Targeted delivery of multivalent phage display vectors into mammalian cells

Novel peptide motives targeting endocytosing receptors were isolated from p hage display libraries of random peptides by recovering internalized phage from mammalian cells. The peptide-presenting phage selected by internalizat ion in HEp-2 and ECV304 human cells were taken up 1000- to 100 000-fold mor e efficiently than their parent libraries, and from 10 to 100 times faster than phage particles displaying integrin-binding peptides. A high degree of selectivity of phage uptake was observed in these cells: phage selected in ECV304 cells were internalized approximately 100-fold more efficiently in ECV304 cells than in HEp-2 cells. Likewise, phage selected in HEp-2 cells w ere subsequently taken up approximately 40-fold more efficiently by HEp-2 c ells than by ECV304 cells. In multiple independent trials using a cyclic pe ptide library, an identical peptide sequence displayed on phage was interna lized by and recovered from ECV304 cells. These findings indicate that the internalization process is highly selective, and is capable of capturing a specific peptide from 2X10(7) peptide variants. Immunofluorescence microsco py showed juxtanuclear localization of internalized phage. These results de monstrate the feasibility of using multivalent phage-display libraries to i dentify new targeting ligands for the intracellular delivery of macromolecu les. (C) 1999 Elsevier Science B.V. All rights reserved.