Paul M. Sondel

Anti-GD2 Antibody with GM-CSF, Interleukin-2, and Isotretinoin for Neuroblastoma

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. (Funded by the National Institutes of Health and the Food and Drug Administration; ClinicalTrials.gov number, NCT00026312.)

Differential function of major histocompatibility complex antigens in t-lymphocyte activation.

The antigenic systems of the major histocompatibility complex can be subdivided into those which are serologically detectable and those which are detected in tests with mixed lymphocytes. The two systems have different roles in the activation of separate populations of T lymphocytes.

Cell Mediated Immunity: Separation of Cells Involved in Recognitive and Destructive Phases

The mixed leukocyte culture (MLC) and the cell mediated lympholysis (CML) assays are used as in vitro models of the afferent, or recognitive, and efferent, or destructive, phases of the homograft reaction. Activity in both of these tests has been related to differences at the major histocompatibility complex, HL-A in man and H-2 in mouse. Recent evidence suggests that the presumed cell surface differences which lead to cell proliferation in MLC are different from those which act as a target for CML. Data are presented providing further support for this hypothesis; in addition separate cell populations may respond to the differences which activate cells in MLC and to the differences which serve as targets for CML. There thus appears to be a dichotomy both for genetic control of, and cell populations involved in, the recognitive and destructive phases of cell mediated immunity.

Antitumor Activity of Hu14.18-IL2 in Patients With Relapsed/Refractory Neuroblastoma: A Children's Oncology Group (COG) Phase II Study

PURPOSE The hu14.18-IL2 fusion protein consists of interleukin-2 molecularly linked to a humanized monoclonal antibody that recognizes the GD2 disialoganglioside expressed on neuroblastoma cells. This phase II study assessed the antitumor activity of hu14.18-IL2 in two strata of patients with recurrent or refractory neuroblastoma. PATIENTS AND METHODS Hu14.18-IL2 was given intravenously (12 mg/m(2)/daily) for 3 days every 4 weeks for patients with disease measurable by standard radiographic criteria (stratum 1) and for patients with disease evaluable only by [(123)I]metaiodobenzylguanidine (MIBG) scintigraphy and/or bone marrow (BM) histology (stratum 2). Response was established by independent radiology review as well as BM histology and immunocytology, and durability was assessed by repeat evaluation after more than 3 weeks. RESULTS Thirty-nine patients were enrolled (36 evaluable). No responses were seen in stratum 1 (n = 13). Of 23 evaluable patients in stratum 2, five patients (21.7%) responded; all had a complete response (CR) of 9, 13, 20, 30, and 35+ months duration. Grade 3 and 4 nonhematologic toxicities included capillary leak, hypoxia, pain, rash, allergic reaction, elevated transaminases, and hyperbilirubinemia. Two patients required dopamine for hypotension, and one patient required ventilatory support for hypoxia. Most toxicities were reversible within a few days of completing a treatment course and were expected based on phase I results. CONCLUSION Patients with disease evaluable only by MIBG and/or BM histology had a 21.7% CR rate to hu14.8-IL2, whereas patients with bulky disease did not respond. Hu14.18-IL2 warrants further testing in children with nonbulky high-risk neuroblastoma.

Interferon-Induced Indoleamine 2,3-Dioxygenase Activity in Human Mononuclear Phagocytes

Interferon (IFN)‐induced tryptophan degradation, catalyzed by indoleamine 2,3‐dioxygenase (IDO), has been shown to mediate antimicrobial activity in epithelial cells. IDO activity has also been augmented in peripheral blood mononuclear cells (PBMC) treated with IFN or interleukin‐2 (IL‐2). The effector cells in this population have now been further characterized. PBMCs were isolated from normal donors, separated into monocyte and lymphocyte populations by plastic adherence, treated with IFN or IL‐2, and cultivated in medium supplemented with [3H]tryptophan. Culture supernatants were collected after a 48‐h incubation and fractionated by high‐performance liquid chromatography; radioactivity was determined in fractions corresponding to tryptophan and its metabolites. IFN‐γ and IFN‐β induced IDO activity only in monocytes (plastic‐adherent, nonspecific esterase‐positive PBMCs). The induction of IDO activity by IL‐2 required both monocytes and lymphocytes. Interaction was required between these populations for induction of IDO by IL‐2, due to production of IFN‐γ by T lymphocytes, with subsequent IFN‐γ‐mediated induction of IDO in monocytes. A number of myeloid cell lines as well as monocyte‐derived macrophages were also tested for their ability to be induced to degrade tryptophan in response to IFN treatment. Monocyte‐derived macrophages were found to retain their capacity to be induced by IFN‐γ and IFN‐β to degrade tryptophan after differentiation, and to possess seven times more IDO activity per cell than IFN‐induced monocytes. However, the presence of lipopolysaccharide (LPS) in the culture medium was required for the maximum induction of IDO activity by IFN‐β. Furthermore, higher concentrations of LPS were sufficient to induce IDO activity in macrophages in the absence of exogenous IFN.

Phase I Study of Chimeric Human/Murine Anti–Ganglioside GD2 Monoclonal Antibody (ch14.18) With Granulocyte-Macrophage Colony-Stimulating Factor in Children With Neuroblastoma Immediately After Hematopoietic Stem-Cell Transplantation: A Children’s Cancer Group Study

PURPOSE 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 AND METHODS 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. RESULTS 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. CONCLUSION 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.

A Phase I Clinical Trial of the hu14.18-IL2 (EMD 273063) as a Treatment for Children with Refractory or Recurrent Neuroblastoma and Melanoma: a Study of the Children’s Oncology Group

Purpose: Evaluate the clinical safety, toxicity, immune activation/modulation, and maximal tolerated dose of hu14.18-IL2 (EMD 273063) in pediatric patients with recurrent/refractory neuroblastoma and other GD2-positive solid tumors. Experimental Design: Twenty-seven pediatric patients with recurrent/refractory neuroblastoma and one with melanoma were treated with a humanized anti-GD2 monoclonal antibody linked to human interleukin 2 (IL-2). Cohorts of patients received hu14.18-IL2, administered i.v. over 4 hours for three consecutive days, at varying doses. Patients with stable disease, partial, or complete responses were eligible to receive up to three additional courses of therapy. Results: Most of the clinical toxicities were anticipated and similar to those reported with IL-2 and anti-GD2 monoclonal antibody therapy and to those noted in the initial phase I study of hu14.18-IL2 in adults with metastatic melanoma. The maximal tolerated dose was determined to be 12 mg/m2/d, with agent-related dose-limiting toxicities of hypotension, allergic reaction, blurred vision, neutropenia, thrombocytopenia, and leukopenia. Three patients developed dose-limiting toxicity during course 1; seven patients in courses 2 to 4. Two patients required dopamine for hypotension. There were no treatment-related deaths, and all toxicity was reversible. Treatment with hu14.18-IL2 led to immune activation/modulation as evidenced by elevated serum levels of soluble IL-2 receptor α (sIL2Rα) and lymphocytosis. The median half-life of hu14.18-IL2 was 3.1 hours. There were no measurable complete or partial responses to hu14.18-IL2 in this study; however, three patients did show evidence of antitumor activity. Conclusion: Hu14.18-IL2 (EMD 273063) can be administered safely with reversible toxicities in pediatric patients at doses that induce immune activation. A phase II clinical trial of hu14.18-IL2, administered at a dose of 12 mg/m2/d × 3 days repeated every 28 days, will be done in pediatric patients with recurrent/refractory neuroblastoma.

Phase I Clinical Trial of the Immunocytokine EMD 273063 in Melanoma Patients

PURPOSE To evaluate the safety, toxicity, in vivo immunologic activation, and maximum-tolerated dose (MTD) of EMD 273063 (hu14.18-IL-2) in patients with metastatic melanoma. PATIENTS AND METHODS Thirty-three patients were treated with EMD 273063, a humanized anti-GD2 monoclonal antibody (mAb) linked to interleukin-2 (IL-2). EMD 273063 was given as a 4-hour intravenous infusion on days 1, 2, and 3 of week 1. Patients with stabilization or regression of disease could receive a second course of treatment at week 5. Dose levels evaluated were 0.8, 1.6, 3.2, 4.8, 6.0, and 7.5 mg/m2/d. RESULTS Nineteen of 33 patients completed course 1 with stable disease and went on to receive course 2. Eight patients had stable disease on completion of course 2. Grade 3 adverse events included hypophosphatemia (11 patients), hyperglycemia (three patients), hypotension (two patients), thrombocytopenia (one patient), hypoxia (three patients), elevated hepatic transaminases (two patients), and hyperbilirubinemia (one patient). Opioids were required for treatment-associated arthralgias and/or myalgias during 17 of 52 treatment courses. No grade 4 adverse events were observed. Dose-limiting toxicities at the MTD included hypoxia, hypotension, and elevations in AST/ALT. Grade 3 toxicities were anticipated based on prior studies of IL-2 or anti-GD2 mAbs, and all resolved. Immune activation was induced, as measured by lymphocytosis, increased peripheral-blood natural killer activity, and cell numbers, and increased serum levels of the soluble alpha chain of the IL-2 receptor complex. CONCLUSION Treatment with the immunocytokine EMD 273063 induced immune activation and was associated with reversible clinical toxicities at the MTD of 7.5 mg/m2/d in melanoma patients.

A multigene family on human chromosome 12 encodes natural killer-cell lectins

We previously isolated a series of cDNA clones designated NKG2-A, B, C, and D from a human natural killer (NK) cell library. These transcripts encode a family of type II integral membrane proteins having an extracellular Ca2+-dependent lectin domain. The predicted peptides share structural similarities and amino acid sequence similarity with known receptor molecules. In this report, the genomic organization and mRNA expression of each of the genes were studied by using transcript-specific probes. Southern blot experiments reveal that the probes cross-hybridize with a maximum of five genes at high stringency. By probing a Southern blot prepared from a series of hamster/human hybrid somatic cell lines, we demonstrated that all of the hybridizing fragments occur on human chromosome 12. No gene rearrangement and little restriction fragment length polymorphism (RFLP) was observed with these probes. mRNA expression of the NKG2 genes occured in NK cells and some T cells but not in other hematopoietic cell types or in other tissues tested. Each of the transcripts occurred in all three of the NK cell lines tested: however, the genes were differentially regulated in T cells. NKG2-D was expressed in nine of fourteen T-cell clones or lines in the panel, whereas NKG2-A/B was expressed in three and NKG2-C was expressed in only one. Expression of each of the transcripts was upregulated following T-cell growth factor (TCGF)-induced activation of a cloned NK cell. The limited distribution of these proteins and their sequence similarity with known receptor molecules suggest that they may function as receptors of human NK cells.

Anti-CD40 Antibody Induces Antitumor and Antimetastatic Effects: The Role of NK Cells

We assessed the effect of the stimulatory anti-CD40 Ab on NK cell activation in vivo and the therapeutic potential of activated NK cells in tumor-bearing mice. Single-dose i.p. injection of the anti-CD40 Ab resulted in production of IL-12 and IFN-γ in vivo, followed by a dramatic increase in NK cell cytolytic activity in PBLs. NK cell activation by anti-CD40 Ab was also observed in CD40 ligand knockout mice. Because NK cells express CD40 ligand but not CD40, our results suggest that NK activation is mediated by increased cytokine production upon CD40 ligation of APCs. Treatment of tumor-bearing mice with anti-CD40 Ab resulted in substantial antitumor and antimetastatic effects in three tumor models. Depletion of NK cells with anti-asialo GM1 Ab reduced or abrogated the observed antitumor effects in all the tested models. These results indicate that a stimulatory CD40 Ab indirectly activates NK cells, which can produce significant antitumor and antimetastatic effects.