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Total Antibody Quantification for MMAE-Conjugated Antibody–Drug Conjugates: Impact of Assay Format and Reagents
journal contribution
posted on 2013-05-15, 00:00 authored by Katherine R. Kozak, Siao Ping Tsai, Aimee Fourie-O’Donohue, Josefa dela Cruz Chuh, Leslie Roth, Ryan Cook, Elton Chan, Pamela Chan, Martine Darwish, Rachana Ohri, Helga Raab, Crystal Zhang, Kedan Lin, Wai Lee
T. WongAntibody–drug conjugates (ADCs)
are target-specific anticancer
agents consisting of cytotoxic drugs covalently linked to a monoclonal
antibody. The number of ADCs in the clinic is growing, and therefore
thorough characterization of the quantitative assays used to measure
ADC concentrations in support of pharmacokinetic, efficacy, and safety
studies is of increasing importance. Cytotoxic drugs such as the tubulin
polymerization inhibiting auristatin, monomethyl auristatin E, have
been conjugated to antibodies via cleavable linkers (MC-vc-PAB) through
internal cysteines. This results in a heterogeneous mixture of antibody
species with drug-to-antibody ratios (DAR) ranging from 0 to 8. In
order to characterize the assays used to quantitate total MC-vc-PAB-MMAE
ADCs (conjugated and unconjugated antibody), we used purified fractions
with defined DARs from 6 therapeutic antibodies to evaluate different
assay formats and reagents. Our investigations revealed that for quantitation
of total antibody, including all unconjugated and conjugated antibody
species, sandwich ELISA formats did not always allow for recovery
of all purified DAR fractions (DAR 0–8) to within ±20%
of the expected values at the reagent concentrations tested. In evaluating
alternative approaches, we found that the recovery of DAR fractions
with semihomogeneous assay (SHA) formats, in which sample, capture,
and detection reagents are preincubated in solution, were less affected
by the antibody’s MMAE drug load as compared to traditional
stepwise sandwich ELISAs. Thus, choosing the optimal assay format
and reagents for total antibody assays is valuable for developing
accurate quantitative assays.