anti-GD2 Antibody Research Articles

Abstract 2411: Di-ganglioside GD2 expression and role in promoting tumorigenicity in prostate cancer

Abstract Background & Significance: Prostate cancer (PCa) is the second leading cause of cancer deaths (~34,000 in 2021 (ACS)) in American men. Castration resistance and resistance to the next-gen androgen receptor (AR) targeted drugs are major challenges. Castration resistance involves multiple mechanisms, including androgen-independent signaling by androgen receptor (AR)- or its variants, and lineage plasticity (LP) with AR-indifferent neuroendocrine (NE) differentiation. Identifying new vulnerabilities across these multitude mechanisms could provide new therapeutic avenues against castration-resistant PCa (CRPC). The cell-surface di-ganglioside GD2 is overexpressed in neural crest cell tumors such as neuroblastoma & melanoma and chimeric (Dinutuximab) or humanized (Naxitamab) anti-GD2 antibodies are now FDA-approved for high-risk neuroblastoma therapy. GD2 expression is reported in other cancers such as breast cancer and glioma and is linked to cancer stem cell behavior. While limited prior studies have detected GD2 expression in PCa cell lines or tumor tissues, nothing is known about the functional role of GD2 in PCa. Objectives: We hypothesized that GD2 overexpression in PCa could play a pro-tumorigenic role and that linkage of GD2 overexpression with CRPC progression may reveal the potential of targeting GD2 for CRPC therapy. Study Design & Results: Immunohistochemical analysis of PCa patient and patient-derived xenograft tissue microarrays (TMAs) revealed GD2 expression in a subset of tumor cells. Fluorescence-activated cell sorter analysis of PCa cell lines showed strong constitutive GD2 expression on murine CRPC cell line RM-1 (derived from mutant Ras and c-Myc overexpressing prostatic epithelial cells) and human PCa line 22Rv1 (overexpresses wild-type AR and ARv7 splice variant). GD2 expression was induced de novo upon induction of lineage plasticity in GD2-negative LNCaP C4-2 prostate adenocarcinoma cell line by shRNA knockdown (KD) of RB1 or TP53. High GD2 expression was also induced when C4-2B cells were made enzalutamide resistant (C4-2BER). Induction of GD2 expression correlated with increased expression of rate-limiting GD2 biosynthetic pathway enzyme GD3 synthase (GD3S). CRISPR-Cas9 mediated stable GD3S knockout (KO) in the RM1 cell line led to the loss of GD2 expression. The GD3S-KO RM1 cells exhibited reduced proliferation, migration, invasion, and tumor sphere forming ability compared to the control cells. Intratibial injections in castrated male C57BL/6 mice showed a significant reduction in tumor development by GD3S KO RM1 cells compared to control cells. Conclusions: Our studies demonstrate that GD2 is expressed in a subset of prostate cancers. Cell line-based studies show that GD2 expression promotes pro-tumorigenic traits. Future studies will assess the biological roles of GD2 in PCa and the potential of targeting GD2+ CRPC with antibody-based approved therapeutic agents. Citation Format: Aaqib M. Bhat, Bhopal C. Mohapatra, Insha Mushtaq, Sukanya Chakraborty, Samikshan Dutta, Sameer Mirza, Matthew D. Storck, Subodh M. Lele, Ming-Fong Lin, Bruce J. Trock, Karen S. Sfanos, Colm Morrissey, Eva Corey, Jonathan Melamed, Leah Cook, Kaustubh Datta, Jane Meza, Jawed Siddiqui, Surinder K. Batra, Vimla Band, Hamid Band. Di-ganglioside GD2 expression and role in promoting tumorigenicity in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2411.

Abstract 4216: Tipifarnib inhibits the secretion of tumor-derived small extracellular vesicles and enhances the immunotherapeutic efficacy of dinutuximab in neuroblastoma

Abstract Anti-GD2 monoclonal antibody (Dinutuximab) immunotherapy has significantly improved the overall survival rate for high-risk neuroblastoma patients. However, 40% of patients fail to respond or develop resistance to the treatment, and the molecular mechanisms by which this occurs remain poorly understood. Here we utilize the syngeneic 9464D-GD2 mouse model to investigate the role of neuroblastoma tumor-derived small extracellular vesicles (sEVs) in developing resistance to the anti-GD2 monoclonal antibody Dinutuximab. Neuroblastoma-derived sEVs significantly attenuated the efficacy of Dinutuximab treatment in vivo. Mechanistically, both RNA-sequencing and flow cytometry analysis of whole tumors demonstrated that neuroblastoma-derived sEVs modulate immune cells tumor infiltration upon Dinutuximab treatment to create an immunosuppressive tumor microenvironment, which contains more tumor-associated macrophages (TAMs) and fewer tumor-infiltrating NK cells, in addition to suppression of splenic NK cell maturation in vivo. Moreover, neuroblastoma-derived sEVs suppressed Dinutuximab-mediated NK cell antibody-dependent cellular cytotoxicity in vitro. Importantly, Tipifarnib, a farnesyltransferase inhibitor that inhibits sEV secretion, drastically enhanced the efficacy of Dinutuximab in vivo and reversed the immunosuppressive effects of neuroblastoma-derived sEVs. Notably, Tipifarnib modulated immature myeloid cells in the bone marrow to disfavor the formation of CD11b+Ly6ChighLy6Glow subpopulations, which are the precursors for TAMs. Taken together, these preclinical findings uncover a novel mechanism by which neuroblastoma-derived sEVs modulate the immune system to promote resistance to Dinutuximab and suggest that Tipifarnib-mediated inhibition of sEV secretion may serve as a viable treatment strategy to improve immunotherapy in high-risk patients. Citation Format: Xiaoming Liu, Carson A. Wills, Longgui Chen, Jiawen Zhang, Yuanjun Zhao, Mi Zhou, Jeffrey M. Sundstrom, Todd D. Schell, Vladimir S. Spiegelman, Megan M. Young, Hong-Gang Wang. Tipifarnib inhibits the secretion of tumor-derived small extracellular vesicles and enhances the immunotherapeutic efficacy of dinutuximab in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4216.

Abstract 5214: Copper chelation overcomes the immunosuppressive tumor microenvironment in neuroblastoma

Abstract Immunotherapy remains a promising approach in the treatment of high-risk neuroblastoma, however efficacy is hampered by the immunosuppressive tumor microenvironment. We previously reported a link between intratumoral copper levels and Programmed Death-Ligand 1 (PD-L1) expression and therefore sought to elucidate the wider impact of copper chelation on immune evasion strategies (Voli et al 2020). Here we profile key elements of the neuroblastoma tumor microenvironment which work in concert to promote immunosuppression and tumor progression. Using the murine TH-MYCN model, animals were treated daily with the copper chelation agent tetraethylenepentamine (TEPA) for seven days before tumor resection and processing. Transcriptome analyses using single-cell RNA-sequencing revealed copper chelation positively skewed the functional status of tumor, immune and stromal compartments to enhance the anti-tumor immune response. This was supported by a tissue microarray screen using NanoString Digital Spatial Profiling which identified increased immune cell infiltration and regions of active tumor killing. High-resolution mass-spectrometry detected upregulation of major histocompatibility complex (MHC) proteins and exclusive antigenic signatures in peptide repertoires. A multiplex cytokine array indicated an improved anti-tumorigenic response underscored by T cell and Natural Killer cell activation. Importantly, combination of copper chelation and anti-GD2 antibody therapy led to tumor regression and significantly improved survival in vivo. Collectively, this study demonstrates the ability of copper chelation to successfully mitigate tumoral immune evasion strategies while providing remarkable insight into neuroblastoma biology. Findings provide crucial evidence in support of combining clinically approved copper chelators with immunotherapy to improve outcomes for neuroblastoma patients. Citation Format: Jourdin R. Rouaen, Daniele Mercatelli, Federica Saletta, Ensieh M. Poursani, Jayne E. Murray, Nicodemus Tedla, Federico M. Giorgi, Orazio Vittorio. Copper chelation overcomes the immunosuppressive tumor microenvironment in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5214.

Abstract 1377: Endogenous antibodies recognize multiple proteins on B78 melanoma in mice cured via immunotherapy

Abstract Introduction: Antibodies can play an important role in innate and adaptive immune responses against cancer. By using a high-density whole mouse proteome peptide array, we were able to assess potential protein-targets for antibodies present in mice cured of their melanoma by a combination immunotherapy regimen of 12Gy local radiation and intratumoral administration of a tumor-specific immunocytokine (anti-GD2 antibody linked to IL-2). This regimen can induce an “in situ vaccine” effect (ISV) enabling mice to be cured of their tumors with long-term immune memory. Methods: Immunocompetent C57/BL6 mice bearing large B78 (GD2-expressing) melanoma tumors were treated with ISV. Sera were collected at different timepoints (before tumor implantation, after implantation before treatment, after completion of treatment, after the mouse was rendered tumor-free and after a rejected rechallenge). Tumor cell surface binding of these serum samples was assessed via flow cytometry and showed a strong increase in binding of sample obtained following immunotherapy, and after rejection of a tumor rechallenge. Binding was assessed against the original tumor line, B78, as well as B16 (parental to B78 without GD2 expression).We used the naïve and immune sera on a Nimble Therapeutics whole mouse proteome peptide array to determine specific antibody-binding sites. Whole proteome data was analyzed using a newly developed algorithm which scans adjacent peptides to detect differential binding between naïve and immune samples. Results: Multiple proteins were selectively identified by immune sera which were not well detected by sera from naïve mice. Hundreds of proteins were targeted by 3 or more of 6 mice and exhibited strong antibody binding only by immune sera. Some proteins were recognized via the same binding epitopes, others via different epitopes in separate mice. Some of the whole protein peptide array results were validated via JPT multi-well peptide array and some via independent peptide ELISA. Results from Nimble peptide array and ELISA validated well. Conclusions: We were able to detect antibody in immune sera binding selectively to specific linear epitopes and identify proteins of interest for further investigation as potential targets for antibody-based or cellular therapies. Results from our whole proteome peptide array were validated in separate assays via peptide ELISA. We are also exploring if some of the identified tumor-specific endogenous antibodies could be used as biomarkers to predict response to our ISV regimen and potentially other immunotherapy treatments. Citation Format: Anna Hoefges, Sean J. McIlwain, Amy K. Erbe, Trang Q. Le, Kaitlin Tetreault, Nicholas Mathers, Alexander L. Rakhmilevich, Jacquelyn A. Hank, Jigar Patel, Brad Garcia, Zachary S. Morris, Irene M. Ong, Paul M. Sondel. Endogenous antibodies recognize multiple proteins on B78 melanoma in mice cured via immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1377.

Use of anti-GD2 (Dinutuximab) as a target for CAR-T cells immunotherapy in neuroblastoma

Treating solid tumours with chimeric antigen receptors-modified T cells (CAR-T cells) has shown limited efficacy due to the lack of cancer-type specific antigens. Dinutuximab is a monoclonal antibody that recognizes a sphingolipid, disialoganglioside GD2, which has limited expression in normal tissues but is overexpressed in paediatric tumours, mainly neuroblastoma (NB) and diffuse intrinsic pontine glioma (DIPG) and other histone H3 K27M (H3K27M) mutated diffuse midline gliomas. Dinutuximab, an anti-GD2 antibody, is currently standard of care in the treatment of NB. We are contemplating the possibility of using Dinutuximab as target element for antiNB-CAR-T cells. Our group have explored the feasibility of producing CAR-T cells from peripheral blood (PB), post-apheresis CD45RA+ fraction and cord blood (CB) using similar protocols of T cell transduction and expansion ability as a way to favour the persistence of CAR-T cells in the host. Afterward, we performed a deeply characterization of phenotype and functionality of anti-FITC-CAR-T cells derived from different sources, and the cytotoxic effect against anti-GD2-FITC- NB labelled cell line. T cells purified from PB, CB and 45RA fraction after apheresis were cultured in vitro with cytokines IL-7+IL-15+IL- 21 to maintain early memory phenotypes as Naïve T cells (TN). A second generation lentiviral expression vector (LV) was used to generate second generation CARs (FITC-L-BBz CAR). After 48h of activation in culture with antiCD3/CD28 and cytokines, T cells were transduced with viral supernatants. Next, we performed a characterization of phenotype of anti-FITC-CAR-T cells by flow cytometry studying how the culture conditions enhanced a TN phenotype expressing CD45RA+ and CCR7+. Cytotoxic function of CAR-T cells was analysed in cocultures with LAN-1 NB cell line labelled with Dinutuximab conjugated with FITC. Preliminary results showed that culture condition with IL-7/15/21 maintained more primitive phenotypes in 45RA (71% TN CD4+ and 66% TN CD8+) and CB (60% of TN CD4+ and 77% of TN CD8+) derived T cells than PB T cells (only a 20% of TN CD4+ and TN CD8+). PB and CB-derived CAR-T cells showed higher cytolitic function than 45RA fraction, resulting in a 30% of dead cells in both cases in a 24h-culture assay. Both, PB and CB derived CAR-T cells showed the best CAR-T activation capacity with 19,6% of cells from PB and 33,9% of cells from CB expressing early activation markers as CD25 and CD134. In vivo experiments are currently on-going to test the efficacy of CAR-T cells in combination with GD2 treatment. Our strategy may complement the current use of Dinutuximab in the treatment of NB through its combination with a targeted CAR-T cell approach.

Naxitamab-based chemoimmunotherapy for resistant high-risk neuroblastoma: Results of "HITS" phase II study.

10028 Background: Chemoresistant disease is an obstacle for cure of high-risk neuroblastoma (HR-NB). Anti-GD2 monoclonal antibodies (MoAb) dinutuximab and naxitamab in combination with cytokines are FDA-approved to consolidate remission and for chemorefractory osteomedullary HR-NB, but responses in progressive disease (PD) are rare. We investigated the combination of Humanized anti-GD2 MoAb naxitamab (Hu3F8), Irinotecan, Temozolomide and Sargramostim (GMCSF) in a phase II "HITS" protocol against resistant HR-NB (NCT03189706). Noteworthy differences between HITS and COG protocol ANBL 1221 included higher MoAb and temozolomide dosage and overlap of naxitamab with GMCSF. Methods: Patients were treated at Memorial Sloan Kettering (MSK) on protocol and at Hospital Sant Joan de Déu (HJSD) per protocol on compassionate basis. Salient eligibility criteria included evaluable or measurable chemoresistant disease. Prior anti-GD2 MoAb or irinotecan/temozolomide (IT) therapy was permitted. Each cycle, administered 3-5 weeks apart, comprised irinotecan 50 mg/m2/day intravenously (IV) plus temozolomide 150 mg/m2/day IV or orally (days 1-5); naxitamab 2.25 mg/kg/day IV, days 2,4,8 and 10, and GMCSF 250 mg/m2/day subcutaneously, days 6-10. Toxicity was measured by CTCAE v4.0 and responses by International Neuroblastoma Response Criteria. Objective responses (OR) were also noted. The primary endpoint of the phase II trial was complete (CR) and partial response (PR) after 4 cycles with a desirable rate of 40%; type I and II errors of 10% (undesirable=20%). Results: Of 90 heavily prior-treated patients (38 at MSK evaluated on trial, 52 at HJSD), 8 had HR-NB refractory to induction chemotherapy while 82 had up to 6 prior relapses (median=1). 503 cycles (median 5/patient) were administered. Toxicities included myelosuppression and diarrhea expected with IT, pain and hypertension expected with naxitamab, plus febrile neutropenia in 4%. No other >grade 2 unexpected toxicities occurred; treatment was outpatient. Primary endpoint was reached in the phase II trial: INRC response = 30.6%, lower boundary = 20.4%. In the entire cohort, best responses were CR (26%), PR (11%), mixed response (9%), stable disease (27%) and PD (27%). OR were noted in 64%, with soft tissue (48%) and skeletal MIBG uptake (66%). CR in BM was seen in 57%. OR occurred in patients with MYCN-amplified (25%), refractory (100%) and relapsed (61%) HR-NB; and patients who had previously received I/T (64%) or naxitamab (68%). In patients who had previously received dinutuximab/IT, OR rate to HITS was 42% (5/12). Human anti-human antibody did not develop in any patient (n=50). Conclusions: Naxitamab-based chemoimmunotherapy was safe without immunogenicity. It was effective against chemoresistant HR-NB in all disease compartments even in patients with multiple prior relapses, and in patients who previously received anti-GD2 MoAbs and/or IT.

Naxitamab and granulocyte macrophage colony stimulating factor (GM-CSF) in combination with irinotecan and temozolomide in patients with high-risk neuroblastoma with primary refractory disease or in first relapse: An international, single-arm, multicenter phase 2 trial.

TPS10061 Background: Neuroblastoma (NB) is the commonest extracranial solid tumor in children. At diagnosis, > 50% have high-risk (HR) disease with poor prognosis despite intensive therapy; thus, there is a high unmet medical need. Naxitamab is an anti-GD2 monoclonal antibody indicated under accelerated approval in the US with GM-CSF for the treatment of relapsed/refractory HR NB in the bone and/or bone marrow in patients (pts) ≥1 year of age with a partial response, minor response, or stable disease to prior therapy. Irinotecan and temozolomide (IT) are commonly used salvage chemotherapies following failure of frontline therapy. The objective of this study is to investigate the efficacy and safety of naxitamab plus IT in pts with HR NB with primary refractory disease or first relapse. Methods: Trial 203 (NCT04560166) is an international, single-arm, multicenter phase 2 trial for HR NB patients ≥12 mo of age, with either evaluable primary refractory disease or first relapse following induction and consolidation therapy. The primary endpoint of the study is the centrally-assessed overall response rate (ORR) (complete [CR] or partial response) at or before completion of 4 cycles of treatment. Key secondary endpoints include ORR after 2 cycles of treatment, duration of response (DoR), CR rate, time to first subsequent therapy or death, progression-free survival (PFS), and overall survival (OS). 52 pts will be enrolled to provide 82% power to demonstrate that response rate is significantly higher than 20%. Key inclusion criteria are documented HR NB at time of diagnosis; received frontline induction/consolidation therapy; active disease despite previous aggressive chemotherapy; and measurable tumor (CT/MRI) that is either MIBG-avid/PET-positive or have MIBG scan with uptake at ≥1 site. Key exclusion criteria include prior treatment with duration ≤3 wks (chemotherapy), ≤6wks (autologous stem cell transplant or therapeutic 131I-MIBG), ≤4wks (radiation therapy), or progressed while on anti-GD2; residual NB in the bone marrow only; and CNS/leptomeningeal disease in the prior 6 mo. In addition, there should not be < 50% performance status per Lansky or Karnosky scale and life expectancy < 6 mo. Pts will receive irinotecan 50 mg/m2/day IV and temozolomide 100 mg/m2/day orally (both on Days 1-5) in combination with naxitamab 2.25 mg/kg/day IV (Days 2, 4, 8 and 10) and GM-CSF 250 ug/m2/day sc (Days 6-10) in 21 days cycle. Response rates will be assessed using the 2-sided binomial test at the 5% significance level; DoR, PFS, and OS will be estimated using Kaplan-Meier analysis. Adverse events will be graded per CTCAE. Clinical trial information: NCT04560166.

Novel infusion strategy to reduce major side effects caused by anti-GD2 monoclonal antibody naxitamab without affecting its pharmacokinetics.

e14502 Background: Intravenous administration of anti-ganglioside GD2 monoclonal antibodies (mAbs) is commonly associated with adverse events (AEs) such as pain and hypertension. Naxitamab is an anti-GD2 mAb intended for outpatient use with a short (30-60 minutes) administration time with 60%-70% of patients in clinical studies experiencing grade 3-4 pain or hypotension. To reduce this risk we assessed a novel administration protocol (StU) that modulates the pharmacodynamics (PD) of naxitamab and mitigates infusion-related reactions. Methods: High-Risk Neuroblastoma (HR-NB) patients in complete remission (CR) received naxitamab as consolidation. Naxitamab cycles comprised priming doses of sc GM-CSF for 5 days at 250 μg/m2/day followed by naxitamab + sc GM-CSF for 5 days at 500 μg/m2/day (days 1-5). Standard naxitamab protocol infusion is provided over 60 minutes at 3 mg/kg/day on day 1 and over 30 minutes on days 3 and 5. Infusions in the StU protocol day 1 were initiated at 1 ml/h with doubling of infusion rate every 15 minutes over 75 minutes (16% of the total dose) and completing the infusion to a final cumulative dose of 3 mg/kg over the remaining 45 minutes; total infusion time of 2h (Table). A faster program (30% of the dose administered in 60 minutes and completed with further 30 minutes) was used for days 3 and 5 infusions. Treatment cycles were repeated every 4 weeks for a goal of 5 cycles. Pharmacokinetics was studied by quantifying serum naxitamab concentrations by ELISA. Infusion related adverse events (AEs) were graded according to the CTCAE v 4.0. Results: 42 HR-NB patients were treated during 2021, 19 with the standard, 15 using the StU, and 8 with both, for a total of 159 cycles (77 StU including 20 cycle one), 477 (231 StU) infusions. All pts presented non-serious CTCAE G1-2 AEs and 7 (37%) CTCAE G3/4 AEs (hypertension x1, hypotension x4, pain x3, airway constriction x5) on the standard protocol. Among the 15 pts on the StU protocol, 1 (6.7%) had CTCAE G3 hypertension. Of the 8 pts who received both types of cycles, 5 (62.5%) had CTCAE G3/4 toxicities (laryngospasm x3; pain x2), 3 with the standard regimen and 2 with the StU protocol. When protocols were compared, the standard regimen cycles (22 cycle one) generated 14.6% (12 of 82) G3/4 AEs whereas the StU 3.9% (3 of 77). Using mixed effects logistic regression analysis, an odds ratio of 0.23 with 95% CI=(0.05, 0.96) and p=0.045 is obtained with the StU cycles reducing the chances to develop G3/4 AEs in 77% compared to the standard regimen. The median serum naxitamab levels pre-StU infusion is 11.46 ug/mL and post infusion 100.95 ug/mL, within the same range as the standard protocol. Conclusions: A pharmacodynamics guided protocol of infusion significantly decreased the severe AEs permitting more tolerable infusions of naxitamab.[Table: see text]

Phase I study of 131I-MIBG with dinutuximab for patients with relapsed or refractory neuroblastoma: A report from the new approaches to neuroblastoma therapy (NANT) consortium.

10038 Background: 131I-metaiodobenzylguanidine (MIBG) is one of the most active salvage therapies for patients with relapsed or refractory (R/R) high-risk neuroblastoma (HRNB). Preclinical neuroblastoma studies show cooperative effects when radiation is combined with anti-GD2 monoclonal antibody (mAb). We hypothesized that MIBG would synergize with the anti-GD2 mAb dinutuximab to provide improved anti-tumor responses. The primary aims of Part A of this study were to determine the maximum tolerated dose (MTD) and/or recommended Phase II dose (RP2D) of MIBG administered with dinutuximab in children with R/R HRNB and to define and describe the toxicities. Methods: Patients 1-29 years of age with R/R HRNB who had MIBG uptake in ≥ 1 site were eligible. Prior anti-GD2 mAb therapy was allowed provided it was not administered with MIBG and not permanently discontinued due to toxicity. One prior MIBG therapy was allowed. MIBG was administered on day 1 at one of three dose levels (DLs): 12, 15, and 18 mCi/kg (DL1-DL3, respectively) with an expansion cohort at the RP2D. Doses were escalated using a rolling six design starting at DL1. The primary endpoint was dose-limiting toxicity (DLT) during course 1. Dinutuximab (17.5 mg/m2/dose) was administered intravenously on days 8-11 and 29-32 and GM-CSF (250 mcg/m2/dose) subcutaneously on days 8-17 and 29-38. Autologous peripheral blood stem cells were infused to all patients on day 15 (+/- 2 days). A maximum of 2 courses per patient were allowed. Response rate was defined as the proportion of patients with a complete or partial response. Results: Thirty-one patients were enrolled. Fourteen were evaluable for dose escalation (4 on DL1, 4 on DL2, and 6 on DL3); 5 evaluable patients were treated in the DL3 expansion. The median age was 7.4 years (range: 3.1 – 22.0) and 20 (65%) were male. Twenty-seven (87%) patients had previously received a median of 8.5 cycles of chemoimmunotherapy (range: 2 – 21). Eight patients previously progressed while receiving anti-GD2 mAb including 7 in DL3. Five (16%) patients had previously received MIBG. No patient at any dose level experienced DLT. Common grade 3/4 treatment-related toxicities were expected hematologic toxicities attributable to MIBG and non-hematologic toxicities attributable to dinutuximab or GM-CSF. Among 26 response-evaluable patients, the centrally-confirmed response rate was 31% across all dose levels: 2/6 (33%) in DL1, 3/5 (60%) in DL2, and 3/15 (20%) in DL3. There were 3 minor responses, 1 in DL2 and 2 in DL3. Conclusions: The RP2D of MIBG in combination with standard doses of dinutuximab and GM-CSF is 18 mCi/kg. This radioimmunotherapy regimen is well-tolerated without additive toxicity. Preliminary efficacy data are encouraging in this heavily pre-treated patient population. A phase 2 trial of this regimen is planned in patients with R/R HRNB. Clinical trial information: NCT03332667.

First-line Anti-GD2 Therapy Combined With Consolidation Chemotherapy in 3 Patients With Newly Diagnosed Metastatic Ewing Sarcoma or Ewing-like Sarcoma.

Despite multimodal therapy, the prognosis of patients with metastatic Ewing sarcoma (ES) remains poor, with new treatments urgently needed. The disialoganglioside GD2, a well-established tumor-associated antigen, is expressed in 40% to 90% of ES cells, making it a suitable therapeutic target. Here we report 3 cases with newly diagnosed, metastatic, GD2-positive ES or Ewing-like sarcoma treated with the anti-GD2 antibody dinutuximab beta in addition to standard chemotherapeutic regimens. Treatment was well-tolerated, and all patients achieved complete remission, without evidence of relapse. First-line anti-GD2 immunotherapy in patients with metastatic, GD2-positive ES or Ewing-like sarcoma represents a promising therapeutic option that warrants further clinical evaluation.

Advancing therapy for neuroblastoma.

Neuroblastomas are tumours of sympathetic origin, with a heterogeneous clinical course ranging from localized or spontaneously regressing to widely metastatic disease. Neuroblastomas recapitulate many of the features of sympathoadrenal development, which have been directly targeted to improve the survival outcomes in patients with high-risk disease. Over the past few decades, improvements in the 5-year survival of patients with metastatic neuroblastomas, from <20% to >50%, have resulted from clinical trials incorporating high-dose chemotherapy with autologous stem cell transplantation, differentiating agents and immunotherapy with anti-GD2 monoclonal antibodies. The next generation of trials are designed to improve the initial response rates in patients with high-risk neuroblastomas via the addition of immunotherapies, targeted therapies (such as ALK inhibitors) and radiopharmaceuticals to standard induction regimens. Other trials are focused on testing precision medicine strategies for patients with relapsed and/or refractory disease, enhancing the antitumour immune response and improving the effectiveness of maintenance regimens, in order to prolong disease remission. In this Review, we describe advances in delineating the pathogenesis of neuroblastoma and in identifying the drivers of high-risk disease. We then discuss how this knowledge has informed improvements in risk stratification, risk-adapted therapy and the development of novel therapies.

TH-MYCN tumors, but not tumor-derived cell lines, are adrenergic lineage, GD2+, and responsive to anti-GD2 antibody therapy

ABSTRACT Neuroblastoma is a commonly lethal solid tumor of childhood and intensive chemoradiotherapy treatment cures ~50% of children with high-risk disease. The addition of immunotherapy using dinutuximab, a monoclonal antibody directed against the GD2 disialoganglioside expressed on neuroblasts, improves survival when incorporated into front-line therapy and shows robust activity in regressing relapsed disease when combined with chemotherapy. Still, many children succumb to neuroblastoma progression despite receiving dinutuximab-based immunotherapy, and efforts to counteract the immune suppressive signals responsible are warranted. Animal models of human cancers provide useful platforms to study immunotherapies. TH-MYCN transgenic mice are immunocompetent and develop neuroblastomas at autochthonous sites due to enforced MYCN expression in developing neural crest tissues. However, GD2-directed immunotherapy in this model has been underutilized due to the prevailing notion that TH-MYCN neuroblasts express insufficient GD2 to be targeted. We demonstrate that neuroblasts in TH-MYCN-driven tumors express GD2 at levels comparable to human neuroblastomas but rapidly lose GD2 expression when explanted ex vivo to establish tumor cell lines. This occurs in association with a transition from an adrenergic to mesenchymal differentiation state. Importantly, not only is GD2 expression retained on tumors in situ, treatment with a murine anti-GD2 antibody, 14G2a, markedly extends survival in such mice, including durable complete responses. Tumors in 14G2a-treated mice have fewer macrophage and myeloid-derived suppressor cells in their tumor microenvironment. Our findings support the utility of this model to inform immunotherapy approaches for neuroblastoma and potential opportunities to investigate drivers of adrenergic to mesenchymal fate decisions.

Immunotherapy of Neuroblastoma: Facts and Hopes.

While the adoption of multimodal therapy including surgery, radiation, and aggressive combination chemotherapy has improved outcomes for many children with high-risk neuroblastoma, we appear to have reached a plateau in what can be achieved with cytotoxic therapies alone. Most children with cancer, including high-risk neuroblastoma, do not benefit from treatment with immune checkpoint inhibitors (ICI) that have revolutionized the treatment of many highly immunogenic adult solid tumors. This likely reflects the low tumor mutation burden as well as the downregulated MHC-I that characterizes most high-risk neuroblastomas. For these reasons, neuroblastoma represents an immunotherapeutic challenge that may be a model for the creation of effective immunotherapy for other "cold" tumors in children and adults that do not respond to ICI. The identification of strong expression of the disialoganglioside GD2 on the surface of nearly all neuroblastoma cells provided a target for immune recognition by anti-GD2 mAbs that recruit Fc receptor-expressing innate immune cells that mediate cytotoxicity or phagocytosis. Adoption of anti-GD2 antibodies into both upfront and relapse treatment protocols has dramatically increased survival rates and altered the landscape for children with high-risk neuroblastoma. This review describes how these approaches have been expanded to additional combinations and forms of immunotherapy that have already demonstrated clear clinical benefit. We also describe the efforts to identify additional immune targets for neuroblastoma. Finally, we summarize newer approaches being pursued that may well help both innate and adaptive immune cells, endogenous or genetically engineered, to more effectively destroy neuroblastoma cells, to better induce complete remission and prevent recurrence.

Clinical Phenotype and Management of Severe Neurotoxicity Observed in Patients with Neuroblastoma Treated with Dinutuximab Beta in Clinical Trials.

Simple SummaryNeurotoxicity is an on-target side effect of GD2-directed immunotherapy due to the expression of GD2 on healthy cells. Patients with high-risk neuroblastoma who receive treatment with anti-GD2 immunotherapy, therefore, require close observation and supportive management to improve treatment tolerance and avoid the persistence of neurological symptoms. This study reports on the incidence, clinical course and management of patients who experienced neurotoxicity due to treatment with the anti-GD2 antibody dinutuximab beta, given with or without interleukin-2, in two clinical trials. The majority of severe neurotoxic events were observed in patients treated with dinutuximab beta combined with interleukin-2, with most patients recovering following supportive management. Given the increased risk of neurotoxic events and the lack of clinical benefit reported for the combination treatment in clinical trials, adding interleukin-2 to dinutuximab beta therapy is not recommended. The clinical experiences described here may aid clinicians in managing neurotoxicity associated with dinutuximab beta more effectively.Neurotoxicity is an off-tumour, on-target side effect of GD2-directed immunotherapy with monoclonal antibodies. Here, we report the frequency, management and outcome of patients enrolled in two prospective clinical trials who experienced severe neurotoxicity during immunotherapy with the anti-GD2 antibody dinutuximab beta (DB) administered as short-term infusion (HR-NBL1/SIOPEN study, randomisation R2, EudraCT 2006-001489-17) or as long-term infusion (HR-NBL1/SIOPEN study, randomisation R4, EudraCT 2006-001489-17 and LTI/SIOPEN study, EudraCT 2009-018077-31), either alone or with subcutaneous interleukin-2 (scIL-2). The total number of patients included in this analysis was 1102. Overall, 44/1102 patients (4.0%) experienced Grade 3/4 neurotoxicities (HR-NBL1 R2, 21/406; HR-NBL1 R4, 8/408; LTI study, 15/288), including 27 patients with severe neurotoxicities (2.5%). Events occurred predominantly in patients receiving combined treatment with DB and scIL-2. Neurotoxicity was treated using dexamethasone, prednisolone, intravenous immunoglobulins and, in two patients, plasmapheresis, which was highly effective. While neurological recovery was observed in 16 of 21 patients with severe neurotoxicities, 5/1102 (0.45%) patients experienced persistent and severe neurological deficits. In conclusion, severe neurotoxicity is most commonly observed in patients receiving DB with scIL-2. Considering the lack of clinical benefit for IL-2 in clinical trials so far, the administration of IL-2 alongside DB is not recommended.

Small extracellular vesicles induce resistance to anti-GD2 immunotherapy unveiling tipifarnib as an adjunct to neuroblastoma immunotherapy

BackgroundAnti-GD2 monoclonal antibody immunotherapy has significantly improved the overall survival rate for high-risk neuroblastoma patients. However, 40% of patients fail to respond or develop resistance to treatment, and the molecular...

Randomized phase 3 study of the anti-disialoganglioside antibody dinutuximab and irinotecan vs irinotecan or topotecan for second-line treatment of small cell lung cancer

IntroductionTopotecan is approved as second-line treatment for small cell lung cancer (SCLC). Irinotecan is also frequently used given its more convenient schedule and superior tolerability. Preclinical studies support disialoganglioside (GD2) as an SCLC target and the combination of dinutuximab, an anti-GD2 antibody, plus irinotecan in this setting. We tested dinutuximab/irinotecan versus irinotecan or topotecan as second-line therapy in relapsed/refractory (RR) SCLC. Materials and methodsPatients with RR SCLC and Eastern Cooperative Oncology Group performance status 0–1 were randomized 2:2:1 to receive dinutuximab 16–17.5 mg/m2 intravenous (IV)/irinotecan 350 mg/m2 IV (day 1), irinotecan 350 mg/m2 IV (day 1), or topotecan 1.5 mg/m2 IV (days 1–5) in 21-day cycles. The primary endpoint was overall survival (OS); secondary endpoints were progression-free survival (PFS), objective response rate (ORR; complete response [CR] + partial response [PR]), and clinical benefit rate (CBR; CR + PR + stable disease). Safety/tolerability were also assessed. ResultsA total of 471 patients were randomized to dinutuximab/irinotecan (n = 187), irinotecan (n = 190), or topotecan (n = 94). Age, sex, performance status, prior therapies, and metastatic disease sites were similar between groups. Survival and response rates were not improved for patients receiving dinutuximab/irinotecan versus those receiving irinotecan or topotecan (median OS 6.9 vs 7.0 vs 7.4 months [p = 0.3132]; median PFS 3.5 vs 3.0 vs 3.4 months [p = 0.3482]; ORR confirmed 17.1% vs 18.9% vs 20.2% [p = 0.8043]; and CBR 67.4% vs 58.9% vs 68.1% [p = 0.0989]), respectively. Grade 3/4 adverse events (≥5% receiving dinutuximab/irinotecan) included neutropenia, anemia, diarrhea, and asthenia. ConclusionsDinutuximab/irinotecan treatment did not result in improved OS in RR SCLC versus irinotecan alone. Irinotecan administered every 21 days demonstrated comparable activity to topotecan administered daily × 5 every 21 days. ClinicalTrials.gov Identifier. NCT03098030.

Dinutuximab beta in the treatment of high-risk neuroblastoma: A follow-up of a case series in Bratislava.

Despite therapeutic advances, high-risk neuroblastoma is still associated with a poor long-term prognosis. Immunotherapy with the anti-GD2 antibody dinutuximab beta has recently been added to the standard of care for patients with high-risk neuroblastoma in our center in Bratislava, and our initial experience with dinutuximab beta has been reported previously. Here we provide a follow-up on the outcomes of 7 patients who were treated with dinutuximab beta under clinical practice conditions at our center.Medical records of 31 patients diagnosed with neuroblastoma between 2017 and 2020 at the Children's Hematology and Oncology Clinic in Bratislava were retrospectively reviewed and 7 patients with high-risk neuroblastoma who were treated with dinutuximab beta were identified. All 7 patients received dinutuximab beta as continuous infusion over 10 days at a dose of 10 mg/m2/day for 5 cycles, following induction and consolidation therapy. Supportive therapy was administered to manage adverse events. Clinical outcomes and treatment tolerance were evaluated.Six of 7 patients treated with dinutuximab beta achieved complete remission, with a median duration of response of 21.5 months as of January 2022, and 1 displayed stable disease 21 months after treatment completion. Treatment was tolerable in most patients, with the majority of adverse events managed with supportive care.Dinutuximab beta is an effective immunotherapy for patients with high-risk neuroblastoma in routine clinical practice when coupled with optimal supportive management of adverse events.

Targeting Tumor Glycans for Cancer Therapy: Successes, Limitations, and Perspectives.

Simple SummaryAberrant glycosylation is a common feature of many cancers, and it plays crucial roles in tumor development and biology. Cancer progression can be regulated by several physiopathological processes controlled by glycosylation, such as cell–cell adhesion, cell–matrix interaction, epithelial-to-mesenchymal transition, tumor proliferation, invasion, and metastasis. Different mechanisms of aberrant glycosylation lead to the formation of tumor-associated carbohydrate antigens (TACAs), which are suitable for selective cancer targeting, as well as novel antitumor immunotherapy approaches. This review summarizes the strategies developed in cancer immunotherapy targeting TACAs, analyzing molecular and cellular mechanisms and state-of-the-art methods in clinical oncology.Aberrant glycosylation is a hallmark of cancer and can lead to changes that influence tumor behavior. Glycans can serve as a source of novel clinical biomarker developments, providing a set of specific targets for therapeutic intervention. Different mechanisms of aberrant glycosylation lead to the formation of tumor-associated carbohydrate antigens (TACAs) suitable for selective cancer-targeting therapy. The best characterized TACAs are truncated O-glycans (Tn, TF, and sialyl-Tn antigens), gangliosides (GD2, GD3, GM2, GM3, fucosyl-GM1), globo-serie glycans (Globo-H, SSEA-3, SSEA-4), Lewis antigens, and polysialic acid. In this review, we analyze strategies for cancer immunotherapy targeting TACAs, including different antibody developments, the production of vaccines, and the generation of CAR-T cells. Some approaches have been approved for clinical use, such as anti-GD2 antibodies. Moreover, in terms of the antitumor mechanisms against different TACAs, we show results of selected clinical trials, considering the horizons that have opened up as a result of recent developments in technologies used for cancer control.

Global Neuroblastoma Network: An international multidisciplinary neuroblastoma tumor board for resource-limited countries.

Tumor boards are part of standard care of patients with complex cancers, but appropriate multidisciplinary expertise and infrastructure are often not available in low- and middle-income countries (LMIC) for pediatric cancers, such as neuroblastoma. Our goal was to review results of a Global Neuroblastoma Network (GNN) tumor board accessible to LMIC. De-identified clinical cases presented via internet conference during a weekly GNN virtual tumor board from 2010 through 2020 were evaluated in a standardized format, including diagnostic imaging, pathology, therapy information, resource limitations, and questions for discussion. Information summarized included the presentations, a survey of the impact on care, and a resource questionnaire. Registered GNN participants included 575 individuals from 77 countries, with a median of 39 participants per session. Total 412 cases were presented from 32 countries, including 351 unique neuroblastoma patients, 52 follow-up cases, and nine non-neuroblastoma diagnoses. Twenty-eight educational sessions were presented. Limited critical resources for diagnostics and staging of cases included MYCN analysis (54.7%), metaiodobenzylguanidine (MIBG) scans (38.7%), and International Neuroblastoma Pathology Classification (49%). Therapies were also limited, with markedly decreased use of radiation and autologous stem cell transplant for high-risk cases, and no availability of anti-GD2 antibody in LMIC. Limited sampling with a post-presentation survey showed that 100% found the GNN helpful, and 70% altered the care plan based on the discussion. This report shows the utility of an international tumor board for LMIC focused on a challenging solid tumor where local expertise may be limited, with international multidisciplinary expert participation and educational sessions.

Synthesis, characterization, and application of gene conjugated polymerized nitrogen-doped graphene quantum dots carriers for in vivo bio-targeting in neuroblastoma treatment

BackgroundNeuroblastoma is the most prevalent cancer that affects infants. It arises from neural crest elements of the sympathetic nervous system. Accurate medical diagnostic techniques are required to achieve targeted treatment. MethodsNitrogen-doped graphene quantum dots (N-GQDs) were synthesized using a classic hydrothermal method. The N-GQDs were further coated with polyethylene glycol (PEG): branched polyethylenimine (b-PEI) to form PEG:b-PEI@N-GQDs (P@N-GQDs). The P@N-GQDs were coupled with an anti-GD2 antibody to form Ab-GD2@P@N-GQDs, which were then visualized using an in vivo imaging system (IVIS). The structure, optical properties, and in vivo imaging performance of the fluorescent markers were investigated. Significant findingsThe P@N-GQDs had an irregular circular shape with 10.6 ± 1.7 nm diameter. The PL emission of P@N-GQDs was extended from 450 ± 10 to 550 ± 10 nm. The visibility of P@N-GQDs in the IVIS spectrum was also improved, enhancing the signal to 1.83 times that achieved by N-GQDs in vivo. The Ab-GD2-P@N-GQDs accumulated in cancer cells, and the maximum fluorescence intensity in the dissected tumor was 6.72 × 107. The proposed probe successfully targeted neuroblastoma and has great potential as a gene carrier.