PB0588 - Epitope Mapping of the α-FXIIa Allosteric Inhibitor Monoclonal Antibody 5C12

Monday, June 26, 2023

18:30 – 19:30 ET

Room: Exhibition

Presenting Author(s)

Sadiq Silbak, BS, MS

Pre-doctoral student
Department of Pathology, Case Western Reserve University
Cleveland, Ohio, United States

Co-Author(s)

Alessandro Pinheiro, PhD (he/him/his)

Postdoctoral
Division of Hematology and Oncology Department of Medicine Case Western Reserve University
Cleveland, Ohio, United States
Michael Wallisch, PhD (he/him/his)

Principal Scientist
Aronora, Inc.
Portland, Oregon, United States
AM

Alona Merkulova

Research Assistant
Division of Hematology and Oncology Department of Medicine Case Western Reserve University
Cleveland, Ohio, United States
TH

Tracey He

Research Assistant
Department of Pathology, Microbiology and Immunology Vanderbilt University Medical Center
Nashville, Tennessee, United States
SD

Stephen Dickeson, PhD

Assistant Research Professor
Department of Pathology, Microbiology and Immunology Vanderbilt University Medical Center
Nashville, Tennessee, United States
David Gailani, MD

Professor
Vanderbilt University Medical Center
Nashville, Tennessee, United States
Owen J.T McCarty, PhD, FAHA

Professor and Chair of Biomedical Engineering
Oregon Health & Science University
Portland, Oregon, United States
AS

Alvin Schmaier

Professor
Division of Hematology and Cell Therapy, Department of Medicine, University Hospitals Cleveland Medical Center
Cleveland, Ohio, United States

Background: Engineered blood-contacting medical devices are essential for patient management. However, medical device-associated thrombosis is common. Inhibitors to FXIIa have been proposed as an alternative to heparin to prevent medical device-associated thrombosis without increasing the risk of bleeding. The monoclonal antibody (Mab), 5C12 is a potent anti-FXIIa antibody in development to prevent medical device-associated thrombosis. We mapped the epitope of 5C12 on α-FXIIa.

Aims: Identify the FXIIa epitope of 5C12 to provide insights into both mechanism of action and identification of druggable targets to prevent medical device-associated thrombosis.

Methods: We mapped 5C12’s binding region using peptides from FXII designed in an iterative, deductive fashion using functional and competition inhibition binding assays.

Results: 5C12 is a conformationally specific Mab. It immunoblots FXII and FXIIa only on non-reduced samples; 5C12 preferentially binds to α-FXIIa over FXII. α-FXIIa blocks 5C12 binding to α-FXIIa linked to microtiter plates with an IC50 of 11.78 nM and 5C12 inhibits α-FXIIa hydrolysis of H-D-Pro-Phe-Arg-pNA·2HCl with an IC50 of 3.5 nM. Through a series of iterative peptide preparations using the crystal structure of FXIIa as a guide, we determined that 5C12 binds to a 5 amino acid external loop I383APCW on FXIIa that is near the active-site H393. The C386W387 sequence is highly conserved in the FXII family of proteins: tPA, uPA, and HGFA. Competition inhibition assays using peptides IAPAW and IAPCA reveal that the cysteine and tryptophan in α-FXIIa are critical for 5C12 binding. Last, 5C12 is unable to immunoblot a FXII C386-C456 mutant.

Conclusion(s): We postulate that 5C12 binding to FXIIa causes a change in the orientation of the active site histidine, incapacitating the catalytic triad. Use of anti-FXIIa Mabs may reduce or eliminate the need for heparin with medical devices. Presently, there is no agent like this approved for this therapeutic indication. [Research Support: NIH grants HL144113, HL157405, and HL143402].