Intracellular delivery of radioimmunoconjugates that target the cancer testis antigen, NY-ESO-1

Objectives Cancer testis antigens (CTA) represent attractive targets for targeted radiotherapy and imaging as their expression is restricted to cancer and germ cells. NY-ESO-1, a member of the CTA family, is highly immunogenic and expressed in multiple tumor types. The aim of this study was to develop radioimmunoconjugates (RIC) to target NY-ESO-1 protein in cancer cells.

Methods Anti-NY-ESO-1 antibodies were modified by addition of DTPA for ¹¹¹In-labelling or, in the presence of Iodogen, were ¹²³I-labelled. Western blotting and immunofluorescence microscopy confirmed that NY-ESO-1 is expressed in SK-MEL-37 melanoma cell lines. Delivery of RICs across the cell membrane was achieved using a protein transfection (PT) reagent (SAINT-PhD) and by chemical linkage with the cell-penetrating peptide, TAT (YGRKKRRQRRR). Cellular internalization, distribution and efflux of ¹¹¹In-DTPA-anti-NY-ESO-1-TAT-PT and ¹²³I-anti-NY-ESO-1-TAT-PT were investigated in cell fractionation and retention assays. Clonogenic survival of SK-MEL-37 cells exposed to ¹¹¹In-DTPA-anti-NY-ESO-1-tat-PT was determined.

Results Radiochemical purity of ¹¹¹In-DTPA-anti-NY-ESO-1-TAT was ≥95%. The amount of radioactivity that internalized in SK-MEL-37 cells following exposure to ¹²³I-anti-NY-ESO-1-TAT-PT or ¹¹¹In-DTPA-anti-NY-ESO-1-TAT-PT, expressed as % of total administered radioactivity, was 18.9% and 1.47% respectively. The presence of TAT improved nuclear uptake of ¹²³I-anti-NY-ESO-1-TAT-PT by 25% (P <0.001). ¹²³I-anti-NY-ESO-1-TAT-PT was retained 60% longer in comparison to a non-specific control RIC (¹²³I-mIgG-TAT-PT), (P <0.005). In clonogenic assays ¹¹¹In-DTPA-anti-NY-ESO-1-TAT-PT (1MBq/μg) reduced surviving fraction of SK-MEL-37 cells to 17%. Cytotoxicity was inversely proportional to specific activity (range 0.2-2 MBq/µg).

Conclusions Protein transfection reagent promoted internalization and nuclear accumulation of RICs. NY-ESO-1-targeting RICs are cytotoxic to SK-MEL-37 cells and represents a novel approach to the treatment of CTA-expressing cancers