515. Targeting Hypoglycosylation with a Chimeric Antigen Receptor

Abstract

Genetically-engineering human T cells to target endogenously-expressed cell markers associated with both normal and malignant cells, whether upregulated or not, has shown tremendous efficacy in targeting CD 19-expressing leukemia and lymphoma cells and will undoubtedly demonstrate promise in other hematological malignancies. However, many of the tumor-associated antigens in epithelial malignancies are upregulated in malignant tissue but also expressed at lower-levels in normal tissue. As observed by the targeting of Her2 with CAR-T cells, targeting these antigens can present on-target, off-site toxicity and emphasizes the need for cancer cell-specific epitopes in CAR T cell therapy. Here, we have taken advantage of the disrupted glycosylation program that occurs during cancer transformation, which exposes cancer-specific glycoantigens that can be attractive targets for CAR development. Mucin-1 (Muc1) is a tumor-associated oncogene that is highly upregulated in many cancers and has been the target of many vaccine studies; less frequently targeted are the glycosylation changes of Muc1 that occurs during cancer transformation. Muc1 is heavily O- and N-glycosylated and transformed cells often fail to construct the complex oligosaccharide chains, exposing hypoglycosylated epitopes such as an O-linked GalNac (Tn antigen). We developed a CAR using the scFv sequence derived from the 5E5 mAb, which was developed against the Tn antigen of Mucin-1 (Muc1), and also using the endodomains of CD3zeta and 4- 1BB that promote T cell activation and co-stimulation, respectively. The resultant Muc1 Tn-antigen specific CAR (5E5CAR) was expressed in a lentiviral vector and we transduced normal human T cells. We showed that the 5E5 mAb and the 5E5CAR are specific to the hypoglycosylated epitope of Muc1, but not normal Muc1. In addition, we developed a safety profile of the 5E5 mAb against normal pediatric and adult human tissue. We demonstrate that human T cells expressing the 5E5CAR rapidly clear established Jurkat and Hs766T tumors in vitro and in NSG mouse xenograft models, as assessed by serial bioluminescence imaging and prolonged survival compared to mice expressing control CD19-specific CAR T cells (P<0.005). Restoring expression of wildtype Cosmc in Jurkat T cells abolishes the expression of the hypoglycosylated form of Muc1 and also abrogrates the cytotoxicity exhibited by 5E5CAR T cells, further highlighting the specificity of the 5E5CAR for the O-linked GalNac. In addition to the T cell leukemia and pancreatic cancer xenograft models, we also demonstrate that the 5E5CAR T cells are responsive to ovarian, breast, and lung cancers, among others. These results demonstrate that the 5E5CAR is specific for the Muc1 Tn antigen and provides a potent and safe cancer-specific immunotherapy for the targeting of malignancies.