The impact of proline isomerization on antigen binding and the analytical profile of a trispecific anti-HIV antibody

Abstract

Proline cis-trans conformational isomerization is a mechanism that affects different types of protein functions and behaviors. Using analytical characterization, structural analysis, and molecular dynamics simulations, we studied the causes of an aberrant two-peak size-exclusion chromatography profile observed for a trispecific anti-HIV antibody. We found that proline isomerization in the tyrosine-proline-proline (YPP) motif in the heavy chain complementarity-determining region (CDR)3 domain of one of the antibody arms (10e8v4) was a component of this profile. The pH effect on the conformational equilibrium that led to these two populations was presumably caused by a histidine residue (H147) in the light chain that is in direct contact with the YPP motif. Finally, we demonstrated that, due to chemical equilibrium between the cis and trans proline conformers, the antigen-binding potency of the trispecific anti-HIV antibody was not significantly affected in spite of a potential structural clash of 10e8v4 YP trans P trans conformers with the membrane-proximal ectodomain region epitope in the GP41 antigen. Altogether, these results reveal at mechanistic and molecular levels the effect of proline isomerization in the CDR on the antibody binding and analytical profiles, and support further development of the trispecific anti-HIV antibody.

The influence of proline isomerization on potency and stability of anti-HIV antibody 10E8

“…During the submission stage of this manuscript Masiero et al, reported their findings of proline isomerization within a trispecific mAb that included 10E8v4 Fab (the same 10E8/iMab construct examined here) and the effect was localized to the same YPP motif we have implicated here 40 . Based on molecular dynamics simulations the authors concluded that the two observed conformers reflect the Y trans P trans P and Y trans P cis P forms.…”

Investigation of anomalous charge variant profile reveals discrete pH-dependent conformations and conformation-dependent charge states within the CDR3 loop of a therapeutic mAb

“…34 Most recently, Masiero et al determined that the pH dependence of two interconverting peaks observed in the size exclusion chromatography profile of a trispecific anti-HIV antibody resulted from a conformational equilibria involving proline isomerization in the HC-CDR3 and a neighboring histidine in the light chain. 46 While each of these reports describes one or more of the phenomena we observed during our investigation of mAb-1, none of them account for the totality of our observations.…”

Polarity effects in 4-fluoro- and 4-(trifluoromethyl)prolines

“…Amide-bond rotation is known as an intrinsically slow process, which contributes to the rate-limiting steps in the protein folding [ 91 ] and molecular timing phenomena [ 92 ]. In peptidyl-prolyl bonds, the transition between the rotational states cis -amide and trans -amide usually occurs in the mHz scale, with the barriers of rotation around 80–90 kJ mol −1 (in water).…”