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Generation of HER2 monoclonal antibodies using epitopes of a rabbit polyclonal antibody
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0002-1389-5371
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0001-8993-048X
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0002-9977-5724
2014 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 31, no 1, 35-43 p.Article in journal (Refereed) Published
Abstract [en]

One of the issues in using polyclonal antibodies is the limited amount of reagent available from an immunisation, leading to batch-to-batch variation and difficulties in obtaining the same antibody performance when the same antigen is re-immunised into several separate animals. This led to the development of hybridoma technology allowing, at least theoretically, for an unlimited production of a specific binder. Nevertheless, polyclonal antibodies are widely used in research and diagnostics and there exists a need for robust methods to convert a polyclonal antibody with good binding performance into a renewable monoclonal with identical or similar binding specificity. Here we have used precise information regarding the functional recognition sequence (epitope) of a rabbit polyclonal antibody with attractive binding characteristics as the basis for generation of a renewable mouse monoclonal antibody. First, the original protein fragment antigen was used for immunisation and generation of mouse hybridoma, without obtaining binders to the same epitope region. Instead a peptide designed using the functional epitope and structural information was synthesised and used for hybridoma production. Several of the monoclonal antibodies generated were found to have similar binding characteristics to those of the original polyclonal antibody. These monoclonal antibodies detected native HER2 on cell lines and were also able to stain HER2 in immunohistochemistry using xenografted mice, as well as human normal and cancer tissues.

Place, publisher, year, edition, pages
2014. Vol. 31, no 1, 35-43 p.
Keyword [en]
Batch-to-batch variations, Binding characteristics, Binding specificities, Hybridoma technology, Immunohistochemistry, Polyclonal antibody, Recognition sequence, Structural information
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-139254DOI: 10.1016/j.nbt.2013.10.002ISI: 000328131200004Scopus ID: 2-s2.0-84889568450OAI: oai:DiVA.org:kth-139254DiVA: diva2:685945
Funder
EU, FP7, Seventh Framework Programme, 241481VinnovaKnut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20140110

Available from: 2014-01-10 Created: 2014-01-08 Last updated: 2017-12-06Bibliographically 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

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Uhlén, Mathias

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