Cloning and paratope analysis of an antibody fragment, a rational approach for the design of a PAI-1 inhibitor.
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 x 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 x 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.
Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium.
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K
Verbeke
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A
Gils
P J
PJ
Declerck
D000915:Antibody Affinity / Q000235:genetics; D001666:Binding Sites, Antibody; D003001:Cloning, Molecular; D022801:Complementarity Determining Regions / Q000235:genetics; D015195:Drug Design; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D017395:Plasminogen Activator Inhibitor 1 / Q000276:immunology*
D022801:Complementarity Determining Regions; D007128:Immunoglobulin Fragments; D017395:Plasminogen Activator Inhibitor 1; C055966:immunoglobulin Fv