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Abstract 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

Abstract 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.

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.

Desmoplastic small round cell tumor (DSRCT) is a rare pediatric cancer caused by the EWSR1-WT1 fusion oncogene. Despite initial response to chemotherapy, DSRCT has a recurrence rate of over 80% leading to poor patient prognosis with a 5-year survival rate of only 15–25%. Owing to the rarity of DSRCT, sample scarcity is a barrier in understanding DSRCT biology and developing effective therapies. Utilizing a novel pair of primary and recurrent DSRCTs, we present the first map of DSRCT genomic breakpoints and the first comparison of gene expression alterations between primary and recurrent DSRCT. Our genomic breakpoint map includes the lone previously published DSRCT genomic breakpoint, the breakpoint from our novel primary/recurrent DSRCT pair, as well as the breakpoints of five available DSRCT cell lines and five additional DSRCTs. All mapped breakpoints were unique and most breakpoints included a 1–3 base pair microhomology suggesting microhomology-mediated end-joining as the mechanism of translocation fusion and providing novel insights into the etiology of DSRCT. Through RNA-sequencing analysis, we identified altered genes and pathways between primary and recurrent DSRCTs. Upregulated pathways in the recurrent tumor included several DNA repair and mRNA splicing-related pathways, while downregulated pathways included immune system function and focal adhesion. We further found higher expression of the EWSR1-WT1 upregulated gene set in the recurrent tumor as compared to the primary tumor and lower expression of the EWSR1-WT1 downregulated gene set, suggesting the EWSR1-WT1 fusion continues to play a prominent role in recurrent tumors. The identified pathways including upregulation of DNA repair and downregulation of immune system function may help explain DSRCT’s high rate of recurrence and can be utilized to improve the understanding of DSRCT biology and identify novel therapies to both help prevent recurrence and treat recurrent tumors.

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.

The cure rate for metastatic or relapsed osteosarcoma has not substantially improved over the past decades despite the exploitation of multimodal treatment approaches, allowing long-term survival in less than 30% of cases. Patients with osteosarcoma often develop resistance to chemotherapeutic agents, where personalized targeted therapies should offer new hope. T cell immunotherapy as a complementary or alternative treatment modality is advancing rapidly in general, but its potential against osteosarcoma remains largely unexplored. Strategies incorporating immune checkpoint inhibitors (ICIs), chimeric antigen receptor (CAR) modified T cells, and T cell engaging bispecific antibodies (BsAbs) are being explored to tackle relapsed or refractory osteosarcoma. However, osteosarcoma is an inherently heterogeneous tumor, both at the intra- and inter-tumor level, with no identical driver mutations. It has a pro-tumoral microenvironment, where bone cells, stromal cells, neovasculature, suppressive immune cells, and a mineralized extracellular matrix (ECM) combine to derail T cell infiltration and its anti-tumor function. To realize the potential of T cell immunotherapy in osteosarcoma, an integrated approach targeting this complex ecosystem needs smart planning and execution. Herein, we review the current status of T cell immunotherapies for osteosarcoma, summarize the challenges encountered, and explore combination strategies to overcome these hurdles, with the ultimate goal of curing osteosarcoma with less acute and long-term side effects.

Yu et al. identify marked differences in the telomere and immunologic profiles of neuroblastoma cell lines displaying MES and ADRN lineage characteristics. They show that pharmacologically converting ADRN into MES cells triggers associated switch in telomere/immunologic protein profiles whereas inhibiting telomerase activity in ADRN cells induces reversible conversion into MES cells.

Desmoplastic Small Round Cell Tumor (DSRCT) is a rare and aggressive pediatric cancer driven by the EWSR1-WT1 fusion oncogene. Combinations of chemotherapy, radiation and surgery are not curative, and the 5-years survival rate is less than 25%. One potential explanation for refractoriness is the existence of a cancer stem cell (CSC) subpopulation able escape current treatment modalities. However, no study to-date has examined the role of CSCs in DSRCT or established in vitro culture conditions to model this subpopulation. In this study, we investigated the role of stemness markers in DSRCT survival and metastasis, finding that elevated levels of SOX2 and NANOG are associated with worse survival in sarcoma patients and are elevated in metastatic DSRCT tumors. We further develop the first in vitro DSRCT CSC model which forms tumorspheres, expresses increased levels of stemness markers (SOX2, NANOG, KLF4, and OCT4), and resists doxorubicin chemotherapy treatment. This model is an important addition to the DSRCT tool kit and will enable investigation of this critical DSRCT subpopulation. Despite lower sensitivity to chemotherapy, the DSRCT CSC model remained sensitive to knockdown of the EWSR1-WT1 fusion protein, suggesting that future therapies directed against this oncogenic driver have the potential to treat both DSRCT bulk tumor and CSCs.

Abstract Background Tumor 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. Methods We 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. Results BsAb-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. Conclusion Reducing 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.

Abstract 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.

Genomic analysis of neuroblastoma has revealed important disease etiology. In this study, the authors assembled whole genome, exome and transcriptome data from over 700 neuroblastomas and identified molecular signatures correlated with age, and rare, potentially targetable variants overlooked in smaller cohorts.

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.

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

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.

Abstract: Acute myeloid leukemia (AML), the most common acute leukemia in adults and the second most common cancer in children, is still a lethal disease in the majority of patients, but immunologic approaches have improved outcome. Bispecific antibodies (BsAbs) are novel immunotherapeutics that can redirect immune cells against AML. We now report a tetravalent (2+2) humanized BsAb in the immunoglobulin G light chain single chain fragment variable [IgG(L)-scFv] format to engage polyclonal T cells to kill CD33+ AML targets. In vitro, this BsAb demonstrated strong antigen-specific T-cell–dependent cell-mediated cytotoxicity (TDCC) with an 50% effective concentration (EC50) in the femtomolar range that translated into treatment of established human AML IV xenografts in vivo. Importantly, it could redirect intraperitoneally injected T cells to ablate established and rapidly growing extramedullary subcutaneous AML xenografts in vivo. Furthermore, internalization of CD33 upon BsAb binding was identical to that of a bivalent (1+1) heterodimer, both being substantially less than anti-CD33 IgG. In contrast to the heterodimer, the tetravalent IgG-scFv BsAb was >10-fold more efficient in TDCC of AML cells in vitro and in vivo. This BsAb did not react with and did not kill CD38–CD34+ hematopoietic stem cells from cord blood. We conclude that the novel anti-CD33 IgG(L)-scFv BsAb construct reported here is a potential candidate for clinical development.

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.

Gangliosides have been considered to modulate cell signals in the microdomain of the cell membrane, lipid/rafts, or glycolipid-enriched microdomain/rafts (GEM/rafts). In particular, cancer-associated gangliosides were reported to enhance the malignant properties of cancer cells. In fact, GD2-positive (GD2+) cells showed increased proliferation, invasion, and adhesion, compared with GD2-negative (GD2−) cells. However, the precise mechanisms by which gangliosides regulate cell signaling in GEM/rafts are not well understood. In order to analyze the roles of ganglioside GD2 in the malignant properties of melanoma cells, we searched for GD2-associating molecules on the cell membrane using the enzyme-mediated activation of radical sources combined with mass spectrometry, and integrin β1 was identified as a representative GD2-associating molecule. Then, we showed the physical association of GD2 and integrin β1 by immunoprecipitation/immunoblotting. Close localization was also shown by immuno-cytostaining and the proximity ligation assay. During cell adhesion, GD2+ cells showed multiple phospho-tyrosine bands, i.e., the epithelial growth factor receptor and focal adhesion kinase. The knockdown of integrin β1 revealed that the increased malignant phenotypes in GD2+ cells were clearly cancelled. Furthermore, the phosphor-tyrosine bands detected during the adhesion of GD2+ cells almost completely disappeared after the knockdown of integrin β1. Finally, immunoblotting to examine the intracellular distribution of integrins during cell adhesion revealed that large amounts of integrin β1 were localized in GEM/raft fractions in GD2+ cells before and just after cell adhesion, with the majority being localized in the non-raft fractions in GD2− cells. All these results suggest that GD2 and integrin β1 cooperate in GEM/rafts, leading to enhanced malignant phenotypes of melanomas.

Longevity of antibody responses has been attributed to persistence of plasma cells in mice. Here the authors provide human data in support of this model by immunoglobulin sequencing bone marrow sections from two human donors over 6.5 years to show temporal stability of plasma cell clonotypes, but not other B cells.

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.

Natural killer (NK) cell-mediated antibody-dependent toxicity is a potent mechanism of action of the anti-GD2 murine monoclonal antibody 3F8 (m3F8). Killer immunoglobulin-like receptor (KIR) and HLA genotypes modulate NK activity and are key prognostic markers in m3F8-treated patients with neuroblastoma. Endogenous NK-cells are suppressed in the setting of high tumor burden and chemotherapy. Allogeneic NK-cells however, demonstrate potent anti-neuroblastoma activity. We report on the results of a phase I clinical trial of haploidentical NK-cells plus m3F8 administered to patients with high-risk neuroblastoma after conditioning chemotherapy. The primary objective was to determine the maximum tolerated NK-cell dose (MTD). Secondary objectives included assessing anti-neuroblastoma activity and its relationship to donor-recipient KIR/HLA genotypes, NK function, and donor NK chimerism. Patients received a lymphodepleting regimen prior to infusion of haploidentical CD3-CD56+ NK-cells, followed by m3F8. Overall and progression free survival (PFS) were assessed from the time of first NK-cell dose. Univariate Cox regression assessed relationship between dose and outcomes. Thirty-five patients received NK-cells at one of five dose levels ranging from 10×106 CD56+cells/kg had improved PFS (HR: 0.36, 95%CI: 0.15–0.87, p = 0.022). Patient NK-cells displayed high NKG2A expression, leading to inhibition by HLA-E-expressing neuroblastoma cells. Adoptive NK-cell therapy in combination with m3F8 is safe and has anti-neuroblastoma activity at higher cell doses.

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

Neuroblastoma, a malignancy of multipotent embryonic neural crest cells, is the most common extracranial solid cancer in childhood and most common cancer in infancy. Cellular phenotype has been shown to be an important determinant of the malignant potential in human neuroblastoma cells and tumors. Whereas neuroblastic (N-type) are moderately malignant and nonneuronal (S-type) cells are nonmalignant, I-type stem cells are highly tumorigenic, irrespective of N-myc amplification status. In the present study, we sought to determine which genes were overexpressed in the I-type cells which might characterize and maintain the stem cell state and/or malignancy of human neuroblastoma cancer stem cells. We used a microarray platform to compare the steady-state expression levels of mRNAs from 13 human neuroblastoma cell lines representing the three cellular phenotypes. Using qRT-PCR and Western blot analyses, we identified seven genes whose expression is consistently elevated exclusively in neuroblastoma cancer stem cells: CD133, KIT, NOTCH1, GPRC5C, PIGF2, TRKB, and LNGFR. Moreover, we show that the genes are phenotype specific, as differentiation of I-type BE(2)-C cells to either an N- or S-type morphology results in significantly reduced mRNA expression. Finally, we show that NOTCH1 plays an important role in maintaining the stem cell phenotype. The identification and characterization of these genes, elevated in highly malignant neuroblastoma stem cells, could provide the basis for developing novel therapies for treatment of this lethal childhood cancer.

Chimeric antigen receptors (CAR) and bispecific antibodies (BsAb) are two powerful immunotherapy approaches for retargeting lymphocytes toward cancer cells. Despite their success in lymphoblastic leukemia, solid tumors have been more recalcitrant. Identifying therapeutic barriers facing CAR-modified (CART) or BsAb-redirected T (BsAb-T) cells should facilitate their clinical translation to solid tumors. Novel lentiviral vectors containing low-affinity or high-affinity 4-1BB second-generation anti-GD2 (disialoganglioside) CARs were built to achieve efficient T cell transduction. The humanized anti-GD2 × CD3 BsAb using the IgG-scFv platform was described previously. CART and BsAb-engaged T cells were tested for viability, proliferation, and activation/exhaustion marker expression, and in vitro cytotoxicity against GD2(+) tumor cells. The antitumor effect of CAR-grafted and BsAb-T cells was compared in a human melanoma xenograft model. The majority of high CAR density T cells were depleted upon exposure to GD2(+) target cells while the BsAb-T cells survived. The in vitro cytotoxicity of the surviving CART cells was inferior to that of the BsAb-T cells. Using low-affinity CARs, inclusion of the 4-1BB co-stimulatory domain or exclusion of a co-stimulatory domain, or blocking PD1 did not prevent CART cell depletion. Both CART cells and BsAb-T cells penetrated established subcutaneous human melanoma xenografts; while both induced tumor regression, BsAb was more efficient. The fate of T cells activated by BsAb differs substantially from that by CAR, translating into a more robust antitumor effect both in vitro and in vivo.

T-cell-based therapies have emerged as one of the most clinically effective ways to target solid and non-solid tumors. HER2 is responsible for the oncogenesis and treatment resistance of several human solid tumors. As a member of the HER family of tyrosine kinase receptors, its over-activity confers unfavorable clinical outcome. Targeted therapies directed at this receptor have achieved responses, although development of resistance is common. We explored a novel HER2/CD3 bispecific antibody (HER2-BsAb) platform that while preserving the anti-proliferative effects of trastuzumab, it recruits and activates non-specific circulating T-cells, promoting T cell tumor infiltration and ablating HER2(+) tumors, even when these are resistant to standard HER2-targeted therapies. Its in vitro tumor cytotoxicity, when expressed as EC50, correlated with the surface HER2 expression in a large panel of human tumor cell lines, irrespective of lineage or tumor type. HER2-BsAb-mediated cytotoxicity was relatively insensitive to PD-1/PD-L1 immune checkpoint inhibition. In four separate humanized mouse models of human breast cancer and ovarian cancer cell line xenografts, as well as human breast cancer and gastric cancer patient-derived xenografts (PDXs), HER2-BsAb was highly effective in promoting T cell infiltration and suppressing tumor growth when used in the presence of human peripheral blood mononuclear cells (PBMC) or activated T cells (ATC). The in vivo and in vitro antitumor properties of this BsAb support its further clinical development as a cancer immunotherapeutic.

Cellular heterogeneity is a hallmark of human neuroblastoma tumors and cell lines. Within a single neuroblastoma are cells from distinct neural crest lineages whose relative abundance is significant for prognosis. We postulate that a self-renewing multipotent tumor stem cell, which gives rise to diverse cell lineages, is the malignant progenitor of this cancer. To test this hypothesis, we have established 22 cloned, phenotypically homogeneous populations of the three major cell types from 17 neuroblastoma cell lines. In vitro, malignant neuroblastoma stem cells, termed 1-type (intermediate type), have distinct morphologic, biochemical, differentiative, and tumorigenic properties. I-type cells express features of both neuroblastic (N) cells (scant cytoplasm, neuritic processes, neurofilaments, pseudoganglia, and granin and neurotransmitter enzyme expression) and substrate-adherent (S) cells (extensive cytoplasm and vimentin and CD44 expression). Moreover, they show bidirectional differentiation to either N or S cells when induced by specific agents. I-type cells are significantly more malignant than N- or S-type cells, with four- to five-fold greater plating efficiencies in soft agar and six-fold higher tumorigenicity in athymic mice. Differences in malignant potential are unrelated to N-myc amplification/overexpression or the ability to digest and migrate through the extracellular matrix. Immunocytochemical analyses of a small series of tumors reveal that frequency of cells coexpressing N and S cell markers correlates with poor prognosis. Thus, I-type stem cells may be instrumental in the genesis and growth of tumors in the patient. Their unique biology deserves attention and further investigation.