Impact of N-Terminal PEGylation on Synthesis and Purification of Peptide-Based Cancer Epitopes for Pancreatic Ductal Adenocarcinoma (PDAC)

Abstract

Peptide-based cancer vaccines have shown promising results in preclinical trials focusing on tumor immunotherapy. However, the presence of hydrophobic amino acid segments within these peptide sequences poses challenges in their synthesis, purification, and solubility, thereby hindering their potential use as cancer vaccines. In this study, we successfully synthesized peptide sequences derived from mesothelin (MSLN), a tumor-associated antigen overexpressed in pancreatic ductal adenocarcinoma (PDAC) by conjugating them with monodisperse polyethylene glycol (PEG). By PEGylating mesothelin epitopes of varying lengths (ranging from 9 to 38 amino acids) and hydrophobicity (60-90%), we achieved an effective method to improve the peptide yield and facilitate the processes of synthesis and purification. PEGylation significantly enhanced the solubility, facilitating the single-step purification of lengthy hydrophobic peptides. Most importantly, PEGylation did not compromise cell viability and had little to no effect on the immunogenicity of the peptides. In contrast, the addition of a palmitoyl group to increase immunogenicity led to reduced yield and solubility. Overall, PEGylation proves to be an effective technique for enhancing the solubility and broadening the range of utility of diverse long hydrophobic peptides.

Figure 1

PEGylated peptide-based cancer vaccines for PDAC immunotherapy.

Figure 2

HPLC chromatogram and MS spectrum (inset) of Fmoc-PEG 23 propionic acid 4. Retention time at 7.5 min in a 5 to 75% B gradient (A: 0.1% TFA in H₂O and B: CH₃CN) for 10 min. ESI-MS: [M + H]⁺ = 1368.7, [M + 2H]²⁺ = 685.1, [M + 3H]³⁺ = 457.1.

Figure 3

Analysis of amino-PEG 23 propionic acid oligomer purity. Linearity of different injection volumes (0.1, 0.3, 0.6, 1.0, 3.0, 6.0, and 10.0 μL) and PEG oligomer concentrations (area under the curve of chromatogram).

Figure 4

Chromatograms of (A) P6 (Palm-MSLN 2), (B) P13 (Palm-PEG11-MSLN 2), and (C) P14 (Palm-PEG23-MSLN 2) in 60–100% A: 0.045% TFA in H₂O and B: 0.036% TFA in CH₃CN for 8 min.

Figure 5

Effect of cell stimulation with PEGylated peptides. Splenocytes from vaccinated mice (adjuvants in PLGA nanoparticle vaccines) were stimulated with unmodified and PEGylated peptides for 48 h. (A) PEGylation of the peptides did not have influence on the viability of the cells. Data was normalized to the absorbance levels of nonstimulated cells. Cell proliferation was measured by the XTT assay. (B) PEGylation of the peptides did not change the levels of IFN-γ in the supernatant measured by ELISA except for MSLN 2.