Bjelke, J. R. | bioRxiv Science

bioRxiv2023

Non-specificity fingerprints for clinical stage antibodies in solution

Monoclonal antibodies (mAbs) have successfully been developed for the treatment of a wide range of diseases. The clinical success of mAbs, does not solely rely on optimal potency and safety, but also require good biophysical properties to ensure high developability potential. In particular, non-specific interactions are a key developability measure to monitor during discovery. Despite an increased focus on the detection of non-specific interactions, their physicochemical origins remain poorly understood. Here, we employ solution-based microfluidic technologies to characterise a set of clinical stage mAbs and their interactions with commonly used non-specificity ligands to generate non-specificity fingerprints, providing quantitative data on the underlying physical chemistry. Furthermore, the solution-based analysis enables us to evaluate the contribution of avidity in non-specific binding by mAbs. Based on our findings, we propose a quantitative solution-based non-specificity score, which can be exploited in the development of biological therapeutics and more widely in protein engineering.

biophysics↗

bioRxiv2021

Prolonging coagulant activity of factor Xa under hemophilic conditions by site-specific N-glycosylation of the surface-exposed autolysis loop

The regulation of Factor X (FX) is critical to maintain hemostasis. To gain insights to the regulation of the active and zymogen form of coagulation FX, we probed specific molecular interactions by introducing novel N-linked glycosylations on the surface-exposed loop spanning residues 143-150 (chymotrypsin numbering) of FX. Introduction of N-glycans in the autolysis loop of these FX variants decreased Factor VIIa (FVIIa)-mediated activation ~3-fold and prothrombin activation 2- to 10-fold presumably through steric hinderance. Prothrombin activation was, however, recovered in presence of cofactor Factor Va (FVa) despite a reduced prothrombinase assembly. The introduced N-glycans exhibited position-specific effects on the interaction with two FXa inhibitors: tissue factor pathway inhibitor (TFPI) and antithrombin (ATIII). Ki for the inhibition by full-length TFPI of these FXa variants was increased by 7- to 1150-fold, while ATIII inhibition in the presence of the heparin-analogue Fondaparinux was modestly increased by 2- to 15-fold compared to wild type. To probe the in vitro hemostatic effect of the FX variants, the thrombin generation potential in FX-depleted plasma was evaluated. When supplemented in zymogen form, the FX variants exhibited reduced thrombin generation activity relative to wild-type FX, whereas enhanced procoagulant activity was measured for activated FX variants with N-glycosylation at positions 148-150. These results indicate that residues of the surface-exposed autolysis loop and residues close by participate in FX activation, proteolytic activity and inhibition of FXa by TFPI and ATIII. In plasma-based assays, a modest decrease in FX-activation rate appeared to compensate for the collective reduction in inhibitor interactions.

biochemistry↗