Establishment of a novel anti-mouse CD73 monoclonal antibody C73Mab-9 by the Cell-Based Immunization and Screening method

Cluster of differentiation 73 (CD73), also known as ecto-5’-nucleotidase, is a glycosyl-phosphatidylinositol-anchored cell-surface molecule that belongs to the 5’-nucleosidase family. CD73 metabolizes extracellular adenosine (ADO) 5’-monophosphate into ADO and inorganic phosphate. CD73 plays a vital role in cancer-immune evasion by facilitating ADO production. Therefore, the development of specific anti-CD73 antibodies is desired to clarify the biological and pathological functions of CD73. Moreover, developing antibodies against mouse CD73 (mCD73) is also essential for pre-clinical experiments. This study successfully established an anti-mCD73 monoclonal antibody (clone C73Mab-9, rat IgG2a, and lambda) using the Cell-Based Immunization and Screening (CBIS) method. In flow cytometric analysis, C73Mab-9 was confirmed to recognize mCD73 in mCD73-overexpressed CHO-K1 (CHO/mCD73), MUSS, and NMuMG cells. The dissociation constant values of C73Mab-9 were 7.6 × 10−9 M for CHO/mCD73 and 1.2 × 10−9 M for MUSS cells, respectively. Furthermore, C73Mab-9 detected mCD73 in Western blot analysis. Therefore, C73Mab-9, established by the CBIS method, is useful for basic research and is expected to contribute to pre-clinical studies.