Examination of Camelid Nanobodies in Human Performance Utilities

摘要

With the increasing threat of bioterrorism, the development of diagnostic and therapeutic tools for pathogens which can potentially be used as biological weapons (BW) deserves serious attention. The real usefulness of any diagnostic tool depends on its sensitivity and specificity, and in regard to therapeutics, parameters such as toxicity, immunogenicity and efficacy after administration largely determine their values. Moreover, due to their intended use in extreme conditions of the field, and the possible necessity to treat large number of people in the event of a bioterrorism attack, the importance of the shelf life and the production costs of BW-related diagnostics and therapeutics should be considered. Yersinia pestis is one of the pathogens considered as BW candidates (1). The Y. pestis LcrV, YscF and F1 proteins are of particular interest as targets for diagnosis and therapy (2-6). Nanobodies are the smallest (15 kDa, 2.2 nm diameter, 4 nm height) in vivo affinity- matured functional antigen-binding entities derived from camelid heavy chain antibodies. Due to their small size, recognition of unique epitopes, low immunogenicity, high affinity, high specificity, high stability, high solubility, high expression yields in microorganisms and the ease to tailor, Nanobodies are attractive affinity reagents for various applications including diagnosis and therapy. In the present work, we immunized alpacas, generated Nanobody libraries and retrieved after phage display 14, 7 and 18 Nanobodies with specificity against Yersinia pestis LcrV, YscF and F1 proteins, respectively. Some of these Nanobodies share a homologous third antigen binding loop which predicts that they will target the same epitope on their cognate antigen, albeit with possible variable affinity. Using this information we delivered 6, 7 and 10 independent Nanobody clones for the LcrV, YscF and F1 antigens.