Abstract
Monoclonal antibody (mAb) 10E8 recognizes a highly conserved epitope on HIV and is capable of neutralizing > 95% of circulating viral isolates making it one of the most promising Abs against HIV. Solution instability and biochemical heterogeneity of 10E8 has hampered its development for clinical use. We identify the source of 10E8 heterogeneity being linked to cis/trans isomerization at two prolines within the YPP motif in the CRD3 loop that exists as two predominant conformers that interconvert on a slow timescale. The YtransP conformation conformer can bind the HIV gp41 epitope, while the YcisP is not binding competent and shows a higher aggregation propensity. The high barrier of isomerization and propensity to adopt non-binding competent proline conformers provides novel insight into the slow binding kinetics, low potency, and poor solubility of 10E8. This study highlights how proline isomerization should be considered a critical quality attribute for biotherapeutics with paratopes containing potential cis proline amide bonds.
Highlights
Proline residues are unique amino acids in that they can adopt a stable cis amide bond, with a high barrier of rotation that can act as a conformational switch and plays a critical role in the folding and function of many proteins[10]
We demonstrate that the cis/trans isomerization state of a pair of prolines within the complementarity determining region 3 of the heavy chain (CDRH3) of 10E8 is responsible for the apparent size-exclusion chromatography (SEC) heterogeneity observed by multiple studies
Previous studies have shown that the SEC resolved 10E8 peaks corresponded to single IgGs that are in a slow dynamic equilibrium[7,8,22]
Summary
Proline residues are unique amino acids in that they can adopt a stable cis amide bond, with a high barrier of rotation that can act as a conformational switch and plays a critical role in the folding and function of many proteins[10]. We demonstrate that the cis/trans isomerization state of a pair of prolines within the complementarity determining region 3 of the heavy chain (CDRH3) of 10E8 is responsible for the apparent SEC heterogeneity observed by multiple studies.
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