Effective targeted cytotoxicity of neuroblastoma cells

Background/Purpose: Despite aggressive treatment with surgery, chemotherapy, and radiotherapy, the prognosis for many children with neuroblastoma remains poor. Targeted toxins represent novel cancer therapeutics designed to selectively target and kill cancer cells. The authors have developed a novel fusion toxin, DT5F11, consisting of truncated diphtheria toxin (DT_A) linked to a single chain antibody (sc5F11) targeting the GD₂ antigen found on most neuroblastoma cells. This report describes the construction, expression, and in vitro function of DT5F11. Methods: Utilizing restriction enzyme digestion, polymerase chain reaction amplification, and gel electrophoresis, the prkDTL5F11 plasmid was created by the fusion of distinct coding sequences for a single-chain GD₂ targeting antibody (sc5F11) and truncated diphtheria toxin (DT_A). DH5α Escherichi coli-competent cells were transformed with prkDTL5F11; DNA was amplified, isolated, and sequenced. The fusion protein was expressed and assayed by Western blot. Targeted cytotoxicity was analyzed on GD₂-positive (SK-N-AS, IMR-32, SK-N-MC, LAN-1) and GD₂-negative (HeLa) cells. Results: Fluorescent dye-labeled cycle sequencing identified the constructed fusion toxin gene. Western blot analysis using a mouse antihuman DTA antibody showed a 69-kD band identifying the fusion toxin, DT5F11. Targeted cell killing with DT5F11 was seen only in GD₂ positive cells. Conclusions: This study demonstrates creation of a novel fusion toxin with effective GD₂-targeted cellular toxicity. Further investigation of this fusion toxin as a therapeutic agent in the management of neuroblastoma is warranted.