anti-GD2 Antibody Research Articles

Prospects of anti-GD2 immunotherapy for retinoblastoma

Retinoblastoma is the most common type of eye tumor in infants and children. Current treatments for retinoblastoma include intravenous chemotherapy, intra-arterial chemotherapy, intravitreal chemotherapy, cryotherapy, radiotherapy, and surgery. However, these treatments come accompanied by adverse effects such as the toxic side effects of chemotherapeutic drugs, post-operative complications including blindness after surgery, or other complications caused by radiotherapy. Immunotherapy is more promising for its low toxicity on normal cells and effectively improves the quality of life of patients. Disialoganglioside (GD2), a sphingolipid expressed on the surface of retinoblastoma, is a potential therapeutic target for retinoblastoma. We summarized immunotherapeutic approaches for both preclinical studies and clinical trials of GD2. An anti-GD2 monoclonal antibody (Dinutuximab), which has been approved for the treatment of high-risk neuroblastomas, has shown promising efficacy in improving patients’ prognosis. Additionally, chimeric antigen receptors (CAR)-T therapy, GD2 vaccines and nanoparticles are also potential therapeutics. Finally, we discuss the prospects and current limitations of these immunotherapeutic approaches for treating retinoblastoma, as well as how to address these problems.

Tuning the Protonation Sensitivity of Weak Acidic Groups on a Zwitterionic Dendrimer for Selectively Targeting GD2-Overexpressed Tumor Cells in an Acidic Tumor Microenvironment.

Disialoganglioside (GD2) is one of the most popular overexpressed antigens for tumor cell targeting. However, GD2-specific antibodies often show unintended targeting to GD2-expressing health-maintaining cells due to the comparable binding affinities both at physiological pH and in a slightly acidic tumor microenvironment (TME). In this work, an affinity-switchable zwitterionic PAMAM G5 dendrimer (G5-3S) is developed for selective binding to GD2 only in a slightly acidic TME. It has 3 sulfonic groups, 128 carboxylic groups, and 125 amino groups on the surface. This affinity switch is realized by multiple hydrogen bond (H-bond) formation between protonated carboxylic groups surrounding a sulfonic group and overexpressed GD2 clusters on the tumor cell membrane in the slightly acidic TME, whereas there is no stable H-bond formation at physiological pH. Thus, G5-3S shows superior selectivity to GD2-overexpressed tumor cells over anti-GD2 antibodies by avoiding targeting GD2-expressing health-maintaining cells at physiological pH. This suggests that G5-3S is a promising candidate for GD2-overexpressed cancer treatment.

Dual-labeled anti-GD2 targeted probe for intraoperative molecular imaging of neuroblastoma

BackgroundSurgical resection is integral for the treatment of neuroblastoma, the most common extracranial solid malignancy in children. Safely locating and resecting primary tumor and remote deposits of disease remains a significant challenge, resulting in high rates of complications and incomplete surgery, worsening outcomes. Intraoperative molecular imaging (IMI) uses targeted radioactive or fluorescent tracers to identify and visualize tumors intraoperatively. GD2 was selected as an IMI target, as it is highly overexpressed in neuroblastoma and minimally expressed in normal tissue.MethodsGD2 expression in neuroblastoma cell lines was measured by flow cytometry. DTPA and IRDye® 800CW were conjugated to anti-GD2 antibody to generate DTPA-αGD2-IR800. Binding affinity (Kd) of the antibody and the non-radiolabeled tracer were then measured by ELISA assay. Human neuroblastoma SK-N-BE(2) cells were surgically injected into the left adrenal gland of 3.5-5-week-old nude mice and the orthotopic xenograft tumors grew for 5 weeks. 111In-αGD2-IR800 or isotype control tracer was administered via tail vein injection. After 4 and 6 days, mice were euthanized and gamma and fluorescence biodistributions were measured using a gamma counter and ImageJ analysis of acquired SPY-PHI fluorescence images of resected organs (including tumor, contralateral adrenal, kidneys, liver, muscle, blood, and others). Organ uptake was compared by one-way ANOVA (with a separate analysis for each tracer/day combination), and if significant, Sidak’s multiple comparison test was used to compare the uptake of each organ to the tumor. Handheld tools were also used to detect and visualize tumor in situ, and to assess for residual disease following non-guided resection.Results111In-αGD2-IR800 was successfully synthesized with 0.75-2.0 DTPA and 2–3 IRDye® 800CW per antibody and retained adequate antigen-binding (Kd = 2.39 nM for aGD2 vs. 21.31 nM for DTPA-aGD2-IR800). The anti-GD2 tracer demonstrated antigen-specific uptake in mice with human neuroblastoma xenografts (gamma biodistribution tumor-to-blood ratios of 3.87 and 3.88 on days 4 and 6 with anti-GD2 tracer), while isotype control tracer did not accumulate (0.414 and 0.514 on days 4 and 6). Probe accumulation in xenografts was detected and visualized using widely available operative tools (Neoprobe® and SPY-PHI camera) and facilitated detection ofputative residual disease in the resection cavity following unguided resection.ConclusionsWe have developed a dual-labeled anti-GD2 antibody-based tracer that incorporates In-111 and IRDye® 800CW for radio- and fluorescence-guided surgery, respectively. The tracer adequately binds to GD2, specifically accumulates in GD2-expressing xenograft tumors, and enables tumor visualization with a hand-held NIR camera. These results encourage the development of 111In-αGD2-IR800 for future use in children with neuroblastoma, with the goal of improving patient safety, completeness of resection, and overall patient outcomes.

Local Sustained Dinutuximab Delivery and Release From Methacrylated Chondroitin Sulfate.

Neuroblastoma (NB) is the most common pediatric extracranial solid tumor. High-risk NB is a subset of the disease that has poor prognosis and requires multimodal treatment regimens, with a 50% rate of recurrence despite intervention. There is a need for improved treatment strategies to reduce high-risk patient mortality. Dinutuximab is an anti-GD2 antibody ideal for targeting GD2 expressing NB cells, but binding of the antibody to peripheral nerve fibers leads to severe pain during systemic administration. Intratumoral delivery of the anti-GD2 antibody would allow for increased local antibody concentration, without increasing systemic toxicity. Chondroitin Sulfate (CS) is a biocompatible glycosaminoglycan that can be methacrylated to form CSMA, a photocrosslinkable hydrogel that can be loaded with therapeutic agents. The methacrylation reaction time can be varied to achieve different degrees of substitution, resulting in different release and degradation profiles. In this work, 4 and 24 h reacted CSMA was used to create hydrogels at 10% and 20% CSMA. Sustained in vitro release of dinutuximab from these formulations was observed over a 24-day period, and 4 h reacted 10% CSMA hydrogels had the highest overall dinutuximab release over time. An orthotropic mouse model was used to evaluate in vivo response to dinutuximab loaded 4 h methacrylated 10% CSMA hydrogels as compared to bolus tail vein injections. Tumor growth was monitored, and there was a statistically significant increase in the days to reach specific tumor size for tumors treated with intratumoral dinutuximab-loaded hydrogel compared to those treated with dinutuximab solution through tail vein injection. This supports the concept that locally delivering dinutuximab within the hydrogel formulation slowed tumor growth. The CSMA hydrogel-only treatment slowed tumor growth as well, an interesting effect that may indicate interactions between the CSMA and cell adhesion molecules in the tumor microenvironment. These findings demonstrate a potential avenue for local sustained delivery of dinutuximab for improved anti-tumoral response in high-risk NB.

Persistence of pupillary abnormalities in a child with neuroblastoma treated with anti-GD2 antibody

We report the case of a child who, to the best of our knowledge, was the first child in India to receive an anti-GD2 antibody for high-risk neuroblastoma, who presented to us with light-near dissociation and bilateral Adie’s-like pupils, which persisted even on discontinuation of the drug.

Current Knowledge and Perspectives of Immunotherapies for Neuroblastoma.

Neuroblastoma (NBL) cells highly express disialoganglioside GD2, which is restricted and weakly expressed in selected healthy cells, making it a desirable target of immunotherapy. Over the past two decades, application of dinutuximab, an anti-GD2 monoclonal antibody (mAb), has been one of the few new therapies to substantially improve outcomes to current levels. Given the persistent challenge of relapse and therapeutic resistance, there is an urgent need for new effective and tolerable treatment options for high-risk NBL. Recent breakthroughs in immune checkpoint inhibitor (ICI) therapeutics have not translated into high-risk NBL, like many other major pediatric solid tumors. Given the suppressed tumor microenvironment (TME), single ICIs like anti-CTLA4 and anti-PD1 have not demonstrated significant antitumor response rates. Meanwhile, emerging studies are reporting novel advancements in GD2-based therapies, targeted therapies, nanomedicines, and other immunotherapies such as adoptive transfer of natural killer (NK) cells and chimeric antigen receptors (CARs), and these hold interesting promise for the future of high-risk NBL patient care. Herein, we summarize the current state of the art in NBL therapeutic options and highlight the unique challenges posed by NBL that have limited the successful adoption of immune-modifying therapies. Through this review, we aim to direct the field's attention to opportunities that may benefit from a combination immunotherapy strategy.

Validating process mining in pediatric oncology: Demonstrating protocol adherence and variability in neuroblastoma treatment.

166 Background: Neuroblastoma is the most common extracranial tumor in children. The mortality rate for patients with metastatic disease is over 50%. In the Netherlands, 30 new patients are diagnosed annually. Current clinical treatment guidelines are complex and include multiple treatment arms consisting of chemotherapy, surgery, radiotherapy, and immunotherapy (IT). Although poor patient health and complications are known to cause adaptations to the otherwise standardized treatment regimen, the extent to which these variations occur is unknown. This study aimed to validate the use of process mining, a data-driven technique, to retrospectively examine protocol adherence and variability within current clinical guidelines based upon the GPOH-DCOG NBL2009 neuroblastoma treatment protocol. Methods: Data was extracted from electronic health records, as well as laboratory, pathology, and pharmaceutical databases, to create individual event logs for each patient. Process mining was performed using a fuzzy mining algorithm. The treatment courses for all neuroblastoma patients were mapped. Commonalities and variations were highlighted, and the throughput time for key treatment phases was calculated. Subsequently, conformance with protocol was determined. Results: Analysis of 70 patients treated between 2018-2022 showed 62 distinct treatment processes, revealing significant variations in patient care. Patients with high-risk disease showed the least variation (n=47, 42 variants). Notably, 17 variants were identified among 18 patients who died, indicating a highly personalized approach in the advanced stages of the disease. Subgroup analysis of 17 high-risk patients who received IT consisting of alternating cycles of anti-GD2 antibody and retinoic acid showed 6 variants. 12/17 patients (70.6%) completed the full IT regimen. Other treatment adjustments included shortened anti-GD2 infusions for two patients (5 and 8 days versus the prescribed 10 days, respectively), potentially indicating treatment toxicity. One patient received a 15-day infusion, exceeding the prescribed limit of 11 days. The recorded interval between the administration of anti-GD2 and retinoic acid was a median 5.5 hours (range: 60 seconds – 6.4 days), deviating from the prescribed interval of 24 hours and warranting further investigation. Conclusions: Process mining is a valuable tool for analyzing protocol performance and adherence in neuroblastoma treatment. Despite standardized treatment protocols, significant heterogeneity was observed in the treatment of neuroblastoma patients, especially those with poor prognosis. Throughput time analysis revealed variabilities in the IT regimen that warrant further investigation. This study underscores the potential of data-driven approaches to optimize treatment protocols and support evidence-based practices in clinical oncology.

Combined use of niraparib enhanced the inhibitory effect of Anti-GD2 antibody on osteosarcoma cells

PurposeThis study aims to investigate the effect of Niraparib in combination with an Anti-GD2 Antibody on osteosarcoma cells.MethodsScratch test was utilized to assess cell migration capacity, while the Transwell experiment was utilized to evaluate cell invasion potential. Cell proliferation was measured using the CCK8 experiment. The affinity between the anti-GD2 antibody and its antigen was determined via ELISA. Tumor growth was evaluated through animal experiments. Western blotting, QRT-PCR, and histological analysis were conducted to examine the expression of relevant proteins and mRNAs.ResultsMG63 cell line was used for an example. The scratch test showed that the migration rate of osteosarcoma cells in Niraparib + Anti-GD2 group was 1.07 ± 0.04 after 48 h, and 0.34 ± 0.04 in the Control group. Transwell experiment showed that the invasion ability of osteosarcoma cells in Niraparib + Anti-GD2 group was 21.0 ± 1.5, and that in Control group was 87.7 ± 2.9. CCK8 experiment showed that the absorbance value of Niraparib + Anti-GD2 group was 0.16 ± 0.10 on day 5, and that of the Control group was 0.76 ± 0.09. Western blotting showed that the expression levels of BALP and CICP in Niraparib + Anti-GD2 group were 0.751 ± 0.135 and 1.086 ± 0.115, respectively, and those in Control group were 1.025 ± 0.143 and 1.216 ± 0.168, respectively. QRT-PCR results showed that the absorbance values of Niraparib + Anti-GD2 group were 0.173 ± 0.065 and 0.170 ± 0.078 on day 14. The results of animal experiments showed that on day 5, the tumor volume of the Control group was 2433 ± 391, and that of the Niraparib + Anti-GD2 group was 1137 ± 148. Histological analysis showed that the mean density values of Niraparib + Anti-GD2 group were 0.19 ± 0.08 and 0.22 ± 0.07, and those of Control group were 0.26 ± 0.09 and 0.29 ± 0.10.ConclusionThe combination of Niraparib and Anti-GD2 antibody significantly inhibits Osteosarcoma cells.

Patterns of recurrence after radiotherapy for high-risk neuroblastoma: Implications for radiation dose and field

BackgroundPrognosis for patients with high-risk neuroblastoma (HR-NBL) is guarded despite aggressive therapy, and few studies have characterized outcomes after radiotherapy in relation to radiation treatment fields. MethodsMulti-institutional retrospective cohort of 293 patients with HR-NBL who received autologous stem cell transplant (ASCT) and EBRT between 1997–2021. LRR was defined as recurrence at the primary site or within one nodal echelon beyond disease present at diagnosis. Follow-up was defined from the end of EBRT. Event-free survival (EFS) and OS were analyzed by Kaplan-Meier method. Cumulative incidence of locoregional progression (CILP) was analyzed using competing risks of distant-only relapse and death with Gray’s test. ResultsMedian follow-up was 7.0 years (range: 0.01–22.4). Five-year CILP, EFS, and OS were 11.9 %, 65.2 %, and 77.5 %, respectively. Of the 31 patients with LRR and imaging review, 15 (48.4 %) had in-field recurrences (>12 Gy), 6 (19.4 %) had marginal failures (≤12 Gy), and 10 (32.3 %) had both in-field and marginal recurrences. No patients receiving total body irradiation (12 Gy) experienced marginal-only failures (p = 0.069). On multivariable analyses, MYCN amplification had higher risk of LRR (HR: 2.42, 95 % CI: 1.06–5.50, p = 0.035) and post-consolidation isotretinoin and anti-GD2 antibody therapy (HR: 0.42, 95 % CI: 0.19–0.94, p = 0.035) had lower risk of LRR. ConclusionsDespite EBRT, LRR remains a contributor to treatment failure in HR-NBL with approximately half of LRRs including a component of marginal failure. Future prospective studies are needed to explore whether radiation fields and doses should be defined based on molecular features such as MYCN amplification, and/or response to chemotherapy.

Naxitamab chemo-immunotherapy regimens other than with irinotecan/temozolomide for patients with relapsed/refractory high-risk neuroblastoma.

10037 Background: Patients with relapsed/refractory (R/R) high-risk neuroblastoma (HR-NB) have dismal prognosis with chemotherapy-only salvage regimens. Naxitamab, a humanized anti-GD2 monoclonal antibody (mAb), when combined with Irinotecan and Temozolomide (I/T), has demonstrated clinically meaningful efficacy in R/R HR-NB patients (NCT03189706). We aimed to investigate the potential synergy of naxitamab with other cytotoxics. Methods: In this retrospective analysis, we examined patients treated at SJD with chemo-refractory disease, who received naxitamab in combination with chemotherapeutics other than I/T. Each cycle comprised naxitamab 2.25 mg/kg/day IV over 30 minutes, days 2, 4, 9 and 11 (total 9 mg/kg or 270 mg/m2 per cycle), and GM-CSF 250 mg/m2/day subcutaneously, days 6-10 combined with either a) cyclophosphamide (250mg/m2) and topotecan (0.75mg/m2) D1 to D5 (C/T), b) ifosfamide (1500mg/m2), etoposide (100mg/m2) D1-D3 and carboplatin (400mg/m2) D1 (ICE) or c) doxorubicin 37.5mg/m2 D1-D2 and cyclophosphamide (250mg/m2) D1-D3 (C/D). Toxicity was measured by CTCAE v4.0 and responses by INRC. Results: Twenty-nine patients received naxitamab plus GM-CSF with C/T (n=23), ICE (n=6) and C/D (n=1). One patient received both ICE and C/T. Seventeen patients had a variable number of prior relapses (1 n=14, 2 n=1, and 3 n=2). Twelve patients had refractory disease, most having received additional therapy post-induction. All but 4 patients had received naxitamab and I/T (n=20), and/or ICE (n=3/n=1), and dinutuximab-beta and I/T (n=1). Toxicities of the new regimens included myelosuppression expected with chemotherapy, and pain and hypertension expected with naxitamab. Hemorrhagic cystitis occurred in 2 patients treated with C/T, BK infection documented in one. A total of 113 cycles (median 2; 1-8) were administered, outpatient. Out of the 30 treatments, 5 achieved complete response (CR), 2 partial response (PR), and 2 mixed response (MR), providing an objective response (OR) of 30%. Eight patients achieved stable disease (SD) and 13 progressed on treatment. Disease control rate (DCR) (OR or SD) after 2 cycles was 56.6%. Patients who never responded with prior chemo-immunotherapy progressed through the new combinations. Contrary, patients who showed initial stabilization with I/T or ICE but ultimately progressed, switching drugs resulted in 60% DCR and 30% OR. Most favourable OR (75%) was seen in patients who achieved CR with prior chemoimmunotherapy and relapsed (3 out of 4). Conclusions: Naxitamab-based chemo-immunotherapy with regimens other than I/T exhibited similar and manageable toxicity profiles. Switching chemotherapeutics provided objective responses only to patients who had previously shown efficacy with I/T chemo-immunotherapy.

Naxitamab-related adverse events within and across treatment cycles in patients with relapsed/refractory (R/R) high-risk neuroblastoma.

10032 Background: Anti-GD2 monoclonal antibodies, including naxitamab, for the treatment of high-risk neuroblastoma (HRNB) are associated with adverse events (AEs), though few studies have characterized their frequency and patterns of presentation. A practical understanding of AEs can help clinicians expect and manage those requiring intervention. In this post hoc analysis, we report the frequency and severity of naxitamab-related AEs across infusions and treatment cycles, based on data from a prespecified interim analysis of Trial 201. Methods: Trial 201 (phase 2, NCT03363373) is investigating naxitamab plus granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with R/R HRNB with residual disease in bone/bone marrow only. Patients with soft tissue or actively progressing disease were excluded. Naxitamab was infused at 3 mg/kg/dose IV on Days (D) 1/3/5 with GM-CSF administered SC on Days -4 to 5 (monthly cycles [C]). Patients (N=74) were evaluated for safety (CTCAE v4.0), with frequency defined as % of patients reporting >1 events. Frequency of AEs of Grade ≥3 were evaluated for C1-C5. Results: Most (81%) naxitamab-related AEs were Grade 1 or 2. Grade ≥3 AEs reported in ≥10% of patients were hypotension (60% of patients), pain (54%), urticaria (19%), bronchospasm (18%), and abdominal pain (16%). The frequency of related hypotension Grade ≥3 (Grade 4, n=3) decreased across cycles, from C1 (47%) to C5 (33%) and across infusions, from C1D1 (43%) to C1D3 (18%) and C1D5 (11%). Similar decreases were seen during C2 to C5. Among patients with Grade ≥3 hypotension, 73% had their first event C1D1, of whom 34% and 31% had their second event C1D3 or C2D1, respectively. The frequency of related pain Grade 3 AEs (all preferred terms with word “pain”) decreased from C1 (53%) to C2 (37%), generally stabilizing thereafter with frequencies consistent across infusions. Most (77%) had their first event C1D1, of whom 82% had a second event C1D3. The frequency of related bronchospasm Grade 3 (no Grade 4 AEs) was relatively stable around 7% from C1 to C3, decreasing to 4% and 2% in C4 and C5, respectively. Patients generally experienced these events on D1 infusions with few reported D3 or D5. No consistent pattern was seen for Grade 3 urticaria. None of the Grade ≥3 AEs of hypotension, urticaria, pain or bronchospasm resulted in treatment discontinuation. Conclusions: The frequency of naxitamab related Grade ≥3 AEs changed over the course of therapy. Hypotension was most frequent C1D1, decreasing across infusions and cycles. Pain frequency peaked C1 and decreased in subsequent cycles, remaining stable across infusions. Bronchospasm events, though relatively rare, generally occurred D1 and decreased across infusions. The results underline the importance of initial and ongoing monitoring within and across cycles to ensure appropriate and timely management. Clinical trial information: NCT03363373 .

Patterns of improvement following initial response in patients treated with naxitamab for relapsed/refractory high-risk neuroblastoma.

10033 Background: Treatment of high-risk neuroblastoma (HRNB), the most common extracranial solid tumor in pediatric patients, is associated with suboptimal efficacy outcomes. While monitoring responses to naxitamab and other anti-GD2 monoclonal antibodies is essential, there is limited evidence on the timing and magnitude of improved responses following first assessment. Here, we report the patterns of improvement following the initial response to naxitamab therapy, based on the results of a prespecified interim analysis of Trial 201. Methods: Trial 201 (phase 2, NCT03363373) is investigating naxitamab plus granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with refractory/relapsed HRNB with residual disease in bone and/or bone marrow (BM) only. “Refractory” and “relapsed” disease comprised those with an incomplete response (partial response [PR], minor response [MR], or stable disease [SD]) per International Neuroblastoma Response Criteria (INRC) to induction therapy or to treatment for actively progressing or relapsed disease, respectively. Patients with soft tissue or actively progressing disease were excluded. Naxitamab was infused at 3 mg/kg/dose IV on Days 1/3/5 with GM-CSF administered SC on Days -4 to 5 (monthly cycles [C]). The primary endpoint was overall response rate (ORR), comprising those with a complete response (CR) or PR per INRC. Response assessments occurred between C2 and C3 and at prespecified timepoints thereafter. Results: The ORR in Trial 201 was 50% (26/52). At first assessment, the ORR was 37% (n=19), with 14 patients achieving CR. An additional 6/26 patients (23%) achieved a CR or PR after C3, of whom 4 went from MR to CR, and 2 from SD to PR. (One patient whose disease was not evaluable at first assessment and who later had a PR was included in the ORR but excluded from this response improvement analysis.) In addition to the 6 patients who achieved a CR or PR after C3, 2 patients with PR at initial assessment later achieved a CR, and 1 patient with initial SD achieved MR, totaling 9 patients with improved post-C3 responses, or 17% of the overall efficacy population. Among those with baseline disease in bone, post-C3 responses in the bone compartment improved in 6/50 patients (12%). Of these 6 patients, 5 had initial SD in bone and achieved a post-C3 CR or PR. Post-C3 responses in the BM compartment improved in 6/23 patients (26%) with baseline BM disease, of whom 5 had initial SD in BM and achieved a post-C3 CR. Naxitamab safety (N=74) has been reported previously. Conclusions: A considerable proportion (23%) of patients achieved a CR or PR only after C3. Among these patients, most had initial SD within specific bone or BM compartments before achieving a post-C3 CR/PR. Taken together, the results support the rationale for continued treatment of patients not achieving CR/PR at first assessment. Clinical trial information: NCT03363373 .

N9: Pilot study of novel shortened induction for high-risk neuroblastoma.

10052 Background: N9 induction for high-risk neuroblastoma was designed to limit non-hematological toxicity (especially ototoxicity) and to prevent relapse in the central nervous system (CNS). N9 builds on 2 novel regimens: high-dose cyclophosphamide-topotecan-vincristine (CTV) and ifosfamide-carboplatin-etoposide (ICE), each with good penetration across the blood-brain barrier. Methods: N9 is a pilot study (Clinicaltrials.gov.NCT04947501) to assess feasibility and safety. Secondary/exploratory objectives include response, collection of peripheral blood stem cells (PBSCs; ≥5x106/kg CD34(+) cells sufficient for ≥2 rescues), tumor resection, and assessment of central nervous system relapse. Early stopping rules centered on excessive delay in timing of chemotherapy. Eligibility criteria included age >1-to-<13 years; 1 prior chemotherapy cycle was allowed. CTCAE Version 5.0 and International Neuroblastoma Response Criteria are used.N9 comprises 4 cycles of chemotherapy. Cycles start after absolute neutrophil count is >500/μL, platelets >100,000/μL, and non-hematologic toxicities grade ≤2. Intervals of 21-28 days between start of cycles are foreseen. PBSC collection and surgery follow >3 cycles. Cycles #1 and #4 (CTV): cyclophosphamide 70mg/kg/day, days 1-2, topotecan 2mg/m2/day, days 1-4, and vincristine 0.067mg/kg, day 1. Cycle #2 (ICE): ifosfamide 1500mg/m2/day, days 1-5, carboplatin 400mg/m2/day, days 1-2, and etoposide 100mg/m2/day, days 1-5. Cycle #3: cyclophosphamide (as in CTV) and 72-hr infusions of doxorubicin 75mg/m2 and vincristine 0.067mg/kg. Results: The target number of 15 patients were enrolled: 10/2021-9/2023; age 1.5–9.8 (median 3.3) years; 14 stage M, 1 stage L2; and 10 post-1 cycle of other chemotherapy. They completed N9 without undue toxicity: all cycles started on time, organ functions remained intact, 11/11 patients tested had no ototoxicity, and acute toxicities were typical for myelosuppression (including uncomplicated fever/neutropenia). Responses were complete (n=6), partial (n=5), and stable disease (n=4). The target number of PBSCs was collected in 12 patients (9-75, median 14 x106/kg CD34(+) cells), 4x106/kg in 2 patients, and pending in 1. All patients had gross total resections of the primary tumor. Post-N9, patients did not undergo transplant, but proceeded to immunotherapy (naxitamab) or chemoimmunotherapy (naxitamab+irinotecan-temozolomide). Of 9 with residual disease post-N9, 7 achieved CR (median of 5.6 months from study entry) and 1 achieved metabolic CR (16 months), though 3 subsequently relapsed (no CNS). Conclusions: N9 shows promise for reducing chemotherapy and long-term toxicity. Less chemotherapy without compromising survival is a realistic goal by virtue of the advances with anti-GD2 monoclonal antibodies which, in combination with GM-CSF+low-dose chemotherapy, are highly effective against chemo-resistant disease in bone/bone marrow. Clinical trial information: NCT04947501 .

Efficacy and safety of dinutuximab in the management of high-risk neuroblastoma: A systematic review and meta-analysis.

e22000 Background: Neuroblastoma (NB) is a malignant tumor of the sympathetic nervous system that usually occurs in children below 5 years of age. High-risk neuroblastoma (HR-NB) has a poor prognosis despite a number of treatment strategies. Dinutuximab, an anti-GD2 monoclonal antibody, has been recently added to the standard of care due to its improved prognosis. We aimed to systematically assess the outcomes of HR-NB patients treated with dinutuximab and compare them with those on other treatment regimens. Methods: A comprehensive search strategy was used to search PubMed and the Cochrane Library for articles investigating the effect of dinutuximab on the outcomes of patients with HR-NB. Eligibility criteria included: 1) Diagnosis of HR-NB based on INRG and INSS staging and MYCN status 2) Dinutuximab as the primary agent used in the intervention group 3) Mean/median follow-up time greater than 6 months. Three investigators independently reviewed and extracted relevant articles. Any disagreements were addressed through consultation with other authors. The risk of bias assessment of the selected articles was conducted using the Cochrane Risk of Bias Tool for randomized controlled trials (RCTs) and the Newcastle-Ottawa scale for observational studies. Review Manager software was used to obtain and display the meta-analysis estimates in forest plots. Random effects models were used to calculate the mean difference and overall estimated effects for continuous variables. The primary outcomes were all-cause mortality and 5-year event-free survival (5-year-EFS). Results: Five studies, including two RCTs, two secondary analyses of clinical trials, and one retrospective cohort study, comprising 1,393 participants, were included in the analysis. 686 of the patients received dinutuximab, while the remaining 707 patients were assigned to other therapies as controls. Dinutuximab was associated with lower all-cause mortality as compared to control [pooled RR, 0.41; 95% CI, 0.22-0.75, P = 0.004, I2 = 31%]. 5-year-EFS was also greater for patients treated with dinutuximab [MD: 0.12; 95% CI, 0.09-0.16; P < 0.001, I2= 98%]. Conclusions: Our findings suggest that dinutuximab is linked to a significant reduction in overall mortality and a noteworthy improvement in the 5-Year-EFS for patients with HR-NB, when compared to other treatment options.

A pilot study of post-consolidation chemoimmunotherapy for high-risk neuroblastoma (ANBL19P1): A report from the Children’s Oncology Group.

10004 Background: Post-consolidation anti-GD2 antibody-based immunotherapy has improved survival for newly diagnosed patients (pts) with high-risk neuroblastoma (HRNBL). However, nearly half of newly diagnosed pts relapse. Based upon the efficacy of anti-GD2 antibody dinutuximab given with chemotherapy (chemoimmunotherapy) in the relapse setting, we hypothesized that post-consolidation chemoimmunotherapy may further improve frontline outcomes. ANBL19P1 first assessed the feasibility of delivering chemoimmunotherapy after tandem high-dose chemotherapy with autologous stem cell transplant (ASCT). Methods: Pts <31 years old with HRNBL who received 4-6 cycles of Induction chemotherapy +/- up to 4 cycles of post-Induction chemotherapy or chemoimmunotherapy, underwent tandem ASCT, had no evidence of progressive disease (PD), and met organ function criteria were eligible. Therapy, administered every 28 days, consisted of temozolomide and irinotecan on Days 1-5, dinutuximab on Days 2-5, and sargramostim on Days 6-12 during Cycles 1-5; isotretinoin on Days 8-21 during Cycles 1-6. Therapy was deemed feasible if the 95% confidence interval (CI) placed on the percentage of pts that completed 5 cycles of dinutuximab + chemotherapy without PD within 30 weeks contained 75%, and if the interim monitoring rules for feasibility and excessive toxicity were not triggered. Event-free (EFS) and overall survival (OS) were determined from time of enrollment. Results: From 11/30/20-6/30/23, 40 eligible pts enrolled and started protocol therapy. 87.5% (n=35) were ≥18 months old and 97.5% (n=39) had INRG Stage M disease, at diagnosis. 87.5% (95% CI 73.9%, 94.5%) completed 5 cycles of dinutuximab + chemotherapy without PD within 30 weeks, exceeding the feasibility benchmark of 75%. Feasibility and monitoring rules were not triggered. Five pts were removed from protocol therapy prior to Cycle 4 [physician determination (n=2), pt/parent refusal of further therapy (n=2), and PD (n=1)]. No unacceptable toxicities or deaths on protocol therapy were reported. Toxicities of interest are summarized (Table). One-year EFS and OS were 90.0±5.0% and 97.5±2.7%, respectively (median follow-up time for pts without event=1.2 years). Conclusions: Administration of post-consolidation chemoimmunotherapy in pts who underwent tandem ASCT is tolerable and met pre-defined feasibility criteria. The impact of this approach on survival outcomes will be studied in a future COG trial. Clinical trial information: NCT04385277 . [Table: see text]

Preliminary experience of ex-vivo expanded allogeneic universal donor TGFβi NK cell infusions in combination with irinotecan, temozolomide, and dinutuximab in patients with relapsed or refractory neuroblastoma: The Allo - STING trial.

10005 Background: Neuroblastoma (NBL) is the most common extra-cranial solid tumor in children and outcomes for children with relapsed/refractory (RR) NBL remain dismal. While the combination of chemoimmunotherapy (CIT) with the anti-GD2 antibody dinutuximab as per COG protocol ANBL1221 has improved responses, this regimen is still not curative. Dinutuximab can recruit NK cells for anticancer activity, however, NK cells are depleted in NBL patients undergoing chemotherapy. We devised a method to expand allogeneic NK cells ex vivo to improve effector function and render the NK cells resistant to TGFβ-induced suppression (TGFβi). These TGFβi NK cells are expanded from a universal donor (UD) pool, enabling multiple cycles of “off-the-shelf” high-dose therapy. We hypothesize that adoptive transfer of UD-TGFβi NK cells to patients with RR NBL sequentially following CIT is safe and improves outcomes compared to CIT alone. Methods: This Phase I/II study evaluates the safety and tolerability of UD-TGFβi NK cells in combination with CIT and compares anti-cancer efficacy to CIT alone. Eligibility includes age < 30 years, RR or progressive NBL, and prior treatment with at least 4 cycles of induction chemotherapy. The treatment protocol consists of 21-day cycles of CIT as per COG protocol ANBL1221 with UD-TGFβi NK cells (1x108 cells/kg) infused on day 8. Up to six cycles of treatment are permitted. Overall response rates are defined using the Revised International NBL Response Criteria, with 95% confidence intervals compared to the results from ANBL1221. Progression-free and overall survival will be estimated by the Kaplan Meier method. Results: Four subjects (7 – 11 years) have been enrolled. All patients presented with high risk NBL: three with relapsed disease and one with refractory disease. All received at least one prior salvage therapy. Three patients have completed all 6 cycles of protocol therapy, and one remains on treatment. 20 NK cell infusions have been administered to date. All NK cell infusions occurred within the protocol specified window. One subject experienced grade 1 fever with two NK cell infusions. There have been no other adverse events attributable to the NK cells. Adverse events attributable to CIT were similar to those described in COG ANBL 1221. Treatment delays for cytopenias due to CIT occurred in two patients. Three patients achieved a partial response. A fourth patient attained prolonged stable disease (9 months) but experienced a skeletal recurrence. Conclusions: UD allo-TGFβi NK cells can be safely and feasibly administered to children with RR NBL after treatment with CIT, with early objective responses observed in the preliminary cohort of patients. Ongoing studies will assess markers of response, NK cell persistence, pharmacokinetics and pharmacodynamics. Clinical trial information: NCT04211675 .

Hu14.18K.322A Causes Direct Cell Cytotoxicity and Synergizes with Induction Chemotherapy in High-Risk Neuroblastoma.

The disialoganglioside, GD2, is a promising therapeutic target due to its overexpression in certain tumors, particularly neuroblastoma (NB), with limited expression in normal tissues. Despite progress, the intricate mechanisms of action and the full spectrum of the direct cellular responses to anti-GD2 antibodies remain incompletely understood. In this study, we examined the direct cytotoxic effects of the humanized anti-GD2 antibody hu14.18K322A (hu14) on NB cell lines, by exploring the associated cell-death pathways. Additionally, we assessed the synergy between hu14 and conventional induction chemotherapy drugs. Our results revealed that hu14 treatment induced direct cytotoxic effects in CHLA15 and SK-N-BE1 cell lines, with a pronounced impact on proliferation and colony formation. Apoptosis emerged as the predominant cell-death pathway triggered by hu14. Furthermore, we saw a reduction in GD2 surface expression in response to hu14 treatment. Hu14 demonstrated synergy with induction chemotherapy drugs with alterations in GD2 expression. Our comprehensive investigation provides valuable insights into the multifaceted effects of hu14 on NB cells, shedding light on its direct cytotoxicity, cell-death pathways, and interactions with induction chemotherapy drugs. This study contributes to the evolving understanding of anti-GD2 antibody therapy and its potential synergies with conventional treatments in the context of NB.

Targeting both GD2 and B7-H3 using bispecific antibody improves tumor selectivity for GD2-positive tumors.

Disialoganglioside 2 (GD2), overexpressed by cancers such as melanoma and neuroblastoma, is a tumor antigen for targeted therapy. The delivery of conventional IgG antibody technologies targeting GD2 is limited clinically by its co-expression on nerves that contributes to toxicity presenting as severe neuropathic pain. To improve the tumor selectivity of current GD2-targeting approaches, a next-generation bispecific antibody targeting GD2 and B7-H3 (CD276) was generated. Differential expression of human B7-H3 (hB7-H3) was transduced into GD2+ B78 murine melanoma cells and confirmed by flow cytometry. We assessed the avidity and selectivity of our GD2-B7-H3 targeting bispecific antibodies (INV34-6, INV33-2, and INV36-6) towards GD2+/hB7-H3- B78 cells relative to GD2+/hB7-H3+ B78 cells using flow cytometry and competition binding assays, comparing results an anti-GD2 antibody (dinutuximab, DINU). The bispecific antibodies, DINU, and a non-targeted bispecific control (bsAb CTRL) were conjugated with deferoxamine for radiolabeling with Zr-89 (t1/2 = 78.4 h). Using positron emission tomography (PET) studies, we evaluated the in vivo avidity and selectivity of the GD2-B7-H3 targeting bispecific compared to bsAb CTRL and DINU using GD2+/hB7-H3+ and GD2+/hB7-H3- B78 tumor models. Flow cytometry and competition binding assays showed that INV34-6 bound with high avidity to GD2+/hB7-H3+ B78 cells with high avidity but not GD2+/hB7-H3+ B78 cells. In comparison, no selectivity between cell types was observed for DINU. PET in mice bearing the GD2+/hB7-H3- and GD2+/hB7-H3+ B78 murine tumor showed similar biodistribution in normal tissues for [89Zr]Zr-Df-INV34-6, [89Zr]Zr-Df-bsAb CTRL, and [89Zr]Zr-Df-DINU. Importantly, [89Zr]Zr-Df-INV34-6 tumor uptake was selective to GD2+/hB7-H3+ B78 over GD2+/hB7-H3- B78 tumors, and substantially higher to GD2+/hB7-H3+ B78 than the non-targeted [89Zr]Zr-Df-bsAb CTRL control. [89Zr]Zr-Df-DINU displayed similar uptake in both GD2+ tumor models, with uptake comparable to [89Zr]Zr-Df-INV34-6 in the GD2+/hB7-H3+ B78 model. The GD2-B7-H3 targeting bispecific antibodies successfully improved selectivity to cells expressing both antigens. This approach should address the severe toxicities associated with GD2-targeting therapies by reducing off-tumor GD2 binding in nerves. Continued improvements in bispecific antibody technologies will continue to transform the therapeutic biologics landscape.

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

The carbohydrate ganglioside GD2/GD3 cancer vaccine adjuvanted by β-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 β-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.

Desensitizing the autonomic nervous system to mitigate anti-GD2 monoclonal antibody side effects.

Anti-GD2 monoclonal antibodies (mAbs) have shown to improve the overall survival of patients with high-risk neuroblastoma (HR-NB). Serious adverse events (AEs), including pain, within hours of antibody infusion, have limited the development of these therapies. In this study, we provide evidence of Autonomic Nervous System (ANS) activation as the mechanism to explain the main side effects of anti-GD2 mAbs. Through confocal microscopy and computational super-resolution microscopy experiments we explored GD2 expression in postnatal nerves of infants. In patients we assessed the ANS using the Sympathetic Skin Response (SSR) test. To exploit tachyphylaxis, a novel infusion protocol (the Step-Up) was mathematically modelled and tested. Through confocal microscopy, GD2 expression is clearly visible in the perineurium surrounding the nuclei of nerve cells. By computational super-resolution microscopy experiments we showed the selective expression of GD2 on the cell membranes of human Schwann cells in peripheral nerves (PNs) significantly lower than on NB. In patients, changes in the SSR were observed 4 minutes into the anti-GD2 mAb naxitamab infusion. SSR latency quickly shortened followed by gradual decrease in the amplitude before disappearance. SSR response did not recover for 24 hours consistent with tachyphylaxis and absence of side effects in the clinic. The Step-Up protocol dissociated on-target off-tumor side effects while maintaining serum drug exposure. We provide first evidence of the ANS as the principal non-tumor target of anti-GD2 mAbs in humans. We describe the development and modeling of the Step-Up protocol exploiting the tachyphylaxis phenomenon we demonstrate in patients using the SSR test.