Synergistic Effect of a Mesothelin-Targeted 227Th Conjugate in Combination with DNA Damage Response Inhibitors in Ovarian Cancer Xenograft Models

Abstract

Targeted ²²⁷Th conjugates (TTCs) represent a new class of therapeutic radiopharmaceuticals for targeted α-therapy. They comprise the α-emitter ²²⁷Th complexed to a 3,2-hydroxypyridinone chelator conjugated to a tumor-targeting monoclonal antibody. The high energy and short range of the α-particles induce antitumor activity, driven by the induction of complex DNA double-strand breaks. We hypothesized that blocking the DNA damage response (DDR) pathway should further sensitize cancer cells by inhibiting DNA repair, thereby increasing the response to TTCs. Methods: This article reports the evaluation of the mesothelin (MSLN)-TTC conjugate (BAY 2287411) in combination with several DDR inhibitors, each of them blocking different DDR pathway enzymes. MSLN is a validated cancer target known to be overexpressed in mesothelioma, ovarian, lung, breast, and pancreatic cancer, with low expression in normal tissue. In vitro cytotoxicity experiments were performed on cancer cell lines by combining the MSLN-TTC with inhibitors of ataxia telangiectasia mutated, ataxia telangiectasia and Rad3-related (ATR), DNA-dependent protein kinase, and poly[adenosine diphosphate ribose] polymerase (PARP) 1/2. Further, we evaluated the antitumor efficacy of the MSLN-TTC in combination with DDR inhibitors in human ovarian cancer xenograft models. Results: Synergistic activity was observed in vitro for all tested inhibitors (inhibitors are denoted herein by the suffix "i") when combined with MSLN-TTC. ATRi and PARPi appeared to induce the strongest increase in potency. Further, in vivo antitumor efficacy of the MSLN-TTC in combination with ATRi or PARPi was investigated in the OVCAR-3 and OVCAR-8 xenograft models in nude mice, demonstrating synergistic antitumor activity for the ATRi combination at doses demonstrated to be nonefficacious when administered as monotherapy. Conclusion: The presented data support the mechanism-based rationale for combining the MSLN-TTC with DDR inhibitors as new treatment strategies in MSLN-positive ovarian cancer.

Keywords: DNA damage response; MSLN-TTC; monoclonal antibodies; radionuclide therapy; targeted alpha therapy; thorium-227.

FIGURE 1.

Synergistic effect of MSLN-TTC and DDRi on ovarian cancer cell line OVCAR-3. Half-maximal inhibitory concentration (IC₅₀)-isobolograms with MSLN-TTC and ATMi AZD0156 (A), ATRi BAY 1895344 (B), DNA-PKi VX-984 (C), and PARPi olaparib (D) on OVCAR-3 cells. CI (mean, n = 3) was determined according to median-effect model of Chou (17), with CI < 0.8 defined as synergistic effect, 0.8 < CI > 1.2 defined as additive effect, and CI > 1.2 defined as antagonistic effect.

FIGURE 2.

In vitro mechanistic experiments from MSLN-TTC ± ATRi BAY 1895344 or PARPi olaparib on OVCAR-3. (A and B) Cell cycle analysis (A) and γ-H2A.X (B) determined after treatment with MSLN-TTC (10 kBq/mL) and ATRi (10 nM) for 3 d. (C and D) Cell cycle analysis (C) and γ-H2A.X (D) determined after treatment with MSLN-TTC (1 kBq/mL) and PARPi (0.5 μM) for 3 d. Error bars represent SD of mean, n = 3.

FIGURE 3.

In vivo characterization of monotreatment with MSLN-TTC in OVCAR-3 xenograft model. (A) Tumor size determined after single dose (100, 250, or 500 kBq/kg, 0.14 mg/kg, intravenous) of MSLN-TTC, isotype control (250 kBq/kg, 0.14 mg/kg, intravenous), or vehicle control. Statistical analysis was performed using 1-way ANOVA followed by Tukey test. ****P < 0.0001. (B) Immunohistochemistry of γH2A.X on tumors after single dose (500 kBq/kg, 0.14 mg/kg, intravenous) of MSLN-TTC or vehicle control.

FIGURE 4.

In vivo efficacy of MSLN-TTC in combination with ATRi or PARPi in OVCAR-3 xenograft model. (A) Tumor size determined after single dose of MSLN-TTC (100 kBq/kg, 0.14 mg/kg, intravenous) and ATRi (40 mg/kg dosed twice daily, 3 d on/4 d off, 4 wk). (B) Tumor size determined after single dose of MSLN-TTC (100 kBq/kg, 0.14 mg/kg, intravenous) and PARPi (50 mg/kg once daily, 4 wk). Statistical analysis was performed using 1-way ANOVA followed by Tukey test. **P < 0.01. ***P < 0.001. ****P < 0.0001. T/C = treatment-to-control ratio.

FIGURE 5.

In vivo efficacy of MSLN-TTC in combination with ATRi in OVCAR-8 xenograft model. (A and B) Tumor size (A) and white blood cells and platelets (B) determined at endpoint of study after 3 (intravenous) injections of MSLN-TTC (200 kBq/kg, 0.14 mg/kg) and ATRi (40 mg/kg dosed twice daily, 2 d on/5 d off). Statistical analysis was performed using 1-way ANOVA followed by Tukey test. ***P < 0.001. ****P < 0.0001. T/C = treatment-to-control ratio.