Selection and characterization of camelid nanobodies towards urokinase-type plasminogen activator.
Urokinase-type plasminogen activator (uPA) is a trypsin-like serine protease that plays a vital role in extracellular conversion of inactive plasminogen into catalytically active plasmin. Activated plasmin facilitates the release of several proteolytic enzymes, which control processes like pericellular proteolysis and remodeling of ECM. uPA and the receptor uPAR, are overexpressed in a number of malignant tumours and uPA/uPAR play major roles in adhesion, migration, invasion and metastasis of cancer cells. Elevated levels of uPA have been reported as a risk biomarker for disease relapse, increased cancer malignancy and poor survival prognosis. For these reasons uPA is considered an important target for anticancer drug therapy. In this study we isolated two camel single domain antibodies (nanobodies) from a naïve library by phage display. The nanobody sequences were sequence-optimized for Escherichia coli expression, cloned into the pET22-B(+) vector system, expressed in BL-21 cells and purified from the periplasmic fraction by IMAC. ELISA tests demonstrated that the purified nanobodies were specific for uPA when tested towards other trypsin-like serine proteases. The apparent affinities of the nanobodies were determined by competitive ELISA to 80 nM and 522 nM, respectively. The best binder did not inhibit uPA (nAb-C3), however the lowest affinity binder (nAb-C8) was able to inhibit the uPA-mediated cleavage of the substrate S-2444. The results validate the naïve library as a resource for retrieval of relevant lead molecules and the novel uPA-nanobodies can be useful pharmacological tools to study uPA structure-function relationships.
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
Jakub Zbigniew
JZ
Kaczmarek
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark. Electronic address: peter.durand.skottrup@regionh.dk.
Peter Durand
PD
Skottrup
D000818:Animals; D000918:Antibody Specificity / Q000235:genetics*; D002162:Camelus; D002460:Cell Line; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D009842:Oligopeptides / Q000737:chemistry*; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000276:immunology
D009842:Oligopeptides; D061905:Single-Domain Antibodies; C028190:5-oxo-prolyl-glycyl-arginine-4-nitroanilide; D014568:Urokinase-Type Plasminogen Activator
Antibody technology; Nanobody; Phage display; Serine protease; Single domain antibody