anti-GD2 Monoclonal Antibody Research Articles

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.

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.

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.

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.

Management of High-Risk Neuroblastoma with Soft-Tissue-Only Disease in the Era of Anti-GD2 Immunotherapy.

Neuroblastoma presents with two patterns of disease: locoregional or systemic. The poor prognostic risk factors of locoregional neuroblastoma (LR-NB) include age, MYCN or MDM2-CDK4 amplification, 11q, histology, diploidy with ALK or TERT mutations, and ATRX aberrations. Anti-GD2 immunotherapy has significantly improved the outcome of high-risk (HR) NB and is mostly effective against osteomedullary minimal residual disease (MRD), but less so against soft tissue disease. The question is whether adding anti-GD2 monoclonal antibodies (mAbs) benefits patients with HR-NB compounded by only soft tissue. We reviewed 31 patients treated at SJD for HR-NB with no osteomedullary involvement at diagnosis. All tumors had molecular genetic features of HR-NB. The outcome after first-line treatment showed 25 (80.6%) patients achieving CR. Thirteen patients remain in continued CR, median follow-up 3.9 years. We analyzed whether adding anti-GD2 immunotherapy to first-line treatment had any prognostic significance. The EFS analysis using Cox models showed a HR of 0.20, p = 0.0054, and an 80% decrease in the risk of relapse in patients treated with anti-GD2 immunotherapy in the first line. Neither EFS nor OS were significantly different by CR status after first-line treatment. In conclusion, adding treatment with anti-GD2 mAbs at the stage of MRD helps prevent relapse that unequivocally portends poor survival.

Transition to a mesenchymal state in neuroblastoma may be characterized by a high expression of GD2 and by the acquisition of immune escape from NK cells.

Neuroblastoma (NB) is characterized by both adrenergic (ADRN) and undifferentiated mesenchymal (MES) subsets. The ganglioside sialic acid-containing glycosphingolipid (GD2) is widely overexpressed on tumors of neuroectodermal origin promoting malignant phenotypes. MES cells are greatly enriched in post-therapy and relapsing tumors and are characterized by decreased expression of GD2. This event may cause failure of GD2-based immunotherapy. NK cells represent a key innate cell subset able to efficiently kill tumors. However, the tumor microenvironment (TME) that includes tumor cells and tumor-associated (TA) cells could inhibit their effector function. We studied eight NB primary cultures that, in comparison with commercial cell lines, more faithfully reflect the tumor cell characteristics. We studied four primary NB-MES cell cultures and two pairs of MES/ADRN (691 and 717) primary cultures, derived from the same patient. In particular, in the six human NB primary cultures, we assessed their phenotype, the expression of GD2, and the enzymes that control its expression, as well as their interactions with NK cells, using flow cytometry, RT-qPCR, and cytotoxicity assays. We identified mature (CD105+/CD133-) and undifferentiated (CD133+/CD105-) NB subsets that express high levels of the MES transcripts WWTR1 and SIX4. In addition, undifferentiated MES cells display a strong resistance to NK-mediated killing. On the contrary, mature NB-MES cells display an intermediate resistance to NK-mediated killing and exhibit some immunomodulatory capacities on NK cells but do not inhibit their cytolytic activity. Notably, independent from their undifferentiated or mature phenotype, NB-MES cells express GD2 that can be further upregulated in undifferentiated NB-MES cells upon co-culture with NK cells, leading to the generation of mature mesenchymal GD2bright neuroblasts. Concerning 691 and 717, they show high levels of GD2 and resistance to NK cell-mediated killing that can be overcome by the administration of dinutuximab beta, the anti-GD2 monoclonal antibody applied in the clinic. NB is a heterogeneous tumor representing a further hurdle in NB immunotherapy. However, different from what was reported with NB commercial cells and independent of their MES/ADRN phenotype, the expression of GD2 and its displayed sensitivity to anti-GD2 mAb ADCC indicated the possible effectiveness of anti-GD2 immunotherapy.

Abstract 2672: GD3 synthase (ST8SIA1) is a key immunomodulator in GD2+ breast cancer cells

Abstract Background: Gangliosides are acidic glycosphingolipids involved in cell adhesion, proliferation, and modulation of signal transduction pathways. It has been reported that tumor-shed gangliosides influence the activity of immune cells, including macrophages, NK cells, and T cells. GD3 synthase (GD3S) is the key enzyme that regulates the biosynthesis of the b and c-series gangliosides, particularly GD3 and GD2, and studies have shown that GD3S is upregulated in most tumors and plays a role in tumor progression. Similarly, we have found GD3S to be significantly upregulated in GD2+ breast cancer stem cells (BCSC) compared to GD2− cells, and the knockdown of this gene prevented tumor formation in mice. Here, we hypothesize that GD3 synthase has an immunomodulatory function in breast cancer (BC) cells through the upregulation of GD2. Methods: We stably overexpressed GD3S in BT549 and MCF-7 breast cancer cell lines by lentiviral transfection and performed CRISPR-Cas9 knockout of GD3S in BT549 and SUM 159 cell lines. We examined the effect of GD3 synthase on macrophage-mediated phagocytosis, NK cell-mediated killing, and T-cell cytotoxicity of BC cells using the live cell imaging system, IncuCyte®. Naxitamab is a US FDA-approved fully humanized anti-GD2 monoclonal antibody for the treatment of neuroblastoma. We evaluated the effects of naxitamab in GD2+ BC cell lines (HCC1395, Hs578T) using the Incucyte, and also examined its effect on in vivo tumor growth using TNBC PDX models. Results: We observed a highly significant decrease in macrophage-mediated phagocytosis (p<0.005; p<0.0001) and NK-mediated killing (p<0.0001; p<0.0001) in BT549 and MCF-7 cell overexpressing GD3S compared to the control cells. Interestingly, we observed a significant increase in phagocytosis in GD3S knockout BT549 and SUM 159 cells (p<0.05; p<0.005) compared to their wild-type counterparts. Moreover, the knockout of GD3S enhanced NK cell-mediated apoptosis in BT549 cells (p<0.001) and T cell killing in BT549 and SUM 159 cells (p<0.0001; p<0.0001). Finally, we found that naxitamab dose-dependently enhances macrophage-mediated phagocytosis and NK cell-mediated apoptosis in HCC1395 and Hs578T BC cells, with 20µg/ml being the most effective concentration in comparison to IgG control (p<0.0001; p<0.0001). In addition, we observed a reduction in tumor volume in naxitamab-treated mice compared to IgG control (p<0.05) Conclusion: GD3 synthase regulates macrophage-mediated phagocytosis, NK cell-induced apoptosis, and T-cell cytotoxicity in BC cells. Naxitamab enhances immune cell killing of breast cancer cells with high GD2 expression. Furthermore, naxitamab inhibited tumor growth in GD2+ TNBC PDX models. Citation Format: Bolutyfe Oderinde, Vivek Anand, Maryam Siddiqui, Anudishi Tyagi, Borgman Jenny, Venkata Lokesh Battula. GD3 synthase (ST8SIA1) is a key immunomodulator in GD2+ breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2672.

Targeting refractory/recurrent neuroblastoma and osteosarcoma with anti-CD3×anti-GD2 bispecific antibody armed T cells

BackgroundThe survival benefit observed in children with neuroblastoma (NB) and minimal residual disease who received treatment with anti-GD2 monoclonal antibodies prompted our investigation into the safety and potential clinical benefits...

Targeted therapies in retinoblastoma: GD2-directed immunotherapy following autologous stem cell transplantation and evaluation of alternative target B7-H3

BackgroundGD2-directed immunotherapy is highly effective in the treatment of high-risk neuroblastoma (NB), and might be an interesting target also in other high-risk tumors.MethodsThe German-Austrian Retinoblastoma Registry, Essen, was searched for patients, who were treated with anti-GD2 monoclonal antibody (mAb) dinutuximab beta (Db) in order to evaluate toxicity, response and outcome in these patients. Additionally, we evaluated anti-GD2 antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in retinoblastoma cell lines in vitro. Furthermore, in vitro cytotoxicity assays directed against B7-H3 (CD276), a new identified potential target in RB, were performed.ResultsWe identified four patients with relapsed stage IV retinoblastoma, who were treated with Db following autologous stem cell transplantation (ASCT). Two out of two evaluable patients with detectable tumors responded to immunotherapy. One of these and another patient who received immunotherapy without residual disease relapsed 10 and 12 months after start of Db. The other patients remained in remission until last follow-up 26 and 45 months, respectively. In vitro, significant lysis of RB cell lines by ADCC and CDC with samples from patients and healthy donors and anti-GD2 and anti-CD276-mAbs were demonstrated.ConclusionAnti-GD2-directed immunotherapy represents an additional therapeutic option in high-risk metastasized RB. Moreover, CD276 is another target of interest.

Toxicity Spectrum of Anti-GD2 Immunotherapy: A Real-World Study Leveraging the US Food and Drug Administration Adverse Event Reporting System.

Anti-disialoganglioside (anti-GD2) monoclonal antibodies are effective immunotherapeutic drugs for treating neuroblastoma, yet their toxicity spectrum is unclear. This study aimed to assess the toxicity profiles of three anti-GD2 monoclonal antibodies (dinutuximab, dinutuximab β, and naxitamab) in clinical applications by mining and evaluating the adverse drug reaction (ADR) signals from the US Food and Drug Administration Adverse Event Reporting System. Data in the US Food and Drug Administration Adverse Event Reporting System from the time anti-GD2 monoclonal antibodies became available in the market to the first quarter of 2023 were searched. The signals of anti-GD2 monoclonal antibody-associated ADRs were quantified using four types of algorithms, including the reporting odds ratio, the proportional reporting ratio, the combination of the proportional reporting ratio and χ2 statistic method used by the UK Medicines and Healthcare Products Regulatory Agency, and the Bayesian confidence propagation neural network. The ADRs were categorized by System Organ Class based on the Medical Dictionary for Regulatory Activities, and were sorted according to the frequency and signal strength of ADRs. A total of 370 adverse drug event reports with anti-GD2 monoclonal antibodies listed as the 'primary suspected drugs' were identified, with 116 ADR signals detected, of which 22 were not in the drug labels. Among the adverse drug event reports, 276 reports concerned dinutuximab/dinutuximab β as primary suspected drugs with 90 ADR signals, involving 19 System Organ Classes, of which 21 signals were not in the label; 94 adverse drug event reports concerned naxitamab as the primary suspected drug with 26 ADR signals, involving 11 System Organ Classes, of which one was not in the label. For dinutuximab/dinutuximab β-related ADRs, the top five most frequent were "fever", "abdominal pain", "elevated aspartate aminotransferase (AST)", "elevated alanine aminotransferase (ALT)" and "hypotension"; the top five most intensive signals were "hypoalbuminemia", "elevated AST", "capillary leakage syndrome", "hypoxia" and "elevated ALT". For naxitamab-related ADRs, the top five most frequent were "hypotension", "pain", "urticarial", "hypertension" and "rash"; the top five most intensive signals were "hypotension", "urticaria", "hypoxemia", "bronchospasm" and "hypertension". Involved System Organ Classes included "investigations" and "respiratory, thoracic and mediastinal disorders" containing the most types of ADR signals in dinutuximab/dintuximab β-related ADRs and naxitamab-related ADRs, respectively. Our study comprehensively analyzed the toxicity profiles of anti-GD2 monoclonal antibodies and provides an important reference for clinical monitoring and ADR identification of these drugs.

Tumor-specific targeting of polymer drug delivery systems with recombinant proteins bound via tris(nitrilotriacetic acid)

Antibody-mediated targeting is an efficient strategy to enhance the specificity and selectivity of polymer nanomedicines towards the target site, typically a tumor. However, direct covalent coupling of an antibody with a polymer usually results in a partial damage of the antibody binding site accompanied with a compromised biological activity. Here, an original solution based on well-defined non-covalent interactions between tris-nitrilotriacetic acid (trisNTA) and hexahistidine (His-tag) groups, purposefully introduced to the structure of each macromolecule, is described. Specifically, trisNTA groups were attached along the chains of a hydrophilic statistical copolymer based on N-(2-hydroxypropyl)methacrylamide (HPMA), and at the end or along the chains of thermo-responsive di-block copolymers based on N-isopropylmethacrylamide (NIPMAM) and HPMA; His-tag was incorporated to the structure of a recombinant single chain fragment of an anti-GD2 monoclonal antibody (scFv-GD2). Static and dynamic light scattering analyses confirmed that mixing of polymer with scFv-GD2 led to the formation of polymer/scFv-GD2 complexes; those prepared from thermo-responsive polymers formed stable micelles at 37 °C. Flow cytometry and fluorescence microscopy clearly demonstrated antigen-specific binding of the prepared complexes to GD2 positive murine T-cell lymphoma cells EL-4 and human neuroblastoma cells UKF-NB3, while no interaction with GD2 negative murine fibroblast cells NIH-3T3 was observed. These non-covalent polymer protein complexes represent a new generation of highly specific actively targeted polymer therapeutics or diagnostics.

Diagnostic problems in pediatrics on the example of a clinical case of neuroblastoma

Neuroblastoma (NB) is a malignant extracranial solid tumor of childhood. The peak incidence occurs in the first year of life. In 50% of cases, the disease already has signs of metastasis at the time of diagnosis. The clinical picture in neuroblastoma is varied; the symptoms are not specific and depend on the localization. The diagnosis is confirmed by histological examination of the biopsy of the primary tumor and metastasis. A specific method for the topical diagnosis of neuroblastoma is 123I-labeled metaiodobenzylguanidine scintigraphy, which makes it possible to detect the primary tumor, the presence of regional and distant metastases. Combinations of high-dose chemotherapy and transplantation of autologous stem cells and differentiating agents, as well as immunotherapy with anti-GD2 monoclonal antibodies, can increase the life expectancy of patients.

Targeting GD2 after allogeneic SCT: effector cell composition defines the optimal use of ch14.18 and the bispecific antibody construct NG-CU (GD2-CD3).

We investigated whether T cell-recruiting bispecific anti-CD3/GD2 antibody NG-CU might be an alternative to therapeutic anti-GD2 monoclonal antibody (mAb) ch14.18, mediating complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) through natural killer (NK) cells for immunotherapy in high-risk/relapsed neuroblastoma after autologous/allogeneic stem cell transplantation (auto/alloSCT). Different antibody concentrations and effector-to-target ratios (E:T) were evaluated using xCELLigence RTCA system, peripheral blood mononuclear cells (PBMCs) (healthy donors and patients after alloSCT), and neuroblastoma cell lines (LS/LAN-1). Mean specific lysis of LS cells utilizing PBMCs from healthy donors and ch14.18 (1µg/ml) was 40/66/75% after 12/24/48 h compared to 66/93/100% in the presence of NG-CU (100 ng/ml). NG-CU showed enhanced cytotoxicity compared to ch14.18, even at lower concentrations and E:T ratios, and completely eradicated LS cells after 72 h. To decipher the influence of effector cell subsets on lysis, different ratios of T and NK cells were tested. At a ratio of 1:1, ch14.18 was more effective than NG-CU. Using patient PBMCs taken at different time points posttransplant, significant lysis with both constructs was detectable depending on percentages and total numbers of T and NK cells; in the early posttransplant phase, NK cells were predominant and ch14.18 was superior, whereas later on, T cells represented the majority of immune cells and NG-CU was more effective. Our study highlights the importance of analyzing effector cell subsets in patients before initiating antibody-based therapy. Consequently, we propose an adjusted administration of both antibody constructs, considering the state of posttransplant immune recovery, to optimize anti-tumor activity.

Treatment of High-Risk Neuroblastoma with Dinutuximab and Chemotherapy Administered in all Cycles of Induction.

Administration of chemoimmunotherapy using concurrent chemotherapy and an anti-GD2 monoclonal antibody (mAb), dinutuximab (DIN), demonstrated efficacy for the treatment of relapsed and refractory neuroblastoma. Chemoimmunotherapy, using a humanized anti-GD2 mAb, demonstrated a signal of activity in a phase 2 study for the treatment of patients with newly diagnosed high-risk neuroblastoma (HRNBL). In this single-institution retrospective study, patients with HRNBL received an Induction chemotherapy regimen plus DIN in all Induction cycles. Toxicity and response data were abstracted from the electronic medical record. Toxicities were graded by CTCAE v.5.0. The end of Induction (EOI) objective response rate was determined using the Revised International Neuroblastoma Response Criteria. Twenty-seven patients with HRNBL (23 newly diagnosed, 16 females, median age 3.9 years) started Induction chemoimmunotherapy from 27 January 2017 to 28 December 2022. All patients received DIN with all cycles of Induction therapy, and all but one patient completed Induction therapy. The most common non-hematologic grade ≥ 3 toxicities included fever (44%), hypoxemia (20%), and hypoalbuminemia (11%). End of Induction responses included eighteen with a complete response (CR), seven with a partial response (PR), one with progressive disease (PD), and zero with a minor response or stable disease. Twenty-six of twenty-seven patients (96%) completed all Induction cycles and were evaluable for a response. The EOI response of PR or better in the evaluable cohort was 96%. Dinutuximab was well tolerated with all Induction cycles, demonstrated an encouraging EOI response rate, and should be evaluated in a randomized study.

Stage 4N neuroblastoma before and during the era of anti-GD2 immunotherapy.

Patients with stage 4N neuroblastoma (distant metastases limited to lymph nodes) stand out as virtually the only survivors of high-risk neuroblastoma (HR-NB) before myeloablative therapy (MAT) and immunotherapy with anti-GD2 monoclonal antibodies (mAbs) became standard. Because no report presents more recent results with 4N, we analyzed our large 4N experience. All 51 pediatric 4N patients (<18 years old) diagnosed 1985 to 2021 were reviewed. HR-NB included MYCN-nonamplified 4N diagnosed at age ≥18 months and MYCN-amplified 4N. Among 34 MYCN-nonamplified high-risk patients, 20 are relapse-free 1.5+ to 37.5+ (median 12.5+) years post-diagnosis, including 13 without prior MAT and 5 treated with little (1 cycle; n = 2) or no mAb (n = 3), while 14 patients (7 post-MAT, 8 post-mAbs) relapsed (all soft tissue). Of 15 MYCN-amplified 4N patients, 7 are relapse-free 2.1+ to 26.4+ (median 11.6+) years from the start of chemotherapy (all received mAbs; 3 underwent MAT) and 4 are in second remission 4.2+ to 21.8+ years postrelapse (all soft tissue). Statistical analyses showed no significant association of survival with either MAT or mAbs for MYCN-nonamplified HR-NB; small numbers prevented these analyses for MYCN-amplified patients. The two patients with intermediate-risk 4N (14-months-old) are relapse-free 7+ years postresection of primary tumors; distant disease spontaneously regressed. The natural history of 4N is marked by NB confined to soft tissue without early relapse in bones or bone marrow, where mAbs have proven efficacy. These findings plus curability without MAT, as seen elsewhere and at our center, support consideration of treatment reduction for MYCN-nonamplified 4N.

Stage 4N neuroblastoma before and during the era of anti-GD2 immunotherapy.

10037 Background: Patients with stage 4N neuroblastoma (distant metastases limited to lymph nodes) stand out as virtually the only survivors of high-risk neuroblastoma (HR-NB) before myeloablative therapy (MAT) and immunotherapy with anti-GD2 monoclonal antibodies (mAb) became standard. No report presents results with contemporary treatment programs for 4N. The only large study to date (from the International Neuroblastoma Risk Group) analyzed patients diagnosed 1990-2002, found 4N to have significantly better event-free (EFS) and overall survival (OS) compared to other stage 4 patients, and recommended considering less intensive therapy for 4N. Methods: We conducted a retrospective analysis of patients treated at our center from 1985-2021 to analyze our large experience to fill the gap in the literature on treatment of 4N, namely, results with contemporary multi-modality therapy including MAT and anti-GD2 mAbs. Results: We found and reviewed 48 pediatric 4N patients ( &lt; 18 years old) treated 1985-2021 at our center. Biological features included segmental chromosomal aberrations in 21/21 MYCN-non-amplified patients, mutations of ALK in 4/22 (18%), ATRX in 4/16 (25%), and TERT in 4/12 (33%) patients. Among 33 MYCN-non-amplified high-risk patients ( &gt; 18 months old), 19 are relapse-free 1.6+-to-36.9+ (median 12.2+) years post-diagnosis, including 13 without prior MAT and 3 with no mAb, while 14 patients (7 without prior immunotherapy) relapsed (all in soft tissue initially). Eleven of the 14 died, including 3 who developed late osteomedullary metastases after 3-5 relapses in soft tissue alone. Of 13 MYCN-amplified 4N patients, 6 are relapse-free 4.5+-to-25.8+ (median 11.7+) post-diagnosis (all received mAbs; 3 underwent MAT) and 3 are in 2nd remission 4.4+-to-21.1+ years post-relapse (all soft tissue). For all high-risk 4N patients, 5/10-year EFS was 55%/52%, and OS was 76%/64%. MAT was not prognostic. The 2 patients with intermediate-risk 4N (14 months old) are relapse-free 7+ years post-resection of primary tumors; distant disease spontaneously regressed. Conclusions: The natural history of 4N is marked by NB confined to soft tissue without early relapse in bones or bone marrow, sites where mAbs have proven efficacy. These findings plus the possibility of survival without MAT and/or mAb immunotherapy, as seen elsewhere and at our center, support consideration of treatment reduction for selected MYCN-non-amplified 4N patients.

Novel infusion strategy reduces severe adverse events caused by the anti-GD2 monoclonal antibody naxitamab.

Anti-disialoganglioside 2 (anti-GD2) monoclonal antibodies (mAbs) are associated with Grade ≥3 (≥G3) adverse events (AEs) such as severe pain, hypotension, and bronchospasm. We developed a novel method of administering the GD2-binding mAb naxitamab, termed "Step-Up" infusion (STU), to reduce the risk of AEs of severe pain, hypotension, and bronchospasm. Forty-two patients with GD2-positive tumors received naxitamab under "compassionate use" protocols and administered via either the standard infusion regimen (SIR) or the STU regimen. The SIR comprises a 60-min infusion of 3 mg/kg/day on Day 1 of cycle 1 and a 30- to 60-min infusion on Day 3 and Day 5, as tolerated. The STU regimen uses a 2-h infusion on Day 1, initiated at a rate of 0.06 mg/kg/h during 15min (0.015 mg/kg) and which increases gradually to a cumulative dose of 3 mg/kg; on Days 3 and 5, the 3-mg/kg dose is initiated at 0.24 mg/kg/h (0.06 mg/kg) and delivered in 90min according to the same gradual-increase strategy. AEs were graded according to Common Terminology Criteria for Adverse Events version 4.0. The frequency of infusions with an associated G3 AE was reduced from 8.1% (23/284 infusions) with SIR to 2.5% (5/202 infusions) with STU. The odds of an infusion being associated with a G3 AE reduced by 70.3% with STU vs. SIR (odds ratio: 0.297; p = 0.037). Mean serum naxitamab levels pre- and post-STU (11.46 µg/ml pre-infusion; 100.95 µg/ml post-infusion) were within the range reported for SIR. The comparable pharmacokinetics of naxitamab during SIR and STU may indicate that switching to STU reduces G3 AEs without impact on efficacy.