Targeted Thorium-227 Conjugates Demonstrate Synergistic Activity In Combination With Pd-L1 Inhibitors

Targeted thorium-227 conjugates (TTCs) represent a new class of targeted alpha therapy (TAT). TTCs consist of an antigen targeting moiety, which is covalently attached to a 3,2-HOPO chelator, enabling specific complexation and delivery of the alpha particle emitter thorium-227 to tumor cells. TTCs have demonstrated potent preclinical in vivo activity in monotherapy and in combination with DNA damage inhibitors. In the present work, the immunostimulatory effects of the mesothelin (MSLN) targeted thorium-227 conjugate (MSLN-TTC) was studied in immunocompetent mice, bearing human MSLN-transfected MC38 cells, in monotherapy and in combination with the PD-L1 inhibitor (PD-L1i) atezolizumab. Surface expression of DAMPs (calreticulin, HMGB1, HSP70 and HSP90), immunoinhibitory markers (PD-L1 and CTLA-4) and immunostimulatory markers (IFNAR, B7H2 and ICOS-L) was analyzed on murine, human MSLN-transfected MC38 colorectal cancer cells (MC38-hMSLN cells) after a 3-day exposure to MSLN-TTC. In vivo, C57/Bl6 mice were inoculated s.c. with MC38-hMSLN cells and treated with a single i.v. dose of non-radiolabeled MSLN-conjugate (0.14 mg/kg), isotype control-TTC (250 kBq/kg; 0.14 mg/kg), MSLN-TTC (250 kBq/kg; 0.14 mg/kg), PD-L1i (1.5 mg/kg; Q3D4; i.p.) or MSLN-TTC/PD-L1i combination. Tumors, lymph nodes, spleen and plasma were analyzed for MSLN and molecular markers. Tumor-free survivors were re-challenged with MC38-hMSLN or vector cells 125 d post-treatment. In a parallel arm of the experiment, CD8+ T cells were depleted using YTS169.4 antibody. MSLN-TTC treatment resulted in a specific dose-dependent increase of IFNAR, PD-L1 and HMGB1 in MC38-hMSLN cells in vitro. MSLN-TTC (T/C 0.38; Day 22) and PD-L1i (T/C 0.0.42; Day 22) monotherapies showed specific antitumor activity (T/C of radiolabeled isotype control was 0.76, Day 22) and their combination was synergistic (T/C 0.08; Day 22) with increased numbers of tumor free survivors. All three treatments increased HMGB1 levels in plasma. Induction of DSBs was observed in MSLN-TTC (+/- PD-L1i) treated tumors. Re-challenge of tumor-free survivors treated with MSLN-TTC, PD-L1i or MSLN-TTC/PD-L1i resulted in rejection of MC38-hMSLN, but not vector-cells. Infiltration of CD8+ T cells into tumors and their increase in tumor-draining lymph nodes were observed in MSLN-TTC, PD-L1i and MSLN-TTC/PD-L1i-treated groups but not in the vehicle group. Depletion of CD8+ T cells decreased the efficacy of PD-L1i and to a lower extent, MSLN-TTC and MSLN-TTC/PD-L1i. These results suggest that the efficacy of MSLN-TTC may involve activation of CD8+ T cells. These findings support the hypothesis that upon binding to antigen-positive cancer cells, TTC treatment induces DNA double strand breaks, leading to apoptosis and potentially immunogenic cell death, resulting in activation of the adaptive immune system. Citation Format: Urs B. Hagemann, Pascale Lejeune, Lisa Bartnitzky, Kathleen Stadelmann, Veronique Cruciani, Roger M. Bjerke, Sandra Berndt, Claudia Kamfenkel, Manuela Brand, Manuela Steinbach, Stefan Stargard, Christoph A. Schatz, Jenny Karlsson, Hartwig Hennekes, Alan S. Cuthbertson, Dominik Mumberg. Targeted thorium-227 conjugates demonstrate synergistic activity in combination with PD-L1 inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2257.