The induction of tumor protective immunity against neuroblastoma remains a major challenge for active immunotherapy. Fractalkine is a unique Th1 CX3C chemokine known to induce adhesion and migration of leukocytes mediated by both, membrane-bound and soluble form, respectively. Here, we tested the hypothesis that chemokine gene therapy with fractalkine (FKN) induces an effective anti-neuroblastoma immune response amplified by targeted IL-2 using the anti-GD2 antibody ch14.18 fused with IL-2 (ch14.18-IL-2). For this purpose, NXS2 cells were genetically engineered to stably produce murine FKN (NXS2-FKN). Transcription and expression of the mFKN gene in tumor tissue of mice inoculated with NXS2-FKN cells were demonstrated in vivo. Importantly, mFKN exhibited a reduction in primary tumor growth and spontaneous liver metastases in syngenic A/J mice. This effect was boosted by targeted IL-2 using small non-curative doses of ch14-18-IL-2. The amplification of the FKN induced immune response was specific, since a non-specific antibody-IL-2 fusion protein ch225-IL-2 was ineffective. In summary, we demonstrated for the first time that chemokine gene therapy is amplified by targeted IL-2 suggesting a combination of both strategies as an adjuvant therapy for neuroblastoma.

Tumor directed cytotoxic therapy is one of the major challenges for the success of chemotherapy. In order to accomplish this goal in neuroblastoma, we rationally designed a prodrug of etoposide as substrate for tyrosine hydroxylase, a well established neuroblastoma associated enzyme. Here, we report synthesis and characterization of a 3,4 dihydroxy-phenyl carbamate derivative of etoposide. In order to demonstrate activation by tyrosine hydroxylase, the coding sequence of murine tyrosine hydroxylase was generated by reverse transcriptase-polymerase chain reaction from NXS2 neuroblastoma cells and cloned into the pRSET-A bacterial expression vector. The enzyme was expressed in Escherichia coli, characterized by Western blot and enzymatic activity was demonstrated by conversion of tyrosine into DOPA in the presence of cofactors using reversed phase high-performance liquid chromatography. Under these enzymatic conditions, we demonstrate conversion of 3,4 dihydroxyphenyl carbamate prodrug into free etoposide. This effect was clearly mediated by the enzyme since bacteria transformed with the empty vector were ineffective of prodrug activation. Furthermore, tyrosine hydroxylase positive cells exposed to the etoposide prodrug were effectively killed in contrast to tyrosine hydroxylase negative controls. These findings demonstrate that etoposide can be designed as a prodrug substrate for tyrosine hydroxylase and thereby establish proof of concept for neuroblastoma directed enzyme prodrug therapy.