Targeting platelet GPVI with glenzocimab: a novel mechanism for inhibition

Abstract

Platelet glycoprotein VI (GPVI) is attracting interest as a potential target for the development of new antiplatelet molecules with a low bleeding risk. GPVI binding to vascular collagen initiates thrombus formation and GPVI interactions with fibrin promote the growth and stability of the thrombus. In this study, we show that glenzocimab, a clinical stage humanized antibody fragment (Fab) with a high affinity for GPVI, blocks the binding of both ligands through a combination of steric hindrance and structural change. A cocrystal of glenzocimab with an extracellular domain of monomeric GPVI was obtained and its structure determined to a resolution of 1.9 Å. The data revealed that (1) glenzocimab binds to the D2 domain of GPVI, GPVI dimerization was not observed in the crystal structure because glenzocimab prevented D2 homotypic interactions and the formation of dimers that have a high affinity for collagen and fibrin; and (2) the light variable domain of the GPVI-bound Fab causes steric hindrance that is predicted to prevent the collagen-related peptide (CRP)/collagen fibers from extending out of their binding site and preclude GPVI clustering and downstream signaling. Glenzocimab did not bind to a truncated GPVI missing loop residues 129 to 136, thus validating the epitope identified in the crystal structure. Overall, these findings demonstrate that the binding of glenzocimab to the D2 domain of GPVI induces steric hindrance and structural modifications that drive the inhibition of GPVI interactions with its major ligands.

Graphical abstract

Figure 1.

Inhibitory properties of glenzocimab. (A) Glenzocimab inhibits collagen-induced platelet activation. Washed human platelets were preincubated with glenzocimab or the 3J24 Fab (50 μg.mL⁻¹) before activation was triggered by the addition of collagen (25 μg.mL⁻¹) during 15 minutes at room temperature. Exposure of P-selectin was assessed by flow cytometry using a fluorescein isothiocyanate-coupled anti–P-selectin. Mean fluorescence intensities are shown. Data are the mean ± SD of 3 experiments made in triplicate. Statistical analysis was performed using one-way analysis of variance followed by a Tukey multiple comparisons test; ∗∗∗P < .001. (B) Glenzocimab inhibits GPVI dimerization and clustering. Box plot showing the effect of glenzocimab (50 μg.mL⁻¹) on the molecular brightness (cpm s⁻¹) of GPVI-eGFP alongside the molecular brightness (cpm s⁻¹) of monomeric CD86-eGFP and dimeric CD28-eGFP control receptors in transfected HEK293T cells. For all box plots, center lines represent the median; box limits indicate the 25th and 75th percentiles and whiskers extend to minimum and maximum points. Significance was measured with Kruskal-Wallis with a Dunn post-hoc test in which P ≤ .05. FCS measurements were taken in 35 to 49 cells from 3 independent experiments. (C) Glenzocimab inhibits fibrin-induced platelet aggregation. Washed human platelets were preincubated with vehicle (red curve), 9μM eptifibatide (green curve), or glenzocimab (50 μg.mL⁻¹) before aggregation was initiated by the addition of a solubilized fibrin (200 μg.mL⁻¹). NS, not significant; SD, standard deviation.

Figure 2.

Delineation of the glenzocimab epitope based on GPVIex/glenzocimab crystal analysis. (A) The heavy chain (VH-CH1) of glenzocimab is depicted in red and the light chain (VL-CL) in blue. GPVI domain 2 (D2) bound to glenzocimab is represented in green as is GPVI domain 1 (D1). The D2 C-C′ loop (residues 131-137) is shown in cyan. (B) Hydrophobic contacts made between the D2 C-C′ loop (shown in green, and the heavy and light variable chains of glenzocimab in red and light blue, respectively). Hydrophobic contacts are shown as red dotted lines and represent residues that are within 4 Å or less from one another. (C) Hydrogen bonds between glenzocimab light chain CDRs (gray) and GPVI (green) according to Qt-PISA. (D) Representation of the glenzocimab epitope on the GPVIex sequence (top strand corresponds to D1 domain, bottom strand to D2 domain); the glenzocimab epitope is discontinuous. In red and in blue: residues having atoms within a distance of 4 Å from the VL and VH CDRs, respectively. In purple: residues with atoms within a distance of 4 Å from both VL and VH CDRs. Underlined are GPVI residues involved in hydrogen bonds with glenzocimab. The solid red box indicates the GPVI 129 to 136 truncation that results in the loss of glenzocimab binding and decreased binding to collagen. Red dashed boxes indicate GPVI residues truncated in the 5OU7, 5OU8, and 5OU9 crystals. Asterisk (∗) indicates N72.

Figure 3.

Structural comparison between GPVIex/glenzocimab and 2GI7 crystals. (A) Overview of the superimposed dimeric GPVIex (PDB ID: 2GI7) on the GPVIex/glenzocimab complex (PDB ID: 7R58). The heavy chain (VH-CH1) of glenzocimab is depicted in red and the light chain (VL-CL) in blue. GPVI domain 2 (D2) bound to glenzocimab is represented in green and GPVI without any ligand (PDB ID: 2GI7) in purple. (B) Close up view of the overlayed D2 domains of dimeric GPVI (PDB:2GI7) (pale cyan) and glenzocimab-bound GPVI (green) highlighting the rearrangements within D2 because of clashes between the βA-βB loop of GPVI and the light chain of glenzocimab (light blue). Contacts with the light chain of glenzocimab cause a conformational change βA-βB loop resulting in the formation of a new β-strand, βA′, (shown in yellow). (C) Zoomed-in view of the dimeric interface formed between 2 βG strands from 2 GPVI subunits in the dimeric crystal structure (PDB:2GI7). Subunits are colored pale cyan and light pink, respectively. Polar contacts between each strand are shown as red dashed lines with binding resides shown as sticks. (D) Zoomed-in view of the βG strand of glenzocimab-bound GPVI (green) superimposed with the alternate GPVI subunit found in the dimeric structure (light pink). The conformational changes in the glenzocimab-bound D2 domain result in the formation of a new βA′ strand, which blocks the interaction between the 2 βG strands. The formation of βA′ also results in a shift in the βG strand resulting in an increased gap of 0.6 Å between the 2 βG strands. Combined, these factors explain how GPVI dimerization is blocked by glenzocimab.

Figure 4.

Glenzocimab inhibits CRP/collagen binding through steric hindrance. (A) D1 domain of GPVI (gray) in complex with CRP (magenta) (PDB ID: 5OU8). (B) D1 domain of GPVI (green) in complex with glenzocimab (PDB ID: 7R58). The VL domain of glenzocimab (light blue) at the front creates a barrier in the way of the CRP indicated by the arrow (magenta). (C) Surface representation of the glenzocimab-bound structure of GPVI (PDB ID: 7R58) with CRP binding superimposed from PDB ID: 5OU8. GPVI, CRP, and glenzocimab heavy and light chains are colored in green, magenta, orange, and blue, respectively. The binding of glenzocimab to the D2 domains means the light variable region of the Fab lies directly in the way of the bound CRP chain. Larger CRP and collagen chains would be prevented from binding owing to the obstruction by the light chain. (D) Stereo view of panel C.

Figure 5.

The CRP-binding groove in the GPVIex/glenzocimab complex. (A) Zoomed-in view of the CRP-binding groove of the CRP-bound GPVI (PDB ID: 5OU8) (A) and glenzocimab-bound GPVI (PDB ID: 7R58) (B); structures shown in gray and green, respectively, with CRP shown in magenta. In (C) both structures are superimposed: the CRP-binding groove is largely made from the βC and βF strands within D1. In the glenzocimab-bound structure the R38 shifts 3.5 Å toward CRP and clashes with a CRP hydroxyproline residue. The side chain of R38 in both structures is shown as a stick, and the shift is shown using a red dotted line.

Figure 6.

Truncation of GPVI Δ129 to 136 disrupts the epitope of glenzocimab. GPVI-Fc Δ129 to 136 was compared with GPVI-Fc regarding interactions with glenzocimab in solid-phase assays. (A) Dose-dependent binding of glenzocimab to immobilized GPVI-Fc (blue circles) and GPVI-Fc Δ129 to 136 (red squares). The EC50 value of GPVI-Fc was of 85 ng.mL⁻¹ (1.88 nM), in line with the K_D value. No binding between glenzocimab and GPVI-Fc Δ129 to 136 could be observed even at 1 μg.mL⁻¹, which is more than 10-fold the EC50 of GPVI-Fc. (B) Dose-dependent binding of GP-VI-Fc (blue circles) and GPVI-Fc Δ129 to 136 (red squares) to immobilized glenzocimab confirms defective interactions between glenzocimab and GPVI-Fc Δ129 to 136. Results are mean ± standard error of the mean of 3 experiments performed in triplicate. (C) Inhibition of GPVI-Fc binding to immobilized collagen by increasing concentrations of glenzocimab. Binding of GPVI-Fc at 2 μg.mL⁻¹ (blue circles) or at 20 μg.mL⁻¹ (red squares) to collagen was dose-dependently inhibited by glenzocimab with IC50 values of 10.25 and 113.7 μg.mL⁻¹, respectively. In contrast, glenzocimab at 20 μg.mL⁻¹ had no effect on the binding of GPVI-Fc Δ129 to 136 to collagen (green triangles).