Cloning and paratope analysis of an antibody fragment, a rational approach for the design of a PAI-1 inhibitor

Abstract

This study reports the cloning, characterization and paratope analysis of the plasminogen activator inhibitor-1 (PAI-1) neutralizing single-chain variable fragment 56A7C10 (scFv-56A7C10). ScFv-56A7C10-wt exhibits a similar affinity (KA = 1.01 ± 0.3 × 109 M-1) and PAI-1 inhibitory capacity (90 ± 6% PAI-1 inhibition at a 16-fold molar excess and IC50 = 44 ± 14 ng mL-1) as MA-56A7C10 (KA = 1.43 ± 0.4 × 109 M-1, 90 ± 2% PAI-1 inhibition at a 16-fold molar excess and IC50 = 122 ± 26 ng mL-1). Subsequently, alanine scanning of the six complementarity determining regions (CDRs) was performed and the scFv-56A7C10-mutants (n = 26) were analyzed for their PAI-1 binding and PAI-1 inhibitory properties. Mutation of the residues Y32 and V33 in the CDR1 of the heavy chain (HCDR1) and the residues R98, H99, W100 or F100a (HCDR3) resulted in reduced PAI-1 inhibitory capacities (IC50 ≥ 418 ng mL-1), confirmed by reduced affinities (14-, 17-, 7-, 9- and 16-fold reduced, respectively, vs. scFv-56A7C10-wt). In the light chain, mutation of the residues W50 (LCDR2), H91, Y92, D93, or W96 (LCDR3) resulted in reduced PAI-1 inhibitory properties (IC50 ≥ 160 ng mL-1) and decreased affinities (i.e. 4-, 9-, 3-, 3- and 2-fold reduced affinity, respectively, vs. scFv-56A7C10-wt). Furthermore, an overlapping peptide scan confirmed the importance of the HCDR3 region. These data, combined with a three-dimensional model of scFv-56A7C10, reveal the molecular and structural properties of the paratope and contribute to the rational design of PAI-1 neutralizing compounds. © 2004 International Society on Thrombosis and Haemostasis.