Contributing to the Development of New Anti-Thrombotic Biotherapeutics
Two Peak/Sygnature scientists, Juli Warwicker and Mark Abbott, have recently co-authored a paper on a new family of anti-thrombotic biotherapeutics. The paper is from the lab of Dr. Yotis Senis, Research Director, INSERM, Strasbourg, France. The Senis lab have christened the term CAPRIs (cis-acting platelet receptor inhibitors). CAPRIs are bivalent single chain Fvs (scFv) that act by clustering the platelet inhibitory receptor G6b-B (G6B, MPIG6B) with the platelet stimulatory receptors GPVI-FcR γ-chain or FcγRIIA. When this clustering occurs platelet activation is blocked in a number of different cellular assays, thus laying the foundation for a novel class of anti-thrombotics with potential for greater efficacy (Figure. 1).
Figure 1: Structural models of ‘receptor bridging complexes’ involving G6b-B, FcγRIIA and their CAPRI (modelling by Dr. Klaus Fütterer, Biosciences, University of Birmingham, UK)
Peak/Sygnature designed and produced these bivalent scFvs. (Example in Figure. 2 below).
Figure 2: Example of the construct design, the G6b-B-GPVI bi-specific ScFv
The design was of a VL1-VH1—VH2-VL2 with a long Gly-Ser linker between the light and heavy variable domains of each scFv and a shorter Gly-Ser linker between the two VH domains. They were designed as a single polypeptide. A 6His tag was placed at the C-terminus to facilitate purification and the proteins produced by transient expression from HEK293 cells. In addition a series of control reagents were also produced as either Fabs or scFvs. Yields were quite variable amongst the different Bi-scFvs and control reagents but without any optimisation the best reached levels over 100 mg/L showing the use of transient mammalian expression to quickly generate useful quantities of protein for research.
Peak/Sygnature has previously worked with Professor Senis to solve the crystal structure of G6b in the presence of a Fab (one of the antibodies used in the current paper) and an endogenous heparin ligand. (PDB 6R0X).
References
Mazharian, Alexandra, et al. “G6b-B antibody-based cis-acting platelet receptor inhibitors (CAPRIs) as a new family of anti-thrombotic therapeutics.” bioRxiv (2024): 2024-05.
Vögtle, Timo, et al. “Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B.” Elife 8 (2019): e46840.
