Abstract 1907: Claudin 6 is a carcinoembryonic antigen with cancer stem cell marker features
Authors: Türeci Özlem , Wagner Meike , Paret Claudia , Kreuzberg Maria M. , Wöll Stefan , Walter Korden , Häcker Sabine C. , Nakajo Ikumi , Yamada Tomohiro , Sahin Ugur 
Published: August 2018

Abstract
Background Claudin 6 (CLDN6) is a tight junction membrane protein whose expression in normal tissue is confined to embryonic cells, but is aberrantly expressed in various human cancers, such as ovarian cancer (OC) and testicular cancer (TC). A monoclonal antibody against CLDN6, IMAB027, has shown promising antitumor activity in preclinical human CLDN6-positive (CLDN6+) cancer models. In this series of nonclinical studies, we investigated CLDN6 expression in normal and cancer tissues, as well as the localization and possible function of CLDN6 in cancer cells.
Methods Expression of CLDN6 was assessed in a wide range of human tissues (eg, lung, colon, skin, ovary) and cultured cells by quantitative RT-PCR, immunohistochemistry (IHC), flow cytometry, and western blotting. To investigate the effect of dedifferentiation on CLDN6 expression, human-induced pluripotent cells were generated by transfecting foreskin fibroblasts with a reprogramming cocktail, and then CLDN6 expression was evaluated. To characterize CLDN6 as a potential novel marker to identify cancer stem cells (CSCs) in OC, coexpression of CLDN6 with known CSC surface markers were analyzed by flow cytometry, and CLDN6+ and CLDN6-negative cells were tested in colony formation and sphere formation assay. Human OC cell lines were transplanted intraperitoneally into nude mice and assessed for metastasis to investigate tumorigenecity of CLDN6+ cells.
Results Except for low mRNA levels measured in placenta, testis, umbilical cord, cerebellum, and lung samples, no CLDN6 (mRNA or protein) was detected in the vast majority of normal tissues. Additionally, there was also a lack of CLDN6 protein expression in tissue zones where stem cells for tissue homeostasis would normally be found as determined by IHC with an anti-CLDN6 antibody. CLDN6 was expressed on the cell surface of several solid tumors, including ovarian, testicular, uterine, and lung cancer tissues; OC and TC samples had high level expression. CLDN6 expression was strongly activated in human-induced pluripotent stem cells generated from fibroblasts. CLDN6 showed selective coexpression with known CSC markers such as CD44, CD24, and CD90 in OC and TC cell lines. In addition, some CLDN6+ OC cells exhibited CSC-like behavior in vitro: CLDN6+ populations were clonogenic and formed well-defined spheres in low attachment conditions; these spheres had the ability to self-renew into secondary spheres. Analysis of OC metastases in mouse xenografts showed when xenografts were generated by OC cells that had &lt;10% of CLDN6+ cells, the metastases were enriched in CLDN6+ cells, suggesting CLDN6+ cells had selective growth advantage.
Conclusions CLDN6 is a cancer cell-specific surface molecule aberrantly expressed in several cancers, and its expression may be an identifier for cells with CSC-like traits. These characteristics make CLDN6 an attractive target candidate for tumor-specific therapeutic antibodies.
Citation Format: Özlem Türeci, Meike Wagner, Claudia Paret, Maria M. Kreuzberg, Stefan Wöll, Korden Walter, Sabine C. Häcker, Ikumi Nakajo, Tomohiro Yamada, Ugur Sahin. Claudin 6 is a carcinoembryonic antigen with cancer stem cell marker features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1907.