Therapeutic enzyme deimmunization by combinatorial T-cell epitope removal using neutral drift

Abstract

A number of heterologous enzymes have been investigated for cancer treatment and other therapeutic applications; however, immunogenicity issues have limited their clinical utility. Here, a new approach has been created for heterologous enzyme deimmunization whereby combinatorial saturation mutagenesis is coupled with a screening strategy that capitalizes on the evolutionary biology concept of neutral drift, and combined with iterative computational prediction of T-cell epitopes to achieve extensive reengineering of a protein sequence for reduced MHC-II binding propensity without affecting catalytic and pharmacological properties. Escherichia coli L-asparaginase II (EcAII), the only nonhuman enzyme approved for repeated administration, is critical in treatment of childhood acute lymphoblastic leukemia (ALL), but elicits adverse antibody responses in a significant fraction of patients. The neutral drift screening of combinatorial saturation mutagenesis libraries at a total of 12 positions was used to isolate an EcAII variant containing eight amino acid substitutions within computationally predicted T-cell epitopes-of which four were nonconservative-while still exhibiting k cat ∕K M ¼ 10 6 M −1 s −1 for L-Asn hydrolysis. Further, immunization of HLA-transgenic mice expressing the ALL-associated DRB1*0401 allele with the engineered variant resulted in significantly reduced T-cell responses and a 10-fold reduction in anti-EcAII IgG titers relative to the existing therapeutic. This significant reduction in the immunogenicity of EcAII may be clinically relevant for ALL treatment and illustrates the potential of employing neutral drift screens to achieve large jumps in sequence space as may be required for the deimmunization of heterologous proteins. directed evolution | protein engineering

Effect of Premedications in a Murine Model of Asparaginase Hypersensitivity

“…Several previous studies have sensitized mice to asparaginase and demonstrated the development of anti-asparaginase antibodies (Roberts et al, 1966;Baechtel and Prager, 1973;Goldberg et al, 1973;Uren and Ragin, 1979;Kamisaki et al, 1981;Yagura et al, 1981;Fernandes and Gregoriadis, 2001;Cantor et al, 2011) and demonstrated delayed hypersensitivity reactions to asparaginase (Kawamura et al, 1985;Gaspar et al, 1996). Our model is the first to show the development of acute symptomatic allergies in sensitized mice and to identify methods of reducing the severity of the symptoms and restoring asparaginase activity.…”

Prevention of influenza by targeting host receptors using engineered proteins

“…S7). Immunogenicity of the mCBMs may be an issue with repeat use, but the proteins could be modified to be less immunogenic if necessary (21).…”

Removing T-cell epitopes with computational protein design

“…We next attempted to recapitulate the results of previous experimentally validated protein deimmunization efforts where immunogenicity was reduced without disrupting biological function. Cantor et al (16) sought to remove T-cell epitopes from Escherichia coli L-asparaginase II, an enzyme approved for treatment of acute lymphoblastic leukemia, while maintaining native-like enzymatic activity and protein stability. Cantor et al (16) first used a neutral drift selection scheme to identify allowable mutations in each of three predicted HLA-DRB1*04:01 epitopes and then combined mutations in each epitope to produce a fully functional enzyme with reduced immunogenicity in vivo.…”