title: Site-Specific Tandem Knoevenagel Condensation-Michael Addition To Generate Antibody-Drug Conjugates.
abstract: Expanded ligation techniques are sorely needed to generate unique linkages for the growing field of functionally enhanced proteins. To address this need, we present a unique chemical ligation that involves the double addition of a pyrazolone moiety with an aldehyde-labeled protein. This ligation occurs via a tandem Knoevenagel condensation-Michael addition. A pyrazolone reacts with an aldehyde to generate an enone, which undergoes subsequent attack by a second pyrazolone to generate a bis-pyrazolone species. This rapid and facile ligation technique is performed under mild conditions in the absence of catalyst to generate new architectures that were previously inaccessible via conventional ligation reactions. Using this unique ligation, we generated three site-specifically labeled antibody-drug conjugates (ADCs) with an average of four drugs to one antibody. The in vitro and in vivo efficacies along with pharmacokinetic data of the site-specific ADCs are reported.
authors.affiliation: Catalent Biologics West , 5703 Hollis Street, Emeryville, California 94530, United States.
authors.forename: Romas A
authors.initials: RA
authors.lastname: Kudirka
authors.affiliation: Catalent Biologics West , 5703 Hollis Street, Emeryville, California 94530, United States.
authors.forename: Robyn M
authors.initials: RM
authors.lastname: Barfield
authors.affiliation: Catalent Biologics West , 5703 Hollis Street, Emeryville, California 94530, United States.
authors.forename: Jesse M
authors.initials: JM
authors.lastname: McFarland
authors.affiliation: Catalent Biologics West , 5703 Hollis Street, Emeryville, California 94530, United States.
authors.forename: Penelope M
authors.initials: PM
authors.lastname: Drake
authors.affiliation: Catalent Biologics West , 5703 Hollis Street, Emeryville, California 94530, United States.
authors.forename: Adam
authors.initials: A
authors.lastname: Carlson
authors.affiliation: Catalent Biologics West , 5703 Hollis Street, Emeryville, California 94530, United States.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Bañas
authors.affiliation: Catalent Biologics West , 5703 Hollis Street, Emeryville, California 94530, United States.
authors.forename: Wes
authors.initials: W
authors.lastname: Zmolek
authors.affiliation: Catalent Biologics West , 5703 Hollis Street, Emeryville, California 94530, United States.
authors.forename: Albert W
authors.initials: AW
authors.lastname: Garofalo
authors.affiliation: Catalent Biologics West , 5703 Hollis Street, Emeryville, California 94530, United States.
authors.forename: David
authors.initials: D
authors.lastname: Rabuka
keywords: Antibody−drug conjugate (ADC); bivalent drugs; site-specific bioconjugation; tandem reactions