Total Antibody Quantification for MMAE-Conjugated Antibody鈥揇rug Conjugates: Impact of Assay Format and Reagents

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• 作者：Katherine R. Kozak ; Siao Ping Tsai ; Aimee Fourie-O鈥橠onohue ; 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. Wong
• 刊名：Bioconjugate Chemistry
• 出版年：2013
• 出版时间：May 15, 2013
• 年：2013
• 卷：24
• 期：5
• 页码：772-779
• 全文大小：403K
• 年卷期：v.24,no.5(May 15, 2013)
• ISSN：1520-4812

文摘

Antibody鈥揹rug 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鈥?) to within 卤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鈥檚 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.