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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) = 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.

SUBMITTER: Cantor JR

PROVIDER: S-EPMC3029727 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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

Cantor Jason R JR Yoo Tae Hyeon TH Dixit Aakanksha A Iverson Brent L BL Forsthuber Thomas G TG Georgiou George G

Proceedings of the National Academy of Sciences of the United States of America 20110105 4

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 extens ...[more]

PMID: 21209329

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Project description:Therapeutic proteins continue to yield revolutionary new treatments for a growing spectrum of human disease, but the development of these powerful drugs requires solving a unique set of challenges. For instance, it is increasingly apparent that mitigating potential anti-therapeutic immune responses, driven by molecular recognition of a therapeutic protein's peptide fragments, may be best accomplished early in the drug development process. One may eliminate immunogenic peptide fragments by mutating the cognate amino acid sequences, but deimmunizing mutations are constrained by the need for a folded, stable, and functional protein structure. These two concerns may be competing, as the mutations that are best at reducing immunogenicity often involve amino acids that are substantially different physicochemically. We develop a novel approach, called EpiSweep, that simultaneously optimizes both concerns. Our algorithm identifies sets of mutations making such Pareto optimal trade-offs between structure and immunogenicity, embodied by a molecular mechanics energy function and a T-cell epitope predictor, respectively. EpiSweep integrates structure-based protein design, sequence-based protein deimmunization, and algorithms for finding the Pareto frontier of a design space. While structure-based protein design is NP-hard, we employ integer programming techniques that are efficient in practice. Furthermore, EpiSweep only invokes the optimizer once per identified Pareto optimal design. We show that EpiSweep designs of regions of the therapeutics erythropoietin and staphylokinase are predicted to outperform previous experimental efforts. We also demonstrate EpiSweep's capacity for deimmunization of the entire proteins, case analyses involving dozens of predicted epitopes, and tens of thousands of unique side-chain interactions. Ultimately, Epi-Sweep is a powerful protein design tool that guides the protein engineer toward the most promising immunotolerant biotherapeutic candidates.

Project description:BACKGROUND: To develop protein therapeutics from exogenous sources, it is necessary to mitigate the risks of eliciting an anti-biotherapeutic immune response. A key aspect of the response is the recognition and surface display by antigen-presenting cells of epitopes, short peptide fragments derived from the foreign protein. Thus, developing minimal-epitope variants represents a powerful approach to deimmunizing protein therapeutics. Critically, mutations selected to reduce immunogenicity must not interfere with the protein's therapeutic activity. RESULTS: This paper develops methods to improve the likelihood of simultaneously reducing the anti-biotherapeutic immune response while maintaining therapeutic activity. A dynamic programming approach identifies optimal and near-optimal sets of conservative point mutations to minimize the occurrence of predicted T-cell epitopes in a target protein. In contrast with existing methods, those described here integrate analysis of immunogenicity and stability/activity, are broadly applicable to any protein class, guarantee global optimality, and provide sufficient flexibility for users to limit the total number of mutations and target MHC alleles of interest. The input is simply the primary amino acid sequence of the therapeutic candidate, although crystal structures and protein family sequence alignments may also be input when available. The output is a scored list of sets of point mutations predicted to reduce the protein's immunogenicity while maintaining structure and function. We demonstrate the effectiveness of our approach in a number of case study applications, showing that, in general, our best variants are predicted to be better than those produced by previous deimmunization efforts in terms of either immunogenicity or stability, or both factors. CONCLUSIONS: By developing global optimization algorithms leveraging well-established immunogenicity and stability prediction techniques, we provide the protein engineer with a mechanism for exploring the favorable sequence space near a targeted protein therapeutic. Our mechanism not only helps identify designs more likely to be effective, but also provides insights into the interrelated implications of design choices.

Project description:Unspecific peroxygenase (UPO) is a highly promiscuous biocatalyst, and its selective mono(per)oxygenase activity makes it useful for many synthetic chemistry applications. Among the broad repertory of library creation methods for directed enzyme evolution, genetic drift allows neutral mutations to be accumulated gradually within a polymorphic network of variants. In this study, we conducted a campaign of genetic drift with UPO in Saccharomyces cerevisiae, so that neutral mutations were simply added and recombined in vivo With low mutational loading and an activity threshold of 45% of the parent's native function, mutant libraries enriched in folded active UPO variants were generated. After only eight rounds of genetic drift and DNA shuffling, we identified an ensemble of 25 neutrally evolved variants with changes in peroxidative and peroxygenative activities, kinetic thermostability, and enhanced tolerance to organic solvents. With an average of 4.6 substitutions introduced per clone, neutral mutations covered approximately 10% of the protein sequence. Accordingly, this study opens new avenues for UPO design by bringing together neutral genetic drift and DNA recombination in vivoIMPORTANCE Fungal peroxygenases resemble the peroxide shunt pathway of cytochrome P450 monoxygenases, performing selective oxyfunctionalizations of unactivated C-H bonds in a broad range of organic compounds. In this study, we combined neutral genetic drift and in vivo DNA shuffling to generate highly functional peroxygenase mutant libraries. The panel of neutrally evolved peroxygenases showed different activity profiles for peroxygenative substrates and improved stability with respect to temperature and the presence of organic cosolvents, making the enzymes valuable blueprints for emerging evolution campaigns. This association of DNA recombination and neutral drift is paving the way for future work in peroxygenase engineering and, from a more general perspective, to any other enzyme system heterologously expressed in S. cerevisiae.

Dataset Information

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

Publications

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

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