Treatment of high-risk neuroblastoma (NB) represents a major challenge in paediatric oncology. Alternative therapeutic strategies include antibodies targeting the disialoganglioside GD2, which is expressed at high levels on NB cells, and infusion of donor-derived natural killer (NK) cells. To combine specific antibody-mediated recognition of NB cells with the potent cytotoxic activity of NK cells, here we generated clonal derivatives of the clinically applicable human NK cell line NK-92 that stably express a GD2-specific chimeric antigen receptor (CAR) comprising an anti-GD2 ch14.18 single chain Fv antibody fusion protein with CD3-ζ chain as a signalling moiety. CAR expression by gene-modified NK cells facilitated effective recognition and elimination of established GD2 expressing NB cells, which were resistant to parental NK-92. In the case of intrinsically NK-sensitive NB cell lines, we observed markedly increased cell killing activity of retargeted NK-92 cells. Enhanced cell killing was strictly dependent on specific recognition of the target antigen and could be blocked by GD2-specific antibody or anti-idiotypic antibody occupying the CAR’s cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD2-specific NK cells was also found against primary NB cells and GD2 expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells.

We generated and characterized a novel anti-idiotype antibody ganglidiomab and demonstrated activity against neuroblastoma.

Disialoganglioside GD2 is an established target for immunotherapy in neuroblastoma. We tested the hypothesis that active immunization against the glycolipid GD2 using DNA vaccines encoding for cyclic GD2-mimicking decapeptides (i.e., GD2 mimotopes) is effective against neuroblastoma. For this purpose, two GD2 peptide mimotopes (MA and MD) were selected based on docking experiments to anti-GD2 antibody ch14.18 (binding free energy: À41.23 kJ/mol for MA and À48.06 kJ/mol for MD) and Biacore analysis (mol/L for MA and 5.3 Â 10 À5 mol/L for MD), showing a higher affinity of MD over MA. These sequences were selected for DNA vaccine design based on pSecTag2-A (pSA) also including a T-cell helper epitope. GD2 mimicry was shown following transfection of CHO-1 cells with pSA-MA and pSA-MD DNA vaccines, with twice-higher signal intensity for cells expressing MD over MA. Finally, these DNA vaccines were tested for induction of tumor protective immunity in a syngeneic neuroblastoma model following oral DNA vaccine delivery with attenuated Salmonella typhimurium (SL 7207). Only mice receiving the DNA vaccines revealed a reduction of spontaneous liver metastases. The highest anti-GD2 humoral immune response and natural killer cell activation was observed in mice immunized with the pSA-MD, a finding consistent with superior calculated binding free energy, dissociation constant, and GD2 mimicry potential for GD2 mimotope MD over MA. In summary, we show that DNA immunization with pSA-MD may provide a useful strategy for active immunization against neuroblastoma. (Cancer Res 2006; 66(21): 10567-75)

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 ...

Therapeutic vaccination against tumor antigens without induction of autoimmunity remains a major challenge in cancer immunotherapy. Here, we show for the first time effective therapeutic vaccination followed by suppression of established spontaneous neuroblastoma metastases using a tyrosine hydroxylase (TH) DNA minigene vaccine. We identified three novel mouse TH (mTH3) derived peptides with high predicted binding affinity to MHC class I antigen H2-K k according to the prediction program SYFPEITHI and computer modeling of epitopes into the MHC class I antigen binding groove. Subsequently, a DNA minigene vaccine was generated based on the expression vector pCMV-F3Ub encoding mutated ubiquitin (Gly 76 to Ala 76 ) and mTH3. Prophylactic and therapeutic efficacies of this vaccine were established following oral delivery with attenuated Salmonella typhimurium SL7207. Only mice immunized with mTH3 were free of spontaneous liver metastases. This effect was clearly dependent on ubiquitin and high affinity of the mTH epitopes to MHC class I antigens. Specifically, we showed a crucial role for minigene expression as a stable ubiquitin-Ala 76 fusion peptide for vaccine efficacy. The immune response following the mTH3 DNA minigene vaccination was mediated by CD8 + T cells as indicated by infiltration of primary tumors and TH-specific cytolytic activity in vitro. Importantly, no cell infiltration was detectable in TH-expressing adrenal medulla, indicating the absence of autoimmunity. In summary, we show effective therapeutic vaccination against neuroblastoma with a novel rationally designed TH minigene vaccine without induction of autoimmunity providing an important baseline for future clinical application of this strategy.