Rapid Fine Conformational Epitope Mapping Using Comprehensive Mutagenesis and Deep Sequencing

Kowalsky, Caitlin A.; Faber, Matthew S.; Nath, Aritro; Dann, Hailey E.; Kelly, Vince W.; Liu, Li; Shanker, P.; Wagner, Ellen K.; Maynard, Jennifer A.; Chan, Christina; Whitehead, Timothy A.

doi:10.1074/jbc.m115.676635

Cited by 76 publications

(103 citation statements)

References 46 publications

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“…1B). The hu1B7 epitope has been defined experimentally: it localizes primarily to the S1 subunit (21) and was finely resolved by use of a combined yeast display/ high-throughput sequencing approach (34). Although experimental epitope mapping of hu11E6 has not been performed, the murine antibody has been reported to bind epitopes present in the homologous S2 and S3 subunits, thereby inhibiting PTx from binding glycosylated and sialylated cellular receptors (33).…”

Section: Resultsmentioning

confidence: 99%

Synergistic Neutralization of Pertussis Toxin by a Bispecific Antibody In Vitro and In Vivo

Wagner

Wang

Bui

et al. 2016

Clin Vaccine Immunol

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Bispecific antibodies are a rapidly growing class of therapeutic molecules, originally developed for the treatment of cancer but recently explored for the treatment of autoimmune and infectious diseases. Bordetella pertussis is a reemerging pathogen, and several of the key symptoms of infection are caused by the pertussis toxin (PTx). Two humanized antibodies, hu1B7 and hu11E6, bind distinct epitopes on PTx and, when coadministered, mitigate disease severity in murine and baboon models of infection. Here we describe the generation of a bispecific human IgG1 molecule combining the hu1B7 and hu11E6 binding sites via a knobs-in-holes design. The bispecific antibody showed binding activity equivalent to that of the antibody mixture in a competition enzyme-linked immunosorbent assay (ELISA). A CHO cell neutralization assay provided preliminary evidence for synergy between the two antibodies, while a murine model of PTx-induced leukocytosis definitively showed synergistic neutralization. Notably, the bispecific antibody retained the synergy observed for the antibody mixture, supporting the conclusion that synergy is due to simultaneous blockade of both the catalytic and receptor binding activities of pertussis toxin. These data suggest that a hu1B7/hu11E6 bispecific antibody is a viable alternative to an antibody mixture for pertussis treatment. Despite vaccination, pertussis infection continues to cause ϳ195,000 deaths worldwide, primarily of infants (1). Of the estimated 16 million cases of pertussis each year, ϳ95% occur in the developing world. Even in developed countries, the disease incidence has increased dramatically over the last decade, reaching prevaccination levels in some countries (2, 3). This rise has been attributed to shortcomings of the current acellular vaccine (4) as well as pathogen adaptation (5). In both cases, high levels of circulating disease place young infants at risk, as this population is the most susceptible to severe disease. An antibody therapeutic could be used to treat seriously ill infants in the developing world and to prevent disease in high-risk areas.Pertussis toxin (PTx) is one of several virulence factors secreted by the Gram-negative bacterium Bordetella pertussis. PTx is directly responsible for suppression of the innate immune system (6) and for systemic leukocytosis, which is the key clinical indicator of severe disease and appears to be directly responsible for pulmonary hypertension and organ failure (7). In addition, low titers of PTx-neutralizing antibodies correlate with susceptibility to clinical infection (8). We previously developed a binary mixture of two humanized anti-pertussis toxin antibodies which was able to mitigate whooping cough in mouse and baboon models of infection (9). The antibody hu11E6 blocks binding of the toxin to host cells, while the antibody hu1B7 interferes with the catalytic pathway. At a dose selected to demonstrate efficacy but not synergy, the individual antibodies and the mixture were able to completely suppress leukocytosis in a murine ...

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Section: Resultsmentioning

confidence: 99%

Synergistic Neutralization of Pertussis Toxin by a Bispecific Antibody In Vitro and In Vivo

Wagner

Wang

Bui

et al. 2016

Clin Vaccine Immunol

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“…Examples include the insertion of post-translational modification sites (glycosylation, acetylation, phosphorylation, etc. ), enhancement of kinetic characteristics by modification of the active site environment, and modification of protein aggregation paths 25, 26, 27, 28. (Fig.…”

Section: Introductionmentioning

confidence: 99%

Biopharmaceuticals from microorganisms: from production to purification

Jozala

Geraldes

Tundisi

et al. 2016

Brazilian Journal of Microbiology

147

105

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The use of biopharmaceuticals dates from the 19th century and within 5–10 years, up to 50% of all drugs in development will be biopharmaceuticals. In the 1980s, the biopharmaceutical industry experienced a significant growth in the production and approval of recombinant proteins such as interferons (IFN α, β, and γ) and growth hormones. The production of biopharmaceuticals, known as bioprocess, involves a wide range of techniques. In this review, we discuss the technology involved in the bioprocess and describe the available strategies and main advances in microbial fermentation and purification process to obtain biopharmaceuticals.

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“…Most previously described studies have used targeted library screening and deep sequencing for epitope mapping or to identify which positions in a protein are functionally important (27,28,30). Some also used deep mutational scanning with the explicit goal of improving the affinity, however.…”

Section: Discussionmentioning

confidence: 99%

“…affinity) in which subtle effects of individual mutations can be discerned by the degree to which they are enriched or depleted by the selection. Deep mutational scanning has been applied successfully in combination with computational protein design for the engineering of enzyme inhibitors (20) and antibody binding proteins (21), for the evolution of antibodies (22)(23)(24) and T-cell receptors (25,26), for epitope mapping (27,28), for engineering immune co-receptors (29), as well as for establishing protein structure-function relationships (30,31).…”

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confidence: 99%

Affinity Maturation of a Cyclic Peptide Handle for Therapeutic Antibodies Using Deep Mutational Scanning

Rosmalen

Janssen

Hendrikse

et al. 2017

Journal of Biological Chemistry

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Edited by Luke O'NeillMeditopes are cyclic peptides that bind in a specific pocket in the antigen-binding fragment of a therapeutic antibody such as cetuximab. Provided their moderate affinity can be enhanced, meditope peptides could be used as specific non-covalent and paratope-independent handles in targeted drug delivery, molecular imaging, and therapeutic drug monitoring. Here we show that the affinity of a recently reported meditope for cetuximab can be substantially enhanced using a combination of yeast display and deep mutational scanning. Deep sequencing was used to construct a fitness landscape of this protein-peptide interaction, and four mutations were identified that together improved the affinity for cetuximab 10-fold to 15 nM. Importantly, the increased affinity translated into enhanced cetuximab-mediated recruitment to EGF receptor-overexpressing cancer cells. Although in silico Rosetta simulations correctly identified positions that were tolerant to mutation, modeling did not accurately predict the affinity-enhancing mutations. The experimental approach reported here should be generally applicable and could be used to develop meditope peptides with low nanomolar affinity for other therapeutic antibodies.

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Rapid Fine Conformational Epitope Mapping Using Comprehensive Mutagenesis and Deep Sequencing

Cited by 76 publications

References 46 publications

Synergistic Neutralization of Pertussis Toxin by a Bispecific Antibody In Vitro and In Vivo

Synergistic Neutralization of Pertussis Toxin by a Bispecific Antibody In Vitro and In Vivo

Biopharmaceuticals from microorganisms: from production to purification

Affinity Maturation of a Cyclic Peptide Handle for Therapeutic Antibodies Using Deep Mutational Scanning

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