-PD-1 mAb followed by tryptic digestion and LC-MS/MS analysis of the PD-1 surrogate peptide, LAAFPEDR. Preliminary quantitative results for extracellular, intracellular and cell surface PD-1 for transfected mouse T cell line 3A9 indicated that the established isolation and quantitation procedure is reliable and reproducible. Future work will focus on the determination of cell surface and intracellular PD-1 using human primary T cells or single cell suspensions generated from human tumor tissue samples. It is expected that the data generated from human primary T cells are more representative for better estimation of tissue RO in clinical studies.
The presentation will describe the developed methodology to isolate cell surface, extracellular and intracellular proteins for hybrid LC-MS/MS analysis.
Discuss and learn from each other by sharing audience’s experience and considerations to further optimize the methodology to isolate and quantify cell surface, extracellular and intracellular proteins.
The presentation will propose an approach for accurate assessment of RO by differentiating the origins of the target proteins for better PK/PD analysis.