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.