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BackgroundDevelopment of successful antibody-based immunotherapeutic and radioimmunotherapeutic strategies rely on the identification of cell surface tumor-associated antigens (TAA) with restricted expression on normal tissues. Desmoplastic small round cell tumor (DSRCT) is a rare and generally neglected malignancy that primarily affects adolescent and young adult males. New therapies capable of treating disseminated disease are needed for DSRCT, which is often widespread at diagnosis.MethodsWe used immunohistochemistry (IHC) on fresh frozen surgical specimens and patient-derived xenograft (PDX) tumors and flow cytometry on DSRCT cell lines to evaluate expression of TAAs in these tumors. In vitro cytotoxicity assays were used to evaluate the efficacy of T cell-engaging bispecific antibodies (T-BsAbs) directed at these targets. In vivo, we used an intraperitoneal xenograft mouse model of DSRCT to test T-BsAbs against several TAAs.ResultsIn DSRCT specimens we found widespread expression of B7-H3, EGFR, GD2, HER2, mesothelin, and polysialic acid, clinical targets for which specific antibody therapeutics are available. The expression of B7-H3, EGFR, HER2, and mesothelin was confirmed on the cell surface of DSRCT cell lines. In vitro cytotoxicity assays confirmed the efficacy of T cell-engaging bispecific antibodies (T-BsAbs) directed at these targets against DSRCT cells. Remarkably, a HER2xCD3 T-BsAb was capable of completely shrinking established tumors in an intraperitoneal mouse model of DSRCT.ConclusionsWe propose that these TAAs should be further investigated in preclinical models as targets for immunotherapy and radioimmunotherapy with the hope of providing a rationale to extend these therapies to patients with advanced DSRCT.

Peritoneal carcinomatosis (PC) is considered incurable, and more effective therapies are needed. Herein we test the hypothesis that GPA33-directed intracompartmental pretargeted radioimmunotherapy (PRIT) can cure colorectal peritoneal carcinomatosis. Nude mice were implanted intraperitoneally with luciferase-transduced GPA33-expressing SW1222 cells for aggressive peritoneal carcinomatosis (e.g., resected tumor mass 0.369 ± 0.246 g; n = 17 on day 29). For GPA33-PRIT, we administered intraperitoneally a high-affinity anti-GPA33/anti-DOTA bispecific antibody (BsAb), followed by clearing agent (intravenous), and lutetium-177 (Lu-177) or yttrium-86 (Y-86) radiolabeled DOTA-radiohapten (intraperitoneal) for beta/gamma-emitter therapy and PET imaging, respectively. The DOTA-radiohaptens were prepared from S-2-(4-aminobenzyl)-1,4,7, 10-tetraazacyclododecane tetraacetic acid chelate (DOTA-Bn). Efficacy and toxicity of single- versus three-cycle therapy were evaluated in mice 26–27 days post-tumor implantation. Single-cycle treatment ([177Lu]LuDOTA-Bn 111 MBq; tumor dose: 4,992 cGy) significantly prolonged median survival (MS) approximately 2-fold to 84.5 days in comparison with controls (P = 0.007). With three-cycle therapy (once weekly, total 333 MBq; tumor dose: 14,975 cGy), 6/8 (75%) survived long-term (MS > 183 days). Furthermore, for these treated long-term survivors, 1 mouse was completely disease free (microscopic “cure”) at necropsy; the others showed stabilized disease, which was detectable during PET-CT using [86Y]DOTA-Bn. Treatment controls had MS ranging from 42–52.5 days (P < 0.001) and 19/20 mice succumbed to progressive intraperitoneal disease by 69 days. Multi-cycle GPA33 DOTA-PRIT significantly prolongs survival with reversible myelosuppression and no chronic marrow (929 cGy to blood) or kidney (982 cGy) radiotoxicity, with therapeutic indices of 12 for blood and 12 for kidneys. MTD was not reached.

PURPOSE The tyrosine kinase receptor anaplastic lymphoma kinase (ALK) can be abnormally activated in neuroblastoma, and somatic ALK mutations occur in 6%-10% of patients. The differential clinical impact of these mutations has not been clearly elucidated. METHODS Data on patients with neuroblastoma harboring ALK mutations were retrospectively analyzed. ALK sequencing was performed by whole-genome sequencing, hybrid-based capture of targeted exomes, or hotspot ALK mutation profiling. The differential impact of ALK mutation site on clinical characteristics, response to treatment, and survival was analyzed. In a subgroup of patients with locoregional neuroblastoma diagnosed after 2014, the impact of all ALK mutations was compared with wild-type ALK. RESULTS Of 641 patients with neuroblastoma with ALK status analyzed on at least one tumor sample, 103 (16%) had tumors harboring ALK mutations. Mutations existed across all ages (birth to 67.8 years), stages (30% locoregional and 70% metastatic), and risk groups (20%, 11%, and 69% with low-, intermediate-, and high-risk disease, respectively). Mutation sites included F1174 (51%), R1275 (29%), R1245 (10%), and others (10%). Mutation site was not prognostic for progression-free survival or overall survival in the entire cohort, high-risk subgroup, or locoregional subgroup. Locoregional tumors with any ALK mutation were generally invasive: L2 by International Neuroblastoma Research Group staging in 30/31 patients with a 2-year progression-free survival (59%, 95% CI, 37.4 to 80.5) that was inferior to historical controls. This observation was corroborated in the post-2014 subgroup in which gross total resection was less likely for ALK-mutated tumors. CONCLUSION Somatic ALK mutations are present across all stages and risk groups of neuroblastoma. No specific mutation carries differential prognostic significance. Locoregional neuroblastoma has an invasive phenotype when harboring somatic ALK mutations in this population.

IntroductionWhile subcutaneous metastases are often observed with stage MS neuroblastoma, an entity that usually resolves spontaneously, skeletal muscle metastases (SMM) have been rarely described. The purpose of this retrospective study was to investigate the significance of SMM in neuroblastoma.Patients and methodsSeventeen patients with neuroblastoma SMM were diagnosed at a median age of 4.3 (0.1-15.6) months. All had SMM at diagnosis and metastases at other sites. Fifteen (88%) had ≥ 2 SMM in disparate muscle groups. One, 14, and 2 patients had low, intermediate, and high-risk disease respectively. Fifteen tumors had favorable histology without MYCN amplification, and 2 were MYCN-amplified. Most SMM (80%; n=12/15 evaluated) were MIBG-avid.ResultsOnly 1 patient (with MYCN-non-amplified neuroblastoma) had disease progression. All survive at median follow-up of 47.9 (16.9-318.9) months post-diagnosis. Biological markers (histology, chromosomal and genetic aberrations) were not prognostic. Whole genome sequencing of 3 matched primary and SMM lesions suggested that both primary and metastatic tumors arose from the same progenitor. SMM completely resolved in 10 patients by 12 months post-diagnosis. Of 4 patients managed with watchful observation alone without any cytotoxic therapy, 3 maintain complete remission with SMM resolving by 5, 13, and 21 months post-diagnosis respectively.ConclusionsChildren with neuroblastoma SMM have an excellent prognosis, with a clinical course suggestive of stage MS disease. Based on these results, the initial management of infants with non-MYCN-amplified NB with SMM could be watchful observation, which could eliminate or reduce exposure to genotoxic therapy.

Background The prognosis for metastatic and recurrent tumors of the central nervous system (CNS) remains dismal, and the need for newer therapeutic targets and modalities is critical. The cell surface glycoprotein B7H3 is expressed on a range of solid tumors with a restricted expression on normal tissues. We hypothesized that compartmental radioimmunotherapy (cRIT) with the anti-B7H3 murine monoclonal antibody omburtamab injected intraventricularly could safely target CNS malignancies. Patients and methods We conducted a phase I trial of intraventricular 131I-omburtamab using a standard 3 + 3 design. Eligibility criteria included adequate cerebrospinal fluid (CSF) flow, no major organ toxicity, and for patients > dose level 6, availability of autologous stem cells. Patients initially received 74 MBq radioiodinated omburtamab to evaluate dosimetry and biodistribution followed by therapeutic 131I-omburtamab dose-escalated from 370 to 2960 MBq. Patients were monitored clinically and biochemically for toxicity graded using CTCAEv 3.0. Dosimetry was evaluated using serial CSF and blood sampling, and serial PET or gamma-camera scans. Patients could receive a second cycle in the absence of grade 3/4 non-hematologic toxicity or progressive disease. Results Thirty-eight patients received 100 radioiodinated omburtamab injections. Diagnoses included metastatic neuroblastoma (n = 16) and other B7H3-expressing solid tumors (n = 22). Thirty-five patients received at least 1 cycle of treatment with both dosimetry and therapy doses. Acute toxicities included Conclusions cRIT with 131I-omburtamab is safe, has favorable dosimetry and may have a therapeutic benefit as adjuvant therapy for B7-H3-expressing leptomeningeal metastases. Trial registration: clinicaltrials.gov NCT00089245, August 5, 2004.

In high-risk neuroblastoma, multimodality therapy including craniospinal irradiation (CSI) is effective for central nervous system (CNS) relapse. Management of post-CSI CNS relapse is not clearly defined.Pediatric patients with neuroblastoma treated with CSI between 2000 and 2019 were identified. Treatment of initial CNS disease (e.g., CSI, intraventricular compartmental radioimmunotherapy [cRIT] withOf 128 patients (65% male, median age 4 years), 19 (15%) received CSI with protons and 115 (90%) had a boost. Most (103, 81%) received cRIT, associated with improved OS (hazard ratio [HR] 0.3, 95% confidence interval [CI]: 0.1-0.5, p .001). Forty (31%) developed a second CNS relapse, associated with worse OS (1-year OS 32.5%, 95% CI: 19-47; HR 3.8; 95% CI: 2.4-6.0, p .001), and more likely if the leptomeninges were initially involved (HR 2.5, 95% CI: 1.3-4.9, p = .006). Median time to second CNS relapse was 6.8 months and 51% occurred outside the CSI boost field. Twenty-five (63%) patients underwent reirradiation, most peri-operatively (18, 45%) with focal hypofractionation. Eight (20%) patients with second CNS relapse received cRIT, associated with improved OS (HR 0.1; 95% CI: 0.1-0.4, p .001).CNS relapse after CSI for neuroblastoma portends a poor prognosis. Surgery with hypofractionated radiotherapy was the most common treatment. Acknowledging the potential for selection bias, receipt of cRIT both at first and second CNS relapse was associated with improved survival. This finding necessitates further investigation.

A cardinal feature that distinguishes clinically high-risk neuroblastoma from low-risk tumors is telomere maintenance. Specifically, neuroblastoma tumors with either active telomerase or alternative lengthening of telomeres exhibit aggressive growth characteristics that lead to poor outcomes, whereas tumors without telomere maintenance can be managed with observation or minimal treatment. Even though the need for cancer cells to maintain telomere DNA—in order to sustain cell proliferation—is well established, recent studies suggest that the neural crest origin of neuroblastoma may enforce unique relationships between telomeres and tumor malignancy. Specifically in neuroblastoma, telomere structure and telomerase activity are correlated with the adrenergic/mesenchymal differentiation states, and manipulating telomerase activity can trigger tumor cell differentiation. Both findings may reflect features of normal neural crest development. This review summarizes recent advances in the characterization of telomere structure and telomere maintenance mechanisms in neuroblastoma and discusses the findings in the context of relevant literature on telomeres during embryonic and neural development. Understanding the canonical and non-canonical roles of telomere maintenance in neuroblastoma could reveal vulnerabilities for telomere-directed therapies with potential applications to other pediatric malignancies.

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

Neuroblastoma (NB) is a malignant embryonal tumor of the sympathetic nervous system that is most commonly diagnosed in the abdomen, often presenting with signs and symptoms of metastatic spread. Three decades ago, high-risk NB metastatic to bone and bone marrow in children was not curable. Today, with multimodality treatment, 50% of these patients will survive, but most suffer from debilitating treatment-related complications. Novel targeted therapies to improve cure rates while minimizing toxicities are urgently needed. Recent molecular discoveries in oncology have spawned the development of an impressive array of targeted therapies for adult cancers, yet the paucity of recurrent somatic mutations or activated oncogenes in pediatric cancers poses a major challenge to the evolving paradigm of personalized medicine. Although low tumor mutational burden is a major hurdle for immune checkpoint inhibitors, an immature or impaired immune system and inhibitory tumor microenvironment can further complicate the prospects for successful immunotherapy. In this regard, despite the poor immunogenic properties of NB, the success of antibody-based immunotherapy and radioimmunotherapy directed at single targets (eg, GD2 and B7-H3) is both encouraging and surprising, given that most solid tumor antibodies that use Fc-dependent mechanisms or radioimmunotargeting have largely failed. Here, we summarize the current information on the immunologic properties of this tumor, its potential immunotherapeutic targets, and novel antibody-based strategies on the horizon.

BackgroundSuccess of T cell immunotherapy hinges on the tumor microenvironment (TME), and abnormal tumor vasculature is a hallmark of most solid tumors and associated with immune evasion. The efficacy of T cell engaging bispecific antibody (BsAb) treatment relies on the successful trafficking and cytolytic activity of T cells in solid tumors. Normalization of tumor vasculature using vascular endothelial growth factor (VEGF) blockades could improve efficacy of BsAb-based T cell immunotherapy.MethodsAnti-human VEGF (bevacizumab, BVZ) or anti-mouse VEGFR2 antibody (DC101) was used as VEGF blockade, and ex vivo armed T cells (EATs) carrying anti-GD2, anti-HER2, or anti-glypican3 (GPC3) IgG-(L)-scFv platformed BsAb were used. BsAb-driven intratumoral T cell infiltration and in vivo antitumor response were evaluated using cancer cell line-derived xenografts (CDXs) or patient-derived xenografts (PDXs) carried out in BALB-Rag2-/-IL-2R-γc-KO (BRG) mice. VEGF expression on human cancer cell lines was analyzed by flow cytometry, and VEGF levels in mouse serum were measured using VEGF Quantikine ELISA Kit. Tumor infiltrating lymphocytes (TILs) were evaluated using flow cytometry and by bioluminescence; both TILs and tumor vasculature were studied using immunohistochemistry.ResultsVEGF expression on cancer cell lines increased with seeding density in vitro. BVZ significantly reduced serum VEGF levels in mice. BVZ or DC101 increased high endothelial venules (HEVs) in the TME and substantially enhanced (2.1–8.1 fold) BsAb-driven T cell infiltration into neuroblastoma and osteosarcoma xenografts, which was preferential for CD8(+) TILs versus CD4(+) TILs, leading to superior antitumor effects in multiple CDX and PDX tumor models without added toxicities.ConclusionsVEGF blockade using specific antibodies against VEGF or VEGFR2 increased HEVs in the TME and cytotoxic CD8(+) TILs, significantly improving the therapeutic efficacy of EAT strategies in preclinical models, supporting the clinical investigation of VEGF blockades to further enhance BsAb-based T cell immunotherapies.

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

BACKGROUND: Median survival from DIPG is less than one year. In a phase 1 dose escalation study (clinicaltrials.gov NCT01502917) 124I-omburtamab targeting B7-H3 was administered intratumorally using CED. METHODS: CED was performed between 4-14 weeks post radiation therapy. Using a 3 + 3 design, 124I-omburtamab was escalated from 0.25-10.0 mCi and infusion volumes (Vi) from 250-10,000 µl with serial 124I PET/CT performed up to ~1 week post-administration. Toxicities were assessed for 30 days. Dose escalation safety was evaluated. Survival was calculated using Kaplan-Meier statistics. RESULTS: 46 children were treated and evaluable for toxicity and survival;4 patients received partial doses and were evaluable for toxicity only. Three patients experienced dose limiting toxicities. Eleven patients had transient treatment related grade 3 toxicities with no grade 4 or 5 toxicities. Grade 3 nervous system toxicities included: muscular weakness(n=8), dysarthria(n=4), ataxia(n=3), dysphagia(n=3), and gait disturbance(n=1). Lesion absorbed doses ranged from 1,000-1,500cGy/mCi, with lesion-to-whole body radiation absorbed-dose ratios of ~900. A dose of 8mCi and infusion volume of 8,000 µl is safe and may provide a distribution volume up to 20cm3. Median survival was 15.3 months (n =46, 95% CI 12.7, 17.3). Survival rate estimates (95% CI) at 1, 2, 3 and 5 years were 0.67 (0.55;0.82); 0.18 (0.09;0.35); 0.10 (0.04;0.26); and 0.05 (0.01;0.20). Four patients survived >3 years; two remain alive (61+ and 106+ months);two have died (44 and 53 month) with distant CNS disease and one with extra-CNS metastasis. CONCLUSION: Administration of escalating doses and volumes of 124I-omburtamab via CED was a viable option for this patient subgroup. The median overall survival was increased 3-4 months compared to historical controls. Anecdotal long-term survival if validated with a planned phase 2 trial would support the concept of whole neuroaxis treatment in combination with CED in a subset of DIPG patients.

Twelve patients without therapy-related leukemia were studied after completing TOP2 poison chemotherapy in a high-risk neuroblastoma regimen. One patient harbored an inv(11) that was a KMT2A rearrangement. The KMT2A-MAML2 transcript was expressed at low level. The patient was prospectively followed. The inv(11) was undetectable in ensuing samples. Leukemia never developed after a 12.8-year follow-up period. Enriched etoposide-induced TOP2A cleavage in the relevant MAML2 genomic region supports a TOP2A DNA damage mechanism. After completing TOP2 poison chemotherapies, covert KMT2A-R clones may occur in a small minority of patients; however, not all KMT2A rearrangements herald a therapy-related leukemia diagnosis.

BackgroundTumor microenvironment (TME) is a dynamic cellular milieu to promote tumor angiogenesis, growth, proliferation, and metastasis, while derailing the host anti-tumor response. TME impedes bispecific antibody (BsAb) or chimeric antigen receptor (CAR)-driven T cells infiltration, survival, and cytotoxic efficacy. Modulating tumor infiltrating myeloid cells (TIMs) could potentially improve the efficacy of BsAb.MethodsWe evaluated the effects of TIM modulation on BsAb-driven T cell infiltration into tumors, their persistence, and in vivo anti-tumor response. Anti-GD2 BsAb and anti-HER2 BsAb built on IgG-[L]-scFv platform were tested against human cancer xenografts in BALB-Rag2−/−IL-2R-γc-KO (BRG) mice. Depleting antibodies specific for polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC), monocytic MDSC (M-MDSC), and tumor associated macrophage (TAM) were used to study the role of each TIM component. Dexamethasone, an established anti-inflammatory agent, was tested for its effect on TIMs.ResultsBsAb-driven T cells recruited myeloid cells into human tumor xenografts. Each TIM targeting therapy depleted cells of interest in blood and in tumors. Depletion of PMN-MDSCs, M-MDSCs, and particularly TAMs was associated with enhanced T cell infiltration into tumors, significantly improving tumor control and survival in multiple cancer xenograft models. Dexamethasone premedication depleted monocytes in circulation and TAMs in tumors, enhanced BsAb-driven T cell infiltration, and anti-tumor response with survival benefit.ConclusionReducing TIMs markedly enhanced anti-tumor effects of BsAb-based T cell immunotherapy by improving intratumoral T cell infiltration and persistence. TAM depletion was more effective than PMN- or M-MDSCs depletion at boosting the anti-tumor response of T cell engaging BsAb.

Desmoplastic small round cell tumor (DRSCT) is a highly aggressive primitive sarcoma that primarily affects adolescent and young adult males. The 5-year survival rate is 15-30% and few curative treatment options exist. Although there is no standard treatment for DSRCT, patients are most often treated with a combination of aggressive chemotherapy, radiation, and surgery. Targeted therapy inhibitors of PDGFA and IGF-1R, which are almost uniformly overexpressed in DSRCT, have largely failed in clinical trials. As in cancer in general, interest in immunotherapy to treat DSRCT has increased in recent years. To that end, several types of immunotherapy are now being tested clinically, including monoclonal antibodies, radionuclide-conjugated antibodies, chimeric antigen receptor T cells, checkpoint inhibitors, and bispecific antibodies (BsAbs). These types of therapies may be particularly useful in DSRCT, which is frequently characterized by widespread intraperitoneal implants, which are difficult to completely remove surgically and are the frequent cause of relapse. Successful treatment with immunotherapy or radioimmunotherapy following debulking surgery could eradiate these micrometasteses and prevent relapse. Although there has been limited success to date for immunotherapy in pediatric solid tumors, the significant improvements in survival seen in the treatment of other pediatric solid tumors, such as metastatic neuroblastoma and its CNS spread, suggest a potential of immunotherapy and specifically compartmental immunotherapy in DSRCT.

BackgroundTumorous heterogeneity is a hallmark of tumor evolution and cancer progression, being a longstanding challenge to targeted immunotherapy. Ex vivo armed T cells (EATs) using IgG-(L)-scFv bispecific antibodies (BsAbs) are potent tumor-specific cytotoxic effectors. To improve the anti-tumor efficacy of EATs against heterogeneous solid tumors, we explored multi-antigen targeting approaches.MethodsEx vivo expanded T cells were armed with BsAbs built on the IgG-(L)-scFv platform, where an anti-CD3 (huOKT3) scFv was attached to the carboxyl end of both light chains of a tumor specific IgG. Multispecificity was created by combining monospecific EATs, combining BsAbs on the same T cell, or combining specificities on the same antibody. Three multi-antigens targeting EAT strategies were tested: (1) pooled-EATs (EATs each with unique specificity administered simultaneously) or alternate-EATs (EATs each with unique specificity administered in an alternating schedule), (2) dual-EATs or multi-EATs (T cells simultaneously armed with ≥2 BsAbs), and (3) TriAb-EATs (T cells armed with BsAb specific for two targets besides CD3 (TriAb)). The properties and efficiencies of these three strategies were evaluated by flow cytometry, in vitro cytotoxicity, cytokine release assays, and in vivo studies performed in BALB-Rag2-/-IL-2R-γc-KO (BRG) mice xenografted with cancer cell line (CDX) or patient-derived tumor (PDX).ResultsMulti-EATs retained target antigen specificity and anti-tumor potency. Cytokine release with multi-EATs in the presence of tumor cells was substantially less than when multiple BsAbs were mixed with unarmed T cells. When tested against CDXs or PDXs, dual-EATs or multi-EATs effectively suppressed tumor growth without clinical toxicities. Most importantly, dual-EATs or multi-EATs were highly efficient in preventing clonal escape while mono-EATs or TriAb- EATs were not as effective.ConclusionsMulti-EATs have the potential to increase potency, reduce toxicity, and overcome tumor heterogeneity without excessive cytokine release. Arming T cells with multiple BsAbs deserves further exploration to prevent or to treat cancer resistance.

BackgroundThe prognosis for metastatic Ewing sarcoma family of tumors (EFT) is still poor despite high-dose chemotherapy and radiation treatment. Immunotherapies hold promise, but cancer antigen-targeting immunotherapies have largely failed to induce effective T cell receptor-mediated antitumor response. However, T cell-engaging bispecific antibodies (T-BsAbs) have yet to be adequately explored.MethodsRehumanized STEAP1-IgG was used to build T-BsAb (named BC261) using the 2+2 IgG-[L]-scFv platform carrying the anti-CD3 huOKT3 scFv as the second specificity. Its binding epitope mapping, species cross-reactivity, tumor cell line staining, and in vitro cytotoxicity were investigated thoroughly. Its potency in driving tumor-infiltrating lymphocytes (TILs) was quantified using bioluminescence, correlated with in vivo antitumor response against cell line-derived or patient-derived xenografts (CDXs or PDXs) and compared with anti-STEAP1 T-BsAbs built on representative antibody platforms.ResultsBC261 binding epitope was mapped to its second extracellular domain of STEAP1 shared among canine and primate orthologs. BC261 induced potent cytotoxicity against panels of EFT, prostate cancer, and canine osteosarcoma cell lines despite their low antigen density. BC261 drove significantly more TILs into tumors (30-fold) and exerted superior antitumor effects compared with the other standard BsAb platforms. The antitumor efficacy of BC261 was consistent against EFT and prostate cancer CDXs and PDXs.ConclusionsBC261 was highly efficient in driving T cell infiltration and tumor ablation. Either as stand-alone therapeutics or for ex vivo armed T cells, this novel anti-STEAP1 T-BsAb BC261 has therapeutic potential.

PURPOSE: We explored the use of intraventricular (131)I-8H9 targeting B7-H3 in patients with ETMR. METHODS: Patients were enrolled in an IRB approved, phase 1, 3+3 dose escalation trial. Patients with CNS disease expressing the antibody target antigen B7-H3, were eligible. We report on a cohort of 3 patients with ETMR who were enrolled on the study. Three symptomatic children (ages 14 months, 3 and 3.5 years) had large parietal masses confirmed to be B7-H3-reactive ETMR. Patients received 2 mCi (131)I-Omburtamab as a tracer followed by 1 or 2 therapeutic (131)I-Omburtamab injections. Dosimetry was based on serial CSF, blood samplings and region of interest (ROI) on nuclear scans. Brain and spine MRIs and CSF cytology were done at baseline, 5 weeks after (131)I-Omburtamab, and approximately every 3 months thereafter. Acute toxicities and survival were noted. RESULTS: Patients received surgery, focal radiation, and high dose chemotherapy. Patients 1 and 2 received (131)I-Omburtamab (80 and 53 mCi, respectively). Patient 3 had a local recurrence prior to (131)I-Omburtamab treated with surgery, external beam radiation, chemotherapy, then (131)I-Omburtamab (36 mCi). (131)I-Omburtamab was well-tolerated. Mean dose delivered by (131)I-Omburtamab was 68.4 cGy/mCi to CSF and 1.95 cGy/mCi to blood. Mean ROI doses were 230.4 (ventricular) and 58.2 (spinal) cGy/mCi. Patients 1 and 2 remain in remission 6.8 years and 2.3 years after diagnosis, respectively; patient 3 died of progressive disease 7 month after therapy (2 years after diagnosis). CONCLUSIONS: (131)I-Omburtamab appears safe with favorable dosimetry therapeutic index. When used as consolidation following surgery and chemoradiation therapy, (131)I-Omburtamab may have therapeutic benefit for patients with ETMR.

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

BackgroundT cell-based immunotherapies using chimeric antigen receptors (CAR) or bispecific antibodies (BsAb) have produced impressive responses in hematological malignancies. However, major hurdles remained, including cytokine release syndrome, neurotoxicity, on-target off-tumor effects, reliance on autologous T cells, and failure in most solid tumors. BsAb armed T cells offer a safe alternative.MethodsWe generated ex vivo armed T cells (EATs) using IgG-[L]-scFv-platformed BsAb, where the anti-CD3 (huOKT3) scFv was attached to the light chain of a tumor-binding IgG. BsAb density on EAT, in vitro cytotoxicity, cytokine release, in vivo trafficking into tumors, and their antitumor activities were evaluated in multiple cancer cell lines and patient-derived xenograft mouse models. The efficacy of EATs after cryopreservation was studied, and gamma delta (γδ) T cells were investigated as unrelated alternative effector T cells.ResultsThe antitumor potency of BsAb armed T cells was substantially improved using the IgG-[L]-scFv BsAb platform. When compared with separate BsAb and T cell injection, EATs released less TNF-α, and infiltrated tumors faster, while achieving robust antitumor responses. The in vivo potency of EAT therapy depended on BsAb dose for arming, EAT cell number per injection, total number of EAT doses, and treatment schedule intensity. The antitumor efficacy of EATs was preserved following cryopreservation, and EATs using γδ T cells were safe and as effective as αβ T cell-EATs.ConclusionsEATs exerted potent antitumor activities against a broad spectrum of human cancer targets with remarkable safety. The antitumor potency of EATs depended on BsAb dose, cell number and total dose, and schedule. EATs were equally effective after cryopreservation, and the feasibility of third-party γδ-EATs offered an alternative for autologous T cell sources.

Background Humanized 3F8-bispecific antibody (hu3F8-BsAb) using the IgG(L)-scFv format (where scFv is single-chain variable fragment), where the anti-CD3 huOKT3 scFv is fused with the carboxyl end of the hu3F8 light chain, has potent antitumor cytotoxicity against GD2(+) tumors. To overcome the insufficient number and function of T cells in cancer patients, they can be rejuvenated and expanded ex vivo before arming with hu3F8-BsAb for adoptive transfer, potentially reducing toxic side effects from direct BsAb administration. Procedure T cells from normal volunteers were expanded and activated ex vivo using CD3/CD28 beads for 8 days. Activated T cells (ATCs) were harvested and co-incubated with a Good Manufacturing Practice grade hu3F8-BsAb at room temperature for 20 min. These armed ATCs were tested for cytotoxicity in vitro and in vivo against human GD2(+) cell lines and patient-derived xenografts in BALB-Rag2-/- IL-2R-γc-KO mice. Results Hu3F8-BsAb armed ATCs showed robust antigen-specific tumor cytotoxicity against GD2(+) tumors in vitro. In vivo, T cells armed with hu3F8-BsAb were highly cytotoxic against GD2(+) melanoma and neuroblastoma xenografts in mice, accompanied by T-cell infiltration without significant side effects. Only zeptomole (10-21 ) quantities of BsAb per T cell was required for maximal antitumor effects. Tumor response was a function of T-cell dose. Conclusion BsAb armed T cells may have clinical utility as the next generation of cytotherapy combined with recombinant BsAb against human tumors for both adult and pediatrics, if autologous T cells can be activated and expanded ex vivo.

The role of T cells in controlling human cancers is well known. Their success requires continued persistence in vivo and efficient trafficking to tumor sites, requirements shared by other effectors such as Natural Killer (NK) cells. To date, cytokine IL2 remains the only clinically approved cytokine therapy available to expand, maintain, and activate these effector lymphoid cells, but toxicities can be severe. Cytokine IL15 offers similar T cell proliferation and activation properties, but without the unwanted side-effects seen with IL2. Several IL15-cytokine fusion proteins have been developed to improve their in vivo function, typically exploiting the IL15Rα to complex with IL15, to extend serum half-life and increase affinity for IL15β receptor on immune cells. Here we describe a novel IL15 complex incorporating the full-length IL15Rα to complex with wild type IL15 to form spontaneous trimers of dimers (6 IL15 + 6 IL15Rα) during co-expression, resulting in a substantial increase in serum half-life and enhancement of in vivo cytokine effect on IgG or T cell engaging antibody-dependent cell-mediated cytotoxicities, when compared to alternative strategies.

Introduction: GD2-SADA is a pre-targeted radioimmunotherapy drug-candidate designed to target radioactive lutetium-177 (in the form of 177Lu-DOTA) to GD2-expressing tumor cells (GD2-SADA:177Lu-DOTA Drug Complex). GD2-SADA is comprised of an anti-GD2 single-chain variable fragment (scFv), an anti-DOTA scFv that binds specifically to DOTA metal chelates, such as 177Lu-DOTA, and a SADA domain based on the human P53 tetramerization sequence, which can self-assemble from a monomeric 60-kDa polypeptide into 240-kDa tetrameric protein. We have previously demonstrated (1) that GD2-SADA has a unique clearance profile that allowed for the delivery of 177Lu-DOTA to tumors with minimal exposure to normal tissues like the bone marrow or kidneys and shrank established neuroblastoma in preclinical mouse models. We now provide additional mechanistic data demonstrating the importance of the SADA domain in providing this potent anti-tumor function. Methods: To study the role of the SADA domain, we designed a modified version of GD2-SADA that could not self-assemble into a tetrameric state by removing the entire SADA domain. The resulting P53(-/-)GD2-SADA protein retained binding to GD2 and Lu-DOTA but remained in a 60 kDa monomeric state in assays. P53(-/-)GD2-SADA and GD2-SADA were compared using SPR to measure antigen binding affinities, flow cytometry to evaluate cell binding, and in preclinical mouse models to evaluate tumor uptake and anti-tumor efficacy. Results: GD2-SADA demonstrated stronger binding affinity to GD2 antigen compared to P53(-/-)GD2-SADA, but comparable binding affinity to Lu-DOTA. In SPECT/CT imaging studies using tumor bearing mice, GD2-SADA demonstrated substantially higher uptake and persistence in GD2-expressing tumors compared with P53(-/-)GD2-SADA. Finally, treatment of tumor bearing mice with 3 cycles of GD2-SADA or P53(-/-)GD2-SADA and 177Lu-DOTA (once per week each for 3 weeks) resulted in potent anti-tumor responses from GD2-SADA, with only modest anti-tumor efficacy from P53(-/-)GD2-SADA at two different dose levels corresponding to equal molar doses (1 GD2-SADA to 1 P53(-/-)GD2-SADA) or equal mass doses (1 GD2-SADA to 4 P53(-/-)GD2-SADA i.e. same number of binding sites). Conclusions: The tetramerizing function of the SADA domain is critically important to the binding activity and anti-tumor efficacy of GD2 SADA. These data confirm that the SADA domain increases tumor antigen binding, uptake and persistence in tumor tissue, and markedly improves anti-tumor responses in preclinical models. References: Santich BH, Cheal SM, Ahmed M, McDevitt MR, Ouerfelli O, Yang G, et al. A Self-Assembling and Disassembling (SADA) Bispecific Antibody (BsAb) Platform for Curative Two-step Pretargeted Radioimmunotherapy. Clin Cancer Res Off J Am Assoc Cancer Res. 2021 Jan 15;27(2):532-41. Citation Format: Brian H. Santich, Stine Louise Kjellev, Sarah Cheal, Linlin Wang, Mette Gillberg, Mallika Vadlamudi, Amadou A. Ouattara, Nico Liebenberg, Lone Frost Larsen, Darren R. Veach, Shin Seo, Nai-Kong V. Cheung, Steven M. Larson, Steen Lisby. Self-assembling and disassembling (SADA) domain is critical to the binding, and anti-tumor efficacy of GD2-SADA [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 3309.

Background and Aim: Ganglioside GD2/GD3 vaccine stimulated robust antibody response among patients with high-risk metastatic neuroblastoma (HR-NB) who had prior disease progression (J Clin Oncol 39:215-226, 2020). High anti-GD2-IgG1antibody titer was associated with improved progression-free survival and overall survival in multivariable analyses. A gel formulation of yeast beta-glucan was used as an oral vaccine adjuvant, but its importance remained unproven. Patients and Methods: In a follow-up randomized Phase II trial (Clinicaltrials.gov identifier: NCT00911560), seven vaccine injections at 1, 2, 3, 8, 20, 32 and 52 weeks were administered to patients with HR-NB during their first or subsequent remissions. Each subcutaneous vaccine injection consisted of 30 ug each of GD2 and GD3, which was lactonized and conjugated to keyhole limpet hemocyanin and mixed with the subcutaneous saponin OPT-821 adjuvant at 150 µg/m2. No patients in this analysis received prior ganglioside vaccine. They were randomized to Arm 1 (n=54) receiving no glucan, or Arm 2 (n=53) receiving oral beta-glucan regimen (40 mg/kg/day, 14 days on/14 days off) starting at week 1. From week 6 onwards, all 107 patients received oral beta-glucan regimen through year end or up to disease progression. Serum IgG1 against GD2 and GD3, and IgM against GD2 were measured by ELISA at (right before) each vaccine injection. Wilcoxon rank sum test was used to compare antibody titers between the 2 arms. Results: In both arms, patients had comparable disease status at study entry, with 70% each in first remission. The remaining patients were in second remission after one prior disease progression. Consistently higher antibody response was observed among Arm 2 patients. The primary endpoint was met for anti-GD2-IgG1 titer at vaccine injection #6 at 32 weeks (p=0.08). Per protocol design, statistical significance (p Conclusion: Adding oral yeast beta-glucan as vaccine adjuvant during the first 6 weeks of immunization significantly enhanced the anti-GD2-IgG1 antibody response without added toxicities. Its impact on patient survival will require a longer clinical follow-up. Citation Format: Irene Y. Cheung, Audrey Mauguen, Yi Feng, Govind Ragupathi, Ellen Basu, Stephen S. Roberts, Shakeel Modak, Brian H. Kushner, Nai-Kong V. Cheung. Oral beta-glucan enhanced anti-ganglioside antibody titer after vaccination against high-risk neuroblastoma: Results of a randomized trial [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 5218.

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]

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.

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

Background The cure rate for metastatic osteosarcoma has not substantially improved over the past decades. Clinical trials of anti-HER2 trastuzumab or anti-GD2 dinutuximab for metastatic or refractory osteosarcoma were not successful, and neither was immune checkpoint inhibitors (ICIs). Methods We tested various target antigen expressions on osteosarcoma cell lines using flow cytometry and analyzed in vitro T cell engaging BsAb (T-BsAb)-dependent T cell-mediated cytotoxicity using 4-h 51Cr release assay. We tested in vivo anti-tumor activities of T-BsAb targeting GD2 or HER2 in established osteosarcoma cell line or patient-derived xenograft (PDX) mouse models carried out in BALB-Rag2−/−IL-2R-γc-KO (BRG) mice. We also generated ex vivo BsAb-armed T cells (EATs) and studied their tumor-suppressive effect against osteosarcoma xenografts. In order to improve the anti-tumor response, ICIs, anti-human PD-1 (pembrolizumab) or anti-human PD-L1 (atezolizumab) antibodies were tested their synergy with GD2- or HER2-BsAb against osteosarcoma. Results GD2 and HER2 were chosen from a panel of surface markers on osteosarcoma cell lines and PDXs. Anti-GD2 BsAb or anti-HER2 BsAb exerted potent anti-tumor effect against osteosarcoma tumors in vitro and in vivo. T cells armed with anti-GD2-BsAb (GD2-EATs) or anti-HER2-BsAb (HER2-EATs) showed significant anti-tumor activities as well. Anti-PD-L1 combination treatment enhanced BsAb-armed T cell function in vivo and improved tumor control and survival of the mice, when given sequentially and continuously. Conclusion Anti-GD2 and anti-HER2 BsAbs were effective in controlling osteosarcoma. These data support the clinical investigation of GD2 and HER2 targeted T-BsAb treatment in combination with immune checkpoint inhibitors, particularly anti-PD-L1, in patients with osteosarcoma to improve their treatment outcome.

Purpose: Many cancer treatments suffer from dose-limiting toxicities to vital organs due to poor therapeutic indices. To overcome these challenges we developed a novel multimerization platform that rapidly removes tumor-targeting proteins from the blood to substantially improve therapeutic index. Experimental Design: The platform was designed as a fusion of a self-assembling and disassembling (SADA) domain to a tandem single-chain bispecific antibody (BsAb, anti-ganglioside GD2 × anti-DOTA). SADA–BsAbs were assessed with multiple in vivo tumor models using two-step pretargeted radioimmunotherapy (PRIT) to evaluate tumor uptake, dosimetry, and antitumor responses. Results: SADA–BsAbs self-assembled into stable tetramers (220 kDa), but could also disassemble into dimers or monomers (55 kDa) that rapidly cleared via renal filtration and substantially reduced immunogenicity in mice. When used with rapidly clearing DOTA-caged PET isotopes, SADA–BsAbs demonstrated accurate tumor localization, dosimetry, and improved imaging contrast by PET/CT. When combined with therapeutic isotopes, two-step SADA-PRIT safely delivered massive doses of alpha-emitting (225Ac, 1.48 MBq/kg) or beta-emitting (177Lu, 6,660 MBq/kg) S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA) payloads to tumors, ablating them without any short-term or long-term toxicities to the bone marrow, kidneys, or liver. Conclusions: The SADA–BsAb platform safely delivered large doses of radioisotopes to tumors and demonstrated no toxicities to the bone marrow, kidneys, or liver. Because of its modularity, SADA–BsAbs can be easily adapted to most tumor antigens, tumor types, or drug delivery approaches to improve therapeutic index and maximize the delivered dose. See related commentary by Capala and Kunos, p. 377

Purpose In patients with high-risk neuroblastoma, there is an increased recognition of relapse in the central nervous system (CNS). Craniospinal irradiation (CSI) has been an effective treatment but carries significant long-term complications. It is unclear whether reducing the CSI dose from 21 to 18 Gy can achieve similar CNS tumor control. Patients and methods A retrospective review of pediatric patients with CNS-relapsed neuroblastoma treated with CSI and boost to parenchymal lesions between 2003 and 2019 was performed. The goal was to assess CNS control comparing 18 Gy and 21 Gy regimens. Results Ninety-four patients with CNS-relapsed neuroblastoma were treated with CSI followed by intraventricular compartmental radioimmunotherapy. Median age at the time of CNS disease was 4 years (range 1-13 years). Forty-one patients (44%) received 21 Gy CSI prior to an institutional decision to lower the dose; 53 patients (56%) received 18 Gy CSI. Seventy-nine patients (84%) received additional boosts. With a median follow up of 4.1 years for surviving patients, 2-year CNS relapse-free survival was 74% for 18 Gy group versus 77% for 21 Gy group, and 5-year CNS relapse-free survival was 66% for 18 Gy versus 72% for 21 Gy group, respectively (P = .40). Five-year overall survival rate was 43% in 18 Gy group versus 47% in 21 Gy group (P = .72). Conclusion For patients with CNS-relapsed neuroblastoma, CNS disease control is comparable between 18 Gy and 21 Gy CSI dose regimens, in conjunction with radioimmunotherapy and CNS penetrating chemotherapy. More than 65% of the patients remain CNS disease free after 5 years. The findings support 18 Gy as the new standard CSI dose for CNS-relapsed neuroblastoma.

Objectives Epithelial ovarian carcinoma (EOC) is a common and lethal gynecologic malignancy that frequently presents as advanced staged disease, such as peritoneal carcinomatosis (PC). We previously reported cures in BT-474 murine xenografts with Pretargeted Radioimmunotherapy (PRIT) using 177Lu radiohaptens targeting Her2 via a bispecific antibody (BsAb). Here-in we report the use of PRIT with alpha emitter 225Ac labelled PrDOTA to treat a PC model of SKOV3, a Her2+ cell line of EOC. Methods 6 wk old female athymic nude mice inoculated IP with 1E5 luciferase/GFP-transfected SKOV3 cells were separated into 5 groups (n=10). Treatment mice received 1 or 2 cycles of Anti-Her2-C825 BsAb + [225Ac]PrDOTA (Her2-Targeted), at 14 and 21 days after inoculation, respectively. Control groups received Anti-Her2 BsAb only, Anti-GPA33 BsAb + [225Ac]PrDOTA (Off-Targeted) or no treatment. On cycle day 1, the mice were injected IP with 0.25mg (1.19nmol) BsAb. On cycle day 2, 25µg (2.76nmol) CCA16-DOTAY clearing agent (CA) was given IV 22h from BsAb. Mice in therapy groups were injected IP with 1µCi (0.74-0.79nmol) [225Ac]PrDOTA-Bn 4h after CA. Weekly weights and BLIs with IP cavity ROIs were obtained and normalized to the respective values for each mouse at week 0 pre-treatment. End points: weight loss >20% baseline, moribund, or severe abdominal distension. At 154 days, 15 surviving treatment and 1 untreated control mice were submitted for hematology and histopathology. Results Histologic cures and prolonged survival were demonstrated in treatment mice (17/20 at 133 days) as compared to control mice (12/27 at 133 d, Logrank p0.05; all weeks). BLI of mice treated with 1 and 2 cycle of Her2 PRIT decreased 47% when compared to baseline within 1 week (T test p=0.04), suggesting treatment effects as early as 1 week. There were no differences in weights when compared to baseline (2-way ANOVA p>0.05). While the untreated mouse had high peritoneal adenocarcinoma tumor burden, there was no histologic evidence of viable neoplasia in 15/15 submitted treatment mice. Treatment mice had moderate renal tubular degenerative lesions on histology, but this did not affect renal function based on serum BUN or Cr. All hematologic parameters were within normal limits for treated mice. Conclusions 1 and 2 cycles of [225Ac]PrDOTA-PRIT against Her2 resulted in histologic cures and prolonged survival in IP SKOV3 xenografts with minimal toxicity. The anti-Her2 PrDOTA-PRIT system is a promising theranostic approach for otherwise incurable PC. Citation Format: Sebastian K. Chung, Christopher S. Chandler, Daniela Burnes Vargas, Shin H. Seo, Michael R. McDevitt, Darren Veach, Blesida Punzalan, Xu Hong, Hong-fen Guo, Garrett M. Nash, Andrea Cercek, Nai-Kong V. Cheung, Steven M. Larson, Sarah M. Cheal. Pretargeted radioimmunotherapy using 225Ac for intraperitoneal Her2-expressing epithelial ovarian carcinoma xenografts [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P168.

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

Radioimmunotherapy of solid tumors using antibody-targeted radionuclides has been limited by low therapeutic indices (TIs). We recently reported a novel 3-step pretargeted radioimmunotherapy (PRIT) strategy based on a glycoprotein A33 (GPA33)–targeting bispecific antibody and a small-molecule radioactive hapten, a complex of 177Lu and S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (177Lu-DOTA-Bn), that leads to high TIs for radiosensitive tissues such as blood (TI = 73) and kidney (TI = 12). We tested our hypothesis that a fractionated anti-GPA33 DOTA-PRIT regimen calibrated to deliver a radiation absorbed dose to tumor of more than 100 Gy would lead to a high probability of tumor cure while being well tolerated by nude mice bearing subcutaneous GPA33-positive SW1222 xenografts. Methods: We treated groups of nude mice bearing 7-d-old SW1222 xenografts with a fractionated 3-cycle anti-GPA33 DOTA-PRIT regimen (total administered 177Lu-DOTA-Bn activity, 167 MBq/mouse; estimated radiation absorbed dose to tumor, 110 Gy). In randomly selected mice undergoing treatment, serial SPECT/CT imaging was used to monitor treatment response and calculate radiation absorbed doses to tumor. Necropsy was done on surviving animals 100–200 d after treatment to determine frequency of cure and assess select normal tissues for treatment-related histopathologies. Results: Rapid exponential tumor progression was observed in control treatment groups (i.e., no treatment or 177Lu-DOTA-Bn only), leading to euthanasia due to excessive tumor burden, whereas 10 of 10 complete responses were observed for the DOTA-PRIT–treated animals within 30 d. Treatment was well tolerated, and 100% histologic cure was achieved in 9 of 9 assessable animals without detectable radiation damage to critical organs, including bone marrow and kidney. Radiation absorbed doses to tumor derived from SPECT/CT (102 Gy) and from biodistribution (110 Gy) agreed to within 6.9%. Of the total dose of approximately 100 Gy, the first dose contributes 30%, the second dose 60%, and the third dose 10%. Conclusion: In a GPA33-positive human colorectal cancer xenograft mouse model, we validated a SPECT/CT-based theranostic PRIT regimen that led to 100% complete responses and 100% cures without any treatment-related toxicities, based on high TIs for radiosensitive tissues. These studies support the view that anti-GPA33 DOTA-PRIT will be a potent radioimmunotherapy regimen for GPA33-positive colorectal cancer tumors in humans.

Immune checkpoint inhibitors (ICI) have shown great promise in a wide spectrum of adult solid and hematological malignancies, achieving objective tumor responses and prolonging survival. However, there is limited clinical success amongst pediatric patients. In this review, we summarize the current understanding of ICI and present an up-to-date overview of recent and ongoing clinical trials of ICI in pediatric malignancies. In addition, we will discuss immunologic and clinical difficulties in this young population, as well as future prospects for combination of ICI with other immune-based and conventional treatments.

Ewing sarcoma (ES) family of tumors includes bone and soft tissue tumors that are often characterized by a specific translocation between chromosome 11 and 22, resulting in the EWS-FLI1 fusion gene. With the advent of multi-modality treatment including cytotoxic chemotherapy, surgery, and radiation therapy, the prognosis for patients with ES has substantially improved. However, a therapeutic plateau is now reached for both localized and metastatic disease over the last two decades. Burdened by the toxicity limits associated with the current frontline systemic therapy, there is an urgent need for novel targeted therapeutic strategies. In this review, we discuss the current treatment paradigm of ES, and explore preclinical evidence and emerging treatments directed at tumor signaling pathways and immune targets.

IMPORTANCE: Embryonal Tumor/Multilayered Rosettes (ETMR) is an aggressive, lethal, CNS tumor in young childr. B7-H3 is an inhibitory ligand for natural killer cells and T cell overexpressed in a number of embryonal tumors. 131-Omburtomab (8H9) is a radiolabeled monoclonal antibody under investigation for intraventricular administration for primary and metastatic CNS disease. We explored the use of intraventricular (131)I-8H9 targeting B7-H3 in patients with ETMR. DESIGN: Patients were enrolled in an IRB approved trial. Three symptomatic children (ages 14 months, 3 and 3.5 years) had large parietal masses confirmed to be B7-H3-reactive ETMR Patients received 2 mCi (131)I-Omburtamab as a tracer followed by 1 or 2 therapeutic (131)I-Omburtamab injections. Dosimetry was based on serial CSF, blood samplings and region of interest (ROI) on nuclear scans. Brain and spine MRIs and CSF cytology were done at baseline, 5 weeks after (131)I-Omburtamab, and approximately every 3 months thereafter. Acute toxicities and survival were noted. RESULTS: Patients received surgery, focal radiation, and high dose chemotherapy. Patients 1 and 2 received (131)I-Omburtamab (80 and 53 mCi, respectively). Patient 3 had a local recurrence prior to (131)I-Omburtamab treated with surgery, external beam radiation, chemotherapy, then (131)I-Omburtamab (36 mCi). (131)I-Omburtamab was well-tolerated. Mean dose delivered by (131)I-Omburtamab was 68.4 cGy/mCi to CSF and 1.95 cGy/mCi to blood. Mean ROI doses were 230.4 (ventricular) and 58.2 (spinal) cGy/mCi. Patients 1 and 2 remain in remission 6.5 years and 2 years after diagnosis, respectively; patient 3 died of progressive disease 7 month after therapy, 2 years after diagnosis. CONCLUSIONS: (131)I-Omburtamab appears safe with favorable dosimetry therapeutic index. When used as consolidation following surgery and chemoradiation therapy, (131)I-Omburtamab may have therapeutic benefit for patients with ETMR.

BACKGROUND/OBJECTIVES: Survivors of high-risk neuroblastoma (NB) are exposed to multimodality therapies early in life and confront late therapy-related toxicities. This study assessed respiratory symptoms, exercise capacity, and longitudinal changes in pulmonary function tests (PFTs) among survivors. DESIGN/METHODS: Survivors of high-risk NB followed in the Long-term Follow-up clinic at Memorial Sloan Kettering Cancer Center were enrolled. Symptom and physical activity questionnaires were completed. Medical records were reviewed for treatments and co-morbidities. Participants completed spirometry, plethysmography, diffusion capacity of the lung for carbon monoxide, 6 Minute Walk Tests (6MWTs), and cardiopulmonary exercise testing. Questionnaires and PFTs were repeated at least one year after enrollment. RESULTS: Sixty-two survivors participated (median age at study: 10.92years; median age at diagnosis: 2.75years; median time since completion of therapy: 5.29years). Thirty-two percent had chronic respiratory symptoms. Seventy-seven percent had PFT abnormalities, mostly mild to moderate severity. Thirty-three completed 6MWTs (median 634.3meters); eight completed cardiopulmonary exercise tests (mean VO(2) max: 63 percent predicted); twenty-three completed a second PFT revealing declines over a median 2.97years (mean percent predicted forced vital capacity: 79.9 to 70.0; mean forced expiratory volume in 1 second: 81.6 to 69.9). Risks for abnormalities included thoracic surgery, chest radiation therapy (RT), thoracic surgery plus chest RT, and hematopoietic stem cell transplant. CONCLUSIONS: In this cohort of survivors of high-risk NB, PFT abnormalities were common but mostly mild or moderate. Maximal exercise capacity may be affected by respiratory limitations and declines in lung function may occur over time. Continued pulmonary surveillance of this at-risk population is warranted.

Purpose For patients with high-risk neuroblastoma (HR-NB), a dose of 21 Gy to the primary tumor site after gross total resection (GTR) provides excellent local control. However, no clinical trial has specifically evaluated the optimal dose of radiation therapy (RT), and RT-related long-term toxicities are of increasing concern. We sought to assess local control, survival outcomes, and toxicity after a reduction in dose to the primary site from 21 Gy to 18 Gy. Methods and Materials After induction chemotherapy and GTR, patients with HR-NB were enrolled and treated on an RT dose-reduction prospective trial with 18 Gy hyperfractionated RT given in twice-daily fractions of 1.5 Gy each. Results The 25 study subjects were 1.6 to 9.5 (median, 4.3) years old at enrollment and included 23 (92%) with stage IV and II (8%) with MYCN-amplified stage III disease. Eleven (44%) were in complete remission (CR), and 14 (56%) had persistence of osteomedullary disease postinduction. Three patients (12%) received proton therapy, and the rest received intensity modulated photon therapy. After a follow-up of 1.8 to 4.2 (median, 3.5) years from initiation of RT, no failures occurred within the RT field; 3 patients had marginal recurrences. The respective 3-year progression-free and overall survival rates were 54.5% and 90.9% for patients in first CR and 42.9% and 76.2% for patients not in metastatic CR. Acute toxicity was negligible. Conclusions Reduced-dose RT with 18 Gy did not compromise local control or survival outcomes in our cohort of patients with HR-NB after GTR. These findings support assessing further RT dose reduction and validation on a larger, multi-institutional trial.

2010 Background: The prognosis of diffuse intrinsic pontine glioma (DIPG) is dire with a median overall survival less than one-year. 124I-omburtamab is a radiolabeled monoclonal antibody that targets B7-H3 epitope. We evaluated the safety of administering escalating doses and volumes of 124I-omburtamab via convection-enhanced delivery (CED) in children with DIPG. Methods: MSKCC 11-011 trial is a standard 3+3 phase 1, open-label, dose escalation study in patients with non-progressive DIPG. CED of 124I-omburtamab was performed between 4-14 weeks post-external radiation therapy. Nine dose levels of a single injection of 124I-omburtamab (Y-mAbs Therapeutics, USA) (range 0.25 to 8.0 mCi; and volume of infusion (Vi) from 250 to 8,000 µl) have been evaluated so far. Patients were assessed weekly for 30 days. Results: 46 children were evaluable for primary and secondary endpoints. The median age at enrolment was 6.5 years (range 2-17). Two patients have experienced AEs CTCAE grade 3 that were categorized as dose limiting toxicities (DLTs), which led to inclusion of three more patients at both the 4 and 6 mCi dose levels. Eight patients have reported transient AEs of grade 3 considered related to 124I-omburtamab. The acute grade 3 AEs were generally indicative of nervous system effects due to volume intolerance or radiation injury, and included hemiparesis (n = 3), dysarthria (n = 3), ataxia (n = 3), dysphagia (n = 2), muscular weakness (n = 2) and gait disturbance (n = 1). There were no related AEs CTCAE grade 4 or 5. Estimations of distribution volumes based on T2-weighted imaging were linearly related to volume with a mean volume of distribution/volume of infusion ratio (Vd/Vi) between 3 and 3.5. The mean ratio of lesion-to-whole body absorbed dose was ̃1000. Median overall survival from diagnosis across all cohorts was 14.8 months (n = 46, 95% CI 11.5, 16.8) and the survival rate estimates (with 95% confidence intervals) at 1, 2, 3 and 5 years were 0.63 (0.46;0.76); 0.13 (0.05;0.26); 0.08 (0.02;0.19); and 0.04 (0.00;0.16), respectively. Four patients have survived > 3 years; two remain alive at 46 and 96 months and two have died at 43 and 53 months, both with CNS disease outside of the treatment field and one with extra-CNS metastases. Conclusions: 124I-omburtamab via CED into the brain stem of children with DIPG and previously irradiated provides a possibility for improved treatment of DIPG. A dose of 8mCi and an infusion volume of 8,000 µl is considered safe and may provide a distribution volume large enough to cover tumor volumes up to 20 cm3. The median overall survival of all patients included in the trial appears to be increased with 3-4 months compared to historical control data from consortia trials. A phase 2 trial aiming at investigating the efficacy of radiolabeled omburtamab administered via CED is being planned. Clinical trial information: NCT01502917.

BackgroundAcute myeloid leukemia (AML) remains one of the most challenging hematological malignancies. Despite progress in therapeutics, majority of patients succumb to this neoplasm. CD33 is a proven therapeutic target, given its expression on most AML cells. Almost all anti-CD33 antibodies target the membrane distal immunoglobulin V (IgV) domain of the CD33 extracellular domain.MethodsIn this manuscript, we present data on three bispecific antibodies (BsAbs) against the CD33 IgV and membrane proximal immunoglobulin C (IgC) domains. We use in vitro binding and cytotoxicity assays to show the effect of these BsAbs on AML cell lines. We also use immunodeficient mice-bearing leukemias from cell lines and patient-derived xenografts to show the effect of these BsAbs in vivo.ResultsIn vitro, the IgV-targeting BsAb had higher binding to AML cell lines using flow cytometry and delivered more potent cytotoxicity in T-cell-dependent cytotoxicity assays; importantly, the IgC domain-targeting outperformed the IgV domain-targeting BsAb in medullary and extramedullary leukemia animal models.ConclusionsThese data support further clinical development of this BsAb for first-in-human phase I clinical trial.

Purpose To evaluate local control after 21-Gy radiation therapy (RT) to the primary site in patients with high-risk neuroblastoma. Methods and Materials After receiving dose-intensive chemotherapy and gross total resection (GTR), 246 patients (aged 1.2-17.9 years, median 4.0 years) with high-risk neuroblastoma underwent RT to the primary site at Memorial Sloan Kettering from 2000 to 2014. Radiation therapy consisted of 21 Gy in twice-daily fractions of 1.5 Gy each. Local failure (LF) was correlated with biologic prognostic factors and clinical findings at the time of diagnosis and start of RT. Results Median follow-up of surviving patients was 6.4 years. Cumulative incidence of LF was 7.1% at 2 years after RT and 9.8% at 5 years after RT. The isolated LF rate was 3.0%. Eighty-six percent of all local failures were within the RT field. Local control was worse in patients who required more than 1 surgical resection to achieve GTR (22.4% vs 8.3%, P =.01). There was also a trend toward inferior local control with MYCN- amplified tumors or serum lactate dehydrogenase ≥1500 U/L ( P =.09 and P =.06, respectively). Conclusion After intensive chemotherapy and maximal surgical debulking, hyperfractionated RT with 21 Gy in high-risk neuroblastoma results in excellent local control. Given the young patient age, concern for late effects, and local control >90%, dose reduction may be appropriate for patients without MYCN amplification who achieve GTR.

AKT plays a pivotal role in driving the malignant phenotype of many cancers, including high-risk neuroblastoma (HR-NB). AKT signaling, however, is active in normal tissues, raising concern about excessive toxicity from its suppression. The oral AKT inhibitor perifosine showed tolerable toxicity in adults and in our phase I trial in children with solid tumors (clinicaltrials.gov NCT00776867). We now report on the HR-NB experience. HR-NB patients received perifosine 50-75mg/m2/day after a loading dose of 100-200mg/m2 on day 1, and continued on study until progressive disease. The 27 HR-NB patients included three treated for primary refractory disease and 24 with disease resistant to salvage therapy after 1-5 (median 2) relapses; only one had MYCN-amplified HR-NB. Pharmacokinetic studies showed μM concentrations consistent with cytotoxic levels in preclinical models. Nine patients (all MYCN-non-amplified) remained progression-free through 43+ to 74+ (median 54+) months from study entry, including the sole patient to show a complete response and eight patients who had persistence of abnormal 123I-metaiodobenzylguanidine skeletal uptake but never developed progressive disease. Toxicity was negligible in all 27 patients, even with the prolonged treatment (11-to-62 months, median 38) in the nine long-term progression-free survivors. The clinical findings 1) confirm the safety of therapeutic serum levels of an AKT inhibitor in children; 2) support perifosine for MYCN-non-amplified HR-NB as monotherapy after completion of standard treatment or combined with other agents (based on preclinical studies) to maximize antitumor effects; and 3) highlight the welcome possibility that refractory or relapsed MYCN-non-amplified HR-NB is potentially curable. This article is protected by copyright. All rights reserved.

Immune checkpoints have been a focus of immunotherapy in the recent decade. Killer cell immunoglobulin-like receptors (KIR) and their cognate human leukocyte antigen (HLA) class I ligands have evolved as checkpoints to ensure self-tolerance of natural killer cells. Both KIR and HLA genetic profiles are potential biomarkers of immunotherapy outcome. Clin Cancer Res; 24(1); 3–5. ©2017 AACR. See related article by Erbe et al., p. 189

Background and Aim: High-risk neuroblastoma (HR-NB) relapse carries a dismal prognosis. Recent experience, however, suggests that cure is possible when remission is achieved with salvage therapy that includes anti-GD2 monoclonal antibody (mAb). The long-term survival and the favorable safety profile of gangliosides GD2 and GD3 vaccine among these patients in a Phase I trial (n=15, Clinical Cancer Res 2014) need to be confirmed. Moreover, the kinetics of seroconversion (mounting anti-vaccine antibody response) and its prognostic impact on survival need to be explored. Patients and Methods: In this Phase II trial (Clinicaltrials.gov NCT00911560), 102 HR-NB patients who achieved complete remission after salvage therapy were enrolled. They received 7 subcutaneous vaccine injections (week 1-2-3-8-20-32-52) plus oral beta-glucan (starting in week 6 at 40 mg/kg/day, 14 days on/14 days off). Each subcutaneous vaccine injection consisted of 30 µg each of GD2 and GD3, lactonized and conjugated to keyhole limpet hemocyanin and mixed with the saponin OPT-821 adjuvant. Serum anti-vaccine antibody titers were quantified by ELISA. Kaplan-Meier statistics and landmark Cox Regression models were used for survival estimates and prognostic impact analyses. Results: Among 102 patients, 63% had one, 21% had 2, and 16% had 3-6 prior disease progressions. 83/101 patients had failed prior anti-GD2 mAb (m3F8, dinutuximab, or naxitamab) therapy before vaccine: one mAb (n=62), two mAbs (n=15), or all three (n=6). Common toxicities were self-limited injection-related local reactions and fever. No pain, neuropathy, or grade 3/4 toxicities occurred during or post treatment. Progression-free survival (PFS) was 44%±5% and overall survival (OS) was 88%±4% at 2 years, and 36%±7% and 70%±8% at 5 years, respectively. Serum anti-GD2 (IgG1 and IgM) and anti-GD3 (IgG1) titers had marked increases following the initiation of beta-glucan at week 6. In univariate analyses, favorable prognostic factors included: one versus ≥2 prior disease progressions (PFS p=0.005, OS p=0.04), none versus any prior anti-GD2 mAb failures (PFS p=0.004, OS p=0.01); and the induction of ≥150 ng/ml anti-GD2-IgG1 titers by week 8 (PFS p=0.02, OS p=0.06). Factors not prognostic included: time to first NB progression, MYCN amplification status, anti-GD2-IgM, anti-GD3-IgG1 or anti-KLH-IgG1 titers, and treatment with anti-GD2 mAb right before vaccine. In multivariate analyses, week 8 anti-GD2-IgG1 titer ≥150 ng/ml yielded a hazard ratio (HR) of 0.41 [0.20, 0.83], p=0.01 for PFS, and HR=0.15 [0.02, 1.12], p=0.06 for OS. The second independent prognostic variable was the number of prior disease progressions (≥2 vs 1), yielding HR of 2.12 [1.26, 3.58], p=0.005 for PFS, and HR=2.77 (1.03, 7.47), p=0.04 for OS. Conclusions: Even with prior disease progressions, anti-GD2 (though not anti-GD3) seroconversion was associated with notable long-term survival among HR-NB patients previously thought to be unsalvageable. A randomized trial to assess the role of beta-glucan in seroconversion is actively accruing patients. Citation Format: Irene Y. Cheung, Nai-Kong V. Cheung, Shakeel Modak, Audrey Mauguen, Ellen Basu, Stephen S. Roberts, Govind Ragupathi, Brian H. Kushner. Survival impact of anti-GD2 antibody response - A phase II ganglioside vaccine trial in relapsed neuroblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-092.

ATRX alterations occur at high frequency in neuroblastoma of adolescents and young adults. Particularly intriguing are the large N-terminal deletions of ATRX (Alpha Thalassemia/Mental Retardation, X-linked) that generate in-frame fusion (IFF) proteins devoid of key chromatin interaction domains, while retaining the SWI/SNF-like helicase region. We demonstrate that ATRX IFF proteins are redistributed from H3K9me3-enriched chromatin to promoters of active genes and identify REST as an ATRX IFF target whose activation promotes silencing of neuronal differentiation genes. We further show that ATRX IFF cells display sensitivity to EZH2 inhibitors, due to derepression of neurogenesis genes, including a subset of REST targets. Taken together, we demonstrate that ATRX structural alterations are not loss-of-function and put forward EZH2 inhibitors as a potential therapy for ATRX IFF neuroblastoma.

Despite progress in the treatment of colorectal cancer, curing metastatic colorectal cancer remains a major unmet medical need worldwide. Here, we describe a T-cell–engaging bispecific antibody (T-BsAb) to redirect polyclonal cytotoxic T cells to eradicate colorectal cancer. A33, a murine antibody specific for GPA33, was humanized to huA33 and reformatted to huA33-BsAb, based on a novel IgG(L)–scFv platform by linking the anti-CD3 huOKT3 scFv to the carboxyl end of the light chain. This T-BsAb was stably expressed in CHO cells and purified as a stable monomer by HPLC, retaining immunoreactivity by FACS through 30 days of incubation at 37°C. In vitro, it induced activation and expansion of unstimulated T cells and elicited potent T-cell–dependent cell-mediated cytotoxicity against colon and gastric cancer cells in an antigen-specific manner. In vivo, huA33-BsAb inhibited the colon and gastric cancer xenografts, in both subcutaneous and intraperitoneal tumor models. More importantly, both microsatellite instable and microsatellite stable colorectal cancer were effectively eliminated by huA33-BsAb. These preclinical results provide further support for the use of IgG(L)–scFv platform to build BsAb, and especially one targeting GPA33 for colorectal cancer. These preclinical results also support further development of huA33-BsAb as a potential immunotherapeutic. Mol Cancer Ther; 17(10); 2164–75. ©2018 AACR.

PURPOSE: Novel treatment strategies are needed for children with recurrent ependymoma. We explored the use of intraventricular targeted immunotherapy with monoclonal antibodies (cRIT-mAb) using (131)I-8H9 and (131)I-3F8 targeting B7-H3 or G(D2), respectively. METHODS: Patients received 2 mCi cRIT- (131)I- mAb for dosimetry with serial CSF sampling and region of interest (ROI) on nuclear scans. Therapy injections were 40 -100 mCi (131)I-8H9 or up to 40 mCi (131)I-3F8. Brain and spine MRIs, CSF cytology were routinely assessed. Acute toxicities and survival were noted. RESULTS: 11 patients with anaplastic ependymoma (median age 8.5 years, 1–32) suffered 1- 7 recurrences (median 2.9); median age at cRIT was 12.3 years (3–35). 39 injections were delivered (13 (131)I-3F8; 26 (131)I-8H9). Transient headache, nausea and vomiting was observed following (131)I-3F8; (131)I-8H9 was well-tolerated. Mean CSF cGy/mCi by sampling was 37.6 (131)I-8H9 and 40.1 (131)I-3F8. Mean ventricular ROI dose was 75.3 cGy/mCi (131)I-8H9 and 111 cGy/mCi (131)I-3F8 and to spine 20.2 cGy/mCi (131)I-8H9 and 18.9 cGy/mCi (131)I-3F8. 4/5 patients with radiographic evidence of disease progressed; 1 had spinal disease improvement. Of 5 patients treated in radiographic remission, 2 remain in remission 2.5 and 8 years since cRIT, 1 is alive with disease 2.5 years post; 2 patients died of disease 7 and 13 years post cRIT. CONCLUSIONS: cRIT mAb appear safe with a favorable dosimetry therapeutic index and may be considered as a viable therapeutic option for patients with recurrent ependymoma.

WT1126 (RMFPNAPYL) is a human leukocyte antigen-A2 (HLA-A2) restricted peptide derived from Wilms tumor protein (WT1), which is widely expressed in a broad spectrum of leukemias, lymphomas and solid tumors. A novel T-cell-receptor (TCR)-like single chain variable fragment (scFv) antibody specific for the T cell epitope consisting of the WT1/HLA-A2 complex was isolated from a human scFv phage library. This scFv was affinity-matured by mutagenesis combined with yeast display, and structurally analyzed using a homology model. This monovalent scFv showed a 100-fold affinity improvement (dissociation constant [KD]= 3nM) and exquisite specificity towards its targeted epitope or HLA-A2+/WT1+ tumor cells. Bivalent scFv-huIgG1-Fc fusion protein demonstrated an even higher avidity (KD = 2pM) binding to the T cell epitope and to tumor targets, and was capable of mediating antibody-dependent cell-mediated cytotoxicity or tumor lysis by chimeric antigen receptor (CAR)-expressing human T or NK-92-MI transfected cells. This antibody demonstrated specific and potent cytotoxicity in vivo towards WT1-positive leukemia xenograft that was HLA-A2 restricted. In summary, T cell epitopes can provide novel targets for antibody-based therapeutics. By combining phage and yeast displays and scFv-Fc fusion platforms, a strategy for developing high affinity TCR-like antibodies could be rapidly explored for potential clinical development.