Engineering Dual Variable Domains for the Generation of Site-Specific Antibody–Drug Conjugates

Abstract

Site-specific antibody–drug conjugate (ADC) technologies are highly desirable for the production of therapeutics with well-defined biochemical and pharmacological characteristics. We have developed a strategy to produce site-specific ADCs using a highly reactive lysine residue embedded in a dual-variable-domain (DVD) format. Here we provide protocols for the engineering, expression, and purification of the DVDs used for this strategy. We also provide a protocol for DVD–drug conjugation and describe methods for their biochemical characterization, including a catalytic assay to monitor conjugation efficiency.