Multiple Bacteriophage Selection Strategies for Improved Affinity of a Peptide Targeting ERBB2

Benjamin M. Larimer, Jeanne M. Quinn, Kevin Kramer, Andrey Komissarov, Susan L. Deutscher

2015 December

ABSTRACT

Due to the heterogeneity of ERBB2-expression between tumors and over the course of treatment, a non-invasive molecular imaging agent is needed to accurately detect overall ERBB2 status. Peptides are a highly advantageous platform for molecular imaging, since they have excellent tumor penetration and rapid pharmacokinetics. One limitation of peptides however, is their traditionally low target affinity, and consequently, tumor uptake. The peptide KCCYSL was previously selected from a bacteriophage (phage) display library to bind ERBB2 and did so with moderate affinity of 295 nM. In order to enhance tumor uptake and clinical utility of the peptide, a novel phage microlibrary was created by flanking the parent sequence with random amino acids, followed by reselection using parallel strategies for high affinity and specific ERBB2 binding in an attempt to affinity maturate the peptide. One limitation of traditional phage display selections is difficulty in releasing the highest affinity phages from the target by incubation of acidic buffer. In an attempt to recover high affinity second-generation peptides from the ERBB2 microlibrary, two elution strategies, sonication and target elution, were undertaken. Sonication resulted in an approximately 50-fold enhancement in recovered phage per round of selection in comparison to target elution. Despite the differences in elution efficiency, the affinities of phage-displayed peptides selected from either strategy were relatively similar. Although both selections yielded peptides with significantly improved affinity in comparison to KCCYSL, the improvements were modest, most likely because the parental peptide binding cannot be improved by additional amino acids.

KEYWORDS

• Affinity maturation 
• ERBB2
•  Peptides 
• Phage display 

ABBREVIATIONS

ELISA – Enzyme linked immunosorbant assay

FMOC – Fluorenylmethyloxycarbonyl

GSG – Gly-Ser-Gly

K_{D –} Dissociation constant

TBS – Tris buffered saline

TBST – Tris buffered saline and tween

TU – Tetracycline transducing units

ACKNOWLEDGMENTS

The authors would like to thank Marie Dickerson, Jessica Newton-Northup and Fabio Gallazzi for their technical assistance. This work is supported by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development, Clinical Sciences Research and Development including the Cooperative Studies Program, Rehabilitation Research and Development Service, and Health Services Research and Development through a VA Merit Award [I01BX000964]. Additional support provided by an National Institute of Biomedical Imaging and Bioengineering Training Grant [NIBIB 5 T32 EB004822] and an NIH R21 Grant [5R21C179069].

CONFLICT OF INTEREST

HUMAN AND ANIMAL RIGHTS AND INFORMED CONSENT

This article does not contain any studies with human or animal subjects performed by any of the authors.

REFERENCES