Induced cross-resistance of BRAFV600E melanoma cells to standard chemotherapeutic dacarbazine after chronic PLX4032 treatment.

The maximum response and 10-year survival rate for metastatic melanoma patients treated with standardised chemotherapy is still less than 15% and 10%, respectively. In contrast, oncogene targeting was found a promising tool for killing of BRAFV600 mutated melanoma cells. Nevertheless, despite improved response and survival rates, resistance acquisition remains an ongoing problem. In this context, the impact of chronic BRAF inhibition on the efficacy of commonly applied cytostatics is still unknown. In our study, human melanoma cells with BRAFV600E mutation were treated with chemotherapeutics and a BRAF inhibitor. Resistance patterns were analysed by microelectrode array-based impedance spectroscopy, XTT and flow cytometric apoptosis/proliferation assay. BRAFV600E melanoma cells acquired a time- and concentration-dependent desensitisation up to 100-fold towards oncogene-specific PLX4032 and chemotherapeutic dacarbazine after twelve months treatment. The impact of multiple drug insensitivity on molecular melanoma characteristics was elaborated via mRNA and protein quantification. Following BRAFV600E targeting, melanoma cells developed an increasingly aggressive, dacarbazine-insensitive phenotype. Thereby, hyperactivated canonical alternative MAPK and bypass PI3K/AKT signalling caused cross-resistance of differently acting drugs. With these results, we are the first to show that long-term melanoma therapy with BRAF inhibitors can prevent further therapeutic success with dacarbazine due to acquisition of cross-resistance.

A multidimensional impedance platform for the real-time analysis of single and combination drug pharmacology in patient-derived viable melanoma models.

In today's development of anticancer drugs, there is an enormous demand for sensitive, non-invasive real-time screening technologies to identify pharmacodynamics/-kinetics of single and combined drugs with high precision. The combination of sophisticated drug sensitivity testing with advanced in vitro tumor models reflecting heterogeneous tumor behavior in vivo is needed to more reasonably predict therapeutic outcome in vivo. In this study, the benefits of our real-time, non-invasive multidimensional impedance platform over standard in vitro drug sensitivity assays were demonstrated quantitatively using an advanced melanoma model. Detailed pharmacological profiles of clinically established targeted therapeutics in single and combination treatment have been identified in patient tissue and isolated 2D/3D cell line cultures. Impedance spectroscopy revealed significant differences in tissue structure responsible for BRAF inhibitor pharmacokinetics in BRAFV600E tumor microfragments and cell lines. Remarkably, BRAF-/MEK inhibitor combination treatment of direct patient-derived tissue, but not melanoma cell lines, resulted in short-term antagonistic effects consistent with in vivo findings. In contrast, the clinically validated resistance delay and thus long-term synergy of targeted therapeutics in advanced melanoma models has been demonstrated using impedance technology. The results demonstrate limited clinical transferability of 2D/3D cancer cell line-based chemosensitivity data and underline the importance of in vivo-like direct patient-derived tissue for predictive drug studies. Our non-invasive and highly sensitive multidimensional impedance platform offers great potential for quantifying short- and long-term drug kinetics and synergies to identify the most effective drug combinations in advanced cancer models, thereby improving personalized drug development and treatment planning and ultimately, overall patient outcomes.

In vitro field potential monitoring on a multi-microelectrode array for the electrophysiological long-term screening of neural stem cell maturation.

Due to the lack of appropriate cell models as well as automated electrophysiology monitoring technologies, the standardized identification of neurotoxic or protective effects in vitro remains a major problem in today's pharmaceutical ingredient development. Over the past few years, in vivo-like human pluripotent stem cell-derived neuronal networks have turned out to be a promising physiological cell source, if the establishment of robust and time-saving functional maturation strategies based on stable and expandable neural progenitor populations can be achieved. Here, we describe a multi-microelectrode array (MMEA)-based bioelectronics platform that was optimized for long-term electrophysiological activity monitoring of neuronal networks via field potential measurements. Differentiation of small molecule-based neuronal progenitors on MMEAs led to functional neurons within 15 days. More strikingly, these functional neuronal cultures could remain electrophysiologically stable on the MMEAs for more than four weeks. The observed electrophysiological properties correlated with the expression of typical neuron subtype markers and were further validated by specific neurotransmitter applications. With our established monitoring platform, we could show for the first time the long-term stability of the neural stem cell-like progenitor population to differentiate to electrophysiologically active dopaminergic neuronal networks for more than 80 passages. In conclusion, we provide a comprehensive long-term stable field potential monitoring platform based on stem cell-derived human neuronal networks that can be automated and up-scaled for standardized high-content screening applications e.g. in the field of neurotoxic and neuroprotective therapeutics identification.

A novel 384-multiwell microelectrode array for the impedimetric monitoring of Tau protein induced neurodegenerative processes.

Over the last decades, countless bioelectronic monitoring systems were developed for the analysis of cells as well as complex tissues. Most studies addressed the sensitivity and specificity of the bioelectronic detection method in comparison to classical molecular biological assays. In contrast, the up scaling as a prerequisite for the practical application of these novel bioelectronic monitoring systems is mostly only discussed theoretically. In this context, we developed a novel 384-multiwell microelectrode array (MMEA) based measurement system for the sensitive label-free real-time monitoring of neurodegenerative processes by impedance spectroscopy. With respect to the needs of productive screening systems for robust and reproducible measurements on high numbers of plates, we focused on reducing the critical contacting of more than 400 electrodes for a 384-MMEA. Therefore, we introduced an on top array of immersive counter electrodes that are individually addressed by a multiplexer and connected all measurement electrodes on the 384-MMEA to a single contact point. More strikingly, our novel approach provided a comparable signal stability and sensitivity similar to an array with integrated counter electrodes. Next, we optimized a SH-SY5Y cell based tauopathy model by introducing a novel 5-fold Tau mutation eliminating the need of artificial tauopathy induction. In combination with our novel 384-MMEA based measurement system, the concentration and time dependent neuroregenerative effect of the kinase inhibitor SRN-003-556 could be quantitatively monitored. Thus, our novel screening system could be a useful tool to identify and develop potential novel therapeutics in the field of Tau-related neurodegenerative diseases.

Impedimetric real-time monitoring of neural pluripotent stem cell differentiation process on microelectrode arrays.

In today's neurodevelopment and -disease research, human neural stem/progenitor cell-derived networks represent the sole accessible in vitro model possessing a primary phenotype. However, cultivation and moreover, differentiation as well as maturation of human neural stem/progenitor cells are very complex and time-consuming processes. Therefore, techniques for the sensitive non-invasive, real-time monitoring of neuronal differentiation and maturation are highly demanded. Using impedance spectroscopy, the differentiation of several human neural stem/progenitor cell lines was analyzed in detail. After development of an optimum microelectrode array for reliable and sensitive long-term monitoring, distinct cell-dependent impedimetric parameters that could specifically be associated with the progress and quality of neuronal differentiation were identified. Cellular impedance changes correlated well with the temporal regulation of biomolecular progenitor versus mature neural marker expression as well as cellular structure changes accompanying neuronal differentiation. More strikingly, the capability of the impedimetric differentiation monitoring system for the use as a screening tool was demonstrated by applying compounds that are known to promote neuronal differentiation such as the γ-secretase inhibitor DAPT. The non-invasive impedance spectroscopy-based measurement system can be used for sensitive and quantitative monitoring of neuronal differentiation processes. Therefore, this technique could be a very useful tool for quality control of neuronal differentiation and moreover, for neurogenic compound identification and industrial high-content screening demands in the field of safety assessment as well as drug development.

Generation and Characterization of a Human/Mouse Chimeric GD2-Mimicking Anti-Idiotype Antibody Ganglidiximab for Active Immunotherapy against Neuroblastoma.

Vaccination with proteins mimicking GD2 that is highly expressed on neuroblastoma (NB) cells is a promising strategy in treatment of NB, a pediatric malignancy with poor prognosis. We previously showed efficacy of ganglidiomab in vivo, a murine anti-idiotype (anti-Id) IgG1. In order to tailor immune responses to variable regions, we generated a new human/mouse chimeric anti-Id antibody (Ab) ganglidiximab by replacing murine constant fragments with corresponding human IgG1 regions. DNA sequences encoding for variable regions of heavy (VH) and light chains (VL) were synthesized by RT-PCR from total RNA of ganglidiomab-producing hybridoma cells and further ligated into mammalian expression plasmids with coding sequences for constant regions of human IgG1 heavy and light chains, respectively. We established a stable production cell line using Chinese hamster ovarian (CHO) cells co-transfected with two expression plasmids driving the expression of either ganglidiximab heavy or light chain. After purification from supernatants, anti-idiotypic characteristics of ganglidiximab were demonstrated. Binding of ganglidiximab to anti-GD2 Abs of the 14.18 family as well as to NK-92tr cells expressing a GD2-specific chimeric antigen receptor (scFv(ch14.18)-zeta) was shown using standard ELISA and flow cytometry analysis, respectively. Ganglidiximab binding affinities to anti-GD2 Abs were further determined by surface plasmon resonance technique. Moreover, binding of anti-GD2 Abs to the nominal antigen GD2 as well as GD2-specific Ab-mediated cytotoxicity (ADCC, CDC) was competitively inhibited by ganglidiximab. Finally, ganglidiximab was successfully used as a protein vaccine in vivo to induce a GD2-specific humoral immune response. In summary, we report generation and characterization of a new human/mouse chimeric anti-Id Ab ganglidiximab for active immunotherapy against NB. This Ab may be useful to tailor immune responses to the paratope regions mimicking GD2 overexpressed in NB.

Pharmacokinetics and pharmacodynamics of ch14.18/CHO in relapsed/refractory high-risk neuroblastoma patients treated by long-term infusion in combination with IL-2.

Ch14.18 manufactured in Chinese hamster ovary (CHO) cells is currently being evaluated in clinical trials. Short-term infusion (STI) (8-20 h/day; 4-5 days) of 100 mg/m2 ch14.18/CHO (dinutiximab ß) per cycle in combination with cytokines is standard treatment of neuroblastoma (NB) patients. As pain is a limiting factor, we investigated a novel delivery method by continuous long-term infusion (LTI) of 100 mg/m2 over 10 days. 53 NB patients were treated with 5-6 cycles of 6 × 106 IU/m2 subcutaneous interleukin-2 (d 1-5, 8-12), LTI of 100 mg/m2 ch14.18/CHO (d 8-18) and 160 mg/m2 oral 13-cis-retinoic acid (d 22-35). Human anti-chimeric antibody (HACA), antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity were determined. With LTI, we observed a maximum concentration of ch14.18/CHO (Cmax) of 12.56 ± 0.68 µg/ml and a terminal half-life time (t1/2 ß) of 32.7 ± 16.2 d. The clearance values for LTI and STI of 0.54 ± 0.13 and 0.41 ± 0.29 L/d m2 and area under the serum concentration-time curve (AUC) values of 189.6 ± 41.4 and 284.8 ± 156.8 µg×d/ml, respectively, were not significantly different. Importantly, we detected ch14.18/CHO trough concentration of ≥ 1 µg/ml at time points preceding subsequent antibody infusions after cycle 1, allowing a persistent activation of antibody effector mechanisms over the entire treatment period of 6 months. HACA responses were observed in 10/53 (19%) patients, similar to STI (21%), indicating LTI had no effect on the immunogenicity of ch14.18/CHO. In conclusion, LTI of ch14.18/CHO induced effector mechanisms over the entire treatment period, and may therefore emerge as the preferred delivery method of anti-GD2 immunotherapy to NB patients.

Targeting of MYCN by means of DNA vaccination is effective against neuroblastoma in mice.

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.

Disialoganglioside-specific human natural killer cells are effective against drug-resistant neuroblastoma.

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.

Functional bioassays for immune monitoring of high-risk neuroblastoma patients treated with ch14.18/CHO anti-GD2 antibody.

Effective treatment of high-risk neuroblastoma (NB) remains a major challenge in pediatric oncology. Human/mouse chimeric monoclonal anti-GD2 antibody (mAb) ch14.18 is emerging as a treatment option to improve outcome. After establishing a production process in Chinese hamster ovary (CHO) cells, ch14.18/CHO was made available in Europe for clinical trials. Here, we describe validated functional bioassays for the purpose of immune monitoring of these trials and demonstrate GD2-specific immune effector functions of ch14.18/CHO in treated patients. Two calcein-based bioassays for complement-dependent- (CDC) and antibody-dependent cellular cytotoxicity (ADCC) were set up based on patient serum and immune cells tested against NB cells. For this purpose, we identified LA-N-1 NB cells as best suited within a panel of cell lines. Assay conditions were first established using serum and cells of healthy donors. We found an effector-to-target (E:T) cell ratio of 20:1 for PBMC preparations as best suited for GD2-specific ADCC analysis. A simplified method of effector cell preparation by lysis of erythrocytes was evaluated revealing equivalent results at an E:T ratio of 40:1. Optimal results for CDC were found with a serum dilution at 1:8. For validation, both within-assay and inter-assay precision were determined and coefficients of variation (CV) were below 20%. Sample quality following storage at room temperature (RT) showed that sodium-heparin-anticoagulated blood and serum are stable for 48 h and 96 h, respectively. Application of these bioassays to blood samples of three selected high-risk NB patients treated with ch14.18/CHO (100 mg/m(2)) revealed GD2-specific increases in CDC (4.5-9.4 fold) and ADCC (4.6-6.0 fold) on day 8 compared to baseline, indicating assay applicability for the monitoring of multicenter clinical trials requiring sample shipment at RT for central lab analysis.

Validated detection of human anti-chimeric immune responses in serum of neuroblastoma patients treated with ch14.18/CHO.

Human/mouse chimeric monoclonal antibody (mAb) ch14.18/CHO is directed against disialoganglioside GD2. Activity and efficacy of this mAb are currently determined in ongoing clinical Phase II and -III studies in high-risk neuroblastoma (NB). Based on the chimeric nature of this mAb, some patients may develop a human anti-chimeric immune response (Mirick et al., 2004) which impacts on pharmacokinetics and may induce anti-anti-idiotype (Id) mAb with a potential survival benefit. Therefore, a validated method of quantitative detection of human anti-chimeric antibodies (HACA) in serum samples of NB patients treated with ch14.18/CHO is an important tool for monitoring of clinical trials. Here, we report a validated sandwich enzyme-linked immunosorbent assay (ELISA) according to the one arm binding principle using ch14.18/CHO as a capture mAb and biotinylated ch14.18/CHO mAb for detection. Ganglidiomab, a monoclonal anti-Id Ab to ch14.18/CHO (Lode et al., 2013), was used as a standard for assay validation and HACA quantification. Systematic evaluation of the established ELISA procedure revealed an optimal serum sample dilution factor of 1:160. Assay validation was accomplished with a set of tailored quality controls (QC) containing distinct concentrations of ganglidiomab (3 and 15µg/ml). The coefficients of variation (CV) for all within-assay and inter-assay measurements using QCs were under 20% and the limit of detection (LOD) was 1.1µg/ml. Three patients (P1, P2, P3) treated with a 10day continuous infusion of 100mg/m(2) of ch14.18/CHO were selected for analysis with this assay. Selection was based on ch14.18/CHO drug level on day 8 in cycle 2 of >10µg/ml (expected) (P1) and of <2µg/ml (unexpected) (P2 and P3). Both patients with unexpected low ch14.18/CHO levels revealed a strong signal in the HACA ELISA. Interestingly, ch14.18/CHO-mediated complement-dependent cytotoxicity (CDC) could not be detected in P2 in contrast to P3 suggesting anti-NB activity even in the presence of HACA. We showed that neither eight freeze-thaw cycles nor storage at room temperature for up to 168h affected HACA stability in serum. In summary, we describe a validated ELISA method suitable for the assessment of HACA in NB patients treated with ch14.18/CHO.

Validated detection of anti-GD2 antibody ch14.18/CHO in serum of neuroblastoma patients using anti-idiotype antibody ganglidiomab.

Human/mouse chimeric monoclonal antibody (mAb) ch14.18 is directed against disialoganglioside GD2 and has demonstrated activity and efficacy in high-risk neuroblastoma (NB). For the purpose of industrial production, ch14.18 was manufactured in Chinese hamster ovarian cells (ch14.18/CHO) in order to facilitate clinical trials in Europe. To determine immunopharmacological effects of ch14.18 in preclinical models and clinical trials, a validated method of quantitative detection of ch14.18/CHO in serum is an important tool. We recently described the generation and characterization of ganglidiomab, a monoclonal anti-idiotype Ab (AIT) of ch14.18 (Lode et al., 2013), which was used to establish quantitative and validated enzyme-linked immunosorbent assay (ELISA) methods using ganglidiomab as a capture mAb. With these ELISA methods, we first demonstrated binding of ch14.18/CHO to ganglidiomab to a similar extent as to the nominal antigen GD2 and in contrast to GD1b and GM2 precursor and metabolite gangliosides, used as negative controls. In order to determine both low (0.5-3.1 µg/ml) and high levels of ch14.18/CHO (1.0-25 µg/ml) in the serum of NB patients treated with ch14.18/CHO, we established two ELISA methods with high and low sensitivity using 1/1001, and 1/5126 sample dilutions, respectively. For validation, we used a set of tailored quality controls (QC) containing distinct concentrations of ch14.18/CHO (1.0, 2.0, 7.0, and 20.0 µg/ml). We determined the limit of detection (LOD) for both ELISA methods to be 0.50 µg/ml for the high sensitivity and 1.02 µg/ml for low sensitivity ELISA. The within-assay precision was 12% for high and 4% for low sensitivity ELISA, and the coefficients of variation (CV) were under 20% for all assays (3% for QC-1.0, 5% for QC-2.0, 7% for QC-7, and 3% for QC-20). With this method, we showed that neither eight freeze-thaw cycles nor storage at room temperature for up to 168 h affected ch14.18/CHO stability in serum. Finally, we analyzed ch14.18 Ab serum levels in selected NB patients receiving ch14.18/CHO as a continuous or bolus infusion with a peak concentration at the last day of Ab application (17.14 ± 7.20mg/ml with continuous and 19.78 ± 2.26 mg/ml with bolus infusion). In summary, we describe validated ELISA methods using ganglidiomab as a capture mAb suitable for the pharmacological evaluation of ch14.18/CHO in NB patients.

A novel 3D label-free monitoring system of hES-derived cardiomyocyte clusters: a step forward to in vitro cardiotoxicity testing.

Unexpected adverse effects on the cardiovascular system remain a major challenge in the development of novel active pharmaceutical ingredients (API). To overcome the current limitations of animal-based in vitro and in vivo test systems, stem cell derived human cardiomyocyte clusters (hCMC) offer the opportunity for highly predictable pre-clinical testing. The three-dimensional structure of hCMC appears more representative of tissue milieu than traditional monolayer cell culture. However, there is a lack of long-term, real time monitoring systems for tissue-like cardiac material. To address this issue, we have developed a microcavity array (MCA)-based label-free monitoring system that eliminates the need for critical hCMC adhesion and outgrowth steps. In contrast, feasible field potential derived action potential recording is possible immediately after positioning within the microcavity. Moreover, this approach allows extended observation of adverse effects on hCMC. For the first time, we describe herein the monitoring of hCMC over 35 days while preserving the hCMC structure and electrophysiological characteristics. Furthermore, we demonstrated the sensitive detection and quantification of adverse API effects using E4031, doxorubicin, and noradrenaline directly on unaltered 3D cultures. The MCA system provides multi-parameter analysis capabilities incorporating field potential recording, impedance spectroscopy, and optical read-outs on individual clusters giving a comprehensive insight into induced cellular alterations within a complex cardiac culture over days or even weeks.

Vaccination with anti-idiotype antibody ganglidiomab mediates a GD(2)-specific anti-neuroblastoma immune response.

PURPOSE: 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. EXPERIMENTAL DESIGN AND RESULTS: 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. CONCLUSION: We generated and characterized a novel anti-idiotype antibody ganglidiomab and demonstrated activity against neuroblastoma.

Fenretinide sensitizes multidrug-resistant human neuroblastoma cells to antibody-independent and ch14.18-mediated NK cell cytotoxicity.

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Combining passive immunotherapy with an antibody to the disialoganglioside GD2 (ch14.18/SP2/0) and cytokines with 13-cis-retinoic acid for post-myeloablative maintenance therapy increased survival in high-risk NB, but the overall prognosis for these children is still in need of improvement. Fenretinide (4-HPR) is a synthetic retinoid that has shown clinical activity in recurrent NB and is cytotoxic to a variety of cancer cells, in part via the accumulation of dihydroceramides, which are precursors of GD2. We investigated the effect of 4-HPR on CHO-derived, ch14.18-mediated anti-NB effector functions, complement-dependent cytotoxicity (CDC), and antibody-dependent and antibody-independent cellular cytotoxicity (ADCC and AICC, respectively). Here, we demonstrate for the first time that pretreatment of fenretinide-resistant NB cells with 4-HPR significantly enhanced ch14.18/CHO-mediated CDC and ADCC and AICC by both human natural killer cells and peripheral blood mononuclear cells. Treatment with 4-HPR increased GD2 and death receptor (DR) expression in resistant NB cells and induced an enhanced granzyme B and perforin production by effector cells. Blocking of ganglioside synthesis with a glucosylceramide synthase inhibitor abrogated the increased ADCC response but had no effect on the AICC, indicating that GD2 induced by 4-HPR mediates the sensitization of NB cells for ADCC. We also showed that 4-HPR induced increased GD2 and DR expression in a resistant NB xenograft model that was associated with an increased ADCC and AICC response using explanted tumor target cells from 4-HPR-treated mice. In summary, these findings provide an important baseline for the combination of 4-HPR and passive immunotherapy with ch14.18/CHO in future clinical trials for high-risk NB patients.

Induced tauopathy in a novel 3D-culture model mediates neurodegenerative processes: a real-time study on biochips.

Tauopathies including Alzheimer's disease represent one of the major health problems of aging population worldwide. Therefore, a better understanding of tau-dependent pathologies and consequently, tau-related intervention strategies is highly demanded. In recent years, several tau-focused therapies have been proposed with the aim to stop disease progression. However, to develop efficient active pharmaceutical ingredients for the broad treatment of Alzheimer's disease patients, further improvements are necessary for understanding the detailed neurodegenerative processes as well as the mechanism and side effects of potential active pharmaceutical ingredients (API) in the neuronal system. In this context, there is a lack of suitable complex in vitro cell culture models recapitulating major aspects of taupathological degenerative processes in sufficient time and reproducible manner.Herewith, we describe a novel 3D SH-SY5Y cell-based, tauopathy model that shows advanced characteristics of matured neurons in comparison to monolayer cultures without the need of artificial differentiation promoting agents. Moreover, the recombinant expression of a novel highly pathologic fourfold mutated human tau variant lead to a fast and emphasized degeneration of neuritic processes. The neurodegenerative effects could be analyzed in real time and with high sensitivity using our unique microcavity array-based impedance spectroscopy measurement system. We were able to quantify a time- and concentration-dependent relative impedance decrease when Alzheimer's disease-like tau pathology was induced in the neuronal 3D cell culture model. In combination with the collected optical information, the degenerative processes within each 3D-culture could be monitored and analyzed. More strikingly, tau-specific regenerative effects caused by tau-focused active pharmaceutical ingredients could be quantitatively monitored by impedance spectroscopy.Bringing together our novel complex 3D cell culture taupathology model and our microcavity array-based impedimetric measurement system, we provide a powerful tool for the label-free investigation of tau-related pathology processes as well as the high content analysis of potential active pharmaceutical ingredient candidates.

NK cells engineered to express a GD2 -specific antigen receptor display built-in ADCC-like activity against tumour cells of neuroectodermal origin.

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.