Novel beta-lactamase-random peptide fusion libraries for phage display selection of cancer cell-targeting agents suitable for enzyme prodrug therapy.
Novel phage-displayed random linear dodecapeptide (X(12)) and cysteine-constrained decapeptide (CX(10)C) libraries constructed in fusion to the amino-terminus of P99 beta-lactamase molecules were used for identifying beta-lactamase-linked cancer cell-specific ligands. The size and quality of both libraries were comparable to the standards of other reported phage display systems. Using the single-round panning method based on phage DNA recovery, we identified several beta-lactamase fusion peptides that specifically bind to live human breast cancer MDA-MB-361 cells. The beta-lactamase fusion to the peptides helped in conducting the enzyme activity-based clone normalization and cell-binding screening in a very time- and cost-efficient manner. The methods were suitable for 96-well readout as well as microscopic imaging. The success of the biopanning was indicated by the presence of approximately 40% cancer cell-specific clones among recovered phages. One of the binding clones appeared multiple times. The cancer cell-binding fusion peptides also shared several significant motifs. This opens a new way of preparing and selecting phage display libraries. The cancer cell-specific beta-lactamase-linked affinity reagents selected from these libraries can be used for any application that requires a reporter for tracking the ligand molecules. Furthermore, these affinity reagents have also a potential for their direct use in the targeted enzyme prodrug therapy of cancer.
Shukla
Girja S
GS
Department of Surgery and Vermont Cancer Center, University of Vermont College of Medicine, Burlington, VT 05405, USA. Girja.Shukla@uvm.edu
Krag
David N
DN
D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001483:Base Sequence; D001943:Breast Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004247:DNA; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D006801:Humans; D009842:Oligopeptides; D019151:Peptide Library; D010455:Peptides; D011355:Prodrugs / Q000494:pharmacology*
D000970:Antineoplastic Agents; D004792:Enzyme Precursors; D009842:Oligopeptides; D019151:Peptide Library; D010455:Peptides; D011355:Prodrugs; D004247:DNA; C085706:FLAG peptide