The affibody is a 58-amino acid three-helix bundle ligand scaffold for clinical molecular imaging probes, targeted therapy, and biotechnology. A challenge in discovery and development of affibodies, and all protein ligands, is the inconsistency of binders evolved against recombinant target to translate to genuine intact cellular target. Selection of yeast-displayed antibody libraries on adherent mammalian cell monolayers has been previously used to address this limitation. To enhance the robustness and selective control of cellular panning, we systematically elucidated the elements that dictate ligand enrichment. An extended PAS peptide between the cell-tethering protein and the displayed ligand enhances enrichment 44-fold. Also, controlled chemical cleavage of displayed ligands controls valency over two orders of magnitude, which empowers broadly titratable affinity discrimination. 20 ligand discovery campaigns were competed, in two scaffolds (affibody and fibronectin domain) and multiple targets, to compare selection strategies. Direct cell panning enhances discovery of translatable hits four-fold versus campaigns using initial enrichment on magnetic beads coated in recombinant target followed by flow cytometry with recombinant target or biotinylated cell lysate. Competitive selections were optimized to enable depletion of non-specific ligands. Deep sequencing an epitope-diverse pool (60,000 unique variants) of evolved binders identified amino acid motifs consistent with evolutionary efficacy, which revealed a non-spatial gradient of diversity (Shannon entropy: 1.4-4.0) in an optimized paratope library. Implementation of these sitewise amino acid distributions yielded a 50-fold increase in binder discovery. Affibodies from this constrained library exhibit dramatically higher thermal stability (24±12 C higher Tm) than naïve binders. The affibody framework was engineered for reduced charge while retaining stability, solubility, and binding. A synthetic consensus design strategy – merging directed evolution and bioinformatics of evolutionarily tolerant residues – yielded better variants than directed evolution or informatics alone. Neutralization of three acidic and three basic residues resulted in a 39±10% reduction in non-specific renal retention while increasing tumor targeting 21±5% in murine xenograft models. Enhancements in library design, ligand selection, and framework design improve the ability to engineer translatable affibody ligands.

Translatable affibody ligand discovery: Engineered biopanning, deep sequence-guided library design, and pharmacokinetic modulation

253rd National Meeting (2017)

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