Therapeutic protein deimmunization by T‐cell epitope removal: antigen‐specific immune responses in vitro and in vivo

Abstract

Hirudin III is an effective anti-coagulant; however, in 40% of treated patients, a high-titer of anti-Hirudin III IgG antibodies is observed. Development of antibody responses requires the activation of helper T lymphocyte (HTL), which is dependent on peptide epitopes binding to HLA class II molecules. Based on computational prediction softwares, four new mutants of Hirudin III, T4K, S9G, V21G, and V21K, had been designed with the aim of reducing the binding affinity of these HTL epitopes. The constructed mutants have been purified and assayed for bioactivity. Finally in vitro and in vivo cell-mediated responses were assessed and humoral immune assays were performed. All modified forms of Hirudin III were active, and showed significantly reduced human T-cell responses. All mutants indicated lower human IFN-γ level compared to native Hirudin, and V21K indicated lowest IFN-γ level. Mice immunized with T4K and V21K showed a significant reduction in total antibody responses and mouse IFN-γ levels. Mice immunized with V21K after 3rd immunization had lower T-cell proliferation compared to native Hirudin and other mutants. Based on these results, V21K is proposed as the best alternate Hirudin III candidate with lowest antigenicity. These findings validate our rational design strategy aimed at providing new active analogs of therapeutic proteins with reduced immunogenicity.

Immunogenicity assessment of fungal l-asparaginases: an in silico approach

“…The identification of immunogenic epitopes in the protein structure by computational tools has been a strategy used to achieve a reduction in the immunogenicity of several proteins [78][79][80]. It is also the case of ASNase from E. coli, where the use of bioinformatics techniques has allowed engineering the enzyme with the aim of obtaining an improved therapeutic agent [58,81,82].…”

Development of a strategy and computational application to select candidate protein analogues with reduced HLA binding and immunogenicity

“…Previous studies have reported different strategies to generate amino acid substitutions for the purpose of reducing immunogenicity (for recent studies see refs ). In several studies, alanine replacements have been used (see refs ).…”

“…Other de‐immunization studies also based on introducing single amino acid substitutions into known epitopes have been reported in the literature (see, for example, refs , as cited above). In those studies, mild and conservative substitution strategies, such as ‘alanine scans’, were shown to be effective.…”