Presentation Details
| Anti-Factor XI Antibodies Targeting the Apple 2 Domain and the Catalytic Domain Exert Distinct In Vitro Inhibitory Profiles on Coagulation Function Dan Chalothorn, KehDih Lai, Weizhen Wu, Holly Sorenson, George Ehrlich, Ashique Rafique, Ishita Chatterjee, Kei Saotome, Matthew Franklin, Lori Morton. Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA |
Abstract
Background: Acute venous and arterial thromboses (i.e., abnormal clotting of blood within vessels) are common causes of morbidity and mortality in developed countries. Although several classes of antithrombotic agents are currently approved, an unmet medical need exists for antithrombotics that do not increase bleeding risk. A combination of genetic and pharmacological evidence from human and animal studies suggests that inhibition of coagulation factor XI (FXI) can be effective at preventing thrombosis with little-to-no increased bleeding risk. We have generated two monoclonal antibodies (mAbs) that bind to different domains on FXI/FXIa and exert distinct inhibitory profiles on coagulation function. Objectives: To characterize epitope binding sites, affinities for FXI and FXIa, and inhibitory effects on coagulation pathway function for each anti-FXI/FXIa mAb. Methods: Epitope binding sites on FXI/FXIa were mapped using cryogenic electron microscopy (cryo-EM), and binding affinities were determined using surface plasmon resonance techniques. Functional effects on coagulation activities were assessed using the clinical method of activated partial thromboplastin time (aPTT), which reflects factor function in both intrinsic and common pathways. Prothrombin time (PT), which reflects factor function in both extrinsic and common pathways, was also assessed. Functional effects on thrombin generation, which is mediated by intrinsic or extrinsic pathway activators, were assessed using a thrombin generation assay. Results: High resolution cryo-EM revealed that one mAb is an Apple 2 domain binder (A2-mAb) and the other a catalytic domain binder (Cat-mAb), which interfere with FXI/FXIa interactions with high-molecular-weight kininogen and coagulation factor IX, respectively. Both mAbs have sub-nanomolar binding affinities for FXI and FXIa (A2-mAb: 23.0 pM and 150 pM, respectively; Cat-mAb: 4.0 pM and 7.0 pM, respectively) at 37°C. In functional dose–response analyses, A2-mAb had an aPTT ratio of 2.0 at 32 nM and a maximum aPTT ratio of 3.0 at ≥320 nM; Cat-mAb had an aPTT ratio of 2.0 at 24 nM and a maximum aPTT ratio of 4.3 at ≥75 nM. Neither mAb affected PT up to the maximum tested concentration of 25 µM. In the thrombin generation assay using pooled healthy human plasma, A2-mAb reduced intrinsic pathway-triggered thrombin generation in a dose-dependent manner and completely abolished thrombin generation at doses ≥250 nM, without affecting extrinsic pathway-triggered thrombin generation. Cat-mAb reduced intrinsic pathway-triggered thrombin generation to 38% of baseline at a dose of 16 nM and completely abolished thrombin generation at doses ≥31 nM; extrinsic pathway-triggered thrombin generation was reduced by 40–50% of baseline at doses ≥16 nM, likely due to the mAb interfering with the thrombin–FXI feed-forward loop. Both mAbs reduced thrombin generation in a contact-mediated thrombosis setting when plasma was incubated with non-biological surfaces (hemodialysis fibers or mechanical heart valve components). Conclusions: A2-mAb and Cat-mAb bind to different domains of FXI/FXIa and consequently exert distinct inhibitory profiles on coagulation function. A2-mAb is a moderately potent inhibitor of intrinsic, but not extrinsic, pathway-triggered activity. Cat-mAb is a more potent/efficacious inhibitor of intrinsic pathway-triggered activity, with moderate effects on propagation/amplification of the thrombin–FXI feed-forward loop, as evidenced by alterations in extrinsic pathway-triggered thrombin generation.
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