The unparalleled specificity and activity of therapeutic proteins has reshaped many aspects of modern clinical practice, and aggressive development of new protein drugs promises a continued revolution in disease therapy. As a result of their biological origins, however, therapeutic proteins present unique design challenges for the biomolecular engineer. For example, protein drugs are subject to immune surveillance within the patient's body; this anti-drug immune response can compromise therapeutic efficacy and even threaten patient safety. Thus, there is a growing demand for broadly applicable protein deimmunization strategies. We have recently developed optimization algorithms that integrate computational prediction of T-cell epitopes and bioinformatics-based assessment of the structural and functional consequences of epitope-deleting mutations. Here, we describe the first experimental validation of our deimmunization algorithms using Enterobacter cloacae P99 β-lactamase, a component of antibody-directed enzyme prodrug cancer therapies. Compared with wild-type or a previously deimmunized variant, our computationally optimized sequences exhibited significantly less in vitro binding to human type II major histocompatibility complex immune molecules. At the same time, our globally optimal design exhibited wild-type catalytic proficiency. We conclude that our deimmunization algorithms guide the protein engineer towards promising immunoevasive candidates and thereby have the potential to streamline biotherapeutic development.

H. pylori persists in the human stomach over decades and promotes several adverse clinical sequelae including gastritis, peptic ulcers and gastric cancer that are linked to the induction and subsequent evasion of chronic gastric inflammation. Emerging evidence indicates that H. pylori infection may also protect against asthma and some other immune-mediated conditions through regulatory T cell effects outside the stomach. To characterize the complexity of the CD4+ T cell response generated during H. pylori infection, computational methods were previously used to generate a panel of 90 predicted epitopes conserved among H. pylori genomes that broadly cover HLA Class II diversity for maximum population coverage. Here, these sequences were tested individually for their ability to induce in vitro responses in peripheral blood mononuclear cells by interferon-γ ELISpot assay. The average number of spot-forming cells/million PBMCs was significantly elevated in H. pylori-infected subjects over uninfected persons. Ten of the 90 peptides stimulated IFN-γ secretion in the H. pylori-infected group only, whereas two out of the 90 peptides elicited a detectable IFN-γ response in the H. pylori-uninfected subjects but no response in the H. pylori-infected group. Cytokine ELISA measurements performed using in vitro PBMC culture supernatants demonstrated significantly higher levels of TNF-α, IL-2, IL-4, IL-6, IL-10, and TGF-β1 in the H. pylori-infected subjects, whereas IL-17A expression was not related to the subjects H. pylori-infection status. Our results indicate that the human T cell responses to these 90 peptides are generally increased in actively H. pylori-infected, compared with H. pylori-naïve, subjects. This information will improve understanding of the complex immune response to H. pylori, aiding rational epitope-driven vaccine design as well as helping identify other H. pylori epitopes with potentially immunoregulatory effects.

No abstract