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

Abstract

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.

Chemical Diversification of Simple Synthetic Antibodies

“…The binding and reactive properties of clones identified in this study highlight the potentially broad scope of antibody modifications achievable with additional chemical functionalization. However, we recognize that there are numerous effective bioconjugation strategies that may be applied to prepare chemically diversified antibodies, including thiol modification strategies, 22,78 enzymatic modification strategies, 23 or emerging conjugation reactions at other canonical amino acids. 79,80 In this regard, the simple synthetic antibodies and characterization strategies elucidated in this work could facilitate side-by-side evaluations of multiple chemical modification strategies.…”

“…32,[35][36][37][38] Numerous approaches to antibody chemical diversification have been explored extensively within the context of antibody-drug conjugates (ADCs). [39][40][41][42][43][44][45][46] However, most ADC development strategies focus on modification sites located in antibody constant regions that have little or no effect on antigen binding; [46][47][48][49][50][51][52] the result is modular addition of new chemistries that leave antibody binding function unchanged. In contrast, only sparse studies exist that examine the effects of adding chemical functionality near antibody complementarity determining regions (CDRs).…”

“…Several strategies for expanding the chemistries within antibodies are feasible, , including post-translational modification of antibody structures containing canonical amino acids − and the introduction of genetically encoded noncanonical amino acids (ncAAs). , Numerous approaches to antibody chemical diversification have been explored extensively within the context of antibody–drug conjugates (ADCs). − However, most ADC development strategies focus on modification sites located in antibody constant regions that have little or no effect on antigen binding; − the result is modular addition of new chemistries that leave antibody binding function unchanged. In contrast, only sparse studies exist that examine the effects of adding chemical functionality near antibody CDRs.…”

Impact of Drug Conjugation on Thermal and Metabolic Stabilities of Aglycosylated and N-Glycosylated Antibodies

“…Our recent screening led to the identification of several genetic variants containing the engineered glutamine tag that permits coupling of linker payload into the C’E loop where N -glycosylation occurs. 18 Using these constructs, we tested the impact of loading drug linkers on the thermal stability of glycosylated and aglycosylated antibodies. In general, conjugating the drug to a glycosylated antibody resulted in decreased thermal stability, whereas attaching the drug to an aglycosylated antibody led to an increased melting temperature for some of the constructs.…”

Site-selective modification strategies in antibody–drug conjugates

“…Yamazoe et al have recently reported a high-throughput protocol for assisting the determination of optimal locations for drug conjugation. 290 mTG-conjugation was utilised to generate a library of ADCs from a pool of mutated antibodies, which were analysed and validated via MS. The screening allowed for several new tags and sites to be identified and may prove to be an important tool for future development and optimisation of homogeneous ADCs.…”