Denk, M. | bioRxiv Science

bioRxiv2020

Antigens in water-in-oil emulsion: a simple antigen extraction method for analysis and proof of equal antigen distribution in vaccination syringe after mixture

Therapeutic vaccination of antigens in oil emulsions is an approach becoming increasingly popular. Water-in-oil emulsions enable the formation of a depot, a slow passive antigen release and a decelerated antigen degradation. Furthermore, particularly advantageous for peptide vaccinations with a low clinical response rate is the increased immunogenicity and activation of CD8+ and CD4+ T-cells. Therefore, the use of personalized peptide vaccination cocktails in oil emulsions are tested in the treatment of cancer patients. An equal peptide distribution in the emulsion is striking for a successful depot effect to ensure the optimal efficacy and additionally, to enable reliable drug release analytics. First, the stability of the generated peptide-Montanide ISA51 emulsion was demonstrated with a cocktail of heterogeneous peptides for 24 h in room temperature. Furthermore, we developed a simple peptide extraction method to investigate the equal peptide distribution in a Montanide ISA51 emulsion in the syringe after mixing. Peptides were successfully extracted from a peptide-Montanide ISA51 vaccination cocktail in sufficient volume for analysis and an equal peptide distribution in the syringe was verified via HPLC.

pharmacology and toxicology↗

bioRxiv2019

Validation of a high-performance liquid chromatography-tandem mass spectrometry immunopeptidomics assay for the identification of HLA class I ligands suitable for pharmaceutical therapies

For more than two decades naturally presented, human leukocyte antigen (HLA)-restricted peptides (immunopeptidome) have been eluted and sequenced using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Since, identified disease-associated HLA ligands have been characterized and evaluated as potential active substances. Treatments based on HLA-presented peptides have shown promising results in clinical application as personalized T cell-based immunotherapy. Peptide vaccination cocktails are produced as investigational medicinal products under GMP conditions. In order to support clinical trials based on HLA-presented tumor-associated antigens, in this study the sensitive LC-MS/MS HLA class I antigen identification pipeline was fully validated for our technical equipment according to the current US Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines.\n\nThe immunopeptidomes of JY cells with or without spiked-in, isotope labeled peptides, of peripheral blood mononuclear cells of healthy volunteers as well as a chronic lymphocytic leukemia and a bladder cancer sample were reliably identified using a data-dependent acquisition method. As the LC-MS/MS pipeline is used for identification purposes, the validation parameters include accuracy, precision, specificity, limit of detection and robustness.

immunology↗