Novel β-lactamase-random peptide fusion libraries for phage display selection of cancer cell-targeting agents suitable for enzyme prodrug therapy

Abstract

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 β-lactamase molecules were used for identifying β-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 singleround panning method based on phage DNA recovery, we identified severalβ-lactamase fusion peptides that specifically bind to live human breast cancer MDA-MB-361 cells. The β-lactamase fusion to the peptides helped in conducting the enzyme activity-based clone normalization and cellbinding 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 ~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 β-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.

Nanobody: A Small Antibody with Big Implications for Tumor Therapeutic Strategy

“…53 This antibody-dependent enzyme prodrug therapy system can cause specific transplant tumor decline or cure, and has a strong targeting for tumor therapy. 54 It has been proved by experiment that this conjugate can establish a nonadverse effect tumor treatment method by selectively activating anticancer precursors. 53 In addition, CEA Nb is more sensitive in clinical diagnosis and treatment of lung cancer than traditional antibodies.…”

Bioinformatics Resources and Tools for Phage Display

“…The DOS based applications were then converted into web based applications by creating COM+ wrappers around the legacy code. As complicated software package online, RELIC, especially its tools for population analysis, motif identification and sequence alignment are extensively used by the phage display community [ 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 ].…”

Cancer cell-specific internalizing ligands from phage displayed -lactamase-peptide fusion libraries

“…The nnk-scheme (where, N presents A, G, C or T and K presents T or G) is an extensively used conventional method in library construction, which eliminates the potential for opal (tga) and ochre (taa) stop codons and still encodes all 20 amino acids using 32 codons. The construction of the libraries were done using the approach reported earlier (Shukla and Krag, 2010). In order to avoid clones without an insert, the randomization was achieved in two steps.…”

“…The cell-binding studies with several cancer and noncancer cells were also conducted by the microscopic technique (Shukla and Krag, 2010). The procedure was the same as described above, except that the cell-bound b-lactamase was visualized (Ex 345 nm/Em 530 nm) with the help of the precipitating type Fluorocillin TM Green substrate (Invitrogen) using Nikon TE2000-U inverted fluorescence microscope (Nikon Corp., Kanagawa, Japan).…”