Here, we describe the isolation of human antibody fragments against the human ED‐B domain that bind to human, mouse and chicken B‐FN. As it has proved difficult to obtain such antibodies by hybridoma technology, we used phage display technology. In principle, antibodies directed against the human ED‐B domain should provide pan‐species markers for angiogenesis as the sequence of this domain is highly conserved in different species (and identical in humans and mice). However, this MAb does not directly recognise the human ED‐B domain nor does it recognise B‐FN of other species therefore, it cannot be used as a marker of angiogenesis in animal models. This isoform, therefore, represents a promising marker for angiogenesis, as already shown using the mouse monoclonal antibody (MAb) BC‐I directed against an epitope on human B‐FN. The B‐FN isoform (with ED‐B domain inserted by splicing) is present in the stroma of foetal and neoplastic tissues and in adult and neoplastic blood vessels during angiogenesis but is undetectable in mature vessels. Phage antibodies with pan‐species recognition of the oncofoetal angiogenesis marker fibronectin ED‐B domain PDFīarbara Carnemolla Dario Neri Patrizia Castellani Alessandra Leprini Giovanni Neri Alessandro Pini Greg Winter Luciano Zardiįibronectin (FN) exists in several polymorphic forms due to alternative splicing. The resulting antibody, L19, bound to the ED-B domain of fibronectin with very high affinity (K d = 54 pm), as determined by real-time interaction analysis with surface plasmon resonance detection, band shift analysis, and by competition experiments with electrochemiluminescent detection.
A further 28-fold affinity improvement could be achieved by mutating residues 32 and 50 of the light chain. The affinity of one anti-ED-B antibody was improved by 27-fold by combinatorially mutating six strategically selected residues in the heavy chain variable domain. These antibodies recognize the native antigen with affinities in the 107–108 m −1 range, and perform well in immunosorbent assays, in two-dimensional Western blotting and in immunohistochemistry. Using only 0.3 μg of antigen eluted from a two-dimensional gel spot, we isolated binders specific for the ED-B domain of fibronectin, a marker of angiogenesis.
In addition, the library was tested by selecting antibodies against six biologically relevant antigens. With this strategy we concentrated sequence diversity in regions of the antibody structure that are centrally located in the antigen binding site, while leaving residues in more peripheral positions available for further mutagenesis aimed at improving the affinity of the selected antibodies. A large repertoire of functional antibodies with similar properties was produced by appending short variable complementarity-determining region 3 (CDR3) onto the two antibody germ line segments most frequently found in human antibodies. We report the construction and the use of a phage display human antibody library (>3 × 108clones) based on principles of protein design. Design and Use of a Phage Display Library HUMAN ANTIBODIES WITH SUBNANOMOLAR AFFINITY AGAINST A MARKER OF ANGIOGENESIS ELUTED FROM A TWO-DIMENSIONAL GEL PDFĪlessandro Pini Francesca Viti Annalisa Santucci Barbara Carnemolla Luciano Zardi Paolo Neri Dario Neri
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