PB0334 - Synthetic llama-derived single-domain antibodies blocking tissue factor pathway inhibitor increase thrombin generation in FVIII-, FIX- and FXI-deficient plasma

Sunday, June 23, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

Claire Auditeau, PharmD

PharmD, PhD Student
Hémostase Inflammation Thrombose (HITh) UMR-S1176 INSERM Université Paris-Saclay Le Kremlin-Bicêtre France
Kremlin-Bicêtre, Ile-de-France, France

Co-Author(s)

IP

Ivan Peyron

Research Engineer
INSERM U1176, Université Paris Saclay, Le Kremlin-Bicêtre, France
Le Kremlin-Bicêtre, Ile-de-France, France
AR

Andréa Ruty

Assistant Engineer
INSERM U1176, Université Paris Saclay, Le Kremlin-Bicêtre, France
Le Kremlin-Bicêtre, Ile-de-France, France
PS

Pradtahna Saenjamsai

Assistant Engineer
INSERM U1176, Université Paris Saclay, Le Kremlin-Bicêtre, France
Le Kremlin-Bicêtre, Ile-de-France, France
EB

Elsa Bianchini

Associate Professor
INSERM U1176, Université Paris Saclay, Le Kremlin-Bicêtre, France
Le Kremlin-Bicêtre, Ile-de-France, France
MK

Mariem Khamari

Technician
INSERM U1176, Université Paris Saclay, Le Kremlin-Bicêtre, France
Le Kremlin-Bicêtre, Ile-de-France, France
Tilman M. Hackeng, PhD

Professor
Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University
Maastricht, Netherlands
Olivier D. Christophe, PhD

Researcher
INSERM U1176, Université Paris Saclay, Le Kremlin-Bicêtre, France
Le Kremlin-Bicêtre, Ile-de-France, France
Cécile V. Denis

Researcher
INSERM U1176, Université Paris Saclay, Le Kremlin-Bicêtre, France
Le Kremlin-Bicêtre, Ile-de-France, France
DB

Delphine Borgel

Professor
INSERM U1176, Université Paris Saclay, Le Kremlin-Bicêtre, France & Assistance Publique Hôpitaux de Paris, Paris, France
Le Kremlin-Bicêtre, Ile-de-France, France
Peter J. Lenting, PhD

Researcher
INSERM U1176, Université Paris Saclay, Le Kremlin-Bicêtre, France
Le Kremlin-Bicêtre, Ile-de-France, France
FS

François Saller

Associate Professor
INSERM U1176, Université Paris Saclay, Le Kremlin-Bicêtre, France
Le Kremlin-Bicêtre, Ile-de-France, France

Background: Tissue factor pathway inhibitor (TFPI) is an important determinant of thrombin generation in the plasma of patients with deficiency in FVIII, FIX and FXI. As such, anti-TFPI monoclonal antibodies such as Concizumab have been developed to treat hemophilia.

Aims: To generate llama-derived single-domain antibodies (sdAbs) as a novel class of pharmacological agents blocking TFPI and increasing thrombin generation in the plasma of patients with severe FVIII, FIX, and FXI deficiency.

Methods: A large synthetic library of llama-derived sdAbs was generated and selected by phage-display with three rounds of biopanning on recombinant human TFPIα (rhTFPIα) immobilized onto ELISA wells. Thrombin generation induced by 1 pM TF was measured on commercial plasmas from patients with severe ( < 1%) deficiency in FVIII (HA), FIX (HB), or FXI by Calibrated Automated Thrombography (CAT). The functional effects of sdAbs on the inhibitory activity of rhTFPIα towards the amidolytic activity of FXa and TF/FVIIa complex were tested in purified systems. Their binding to maltose-binding protein (MBP)-fusion proteins with the individual Kunitz (K1, K2 and K3) domains of TFPIα were measured by ELISA.

Results: 21 out of 188 screened sdAbs specifically bound to TFPIα, and two of them (26E5 and 26E8) were found to dose-dependently increase thrombin generation in the plasma of patients with HB (Figure 1A). These sdAbs also efficiently increased thrombin generation in HA and FXI-deficient plasmas (Figure 1B). 26E5 and 26E8 impaired the ability of rhTFPIα to inhibit the amidolytic activity of FXa and TF/FVIIa complex. Furthermore, 26E5 specifically bound to MBP-K1, suggesting that this sdAb was directed against the K1 domain of TFPI, while the epitope for 26E8 could not be localized using this assay.

Conclusion(s): Our large synthetic library could be used for readily generating diverse and functionally relevant llama-derived monovalent anti-TFPI sdAbs that might be therapeutically attractive.