Long Prime-Boost Interval and Heightened Anti-GD2 Antibody Response to Carbohydrate Cancer Vaccine.

The carbohydrate ganglioside GD2/GD3 cancer vaccine adjuvanted by ß-glucan stimulates anti-GD2 IgG1 antibodies that strongly correlate with improved progression-free survival (PFS) and overall survival (OS) among patients with high-risk neuroblastoma. Thirty-two patients who relapsed on the vaccine (first enrollment) were re-treated on the same vaccine protocol (re-enrollment). Titers during the first enrollment peaked by week 32 at 751 ± 270 ng/mL, which plateaued despite vaccine boosts at 1.2-4.5 month intervals. After a median wash-out interval of 16.1 months from the last vaccine dose during the first enrollment to the first vaccine dose during re-enrollment, the anti-GD2 IgG1 antibody rose to a peak of 4066 ± 813 ng/mL by week 3 following re-enrollment (p < 0.0001 by the Wilcoxon matched-pairs signed-rank test). Yet, these peaks dropped sharply and continually despite repeated boosts at 1.2-4.5 month intervals, before leveling off by week 20 to the first enrollment peak levels. Despite higher antibody titers, patients experienced no pain or neuropathic side effects, which were typically associated with immunotherapy using monoclonal anti-GD2 antibodies. By the Kaplan-Meier method, PFS was estimated to be 51%, and OS was 81%. The association between IgG1 titer during re-enrollment and ß-glucan receptor dectin-1 SNP rs3901533 was significant (p = 0.01). A longer prime-boost interval could significantly improve antibody responses in patients treated with ganglioside conjugate cancer vaccines.

Clonal evolution during metastatic spread in high-risk neuroblastoma.

Patients with high-risk neuroblastoma generally present with widely metastatic disease and often relapse despite intensive therapy. As most studies to date focused on diagnosis-relapse pairs, our understanding of the genetic and clonal dynamics of metastatic spread and disease progression remain limited. Here, using genomic profiling of 470 sequential and spatially separated samples from 283 patients, we characterize subtype-specific genetic evolutionary trajectories from diagnosis through progression and end-stage metastatic disease. Clonal tracing timed disease initiation to embryogenesis. Continuous acquisition of structural variants at disease-defining loci (MYCN, TERT, MDM2-CDK4) followed by convergent evolution of mutations targeting shared pathways emerged as the predominant feature of progression. At diagnosis metastatic clones were already established at distant sites where they could stay dormant, only to cause relapses years later and spread via metastasis-to-metastasis and polyclonal seeding after therapy.

Effect of Oral ß-Glucan on Antibody Response to Ganglioside Vaccine in Patients With High-Risk Neuroblastoma: A Phase 2 Randomized Clinical Trial.

Importance: Among patients with high-risk relapsed metastatic neuroblastoma, oral ß-glucan adjuvant during GD2/GD3 ganglioside vaccine boost has stimulated IgG antibody response, which was associated with improved survival; however, the effectiveness of oral ß-glucan during the vaccine priming phase remains unproven. Objective: To isolate the adjuvant effect of oral ß-glucan on antibody response to GD2/GD3 ganglioside vaccine in patients with high-risk neuroblastoma. Design, Setting, and Participants: In this phase 2 randomized clinical trial, enrolled patients with high-risk neuroblastoma were randomized to 2 groups to receive the GD2/GD3 vaccine at a large cancer center in a major metropolitan area from October 2018 to September 2020. Data were analyzed from October 7, 2021, to February 28, 2022. Interventions: Eligible patients receiving GD2/GD3 vaccine were randomly assigned to group 1 (n = 54) to receive no ß-glucan or group 2 (n = 53) to receive an oral ß-glucan regimen during the first 5 weeks of vaccine priming. From week 6 onwards, all 107 patients received oral ß-glucan during vaccine boost for 1 year or until disease progression. Main Outcomes and Measures: Primary end point was comparison of anti-GD2 IgG1 response before vaccine injection 6 (week 32) in group 1 vs group 2. Seroconversion rate and the association of antibody titer with ß-glucan receptor dectin-1 single nucleotide polymorphism (SNP) rs3901533 were also assessed. Results: In all, 107 patients with high-risk neuroblastoma were randomized to the 2 groups: 54 patients (median [range] age, 5.2 [1.0-17.3] years; 28 [52%] male and 26 [48%] female) in group 1; and 53 patients (median [range] age, 6.2 [1.9-18.4] years; 25 [47%] male and 28 [53%] female) in group 2; both groups were also comparable in their first remission status at study entry (70% vs 70%). Adding oral ß-glucan during the first 5 weeks of vaccine priming elicited a higher anti-GD2 IgG1 antibody response in group 2 (1.80; 90% CI, 0.12-3.39; P = .08; planned type I error, 0.10). Anti-GD2 IgG1 titer of 230 ng/mL or greater by week 8 was associated with statistically favorable PFS. Antibody titer correlated significantly with dectin-1 SNP. The genotype frequency, seroconversion rates, and vaccine-related toxic effects were similar in the 2 groups. Conclusions and Relevance: This phase 2 randomized clinical trial found that adding oral ß-glucan during vaccine priming increased anti-GD2 IgG1 titer among genetic responders without added toxic effects. Because responder dectin-1 SNP was identical in the 2 randomized groups, no difference was detected in seroconversion rates. Alternative or additional adjuvants may be needed to enhance seroconversion. Trial Registration: ClinicalTrials.gov Identifier: NCT00911560.

Bone Marrow Surveillance of Pediatric Cancer Survivors Identifies Clones that Predict Therapy-Related Leukemia.

PURPOSE: Therapy-related myelodysplastic syndrome and acute leukemias (t-MDS/AL) are a major cause of nonrelapse mortality among pediatric cancer survivors. Although the presence of clonal hematopoiesis (CH) in adult patients at cancer diagnosis has been implicated in t-MDS/AL, there is limited published literature describing t-MDS/AL development in children. EXPERIMENTAL DESIGN: We performed molecular characterization of 199 serial bone marrow samples from 52 patients treated for high-risk neuroblastoma, including 17 with t-MDS/AL (transformation), 14 with transient cytogenetic abnormalities (transient), and 21 without t-MDS/AL or cytogenetic alterations (neuroblastoma-treated control). We also evaluated for CH in a cohort of 657 pediatric patients with solid tumor. RESULTS: We detected at least one disease-defining alteration in all cases at t-MDS/AL diagnosis, most commonly TP53 mutations and KMT2A rearrangements, including involving two novel partner genes (PRDM10 and DDX6). Backtracking studies identified at least one t-MDS/AL-associated mutation in 13 of 17 patients at a median of 15 months before t-MDS/AL diagnosis (range, 1.3-32.4). In comparison, acquired mutations were infrequent in the transient and control groups (4/14 and 1/21, respectively). The relative risk for development of t-MDS/AL in the presence of an oncogenic mutation was 8.8 for transformation patients compared with transient. Unlike CH in adult oncology patients, TP53 mutations were only detectable after initiation of cancer therapy. Last, only 1% of pediatric patients with solid tumor evaluated had CH involving myeloid genes. CONCLUSIONS: These findings demonstrate the clinical relevance of identifying molecular abnormalities in predicting development of t-MDS/AL and should guide the formation of intervention protocols to prevent this complication in high-risk pediatric patients.

Phase I Trial of Oral Yeast-Derived ß-Glucan to Enhance Anti-GD2 Immunotherapy of Resistant High-Risk Neuroblastoma.

Beta glucans, complex polysaccharides, prime leukocyte dectin-1 and CR3-receptors and enhance anti-tumor cytotoxicity of complement-activating monoclonal antibodies. We conducted a phase I study (clinicaltrials.gov NCT00492167) to determine the safety of the combination of yeast-derived beta glucan (BG) and anti-GD2 murine monoclonal antibody 3F8 in patients with relapsed or refractory high-risk neuroblastoma. Patients received intravenous 3F8 (fixed dose of 10 mg/m2/day × 10 days) and oral BG (dose-escalated from 10-200 mg/kg/day × 17 days in cohorts of 3-6 patients each). Forty-four patients completed 141 cycles. One patient developed DLT: transient self-limiting hepatic transaminase elevation 5 days after starting BG (120 mg/kg/day). Overall, 1, 3, 12 and 24 evaluable patients had complete response, partial response, stable and progressive disease, respectively, at the end of treatment. Positive human anti-mouse antibody response and dectin-1 rs3901533 polymorphism were associated with better overall survival. BG dose level and serum BG levels did not correlate with response. Progression-free and overall survival at 2 years were 28% and 61%, respectively. BG lacked major toxicity. Treatment with 3F8 plus BG was associated with anti-neuroblastoma responses in patients with resistant disease. Although the maximal tolerated dose for yeast BG was not reached, considering the large volume of oral BG, we recommended 40 mg/kg/day as the phase II dose.

Survival Impact of Anti-GD2 Antibody Response in a Phase II Ganglioside Vaccine Trial Among Patients With High-Risk Neuroblastoma With Prior Disease Progression.

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 ß-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 ß-glucan at week 6. There was an association between IgG1 titer and SNP rs3901533 of dectin-1, the ß-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 ß-glucan elicited robust antibody responses in patients with HR-NB with prior PD. Higher anti-GD2-IgG1 titer was associated with improved survival.

B7H3-Directed Intraperitoneal Radioimmunotherapy With Radioiodinated Omburtamab for Desmoplastic Small Round Cell Tumor and Other Peritoneal Tumors: Results of a Phase I Study.

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.

Telomere Trimming and DNA Damage as Signatures of High Risk Neuroblastoma.

Telomeres play important roles in genome stability and cell proliferation. High risk neuroblastoma (HRNB), an aggressive childhood cancer, is especially reliant on telomere maintenance. Three recurrent genetic aberrations in HRNB (MYCN amplification, TERT re-arrangements, and ATRX mutations) are mutually exclusive and each capable of promoting telomere maintenance mechanisms (i.e., through telomerase or ALT). We analyzed a panel of 5 representative HRNB cell lines and 30 HRNB surgical samples using assays that assess average telomere lengths, length distribution patterns, single-stranded DNA on the G- and C-strand, as well as extra-chromosomal circular telomeres. Our analysis pointed to substantial and variable degrees of telomere DNA damage in HRNB, including pervasive oxidative lesions. Moreover, unlike other cancers, neuroblastoma consistently harbored high levels of C-strand ssDNA overhangs and t-circles, which are consistent with active "telomere trimming". This feature is observed in both telomerase- and ALT-positive tumors and irrespective of telomere length distribution. Moreover, evidence for telomere trimming was detected in normal neural tissues, raising the possibility that TMMs in HRNB evolved in the face of a canonical developmental program of telomere shortening. Telomere trimming by itself appears to distinguish neuroectodermal derived tumors from other human cancers, a distinguishing characteristic with both biologic and therapeutic implications.

Humanized 3F8 Anti-GD2 Monoclonal Antibody Dosing With Granulocyte-Macrophage Colony-Stimulating Factor in Patients With Resistant Neuroblastoma: A Phase 1 Clinical Trial.

Importance: Chimeric and murine anti-GD2 antibodies are active against neuroblastoma, but the development of neutralizing antibodies can compromise efficacy. To decrease immunogenicity, hu3F8, a humanized anti-GD2 antibody, was constructed. Objective: To find the maximum-tolerated dose of hu3F8 with granulocyte-macrophage colony-stimulating factor. Design, Setting, and Participants: This phase 1 clinical trial used a 3 + 3 dose-escalation design in a single referral center (Memorial Sloan Kettering Cancer Center, New York, New York). Participants were enrolled from December 24, 2012, through May 3, 2016, with follow-up and analyses through February 28, 2018. Eligibility criteria included older than 1 year and resistant or recurrent neuroblastoma regardless of the number or kinds of prior treatments. All 57 participants met the eligibility criteria, received treatment according to the protocol, and were included in all analyses. Interventions: Treatment cycles were monthly, if human antihuman antibody remained negative. Each cycle comprised hu3F8 infused intravenously for 30 minutes on Monday, Wednesday, and Friday as well as granulocyte-macrophage colony-stimulating factor administered subcutaneously daily from 5 days before infusion through the last day of infusion. After cycle 2, hu3F8 was increased to the highest dose level that had been confirmed as safe. Main Outcomes and Measures: Toxicity, pharmacokinetics, immunogenicity, and disease response. Results: Of the 57 participants, 34 (60%) were male and 23 (40%) were female (male-to-female ratio of 1.5), with a median (range) age of 6.8 (2.4-31.3) years at enrollment and a median (range) time of 3.1 (0.6-9.0) years since initial chemotherapy. Participants received a median (range) of 4 (1-15) cycles. Treatment was outpatient with reversible neuropathic pain and without unexpected toxic effects. No maximum-tolerated dose was identified. Dose escalation was associated with increased serum levels and proceeded through dosage of 9.6 mg/kg/cycle (approximately 288 mg/m2), which is more than 2.5 times higher than the standard dosage of 75 mg/m2/cycle or 100 mg/m2/cycle of dinutuximab and m3F8. Human antihuman antibody positivity developed in 5 of 57 patients (9%) after cycle 1, including in 1 of 10 patients (10%) not previously treated with anti-GD2 antibody and in 4 of 47 patients (9%) previously exposed to 1 or 2 anti-GD2 antibodies. Antineuroblastoma activity included major responses associated with higher dosing and prolonged progression-free survival despite a history of relapses. Conclusions and Relevance: This phase 1 clinical trial found hu3F8 to be associated with modest toxic effects, low immunogenicity, and substantial antineuroblastoma activity; phase 2 trials are in progress. Trial Registration: ClinicalTrials.gov identifier: NCT01757626.

Adoptive immunotherapy with haploidentical natural killer cells and Anti-GD2 monoclonal antibody m3F8 for resistant neuroblastoma: Results of a phase I study.

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 <1×106 to 50×106 CD3-CD56+cells/kg. One patient experienced grade 3 hypertension and grade 4 pneumonitis. MTD was not reached. Ten patients (29%) had complete or partial response; 17 (47%) had no response; and eight (23%) had progressive disease. No relationship was found between response and KIR/HLA genotype or between response and FcγRIII receptor polymorphisms. Patients receiving >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.

MYCN-amplified stage 2/3 neuroblastoma: excellent survival in the era of anti-GD2 immunotherapy.

High-risk neuroblastoma (HR-NB) includes MYCN-amplified stage 2/3, but reports covering anti-GD2 immunotherapy, which recently became standard for HR-NB, do not provide details on this subset. We now report on all 20 MYCN-amplified stage 2/3 patients who received induction chemotherapy at our center during the era of consolidation with anti-GD2 antibody 3F8/ granulocyte-macrophage colony-stimulating factor (GM-CSF) (2000-2015). Early in this period, consolidation included autologous stem-cell transplantation (ASCT). Event-free survival (EFS) and overall survival (OS) were estimated using Kaplan-Meier analyses. With induction, 19/20 (95%) patients achieved complete/very good partial remission (CR/VGPR) but one had progressive disease with early death. One responder did not receive consolidation and died of relapse. Five-year post-diagnosis EFS/OS rates for all 20 patients were 72%/84%. The 18 CR/VGPR patients who received consolidation had EFS/OS 81%/94% at five years from starting 3F8/GM-CSF: 4/4 ASCT patients remained relapse-free, while 11/14 non-ASCT patients remained relapse-free and two of the three relapsed patients achieved 2nd CR (consolidated by retreatment with 3F8/GM-CSF) and remained in 2nd CR at 36+ and 95+ months post-relapse. The 14 non-ASCT patients had EFS/OS 73.5%/93% at five years from starting 3F8/GM-CSF. This subset appears to have a good prognosis with contemporary multi-modality therapy, possibly even without ASCT.

Phase I trial of anti-GD2 monoclonal antibody hu3F8 plus GM-CSF: Impact of body weight, immunogenicity and anti-GD2 response on pharmacokinetics and survival.

Fifty-seven stage 4 patients with refractory/relapsed neuroblastoma were enrolled in a phase I trial (Clinicaltrials.gov NCT01757626) using humanized anti-GD2 monoclonal antibody hu3F8 in combination with granulocyte-macrophage colony-stimulating factor. The influence of body weight and human anti-human antibody (HAHA) on the pharmacokinetics (PK) of hu3F8, and the effect of de novo anti-GD2 response on patient outcome were explored. Serum samples before hu3F8 infusion, and serially up to day 12 during treatment cycle #1, and at 5 min after each hu3F8 infusion for all subsequent cycles were collected. PK was analyzed using non-compartmental modeling. Immunogenicity was assayed by HAHA response, and vaccination effect by induced host anti-GD2 response measured periodically until disease progression or last followup. Progression-free and overall survival was estimated by the Kaplan-Meier method. Despite dosing being based on body weight, smaller patients had consistently lower area-under-the-curve and faster clearance over the 15 dose levels (0.9 to 9.6 mg/kg per treatment cycle) in this trial. Positive HAHA, defined by the upper limit of normal, when measured within 10 days from the last hu3F8 dose received, was associated with significantly lower serum hu3F8. Despite prior sensitization to other anti-GD2 antibody, e.g. mouse 3F8 or ch14.18, 75% of the patients never developed HAHA response even after getting more treatment cycles. Hu3F8 induced a de novo anti-GD2 response in patients, which was prognostic of progression-free survival. We conclude that hu3F8 had low immunogenicity. During treatment, positive HAHA and low body weight affected PK adversely, whereas induced anti-GD2 response was an outcome predictor.

Oncotargets GD2 and GD3 are highly expressed in sarcomas of children, adolescents, and young adults.

BACKGROUND: GD2 and GD3 are the tumor-associated glycolipid antigens found in a broad spectrum of human cancers. GD2-specific antibody is currently a standard of care for high-risk neuroblastoma therapy. In this study, the pattern of GD2 and GD3 expression among pediatric/adolescent or young adult tumors was determined, providing companion diagnostics for targeted therapy. METHODS: Ninety-two specimens of human osteosarcoma (OS), rhabdomyosarcoma (RMS), Ewing family of tumors, desmoplastic small round cell tumor (DSRCT), and melanoma were analyzed for GD2/GD3 expression by immunohistochemistry. Murine monoclonal antibody 3F8 was used for GD2 staining, and R24 for GD3. Staining was scored according to both intensity and percentage of positive tumor cells from 0 to 4. RESULTS: Both gangliosides were highly prevalent in OS and melanoma. Among other tumors, GD3 expression was higher than GD2 expression. Most OS samples demonstrated strong staining for GD2 and GD3, whereas expression for other tumors was highly variable. Mean intensity of GD2 expression was significantly more heterogeneous (P < 0.001) when compared to GD3 across tumor types. When assessing the difference between GD2 and GD3 expression in all tumor types combined, GD3 expression had a significantly higher score (P = 0.049). When analyzed within each cancer, GD3 expression was significantly higher only in DSRCT (P = 0.002). There was no statistical difference in either GD2 or GD3 expression between primary and recurrent sarcomas. CONCLUSION: GD2/GD3 expression among pediatric solid tumors is common, albeit with variable level of expression. Especially for patients with sarcoma, these gangliosides can be potential targets for antibody-based therapies.

Antitumor Efficacy of Anti-GD2 IgG1 Is Enhanced by Fc Glyco-Engineering.

The affinity of therapeutic antibodies for Fcγ receptors (FcγRs) strongly influences their antitumor potency. To generate antibodies with optimal binding and immunologic efficacy, we compared the affinities of different versions of an IgG1 Fc region that had an altered peptide backbone, altered glycans, or both. To produce IgG1 with glycans that lacked α1,6-fucose, we used CHO cells that were deficient in the enzyme UDP-N-acetylglucosamine: α-3-d-mannoside-ß-1,2-N-acetylglucosaminyltransferase I (GnT1), encoded by the MGAT1 gene. Mature N-linked glycans require this enzyme, and without it, CHO cells synthesize antibodies carrying only Man5-GlcNAc2, which were more effective in antibody-dependent cell-mediated cytotoxicity (ADCC). Our engineered IgG1, hu3F8-IgG1, is specific for GD2, a neuroendocrine tumor ganglioside. Its peptide mutant is IgG1-DEL (S239D/I332E/A330L), both produced in wild-type CHO cells. When produced in GnT1-deficient CHO cells, we refer to them as IgG1n and IgG1n-DEL, respectively. Affinities for human FcγRs were measured using Biacore T-100 (on CD16 and CD32 polymorphic alleles), their immunologic properties compared for ADCC and complement-mediated cytotoxicity (CMC) in vitro, and pharmacokinetics and antitumor effects were compared in vivo in humanized mice. IgG1n and IgG1n-DEL contained only mannose and acetylglucosamine and had preferential affinity for activating CD16s, over inhibitory CD32B, receptors. In vivo, the antitumor effects of IgG1, IgG1-DEL, and IgG1n-DEL were similar but modest, whereas IgG1n was significantly more effective (P < 0.05). Thus, IgG1n antibodies produced in GnT1-deficient CHO cells may have potential as improved anticancer therapeutics. Cancer Immunol Res; 4(7); 631-8. ©2016 AACR.

Lack of survival advantage with autologous stem-cell transplantation in high-risk neuroblastoma consolidated by anti-GD2 immunotherapy and isotretinoin.

Since 2003, high-risk neuroblastoma (HR-NB) patients at our center received anti-GD2 antibody 3F8/GM-CSF + isotretinoin - but not myeloablative therapy with autologous stem-cell transplantation (ASCT). Post-ASCT patients referred from elsewhere also received 3F8/GM-CSF + isotretinoin. We therefore accrued a study population of two groups treated during the same period and whose consolidative therapy, aside from ASCT, was identical. We analyzed patients enrolled in 1st complete/very good partial remission (CR/VGPR). Their event-free survival (EFS) and overall survival (OS) were calculated from study entry. Large study size allowed robust statistical analyses of key prognosticators including MYCN amplification, minimal residual disease (MRD), FCGR2A polymorphisms, and killer immunoglobulin-like receptor genotypes of natural killer cells. The 170 study patients included 60 enrolled following ASCT and 110 following conventional chemotherapy. The two cohorts had similar clinical and biological features. Five-year rates for ASCT and non-ASCT patients were, respectively: EFS 65% vs. 51% (p = .128), and OS 76% vs. 75% (p = .975). In multivariate analysis, ASCT was not prognostic and only MRD-negativity after two cycles of 3F8/GM-CSF correlated with significantly improved EFS and OS. Although a trend towards better EFS is seen with ASCT, OS is near identical. Cure rates may be similar, as close surveillance detects localized relapse and effective salvage treatments are applied. ASCT may not be needed to improve outcome when anti-GD2 immunotherapy is used for consolidation after dose-intensive conventional chemotherapy.

Human derived dimerization tag enhances tumor killing potency of a T-cell engaging bispecific antibody.

Bispecific antibodies (BsAbs) have proven highly efficient T cell recruiters for cancer immunotherapy by virtue of one tumor antigen-reactive single chain variable fragment (scFv) and another that binds CD3. In order to enhance the antitumor potency of these tandem scFv BsAbs (tsc-BsAbs), we exploited the dimerization domain of the human transcription factor HNF1α to enhance the avidity of a tsc-BsAb to the tumor antigen disialoganglioside GD2 while maintaining functional monovalency to CD3 to limit potential toxicity. The dimeric tsc-BsAb showed increased avidity to GD2, enhanced T cell mediated killing of neuroblastoma and melanoma cell lines in vitro (32-37 fold), exhibited a near 4-fold improvement in serum half-life, and enhanced tumor ablation in mouse xenograft models. We propose that the use of this HNF1α-derived dimerization tag may be a novel and effective strategy to increase the potency of T-cell engaging antibodies for clinical cancer immunotherapy.

Prolonged progression-free survival after consolidating second or later remissions of neuroblastoma with Anti-GD2 immunotherapy and isotretinoin: a prospective Phase II study.

Relapse of high-risk neuroblastoma (HR-NB) is deemed invariably fatal yet increasing numbers of HR-NB patients achieve a second complete/very good partial remission (CR/VGPR), hence the urgency to find a successful consolidative therapy. Identifying efficacy in patients without assessable disease, however, is problematic. We report the first study providing outcome data for this group of patients with poor prognosis. To prevent another relapse, HR-NB patients in second or later CR/VGPR received the anti-GD2 murine antibody 3F8 plus granulocyte-macrophage colony-stimulating factor plus isotretinoin in a Phase II trial. Upon meeting the target aim for progression-free survival (PFS) in the initial cohort of 33 patients, the trial was amended to allow patients who developed human anti-mouse antibody (HAMA) to receive rituximab to ablate HAMA with or without low-dose maintenance chemotherapy until immunotherapy could resume. For the total of 101 study patients, 5-year PFS and overall survival (OS) rates were 33% ± 5% and 48% ± 5%, respectively. Among the 33 long-term progression-free survivors, 19 had MYCN amplification, 19 had previously received anti-GD2 immunotherapy plus isotretinoin (as first-line therapy), and 15 never received maintenance chemotherapy. In a multivariate analysis of prognostic factors, only absence of minimal residual disease in bone marrow after 2 cycles of immunotherapy and before initiation of isotretinoin or anti-HAMA therapy was significantly favorable for both PFS and OS. Therefore, long-term PFS is possible for HR-NB patients who achieve at least a second CR/VGPR and receive consolidation that includes anti-GD2 immunotherapy plus isotretinoin, even if the patients received these biological treatments before relapse. Results from this prospective study will aid in the development of future Phase II studies for this growing ultra high-risk patient population.

Alteration of Electrostatic Surface Potential Enhances Affinity and Tumor Killing Properties of Anti-ganglioside GD2 Monoclonal Antibody hu3F8.

Ganglioside GD2 is highly expressed on neuroectodermal tumors and an attractive therapeutic target for antibodies that have already shown some clinical efficacy. To further improve the current antibodies, which have modest affinity, we sought to improve affinity by using a combined method of random mutagenesis and in silico assisted design to affinity-mature the anti-GD2 monoclonal antibody hu3F8. Using yeast display, mutants in the Fv with enhanced binding over the parental clone were FACS-sorted and cloned. In silico modeling identified the minimal key interacting residues involved in the important charged interactions with the sialic acid groups of GD2. Two mutations, D32H (L-CDR1) and E1K (L-FR1) altered the electrostatic surface potential of the antigen binding site, allowing for an increase in positive charge to enhance the interaction with the negatively charged GD2-pentasaccharide headgroup. Purified scFv and IgG mutant forms were then tested for antigen specificity by ELISA, for tissue specificity by immunohistochemistry, for affinity by BIACORE, for antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity in vitro, and for anti-tumor efficacy in xenografted humanized mice. The nearly 7-fold improvement in affinity of hu3F8 with a single D32H (L-CDR1) mutation translated into a ∼12-fold improvement in NK92MI-transfected CD16-mediated ADCC, a 6-fold improvement in CD32-mediated ADCC, and a 2.5-fold improvement in complement-mediated cytotoxicity while maintaining restricted normal tissue cross-reactivity and achieving substantial improvement in tumor ablation in vivo. Despite increasing GD2 affinity, the double mutation D32H (L-CDR1) and E1K (L-FR1) did not further improve anti-tumor efficacy.

Bone marrow minimal residual disease was an early response marker and a consistent independent predictor of survival after anti-GD2 immunotherapy.

PURPOSE: Immunotherapy is a standard of care for children with high-risk neuroblastoma, where bone marrow (BM) is the predominant metastatic site. Early response markers of minimal residual disease (MRD) in the BM that are also predictive of survival could help individualize patient therapies. PATIENTS AND METHODS: After achieving first remission (n = 163), primary refractory disease (n = 102), or second remission (n = 95), children with stage 4 neuroblastoma received anti-GD2 3F8 antibody immunotherapy. BM MRD before 3F8 treatment and after cycle 2 (postMRD) was measured using a four-marker panel (B4GALNT1, PHOX2B, CCND1, and ISL1) by quantitative reverse transcription polymerase chain reaction. Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method. Prognostic variables were tested in both univariable and multivariable analyses, and MRD markers were further assessed individually and in combination as binary composite (postMRD: 0 and 1) and as equal sum (postMRDSum: 0 to 4) using the Cox regression models, and their predictive accuracy was determined by the concordance index. RESULTS: When BM was evaluated after cycle 2, individual markers were highly predictive of PFS and OS. The prediction accuracy improved when they were combined in postMRDSum. A multivariable model taking into account all the variables significant in the univariable analyses identified postMRDSum to be independently predictive of PFS and OS. When the model for OS also included missing killer immunoglobulin-like receptor ligand, human antimouse antibody response, and the enrollment disease status, the concordance index was 0.704. CONCLUSION: BM MRD after two cycles of immunotherapy was confirmed as an early response marker and a consistent independent predictor of survival.

Deep MicroRNA sequencing reveals downregulation of miR-29a in neuroblastoma central nervous system metastasis.

Central nervous system (CNS) is an increasingly common site of isolated metastasis for patients with Stage 4 neuroblastoma. To explore the microRNA (miRNA) profile of this metastatic process, miRNA sequencing was performed to identify miRNA sequence families with differential expression between tumor pairs (pre-CNS primary and CNS metastasis) from 13 patients with Stage 4 neuroblastoma. Seven miRNA sequence families had distinct expression in CNS metastases when compared with their corresponding pre-CNS primaries. MiR-7 was upregulated (3.75-fold), and miR-21, miR-22, miR-29a, miR-143, miR-199a-1-3p, and miR-199a-1-5p were downregulated (3.5-6.1-fold), all confirmed by quantitative reverse transcription-PCR. MiR-29a, previously shown to be downregulated in a broad spectrum of solid tumors including neuroblastoma, had the most significant decrease in all 13 CNS metastases (P = 0.001). Its known onco-targets CDC6, CDK6, and DNMT3A, as well as B7-H3, an inhibitory ligand for T cells, and natural killer cells, were found to have higher differential expression in these 13 CNS metastases when compared with their paired primaries. Additionally, miR-29a expression in primary tumors was significantly lower among patients who eventually relapsed in the CNS. Irrespective of the amplification status of MYCN, which is known to be associated with metastasis, pre-CNS primaries, and CNS metastases had significantly lower miR-29a expression than non-CNS primary tumors. Among MYCN amplified cell lines, those from CNS relapse also had lower miR-29a expression than non-CNS relapse. These findings raised the hypothesis that miR-29a could be a biomarker for neuroblastoma CNS metastasis, and its downregulation may play a pivotal role in CNS progression.