Abstract 5771: Molecular radiation therapy: Targeting DNA damage response proteins

Abstract

DNA damage is an early event after exposure of cells to many anti-cancer agents. Auger electron-emitting radiopharmaceuticals that localize at sites of DNA-double strand breaks (dsb) cause added damage, thus stimulating the formation of their own target and converting sublethal DNA lesions into irreparable damage. The histone H2A variant, H2AX, is phosphorylated on Ser139 (γH2AX) and forms foci at DNA-dsb. γH2AX foci are generally more numerous and resolve more slowly in cancer cells which are genomically unstable compared to normal cells. The aim of this study was to target γH2AX foci for molecular radiation therapy.

Methods: Anti-γH2AX (aγH2AX) or anti-mouse IgG (amIgG) monoclonal antibodies were modified by addition of a nuclear localizing signal (NLS)-containing cell penetrating peptide, TAT, through N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling. Cyclic diethylenetriaminepentaacetic dianhydride (cDTPA) was added for radiolabeling with 111-indium. Fluorophore AlexaFluor 488-labelled variants of both radioimmunoconjugates (RICs) were also prepared.

Results: Using confocal microscopy, AF488-aγH2AX-TAT but not AF488-amIgG-TAT was found to colocalize with γH2AX-foci in cells exposed to X-Ray irradiation (IR) (4 Gy). To evaluate cellular retention of RICs, MDA-MB-468 or MDA-MB-231/H2N human breast cancer cells were treated with ¹¹¹In-aγH2AX-TAT or ¹¹¹In-amIgG-TAT (0.5 µg, 0.1 MBq/µg) and exposed to IR (4 Gy) or bleomycin (20 µg/mL). ¹¹¹In was retained 16-fold longer in IR- or bleomycin-treated cells exposed to ¹¹¹In-aγH2AX-TAT compared to control cells exposed to ¹¹¹In-aγH2AX-TAT. ¹¹¹In-aγH2AX-TAT (6 MBq/µg) caused amplification of both IR-induced DNA-dsb (evaluated by γH2AX foci number and the neutral comet assay [NCA]) and clustered DNA damage (evaluated by NCA plus glycosylases). In clonogenic assays, the combination of ¹¹¹In-aγH2AX-TAT plus IR or bleomycin was significantly more cytotoxic than either agent alone and more cytotoxic than ¹¹¹In-amIgG-Tat plus IR or bleomycin alone. In in vivo experiments, uptake of ¹¹¹In-aγH2AX-TAT, but not ¹¹¹In-amIgG-TAT, was up to 6-fold greater in MDA-MB-231/H2N xenografts exposed to IR (4 or 10 Gy) and in bleomycin-treated mice compared to untreated controls. The volume doubling time of MDA-MB-231/H2N xenografts increased 13-fold (p < 0.05) in animals treated with IR (10 Gy) plus ¹¹¹In-aγH2AX-TAT (10 µg, 6 MBq/µg) compared to those exposed to IR or ¹¹¹In-αγH2AX-TAT alone.

Conclusion: ¹¹¹In-aγH2AX-TAT is a novel radiopharmaceutical that amplifies DNA-damage and enhances the cytotoxicity of IR and chemotherapy in vitro and in vivo.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5771.