Abstract
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.
Highlights
The use of combinatorial phage display single chain fragment variable (scFv) libraries for generation of therapeutic antibodies is well established and has resulted in clinically valuable reagents [1,2]
Library design Our rationale was to introduce a structural selectivity to a phage display library where the three-dimensional (3D) avb6 ligand recognition motif (RGD-helix) was genetically encoded into the antibody binding pocket at the variable heavy (VH)-CDR3
The a-helix template VH-CDR3 algorithm was: E1P2R3G4D5L6X7X8L9A10A11R12Z13K14R15Z16F17N18E19 Z20L21A22Z23L24Q25E26K27G28I29 where Z and X were random amino-acid residues introduced into the same quadrant as the leucine residues of the RGDLXXL motif: at position 13, 16, 20 and 23 in the a-helix based on the helical wheel (3.6 residues per turn of helix)
Summary
The use of combinatorial phage display scFv libraries for generation of therapeutic antibodies is well established and has resulted in clinically valuable reagents [1,2]. Since receptor:ligand interactions must be considered as interacting topographical maps we wondered if it were possible to generate a target-selective library by incorporating a panel of specific three-dimensional shapes into the CDR3 of the variable heavy (VH-CDR3) If such a library used stereochemical shapes that corresponded to a ligand-binding interface it would more likely generate scFv(s) that will block the ligand:receptor interaction than would a conventional random library. To test this hypothesis we considered a therapeutically relevant target, the integrin avb, which represents a novel and important tumourselective target that is expressed on the surface of cancer cells. Strong expression of avb correlates with poor prognosis in multiple cancers [11,12,13] and human therapeutic antibodies to this integrin are likely to have a significant therapeutic value
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