High-Throughput Platform to Identify Antibody Conjugation Sites from Antibody–Drug Conjugate Libraries

Antibody–drug
conjugates (ADCs) are a therapeutic modality
that traditionally enable the targeted delivery of highly potent cytotoxic
agents to specific cells such as tumor cells. More recently, antibodies
have been used to deliver molecules such as antibiotics, antigens,
and adjuvants to bacteria or specific immune cell subsets. Site-directed
mutagenesis of proteins permits more precise control over the site
and stoichiometry of their conjugation, giving rise to homogeneous
chemically defined ADCs. Identification of favorable sites for conjugation
in antibodies is essential as reaction efficiency and product stability
are influenced by the tertiary structure of immunoglobulin G (IgG).
Current methods to evaluate potential conjugation sites are time-consuming
and labor intensive, involving multistep processes for individually
produced reactions. Here, we describe a highly efficient method for
identification of conjugatable genetic variants by analyzing pooled
ADC libraries using mass spectrometry. This approach provides a versatile
platform to rapidly uncover new conjugation sites for site-specific
ADCs.