Design and generation of humanized single-chain Fv derived from mouse hybridoma for potential targeting application
บทความในวารสาร
ผู้เขียน/บรรณาธิการ
กลุ่มสาขาการวิจัยเชิงกลยุทธ์
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รายละเอียดสำหรับงานพิมพ์
รายชื่อผู้แต่ง: Khantasup K., Chantima W., Sangma C., Poomputsa K., Dharakul T.
ผู้เผยแพร่: Mary Ann Liebert Inc.
ปีที่เผยแพร่ (ค.ศ.): 2015
Volume number: 34
Issue number: 6
หน้าแรก: 404
หน้าสุดท้าย: 417
จำนวนหน้า: 14
นอก: 2167-9436
eISSN: 2167-9436
ภาษา: English-Great Britain (EN-GB)
บทคัดย่อ
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. ฉ Mary Ann Liebert, Inc. 2015.
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