Intratumoral Copper Modulates PD-L1 Expression and Influences Tumor Immune Evasion.

Therapeutic checkpoint antibodies blocking programmed death receptor 1/programmed death ligand 1 (PD-L1) signaling have radically improved clinical outcomes in cancer. However, the regulation of PD-L1 expression on tumor cells is still poorly understood. Here we show that intratumoral copper levels influence PD-L1 expression in cancer cells. Deep analysis of the The Cancer Genome Atlas database and tissue microarrays showed strong correlation between the major copper influx transporter copper transporter 1 (CTR-1) and PD-L1 expression across many cancers but not in corresponding normal tissues. Copper supplementation enhanced PD-L1 expression at mRNA and protein levels in cancer cells and RNA sequencing revealed that copper regulates key signaling pathways mediating PD-L1-driven cancer immune evasion. Conversely, copper chelators inhibited phosphorylation of STAT3 and EGFR and promoted ubiquitin-mediated degradation of PD-L1. Copper-chelating drugs also significantly increased the number of tumor-infiltrating CD8+ T and natural killer cells, slowed tumor growth, and improved mouse survival. Overall, this study reveals an important role for copper in regulating PD-L1 and suggests that anticancer immunotherapy might be enhanced by pharmacologically reducing intratumor copper levels. SIGNIFICANCE: These findings characterize the role of copper in modulating PD-L1 expression and contributing to cancer immune evasion, highlighting the potential for repurposing copper chelators as enhancers of antitumor immunity. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/19/4129/F1.large.jpg.

Prussian blue nanoparticle-based antigenicity and adjuvanticity trigger robust antitumor immune responses against neuroblastoma.

We describe the synthesis of CpG oligodeoxynucleotide-coated Prussian blue nanoparticles (CpG-PBNPs) that function as a nanoimmunotherapy for neuroblastoma, a common childhood cancer. These CpG-PBNPs increase the antigenicity and adjuvanticity of the treated tumors, ultimately driving robust antitumor immunity through a multi-pronged mechanism. CpG-PBNPs are synthesized using a facile layer-by-layer coating scheme resulting in nanoparticles that exhibit monodisperse size distributions and multiday stability without cytotoxicity. The strong intrinsic absorption of PBNPs in the CpG-PBNPs enables ablative photothermal therapy (CpG-PBNP-PTT) that triggers tumor cell death, as well as the release of tumor antigens to increase antigenicity. Simultaneously, the CpG coating functions as an exogenous molecular adjuvant that complements the endogenous adjuvants released by the CpG-PBNP-PTT (e.g. ATP, calreticulin, and HMGB1). In cell culture, coating NPs with CpG increases immunogenicity while maintaining the photothermal activity of PBNPs. When administered in a syngeneic, Neuro2a-based, murine model of neuroblastoma, CpG-PBNP-PTT results in complete tumor regression in a significantly higher proportion (70% at 60 days) of treated animals relative to controls. Furthermore, the long-term surviving, CpG-PBNP-PTT-treated animals reject Neuro2a rechallenge, suggesting that this therapy generates immunological memory. Our findings point to the importance of simultaneous cytotoxicity, antigenicity, and adjuvanticity to generate robust and persistent antitumor immune responses against neuroblastoma.

Novel Immunotherapeutic Approaches for Neuroblastoma and Malignant Melanoma.

Neuroblastoma (NB) and malignant melanoma (MM), tumors of pediatric age and adulthood, respectively, share a common origin, both of them deriving from the neural crest cells. Although NB and MM have a different behavior, in respect to age of onset, primary tissue involvement and metastatic spread, the prognosis for high stage-affected patients is still poor, in spite of aggressive treatment strategies and the huge amount of new discovered biological knowledge. For these reasons researchers are continuously attempting to find out new treatment options, which in a near future could be translated to the clinical practice. In the last two decades, a strong effort has been spent in the field of translational research of immunotherapy which led to satisfactory results. Indeed, several immunotherapeutic clinical trials have been performed and some of them also resulted beneficial. Here, we summarize preclinical studies based on immunotherapeutic approaches applied in models of both NB and MM.

Interleukin 2 with anti-GD2 antibody ch14.18/CHO (dinutuximab beta) in patients with high-risk neuroblastoma (HR-NBL1/SIOPEN): a multicentre, randomised, phase 3 trial.

BACKGROUND: Immunotherapy with the chimeric anti-GD2 monoclonal antibody dinutuximab, combined with alternating granulocyte-macrophage colony-stimulating factor and intravenous interleukin-2 (IL-2), improves survival in patients with high-risk neuroblastoma. We aimed to assess event-free survival after treatment with ch14.18/CHO (dinutuximab beta) and subcutaneous IL-2, compared with dinutuximab beta alone in children and young people with high-risk neuroblastoma. METHODS: We did an international, open-label, phase 3, randomised, controlled trial in patients with high-risk neuroblastoma at 104 institutions in 12 countries. Eligible patients were aged 1-20 years and had MYCN-amplified neuroblastoma with stages 2, 3, or 4S, or stage 4 neuroblastoma of any MYCN status, according to the International Neuroblastoma Staging System. Patients were eligible if they had been enrolled at diagnosis in the HR-NBL1/SIOPEN trial, had completed the multidrug induction regimen (cisplatin, carboplatin, cyclophosphamide, vincristine, and etoposide, with or without topotecan, vincristine, and doxorubicin), had achieved a disease response that fulfilled prespecified criteria, had received high-dose therapy (busulfan and melphalan or carboplatin, etoposide, and melphalan) and had received radiotherapy to the primary tumour site. In this component of the trial, patients were randomly assigned (1:1) to receive dinutuximab beta (20 mg/m2 per day as an 8 h infusion for 5 consecutive days) or dinutuximab beta plus subcutaneous IL-2 (6 × 106 IU/m2 per day on days 1-5 and days 8-12 of each cycle) with the minimisation method to balance randomisation for national groups and type of high-dose therapy. All participants received oral isotretinoin (160 mg/m2 per day for 2 weeks) before the first immunotherapy cycle and after each immunotherapy cycle, for six cycles. The primary endpoint was 3-year event-free survival, analysed by intention to treat. This trial was registered with ClinicalTrials.gov, number NCT01704716, and EudraCT, number 2006-001489-17, and recruitment to this randomisation is closed. FINDINGS: Between Oct 22, 2009, and Aug 12, 2013, 422 patients were eligible to participate in the immunotherapy randomisation, of whom 406 (96%) were randomly assigned to a treatment group (n=200 to dinutuximab beta and n=206 to dinutuximab beta with subcutaneous IL-2). Median follow-up was 4·7 years (IQR 3·9-5·3). Because of toxicity, 117 (62%) of 188 patients assigned to dinutuximab beta and subcutaneous IL-2 received their allocated treatment, by contrast with 160 (87%) of 183 patients who received dinutuximab beta alone (p<0·0001). 3-year event-free survival was 56% (95% CI 49-63) with dinutuximab beta (83 patients had an event) and 60% (53-66) with dinutuximab beta and subcutaneous IL-2 (80 patients had an event; p=0·76). Four patients died of toxicity (n=2 in each group); one patient in each group while receiving immunotherapy (n=1 congestive heart failure and pulmonary hypertension due to capillary leak syndrome; n=1 infection-related acute respiratory distress syndrome), and one patient in each group after five cycles of immunotherapy (n=1 fungal infection and multi-organ failure; n=1 pulmonary fibrosis). The most common grade 3-4 adverse events were hypersensitivity reactions (19 [10%] of 185 patients in the dinutuximab beta group vs 39 [20%] of 191 patients in the dinutuximab plus subcutaneous IL-2 group), capillary leak (five [4%] of 119 vs 19 [15%] of 125), fever (25 [14%] of 185 vs 76 [40%] of 190), infection (47 [25%] of 185 vs 64 [33%] of 191), immunotherapy-related pain (19 [16%] of 122 vs 32 [26%] of 124), and impaired general condition (30 [16%] of 185 vs 78 [41%] of 192). INTERPRETATION: There is no evidence that addition of subcutaneous IL-2 to immunotherapy with dinutuximab beta, given as an 8 h infusion, improved outcomes in patients with high-risk neuroblastoma who had responded to standard induction and consolidation treatment. Subcutaneous IL-2 with dinutuximab beta was associated with greater toxicity than dinutuximab beta alone. Dinutuximab beta and isotretinoin without subcutaneous IL-2 should thus be considered the standard of care until results of ongoing randomised trials using a modified schedule of dinutuximab beta and subcutaneous IL-2 are available. FUNDING: European Commission 5th Frame Work Grant, St. Anna Kinderkrebsforschung, Fondation ARC pour la recherche sur le Cancer.

Anti-GD2 mAbs and next-generation mAb-based agents for cancer therapy.

Tumor-specific monoclonal antibodies (mAbs) have demonstrated efficacy in the clinic, becoming an important approach for cancer immunotherapy. Due to its limited expression on normal tissue, the GD2 disialogangloside expressed on neuroblastoma cells is an excellent candidate for mAb therapy. In 2015, dinutuximab (an anti-GD2 mAb) was approved by the US FDA and is currently used in a combination immunotherapeutic regimen for the treatment of children with high-risk neuroblastoma. Here, we review the extensive preclinical and clinical development of anti-GD2 mAbs and the different mechanisms by which they mediate tumor cell killing. In addition, we discuss different mAb-based strategies that capitalize on the targeting ability of anti-GD2 mAbs to potentially deliver, as monotherapy, or in combination with other treatments, improved antitumor efficacy.

Results of induction chemotherapy in children older than 18 months with stage-4 neuroblastoma treated with an adaptive-to-response modified N7 protocol (mN7)

Purpose. We report the response rate in children older than 18 months with stage 4 Neuroblastoma, using a modified dose-intensive, response-adaptive, induction mN7 protocol. Methods. From 2005 to 2012, 24 patients were treated with the mN7 protocol. Phase 1 included five MSKCC N7 cycles and surgery and two high-dose cyclophosphamide-topotecan (HD-CT) cycles for those who did not achieve complete remission (CR) and negative bone marrow (BM) minimal residual disease (MRD) status (CR+MRD-). Phase 2 consisted of myeloablative doses of topotecan, thiotepa and carboplatin plus hyperfractionated RT. Phase 3 included isotretinoin and 3F8 immunotherapy plus GM-CSF. BM MRD was monitored using GD2 synthase, PHOX2B and cyclin D1 mRNAs. Results. After 3 cycles, all patients showed BM complete histological clearance and 6 (25 %) were MRD-. Twenty of 21 s-look surgeries achieved macroscopic complete resection. After 5 cycles and surgery, 123I-MIBG scan was negative in 15 (62.5 %) cases, BM disease by histology was negative in 23 (96 %) and 10 (42 %) patients were MRD-. Twelve (50 %) pts were in CR, 2 in very good partial response (VGPR), 9 partial response (PR) and one had progressive disease. With 2 HD-CT extra cycles, 17 (71 %) pts achieved CR+MRD- status moving to phase 2. Overall and event-free survival at 3 years for the 17 patients who achieved CR+MRD- is 65 and 53 %, respectively, median follow-up 47 months. Seven (29 %) patients never achieved CR+MRD-. Univariate Cox regression analysis shows CR+MRD- status after mN7 induction as the only statistically significant prognostic factor to predict overall survival. Conclusions. mN7 induction regimen produced a CR+MRD- rate of 71 %. CR+MRD- status following induction was the only predictive marker of long-term survival (AU)

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Veno-occlusive disease of the liver in the absence of elevation in bilirubin in pediatric patients after hematopoietic stem cell transplantation.

Veno-occlusive disease (VOD) of the liver is a well-described and significant complication of hematopoietic stem cell transplantation (HSCT), with limited successful therapeutic options in severe cases. Prompt diagnosis and initiation of treatment is crucial to restrict the extent of disease. However, a subset of patients may not meet all current diagnostic criteria at presentation, and waiting for these to be met may delay therapy. We retrospectively reviewed 794 HSCT patients treated at our institution between 2003 and 2013, identifying 17 (2.1%) who developed VOD. Of these, 5 (29%) were noted to have an absence of elevated bilirubin at the time of VOD diagnosis and reversal of portal venous flow on ultrasound. Median total and conjugated bilirubin at VOD diagnosis were 1.0 and 0.2 mg/dL, respectively. All 5 patients were subsequently diagnosed with multiorgan failure associated with VOD, including 1 with encephalopathy. Four were treated with intravenous high-dose methylprednisolone (500 mg/m(2) per dose every 12 hours for 6 doses). One patient received defibrotide therapy in addition to steroids and another supportive care alone. VOD resolved in 4 of 5 patients, with median time to resolution of VOD, defined as recovery of all organ function and normalization of bilirubin and portal venous flow, of 8 days. Two patients died later from progressive primary disease and chronic graft-versus-host disease, respectively. We conclude that a high index of suspicion for VOD should be maintained in patients despite lack of bilirubin elevation in the presence of other diagnostic criteria such as hepatomegaly, abdominal pain, ascites, or weight gain. Early ultrasound evaluation in these patients may lead to more timely diagnosis and therapeutic interventions.

Mechanisms of the antitumor activity of human Vγ9Vδ2 T cells in combination with zoledronic acid in a preclinical model of neuroblastoma.

Low expression of surface major histocompatibility complex (MHC) class I molecules and defects in antigen processing machinery make human neuroblastoma (NB) cells appropriate targets for MHC unrestricted immunotherapeutic approaches. Human T-cell receptor (TCR) Vγ9Vδ2 lymphocytes exert MHC-unrestricted antitumor activity and are activated by phosphoantigens, whose expression in cancer cells is increased by aminobisphosphonates. With this background, we have investigated the in vivo anti-NB activity of human Vγ9Vδ2 lymphocytes and zoledronic acid (ZOL). SH-SY-5Y human NB cells were injected in the adrenal gland of immunodeficient mice. After 3 days, mice received ZOL or human Vγ9Vδ2 T cells or both agents by intravenous administration once a week for 4 weeks. A significantly improved overall survival was observed in mice receiving Vγ9Vδ2 T cells in combination with ZOL. Inhibition of tumor cell proliferation, angiogenesis and lymphangiogenesis, and increased tumor cell apoptosis were detected. Vγ9Vδ2 T lymphocytes were attracted to NB-tumor masses of mice receiving ZOL where they actively modified tumor microenvironment by producing interferon-γ (IFN-γ), that in turn induced CXCL10 expression in NB cells. This study shows that human Vγ9Vδ2 T cells and ZOL in combination inhibit NB growth in vivo and may provide the rationale for a phase I clinical trial in patients with high-risk NB.

Polyphenon [corrected] E enhances the antitumor immune response in neuroblastoma by inactivating myeloid suppressor cells.

PURPOSE: Neuroblastoma is a rare childhood cancer whose high risk, metastatic form has a dismal outcome in spite of aggressive therapeutic interventions. The toxicity of drug treatments is a major problem in this pediatric setting. In this study, we investigated whether Polyphenon E, a clinical grade mixture of green tea catechins under evaluation in multiple clinical cancer trials run by the National Cancer Institute (Bethesda, MD), has anticancer activity in mouse models of neuroblastoma. EXPERIMENTAL DESIGN: We used three neuroblastoma models: (i) transgenic TH-MYCN mouse developing spontaneous neuroblastomas; (ii) nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenotransplanted with human SHSY5Y cells; and (iii) A/J mice transplanted with syngeneic Neuro 2A cells. Mice were randomized in control and Polyphenon E-drinking groups. Blood from patients with neuroblastoma and normal controls was used to assess the phenotype and function of myeloid cells. RESULTS: Polyphenon E reduced the number of tumor-infiltrating myeloid cells, and inhibited the development of spontaneous neuroblastomas in TH-MYCN transgenic mice. In therapeutic models of neuroblastoma in A/J, but not in immunodeficient NOD/SCID mice, Polyphenon E inhibited tumor growth by acting on myeloid-derived suppressor cells (MDSC) and CD8 T cells. In vitro, Polyphenon E impaired the development and motility of MDSCs and promoted differentiation to more neutrophilic forms through the 67 kDa laminin receptor signaling and induction of granulocyte colony-stimulating factor. The proliferation of T cells infiltrating a patient metastasis was reactivated by Polyphenon E. CONCLUSIONS: These findings suggest that the neuroblastoma-promoting activity of MDSCs can be manipulated pharmacologically in vivo and that green tea catechins operate, at least in part, through this mechanism.

High-dose iodine-131-metaiodobenzylguanidine with haploidentical stem cell transplantation and posttransplant immunotherapy in children with relapsed/refractory neuroblastoma.

We evaluated the feasibility and efficacy of using high-dose iodine-131-metaiodobenzylguanidine ((131)I-MIBG) followed by reduced-intensity conditioning (RIC) and transplantation of T cell-depleted haploidentical peripheral blood stem cells (designated haplo-SCT) to treat relapsing/refractory neuroblastoma (RRNB). Five RRNB patients were enrolled: 4 with relapse (3 after autologous SCT) and 1 with induction therapy failure. The preparative regimen included high-dose (131)I-MIBG on day -20, followed by fludarabine (Flu), thiotepa, and melphalan (Mel) from day -8 to -1. Granulocyte-colony stimulating factor (G-CSF)-mobilized, T cell-depleted haploidentical paternal stem cells were infused on day 0 together with cultured donor mesenchymal stem cells. A single dose of rituximab was given on day +1. After cessation of short immunosuppression (mycophenolate, OKT3), 4 children received donor lymphocyte infusion (DLI). (131)I-MIBG infusion and RIC were well tolerated. All patients engrafted. No primary acute graft-versus-host disease (aGVHD) was observed. Four children developed aGVHD after DLI and were successfully treated. Analysis of immunologic recovery showed fast reappearance of potentially immunocompetent natural killer (NK) and T cells, which might have acted as effector cells responsible for the graft-versus-tumor (GVT) effect. Two children are alive and well, with no evidence of disease 40 and 42 months after transplantation. One patient experienced late progression with new bone lesions (sternum) 38 months after haplo-SCT, and is being treated with local irradiation and reinstituted DLI. One patient rejected the graft, was rescued with autologous backup, and died of progressive disease 5 months after transplantation. Another child relapsed 7 months after transplantation and died 5 months later. High-dose (131)I-MIBG followed by RIC and haplo-SCT for RRNB is feasible and promising, because 2 of 5 children on that regimen achieved long-lasting remission. Further studies are needed to evaluate targeted therapy and immune-mediated tumor control in high-risk neuroblastoma.

Long term survival in anti-Hu associated adult neuroblastoma.

We report on a young lady suffering from adult neuroblastoma and anti-Hu associated paraneoplastic encephalomyelitis (PEM) with a tumour free survival of nine years up to now. Treatment included tumour surgery, radiation, high dose chemotherapy, and stem cell transplantation. Serological testing demonstrated a marked decline in anti-Hu antibody titres under therapy, and subsequent disappearance of the antibody 31 months after second tumour resection.

[Immunotherapy of poor-prognosis neuroblastoma in children: from bench to bedside]. / Immunothérapie des neuroblastomes de mauvais pronostic chez l'enfant: depuis les concepts fondamentaux jusqu'aux essais cliniques de phase I-II.

During the last two decades, improvements in the induction and consolidation treatment phases in patients with high-risk neuroblastoma have not translated into significant increases in survival rates. Efforts to improve outcome have used high-dose chemotherapy with stem cell rescue and more recently, differentiating (retinoids) and antiangiogenic agents. In parallel, immunotherapy has become an increasingly important part of the treatment of high-risk neuroblastoma. We review here the biological concepts underlying these new approaches and their clinical applications, with a particular emphasis on applications that manipulate the immune system, including monoclonal antibodies, gene-modified tumor cells (vaccines) or immune effectors.

Detection of microscopic disease: comparing histology, immunocytology, and RT-PCR of tyrosine hydroxylase, GAGE, and MAGE.

BACKGROUND: We first explored the use of multiple molecular markers to overcome tumor heterogeneity. Sixty-seven neuroblastoma (NB) tumors were tested for the expression of GAGE, MAGE-2, MAGE-2, MAGE-3, and MAGE-4 by RT-PCR and then chemiluminescence; 82% of tumors had detectable GAGE, and 88% expressed at least one of the four MAGE genes. PROCEDURE AND RESULTS: By combining GAGE and MAGE, 64 of 67 (95%) of tumors became detectable; 17 of 67 coexpressed all five molecular markers. Neither GAGE nor MAGE expression correlated with stage. GAGE was found to have the broadest (18 of 18) expression among stage 4 tumors. Two hundred fifty-nine bone marrows from 99 patients were then studied for NB positivity by four detection methods: histology, immunocytology, and molecular detection by GAGE and tyrosine hydroxylase (TH) mRNA. Two hundred seven samples were NB-positive by one detection method. All four techniques were comparable in detecting tumor cells at diagnosis and at relapse. GAGE and immunocytology were far more sensitive than histology and TH mRNA when marrows were sampled during chemotherapy and at the time of clinical remission. CONCLUSIONS: By combining multiple molecular markers and independent screening techniques, we may be able to overcome tumor heterogeneity and expedite the detection of microscopic disease in the clinical management of neuroblastoma.

Engineered herpes simplex virus expressing IL-12 in the treatment of experimental murine brain tumors.

Genetically engineered, neuroattenuated herpes simplex viruses (HSVs) expressing various cytokines can improve survival when used in the treatment of experimental brain tumors. These attenuated viruses have both copies of gamma(1)34.5 deleted. Recently, we demonstrated increased survival of C57BL/6 mice bearing syngeneic GL-261 gliomas when treated with an engineered HSV expressing IL-4, as compared with treatment with the parent construct (gamma(1)34. 5(-)) alone or with a virus expressing IL-10. Herein, we report construction of a conditionally replication-competent mutant expressing both subunits of mIL-12 (M002) and its evaluation in a syngeneic neuroblastoma murine model. IL-12 induces a helper T cell subset type 1 response, which may induce more durable antitumor effects. In vitro studies showed that, when infected with M002, both Vero cells and murine Neuro-2a neuroblastoma cells produced physiologically relevant levels of IL-12 heterodimers, as determined by ELISA. M002 was cytotoxic for Neuro-2a cells and human glioma cell lines U251MG and D54MG. Neurotoxicity studies, as defined by plaque-forming units/LD(50), performed in HSV-1-sensitive A/J strain mice found that M002 was not toxic even at high doses. When evaluated in an intracranial syngeneic neuroblastoma murine model, median survival of M002-treated animals was significantly longer than the median survival of animals treated with R3659, the parent gamma(1)34.5(-) mutant lacking any cytokine gene insert. Immunohistochemical analysis of M002-treated tumors identified a pronounced influx of CD4(+) T cells and macrophages as well as CD8(+) cells when compared with an analysis of R3659-treated tumors. We conclude that M002 produced a survival benefit via oncolytic effects combined with immunologic effects meditated by helper T cells of subset type 1.

Corticosteroid administration does not affect viral oncolytic activity, but inhibits antitumor immunity in replication-competent herpes simplex virus tumor therapy.

A multimutated, conditionally replicating herpes simplex virus type 1, G207, has been developed as an effective means of treating human malignant brain tumors. We have shown that intraneoplastic inoculation of G207 induces a specific and systemic antitumor immune response that plays an important role in the antitumor activity, in addition to the direct oncolytic action of G207. Since a large number of malignant brain tumor patients are treated with corticosteroids, it is important to evaluate whether the therapeutic efficacy of G207 is affected by corticosteroid-induced immunosuppression. For a tumor model, we used G207-permissive N18 murine neuroblastoma cells implanted subcutaneously in syngeneic A/J mice. Intraneoplastic inoculation of G207 (10(7) PFU) induced significant suppression of tumor growth whether or not dexamethasone (5 mg/kg) was given. When dexamethasone was given for an extensive time (16 days starting on day -2), all G207-treated mice showed tumor growth despite initial shrinkage, whereas in the saline group, four of eight of the G207-treated mice were cured. Dexamethasone administration significantly reduced serum neutralizing antibodies against G207 at 14 and 21 days after intraneoplastic G207 inoculation. However, there was no difference between the dexamethasone and saline groups in terms of the amount of infectious G207 isolated from tumors. Dexamethasone administration completely abolished G207-induced cytotoxic T lymphocyte activity against N18 cells. These results indicate that the oncolytic activity of G207 is retained under corticosteroid administration. However, intensive immunosuppression may diminish the long-term efficacy of G207 owing to suppression of tumor-specific cytotoxic T lymphocyte induction.

Enhancement of interleukin-2 immunotherapy with L-arginine.

Nutrient substrates have been shown to enhance cell-mediated immunity, but their role as adjuvants to immunotherapy has not been previously determined. This study evaluated L-arginine as an essential substrate for optimal generation of lymphokine-activated killer (LAK) cells. This experiment also assessed supplemental dietary L-arginine as a means to potentiate the host antitumor response to interleukin-2 (IL-2) in a murine neuroblastoma (NRB) model. A/J mice received 1% arginine or isonitrogenous 1.7% glycine in addition to a regular diet 14 days before subcutaneous inoculation with C1300 NRB cells. Twenty-four hours later, animals received low (1 x 10(6) U/kg three times a day) or high (3 x 10(6) U/kg three times a day) doses of IL-2 or saline intraperitoneally for 4 days. On days 4 and 10 post-C1300 NRB inoculation, mice were killed for assessment of natural killer cell and tumor specific cytotoxicity. Remaining animals were followed for tumor incidence, tumor growth, and duration of host survival. Interleukin-2 therapy in mice receiving dietary arginine compared with those receiving glycine resulted in significantly augmented natural killer cell cytotoxicity (day 4) and generation of specific tumoricidal mechanisms (day 10). The addition of dietary arginine to low-dose IL-2 therapy significantly diminished C1300 NRB engraftment (p less than 0.05) and growth (p less than 0.001) and prolonged the duration of host survival (p less than 0.05) compared with the glycine treatment group. In vitro studies demonstrated that L-arginine is an essential substrate for optimal generation of LAK cells. Thus, supplemental dietary L-arginine enhances lymphocyte cytotoxic mechanisms and potentiates IL-2 immunotherapy.

Immunologic effects of arginine supplementation in tumor-bearing and non-tumor-bearing hosts.

Supplemental dietary arginine has anti-tumor properties but the degree and mechanisms are unclear. In non-tumor-bearing CBA/J mice (n = 60), 1% arginine supplementation significantly enhanced thymic weight, spleen cell mitogenesis, and interferon-activated natural killer cell activity; no further enhancement was observed with 2% or 4% supplementation. Supplemental 1% arginine, when compared with 1.7% glycine, enhanced interferon-induced natural killer cell activity, lymphokine-activated killer cell generation, and macrophage cytotoxicity. In A/J mice (n = 420), bearing either a moderately immunogenic (C1300) or weakly immunogenic (TBJ) murine neuroblastoma, 1% arginine significantly (p less than 0.05) retarded tumor growth and prolonged median survival time compared with glycine or no supplementation. Dietary arginine enhanced T-cell function and significantly increased responsiveness to autologous C1300 tumor in a mixed lymphocyte tumor cell culture (MLTC). The immunomodulatory effects of arginine provide nutritional and immunologic support of the tumor-bearing host and may be helpful when given concommitant with immunotherapy.

Arginine, protein malnutrition, and cancer.

The amino acid arginine has anabolic and immunostimulatory properties. This study evaluated the potency of arginine in limiting the severe nutritional and immunological insults of protein calorie malnutrition and increasing tumor load. In protein-depleted A/J mice (n = 340) bearing either an immunogenic (C1300) or poorly immunogenic (TBJ) neuroblastoma, arginine supplementation [1%] significantly augmented T lymphocyte responses (mitogenesis, interleukin-2 production) compared with both a glycine-supplemented and nonsupplemented group. Arginine supplementation significantly retarded the growth of C1300 and prolonged median host survival. These results correlated with augmented autologous mixed lymphocyte tumor cell responses and enhanced specific cytotoxicity. This anti-tumor effect was not demonstrated in mice bearing TBJ where both arginine and glycine stimulated tumor growth compared with nonsupplemented mice. There was no significant difference between arginine and glycine in preservation of carcass weight. These studies suggest that the immunostimulatory effects of arginine are not due to supplemental nitrogen and that an associated antitumor effect is dependent on tumor antigenicity.