Immunoglobulin Fc-Fused Peptide without C‑Terminal Arg or Lys Residue Augments Neuropilin-1-Dependent Tumor Vascular Permeability

• Published in: Molecular pharmaceutics Vol. 15; no. 2; pp. 394 - 402
• Main Authors: Baek, Du-San, Kim, Jeong-Ho, Kim, Ye-Jin, Kim, Yong-Sung
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 05.02.2018
• Subjects: Animals; Capillary Permeability - drug effects; Carcinogenesis - drug effects; Cell Line, Tumor; Female; Humans; Immunoglobulin Fc Fragments - genetics; Immunoglobulin Fc Fragments - isolation & purification; Immunoglobulin Fc Fragments - pharmacology; Immunoglobulin Fc Fragments - therapeutic use; Ligands; Mice; Mice, Inbred BALB C; Mice, Nude; Mutagenesis; Neoplasms - blood supply; Neoplasms - drug therapy; Neuropilin-1 - genetics; Neuropilin-1 - isolation & purification; Neuropilin-1 - metabolism; Peptides - genetics; Peptides - isolation & purification; Peptides - pharmacology; Peptides - therapeutic use; Protein Binding; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - pharmacology; Recombinant Fusion Proteins - therapeutic use; Vascular Endothelial Growth Factor A - metabolism; Xenograft Model Antitumor Assays; C-end rule; vascular permeability; yeast surface display; neuropilin-1; peptide engineering
• ISSN: 1543-8384; 1543-8392
• DOI: 10.1021/acs.molpharmaceut.7b00761

Neuropilin-1 (NRP1), which functions as a coreceptor for vascular endothelial growth factor (VEGF) and is implicated in vascular permeability and tumorigenesis, has been targeted by peptides that specifically bind to the VEGF-binding region on NRP1. Like natural VEGF ligands, all known peptides with NRP1-binding activity bind only through a carboxy (C)-terminal R/K-x-x-R/K sequence motif (x stands for any amino acids); this strict requirement is called the C-end rule (CendR). Here, we report immunoglobulin Fc-fused NRP1-specific peptides deviating from CendR. We screened a yeast surface-displayed Fc-fused non-CendR peptide library against NRP1 and isolated Fc-V12, wherein V12 peptide comprising 12 amino acids has a PPRV sequence at its C-terminal end. Although Fc-V12 lacked the CendR motif, it showed selective binding to the VEGF-binding region of NRP1 and triggered cellular internalization of NRP1, which resulted in enhanced extravasation into tumor tissues and tumor tissue penetration of the Fc-fused peptide along with the coinjected chemical drug in tumor-bearing mice. Through a saturation mutagenesis study, we identified that the Val residue at the C-terminus of Fc-V12 is crucial for NRP1 binding. We further improved NRP1 affinity of Fc-V12 (K D = ∼761 nM) through directed evolution of the upstream sequence of PPRV to obtain Fc-V12–33 (K D = ∼17.4 nM), which exhibited enhanced NRP1-mediated vascular permeability as compared with Fc-V12. Our results provide functional Fc-fused non-CendR peptides, which bind to the VEGF-binding region of NRP1 and enhance vascular permeability, expanding the sequence space of NRP1-targeting peptides.