Landscape phages and their fusion proteins targeted to breast cancer cells

Abstract

Breast cancer is a leading cause of death among women in the USA. The efficacy of existing anticancer therapeutics can be improved by targeting them through conjugation with ligands binding to cellular receptors. Recently, we developed a novel drug targeting strategy based on the use of pre-selected cancer-specific 'fusion pVIII proteins' (fpVIII), as targeting ligands. To study the efficiency of this approach in animal models, we developed a panel of breast cancer cell-binding phages as a source of targeted fpVIIIs. Two landscape phage peptide libraries (8-mer f8/8 and 9-mer f8/9) were screened to isolate 132 phage variants that recognize breast carcinoma cells MCF-7 and ZR-75-1 and internalize into the cells. When tested for their interaction with the breast cancer cells in comparison with liver cancer cells HepG2, human mammary cells MCF-10A cells and serum, 16 of the phage probes selectively interacted with the breast cancer cells whereas 32 bound both breast and liver cancer cells. The most prominent cancer-specific phage DMPGTVLP, demonstrating sub-nanomolar Kd in interaction with target cells, was used for affinity chromatography of cellular membrane molecules to reveal its potential binding receptor. The isolated protein was identified by direct sequencing as cellular surface nucleolin. This conclusion was confirmed by inhibition of the phage-cell interaction with nucleolin antibodies. Other prominent phage binders VPTDTDYS, VEEGGYIAA, and DWRGDSMDS demonstrate consensus motifs common to previously identified cancer-specific peptides. Isolated phage proteins exhibit inherent binding specificity towards cancer cells, demonstrating the functional activity of the selected fused peptides. The selected phages, their peptide inserts and intact fusion proteins can serve as promising ligands for the development of targeted nanomedicines and their study in model mice with xenograft of human cells MCF-7 and ZR-75-1.

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

“…Also a few common motifs between both libraries were identified, including EPG[Q/E], NGR, RGD and DGR, most families were unique within their respective library of origin due to the differences in amino acid diversity in each library before selection. We isolated a family of sequences containing the motif DxDY[S/T] that had been identified in previous screening of an 8-mer phage library against the MCF-7 breast cancer cell line [34] suggesting a possible common receptor between the two types of cancer. We also selected a phage clone, VEEGGYIAA, isolated during selection against lung cancer cells, that had also been reported previously in selection against breast cancer [34] and pancreatic cancer cells [36] using the same library suggesting a common receptor between all three cancer types.…”

“…The ability of unique phage clones to bind to their target lung cancer cell line, Calu-3, in comparison to other control cells and serum was studied by a phage capture assay described previously [34]. In this assay, small airway epithelial cells were used as phenotypically normal lung epithelial cells and MCF-7 breast cancer cells were used as non-related cancer cells.…”

Autonomous self-navigating drug-delivery vehicles: From Science Fiction to Reality

“…Since its discovery, promiscuous RGD motif-containing peptides, which bind a majority of known integrins [33] of tumor endothelium and cancer cells have been widely used in laboratory experiments to study targeted delivery of nanomedicines both in vitro and in vivo [34,35]. A promiscuous peptide of the second type -surface nucleolin-binding octamer DMPGTVLP fused to the N-terminus of the p8 phage protein, was isolated from the landscape phage library f8/8 [36] by its screening against breast cancer cells MCF-7 [37]. Nucleolin is a multifaceted and ubiquitous nonhistone nucleolar phosphoprotein, which shuttles between the nucleus and the cytoplasm [38].…”

“…Their isolation in pure form is very fast, simple and inexpensive [73]. It is important to note that capability of the selected landscape phage to specifically and selectively bind and penetrate into cancer cells is translated both to their individual proteins [37] and to corresponding phage protein-navigated nanomedicines. Combinatorial approach allowed us to screen dozens of phage-driven nanomedicines in one experiment [73].…”

“…Phages with different profiles of interaction with cancer cells have been isolated using different methods of their extraction. For example, acid extraction is used for isolation of surface-binding phage; pH change -for isolation of phages from cytoplasm, and mild detergents -for extraction of membranebound phages [37,43]. The p8-expressing landscape phage libraries allow selecting peptides with lower affinities, as their dense arrangement on the virion's surface results in a stronger binding due to avidity effect [74].…”