Engineering strategies for ABD-derived affinity proteins for therapeutic and diagnostic applications

Detta är en avhandling från Stockholm : KTH Royal Institute of Technology

Sammanfattning: Small stable protein domains are attractive scaffolds for engineering affinity proteins due to their high tolerance to mutagenesis without loosing structural integrity. The albuminbinding domain is a 5 kDa three-helix bundle derived from the bacterial receptor Protein G with low-nanomolar affinity to albumin. In this thesis, the albumin-binding domain is explored as a scaffold for engineering novel affinity proteins with the possible benefit of combining a prolonged serum half-life with specific targeting in a single small scaffold protein. Previously, a library was created by randomizing surface-exposed residues in order to engineer affinity to a new target antigen in addition to the inherent albumin affinity. Here, phage display selections were separately performed against the tumor antigens ERBB2 and ERBB3. The ERBB3 selection resulted in a panel of candidates that were found to have varying affinities to ERBB3 in the nanomolar range, while still retaining a high affinity to albumin. Further characterization concluded that the clones also competed for binding to ERBB3 with the natural activating ligand Heregulin. The selections against ERBB2 resulted in sub-nanomolar affinities to ERBB2 where the binding site was found to overlap with the antibody Trastuzumab. The binding sites on ABD to albumin and either target were found in both selections to be mutually exclusive, as increased concentrations of albumin reduced the level of binding to ERBB2 or ERBB3. An affinity-matured ERBB2 binder, denoted ADAPT6, which lacked affinity to albumin was evaluated as a radionuclide-labeled imaging tracer for diagnosing ERBB2-positive tumors. Biodistribution studies in mice showed a high renal uptake consistent with affinity proteins in the same size range and the injected ADAPT quickly localized to the implanted tumor. High contrast images could be generated and ERBB2-expressing tissue could be distinguished from normal tissue with high contrast, demonstrating the feasibility of the scaffold for use as diagnostic tool. In a fourth study, affinity maturation strategies using staphylococcal cell-surface display were evaluated by comparing two replicate selections and varying the stringency. A sub-nanomolar target concentration was concluded to be inappropriate for equilibrium selection as the resulting output was highly variable between replicates. In contrast, equilibrium sorting at higher concentrations followed by kinetic-focused off-rate selection resulted in high output overlap between attempts and a clear correlation between affinity and enrichment.

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