Carcinoembryonic Antigen Related Cell Adhesion Molecule 6 (CEACAM6) is overexpressed in many types of human cancers such as breast, pancreatic, colorectal, and non-small-cell lung adenocarcinoma, and is an independent predictor of overall survival and disease free survival. Targeting this molecule by antibodies has slowed tumor progression in certain animal models. 2A3 is a camelid single domain antibody isolated from a whole cancer cell immunized llama library. The antibody binds specifically to the CEACAM6 antigen with high affinity (5nM as measured by SPR) and inhibits the proliferation of CEACAM6-expressing cancer cells in vitro. In this study, Chimeric-Antigen Receptor (CAR) T cells were engineered to target human CEACAM6 antigen by transducing the 2A3 antibody sequence to generate a modified chimeric CD28 signaling domain fused to chimeric CD3-zeta. Transduction efficiency and expression of 2A3 antibody were verified by flow cytometry. Co-incubation of CEACAM6-specific CAR-T (CEACAM6-CAR-T) cells with the CEACAM6-expressing pancreatic cell line BxPC-3 resulted in augmented cytotoxicity and cytokines (IL-2 and IFN-γ) release, suggesting potential anti-cancer activity of the CAR-T cells. Data from real-time cell analysis showed a significant increase in BxPC-3 cell cytotoxicity by CEACAM6-CAR-T cells, as compared to native T cells. However, CEACAM6-CAR-T cells showed much lower cytotoxic activity on negative control cell lines. The efficacy of CEACAM6-CAR-T cells in xenograft model was examined in vivo. BxPC-3 cells were inoculated subcutaneously into the hind flank of CIEA NOG mice. Three groups of mice then received intravenous injection of either PBS, native T cells, or CEACAM6-CAR-T cells, respectively, at day 1, 8, and 15. The data demonstrated very high efficacy of CEACAM6-CAR-T cells against the pancreatic cancer xenograft. CEACAM6-CAR-T cells significantly decreased the growth of the BxPC-3 xenograft as compared to that of native T cells (p-value=0.00025) and PBS (p-value=7.91x106). No toxic effect was observed based on body weight measurement. The results strongly support that CEACAM6-CAR-T cells can be used as an effective immunotherapy agent against CEACAM6-expressing cancers, and that camelid single domain antibodies can be easily adopted for CAR-T type therapies.

Citation Format: Wah Yau Wong, Jamshid Tanha, Lakshmi Krishnan, Baomin Tian, Praveen Kumar, Kim Gaspar, Steve Demas, Sven Rohmann, Heman Chao. CAR-T cells harboring camelid single domain antibody as targeting agent to CEACAM6 antigen in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A74.