10.1021/bc300491k.s001
Katherine R. Kozak
Katherine R.
Kozak
Siao Ping Tsai
Siao Ping
Tsai
Aimee Fourie-O’Donohue
Aimee
Fourie-O’Donohue
Josefa dela Cruz Chuh
Josefa
dela Cruz Chuh
Leslie Roth
Leslie
Roth
Ryan Cook
Ryan
Cook
Elton Chan
Elton
Chan
Pamela Chan
Pamela
Chan
Martine Darwish
Martine
Darwish
Rachana Ohri
Rachana
Ohri
Helga Raab
Helga
Raab
Crystal Zhang
Crystal
Zhang
Kedan Lin
Kedan
Lin
Wai Lee
T. Wong
Wai Lee
T.
Wong
Total Antibody
Quantification for MMAE-Conjugated
Antibody–Drug Conjugates:
Impact of Assay Format and Reagents
American Chemical Society
2013
assay
reagent
monomethyl auristatin E
MMAE
sandwich ELISA formats
SHA
cytotoxic drugs covalently
measure ADC concentrations
antibody species
DAR fractions
Total Antibody Quantification
2013-05-15 00:00:00
Journal contribution
https://acs.figshare.com/articles/journal_contribution/Total_Antibody_Quantification_for_MMAE_Conjugated_Antibody_Drug_Conjugates_Impact_of_Assay_Format_and_Reagents/2414677
Antibody–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.