The clinical efficacy of the first approved alpha pharmaceutical, Xofigo (radium-223 dichloride, 223 RaCl 2 ), has stimulated significant interest in the development of new alpha-particle emitting drugs in oncology. Unlike radium-223 ( 223 Ra), the parent radionuclide thorium-227 ( 227

Targeted alpha therapy with 227Th-trastuzumab of human SKOV3-luc-D3 cells growing intraperitoneally in nude mice was clearly superior to unlabeled trastuzumab therapy. The results warrant further studies of 227Th-radioimmunotherapy used as adjuvant treatment and for metastatic cancer.

Mesothelin (MSLN) is a 40 kDa membrane-anchored glycoprotein, involved in mediating cell-cell adhesion, metastatic spread, promotion of cell proliferation and resistance to apoptosis. Overexpression of MSLN is most prominent in mesothelioma, ovarian, lung, triple-negative breast (TNBC) and pancreatic cancers, while in healthy tissue, MSLN is confined mainly to the mesothelial cells of the peritoneum and pericardium. Several MSLN-targeting approaches are currently being investigated, including antibody drug conjugates. We describe herein a high energy, alpha-particle emitting MSLN Targeted Thorium Conjugate (MSLN-TTC). Thorium-227 (227Th) has a half-life of 18.7 days and decays via emission of an alpha particle to radium-223 (half-life 11.4 days), a calcium-mimetic used in the treatment of CRPC [1]. The MSLN-TTC comprises an anti-mesothelin monoclonal antibody covalently linked via an amide bond to a chelator moiety possessing high affinity for thorium-227. We present data from in vitro cytotoxicity assays demonstrating selective cell killing on MSLN positive cell-lines as well as in vivo efficacy in a mouse orthotopic bone xenograft model using NCI-H226 luciferase labeled cells. Experimental procedures: MSLN-TTC was prepared in high radiochemical yields and purity. In vitro cytotoxicity experiments were performed on the mesothelin-positive cell lines Ovcar-3 (ovarian), NCI-H226 (lung mesothelioma) and mesothelin-transfected HT29 (HT29MSLN/colorectal) cells. An in vivo model was established by orthotopic intratibial inoculation of luciferase-transfected NCI-H226 cells in athymic mice. Development of bone disease was monitored by luciferase activity and the extent of bone lesions determined by x-ray imaging and microCT. Results: MSLN-TTC induced specific in vitro cytotoxicity via induction of DNA double strand breaks as determined by phosphorylated histone protein H2AX. MSLN-TTC demonstrated statistical significant in vivo potency administered as a single dose of either 250 or 500 kBq/ kg in the orthotopic bone xenograft model. Animals treated with MSLN-TTC showed a) significantly reduced disease in the bone metastatic lesions b) decreased metastatic disease in the lungs and c) significant reduction in osteolytic/ osteoblastic bone lesions as evidenced by X-Ray and microCT compared to the vehicle control group. Furthermore, no significant loss in body weight was observed during the course of the study demonstrating that the MSLN-TTC was well tolerated. The data presented support the further investigation of the MSLN-TTC in bone metastatic disease. References: 1. Henriksen, G., et al., Targeting of osseous sites with alpha-emitting 223Ra: comparison with the beta-emitter 89Sr in mice. J Nucl Med, 2003. 44(2): p. 252-9. Citation Format: Urs B. Hagemann, Else-Marie Hagelin, Katrine Wickstroem, Kristine Sponheim, Roger Smeets, Jenny Karlsson, Roger M. Bjerke, Mari I. Suominen, Yvonne Konkol, Jenni Bernoulli, Jukka Rissanen, Jussi Halleen, Liv-Ingrid Oedegaardstuen, Alan Cuthbertson. A novel high energy alpha-pharmaceutical: In vitro and in vivo potency of a mesothelin-targeted thorium-227 conjugate (TTC) in a model of bone disease. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 591.

<div>Abstract<p>The clinical efficacy of the first approved alpha pharmaceutical, Xofigo (radium-223 dichloride, ²²³RaCl₂), has stimulated significant interest in the development of new alpha-particle emitting drugs in oncology. Unlike radium-223 (²²³Ra), the parent radionuclide thorium-227 (²²⁷Th) is able to form highly stable chelator complexes and is therefore amenable to targeted radioimmunotherapy. We describe the preparation and use of a CD33-targeted thorium-227 conjugate (CD33-TTC), which binds to the sialic acid receptor CD33 for the treatment of acute myeloid leukemia (AML). A chelator was conjugated to the CD33-targeting antibody lintuzumab via amide bonds, enabling radiolabeling with the alpha-emitter ²²⁷Th. The CD33-TTC induced <i>in vitro</i> cytotoxicity on CD33-positive cells, independent of multiple drug resistance (MDR) phenotype. After exposure to CD33-TTC, cells accumulated DNA double-strand breaks and were arrested in the G₂ phase of the cell cycle. <i>In vivo</i>, the CD33-TTC demonstrated antitumor activity in a subcutaneous xenograft mouse model using HL-60 cells at a single dose regimen. Dose-dependent significant survival benefit was further demonstrated in a disseminated mouse tumor model after single dose injection or administered as a fractionated dose. The data presented support the further development of the CD33-TTC as a novel alpha pharmaceutical for the treatment of AML. <i>Mol Cancer Ther; 15(10); 2422–31. ©2016 AACR</i>.</p></div>

<p>Binding of CD33 antibody and CD33 antibody chelator-conjugate to CD33-positive HL-60 cells by FACS staining in comparison to an isotype control. Bound antibodies were detected using secondary PE-labeled anti-human antibody. Median fluorescence intensity (MFI) was recorded and plotted against antibody concentrations. EC50 values were determined by fitting data using Graph Pad software.</p>