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

Abstract

critical quality attribute for both characterization and engineering of mAbs that, based on available structures, is relevant to many potential therapeutic antibodies. Results & discussion To investigate the apparent heterogeneity of 10E8 we performed SEC under a series of conditions. As reported previously 7,8,22 , 10E8 showed elution profiles with multiple peaks. When performing SEC with a monolayer coated silica matrix (Sepax, SRT), three baseline resolved peaks (here denoted as "SEC peaks 1-3") were observed that eluted much later than expected based on standard protein molecular weight markers (Fig. 1A). Using a lay-down monolayer coated silica SEC column (Sepax, SRT-C) alleviated the extensive column retention, but still showed three partially resolved peaks (Fig. 1B). Previous studies have shown that the SEC resolved 10E8 peaks corresponded to single IgGs that are in a slow dynamic equilibrium 7,8,22. To confirm that our 10E8 was similarly behaved we performed SEC with multi-angle light scattering (MALS) and observed three peaks, each of which had a molecular mass near 150 kDa consistent with an intact IgG, as reported previously (Fig. S1). To quantitatively investigate the dynamic equilibrium among the isomers, we performed preparative SEC to collect individual species and reinject them over SEC after a range of delays to measure the kinetics of isomerization. When the last eluting peak ("peak 3") was reinjected, after 30 min it only partially re-equilibrated, but after 2.5 h it yielded a SEC profile identical to the starting material (Fig. S2). Conversely, peak 1 or peak 2 reinjected after 12 h also yielded an identical SEC profile as the starting material. From the time points collected, the rate of inter-conversion at 25 °C from isolated peak 3 back to the equilibrium distribution was found to be 0.00034 ± 0.00004 s-1 , corresponding to a half-life of 34 min. To test for any biologically relevant phenotypic differences among the SEC resolved isomers we isolated each fraction and performed a rapid ELISA against HIV gp41 (see "Methods"). Being mindful of the isomerization

Combination of IM-Based Approaches to Unravel the Coexistence of Two Conformers on a Therapeutic Multispecific mAb

“…Guttman et al also demonstrated that this YPP motif could be responsible for the SEC heterogeneity. 14 For the tsAb studied in the present work, the YPP motif was shown to be essential for the optimal antigen binding as various mutations within the motif led to significant loss of affinity to the target. 11 Besides, previous work demonstrated that, despite a fast sequestration of the more affine cis conformer by the antigen and a much slower binding for the trans conformer, the potency of the molecule was not impacted by the presence of two isomers due to consequent re-equilibrium of the conformers.…”

“…First results revealed a conformational switching in complementarity determining regions (CDR) of the CODV arm due to a specific motif containing proline residues known to induce cis and trans isomers. − More specifically, the tyrosine–proline–proline (YPP) motif of the heavy chain CDR3 (variable part targeting MPER epitope) was evidenced as playing a key role in isomerization through the interaction with a histidine residue of the light chain. Guttman et al also demonstrated that this YPP motif could be responsible for the SEC heterogeneity . For the tsAb studied in the present work, the YPP motif was shown to be essential for the optimal antigen binding as various mutations within the motif led to significant loss of affinity to the target .…”

Synthesis of fluorescence labelled aptamers for use as low‑cost reagents in HIV/AIDS research and diagnostics

“…To achieve this, a HIV-1 capture assay was performed ( 32 ) with three HIV-1 subtype C pseudoviruses, ZM53M.PB12, DU156.12 and CAP210.E8, known to interact with CSIR1.1( 10 ). The virus was captured with the 10E8 mAb ( 38 ) that binds gp41 and detected using different concentrations of the FITC-CSIR1.1 aptamer ranging from 0.00-0.05 mg/ml. The choice of 10E8 mAb for capture was because it binds the membrane-proximal external region of the viral glycoprotein, thus making it less likely to compete with the aptamer for binding to the envelope ( 39 ).…”

Early-Stage Oligomerization of Prion-like Polypeptides Reveals the Molecular Mechanism of Amyloid-Disrupting Capacity by Proline Residues

“…The influence of such conformational change on protein binding kinetics is found to be comprehensive; it changes the rate of binding, the kinetic pathway, as well as the global geometry . Recent studies have also shown that proline cis – trans isomerization plays a key role in dimerization of cyclopeptides, amyloid aggregation, , and the development of antibody biotheurapeutics . These studies all highlight the effect of torsional conformational constraints on the shape-complementarity between protein partners, which is able to propagate to a protein global property change via mechanical coupling, thereby modulating binding thermodynamics and kinetics.…”