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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 deliv-ery 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 homogenous 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 multi-step process-es 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 plat-form to rapidly uncover new conjugation sites for site-specific ADCs.