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Optimization of production of the anti-keratin 8 single-chain Fv TS1-218 in Pichia Pastoris using design of experiments
Karlstad University, Faculty of Technology and Science, Department of Chemistry and Biomedical Sciences.
Karlstad University, Faculty of Technology and Science, Department of Chemistry and Biomedical Sciences.
Karlstad University, Faculty of Technology and Science, Department of Chemistry and Biomedical Sciences.
2011 (English)In: Microbial Cell Factories, ISSN 1475-2859, Vol. 10, 34- p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2011. Vol. 10, 34- p.
National Category
Medical Biotechnology
Identifiers
URN: urn:nbn:se:kau:diva-6640DOI: 10.1186/1475-2859-10-34ISI: 000291855800001OAI: oai:DiVA.org:kau-6640DiVA: diva2:373179
Available from: 2010-11-30 Created: 2010-11-30 Last updated: 2013-10-17Bibliographically approved
In thesis
1. Construction, expression and evaluation of anti-keratin 8 single-chain antibody fragments
Open this publication in new window or tab >>Construction, expression and evaluation of anti-keratin 8 single-chain antibody fragments
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Antibodies are glycoproteins specifically binding to a variety of antigens and today extensively used in prevention, diagnosis and treatment of diseases. Carcinomas originate from epithelial tissues and are the most common forms of human malignancies. In the necrotic areas of carcinomas significant amounts of keratins (K) are found extracellularly. They remain there due to their low solubility and can be used as targets in immuno-targeting and -therapy. Single-chain fragment variable (scFv) may display several advantages over intact IgGs in various applications but their use in immunotherapy of tumors could be limited. Their small size and monovalency may result in low accumulation of the scFv in the tumors. The targeting efficiency of antibodies needs to be evaluated in vitro prior to in vivo studies in order to exclude poor candidates. In vitro models should preferably resemble the in vivo situation as much as possible.

In the present study a scFv variant (TS1-218) of the anti-keratin 8 (K 8) monoclonal antibody TS1 (mAb TS1) was constructed and characterized using site-directed mutagenesis. In addition, the valency of the TS1-218 and one of its mutants, HE1-Q, were increased by construction of a covalently linked divalent single-chain fragment variable (sc(Fv)2). To improve the yield of the antibody fragments, the expression host was changed from E. coli to P. pastoris and culture conditions were optimized using Design of Experiments (DoE). Furthermore, a HeLa HEp-2 multicellular tumor spheroid (MCTS) in vitro model was established. The functionality of the radiolabeled TS1-218 alone and in immunocomplex with its anti-idiotype scFv, αTS1 scFv, were evaluated in the MCTS model and compared to a tumor xenograft nude mouse model. The targeting efficiency of the scFv and sc(Fv)2s were also investigated using MCTS.

TS1-218 in immunocomplex with αTS1 scFv displayed a significantly higher uptake than the TS1-218 alone in both MCTS and tumor xenografts. The sc(Fv)2s, DiTS1-218 and DiHE1-Q demonstrated a higher functional affinity to K 8 in ELISA and MCTS and were retained to a larger extent in the MCTS than their scFv counterparts, with a 3.9 and 9.4-fold longer half-life, respectively. Furthermore, the yield of the antibody fragments were improved after expression in P. pastoris with an 86-fold improvement for the TS1-218 following optimization using DoE.

Place, publisher, year, edition, pages
Karlstad: Karlstad University, 2010. 78 p.
Series
Karlstad University Studies, ISSN 1403-8099 ; 2010:34
Keyword
Single-chain antibody fragments, keratin 8, site-directed mutagenesis, heterologous expression, P. pastoris, design of experiments, multicellular tumor spheroids, immunotargeting
National Category
Biomedical Laboratory Science/Technology
Research subject
Biomedical Sciences
Identifiers
urn:nbn:se:kau:diva-6502 (URN)978-91-7063-326-3 (ISBN)
Public defence
2010-12-16, 1B306 Fryxellsalen, Karlstads universitet, Karlstad, 10:15 (Swedish)
Opponent
Supervisors
Available from: 2010-12-01 Created: 2010-10-27 Last updated: 2011-10-26Bibliographically approved

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