Anti-neuroblastoma Activity Research Articles

Interferon-β counter-regulates its own pro-apoptotic action by activating p38 MAPK signalling in human SH-SY5Y neuroblastoma cells.

Type I interferons (IFNs) induce apoptosis of neuroblastoma cells, but the molecular mechanisms regulating this event have not been completely elucidated. Here, we investigated the role of p38 mitogen activated protein kinase (MAPK) activity, a key regulator of apoptosis and a known modulator of IFN-induced responses in non-neuronal cells. We show that in SH-SY5Y human neuroblastoma cells IFN-β induced a delayed and sustained increase of p38 MAPK activity through a novel mechanism involving the sequential activation of Janus kinase-signal transducer and activator of transcription-1 signalling, enhanced expression of the NADPH oxidase catalytic subunit gp91(phox), increased reactive oxygen species production and stimulation of the MAPK kinase kinase transforming growth factor-β-activated kinase 1. Either blockade of p38 MAPK by the second generation inhibitors BIRB0796 and VX745 or siRNA knockdown of p38α MAPK enhanced IFN-β-induced apoptosis of neuroblastoma cells. Exposure to IFN-β increased the phosphorylation of the small heat shock protein HSP27 at Ser15, Ser78 and Ser82 with a time course similar to p38 MAPK activation and this response was suppressed by either p38α MAPK depletion or pharmacological inhibition of p38 MAPK and MAPK-activated protein kinase 2 (MK2). Either silencing of HSP27 expression by siRNA or MK2 inhibition potentiated IFN-β-induced apoptotic death. These results indicate that IFN-β-induced apoptosis of human SH-SY5Y neuroblastoma cells is associated with a long-lasting up-regulation of p38 MAPK activity, stimulation of MK2 and phosphorylation of the pro-survival protein HSP27. Moreover, the data show that inhibition of p38 MAPK signalling potentiates the anti-neuroblastoma activity of the cytokine, indicating that this pathway mediates a counter-regulatory response.

Selective inhibition of Histone Deacetylase 8 as a novel therapeutic strategy for the treatment of high-risk neuroblastoma

Aims: Expression studies of primary neuroblastoma samples from the German Neuroblastoma Trial revealed histone deacetylase 8 (HDAC8) to be upregulated in stage 4 patients, associated with a significantly worse prognosis. Here, we investigated the anti-neuroblastoma activity of HDAC8-selective inhibitors (HDAC8i) in vitro and in vivo.

Abstract 2858: Binding characteristics of the immunocytokine hu14.18-IL2 and induction of human effector functions as anticipated mode of action.

Abstract Immunotherapy of neuroblastoma with anti-GD2 antibodies in combination with interleukin-2 (IL2) emerges as an important treatment option as shown in several clinical trials. In consequence, an antibody-IL2 fusion protein (hu14.18-IL2; immunocytokine) was designed that combines the targeting and effector functions of an anti-GD2 antibody and the immune modulation activity of IL2. In addition to mediation of typical effector functions such as CDC and ADCC this immunocytokine targets IL2 to GD2+ tumor cells and thereby links tumor cells to IL2-receptor bearing effector cells via an Fc-gamma independent mechanism. This concept has already shown encouraging results in Phase I/II trials in relapsed/refractory neuroblastoma patients. Here, we report the antigen binding characteristics and in vitro anti-neuroblastoma activity of a new GMP preparation of hu14.18-IL2 (APN301). Binding of APN301 to its nominal antigen GD2 was analyzed by solid phase ELISA and compared to other antibodies of the 14G2a family. Affinities of APN301 to GD2 and to anti-14.18 anti-idiotype antibodies were also determined by Biacore® analysis. GD2 specific ADCC, CDC and whole blood cytotoxicity were analyzed in cytotoxicity assays in vitro (calcein-release, 51Cr release) using GD2+ neuroblastoma target cell lines. We demonstrate similar binding characteristics of APN301 to GD2 compared to parent 14.18 antibodies (murine and chimeric). The anti-14.18 anti-id antibody ganglidiomab fully inhibits binding of APN301 to the nominal antigen GD2, thereby proving internal image properties. The dissociation constants of APN301 to GD2 and ganglidiomab as determined in Biacore analyses are in the 10-9M and 10-7M range, respectively, similar to those determined for the naked chimeric 14.18 antibody. Relevant for the prediction of clinical activity we demonstrate potent specific lysis of GD2+ human neuroblastoma cells mediated by APN301 in a whole blood cytotoxicity assay in vitro, utilizing whole blood of healthy donors as well as whole blood of high-risk neuroblastoma patients in advanced disease stage. Further clinical trials with APN301 in neuroblastoma patients are being planned, taking into account the found potent whole blood cytolytic activity and the pharmakokinetic properties of this immunocytokine. Citation Format: Hans Loibner, Manfred Schuster, Evelyne Janzek, Stefan Stranner, Bernhard Peball, Susanne Wiederkum, Oliver Mutschlechner, Nikolai Siebert, Holger Lode. Binding characteristics of the immunocytokine hu14.18-IL2 and induction of human effector functions as anticipated mode of action. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2858. doi:10.1158/1538-7445.AM2013-2858 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Humanizing murine IgG3 anti-GD2 antibody m3F8 substantially improves antibody-dependent cell-mediated cytotoxicity while retaining targeting in vivo

Murine IgG3 anti-GD2 antibody m3F8 has shown anti-neuroblastoma activity in Phase I/II studies, where antibody-dependent cell-mediated cytotoxicity (ADCC) played a key role. Humanization of m3F8 should circumvent human anti-mouse antibody (HAMA) response and enhance its ADCC properties to reduce dosing and pain side effect. Chimeric 3F8 (ch3F8) and humanized 3F8 (hu3F8-IgG1 and hu3F8-IgG4) were produced and purified by protein A affinity chromatography. In vitro comparison was made with m3F8 and other anti-GD2 antibodies in binding, cytotoxicity, and cross-reactivity assays. In GD2 binding studies by SPR, ch3F8 and hu3F8 maintained KD comparable to m3F8. Unlike other anti-GD2 antibodies, m3F8, ch3F8 and hu3F8 had substantially slower koff.. Similar to m3F8, both ch3F8 and hu3F8 inhibited tumor cell growth in vitro, while cross-reactivity with other gangliosides was comparable to that of m3F8. Both peripheral blood mononuclear cell (PBMC)-ADCC and polymorphonuclear leukocytes (PMN)-ADCC of ch3F8 and hu3F8-IgG1 were more potent than m3F8. This superiority was consistently observed in ADCC assays, irrespective of donors or NK-92MI-transfected human CD16 or CD32, whereas complement mediated cytotoxicity (CMC) was reduced. As expected, hu3F8-IgG4 had near absent PBMC-ADCC and CMC. Hu3F8 and m3F8 had similar tumor-to-non tumor ratios in biodistribution studies. Anti-tumor effect against neuroblastoma xenografts was better with hu3F8-IgG1 than m3F8. In conclusion, humanizing m3F8 produced next generation anti-GD2 antibodies with substantially more potent ADCC in vitro and anti-tumor activity in vivo. By leveraging ADCC over CMC, they may be clinically more effective, while minimizing pain and HAMA side effects. A Phase I trial using hu3F8-IgG1 is ongoing.

Abstract 2493: Enhanced activity of fenretinide/-LYM-X-SORBTM (4-HPR/LXS) oral powder in combination with ketoconazole and vincristine against recurrent neuroblastoma xenografts

Abstract Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. Survival from relapsed NB is low and novel therapies are needed against recurrent NB, which often manifest loss of p53 function and multidrug resistance. Fenretinide (4-HPR) is a synthetic retinoid that achieved multiple complete responses in minimal disease in a phase I clinical trial in recurrent neuroblastoma when formulated as an oral powder using the LYM-X-SORBTM (LXS) organized lipid matrix (4-HPR/LXS). We recently showed that ketoconazole (keto) increased 4-HPR plasma concentrations in mice by > 2-fold over 4-HPR alone (P<0.02) (Br J Pharmacol 163:1263-75, 2011). To explore the anti-neuroblastoma activity in vivo of 4-HPR/LXS given with keto, we tested three human neuroblastoma xenografts established from patients with progressive disease, CHLA-119 and CHLA-90 (both TP53-mutant, multidrug-resistant cell lines) and COG-N-415x (from a progressive disease patient). 4-HPR/LXS was dosed at 240 mg/kg/day, keto at 38 mg/kg/day (both by oral gavagex5d/wk). 4-HPR and metabolite (4-oxo-4-HPR and 4-MPR) tumor concentrations were determined 4 h after last treatment and were quantified by HPLC. In the CHLA-119 xenograft, keto increased the complete response (CR) rate of 4-HPR/LXS from 1/10 mice to 5/10 mice. Survival >350 days with 4-HPR/LXS alone was 10% compared to 50% of mice treated with 4-HPR/LXS+keto (P< 0.005). 4-HPR/LXS+keto increased 4-HPR and 4-oxo-4-HPR tumor concentrations by > 2-fold over 4-HPR alone (P<0.02) and (P<0.04) respectively. We then added intravenous (i.v.) vincristine (VCR) at 0.5 mg/kg given twice a week every other week to the 4-HPR/LXS+keto regimen. In CHLA-119 xenografts, VCR increased the CR rate to 5/10 mice compared to 3/8 with 4-HPR/LXS+keto and 1/10 with VCR alone. Mouse survival for >300 days with 4-HPR/LXS+keto+VCR was 50% vs 38% with 4-HPR/LXS+keto (P<0.01) and 10% for VCR alone (P<0.003). COG-N-415x and CHLA-90 xenografts failed to achieve responses to 4-HPR/LXS or in combination with keto. Addition of VCR to 4-HPR/LXS+keto achieved CR's in 2/9 COG-415x mice compared to 0/9 and 0/8 for 4-HPR/LXS+keto and VCR alone, respectively. Survival of COG-415x mice was >90 days with 4-HPR+keto+VCR for 2/9 mice (22%) vs 0% for the other treatments (P<0.001). In CHLA-90 xenografts, 4-HPR/LXS+keto+VCR achieved 1 CR and 1 stable disease compared to no responses with 4-HPR/LXS+keto or VCR alone. CHLA-90 xenograft mouse survival for >60 days with 4-HPR/LXS+keto+VCR was 2/5 (40%) vs 0% for VCR or 4-HPR/LXS+keto alone. Toxicity of the combination regimens in mice (assessed by body weight) was negligible. These data suggest that if an ongoing phase I study of 4-HPR/LXS+keto is well-tolerated that a subsequent clinical trial exploring that combination+VCR should be undertaken in patients with recurrent NB. Supported by CPRIT RP10072. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2493. doi:1538-7445.AM2012-2493

In vitro anti-neuroblastoma activity of saquinavir and its association with imatinib

Neuroblastoma (NB) has a poor prognosis when in advanced stages, highlighting the need for new therapeutic options. The human immunodeficiency virus (HIV) protease inhibitor saquinavir is active invitro against chronic myeloid leukaemia cells, in synergy with the tyrosine kinase inhibitor imatinib. Here, we evaluated the effects of saquinavir, alone or in association with imatinib, on cell proliferation (count of viable cells after trypan blue exclusion), apoptosis (AnnexinV binding) and invasion (through a transwell membrane coated with Matrigel) in SJ-N-KP, IMR5, AF-8, SK-N-SH and SK-N-BE NB lines, all expressing c-kit and PDGF-R (determined by flow cytometry). Saquinavir showed a dose-dependent anti-proliferative and anti-invasive activity on NB lines, increased by the association with imatinib when the two drugs were utilized at clinically attainable concentrations. The same low saquinavir concentrations inhibited in NB cells the nuclear activation of NF-κB (Western immuno-blotting for nuclear NF-κB p50 and p65). Saquinavir at high concentrations also exerted a pro-apoptotic activity on NB lines, significantly increased by the association with imatinib. In conclusion, saquinavir and imatinib are both drugs utilized for long-term therapies, with good oral bioavailability and a well-known toxicity profile. The anti-NB activity of saquinavir and of its association with imatinib suggests a potential usefulness in the treatment of NB, particularly for remission maintenance.

Enhanced anti-neuroblastoma activity of a fenretinide complexed form after intravenous administration

The major limitation to successful chemotherapy of neuroblastoma (NB) is the toxicity and the poor bioavailability of traditional drugs. We synthesised an amphiphilic dextrin derivative (DX-OL) able to host fenretinide (4-HPR) by complexation. In this study, we have investigated the effects of 4-HPR-loaded amphipilic dextrin (DX-OL/4-HPR) in comparison with 4-HPR alone both in vitro on human NB cells and in vivo in pseudometastatic NB models. The haemolysis assay was used as a measure of the potential damage caused by the pharmaceutical formulation in vivo. Pharmacokinetic experiments were performed to assess drug plasma levels in mice treated with free or complexed 4-HPR. DX-OL/4-HPR exerted a more potent cytotoxic activity on NB cells. Complexed 4-HPR significantly increased the proportion of sub-G1 cells with respect to free 4-HPR. Dextrin derivatives showed no haemolytic activity, indicating their suitability for parenteral administration. DX-OL/4-HPR increased the lifespan and the long-term survival of treated mice over controls. The analysis of drug plasma levels indicates that the complexed drug has a higher AUC due to a reduced clearance from the blood. Our data suggest that DX-OL/4-HPR is an injectable formulation that is able to improve drug aqueous solubility and bioavailability.

Anti-Neuroblastoma Activity of Gold Nanorods Bound with GD2 Monoclonal Antibody under Near-Infrared Laser Irradiation

High-risk neuroblastoma is one of the most common deaths in pediatric oncology. Current treatment of this disease involves a coordinated sequence of chemotherapy, surgery, and radiation. Further advances in therapy will require the targeting of tumor cells in a more selective and efficient way so that survival can be improved without substantially increasing toxicity. To achieve tumor-selective delivery, disialoganglioside (GD2) expressed by almost all neuroblastoma tumors represents a potential molecular target that can be exploited for tumor-selective delivery. In this study, GD2 monoclonal antibody (anti-GD2) was conjugated to gold nanorods (GNRs) which are one of anisotropic nanomaterials that can absorb near-infrared (NIR) laser light and convert it to energy for photothermolysis of tumor cells. Thiolated chitosan, due to its biocompatibility, was used to replace cetyltrimethylammonium bromide (CTAB) originally used in the synthesis of gold nanorods. In order to specifically target GD2 overexpressed on the surface of neuroblastoma stNB-V1 cells, anti-GD2 was conjugated to chitosan modified GNRs (CGNRs). To examine the fate of CGNRs conjugated with anti-GD2 after incubation with neuroblastoma cells, rhadoamine B was labeled on CGNRs functionalized with anti-GD2. Our results illustrated that anti-GD2-conjugated CGNRs were extensively endocytosed by GD2+ stNB-V1 neuroblastoma cells via antibody-mediated endocytosis. In addition, we showed that anti-GD2 bound CGNRs were not internalized by GD2− SH-SY5Y neuroblastoma cells. After anti-GD2-linked CGNRs were incubated with neuroblatoma cells for six hours, the treated cells were further irradiated with 808 nm NIR laser. Post-NIR laser exposure, when examined by calcein-AM dye, stNB-V1 cells all underwent necrosis, while non-GD2 expressing SH-SY5Y cells all remained viable. Based on the in vitro study, CGNRs bound with anti-GD2 has the potential to be utilized as a therapeutic thermal coupling agent that generates heat sufficient to selectively kill neuroblastoma cells under NIR laser light exposure.

Anti-neuroblastoma activity of hu14.18-IL2 against minimal residual disease in a Children’s Oncology Group (COG) phase II study

3002 Background: The hu14.18-IL2 fusion protein consists of IL2 linked to a humanized mAb that recognizes the GD2 disialoganglioside expressed on neuroblastoma (NB) cells. Hu14.18-IL2 has preclinical activity in mouse models of NB and melanoma, particularly when given to mice with a smaller tumor burden. Using the dosing and schedule developed in our phase I study, we determined the antitumor activity of hu14.18-IL2 in two strata of patients with recurrent or refractory NB. Methods: Hu14.18-IL2 was given intravenously at a dose of 12 mg/m2/day over 4 hours for three consecutive days every 28 days. Responses were evaluated every 2 cycles for patients with disease measurable by standard radiographical criteria (stratum 1) and for patients with disease evaluable only by MIBG and/or bone marrow histology (stratum 2). In each stratum, a one-stage design was powered to conclude efficacy with 4 or more responders (CR or PR) out of 20. Response was established by independent radiology review as well as bone marro...

Anti-neuroblastoma activity of Helminthosporium carbonum (HC)-toxin is superior to that of other differentiating compounds in vitro

Treatment of high-risk neuroblastoma (NB) is difficult. Novel therapeutics improving survival rates are urgently required. We have previously shown that the histone deacetylase inhibitor (HDACI) Helminthosporium carbonum (HC)-toxin induces differentiation of neuroblastoma (NB) cells. Here, we show that HC-toxin inhibits the growth of both established NB cell lines and primary cultures with and without amplified MYCN stronger than retinoids (RAs) and other HDACIs (MS-275, n-butyric acid, suberoylanilide hydroxamic acid, trichostatin A, valproic acid). Nanomolar dosages suppress E2F-1, N-myc, Skp2, Mad2 and survivin proteins, found at high levels in high-risk NBs, more efficiently than both RAs and other HDACIs. The level of hypophosphorylated active retinoblastoma (RB) tumor suppressor protein is increased most effectively. HC-toxin’s epoxy group is essential for inhibiting HDACs and promoting anti-NB activity. Without this functional group, those cellular effects are not observed. In conclusion, the anti-NB activity of HC-toxin is superior to that of RAs and that of all other HDACIs tested.

Sodium Thiosulfate Administered Six Hours after Cisplatin Does Not Compromise Antineuroblastoma Activity

We determined if the potentially otoprotective agent sodium thiosulfate (STS) could be given 6 h after cisplatin without diminishing the antineuroblastoma activity of cisplatin in human neuroblastoma cell lines in vitro (including cisplatin-resistant cell lines) and in neuroblastoma xenografts in vivo. We determined the antineuroblastoma activity of cisplatin with or without the addition of STS at 0 or 6 h after cisplatin in six neuroblastoma cell lines, both in standard cell culture conditions (20% O(2)) and in physiologic hypoxia (2% O(2)). Drug cytotoxicity was measured using the DIMSCAN fluorescence/digital imaging microscopy assay. In vivo studies of cisplatin combined with STS used a human neuroblastoma subcutaneous xenograft model (SMS-SAN) in athymic nu/nu mice. A significant protection against cisplatin cytotoxicity was seen when the neuroblastoma cells were exposed to cisplatin directly combined with STS. However, when cisplatin was given first and STS exposure occurred 6 h later, no effect on cisplatin cytotoxicity was observed. In a subcutaneous neuroblastoma xenograft model in nu/nu mice, mice receiving cisplatin alone or cisplatin + STS at 6 h had significantly better progression-free survival rates (P < 0.03) compared with controls or mice treated with cisplatin + STS concurrently. There was no statistically significant difference in outcomes between mice treated with cisplatin alone and the group treated with cisplatin followed by STS 6 h later (P = 0.9). These preclinical data suggest that the use of STS 6 h after cisplatin for otoprotection is unlikely to compromise the antineuroblastoma activity of cisplatin.

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.

Anti-neuroblastoma effect of ch14.18 antibody produced in CHO cells is mediated by NK-cells in mice

Successful treatment of stage 4 neuroblastoma remains a major challenge in pediatric oncology. In order to improve the outcome, passive immunotherapy using human–mouse chimeric monoclonal anti-disialoganglioside GD2 antibody ch14.18 has been evaluated in early phase clinical trials with promising results in progressing stage 4 neuroblastoma patients. In preparation of European phase III clinical trial (HR-NBL-1/ESIOP), the cell line used for production of ch14.18 was changed. Specifically, the plasmid encoding for ch14.18 antibody was recloned into CHO cells. Here, we report the in vitro and in vivo anti-neuroblastoma activity of antibody ch14.18 produced in CHO cells (ch14.18/CHO) compared to that of ch14.18 manufactured from SP2/0 (ch14.18/SP2/0) and NS0 cells (ch14.18/NS0). First, we demonstrate identical binding of ch14.18/CHO to the nominal antigen disialoganglioside GD2 in vitro compared to ch14.18/SP2/0 and ch14.18/NS0. Binding was GD2-specific, since all precursor- and metabolite-gangliosides of GD2 tested were not recognized by ch14.18/CHO. Second, the functional properties of ch14.18/CHO were determined in complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) reactions against GD2 positive neuroectodermal tumor cell lines in vitro. There was no difference in CDC mediated specific tumor cell lysis among the three different ch14.18 antibody preparations. Interestingly, ch14.18/CHO showed superior ADCC activity at low antibody concentrations. Third, the efficacy of ch14.18/CHO was evaluated in the NXS2 neuroblastoma model in vivo. Importantly, the ch14.18/CHO preparation was effective in suppression of experimental liver metastasis in this model. In vivo depletion of NK-cells completely abrogated this effect, suggesting that the mechanism involved in the ch14.18/CHO induced anti-neuroblastoma effect is mediated by NK-dependent ADCC.

Camptothecin analogs (irinotecan or topotecan) plus high-dose cyclophosphamide as preparative regimens for antibody-based immunotherapy in resistant neuroblastoma.

We used high-dose cyclophosphamide plus topotecan/vincristine (CTV) or irinotecan (C/I) in patients with resistant neuroblastoma. The aim was to use a regimen with little risk to major organs to (a) achieve or consolidate remission in heavily treated patients and to (b) induce an immunological state conducive to passive immunotherapy with the murine 3F8 antibody. CTV and C/I included cyclophosphamide 140 mg/kg ( approximately 4200 mg/m(2)). With CTV, topotecan 2 mg/m(2) was infused i.v. (30 min) on days 1-4 (total, 8 mg/m(2)), and vincristine 0.067 mg/kg was injected on day 1. With C/I, irinotecan, 50 mg/m(2) was infused i.v. (1 h) on days 1-5 (total, 250 mg/m(2)). Mesna and granulocyte colony-stimulating factor were used. Twenty-nine patients received 38 courses of CTV, and 26 patients received 38 courses of C/I. All patients had previously received topotecan, a hemopoietic stem-cell transplant, and/or high-dose cyclophosphamide. CTV and C/I caused myelosuppression of comparably prolonged duration as follows: absolute neutrophil counts <500/ micro l lasted 5-12 days in patients who had not previously received transplant and 7-21 days in patients who were post-transplant. Other significant toxicities included typhlitis (two CTV-treated patients, one C/I-treated patient) and hemorrhagic cystitis (one C/I-treated patient). Major responses were seen in 4 (15%) of 26 CTV and 4 (17%) of 24 C/I-treated patients with assessable disease. Bone marrow disease resolved in 5 (28%) of 18 CTV-treated patients and in 4 (27%) of 15 C/I-treated patients. 3F8 after CTV or C/I was not blocked by neutralizing antibodies, consistent with the desired immunosuppressive effect of high-dose cyclophosphamide. CTV and C/I require transfusional and antibiotic support but otherwise entail tolerable morbidity. They have modest antineuroblastoma activity in heavily treated patients and are good preparative regimens for passive immunotherapy with monoclonal antibodies.

Buthionine sulphoximine alone and in combination with melphalan (L-PAM) is highly cytotoxic for human neuroblastoma cell lines.

Buthionine sulphoximine (BSO) selectively inhibits glutathione (GSH) synthesis and may enhance the antineuroblastoma activity of melphalan (L-PAM). We determined the cytotoxicity of BSO (dose range 0-1000 microM) alone and in combination with L-PAM (dose range 0-0 microM) in a panel of 18 human neuroblastoma cell lines. BSO alone was highly cytotoxic with 16/18 neuroblastoma cell lines having IC90 values (range 2.1- > 1000 microM) below the clinically achievable steady-state plasma level of 500 microM BSO. Maximal cell killing correlated with GSH levels decreased to less than 10% baseline, and was partially reversed by the addition of exogenous anti-oxidants (GSH, vitamin E and ascorbate). Fluorocytometric analysis of DNA fragments by the Tunnel method detected 92% of a BSO sensitive cell line in apoptosis after a 48 h exposure to 500 microM BSO. The combination of L-PAM and BSO synergistically enhanced the cell killing of L-PAM alone by > 1-3 logs (combination index < 1). We conclude that BSO has significant single-agent cytotoxicity against neuroblastoma and enhances cell killing when combined with L-PAM.

Mechanisms of in vivo anti-neuroblastoma activity of human natural IgM.

Normal human sera of healthy adults contain natural IgM antibodies which are cytotoxic for human neuroblastoma cells. In this study, we evaluated the anti-neuroblastoma activity of these natural IgM antibodies in nude rats bearing solid human neuroblastoma tumours. A single intravenous (i.v.) injection of purified cytotoxic IgM led to uptake of IgM in the tumours with massive perivascular complement activation and accumulation of neutrophil granulocytes after 24 h. Five consecutive i.v. injections of purified cytotoxic IgM into neuroblastoma-bearing animals resulted in complete growth arrest of even large established solid tumours which lasted for several weeks after discontinuation of the injections, whereas tumours of control animals continued to grow exponentially during the observation period. These studies suggest that natural anti-neuroblastoma IgM may have a potential as a novel therapeutic modality in the treatment of human neuroblastoma.

Effect of different exposures to desferrioxamine on neuroblastoma cell lines.

Desferrioxamine (DFO) has shown anti-proliferative and cytotoxic effects on several tumor cells. DFO is used at present in the treatment of neuroblastoma in combination with chemotherapy (D-CECaT regimen: cyclophosphamide, etoposide, carboplatin, and thiotepa). We compared the effect of continuous or intermittent exposures to DFO on 3H-thymidine uptake, viability, and cell cycle of human neuroblastoma (NB) cell lines. Our results show that continuous exposures to DFO cause dose- and time-dependent cytotoxicity of NB cells, while intermittent exposures result in significant NB cell toxicity only when using high DFO concentrations. By 3H-thymidine uptake, a significant inhibition of proliferation was observed only in continuous exposures. In addition, a consistent arrest in G1 phase was detected only in cultures treated continuously with high DFO concentrations. Our data indicate that 3H-thymidine uptake, viability, and cell cycle changes are proportional to the extent of exposure and concentration of DFO, suggesting that in vivo DFO continuous infusion may improve anti-neuroblastoma activity.