PB0535 - Development of function-blocking antibodies to plasma prekallikrein for the study of the interplay between coagulation and inflammation

Monday, June 26, 2023

18:30 – 19:30 ET

Room: Exhibition

Presenting Author(s)

Samantha A. Moellmer

Graduate Student
Oregon Health & Science University
Hillsboro, Oregon, United States

Co-Author(s)

YZ

Yong-Ping Zhong

Research Assistant
OHSU
Portland, Oregon, United States
MS

Mao-fu Sun

Staff Scientist
Department of Pathology, Immunology and Microbiology, Vanderbilt University Medical Center
Nashville, Tennessee, United States
Cristina Puy, PhD

Research Assistant Professor
Oregon Health & Science University
Portland, Oregon, United States
PS

Philip Streeter

Associate Professor
OHSU
Portland, Oregon, United States
JS

Joseph J. Shatzel

Assistant Professor
Oregon Health & Science University
Portland, Oregon, United States
KN

Karina Nakayama

Assistant Professor
OHSU
Portland, Oregon, 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
ET

Erik I. Tucker

Chief Executive Officer
Aronora, Inc.
Portland, Oregon, United States
CL

Christina U. Lorentz

Director of Clinical Operations
Aronora, Inc
Portland, Oregon, United States

Background: Prekallikrein (PK) is a zymogen in the contact pathway with roles in both coagulation and inflammation. Specifically, PK is involved in the intrinsic pathway of coagulation through activation of factor XII. The activated form of PK, plasma kallikrein, initiates inflammation via liberation of the vasoactive peptide bradykinin from high molecular weight kininogen. Bradykinin is a potent vasodilator and induces vascular permeability. How the bi-directional relationship between coagulation and inflammation within the contact pathway contributes to vascular physiology or vascular disease pathogenesis in select settings remains unclear.

Aims: Develop and validate anti-PK monoclonal antibodies for use in the study of PK in health and disease.

Methods: PK-deficient Balb-C mice were immunized with recombinant mouse or human PK. Antibody producing plasma cells were isolated from the spleen and fused with immortalized myeloma cells to generate hybridomas. IgG antibodies were collected from the hybridomas and evaluated for strength of PK binding by ELISA. Antibodies with high affinity were screened for anticoagulant properties by activated partial thromboplastin time assay (aPTT), using PK-depleted plasma supplemented with mouse or human PK.

Results: Generated from the mouse PK immunization, antibody 4H11 cross reacts with mouse and human PK, and effectively prolonged aPTT by 176 and 105 seconds, respectively. Nine antibodies from this fusion exhibited strong inhibitory effects in a clotting assay using PK-depleted plasma reconstituted with mouse PK. The human PK immunization generated 10 antibodies that prolong clotting in PK-depleted plasma reconstituted with human PK. Structure-function studies, including kallikrein activity and endothelial cell activation, are currently underway and will further characterize the antibodies.

Conclusion(s): These function-blocking anti-PK antibodies will be useful tools for the study of PK in physiology as well as under thrombotic and inflammatory conditions.