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Utilizing Solid Phase Cloning, Surface Display And Epitope Information for Antibody Generation and Characterization
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0002-1389-5371
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 [en]
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: urn:nbn:se:kth:diva-205410ISBN: 9789177293538 (print)OAI: oai:DiVA.org:kth-205410DiVA: diva2:1088901
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
List of papers
1. Generation of HER2 monoclonal antibodies using epitopes of a rabbit polyclonal antibody
Open this publication in new window or tab >>Generation of HER2 monoclonal antibodies using epitopes of a rabbit polyclonal antibody
2014 (English)In: New Biotechnology, ISSN 1871-6784, 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.

Keyword
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:nbn:se:kth:diva-139254 (URN)10.1016/j.nbt.2013.10.002 (DOI)000328131200004 ()2-s2.0-84889568450 (Scopus ID)
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: 2017-04-18Bibliographically approved
2. Stratification of responders towards eculizumab using a structural epitope mapping strategy
Open this publication in new window or tab >>Stratification of responders towards eculizumab using a structural epitope mapping strategy
Show others...
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
Keyword
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:nbn:se:kth:diva-191745 (URN)10.1038/srep31365 (DOI)000381185300001 ()27509843 (PubMedID)2-s2.0-84982176744 (Scopus ID)
External cooperation:
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
3. Phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity binders
Open this publication in new window or tab >>Phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity binders
Show others...
(English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347Article in journal (Other academic) Submitted
Abstract [en]

Surface display couples genotype with a surface exposed phenotype and thereby allows for screening of gene-encoded protein libraries for desired characteristics. Of the various display systems, phage display is by far the most popular, mainly thanks to its ability to harbor large library sizes. Here, we describe the first use of a grampositive host for display of a library of human antibody genes. The method allows for swift generation of binders by combining phage and gram-positive display, for its ease of use for screening, sorting and ranking by flow cytometry. We demonstrate the utility of this method by identifying specific low nanomolar scFv towards human HER2. The ranking and performance of the scFv isolated by flow sorting in surface immobilized form was retained when expressed as soluble scFv and analyzed by biolayer interferometry as well as after expression as full-length antibodies in mammalian cells. We also show the possibility to use gram-positive display to directly improve the affinity of the identified binders via an affinity maturation step using random mutagenesis and flow sorting. We believe this combined approach has the potential for a more complete scan of the antibody repertoire and for swift affinity maturation of human antibody formats.

Keyword
S.carnosus, Flow cytometry, Antibody, HER2, phage display, cell-surface display, affinity maturation
National Category
Other Industrial Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-205555 (URN)
Note

Article first submitted to Scientific Reports.

QC 20170418

Available from: 2017-04-18 Created: 2017-04-18 Last updated: 2017-08-28Bibliographically approved
4. SPUX - A Solid Phase Uracil Excision Method for Antibody Affinity Maturation and Paratope Mapping
Open this publication in new window or tab >>SPUX - A Solid Phase Uracil Excision Method for Antibody Affinity Maturation and Paratope Mapping
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Mutagenesis libraries are the heart of combinatorial protein engineering where proteins such as antibodies are evolved for improved functionality. Despite recent improvements in gene synthesis and selection methodologies, current methods still fail to provide practical means for synthesis of complete antibody scFv and screening of theoretical diversities, hence forcing the user to focused diversity screening and assembly of shorter oligos to avoid synthesis errors and maximize library functionality. Here we demonstrate a way to generate highly functional tailored mutagenesis libraries for efficient antibody affinity maturation using a rapid cell-free solid phase cloning method with single strand diversity oligonucleotides. For this we are utilizing a combination of a high-fidelity polymerase for PCR-based incorporation of Uracil into a wild-type template, bead-based solid-phase technology for elution of single strand DNA, oligonucleotide annealing, extension and automation, and an uracil excision enzyme cocktail for in vitro degradation of template DNA to minimize background. Our method allowed for fast (8 hours) mutagenesis and automated cloning of a complete set of 50 position specific alanine-mutations for mapping of the paratope of a scFv antibody in a single robot run. We further exemplify our method by generating and stratifying a set of antibody scFv affinity maturation libraries with targeted diversity into critical or nonessential paratope positions, as well as by a complete randomization in all positions. The libraries were subjected to bacterial surface display selections and output was followed by Illumina deep sequencing and binding analysis by SPR. The functional quality of our libraries were high, with a yield of >99% functional diversity in the case for two of our libraries. We were further able to target all positions in all loops with diversity, and we could show the ability to target all six loops with diversity at the same time. The comparison of different library focus showed us that scFv libraries with diversity targeted to non-essential enhancing paratope positions more quickly rendered enrichment of improved binders compared to random diversity or paratope-targeted libraries. Surprisingly several of the improved binders from the random library had beneficial mutations in the same positions targeted by the smaller focused non-essential enhancing residue focused library indicating a possible benefit of focusing diversity to these spots. We believe our method for construction of libraries with site directed mutagenesis to be a viable way for generation of functional and diverse genetic libraries, particularly suitable for affinity maturation and paratope mapping of antibodies.

Keyword
Solid phase cloning, Mutagenesis, Antibody library, Affinity maturation, S.carnosus
National Category
Other Industrial Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-205554 (URN)
Note

QC 20170418

Available from: 2017-04-18 Created: 2017-04-18 Last updated: 2017-04-18Bibliographically approved

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