Design and use of a phage display library. Human antibodies with subnanomolar affinity against a marker of angiogenesis eluted from a two-dimensional gel.
We report the construction and the use of a phage display human antibody library (>3 x 10(8) clones) based on principles of protein design. 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. 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. In addition, the library was tested by selecting antibodies against six biologically relevant antigens. Using only 0.3 microg of antigen eluted from a two-dimensional gel spot, we isolated binders specific for the ED-B domain of fibronectin, a marker of angiogenesis. These antibodies recognize the native antigen with affinities in the 10(7)-10(8) M-1 range, and perform well in immunosorbent assays, in two-dimensional Western blotting and in immunohistochemistry. 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. A further 28-fold affinity improvement could be achieved by mutating residues 32 and 50 of the light chain. The resulting antibody, L19, bound to the ED-B domain of fibronectin with very high affinity (Kd = 54 pM), as determined by real-time interaction analysis with surface plasmon resonance detection, band shift analysis, and by competition experiments with electrochemiluminescent detection.
Pini
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Dipartimento di Biologia Molecolare, Sezione di Biochimica, Universita' di Siena, 53'100 Siena, Italy.
Viti
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Santucci
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Carnemolla
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Zardi
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Neri
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Neri
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D
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology*; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D015415:Biomarkers; D003001:Cloning, Molecular; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay; D015723:Gene Library; D018095:Germ-Line Mutation; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018919:Neovascularization, Physiologic / Q000276:immunology*; D019151:Peptide Library
D000906:Antibodies; D015415:Biomarkers; D007128:Immunoglobulin Fragments; D019151:Peptide Library