A total of 41 ch14.18/CHO courses were given (10 × 3 courses, 5 × 2 courses, 1 × 1 course). Side effects were similar in expectedness, frequency and magnitude to those reported for ch14.18/SP2/0. The dose level of 20 mg/m(2)/day was confirmed. Toxicity was reversible and no treatment-related deaths occurred. In children, the peak plasma concentration was 16.51 µg/ml ± 5.9 µg/ml and the half-life was 76.91 h ± 52.5 h. A partial response following ch14.18/CHO was observed in 2/7 patients with residual disease. In mice, the half-lives were 22.7 h ± 1.9h for ch14.18/CHO and 25.0 h ± 1.9 h for ch14.18/SP2/0. The biodistribution of (125)I-ch14.18/CHO in mice with neuroblastoma was identical to (125)I-ch14.18/SP2/0, indicating GD 2 targeting activity in vivo. Ch14.18 produced in CHO cells showed an unchanged toxicity profile and pharmacokinetics in neuroblastoma patients compared with ch14.18 produced in SP2/0 cells, and evidence of clinical activity was observed. In mice, analysis of pharmacokinetics and biodistribution showed comparable results between ch14.18/CHO and ch14.18/SP2/0. Based on these results, ch14.18/CHO was accepted for prospective clinical evaluation.

The inhibitor of apoptosis protein survivin is highly expressed in neuroblastoma (NB) and survivin-specific T cells were identified in Stage 4 patients. Therefore, we generated a novel survivin minigene DNA vaccine (pUS-high) encoding exclusively for survivinderived peptides with superior MHC class I (H2-K k ) binding affinities and tested its efficacy to suppress tumor growth and metastases in a syngeneic NB mouse model. Vaccination was performed by oral gavage of attenuated Salmonella typhimurium SL7207 carrying pUS-high. Mice receiving the pUS-high in the prophylactic setting presented a 48-52% reduction in s.c. tumor volume, weight and liver metastasis level in contrast to empty vector controls. This response was as effective as a survivin full-length vaccine and was associated with an increased target cell lysis, increased presence of CD8 1 T-cells at the primary tumor site and enhanced production of proinflammatory cytokines by systemic CD8 1 T cells. Furthermore, depletion of CD8 1 but not CD4 1 T-cells completely abrogated the pUS-high mediated primary tumor growth suppression, demonstrating a CD8 1 T-cell mediated effect. Therapeutic vaccination with pUS-high led to complete NB eradication in over 50% of immunized mice and surviving mice showed an over 80% reduction in primary tumor growth upon rechallenge in contrast to controls. In summary, survivin-based DNA vaccination is effective against NB and the rational minigene design provides a promising approach to circumvent potentially hazardous effects of using full length antiapoptotic genes as DNA vaccines. ' UICCKey words: surviving; neuroblastoma; DNA vaccine; Salmonella typhimurium Development of an effective treatment against neuroblastoma (NB), the most common solid extracranial tumor during childhood, is one of the major objectives in pediatric oncology. The inhibitor of apoptosis protein survivin emerges as a suitable target for the establishment of an antineuroblastoma immunotherapy. Survivin is required to maintain cancer cell viability 1,2 and its blockade by antisense oligonucleotides, siRNAs, hammerhead ribozymes or small molecule antagonists lead to an increased tumor cell death rate. 3 The survivin gene is mapped to human chromosome 17q25, 4 within the prognostic unfavorable 17 q gain region seen in 90% of advanced stage NB cases. [5][6][7] Increased survivin expression in NB patients was associated with age, stage, unfavorable histology, MYCN amplification and showed to be predictive of recurrent disease and death. [8][9][10][11][12] In addition to the NB-associated survivin overexpression, 9 survivin-specific cytotoxic T cells (CTLs) were detected in NB patients. 13,14 Accordingly, the first step in targeting survivin for antineuroblastoma immunotherapy has been introduced by Coughlin and colleagues. 15 They primed T cells with CD40-activated B cells (CD40-B) transfected with NB-derived mRNA resulting in a T cell response that lysed HLA-matched NB cells, but not autologous benign cells in vitro. Some of the CTLs were survivinspecific ...

Neuroblastoma (NB) is a challenging malignancy of the sympathetic nervous tissue characterized by a very poor prognosis. One important marker for NB is the expression of tyrosine hydroxylase (TH), the first-step enzyme of catecholamine biosynthesis. We could show stable and high TH gene expression in 67 NB samples independent of the clinical stage. Based on this observation, we addressed the question of whether xenogeneic TH DNA vaccination is effective in inducing an anti-NB immune response. For this purpose, we generated three DNA vaccines based on pCMV-F3Ub and pBUD-CE4