Selection of Lung Cancer-Specific Landscape Phage for Targeted Drug Delivery

Abstract

Cancer cell-specific diagnostic or therapeutic tools are commonly believed to significantly increase the success rate of cancer diagnosis and targeted therapies. To extend the repertoire of available cancer cell-specific phage fusion proteins and study their efficacy as navigating moieties, we used two landscape phage display libraries f8/8 and f8/9 displaying an 8- or 9-mer random peptide fusion to identify a panel of novel peptide families that are specific to Calu-3 cells. Using a phage capture assay, we showed that two of the selected phage clones, ANGRPSMT and VNGRAEAP (phage and their recombinant proteins are named by the sequence of the fusion peptide), are selective for the Calu-3 cell line in comparison to phenotypically normal lung epithelial cells and distribute into unique subcellular fractions.

Figure 1

(A) Schematic of f8 type landscape phage display libraries. Diverse fusion derivatives of filamentous phage fd-tet from f8/8 and f8/9 libraries consist of phage with dimensions of 1.2 μm × 6.5 nm and contain a randomized peptide (red) fused to every copy of the N-terminus of the major coat protein pVIII (blue). The major coat proteins cover the length of the single-stranded DNA accommodated at the core of the phage particle. The ends of phage particles are closed by minor coat proteins pIII, pVI, pIX, & pVI. Phage composed of different random peptide fusions create a library of billions of different phage variants with unique surface landscapes. (B) Selection scheme to identify lung cancer cell-specific phage clones. Two input sublibraries were used beginning with second round, identified as an eluate input (solid line) and a lysate input (dashed line). Each input sublibrary was split into two library fractions consisting of eluate and lysate phage that were amplified as individual fractions. In subsequent rounds of selection, the eluate input sublibrary was prepared by combining the eluate fractions generated from both the eluate and lysate output sublibraries from the previous round of selection. A similar scheme was followed for the lysate input.

Figure 2

Recovery of phage (output phage/input phage, %) in (A) eluate selection scheme (solid line from Figure 1) and (B) lysate selection scheme (dashed line from Figure 1), isolated from the depleted f8/8 library (gray bars) and f8/9 library (black bars). (C) Histogram of the change in information content available in each sublibrary generated after four rounds of selection against a NSCLC cell line, Calu-3. Information content from clones in f8/8 (black line) and f8/9 (dashed line) are presented. Positive values indicate an increase in total information content, while negative values indicate a decrease in total information content. More detailed information is given in the results section.

Figure 3

(A) Representative selectivity and specificity assay for select phage clones from the f8/8 library. Individual phage clones were incubated at a constant concentration with: 1) target Calu-3 cells (black), 2) non-related MCF-7 cancer cells (striped), 3) phenotypically normal lung small airway epithelial cells (white), and 4) culture medium with 10% serum (grey). After washing, mammalian cells were lysed and the remaining phage were titered as described in the methods section. Percent recovery was plotted as the number of recovered phage per number of input phage particles for each phage clone. All phage clones binding Calu-3 cells were statistically different from an unrelated phage (P < 0.0001) and also different from paired phage samples with different targets (P < 0.0001). Phage interactions with MCF-7 cells were compared between paired phage samples with different targets and significant interactions were marked (* P < 0.0001). (B–D) Phage mode of interaction with Calu-3 cells after recovery of cell-associated phage by different elution steps with acid and detergent. Cells were split into three general subcellular fractions and the amount of phage associated with each fraction was determined by titering in K91BluKan E. coli. The portion of each fraction was calculated as the part of each fraction per total recovered phage. Three different classes of phage were identified: B) surface bound, C) cytoplasmic, and D) membrane bound.