-
Journal of Personalized Medicine Aug 2021Cancer immunotherapy has transformed the landscape of adult cancer treatment and holds a great promise to treat paediatric malignancies. However, in vitro test coculture...
Cancer immunotherapy has transformed the landscape of adult cancer treatment and holds a great promise to treat paediatric malignancies. However, in vitro test coculture systems to evaluate the efficacy of immunotherapies on representative paediatric tumour models are lacking. Here, we describe a detailed procedure for the establishment of an ex vivo test coculture system of paediatric tumour organoids and immune cells that enables assessment of different immunotherapy approaches in paediatric tumour organoids. We provide a step-by-step protocol for an efficient generation of patient-derived diffuse intrinsic pontine glioma (DIPG) and neuroblastoma organoids stably expressing eGFP-ffLuc transgenes using defined serum-free medium. In contrast to the chromium-release assay, the new platform allows for visualization, monitoring and robust quantification of tumour organoid cell cytotoxicity using a non-radioactive assay in real-time. To evaluate the utility of this system for drug testing in the paediatric immuno-oncology field, we tested our in vitro assay using a clinically used immunotherapy strategy for children with high-risk neuroblastoma, dinutuximab (anti-GD2 monoclonal antibody), on GD2 proficient and deficient patient-derived neuroblastoma organoids. We demonstrated the feasibility and sensitivity of our ex vivo coculture system using human immune cells and paediatric tumour organoids as ex vivo tumour models. Our study provides a novel platform for personalized testing of potential anticancer immunotherapies for aggressive paediatric cancers such as neuroblastoma and DIPG.
PubMed: 34575646
DOI: 10.3390/jpm11090869 -
Paediatric Drugs Nov 2021Neuroblastoma is the most common extracranial solid tumour in children, accounting for 15% of all paediatric cancer deaths. High-risk neuroblastoma is a particularly...
Neuroblastoma is the most common extracranial solid tumour in children, accounting for 15% of all paediatric cancer deaths. High-risk neuroblastoma is a particularly challenging-to-treat form of disease that requires multimodality treatment, consisting of chemotherapy, surgery, high-dose chemotherapy with autologous haematopoietic stem cell rescue, radiotherapy and differentiation therapy. However, despite intense multimodal treatment regimens, the prognosis for this patient population remains poor. In recent years, immunotherapy with anti-disialoganglioside 2 (anti-GD2) antibodies was found to improve survival rates for patients with high-risk neuroblastoma. Based on studies led by the SIOPEN (International Society of Paediatric Oncology European Neuroblastoma) group, the anti-GD2 antibody dinutuximab beta was approved for use in high-risk neuroblastoma by the European Medicines Agency and has been implemented into the standard of care in many countries across Europe. However, immunotherapy with dinutuximab beta is associated with a number of adverse events that may be challenging for clinicians, such as pain, fever, hypersensitivity reactions and capillary leak syndrome. While these adverse events are considered manageable, there are currently no formal guidelines to support clinicians with their management. The aim of this article is to discuss the management of the most common adverse events encountered in clinical practice and to provide practical guidance to assist clinicians in minimising toxicity associated with dinutuximab beta.
Topics: Antibodies, Monoclonal; Humans; Immunologic Factors; Immunotherapy; Neuroblastoma
PubMed: 34541620
DOI: 10.1007/s40272-021-00469-9 -
Journal of Chromatography. A Oct 2021Due to the increasing number of therapeutic monoclonal antibodies (mAbs) used in the clinic, there is an increasing need for robust analytical methods to quantify total...
A generic sample preparation method for the multiplex analysis of seven therapeutic monoclonal antibodies in human plasma or serum with liquid chromatography-tandem mass spectrometry.
Due to the increasing number of therapeutic monoclonal antibodies (mAbs) used in the clinic, there is an increasing need for robust analytical methods to quantify total mAb concentrations in human plasma for clinical studies and therapeutic drug monitoring. We developed an easy, rapid, and robust sample preparation method for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The method was validated for infliximab (IFX), rituximab (RTX), cetuximab (CTX), dupilumab (DPL), dinutuximab (DNX), vedolizumab (VDZ), and emicizumab (EMZ). Saturated ammonium sulfate (AS) was used to precipitate immunoglobulins in human plasma. After centrifugation, supernatant containing albumin was decanted, and the precipitated immunoglobulin fraction was re-dissolved in buffer containing 6M guanidine. This fraction was then completely denatured, reduced, alkylated, and trypsin digested. Finally, signature peptides from the seven mAbs were simultaneously quantified on LC-MS/MS together with their internal standards stable isotopically labeled peptide counterparts. The linear dynamic ranges (1 - 512 mg/L) of IFX, CTX, RTX, and EMZ showed excellent (R2 > 0.999) linearity and those of DPL, DNX, and VDZ showed good (R2 > 0.995) linearity. The method was validated in accordance with the EMA guidelines. EDTA plasma, sodium citrate plasma, heparin plasma, and serum yielded similar results. Prepared samples were stable at room temperature (20°C) and at 5°C for 3 days, and showed no decline in concentration for all tested mAbs. This described method, which has the advantage of an easy, rapid, and robust pre-analytical sample preparation, can be used as a template to quantify other mAbs in human plasma or serum.
Topics: Antibodies, Monoclonal; Chromatography, Liquid; Humans; Infliximab; Plasma; Tandem Mass Spectrometry
PubMed: 34509691
DOI: 10.1016/j.chroma.2021.462489 -
Cancers Aug 2021High-risk neuroblastoma, especially after recurrence, still has a very low survival rate. Immune checkpoint inhibitors targeting T cells have shown remarkable clinical...
High-risk neuroblastoma, especially after recurrence, still has a very low survival rate. Immune checkpoint inhibitors targeting T cells have shown remarkable clinical efficacy in adult solid tumors, but their effects in pediatric cancers have been limited so far. On the other hand, targeting myeloid immune checkpoints, such as CD47-SIPRα, provide the opportunity to enhance antitumor effects of myeloid cells, including that of neutrophils, especially in the presence of cancer-opsonizing antibodies. Disialoganglioside (GD2)-expressing neuroblastoma cells targeted with anti-GD2 antibody dinutuximab are in part eradicated by neutrophils, as they recognize and bind the antibody targeted tumor cells through their Fc receptors. Therapeutic targeting of the innate immune checkpoint CD47-SIRPα has been shown to promote the potential of neutrophils as cytotoxic cells in different solid tumor indications using different cancer-targeting antibodies. Here, we demonstrate that the capacity of neutrophils to kill dinutuximab-opsonized neuroblastoma cells is also controlled by the CD47-SIRPα axis and can be further enhanced by antagonizing CD47-SIRPα interactions. In particular, CD47-SIRPa checkpoint inhibition enhanced neutrophil-mediated ADCC of dinutuximab-opsonized adrenergic neuroblastoma cells, whereas mesenchymal neuroblastoma cells may evade immune recognition by a reduction of GD2 expression. These findings provide a rational basis for targeting CD47-SIRPα interactions to potentiate dinutuximab responsiveness in neuroblastomas with adrenergic phenotype.
PubMed: 34503071
DOI: 10.3390/cancers13174261 -
Frontiers in Immunology 2021Haploidentical stem cell transplantation (haplo SCT) in Stage IV neuroblastoma relapsed patients has been proven efficacious, while immunotherapy utilizing the anti-GD2...
Immunomonitoring of Stage IV Relapsed Neuroblastoma Patients Undergoing Haploidentical Hematopoietic Stem Cell Transplantation and Subsequent GD2 (ch14.18/CHO) Antibody Treatment.
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 testing of patient-derived effector cells and the sera collected during dinutuximab therapy demonstrated both high functionality of the newly established lymphoid immune compartment and provided confidence that the antibody dosing regimen was sufficient over the duration of the dinutuximab therapy (up to nine cycles in a 9-month period). During the course of the dinutuximab therapy, proinflammatory cytokines and markers (sIL2R, TNFa, IL6, and C reactive protein) were significantly elevated indicating a strong anti-GD2 immune response. No impact of FcGR polymorphism on event-free and overall survival was found. Collectively, this study has shown that in-patient functional immunomonitoring is feasible and valuable in contributing to the understanding of anti-cancer combinatorial treatments such as haplo SCT and antibody immunotherapy.
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
PubMed: 34367149
DOI: 10.3389/fimmu.2021.690467 -
Journal For Immunotherapy of Cancer Jul 2021Neuroblastoma is the most common extracranial solid tumor of childhood. Patients with high-risk disease undergo extremely aggressive therapy and nonetheless have cure...
BACKGROUND
Neuroblastoma is the most common extracranial solid tumor of childhood. Patients with high-risk disease undergo extremely aggressive therapy and nonetheless have cure rates below 50%. Treatment with the ch14.18 monoclonal antibody (dinutuximab beta), directed against the GD2 disialoganglioside, improved 5-year event-free survival in high-risk patients when administered in postconsolidation therapy and was recently implemented in standard therapy. Relapse still occurred in 57% of these patients, necessitating new therapeutic options. Bispecific trifunctional antibodies (trAbs) are IgG-like molecules directed against T cells and cancer surface antigens, redirecting T cells (via their CD3 specificity) and accessory immune cells (via their functioning Fc-fragment) toward tumor cells. We sought proof-of-concept for GD2/CD3-directed trAb efficacy against neuroblastoma.
METHODS
We used two GD2-specific trAbs differing only in their CD3-binding specificity: EKTOMUN (GD2/human CD3) and SUREK (GD2/mouse Cd3). This allowed trAb evaluation in human and murine experimental settings. Tumor-blind trAb and the ch14.18 antibody were used as controls. A coculture model of human peripheral blood mononuclear cells (PBMCs) and neuroblastoma cell lines was established to evaluate trAb antitumor efficacy by assessing expression of T-cell surface markers for activation, proinflammatory cytokine release and cytotoxicity assays. Characteristics of tumor-infiltrating T cells and response of neuroblastoma metastases to SUREK treatment were investigated in a syngeneic immunocompetent neuroblastoma mouse model mimicking minimal residual disease.
RESULTS
We show that EKTOMUN treatment caused effector cell activation and release of proinflammatory cytokines in coculture with neuroblastoma cell lines. Furthermore, EKTOMUN mediated GD2-dependent cytotoxic effects in human neuroblastoma cell lines in coculture with PBMCs, irrespective of the level of target antigen expression. This effect was dependent on the presence of accessory immune cells. Treatment with SUREK reduced the intratumor Cd4/Cd8 ratio and activated tumor infiltrating T cells in vivo. In a minimal residual disease model for neuroblastoma, we demonstrated that single-agent treatment with SUREK strongly reduced or eliminated neuroblastoma metastases in vivo. SUREK as well as EKTOMUN demonstrated superior tumor control compared with the anti-GD2 antibody, ch14.18.
CONCLUSIONS
Here we provide proof-of-concept for EKTOMUN preclinical efficacy against neuroblastoma, presenting this bispecific trAb as a promising new agent to fight neuroblastoma.
Topics: Animals; Antibodies, Bispecific; Antibodies, Monoclonal; Antineoplastic Agents; Disease Models, Animal; Female; Humans; Immunotherapy; Male; Mice; Neoplasm Metastasis; Neuroblastoma
PubMed: 34285106
DOI: 10.1136/jitc-2021-002923 -
Journal For Immunotherapy of Cancer Jul 2021Children with recurrent and/or metastatic osteosarcoma (OS), neuroblastoma (NB) and glioblastoma multiforme (GBM) have a dismal event-free survival (<25%). The majority...
Combinatorial immunotherapy of N-803 (IL-15 superagonist) and dinutuximab with ex vivo expanded natural killer cells significantly enhances in vitro cytotoxicity against GD2 pediatric solid tumors and in vivo survival of xenografted immunodeficient NSG mice.
BACKGROUND
Children with recurrent and/or metastatic osteosarcoma (OS), neuroblastoma (NB) and glioblastoma multiforme (GBM) have a dismal event-free survival (<25%). The majority of these solid tumors highly express GD2. Dinutuximab, an anti-GD2 monoclonal antibody, significantly improved event-free survival in children with GD2 NB post autologous stem cell transplantation and enhanced natural killer (NK) cell-mediated antibody-dependent cell cytotoxicity. Thus, approaches to increase NK cell number and activity, improve persistence and trafficking, and enhance tumor targeting may further improve the clinical benefit of dinutuximab. N-803 is a superagonist of an interleukin-15 (IL-15) variant bound to an IL-15 receptor alpha Su-Fc fusion with enhanced biological activity.
METHODS
The anti-tumor combinatorial effects of N-803, dinutuximab and ex vivo expanded peripheral blood NK cells (exPBNK) were performed in vitro using cytoxicity assays against GD2 OS, NB and GBM cells. Perforin and interferon (IFN)-γ levels were measured by ELISA assays. Multiple cytokines/chemokines/growth factors released were measured by multiplex assays. Human OS, GBM or NB xenografted NOD/SCID/IL2rγnull (NSG) mice were used to investigate the anti-tumor combinatorial effects in vivo.
RESULTS
N-803 increased the viability and proliferation of exPBNK. The increased viability and proliferation are associated with increased phosphorylation of Stat3, Stat5, AKT, p38MAPK and the expression of NK activating receptors. The combination of dinutuximab and N-803 significantly enhanced in vitro cytotoxicity of exPBNK with enhanced perforin and IFN-γ release against OS, GBM and NB. The combination of exPBNK+N-803+dinutuximab significantly reduced the secretion of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), platelet-derived growth factor-BB (PDGF-BB), and stem cell growth factor beta (SCGF-β) from OS or GBM tumor cells. Furthermore, OS or GBM significantly inhibited the secretion of regulated on activation, normal T cell expressed and presumably secreted (RANTES) and stromal cell-derived factor-1 alpha (SDF-1α) from exPBNK cells (p<0.001) but significantly enhanced monokine induced by gamma interferon (MIG) secretion from exPBNK cells (p<0.001). N-803 combined with dinutuximab and exPBNK cells significantly extended the survival of OS, GBM or NB xenografted NSG mice.
CONCLUSIONS
Our results provide the rationale for the development of a clinical trial of N-803 in combination with dinutuximab and ex vivo exPBNK cells in patients with recurrent or metastatic GD2 solid tumors.
Topics: Animals; Antibodies, Monoclonal; Antineoplastic Combined Chemotherapy Protocols; Heterografts; Humans; Immunotherapy; Interleukin-15; Killer Cells, Natural; Mice; Mice, Inbred NOD; Neoplasms; Recombinant Fusion Proteins; Survival Analysis
PubMed: 34244307
DOI: 10.1136/jitc-2020-002267 -
Case Reports in Pediatrics 2021Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of all pediatric cancer deaths. High-risk neuroblastoma (HRNB) is a...
Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of all pediatric cancer deaths. High-risk neuroblastoma (HRNB) is a particularly difficult-to-treat form of the disease that requires aggressive multimodality therapy, including induction chemotherapy, consolidation therapy with high-dose chemotherapy and autologous stem cell transplant, and maintenance therapy with dinutuximab beta. Despite treatment advances, the prognosis of these patients remains poor. As a better response to induction therapy has been associated with prolonged survival in patients with HRNB, we hypothesized that early use of dinutuximab beta-post-induction chemotherapy-may improve patient outcomes. We describe here our experience of administering at least one cycle of dinutuximab beta post-induction and prior to surgery in three children with HRNB who did not demonstrate a complete response to induction chemotherapy. All three patients achieved complete remission. Early use of dinutuximab beta may therefore have the potential to improve outcomes in patients with HRNB.
PubMed: 34239748
DOI: 10.1155/2021/6610955 -
Journal of Nuclear Medicine : Official... Feb 2022The tumor-selective ganglioside antigene GD2 is frequently expressed on neuroblastomas and to a lesser extent on sarcomas and neuroendocrine tumors. The aim of our study...
The tumor-selective ganglioside antigene GD2 is frequently expressed on neuroblastomas and to a lesser extent on sarcomas and neuroendocrine tumors. The aim of our study was to evaluate the tumor targeting and biodistribution of I-labeled chimeric GD2-antibody clone 14/18 (I-GD2-ch14.18) in patients with late-stage disease in order to identify eligibility for radioimmunotherapy. Twenty patients (neuroblastoma, = 9; sarcoma, = 9; pheochromocytoma, = 1; and neuroendocrine tumor, = 1) were involved in this study. A 21- to 131-MBq dose (1-2 MBq/kg) of I-GD2-ch14.18 (0.5-1.0 mg) was injected intravenously. Planar scintigraphy was performed within 1 h from injection (day 0) and on days 1, 2, 3, and 6 or 7 to analyze tumor uptake and tracer biodistribution. Serial blood samples were collected in 4 individuals. Absorbed dose to tumor lesions and organs was calculated using OLINDA software. The tumor-targeting rate on a per-patient base was 65% (13/20), with 6 of 9 neuroblastomas showing uptake of I-GD2-ch14.18. Tumor lesions showed maximum uptake at 20-64 h after injection (effective half-life in tumors, 33-192 h). The tumor-absorbed dose varied between 0.52 and 30.2 mGy/MBq (median, 9.08 mGy/MBq; = 13). Visual analysis showed prominent blood-pool activity up to day 2 or 3 after injection. No pronounced uptake was observed in the bone marrow compartment or in the kidneys. Bone marrow dose was calculated at 0.09-0.18 mGy/MBq (median, 0.12 mGy/MBq), whereas blood dose was 1.1-4.7 mGy/MBq. Two patients (1 neuroblastoma and 1 pheochromocytoma) with particularly high tumor uptake underwent radioimmunotherapy using 2.3 and 2.9 GBq of I-GD2-ch14.18, both achieving stable disease. Overall survival was 17 and 6 mo, respectively. I-GD2-ch14.18 is cleared slowly from blood, not resulting in good tumor-to-background contrast until 2 d after application. With acceptable red marrow and organ dose, radioimmunotherapy is an option for patients with high tumor uptake. However, because of the variable GD2 expression, the decision should depend on pretherapeutic dosimetry.
Topics: Adrenal Gland Neoplasms; Antibodies, Monoclonal; Gangliosides; Humans; Iodine Radioisotopes; Neuroblastoma; Neuroendocrine Tumors; Pheochromocytoma; Radioimmunotherapy; Radionuclide Imaging; Tissue Distribution
PubMed: 34049985
DOI: 10.2967/jnumed.120.261854 -
Journal For Immunotherapy of Cancer May 2021Current immunotherapy for patients with high-risk neuroblastoma involves the therapeutic antibody dinutuximab that targets GD2, a ganglioside expressed on the majority... (Comparative Study)
Comparative Study
BACKGROUND
Current immunotherapy for patients with high-risk neuroblastoma involves the therapeutic antibody dinutuximab that targets GD2, a ganglioside expressed on the majority of neuroblastoma tumors. Opsonized tumor cells are killed through antibody-dependent cellular cytotoxicity (ADCC), a process mediated by various immune cells, including neutrophils. The capacity of neutrophils to kill dinutuximab-opsonized tumor cells can be further enhanced by granulocyte-macrophage colony-stimulating factor (GM-CSF), which has been shown in the past to improve responses to anti-GD2 immunotherapy. However, access to GM-CSF (sargramostim) is limited outside of Northern America, creating a high clinical need for an alternative method to stimulate dinutuximab responsiveness in the treatment of neuroblastoma. In this in vitro study, we have investigated whether clinically well-established granulocyte colony-stimulating factor (G-CSF) can be a potentially suitable alternative for GM-CSF in the dinutuximab immunotherapy regimen of patients with neuroblastoma.
METHODS
We compared the capacity of neutrophils stimulated either in vitro or in vivo with GM-CSF or G-CSF to kill dinutuximab-opsonized GD2-positive neuroblastoma cell lines and primary patient tumor material. Blocking experiments with antibodies inhibiting either respective Fc gamma receptors (FcγR) or neutrophil integrin CD11b/CD18 demonstrated the involvement of these receptors in the process of ADCC. Flow cytometry and live cell microscopy were used to quantify and visualize neutrophil-neuroblastoma interactions.
RESULTS
We found that G-CSF was as potent as GM-CSF in enhancing the killing capacity of neutrophils towards neuroblastoma cells. This was observed with in vitro stimulated neutrophils, and with in vivo stimulated neutrophils from both patients with neuroblastoma and healthy donors. Enhanced killing due to GM-CSF or G-CSF stimulation was consistent regardless of dinutuximab concentration, tumor-to-neutrophil ratio and concentration of the stimulating cytokine. Both GM-CSF and G-CSF stimulated neutrophils required FcγRIIa and CD11b/CD18 integrin to perform ADCC, and this was accompanied by trogocytosis of tumor material by neutrophils and tumor cell death in both stimulation conditions.
CONCLUSIONS
Our preclinical data support the use of G-CSF as an alternative stimulating cytokine to GM-CSF in the treatment of high-risk neuroblastoma with dinutuximab, warranting further testing of G-CSF in a clinical setting.
Topics: Adjuvants, Immunologic; Antibodies, Monoclonal; Antineoplastic Agents, Immunological; Antineoplastic Combined Chemotherapy Protocols; CD11b Antigen; CD18 Antigens; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Cytotoxicity, Immunologic; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Neuroblastoma; Neutrophils; Receptors, IgG; Trogocytosis; Tumor Microenvironment
PubMed: 34049929
DOI: 10.1136/jitc-2020-002259