A Generic Approach for Miniaturized Unbiased High-Throughput Screens of Bispecific Antibodies and Biparatopic Antibody–Drug Conjugates

Abstract

The toolbox of modern antibody engineering allows the design of versatile novel functionalities exceeding nature’s repertoire. Many bispecific antibodies comprise heterodimeric Fc portions recently validated through the approval of several bispecific biotherapeutics. While heterodimerization methodologies have been established for low-throughput large-scale production, few approaches exist to overcome the bottleneck of large combinatorial screening efforts that are essential for the identification of the best possible bispecific antibody. This report presents a novel, robust and miniaturized heterodimerization process based on controlled Fab-arm exchange (cFAE), which is applicable to a variety of heterodimeric formats and compatible with automated high-throughput screens. Proof of applicability was shown for two therapeutic molecule classes and two relevant functional screening read-outs. First, the miniaturized production of biparatopic anti-c-MET antibody–drug conjugates served as a proof of concept for their applicability in cytotoxic screenings on tumor cells with different target expression levels. Second, the automated workflow enabled a large unbiased combinatorial screening of biparatopic antibodies and the identification of hits mediating potent c-MET degradation. The presented workflow utilizes standard equipment and may serve as a facile, efficient and robust method for the discovery of innovative therapeutic agents in many laboratories worldwide.

Welding PROxAb Shuttles: A modular approach for generating bispecific antibodies via site-specific protein-protein conjugation

“…38 However, this is a two-step synthesis which requires to eliminate excess reaction compounds in between. Alternative approaches for generating dual binding constructs include intein-based conjugation 39 , the SypTag/SypCatcher approach 40 , SynAbs strategy 41 or controlled Fab arm exchange 42 . Yet, in contrast to our approach, these further technologies are limited to the use of antibodies or Fc fragments equipped with special functionalities and cannot be applied to off-the-shelf antibodies.…”

“…23 These results indicate, that the conjugation of MIC7 via its N-terminus does not affect its affinity to VHL based PROTACs which is in line with literature findings reporting no loss of PROTAC binding after genetic fusion to antibody C-termini. 42, 47, 48…”

Welding PROxAb Shuttles: A Modular Approach for Generating Bispecific Antibodies via Site-Specific Protein–Protein Conjugation

“…However, this is a two-step synthesis that requires eliminating excess reaction compounds in between. Alternative approaches for generating dual binding constructs include intein-based conjugation, the SypTag/SypCatcher approach, the SynAbs strategy, or controlled Fab arm exchange . Yet, in contrast to our approach, these technologies are limited to the use of antibodies or Fc fragments equipped with special functionalities and cannot be applied to off-the-shelf antibodies.…”