Publication: Design and generation of humanized single-chain Fv derived from mouse hybridoma for potential targeting application
Issued Date
2015-12-01
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ISSN
21679436
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2-s2.0-84951771744
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Mahidol University
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SCOPUS
Bibliographic Citation
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy. Vol.34, No.6 (2015), 404-417
Suggested Citation
Kannika Khantasup, Warangkana Chantima, Chak Sangma, Kanokwan Poomputsa, Tararaj Dharakul Design and generation of humanized single-chain Fv derived from mouse hybridoma for potential targeting application. Monoclonal Antibodies in Immunodiagnosis and Immunotherapy. Vol.34, No.6 (2015), 404-417. doi:10.1089/mab.2015.0036 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/36049
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Title
Design and generation of humanized single-chain Fv derived from mouse hybridoma for potential targeting application
Abstract
© Mary Ann Liebert, Inc. 2015. Single-chain variable antibody fragments (scFvs) are attractive candidates for targeted immunotherapy in several human diseases. In this study, a concise humanization strategy combined with an optimized production method for humanizing scFvs was successfully employed. Two antibody clones, one directed against the hemagglutinin of H5N1 influenza virus, the other against EpCAM, a cancer biomarker, were used to demonstrate the validity of the method. Heavy chain (VH) and light chain (VL) variable regions of immunoglobulin genes from mouse hybridoma cells were sequenced and subjected to the construction of mouse scFv 3-D structure. Based on in silico modeling, the humanized version of the scFv was designed via complementarity-determining region (CDR) grafting with the retention of mouse framework region (FR) residues identified by primary sequence analysis. Root-mean-square deviation (RMSD) value between mouse and humanized scFv structures was calculated to evaluate the preservation of CDR conformation. Mouse and humanized scFv genes were then constructed and expressed in Escherichia coli. Using this method, we successfully generated humanized scFvs that retained the targeting activity of their respective mouse scFv counterparts. In addition, the humanized scFvs were engineered with a C-terminal cysteine residue (hscFv-C) for site-directed conjugation for use in future targeting applications. The hscFv-C expression was extensively optimized to improve protein production yield. The protocol yielded a 20-fold increase in production of hscFv-Cs in E. coli periplasm. The strategy described in this study may be applicable in the humanization of other antibodies derived from mouse hybridoma.