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

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 ?v?6, 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 ?v?6 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 ?v?6; inhibition of ?v?6-dependent cell and ligand adhesion, ?v?6-dependent cell internalisation; and selective retention by ?v?6-expressing, but not ?v?6-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.

SUBMITTER: Man YK

PROVIDER: S-EPMC3739823 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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

Man Y K Stella YK DiCara Danielle D Chan Nicole N Vessillier Sandrine S Mather Stephen J SJ Rowe Michelle L ML Howard Mark J MJ Marshall John F JF Nissim Ahuva A

PloS one 20130809 8

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 synth ...[more]

PMID: 23950939

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Project description:Monoclonal antibodies (mAbs) are essential reagents for deciphering gene or protein function and have been a fruitful source of therapeutic and diagnostic agents. However, developing anticarbohydrate antibodies to target glycans for those purposes has been less successful because the molecular basis for glycan-mAb interactions is poorly understood relative to protein- or peptide-binding mAbs. Here, we report our investigation on glycan-mAb interactions by using the unique architectural scaffold of 2G12, an antibody that targets oligomannoses on the HIV-1 glycoprotein gp120, as the template for engineering highly specific mAbs to target glycans. We first analyzed 24 different X-ray structures of antiglycan mAbs from the Protein Data Bank to determine side chain amino acid distributions in of glycan-mAb interactions. We identified Tyr, Arg, Asn, Ser, Asp, and His as the six most prevalent residues in the glycan-mAb contacts. We then utilized this information to construct two phage display libraries ("Lib1" and "Lib2") in which positions on the heavy chain variable domains of 2G12 were allowed to vary in restricted manner among Tyr, Asp, Ser, His, Asn, Thr, Ala and Pro to interrogate the minimal physicochemical requirements for oligomannose recognition. We analyzed the sequences of 39 variants from Lib1 and 14 variants from Lib2 following selection against gp120, the results showed that there is a high degree of malleability within the 2G12 for glycan recognitions. We further characterized five unique phage clones from both libraries that exhibited a gp120-specific binding profile. Expression of two of these variants as soluble mAbs indicated that, while specificity of gp120-binding was retained, the affinity of these mutants was significantly reduced relative to WT 2G12. Nonetheless, the results indicate these is some malleability in the identity of contact residues and provide a novel insight into the nature of glycan-antibody interactions and how they may differ from protein-antibody binding interactions.

Dataset Information

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

Publications

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

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