Abstract
Insufficient membrane penetration of drugs, in particular biotherapeutics and/or low target specificity remain a major drawback in their efficacy. We propose here the rational characterization and optimization of peptides to be developed as vectors that target cells expressing specific receptors involved in endocytosis or transcytosis. Among receptors involved in receptor-mediated transport is the LDL receptor. Screening complex phage-displayed peptide libraries on the human LDLR (hLDLR) stably expressed in cell lines led to the characterization of a family of cyclic and linear peptides that specifically bind the hLDLR. The VH411 lead cyclic peptide allowed endocytosis of payloads such as the S-Tag peptide or antibodies into cells expressing the hLDLR. Size reduction and chemical optimization of this lead peptide-vector led to improved receptor affinity. The optimized peptide-vectors were successfully conjugated to cargos of different nature and size including small organic molecules, siRNAs, peptides or a protein moiety such as an Fc fragment. We show that in all cases, the peptide-vectors retain their binding affinity to the hLDLR and potential for endocytosis. Following i.v. administration in wild type or ldlr-/- mice, an Fc fragment chemically conjugated or fused in C-terminal to peptide-vectors showed significant biodistribution in LDLR-enriched organs. We have thus developed highly versatile peptide-vectors endowed with good affinity for the LDLR as a target receptor. These peptide-vectors have the potential to be further developed for efficient transport of therapeutic or imaging agents into cells -including pathological cells—or organs that express the LDLR.
Highlights
Targeted organ delivery and expedient biotransport of drugs are challenging goals for the pharmaceutical industry and the search for alternative modes of drug delivery has developed as an active field of research
A cell line with stable expression of the human transferrin receptor fused to EGFP was used as alternative control
Drug delivery strategies based on apolipoproteins or full size endogenous ligands may appear difficult to implement at the industrial level and their clinical use may be further limited by saturating levels of the circulating endogenous ligands
Summary
Targeted organ delivery and expedient biotransport of drugs are challenging goals for the pharmaceutical industry and the search for alternative modes of drug delivery has developed as an active field of research. The low-density lipoprotein receptor (LDLR) family is composed of a class of single transmembrane glycoproteins recognized as cell surface endocytic receptors that bind apolipoprotein complexes, that may elicit signal transduction upon binding of extracellular ligands, and that internalize these ligands for intracellular processing and/or degradation by lysosomes [3]. Members of the LDLR family share homology within their extracellular domains, which are highlighted by the presence of clusters of ligandbinding repeats. Among these receptors, the LDLR binds cholesterol-carrying lipoprotein particles such as LDL [4]. Cholesterol plays several structural and metabolic roles that are vital It is found in the plasma membrane of cells, concentrates in rafts and caveolae which are sphingolipid-rich domains, and modulates membrane fluidity [5]. Upon binding to the PCSK9 protein, the LDLR-PCSK9 complex is directed to lysosomes for degradation, thereby leading to LDLR down-regulation [7]
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