Engineering an anti-CD52 antibody for enhanced deamidation stability

Abstract

Deamidation evaluation and mitigation is an important aspect of therapeutic antibody developability assessment. We investigated the structure and function of the Asn-Gly deamidation in a human anti-CD52 IgG1 antibody light chain complementarity-determining region 1, and risk mitigation through protein engineering. Antigen binding affinity was found to decrease about 400-fold when Asn33 was replaced with an Asp residue to mimic the deamidation product, suggesting significant impacts on antibody function. Other variants made at Asn33 (N33H, N33Q, N33H, N33R) were also found to result in significant loss of antigen binding affinity. The co-crystal structure of the antigen-binding fragment bound to a CD52 peptide mimetic was solved at 2.2Å (PDB code 6OBD), which revealed that Asn33 directly interacts with the CD52 phosphate group via a hydrogen bond. Gly34, but sits away from the binding interface, rendering it more amendable to mutagenesis without affecting affinity. Saturation mutants at Gly34 were prepared and subjected to forced deamidation by incubation at elevated pH and temperature. Three mutants (G34R, G34K and G34Q) showed increased resistance to deamidation by LC-MS peptide mapping, while maintaining high binding affinity to CD52 antigen measured by Biacore. A complement -dependent cytotoxicity assay indicated that these mutants function by triggering antibody effector function. This study illustrates the importance of structure-based design and extensive mutagenesis to mitigate antibody developability issues.

Antibody Structure and Function: The Basis for Engineering Therapeutics

“…Protein degradation [220][221][222]; Tertiary changes to Ab structure [223]; Isoaspartic acid [224]; Aggregation [225] Isomerization can affect IgG avidity [226]; Deamidation affects binding [227]; Deamidation affects PK [216] Gln Gln deamidation Slower deamidation than Asn, heterogeneity and stability [228] Biological activity on Fab and Fc *…”

Application of an integrated computational antibody engineering platform to design SARS-CoV-2 neutralizers

“…The mutations in SARS-CoV-2 from SARS-CoV RBD prevent these antibodies, except for CR3022, blocking the SARS-CoV-2 RBD–ACE2 interactions [ 36 , 37 ]. Therefore, we sought to engineer the three antibodies to make them reactive against SARS-CoV-2 by using our structure-based rational engineering platform with publicly available structures of the Fab in complex with RBD [ 38 ]. The epitopes of these three antibodies are located in relatively conserved surfaces on the RBD ( Fig.…”

Application of an integrated computational antibody engineering platform to design SARS-CoV-2 neutralizers

“…The mutations between SARS-CoV-2 and SARS-CoV RBD make these neutralizers not immediately applicable to block the RBD-ACE2 interactions [36,37]. Publicly available high-quality structures of Fab in complex with RBD allow us to quickly design SARS-CoV-2 binders through our structure-based rational engineering platform, which has been serving our cross reactivity and affinity maturation engineering purposes in biologics projects [38]. The epitopes of these three antibodies are located in relatively conserved surfaces on the RBD ( Figure 1A&B).…”