TY - JOUR
T1 - Epitope Mapping of Rituximab Using HisMAP Method
AU - Asano, Teizo
AU - Suzuki, Hiroyuki
AU - Kaneko, Mika K.
AU - Kato, Yukinari
N1 - Funding Information:
DNA encoding the CD20 gene (IRAL012D02) was provided by RIKEN BRC through the National BioResource Project of MEXT, Japan.(29,30) The open reading frame of CD20 was subcloned into a pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Insertions of a 5xHis-tag (5xH*) in the extracellular region of CD20 were performed using the HotStar HiFidelity Polymerase Kit with oligonucleotides containing 5xH* insertions at the selected position. Ile141_5xH*_Lys142 (I141_5xH*_K142) was produced for instance by inserting the 5-histidine sequence between Ile141 and Lys142 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).
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.), 21K07168 (to M.K.K.), and 19K07705 (to Y.K.).
Publisher Copyright:
© Mary Ann Liebert, Inc.
PY - 2022/2/1
Y1 - 2022/2/1
N2 - CD20 is expressed in the B lymphocyte, and an effective target for the detection and treatment of B cell lymphomas. Therefore, CD20 has been studied as a therapeutic target of B cell lymphomas and autoimmune disorders. Specific anti-CD20 monoclonal antibodies (mAbs), such as rituximab, ofatumumab, veltuzumab, and ocaratuzumab, have been developed. Revealing the recognition mechanism of antigen by mAbs could contribute to understanding the function of mAbs and could be useful for the development of vaccine. Rituximab is a mouse-human chimeric anti-CD20 mAb, which was developed and approved for the treatment of the B cell malignancies. Hence, the binding epitope of rituximab for CD20 has been studied. Some reports show that 170-ANPS-173, especially Ala170 and Pro172 of CD20 are important for rituximab binding. However, only phage display results showed that 182-YCYSI-186 of CD20 is also important for rituximab binding to CD20. In this study, we tried to determine the binding epitope of rituximab for CD20 using histidine-tag insertion for epitope mapping (HisMAP) method. The results showed that two regions of CD20 (169-PANPSE-174 and 183-CYSIQ-187) are important for rituximab-binding for CD20.
AB - CD20 is expressed in the B lymphocyte, and an effective target for the detection and treatment of B cell lymphomas. Therefore, CD20 has been studied as a therapeutic target of B cell lymphomas and autoimmune disorders. Specific anti-CD20 monoclonal antibodies (mAbs), such as rituximab, ofatumumab, veltuzumab, and ocaratuzumab, have been developed. Revealing the recognition mechanism of antigen by mAbs could contribute to understanding the function of mAbs and could be useful for the development of vaccine. Rituximab is a mouse-human chimeric anti-CD20 mAb, which was developed and approved for the treatment of the B cell malignancies. Hence, the binding epitope of rituximab for CD20 has been studied. Some reports show that 170-ANPS-173, especially Ala170 and Pro172 of CD20 are important for rituximab binding. However, only phage display results showed that 182-YCYSI-186 of CD20 is also important for rituximab binding to CD20. In this study, we tried to determine the binding epitope of rituximab for CD20 using histidine-tag insertion for epitope mapping (HisMAP) method. The results showed that two regions of CD20 (169-PANPSE-174 and 183-CYSIQ-187) are important for rituximab-binding for CD20.
KW - CD20
KW - Epitope mapping
KW - His tag
KW - Monoclonal antibody
KW - Rituximab
UR - http://www.scopus.com/inward/record.url?scp=85125552720&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85125552720&partnerID=8YFLogxK
U2 - 10.1089/mab.2021.0044
DO - 10.1089/mab.2021.0044
M3 - Article
C2 - 35225667
AN - SCOPUS:85125552720
SN - 2167-9436
VL - 41
SP - 8
EP - 14
JO - Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
JF - Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
IS - 1
ER -
