Background: SS1P and LMB-100 are anti-mesothelin immunotoxins composed of a targeting antibody fragment genetically fused to a truncated pseudomonas exotoxin A. We have previously repotrted a synergistic antitumor effect when SS1P or LMB-100 were injected locally into tumors of murine breast cancer in combination with antibodies that block the immune check point CTLA-4. In this study our goal is to explore if this treatment can be applied in a murine mesothelioma model. Method: The mouse mesothelioma AE17M cell line, expressing human mesothelin, was treated in culture with anti-mesothelin immunotoxins. We collected data on the cytotoxic activity and ability to induce markers of immunogenic cell death. In vivo effects were performed on AE17M tumors growing in C57/Bl6 mice. Results: AE17M cells are sensitive to both SS1P and LMB-100 in culture, but when injected IV, the immunotoxins did not affect AE17M tumor growth rate. To overcome obstacles in drug penetration we injected immunotoxins directly into AE17M tumors and found that they only increased the survival of mice from 16 days to 20 days. In addition, anti-CTLA-4 monotherapy failed to control AE17M tumor growth rate in most mice. But when locally injected SS1P or LMB-100 was combined with i.p. anti-CTLA-4, tumor regressions occurred and the median survival increased to 80 days or longer. In search for possible mechanisms enabling immunotoxins to contribute to anti-CTLA-4 activity, we examined their ability to induce markers of immunogenic cell death. We found that both LMB-100 and SS1P increased release of ATP from AE17M cells. In addition, LMB-100 induced calreticulin expression on the surface of both AE17M cells and KLM-1 cells. These results point to possible pathways allowing immunotoxins to promote anti-tumor immunity and sensitize tumors for anti-CTLA-4 effect. Conclusion: Our findings in the AE17M model provide additional support for the use of local immunotoxins in combination with anti-CTLA-4.

Citation Format: Yasmin Leshem, Emily King, Yoram Reiter, Ira Pastan. Anti-mesothelin immunotoxins induce markers of immunogenic cell death and when injected locally into AE17M mesothelioma tumors enhance the effect of CTLA-4 blockade [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A142.