RT Journal Article SR Electronic T1 Identification of CD73 as the antigen of an antigen-unknown monoclonal antibody established by exosome immunization, and its antibody-drug conjugate exerts an antitumor effect on glioblastoma cell lines JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.09.30.462527 DO 10.1101/2021.09.30.462527 A1 Takahiro anzai A1 Shinji Saijou A1 Hiroki Takashima A1 Misato Hara A1 Shingo Hanaoka A1 Yasuhiro Matsumura A1 Masahiro Yasunaga YR 2021 UL http://biorxiv.org/content/early/2021/10/01/2021.09.30.462527.abstract AB Development of antibodies against the native structure of membrane proteins with multiple transmembrane domains is challenging because it is difficult to prepare antigens with native structures. Previously, we successfully developed a monoclonal antibody against multi-pass membrane protein TMEM180 by exosome immunization in rats. This approach yielded antibod-ies that recognized cancer-specific antigens on the exosome. In this study, we performed im-munoprecipitation using magnetic beads to identify the antigen of one of the rat antibody clones, 0614, as CD73. We then converted antibody 0614 to human chimeric antibody 0614-5 and found that the humanized antibody did not inhibit CD73 enzymatic activity. Glioblastoma (GB) was the cancer type with the highest expression of CD73 in the tumor relative to healthy tissue. An antibody–drug conjugate (ADC) of 0614-5 exerted an antitumor effect on GB cell lines according to expression of CD73. The 0614-5-ADC could be used to treat cancers with high CD73 expression. In addition, our strategy could be used to determine the antigen of any antibody produced by exosome immunization.Competing Interest StatementY.M. is co-founder, shareholder, and Board Member of RIN Institute, Inc. S.S. and S.H. are em-ployees of RIN Institute, Inc. M.H. is an employee of Tamagawa Seiki Co. Ltd. M.Y. is a share-holder of RIN Institute, Inc. The other authors declare no competing interests.