Design, development and characterization of ACT017, a humanized Fab that blocks platelet's glycoprotein VI function without causing bleeding risks

Abstract

Glycoprotein VI is a platelet-specific collagen receptor critical for in vivo formation of arterial thrombosis. It is also considered as an attractive target for the development of anti-thrombotic drugs because blocking glycoprotein (GP)VI inhibits platelet aggregation without inducing detrimental effects on physiologic hemostasis.Here, we present data on the identification, in vitro and ex vivo pharmacology of a humanized Fab fragment designated as ACT017. ACT017 was selected out of 15 humanized variants based upon structural and functional properties. It was produced under GMP-like conditions followed by detailed physico-chemical analysis and functional characterization indicating high antigen-binding specificity and affinity. In addition, we demonstrate, in a dose-escalation study, that ACT017 has a high capacity to specifically inhibit collagen-induced platelet aggregation ex vivo after injection to the macaque without inducing thrombocytopenia, GPVI depletion or bleeding side effects as is the case for conventional anti-platelets. Therefore, ACT017 is a promising therapeutic candidate for the development of a new generation of safe and efficient anti-thrombotic drugs.

Glycoprotein VI in securing vascular integrity in inflamed vessels

“…Consistent with observations made in GPVI‐deficient patients, inhibition, immunodepletion, or genetic deletion of GPVI in animals only had minor consequences on primary hemostasis . In contrast to its limited impact on primary hemostasis, GPVI deficiency conferred remarkable protection against thrombosis in a variety of in vitro and in vivo experimental models, including flow chamber‐based assays using human atherosclerotic plaque material . For these reasons, and despite some controversies, GPVI has been proposed as a promising target for antithrombotic therapy with reduced bleeding risk as compared to current antiplatelet therapies based on administration of aspirin and/or P2Y12 inhibitors …”

“…42,43 In contrast to its limited impact on primary hemostasis, GPVI deficiency conferred remarkable protection against thrombosis in a variety of in vitro and in vivo experimental models, including flow chamber-based assays using human atherosclerotic plaque material. 27,33,[42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58] For these reasons, and despite some controversies, GPVI has been proposed as a promising target for antithrombotic therapy with reduced bleeding risk as compared to current antiplatelet therapies based on administration of aspirin and/or P2Y12 inhibitors. 42,43 The mild bleeding manifestations in patients with GPVI deficiency suggest that either GPVI plays a modest role in hemostasis or, more likely, that its absence is overcome by compensatory and/ or redundant mechanisms.…”

Loxoscelism: Advances and Challenges in the Design of Antibody Fragments with Therapeutic Potential

“…We also successfully engineered a PpL binding motif in order to make the purification of antibody fragments using affinity chromatography possible, without requiring the insertion of an epitope tag. This strategy is essential considering future pharmaceutical developments [38][39][40]. The monomeric scFv was produced in bacteria and characterized from a physico-chemical point of view.…”

“…The decrease in the affinity for the toxin and potentially the non-optimal thermal stability we observed for scFv 15 hLi7 could be related to some of the mutations that also altered the VH/VL packing angle. Recent studies have shown that a high degree of cooperation between the VH/VL is required for mutual stabilization and also that a limited number of residues buried inside the antibody domain are critical to maintain the topography of the antigen-binding site [25,39,60]. The mutation H80 A > R slightly impacts the canonical H2 conformation.…”

Exploration and Modulation of Antibody Fragment Biophysical Properties by Replacing the Framework Region Sequences

“…When re-engineering variable domains, affinity maturation and antibody humanization are carefully considered and worked on [11][12][13][14][15][16]. In some cases, mutations in the antibody complementarity determining regions (CDRs) have shown to improve molecule stability [17,18] thus being able to repair intrinsic flaws in the packing between two V-domains [19].…”