TY - JOUR
T1 - Multiparameter Flow Cytometry Assay for Quantification of Immune Cell Subsets, PD‐1 Expression Levels and PD‐1 Receptor Occupancy by Nivolumab and Pembrolizumab
AU - Pluim, Dick
AU - Ros, Willeke
AU - Miedema, IHC
AU - Beijnen, Jos H.
AU - Schellens, J. H.M.S.
N1 - Publisher Copyright: © 2019 International Society for Advancement of Cytometry Copyright: Copyright 2019 Elsevier B.V., All rights reserved.
PY - 2019/10/1
Y1 - 2019/10/1
N2 - We report the development and validation of a 12 parameter immunofluorescence flow cytometry method for the sensitive determination of cell concentrations, their expression of PD-1, and PD-1 receptor occupancy. Cell subsets include CD4 + and CD8 +-T-cells, B-cells, natural killer cells, classical-, intermediate- and non-classical monocytes, and myeloid- and plasmacytoid dendritic cells. Cells were isolated from peripheral blood by density gradient centrifugation. The validation parameters included specificity, linearity, limit of quantification, precision, biological within- and between subject variations. The lower limit of quantification was 5.0% of PD-1 + cells. Samples were stable for at least 153 days of storage at −80°C. The clinical applicability of the method was demonstrated in 11 advanced cancer patients by the successful determination of immune cell concentrations, relative number of PD-1 + immune cells, and number of PD-1 molecules per immune cell. Shortly after infusion of nivolumab, receptor occupancy on CD8 +-T-cells was 98%. Similar values were found predose cycle 2, suggesting receptor occupancy remained high throughout the entire cycle.
AB - We report the development and validation of a 12 parameter immunofluorescence flow cytometry method for the sensitive determination of cell concentrations, their expression of PD-1, and PD-1 receptor occupancy. Cell subsets include CD4 + and CD8 +-T-cells, B-cells, natural killer cells, classical-, intermediate- and non-classical monocytes, and myeloid- and plasmacytoid dendritic cells. Cells were isolated from peripheral blood by density gradient centrifugation. The validation parameters included specificity, linearity, limit of quantification, precision, biological within- and between subject variations. The lower limit of quantification was 5.0% of PD-1 + cells. Samples were stable for at least 153 days of storage at −80°C. The clinical applicability of the method was demonstrated in 11 advanced cancer patients by the successful determination of immune cell concentrations, relative number of PD-1 + immune cells, and number of PD-1 molecules per immune cell. Shortly after infusion of nivolumab, receptor occupancy on CD8 +-T-cells was 98%. Similar values were found predose cycle 2, suggesting receptor occupancy remained high throughout the entire cycle.
KW - PD-1 receptor occupancy
KW - immune cell subsets
KW - immunotherapy
KW - nivolumab
KW - pembrolizumab
KW - pharmacodynamic
UR - http://www.scopus.com/inward/record.url?scp=85070713072&partnerID=8YFLogxK
U2 - https://doi.org/10.1002/cyto.a.23873
DO - https://doi.org/10.1002/cyto.a.23873
M3 - Article
SN - 1552-4930
VL - 95
SP - 1053
EP - 1065
JO - Cytometry Part A
JF - Cytometry Part A
IS - 10
ER -