Computationally optimized deimmunization libraries yield highly mutated enzymes with low immunogenicity and enhanced activity

Abstract

Therapeutic proteins of wide-ranging function hold great promise for treating disease, but immune surveillance of these macromolecules can drive an antidrug immune response that compromises efficacy and even undermines safety. To eliminate widespread T-cell epitopes in any biotherapeutic and thereby mitigate this key source of detrimental immune recognition, we developed a Pareto optimal deimmunization library design algorithm that optimizes protein libraries to account for the simultaneous effects of combinations of mutations on both molecular function and epitope content. Active variants identified by high-throughput screening are thus inherently likely to be deimmunized. Functional screening of an optimized 10-site library (1,536 variants) of P99 β-lactamase (P99βL), a component of ADEPT cancer therapies, revealed that the population possessed high overall fitness, and comprehensive analysis of peptide-MHC II immunoreactivity showed the population possessed lower average immunogenic potential than the wild-type enzyme. Although similar functional screening of an optimized 30-site library (2.15 × 10 9 variants) revealed reduced population-wide fitness, numerous individual variants were found to have activity and stability better than the wild type despite bearing 13 or more deimmunizing mutations per enzyme. The immunogenic potential of one highly active and stable 14-mutation variant was assessed further using ex vivo cellular immunoassays, and the variant was found to silence T-cell activation in seven of the eight blood donors who responded strongly to wild-type P99βL. In summary, our multiobjective library-design process readily identified large and mutually compatible sets of epitope-deleting mutations and produced highly active but aggressively deimmunized constructs in only one round of library screening.deimmunization | computational protein design | immunogenicity | T-cell epitope | combinatorial library P rotein engineering techniques have enabled targeted modification of biotherapeutics to mitigate the T-cell-mediated immune response that otherwise can undermine clinical applications (1). These approaches mutate specific amino acids in a therapeutic candidate so as to disrupt MHC II binding of constituent peptides processed from the protein following uptake by antigen-presenting cells (APCs). The amino acid substitutions prevent APCs from displaying peptide epitopes to cognate CD4 +

Multifunctional CRISPR/Cas9 with engineered immunosilenced human T cell epitopes

“…Conventional methods of deimmunizing non-human therapeutic proteins rely on trial-and-error mutagenesis or machine learning and often includes deletion of whole regions of the protein [35][36][37][38][39] . Here, as a general principle, we show that alteration of one of the anchor residues of an immunodominant epitope abolished specific T cell recognition.…”

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].…”

A High-Throughput Single-Clone Phage Fluorescence Microwell Immunoassay and Laser-Driven Clonal Retrieval System

“…The immunogenicity of therapeutic antibodies can drive an anti-drug immune response that compromises efficacy and undermines safety [43]. The presence of human T-cell epitopes within the antibody can activate helper T cells, neutralizing the therapeutic effect [44][45][46].…”