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Esser R.,University Hospital | Muller T.,Chemotherapeutisches Forschungsinstitut Georg Speyer Haus | Stefes D.,Chemotherapeutisches Forschungsinstitut Georg Speyer Haus | Kloess S.,University Hospital | And 12 more authors.
Journal of Cellular and Molecular Medicine | Year: 2012

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. © 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd. Source

Yu A.L.,University of California at San Diego | Yu A.L.,Academia Sinica, Taiwan | Gilman A.L.,Levine Childrens Hospital | Ozkaynak M.F.,New York Medical College | And 18 more authors.
New England Journal of Medicine | Year: 2010

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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. Copyright © 2010 Massachusetts Medical Society. All rights reserved.Results:With 61% of the number of expected events observed, the study met the criteria for early stopping of the randomization, on the basis of the superiority of immunotherapy over standard therapy with regard to event-free survival. The 2-year estimate for event-free survival was 66% in the immunotherapy group and 46% in the standard-therapy group. Immunotherapy was also superior to standard therapy with regard to the estimated rate of overall survival (86% vs. 75% at 2 years). The rate of event-free survival was significantly greater in the immunotherapy group than in the standard-therapy group (63% vs. 42% at 2 years). There was also a trend toward improved overall survival with immunotherapy as compared with standard therapy (84% vs. 76% at 2 years). The 2-year estimates for event-free survival and overall survival were 36% (16 events) and 76% (10 deaths, all disease-related), respectively in 25 patients nonrandomly assigned to receive immunotherapy. The event-free survival was worse in patients with disease of INSS stage 4 than in patients with disease of INSS stage 2, 3, or 4S. Diploidy was predictive of worse overall survival than hyperdiploidy. A complete or very good partial response, as compared with a partial response, before autologous stem-cell transplantation was predictive of improved event-free survival and overall survival. The treatment-group comparisons were not influenced by these factors. The effects of most interest reported in the immunotherapy group were pain, hypotension, capillary leak syndrome, and hypersensitivity reactions, with relatively few toxic effects in the standard-therapy group. Pain of grade 3 or 4 was noted in 52% of patients (during 25% of 598 cycles of immunotherapy). Pain reactions in the immunotherapy group were most frequent during cycle 1, occurring in 37% of patients, and decreasing to 14% during cycle 5. The most common site of pain was the abdomen. The capillary leak syndrome was reported in a total of 23% of patients, during 8% of immunotherapy cycles. It occurred more frequently during cycles 2 and 4, which involved Proleukin, with incidences of 11% and 13%, respectively, as compared with 3 to 7% during courses involving GM-CSF (cycles 1, 3, and 5). Grade 3 or 4 hypersensitivity reactions were reported in 25% of patients, during 15% of immunotherapy cycles. Hypersensitivity reactions were more frequent during the two cycles involving Proleukin, with incidences of 26% and 25%, as compared with 5 to 12% during the three cycles involving GM-CSF. Such reactions may be attributable to symptoms and signs that reflect both toxic effects of Proleukin and antibody-related hypersensitivity. Other toxic effects that were common during immunotherapy cycles included fever (39%), hypokalemia (35%), hyponatremia (23%), liver dysfunction (abnormal alanine aminotransferase level, 23%), hypotension (18%), diarrhea (13%), urticaria (13%), and hypoxia (13%). Early in the study, 2 patients were inadvertently given an overdose of the scheduled Proleukin due to a medication error; one of these patients died of Proleukin-related capillary leak and pulmonary edema. All other toxic effects were self-limited and resolved soon after the cessation of treatment and well before the beginning of the subsequent treatment.Patients:226 patients. Standard-therapy group (isotretinoin only): n=113, 4 were <18 months and 109 were ≥18 months. Immunotherapy group (6 cycles of isotretinoin and 5 concomitant cycles of ch14.18 in combination with alternating GM-CSF and Proleukin): n=113, 4 were <18 months and 109 were ≥18 months; 6 did not receive assigned intervention, having declined immunotherapy and received standard therapy. 25 patients (over 18 months of age) were nonrandomly assigned to receive immunotherapy because of biopsy-proven residual disease after autologous stem-cell transplantation. Follow-up: 4 days to 6.9 years (median 2.1 years). Dropouts: n=40 [23 on immunotherapy (17 due to side effects) and 17 on standard therapy].Indications:132 patients with refractory neuroblastoma [International Neuroblastoma Staging System stage (INSS): 2 (n=4), 3 (n=10), 4S (n=2), 4 (n=89) and unknown (n=8)].TypeofStudy:An open, randomized study evaluating whether adding Proleukin or granulocyte-macrophage colony-stimulating factor (GM-CSF) with monoclonal antibody ch14.18 to standard isotretinoin therapy after intensive multimodal therapy would improve outcomes in high-risk neuroblastoma. NCT00026312.DosageDuration:3.0×106 IU per square meter daily was given in cycles 2 and 4 by means of continuous infusion, for 4 days during week 1, for 4 days during week 2 at 4.5×106 IU per square meter daily. Duration: 2 years. 2 patients were inadvertently given an overdose of the scheduled Proleukin (i.e., a dose >20 times the scheduled dose) due to a medication error.AdverseEffects:An unspecified number of patients had neuropathic pain, hypotension, hypoxia, fever without neutropenia, urticaria, infection (any), infection (catheter related), nausea, vomiting, diarrhea, hyponatremia, hypokalemia, increased alanine aminotransferase levels, increased aspartate aminotransferase levels, hypercalcemia, serum sickness, seizure, central nervous system cortical symptoms (encephalopathy, confusion and psychosis). 12 (11%) patients in cycle 2 and 15 (13%) in cycle 4 had capillary leak syndrome; 29 (26%) and 28 (25%), respectively hypersensitivity reactions. 1 patient had drug intoxication characterized by capillary leak and pulmonary edema. 17 patients had adverse events leading to withdrawal.FreeText:Primary endpoint: event-free survival. Secondary endpoint: overall survival. Tumor MYCN status: 52 were not amplified, 36 amplified and 25 unknown. Tumor histologic features: 4 were favorable, 68 unfavorable and 41 unknown. Tumor ploidy: 49 were hyperdiploid, 35 diploid and 29 unknown. 28 patients had ≥1 purged stem cell infusions. Response before autologous stem cell transplantation (ASCT): 40 complete response, 47 very good partial response and 26 partial response. 107 patients had 1 ASCT and 6 had 2 ASCTs. In nonrandomized patients, 23 had INSS stage 4 disease; 6 tumors showed MYCN amplification, 16 had unfavorable histologic features, 12 were diploid, 21 had a partial response before ASCT and only 1 had undergone 2 autologous stem-cell transplantations. Patients had induction therapy, ASCT, and radiotherapy. The monoclonal antibody ch14.18 was given in cycles 1 through 5; GM-CSF was given in cycles 1, 3, and 5; and isotretinoin was given during the last 2 weeks in each of the five cycles and also given by itself during a final 6th cycle. Concomitant drugs: isotretinoin at a dose of 160 mg per square meter daily, monoclonal antibody ch14.18 at a dose of 25 mg per square meter daily for 4 consecutive days.AuthorsConclusions:In summary, the addition of ch14.18, GM-CSF, and interleukin-2 to isotretinoin therapy was associated with improved event-free and overall survival among children with high-risk neuroblastoma who had a response to initial chemotherapy and received immunotherapy within 100 days after autologous stem-cell transplantation. Our data suggest that more routine use of this immunotherapy regimen for such patients may be beneficial. Future avenues of investigation include developing more effective and less toxic ways to stimulate ch14.18-mediated antibody-dependent cell-mediated cytotoxicity and identifying more efficacious GD2-targeted monoclonal antibodies or genetically modified constructs targeting GD2. Source

Gubbels J.A.A.,University of Wisconsin - Madison | Gubbels J.A.A.,Augustana College at Sioux Falls | Gadbaw B.,University of Wisconsin - Madison | Buhtoiarov I.N.,University of Wisconsin - Madison | And 10 more authors.
Cancer Immunology, Immunotherapy | Year: 2011

The huKS-IL2 immunocytokine (IC) consists of IL2 fused to a mAb against EpCAM, while the hu14.18-IL2 IC recognizes the GD2 disialoganglioside. They are under evaluation for treatment of EpCAM+ (ovarian) and GD2 + (neuroblastoma and melanoma) malignancies because of their proven ability to enhance tumor cell killing by antibody-dependent cell-mediated cytotoxicity (ADCC) and by antitumor cytotoxic T cells. Here, we demonstrate that huKS-IL2 and hu14.18-IL2 bind to tumor cells via their antibody components and increase adhesion and activating immune synapse (AIS) formation with NK cells by engaging the immune cells' IL-2 receptors (IL2R). The NK leukemia cell line, NKL (which expresses high affinity IL2Rs), shows fivefold increase in binding to tumor targets when treated with IC compared to matching controls. This increase in binding is effectively inhibited by blocking antibodies against CD25, the α-chain of the IL2R. NK cells isolated from the peritoneal environment of ovarian cancer patients, known to be impaired in mediating ADCC, bind to huKS-IL2 via CD25. The increased binding between tumor and effector cells via ICs is due to the formation of AIS that are characterized by the simultaneous polarization of LFA-1, CD2 and F-actin at the cellular interface. AIS formation of peritoneal NK and NKL cells is inhibited by anti-CD25 blocking antibody and is 50-200% higher with IC versus the parent antibody. These findings demonstrate that the IL-2 component of the IC allows IL2Rs to function not only as receptors for this cytokine but also as facilitators of peritoneal NK cell binding to IC-coated tumor cells. © 2011 Springer-Verlag. Source

Sorkin L.S.,University of California at San Diego | Otto M.,St Jude Childrens Research Hospital | Baldwin III W.M.,Cleveland Clinic | Vail E.,University of California at San Diego | And 7 more authors.
Pain | Year: 2010

Monoclonal antibodies against GD2 ganglioside, such as ch14.18, the human-mouse chimeric antibody, have been shown to be effective for the treatment of neuroblastoma. However, treatment is associated with generalized, relatively opiate-resistant pain. We investigated if a point mutation in ch14.18 antibody (hu14.18K332A) to limit complement-dependent cytotoxicity (CDC) would ameliorate the pain behavior, while preserving antibody-dependent cellular cytotoxicity (ADCC). In vitro, CDC and ADCC were measured using europium-TDA assay. In vivo, allodynia was evaluated by measuring thresholds to von Frey filaments applied to the hindpaws after injection of either ch14.18 or hu14.18K332 into wild type rats or rats with deficient complement factor 6. Other rats were pretreated with complement factor C5a receptor antagonist and tested following ch14.18 injection. The mutation reduces the antibody's ability to activate complement, while maintaining its ADCC capabilities. Injection of hu14.18K322 (1 or 3 mg/kg) produced faster resolving allodynia than that engendered by ch14.18 (1 mg/kg). Injection of ch14.18 (1 mg/kg) into rats with C6 complement deficiency further reduced antibody-induced allodynia, while pre-treatment with complement factor C5a receptor antagonist completely abolished ch14.18-induced allodynia. These findings showed that mutant hu14.18 K322 elicited less allodynia than ch14.18 and that ch14.18-elicited allodynia is due to activation of the complement cascade: in part, to formation of membrane attack complex, but more importantly to release of complement factor C5a. Development of immunotherapeutic agents with decreased complement-dependent lysis while maintaining cellular cytotoxicity may offer treatment options with reduced adverse side effects, thereby allowing dose escalation of therapeutic antibodies. © 2010 International Association for the Study of Pain. Source

Albertini M.R.,University of Wisconsin - Madison | Albertini M.R.,K6 530 Clinical Science Center | Hank J.A.,University of Wisconsin - Madison | Gadbaw B.,University of Wisconsin - Madison | And 8 more authors.
Cancer Immunology, Immunotherapy | Year: 2012

Phase I testing of the hu14.18-IL2 immunocytokine in melanoma patients showed immune activation, reversible toxicities, and a maximal tolerated dose of 7.5 mg/m2/day. In this phase II study, 14 patients with measurable metastatic melanoma were scheduled to receive hu14.18-IL2 at 6 mg/m 2/day as 4-h intravenous infusions on Days 1, 2, and 3 of each 28 day cycle. Patients with stable disease (SD) or regression following cycle 2 could receive two additional treatment cycles. The primary objective was to evaluate antitumor activity and response duration. Secondary objectives evaluated adverse events and immunologic activation. All patients received two cycles of treatment. One patient had a partial response (PR) [1 PR of 14 patients = response rate of 7.1 %; confidence interval, 0.2-33.9 %], and 4 patients had SD and received cycles 3 and 4. The PR and SD responses lasted 3-4 months. All toxicities were reversible and those resulting in dose reduction included grade 3 hypotension (2 patients) and grade 2 renal insufficiency with oliguria (1 patient). Patients had a peripheral blood lymphocytosis on Day 8 and increased C-reactive protein. While one PR in 14 patients met protocol criteria to proceed to stage 2 and enter 16 additional patients, we suspended stage 2 due to limited availability of hu14.18-IL2 at that time and the brief duration of PR and SD. We conclude that subsequent testing of hu14.18-IL2 should involve melanoma patients with minimal residual disease based on compelling preclinical data and the confirmed immune activation with some antitumor activity in this study. © Springer-Verlag 2012. Source

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