TY - JOUR
T1 - Pharmacodynamic assessment of cell-bound natalizumab on PBMC samples stored in liquid nitrogen
AU - ten Brinke, Anja
AU - Claessen, Iris
AU - van Kempen, Zoé L. E.
AU - Killestein, Joep
AU - Rispens, Theo
PY - 2019/10
Y1 - 2019/10
N2 - Natalizumab is a monoclonal IgG4 antibody used for treatment of relapsing remitting MS. Natalizumab interferes with lymphocyte migration by blocking alpha-4 integrin (CD49d). Saturation levels of alpha-4 integrin on circulating T cells by natalizumab have been associated with clinical effectiveness of therapy. However, in most cases, measurements have been carried out using freshly isolated PBMCs. The aim of this study was to set up and evaluate a method to measure relative levels of cell-bound natalizumab using frozen PBMC samples. A new method was set up to measure cell-bound natalizumab by flow cytometry on T cell subsets using fully saturated cells as a 100% reference. A comparison was made between spike samples and samples of natalizumab-treated MS patients freshly isolated and stored in liquid nitrogen. Cell-bound natalizumab could be measured (using an anti-IgG4 antibody) on cells stored in liquid nitrogen. Natalizumab was found to slowly dissociate from the cells during isolation and subsequent sample work-up. This dissociation was more pronounced for monovalent natalizumab resulting from Fab arm exchange (the predominant isoform in patients) than bivalent natalizumab straight from the vial. We established a correction factor to account for this phenomenon. The resulting method has good accuracy compared to assessing fresh cells. The inter-assay precision (%CV) is ca. 12% using frozen cells. In conclusion, we established a method to assess relative levels of cell-bound natalizumab on cells obtained from frozen PBMC samples.
AB - Natalizumab is a monoclonal IgG4 antibody used for treatment of relapsing remitting MS. Natalizumab interferes with lymphocyte migration by blocking alpha-4 integrin (CD49d). Saturation levels of alpha-4 integrin on circulating T cells by natalizumab have been associated with clinical effectiveness of therapy. However, in most cases, measurements have been carried out using freshly isolated PBMCs. The aim of this study was to set up and evaluate a method to measure relative levels of cell-bound natalizumab using frozen PBMC samples. A new method was set up to measure cell-bound natalizumab by flow cytometry on T cell subsets using fully saturated cells as a 100% reference. A comparison was made between spike samples and samples of natalizumab-treated MS patients freshly isolated and stored in liquid nitrogen. Cell-bound natalizumab could be measured (using an anti-IgG4 antibody) on cells stored in liquid nitrogen. Natalizumab was found to slowly dissociate from the cells during isolation and subsequent sample work-up. This dissociation was more pronounced for monovalent natalizumab resulting from Fab arm exchange (the predominant isoform in patients) than bivalent natalizumab straight from the vial. We established a correction factor to account for this phenomenon. The resulting method has good accuracy compared to assessing fresh cells. The inter-assay precision (%CV) is ca. 12% using frozen cells. In conclusion, we established a method to assess relative levels of cell-bound natalizumab on cells obtained from frozen PBMC samples.
KW - CD49d
KW - IgG4
KW - Integrin
KW - Natalizumab
KW - Receptor saturation
UR - https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85069216845&origin=inward
UR - https://www.ncbi.nlm.nih.gov/pubmed/31306641
U2 - https://doi.org/10.1016/j.jim.2019.07.004
DO - https://doi.org/10.1016/j.jim.2019.07.004
M3 - Article
C2 - 31306641
SN - 0022-1759
VL - 473
JO - Journal of immunological methods
JF - Journal of immunological methods
M1 - 112632
ER -