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
Abstract
Treatment of high‐risk neuroblastoma (NB) represents a major challenge in paediatric oncology. Alternative therapeutic strategies include antibodies targeting the disialoganglioside GD
2
, ...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 GD
2
‐specific chimeric antigen receptor (CAR) comprising an anti‐GD
2
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 GD
2
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 GD
2
‐specific antibody or anti‐idiotypic antibody occupying the CAR’s cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD
2
‐specific NK cells was also found against primary NB cells and GD
2
expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells.
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.
The disialoganglioside GD2 is a well-established target antigen for passive immunotherapy in neuroblastoma (NB). Despite the recent success of passive immunotherapy with the anti-GD2 antibody ch14.18 ...and cytokines, treatment of high-risk NB remains challenging. We expanded the approach of GD2-specific, antibody-based immunotherapy to an application of a GD2-specific natural killer (NK) cell line, NK-92-scFv(ch14.18)-zeta. NK-92-scFv(ch14.18)-zeta is genetically engineered to express a GD2-specific chimeric antigen receptor generated from ch14.18. Here, we show that chimeric receptor expression enables NK-92-scFv(ch14.18)-zeta to effectively lyse GD2
+
NB cells also including partially or multidrug-resistant lines. Our data suggest that recognition of GD2 by the chimeric receptor is the primary mechanism involved in NK-92-scFv(ch14.18)-zeta-mediated lysis and is independent of activating NK cell receptor/ligand interactions. Furthermore, we demonstrate that NK-92-scFv(ch14.18)-zeta is able to mediate a significant anti-tumor response in vivo in a drug-resistant GD2
+
NB xenograft mouse model. NK-92-scFv(ch14.18)-zeta is an NB-specific NK cell line that has potential for future clinical development due to its high stability and activity toward GD2
+
NB cell lines.
Highlights ► SL7207 were dismantled as most effective vaccine carrier for survivin-based DNA vaccination in a syngeneic neuroblastoma. ► As a second-best DNA delivery system emerged the application ...of lentiviral-transduced DCs. ► Gene gun application appeared the least effective procedure for DNA vaccination. ► DC vaccination was more effective in inducing a cellular immune response when loaded with peptide epitopes. ► The level of Salmonella-associated side effects was not significant.
Immunotherapy targeting disialoganglioside GD(2) emerges as an important treatment option for neuroblastoma, a pediatric malignancy characterized by poor outcome. Here, we report the induction of a ...GD(2)-specific immune response with ganglidiomab, a new anti-idiotype antibody to anti-GD(2) antibodies of the 14.18 family.
Ganglidiomab was generated following immunization of Balb/c mice with 14G2a, and splenocytes were 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, IgG(1)). This antibody bound to anti-GD(2) 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-GD(2) antibodies to the nominal antigen GD(2) as well as GD(2)-specific lysis of neuroblastoma cells by NK-92-scFv(ch14.18)-zeta cells was competitively inhibited by ganglidiomab, proving GD(2) surrogate function and anti-idiotype characteristics. The dissociation constants of ganglidiomab from anti-GD(2) antibodies ranged from 10.8 ± 5.01 to 53.5 ± 1.92 nM as determined by Biacore analyses. The sequences of framework and complementarity-determining regions of ganglidiomab were identified. Finally, we demonstrated induction of a GD(2)-specific humoral immune response after vaccination of mice with ganglidiomab effective in mediating GD(2)-specific killing of neuroblastoma cells.
We generated and characterized a novel anti-idiotype antibody ganglidiomab and demonstrated activity against neuroblastoma.
Abstract
High-level expression of MycN caused by amplification of the gene characterizes the malignant phenotype of neuroblastoma. Recent studies suggest that MYCN is a suitable target for ...immunotherapy, but up to now, a syngeneic NB mouse model over expressing MYCN is not available to examine immunotherapeutic strategies in vivo.
Here, we report the development of a tetracycline inducible MYCN expressing murine NB cell line syngeneic to A/J mice and a MYCN-DNA vaccine. For this purpose, the murine NB cell line C1300, syngeneic to A/J mice, was stably transfected with a tetracycline inducible MYCN expression vector. Stable transfection was verified by real-time PCR and Western-Blot, revealing high expression levels of MYCN RNA and protein, respectively. Furthermore, a MYCN-DNA vaccine, based on epitopes encoding for three peptides from the murine MYCN protein sequence with high affinity to the A/J mouse MHC class I allele H2-Kk, was designed and tested in vivo for its ability to induce an antigen-specific immune response. Lymphocytes isolated from A/J mice vaccinated with the MYCN vaccine effectively lysed C1300-MYCN +tet cells in cytotoxicity assays in contrast to wild type C1300 cells and lymphocytes from control mice. Lymphocytes from minigene vaccinated mice produced significantly higher amounts of IFN-γ after stimulation with irradiated C1300 cells than lymphocytes from control mice. Interestingly, vaccine induced cytotoxic T lymphocytes also kill parental C1300 tumor cells which show low MYCN expression. We expect that this effect will be enhanced when MYCN-over expressing C1300 cells are employed in a similar cytotoxicity assay which will be reported at the meeting.
In summary, we report the development of a new MYCN-DNA vaccine and a murine tetracycline inducible MYCN NB cell line providing an useful syngeneic mouse model for in vivo evaluation of MYCN directed immunotherapeutic strategies.
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 LB-155. doi:10.1158/1538-7445.AM2011-LB-155
The
MYCN
oncogene is a strong genetic marker associated with poor prognosis in neuroblastoma (NB). Therefore,
MYCN
gene amplification and subsequent overexpression provide a possible target for new ...treatment approaches in NB. We first identified an inverse correlation of MYCN expression with CD45 mRNA in 101 NB tumor samples. KEGG mapping further revealed that
MYCN
expression was associated with immune-suppressive pathways characterized by a down-regulation of T cell activation and up-regulation of T cell inhibitory gene transcripts. We then aimed to investigate whether DNA vaccination against MYCN is effective to induce an antigen-specific and T cell-mediated immune response. For this purpose, we generated a MYCN-expressing syngeneic mouse model by
MYCN
gene transfer to NXS2 cells. MYCN-DNA vaccines were engineered based on the pCMV-F3Ub plasmid backbone to drive ubiquitinated full-length MYCN-cDNA and minigene expression. Vaccines were delivered orally with attenuated
S. typhimurium
strain SL7207 as a carrier. Immunization with both MYCN-DNA vaccines significantly reduced primary tumor growth of MYCN-expressing NB cells in contrast to negative controls. The immune response was mediated by tumor-infiltrating T cells in vivo, which revealed MYCN-specific and MHC class I-restricted lysis of inducible
MYCN
-expressing NB target cells in vitro. Finally, these antigen-specific T cells also killed MYCN-negative mammary carcinoma cells pulsed with MYCN peptides in contrast to controls. In summary, we demonstrate proof of concept that MYCN can be targeted by DNA vaccination, which may provide an approach to overcoming MYCN immune-suppressive activities in patients with
MYCN
-amplified disease.
Abstract
Background:
Drug-resistant neuroblastoma remains a major challenge in pediatric oncology. A human NK cell line NK-92-scFv(ch14.18)-z engineered to express a GD2-specific chimeric antigen ...receptor (CAR) may help to address this problem. We investigated the cytotoxicity of NK-92-scFv(ch14.18)-z in a panel of GD2+ drug-resistant neuroblastoma cell lines and analyzed the anti-tumor efficacy of NK-92-scFv(ch14.18)-z in a drug-resistant neuroblastoma xenograft mouse model.
Methods
Cytotoxic activity of GD2-specific NK-92-scFv(ch14.18)-z towards a panel of GD2+ cell lines (CHLA-20, SK-N-BE(2), CHLA-136, CHLA-79, LA-N-1, LA-N-5), some of which exhibit partial or multidrug resistance, was analyzed in a 51Cr release assay. We investigated the impact of GD2 recognition on NK-92-scFv(ch14.18)-z-mediated lysis by blocking the CAR through the addition of an anti-idiotype antibody (anti-IdAb) and also by downregulating GD2 on target cells induced by the glucosylceramide synthase (GCS) inhibitor PPPP. We then employed ELISA to determine the production of effector molecules granzyme B and perforin in response to activation with immobilized GD2. Anti-tumor efficacy of NK-92-scFv(ch14.18)-z and IL-2 was analyzed in a drug-resistant GD2+ xenograft mouse model by peritumoral injections of the GD2-specific NK cell line.
Results
NK-92-scFv(ch14.18)-z effectively lysed GD2+ drug-resistant NB cell lines. This effect was almost completely abrogated by blocking the CAR with an anti-IdAb. Decreased GD2 expression on target cells also resulted in diminished lysis mediated by NK-92-scFv(ch14.18)-z. Quantification of granzyme B and perforin production with ELISA revealed that the plate-bound antigen GD2 alone was sufficient to induce activation of NK-92-scFv(ch14.18)-z. Importantly, repeated peritumoral subcutaneous injections of a combination of NK-92-scFv(ch14.18)-zeta and IL-2 significantly prolonged survival time of mice challenged with aggressively growing subcutaneous CHLA-20 tumors in a xenograft mouse model.
Conclusions
These encouraging results indicate that GD2-directed immunotherapy with genetically engineered NK cells is an appropriate treatment strategy especially in relapsed NB that exhibit drug resistance.
Citation Format: Diana Seidel, Anastasia Shibina, C. Patrick Reynolds, Winfried S. Wels, Nicole Huebener, Holger N. Lode. GD2-specific genetically engineered NK cell therapy is effective in a drug-resistant neuroblastoma xenograft mouse model. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2808. doi:10.1158/1538-7445.AM2014-2808
Abstract Cancer remains the main cause of disease-related death in childhood. Pediatric tumors are characterized by a low mutational burden and high intertumoral heterogeneity, with multiple subtypes ...compared to their adult counterparts. The lack of access to many innovative therapies remains one of the main challenges in the pediatric oncology, especially for the 25% of patients who experience relapses. In this context, the need for the development of a well characterized collection of pediatric models, to provide large scale preclinical testing, is capital for the subsequent identification and prioritization of promising novel therapeutic options. The EU funded “Innovative Therapies for Children with Cancer-Pediatric Preclinical Proof-of-Concept Platform” (ITCC-P4) consortium is a unique public-private collaborative project consisting of academic and industrial partners that aimed at establishing a collection of >400 patient-derived xenograft (PDX) models representing the most common high-risk pediatric cancers. The project involved various aspects of model development including the thorough molecular and pharmacological characterization. XenTech’s participation was focused on the development and preclinical in vivo drug testing of Ewing sarcoma (n=17), hepatoblastoma (n=10), rhabdoid tumors (n=6), synovial sarcoma (n=2), rhabdomyosarcoma (n=2) and other tumors (n=6), as part of overall cohort. PDXs were obtained by transplantation of post-surgery tumor specimens, either by grafting tumor fragments into the interscapular region or subcutaneously in the right flank of nude, NOD-Scid or NOD-Scid gamma mice. Tumor xenografts were amplified by serial transplantation, and tissue samples were retained at early passages for molecular characterization. Fragments from established PDX models where frozen to generate a revivable ITCC-P4 PDX collection. Then, proof-of-concept drug testing was conducted, in a single mouse trial format: each tumor type (n=X PDX models) was treated with a dedicated panel of Standard-of-Care (SoC;n=3) and novel targeted therapies (n=6), or combinations of 2 or 3 novel targeted therapies; for each PDX model n=1 mouse being included per treatment. All molecular and drug-testing data obtained by the different partners are being centralized in the R2 repository (https://r2.amc.nl), providing a powerful tool for data integration, visualization and interpretation of the results. A unique collection of well characterized pediatric PDX models derived from the most relevant pediatric tumor types was enabled by a strong public-private collaborative project. This large cohort is now available for preclinical testing of novel therapeutic agents within a non-for-profit spinoff company, ITCC-P4 gGmbH (www.itccp4.com), offering new perspectives to the identification of promising treatment options for children with cancer. Citation Format: Emilie Indersie, Sophie Branchereau, Brice Fresneau, Christophe Chardot, Didier Surdez, Alexandra Saint-Charles, Maria Eugénia Marques da Costa, Ángel M. Carcaboso, Katia Scotlandi, Massimo Moro, Heinrich Kovar, Jan-Henning Klusmann, Klaus-Michael Debatin, Simon Bomken, Louis Chesler, Chris Jones, Beat Schäfer, Marco Wachtel, Johannes Gojo, Walter Berger, Christina Guttke, Maureen Hattersley, Frédéric Colland, Ashley Strougo, Dennis Gürgen, Jens Hoffmann, Julia Schueler, Pablo M. Aviles, María José Guillén, Aniello Federico, Apurva Gopisetty, Justyna Anna Wierzbinska, Andreas Schlicker, Sara Colombetti, Olaf Heidenreich, Fatima Iradier, Nicole Huebener, Natalie Jäger, Jan Koster, Marcel Kool, Gudrun Schleiermacher, Jan J. Molenaar, Birgit Geoerger, David J. Shields, Hubert N. Caron, Louis F. Stancato, Stefan M. Pfister, Gilles Vassal, Eva-Maria Rief, Olivier Déas. ITCC-P4, a preclinical proof-of-concept drug testing platform as a tool for pharmacological screening in pediatric tumor models abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5469.
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