Structural guided scaffold phage display libraries as a source of bio-therapeutics

• Published in: PloS one Vol. 8; no. 8; p. e70452
• Main Authors: Man, Y K Stella, DiCara, Danielle, Chan, Nicole, Vessillier, Sandrine, Mather, Stephen J, Rowe, Michelle L, Howard, Mark J, Marshall, John F, Nissim, Ahuva
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 09.08.2013; Public Library of Science (PLoS)
• Subjects: Adhesive strength; Algorithms; Amino acid composition; Amino Acid Sequence; Amino acids; Animals; Antigens, Neoplasm - chemistry; Antigens, Neoplasm - genetics; Antigens, Neoplasm - metabolism; Binding Sites; Biology; Cancer; Cancer therapies; Cell surface; Complementarity-determining region 3; Engineering; Female; Foot & mouth disease; Helices; Humans; Immunoglobulins; Integrins; Integrins - chemistry; Integrins - genetics; Integrins - metabolism; Lead; Libraries; Ligands; Magnetic Resonance Spectroscopy; Medical imaging; Medical screening; Medicine; Mice; Mice, Nude; Models, Molecular; Molecular Sequence Data; NMR; Nuclear magnetic resonance; Oligopeptides - chemistry; Oligopeptides - genetics; Oligopeptides - metabolism; Peptide Library; Peptides; Phage display; Phages; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Proteins; Single-Chain Antibodies - chemistry; Single-Chain Antibodies - metabolism; Single-Chain Antibodies - pharmacology; Spectrum analysis; Structural analysis; Structural Homology, Protein; Studies; Target recognition; Tumor Burden - drug effects; Tumors; Xenograft Model Antitumor Assays; Xenografts; United Kingdom > UK
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0070452

We have developed a structurally-guided scaffold phage display strategy for identification of ligand mimetic bio-therapeutics. As a proof of concept we used the ligand of integrin αvβ6, a tumour cell surface receptor and a major new target for imaging and therapy of many types of solid cancer. NMR structure analysis showed that RGD-helix structures are optimal for αvβ6 ligand-interaction, so we designed novel algorithms to generate human single chain fragment variable (scFv) libraries with synthetic VH-CDR3 encoding RGD-helix hairpins with helices of differing pitch, length and amino acid composition. Study of the lead scFv clones D25scFv and D34scFv and their corresponding VH-CDR3 derived peptides, D25p and D34p, demonstrated: specific binding to recombinant and cellular αvβ6; inhibition of αvβ6-dependent cell and ligand adhesion, αvβ6-dependent cell internalisation; and selective retention by αvβ6-expressing, but not αvβ6-negative, human xenografts. NMR analysis established that both the D25p and D34p retained RGD-helix structures confirming the success of the algorithm. In conclusion, scFv libraries can be engineered based on ligand structural motifs to increase the likelihood of developing powerful bio-therapeutics.