Selection and characterization of camelid nanobodies towards urokinase-type plasminogen activator

• Published in: Molecular immunology 2015-06, Vol.65 (2), p.384-390
• Main Authors: Kaczmarek, Jakub Zbigniew, Skottrup, Peter Durand
• Format: Article
• Language: English
• Subjects: Animals; Antibody Specificity - genetics; Antibody technology; Camelus; Cell Line; Gene Expression; Gene Library; Humans; Nanobody; Oligopeptides - chemistry; Phage display; Serine protease; Single domain antibody; Single-Domain Antibodies - chemistry; Single-Domain Antibodies - genetics; Single-Domain Antibodies - immunology; Urokinase-Type Plasminogen Activator - chemistry; Urokinase-Type Plasminogen Activator - immunology
• ISSN: 0161-5890; 1872-9142
• DOI: 10.1016/j.molimm.2015.02.011

•Selection of nanobodies towards urokinase-type plasminogen activator was achieved.•Nanobodies expression was optimized in E. coli.•Nanobodies were specific for urokinase-type plasminogen activator.•One nanobody was able to inhibit 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 80nM and 522nM, 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.