Purpose: To report on a phase I trial designed to find the maximally tolerated dose in children of the immunologic adjuvant OPT-821 in a vaccine containing neuroblastoma-associated antigens (GD2 and GD3; Clinicaltrials.gov NCT00911560). Secondary objectives were to obtain preliminary data on immune response and activity against minimal residual disease (MRD). Treatment also included the immunostimulant β-glucan.Experimental Design: Patients with neuroblastoma in ≥2nd complete/very good partial remission received vaccine subcutaneously (weeks 1–2–3–8–20–32–52). Vaccine contained 30 μg each of GD2 and GD3 stabilized as lactones and conjugated to the immunologic carrier protein keyhole limpet hemocyanin; and OPT-821, which was dose escalated as 50, 75, 100, and 150 μg/m2 per injection. Oral β-glucan (40 mg/kg/day, 14 days on/14 days off) started week 6.Results: The study was completed with 15 patients because there was no dose-limiting toxicity at 150 μg/m2 of OPT-821 (the dosing used in adults). Thirteen of fifteen patients received the entire protocol treatment, including 12 who remain relapse-free at 24+ to 39+ (median 32+) months and 1 who relapsed (single node) at 21 months. Relapse-free survival was 80% ± 10% at 24 months. Vaccine and β-glucan were well tolerated. Twelve of fifteen patients had antibody responses against GD2 and/or GD3. Disappearance of MRD was documented in 6 of 10 patients assessable for response.Conclusions: This immunotherapy program lacks major toxicity and is transportable to any outpatient clinic. Patient outcome is encouraging but the efficacy is uncertain because of the complexity and heterogeneity of prior therapies. A larger phase II trial is underway. Clin Cancer Res; 20(5); 1375–82. ©2014 AACR.

PURPOSE: Anti-GD2 monoclonal antibody (mAb) has proven efficacy in high-risk neuroblastoma (HR-NB). A small phase I GD2/GD3 vaccine trial (n = 15) described long-term survival and a favorable safety profile among patients with a history of disease progression (PD). The kinetics of mounting antibody response to vaccine and its prognostic impact on survival are now investigated in a phase II study (ClinicalTrials.gov identifier: NCT00911560 ). PATIENTS AND METHODS: One hundred two patients with HR-NB who achieved remission after salvage therapies were enrolled in this trial. They received seven subcutaneous injections of GD2/GD3 vaccine spanning 1 year plus oral β-glucan starting at week 6 after the third dose of vaccine. Serum anti-vaccine antibody titers were quantified by enzyme-linked immunosorbent assay. Single nucleotide polymorphisms (SNPs) were determined by quantitative polymerase chain reaction. Kaplan-Meier and landmark Cox Regression models were used for survival estimates. RESULTS: Patients had a history of one (63%), two (21%), or three to six (16%) episodes of PD. 82% of them progressed following anti-GD2 mAb (m3F8/dinutuximab/naxitamab) therapy. Vaccine-related toxicities were self-limited injection–associated local reactions and fever without any > grade 3 toxicities. The progression-free survival (PFS) was 32% ± 6%, and the overall survival (OS) was 71% ± 7% at 5 years. Serum anti-GD2 (immunoglobulin G1 [IgG1] and IgM) and anti-GD3 (IgG1) titers showed notable increases following the initiation of β-glucan at week 6. There was an association between IgG1 titer and SNP rs3901533 of dectin-1, the β-glucan receptor. Multivariable analyses showed that anti-GD2-IgG1 titer ≥ 150 ng/mL by week 8 was associated with favorable PFS and OS, while having prior episodes of PD and the time from last PD to vaccine were associated with PFS. CONCLUSION: GD2/GD3 vaccine plus β-glucan elicited robust antibody responses in patients with HR-NB with prior PD. Higher anti-GD2-IgG1 titer was associated with improved survival.

PURPOSE: Desmoplastic small round cell tumor (DSRCT), a rare sarcoma of adolescents/young adults primarily involving the peritoneum, has a long-term survival of < 20% despite aggressive multimodality treatment. B7H3 is expressed on DSRCT cell surface, providing a target for antibody-based immunotherapy. PATIENTS AND METHODS: In this phase I study, we evaluated the safety, pharmacokinetics, and biodistribution of intraperitoneal (IP) radioimmunotherapy (RIT) with the anti-B7H3 murine monoclonal antibody ¹³¹I-omburtamab in patients with DSRCT or other B7H3-expressing tumors involving the peritoneum. After thyroid blockade, patients received ¹³¹I-omburtamab as a single IP injection at escalated activities from 1.11 to 3.33/GBq/m². A prior tracer dose of IP 74 MBq¹²⁴I-omburtamab was used for radioimmuno–positron emission tomography imaging. Each injection was followed by IP saline infusion. RESULTS: Fifty-two patients (48, three, and one with DSRCT, peritoneal rhabdomyosarcoma, and Ewing sarcoma, respectively) received IP ¹³¹I-omburtamab administered on an outpatient basis. Maximum tolerated dose was not reached; there were no dose-limiting toxicities. Major related adverse events were transient: grade 4 neutropenia (n = 2 patients) and thrombocytopenia (n = 1), and grade 1 (10%) and grade 2 (52%) pain lasting < 2 hours related to saline infusion. Hypothyroidism was not observed, and antidrug antibody was elicited in 5%. Mean (± SD) projected peritoneal residence time was 22.4 ± 7.9 hours. Mean projected absorbed doses for ¹³¹I-omburtamab based on ¹²⁴I-omburtamab dosimetry to normal organs were low and well within tolerable limits. More than 80% ¹³¹I remained protein bound in blood 66 hours after RIT. On the basis of peritoneal dose and feasibility for outpatient administration, the recommended phase II activity was established at 2.96 GBq/m². Patients with DSRCT receiving standard whole-abdominal radiotherapy after RIT did not experience unexpected toxicity. CONCLUSION: IP RIT ¹³¹I-omburtamab was well tolerated with minimal toxicities. Radiation exposure to normal organs was low, making combination therapy with other anticancer therapies feasible.

Natural killer (NK) cell-mediated antibody-dependent toxicity is a potent mechanism of action of the anti-GD2 murine monoclonal antibody 3F8 (m3F8). Killer immunoglobulin-like receptor (KIR) and HLA genotypes modulate NK activity and are key prognostic markers in m3F8-treated patients with neuroblastoma. Endogenous NK-cells are suppressed in the setting of high tumor burden and chemotherapy. Allogeneic NK-cells however, demonstrate potent anti-neuroblastoma activity. We report on the results of a phase I clinical trial of haploidentical NK-cells plus m3F8 administered to patients with high-risk neuroblastoma after conditioning chemotherapy. The primary objective was to determine the maximum tolerated NK-cell dose (MTD). Secondary objectives included assessing anti-neuroblastoma activity and its relationship to donor-recipient KIR/HLA genotypes, NK function, and donor NK chimerism. Patients received a lymphodepleting regimen prior to infusion of haploidentical CD3-CD56+ NK-cells, followed by m3F8. Overall and progression free survival (PFS) were assessed from the time of first NK-cell dose. Univariate Cox regression assessed relationship between dose and outcomes. Thirty-five patients received NK-cells at one of five dose levels ranging from 10×10⁶ CD56+cells/kg had improved PFS (HR: 0.36, 95%CI: 0.15–0.87, p = 0.022). Patient NK-cells displayed high NKG2A expression, leading to inhibition by HLA-E-expressing neuroblastoma cells. Adoptive NK-cell therapy in combination with m3F8 is safe and has anti-neuroblastoma activity at higher cell doses.

Purpose: Minimal residual disease (MRD) presents a significant hurdle to curing metastatic neuroblastoma. Biological therapies directed against MRD can improve outcome. Evaluating treatment efficacy requires MRD measurement, which serves as surrogate endpoint. Because of tumor heterogeneity, no single marker will likely be adequate. Genome-wide expression profiling can uncover potential MRD markers differentially expressed in tumors over normal marrow/blood. Experimental Design: Gene expression array was carried out on 48 stage 4 tumors and 9 remission marrows using the Affymetrix U95 gene chip. Thirty-four genes with a tumor-to-marrow expression ratio higher than tyrosine hydroxylase were identified. Quantitative reverse transcription-PCR was done on all 34 genes to study the dynamic range of tumor cell detection and the expression of these genes in normal marrow/blood samples and in stage 4 neuroblastoma tumors. Top ranking markers were then tested for prognostic significance in the marrows of stage 4 patients collected from the same treatment protocol after two cycles of immunotherapy. Results: Based on sensitivity assays, 8 top-ranking markers were identified: CCND1, CRMP1, DDC, GABRB3, ISL1, KIF1A, PHOX2B, and TACC2. They were abundantly expressed in stage IV neuroblastoma tumors (n = 20) and had low to no detection in normal marrow/blood samples (n = 20). Moreover, expression of CCND1, DDC, GABRB3, ISL1, KIF1A, and PHOX2B in 116 marrows sampled after two treatment cycles was highly prognostic of progression-free and overall survival (P < 0.001). Conclusions: Marker discovery based on differential gene expression profiling, stringent sensitivity and specificity assays, and well-annotated patient samples can rapidly prioritize and identify potential MRD markers of neuroblastoma.

Purpose: Adjuvant therapy using anti-GD2 monoclonal antibody and granulocyte-macrophage colony-stimulating factor (GM-CSF) has shown treatment success for patients with high-risk neuroblastoma (NB). Although there is ample evidence on how the antibody targets NB, in vivo contribution by GM-CSF remains unclear. This report investigates granulocyte activation and its correlation with treatment outcome.Patients and Methods: Patients enrolled onto NCT00072358 received multiple treatment cycles, each consisting of anti-GD2 antibody 3F8 plus subcutaneous (SC) GM-CSF. Peripheral-blood (PB) samples from 151 patients were collected on day 0 and day 4 of cycle 1. PB from a subgroup of 35 patients had intravenous (IV) instead of SC GM-CSF during cycle 4. Samples were analyzed by flow cytometry for CD11a, CD63, CD87, and CD11b and its activation epitope CBRM1/5.Results: Comparing cycle 1 day 4 PB samples with day 0 PB samples, five of five activation marker–positive granulocytes were significantly higher. The change in frequency and mean fluorescence intensity of CBRM1/5-positive granulocytes correlated with progression-free survival (PFS; P = .024 and P = .008, respectively). A multivariable analysis identified increasing CBRM1/5-positive granulocytes and missing killer immunoglobulin-like receptor ligand as positive independent prognostic factors for PFS, whereas second-line cyclophosphamide-based therapy before protocol entry negatively influenced outcome. Thirty-five patients who received SC GM-CSF at cycle 1 and IV GM-CSF at cycle 4 had significantly less CBRM1/5 activation after IV GM-CSF. In contrast, 63 patients who received SC GM-CSF at both cycles had comparable CBRM1/5 activation.Conclusion: GM-CSF–induced granulocyte activation in vivo is associated with improved patient outcome. This activation was more apparent when GM-CSF was given by the SC route instead of IV route.