Tumour-specific delivery of cleavable radiolabelled antibodies against intranuclear γH2AX

Objectives A bifuntional immunoconjugate which targets epidermal growth factor (EGF) receptor (EGFR) and the nuclear DNA repair signalling protein, γH2AX, was synthesized. EGF was conjugated to a fluorophore- or radioisotope-labelled anti-γH2AX antibody, tagged with a nuclear localisation peptide.

Methods Conjugation was achieved through either a stable amide(A) or an unstable disulphide linker(B), in a 1:1 ratio.

Results Both A and B bound EGFR on MDA-MB-468 cells, and γH2AX in cell extracts from X-irradiated cells. Both AlexaFluor555- and ¹¹¹In-labelled compounds internalized into MDA-MB-468 cells in an EGF-dependent manner. Compound A colocalized with AlexaFluor488-labelled EGF and EGFR during intracellular trafficking. However, intracellular trafficking of cleavable compound B was not dependent on EGF/EGFR trafficking. In irradiated MDA-MB-468 cells, expressing γH2AX, compound B was observed as foci in cell nuclei. Retention of ¹¹¹In labelled compound B, but not A, was prolonged 4-fold in cells expressing γH2AX. Prolonged retention of B was abrogated when the anti-γH2AX moiety was replaced with a nonspecific antibody, or when cells did not express γH2AX or EGFR. Clonogenic survival of cells exposed to the combination of 4 Gy X-irradiation plus ¹¹¹In-labelled compound A and B, was significantly reduced compared to either compound alone, or compared to non-EGFR- or non-γH2AX-binding control compounds, and compared to non-¹¹¹In-labelled compound. In vivo, uptake of compound A in a MDA-BM-468 subcutaneous xenograft was EGFR and γH2AX specific, as measured by small animal SPECT/CT imaging.

Conclusions Tumour-specific delivery of radioisotope or fluorescently labelled antibodies against intranuclear epitopes is possible using cleavable immunoconjugates