Abstract B48: Preclinical development of meta-[211At] astatobenzylguanidine ([211At] MABG) targeted radiotherapy for neuroblastoma
Authors: Batra V , Chacko AM , Gagliardi M , Hou C , Mikitsh J L. , Freifelder R H. , Kachur A , LeGeyt B C. , Schmitz A , Toto L , Vaidyanathan G , Zalutsky M R. , Matthay K K. , Weiss W A. , Gustafson W C. , Pryma D , Maris J M. 
Published: May 2015

Abstract
Background: Neuroblastoma (NB) is a radiosensitive malignancy accounting for 10% of childhood cancer mortality. NB cells frequently express the norepinephrine transporter (NET) providing a specific mechanism for uptake of NET-ligands. Meta-[131I]iodobenzylguanidine ([131I]MIBG) is a NET-ligand radiotherapeutic that shows single-agent response rates in refractory NB of 40-50%. However, due to the long path lengths of 131 I beta (β)-emission, and low biological effectiveness compared to alpha (α)-emitting radionuclides, [131I]MIBG is generally not curative, perhaps due to non-targeting of isolated circulating tumor cells. Here we report our efforts to optimize NET-targeted radiotherapy by developing relevant preclinical models of refractory NB for α-particle therapeutic [211At] MABG therapy.
Methods: We first determined NET (SLC6A2) mRNA and protein expression in 35 human NB cell lines using quantitative RT-PCR and western blotting. We then chose 5 lines with absent to intermediate levels of native NET expression (NB1691, SKNSH, IMR5, NLF and SKNBE2) for dual forced overexpression of human NET and luciferase cDNAs. We used [125I]MIBG for cell-based uptake assays in all isogenic pairs and biodistribution experiments in athymic mice bearing three separate NET-transduced xenografts (N=5 per cell line). These cell lines were also treated with [131I]MIBG and/or external beam radiation (XRT) followed by multi-log cytotoxicity assays. Therapeutic trials of [131I]MIBG (25 mCi/kg) in NB1691 subcutaneous xenograft and metastatic mouse models were also conducted. In parallel, [211At] MABG was synthesized by: (i) cyclotron-production of 211 At via 209 Bi(α,2n)211At reaction (ii) distillation of 211 At from the target, and (iii) solid phase no-carrier-added synthesis of [211At] MABG by radioastato-destannylation. [211At] MABG uptake studies were performed in isogenic NB cell lines.
Results: Unlike primary human NBs, NET expression was low in the majority of 35 cell-lines studied (median normalized expression value = 0.145; range 0.000-1.005), but all transduced lines showed significant overexpression (0.860-1.107) comparable to human primary tumors. Transduced lines showed 4-10 fold higher uptake of [125I]MIBG than non-transduced isogenic parental cell lines in vitro, and demonstrated significant tumor-specific uptake and retention in vivo with tumor-muscle ratios ranging from 13.80 to 29.48. In vitro cytotoxicity experiments using [131I]MIBG showed NET-expressing cell lines to be more susceptible to treatment compared to non-NET expressing pairs (IC50 of 2.937nCi vs. 15.99 nCi). Treatment of mice bearing NB1691-NET xenografts with [131I]MIBG showed tumor growth delay (p=0.0065), but no significant impact on survival, likely due to de novo radioresistance (1200 cGy of XRT had no impact on NB1691 proliferation; IMR-05 showed 97% decreased cell viability). Lastly, we successfully synthesized [211At] MABG, with radiochemical yields of ∼20% and showed NET specific uptake of [211At] MABG into 1691 NET transfected cells.
Conclusions: Development of targeted radiotherapy for neuroblastoma has been limited by the lack of preclinical models and alternative therapeutics. Our development of multiple isogenic pairs with varying NET expression, documentation of de novo radiation sensitivity, and the production of [211At] MABG, will allow for rapid assessment of targeted radiotherapeutic strategies (including combination approaches) to support clinical development of alpha-particle therapeutics in a childhood cancer.
Citation Format: V Batra, AM Chacko, M Gagliardi, C Hou, J L. Mikitsh, R H. Freifelder, A Kachur, B C. LeGeyt, A Schmitz, L Toto, G Vaidyanathan, M R. Zalutsky, K K. Matthay, W A. Weiss, W C. Gustafson, D Pryma, J M. Maris. Preclinical development of meta-[211At] astatobenzylguanidine ([211At] MABG) targeted radiotherapy for neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B48.