Computational design of N-linked glycans for high throughput epitope profiling.

Efficient identification of epitopes is crucial for drug discovery and design as it enables the selection of optimal epitopes, expansion of lead antibody diversity, and verification of binding interface. Although high-resolution low throughput methods like x-ray crystallography can determine epitopes or protein-protein interactions accurately, they are time-consuming and can only be applied to a limited number of complexes. To overcome these limitations, we have developed a rapid computational method that incorporates N-linked glycans to mask epitopes or protein interaction surfaces, thereby providing a mapping of these regions. Using human coagulation factor IXa (fIXa) as a model system, we computationally screened 158 positions and expressed 98 variants to test experimentally for epitope mapping. We were able to delineate epitopes rapidly and reliably through the insertion of N-linked glycans that efficiently disrupted binding in a site-selective manner. To validate the efficacy of our method, we conducted ELISA experiments and high-throughput yeast surface display assays. Furthermore, x-ray crystallography was employed to verify the results, thereby recapitulating through the method of N-linked glycans a coarse-grained mapping of the epitope.

A factor VIIIa-mimetic bispecific antibody, Mim8, ameliorates bleeding upon severe vascular challenge in hemophilia A mice.

Hemophilia A is a bleeding disorder resulting from deficient factor VIII (FVIII), which normally functions as a cofactor to activated factor IX (FIXa) that facilitates activation of factor X (FX). To mimic this property in a bispecific antibody format, a screening was conducted to identify functional pairs of anti-FIXa and anti-FX antibodies, followed by optimization of functional and biophysical properties. The resulting bispecific antibody (Mim8) assembled efficiently with FIXa and FX on membranes, and supported activation with an apparent equilibrium dissociation constant of 16 nM. Binding affinity with FIXa and FX in solution was much lower, with equilibrium dissociation constant values for FIXa and FX of 2.3 and 1.5 µM, respectively. In addition, the activity of Mim8 was dependent on stimulatory activity contributed by the anti-FIXa arm, which enhanced the proteolytic activity of FIXa by 4 orders of magnitude. In hemophilia A plasma and whole blood, Mim8 normalized thrombin generation and clot formation, with potencies 13 and 18 times higher than a sequence-identical analogue of emicizumab. A similar potency difference was observed in a tail vein transection model in hemophilia A mice, whereas reduction of bleeding in a severe tail-clip model was observed only for Mim8. Furthermore, the pharmacokinetic parameters of Mim8 were investigated and a half-life of 14 days shown in cynomolgus monkeys. In conclusion, Mim8 is an activated FVIII mimetic with a potent and efficacious hemostatic effect based on preclinical data.

Mutational scanning of the human serotonin transporter reveals fast translocating serotonin transporter mutants.

The serotonin transporter (SERT) belongs to a family of sodium-chloride-dependent transporters responsible for uptake of amino acids and biogenic amines from the extracellular space. SERT represents a major pharmacological target in the treatment of several clinical conditions, including depression and anxiety. In the present study we have undertaken a mutational scanning of human SERT in order to identify residues that are responsible for individual differences among related monoamine transporters. One mutant, G100A, was inactive in transport. However, ligand binding affinity was similar to wild-type, suggesting that G100A amongst different possible SERT conformations is restrained to a binding conformation. We suggest that the main role of glycine-100 is to confer structural flexibility during substrate translocation. For the two single mutants, T178A and F263C, uptake rates and K(m) values were both several-fold higher than wild-type while binding affinities and inhibitory potencies decreased considerably for several drugs. Ion dependency increased and only at hyperosmotic concentrations were K(m) values partly restored. For the double mutant, T178A/F263C, shifts in uptake kinetics and ligand affinities, as well as ion dependencies, were drastic. Effects were synergistic compared to the corresponding single mutants. In conclusion, we suggest that mutating threonine-178 to an alanine and phenylalanine-263 to a cysteine mainly alter the overall uptake kinetics of SERT by affecting the conformational equilibrium of different transporter conformations.