Synthesis and characterization of a high-affinity α_vβ₆-specific ligand for in vitro and in vivo applications

Inhibition of bacteriophage uptake by different peptide scaffolds synthesized by the convergent strategy. The normalized phage uptake is defined as the ratio of output phage to input phage in the presence of peptide normalized to the output/input ratio of phage with no peptide added (1, 18). The number of input phage particles was held constant. Error bars, SE measurement of a minimum of three replicates. A, comparison of the inhibition of bacteriophage uptake by the H2009.1 20-mer peptides prepared by either convergent or linear synthesis. Synthesis of the peptide by linear synthesis and its ability to block phage uptake has been previously reported (16). B, the truncated 10-mer peptide blocks uptake of the H2009.1 phage clone as efficiently as the full-length 20-mer H2009.1 peptide. Single amino acid deletions from the carboxy terminus reveal that the minimal binding domain is a seven-amino-acid peptide. C, comparison of effect of peptide length on binding when placed on the trilysine core. D, evaluation of the effect of valency on peptide affinity.

The tetrameric 10-mer H2009.1 peptide mediates cellular uptake. A, H2009 cells were incubated for 10 min at 37°C with 1 μmol/L H2009.1 10-mer tetramer-Alexa Fluor 488 conjugate. Images were captured on a Leica TCS SP5 confocal microscope at 400× magnification with 7× optical zoom. Images are presented as an overlay series of 18 frames from the Z-stack acquisition (left). A single z-series slice from the image obtained in frame to the left is shown (middle). The H2009.1 10-mer tetrameric peptide mediates cellular uptake of Qdots in H2009 cells (right). The peptide was conjugated to the Qdot through a streptavidin-biotin interaction. Cells were incubated with 20 nmol/L of the SAQdot605-conjugated tetrameric H2009.1 peptide. In all panels, similar data were collected from distinct areas of the slide. B, H2009.1 cells were treated with H2009.1-Qdot conjugate (right) or H1299.2-Qdot conjugate (left). After incubation, the cells were fixed, permeabilized, and stained for β₆ (green). Within the 10-min incubation time, the H2009.1-Qdot conjugate and the β₆ integrin are internalized. Colocalization of the H2009.1-Qdot conjugate and β₆ is indicated by the yellow color and pointed out by the arrows for several cells. H2009 cells do not mediate uptake of the H1299.2-Qdot conjugate (left). Additionally, the β₆ integrin remains on the cell surface. C, uptake of peptide-conjugated Qdots is independent of cell cycle progression. Cells were cultured overnight in eight-well chamber slides before incubation with 10 nmol/L Qdot-H2009.1 peptide conjugate for 10 min at 37°C. Wells were washed and fixed in PBS-buffered formalin as detailed in Experimental Procedures. Cellular DNA was stained with 4′,6-diamidino-2-phenylindole (top left) and the cell membrane was stained with wheat germ agglutinin-Alexa Fluor 488 (top right). Bottom left, Qdot uptake; bottom right, an overlay of the three images. Arrows, cells captured during mitosis: a, prophase; b, metaphase; c, early anaphase; and d, late telophase/cytokinesis.

The H2009.1 10-mer tetrameric peptide can be used in diagnostic formats. A, formalin-fixed, paraffin-embedded tumor sections were stained with H&E (top left) or H2009.1 10-mer peptide-labeled Qdots (top right and bottom left) or Qdots with no peptide (bottom right). Bottom left (400× magnification), peptide-labeled Qdot staining (red) is associated with intact tumor cells as indicated by the staining of nuclei (blue), whereas unlabeled Qdots exhibit minimal cellular staining. At 100× magnification (top right), the labeled Qdot staining appears uniform throughout the tumor. Similar results were observed with independently prepared tumor specimens. B, the H2009.1 10-mer tetrameric peptide can be used as a flow cytometry reagent to bind to α_vβ₆-positive cells. Cells (α_vβ₆-positive H2009 cells and α_vβ₆-negative H460 cells; ref. 2) were incubated for 1 h with H2009.1 10-mer peptide-Alexa Fluor 488 conjugate or scrambled peptide-dye conjugate, washed, and evaluated for peptide-dye uptake by flow cytometry. Cells were gated by cell volume and side scatter to remove cell fragments and debris from analysis. A total of 10,000 events were evaluated for fluorescence in channel 1 (excitation at 488 nm, emission at 500–550 nm). The data shown were obtained at 10 nmol/L peptide-dye conjugate concentration.