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Stratification of responders towards eculizumab using a structural epitope mapping strategy
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, School of Biotechnology (BIO), Centres, Science for Life Laboratory, SciLifeLab, KTH Center for Applied Proteomics (KCAP).ORCID iD: 0000-0003-1096-9061
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, School of Biotechnology (BIO), Centres, Science for Life Laboratory, SciLifeLab, KTH Center for Applied Proteomics (KCAP).ORCID iD: 0000-0002-1389-5371
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2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, 31365Article in journal (Refereed) Published
Abstract [en]

The complement component 5 (C5)-binding antibody eculizumab is used to treat patients with paroxysmal nocturnal hemoglobinuria (PNH) and atypical haemolytic uremic syndrome (aHUS). As recently reported there is a need for a precise classification of eculizumab responsive patients to allow for a safe and cost-effective treatment. To allow for such stratification, knowledge of the precise binding site of the drug on its target is crucial. Using a structural epitope mapping strategy based on bacterial surface display, flow cytometric sorting and validation via haemolytic activity testing, we identified six residues essential for binding of eculizumab to C5. This epitope co-localizes with the contact area recently identified by crystallography and includes positions in C5 mutated in non-responders. The identified epitope also includes residue W917, which is unique for human C5 and explains the observed lack of cross-reactivity for eculizumab with other primates. We could demonstrate that Ornithodorus moubata complement inhibitor (OmCI), in contrast to eculizumab, maintained anti-haemolytic function for mutations in any of the six epitope residues, thus representing a possible alternative treatment for patients non-responsive to eculizumab. The method for stratification of patients described here allows for precision medicine and should be applicable to several other diseases and therapeutics.

Place, publisher, year, edition, pages
Nature Publishing Group, 2016. Vol. 6, 31365
Keyword [en]
Paroxysmal-Nocturnal Hemoglobinuria, Hemolytic-Uremic Syndrome, Catastrophic Antiphospholipid Syndrome, Antibody-Mediated Rejection, Bacterial Surface Display, Tick Ornithodoros-Moubata, Optica Spectrum Disorders, Complement Inhibitor, Transplant Recipients, Renal-Transplantation
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-191745DOI: 10.1038/srep31365ISI: 000381185300001PubMedID: 27509843Scopus ID: 2-s2.0-84982176744OAI: oai:DiVA.org:kth-191745DiVA: diva2:970945
Funder
Novo NordiskScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20160915

Available from: 2016-09-15 Created: 2016-09-02 Last updated: 2017-08-24Bibliographically approved
In thesis
1. Utilizing Solid Phase Cloning, Surface Display And Epitope Information for Antibody Generation and Characterization
Open this publication in new window or tab >>Utilizing Solid Phase Cloning, Surface Display And Epitope Information for Antibody Generation and Characterization
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Antibodies have become indispensable tools in diagnostics, research and as therapeutics. There are several strategies to generate monoclonal antibodies (mAbs) in order to avoid the drawbacks of polyclonal antibodies (pAbs) for therapeutic use. Moreover, the growing interest in precision medicine requires a well-characterized target and antibody to predict the responsiveness of a treatment. This thesis describes the use of epitope information and display technologies to generate and characterize antibodies. In Paper I, we evaluated if the epitope information of a well-characterized pAb could be used to generate mAbs with retained binding characteristics. In Paper II, the epitope on the complement protein C5 towards Eculizumab was mapped with surface display, the results of which explained the non-responsiveness of Eculizumab treatment among a patient group due to a mutated C5 gene. With this in mind, we showed efficacy in treatment of the mutated C5 variants using a drug binding to another site on C5, suggesting that our approach can be used to guide treatment in precision medicine. In Paper III, a Gram-positive bacterial display platform was evaluated to complement existing platforms for selection of human scFv libraries. When combined with phage display, a thorough library screening and isolation of nano-molar binders was possible. In Paper IV, a solid phase method for directed mutagenesis was developed to generate functional affinity maturation libraries by simultaneous targeting of all six CDRs. The method was also used to create numerous individual mutants to map the paratope of the parent scFv. The paratope information was used to create directed libraries and deep sequencing of the affinity maturation libraries confirmed the viability of the combination approach. Taken together, precise epitope/paratope information together with display technologies have the potential to generate attractive therapeutic antibodies and direct treatment in precision medicine.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2017. 100 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:10
Keyword
antibody engineering, antibody affinity maturation, combinatorial protein engineering, epitope mapping, FACS, HER2, complement C5, Staphylococcal surface display, surface display.
National Category
Other Industrial Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-205410 (URN)9789177293538 (ISBN)
Public defence
2017-05-19, F3, Lindstedtsvägen 26, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20170418

Available from: 2017-04-18 Created: 2017-04-18 Last updated: 2017-05-26Bibliographically approved
2. Cell line and protein engineering tools for production and characterization of biologics
Open this publication in new window or tab >>Cell line and protein engineering tools for production and characterization of biologics
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Our increasing understanding of disease mechanisms coupled with technological advances has facilitated the generation of pharmaceutical proteins, which are able to address yet unmet medical needs. Diseases that were fatal in the past can now be treated with novel biological medications improving and prolonging life for many patients. Pharmaceutical protein production is, however, a complex undertaking, which is by no means problem-free. The demand for more complex proteins and the realization of the importance of post-translational modifications have led to an increasing use of mammalian cells for protein expression. Despite improvements in design and production, the costs required for the development of pharmaceutical proteins still are far greater than those for conventional, small molecule drugs. To render such treatments affordable for healthcare suppliers and assist in the implementation of precision medicine, further progress is needed. In five papers this thesis describes strategies and methods that can help to advance the development and manufacturing of pharmaceutical proteins. Two platforms for antibody engineering have been developed and evaluated, one of which allows for efficient screening of antibody libraries whilst the second enables the straightforward generation of bispecific antibodies. Moreover, a method for epitope mapping has been devised and applied to map the therapeutic antibody eculizumab’s epitope on its target protein. In a second step it was shown how this epitope information can be used to stratify patients and, thus, contribute to the realization of precision medicine. The fourth project focuses on the cell line development process during pharmaceutical protein production. A platform is described combining split-GFP and fluorescence-activated droplet sorting, which allows for the efficient selection of highly secreting cells from a heterogeneous cell pool. In an accompanying study, the split-GFP probe was improved to enable shorter assay times and increased sensitivity, desirable characteristics for high-throughput screening of cell pools. In summary, this thesis provides tools to improve design, development and production of future pharmaceutical proteins and as a result, it makes a contribution to the goal of implementing precision medicine through the generation of more cost-effective biopharmaceuticals for well-characterized patient groups.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2017. 91 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:16
Keyword
Pharmaceutical proteins, precision medicine, antibody engineering, epitope mapping, cell line development, split-GFP
National Category
Pharmaceutical Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-212931 (URN)978-91-7729-497-9 (ISBN)
Public defence
2017-09-29, E3, Osquars backe 14, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20170828

Available from: 2017-08-28 Created: 2017-08-24 Last updated: 2017-08-30Bibliographically approved

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