title: Total antibody quantification for MMAE-conjugated antibody-drug conjugates: impact of assay format and reagents.
abstract: 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.
authors.affiliation: Department of Biochemical and Cellular Pharmacology, Genentech, Inc., South San Francisco, CA 94080, USA. kkozak@gene.com
authors.forename: Katherine R
authors.initials: KR
authors.lastname: Kozak
authors.forename: Siao Ping
authors.initials: SP
authors.lastname: Tsai
authors.forename: Aimee
authors.initials: A
authors.lastname: Fourie-O'Donohue
authors.forename: Josefa
authors.initials: J
authors.lastname: dela Cruz Chuh
authors.forename: Leslie
authors.initials: L
authors.lastname: Roth
authors.forename: Ryan
authors.initials: R
authors.lastname: Cook
authors.forename: Elton
authors.initials: E
authors.lastname: Chan
authors.forename: Pamela
authors.initials: P
authors.lastname: Chan
authors.forename: Martine
authors.initials: M
authors.lastname: Darwish
authors.forename: Rachana
authors.initials: R
authors.lastname: Ohri
authors.forename: Helga
authors.initials: H
authors.lastname: Raab
authors.forename: Crystal
authors.initials: C
authors.lastname: Zhang
authors.forename: Kedan
authors.initials: K
authors.lastname: Lin
authors.forename: Wai Lee T
authors.initials: WL
authors.lastname: Wong
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000493:pharmacokinetics*; D004797:Enzyme-Linked Immunosorbent Assay; D000922:Immunotoxins / Q000737:chemistry / Q000493:pharmacokinetics*; D051379:Mice; D016513:Mice, SCID; D009842:Oligopeptides / Q000737:chemistry / Q000493:pharmacokinetics*; D050257:Tubulin Modulators / Q000737:chemistry / Q000493:pharmacokinetics*
chemical_list: D000970:Antineoplastic Agents; D000922:Immunotoxins; D009842:Oligopeptides; D050257:Tubulin Modulators; C495575:monomethyl auristatin E