Generation and in vitro characterisation of inhibitory nanobodies towards plasminogen activator inhibitor 1.
Plasminogen activator inhibitor 1 (PAI-1) is the principal physiological inhibitor of tissue-type plasminogen activator (t-PA) and has been identified as a risk factor in cardiovascular diseases. In order to generate nanobodies against PAI-1 to interfere with its functional properties, we constructed three nanobody libraries upon immunisation of three alpacas with three different PAI-1 variants. Three panels of nanobodies were selected against these PAI-1 variants. Evaluation of the amino acid sequence identity of the complementarity determining region-3 (CDR3) reveals 34 clusters in total. Five nanobodies (VHH-s-a98, VHH-2w-64, VHH-s-a27, VHH-s-a93 and VHH-2g-42) representing five clusters exhibit inhibition towards PAI-1 activity. VHH-s-a98 and VHH-2w-64 inhibit both glycosylated and non-glycosylated PAI-1 variants through a substrate-inducing mechanism, and bind to two different regions close to αhC and the hinge region of αhF; the profibrinolytic effect of both nanobodies was confirmed using an in vitro clot lysis assay. VHH-s-a93 may inhibit PAI-1 activity by preventing the formation of the initial PAI-1t-PA complex formation and binds to the hinge region of the reactive centre loop. Epitopes of VHH-s-a27 and VHH-2g-42 could not be deduced yet. These five nanobodies interfere with PAI-1 activity through different mechanisms and merit further evaluation for the development of future profibrinolytic therapeutics.
Xiaohua
X
Zhou
Maarten L V
ML
Hendrickx
Gholamreza
G
Hassanzadeh-Ghassabeh
Serge
S
Muyldermans
Paul Declerck, Laboratory for Therapeutic and Diagnostic Antibodies, Department of Pharmaceutical and Pharmacological Sciences, Katholieke Universiteit Leuven, Campus Gasthuisberg, O&N2, PB 820, Herestraat 49, B-3000 Leuven, Belgium, Tel.: +32 16 32 34 31, Fax: +32 16 32 34 60, E-mail: paul.declerck@kuleuven.be.
Paul J
PJ
Declerck
D000595:Amino Acid Sequence; D018604:Epitope Mapping; D005342:Fibrinolysis; D006801:Humans; D017395:Plasminogen Activator Inhibitor 1 / Q000276:immunology*; D017434:Protein Structure, Tertiary; D061905:Single-Domain Antibodies / Q000276:immunology*; D010959:Tissue Plasminogen Activator
D017395:Plasminogen Activator Inhibitor 1; D061905:Single-Domain Antibodies; D010959:Tissue Plasminogen Activator
Cardiovascular diseases; fibrinolysis; nanobodies; neutralising antibody fragments; plasminogen activator inhibitor 1