Multiplexing of receptor occupancy measurements for pharmacodynamic biomarker assessment of biopharmaceuticals

Cytometry. Part B, Clinical Cytometry
Inna VainshteinMeina Liang

Abstract

Receptor occupancy (RO) assays measure drug target engagement, and are used as pharmacodynamic (PD) biomarkers. RO assays are commonly performed by flow cytometry and often require multiplexing for assessment of multiple PD biomarkers when specimen volumes are limited. We present multiplexed RO assays for an IGF1R-EGFR bispecific antibody (Bs-Ab) and a CTLA4-Ig recombinant fusion protein to demonstrate key considerations for accurate RO assessment. RO in cynomolgus monkeys was determined in whole blood using flow cytometry. Free and total receptors were measured using anti-receptor fluorescence-labeled detection reagents, competitive and noncompetitive to drug, respectively. RO of IGF1R was examined as PD for Bs-Ab, since IGF1R was expressed on blood cells. Multiplexed measurements of free and total IGF1R showed that IGF1R expression measured by total receptor was highly variable, impacting interpretation of free-IGF1R. Normalization of free-over-total IGF1R measurements compensated for variability of receptor expression allowing for accurate RO assessment. RO of CTLA4-Ig, a recombinant fusion protein targeting CD80 and CD86 receptors, was multiplexed to simultaneously measure target engagements for both receptors. Both RO meth...Continue Reading

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Citations

Jun 9, 2015·Cytometry. Part B, Clinical Cytometry·Meina LiangLorin K Roskos
Mar 30, 2016·Cytometry. Part B, Clinical Cytometry·Virginia LitwinJennifer J Stewart
Nov 25, 2016·The Journal of Immunology : Official Journal of the American Association of Immunologists·Julie DouthwaiteLutz Jermutus
Dec 2, 2020·Cytometry. Part B, Clinical Cytometry·Ed HiltJennifer J Stewart

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Methods Mentioned

BETA
flow cytometry
ELISA
flow

Software Mentioned

MedImmune

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