ganglioside--gd2 and Neuroblastoma

Neuroblastoma (NBL), the most frequent and lethal pediatric cancer of children in pre-school age, is considered enigmatic in view of its extreme heterogeneity, from spontaneous regression in the IV-S form to incurable disease in approx. 40% of cases (High Risk, HR-NBL). It has an embryonal origin and a very heterogeneous genomic landscape, hampering the success of targeted strategies. The glycosphingolipid GD2 was shown to be expressed on NBL cells and utilized as target for passive immunotherapy with anti-GD2 antibodies (GD2-IMT). An international protocol was established with GD2-IMT, which increases remission length and survival in HR-NBL. By reviewing the different biological and molecular aspects of NBL and GD2-IMT, this mini-review questions the present lack of association between GD2-IMT and the underlying molecular landscape. The alternative model of Micro-Foci inducing virus (MFV) is presented, since MFV infection can induce extensive genomic aberrations (100X NMYC DNA-amplification). Since this family of viruses uses molecules for cell penetration similar to GD2 (i.e., GM2), it is hypothesized that GD2 is the port-of-entry for MFV and that success of anti-GD2 therapies is also associated to inhibition of this clastogenic virus in HR-NBL.

Topics: Antineoplastic Agents, Immunological; Child, Preschool; Clinical Trials as Topic; Gangliosides; Genetic Heterogeneity; Humans; Immunization, Passive; Neuroblastoma

Neuroblastoma (NB) is a neuroectodermal embryonic cancer that originates from primordial neural crest cells, and amongst pediatric cancers with high mortality rates. NB is categorized into high-, intermediate-, and low-risk cases. A significant proportion of high-risk patients who achieve remission have a minimal residual disease (MRD) that causes relapse. Whilst there exists a myriad of advanced treatment options for NB, it is still characterized by a high relapse rate, resulting in a reduced chance of survival. Disialoganglioside (GD2) is a lipo-ganglioside containing a fatty acid derivative of sphingosine that is coupled to a monosaccharide and a sialic acid. Amongst pediatric solid tumors, NB tumor cells are known to express GD2; hence, it represents a unique antigen for subclinical NB MRD detection and analysis with implications in determining a response for treatment. This article discusses NB MRD expression and analytical assays for GD2 detection and quantification as well as computational approaches for GD2 characterization based on high-throughput image processing and genomic data analysis.

Topics: Animals; Antineoplastic Agents; Aptamers, Nucleotide; Gangliosides; Humans; Neoplasm Recurrence, Local; Neoplasm, Residual; Neuroblastoma

The focus of this review is the ganglio-series of glycosphingolipids found in neuroblastoma (NB) and the myriad of unanswered questions associated with their possible role(s) in this cancer. NB is one of the more common solid malignancies of children. Five-year survival for those diagnosed with low risk NB is 90-95%, while that for children with high-risk NB is around 40-50%. Much of the survival rate reflects age of diagnosis with children under a year having a much better prognosis than those over two. Identification of expression of GD2 on the surface of most NB cells led to studies of the effectiveness and subsequent approval of anti-GD2 antibodies as a treatment modality. Despite much success, a subset of patients, possibly those whose tumors fail to express concentrations of gangliosides such as GD1b and GT1b found in tumors from patients with a good prognosis, have tumors refractory to treatment. These observations support discussion of what is known about control of ganglioside synthesis, and their actual functions in NB, as well as their possible relationship to treatment response.

Topics: Child; Disease-Free Survival; Gangliosides; Humans; Neuroblastoma; Risk Factors; Survival Rate

Neuroblastome de haut risque - Place actuelle et perspectives de l’utilisation des anticorps monoclonaux anti-GD2.. Le neuroblatome de haut risque reste un défi thérapeutique de l’oncologie pédiatrique puisque qu’il touche de très jeunes patients (90 % ont moins de 5 ans), dont les chances de survie restent inférieures à 50 % malgré des traitements très lourds. Une immunothérapie par l’anticorps monoclonal anti-GD2 dinutuximab bêta (Qarziba

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents, Immunological; Brain Neoplasms; Child; Child, Preschool; Gangliosides; Humans; Infant; Medical Oncology; Neuroblastoma; Risk Factors

Neuroblastoma (NBL) is the most common extracranial solid tumor in pediatrics, yet overall survival is poor for high-risk cases. Immunotherapy regimens using a tumor-selective antidisialoganglioside (anti-GD2) monoclonal antibody (mAb) have been studied for several decades now, but have only recently been incorporated into standard of care treatment for patients with high-risk NBL with clear benefit. Here we review a brief history of anti-GD2-based immunotherapy, current areas of neuroblastoma research targeting GD2, and potential diagnostic and therapeutic uses targeting GD2.

Topics: Antibodies, Monoclonal; Gangliosides; Humans; Immunotherapy; Molecular Targeted Therapy; Neuroblastoma; Pathology, Molecular; Risk

Neuroblastoma (NB) is a common and deadly malignancy mostly observed in children. Evolution of therapeutic options for NB led to the addition of immunotherapeutic modalities to the previously recruited chemotherapeutic options. Molecular studies of the NB cells resulted in the discovery of many tumor-associated genes and antigens such as

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Child; Clinical Trials as Topic; Gangliosides; Humans; Immunotherapy; Molecular Targeted Therapy; N-Myc Proto-Oncogene Protein; Neuroblastoma; Treatment Outcome

Dinutuximab, an anti-GD2 antibody, specifically targets the high-expression of GD2 on neuroblastoma cells, and its incorporation into maintenance high-risk neuroblastoma therapy has increased event-free survival for this devastating disease. Efficacy of dinutuximab during other phases of therapy or in relapsed and refractory patients remains under investigation. Areas covered: This review looked at available publications (via PubMed search and online abstract catalogs of recent scientific meetings) on pre-clinical safety studies of dinutuximab as well as results and current impressions of pending trials including dinutuximab and other anti-GD2 antibodies. While dinutuximab has become the standard of care for maintenance therapy in many cooperative trials, long-term follow-ups as well as ongoing assessment about timing of antibody use and co-administration of other pharmacologic or immune-modulatory agents remains under study. Expert opinion: Results of these and ongoing trials demonstrate prolonged time to first relapse and potentially overall survival benefit when dinutuximab is used during maintenance therapy. The role cytokines administered in conjunction with dinutuximab remains unclear and may increase toxicity without additional benefit. Current also investigations demonstrate promising efficacy in relapsed and refractory neuroblastoma. Further study is warranted in order to appropriately incorporate dinutuximab into current treatment strategies.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Child; Cytokines; Disease-Free Survival; Gangliosides; Humans; Neuroblastoma; Risk

Neuroblastoma (NB) with various clinical presentation is a known childhood malignancy. Despite significant progress in treatment of NB afflicted patients, high risk disease is usually associated with poor outcome, resulting in long-term survival of less that 50%. Known as a disease most commonly originated form the nerve roots, the variants involved in NB imitation and progression remain to be elucidated. The outcome of low to intermediate risk disease is favorable whereas the high risk NB disease with dismal prognosis, positing the necessity of novel approaches for early detection and prognostication of advanced disease. Tailored immunotherapy approaches have shown significant improvement in high-risk NB patients. It has found a link between Gangliosides and progression of NB. The vast majority of neuroblastoma tumors express elevated levels of GD2, opening new insight into using anti-GD2 drugs as potential treatments for NBs. Implication of anti-GD2 monoclonal antibodies for treatment of high risk NBs triggers further investigation to unearth novel biomarkers as prognostic and response biomarker to guide additional multimodal tailored treatment approaches. A growing body of evidence supports the usefulness of miRNAs to evaluate high risk NBs response to anti-GD2 drugs and further prevent drug-related toxicities in refractory or recurrent NBs. miRNAs and circulating proteins in body fluids (plasma and serum) present as potential biomarkers in early detection of NBs. Here, we summarize various biomarkers involved in diagnosis, prognosis and response to treatment in patients with NB. We further attempted to overview prognostic biomarkers in response to treatment with anti-GD2 drugs.

Topics: Animals; Antineoplastic Agents; Biomarkers, Tumor; Brain Neoplasms; Gangliosides; Humans; Immunotherapy; MicroRNAs; Molecular Diagnostic Techniques; Neuroblastoma; Predictive Value of Tests; Treatment Outcome

Current therapeutic approaches for high-risk neuroblastoma (HR-NB) include high-dose chemotherapy, surgery and radiotherapy; interventions that are associated with long and short-term toxicities. Effective immunotherapy holds particular promise for improving survival and quality of life by reducing exposure to cytotoxic agents. GD2, a surface glycolipid is the most common target for immunotherapy. Areas covered: We review the status of anti-GD2 immunotherapies currently in clinical use for neuroblastomas and novel GD2-targeted strategies in preclinical development. Expert commentary: Anti-GD2 monoclonal antibodies are associated with improved survival in patients in their first remission and are increasingly being used for chemorefractory and relapsed neuroblastoma. As protein engineering technology has become more accessible, newer antibody constructs are being tested. GD2 is also being targeted by natural killer cells and T-cells. Active immunity can be elicited by anti-GD2 vaccines. The rational combination of currently available and soon-to-emerge immunotherapeutic approaches, and their integration into conventional multimodality therapies will require further investigation to optimize their use for HR-NB.

Topics: Animals; Antibodies, Monoclonal; Cancer Vaccines; Gangliosides; Humans; Immunotherapy; Molecular Targeted Therapy; Neoplasm Recurrence, Local; Neuroblastoma; Quality of Life; Survival Rate

Tumor-specific monoclonal antibodies (mAbs) have demonstrated efficacy in the clinic, becoming an important approach for cancer immunotherapy. Due to its limited expression on normal tissue, the GD2 disialogangloside expressed on neuroblastoma cells is an excellent candidate for mAb therapy. In 2015, dinutuximab (an anti-GD2 mAb) was approved by the US FDA and is currently used in a combination immunotherapeutic regimen for the treatment of children with high-risk neuroblastoma. Here, we review the extensive preclinical and clinical development of anti-GD2 mAbs and the different mechanisms by which they mediate tumor cell killing. In addition, we discuss different mAb-based strategies that capitalize on the targeting ability of anti-GD2 mAbs to potentially deliver, as monotherapy, or in combination with other treatments, improved antitumor efficacy.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Clinical Trials as Topic; Drug Evaluation, Preclinical; Drug Therapy, Combination; Gangliosides; Humans; Immunotherapy; Neuroblastoma; Treatment Outcome

The prognosis of high-risk neuroblastoma (NB) is still poor, in spite of aggressive multimodal treatment. Recently, adjuvant immunotherapy with anti-GD2 antibodies combined with IL-2 or GM-CSF has been shown to improve survival. Several other immunotherapy strategies proved efficacy in preclinical models of NB, including different types of vaccines, adoptive cell therapies and combined approaches. The remarkable differences in the immunobiology of syngeneic models and human NB may, at least in part, limit the translation of preclinical therapies to a clinical setting. Nonetheless, several preliminary evidences suggest that new antibodies, cancer vaccines and adoptive transfer of lymphocytes, genetically engineered to acquire NB specificity, may result in clinical benefit, and clinical studies are currently ongoing.

Topics: Animals; Antibodies, Monoclonal; Cancer Vaccines; Chemotherapy, Adjuvant; Combined Modality Therapy; Drug Evaluation, Preclinical; Gangliosides; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunotherapy; Interleukin-2; Neuroblastoma

United Therapeutics Corporation and the National Cancer Institute are developing dinutuximab (Unituxin™; ch14.18), a monoclonal antibody targeting GD2, for the treatment of neuroblastoma. GD2 is a glycolipid found on the surface of tumour cells, which is overexpressed in neuroblastoma. Dinutuximab, an IgG1 human/mouse chimeric switch variant of murine monoclonal antibody 14G2a, binds to GD2 and induces antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The US FDA has recently approved the use of dinutuximab combination therapy for the treatment of high-risk neuroblastoma in paediatric patients. The marketing authorization application for dinutuximab is under regulatory review in the EU, and phase I-III development is underway in several other countries. This article summarizes the milestones in the development of dinutuximab leading to this first approval for use (in combination with granulocyte macrophage colony-stimulating factor, interleukin-2 and 13-cis retinoic acid) in the treatment of paediatric patients with high-risk neuroblastoma who achieve at least partial response to prior first-line multiagent, multimodality therapy.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Combined Chemotherapy Protocols; Child; Drug Approval; Gangliosides; Humans; Neuroblastoma; United States; United States Food and Drug Administration

Neuroblastoma, the most common extracranial solid tumor in children, is derived from neural crest cells. Nearly half of patients present with metastatic disease and have a 5-year event-free survival of <50%. New approaches with targeted therapy may improve efficacy without increased toxicity. In this review we evaluate 3 promising targeted therapies: (i) (131)I-metaiodobenzylguanidine (MIBG), a radiopharmaceutical that is taken up by human norepinephrine transporter (hNET), which is expressed in 90% of neuroblastomas; (ii) immunotherapy with monoclonal antibodies targeting the GD2 ganglioside, which is expressed on 98% of neuroblastoma cells; and (iii) inhibitors of anaplastic lymphoma kinase (ALK), a tyrosine kinase that is mutated or amplified in ~10% of neuroblastomas and expressed on the surface of most neuroblastoma cells. Early-phase trials have confirmed the activity of (131)I-MIBG in relapsed neuroblastoma, with response rates of ~30%, but the technical aspects of administering large amounts of radioactivity in young children and limited access to this agent have hindered its incorporation into treatment of newly diagnosed patients. Anti-GD2 antibodies have also shown activity in relapsed disease, and a recent phase III randomized trial showed a significant improvement in event-free survival for patients receiving chimeric anti-GD2 (ch14.18) combined with cytokines and isotretinoin after myeloablative consolidation therapy. A recently approved small-molecule inhibitor of ALK has shown promising preclinical activity for neuroblastoma and is currently in phase I and II trials. This is the first agent directed to a specific mutation in neuroblastoma, and marks a new step toward personalized therapy for neuroblastoma. Further clinical development of targeted treatments offers new hope for children with neuroblastoma.

Topics: 3-Iodobenzylguanidine; Anaplastic Lymphoma Kinase; Antibodies, Monoclonal; Child; Child, Preschool; Combined Modality Therapy; Crizotinib; Gangliosides; Humans; Molecular Targeted Therapy; Neural Crest; Neuroblastoma; Norepinephrine Plasma Membrane Transport Proteins; Precision Medicine; Pyrazoles; Pyridines; Radiopharmaceuticals; Receptor Protein-Tyrosine Kinases

Cytokines released by cancer cells or by cells of the tumor microenvironment stimulate angiogenesis, act as autocrine or paracrine growth factors for malignant cells, promote tumor cell migration and metastasis or create an immunosuppressive microenvironment. These tumor-promoting effects of cytokines also apply to neuroblastoma (NB), a pediatric neuroectodermal malignancy with frequent metastatic presentation at diagnosis and poor prognosis. IL-6 and VEGF are the best characterized cytokines that stimulated tumor growth and metastasis, while others such as IFN-γ can exert anti-NB activity by inducing tumor cell apoptosis and inhibiting angiogenesis. On the other hand, cytokines are part of the anti-NB therapeutic armamentarium, as exemplified by IL-2 and granulocyte-macrophage colony stimulating factor that potentiate the activity of anti-NB antibodies. These recent results raise hope for more efficacious treatment of this ominous pediatric malignancy.

Topics: Angiogenesis Inhibitors; Animals; Antibodies, Monoclonal, Humanized; Apoptosis; Bevacizumab; Cytokines; Gangliosides; Humans; Immunotherapy; Interferon-gamma; Interleukin-6; Mice; Mice, SCID; Neuroblastoma; Recombinant Fusion Proteins; Tumor Cells, Cultured; Tumor Microenvironment; Vascular Endothelial Growth Factors

Anticancer monoclonal antibodies (mAbs) targeting specific antigens on the tumour surface are increasingly being applied in cancer treatment. Potential advantages include long half-life, low toxicity, high affinity and specificity. In order to develop novel immune therapies for high-risk cancers, finding tumour targets that are not widely shared by normal cells is a goal. GD2-disialoganglioside is one of them. It is expressed on the surface of a variety of tumours with no curative therapies for patients with advanced disease. In childhood, neuroblastoma is the most common GD2-expressing tumour. Because of this tumour-selective expression, it is an attractive target for tumour-specific therapies such as antibody therapy. Over the last two decades, several anti-GD2 antibodies have been developed. To reduce both toxicity and development of human anti-mouse antibodies (HAMA), research efforts have primarily focused on exploring anti-GD2 antibodies that substitute mouse components by human ones. This review will examine antibodies currently undergoing clinical testing as well as the most recent advances to improve antibody therapy for patients with high-risk neuroblastoma.

Topics: Animals; Antibodies, Monoclonal; Gangliosides; Humans; Immunotherapy; Molecular Targeted Therapy; Neuroblastoma; Risk

Liposome encapsulation of anticancer agents results in reduced side effects of the entrapped drug and improved therapeutic efficacy. The external surface of the lipidic envelope can be coupled with antibodies directed against tumor-associated antigens. The resulting immunoliposomes allow to increase the therapeutic index of cytotoxic drugs while minimizing their systemic toxicity. In this regard, the disialoganglioside GD2 is a very promising tumor-associated antigen since it is expressed at high intensity on human neuroblastoma cells, but is detected only in normal cerebellum and peripheral nerves. Immunoliposomes can be used as vectors to deliver antisense oligonucleotides to cancer cells with the aim to modulate oncogene expression. Furthermore, antisense oligonucleotides have attracted much interest because of their ability to stimulate immune responses. Here, we will describe a novel experimental therapeutic approach for neuroblastoma based on anti-GD2 liposomal c-myb-selective antisense oligonucleotides.

Topics: Animals; Disease Models, Animal; Gangliosides; Genes, myb; Humans; Liposomes; Mice; Neuroblastoma; Oligonucleotides, Antisense

The impact of monoclonal antibodies (mAbs) in the treatment of human tumors has greatly increased in recent years. mAb engineering has allowed reducing the immunogenicity of therapeutic antibodies as well as improving their biodistribution. Furthermore, engineered mAbs have been used to vehiculate toxins, drugs and other anti-neoplastic agents to the tumor site. In the case of neuroblastoma (NB), a pediatric malignancy originating from the neural crest, both murine and chimeric antibodies against the tumor associated antigen GD2 have been tested in clinical trials, either alone or in combination with cytokines. A novel promising approach to mAb engineering is the small immuno-protein (SIP) technique, whereby the variable regions of heavy and light chains of a mAb with a given specificity are connected to the dimerizing CH(3) domain of an immunoglobulin molecule. The current status of mAb therapy for NB is discussed together with our preliminary results on the generation of novel anti-GD2 molecules using the SIP technique.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Gangliosides; Humans; Immunotherapy; Mice; Neuroblastoma

Neuroblastoma (NB) is the most common extra-cranial solid tumor in children. Since intensive therapeutic intervention does not prolong the overall disease-free survival rate for this tumor, novel therapeutic strategies are required. NB tumor, but not normal tissues, over-express the disialoganglioside (GD(2)) at the cell surface. In this study we developed a novel immunoliposomal formulation by covalently coupled Fab' fragments of the monoclonal antibody anti-GD(2) to Stealth liposomes (Fab'-SIL). In vitro experiments showed specific, competitive binding to, and uptake by various NB cell lines. Moreover, doxorubicin-loaded immunoliposomes (Fab'-SIL[DXR]) presented increased selectivity and efficacy in inhibiting NB cell proliferation compared to free drug and non-targeted liposomes (SL[DXR]). The in vivo cytotoxic effectiveness of different liposomal formulations encapsulating DXR was tested against an experimental metastatic model of human NB in nude mice. Long term survivors were obtained in mice treated with Fab'-SIL[DXR], but not in untreated animals or those treated with free anti-GD(2) Fab' fragments, Fab'-SIL (no drug), free-DXR or SL[DXR] (P<0.0001). Fab'-SIL[DXR] prevented the establishment and the metastatic growth of the tumor cells in all organs examined. In conclusion, Fab'-SIL[DXR] formulations should receive clinical evaluation as adjuvant therapy of neuroblastoma.

Topics: Animals; Antibiotics, Antineoplastic; Cell Division; Cell Survival; Doxorubicin; Gangliosides; Humans; Immunoglobulin Fab Fragments; Liposomes; Mice; Mice, Nude; Neuroblastoma; Survival Rate; Tumor Cells, Cultured

In spite of the satisfactory frequency of clinical response to first-line therapy in neuroblastoma (NB), complete eradication of NB cells is rarely achieved. As a consequence, the majority of patients with advanced stage NB undergo relapse, which is often resistant to conventional treatment and rapidly overwhelming. Thus, after induction of the apparent remission, new therapeutic strategies are needed to completely eradicate the small number of surviving NB cells and to prevent relapse. We explored the potential of different doses of the anti-GD2 monoclonal antibody (mAb) 14G2a in an experimental metastatic model where a limited number of HTLA-230 human NB cells are injected i.v. into nude mice, leading to extensive metastases and death of animals within 7-8 weeks. Treatment with 14G2a mAb (1-4 mg/kg cumulative dose given as five i.v. daily administrations) dramatically reduced the metastatic spread of NB cells and prolonged the long-term survival of treated mice in a dose-dependent manner. Neither macrophages nor NK cells appeared to contribute to the protective effect of antibody treatment in vivo, suggesting either an involvement of granulocytes or a complement-mediated cytotoxicity towards NB cells. Whatever the effecting mechanism(s) involved, these results strongly support the clinical use of anti-GD2 mAbs after first-line induction regimens.

Topics: Animals; Antibodies, Monoclonal; Gangliosides; Humans; Immunotherapy; Mice; Neoplasm Recurrence, Local; Neuroblastoma; Survival Rate; Tumor Cells, Cultured

Neuroblastoma (NB) is the most common neuroectoderma derived solid tumour of paediatric age. Since conventional treatments are often inefficient, novel therapeutic interventions are required. Among these, the use of antisense oligonucleotides (asODNs) as therapeutic antineoplastic agents has been recently investigated. Oligonucleotide in vivo applicability is impaired from their high sensitivity to cellular nuclease degradation. Encapsulating them within liposomes could nevertheless increase their stability. C-myb gene expression has been reported in several solid tumours of different embryonic origin, including NB, where it is linked to cell proliferation and/or differentiation. We performed a new technique to encapsulate c-myb antisense oligonucleotides within lipid particles. Liposomes resulting from this technique were called coated cationic liposomes (CCLs), since they were made up of a central core of a cationic phospholipid bound to myb-asODNs, and an outer shell of neutral lipids. A monoclonal antibody (mAb) specific for the neuroectoderma antigen disialoganglioside GD(2), has been covalently coupled to their external surface. The resulting anti-GD(2)-targeted CCLs showed high loading efficiency for the asODNs, small particle size and good stability. In vitro, they were able to deliver myb-asODNs selectively to GD(2)-positive NB cell lines more efficiently than non-targeted liposomes or free asODNs. Consequently, targeted formulations showed greater inhibition of cell proliferation than non-targeted formulations or free asODNs. Furthermore, we demonstrated that the inhibition of cell proliferation was dependent on the down-modulation of c-myb protein expression. Pharmacokinetic studies showed that these targeted liposomal formulations were long circulating in blood. Biodistribution studies presented differences between the free and the encapsulated myb-as ODN profiles, as well. While free myb-as ODNs are widely distributed (mainly liver, kidney and spleen) even after 30 min post-injection, myb-as ODN entrapped into CCL or anti-GD(2)-CCL presents only an accumulation in the spleen after 24 h. Future studies will be performed to evaluate the antitumour efficacy of the above formulations in animal models.

Topics: Antibodies, Monoclonal; Drug Delivery Systems; Gangliosides; Gene Expression Regulation, Neoplastic; Humans; Liposomes; Neuroblastoma; Oligodeoxyribonucleotides, Antisense; Proto-Oncogene Proteins c-myb; Tumor Cells, Cultured

Despite the use of aggressive chemotherapy, stage 4 high risk neuroblastoma still has a very poor prognosis, which is estimated at 25%. Therefore, novel treatment approaches are needed. Increasing number of reports has been concerned with the use of novel treatment modalities. Literature regarding intensive induction, local therapy, myeloablative therapy and immunotherapy and biotherapy was reviewed in order to draw conclusions and recommendations for the management of children with high risk neuroblastic tumors.

Topics: Antibodies, Monoclonal; Bone Marrow Purging; Bone Marrow Transplantation; Child; Gangliosides; Humans; Isotretinoin; Neuroblastoma

Topics: Animals; Antibodies, Monoclonal; Cytotoxicity, Immunologic; Gangliosides; Humans; Immunotherapy; Melanoma; Melanoma, Experimental; Mice; Mice, Nude; Neoplasm Transplantation; Neuroblastoma; Protein Engineering; Species Specificity; Tissue Distribution; Transplantation, Heterologous

Topics: Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Gangliosides; Humans; Immunotherapy; Neuroblastoma

Neuroblastomas from children presenting with tumors at various ages and different primary sites (abdominal, adrenals, pelvic, and thoracic) were studied. Analysis of the ganglioside patterns of 53 tumors indicated that patients who were either disease positive 2 yr following surgery or dead of disease, had significantly (p less than 0.005) less GT1b plus GD1b than tumors from patients that were disease free 2 yr post surgery. The presence of GD2 in 45 of the tumors correlates well with the suggestion that it can be used as a marker in neuroblastoma diagnosis. Children with thoracic neuroblastomas have a significantly better prognosis than children with tumors in other anatomic sites. Analysis of the ganglioside composition of these tumors only, indicated that they had a significantly higher (p less than 0.005) concentration of GT1b and GD1b and a significantly lower concentration (p less than 0.025) of monosialogangliosides than those patients who were dead of disease or had persistent disease. These results suggest that low levels of GT1b and GD1b correlate with a poor prognosis. The thoracic neuroblastomas may be comprised of more "differentiated" neuroblastoma cells (ganglioside patterns more similar to the CNS), and this may contribute to the fact that about 85% of children with thoracic neuroblastoma recover. To understand why the ganglioside pattern may serve as a prognostic indicator for neuroblastoma, it is necessary to know whether gangliosides have specific roles in neuronal differentiation. Our approach to this question is to compare the effect(s) of added ganglioside or the corresponding oligosaccharide on neuroblastoma cells. Results obtained suggest that the oligosaccharide from GM1 is able to enhance neuritogenesis by S20Y murine neuroblastoma cells to the same extent that GM1 does.

Topics: Cell Division; Cells, Cultured; Child, Preschool; Gangliosides; Humans; Infant; Microscopy, Electron, Scanning; Neuroblastoma; Oligosaccharides; Prognosis; Thoracic Neoplasms

Among patients with high-risk relapsed metastatic neuroblastoma, oral β-glucan adjuvant during GD2/GD3 ganglioside vaccine boost has stimulated IgG antibody response, which was associated with improved survival; however, the effectiveness of oral β-glucan during the vaccine priming phase remains unproven.. To isolate the adjuvant effect of oral β-glucan on antibody response to GD2/GD3 ganglioside vaccine in patients with high-risk neuroblastoma.. In this phase 2 randomized clinical trial, enrolled patients with high-risk neuroblastoma were randomized to 2 groups to receive the GD2/GD3 vaccine at a large cancer center in a major metropolitan area from October 2018 to September 2020. Data were analyzed from October 7, 2021, to February 28, 2022.. Eligible patients receiving GD2/GD3 vaccine were randomly assigned to group 1 (n = 54) to receive no β-glucan or group 2 (n = 53) to receive an oral β-glucan regimen during the first 5 weeks of vaccine priming. From week 6 onwards, all 107 patients received oral β-glucan during vaccine boost for 1 year or until disease progression.. Primary end point was comparison of anti-GD2 IgG1 response before vaccine injection 6 (week 32) in group 1 vs group 2. Seroconversion rate and the association of antibody titer with β-glucan receptor dectin-1 single nucleotide polymorphism (SNP) rs3901533 were also assessed.. In all, 107 patients with high-risk neuroblastoma were randomized to the 2 groups: 54 patients (median [range] age, 5.2 [1.0-17.3] years; 28 [52%] male and 26 [48%] female) in group 1; and 53 patients (median [range] age, 6.2 [1.9-18.4] years; 25 [47%] male and 28 [53%] female) in group 2; both groups were also comparable in their first remission status at study entry (70% vs 70%). Adding oral β-glucan during the first 5 weeks of vaccine priming elicited a higher anti-GD2 IgG1 antibody response in group 2 (1.80; 90% CI, 0.12-3.39; P = .08; planned type I error, 0.10). Anti-GD2 IgG1 titer of 230 ng/mL or greater by week 8 was associated with statistically favorable PFS. Antibody titer correlated significantly with dectin-1 SNP. The genotype frequency, seroconversion rates, and vaccine-related toxic effects were similar in the 2 groups.. This phase 2 randomized clinical trial found that adding oral β-glucan during vaccine priming increased anti-GD2 IgG1 titer among genetic responders without added toxic effects. Because responder dectin-1 SNP was identical in the 2 randomized groups, no difference was detected in seroconversion rates. Alternative or additional adjuvants may be needed to enhance seroconversion.. ClinicalTrials.gov Identifier: NCT00911560.

Topics: Antibody Formation; beta-Glucans; Cancer Vaccines; Child; Child, Preschool; Female; Gangliosides; Humans; Immunoglobulin G; Male; Neuroblastoma

Haploidentical stem cell transplantation (haplo SCT) in Stage IV neuroblastoma relapsed patients has been proven efficacious, while immunotherapy utilizing the anti-GD2 antibody dinutuximab beta has become a standard treatment for neuroblastoma. The combinatorial therapy of haplo SCT and dinutuximab may potentiate the efficacy of the immunotherapy. To gain further understanding of the synergistic effects, functional immunomonitoring was assessed during the clinical trial CH14.18 1021 Antibody and IL2 After haplo SCT in Children with Relapsed Neuroblastoma (NCT02258815). Rapid immune reconstitution of the lymphoid compartment was confirmed, with clinically relevant dinutuximab serum levels found in all patients over the course of treatment. Only one patient developed human anti-chimeric antibodies (HACAs). In-patient monitoring revealed highly functional NK cell posttransplant capable of antibody-dependent cellular cytotoxicity (ADCC). Degranulation of NK cell subsets revealed a significant response increased by dinutuximab. This was irrespective of the KIR receptor-ligand constellation within the NK subsets, defined by the major KIR receptors CD158a, CD158b, and CD158e. Moreover, complement-dependent cytotoxicity (CDC) was shown to be an extremely potent effector-cell independent mechanism of tumor cell lysis, with a clear positive correlation to GD2 expression on the cancer cells as well as to the dinutuximab concentrations. The

Topics: Antibodies, Monoclonal; Antineoplastic Agents, Immunological; Cytokines; Feasibility Studies; Gangliosides; Hematopoietic Stem Cell Transplantation; Humans; Inflammation Mediators; Monitoring, Immunologic; Neoplasm Recurrence, Local; Neoplasm Staging; Neuroblastoma; Predictive Value of Tests; Prospective Studies; Time Factors; Transplantation, Haploidentical; Treatment Outcome

To determine the incidence of and factors associated with the development of mydriasis and impaired accommodation in patients with refractory or recurrent neuroblastoma receiving the anti-GD2 antibody hu14.18K322A.. The medical records of eligible patients with refractory or recurrent neuroblastoma who received escalating doses of hu14.18K322A, ranging from 2 to 70 mg/m(2)/dose for 4 consecutive days every 28 days, were retrospectively reviewed to identify ocular abnormalities arising during the treatment period.. A total of 38 patients (median age, 7 years; 23 males) were included. All patients underwent comprehensive eye examinations prior to each course of therapy. Mydriasis was seen in 13 patients (34%), and impaired accommodation was seen in 9 (24%), indicating a dose-related effect between hu14.18K322A and both mydriasis (P = 0.021) and impaired accommodation (P = 0.029). Age and sex were not associated with ocular abnormalities. Ocular symptoms resolved in the majority of patients after the drug was discontinued.. Side effects of mydriasis and impaired accommodation have a dose-dependent relationship with hu14.18K322A. These side effects do not warrant discontinuation of treatment, as they usually resolve after completion of therapy. Management of ocular side effects should focus on treating symptoms with manifest refraction, bifocals, or tinted spectacles.

Topics: Accommodation, Ocular; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Brain Neoplasms; Child; Dose-Response Relationship, Drug; Female; Gangliosides; Humans; Incidence; Infusions, Intravenous; Male; Mydriasis; Neoplasm Recurrence, Local; Neuroblastoma; Retrospective Studies; Risk Factors

Anti-G(D2) murine antibody 3F8 plus subcutaneously (sc) administered granulocyte-macrophage colony-stimulating factor (GM-CSF) was used against primary refractory neuroblastoma in metastatic osteomedullary sites. Large study size and long follow-up allowed assessment of prognostic factors in a multivariate analysis not reported with other anti-G(D2) antibodies. In a phase II trial, 79 patients without prior progressive disease were treated for persistent osteomedullary neuroblastoma documented by histology and/or metaiodobenzyl-guanidine (MIBG) scan. In the absence of human antimouse antibody, 3F8 + scGM-CSF cycles were repeated up to 24 months. Minimal residual disease (MRD) in bone marrow was measured by quantitative reverse transcription-polymerase chain reaction pre-enrollment and post-cycle #2, before initiation of 13-cis-retinoic acid. Study endpoints were: (i) progression-free survival (PFS) compared with the predecessor trial of 3F8 plus intravenously administered (iv) GM-CSF (26 patients) and (ii) impact of MRD on PFS. Using all 105 patients from the two consecutive 3F8 + GM-CSF trials, prognostic factors were analyzed by multivariate Cox regression model. Complete response rates to 3F8 + scGM-CSF were 87% by histology and 38% by MIBG. Five-year PFS was 24 ± 6%, which was significantly superior to 11 ± 7% with 3F8 + ivGM-CSF (p = 0.002). In the multivariate analysis, significantly better PFS was associated with R/R or H/R FCGR2A polymorphism, sc route of GM-CSF and early MRD response. MYCN amplification was not prognostic. Complement consumption was similar with either route of GM-CSF. Toxicities were manageable, allowing outpatient treatment. 3F8 + scGM-CSF is highly active against chemoresistant osteomedullary neuroblastoma. MRD response may be an indicator of tumor sensitivity to anti-G(D2) immunotherapy. Correlative studies highlight the antineoplastic potency of myeloid effectors.

Topics: Adolescent; Adult; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antineoplastic Combined Chemotherapy Protocols; Bone Neoplasms; Child; Child, Preschool; Drug Resistance, Neoplasm; Female; Follow-Up Studies; Gangliosides; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunoglobulin G; Infant; Isotretinoin; Male; Myeloid Cells; Neoplasm Staging; Neoplasm, Residual; Neuroblastoma; Prognosis; Survival Rate; Young Adult

Ch14.18 improves survival in children with high-risk neuroblastoma but is associated with substantial toxicity. Ch14.18 pharmacokinetics were previously reported to be highly variable and characterized by a higher clearance in children than in adults, and a large volume of distribution. Identifying factors responsible for its variability could lead to alternative dosing strategies that reduce toxicity.. Plasma sampling was performed prior to, during, and for 25 days after four daily 10-h infusions of 25 mg/m(2) of ch14.18 administered with sargramostim. Ch14.18 concentrations were quantified with an electrochemiluminescence immunoassay, and pharmacokinetic parameters were derived using non-compartmental methods and from fitting a two-compartment model. Human anti-chimeric antibody (HACA) was measured before each course.. Fourteen subjects (median age, 4.3 years) were enrolled; seven had sampling on two courses to assess intra-subject variability. Mean peak ch14.18 plasma concentration was 11 µg/mL, and disappearance was biexponential with half-life of 7 days. Mean trough (day 28) concentration was 0.2 µg/mL. Mean AUC0-∞ was 1,380 µg h/mL and was less variable than previously reported (CV 29 %). Intra-patient variability was also minimal, but one subject who developed HACA had a 41 % decrease in AUC 0-tlast from courses 1 to 3. Clearance (2 L/day m(2)) was fourfold higher in children than in adults and appeared to be age dependent. Steady state volume of distribution was 0.4 L/kg. Two-compartment model parameters were used to simulate alternative dosing schedules.. Ch14.18 disposition in children is less variable than previously reported. Clearance is age dependent and more rapid in younger children.

Topics: Algorithms; Antibodies, Monoclonal; Area Under Curve; Child; Child, Preschool; Female; Gangliosides; Humans; Immunoassay; Infant; Male; Metabolic Clearance Rate; Models, Biological; Neuroblastoma; Recombinant Fusion Proteins; Risk Factors; Treatment Outcome

Pain can hinder immunotherapy with anti-G(D2) monoclonal antibodies (MoAbs) like 3F8. Heat-modified 3F8 (HM3F8) lacks effector functions and could mask G(D2) or cross-reactive epitopes on nerves, thereby preventing a subsequent dose of unmodified 3F8 from activating pain fibers. We hypothesized that 3F8 dose escalation is possible without increased analgesic requirements in patients pretreated with HM3F8.. Thirty patients with resistant neuroblastoma (NB) received one to two cycles of 3F8 plus granulocyte-macrophage colony-stimulating factor. 3F8 dosing began at 20 mg/m(2)/d and increased by 20 mg/m(2)/d in the absence of dose-limiting toxicity (DLT). Premedication included analgesics, antihistamines, and 5-minute infusions of HM3F8. On the basis of experience with 3F8 10 mg/m(2)/d in prior protocols, the DLT of pain was defined as more than seven doses of opioids administered within 2 hours. Opioid use was compared with a contemporary control group treated with 3F8 20 mg/m(2)/d but no HM3F8. Disease response was assessed.. Treatment was administered in the outpatient setting. Dose escalation stopped at 160 mg/m(2)/d because of drug supply limitations; even through this dosage level, analgesic requirements were similar to historical controls, and there were no DLTs. Analgesic requirements at 3F8 dosage levels through 80 mg/m(2)/d were significantly less compared with controls. Anti-NB activity occurred at all dosages.. Multifold dose escalation of 3F8 is feasible. The findings can be interpreted as compatible with the possibility that HM3F8 can modify toxicity without blunting anti-NB activity. This pain control strategy may help achieve dose escalation with other anti-G(D2) MoAbs.

Topics: Adolescent; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Bone Marrow Neoplasms; Bone Neoplasms; Child; Child, Preschool; Dose-Response Relationship, Immunologic; Female; Gangliosides; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunotherapy; Infant; Male; Maximum Tolerated Dose; Neuroblastoma; Prospective Studies; Survival Rate; Treatment Outcome

We generated MHC-independent chimeric antigen receptors (CARs) directed to the GD2 antigen expressed by neuroblastoma tumor cells and treated patients with this disease. Two distinguishable forms of this CAR were expressed in EBV-specific cytotoxic T lymphocytes (EBV-CTLs) and activated T cells (ATCs). We have previously shown that EBV-CTLs expressing GD2-CARs (CAR-CTLs) circulated at higher levels than GD2-CAR ATCs (CAR-ATCs) early after infusion, but by 6 weeks, both subsets became low or undetectable. We now report the long-term clinical and immunologic consequences of infusions in 19 patients with high-risk neuroblastoma: 8 in remission at infusion and 11 with active disease. Three of 11 patients with active disease achieved complete remission, and persistence of either CAR-ATCs or CAR-CTLs beyond 6 weeks was associated with superior clinical outcome. We observed persistence for up to 192 weeks for CAR-ATCs and 96 weeks for CAR-CTLs, and duration of persistence was highly concordant with the percentage of CD4(+) cells and central memory cells (CD45RO(+)CD62L(+)) in the infused product. In conclusion, GD2-CAR T cells can induce complete tumor responses in patients with active neuroblastoma; these CAR T cells may have extended, low-level persistence in patients, and such persistence was associated with longer survival. This study is registered at www.clinialtrials.gov as #NCT00085930.

Topics: Adolescent; Adoptive Transfer; Antigens, Tumor-Associated, Carbohydrate; Bone Marrow Neoplasms; Bone Neoplasms; Cell Line, Tumor; Child; Child, Preschool; Female; Follow-Up Studies; Gangliosides; Genetic Therapy; Humans; Immunologic Memory; Male; Neoplasm, Residual; Neuroblastoma; Receptors, Antigen, T-Cell; Recombinant Fusion Proteins; T-Lymphocytes, Cytotoxic; Young Adult

Preclinical and preliminary clinical data indicate that ch14.18, a monoclonal antibody against the tumor-associated disialoganglioside GD2, has activity against neuroblastoma and that such activity is enhanced when ch14.18 is combined with granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-2. We conducted a study to determine whether adding ch14.18, GM-CSF, and interleukin-2 to standard isotretinoin therapy after intensive multimodal therapy would improve outcomes in high-risk neuroblastoma.. Patients with high-risk neuroblastoma who had a response to induction therapy and stem-cell transplantation were randomly assigned, in a 1:1 ratio, to receive standard therapy (six cycles of isotretinoin) or immunotherapy (six cycles of isotretinoin and five concomitant cycles of ch14.18 in combination with alternating GM-CSF and interleukin-2). Event-free survival and overall survival were compared between the immunotherapy group and the standard-therapy group, on an intention-to-treat basis.. A total of 226 eligible patients were randomly assigned to a treatment group. In the immunotherapy group, a total of 52% of patients had pain of grade 3, 4, or 5, and 23% and 25% of patients had capillary leak syndrome and hypersensitivity reactions, respectively. With 61% of the number of expected events observed, the study met the criteria for early stopping owing to efficacy. The median duration of follow-up was 2.1 years. Immunotherapy was superior to standard therapy with regard to rates of event-free survival (66±5% vs. 46±5% at 2 years, P=0.01) and overall survival (86±4% vs. 75±5% at 2 years, P=0.02 without adjustment for interim analyses).. Immunotherapy with ch14.18, GM-CSF, and interleukin-2 was associated with a significantly improved outcome as compared with standard therapy in patients with high-risk neuroblastoma. (Funded by the National Institutes of Health and the Food and Drug Administration; ClinicalTrials.gov number, NCT00026312.)

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Child; Child, Preschool; Combined Modality Therapy; Drug Therapy, Combination; Gangliosides; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunotherapy; Infant; Intention to Treat Analysis; Interleukin-2; Isotretinoin; Neuroblastoma; Stem Cell Transplantation; Survival Analysis

We previously reported a high response rate with a dose-intensive chemotherapy regimen in 24 children with high-risk neuroblastoma (NB). We now describe similar results with changes that reduce toxicity (fewer cycles, less vincristine, use of granulocyte colony-stimulating factor).. Eighty-seven consecutive newly diagnosed children with high-risk NB underwent induction that initially had seven cycles (57 patients) but was later limited to five (30 patients). Cycles 1, 2, 4, and 6 used cyclophosphamide (140 mg/kg)/doxorubicin (75 mg/m(2))/vincristine (0.15 mg/kg in the first 27 patients, 0.067 mg/kg subsequently). Cycles 3, 5, and 7 used cisplatin (200 mg/m(2))/etoposide (600 mg/m(2)). Tumor resection followed a minimum of three cycles. The induction was eventually modified to include anti-G(D2) immunotherapy after each of the last three cycles (38 patients).. Bone marrow disease resolved in 70 (91%) of 77 patients and was not detected pre- and postinduction in 10 patients. After cycle 3 or 4, 86% of primary tumors were more than 50% smaller. Postinduction metaiodobenzylguanidine scans showed normal radiotracer distribution in metastatic sites in 74 (87%) of 85 patients. Overall results were: 68 (79%) complete/very good partial responses (CR/VGPR); 14 (16%) partial responses (PR); three (3%) less than PR; one (1%) death from infection; and one patient not assessable for response. Five cycles yielded a CR/VGPR rate of 83%, compared with a 77% rate from seven cycles. Side effects were myelosuppression, mucositis, and hearing deficits; neurotoxicity was insignificant with the lower vincristine dosage. Four patients (each received seven cycles) developed myelodysplasia/leukemia.. Five cycles of this induction regimen, plus surgery, suffice to achieve CR/VGPR in approximately 80% of children with high-risk NB.

Topics: Antibodies; Antineoplastic Combined Chemotherapy Protocols; Child; Child, Preschool; Cisplatin; Cyclophosphamide; Dose-Response Relationship, Drug; Doxorubicin; Etoposide; Female; Gangliosides; Granulocyte Colony-Stimulating Factor; Humans; Immunotherapy; Infant; Male; Neuroblastoma; Risk Factors; Treatment Outcome; Vincristine

To describe oncolytic effects of treatment with anti-G(D2) monoclonal antibody 3F8 plus granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with neuroblastoma (NB).. Patients were eligible for 3F8/GM-CSF if intensive therapy had not eradicated potentially lethal NB. One cycle consisted of GM-CSF (subcutaneous bolus) on days 1 through 5, 11, and 12, and GM-CSF (2-hour intravenous [IV] infusion) followed after a 1-hour interval by 3F8 (1.5-hour IV infusion) on days 6 through 10 and 13 through 17. GM-CSF was dosed at 250 microg/m(2)/d on days 1 through 7 and at 500 microg/m(2)/d on days 8 through 17. 3F8 was dosed at 10 mg/m(2)/d (100 mg/m(2)/cycle). 3F8 was given with an opiate and an antihistamine. Patients without progressive disease (PD) or elevated human antimouse antibody titers could be treated again beginning 3 weeks after completion of a cycle.. Among 19 patients treated for NB resistant to induction therapy, 12 of 15 had complete remission (CR) of bone marrow (BM) disease, and three others who had less than partial responses achieved prolonged progression-free survival (one remains on study at 21+ months, two had PD at 12 and 17 months). Among patients treated for recurrent NB resistant to retrieval therapy, five of 10 had CR in BM. The 15 patients treated for PD fared poorly, although two had scintigraphic findings suggestive of a short-term response. Side effects were limited to readily manageable pain and, less commonly, rash of short duration; hence, patients were treated as outpatients.. 3F8/GM-CSF is well tolerated and shows promise for treatment of minimal residual NB in BM.

Topics: Adolescent; Adult; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antibody-Dependent Cell Cytotoxicity; Bone Marrow Neoplasms; Bone Neoplasms; Child; Child, Preschool; Disease Progression; Drug Therapy, Combination; Female; Gangliosides; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunoglobulin G; Infusions, Intravenous; Male; Neuroblastoma

To improve autologous leukapheresis strategies in high-risk neuroblastoma (NB) patients with extensive bone marrow involvement at diagnosis.. Anti-G(D2) immunocytochemistry (sensitivity, 1 in 10(5) to 10(6) leukocytes) was used to evaluate blood and bone marrow disease at diagnosis and during the recovery phase of the first six chemotherapy cycles in 57 patients with stage 4 NB and bone marrow disease at diagnosis. A total of 42 leukapheresis samples from the same patients were evaluated with immunocytology, and in 24 of these patients, an anti-G(D2) immunomagnetic enrichment step was used to enhance tumor-cell detection.. Tumor cytoreduction was much faster in blood compared with bone marrow (3.2 logs after the first cycle and 2.1 logs after the first two cycles, respectively). Bone marrow disease was often detectable throughout induction, with a trend to plateau after the fourth cycle. By direct anti-G(D2) immunocytology, a positive leukapheresis sample was obtained in 7% of patients after either the fifth or sixth cycle; when NB cell immunomagnetic enrichment was applied, 25% of patients had a positive leukapheresis sample (sensitivity, 1 in 10(7) to 10(8) leukocytes).. Standard chemotherapy seems to deliver most of its in vivo purging effect within the first four cycles. In patients with overt marrow disease at diagnosis, postponing hematopoietic stem-cell collection beyond this point may not be justified. Tumor-cell clearance in blood seems to be quite rapid, and earlier collections via peripheral-blood leukapheresis might be feasible. Immunomagnetically enhanced NB cell detection can be highly sensitive and can indicate whether ex vivo purging should be considered.

Topics: Adolescent; Antibodies, Monoclonal; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow Neoplasms; Bone Marrow Purging; Child; Child, Preschool; Gangliosides; Hematopoietic Stem Cell Transplantation; Humans; Immunomagnetic Separation; Infant; Leukapheresis; Neoplastic Cells, Circulating; Neuroblastoma

Ganglioside G(D2) is strongly expressed on the surface of human neuroblastoma cells. It has been shown that the chimeric human/murine anti-G(D2) monoclonal antibody (ch14.18) can induce lysis of neuroblastoma cells by antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. The purposes of the study were (1) to determine the maximum-tolerated dose (MTD) of ch14.18 in combination with standard dose granulocyte-macrophage colony-stimulating factor (GM-CSF) for patients with neuroblastoma who recently completed hematopoietic stem-cell transplantation (HSCT), and (2) to determine the toxicities of ch14.18 with GM-CSF in this setting.. Patients became eligible when the total absolute phagocyte count (APC) was greater than 1, 000/microL after HSCT. ch14.18 was infused intravenously over 5 hours daily for 4 consecutive days. Patients received GM-CSF 250 microg/m(2)/d starting at least 3 days before ch14.18 and continued for 3 days after the completion of ch14.18. The ch14.18 dose levels were 20, 30, 40, and 50 mg/m(2)/d. In the absence of progressive disease, patients were allowed to receive up to six 4-day courses of ch14.18 therapy with GM-CSF. Nineteen patients with neuroblastoma were treated.. A total of 79 courses were administered. No toxic deaths occurred. The main toxicities were severe neuropathic pain, fever, nausea/vomiting, urticaria, hypotension, mild to moderate capillary leak syndrome, and neurotoxicity. Three dose-limiting toxicities were observed among six patients at 50 mg/m(2)/d: intractable neuropathic pain, grade 3 recurrent urticaria, and grade 4 vomiting. Human antichimeric antibody developed in 28% of patients.. ch14.18 can be administered with GM-CSF after HSCT in patients with neuroblastoma with manageable toxicities. The MTD is 40 mg/m(2)/d for 4 days when given in this schedule with GM-CSF.

Topics: Adolescent; Animals; Antibodies, Monoclonal; Antibody Formation; Antibody-Dependent Cell Cytotoxicity; Child; Child, Preschool; Combined Modality Therapy; Disease Progression; Dose-Response Relationship, Immunologic; Drug Administration Schedule; Female; Follow-Up Studies; Gangliosides; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Stem Cell Transplantation; Humans; Male; Mice; Neuroblastoma; Recombinant Fusion Proteins

3F8 is an IgG3 murine monoclonal antibody directed against the ganglioside GD2. In a phase II study, 3F8 was administered i.v. to 16 patients (pts) who had stage 4 neuroblastoma. Response was seen in bony lesions (2 of 7 pts) and marrow (3 of 8 pts). Acute toxicities of pain, fever, urticaria, hypertension, hypotension and anaphylactoid reactions were self-limited and manageable. Three pts are long-term survivors between 79-130+ months after 3F8 treatment without additional systemic therapy and no delayed neurological complications. The potential benefits of 3F8 when added to chemoradio-therapy warrant further investigation.

Topics: Abdominal Pain; Adolescent; Antibodies, Monoclonal; Antibody Formation; Antineoplastic Agents; Child; Child, Preschool; Fever; Gangliosides; Humans; Hypertension; Hypotension; Immunotherapy; Infant; Infant, Newborn; Neuroblastoma; Respiration Disorders; Treatment Outcome; Urticaria

To evaluate the toxicity, immunogenicity, and pharmacokinetics of a human-mouse chimeric monoclonal antibody (mAb) ch 14.18 directed against disialoganglioside (GD2) and to obtain preliminary information on its clinical efficacy, we conducted a phase I trial in 10 patients with refractory neuroblastoma and one patient with osteosarcoma.. Eleven patients were entered onto this phase I trial. They received 20 courses of mAb ch 14.18 at dose levels of 10, 20, 50, 100, and 200 mg/m2. Dose escalation was performed in cohorts of three patients; intrapatient dose escalation was also permitted.. The most prevalent toxicities were pain, tachycardia, hypertension, fever, and urticaria. Most of these toxicities were dose-dependent and rarely noted at dosages of 20 mg/m2 and less. Although the maximum-tolerated dose was not reached in this study, clinical responses were observed. These included one partial (PR) and four mixed responses (MRs) and one stable disease (SD) among 10 assessable patients. Biologic activity of ch 14.18 in vivo was shown by binding of ch 14.18 to tumor cells and complement-dependent cytotoxicity of posttreatment sera against tumor target cells. An anti-ch 14.18 immune response was detectable in seven of 10 patients studied.. In summary, with the dose schedule used, ch 14.18 appears to be clinically safe and effective, and repeated mAb administration was not associated with increased toxicities. Further clinical trials of mAb ch 14.18 in patients with neuroblastoma are warranted.

Topics: Adult; Animals; Antibodies, Monoclonal; Child; Child, Preschool; Complement C3; Complement C4; Complement Hemolytic Activity Assay; Female; Fluorescent Antibody Technique, Indirect; Gangliosides; Humans; Infusions, Intravenous; Male; Mice; Neuroblastoma; Osteosarcoma; Pain; Recombinant Fusion Proteins; Treatment Outcome

To eradicate minimal residual disease with anti-G(D2) monoclonal antibody 3F8 in stage 4 neuroblastoma (NB) diagnosed at more than 1 year of age.. Thirty-four patients were treated with 3F8 at the end of chemotherapy. Most had either bone marrow (n=31) or distant bony metastases (n=29). Thirteen patients were treated at second or subsequent remission (group I) and 12 patients in this group had a history of progressive/persistent disease after bone marrow transplantation (BMT); 21 patients were treated in first remission following N6 chemotherapy (group II).. Before 3F8 treatment, 23 patients were in complete remission CR, eight in very good partial remission (VGPR), one in partial remission (PR), and two had microscopic foci in marrow. Twenty-five had evidence of NB by at least one measurement of occult/minimal tumor (iodine 131[(131)I]-3F8 imaging, marrow immunocytology, or marrow reverse-transcriptase polymerase chain reaction [RT-PCR]). Acute self-limited toxicities of 3F8 treatment were severe pain, fever, urticaria, and reversible decreases in blood counts and serum complement levels. There was evidence of response by immunocytology (six of nine), by GAGE RT-PCR (seven of 12), and by (131)I-3F8 scans (six of six). Fourteen patients are alive and 13 (age 1.8 to 7.4 years at diagnosis) are progression-free (40 to 130 months from the initiation of 3F8 treatment) without further systemic therapy, none with late neurologic complications. A transient anti-mouse response or the completion of four 3F8 cycles was associated with significantly better survival.. Despite high-risk nature of stage 4 NB, long-term remission without autologous (A)BMT can be achieved with 3F8 treatment. Its side effects were short-lived and manageable. The potential benefits of 3F8 in consolidating remission warrant further investigations.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Bone Marrow Neoplasms; Bone Neoplasms; Child; Child, Preschool; Combined Modality Therapy; Female; Gangliosides; Humans; Immunotherapy; Infant; Male; Neoplasm Staging; Neoplasm, Residual; Neuroblastoma; Treatment Outcome

Ganglioside GD2 is abundant on human neuroblastoma (NB). Monoclonal antibody 3F8 targeted to GD2 may have imaging and therapeutic potential. Antigen-negative clones can escape immune-mediated attack, leading to clinical resistance or recurrence. Among 95 evaluable patients treated i.v. with 3F8 (94 stage 4 and 1 stage 3), 66 received nonradiolabeled 3F8, 11 received 131I-labeled 3F8 (8-28 mCi/kg) with autologous bone marrow rescue, and 18 received both forms of treatment. Prior to treatment, 91 patients tested positive for GD2 reactivity by bone marrow immunofluorescence (n = 68), tumor immunohistochemistry (n = 20), or diagnostic radioimmunoscintigraphy only (n = 3). Of 62 patients who had refractory or recurrent NB following 3F8 treatment, 61 (98%) tested positive for GD2 reactivity by bone marrow immunofluorescence (n = 51) or tumor immunohistochemistry (n = 10). The sole tumor that lost GD2 expression underwent phenotypic transformation into a pheochromocytoma-like tumor. The persistence of GD2 expression in refractory or recurrent NB suggests that complete antigen loss is an uncommon event and cannot account for treatment failure.

Topics: Antibodies, Monoclonal; Child, Preschool; Female; Fluorescent Antibody Technique; Gangliosides; Humans; Immunotherapy; Male; Neuroblastoma

The murine monoclonal antibody (MoAb) 14.G2a recognizes GD2, a disialoganglioside expressed in tumors of neuroectodermal origin, and facilitates antibody dependent cellular cytotoxicity (ADCC) in vitro. When given in vivo, interleukin-2 (IL-2) can increase ADCC by enhancing the activity and number of circulating lymphocytes.. Thirty-three pediatric patients with GD2 positive malignancies, ranging in age from 2 to 17 years (median, 9.9 years), received IL-2 and 14.G2a in this Phase I/IB study of the Children's Cancer Group (CCG) and were monitored for toxicities and response to therapy. Seven of these patients also received granulocyte-macrophage-colony stimulating factor.. The maximum tolerated dose (MTD) of 14.G2a with IL-2 was 15 mg/m2/day. The most prevalent Grade 3-4 toxicities were generalized pain (n = 14 [42%]) and fever without documented infection (n = 17 [52%]). IL-2 was thought to be the causative agent in most cases of fever. Toxicities attributed to 14.G2a included pain, allergic or anaphylactic reactions, and rash. Human antimouse antibodies were demonstrated in 9 of 21 evaluated patients. One patient with neuroblastoma had a partial response, and one patient with osteosarcoma had a complete response. Immunocytology demonstrated that the number of neuroblastoma cells in bone marrow decreased in three patients.. The murine MoAb 14.G2a was well tolerated at the MTD and appeared to have some antitumor activity. Further development of this approach will involve additional engineered forms of the antibody as well as testing in the adjuvant and minimal residual disease setting.

Topics: Adjuvants, Immunologic; Adolescent; Animals; Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Bone Marrow; Child; Child, Preschool; Female; Gangliosides; Humans; Interleukin-2; Male; Mice; Neoplasm Proteins; Neuroblastoma; Recombinant Proteins; Remission Induction

A phase I trial of a murine anti-ganglioside (GD2) monoclonal antibody (mAb) 14G2a was conducted in 14 neuroblastoma patients and 1 osteosarcoma patient to assess its safety, toxicity and pharmacokinetics in pediatric patients. The pharmacokinetics of mAb 14G2a were biphasic with a t alpha 1/2 of 2.8 +/- 2.8 h and a t beta 1/2 of 18.3 +/- 11.8 h. In general, t beta 1/2 was dose-dependent with a level of significance of P = 0.036, and it reached a plateau at doses of 250 mg/m2 or more. Overall the peak serum levels were dose-dependent at P < 0.001. However, they demonstrated an abrupt increase between doses of 100 mg/m2 and 250 mg/m2. The latter two suggest a saturable mechanism for mAb elimination. In addition, peak serum concentrations were observed earlier at higher mAb doses, which indicates the achievement of a steady state. The t beta 1/2 of mAb 14G2a in children appears to be shorter than in adults. Furthermore, 2 patients demonstrated a considerable decrease in t beta 1/2 following retreatment with 14G2a. This was paralleled by high human anti-(mouse Ig) antibody levels. This study represents the first comprehensive analysis of murine mAb pharmacokinetics in children and will be useful in the future design of mAb therapy.

Topics: Adolescent; Age Factors; Antibodies, Monoclonal; Child; Dose-Response Relationship, Immunologic; Female; Gangliosides; Humans; Male; Metabolic Clearance Rate; Neuroblastoma; Osteosarcoma

9 patients with stage IV neuroblastoma were treated with 19 courses of human/mouse chimeric monoclonal antiganglioside GD2 antibody ch14.18 at dose levels of 30, 40 and 50 mg/m2/day for 5 days per course. The maximum tolerated dose (MTD) per injection was 50 mg/m2/day. 7 patients received more than one course of treatment, and none revealed any human anti-mouse antibody (HAMA) response. Clinical side-effects of patients treated with ch14.18 were abdominal and joint pains, pruritus and urticaria. One patient presented with a transient pupillatonia, while 2 others showed a unilateral atrophy of the optical nerve that was probably attributable to prior therapies. A complete remission was seen in 2 patients, partial remission in 2 patients, a minor response in 1 patient and stable disease in 1 patient. 3 patients showed tumour progression. Thus, our results indicate that treatment with chimeric MAb ch14.18 can elicit some complete and partial tumour responses in neuroblastoma patients.

Topics: 3-Iodobenzylguanidine; Animals; Antibodies, Monoclonal; Child; Child, Preschool; Female; Gangliosides; Hemolysis; Humans; Iodine Radioisotopes; Iodobenzenes; Male; Mice; Neuroblastoma; Radionuclide Imaging

Topics: Antibodies, Monoclonal; Antineoplastic Combined Chemotherapy Protocols; Child; Child, Preschool; Combined Modality Therapy; Female; Gangliosides; Humans; Immunoglobulin G; Infant; Infusions, Intravenous; Male; Neoplasm Staging; Neuroblastoma; Remission Induction; Retrospective Studies; Survival Analysis

Nine patients with neuroblastoma stage IV were treated with the murine monoclonal antibody 14.G2a, directed against disialoganglioside GD2. The antibody was injected daily for 5-10 days and the total applied dosage ranged between 100 mg/m2 and 400 mg/m2. The peak serum levels of mAb 14.G2a ranged from 28 micrograms/ml to 61 micrograms/ml. Pharmacokinetic data obtained in three patients indicated that the serum elimination of mAb 14.G2a fits a two-compartment model, with an alpha-half-time (t1/2 alpha) between 0.66 h and 1.98 h and a beta-half-time (t1/2 beta) between 30.13 h and 53.33 h. All patients presented with a human anti-(mouse IgG) antibody response either during or shortly after therapy. Eight patients showed a continuous decrease in complement component C4 during therapy, as well as an initial decrease in C3c and an initial increase in C3a, all suggesting an activation of the complement cascade. Side-effects consisted of allergic reactions like pruritus, exanthema, urticaria and of severe pain, predominantly located in the abdomen and lower extremities, which required the use of continuous intravenous morphine. Four patients additionally developed a transient hypertension and one patient experienced a transient nephrotic syndrome. Three patients were treated in an adjuvant setting and are not evaluable for tumor response. Of the remaining six patients, two had a complete remission, two showed a partial remission, and two patients did not respond to treatment.

Topics: Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Child; Child, Preschool; Complement Activation; Female; Gangliosides; Humans; Male; Neuroblastoma

Immunotherapy with chimeric antigen receptor (CAR)-expressing T cells that target the disialoganglioside GD2 expressed on tumor cells may be a therapeutic option for patients with high-risk neuroblastoma.. In an academic, phase 1-2 clinical trial, we enrolled patients (1 to 25 years of age) with relapsed or refractory, high-risk neuroblastoma in order to test autologous, third-generation GD2-CAR T cells expressing the inducible caspase 9 suicide gene (GD2-CART01).. A total of 27 children with heavily pretreated neuroblastoma (12 with refractory disease, 14 with relapsed disease, and 1 with a complete response at the end of first-line therapy) were enrolled and received GD2-CART01. No failure to generate GD2-CART01 was observed. Three dose levels were tested (3-, 6-, and 10×10. The use of GD2-CART01 was feasible and safe in treating high-risk neuroblastoma. Treatment-related toxic effects developed, and the activation of the suicide gene controlled side effects. GD2-CART01 may have a sustained antitumor effect. (Funded by the Italian Medicines Agency and others; ClinicalTrials.gov number, NCT03373097.).

Topics: Caspase 9; Child; Humans; Immunotherapy, Adoptive; Neoplasm Recurrence, Local; Neuroblastoma; Receptors, Chimeric Antigen

Topics: Age Factors; Antineoplastic Agents, Immunological; Commerce; Cost-Benefit Analysis; Diffusion of Innovation; Drug Approval; Drug Costs; Drug Development; Gangliosides; Humans; Immunotherapy; Neuroblastoma; Patient Advocacy; Rare Diseases; Therapies, Investigational; Treatment Outcome

The disialoganglioside G. Leftover plasma samples taken for routine clinical laboratory tests from children without cancer were collected and assayed for the 18-carbon fatty acid chain length lipoform of G. G. Age-dependent reference intervals were defined for circulating G

Topics: Age Factors; Biomarkers, Tumor; Child; Child, Preschool; Female; Gangliosides; Humans; Infant; Infant, Newborn; Male; Neuroblastoma; Reference Values

Neuroblastoma is the most common extracranial solid tumor of childhood and is associated with poor survival in high risk patients. Recently, dinutuximab (DNX) has emerged as an effective immunotherapy to treat patients with high risk neuroblastoma. DNX works through the induction of cell lysis via complement-dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC). However, one third of patients who undergo DNX treatment exhibit tumor relapse and the therapy is dose limited by side effects such as severe pain. To overcome delivery challenges of DNX, including large size and dose limiting side effects, we fabricated a delivery system capable of sustained local delivery of bioactive DNX utilizing silk fibroin. We evaluated the impact of silk properties (MW, crystallinity, and concentration) on release properties and confirmed the bioactivity of the release product. Additionally, we observed that the effectiveness of CDC induction by DNX could be correlated to the GD2 expression level of the target cells, with both the intravenous DNX formulation and the released DNX. Collectively, these data highlights a strategy to overcome delivery challenges and potentially improve therapeutic efficacy in cells expressing heterogenous levels of GD2.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents, Immunological; Bombyx; Cell Death; Cell Line, Tumor; Delayed-Action Preparations; Drug Delivery Systems; Fibroins; Gangliosides; Neuroblastoma

The ability to utilize preclinical models to predict the clinical toxicity of chimeric antigen receptor (CAR) T cells in solid tumors is tenuous, thereby necessitating the development and evaluation of gated systems. Here we found that murine GD2 CAR-T cells, specific for the tumor-associated antigen GD2, induce fatal neurotoxicity in a costimulatory domain-dependent manner. Meanwhile, human B7H3 CAR-T cells exhibit efficacy in preclinical models of neuroblastoma. Seeking a better CAR, we generated a SynNotch gated CAR-T, GD2-B7H3, recognizing GD2 as the gate and B7H3 as the target. GD2-B7H3 CAR-T cells control the growth of neuroblastoma in vitro and in metastatic xenograft mouse models, with high specificity and efficacy. These improvements come partly from the better metabolic fitness of GD2-B7H3 CAR-T cells, as evidenced by their naïve T-like post-cytotoxicity oxidative metabolism and lower exhaustion profile.

Topics: Animals; Cell Line, Tumor; Cell Survival; Cytotoxicity, Immunologic; Gangliosides; Humans; Immunotherapy, Adoptive; Mice, 129 Strain; Mice, Inbred C57BL; Neoplasm Metastasis; Neuroblastoma; Receptors, Antigen, T-Cell; Receptors, Chimeric Antigen; Tumor Burden; Xenograft Model Antitumor Assays

Immune escape mechanisms employed by neuroblastoma (NB) cells include secretion of immunosuppressive factors disrupting effective antitumor immunity. The use of cellular therapy to treat solid tumors needs to be implemented. Killing activity of anti-GD2 Chimeric Antigen Receptor (CAR) T or natural killer (NK) cells against target NB cells was assessed through coculture experiments and quantified by FACS analysis. ELISA assay was used to quantify interferon-γ (IFNγ) secreted by NK and CAR T cells. Real Time PCR and Western Blot were performed to analyze gene and protein levels modifications. Transcriptional study was performed by chromatin immunoprecipitation and luciferase reporter assays on experiments of mutagenesis on the promoter sequence. NB tissue sample were analyzed by IHC and Real Time PCR to perform correlation study. We demonstrate that Indoleamine-pyrrole 2,3-dioxygenase1 (IDO1), due to its ability to convert tryptophan into kynurenines, is involved in NB resistance to activity of immune cells. In NB, IDO1 is able to inhibit the anti-tumor effect displayed by of both anti-GD2 CAR (GD2.CAR) T-cell and NK cells, mainly by impairing their IFNγ production. Furthermore, inhibition of MYCN expression in NB results into accumulation of IDO1 and consequently of kynurenines, which negatively affect the immune surveillance. Inverse correlation between IDO1 and MYCN expression has been observed in a wide cohort of NB samples. This finding was supported by the identification of a transcriptional repressive role of MYCN on IDO1 promoter. The evidence of IDO1 involvement in NB immune escape and its ability to impair NK and GD2.CAR T-cell activity contribute to clarify one of the possible mechanisms responsible for the limited efficacy of these immunotherapeutic approaches. A combined therapy of NK or GD2.CAR T-cells with IDO1 inhibitors, a class of compounds already in phase I/II clinical studies, could represent a new and still unexplored strategy capable to improve long-term efficacy of these immunotherapeutic approaches.

Topics: Cell Line, Tumor; Coculture Techniques; Gangliosides; Gene Expression Regulation, Neoplastic; Humans; Immunotherapy, Adoptive; Indoleamine-Pyrrole 2,3,-Dioxygenase; Interferon-gamma; Killer Cells, Natural; Lymphocyte Activation; N-Myc Proto-Oncogene Protein; Neuroblastoma; Receptors, Chimeric Antigen; Signal Transduction; T-Lymphocytes; Tumor Escape; Tumor Microenvironment

High-risk neuroblastomas (HR-NBs) are rare, aggressive pediatric cancers characterized by resistance to therapy and relapse in more than 30% of cases, despite using an aggressive therapeutic protocol including targeting of GD2. The mechanisms responsible for therapy resistance are unclear and might include the presence of GD2neg/low NB variants and/or the expression of immune checkpoint ligands such as B7-H3.. No false positive are detected, and MFC shows high sensitivity (0.0005%). Optimized MFC identifies CD45negCD56pos NB cells in 11 out of 12 (91.6%) of BM indicated as infiltrated by CA, 7 of which coexpress high levels of GD2 and B7-H3. MFC detects CD45negCD56posGD2neg/low NB variants expressing high surface levels of B7-H3 in two patients with HR-NB (stage M) diagnosed at 53 and 139 months of age. One of them has a non-MYCN amplified tumor with unusual THpos PHOX2Bneg phenotype, which relapsed 141 months post-diagnosis with BM infiltration and a humerus lesion. All GD2neg/low NB variants are detected in patients at relapse. Kaplan-Meier analysis highlights an interesting dichotomous prognostic value of MML5, ULBPs, PVR, B7-H6, and CD47, ligands involved in NB recognition by the immune system.. Our study validates a sensitive MFC analysis providing information on GD2 and B7-H3 surface expression and allowing fast, specific and sensitive evaluation of BM tumor burden. With other routinely used diagnostic and prognostic tools, MFC can improve diagnosis, prognosis, orienting novel personalized treatments in patients with GD2low/neg NB, who might benefit from innovative therapies combining B7-H3 targeting.

Topics: Adolescent; B7 Antigens; Biomarkers, Tumor; Cell Line, Tumor; Child; Child, Preschool; Flow Cytometry; Gangliosides; Humans; Infant; Male; Neuroblastoma; Predictive Value of Tests; Progression-Free Survival; Reproducibility of Results; Time Factors

Disialoganglioside (GD2)-specific chimeric antigen receptor (CAR)-T cells (GD2-CAR-T cells) have been developed and tested in early clinical trials in patients with relapsed/refractory neuroblastoma. However, the effectiveness of immunotherapy using these cells is limited, and requires improvement. Combined therapy with CAR-T cells and molecular targeted drugs could be a promising strategy to enhance the antitumor efficacy of CAR T cell immunotherapy. Here, we generated GD2-CAR-T cells through piggyBac transposon (PB)-based gene transfer (PB-GD2-CAR-T cells), and analyzed the combined effect of these cells and a MEK inhibitor in vitro and in vivo on neuroblastoma. Trametinib, a MEK inhibitor, ameliorated the killing efficacy of PB-GD2-CAR-T cells in vitro, whereas a combined treatment of the two showed superior antitumor efficacy in a murine xenograft model compared to that of PB-GD2-CAR-T cell monotherapy, regardless of the mutation status of the MAPK pathway in tumor cells. The results presented here provide new insights into the feasibility of combined treatment with CAR-T cells and MEK inhibitors in patients with neuroblastoma.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Combined Modality Therapy; Coumarins; DNA Transposable Elements; Drug Resistance, Neoplasm; Female; Gangliosides; Genetic Therapy; Humans; Immunotherapy, Adoptive; Mice; Mice, SCID; Mitogen-Activated Protein Kinase Kinases; Mutation; Neoplasm Recurrence, Local; Neuroblastoma; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; ras Proteins; Receptors, Chimeric Antigen; T-Lymphocytes; Xenograft Model Antitumor Assays

Vα24-invariant natural killer T cells (NKT) are attractive carriers for chimeric antigen receptors (CAR) due to their inherent antitumor properties and preferential localization to tumor sites. However, limited persistence of CAR-NKTs in tumor-bearing mice is associated with tumor recurrence. Here, we evaluated whether coexpression of the NKT homeostatic cytokine IL15 with a CAR enhances the. Human primary NKTs were. Coexpression of IL15 with either costimulatory domain increased CAR-NKT absolute numbers. However, constructs containing 4-1BB induced excessive activation-induced cell death and reduced numeric expansion of NKTs compared with respective CD28-based constructs. Further evaluation of CD28-based GD2.CAR and GD2.CAR.15 showed that coexpression of IL15 led to reduced expression levels of exhaustion markers in NKTs and increased multiround. Our results informed selection of the CD28-based GD2.CAR.15 construct for clinical testing and led to initiation of a first-in-human CAR-NKT cell clinical trial (NCT03294954).

Topics: Animals; Apoptosis; Cell Proliferation; Cytotoxicity, Immunologic; Gangliosides; Humans; Immunotherapy, Adoptive; Interleukin-15; Lymphocyte Activation; Mice; Mice, Inbred NOD; Mice, SCID; Natural Killer T-Cells; Neuroblastoma; Receptors, Antigen, T-Cell; Receptors, Chimeric Antigen; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Originating from the abnormal growth of neuroblasts, pediatric neuroblastoma affects the age group below 15 years. It is an aggressive heterogenous cancer with a high morbidity rate. Biological marker GD2 synthesised by the GD2 gene acts as a powerful predictor of neuroblastoma cells. GD2 gangliosides are sialic acid-containing glycosphingolipids. Differential expression during brain development governs the function of the GD2. The present study explains the interaction of the GD2 with its established inhibitors and discovers the compound having a high binding affinity against the target protein. Technically, during the development of new compounds through docking studies, the best drug among all pre-exist inhibitors was filtered. Hence in reference to the best docked compound, the study proceeded further.. The In silico approach provides a platform to determine and establish potential inhibitor against GD2 in Pediatric neuroblastoma. The 3D structure of GD2 protein was modelled by homology base fold methods using Smith-Watermans' Local alignment. A total of 18 established potent compounds were subjected to molecular docking and Etoposide (CID: 36462) manifested the highest affinity. The similarity search presented 336 compounds similar to Etoposide.. Through virtual screening, the compound having PubChem ID 10254934 showed a better affinity towards GD2 than the established inhibitor. The comparative profiling of the two compounds based on various interactions such as H-bond interaction, aromatic interactions, electrostatic interactions and ADMET profiling and toxicity studies were performed using various computational tools.. The docking separated the virtual screened drug (PubChemID: 10254934) from the established inhibitor with a better re-rank score of -136.33. The toxicity profile of the virtual screened drug was also lesser (less lethal) than the established drug. The virtual screened drug was observed to be bioavailable as it does not cross the blood-brain barrier. Conclusively, the virtual screened compound obtained in the present investigation is better than the established inhibitor and can be further augmented by In vitro analysis, pharmacodynamics and pharmacokinetic studies.

Topics: Adolescent; Amino Acid Sequence; Antineoplastic Agents; Child; Child, Preschool; Computer Simulation; Drug Screening Assays, Antitumor; Gangliosides; Humans; Infant; Molecular Docking Simulation; Neuroblastoma; Sequence Homology, Amino Acid

A delay in encountering the cognate antigen while in the circulation, and the suboptimal costimulation received at the tumor site are key reasons for the limited activity of chimeric antigen receptor-redirected T cells (CAR-T) in solid tumors. We have explored the benefits of incorporating the IL15 cytokine within the CAR cassette to provide both a survival signal before antigen encounter, and an additional cytokine signaling at the tumor site using a neuroblastoma tumor model.. We optimized the construct for the CAR specific for the NB-antigen GD2 without (GD2.CAR) or with IL15 (GD2.CAR.15). We then compared the expansion, phenotype, and antitumor activity of T cells transduced with these constructs against an array of neuroblastoma cell lines. We observed that optimized GD2.CAR.15-Ts have reduced expression of the PD-1 receptor, are enriched in stem cell-like cells, and have superior antitumor activity upon repetitive tumor exposures. Our results guide new therapeutic options for GD2.CAR-Ts in patients with neuroblastoma, and CAR-T development for a broad range of solid tumors.

Topics: Animals; Antigens, Neoplasm; Apoptosis; Cell Proliferation; Female; Gangliosides; Humans; Immunotherapy; Interleukin-15; Male; Mice; Mice, Inbred NOD; Mice, SCID; Neuroblastoma; Protein Engineering; Receptors, Chimeric Antigen; T-Lymphocytes, Cytotoxic; Transduction, Genetic; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Neuroblastoma cells highly express the disialoganglioside GD2, a tumor-associated carbohydrate antigen, which is only sparsely expressed on healthy tissue. GD2 is a primary target for the development of immunotherapy for neuroblastoma. Immunotherapy with monoclonal anti-GD2 antibodies has proven safety and efficacy in clinical trials and is included in the standard treatment for children with high-risk neuroblastoma. Strategies to modulate GD2 expression in neuroblastoma could further improve anti-GD2-targeted immunotherapy. Here, we report that the cellular sialylation pathway, as well as epigenetic reprogramming, strongly modulates GD2 expression in human and mouse neuroblastoma cell lines. Recognition of GD2 by the 14G2a antibody is sialic acid-dependent and was blocked with the fluorinated sialic acid mimetic Ac

Topics: Animals; Antigens, Neoplasm; Cell Line, Tumor; Gangliosides; Histone Deacetylase Inhibitors; Immunotherapy; Mice; N-Acetylneuraminic Acid; Neuroblastoma; Sialyltransferases; Up-Regulation

We determined whether elimination of CD105. The effect of MSCs and monocytes on antibody-dependent cellular cytotoxicity (ADCC) mediated by dinutuximab with aNK cells against neuroblastoma cells was determined. ADCC mediated by dinutuximab with aNK cells against neuroblastoma cells. Immunotherapy of neuroblastoma with anti-GD2 antibody dinutuximab and aNK cells is suppressed by CD105

Topics: Animals; Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Antineoplastic Agents; Cell Line, Tumor; Endoglin; Gangliosides; Humans; Immunotherapy; Killer Cells, Natural; Mice; Mice, Inbred NOD; Mice, SCID; Neuroblastoma; Xenograft Model Antitumor Assays

The GD2 ganglioside, which is abundant on the surface of neuroblastoma cells, is targeted by an FDA-approved therapeutic monoclonal antibody and is an attractive tumor-associated antigen for cellular immunotherapy. Chimeric antigen receptor (CAR)-modified T cells can have potent antitumor activity in B-cell malignancies, and trials to harness this cytolytic activity toward GD2 in neuroblastoma are under way. In an effort to enhance the antitumor activity of CAR T cells that target GD2, we generated variant CAR constructs predicted to improve the stability and the affinity of the GD2-binding, 14G2a-based, single-chain variable fragment (scFv) of the CAR and compared their properties

Topics: Animals; CD3 Complex; Cell Line, Tumor; Cell Proliferation; Cytotoxicity, Immunologic; Disease Models, Animal; Encephalitis; Gangliosides; Gene Order; Genetic Vectors; Humans; Immunotherapy, Adoptive; Mice; Neuroblastoma; Receptors, Antigen, T-Cell; Receptors, Chimeric Antigen; Single-Chain Antibodies; T-Lymphocytes; Tumor Necrosis Factor Receptor Superfamily, Member 9; Xenograft Model Antitumor Assays

Although anti-disialoganglioside (GD2) antibodies are successfully used for neuroblastoma therapy, a third of patients with neuroblastoma experience treatment failure or serious toxicity. Various strategies have been employed in the clinic to improve antibody-dependent cell-mediated cytotoxicity (ADCC), such as the addition of interleukin (IL)-2 to enhance natural killer (NK) cell function, adoptive transfer of allogeneic NK cells to exploit immune surveillance, and retinoid-induced differentiation therapy. Nevertheless, these mechanisms are not fully understood. We developed a quantitative assay to test ADCC induced by the anti-GD2 antibody Hu14.18K322A in nine neuroblastoma cell lines and dissociated cells from orthotopic patient-derived xenografts (O-PDXs) in culture. IL-2 improved ADCC against neuroblastoma cells, and differentiation with all-trans retinoic acid stabilized GD2 expression on tumor cells and enhanced ADCC as well. Degranulation was highest in licensed NK cells that expressed CD158b (P < 0.001) and harbored a killer-cell immunoglobulin-like receptor (KIR) mismatch against the tumor-specific human leukocyte antigen (HLA; P = 0.016). In conclusion, IL-2 is an important component of immunotherapy because it can improve the cytolytic function of NK cells against neuroblastoma cells and could lower the antibody dose required for efficacy, thereby reducing toxicity. The effect of IL-2 may vary among individuals and a biomarker would be useful to predict ADCC following IL-2 activation. Sub-populations of NK cells may have different levels of activity dependent on their licensing status, KIR expression, and HLA-KIR interaction. Better understanding of HLA-KIR interactions and the molecular changes following retinoid-induced differentiation is necessary to delineate their role in ADCC.

Topics: Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Antineoplastic Combined Chemotherapy Protocols; Combined Modality Therapy; Gangliosides; Humans; Interleukin-2; Killer Cells, Natural; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

Sepantronium bromide (YM155) is a hydrophilic quaternary compound that cannot be administered orally due to its low oral bioavailability; it is furthermore rapidly eliminated via the kidneys. The current study aims at improving the pharmacokinetic profile of YM155 by its formulation in immunoliposomes that can achieve its enhanced delivery into tumor tissue and facilitate uptake in neuroblastoma cancer cells.. PEGylated YM155 loaded liposomes composed of DPPC, cholesterol and DSPE-PEG. YM155 loaded immunoliposomes had a size of 170 nm and zeta potential of -10 mV, with an antibody coupling efficiency of 60% andYM155 encapsulation efficiency of14%. Targeted and control liposomal formulations were found to have similar YM155 release rates in a release medium containing 50% serum. An in-vitro toxicity study on KCNR cells showed less toxicity for immunoliposomes as compared to free YM155. In-vivo pharmacokinetic evaluation of YM155 liposomes showed prolonged blood circulation and significantly increased half-lives of liposomal YM155 in tumor tissue, as compared to a bolus injection of free YM155.. YM155 loaded immunoliposomes were successfully formulated and characterized, and initial in-vivo results show their potential for improving the circulation time and tumor accumulation of YM155.

Topics: Animals; Antibodies; Antineoplastic Agents; Cell Line, Tumor; Drug Compounding; Drug Liberation; Drug Stability; Female; Gangliosides; Half-Life; Humans; Hydrophobic and Hydrophilic Interactions; Imidazoles; Injections, Intravenous; Liposomes; Mice; Mice, Nude; Naphthoquinones; Neuroblastoma; Pilot Projects; Polyethylene Glycols; Survivin; Xenograft Model Antitumor Assays

Adult-onset neuroblastoma is rare and little is known about its biology and clinical course. There is no established therapy for adult-onset neuroblastoma. Anti-GD2 immunotherapy is now standard therapy in children with high-risk neuroblastoma; however, its use has not been reported in adults. Forty-four adults (18-71 years old) diagnosed with neuroblastoma between 1979 and 2015 were treated at Memorial Sloan Kettering Cancer Center. Five, 1, 5 and 33 patients had INSS stage 1, 2, 3 and 4 diseases, respectively. Genetic abnormalities included somatic ATRX (58%) and ALK mutations (42%) but not MYCN-amplification. In the 11 patients with locoregional disease, 10-year progression-free (PFS) and overall survival (OS) was 35.4 ± 16.1% and 61.4 ± 15.3%, respectively. Among 33 adults with stage 4 neuroblastoma, 7 (21%) achieved complete response (CR) after induction chemotherapy and/or surgery. Seven patients with primary refractory neuroblastoma (all with osteomedullary but no soft tissue disease) received anti-GD2 antibodies, mouse or humanized 3F8. Antibody-related adverse events were similar to those in children, response rate being 71.4%. In patients with stage 4 disease at diagnosis, 5-year PFS was 9.7± 5.3% and most patients who were alive with disease at 5 years died of neuroblastoma over the next 5 years, 10-year OS being only 19.0 ± 8.2%. Patients who achieved CR after induction had superior PFS and OS (p = 0.006, p = 0.031, respectively). Adult-onset neuroblastoma appeared to have different biology from pediatric or adolescent NB, and poorer outcome. Complete disease control appeared to improve long-term survival. Anti-GD2 immunotherapy was well tolerated and might be beneficial.

Topics: Adolescent; Adult; Aged; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Cohort Studies; Female; Follow-Up Studies; Gangliosides; Humans; Immunoglobulin G; Immunotherapy; Male; Middle Aged; Neoplasm Recurrence, Local; Neuroblastoma; Survival Rate; Treatment Outcome; Young Adult

Topics: Antibodies; Child, Preschool; Gangliosides; Humans; Magnetic Resonance Imaging; Male; Neuroblastoma; Spinal Cord Neoplasms; Spinal Nerve Roots

The process of autophagy and its role in survival of human neuroblastoma cell cultures was studied upon addition of an anti-GD2 ganglioside (GD2) 14G2a mouse monoclonal antibody (14G2a mAb) and an aurora A kinase specific inhibitor, MK-5108. It was recently shown that combination of these agents significantly potentiates cytotoxicity against IMR-32 and CHP-134 neuroblastoma cells in vitro, as compared to the inhibitor used alone. In this study we gained mechanistic insights on autophagy in the observed cytotoxic effects exerted by both agents using cytotoxicity assays, RT-qPCR, immunoblotting, and autophagy detection methods. Enhancement of the autophagy process in the 14G2a mAb- and MK-5108-treated IMR-32 cells was documented by assessing autophagic flux. Application of a lysosomotropic agent-chloroquine (CQ) affected the 14G2a mAb- and MK-5108-stimulated autophagic flux. It is our conclusion that the 14G2a mAb (40 μg/ml) and MK-5108 inhibitor (0.1 μM) induce autophagy in IMR-32 cells. Moreover, the combinatorial treatment of IMR-32 cells with the 14G2a mAb and CQ significantly potentiates cytotoxic effect, as compared to CQ used alone. Most importantly, we showed that interfering with autophagy at its early and late step augments the 14G2a mAb-induced apoptosis, therefore we can conclude that inhibition of autophagy is the primary mechanism of the CQ-mediated sensitization to the 14G2a mAb-induced apoptosis. Although, there was no virtual stimulation of autophagy in the 14G2a mAb-treated CHP-134 neuroblastoma cells, we were able to show that PHLDA1 protein positively regulates autophagy and this process exists in a mutually exclusive manner with apoptosis in PHLDA1-silenced CHP-134 cells.

Topics: Animals; Antibodies, Monoclonal; Apoptosis; Aurora Kinase A; Autophagy; Cell Line, Tumor; Cell Proliferation; Cyclohexanecarboxylic Acids; Gangliosides; Humans; Mice; Neuroblastoma; Thiazoles; Transcription Factors

Children diagnosed with high risk neuroblastoma have poor prognosis which stimulates efforts to broaden therapies of the neoplasm. GD2-ganglioside (GD2) marks neuroblastoma cells and is a target for monoclonal antibodies. We have recently shown that some neuroblastoma cell lines are sensitive to direct cytotoxicity of the anti-GD2 mouse monoclonal antibody 14G2a (mAb). For IMR-32 and LA-N-1 cell lines, treatment with the 14G2a mAb induced evident changes in appearance such as cell rounding, aggregation, loose contact with culture plastic, or detachment. Such findings prompted us to investigate whether modulation of attachment of neuroblastoma cells to extracellular matrix (ECM) proteins can affect their sensitivity to the 14G2a mAb treatment. First, using ultra-low attachment plates, we show that survival of the IMR-32, LA-N-1, LA-N-5, CHP-134 and Kelly cells depends on attachment. Next, we compared cellular ATP levels of the cell lines treated with the 14G2a mAb using uncoated, fibronectin-, collagen IV-coated surfaces to show that the ECM proteins slightly modulate sensitivity of the cell lines to the mAb. Then, we characterized presence of selected integrin subunits or their complexes on the cell surface. Finally, we applied small molecule inhibitors of selected integrin complexes: obtustatin (inhibiting α1β1 heterodimer), BIO 1211 (inhibiting active α4β1 heterodimer), cilengitide and SB273005 (inhibitors of αVβ3, αVβ5 heterodimers) to verify their effects on attachment of cell lines, cellular ATP levels, and in some experiments activities of apoptosis-executing caspase-3 and -7, for the compounds used alone or in combination with the 14G2a mAb. We characterized levels of total FAK (focal adhesion kinase), p-FAK (Tyr397) in IMR-32 cells treated with BIO 1211, and in LA-N-5, Kelly and SK-N-SH cells treated with SB273005. Our results extend knowledge on factors influencing cytotoxicity of 14G2a.

Topics: Adenosine Triphosphate; Animals; Antibodies, Monoclonal; Apoptosis; Bridged Bicyclo Compounds, Heterocyclic; Caspase 3; Cell Line, Tumor; Diterpenes; Extracellular Matrix Proteins; Gangliosides; Humans; Mice; Neuroblastoma; Small Molecule Libraries; Snake Venoms

Loss of disialoganglioside 2 (GD2) expression in neuroblastoma (NB) bone marrow cells has been reported in rare cases. This study investigated prospectively the frequency and the patterns of visible GD2 loss at diagnosis, during treatment, and at recurrence.. Bone marrow aspirates of patients with new or recurrent stage 4 and 4S NB diagnosed between January 1, 2002 and August 31, 2013 were investigated in parallel by cytology and GD2 immunocytology. Complete negative immunostaining was defined if staining was absent in all and partial if absent in a portion and/or in case of atypical faint staining.. Of 1,261 investigated trial patients of all stages, 474 had unequivocal cytological bone marrow infiltration at initial diagnosis. Thirty-seven patients had tumor cells with complete or partial negative GD2 staining at initial diagnosis, nine during chemotherapy, and 11 at recurrence (altogether 12.0%). The percentage of GD2 negativity in stages 4 and 4S were similar (13% and 9%, respectively). Complete negativity was seen in 14 and partial in 43 cases. Twenty-one cases changed from positive to negative (15 to partial and six to complete) and three cases from negative to positive staining (two to partial and one to complete). The GD2 negative and positive groups were not different regarding tumor sites, molecular characteristics, histology, and tumor markers. Children with stage 4 and GD2 negativity tended to be older at diagnosis (42 vs. 32 months, P = 0.056). Event-free survival and overall survival comparing negative versus positive staining did not show any differences.. Complete or partial lack of GD2 staining on NB cells in bone marrow is more frequent than currently recognized.

Topics: Adolescent; Adult; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Bone Marrow Neoplasms; Child; Child, Preschool; Female; Follow-Up Studies; Gangliosides; Humans; Immunoenzyme Techniques; Male; Neoplasm Recurrence, Local; Neoplasm Staging; Neuroblastoma; Prognosis; Prospective Studies; Survival Rate; Young Adult

Since 2003, high-risk neuroblastoma (HR-NB) patients at our center received anti-GD2 antibody 3F8/GM-CSF + isotretinoin - but not myeloablative therapy with autologous stem-cell transplantation (ASCT). Post-ASCT patients referred from elsewhere also received 3F8/GM-CSF + isotretinoin. We therefore accrued a study population of two groups treated during the same period and whose consolidative therapy, aside from ASCT, was identical. We analyzed patients enrolled in 1st complete/very good partial remission (CR/VGPR). Their event-free survival (EFS) and overall survival (OS) were calculated from study entry. Large study size allowed robust statistical analyses of key prognosticators including MYCN amplification, minimal residual disease (MRD), FCGR2A polymorphisms, and killer immunoglobulin-like receptor genotypes of natural killer cells. The 170 study patients included 60 enrolled following ASCT and 110 following conventional chemotherapy. The two cohorts had similar clinical and biological features. Five-year rates for ASCT and non-ASCT patients were, respectively: EFS 65% vs. 51% (p = .128), and OS 76% vs. 75% (p = .975). In multivariate analysis, ASCT was not prognostic and only MRD-negativity after two cycles of 3F8/GM-CSF correlated with significantly improved EFS and OS. Although a trend towards better EFS is seen with ASCT, OS is near identical. Cure rates may be similar, as close surveillance detects localized relapse and effective salvage treatments are applied. ASCT may not be needed to improve outcome when anti-GD2 immunotherapy is used for consolidation after dose-intensive conventional chemotherapy.

Topics: Adolescent; Antibodies, Monoclonal; Child; Child, Preschool; Combined Modality Therapy; Dermatologic Agents; Female; Gangliosides; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunoenzyme Techniques; Immunotherapy; Infant; Infant, Newborn; Isotretinoin; Male; Neoplasm Recurrence, Local; Neoplasm Staging; Neuroblastoma; Prognosis; Stem Cell Transplantation; Survival Rate; Transplantation, Autologous

In patients with neuroblastoma (NB), treatment with anti-GD2 monoclonal antibody (mAb) directs natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) against tumor cells. However, tumor cytotoxicity is attenuated by ligation of inhibitory killer immunoglobulin-like receptors (KIRs) by HLA class I molecules. KIR3DL1 polymorphism influences its ability to engage HLA-Bw4 ligands. We tested the hypothesis that poorly interacting combinations of KIR3DL1 and HLA ligands are more permissive of mAb-mediated antitumor effect.. KIR3DL1 and HLA-B subtyping were performed with a multiplex intermediate-resolution polymerase chain reaction assay for a cohort of 245 patients who were treated with antibody 3F8 for high-risk NB. Patient outcomes were analyzed according to expected degree of interaction between KIR3DL1 and HLA-B subtypes and grouped as strong, weak, or noninteractors. A comparison of NK response to 3F8 mAb opsonized NB cells between strong- and noninteracting donors was performed by flow cytometry.. KIR3DL1 and HLA-B subtype combinations associated with noninteraction as a result of lack of receptor expression [KIR3DL1(-)], failure of interaction with inhibitory ligands [KIR3DS1(+)], or absence of KIR ligands resulted in significantly improved overall and progression-free survival. Patients with KIR3DL1 and HLA-B subtype combinations that were predictive of weak interaction had superior outcomes compared with those that were predictive of strong interaction; however, both groups were inferior to those with noninteracting subtype combinations. In vitro analysis of 3F8-mediated ADCC showed that KIR3DL1(-) and 3DS1(+) NK cells were insensitive to inhibition by HLA-Bw4-expressing NB targets.. We conclude that KIR3LD1 and HLA-B allele combinations can have a prognostic impact on patient survival after treatment with anti-GD2 mAb that relies on NK-ADCC. The survival advantage seen in noninteracting combinations supports the therapeutic disinhibition of individuals with strongly interacting KIR and ligand pairs.

Topics: Adolescent; Adult; Alleles; Antibodies, Monoclonal; Child; Child, Preschool; Epitopes; Gangliosides; HLA-B Antigens; Humans; Killer Cells, Natural; Neuroblastoma; Polymorphism, Genetic; Receptors, KIR3DL1; Retrospective Studies

KIR3DL1 and HLA-B allele combinations predict response to anti-GD2 mAb in neuroblastoma.

Topics: Antibodies, Monoclonal; Gangliosides; HLA-B Antigens; Humans; Neuroblastoma; Protein Binding; Receptors, KIR3DL1

Genetically engineered T cells expressing CD19-specific chimeric antigen receptors (CAR) have shown impressive activity against B-cell malignancies, and preliminary results suggest that T cells expressing a first-generation disialoganglioside (GD2)-specific CAR can also provide clinical benefit in patients with neuroblastoma. We sought to assess the potential of GD2-CAR therapies to treat pediatric sarcomas. We observed that 18 of 18 (100%) of osteosarcomas, 2 of 15 (13%) of rhabdomyosarcomas, and 7 of 35 (20%) of Ewing sarcomas expressed GD2. T cells engineered to express a third-generation GD2-CAR incorporating the 14g2a-scFv with the CD28, OX40, and CD3ζ signaling domains (14g2a.CD28.OX40.ζ) mediated efficient and comparable lysis of both GD2

Topics: Animals; Cell Line, Tumor; Child; Combined Modality Therapy; Gangliosides; Humans; Immunotherapy, Adoptive; Mice, Inbred NOD; Myeloid-Derived Suppressor Cells; Neuroblastoma; Receptors, Antigen, T-Cell; Sarcoma; T-Lymphocytes; Treatment Outcome; Tretinoin; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Antibody-based immunotherapy has proven efficacy for patients with high-risk neuroblastoma. However, despite being the most efficient tumoricidal effectors, T cells are underutilized because they lack Fc receptors. Using a monovalent single-chain fragment (ScFv) platform, we engineered tandem scFv bispecific antibodies (BsAbs) that specifically target disialoganglioside (GD2) on tumor cells and CD3 on T cells. Structural variants of BsAbs were constructed and ranked based on binding to GD2, and on competency in inducing T-cell-mediated tumor cytotoxicity. In vitro thermal stability and binding measurements were used to characterize each of the constructs, and in silico molecular modeling was used to show how the orientation of the variable region heavy (VH) and light (VL) chains of the anti-GD2 ScFv could alter the conformations of key residues responsible for high affinity binding. We showed that the VH-VL orientation, the (GGGGS)3 linker, disulfide bond stabilization of scFv, when combined with an affinity matured mutation provided the most efficient BsAb to direct T cells to lyse GD2-positive tumor cells. In vivo, the optimized BsAb could efficiently inhibit melanoma and neuroblastoma xenograft growth. These findings provide preclinical validation of a structure-based method to assist in designing BsAb for T-cell-mediated therapy.

Topics: Animals; Antibodies, Bispecific; Antibodies, Monoclonal; Blotting, Western; CD3 Complex; Cell Proliferation; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Gangliosides; Humans; Immunotherapy; Lymphocyte Activation; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Models, Molecular; Neuroblastoma; Single-Chain Antibodies; T-Lymphocytes; Tumor Cells, Cultured

Treatment of neuroblastoma with targeted immunotherapy using chimeric anti-GD2 monoclonal antibodies (ch14.18) is associated with significant pain requiring management with a high-dose opioid infusion. We present a case series of six children, for whom dexmedetomidine and hydromorphone infusions safely and effectively reduced the pain of ch14.18 therapy in the oncology ward setting.. The ch14.18 infusion is administered for ≥ 10 hr over four consecutive days in each of 5 cycles. Hydromorphone (2-8 mcg.kg(-1) .hr(-1) ) and dexmedetomidine (0.1-0.6 mcg.kg(-1) .hr(-1) ) infusions were commenced 1 hr before starting ch14.18 immunotherapy and titrated to optimal clinical effect. One hour after stopping the ch14.18 infusion, dexmedetomidine was discontinued and hydromorphone weaned as tolerated. This strategy was supervised by the Acute Pain Service with strict monitoring and nursing policies. Patient charts were reviewed for evidence of side effects and quality of analgesia.. Data from six patients, with median (range) age of 3.5 (2-12) years are reported. Median (range) utilization of hydromorphone was 2.9 (2.0-4.7) mcg.kg(-1) .hr(-1) , and of dexmedetomidine was 0.17 (0.10-0.20) mcg.kg(-1) .hr(-1) . A drop in mean arterial pressure ≥ 30% below baseline was identified during 30% of treatment days, but only prompted interrupting or reducing the ch14.18 infusion on 7% of treatment days; only one episode of grade 3 hypotension was recorded during this series. Hypoxemia occurred during 8% and bradycardia during 4% of treatment days.. Dexmedetomidine infusion may be an effective and safe pain management adjunct to opioid therapy for the pain of ch14.18 infusion.

Topics: Analgesics, Non-Narcotic; Analgesics, Opioid; Antibodies, Monoclonal; Child; Child, Preschool; Dexmedetomidine; Drug Therapy, Combination; Female; Follow-Up Studies; Gangliosides; Humans; Hydromorphone; Immunotherapy; Male; Neuroblastoma; Pain; Pain Management; Prognosis

Addition of anti-GD2 antibody ch14.18 to the treatment of neuroblastoma has improved outcomes. The most common side effect of ch14.18 is neuropathic pain, which may in part be complement-mediated. Hu14.18K322A is a humanized anti-GD2 antibody designed to diminish complement activation and induce less pain. We compare the pain outcomes in patients treated with ch14.18 and those treated with hu14.18K322A, and explore dose-dependent relationships between pain scores, opioid requirements, and complement levels in patients treated with hu14.18K322A.. Opioid (morphine equivalent mg/kg) and anxiolytic requirements during course 1 (4 days) in patients treated with hu14.18K322A and ch14.18 were reviewed. Correlations between antibody dose and pain scores, opioid requirements, and complement levels were examined for patients receiving hu14.18K322A.. Patients treated with hu14.18K322A (n = 19) had lower opioid requirements than those who received ch14.18 (n = 9). The differences in median opioid requirements (mg/kg) were statistically significant for the overall course (1.57 vs. 2.41, P = 0.019) as well as for Days 3 (0.34 vs. 0.65, P = 0.005), and 4 (0.32 vs. 0.64, P = 0.010). No difference in anxiolytic use was observed between the two groups. In the group treated with hu14.18K322A, we found a positive correlation between antibody dose administered and pain scores, but no correlation between antibody dose and opioid requirements or changes in complement levels.. In this retrospective analysis, hu14.18K322A induced less pain than ch14.18 based on opioid requirements. Pediatr Blood Cancer 2015;62:224-228. © 2014 Wiley Periodicals, Inc.

Topics: Adolescent; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Child; Child, Preschool; Complement Activation; Complement Inactivator Proteins; Complement System Proteins; Female; Gangliosides; Humans; Immunotherapy; Infant; Male; Neuralgia; Neuroblastoma; Retrospective Studies; Treatment Outcome

Anti-disialoganglioside GD2 IgG antibodies have shown clinical efficacy in solid tumors that lack human leukocyte antigens (e.g., neuroblastoma) by relying on Fc-dependent cytotoxicity. However, there are pain side effects secondary to complement activation. T-cell retargeting bispecific antibodies (BsAb) also have clinical potential, but it is thus far only effective against liquid tumors. In this study, a fully humanized hu3F8-BsAb was developed, in which the anti-CD3 huOKT3 single-chain Fv fragment (ScFv) was linked to the carboxyl end of the anti-GD2 hu3F8 IgG1 light chain, and was aglycosylated at N297 of Fc to prevent complement activation and cytokine storm. In vitro, hu3F8-BsAb activated T cells through classic immunologic synapses, inducing GD2-specific tumor cytotoxicity at femtomolar EC50 with >10⁵-fold selectivity over normal tissues, releasing Th1 cytokines (TNFα, IFNγ, and IL2) when GD2⁺ tumors were present. In separate murine neuroblastoma and melanoma xenograft models, intravenous hu3F8-BsAb activated T cells in situ and recruited intravenous T cells for tumor ablation, significantly prolonging survival from local recurrence or from metastatic disease. Hu3F8-BsAb, but not control BsAb, drove T cells and monocytes to infiltrate tumor stroma. These monocytes were necessary for sustained T-cell proliferation and/or survival and contributed significantly to the antitumor effect. The in vitro and in vivo antitumor properties of hu3F8-BsAb and its safety profile support its further clinical development as a cancer therapeutic, and provide the rationale for exploring aglycosylated IgG-scFv as a structural platform for retargeting human T cells.

Topics: Animals; Antibodies, Bispecific; CD3 Complex; Cell Line, Tumor; Cell Proliferation; Gangliosides; Humans; Immunoglobulin G; Melanoma; Mice; Mice, Inbred BALB C; Mice, Knockout; Monocytes; Neuroblastoma; Single-Chain Antibodies; T-Lymphocytes; Xenograft Model Antitumor Assays

Immunotherapy is a standard of care for children with high-risk neuroblastoma, where bone marrow (BM) is the predominant metastatic site. Early response markers of minimal residual disease (MRD) in the BM that are also predictive of survival could help individualize patient therapies.. After achieving first remission (n = 163), primary refractory disease (n = 102), or second remission (n = 95), children with stage 4 neuroblastoma received anti-GD2 3F8 antibody immunotherapy. BM MRD before 3F8 treatment and after cycle 2 (postMRD) was measured using a four-marker panel (B4GALNT1, PHOX2B, CCND1, and ISL1) by quantitative reverse transcription polymerase chain reaction. Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method. Prognostic variables were tested in both univariable and multivariable analyses, and MRD markers were further assessed individually and in combination as binary composite (postMRD: 0 and 1) and as equal sum (postMRDSum: 0 to 4) using the Cox regression models, and their predictive accuracy was determined by the concordance index.. When BM was evaluated after cycle 2, individual markers were highly predictive of PFS and OS. The prediction accuracy improved when they were combined in postMRDSum. A multivariable model taking into account all the variables significant in the univariable analyses identified postMRDSum to be independently predictive of PFS and OS. When the model for OS also included missing killer immunoglobulin-like receptor ligand, human antimouse antibody response, and the enrollment disease status, the concordance index was 0.704.. BM MRD after two cycles of immunotherapy was confirmed as an early response marker and a consistent independent predictor of survival.

Topics: Adolescent; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antineoplastic Agents; Bone Marrow; Child; Child, Preschool; Female; Gangliosides; Humans; Immunoglobulin G; Immunotherapy; Infant; Kaplan-Meier Estimate; Male; Neoplasm, Residual; Neuroblastoma; Predictive Value of Tests; Prognosis; Time Factors

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.

Topics: Animals; Antibodies, Anti-Idiotypic; Cell Line; Cytotoxicity, Immunologic; Drug Resistance, Neoplasm; Female; Gangliosides; Genetic Engineering; Humans; Immunotherapy, Adoptive; Killer Cells, Natural; Mice; Mice, Inbred NOD; Neoplasm Transplantation; Neuroblastoma; Receptors, Antigen; Single-Chain Antibodies

Ganglioside GD2 is highly expressed on neuroectodermal tumors and an attractive therapeutic target for antibodies that have already shown some clinical efficacy. To further improve the current antibodies, which have modest affinity, we sought to improve affinity by using a combined method of random mutagenesis and in silico assisted design to affinity-mature the anti-GD2 monoclonal antibody hu3F8. Using yeast display, mutants in the Fv with enhanced binding over the parental clone were FACS-sorted and cloned. In silico modeling identified the minimal key interacting residues involved in the important charged interactions with the sialic acid groups of GD2. Two mutations, D32H (L-CDR1) and E1K (L-FR1) altered the electrostatic surface potential of the antigen binding site, allowing for an increase in positive charge to enhance the interaction with the negatively charged GD2-pentasaccharide headgroup. Purified scFv and IgG mutant forms were then tested for antigen specificity by ELISA, for tissue specificity by immunohistochemistry, for affinity by BIACORE, for antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity in vitro, and for anti-tumor efficacy in xenografted humanized mice. The nearly 7-fold improvement in affinity of hu3F8 with a single D32H (L-CDR1) mutation translated into a ∼12-fold improvement in NK92MI-transfected CD16-mediated ADCC, a 6-fold improvement in CD32-mediated ADCC, and a 2.5-fold improvement in complement-mediated cytotoxicity while maintaining restricted normal tissue cross-reactivity and achieving substantial improvement in tumor ablation in vivo. Despite increasing GD2 affinity, the double mutation D32H (L-CDR1) and E1K (L-FR1) did not further improve anti-tumor efficacy.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Antibody-Dependent Cell Cytotoxicity; Cell Proliferation; Computer Simulation; Flow Cytometry; Gangliosides; Humans; Immunoenzyme Techniques; Immunoglobulin G; Mice; Mice, Inbred BALB C; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Neuroblastoma; Protein Conformation; Static Electricity; Surface Properties; Tumor Cells, Cultured; Two-Hybrid System Techniques; Xenograft Model Antitumor Assays

Neuroblastoma is the most common extracranial solid tumor of childhood, and survival remains poor for patients with advanced disease. Novel immune therapies are currently in development, but clinical outcomes have not matched preclinical results. Here, we describe key mechanisms in which neuroblastoma inhibits the immune response. We show that murine and human neuroblastoma tumor cells suppress T-cell proliferation through increased arginase activity. Arginase II is the predominant isoform expressed and creates an arginine-deplete local and systemic microenvironment. Neuroblastoma arginase activity results in inhibition of myeloid cell activation and suppression of bone marrow CD34(+) progenitor proliferation. Finally, we demonstrate that the arginase activity of neuroblastoma impairs NY-ESO-1-specific T-cell receptor and GD2-specific chimeric antigen receptor-engineered T-cell proliferation and cytotoxicity. High arginase II expression correlates with poor survival for patients with neuroblastoma. The results support the hypothesis that neuroblastoma creates an arginase-dependent immunosuppressive microenvironment in both the tumor and blood that leads to impaired immunosurveillance and suboptimal efficacy of immunotherapeutic approaches.

Topics: Animals; Antigens, Neoplasm; Arginase; Arginine; Cell Proliferation; Gangliosides; Humans; Lymphocyte Activation; Membrane Proteins; Mice; Neoplasms, Experimental; Neuroblastoma; Receptors, Antigen, T-Cell; Recombinant Proteins; Tumor Microenvironment

Mechanisms leading to inhibitory effects of an anti-GD2 ganglioside (GD2) 14G2a mouse monoclonal antibody (mAb) and PI3K/Akt/mTOR pathway inhibitors on human neuroblastoma cell survival were studied in vitro. We have recently shown on IMR-32, CHP‑134, and LA-N-1 neuroblastoma cells that targeting GD2 with the mAb decreases cell viability of the cell lines. In this study we used cytotoxicity assays, proteomic arrays and immunoblotting to evaluate the response of the three cell lines to the anti‑GD2 14G2a mAb and specific PI3K/Akt/mTOR pathway inhibitors. We show here that the mAb modulates intracellular signal transduction through changes in several kinases and their substrates phosphorylation. More detailed analysis of the PI3K/Akt/mTOR pathway showed significant decrease in activity of Akt, mTOR, p70 S6 and 4E-BP1 proteins and transient increase in PTEN (a suppressor of the pathway), leading to inhibition of the signaling network responsible for stimulation of translation and proliferation. Additionally, combining the GD2-specific 14G2a mAb with an Akt inhibitor (perifosine), dual mTOR/PI3K inhibitors (BEZ-235 and SAR245409), and a pan-PI3K inhibitor (LY294002) was shown to enhance cytotoxic effects against IMR-32, CHP‑134 and LA-N-1 cells. Our study extends knowledge on mechanisms of action of the 14G2a mAb on the neuroblastoma cells. Also, it stresses the need for further delineation of molecular signal orchestration aimed at more reasonable selection of drugs to target key cellular pathways in quest for better cure for neuroblastoma patients.

Topics: Antibodies, Monoclonal; Apoptosis; Cell Line, Tumor; Drug Synergism; Gangliosides; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Neuroblastoma; Phosphatidylinositol 3-Kinases; Phosphorylcholine; Proto-Oncogene Proteins c-akt; Quinolines; Quinoxalines; Signal Transduction; Sulfonamides; TOR Serine-Threonine Kinases

Neuroblastoma is the most common extra-cranial solid tumor of childhood and it is characterized by the presence of a glycosphingolipid, GD2 ganglioside. Monoclonal antibodies targeting the antigen are currently tested in clinical trials. Additionally, several research groups reported results revealing that ganglioside-specific antibodies can affect cellular signaling and cause direct cytotoxicity against tumor cells. To shed more light on gene expression signatures of tumor cells, we used microarrays to analyze changes of transcriptome in IMR-32 human neuroblastoma cell cultures treated with doxorubicin (DOX) or a mouse monoclonal antibody binding to GD2 ganglioside 14G2a (mAb) for 24 h. The obtained results highlight that disparate cellular pathways are regulated by doxorubicin and 14G2a. Next, we used RT-PCR to verify mRNA levels of selected DOX-responsive genes such as RPS27L, PPM1D, SESN1, CDKN1A, TNFSF10B, and 14G2a-responsive genes such as SVIL, JUN, RASSF6, TLX2, ID1. Then, we applied western blot and analyzed levels of RPS27L, PPM1D, sestrin 1 proteins after DOX-treatment. Additionally, we aimed to measure effects of doxorubicin and topotecan (TPT) and 14G2a on expression of a novel human NDUFAF2 gene encoding for mimitin protein (MYC-induced mitochondrial protein) and correlate it with expression of the MYCN gene. We showed that expression of both genes was concomitantly decreased in the 14G2a-treated IMR-32 cells after 24 h and 48 h. Our results extend knowledge on gene expression profiles after application of DOX and 14G2a in our model and reveal promising candidates for further research aimed at finding novel anti-neuroblastoma targets.

Topics: Animals; Antibiotics, Antineoplastic; Antibodies, Monoclonal; Cell Line, Tumor; Cluster Analysis; Dose-Response Relationship, Drug; Doxorubicin; Gangliosides; Gene Expression Regulation, Neoplastic; Humans; Mice; Mitochondrial Proteins; Molecular Chaperones; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Oncogene Proteins; Software; Topotecan; Transcriptome

Chimeric antigen receptor (CAR)-expressing T cells are a promising therapeutic option for patients with cancer. We developed a new CAR directed against the disialoganglioside GD2, a surface molecule expressed in neuroblastoma and in other neuroectoderm-derived neoplasms. The anti-GD2 single-chain variable fragment (scFv) derived from a murine antibody of IgM class was linked, via a human CD8α hinge-transmembrane domain, to the signaling domains of the costimulatory molecules 4-1BB (CD137) and CD3-ζ. The receptor was expressed in T lymphocytes by retroviral transduction and anti-tumor activities were assessed by targeting GD2-positive neuroblastoma cells using in vitro cytotoxicity assays and a xenograft model. Transduced T cells expressed high levels of anti-GD2 CAR and exerted a robust and specific anti-tumor activity in 4- and 48-hour cultures with neuroblastoma cells. Cytotoxicity was associated with the release of pro-apoptotic molecules such as TRAIL and IFN-γ. These results were confirmed in a xenograft model, where anti-GD2 CAR T cells infiltrating tumors and persisting into blood circulation induced massive apoptosis of neuroblastoma cells and completely abrogated tumor growth. This anti-GD2 CAR represents a powerful new tool to redirect T cells against GD2. The preclinical results of this study warrant clinical testing of this approach in neuroblastoma and other GD2-positive malignancies.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cytotoxicity, Immunologic; Flow Cytometry; Gangliosides; Green Fluorescent Proteins; HEK293 Cells; Humans; Immunotherapy, Adoptive; Interferon-gamma; Mice, Inbred NOD; Mice, SCID; Microscopy, Fluorescence; Neuroblastoma; Receptors, Antigen, T-Cell; Single-Chain Antibodies; T-Lymphocytes; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor Receptor Superfamily, Member 9; Xenograft Model Antitumor Assays

Current multimodal treatments for patients with neuroblastoma (NBL), including anti-disialoganglioside (GD2) monoclonal antibody (mAb) based immunotherapy, result in a favorable outcome in around only half of the patients with advanced disease. To improve this, novel immunocombinational strategies need to be developed and tested in autologous preclinical NBL models. A genetically well-explored autologous mouse model for NBL is the TH-MYCN model. However, the immunobiology of the TH-MYCN model remains largely unexplored. We developed a mouse model using a transplantable TH-MYCN cell line in syngeneic C57Bl/6 mice and characterized the immunobiology of this model. In this report, we show the relevance and opportunities of this model to study immunotherapy for human NBL. Similar to human NBL cells, syngeneic TH-MYCN-derived 9464D cells endogenously express the tumor antigen GD2 and low levels of MHC Class I. The presence of the adaptive immune system had little or no influence on tumor growth, showing the low immunogenicity of the NBL cells. In contrast, depletion of NK1.1+ cells resulted in enhanced tumor outgrowth in both wild-type and Rag1(-/-) mice, showing an important role for NK cells in the natural anti-NBL immune response. Analysis of the tumor infiltrating leukocytes ex vivo revealed the presence of both tumor associated myeloid cells and T regulatory cells, thus mimicking human NBL tumors. Finally, anti-GD2 mAb mediated NBL therapy resulted in ADCC in vitro and delayed tumor outgrowth in vivo. We conclude that the transplantable TH-MYCN model represents a relevant model for the development of novel immunocombinatorial approaches for NBL patients.

Topics: Animals; Antibodies, Monoclonal; Blotting, Western; CD8-Positive T-Lymphocytes; Cell Proliferation; Disease Models, Animal; Female; Flow Cytometry; Fluorescent Antibody Technique; Gangliosides; Homeodomain Proteins; Humans; Immunotherapy; Killer Cells, Natural; Mice; Mice, Inbred C57BL; Mice, Transgenic; N-Myc Proto-Oncogene Protein; Neuroblastoma; Proto-Oncogene Proteins; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; T-Lymphocytes, Regulatory; Transgenes; Tumor Cells, Cultured

Nonpharmacologic, mind-body interventions are used to reduce anxiety in pediatric patients. Anti-ganglioside GD2 monoclonal antibody (anti-GD2 MoAb 3F8) therapy is the standard of care for high-risk neuroblastoma and pain is its major side effect. We performed a retrospective analysis of children undergoing anti-GD2 MoAb 3F8 treatment who received guided meditation. Meditation involved concentrating on the repetition of rhythmic, melodic sounds purported to slow breathing and induce a relaxation response. A total of 71% patients completed a session at first (n=19) or second attempt (n=5). Patients received fewer analgesic doses to manage anti-GD2 MoAb 3F8-induced pain when participating in meditation (n=17, mean=-0.4 dose, P<0.01). Mantram meditation is a feasible outpatient intervention associated with reduced analgesic requirements.

Topics: Adolescent; Antibodies, Monoclonal; Antineoplastic Agents; Child; Child, Preschool; Female; Gangliosides; Humans; Male; Meditation; Neuroblastoma; Pain; Retrospective Studies

Survival outcomes for patients with osteosarcoma have remained stagnant over the past 30 years. Targeting of ganglioside GD2, a glycosphingolipid on the cell surface of some tumors, with immunotherapy has resulted in improved outcomes for patients with neuroblastoma. In the current study, the expression pattern of GD2 was examined in osteosarcoma.. Immunohistochemistry was performed on osteosarcoma samples from patients at the time of initial biopsy, definitive surgery, and disease recurrence. The intensity and location of staining were scored. Cell-based enzyme-linked immunoadsorbent assay was performed on osteosarcoma cell lines to quantitate the level of GD2 expression.. Forty-four osteosarcoma samples were evaluated by immunohistochemistry, including 8 samples from the initial biopsy, 28 samples from the definitive surgery, and 8 samples from the time of disease recurrence. GD2 was expressed on all 44 osteosarcoma samples. Osteosarcoma tissue obtained at the time of disease recurrence demonstrated a higher intensity of staining compared with samples obtained at initial biopsy and definitive surgery (P = .016). The majority of osteosarcoma cell lines expressed GD2 at higher levels than the neuroblastoma cell line BE(2)-C.. Ganglioside GD2 is highly expressed on osteosarcomas. Clinical trials are needed to assess the efficacy of targeting GD2 in patients with osteosarcoma.

Topics: Antibodies, Monoclonal; Biopsy; Bone Neoplasms; Gangliosides; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Recurrence, Local; Neuroblastoma; Osteosarcoma; Primary Cell Culture; Radioimmunotherapy

Anti-GD2 antibody is a proven therapy for GD2-positive neuroblastoma. Monoclonal antibodies against GD2, such as chimeric mAb ch14.18, have become benchmarks for neuroblastoma therapies. Pain, however, can limit immunotherapy with anti-GD2 therapeutic antibodies like ch14.18. This adverse effect is attributed to acute inflammation via complement activation on GD2-expressing nerves. Thus, new strategies are needed for the development of treatment intensification strategies to improve the outcome of these patients.. We established the mouse-human chimeric antibody c.8B6 specific to OAcGD2 in order to reduce potential immunogenicity in patients and to fill the need for a selective agent that can kill neuroblastoma cells without inducing adverse neurological side effects caused by anti-GD2 antibody immunotherapy. We further analyzed some of its functional properties compared with anti-GD2 ch14.18 therapeutic antibody. With the exception of allodynic activity, we found that antibody c.8B6 shares the same anti-neuroblastoma attributes as therapeutic ch14.18 anti-GD2 mAb when tested in cell-based assay and in vivo in an animal model.. The absence of OAcGD2 expression on nerve fibers and the lack of allodynic properties of c.8B6-which are believed to play a major role in mediating anti-GD2 mAb dose-limiting side effects-provide an important rationale for the clinical application of c.8B6 in patients with high-risk neuroblastoma.

Topics: Acetylation; Animals; Antibodies, Monoclonal; Antibody Specificity; Flow Cytometry; Gangliosides; Humans; Hyperalgesia; Injections, Intravenous; Mice; Neuroblastoma; Protein Binding; Rats; Rats, Sprague-Dawley

Neuroblastoma, a childhood tumour of neuroectodermal origin, accounts for 15 % of paediatric cancer deaths, which is often metastatic at diagnosis and despite aggressive therapies, it has poor long-term prognosis with high risk of recurrence. Monoclonal antibody (mAb) therapy targeting GD2, a disialoganglioside expressed on neuroblastoma, has shown promise in recent trials with natural killer cell (NK)-mediated antibody-dependent cellular cytotoxicity (ADCC) thought to be central to efficacy, although other immune effectors may be important. To further enhance therapy, immunomonitoring of patients is essential to elucidate the in vivo mechanisms of action and provides surrogate end points of efficacy for future clinical trials. Our aim was to establish a 'real-time' ex vivo wholeblood (WB) immunomonitoring strategy to perform within the logistical constraints such as limited sample volumes, anticoagulant effects, sample stability and shipping time. A fluorescent dye release assay measuring target cell lysis was coupled with flow cytometry to monitor specific effector response. Significant target cell lysis with anti-GD2 antibody (p < 0.05) was abrogated following NK depletion. NK up-regulation of CD107a and CD69 positively correlated with target cell lysis (r > 0.6). The ADCC activity of WB correlated with peripheral blood mononuclear cells (r > 0.95), although WB showed overall greater target cell lysis attributed to the combination of NK-mediated ADCC, CD16+ granulocyte degranulation and complement- dependent cytotoxicity. Response was maintained in heparinised samples stored for 24 h at room temperature, but not 4 °C. Critically, the assay showed good reproducibility (mean % CV < 6.4) and was successfully applied to primary neuroblastoma samples. As such, WB provides a resourceful analysis of multiple mechanisms for efficient end point monitoring to correlate immune modulation with clinical outcome.

Topics: Adult; Antibody-Dependent Cell Cytotoxicity; Flow Cytometry; Gangliosides; Humans; Leukocytes, Mononuclear; Neuroblastoma; Tumor Cells, Cultured

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.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Clinical Trials, Phase II as Topic; Clinical Trials, Phase III as Topic; Enzyme-Linked Immunosorbent Assay; Europe; Gangliosides; Humans; Immunity, Humoral; Immunotherapy; Mice; Neuroblastoma; Quality Control

Advances in the design of chimeric antigen receptors (CARs) have improved the antitumor efficacy of redirected T cells. However, functional heterogeneity of CAR T cells limits their therapeutic potential and is associated with toxicity. We proposed that CAR expression in Vα24-invariant natural killer T (NKT) cells can build on the natural antitumor properties of these cells while their restriction by monomorphic CD1d limits toxicity. Primary human NKT cells were engineered to express a CAR against the GD2 ganglioside (CAR.GD2), which is highly expressed by neuroblastoma (NB). We compared CAR.GD2 constructs that encoded the CD3ζ chain alone, with CD28, 4-1BB, or CD28 and 4-1BB costimulatory endodomains. CAR.GD2 expression rendered NKT cells highly cytotoxic against NB cells without affecting their CD1d-dependent reactivity. We observed a striking T helper 1-like polarization of NKT cells by 4-1BB-containing CARs. Importantly, expression of both CD28 and 4-1BB endodomains in the CAR.GD2 enhanced in vivo persistence of NKT cells. These CAR.GD2 NKT cells effectively localized to the tumor site had potent antitumor activity, and repeat injections significantly improved the long-term survival of mice with metastatic NB. Unlike T cells, CAR.GD2 NKT cells did not induce graft-versus-host disease. These results establish the potential of NKT cells to serve as a safe and effective platform for CAR-directed cancer immunotherapy.

Topics: 4-1BB Ligand; Animals; Antigens, CD1d; Cancer Vaccines; CD28 Antigens; Cell Line; Cell Proliferation; Cytokines; Cytotoxicity, Immunologic; Gangliosides; Graft vs Host Disease; Humans; Immunotherapy; Lymphocyte Activation; Macrophages; Mice; Natural Killer T-Cells; Neoplasm Metastasis; Neuroblastoma; Protein Structure, Tertiary; Receptors, Antigen; Retroviridae; Transduction, Genetic; Treatment Outcome

Chimeric antigen receptor (CAR) therapy has begun to demonstrate success as a novel treatment modality for hematologic malignancies. The success observed thus far has been with T cells permanently engineered to express chimeric receptors. T cells engineered using RNA electroporation represent an alternative with the potential for similar efficacy and greater safety when initially targeting novel antigens. Neuroblastoma is a common pediatric solid tumor with the potential to be targeted using immunotherapy. We performed xenograft studies in NSG mice in which we assessed the efficacy of both permanently modified and transiently modified CAR T cells directed against the neuroblastoma antigen GD2 in both local and disseminated disease models. Disease response was monitored by tumor volume measurement and histologic examination, as well as in vivo bioluminescence. RNA-modified GD2 CAR T cells mediated rapid tumor destruction when delivered locally. A single infusion of lentivirally modified GD2 CAR T cells resulted in long-term control of disseminated disease. Multiple infusions of RNA GD2 CAR T cells slowed the progression of disseminated disease and improved survival, but did not result in long-term disease control. Histologic examination revealed that the transiently modified cells were unable to significantly penetrate the tumor environment when delivered systemically, despite multiple infusions of CAR T cells. Thus, we demonstrate that RNA-modified GD2 CAR T cells can mediate effective antitumor responses in vivo, and permanently modified cells are able to control disseminated neuroblastoma in xenograft mice. Lack of long-term disease control by RNA-engineered cells resulted from an inability to penetrate the tumor microenvironment.

Topics: Animals; Antigens, Neoplasm; Gangliosides; Humans; Immunohistochemistry; Immunotherapy; Mice; Mice, Inbred NOD; Mice, SCID; Neuroblastoma; Protein Engineering; Receptors, Antigen, T-Cell; T-Lymphocytes, Cytotoxic; Transduction, Genetic; Xenograft Model Antitumor Assays

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.

Topics: Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Antigens, Surface; Antineoplastic Agents; Cell Line, Tumor; Complement System Proteins; Cytotoxicity Tests, Immunologic; Cytotoxicity, Immunologic; Gangliosides; Humans; Immunophenotyping; Leukocyte Count; Monitoring, Immunologic; Neuroblastoma; Phenotype; Quality Control; Reproducibility of Results

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.

Topics: Amino Acid Sequence; Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Base Sequence; Binding, Competitive; Cancer Vaccines; Cell Line, Tumor; Gangliosides; Humans; Kinetics; Mice; Molecular Sequence Data; Neuroblastoma; Protein Binding; Sequence Alignment

The mechanism of the inhibitory effect of anti-GD2 ganglioside (GD2) 14G2a mouse monoclonal antibody (mAb) on human neuroblastoma cells survival was studied in vitro. It was recently shown in IMR-32 cells that death induced by this antibody exhibited several characteristics typical of apoptosis. In this study we used cytotoxixity assays, qRT-PCR and immunoblotting to evaluate the response of several human neuroblastoma cell lines to the anti-GD2 14G2a mAb. We showed that the mAb decreases all three aurora kinases expression and phosphorylation in IMR-32 and LA-N-1 cells. Most importantly, we show, that MK-5108 specific aurora A kinase inhibitor decreases neuroblastoma cell survival, and when used in combination with the mAb, significantly potentiates cytotoxicity against IMR-32, CHP-134, and LA-N-5 neuroblastoma cells in vitro. It was shown that downregulation of aurora A kinase by the therapeutic antibody is associated with decreased levels of MYCN protein in cytoplasm, and induced expression of PHLDA1 and P53 proteins.

Topics: Animals; Antibodies, Monoclonal; Aurora Kinase A; Caspase 3; Caspase 7; Cell Line, Tumor; Cell Survival; Cyclohexanecarboxylic Acids; Flow Cytometry; Gangliosides; Gene Expression; Humans; Immunoblotting; Mice; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Phosphorylation; Protein Kinase Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; Thiazoles; Transcription Factors; Tumor Suppressor Protein p53

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.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Cricetinae; Enzyme-Linked Immunosorbent Assay; Gangliosides; Humans; Neuroblastoma; Sensitivity and Specificity

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood, with grim prognosis in a half of patients. Exosomes are nanometer-sized membrane vesicles derived from the multivesicular bodies (MVBs) of the endocytic pathway and released by normal and neoplastic cells. Tumor-derived exosomes have been shown in different model systems to carry molecules that promote cancer growth and dissemination. In this respect, we have here performed the first characterization and proteomic analysis of exosomes isolated from human NB cell lines by filtration and ultracentrifugation. Electron microscopy demonstrated that NB-derived exosomes exhibited the characteristic cup-shaped morphology. Dynamic light scattering studies showed a bell-shaped curve and a polydispersity factor consistent with those of exosomes. Zeta potential values suggested a good nanoparticle stability. We performed proteomic analysis of NB-derived exosomes by two dimension liquid chromatography separation and mass spectrometry analyses using the multidimensional protein identification technology strategy. We found that the large majority of the proteins identified in NB derived exosomes are present in Exocarta database including tetraspanins, fibronectin, heat shock proteins, MVB proteins, cytoskeleton-related proteins, prominin-1 (CD133), basigin (CD147) and B7-H3 (CD276). Expression of the CD9, CD63 and CD81 tetraspanins, fibronectin, CD133, CD147 and CD276 was validated by flow cytometry. Noteworthy, flow cytometric analysis showed that NB-derived exosomes expressed the GD2 disialoganglioside, the most specific marker of NB. In conclusion, this study shows that NB-derived exosomes express a discrete set of molecules involved in defense response, cell differentiation, cell proliferation and regulation of other important biological process. Thus, NB-derived exosomes may play an important role in the modulation of tumor microenvironment and represent potential tumor biomarkers.

Topics: Cell Line, Tumor; Disease Progression; Exosomes; Flow Cytometry; Gangliosides; Humans; Neuroblastoma; Proteome; Proteomics; Reproducibility of Results

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.

Topics: Animals; Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Antineoplastic Agents; Cell Line, Tumor; Coculture Techniques; Complement System Proteins; Drug Resistance, Neoplasm; Female; Fenretinide; Gangliosides; Humans; Killer Cells, Natural; Mice; Neuroblastoma; Receptors, Death Domain; Xenograft Model Antitumor Assays

Immunocytokines (ICKs) targeting cytokines to the tumor environment using antibodies directed against a tumor-associated antigen often have a higher therapeutic index than the corresponding unconjugated cytokines. Various ICKs displaying significant antitumoral effects in several murine tumor models have already been developed, and some of them, in particular interleukin (IL)-2-based ICKs, are in Phase II clinical trials. Although sharing common biological activities with IL-2 in vitro, IL-15 is now considered as having a better potential in antitumor immunotherapeutical strategies and has been shown to be less toxic than IL-2 in preclinical studies. We previously developed the fusion protein RLI, linking a soluble form of human IL-15Rα-sushi+ domain to human IL-15. RLI showed better biological activities than IL-15 in vitro as well as higher antitumoral effects in vivo in murine and human cancer models. Here, we investigated, in the context of an ICK, the effect of associating RLI with an antibody targeting the GD2 ganglioside, a validated tumoral target expressed on many neurectodermal tumors. Anti-GD2-RLI fully retained the cytokine potential of RLI and the antibody effector functions (antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity). It displayed strong antitumor activities in two syngeneic cancer models in immunocompetent mice (subcutaneous EL4 and metastatic NXS2). Its therapeutic potency was higher than those of RLI and anti-GD2 alone or in combination. We suggest that this is related to its bifunctional (cytokine and antibody) nature.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neoplasm; Antibody-Dependent Cell Cytotoxicity; Antigens, Neoplasm; Cell Line, Tumor; Cell Proliferation; Female; Gangliosides; Humans; Immunotherapy; Interleukin-15; Interleukin-15 Receptor alpha Subunit; Liver Neoplasms; Lymphoma, T-Cell; Male; Mice; Mice, Inbred A; Mice, Inbred C57BL; Neoplasm Metastasis; Neuroblastoma; Protein Binding; Recombinant Fusion Proteins

The bone marrow (BM) TH, ELAVL4 and GD2 genes expression was evaluated in 331 samples from 57 different stage neuroblastoma (NB) patients, 26 BM samples from patients without NB and samples from 2 NB cell lines (IMR-32, Kelly) by real-time PCR. BM samples were considered NB-positive if PHOX2B expression was found or tumor cells were detected in BM smears. TH expression was not revealed in normal BM and was significantly lower in NB-negative samples. Expression of PHOX2B, TH and GD2 remained stable throughout NB treatment, while ELAVL4 expression was down-modulated. ROC-analysis revealed similar initial and follow-up values of TH and PHOX2B in NB patients' bone marrow making it possible to be used for disease detection and monitoring. The test prediction value was 0.994 and 0.952, respectively. The additional test for TH didn't increase the test effectiveness in comparison with PHOX2B test. ELAVL4 and GD2 assessment didn't add diagnostic value for BM involvement monitoring in NB patients.

Topics: Biomarkers, Tumor; Bone Marrow Neoplasms; Cell Line, Tumor; ELAV Proteins; ELAV-Like Protein 4; Gangliosides; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Neoplasm Staging; Nerve Tissue Proteins; Neuroblastoma; Neuronal Apoptosis-Inhibitory Protein; Real-Time Polymerase Chain Reaction; Transcription Factors

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.

Topics: Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; CD3 Complex; Cell Line, Tumor; Child; Gangliosides; Gene Expression; Genetic Engineering; Genetic Vectors; Humans; Immunotherapy, Adoptive; Jejunal Neoplasms; Jejunum; Killer Cells, Natural; Neuroblastoma; Receptors, Antigen; Recombinant Fusion Proteins; Retroviridae; Transduction, Genetic

Topics: Antibodies, Monoclonal; Child; Combined Modality Therapy; Gangliosides; Humans; Immunotherapy; Monitoring, Physiologic; Neoplasm Recurrence, Local; Neuroblastoma; Nursing Assessment

Neuroblastoma is one of the most challenging malignancies of childhood, being associated with the highest death rate in paediatric oncology, underlining the need for novel therapeutic approaches. Typically, patients with high risk disease undergo an initial remission in response to treatment, followed by disease recurrence that has become refractory to further treatment. Here, we demonstrate the first silica nanoparticle-based targeted delivery of a tumor suppressive, pro-apoptotic microRNA, miR-34a, to neuroblastoma tumors in a murine orthotopic xenograft model. These tumors express high levels of the cell surface antigen disialoganglioside GD2 (GD(2)), providing a target for tumor-specific delivery.. Nanoparticles encapsulating miR-34a and conjugated to a GD(2) antibody facilitated tumor-specific delivery following systemic administration into tumor bearing mice, resulted in significantly decreased tumor growth, increased apoptosis and a reduction in vascularisation. We further demonstrate a novel, multi-step molecular mechanism by which miR-34a leads to increased levels of the tissue inhibitor metallopeptidase 2 precursor (TIMP2) protein, accounting for the highly reduced vascularisation noted in miR-34a-treated tumors.. These novel findings highlight the potential of anti-GD(2)-nanoparticle-mediated targeted delivery of miR-34a for both the treatment of GD(2)-expressing tumors, and as a basic discovery tool for elucidating biological effects of novel miRNAs on tumor growth.

Topics: Animals; Apoptosis; Cell Line, Tumor; Gangliosides; Gene Expression; Gene Expression Profiling; Humans; Mice; Mice, SCID; MicroRNAs; N-Myc Proto-Oncogene Protein; Nanoconjugates; Neovascularization, Pathologic; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Xenograft Model Antitumor Assays

The ganglioside GD2 is an attractive target for immunotherapy of neuroectodermal tumors. We tested a unique bispecific antibody anti-CD3 × anti-GD2 (3F8BiAb) for its ability to redirect activated T cells (ATC) to target GD2-positive neuroblastomas.. ATC were generated from normal human peripheral blood mononuclear cells (PBMC) by stimulating the PBMC with OKT3 and expanding the T cells in the presence of interleukin 2 (IL-2) for 14 days. ATC were armed with 3F8BiAb (100 ng/10(6)  cells) or Her2BiAb (50 ng/10(6)  cells) prior to use. 3F8 BiAb were tested for its dual-binding specificity to GD2 expressed on cancer cell lines and CD3 expressed on ATC. 3F8BiAb-armed ATC were further tested ex vivo for their cytotoxicity against GD2 positive tumor targets and its ability to induce cytokine response upon binding to targets.. GD2 expression in neuroblastoma cells was confirmed by FACS analysis. Specific binding of 3F8BiAb to the tumor targets as well as to ATC was confirmed by FACS analysis. 3F8BiAb-armed ATC exhibited specific killing of GD2 positive neuroblastoma cell lines significantly above unarmed ATC (P < 0.001). GD2BiAb-armed ATC secreted significantly higher levels of Th(1) cytokines and chemokines compared to unarmed ATC (P < 0.001).. These preclinical findings support the potential of a novel immunotherapeutic approach to target T cells to neuroblastoma.

Topics: Antibodies, Bispecific; Binding Sites, Antibody; CD3 Complex; Cell Line, Tumor; Cytokines; Cytotoxicity Tests, Immunologic; Gangliosides; Humans; Immunotherapy; Lymphocyte Activation; Neuroblastoma; Receptor, ErbB-2; T-Lymphocytes, Cytotoxic

Polyethylene glycol-grafted polyethylenimine (PEG-g-PEI) which was functionalized with a neuroblastoma cell-specific ligand, the GD2 single chain antibody (scAb(GD2)), was synthesized in order to effectively deliver Bcl-2 siRNA into neuroblastoma cells. This polymer was complexed first with superparamagnetic iron oxide nanoparticle (SPION) to get a MRI-visible targeted non-viral vector (scAb(GD2)-PEG-g-PEI-SPION) and then with Bcl-2 siRNA to form nanoparticles showing low cytotoxicity. The targeting capacity of scAb(GD2)-PEG-g-PEI-SPION was successfully verified in vivo and in vitro by magnetic resonance imaging. The single chain antibody encoded targeted polyplex was more effective in transferring Bcl-2 siRNA than the nontargeting one in SK-N-SH cells, a human neuroblastoma cell line, resulting in a 46.34% inhibition in the expression of Bcl-2 mRNA. Consequently, a high level of cell apoptosis up to 50.76% and a significant suppression of tumor growth were achieved, which indicates that scAb(GD2)-PEG-g-PEI-SPION is a promising magnetic resonance imaging-visible non-viral vector for targeted neuroblastoma siRNA therapy and diagnosis.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; Drug Carriers; Electrophoretic Mobility Shift Assay; Female; Gangliosides; Genetic Vectors; Humans; Immunohistochemistry; Magnetic Resonance Imaging; Magnetite Nanoparticles; Mice; Mice, Nude; Microscopy, Confocal; Neoplasms, Experimental; Neuroblastoma; Particle Size; Polyethylene Glycols; Polyethyleneimine; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Single-Chain Antibodies; Transfection; Xenograft Model Antitumor Assays

Survival outcomes for patients with high-risk neuroblastoma (NB) have significantly improved with anti-disialoganglioside GD2 mAb therapy, which promotes NK cell activation through antibody-dependent cell-mediated cytotoxicity. NK cell activation requires an interaction between inhibitory killer cell immunoglobulin-like receptors (KIRs) and HLA class I ligands. NK cells lacking KIRs that are specific for self HLA are therefore "unlicensed" and hyporesponsive. mAb-treated NB patients lacking HLA class I ligands for their inhibitory KIRs have significantly higher survival rates, suggesting that NK cells expressing KIRs for non-self HLA are mediating tumor control in these individuals. We found that, in the presence of mAb, both licensed and unlicensed NK cells are highly activated in vitro. However, HLA class I expression on NB cell lines selectively inhibited licensed NK cell activity, permitting primarily unlicensed NK cells to mediate antibody-dependent cell-mediated cytotoxicity. These results indicate that unlicensed NK cells play a key antitumor role in patients undergoing mAb therapy via antibody-dependent cell-mediated cytotoxicity, thus explaining the potent "missing KIR ligand" benefit in patients with NB.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antibody-Dependent Cell Cytotoxicity; Antineoplastic Agents; Cell Line, Tumor; Cells, Cultured; Coculture Techniques; Disease-Free Survival; Gangliosides; Histocompatibility Antigens Class I; Humans; Immunoglobulin G; Infant; Interferon-gamma; Kaplan-Meier Estimate; Killer Cells, Natural; Leukocytes, Mononuclear; Lymphocyte Activation; Neuroblastoma; Receptors, KIR

We investigated the anti-tumor effect of peritumoral resveratrol in combination with immunotherapy in vivo in neuroblastoma-bearing mice. Subcutaneous NXS2 tumors were induced in A/J mice. On day 10, some mice received 15 mcg of intravenous immunocytokine for 5 days, mice received 20 mg of peritumoral resveratrol twice a week (starting on day 12) for a total of 5 injections, and a separate group received a combination of both regimens. Tumor progression and survival were assessed every 3-4 days. Blood and primary tumor tissue samples were collected on day 20 for Complete Blood Count and CD45 immunohistochemistry and histology, respectively. The primary tumor regressed in all mice receiving peritumoral resveratrol. Most of these mice receiving peritumoral resveratrol alone developed metastatic tumors and recurrence of the primary tumor after cessation of therapy. When resveratrol and immunocytokine regimens were combined, 61% of the mice receiving this combination therapy resolved their primary tumors and survived without developing metastatic tumors, compared to 15 and 13% receiving resveratrol or immunocytokine alone, respectively. None of the therapeutic regimes prevented lymphocyte infiltration or affected the complete blood count. Greater necrosis was observed microscopically in tumors from mice receiving the combination therapy. These results demonstrate that the combination therapy of peritumoral resveratrol plus intravenous immunocytokine provides better anti-tumor effects in this model than either therapy alone.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Combined Modality Therapy; Flow Cytometry; Gangliosides; Immunoglobulin G; Immunotherapy; Interleukin-2; Leukocyte Common Antigens; Leukocytes; Mice; Mice, Inbred A; Neoplasm Recurrence, Local; Neuroblastoma; Resveratrol; Stilbenes; Survival; Tumor Burden

Overexpression of the GD2 ganglioside (GD2) is a hallmark of neuroblastoma. The antigen is used in neuroblastoma diagnosis and to target newly developed therapies to cancer cells. Peptide mimetics are novel approaches in the design of antigens for vaccine development. We previously reported the isolation of five GD2-mimicking peptides from the LX-8 phage display library with the monoclonal antibody (mAb) 14G2a. The goal of our current study was to analyze and optimize the binding of the peptide mimetics to the mAb 14G2a. Therefore, we performed further experiments and supported them with molecular modeling to investigate structure-activity relationships that are the basis for the observed mimicry of GD2 by our peptides. Here, we show that the peptides have overlapping binding sites on the mAb, 14G2a and restricted specificity, as they did not crossreact with other ganglioside-specific antibodies tested. In addition we demonstrate that the phage environment was involved in the process of selection of our peptides. The AAEGD sequence taken from the viral major coat protein, p8, and added to the C-termini of the peptides #65, #85 and #94 significantly improved their binding to the mAb, 14G2a. By application of analogs with amino acid substitutions and sequence truncations, we elucidated the structure-activity relationships necessary for the interactions between the 14G2a mAb and the peptide #94 (RCNPNMEPPRCF). We identified amino acids indispensable for the observed GD2-mimicry by #94 and confirmed a pivotal role of the disulphide bridge between the cysteine residues of #94 for binding to the mAb 14G2a. More importantly, we report five new peptides demonstrating a significant improvement of mAb 14G2a binding. The experimental data were supported and expanded with molecular modeling tools. Taken together, the experimental results and the in silico data allowed us to probe in detail the mechanism of the molecular mimicry of GD2 by the peptides. Additionally, we significantly optimized binding of the leading peptide sequence #94 to the mAb 14G2a. We can conclude that our findings add to the knowledge on factors governing selections of peptide mimetics from phage-display libraries.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Binding Sites; Cell Line, Tumor; Gangliosides; Humans; Models, Molecular; Molecular Mimicry; Molecular Sequence Data; Neuroblastoma; Peptide Library; Peptides; Structure-Activity Relationship

To optimise and simplify preparation of targeted liposomes for efficient siRNA delivery to neuroblastoma, the most common solid tumour in early childhood.. Liposomes containing siRNA were prepared by combining the novel dual asymmetric centrifugation (DAC) method and the recently optimised sterol-based post-insertion technique (SPIT) to couple anti-GD2 antibody for selective interaction with neuroblastoma cells. Cultured human neuroblastoma cell lines were used to evaluate the efficiency of siRNA delivery.. The size of liposomes prepared by DAC ranged from 190 to 240 nm; siRNA encapsulation efficiency was up to 50%. An average of 70 and 100 molecules of anti-GD2 antibody per particle were coupled. A significant association of liposomes with neuroblastoma cells as well as effective siRNA delivery was observed only when anti-GD2 antibody was coupled. Preliminary data suggest delivery of siRNA using anti-GD2-liposomes occurs via GD2-mediated endocytosis. Vascular endothelial growth factor A (VEGF-A) was down-regulated using siRNA delivered by anti-GD2-liposomes.. DAC and SPIT allow for the straightforward preparation of liposomes for the targeted delivery of siRNA. Anti-GD2-liposomes thus produced can serve as versatile carriers of siRNA to neuroblastoma cells.

Topics: Antibodies, Monoclonal; Cell Line, Tumor; Centrifugation; Drug Compounding; Gangliosides; Gene Expression Regulation, Neoplastic; Gene Transfer Techniques; Humans; Neuroblastoma; Real-Time Polymerase Chain Reaction; RNA, Small Interfering; Sterols

The anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor that is involved in the pathogenesis of different types of human cancers, including neuroblastoma (NB). In NB, ALK overexpression, or point mutations, are associated with poor prognosis and advanced stage disease. Inhibition of ALK kinase activity by small-molecule inhibitors in lung cancers carrying ALK translocations has shown therapeutic potential. However, secondary mutations may occur that, generate tumor resistance to ALK inhibitors. To overcome resistance to ALK inhibitors in NB, we adopted an alternative RNA interference (RNAi)-based therapeutic strategy that is able to knockdown ALK, regardless of its genetic status [mutated, amplified, wild-type (WT)]. NB cell lines, transduced by lentiviral short hairpin RNA (shRNA), showed reduced proliferation and increased apoptosis when ALK was knocked down. In mice, a nanodelivery system for ALK-specific small interfering RNA (siRNA), based on the conjugation of antibodies directed against the NB-selective marker GD(2) to liposomes, showed strong ALK knockdown in vivo in NB cells, which resulted in cell growth arrest, apoptosis, and prolonged survival. ALK knockdown was associated with marked reductions in vascular endothelial growth factor (VEGF) secretion, blood vessel density, and matrix metalloproteinases (MMPs) expression in vivo, suggesting a role for ALK in NB-induced neoangiogenesis and tumor invasion, confirming this gene as a fundamental oncogene in NB.

Topics: Anaplastic Lymphoma Kinase; Animals; Apoptosis; Blotting, Western; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Female; Gangliosides; HeLa Cells; Humans; Immunoenzyme Techniques; Liposomes; Matrix Metalloproteinases; Mice; Mice, Nude; Mice, SCID; Mutation; Neovascularization, Pathologic; Neuroblastoma; Phosphorylation; Real-Time Polymerase Chain Reaction; Receptor Protein-Tyrosine Kinases; RNA Interference; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Survival Rate

Neuroblastic tumors (NBT) are composed by neuroblasts and Schwannian-like stroma. The origin of these two cell subtypes remains unclear. In this study, we describe, a neuroblastic-like subpopulation in neuroblastoma (NB) coexpressing GD2 and S100A6, neuroblastic and glial lineage markers respectively. The GD2(+)/S100A6(+) neuroblastic subpopulation was found to be enriched in low risk NB, distributed around the perivascular niche. Some stromal bundles showed GD2(+)/S100A6 costaining. Metastatic bone marrow specimens also showed GD2(+)/S100A6(+) cells. During in vitro retinoic acid induced differentiation of NB cell lines, rare GD2(+)/S100A6 neuroblatic cells appeared. We conclude that GD2(+)/S100A6(+) neuroblasts may represent a tumoral glial precursor subpopulation in NBT.

Topics: Antigens, Differentiation; Cell Cycle Proteins; Cell Differentiation; Cell Lineage; Gangliosides; Glioma; Humans; Neoplastic Stem Cells; Neural Stem Cells; Neuroblastoma; Neuroglia; S100 Calcium Binding Protein A6; S100 Proteins; Schwann Cells

Monoclonal antibodies (mAb) against GD2 ganglioside have been shown to be effective for the treatment of neuroblastoma. Beneficial actions are, however, associated with generalized pain due to the binding of anti- GD2 mAbs to peripheral nerve fibers followed by complement activation. Neuroblastoma cells that express GD2 also express its O-acetyl derivative, O-acetyl- GD2 ganglioside (OAcGD2). Hence, we investigated the distribution of OAcGD2 in human tissues using mAb 8B6 to study the cross-reactivity of mAb 8B6 with human tissues.. The distribution of OAcGD2 was performed in normal and malignant tissues using an immunoperoxydase technique. Anti-tumor properties of mAb 8B6 were studied in vitro and in vivo in a transplanted tumor model in mice. We found that OAcGD2 is not expressed by peripheral nerve fibers. Furthermore, we demonstrated that mAb 8B6 was very effective in the in vitro and in vivo suppression of the growth of tumor cells. Importantly, mAb 8B6 anti-tumor efficacy was comparable to that of mAb 14G2a specific to GD2.. Development of therapeutic antibodies specific to OAcGD2 may offer treatment options with reduced adverse side effects, thereby allowing dose escalation of antibodies.

Topics: Antibodies, Monoclonal; Apoptosis; Cell Line, Tumor; Cell Survival; Gangliosides; Humans; In Vitro Techniques; Neuroblastoma; Peripheral Nervous System

The presence of disseminated neuroblastoma cells in bone marrow in children over 1 year old is important for clinical staging and risk assessment at diagnosis and for therapy monitoring. Reliable detection of single tumor cells in bone marrow may be a factor of great prognostic significance. Currently disseminated NBL cells are detected by conventional cytomorphological examination of bone marrow smears but this method is not sensitive enough to detect single tumor cells. The development of more sensitive methods of evaluation bone marrow is needed. For this purpose Neuroblastoma Bone Marrow Committee developed standard immunocytochemical assay based on detection of the neuroblastoma-specific antigen. Disialoganglioside GD2 is a surface antigen expressed on neuroblastoma cells but not detectable on the surface of normal bone marrow cells. This article describes significance of immunoctochemical method of identification neuroblastoma cells in bone marrow.

Topics: Biomarkers, Tumor; Bone Marrow; Bone Marrow Cells; Gangliosides; Humans; Immunohistochemistry; Neoplasm Staging; Neuroblastoma; Prognosis

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. The majority of children suffers from high risk neuroblastoma and has disseminated disease at the time of diagnosis. Despite recent advances in chemotherapy, the prognoses for children with high risk NB remain poor. Therefore, new treatment modalities are urgently needed. GD2 ganglioside is an antigen that is highly expressed on NB cells with only limited distribution on healthy tissues. Consequently, it appears to be an ideal target for both active and passive immunotherapy. The immunological effector mechanisms mediated by anti-GD2 monoclonal antibodies (mAbs) have been already well characterized. However, a growing number of reports suggest that GD2-specific antibodies may exhibit anti-proliferative effects without the immune system involvement. Here, we have shown that anti-GD2 14G2a mAb is capable of decreasing survival of IMR-32 human neuroblastoma cells in a dose-dependent manner. Death induced by this antibody exhibited several characteristics typical for apoptosis such as increased number of Annexin V- and propidium iodide-positive cells, cleavage of caspase 3 and prominent rise in caspase activity. The use of a pan caspase inhibitor Z-VAD-fmk suggested that the killing potential of this mAb is partially caspase-dependent. 14G2a mAb was rapidly endocytosed upon antigen binding. Employment of chloroquine, an inhibitor of lysosomal degradation, did not rescue IMR-32 cells from antibody-induced cell death suggesting lack of ceramide involvement in the observed effect. Most importantly, our studies showed that at particular drug concentrations 14G2a mAb exerts a synergistic effect with doxorubicin and topotecan, as well as an additive effect with carboplatin in killing IMR-32 cells in vitro. Our results provide guidance regarding how to best combine GD2-specific 14G2a antibody with existing cancer therapeutic agents to improve available treatment modalities for neuroblastoma.

Topics: Antibodies, Monoclonal; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Carboplatin; Cell Adhesion; Cell Line, Tumor; Doxorubicin; Drug Synergism; Flow Cytometry; Gangliosides; Humans; Immunotherapy; Microscopy, Fluorescence; Neuroblastoma; Topotecan

High-risk Neuroblastoma (NB) has still a poor prognosis. Liposomes targeted to NB cells and encapsulating antisense CpG-containing oligonucleotides (TL-asCpG) had increased anti-tumour efficacy in NB xenografts compared to free asCpG. Interleukin 10 (IL-10) suppresses antigen presenting cell activation contributing to tumour-mediated immune suppression. In principle, combination of TL-asCpG and antibodies against IL-10 receptor (aIL-10R) could prolong immune system activation, leading to better therapeutic results. Mice treated with TL-asCpG 4 h after human NB cell inoculation survived significantly longer than controls. An increased life span was achieved also in mice receiving TL-asCpG 24 and 72 h after NB cell challenge. The addition of aIL-10R to TL-asCpG in the 4-h protocol significantly increased the percentage of long term survivors compared to TL-asCpG only. Surviving mice treated with the combined strategy were completely cured. In contrast, long term surviving mice treated only with TL-asCpG presented lymph node infiltration with NB cells. TL-asCpG plus aIL-10R treatment was significantly superior to TL-asCpG alone also for the 24-h protocol. Ex vivo experiments demonstrated that the combined therapy evoked a stronger and more prolonged immune system activation compared to monotherapy. These results support the feasibility of a clinical trial with TL-asCpG and aIL-10R in advanced NB patients.

Topics: Animals; Antibodies, Monoclonal; Cell Line, Tumor; Cell Proliferation; CpG Islands; Female; Gangliosides; Humans; Immunity, Innate; Interleukin-10; Liposomes; Mice; Mice, Nude; Neoplasm Transplantation; Neuroblastoma; Oligonucleotides, Antisense; Proto-Oncogene Proteins c-myb; Receptors, Interleukin-10; Xenograft Model Antitumor Assays

Despite aggressive multimodality therapy, most neuroblastoma-bearing patients relapse and survival rate remains poor. Exploration of alternative therapeutic modalities is needed. Carbon nanotubes (CNTs), revealing optical absorbance in the near-infrared region, warrant their merits in photothermal therapy. In order to specifically target disialoganglioside (GD2) overexpressed on the surface of neuroblastoma stNB-V1 cells, GD2 monoclonal antibody (anti-GD2) was conjugated to acidified CNTs. To examine the fate of anti-GD2 bound CNTs after incubation with stNB-V1 cells, rhodamine B was labeled on carboxylated CNTs functionalized with and without anti-GD2. Our results illustrated that anti-GD2-linked CNTs were extensively internalized by neuroblastoma cells via GD2-mediated endocytosis. In addition, we showed that anti-GD2 bound CNTs were not ingested by PC12 cells without GD2 expression. After anti-GD2 conjugated CNTs were incubated with neuroblastoma cells for 6 h and endocytosed by the cells, CNT-laden neuroblastoma cells were further irradiated with an 808 nm near-infrared (NIR) laser with intensity ramping from 0.6 to 6 W cm(-2) for 10 min which was then maintained at 6 W cm(-2) for an additional 5 min. Post-NIR laser exposure, and after being examined by calcein-AM dye, stNB-V1 cells were all found to undergo necrosis, while non-GD2 expressing PC12 cells all remained viable. Based on the in vitro study, CNTs bound with anti-GD2 have 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.

Topics: Animals; Antibodies, Monoclonal; Cell Line, Tumor; Cell Survival; Fluorescent Dyes; Gangliosides; Humans; Hyperthermia, Induced; Immunoconjugates; Microscopy, Electron, Transmission; Nanotubes, Carbon; Neuroblastoma; PC12 Cells; Rats; Rhodamines

Topics: Antibodies, Monoclonal; Clinical Trials, Phase III as Topic; Gangliosides; Humans; Immunotherapy; Neuroblastoma; Survival Rate; Treatment Outcome

Neuroblastoma, the most common extracranial solid tumour of childhood, is a malignancy of unknown origin and non-specific symptoms. One of the markers of the disease is GD2 ganglioside (disialoganglioside), which is abundantly expressed on the surface of neuroblastoma cells. Gangliosides are known to be shed by tumour cells and this phenomenon can be significant in cancer progression as they inhibit a number of immune responses both in vitro and in vivo. In search for novel markers useful in monitoring and prognosis of neuroblastoma, we developed and validated a new quantitative method of GD2 ganglioside analysis in human blood plasma. We evaluated the level of gangliosides in blood serum of 34 neuroblastoma patients using high-performance liquid chromatography. The technique was used to detect fluorescently labelled oligosaccharides derived from serum glycosphingolipids by enzymatic digestion with ceramide glycanase. The developed method allowed determination of GD2 concentrations at the picomole level and required only 40 microl of plasma, which should be particularly useful when the quantity of clinical material is limiting. Moreover, this method can be applied to study concentration of other gangliosides, as shown for GD3 ganglioside. Analysis of plasma samples from the 34 neuroblastoma patients did not reveal any correlations between the concentration of GD2 ganglioside and clinical parameters, including the results of therapy; it showed, however, that the concentration of GD2 ganglioside in the plasma of neuroblastoma patients decreased substantially in the course of treatment.

Topics: Case-Control Studies; Cell Line, Tumor; Child; Child, Preschool; Chromatography, High Pressure Liquid; Female; Flow Cytometry; Gangliosides; Humans; Infant; Male; Neuroblastoma

A major challenge for inducing antitumor immune responses with native or modified tumor/self-Ags in tumor-bearing hosts relates to achieving efficient uptake and processing by dendritic cells (DCs) to activate immune effector cells and limit the generation of regulatory T cell activity. We analyzed the ability of therapeutic DC vaccines expressing a CD166 cross-reactive mimotope of the GD2 ganglioside, 47-LDA, to selectively expand adoptively transferred, tumor-specific T cells in NXS2 neuroblastoma tumor-bearing syngeneic mice. Before the adoptive cell transfer and DC vaccination, the tumor-bearing mice were lymphodepleted by nonmyeloablative total body irradiation or a myeloablative regimen that required bone marrow transplantation. The 47-LDA mimotope was presented to DCs either as a linear polypeptide in conjunction with universal Th epitopes or as a fusion protein with the murine IgG2a Fc fragment (47-LDA-Fcgamma2a) to deliver the antigenic cassette to the activating Fcgamma receptors. We demonstrate that immunization of adoptively transferred T cells in tumor-bearing mice with the 47-LDA mimotope expressed in the context of the activating Fc fusion protein induced higher levels of antitumor immune responses and protection than the 47-LDA polypeptide-DC vaccine. The antitumor efficacy of the therapeutic 47-LDA-Fcgamma2a-DC vaccine was comparable to that achieved by a virotherapy-associated cancer vaccine using a recombinant oncolytic vaccinia virus expressing the 47-LDA-Fcgamma2a fusion protein. The latter treatment, however, did not require total body irradiation or adoptive cell transfer and resulted in induction of antitumor immune responses in the setting of established tolerance, paving the way for testing novel anticancer treatment strategies.

Topics: Activated-Leukocyte Cell Adhesion Molecule; Adoptive Transfer; Animals; Cancer Vaccines; CD8-Positive T-Lymphocytes; Dendritic Cells; Epitopes; Female; Gangliosides; Kaplan-Meier Estimate; Mice; Mice, Inbred A; Neuroblastoma; Oncolytic Virotherapy; Receptors, IgG; Recombinant Fusion Proteins; Whole-Body Irradiation

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood and the most frequently diagnosed neoplasm during infancy. Despite of aggressive treatment strategies, the 5-year survival rate for metastatic disease is still less than 60% and, consequently, novel therapeutic approaches are needed. For increasing the therapeutic index of anticancer drugs, while reducing side effects, one of the most promising strategies in modern chemotherapy is based on the development of innovative drug delivery systems, such as liposomes. "Anticancer drug"-loaded liposomes have demonstrated enhanced ability to target to the affected area, as well as increased antitumor efficacy compared to conventional drugs. Liposomes tend to extravasate preferentially and to accumulate into tumor interstitial fluids, due to the defective structure of the new angiogenic vessels within the tumor masses. This inherent tumor selectivity can be increased further by coupling tumor-specific antibodies or other targeting moieties to the surface of the lipid envelope. Here, we describe the methodology used in these studies, as well as the antitumor results obtained by the use of several "anticancer drugs," encapsulated into antibody- and peptide-targeted liposomal formulations, against NB.

Topics: Animals; Antineoplastic Agents; Doxorubicin; Drug Delivery Systems; Fenretinide; Gangliosides; Gold; Liposomes; Mice; Microscopy, Electron, Transmission; Neoplasm Transplantation; Neovascularization, Pathologic; Neuroblastoma

The GD2 ganglioside expressed on neuroectodermal tumor cells has been used as a target for passive and active immunotherapy in patients with malignant melanoma and neuroblastoma. We have reported that immunization of mice with a 47-LDA mimotope of GD2, isolated from a phage display peptide library with anti-GD2 mAb 14G2a, induces MHC class I-restricted CD8(+) T cell responses to syngeneic neuroblastoma tumor cells. The cytotoxic activity of the vaccine-induced CTLs was independent of GD2 expression, suggesting recognition of a novel tumor-associated Ag cross-reacting with 47-LDA. Glycan microarray and immunoblotting studies using 14G2a mAb demonstrated that this Ab is highly specific for the entire carbohydrate motif of GD2 but also cross-reacts with a 105 kDa glycoprotein expressed by GD2(+) and GD2(-) neuroblastoma and melanoma cells. Functional studies of tumor cells grown in three-dimensional collagen cultures with 14G2a mAb showed decreases in matrix metalloproteinase-2 activation, a process regulated by the 105 kDa-activated leukocyte cell adhesion molecule (ALCAM/CD166). A recombinant CD166 glycoprotein was shown to be recognized by 14G2a Ab and inhibition of CD166 expression by RNA interference ablated the cell sensitivity to lysis by 47-LDA-induced CD8(+) T cells in vitro and in vivo. The binding of 14G2a to CD166 was not disruptable by a variety of exo- and endo-glycosidases, implying recognition of a non-glycan epitope on CD166. These results suggest that the vaccine-induced CTLs recognize a 47-LDA cross-reactive epitope expressed by CD166, and reveal a novel mechanism of induction of potent tumor-specific cellular responses by mimotopes of tumor-associated carbohydrate Ags.

Topics: Activated-Leukocyte Cell Adhesion Molecule; Animals; Antigen Presentation; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cells, Cultured; Epitopes, T-Lymphocyte; Female; Ganglia, Spinal; Ganglioglioma; Gangliosides; Humans; Immunotherapy, Adoptive; Lymphocyte Activation; Melanoma; Mice; Mice, Inbred A; Molecular Mimicry; Neuroblastoma

Aberrant glycosylation is a universal feature of cancer cells. There are quantitative and qualitative changes in expression of gangliosides observed in tumors of a neuroectodermal origin such as neuroblastoma, melanoma and astrocytoma. The presence of large amounts of GD2 ganglioside on neuroblastoma cells, as compared to normal cells, opens the possibilities to use the tumor-associated carbohydrate antigen in diagnosis and immunotherapeutic approaches. In the quest for immunogens potentially capable of eliciting anti-GD2 ganglioside immune responses, we performed affinity purification of phage-displayed peptides from the LX-8 library (12-mer containing disulphide bridge). The library was screened with the biotinylated anti-GD2 ganglioside 14G2a mAb monoclonal antibody. Our goal was to isolate and characterize peptide mimics of GD2 ganglioside. Numerous individual phage clones that bound 14G2a mAb were identified with the application of immunoblotting technique in the phage pools yielded from the pannings. The phage-borne peptides were tested for their anti-GD2 ganglioside antibody binding ability using ELISA. Among these clones five different phage-displayed peptide sequences were identified. Moreover, we showed that the secondary structure of the peptides, stabilized by the disulfide bridging between cysteine residues at positions 2 and 11, was crucial for the binding of the peptides to 14G2a mAb. In a separate set of experiments, we observed a competition of the peptides, expressed on phages as well as in their synthetic form, with the nominal antigen GD2 ganglioside expressed on IMR-32 neuroblastoma cells for binding to 14G2a mAb. Based on the obtained results we concluded that all of these 5 peptides were mimics of the GD2 ganglioside.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Binding, Competitive; Cell Line, Tumor; Clone Cells; Disulfides; Enzyme-Linked Immunosorbent Assay; Gangliosides; Humans; Immunoblotting; Molecular Mimicry; Molecular Sequence Data; Neuroblastoma; Peptide Library; Peptides; Protein Binding; Sequence Analysis, Protein

The GD2 ganglioside expressed on neuroectodermal tumor cells is weakly immunogenic in tumor-bearing patients and induces predominantly IgM antibody responses in the immunized host. Using a syngeneic mouse challenge model with GD2-expressing NXS2 neuroblastoma, we investigated novel strategies for augmenting the effector function of GD2-specific antibody responses induced by a mimotope vaccine. We demonstrated that immunization of A/J mice with DNA vaccine expressing the 47-LDA mimotope of GD2 in combination with IL-15 and IL-21 genes enhanced the induction of GD2 cross-reactive IgG2 antibody responses that exhibited cytolytic activity against NXS2 cells. The combined immunization regimen delivered 1 day after tumor challenge inhibited subcutaneous (s.c.) growth of NXS2 neuroblastoma in A/J mice. The vaccine efficacy was reduced after depletion of NK cells as well as CD4(+) and CD8(+) T lymphocytes suggesting involvement of innate and adaptive immune responses in mediating the antitumor activity in vivo. CD8(+) T cells isolated from the immunized and cured mice were cytotoxic against syngeneic neuroblastoma cells but not against allogeneic EL4 lymphoma, and exhibited antitumor activity after adoptive transfer in NXS2-challenged mice. We also demonstrated that coimmunization of NXS2-challenged mice with the IL-15 and IL-21 gene combination resulted in enhanced CD8(+) T cell function that was partially independent of CD4(+) T cell help in inhibiting tumor growth. This study is the first demonstration that the mimotope vaccine of a weakly immunogenic carbohydrate antigen in combination with plasmid-derived IL-15 and IL-21 cytokines induces both innate and adaptive arms of the immune system leading to the generation of effective protection against neuroblastoma challenge.

Topics: Animals; Cancer Vaccines; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line; Dendritic Cells; Female; Gangliosides; Genetic Vectors; Immunotherapy, Adoptive; Interferon-gamma; Interleukin-15; Interleukins; Mice; Neuroblastoma; T-Lymphocyte Subsets

The disialoganglioside GD2, a carbohydrate antigen, is expressed on all tumors of neuroectodermal origin, including melanoma, neuroblastoma, sarcoma and small cell lung cancer. Due to its specific expression on tumor surfaces, GD2 is an attractive target for immunotherapies. The mouse/human chimeric anti-GD2 mAb ch14.18 is already applied in melanoma and neuroblastoma trials as a passive immunotherapy. To establish an active immunotherapy alternative, we aimed to replace the poorly immunogenic ganglioside with immunogenic peptides. Previously, we used the ch14.18 antibody to select GD2 peptide mimics from a phage display library. In the present study, two mimics of the ch14.18 epitope were coupled to keyhole limpet hemocyanin and used for immunizing BALB/c mice. Induction of a specific humoral immune response towards the original antigen GD2, both purified and expressed on neuroblastoma and melanoma cells, could be demonstrated in ELISA, Western blot, and immunofluorohistochemistry. As the elicited antibodies were of the IgG isotype, the mimotope conjugates were capable of recruiting T cell help and inducing memory phenomena. In conclusion, we show that an epitope of the carbohydrate antigen GD2 can successfully be translated into immunogenic peptide mimotopes. Our immunization experiments indicate that GD2 mimotopes are suitable for active immunotherapy of GD2-expressing tumors.

Topics: Animals; Antibodies, Neoplasm; Antigens, Neoplasm; Cancer Vaccines; Cell Line, Tumor; Cross Reactions; Gangliosides; Humans; Immunoglobulin G; Melanoma; Mice; Mice, Inbred BALB C; Neuroblastoma; Vaccination

Neuroblastoma treatment with chimeric antidisialoganglioside GD2 Ab ch14.18 showed objective antitumor responses. Production of anti-idiotypic Abs (Ab2) against ch14.18 (Ab1) in some cases was positively correlated with a more favorable prognosis. According to Jerne's network theory, a subset of anti-idiotypic Abs (Ab2beta) carries an "internal image" of the Ag and induces Abs (Ab3) against the original Ag. The molecular origin of an anti-idiotypic Ab response in tumor patients was not investigated previously. To clone anti-idiotypic Abs, B cells of a ch14.18-treated neuroblastoma patient with Ab2 serum reactivity were used to construct Ab phage display libraries. After repeated biopannings on ch14.18 and its murine relative, anti-GD2 mAb 14G2a, we selected 40 highly specific clones. Sequence analysis revealed at least 10 of 40 clones with different Ig genes. Identities to putative germline genes ranged between 94.90 and 100% for V(H) and between 93.90 and 99.60% for V(L). An overall high rate of replacement mutations suggested a strong Ag-driven maturation of the anti-idiotypic Abs. Two clones that were analyzed further, GK2 and GK8, inhibited binding of ch14.18 to GD2 just as the patient's serum did. GK8 alone inhibited >80% of the patient's anti-idiotypic serum Abs in binding to ch14.18. Rabbits vaccinated with GK8 or GK2 (weaker) produced Ab3 against the original target Ag GD2. GK8 may be useful as a tumor vaccine for GD2-positive [corrected] tumors.

Topics: Amino Acid Sequence; Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Bacteriophage M13; Binding Sites, Antibody; Binding, Competitive; Cancer Vaccines; Cloning, Molecular; Combinatorial Chemistry Techniques; Gangliosides; Humans; Immunoglobulin kappa-Chains; Immunoglobulin lambda-Chains; Immunoglobulin Variable Region; Mice; Molecular Sequence Data; Neoplasm Recurrence, Local; Neuroblastoma; Neuroectodermal Tumors; Peptide Library

Neuroblastoma, the most common solid tumor of infancy derived from the sympathetic nervous system, continues to present a formidable clinical challenge. Sterically stabilized immunoliposomes (SIL) have been shown to enhance the selective localization of entrapped drugs to solid tumors, with improvements in therapeutic indices. We showed that SIL loaded with doxorubicin (DXR) and targeted to the disialoganglioside receptor GD(2) [aGD(2)-SIL(DXR)] led to a selective inhibition of the metastatic growth of experimental models of human neuroblastoma. By coupling NGR peptides that target the angiogenic endothelial cell marker aminopeptidase N to the surface of DXR-loaded liposomes [NGR-SL(DXR)], we obtained tumor regression, pronounced destruction of the tumor vasculature, and prolonged survival of orthotopic neuroblastoma xenografts. Here, we showed good liposome stability, long circulation times, and enhanced time-dependent tumor accumulation of both the carrier and the drug. Antivascular effects against animal models of lung and ovarian cancer were shown for formulations of NGR-SL(DXR). In the chick embryo chorioallantoic assay, NGR-SL(DXR) substantially reduced the angiogenic potential of various neuroblastoma xenografts, with synergistic inhibition observed for the combination of NGR-SL(DXR) with aGD(2)-SIL(DXR). A significant improvement in antitumor effects was seen in neuroblastoma-bearing animal models when treated with the combined formulations compared with control mice or mice treated with either tumor- or vascular-targeted liposomal formulations, administered separately. The combined treatment resulted in a dramatic inhibition of tumor endothelial cell density. Long-term survivors were obtained only in animals treated with the combined tumor- and vascular-targeted formulations, confirming the pivotal role of combination therapies in treating aggressive metastatic neuroblastoma.

Topics: Animals; Apoptosis; Cell Growth Processes; Cell Line, Tumor; Doxorubicin; Female; Gangliosides; GPI-Linked Proteins; Humans; Lung Neoplasms; Mice; Mice, Nude; Mice, SCID; Myelin Proteins; Neovascularization, Pathologic; Neuroblastoma; Nogo Receptor 1; Ovarian Neoplasms; Peptide Fragments; Receptors, Cell Surface; Tissue Distribution; Xenograft Model Antitumor Assays

Disialoganglioside GD2 is an established target for immunotherapy in neuroblastoma. We tested the hypothesis that active immunization against the glycolipid GD2 using DNA vaccines encoding for cyclic GD2-mimicking decapeptides (i.e., GD2 mimotopes) is effective against neuroblastoma. For this purpose, two GD2 peptide mimotopes (MA and MD) were selected based on docking experiments to anti-GD2 antibody ch14.18 (binding free energy: -41.23 kJ/mol for MA and -48.06 kJ/mol for MD) and Biacore analysis (K(d) = 12.3 x 10(-5) mol/L for MA and 5.3 x 10(-5) mol/L for MD), showing a higher affinity of MD over MA. These sequences were selected for DNA vaccine design based on pSecTag2-A (pSA) also including a T-cell helper epitope. GD2 mimicry was shown following transfection of CHO-1 cells with pSA-MA and pSA-MD DNA vaccines, with twice-higher signal intensity for cells expressing MD over MA. Finally, these DNA vaccines were tested for induction of tumor protective immunity in a syngeneic neuroblastoma model following oral DNA vaccine delivery with attenuated Salmonella typhimurium (SL 7207). Only mice receiving the DNA vaccines revealed a reduction of spontaneous liver metastases. The highest anti-GD2 humoral immune response and natural killer cell activation was observed in mice immunized with the pSA-MD, a finding consistent with superior calculated binding free energy, dissociation constant, and GD2 mimicry potential for GD2 mimotope MD over MA. In summary, we show that DNA immunization with pSA-MD may provide a useful strategy for active immunization against neuroblastoma.

Topics: Animals; Cancer Vaccines; Cell Line, Tumor; CHO Cells; Cricetinae; Gangliosides; Interferon-gamma; Mice; Neoplasm Metastasis; Neuroblastoma; Vaccination; Vaccines, DNA

The disialoganglioside GalAcbeta1-4(NeuAcalpha2-8NeuAcalpha2-3)Galbeta1-4Glcbeta1-1Cer (GD2) is expressed on various tumors, including neuroblastoma, and was defined as a relevant tumor antigen. The monoclonal anti-GD2 antibody 14.18 is widely used for diagnostic purposes in neuroblastoma, and in its mouse/human chimeric form (ch14.18) now enters passive immunotherapeutic regimens in phase II clinical trials. This study aimed to generate structural mimics of the 14.18 epitope of GD2. Therefore, we used the ch14.18 antibody for selecting immunoreactive GD2 peptide mimotopes from a decamer phage display library. In all, 13 GD2 peptide mimics could be determined by biopanning and their specificity was demonstrated by exclusive recognition by the ch14.18 antibody. Furthermore, their nature of being GD2 mimics and their degree of mimicry was confirmed by competition with the natural antigen. When performing a comparative visualization of the GD2 epitope and selected mimotopes using a three-dimensional computer modeling system (BALLView), we demonstrated fitting of the GD2 molecule and the mimotopes in the antigen-binding pouch of a GD2 specific antibody. Moreover, the computer modeling argued for optimal affinity of the GD2 mimotopes. We thus provide evidence that the generation of GD2 peptide mimotopes is successful when using the neuroblastoma antibody ch14.18 for selection, and that this approach might offer a tool to develop a vaccination strategy against this malignant pediatric tumor.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antigens, Tumor-Associated, Carbohydrate; Binding, Competitive; Enzyme-Linked Immunosorbent Assay; Epitopes; Gangliosides; Humans; Immunoglobulin Fab Fragments; Mice; Molecular Mimicry; Molecular Sequence Data; Neuroblastoma; Peptide Library; Peptides; Protein Conformation

Immunocytological bone marrow assessment for contamination with neuroblastoma cells is based on their characteristic GD2 surface staining. Neuroblastoma without GD2 expression have been rarely and only after antibody therapy reported. Conventional cytology was performed using Pappenheim staining. For immunocytology, the APAAP method was utilized with the 14G2a anti-GD2 mouse monoclonal antibody. 7 x 10(5) cells on cytospin preparations were investigated. In 2003, 288 bone marrow samples from 191 neuroblastoma patients were investigated by cytology and immunocytology. Three cases demonstrated GD2 negativity on cytologically unambiguous neuroblastoma cells. Two female cases (94 and 37 months of age) with stage 4 neuroblastoma had GD2 expressing neuroblastoma cells in bone marrow at diagnosis. At 2nd relapse 25 and 23 months after diagnosis and 8 months and 12 months after anti-GD2 antibody treatment (ch14.18), the bone marrow infiltrating neuroblastoma cells lacked GD2 staining. The third patient, a 63-month-old girl with bone marrow replacement by neuroblastoma cells showed at diagnosis a mixture of GD2-unstained tumor clumps and very weakly stained neuroblastoma cells. Neuroblastoma cells may lack GD2 expression at diagnosis and at recurrence. This observation has diagnostic and therapeutic implications.

Topics: Antibodies, Monoclonal; Biomarkers, Tumor; Bone Marrow Neoplasms; Child; Child, Preschool; False Negative Reactions; Female; Gangliosides; Humans; Immunohistochemistry; Neoplasm, Residual; Neuroblastoma

Topics: Antibodies, Monoclonal; Flow Cytometry; Gangliosides; Humans; Immunophenotyping; Neoplasm Staging; Neuroblastoma; Quality Control; Reproducibility of Results

The GD2 ganglioside expressed on neuroectodermally derived tumors, including neuroblastoma and melanoma, is weakly immunogenic in tumor-bearing patients and induces predominantly immunoglobulin (Ig)-M antibody responses in the immunized host. Here, we investigated whether interconversion of GD2 into a peptide mimetic form would induce GD2 cross-reactive IgG antibody responses in mice. Screening of the X(15) phage display peptide library with the anti-GD2 monoclonal antibody (mAb) 14G2a led to isolation of mimetic peptide 47, which inhibited the binding of 14G2a antibody to GD2-positive tumor cells. The peptide was also recognized by GD2-specific serum antibodies from a patient with neuroblastoma, suggesting that it bears an internal image of GD2 ganglioside expressed on the tumor cells. The molecular basis for antigenicity of the GD2 mimetic peptide, established by molecular modeling and mutagenesis studies, led to the generation of a 47-LDA mutant with an increased mimicry to GD2. Immunization of mice with peptide 47-LDA-encoded plasmid DNA elicited GD2 cross-reactive IgG antibody responses, which were increased on subsequent boost with GD2 ganglioside. The vaccine-induced antibodies recognized GD2-positive tumor cells, mediated complement-dependent cytotoxicity, and exhibited protection against s.c. human GD2-positive melanoma growth in the severe combined immunodeficient mouse xenograft model. The results from our studies provide insights into approaches for boosting GD2 cross-reactive IgG antibody responses by minigene vaccination with a protective epitope of GD2 ganglioside.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Binding Sites, Antibody; Cancer Vaccines; Cross Reactions; Female; Gangliosides; Humans; Immunoglobulin G; Immunotherapy, Active; Melanoma; Mice; Mice, Inbred BALB C; Mice, SCID; Molecular Sequence Data; Neuroblastoma; Peptide Library; Peptides; Vaccines, DNA; Xenograft Model Antitumor Assays

A quantitative and precise measure of treatment response is warranted in neuroblastoma patients. We compared three quantitative methods often used for detection of minimal residual disease in such patients. Specificity, sensitivity and concordance of immunocytochemistry, real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry were compared using experimental cell suspensions (n = 8) and clinical samples (n = 126). Neuroblastoma cells were identified by immunocytochemistry and flow cytometry using anti-GD2 (14.G2a) and anti-NCAM (5.1H11) antibodies, whereas tyrosine hydroxylase mRNA was the molecular target for quantitative RT-PCR. The sensitivity using flow cytometry was 1-2 logs less than using immunocytochemistry or quantitative RT-PCR. All control samples (n = 35) tested negative by immunocytochemistry, whereas 2/34 (6%) and 1/14 (7%) were false positive by quantitative RT-PCR and flow cytometry respectively. Concordant results were obtained in 85% of patient samples (n = 116) analyzed in parallel by quantitative RT-PCR and immunocytochemistry, whereas 71% of samples analyzed by flow cytometry and immunocytochemistry were concordant (n = 35). The correlation between tumor cell levels analyzed by quantitative RT-PCR and immunocytochemistry was high (r = 0.78, p < 0.001). Quantitative RT-PCR and immunocytochemistry both reliably detected very low levels of neuroblastoma cells in clinical samples. The agreement and correlation between these methods were high. In comparison, flow cytometry was less sensitive.

Topics: Antibodies; Bone Marrow Cells; Cell Line, Tumor; Cell Survival; Child, Preschool; False Positive Reactions; Flow Cytometry; Gangliosides; Humans; Immunohistochemistry; Infant; Infant, Newborn; Neoplasm, Residual; Neural Cell Adhesion Molecules; Neuroblastoma; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity; Tyrosine 3-Monooxygenase

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.

Topics: Animals; Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Cell Line, Tumor; Child; CHO Cells; Cricetinae; Cytotoxicity, Immunologic; Gangliosides; Humans; Immunization, Passive; Killer Cells, Natural; Liver Neoplasms, Experimental; Mice; Mice, Inbred A; Neuroblastoma; Recombinant Fusion Proteins

To evaluate a combined cellular and humoral immunotherapy regimen in a mouse model of disseminated human neuroblastoma. We tested combinations of clinical-grade, isolated human gammadelta T cells with the humanized anti-GD2 antibody hu14.18 and a novel fusion cytokine, Fc-IL7.. gammadelta T cells were large-scale enriched from leukapheresis product obtained from granulocyte colony-stimulating factor-mobilized donors. gammadelta T cell cytotoxicity was tested in a europium-TDA release assay. The effect of Fc-IL7 on gammadelta T-cell survival in vitro was assessed by flow cytometry. NOD.CB17-Prkdc(scid)/J mice received 1 x 10(6) NB-1691 neuroblastoma cells via the tail vein 5 to 6 days before therapy began. Treatment, for five consecutive weeks, consisted of injections of 1 x 10(6) gammadelta T cells weekly, 1 x 10(6) gammadelta T cells weekly, and 20 microg hu14.18 antibody four times per week, or 1 x 10(6) gammadelta T cells weekly with 20 microg hu14.18 antibody four times per week, and 20 mug Fc-IL7 once weekly.. The natural cytotoxicity of gammadelta T cells to NB-1691 cells in vitro was dramatically enhanced by hu14.18 antibody. Fc-IL7 effectively kept cultured gammadelta T cells viable. Combination therapy with gammadelta T cells and hu14.18 antibody significantly enhanced survival (P = 0.001), as did treatment with gammadelta T cells, hu14.18 antibody, and Fc-IL7 (P = 0.005). Inclusion of Fc-IL7 offered an additional survival benefit (P=0.04).. We have shown a new and promising immunotherapy regimen for neuroblastoma that requires clinical evaluation. Our approach might also serve as a therapeutic model for other malignancies.

Topics: Animals; Antibodies, Monoclonal; Blood Donors; Cell Survival; Cytotoxicity Tests, Immunologic; Cytotoxicity, Immunologic; Female; Flow Cytometry; Gangliosides; Granulocyte Colony-Stimulating Factor; Humans; Immunotherapy; Interleukin-7; Leukapheresis; Mice; Mice, Inbred NOD; Mice, SCID; Neuroblastoma; Receptors, Antigen, T-Cell, gamma-delta; T-Lymphocyte Subsets; T-Lymphocytes, Cytotoxic; Transplantation, Heterologous

Established s.c. NXS2 murine neuroblastoma tumors exhibited transient resolution after suboptimal therapy using the hu14.18-IL2 immunocytokine (IC). The hu14.18-IL2 IC is a fusion protein that has linked a molecule of interleukin 2 (IL-2) to the COOH terminus of each of the IgG heavy chains on the humanized anti-GD(2) monoclonal antibody hu14.18. To induce more potent and longer lasting in vivo antitumor effects, we tested hu14.18-IL2 IC in a regimen combining it with constant infusion IL-2 in NXS2 tumor-bearing mice. The addition of the constant infusion IL-2 augmented the antitumor response induced by treatment with the hu14.18-IL2 IC in animals with experimentally induced hepatic metastases and in animals bearing localized s.c. tumors. The combined treatment induced prolonged tumor eradication in most animals bearing s.c. tumors and involved both natural killer cells and T cells. The enhanced ability of this combined treatment to prevent tumor recurrence was not observed when a larger dose of hu14.18-IL2 IC, similar in IL-2 content to the IC plus systemic IL-2 regimen, was tested as single-agent therapy. Animals showing prolonged tumor eradication of established tumors after the combined hu14.18-IL2 plus IL-2 regimen exhibited a protective T-cell-dependent antitumor memory response against NXS2 rechallenge.

Topics: Animals; Antibodies, Monoclonal; Cell Line, Tumor; Cytotoxicity, Immunologic; Female; Gangliosides; Humans; Interleukin-2; Killer Cells, Natural; Liver Neoplasms, Experimental; Mice; Mice, Inbred Strains; Neoplasm Metastasis; Neuroblastoma; Recombinant Fusion Proteins; Spleen; Time Factors

Expression of the c-myb proto-oncogene in neuroblastoma, the most common extracranial solid tumor of infancy, is linked with cell proliferation and differentiation. Neuroblastoma can be selectively targeted via monoclonal antibodies against the disialoganglioside (GD2) tumor-associated antigen. Liposomes coated with anti-GD2 antibodies (targeted liposomes) and entrapping a c-myb antisense oligonucleotide have antitumor activity. Because antisense oligonucleotides containing CpG motifs can stimulate immune responses, we evaluated the effect of CpG-containing c-myb antisense oligonucleotides encapsulated within targeted liposomes.. Antisense (myb-as) and scrambled (myb-scr) control oligonucleotides with CpG motifs were encapsulated within GD2-targeted and non-targeted liposomes. Two murine (nude and SCID-bg) xenograft models of neuroblastoma were established. Mice (groups of 10) were injected intravenously with various oligonucleotide and liposome formulations, and life span, long-term survival, immune cell activation, and cytokine release were measured over time.. Tumor-bearing mice injected with targeted liposome-CpG-myb-as or targeted liposome-CpG-myb-scr lived longer than mice in any other group, although long-term survival (i.e., more than 120 days) was obtained only in mice injected with targeted liposome-CpG-myb-as. Splenocytes isolated from mice injected with targeted liposome-CpG-myb-as contained activated macrophages, B cells, and natural killer (NK) cells, but only activated NK cells were associated with antitumor cytotoxic activity. In vivo immune cell activation was accompanied by the time-dependent increases in plasma levels of the cytokines interleukin 12 (IL-12; maximum level reached by 2 hours) and interferon gamma (IFN-gamma; maximum level reached by 18 hours) and was dependent on the oligonucleotide CpG motif. Ablation of macrophages or NK cells resulted in a loss of in vivo antitumor activity.. Immune cell activation, involving the time-dependent activation of macrophages and NK cells, contributes to the antitumor activity of targeted liposome-CpG-myb-as against neuroblastoma and could improve the effectiveness of antitumor targeted liposomes.

Topics: Animals; Antineoplastic Agents; B-Lymphocytes; Cell Line, Tumor; CpG Islands; Cytokines; Gangliosides; Gene Expression Regulation, Neoplastic; Genes, myb; Humans; Injections, Intravenous; Killer Cells, Natural; Liposomes; Macrophages; Mice; Mice, Nude; Mice, SCID; Neuroblastoma; Oligonucleotides, Antisense; Proto-Oncogene Mas; Proto-Oncogene Proteins c-myb; Spleen; Transplantation, Heterologous

Because the cytomorphologic examination of bone marrow (BM) aspirates appears not sensitive enough to detect residual neuroblastoma cells, two four-color flow cytometric assays using different combinations of CD9, CD81, CD56, CD45, and anti-GD2 were evaluated.. The sensitivity of the flow cytometric assays was assessed by spiking experiments in normal peripheral blood samples. Twenty-eight BM samples, 12 biopsies, and 3 peripheral blood stem cell (PBSC) preparations from 22 patients with neuroblastoma were analyzed. The results were compared with those of an anti-GD2 immunocytochemical reference assay.. Flow cytometric and immunocytochemical analyses showed residual neuroblastoma cells in four BM samples. One PBSC preparation and 20 BM samples were negative for both assays. Four BM and two PBSC samples scored positive for the immunocytochemical assay but were negative for the flow cytometric tests. This was due to the limited number of cells that were flow cytometrically analyzed. A strong correlation between the flow cytometric and immunocytochemical tests was found (chi2 = 6.4, P = 0.011).. When an equal amount of cells is analyzed, the sensitivity of the flow cytometric assays is to be about 10 times lower than that of the immunocytochemical test. However, the flow cytometric assays can be used to screen for residual cells in clinical samples with a sensitivity of one neuroblastoma cell in 10(4) to 10(5) normal mononuclear cells. Flow cytometry is simple, quick, and cost effective compared with immunocytochemistry. In addition, the flow cytometric assays can be used to screen for residual neuroblastoma cells in case of a GD2-negative primary tumor. Therefore we recommend flow cytometry for the detection of residual neuroblastoma cells.

Topics: Adolescent; Antibodies, Monoclonal; Antigens, CD; Biopsy; Bone Marrow Cells; CD56 Antigen; Cell Line, Tumor; Child; Child, Preschool; Cytoplasm; Female; Flow Cytometry; Gangliosides; Humans; Immunohistochemistry; Infant; Leukocyte Common Antigens; Leukocytes, Mononuclear; Male; Membrane Glycoproteins; Neoplastic Cells, Circulating; Neuroblastoma; Phenotype; Sensitivity and Specificity; Stem Cells; Tetraspanin 28; Tetraspanin 29

Labeling of specific antibodies with bifunctional chelated Actinium-225 ((225)Ac; an alpha generator) allows the formation of new, highly potent and selective alpha-emitting anticancer drugs. We synthesized and evaluated a radioimmunoconjugate based on 3F8, an IgG(3) antibody that specifically binds to ganglioside GD2, which is overexpressed by many neuroectodermal tumors including neuroblastoma. The (225)Ac-1,4,7,10-tetra-azacylododecane (DOTA)-3F8 construct was evaluated for radiochemical purity and sterility, immunoreactivity, cytotoxicity in vitro, induction of apoptosis on GD2-positive cells, as well as for pharmacological biodistribution and metabolism of the (225)Ac generator and its daughters in a nude mouse xenograft model of neuroblastoma. The (225)Ac-3F8 showed an IC(50) of 3 Bq/ml (80 pCi/ml) on the neuroblastoma cell line, NMB7, in vitro. Apoptosis of these cells was not observed. Biodistribution in mice showed specific targeting of a subcutaneous tumor; there was redistribution of the (225)Ac daughter nuclides mainly from blood to kidneys and to small intestine. Toxicity was examined in cynomolgus monkeys. Monkeys injected with 1 to 3 doses of intrathecal (225)Ac-3F8 radioimmunoconjugate (80 to 150 kBq/kg total dose) did not show signs of toxicity based on blood chemistry, complete blood counts, or by clinical evaluations. Therapeutic efficacy of intrathecal (225)Ac-3F8 was studied in a nude rat xenograft model of meningeal carcinomatosis. The (225)Ac-3F8 treatment improved survival 2-fold from 16 to 34 days (P = 0.01). In conclusion, in vivo alpha generators targeted by 3F8 warrant additional study as a possible new approach to the treatment of carcinomatous meningitis.

Topics: Actinium; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Carcinoma; Gangliosides; Haplorhini; Humans; Immunoconjugates; Immunoglobulin G; Injections, Spinal; Meningeal Neoplasms; Mice; Neuroblastoma; Rats; Rats, Nude; Survival Analysis; Tissue Distribution; Transplantation, Heterologous

The sensitive detection of bone marrow involvement is crucial for tumor staging at diagnosis and for monitoring of the therapeutic response in the patient's follow-up. In neuroblastoma, only conventional cytomorphological techniques are presently accepted for the detection of bone marrow involvement, yet since the therapeutic consequences of the bone marrow findings may be far-reaching, the need for highly reliable detection methods has become evident. For this purpose, we developed an automatic immunofluorescence plus FISH (AIPF) device which allows the exact quantification of disseminated tumor cells and the genetic verification in critical cases. In this study, the power of the immunofluorescence technique is compared with conventional cytomorphology. 198 samples from 23 neuroblastoma patients (stages 4 and 4s) at diagnosis and during follow-up were investigated. At diagnosis, 45.6% of the samples (26 of 57) which were positive by AIPF investigation were negative by cytomorphology. During follow-up, 74.2% (49 of 66) of AIPF-positive samples showed no cytological signs of tumor cell involvement. False negative morphological results were found in up to 10% of tumor cell content. A tumor cell infiltrate below 0.1% was virtually not detectable by conventional cytomorphology. Using the sensitive immunofluorescence technique, the analysis of only two instead of four puncture sites did not lead to false negative results. Thus, the immunofluorescence technique offers an excellent tool for reliable detection and quantification of disseminated tumor cells at diagnosis and during the course of the disease.

Topics: Bone Marrow Cells; False Negative Reactions; Fluorescent Antibody Technique; Gangliosides; Humans; In Situ Hybridization, Fluorescence; Neoplasm Staging; Neuroblastoma; Sensitivity and Specificity

Despite aggressive treatment with surgery, chemotherapy, and radiotherapy, the prognosis for many children with neuroblastoma remains poor. Targeted toxins represent novel cancer therapeutics designed to selectively target and kill cancer cells. The authors have developed a novel fusion toxin, DT5F11, consisting of truncated diphtheria toxin (DT(A)) linked to a single chain antibody (sc5F11) targeting the GD(2) antigen found on most neuroblastoma cells. This report describes the construction, expression, and in vitro function of DT5F11.. Utilizing restriction enzyme digestion, polymerase chain reaction amplification, and gel electrophoresis, the prkDTL5F11 plasmid was created by the fusion of distinct coding sequences for a single-chain GD(2) targeting antibody (sc5F11) and truncated diphtheria toxin (DT(A)). DH5alpha Escherichi coli-competent cells were transformed with prkDTL5F11; DNA was amplified, isolated, and sequenced. The fusion protein was expressed and assayed by Western blot. Targeted cytotoxicity was analyzed on GD(2)-positive (SK-N-AS, IMR-32, SK-N-MC, LAN-1) and GD(2)-negative (HeLa) cells.. Fluorescent dye-labeled cycle sequencing identified the constructed fusion toxin gene. Western blot analysis using a mouse antihuman DT(A) antibody showed a 69-kD band identifying the fusion toxin, DT5F11. Targeted cell killing with DT5F11 was seen only in GD(2) positive cells.. This study demonstrates creation of a novel fusion toxin with effective GD(2)-targeted cellular toxicity. Further investigation of this fusion toxin as a therapeutic agent in the management of neuroblastoma is warranted.

Topics: Antibodies, Monoclonal; Antibodies, Neoplasm; Antigens, Neoplasm; Cell Death; Cytotoxicity, Immunologic; Diphtheria Toxin; Gangliosides; HeLa Cells; Humans; Immunoglobulin Fab Fragments; Immunotoxins; Neuroblastoma; Peptide Fragments; Plasmids; Recombinant Fusion Proteins; Tumor Cells, Cultured

Unusual ocular symptoms observed during intravenous treatment with anti-disialoganglioside antibody (Ab) in children suffering from neuroblastoma were analyzed and the results reported. Within the framework of the German Collaborative Neuroblastoma Study NB97, 85 children with high-risk neuroblastoma received anti-GD2 monoclonal antibody ch14.18 intravenously. Side effects were regularly reported to the study center. Ocular symptoms were recorded in clinical detail, duration and development over time. Symptoms of a parasympathetic deficit corresponding to internal ophthalmoplegia, i.e. mydriasis and accommodation deficit, were found in 10 patients. They were uni- or bilateral, began after the termination of Ab infusion and improved or disappeared in all surviving children. They did not reappear or worsen upon repeated Ab infusions. The pathophysiology of these disorders remains poorly understood. It is concluded that during systemic treatment with the anti-GD2 antibody ch14.18, reversible symptoms of parasympathetic denervation of the eye may occur which, however, do not warrant termination of this treatment.

Topics: Accommodation, Ocular; Animals; Antibodies, Monoclonal; Child, Preschool; Female; Gangliosides; Humans; Infant; Male; Mice; Mydriasis; Neuroblastoma; Recombinant Fusion Proteins

The antigen GD2 is selectively expressed on the surface of neuroblastoma cells, and is detected by the monoclonal antibody BW704. In this study, we describe the antitumoral capacity of the immunotoxin BW704dgA (BW704 conjugated to deglycosylated ricin A), and of anti-CD3xanti-GD2 bispecific antibodies that are capable of redirecting cytotoxic T cells towards neuroblastoma cells. We further investigate the in vivo activity of BW704dgA immunotoxins in a human neuroblastoma model in SCID mice.. BW704dgA immunotoxins were injected i.p. as a single close (48 microg/mouse) on day 4 or divided into three doses on day 4, 5, and 6 after i.v. inoculation of the human neuroblastoma cell line IMR5-75.. The mean survival time (MST) of BW704dgA treated animals was significantly increased (MST 49 days) compared to the control animals treated with irrelevant immunotoxin, unconjugated BW704, or control buffer (MST 33 to 39 days, P < 0.0001), without differences in the application schedules. Anti-CD3xanti-NP antibodies and NP-conjugated GD2-antibodies (BW704-NP) were used in a cytotoxicity assay with cytotoxic T-cells as effectors, and tracer labeled neuroblastoma cell line IMR5 as target cells. Anti-CD3xanti-NP antibodies, together with BW704-NP, showed increased cytotoxic activity compared to the incubation with CD3xanti-NP antibodies alone or with unconjugated anti-GD2. Additionally, a dose-dependent effect of NP-conjugated anti-GD2-antibodies upon the lysis of the target cells could be demonstrated. In this report, we describe two immunotherapeutic approaches using GD2 binding BW704 antibodies, modified as immunotoxin and a bispecific antibody, for the targeting and elimination of neuroblastoma cells.. We envisage a combined immunotherapeutic regimen consisting of BW704dgA mediated stem cell purging, followed by a systemic treatment with anti-CD3xanti-GD2 bispecific antibodies in neuroblastoma.

Topics: Animals; Antibodies, Bispecific; Antibodies, Monoclonal; Antibodies, Neoplasm; Antibody Specificity; CD3 Complex; Gangliosides; Haptens; Humans; Immunotherapy; Immunotoxins; Mice; Mice, SCID; Neuroblastoma; Ricin; T-Lymphocytes, Cytotoxic; Trinitrobenzenes; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Rare tumor cells circulating in the hematopoietic system can escape identification. On the other hand, the nature of these cells, positive for an immunologiCal tumor marker, cannot be determined without any genetic information.. To overcome these problems a novel computer assisted scanning system for automatic cell search, analysis, and sequential repositioning was developed. This system allows an exact quantitative analysis of rare tumor cells in the bone marrow and peripheral blood by sequential immunological and molecular cytogenetic characterization.. In that virtually all tumor cells in a mixing experiment could be recovered unambiguously, we can conclude that the sensitivity of this approach is set by the number of cells available for analysis. Sequential FISH analyses of immunologically positive cells improve both the specificity and the sensitivity of the microscopic minimal residual disease detection.

Topics: Biomarkers, Tumor; Blood Cell Count; Bone Marrow Examination; Chromosome Aberrations; Chromosome Deletion; Chromosomes, Human, Pair 1; Chromosomes, Human, Pair 17; Diagnosis, Computer-Assisted; DNA Probes; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Gangliosides; Gene Amplification; Gene Expression Profiling; Genes, myc; Humans; In Situ Hybridization, Fluorescence; Indoles; Neoplasm, Residual; Neoplastic Cells, Circulating; Neuroblastoma; Sensitivity and Specificity; Trisomy; Tumor Cells, Cultured

Gangliosicle GD2 is abundant on human neuroblastoma (NB). Monoclonal antibody 3F8 targeted to GD2 may have imaging and therapeutic potential. Antigen-negative clones can escape immune-mediated attack leading to clinical resistance or recurrence.. Among 95 evaluable patients treated intravenously with 3F8 (94 Stage 4, 1 Stage 3), 66 received nonradiolabeled 3F8, 11 received 131-iodine-labeled-3F8 (8-28 mCi/kg) with autologous bone marrow rescue, and 18 received both forms of treatment. Prior to treatment, 90 patients tested positive for GD2 reactivity by bone marrow immunofluorescence (n = 68), tumor immunohistochemistry (n = 20), or diagnostic radioimmunoscintigraphy (n = 2).. Of 62 patients who had refractory or recurrent neuroblastoma following 3F8 treatment, 61 (98%) tested positive for GD2 reactivity by bone marrow immunofluorescence (n = 51) or tumor immunohistochemistry (n = 10). The sole tumor that lost GD2 expression underwent phenotypic transformation into a pheochromocytoma-like tumor.. The persistence of GD2 expression in refractory or recurrent NB suggests that complete antigen loss is an uncommon event and cannot account for treatment failure.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antibody Specificity; Antigens, Neoplasm; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Bone Marrow Transplantation; Cell Lineage; Child, Preschool; Combined Modality Therapy; Fatal Outcome; Female; Ganglioneuroblastoma; Gangliosides; Humans; Immunization, Passive; Immunoconjugates; Immunoglobulin G; Iodine Radioisotopes; Male; Neoplasm Metastasis; Neuroblastoma; Radioimmunodetection; Radioimmunotherapy; Remission Induction; Retrospective Studies; Transplantation, Autologous

We first explored the use of multiple molecular markers to overcome tumor heterogeneity. Sixty-seven neuroblastoma (NB) tumors were tested for the expression of GAGE, MAGE-2, MAGE-2, MAGE-3, and MAGE-4 by RT-PCR and then chemiluminescence; 82% of tumors had detectable GAGE, and 88% expressed at least one of the four MAGE genes.. By combining GAGE and MAGE, 64 of 67 (95%) of tumors became detectable; 17 of 67 coexpressed all five molecular markers. Neither GAGE nor MAGE expression correlated with stage. GAGE was found to have the broadest (18 of 18) expression among stage 4 tumors. Two hundred fifty-nine bone marrows from 99 patients were then studied for NB positivity by four detection methods: histology, immunocytology, and molecular detection by GAGE and tyrosine hydroxylase (TH) mRNA. Two hundred seven samples were NB-positive by one detection method. All four techniques were comparable in detecting tumor cells at diagnosis and at relapse. GAGE and immunocytology were far more sensitive than histology and TH mRNA when marrows were sampled during chemotherapy and at the time of clinical remission.. By combining multiple molecular markers and independent screening techniques, we may be able to overcome tumor heterogeneity and expedite the detection of microscopic disease in the clinical management of neuroblastoma.

Topics: Algorithms; Antibodies, Monoclonal; Antigens, Neoplasm; Biomarkers, Tumor; Biotinylation; Bone Marrow Examination; Disease-Free Survival; DNA, Complementary; Gangliosides; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Luminescent Measurements; Melanoma-Specific Antigens; Neoplasm Proteins; Neoplasm Staging; Neoplasm, Residual; Neuroblastoma; Remission Induction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Sensitivity and Specificity; Tyrosine 3-Monooxygenase

The coexistence of neuroblastic and Schwannian stromal (SS) cells in differentiating neuroblastoma (NB), and derivation of Schwannian-like cells from neuroblastic clones in vitro, were accepted previously as evidence of a common pluripotent tumor stem line. This paradigm was challenged when SS cells were suggested to be reactive in nature. The advent of microdissection techniques, PCR-based allelic analysis, and in situ fluorescent cytometry made possible the analysis of pure cell populations in fresh surgical specimens, allowing unequivocal determination of clonal origins of various cell subtypes. To overcome the complexity and heterogeneity of three-dimensional tissue structure, we used: (a) Laser-Capture Microdissection to obtain histologically homogeneous cell subtype populations for allelotype analysis at chromosomes 1p36, 11q23, 14q32, and 17q and study of MYCN copy number; (b) multiparametric analysis by Laser-Scanning Cytometry of morphology, DNA content, and immunophenotype of intact cells from touch imprints; and (c) bicolor fluorescence in situ hybridization on touch imprints from manually microdissected neuroblast and stroma-rich areas. Histologically distinct SS and neuroblastic cells isolated by Laser-Capture Microdissection had the same genetic composition in 27 of 28 NB analyzed by allelic imbalance and gene copy number. In all 20 cases studied by Laser-Scanning Cytometry, SS cells identified by morphology and S-100 immunostaining had identical DNA content and GD2-staining pattern as their neuroblastic counterparts. In 7 cases, fluorescence in situ hybridization demonstrated the same chromosomal makeup for SS and neuroblastic cells. These results provide unequivocal evidence that neuroblastic and SS cells in NB are derived from genetically identical neoplastic cells and support the classical paradigm that NB arises from tumoral cells capable of development along multiple lineages.

Topics: Adolescent; Adult; Alleles; Child; Child, Preschool; DNA, Neoplasm; Gangliosides; Gene Dosage; Genes, myc; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Infant; Neoplastic Stem Cells; Neuroblastoma; Ploidies; S100 Proteins; Schwann Cells; Stromal Cells

Since the disialoganglioside GD2 is abundantly present on the surface of neuroblastoma cells, we constructed a new recombinant immunotoxin for possible clinical use in patients with neuroblastoma. A functional 14.18 scFv-phage was obtained by selection of an anti-GD2 hybridoma derived phage antibody mini-library on the neuroblastoma-derived, GD2-expressing cell line IMR5. By insertion into the bacterial expression vector pBM1.1 the selected scFv was fused to a deletion mutant of Pseudomonas exotoxin A (ETA'). Periplasmically expressed 14.18(scFv)-ETA' bound to the GD2 expressing cell line IMR5, but not to the GD2 negative Hodgkin-derived cell line L540Cy as documented by ELISA and flow cytometry. The recombinant immunotoxin (rIT) inhibited cell viability of IMR5 cells by 50% at concentrations (IC(50)) of 0.326 microg/ml. This recombinant immunotoxin will be further investigated in vivo for its value as a new immunotherapeutic agent for the treatment of patients with neuroblastoma.

Topics: ADP Ribose Transferases; Antibodies, Monoclonal; Bacterial Toxins; Binding, Competitive; Cell Membrane; Cell Survival; Cloning, Molecular; Cytotoxicity, Immunologic; Dose-Response Relationship, Drug; Exotoxins; Gangliosides; Humans; Immunoglobulin Fragments; Immunoglobulin Variable Region; Immunotoxins; Neuroblastoma; Protein Binding; Pseudomonas aeruginosa Exotoxin A; Recombinant Fusion Proteins; Tumor Cells, Cultured; Virulence Factors

Genetic engineering of human T lymphocytes to express tumor antigen-specific chimeric immune receptors is an attractive means for providing large numbers of effector cells for adoptive immunotherapy while bypassing major mechanisms of tumor escape from immune recognition. We have applied this strategy to the targeting of a G(D2)-positive tumor, neuroblastoma, which is the commonest extracranial solid tumor of childhood. Chimeric immune receptors were generated by joining an extracellular antigen-binding domain derived from either of the 2 ganglioside G(D2)-specific antibodies sc7A4 and sc14.G2a to a cytoplasmic signaling domain. The variable domains of hybridoma antibody 14.G2a were cloned and selected using a phage display approach. Upon coincubation with G(D2)-expressing tumor cell targets, human T lymphocytes transduced with recombinant retroviruses encoding chimeric receptors based on sc14.G2a, but not sc7A4, secreted significant levels of cytokines in a pattern comparable to the cytokine response obtained by engagement of the CD3 receptor. T cells transduced with the sc14.G2a-based chimeric T-cell receptors also displayed specific lysis of G(D2)-positive neuroblastoma cells, which was blocked in the presence of monoclonal antibody 14.G2a. In the absence of nonspecific stimulation of transduced cells, their functionality declined over time and antigenic stimulation of the chimeric receptor alone did not induce commitment to proliferation. These results support the feasibility of redirecting human T lymphocytes to a tumor-associated ganglioside epitope but emphasize that successful chimeric receptor-mediated adoptive immunotherapy will require additional strategies that overcome functional inactivation of gene-modified primary T lymphocytes.

Topics: Cytokines; Gangliosides; Genetic Engineering; Humans; Immunotherapy, Adoptive; Neuroblastoma; Receptors, Antigen, T-Cell; Recombinant Fusion Proteins; T-Lymphocytes; Transduction, Genetic; Tumor Cells, Cultured

Early and correct diagnosis of local tumor recurrence, occurrence of metastases, and therapy response are essential in patients with neuroblastoma stage IV. The aim of the study was to evaluate the diagnostic value of metaiodobenzylguanidine (mIBG) and a chimeric GD2 antibody in the follow-up of patients with neuroblastoma. In a prospective study, mIBG (N = 31 scans) and immunoscintigraphy were compared with a chimeric antiganglioside antibody, ch14.18 (MAb) (N = 31 scans), labeled with technetium Tc 99m in the follow-up of 18 patients with stage IV neuroblastoma. The findings were compared with histologic findings, other imaging examinations, and clinical changes over the course of 4 to 6 years. For the diagnosis of local tumor recurrences, sensitivity was 80% for MAb and 70% for mIBG. Specificity was 93% for MAb and 72% for mIBG. The MAb was superior for the detection of skeletal metastases, with a sensitivity of 82% compared with 72% for mIBG. Specificity was 100% for both techniques. Also, for soft tissue/lymph node metastases, sensitivity for MAb was higher (50%) than for mIBG (31%). Specificity was 100% for each technique. In sequential studies, metastases were detected earlier with MAb (mean: 2.3 m for skeletal metastases, 3.6 m for soft tissue metastases) than with mIBG. After therapy, tumor uptake was visualized longer with mIBG (mean 6.3 m) than with MAb. The chimeric antibody ch14.18 is likely to be valuable for follow-up examinations and for assessment of therapy response because of earlier detection of new metastases.

Topics: 3-Iodobenzylguanidine; Adolescent; Antibodies, Monoclonal; Bone Neoplasms; Child; Child, Preschool; False Negative Reactions; Female; Fluorescent Antibody Technique; Follow-Up Studies; Gangliosides; Humans; Liver Neoplasms; Male; Neoplasm Recurrence, Local; Neoplasm Staging; Neuroblastoma; Radionuclide Imaging; Radiopharmaceuticals; Recombinant Fusion Proteins; Soft Tissue Neoplasms; Technetium

Neutrophils and mononuclear cells (MNC) can mediate antibody-dependent cellular cytotoxicity (ADCC) against cancer cells. To study cytotoxicity and growth inhibition of neuroblastoma cells by neutrophils and MNC with chimeric anti-disialoganglioside (GD2) monoclonal antibody (mAb) ch14.18, we developed digital image microscopy scanning (DIMSCAN) assays that measure fluorescence of target cells in 96-well plates after 6-18 h (cytotoxicity assay) or 7 days (growth assay). Neuroblastoma cell lines (GD2-positive: SMS-KCN, SMS-LHN, LA-N-1; GD2-negative: SK-N-SH) were preloaded with calcein acetoxymethyl ester for the cytotoxicity assay or labeled in situ after 7 days of culture with fluorescein diacetate in the growth assay. Fluorescence, as quantified by DIMSCAN, was correlated with neuroblastoma cell number in both assays (100-2000 cells/well). In the cytotoxicity test, both neutrophils and MNC effectively mediated ADCC of GD2-positive but not GD2-negative neuroblastoma cell lines. Cytotoxicity of both neutrophils and MNC increased with effector to target cell (E:T) ratio (5-50:1) and mAb ch.14.18 dose (0.1-10 microg/ml). ADCC of neutrophils, but not MNC, increased with addition of GM-CSF. Neutrophils, especially with rhGM-CSF, significantly suppressed growth of GD2-positive cell lines at a high E:T ratio (50:1) and mAb dose (10 microg/ml). Without antibody, neutrophils inhibited growth of one cell line (LA-N-1) but stimulated growth of two others (SMS-KCN, SMS-LHN). If neuroblastoma cells did not express GD2 (SK-N-SH), neutrophils stimulated growth whether or not antibody was present. Neutrophil culture supernatants increased growth of SK-N-SH, LA-N-1, and SMS-KCN cells, and MNC culture supernatants increased growth of SK-N-SH. In conclusion, neutrophils can mediate cytotoxicity and growth inhibition with a chimeric anti-GD2 antibody but also can promote tumor cell growth if antibody is not present or if GD2 is not expressed.

Topics: Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Antigens, Neoplasm; Cell Count; Cell Division; Culture Media, Conditioned; Dose-Response Relationship, Immunologic; Eosine Yellowish-(YS); Fluoresceins; Fluorescent Dyes; Gangliosides; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Image Processing, Computer-Assisted; Microscopy, Fluorescence; Monocytes; Neuroblastoma; Neutrophils; Recombinant Fusion Proteins; Tumor Cells, Cultured

Treatment with anti-G(D2) monoclonal antibody 3F8 (Ab1) at the time of remission may prolong survival for children with stage 4 neuroblastoma. A transient human antimouse antibody (HAMA) response was associated with significantly longer survival (Cheung et al., J. Clin. Oncol., 16: 3053-3060, 1998). Because this response was primarily anti-idiotypic (Ab2), we postulate that the subsequent induction of an idiotype network that included an elevation of anti-anti-idiotypic (Ab3) and anti-G(D2) (Ab3') antibody titers may be responsible for tumor control. Thirty-four patients with stage 4 neuroblastoma diagnosed at >1 year of age were treated with 3F8 at the end of chemotherapy. Most had either bone marrow (31 of 34) or distant bony (29 of 34) metastases at diagnosis. Thirteen patients were treated at second or subsequent remission, and 12 patients in this group had a history of progressive/persistent disease after bone marrow transplantation; 21 patients were treated in the first remission after N6 chemotherapy. Their serum HAMA, Ab3, and Ab3' titers prior to, at 6, and at 14 months after antibody treatment were measured by ELISA. Among these 34 patients, 14 are alive, and 13 (1.8-7.4 years at diagnosis) are progression free (53-143 months from the initiation of 3F8 treatment) without further systemic therapy. Long-term progression-free survival (PFS) and survival correlated significantly with Ab3' (anti-G(D2)) response at 6 months and with Ab3 response at 6 and 14 months. By defining Ab3 threshold ranging from the ratio of 1.1 to 2.6 above pretreatment level, the difference in PFS and survival between the high-Ab3 and low-Ab3 groups became markedly widened. Similarly, increasing the Ab3' threshold at either 6 or 14 months to 300% above pre-3F8 levels also increased the spread between the high versus low Ab3' groups for both PFS and survival curves. Non-idiotype antibody responses (anti-mouse-IgG3 or anti-tumor nuclear HUD antigen) had no apparent impact on PFS or survival. In conclusion, despite the high-risk nature of stage 4 neuroblastoma, long-term remission without myeloablative therapy can be achieved with 3F8 treatment. Ab3 and Ab3' antibody response correlated with prolonged PFS and survival. We postulate that successful induction of an idiotype network in patients may be responsible for long-term tumor control.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Bone Marrow Transplantation; Child; Child, Preschool; Disease-Free Survival; Female; Gangliosides; Humans; Immunoglobulin G; Infant; Male; Mice; Neoplasm Staging; Neuroblastoma; Recurrence; Retrospective Studies; Survival Rate; Survivors; Time Factors

Accurate quantification of disseminated tumor cells in hematological samples is of fundamental importance in clinical oncology. However, even highly standardized protocols allow only a rough estimation of the total analyzed cell number, as sample processing may have adverse effects on the number of cells available for analysis. The fluorescence-based microscopic scanning system (MetaCyte) detects, counts, captures, and relocates immunolabeled tumor cells in hematopoietic samples. We report on a cell-counting approach that has been implemented into the scanning system to precisely quantify the number of cells per slide. The cell-counting function, which was designed to determine the number of all nucleated (DAPI-stained) cells on the slide, allows an accurate counting of the tumor cells and the total number of cells analyzed in the given microscopic sample. The reliability of the cell-counting approach was demonstrated by the analysis of DAPI-stained images with 18-1,363 nucleated cells. A good correlation (r(2) = 0.965) between the manually and automatically gained results was observed. The counting accuracy could even be optimized after implementing a correction factor. To prove or disprove an interslide variation, routine bone marrow cytospin preparations from neuroblastoma patients were immunostained for GD2/FITC and counterstained with DAPI. Automatic cell counting of cytospin preparations from the same patients showed significant differences in the total cell number (up to 67% cell loss during preparation, with a maximum interslide difference of 4.7 x 10(5) mononuclear cells). We conclude that determination of the tumor cell content in hematopoietic samples is only reliable when it is performed together with accurate cell counting.

Topics: Automation; Bone Marrow Cells; Bone Marrow Neoplasms; Cell Count; Fluorescent Antibody Technique; Gangliosides; Humans; Image Processing, Computer-Assisted; Microscopy, Fluorescence; Neuroblastoma; Reproducibility of Results

Except at diagnosis and relapse, when gross disease is present, histologic evaluation is less sensitive than immunocytology (IC) of bone marrow for detecting metastatic neuroblastoma. We examined whether the chance of a positive IC from a single marrow site was comparable to an IC of pooled marrow from multiple sites.. We carried out 47 marrow examinations on 29 patients with high-risk neuroblastoma on therapy. Each examination consisted of histologic evaluation of four aspirates and two biopsies (six sites), IC of a 2.5-5-mL heparinized sample from a single site (the right posterior iliac crest; IC-RPIC), as well as IC of 8-10 mL of heparinized marrow pooled from bilateral anterior and bilateral posterior iliac crests (IC-pooled). IC was performed using a panel of monoclonal antibodies specific for ganglioside GD2.. The number of GD2-positive tumor cells detected by IC-pooled had a strong linear correlation with that by IC-RPIC (R = 0.91), although IC-pooled detected an average of 3.3 times more GD2-positive cells. Of 47 examinations, 15 tested positive by histology (6 sites), 20 by IC- pooled, and 17 by IC-RPIC. Among 29 patients, the level of agreement between IC-RPIC and IC-pooled was generally good (kappa statistic > or = 0.72), giving a false negative rate of < or = 30%.. Compared to conventional histologic examinations, immunocytology of a single marrow aspirate generally agrees with that of marrow pooled from six sites. However, the false negative rate may be too high in the setting of examination prior to bone marrow or stem cell harvest.

Topics: Antibodies, Monoclonal; Biopsy, Needle; Bone Marrow; Bone Marrow Neoplasms; Fluorescent Antibody Technique; Gangliosides; Humans; Immunohistochemistry; Neuroblastoma

Induction, maintenance, and amplification of tumor-protective immunity after cytokine gene therapy is essential for the clinical success of immunotherapeutic approaches. We investigated whether this could be achieved by single-chain IL-12 (scIL-12) gene therapy followed by tumor-targeted IL-2 using a fusion protein containing a tumor-specific recombinant anti-ganglioside GD(2) antibody and IL-2 (ch14.18-IL-2) in a poorly immunogenic murine neuroblastoma model. Herein, we demonstrate the absence of liver and bone marrow metastases after a lethal challenge with NXS2 wild-type cells only in mice (five of six animals) vaccinated with scIL-12-producing NXS2 cells and given a booster injection of low-dose ch14.18-IL-2 fusion protein. This tumor-protective immunity was effective 3 months after initial vaccination, in contrast to control animals treated with a nonspecific fusion protein or an equivalent mixture of antibody and IL-2. Only vaccinated mice receiving the tumor-specific ch14.18-IL-2 fusion protein revealed a reactivation of CD8(+) T cells and subsequent MHC class I-restricted tumor target cell lysis in vitro. The sequential increase in the usage of TCR chains Vbeta11 and -13 in mouse CD8(+) T cells after vaccination and amplification with ch14.18-IL-2 suggests that the initial polyclonal CD8(+) T cell response is effectively boosted by targeted IL-2. In conclusion, we demonstrate that a successful boost of a partially protective memory T cell immune response that is induced by scIL-12 gene therapy could be generated by tumor-specific targeting of IL-2 with a ch14.18-IL-2 fusion protein. This approach could increase success rates of clinical cancer vaccine trials.

Topics: Animals; Antibodies, Neoplasm; Antineoplastic Agents; Cancer Vaccines; CD8-Positive T-Lymphocytes; Disease Models, Animal; Gangliosides; Genetic Therapy; Immunotherapy; Interleukin-12; Interleukin-2; Mice; Neoplasm Transplantation; Neuroblastoma; Receptors, Antigen, T-Cell; Recombinant Fusion Proteins; Tumor Cells, Cultured; Vaccination

3F8 is a murine IgG3 monoclonal antibody (MAb) selective for the ganglioside G(D2). Previous studies using 131I-3F8 have shown great potential in the imaging of neuroectodermal tumors and the therapy of human neuroblastoma. 131I is commonly used in radioimmunodiagnosis, but its relatively long half-life (8 days) and its high energy gamma-emission (364 KeV) are suboptimal for imaging purposes when compared with 99mTc (6 h and 140 KeV, respectively). To label 3F8 with 99mTc, the antibody was first coupled with a heterobifunctional linker, succinimidyl-6-hydrazinonicotinate hydrochloride (SHNH), obtaining a hydrazinonicotinamide-antibody conjugate. Using 99mTc-Tricine as the precursor complex, 3F8-SHNH was coupled efficiently to 99mTc, resulting in >90% radiometal incorporation, with a specific activity >10 mCi/mg and retaining full immunoreactivity. Immunoscintigraphy at 6, 22, and 46 h after intravenous injection of 1 mCi of 99mTc-3F8 showed selective neuroblastoma localization in xenografted nude mice, comparable to that obtained with the injection of 100 microCi of 131I-3F8. Biodistribution studies of 131I-3F8 and 99mTc-3F8 in mice demonstrated comparable %ID/g uptake in tumor (with a T/B ratio: approximately 2.5 at 24 h and approximately 3.5 at 48 h) and normal organs, including blood, except for spleen and liver which had about a three times higher uptake of the 99mTc conjugate. In conclusion, 99mTc can be coupled conveniently at high specific activity to 3F8 without compromising immunoreactivity. SHNH appears to be a useful linker for 99mTc in tumor diagnostic imaging and may have potential utility in coupling other radioisotopes (e.g., 94mTc) for positron imaging and therapy.

Topics: Animals; Antibodies, Monoclonal; Gangliosides; Humans; Iodine Radioisotopes; Mice; Mice, Nude; Neuroblastoma; Niacinamide; Organotechnetium Compounds; Radionuclide Imaging; Radiopharmaceuticals; Succinimides; Tissue Distribution; Transplantation, Heterologous; Tumor Cells, Cultured

Granulocyte/macrophage-colony stimulating factor (GM-CSF) is very effective at enhancing antibody-dependent cellular cytotoxicity (ADCC) mediated by granulocytes and monocytes. Recently, a fusion protein consisting of GM-CSF and chimeric human/mouse anti-ganglioside G(D2) antibody Ch14.18 (Ch14.18-GM-CSF) has been generated to improve the effectiveness of immunotherapy by directing GM-CSF to the tumor microenvironment and prolonging its relatively short half-life. In this study, we examined the ability of this fusion protein to enhance the in vitro killing of G(D2)-expressing human neuroblastoma cells by granulocytes and mononuclear cells, as well as by complement. The Ch14.18-GM-CSF fusion protein was equally effective as the combination of equivalent amounts of free Ch14.18 and GM-CSF in mediating the killing of NMB7 neuroblastoma cells by granulocytes from seven of eight neuroblastoma patients. The fusion protein was also equally effective as the combination of Ch14.18 and GM-CSF in mediating ADCC by neuroblastoma patients' mononuclear cells. In addition, the fusion protein was as effective as Ch14.18 alone in directing complement-dependent cytotoxicity against NMB7 cells. Our results demonstrate that the biological activities expressed by ADCC and complement-dependent cytotoxicity of both monoclonal antibody Ch14.18 and GM-CSF are retained by the Ch14.18-GM-CSF fusion protein and lend further support for future clinical trials of this fusion protein in patients with neuroblastoma.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Complement System Proteins; Cytotoxicity, Immunologic; Gangliosides; Granulocyte-Macrophage Colony-Stimulating Factor; Granulocytes; Humans; Immunoconjugates; Leukocytes, Mononuclear; Mice; Neuroblastoma; Recombinant Fusion Proteins; Tumor Cells, Cultured

Targeted interleukin-2 (IL-2) therapy with a genetically engineered antidisialoganglioside GD2 antibody-IL-2 fusion protein induced a cell-mediated antitumor response that effectively eradicated established bone marrow and liver metastases in a syngeneic model of neuroblastoma. The mechanism involved is exclusively natural killer (NK) cell-dependent, because NK-cell deficiency abrogated the antitumor effect. In contrast, the fusion protein remained completely effective in the T-cell-deficient mice or immunocompetent mice depleted of CD8+ T cells in vivo. A strong stimulation of NK-cell activity was also shown in vitro. Immunohistology of the leukocytic infiltrate of livers from treated mice revealed a strong staining for NK cells but not for CD8+ T cells. The therapeutic effect of the fusion protein was increased when combined with NK-cell-stimulating agents, such as poly I:C or recombinant mouse interferon-gamma. In conclusion, these data show that targeted delivery of cytokines to the tumor microenvironment offers a new strategy to elicit an effective cellular immune response mediated by NK cells against metastatic neuroblastoma. This therapeutic effect may have general clinical implications for the treatment of patients with minimal residual disease who suffer from T-cell suppression after high-dose chemotherapy but are not deficient in NK cells.

Topics: Animals; Antibodies; Bone Marrow Neoplasms; Female; Gangliosides; Histocompatibility Antigens Class I; Immunotherapy; Interleukin-2; Killer Cells, Natural; Liver Neoplasms; Mice; Mice, SCID; Neuroblastoma; Recombinant Fusion Proteins; Tumor Cells, Cultured

A human IgG1.k monoclonal antibody (MAb) designated GMA1 was developed by fusing pooled lymph node lymphocytes from cancer patients with the human lymphoblastoid cell line, SHFP-1. The GMA1 MAb reacted with several melanoma and neuroblastoma cell lines. Normal tissue derived from human brain and tumor-cell lines derived from colon, ovary, and breast were not reactive. FACS analysis performed using live cells demonstrated that the antibody recognizes a cell-surface antigen. Enzyme immunoassay (EIA) and thin layer chromatography (TLC) immunostaining with purified gangliosides indicated that the antibody has specificity for the major tumor associated gangliosides GD3, GM3, and GD2. GMA1 heavy and light chain genes were isolated by RT-PCR and a recombinant derivative of this human antibody was expressed in Chinese hamster ovary (CHO) cells. High-level antibody synthesis and secretion was achieved using a vector designed to maximize expression. FACS analysis and TLC immunostaining indicated recombinant GMA1 reacted with human tumor cell lines and gangliosides GD3, GM3, GD2 in a manner similar to the antibody produced by the hybridoma cell line, demonstrating that the specificity of the antibody was not altered during molecular cloning.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Antibody Specificity; Antigens, Neoplasm; Antigens, Surface; Base Sequence; G(M1) Ganglioside; Gangliosides; Humans; Hybridomas; Immunoglobulin G; Immunoglobulin Heavy Chains; Immunoglobulin Light Chains; Immunoglobulin Variable Region; Melanoma; Molecular Sequence Data; Neuroblastoma; Recombinant Proteins; Tumor Cells, Cultured

We have evaluated the anti-tumor effect of anti-GD2 mouse monoclonal antibody (mAb) 220-51 against human neuroblastoma cell line TGW in vitro and in vivo. The mAb 220-51 was able to mediate complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) using human effector cells. In the presence of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte ADCC was significantly augmented in vitro. When mAb 220-51 was administered to tumor-bearing nude mice, tumor growth was significantly inhibited as compared with untreated controls. Administration of recombinant murine GM-CSF in combination with mAb 220-51 significantly enhanced the anti-tumor effect of mAb in vivo. Recombinant human granulocyte colony-stimulating factor (G-CSF) combined with mAb 220-51 was also able to enhance it, although granulocyte ADCC was not affected by the presence of recombinant human G-CSF in vitro. Moreover, GM-CSF and G-CSF work additively to enhance the anti-tumor effect of mAb 220-51 in vivo. The GM-CSF and G-CSF may have a clinical potency in immunotherapy with anti-GD2 mAb for the treatment of neuroblastoma.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Brain Neoplasms; Cell Line, Tumor; Drug Synergism; Drug Therapy, Combination; Female; Gangliosides; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Granulocytes; Humans; Leukocyte Count; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Neuroblastoma; Recombinant Proteins; Transplantation, Heterologous

To measure the sensitivity of histologic examination in detecting metastatic solid tumor in bone marrow.. A total of 145 patients with stage 4 neuroblastoma underwent 840 marrow examinations, each consisting of six sites (four aspirates and two biopsies), from October 1990 to June 1996 at Memorial Sloan-Kettering Cancer Center. Metastasis was detected by either histology (aspirate by Wright-Giemse and biopsy by Hematoxylin-Eosin stains) or immunostaining of aspirates using anti-G(D2) monoclonal antibodies.. The absence of tumor by histology at a single marrow site was a poor guarantee of the absence of disease. The number of false-negative sites increased as the percent of G(D2)-positive tumor cells in the marrow decreased: zero of six if tumor cell count was > or = 1%, and approximately six of six sites if < or = 0.003%. Sensitivity was comparable between marrow aspirate and biopsy. A lower bound (LB) for the probability of false-negative histology was calculated from the (1) discordance among the six marrow samplings and (2) comparison with immunofluorescence. When disease was extensive (eg, at diagnosis), the LB was 0.13 and 0.3, respectively. After treatment, it increased to 0.37 and 0.8. Examining multiple marrow sites can decrease the LB to 0.15. However, at least three sites have to be negative at relapse, six at diagnosis, and more than 50 during treatment or off-therapy follow-up. The marginal decrease in the LB by additional samplings rapidly diminished to less than 0.05 after two sites.. Except at diagnosis and relapse when gross disease is present, marrow sampling by histology has limited sensitivity. Current practice grossly underestimates the true prevalence of marrow disease.

Topics: Biomarkers, Tumor; Biopsy, Needle; Bone Marrow; Bone Marrow Neoplasms; False Negative Reactions; Gangliosides; Humans; Immunohistochemistry; Neuroblastoma; Predictive Value of Tests; Sensitivity and Specificity

Anti-disialoganglioside (GD2) monoclonal antibodies (MAbs) have been used in vivo for immunolocalization and in phase I and II trials to target disseminated neuroblastoma, the most common extracranial solid tumor in children. However, the efficacy of these first-generation MAbs is likely to be improved by using engineered anti-GD2 antibodies. The generation of single-chain antibody fragments (scFv) could be very helpful as these molecules can be further modified to produce recombinant molecules with pre-defined properties such as immunotoxins, chimeric, or bispecific antibodies. Thus, a scFv directed against GD2 (scFv 7A4) was cloned, sequenced, and expressed. Its binding properties were characterized and compared to that of the parental MAb 7A4. Nucleotide sequence analysis of the scFv 7A4 indicated that its VH region belongs to the V region IIID subgroup and the V kappa to the V region II subgroup. The scFv 7A4 bound to GD2+ neuroblastoma cell lines but not to GD2- cell lines or to GD2- cells isolated from peripheral blood. ELISA and thin-layer chromatography (TLC) indicated that it retained the anti-GD2 specificity, and exhibited a slight cross-reaction with GD3 as the parental MAb. This scFv makes it possible to develop new useful reagents through genetic engineering for adjuvant tumor therapy.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antibody Specificity; Base Sequence; Cloning, Molecular; Cross Reactions; Fluorescent Antibody Technique, Indirect; Gangliosides; Humans; Hybridomas; Immunoglobulin Fragments; Immunoglobulin Variable Region; Mice; Molecular Sequence Data; Neuroblastoma; Recombinant Fusion Proteins; Sequence Alignment; Sequence Analysis, DNA; Tumor Cells, Cultured

Topics: Antibodies, Monoclonal; Child; Child, Preschool; Female; Gangliosides; Humans; Infusions, Intravenous; Lidocaine; Male; Neuroblastoma; Pain

Shedding of neuroblastoma gangliosides is positively correlated with tumour progression in patients with neuroblastoma. In assessing the biological activity of these ganglioside molecules, we recently found that total human neuroblastoma gangliosides inhibit cellular immune responses. Here, we have studied the major neuroblastoma ganglioside, GD2. GD2 was purified by high performance liquid chromatography and structurally characterized by mass spectrometry. Immunoregulatory effects of GD2 in vivo were then determined in an established murine model. GD2 significantly downregulated the local cellular immune response to an allogeneic cell challenge; the usual increase in mass of the lymph node draining the injection site was reduced by 88%, from 1.52 to 0.19 mg (control versus GD2-treated mice; p < 0.01). In parallel, lymphocyte recovery from each node was also reduced from 2.4 to 1.2 x 10(6) cells, and lymphocyte DNA synthesis was reduced to half of the control level. These results show that certain shed tumour gangliosides, such as GD2, function as intercellular signalling molecules, downregulate the cellular immune response, and may thereby enhance tumour formation and progression.

Topics: Animals; Carbohydrate Sequence; Cell Line; Chromatography, High Pressure Liquid; Disease Progression; Gangliosides; Humans; Immunity, Cellular; Lymph Nodes; Lymphocyte Activation; Lymphocytes; Mass Spectrometry; Mice; Molecular Sequence Data; Neuroblastoma; Spectrometry, Mass, Fast Atom Bombardment; Tumor Cells, Cultured

Superantigens such as the staphylococcal enterotoxin A (SEA) are among the most potent T cell activators known. They bind to major histocompatibility complex (MHC) class II molecules and interact with T cells depending on their T cell receptor (TCR) V beta expression. Superantigens also induce a variety of cytokines and trigger a direct cytotoxic effect against MHC-class-II-positive target cells. In order to extend superantigen-dependent cell-mediated cytotoxicity (SDCC) to MHC-class-II-negative neuroblastoma cells, SEA was linked to the anti-ganglioside GD2 human/mouse chimeric monoclonal antibody (mAb) ch14.18. Ganglioside GD2 is expressed on most tumours of neuroectodermal origin but is expressed to a lesser extent on normal tissues. The linkage of ch14.18 to SEA was achieved either with a protein-A-SEA fusion protein or by chemical coupling. Both constructs induced T-cell-mediated cytotoxicity towards GD2-positive neuroblastoma cells in an effector-to-target(E:T)-ratio- and dose-dependent manner in vitro. To reduce the MHC class II affinity of SEA, a point mutation was introduced in the SEA gene (SEAm9) that resulted in 1000-fold less T cell killing of MHC-class-II-expressing cells as compared to native SEA. However, a protein-A-SEAm9 fusion protein mediated cytotoxicity similar to that of protein-A-SEA on ch14.18-coated, MHC-class-II-negative neuroblastoma cells. Taken together, these findings suggest that superantigen-dependent and monoclonal-antibody-targeted lysis may be a potent novel approach for neuroblastoma therapy.

Topics: Antibodies, Monoclonal; Enterotoxins; Flow Cytometry; Gangliosides; Histocompatibility Antigens Class II; Humans; Lymphoma, B-Cell; Neuroblastoma; Staphylococcal Protein A; Superantigens; T-Lymphocytes; Tumor Cells, Cultured

Neuroblastoma is the most frequent tumour of the childhood under the age of 5. The staging and the follow up are achieved by MIBG scintigraphy, considered as the method of reference, but sometimes difficult to interpret . The availability of monoclonal antibodies against the ganglioside GD2, expressed on the cell membrane of neuroblastoma and neuro-endocrine cancers offers novel tools that deserve to be carefully explored. We investigated four mouse monoclonal antibodies (3 IgG3: BW704, 7A4, 60C3, and the IgG1 variant of BW704: MAK704), on nude mice xenografted with a human neuroblastoma (REM). Sixty one nude mice were included. The three former MAbs provided tumour imaging, the best results being obtained with BW704, followed by 7A4 and 60C3. MAK704 was disappointing. A control antiphosphorylcholine antibody (P51-1) did not give any tumour image in the three tested mice. Scintigraphy ratios tumour/liver and tumour/muscle reached 20 and 100 with BW704, respectively, on the 10th day. Good imaging quality was already obtained from the 24th h. The tumour uptake, calculated from radioactivity countings of resected samples, reached 22 +/- 3% of injected dose per gramme. These results let us hope that these antibodies could also provide highly contrasted images in humans and could open the way for therapeutic applications.

Topics: Animals; Antibodies, Monoclonal; Child, Preschool; Female; Gangliosides; Humans; Infant; Iodine Radioisotopes; Mice; Mice, Nude; Neoplasm Transplantation; Neuroblastoma; Radioimmunodetection

A genetically engineered fusion protein consisting of a human/mouse chimeric anti-ganglioside GD2 antibody (ch14.18) and recombinant human interleukin 2 (rhIL-2) was tested for its ability to target rhIL-2 to tumor sites and stimulate immune effector cells sufficiently to achieve effective tumor cell lysis in vivo. The ch14.18-IL-2 fusion protein proved more effective than equivalent doses of rhIL-2 in suppressing dissemination and growth of human neuroblastoma in an experimental hepatic metastases model of scid (severe combined immunodeficiency) mice reconstituted with human lymphokine-activated killer cells. The ch14.18-IL-2 fusion protein was also more proficient than equivalent doses of rhIL-2 in prolonging the life-span of these animals. This recombinant antibody-cytokine fusion protein may prove useful for future treatment of GD2-expressing human tumors in an adjuvant setting.

Topics: Animals; Antibodies; Cytotoxicity, Immunologic; Gangliosides; Humans; Immunotherapy, Adoptive; Interleukin-2; Killer Cells, Lymphokine-Activated; Liver Neoplasms; Mice; Mice, SCID; Neuroblastoma; Recombinant Fusion Proteins; Transplantation, Heterologous

Mouse monoclonal antibodies against tumour-associated gangliosides GD2 (14.G2a) and GD3 (MB 3.6) were tested to mediate antibody-dependent cellular cytotoxicity (ADCC) with various effector cells or complement-dependent cytolysis (CDC). We also evaluated the immunomodulating potential of interferons in combination with cellular cytotoxicity. Using effector:target (E/T) ratios of 40:1, ADCC with effector cells such as granulocytes or mononuclear blood cells was not detectable against melanoma cell lines GR, SK-MEL-28 and G-361 which preferentially express GD3 and bind antibody MB 3.6. Neuroblastoma cell line SK-N-LO, which was used for comparative purposes, mainly expressed GD2 and the tumour cells were killed effectively after labelling with antibody 14.G2a. Granulocytes did not show significant killing of melanoma cells by ADCC, but neuroblastoma cells were killed very efficiently. Peripheral blood mononuclear cells (PBMC) also failed to kill melanoma cells. Interferon-beta slightly stimulated PBMC and increased killing of neuroblastoma cells, but no additive effects with ADCC were detectable. Incubation of target cells with interferons produced no significant differences in susceptibility of the target cells to interferon-activated PBMC cytotoxicity. Despite the lack of effectiveness in mediating cellular cytotoxicity, GD3 antibody MB 3.6 showed strong complement-dependent cytolysis in the presence of human plasma. There were remarkable differences in individual activity and different susceptibility of the melanoma cell lines. We assume that CDC may have more activity against melanoma cells than cytotoxicity associated with various effector cells.

Topics: Animals; Antibodies, Monoclonal; Cell Membrane; Complement Activation; Cytotoxicity, Immunologic; Gangliosides; Granulocytes; Humans; Immunotherapy; Interferon-beta; Killer Cells, Natural; Leukocytes, Mononuclear; Melanoma; Mice; Neuroblastoma; Tumor Cells, Cultured

Therapy of neuroblastoma patients with interleukin (IL)-2 activates effector cells capable of lysing tumor cells in vitro. When tumor cells are pretreated with certain monoclonal antibodies (MoAb), these in vivo activated effectors show augmented tumor lysis via antibody-dependent cellular cytotoxicity (ADCC). This study presents immunological analyses of serial blood samples from two refractory neuroblastoma patients who received combined in vivo therapy with murine anti-ganglioside GD2 monoclonal antibody 14.G2a and IL-2. These studies were designed to determine whether conditions that induce ADCC in vitro can be generated in vivo by combined therapy with IL-2 and MoAb. As shown previously, administration of IL-2 dramatically augments the ability of peripheral blood mononuclear cells (PBMC) to mediate ADCC. In addition, we demonstrate here that sera, obtained 1 h after infusion of 14.G2a, provides an effective source of functional antibody for ADCC mediated by PBMC from healthy donors. Finally, effective ADCC-mediated killing of neuroblastoma target cells was also achieved in vitro following IL-2 plus 14.G2a treatment when patients' effector cells were combined with patients' serum, as the source of 14.G2a antibody. These results indicate that this combination of IL-2 and 14.G2a generates conditions within the peripheral blood of pediatric neuroblastoma patients that enable their own lymphocytes to mediate antibody-dependent cellular cytotoxicity sufficient to effectively kill neuroblastoma cells in vitro.

Topics: Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Cell Line; Child; Child, Preschool; Female; Gangliosides; Humans; Immunotherapy; In Vitro Techniques; Interleukin-2; Male; Neuroblastoma

Molecular determinants of biological activity of gangliosides are generally believed to be carbohydrate in nature. However, our studies of immunomodulation by highly purified naturally occurring tumor gangliosides provide another perspective: while the immunosuppressive activity of gangliosides requires the intact molecule (both carbohydrate and ceramide moieties), ceramide structure strikingly influences ganglioside immunosuppressive activity. Molecular species of human neuroblastoma GD2 ganglioside in which the ceramide contains a shorter fatty acyl chain (C16:0, C18:0) were 6- to 10-fold more active than those with a longer fatty acyl chain (C22:0/C24:1, C24:0). These findings were confirmed in studies of ceramide species of human leukemia sialosylparagloboside and murine lymphoma GalNAcGM1b. Gangliosides that contain shorter-chain fatty acids (and are most immunosuppressive) are known to be preferentially shed by tumor cells. Therefore, the results suggest that the tumor cell is optimized to protect itself from host immune destruction by selective shedding of highly active ceramide species of gangliosides.

Topics: Animals; Brain; Brain Chemistry; Carbohydrate Sequence; Ceramides; Gangliosides; Humans; Immune Tolerance; Immunosuppressive Agents; Mice; Molecular Sequence Data; Neoplasms; Neuroblastoma; Structure-Activity Relationship; Tumor Cells, Cultured

Clinical strategies which modulate the human anti-mouse antibody response (HAMA) in patients may have a profound influence on the idiotype network inducible by murine monoclonal antibodies (MoAb). Prior to myeloablative chemotherapy (ABMT), 9 patients with Stage IV neuroblastoma were imaged with 131I-3F8, a MoAb specific for the ganglioside GD2. Their serum HAMA, anti-idiotypic, anti-GD2, and anti-anti-idiotypic antibodies were assayed by enzyme-linked immunosorbent assay prior to, and at 3 and 6 months postimaging. HAMA and anti-idiotypic levels remained low, in contrast to the high levels in 10 patients imaged with 131I-3F8 without ABMT. Five of the 9 patients are long-term survivors; all had elevated anti-GD2 and anti-anti-idiotypic levels, significantly higher than those who died of disease. Although 131I-3F8 imaging prior to ABMT detected abnormal sites in 4 of 9 patients, 3 of the 4 patients have continued in remission for 24-63 months after ABMT, and all 3 mounted anti-GD2 and anti-anti-idiotypic antibody responses. We conclude that myeloablative therapy strongly suppressed the HAMA/anti-idiotypic response to murine MoAb and that the prognostic significance of host immune response to ganglioside GD2 MoAb deserves further investigation.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Antibody Formation; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow Transplantation; Carmustine; Cisplatin; Enzyme-Linked Immunosorbent Assay; Etoposide; Gangliosides; Humans; Melphalan; Mice; Neoplasm Staging; Neuroblastoma; Prognosis; Radiotherapy Dosage; Survival Analysis; Survival Rate; Thiotepa

Disialoganglioside GD2 is widely expressed among neuroblastomas, melanomas, small-cell lung carcinoma, sarcomas and brain tumors. Immunity directed against this antigen may have anti-tumor utility. Since GD2 is poorly immunogenic, anti-idiotypic antibodies may serve as alternative tumor vaccines. Monoclonal antibody 3F8, a murine IgG3 specific for GD2, has shown excellent tumor-targeting ability in vitro and in vivo. LOU/CN rats were immunized with 3F8 and their spleens were used in somatic-cell hybridization, using SP2/0, P3 and Y3 as fusion partners. Six anti-idiotypic (anti-id) MAbs (C2D8, Idio-2, AIG4, C2H7, C4E4, A2A6) were selected based on their reactivity with 3F8 and non-reactivity with murine IgG3 myelomas. Specificity of each anti-id was demonstrated by using various ELISA: (i) lack of direct binding to solid phase myelomas and serum proteins; (ii) inability of other myelomas to inhibit anti-id binding to 3F8; (iii) absence of cross-reactivity of other myelomas to solid-phase anti-id; (iv) lack of inhibition by anti-id of binding of other ganglioside antibodies to their antigens. Antigen specificity was further examined by inhibition of binding of 3F8 to GD2 on immuno-thin-layer chromatography, and by inhibition of 3F8 immunostaining of neuroblastoma cell lines. These 6 antibodies were demonstrated to be distinct, in view of their cross-reactivity, fusion partners and relative strength of binding to 3F8. Anti-GD2 antibodies were induced after immunization with these anti-id antibodies in C57Bl/6 mice. These rat anti-3F8-idiotypic antibodies with exquisite specificity for anti-GD2 antibodies may be useful in vaccine construction.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Antibodies, Neoplasm; Antigens, Neoplasm; Cross Reactions; Enzyme-Linked Immunosorbent Assay; G(M2) Ganglioside; Gangliosides; Humans; Immunization; Immunotherapy; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Myeloma Proteins; Neuroblastoma; Rats; Rats, Inbred Strains

Murine monoclonal antibody (MAb) 3F8 was previously shown to react with disialoganglioside GD2, but not with GD3, GT1b, GD1b, GD1a, GM1, GM3 and GM4. However, when the base-treatment step was ommitted from the standard neuroblastoma ganglioside extraction procedure, immuno-thin-layer-chromatography (ITLC) using 3F8 and other anti-GD2 MAbs revealed a new ganglioside band, abbreviated as NG (Rf 0.342) besides GD2 (R 0.183). It migrated below GD3 (Rf 0.358) on high-performance (HP) TLC plate and its binding to 3F8 on ITLC could be inhibited by rat anti-3F8 idiotypic antibody Idio-2, while the binding of GD2 to MAb 3F8 was not affected. Immunochemical analysis showed that this new neuroblastoma ganglioside contained alkali-sensitive O-acetylated sialic-acid residues recognized by MAb DI.I. After base treatment, its subsequent identity on ITLC was confirmed to be GD2. Lactonization of GD2 yielded 2 major bands, with Rf values (0.401, 0.583) distinct from that of the new ganglioside band. In addition, MAb DI.I did not bind to any of these GD2 lactones. Of 15 anti-GD2 MAbs studied, 13 reacted strongly with the novel ganglioside NG. By ITLC, this NG was found in ganglioside extracts of fresh surgical tumor specimens (4/4 neuroblastomas, I/I schwannoma and I/I anaplastic astrocytoma), and nude mice/rat xenografts (2/2 neuroblastomas, 2/2 osteogenic sarcomas). These data provided the first evidence that O-acetylated GD2 is a naturally occurring ganglioside derivative in human tumors and that it could cross-react with most anti-GD2 antibodies.

Topics: Acetylation; Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Antigens, Neoplasm; Chromatography, Thin Layer; Gangliosides; Humans; Hydrogen-Ion Concentration; Male; Mice; Mice, Nude; Neuroblastoma; Rats; Rats, Nude

An anti-GD2 ganglioside human/mouse chimeric monoclonal antibody, ch14.18, like its murine counterpart, 14.G2a, was shown to bind to human neuroblastoma cells. This chimeric antibody proved to be more effective than 14.G2a in mediating the lysis of neuroblastoma cells with human effector cells, such as granulocytes and natural killer cells within the peripheral blood mononuclear cell population. A comparison of these two effector cell populations isolated from the same donor revealed granulocytes to be more effective than peripheral blood mononuclear cells in lysing neuroblastoma cells, which were coated with monoclonal antibody ch14.18. Addition of recombinant human granulocyte-macrophage colony-stimulatory factor increased ch14.18-mediated lysis of neuroblastoma cells by granulocytes but not by peripheral blood mononuclear cells. In fact, granulocytes were effective in mediating lysis of neuroblastoma cells coated with ch14.18 irrespective of whether they were obtained from normal adults or from neuroblastoma patients.

Topics: Animals; Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Antigens, Neoplasm; Antigens, Surface; Chimera; Dose-Response Relationship, Immunologic; Gangliosides; Granulocyte-Macrophage Colony-Stimulating Factor; Granulocytes; Humans; Killer Cells, Natural; Leukocytes, Mononuclear; Mice; Neuroblastoma; Recombinant Proteins

Three monoclonal antibodies (IgG2) have been produced from hybridomas obtained by fusion of murine myeloma cells and spleen cells of mice hyperimmunized with gamma-interferon-treated neuroblastoma cells. The 3 MAbs, 7A4, 2A6 and IG8, detected an antigen present on neuroblastoma tumors and cell lines, but also on some neuro-ectoderm-derived tissues and cells. All 3 clones were shown to react with an epitope of the di-sialo-ganglioside GD2 molecules highly expressed by some neuro-ectoderm-derived tumors, mainly neuroblastoma. Whereas MAb IG8 specificity was restricted to GD2 and its o-acylated form, MAb 2A6 and 7A4 were also able to detect GD3 at high concentration of antibody as shown by TLC analysis and immunodetection. The 3 MAbs were able to lyse 100% neuroblastoma cells in the presence of rabbit or human complement. Direct binding assays with 125I-labelled MAbs showed that MAb 7A4 might be a good candidate for in vivo immunolocalization experiments. The high proportion of anti-GD2 MAbs obtained by our fusion and the increased binding of anti-GD2 MAbs on gamma-IFN-treated neuroblastoma cells suggests a modulation of the exposure and an increase in the immunogenicity of GD2 induced by gamma-IFN.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Binding Sites, Antibody; Cell Line; Complement System Proteins; Cytotoxicity, Immunologic; Epitopes; Gangliosides; Humans; Hybridomas; Immunization; Immunoglobulin Isotypes; Interferon-gamma; Mice; Neuroblastoma

A complete family of IgG isotype switch variant hybridomas was generated from the anti-GD2 monoclonal IgG3-producing hybridoma, 14.18, with the aid of the fluorescence-activated cell sorter. The IgG1, IgG2b, and IgG2a monoclonal antibodies (Mabs) produced by respective isotype switch variant hybridomas 14G1, 14G2b, or 14G2a, have binding activities for the biochemically defined GD2 antigen and GD2-expressing neuroblastoma target cell lines identical to that of IgG3 Mabs produced by the 14.18 parent cell line. This permitted us to examine the relative in vitro and in vivo cytotoxic capacities of each of the anti-GD2 antibodies for GD2-expressing neuroblastoma cells independent of antibody binding affinity or specificity. Mabs produced by 14.18, 14G2a, or 14G2b, but not 14G1, can direct efficient complement-dependent cytotoxicity against neuroblastoma tumor cells in the presence of human complement. Mabs produced by the parent 14.18 or by 14G2a are more efficient in directing antibody-dependent cell-mediated cytotoxicity than Mabs produced by 14G2b, and Mabs of 14G1 are inactive. However, despite these noted in vitro differences, antibodies produced by each member of this switch variant family suppress the growth of human neuroblastoma tumor cells in BALB/c athymic nu/nu mice. These studies suggest that a mechanism(s) other than Fc-directed complement-dependent cytotoxicity or antibody-dependent cell-mediated cytotoxicity may account for the in vivo antitumor effects of these particular antibodies.

Topics: Animals; Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Gangliosides; Humans; Immunity, Cellular; Mice; Mice, Inbred BALB C; Mice, Nude; Neuroblastoma; Organ Specificity; Tumor Cells, Cultured

Topics: Antibodies, Monoclonal; Gangliosides; Humans; Immunotherapy; Iodine Radioisotopes; Neoplasm Metastasis; Neuroblastoma

Concentration of gangliotriaose-series glycosphingolipids, including GA2, GM2, GD2 and GT2, was measured in human sera by a thin-layer chromatography/enzyme-immunostaining method. By this method, as little as 5-10 ng/ml of these glycolipids in serum could be determined simultaneously. Although GD2 ganglioside could be consistently detected in normal cord blood (1-2 ng/ml of serum), the ganglioside was never detected in normal adult serum. However, the same ganglioside was found to be present in large quantity in preoperative sera of 6/9 patients with neuroblastomas (25-658 ng/ml of serum). In addition to GD2, gangliosides GM2 and GA2 increased concomitantly than usual. It is concluded that this highly sensitive quantification of the tumor-associated glycolipids circulating in serum of neuroblastoma patients could be useful in their diagnosis.

Topics: Adult; Chromatography, Thin Layer; Fetal Blood; G(M2) Ganglioside; Gangliosides; Glycosphingolipids; Humans; Immunoenzyme Techniques; Neuroblastoma

Substantial concentrations of the cell-surface glycosphingolipid, the disialoganglioside GD2, are uniformly present in human neuroblastoma tumors. This ganglioside can also be detected in the plasma of patients with neuroblastoma by direct thin-layer chromatographic analysis. Among 32 neuroblastoma patients in all clinical stages studied prior to the initiation of treatment, 27 (84%) showed measurably elevated plasma concentrations of GD2 (greater than or equal to 50 pmol/ml). The mean level (545 +/- 108 pmol/ml) was more than 50 times the normal plasma GD2 concentration of less than or equal to 10 pmol/ml. Circulating GD2 was not detected in the plasma of patients with the related, more differentiated tumors, ganglioneuroblastoma and ganglioneuroma, indicating an association of the shedding of this ganglioside with the undifferentiated phenotype. Circulating GD2 diminished in patients in response to therapy, and reappeared in patients whose disease recurred. The results suggest that the sequential determination of circulating GD2 will be of value in monitoring individual patients with neuroblastoma.

Topics: Antibodies, Monoclonal; Bone Marrow Transplantation; Gangliosides; Humans; Neuroblastoma

The murine IgG3 monoclonal antibody (MoAb) 3F8, specific for the ganglioside GD2, activates human complement, is active in antibody-dependent cell-mediated cytotoxicity (ADCC), and can target specifically to human neuroblastoma in patients with metastatic disease. In a phase I study, 3F8 was administered intravenously (IV) to 17 patients with metastatic GD2 positive neuroblastoma or malignant melanoma at doses of 5, 20, 50, and 100 mg/m2. Serum 3F8 levels achieved were proportional to the dose of 3F8 infused. However, serum antimouse antibody levels did not increase with the amount of 3F8 administered. Toxicities included pain, hypertension, urticaria, and complement depletion. All acute side effects were controllable with symptomatic therapy. No long-term side effects were detected in patients observed for more than 14 months. None of the 17 patients received any antitumor therapy postantibody treatment. Antitumor responses occurred in seven of 17 patients. These ranged from complete clinical remissions to mixed responses. The murine monoclonal antibody (MoAb) 3F8 has clinical utility for the diagnosis and therapy of neuroblastoma and melanoma.

Topics: Adolescent; Adult; Antibodies, Monoclonal; Child; Child, Preschool; Drug Evaluation; Female; Gangliosides; Humans; Infant; Male; Melanoma; Middle Aged; Neuroblastoma

The biodistribution of 3F8, and IgG3 murine monoclonal antibody (MoAb) specific for the disialoganglioside GD2, was studied in nude mice xenografted with human neuroblastoma (NB). 3F8 conjugated to radioactive iodine and injected intravenously localized selectively to seven human NB when compared with Ewing's sarcoma and Hela cell xenograft controls. Uptake in NB was shown to be specific for MoAb 3F8 when contrasted with pooled mouse IgG or irrelevant IgG3 MoAb controls. Both small (50 mg) and large tumors greater than 2 g) showed radiolocalization. The percent injected dose uptake per gram tumor ranged from 8 to 50% and was inversely correlated with tumor size. Optimal tumor to normal tissue ratios were reached by 24-48 hr. There was no abnormal uptake in the reticuloendothelial system and the MoAb did not cross the blood-brain barrier. Based on the kinetics of the amount of radioactivity deposited in tissues, the relative radiation dose to normal organs was estimated to be 1% to 20% of the tumor dose. The MoAb 3F8 is useful for targeting radioactivity to human NB in vivo and the nude mice xenograft model may allow optimization of parameters that influence such biodistribution.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Gangliosides; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Neuroblastoma; Tissue Distribution; Transplantation, Heterologous

Human melanoma cells express relatively large amounts of the disialogangliosides GD3 and GD2 on their surface whereas neuroblastoma cells express GD2 as a major ganglioside. Monoclonal antibodies (Mabs) directed specifically to the carbohydrate moiety of GD3 and GD2 inhibit melanoma and neuroblastoma cell attachment to various substrate adhesive proteins, e.g. collagen, vitronectin, laminin, fibronectin, and a heptapeptide, glycyl-L-arginyl-glycyl-L-aspartyl-L-seryl-L-prolyl-L-cysteine, which constitutes the cell attachment site of fibronectin. Cells that are preattached to a fibronectin substrate can also be induced to detach and round up in the presence of purified anti-ganglioside Mab. Moreover, when melanoma cells that contain both GD2 and GD3 are incubated with Mabs directed to both of these molecules an additive inhibition is observed. The specificity of this inhibition is demonstrated since Mabs of various isotypes directed to either protein or carbohydrate epitopes on a number of other major melanoma or neuroblastoma cell surface antigens have no effect on cell attachment. A study of the kinetics involved in this inhibition indicates that significant effects occur during the first 5 min of cell attachment, suggesting an important role for GD2 and GD3 in the initial events of cell-substrate interactions. The role of gangliosides in cell attachment apparently does not directly involve a strong interaction with fibronectin since we could not observe any binding of radiolabeled fibronectin or fragments of the molecule known to contain the cell attachment site to melanoma gangliosides separated on thin-layer chromatograms. An alternative explanation would be that gangliosides may play a role in the electrostatic requirements for cell-substrate interactions. In this regard, controlled periodate oxidation of terminal, unsubstituted sialic acid residues on the cell surface not only specifically destroys the antigenic epitopes on GD2 and GD3 recognized by specific Mabs but also inhibits melanoma cell and neuroblastoma cell attachment. In fact, the periodate-induced ganglioside oxidation and the inhibition of cell attachment are equally dose dependent. These data suggest that cell-substratum interactions may depend in part on the electrostatic environment provided by terminal sialic acid residues of cell surface gangliosides and possibly other anionic glycoconjugates.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Antigens, Surface; Cell Adhesion; Cell Line; Extracellular Matrix; Fibronectins; Gangliosides; Humans; Laminin; Lung Neoplasms; Melanoma; Neuroblastoma; Periodic Acid

Primary neuroblastomas obtained before therapy from 36 patients were studied to determine the frequency of tumors expressing a specific glycosphingolipid, GD2 ganglioside. Total tissue gangliosides were purified by a new partition method, quantitated, and analyzed by high-performance thin-layer chromatography. All 36 neuroblastoma tumors, representing all clinical stages, contained GD2 ganglioside. The mean relative and absolute concentrations of GD2 were substantial (12% of the total tissue gangliosides and 50 nmol/g of tissue) and were independent of the clinical stage of the tumor. In contrast, 6 samples of related but more differentiated tumors (ganglioneuroblastoma and ganglioneuroma) had little or no detectable GD2 (less than or equal to 1.5% of total gangliosides and less than or equal to 4 nmol/g of tissue). These results suggest that GD2 is a sensitive marker for neuroblastoma tissue and may be an excellent target antigen for immunotherapy of this tumor.

Topics: Cell Differentiation; Gangliosides; Humans; Neuroblastoma; Neuroma

Four monoclonal antibodies (3F8, 3A7, 3G6, and 2F7) against human neuroblastoma cells have been suggested to react with surface glycolipids of these cells. In this report these monoclonal antibodies were shown to be specific to the disialoganglioside GD2 using a thin-layer chromatography (TLC)-immunostaining method. When mixed human brain gangliosides were developed by TLC in two different solvent systems and incubated with each of the monoclonal antibodies, only GD2 was stained. These antibodies also reacted with highly purified GD2 on the plate. These findings suggest that GD2 provides an antigenic site on the surface of human neuroblastoma cells.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Brain Chemistry; Chromatography, Thin Layer; Epitopes; Gangliosides; Humans; Mice; Mice, Inbred BALB C; Neuroblastoma

A murine monoclonal antibody (monoclonal antibody 126) produced against cultured human neuroblastoma cells (LAN-1) was found to be specifically directed to a disialoganglioside (GD2) antigen preferentially expressed on both cell lines and tissues derived from melanoma and neuroblastoma. In enzyme-linked immunosorbent assays, monoclonal antibody 126 failed to react with leukemic and lymphoblastoid cells as well as with a variety of carcinoma and sarcoma cell lines. Immunohistological analysis by the immunoperoxidase technique revealed strong reactivity of monoclonal antibody 126 with frozen and formaldehyde-fixed neuroblastoma and melanoma tissues. Tissues from patients with glioma or with small cell cancer of the lung showed faint staining, whereas those from individuals with sarcoma, lymphoma, and a variety of other neoplasms proved to be negative. Sera of neuroblastoma patients showed significantly elevated GD2 levels compared to normal children (p less than 0.001) and children with other tumors (p less than 0.001) as determined by a quantitative competitive enzyme-linked immunosorbent assay. Furthermore, the GD2 serum level of one neuroblastoma patient, when followed serially, was found to correlate with progression of disease, suggesting the potential usefulness of this assay for the diagnosis and monitoring of neuroblastoma.

Topics: Antibodies, Monoclonal; Cell Line; Enzyme-Linked Immunosorbent Assay; Gangliosides; Humans; Neoplasms; Neuroblastoma