Epitope Mapping of an Anti-Mouse CD39 Monoclonal Antibody Using PA Scanning and RIEDL Scanning

Abstract

A cell-surface ectonucleotidase CD39 mediates the conversion of extracellular ATP into immunosuppressive adenosine with another ectonucleotidase CD73. The elevated adenosine in the tumor microenvironment (TME) attenuates antitumor immunity, which promotes tumor cell immunologic escape and progression. Anti-CD39 monoclonal antibodies (mAbs), which suppress the enzymatic activity, can be applied to antitumor therapy. Therefore, an understanding of the relationship between the inhibitory activity and epitope of mAbs is important. We previously established an anti-mouse CD39 (mCD39) mAb, C39Mab-1 using the Cell-Based Immunization and Screening (CBIS) method. In this study, we determined the critical epitope of C39Mab-1 using flow cytometry. We performed the PA tag (12 amino acids)-substituted analysis (named PA scanning) and RIEDL tag (5 amino acids)-substituted analysis (named RIEDL scanning) to determine the critical epitope of C39Mab-1 using flow cytometry. By the combination of PA scanning and RIEDL scanning, we identified the conformational epitope, spanning three segments of 275 th to 279 th , 282 nd to 291 st , and 306 th to 323 rd amino acids of mCD39. These analyses would contribute to the identification of the conformational epitope of membrane proteins.

Establishment of a Highly-sensitive Anti-EphB2 Monoclonal Antibody Eb2Mab-3 for Flow Cytometry

“…The epitopes of anti-EGFR mAbs (EMab-51 and EMab-134) [25,26] and anti-CD44 mAbs (C44Mab-5 and C44Mab-46) [27,28] could be determined using the REMAP method. Furthermore, the epitopes of antimouse CD39 mAb (C39Mab-1) could be determined using both REMAP and PAMAP methods [29]. Therefore, further studies are required to determine the epitope and biological activities of Eb2Mab-3.…”