Chimeric antigen receptors (CARs) have found clinical success in B cell malignancies, but a dearth of potential targets limits their wider clinical application, especially in solid tumours. Here, we describe the development of an anti-annexin A2 CAR, CAR(2448), derived from an antibody found to have activity against epithelial ovarian cancer cell lines. The spacer length of CAR(2448) was optimised based on in vitro cytotoxic activity against ovarian cancer (OC) cell lines via a real-time cytotoxicity assay. The longer spacer CAR(2448)L T cells exhibit significant effector activity, inducing inflammatory cytokine release and cytotoxicity against OC cell lines. Furthermore, CAR(2448)L-BBz T cells induced enhanced survival in an in vivo OC xenograft model and reduced tumour volume by 76.6%. Our preclinical studies of CAR(2448) suggest its potential for the unmet need of novel strategies for the treatment of ovarian cancer.

Chimeric antigen receptors (CARs) have been a significant focus of cancer therapeutic investigation in recent years, due to the remarkably effective responses observed in hematological cancers. However, the utility of CAR T cells has yet to be fully exhibited in solid tumours. One of the key challenges facing the treatment of solid tumors with CAR T cells is the dearth of viable antigen targets, which limits the expansion of this therapeutic space. Here, we describe how the anti-glycan monoclonal antibody 2448, which target annexin A2 on a number of ovarian and breast cancer cell lines, has been successfully reformatted as a CAR T cell construct. Messenger RNA (mRNA) encoding CAR(2448) was transfected into activated T cells, resulting in cytotoxicity and cytokine release upon co-incubation with target cells expressing annexin A2. While CAR(2448) variants with differing spacer chain lengths all mediated cytotoxicity against target cells compared to untransfected T cells, real-time target cell growth monitoring revealed that the long spacer CAR(2448) mediated higher levels of cytotoxicity, consistent with the membrane-proximal location of the annexin A2 target epitope. This optimization of CAR spacer length in the in vitro setting, using mRNA transfected T cells and real-time target cell growth monitoring, suggests that several aspects of CAR construction can be optimized in vitro, thereby lowering the threshold for CAR T cell development. These results suggest the potential for anti-glycan antibodies to be repurposed as CARs, and the development of CAR(2448) is currently being extended to lentiviral vectors for the purpose of developing long-term expressing CAR(2448) T cells, in anticipation of in vivo studies in murine models. This project was funded by the Agency for Science, Technology, and Research (A*STAR), Singapore and the A*STAR Graduate Academy (A*GA). Citation Format: Leonard W. Leong, Simeon Cua, Heng Liang Tan, Andre B. Choo. Development of a chimeric antigen receptor against glycosylated annexin A2 on ovarian tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1436.

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