Nicole Huebener

Fractalkine (CX3CL1)- and interleukin-2-enriched neuroblastoma microenvironment induces eradication of metastases mediated by T cells and natural killer cells.

Fractalkine (FKN) is a unique CX3C chemokine (CX3CL1) known to induce both adhesion and migration of leukocytes mediated by a membrane-bound and a soluble form, respectively. Its function is mediated through CX3C receptor (CX3CR), which is expressed by T(H)1 immune cells including T cells and natural killer (NK) cells. FKN was shown to be expressed in >90% of 68 neuroblastoma samples as determined by cDNA microarray analysis. Here, we characterized the effect of FKN in the neuroblastoma microenvironment using a syngeneic model genetically engineered to secrete FKN. We show FKN-mediated migration, adhesion, and IFN-gamma secretion of immune effector cells, but limited antineuroblastoma activity, in vitro and in vivo. Therefore, we tested the hypothesis that a combined increase of FKN and interleukin-2 (IL-2) in the neuroblastoma microenvironment induces an effective antitumor immune response. For this purpose, IL-2 was targeted to ganglioside GD2, which is highly expressed on neuroblastoma tissue, using an anti-GD2 antibody IL-2 immunocytokine (ch14.18-IL-2). Only mice bearing FKN- and IL-2-enriched neuroblastoma tumors exhibited a reduction in primary tumor growth and a complete eradication of experimental liver metastases. The depletion of T cells and NK cells in vivo abrogated the effect, and these effector cells showed the highest cytolytic activity in vitro. Finally, only the FKN- and IL-2-enriched neuroblastoma microenvironment resulted in T-cell activation and the release of proinflammatory cytokines. In summary, we showed for the first time the immunologic mechanisms by which targeted IL-2 treatment of neuroblastoma with an FKN-rich microenvironment induces an effective antitumor response.

A rationally designed tyrosine hydroxylase DNA vaccine induces specific antineuroblastoma immunity

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-Kk 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 (Gly76 to Ala76) 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-Ala76 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. [Mol Cancer Ther 2008;7(7):2241–51]

Xenogeneic immunization with human tyrosine hydroxylase DNA vaccines suppresses growth of established neuroblastoma

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-CE4.1 plasmids encoding for human (h)THcDNA (A), hTH minigene (B), and hTHcDNA in combination with the proinflammatory cytokine interleukin 12 (C), and tested prophylactic and therapeutic efficacy to suppress primary tumor growth and spontaneous metastasis. Here we report that xenogeneic TH DNA vaccination was effective in eradicating established primary tumors and inhibiting metastasis. Interestingly, this effect could not be enhanced by adding the Th1 cytokine interleukin 12. However, increased IFN-γ production and NB cytotoxicity of effector cells harvested from vaccinated mice suggested the participation of tumor-specific CTLs in the immune response. The depletion of CD8+T cells completely abrogated the hTH vaccine–mediated anti-NB immune response. Furthermore, rechallenging of surviving mice resulted in reduced primary tumor growth, indicating the induction of a memory immune response. In conclusion, xenogeneic immunization with TH-derived DNA vaccines is effective against NB, and may open a new venue for a novel and effective immunotherapeutic strategy against this challenging childhood tumor. [Mol Cancer Ther 2009;8(8):2392–401]

Rationally designed hydrolytically activated etoposide prodrugs, a novel strategy for the treatment of neuroblastoma.

Effective chemotherapy in neuroblastoma is limited by poor anti-tumor efficacy, systemic toxicity and the induction of drug resistance. Here, we provide further evidence that a hydrolytic activated prodrug design may overcome these problems. For this purpose, VP-16 was functionally blocked by a carbonate linker to generate two novel chemically stable prodrugs of VP-16, ProVP-16 I and II. We demonstrate profoundly different biological effects in vitro and in vivo of the prodrugs compared to parental VP-16. First, we established an up to >2 log higher in vitro toxicity of the two prodrugs compared to VP-16 on a panel of neuroblastoma cell lines. The highest increase of prodrug mediated cytotoxicity was observed in multi drug resistant cell lines. Second, in vivo studies showed a maximum tolerated dose (MTD) of ProVP-16 II (60 mg/kg), which was at least threefold higher than that of VP-16 (20 mg/kg). Tests of ProVP-16 II in a syngeneic NXS2 neuroblastoma model indicated that mice treated with this prodrug at 1/3 of the MTD was as effective as VP-16 parental compound used at the MTD in suppression of tumor growth. In summary, the etoposide prodrugs proved effective and less toxic and are therefore highly promising new anti-neuroblastoma compounds.

Eradication of CD19+ leukemia by targeted calicheamicin θ.

Children with relapsed and refractory acute lymphoblastic leukemia (ALL) still face a critical prognosis. We tested the hypothesis that targeted calicheamicin theta (θ) using an anti-CD19-immunoconjugate may provide an effective treatment strategy for CD19(+) ALL. Calicheamicin θ is a rationally designed prodrug of the natural enediyene calicheamicin γ, obtained by total synthesis. It offers the advantage of increased in vivo stability and 1000-fold higher antitumor potency over calicheamicin γ. First, we demonstrate efficacy of calicheamicin θ against primary pre-B leukemic cells and multidrug-resistant leukemia cell lines (IC(50) = 10(-9) to 10(-12) M). Second, conjugation of calicheamicin θ to an internalizing murine anti-CD19 monoclonal antibody was demonstrated to affect neither calicheamicin θ mediated cytotoxicity nor binding of the antibody to the target molecule. Third, anti-CD19-calicheamicin θ immunoconjugate revealed a maximum tolerated dose of 10 μg/kg and CD19-specific and long-lasting eradication of established leukemia was demonstrated in a xenograft model. Finally, we show that the antileukemic effect of anti-CD19-calicheamicin θ is mediated by induction of apoptosis proceeding through the caspase-mediated mitochondrial pathway. On the basis of these results, we conclude that anti-CD19-calicheamicin θ immunoconjugates may offer a novel and effective approach for the treatment of relapsed CD19(+) ALL.

Abstract A28: Generation and characterization of anti-idiotype antibody ganglidiomab as GD2 surrogate for immunotherapy of neuroblastoma.

Purpose: Immunotherapy targeting disialoganglioside GD2 emerges as an important treatment option for neuroblastoma, a pediatric malignancy characterized by poor outcome. Here, we report the generation and characterization of ganglidiomab, a new anti-idiotype antibody to anti-GD2 antibodies of the 14.18 family for monitoring of clinical trials and the development of neuroblastoma vaccines. Experimental Design and Results: Balb/c mice were immunized with 14G2a and splenocytes harvested to generate hybridoma cells. Clones were screened by ELISA for mouse antibody binding to hu14.18. One positive clone was selected to purify and characterize the secreted IgG protein (κ, IgG1). This antibody bound to anti-GD2 antibodies 14G2a, ch14.18/CHO, hu14.18 and to immunocytokines ch14.18-IL2 and hu14.18-IL2 as well as to NK-92 cells expressing scFv(ch14.18)-zeta receptor. Binding of these anti-GD2 antibodies to the nominal antigen GD2 as well as GD2 specific lysis of neuroblastoma cells by NK-92-scFv(ch14.18)-zeta cells was competitively inhibited by ganglidiomab, proving GD2 surrogate function and anti-idiotype characteristics. The dissociation constants of ganglidiomab from anti-GD2 antibodies ranged from 10.8 ± 5.01 to 53.5 ± 1.92 nM as determined by Biacore analyses using “steady state” analysis. The sequences of framework- (FRs) and complementarity determining -regions (CDRs) of ganglidiomab were identified. Finally, we demonstrate induction of a GD2 specific humoral immune response after vaccination of mice with ganglidiomab effective in mediating GD2 specific killing of neuroblastoma cells. Conclusion: We generated and characterized a novel anti-idiotype antibody ganglidiomab for immune monitoring of clinical trials with anti-GD2 antibodies and provide an important baseline for the development of anti-idiotype vaccines against malignancies expressing GD2. Citation Format: Holger N. Lode, Manuela Schmidt, Diana Seidel, Nicole Huebener, Diana Brackrock, Matthias Bleeke, Daniel Reker, Sven Brandt, Hans-Peter Mueller, Christiane Helm, Nikolai Siebert. Generation and characterization of anti-idiotype antibody ganglidiomab as GD2 surrogate for immunotherapy of neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A28.

Abstract 1800: 4-HPR (fenretinide) sensitizes human neuroblastoma cells for antibody-independent and ch14.18-mediated NK cell killing

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: Neuroblastoma (NB) is the most common extracranial solid tumor in children. Recently, the addition of passive immunotherapy with the anti-GD2 antibody ch14.18 provided an increased survival of children with high-risk disease. However, still one third of the patients die from their disease emphasizing the need for improved therapy protocols. 4-HPR is a synthetic retinoid known to induce apoptosis in cancer cells mainly via the accumulation of ceramides. We tested the hypothesis whether 4-HPR and ch14.18 would work additively in NK cell-mediated killing (antibody-dependent and independent cellular cytotoxicity, ADCC and AICC) when applied in combination. Methods: We employed 6 GD2+, drug-resistant human NB cell lines. First, we tested whether 4-HPR treatment would result in an increase of the GD2 binding index by using flow cytometry (FC). Second, we determined the effect of 4-HPR on death receptor (DR) expression on human NB cells (Fas, TRAIL-R1 and TRAIL-R2) by FC. Third, cytotoxicity assays were performed in which 4-HPR-treated, live cells were used with expanded natural killer (NK) cells and peripheral blood mononuclear cells (PBMCs) isolated from healthy donors as effector cells. Forth, we blocked ganglioside synthesis by using the glucosylceramide synthase inhibitor PPPP and determined the effects on ADCC and AICC and GD2 and DR expression. Fifth, we induced s.c. tumors (cell line CHLA-136) in NOD/SCID mice and treated them with 240mg/kg/d 4-HPR by oral gavage for 12 days. Then, we isolated tumor cells and measured GD2 and Fas expression levels by FC. Further, tumor cells were employed ex vivo in cytotoxicity assays using expanded NK cells as effector population. Results: Pre-treatment of drug-resistant NB cells with 4-HPR significantly enhances ch14.18-mediated ADCC as well as AICC by both human NK cells and PBMCs. Interestingly, we also found an increase of the GD2 binding index as well as an increase of DRs in all cell lines tested. Blocking of ganglioside synthesis results in an abrogation of the increased ADCC response by human NK cells but has no effect on the enhanced AICC after 4-HPR pre-treatment. Finally, we could show that tumor xenografts isolated from mice that were treated with 4-HPR exhibit an increased GD2 binding index as well as Fas expression compared to tumors isolated from vehicle treated animals. Importantly, this correlates well with a significantly higher ex vivo ADCC and AICC response of human NK cells towards 4-HPR treated tumors than towards vehicle treated controls. Conclusion: New therapy protocols are needed in order to optimize treatment of children with high-risk NB. We can show here for the first time that 4-HPR treatment increases ADCC and AICC by human NK cells and PBMCs towards drug-resistant human NB cells, thereby providing an important baseline for the combination of 4-HPR and passive immunotherapy with ch14.18 in future clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1800. doi:10.1158/1538-7445.AM2011-1800

Abstract 1808: Neuroblastoma cell lines established from progressive disease that exhibit partial or multi drug resistance are highly sensitive to chimeric receptor scFv(ch14.18)-zeta mediated NK cell killing

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: Neuroblastoma (NB) is a neuroectodermal tumor of childhood characterized by a poor prognosis especially for high-risk patients. To specifically direct the cytotoxic abilities of NK cells towards NB cells expressing the glycolipid antigen ganglioside GD2, the human NK cell line NK-92 was genetically engineered to express a chimeric receptor, consisting of a GD2-specific ch14.18scFv-antibody fragment and the signal transducing zeta-chain of the CD3 complex (NK-92-scFv(ch14.18)-zeta). Recently, we showed that NK-92-scFv(ch14.18)-zeta (NK-92tr) are able to efficiently lyse GD2 expressing NB cells. Furthermore, plate-bound GD2 is sufficient to induce the activation of NK-92tr, indicated by increased granzymeB and perforin production. Both effects can be abrogated by blocking the chimeric receptor with Id14G2A-17-9. The treatment of high risk NB patients is still challenging, as the majority of these patients develops progressive disease after positive initial responses. These relapsed tumors often show resistance towards one or multiple drugs (MDR). Therefore we tested the hypothesis whether NK-92tr are able to efficiently lyse GD2+ tumor cell lines established from progressive disease that exhibit partial or MDR. Aims and Methods: We employed NB cell line pairs from five patients in Cr51 release cytotoxicity assays. Each pair consists of a cell line established at time point of diagnosis (SK-N-BE(1), SMS-KAN, SMS-KCN, CHLA-15, CHLA-122) and another cell line established from progressive disease (SK-N-BE(2), SMS-KANR, SMS-KCNR, CHLA-20, CHLA-136). Two of the relapse cell lines exhibit MDR (SK-N-BE(2), CHLA-20), the others partial drug resistance. Flow cytometry was utilized to analyze GD2 expression on all cell line pairs to analyze whether GD2 expression levels changed during progressive disease after initial chemotherapy. Further, we inhibited ganglioside synthesis in relapse cell lines by using the glucosylceramide synthase inhibitor PPPP to abrogate NK-92tr mediated cell killing. Results and Conclusions: The comparison of cell lines established at different time points from the same patients revealed for all five patients that the relapse cell lines show significantly higher sensitivity towards NK-92tr mediated lysis. This effect correlates with a significantly increased GD2 surface expression on relapse cell lines and can be abrogated by blocking ganglioside synthesis. Our results indicate that relapse cell lines which exhibit partial or MDR are more sensitive towards NK-92tr mediated killing than their counterparts established at time of diagnosis. These interesting and encouraging results provide an important baseline to further investigate the sensitivity of NB relapse cell lines towards GD2-specific NK-92tr and NK cell killing in general. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1808. doi:10.1158/1538-7445.AM2011-1808