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.

Fig 1. Mode of action of VEGF-targeting Peptide-Drug Conjugates (PDCs).

The PDC accumulates around tumor cells, which overexpress VEGF. The PDC-VEGF complex binds to VEGFR on surrounding blood vessels to stimulate receptor-mediated endocytosis, delivering the PDC into the cells. The PDC is broken down in lysosomes to release the drug into the cytosol.

Fig 2. The binding characteristics of M49.

(a) Schematic of the HLH peptide library. Spheres colored in green, red, and orange represent the positions of leucines inside of the helices, randomized residues in the loop region, and randomized residues on the C-terminal helix, respectively. (b) The binding affinity of M49 was determined via SPR. VEGF was immobilized by the amine coupling, and the peptides were injected as an analyte at different concentrations (50–3 nM). The data were fitted with the 1:1 Langmuir model. Black lines indicate the fitting curves. (c) Ternary complex formation consisting of M49, VEGF, and VEGFR-2 in the SPR assay. VEGF was injected at a concentration of 25 nM over immobilized VEGFR-2/Fc fusion protein followed by injection of M49 at a concentration of 250 nM (red line). A sensorgram of M49 (250 nM) for VEGFR-2 is shown as a black line. The injection points were represented as arrows.

Fig 3. The binding specificity of M49 as revealed by a human proteome microarray.

The normalized log₂(M49/Background) were presented for all 6,144 spots (upper panel) and focusing on VEGF family proteins (lower panel).

Fig 4. Confocal microscopy of HUVECs treated with Cy5-labeled M49K.

HUVECs were incubated with different solutions for 6h at 37°C before imaging: (a) M49K-Cy5 and VEGF-Alexa488 conjugate and (b) M49K-Cy5. (c) At 4°C, HUVECs were treated with M49K-Cy5 and VEGF, and VEGFR2 was detected using a mouse anti-VEGFR2 antibody and an Alexa488-anti-mouse IgG antibody. The white square has been enlarged in S10B Fig.

Fig 5. Peptide-drug conjugate structure and cell viability.

(a) Structure of peptide-drug conjugate M49K-vcMMAE. (b) Cell viability assay. HUVECs were treated with VEGF (25 ng/mL) and peptide-drug conjugates. After 24 h, cell viability was measured using WST-1 reagent. The data represent the mean ± standard deviation (n = 3).