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_transP_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.

Keywords: Proline isomerization; antibody conformers; chemical equilibrium; developability.

Figure 1.

(a) UHPLC-SEC of SAR441236 showing the two peaks profile. (b) Apparent mass and hydrodynamic radius (Rh) by SEC-LS of SAR441236. Both peaks show the same apparent molecular weight in SEC-LS = ~200kDa and a Rh = ~6.4 nm. (c) Deconvoluted MS spectrum of deglycosylated SAR441236 in denaturing conditions. (d) SEC profile of each fraction (Fraction 1 – black, Fraction 2 – blue) 30–60 min after collections (left) and after 72 h (right) at 8°C in Dulbecco phosphate-buffered saline (DPBS2X). (e) Kinetics of conformer ratio after 5 times dilution in pH 5.0 buffer and 5 times dilution in pH 7.0 buffer. Conformer I “open” is depicted in blue empty squares, conformer II “closed” is depicted in red empty circles

Figure 2.

(a) Enzymatic cleavage, (b) total ion current (TIC), (c)–(e) deconvoluted spectra of the three major peaks. The peak of each conformer (conformation I – blue and conformation II – red) found in the CODV arm shows the same mass. (b)–(e) The blue arrow on the y-axis is an annotation threshold given by the software representing the lowest threshold to show peaks annotation

Figure 3.

Structural superposition between 10e8v4 CODV and Fab crystal structures. (a) Highlighted difference (red circle) between 10e8v4 HCDR3 of CODV (purple) – “open” conformation (conformation I) and Fab (blue) – “closed” conformation (conformation II). (b) The trans dihedral angle of the CODV P113 (cyan) and the cis dihedral angle of Fab P113 (yellow). (c) Topology of H147 with respect to YPP motif (heavy chain – red) and R207 (light chain – blue)

Figure 4.

ELISA optical density (OD) at different concentrations for (a) reference batch vs conformer I “open”, (b) reference batch vs conformer II “closed” and (c) conformer I “open” vs conformer II “closed”

Figure 5.

Plot of the kinetic of the three peaks (Conformer I “open” – blue/Conformer II “closed” – red/complex mAb-Ag – green) followed by LC/MS, based on the peak area surface

Figure 6.

(a) Heat map of the preferred P112-P113 conformations obtained by 4 × 100 ns accelerated molecular dynamics. The most populated transition state is highlighted within orange-dotted box. A dihedral value of 0° corresponds to the cis conformers, values close to 180° or −180°correspond to trans conformers. (b) Histogram plot of the dihedral angle of P112 (red) and P113 (green) vs the relative dihedral population density

Figure 7.

(a) Native SEC analysis UV trace of the mutants versus the reference material and (b) ELISA binding curves of the reference batch (black), Var1 (blue), Var2 (red), Var3 (green), Var4 (purple) and Var5 (cyan)