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Protein deimmunization via structure-based design enables efficient epitope deletion at high mutational loads.

ABSTRACT: Anti-drug immune responses are a unique risk factor for biotherapeutics, and undesired immunogenicity can alter pharmacokinetics, compromise drug efficacy, and in some cases even threaten patient safety. To fully capitalize on the promise of biotherapeutics, more efficient and generally applicable protein deimmunization tools are needed. Mutagenic deletion of a protein's T cell epitopes is one powerful strategy to engineer immunotolerance, but deimmunizing mutations must maintain protein structure and function. Here, EpiSweep, a structure-based protein design and deimmunization algorithm, has been used to produce a panel of seven beta-lactamase drug candidates having 27-47% reductions in predicted epitope content. Despite bearing eight mutations each, all seven engineered enzymes maintained good stability and activity. At the same time, the variants exhibited dramatically reduced interaction with human class II major histocompatibility complex proteins, key regulators of anti-drug immune responses. When compared to 8-mutation designs generated with a sequence-based deimmunization algorithm, the structure-based designs retained greater thermostability and possessed fewer high affinity epitopes, the dominant drivers of anti-biotherapeutic immune responses. These experimental results validate the first structure-based deimmunization algorithm capable of mapping optimal biotherapeutic design space. By designing optimal mutations that reduce immunogenic potential while imparting favorable intramolecular interactions, broadly distributed epitopes may be simultaneously targeted using high mutational loads.

SUBMITTER: Salvat RS

PROVIDER: S-EPMC4452428 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Protein deimmunization via structure-based design enables efficient epitope deletion at high mutational loads.

Salvat Regina S RS Choi Yoonjoo Y Bishop Alexandra A Bailey-Kellogg Chris C Griswold Karl E KE

Biotechnology and bioengineering 20150223 7

Anti-drug immune responses are a unique risk factor for biotherapeutics, and undesired immunogenicity can alter pharmacokinetics, compromise drug efficacy, and in some cases even threaten patient safety. To fully capitalize on the promise of biotherapeutics, more efficient and generally applicable protein deimmunization tools are needed. Mutagenic deletion of a protein's T cell epitopes is one powerful strategy to engineer immunotolerance, but deimmunizing mutations must maintain protein structu ...[more]

PMID: 25655032

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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:BackgroundSynthetic zinc finger (ZF) proteins can be targeted to desired DNA sequences and are useful tools for gene therapy. We recently developed a ZF transcription repressor (ZF-KOX1) able to bind to expanded DNA CAG-repeats in the huntingtin (HTT) gene, which are found in Huntington's disease (HD). This ZF acutely repressed mutant HTT expression in a mouse model of HD and delayed neurological symptoms (clasping) for up to 3 weeks. In the present work, we sought to develop a long-term single-injection gene therapy approach in the brain.MethodSince non-self proteins can elicit immune and inflammatory responses, we designed a host-matched analogue of ZF-KOX1 (called mZF-KRAB), to treat mice more safely in combination with rAAV vector delivery. We also tested a neuron-specific enolase promoter (pNSE), which has been reported as enabling long-term transgene expression, to see whether HTT repression could be observed for up to 6 months after AAV injection in the brain.ResultsAfter rAAV vector delivery, we found that non-self proteins induce significant inflammatory responses in the brain, in agreement with previous studies. Specifically, microglial cells were activated at 4 and 6 weeks after treatment with non-host-matched ZF-KOX1 or GFP, respectively, and this was accompanied by a moderate neuronal loss. In contrast, the host-matched mZF-KRAB did not provoke these effects. Nonetheless, we found that using a pCAG promoter (CMV early enhancer element and the chicken β-actin promoter) led to a strong reduction in ZF expression by 6 weeks after injection. We therefore tested a new non-viral promoter to see whether the host-adapted ZF expression could be sustained for a longer time. Vectorising mZF-KRAB with a promoter-enhancer from neuron-specific enolase (Eno2, rat) resulted in up to 77 % repression of mutant HTT in whole brain, 3 weeks after bilateral intraventricular injection of 10(10) virions. Importantly, repressions of 48 % and 23 % were still detected after 12 and 24 weeks, respectively, indicating that longer term effects are possible.ConclusionHost-adapted ZF-AAV constructs displayed a reduced toxicity and a non-viral pNSE promoter improved long-term ZF protein expression and target gene repression. The optimized constructs presented here have potential for treating HD.

Dataset Information

Protein deimmunization via structure-based design enables efficient epitope deletion at high mutational loads.

Publications

Protein deimmunization via structure-based design enables efficient epitope deletion at high mutational loads.

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