The Interactions of Peptides with the Innate Immune System Studied with Use of T7 Phage Peptide Display

Abstract

The icosahedral T7 phage (diameter approximately 65 nm) displaying random peptides at the carboxy-terminus of the phage coat proteins was used as a model for drug and gene delivery vehicles containing peptide ligands. We found that displayed peptides were recognized by natural antibodies and induced complement activation. Strikingly, the phage inactivation by complement was peptide-specific that implied the existence of numerous natural antibodies with different peptide specificity. Selection of phage that avoided inactivation by complement allowed the identification of peptides that protected the phage by binding to serum proteins. In rat blood, peptides with carboxy-terminal lysine or arginine residues protected the phage against complement-mediated inactivation by binding C-reactive protein. In human serum, a number of protective peptides with tyrosine residues were selected. The recognition of displayed peptides by natural antibodies appears to represent a universal mechanism for activation of complement at sites that contain identical or homologous proteins with exposed carboxy-termini.

Peptide-mediated targeting of hepatocytes via low density lipoprotein receptor-related protein (LRP)

“…However, the LRP-mediated endocytosis of apoE-enriched chylomicron remnants, beta-VLDL, and LRP antibodies occurs largely in liver, where both hepatocytes and Kupffer cells are accessible to small particles due to vasculature fenestration Herz et al, 1990). The predominant occurrence of the LRP internalization activity in liver is consistent with our observations that up to 95% of T7 phage injected into mice is recovered from liver provided that the phage is rendered 'invisible' to natural IgM antibodies by removing its coat protein C-terminus (Sokoloff et al, 2000(Sokoloff et al, , 2003. Importantly, LRP expression is not observed in normal endothelial cells despite the presence in these cells of some LRP mRNA (Moestrup et al, 1992;Lupu et al, 1994).…”

In Vivo Selection and Validation of Liver-Specific Ligands Using a New T7 Phage Peptide Display System

“…The new peptide display system is based on small phage T7 that easily penetrates through the space of Disse and presents displayed sequences at the p17 site that is evidently accessible to cell surface receptors (Sokoloff et al 2000). Our studies showed that the p17 display site was able to accommodate a wide variety of peptide sequences.…”

“…Therefore, while the substitution of the wild-type p17 sequence by a random sequence in a domain composed of perfectly aligned p17 chains would probably "break" its rod-like configuration and significantly reduce phage viability; the alignment shift is likely to mitigate the impact of such substitution. Peptide sequences are displayed in this system in a constrained configuration, which promotes selection of stable and efficacious ligands (Falciani et al 2005) and prevents the recognition of displayed sequences by natural antibodies (Sokoloff et al 2000). The avoidance of such recognition may be of paramount importance in designing therapeutically relevant delivery vehicles.…”

“…Thus normal human serum contains a large population of natural IgM antibodies that collectively recognize virtually all randomly generated linear C-terminal peptides (Sokoloff et al 2000;Sokoloff et al 2001;Sokoloff et al 2004). This suggests that linear C-terminal peptides as a group may be difficult to adapt as therapeutic ligands, particularly if present as multiple copies.…”

“…The truncation of p10B after its residue Q343 abolished the recognition of phage by natural IgM antibodies that specifically react with protein C-terminal sequences. This prevented phage uptake by Kupffer cells that apparently recognize phage through complement protein fragments attached to the phage surface as a result of complement activation by IgM-phage complexes (Sokoloff et al 2000(Sokoloff et al , 2003(Sokoloff et al , 2004. The phage uptake by hepatocytes was disrupted by a single point mutation (K 211 → E) in the coiled-coil domain of p17 ( Figure 1c) (Sokoloff et al 2003).…”