Formulation and characterization of a ten-peptide single-vial vaccine EP-2101, designed to induce cytotoxic T-lymphocyte responses for cancer immunotherapy

Abstract

Effective vaccines that mediate clinical responses in cancer patients may require generation of broadly specific cytotoxic T lymphocytes (CTL) directed against multiple epitopes and tumor-associated antigens (TAA). Pursuant to this goal we developed a synthetic peptide vaccine, EP-2101, composed of 10 synthetic peptide epitopes and formulated in Montanide® ISA 51 adjuvant. Nine of the HLA-A*0201-restricted CTL epitopes were derived from five well-characterized TAA. The universal HLA-DR binding epitope PADRE® was also included for T-cell help. Herein we describe studies on the formulation and characterization of the EP-2101 vaccine which supports generation of a sterile single-vial emulsion using standardized processes. The physicochemical properties of the peptides were highly disparate and as such, solubilization studies were required to identify a process which supported sterile filtration of the EP-2101 peptide mix. A homogenization-based formulation process with Montanide ISA 51 and 0.5 mg/ml of each peptide was developed to generate a water-in-oil emulsion. Physical studies indicated the vaccine emulsion to be stable, with little change in visual appearance, viscosity and water droplet size for at least 3 months. The physical stability of individual peptides in the vaccine emulsion was demonstrated using HPLC and immunogenicity of the vaccine formulation was confirmed in HLA-A*0201/Kb transgenic mice where T-cell responses could be induced to all epitopes in EP-2101 following vaccination. Our study process is scalable for production of approximately 1.5 liters of potent experimental vaccine for preclinical animal toxicity and phase 1 clinical testing in patients with breast, colon or lung cancer.