OC 64.1 - First-in-class human antibodies targeting protein disulfide isomerase effectively attenuate arterial thrombosis, vascular inflammation, and ischemic stroke in mice

Background: Extracellular protein disulfide isomerase (PDI) promotes platelet and neutrophil adhesive function under thromboinflammatory conditions, indicating that targeting extracellular PDI may be a therapeutic strategy.

Aims: The goal of this study is to characterize the first-in-class human anti-PDI antibodies in vitro and in vivo.

Methods: We used a human phage display library to generate monoclonal anti-PDI antibodies. Biochemical and cell-biological assays, flow cytometry, and mouse models of arterial thrombosis, vascular inflammation, and ischemic stroke were utilized to investigate how those antibodies mitigate thromboinflammatory conditions.

Results: We identified two human IgG1 anti-PDI antibodies with distinct modes of action: 3H3 bound to the PDI a-b-b’ domains (KD = 0.9 nM) and inhibited PDI activity, whereas 2C9 bound to the PDI a-domain (KD = 2.4 nM) and blocked the interaction between PDI and glycoprotein Ibα, a platelet receptor playing a crucial role in thromboinflammation, without perturbing PDI activity. 3H3 and 2C9 inhibited αIIbβ3 integrin activation, but not P-selectin exposure, and impaired platelet aggregation induced by thrombin, collagen-related peptides, or von Willebrand factor. However, such effects disappeared in PDI-null platelets, indicating their specificity. Treatment of mouse and human blood with 3H3 or 2C9 reduced the surface coverage and volume of thrombi over collagen-coated surfaces under 10-40 dyne/cm2. Intravital microscopy revealed that compared with human IgG1, intravenous injection of 3H3 and 2C9 into mice inhibited platelet thrombus formation in laser-induced cremaster arteriolar thrombosis and impaired neutrophil recruitment on postcapillary venules in TNF-α-induced inflammation. Both antibodies effectively reduced FeCl3-induced carotid arterial thrombosis without prolonging tail bleeding times. Eptifibatide, a clinically-used αIIbβ3 antagonist, showed similar inhibitory effects on arterial thrombosis but prolonged tail bleeding times. Further, 3H3 and 2C9 improved neurological deficits and reduced infarct volume in mice subjected to transient middle cerebral artery occlusion.

Conclusion(s): Our results provide strong evidence that our human anti-PDI antibodies may be an effective anti-thromboinflammatory agent.