A “ligand-targeting” peptide-drug conjugate: Targeted intracellular drug delivery by VEGF-binding helix-loop-helix peptides via receptor-mediated endocytosis

Abstract

As a new alternative to antibody-drug conjugates, we generated “ligand-targeting” peptide-drug conjugates (PDCs), which utilize receptor-mediated endocytosis for targeted intracellular drug delivery. The PDC makes a complex with an extracellular ligand and then binds to the receptor on the cell surface to stimulate intracellular uptake via the endocytic pathway. A helix-loop-helix (HLH) peptide was designed as the drug carrier and randomized to give a conformationally constrained peptide library. The phage-displayed library was screened against vascular endothelial growth factor (VEGF) to yield the binding peptide M49, which exhibited strong binding affinity (KD = 0.87 nM). The confocal fluorescence microscopy revealed that peptide M49 formed a ternary complex with VEGF and its receptor, which was then internalized into human umbilical vein endothelial cells (HUVECs) via VEGF receptor-mediated endocytosis. The backbone-cyclized peptide M49K was conjugated with a drug, monomethyl auristatin E, to afford a PDC, which inhibited VEGF-induced HUVEC proliferation. HLH peptides and their PDCs have great potential as a new modality for targeted molecular therapy.

New Class of Drug Modalities: Directed Evolution of a De Novo Designed Helix–Loop–Helix Peptide to Bind VEGF for Tumor Growth Inhibition

“…In the previous work, we isolated a VEGF-targeting HLH peptide, M49, by bio-panning with library ΔPTA-6R-Loop11C in which the binding phage particles were eluted under acidic conditions (glycine-HCl buffer, pH = 2.0). 10 The peptide M49 showed tight binding to VEGF (K D = 0.87 nM), but no inhibitory activity for the VEGF/VEGFR interaction. Therefore, we conjugated M49 with a toxin to generate a peptide− drug conjugate, which made a ternary complex with VEGF and VEGFR to go into cells by receptor-mediated endocytosis.…”

“…Previously, we constructed phagedisplayed libraries of HLH peptides, which were screened against a variety of disease-related proteins to develop molecular targeting HLH peptides with antibody-like affinity and specificity. 9,10 In this work, we demonstrate the generation of an HLH peptide inhibitor for human vascular endothelial growth factor-A (VEGF) from the HLH peptide libraries. VEGF is a wellestablished molecular target for cancer therapy.…”

“…Therefore, random mutations are introduced into the solvent-exposed positions in YT1-S to give a library of peptides with the HLH structure. Previously, we constructed phage-displayed libraries of HLH peptides, which were screened against a variety of disease-related proteins to develop molecular targeting HLH peptides with antibody-like affinity and specificity. , …”

“Human and Mouse Cross-Reactive” Albumin-Binding Helix–Loop–Helix Peptide Tag for Prolonged Bioactivity of Therapeutic Proteins

“… 7 − 10 Due to the rigid constrained conformation, the HLH peptide shows strong binding affinity, high target specificity, and proteolytic resistance. 10 The small molecular size induces no unwanted immunogenic reactions. In addition, HLH peptides, which consist of natural l -amino acids, are easily produced by conventional chemical synthesis, having the advantages of synthetic simplicity and low-cost manufacture.…”

Generation of inhibitory peptides for IKKε from a kinase‐focused phage library of helix‐loop‐helix peptides

“…[ 4,5 ] Using phage or yeast surface display, we obtained HLH peptide ligands against ganglioside GM3, granulocyte colony‐stimulating factor receptor, the Fc domain of IgG, cytotoxic T lymphocyte antigen‐4, and vascular endothelial growth factor. [ 6–10 ] Moreover, we developed the inhibitory peptide cHLHp53‐R for p53–HDM2 interaction inside cells. [ 11 ] The binding activity of the inhibitory peptide to HDM2 was achieved by protein epitope grafting, and the cell membrane permeability was conferred to the peptide by substituting the six solvent‐exposed alanines in the N‐terminal helix to arginines.…”