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Phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity binders
KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.ORCID-id: 0000-0002-1389-5371
KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.ORCID-id: 0000-0003-1096-9061
Department of Immunotechnology, Lund University, Medicon Village (Bldg 406), 223 81 Lund, Sweden..
Department of Immunotechnology, Lund University, Medicon Village (Bldg 406), 223 81 Lund, Sweden..
Vise andre og tillknytning
(engelsk)Inngår i: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347Artikkel i tidsskrift (Annet vitenskapelig) 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.

Emneord [en]
S.carnosus, Flow cytometry, Antibody, HER2, phage display, cell-surface display, affinity maturation
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
URN: urn:nbn:se:kth:diva-205555OAI: oai:DiVA.org:kth-205555DiVA, id: diva2:1089127
Merknad

Article first submitted to Scientific Reports.

QC 20170418

Tilgjengelig fra: 2017-04-18 Laget: 2017-04-18 Sist oppdatert: 2017-08-28bibliografisk kontrollert
Inngår i avhandling
1. Utilizing Solid Phase Cloning, Surface Display And Epitope Information for Antibody Generation and Characterization
Åpne denne publikasjonen i ny fane eller vindu >>Utilizing Solid Phase Cloning, Surface Display And Epitope Information for Antibody Generation and Characterization
2017 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
KTH Royal Institute of Technology, 2017. s. 100
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:10
Emneord
antibody engineering, antibody affinity maturation, combinatorial protein engineering, epitope mapping, FACS, HER2, complement C5, Staphylococcal surface display, surface display.
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-205410 (URN)9789177293538 (ISBN)
Disputas
2017-05-19, F3, Lindstedtsvägen 26, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Merknad

QC 20170418

Tilgjengelig fra: 2017-04-18 Laget: 2017-04-18 Sist oppdatert: 2017-05-26bibliografisk kontrollert

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Volk, Anna-LuisaUhlén, MathiasRockberg, Johan

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Hu, Francis JingxinVolk, Anna-LuisaUhlén, MathiasRockberg, Johan
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