@article{d74d37d207304f30aae739bf675bca70,
title = "Epitope Mapping of the Anti-CD20 Monoclonal Antibodies (C20Mab-11 and 2H7) Using HisMAP Method",
abstract = "CD20, which is expressed on B lymphocytes, has been studied as a therapeutic target for B cell lymphomas and autoimmune disorders. Identifying the binding epitopes of monoclonal antibodies (mAbs) can contribute to our understanding of their functions. We have previously developed an anti-CD20 mAb (clone C20Mab-11) using a Cell-Based Immunization and Screening (CBIS) method. In this study, we aimed to determine the binding epitopes of anti-CD20 mAbs, such as C20Mab-11 and 2H7, using the His-tag insertion for epitope mapping (HisMAP). The results showed that 171-NPSE-174 and 168-EPANPSE-174 in the second loop of CD20 were essential for C20Mab-11 binding and 2H7 binding, respectively. Although we developed many mAbs that recognize conformational epitopes using the CBIS method, there are many difficulties in epitope mapping for these mAbs. HisMAP could be useful for determining the conformational epitopes of other mAbs against membrane proteins.",
keywords = "CD20, Epitope mapping, His tag, Monoclonal antibody",
author = "Hiroyuki Suzuki and Teizo Asano and Tomohiro Tanaka and Kaneko, {Mika K.} and Yukinari Kato",
note = "Funding Information: This research was supported, in part, by Japan Agency for Medical Research and Development (AMED) under Grant Nos. JP21am0401013 (to Y.K.) and JP21am0101078 (to Y.K.), and by the Japan Society for the Promotion of Science ( JSPS) Grants-in-Aid for Scientific Research (KAKENHI) Grant Nos. 21K15523 (to T.A.), 21K20789 (to T.T.), 21K07168 (to M.K.K.), and 19K07705 (to Y.K.). Funding Information: DNA encoding the CD20 gene (IRAL012D02) was provided by RIKEN BRC through the National BioResource Project of MEXT, Japan.(15,16) The open reading frame of CD20 was subcloned into a pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation). Insertions of 5-histidine tag (5 · H*) in the second loop of CD20 were performed using the HotStar HiFidelity Polymerase Kit with oligonucleotides containing 5 · H* insertions at the selected position. Lys142_5 · H*_Ile143 (K142_5 · H*_I143) was produced for instance by inserting the 5-histidine tag between Lys142 and Ile143 of CD20. Polymerase chain reaction fragments bearing the desired mutations were inserted into the pCAG-Ble vector using the In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan). Publisher Copyright: {\textcopyright} Mary Ann Liebert, Inc.",
year = "2022",
month = feb,
day = "1",
doi = "10.1089/mab.2021.0051",
language = "English",
volume = "41",
pages = "20--26",
journal = "Monoclonal Antibodies in Immunodiagnosis and Immunotherapy",
issn = "2167-9436",
publisher = "Mary Ann Liebert Inc.",
number = "1",
}