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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.


Gillies S.D.,EMD Serono, Inc. | Gillies S.D.,Provenance Biopharmaceuticals | Lan Y.,EMD Serono, Inc. | Hettmann T.,EMD Serono, Inc. | And 6 more authors.
Clinical Cancer Research | Year: 2011

Purpose: The goal of the study was to engineer a form of interleukin 2 (IL-2) that, when delivered as a tumor-specific antibody fusion protein, retains the ability to stimulate an antitumor immune response via interaction with the high-affinity IL-2 receptor but has lower toxicity because of the reduced activation of the intermediate-affinity IL-2 receptor. Experimental Design: We investigated changes in the proposed toxin motif of IL-2 by introducing a D20T mutation that has little effect on the activity of free IL-2. We expressed this IL-2 variant as a fusion protein with an antibody (NHS76) that targets the necrotic core of tumors and characterized this molecule (NHS-IL2LT) in vitro and in vivo. Results: NHS-IL2LT was shown to have near normal biological activity in vitro by using T-cell lines expressing the high-affinity IL-2 receptor, but little or no activity by using cell lines expressing only the intermediate IL-2 receptor. Relative to the control antibody fusion protein containing wild-type IL-2, NHSIL2LT retained antitumor activity against established neuroblastoma and non-small cell lung cancer metastases in syngeneic mouse tumor models but was much better tolerated in immune-competent mice and in cynomolgus monkeys. Conclusions: The qualities of low toxicity and single-agent efficacy shown suggest that NHS-IL2LT is a good candidate for therapeutic approaches combining standard cytotoxic and immune therapies. In fact, this molecule (also known as Selectikine or EMD 521873) is currently in phase I clinical trial. ©2011 AACR.


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.


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.


Delgado D.C.,University of Wisconsin - Madison | Hank J.A.,University of Wisconsin - Madison | Kolesar J.,University of Wisconsin - Madison | Lorentzen D.,University of Wisconsin - Madison | And 14 more authors.
Cancer Research | Year: 2010

Response to immunocytokine (IC) therapy is dependent on natural killer cells in murine neuroblastoma (NBL) models. Furthermore, killer immunoglobulin-like receptor (KIR)/KIR-ligand mismatch is associated with improved outcome to autologous stem cell transplant for NBL. Additionally, clinical antitumor response to monoclonal antibodies has been associated with specific polymorphic-FcγR alleles. Relapsed/refractory NBL patients received the hu14.18-IL2 IC (humanized anti-GD2 monoclonal antibody linked to human IL2) in a Children's Oncology Group phase II trial. In this report, these patients were genotyped for KIR, HLA, and FcR alleles to determine whether KIR receptor-ligand mismatch or specific FcγR alleles were associated with antitumor response. DNA samples were available for 38 of 39 patients enrolled: 24 were found to have autologous KIR/KIR-ligand mismatch; 14 were matched. Of the 24 mismatched patients, 7 experienced either complete response or improvement of their disease after IC therapy. There was no response or comparable improvement of disease in patients who were matched. Thus KIR/KIR-ligand mismatch was associated with response/improvement to IC (P = 0.03). There was a trend toward patients with the FcγR2A 131-H/H genotype showing a higher response rate than other FcγR2A genotypes (P = 0.06). These analyses indicate that response or improvement of relapsed/refractory NBL patients after IC treatment is associated with autologous KIR/KIR-ligand mismatch, consistent with a role for natural killer cells in this clinical response. ©2010 AACR.


PubMed | Bristol Myers Squibb, Provenance Biopharmaceuticals, University of Wisconsin - Madison and Biologics
Type: | Journal: Journal of immunology (Baltimore, Md. : 1950) | Year: 2017

Most cancer immunotherapies include activation of either innate or adaptive immune responses. We hypothesized that the combined activation of both innate and adaptive immunity will result in better antitumor efficacy. We have previously shown the synergy of an agonistic anti-CD40 mAb (anti-CD40) and CpG-oligodeoxynucleotides in activating macrophages to induce tumor cell killing in mice. Separately, we have shown that a direct intratumoral injection of immunocytokine (IC), an anti-GD2 Ab linked to IL-2, can activate T and NK cells resulting in antitumor effects. We hypothesized that activation of macrophages with anti-CD40/CpG, and NK cells with IC, would cause innate tumor destruction, leading to increased presentation of tumor Ags and adaptive T cell activation; the latter could be further augmented by anti-CTLA-4 Ab to achieve tumor eradication and immunological memory. Using the mouse GD2


Alderson K.L.,University of Wisconsin - Madison | Luangrath M.,University of Wisconsin - Madison | Elsenheimer M.M.,University of Wisconsin - Madison | Gillies S.D.,Provenance Biopharmaceuticals | And 3 more authors.
Cancer Immunology, Immunotherapy | Year: 2013

Targeted monoclonal antibodies (mAb) can be used therapeutically for tumors with identifiable antigens such as disialoganglioside GD2, expressed on neuroblastoma and melanoma tumors. Anti-GD2 mAbs (αGD2) can provide clinical benefit in patients with neuroblastoma. An important mechanism of mAb therapy is antibody-dependent cellular cytotoxicity (ADCC). Combinatorial therapeutic strategies can dramatically increase the anti-tumor response elicited by mAbs. We combined a novel αGD2 mAb, hu14.18K322A, with an immunostimulatory regimen of agonist CD40 mAb and class B CpG-ODN 1826 (CpG). Combination immunotherapy was more effective than the single therapeutic components in a syngeneic model of GD2-expressing B16 melanoma with minimal tumor burden. NK cell depletion in B6 mice showed that NK cells were required for the anti-tumor effect; however, anti-tumor responses were also observed in tumor-bearing SCID/beige mice. Thus, NK cell cytotoxicity did not appear to be essential. Peritoneal macrophages from anti-CD40 + CpG-treated mice inhibited tumor cells in vitro in an hu14.18K322A antibody-dependent manner. These data highlight the importance of myeloid cells as potential effectors in immunotherapy regimens utilizing tumor-specific mAb and suggest that further studies are needed to investigate the therapeutic potential of activated myeloid cells and their interaction with NK cells. © 2012 Springer-Verlag Berlin Heidelberg.


A novel method for constructing immunocytokines has been developed that utilizes fusion of cytokines to the C-terminus of the Ig light chain, rather than fusing to the heavy chain. Such molecules are expressed well in transfected cells, are very stable in normal buffers and have biological properties that are superior to immunocytokines made by fusion to the heavy chain. These properties include longer circulating half-life, increased uptake following subcutaneous dosing and similar or improved antibody effector activities of antibody-dependent cytotoxic activity and complement-dependent cytotoxicity, respectively. Furthermore, the sequestering effect of this fusion junction allows one to adjust intermediate affinity (βγ) interleukin 2 receptor (IL2R) binding and activation by shortening the N-terminus of IL2 at the fusion point. This appears to limit access of the critical contact residue Asp20 of IL2 to the β-chain of βγ IL2R, while maintaining binding and activation of high-affinity (αβγ) IL2R-expressing cells. Several immunocytokine forms with varying degrees of IL2R specificity have been constructed, and some appear to regain their activity for the βγ IL2R when bound to antigen-coated beads. Such molecules may have reduced toxicity in the circulation and enhanced anti-tumor activity. © The Author 2013. Published by Oxford University Press. All rights reserved.

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