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1.  Smaller Neuroblastoma Tumors in Mice Intratumorally Treated with Hu14.18-IL2 Have More Activated Tumor-Infiltrating Lymphocytes and Better Outcome 
Hu14.18-IL2 is an immunocytokine (IC) consisting of human IL-2 linked to hu14.18 mAb, which recognizes GD2 disialoganglioside. Phase II clinical trials of intravenous-hu14.18-IL2 (IV-IC) in neuroblastoma and melanoma are underway, and have already demonstrated activity in neuroblastoma. In our Phase II trial, lower neuroblastoma burden at the time of treatment was associated with a greater likelihood of clinical response to IV-IC. We have previously shown that intratumoral-hu14.18-IL2 (IT-IC) compared to IV-IC results in enhanced local and systemic antitumor activity in tumor-bearing mice. We utilized a mouse model to investigate the impact of tumor burden on hu14.18-IL2 treatment efficacy in IV vs IT treated animals. Studies presented here describe analyses of tumor burden at initiation of treatment and its effects on treatment efficacy, survival and tumor-infiltrating leukocytes in A/J mice bearing subcutaneous NXS2 neuroblastoma. We show that smaller tumor burden at treatment initiation is associated with increased infiltration of NK and CD8+T cells and increased overall survival. NXS2 tumor shrinkage shortly after completion of the 3 days of hu14.18-IL2 treatment is necessary for long-term survival. This model demonstrates that tumor size is a strong predictor of hu14.18-IL2-induced lymphocyte infiltration and treatment outcome.
doi:10.1007/s00262-013-1430-x
PMCID: PMC3720701  PMID: 23661160
hu14.18-IL2; immunocytokine; tumor-infiltrating leukocytes; NKG2D; neuroblastoma
2.  Antitumor effects of anti-CD40/CpG immunotherapy combined with gemcitabine or 5-fluorouracil chemotherapy in the B16 melanoma model 
International immunopharmacology  2013;17(4):10.1016/j.intimp.2013.10.019.
Our previous studies demonstrated that anti-CD40 mAb (anti-CD40) can synergize with CpG oligodeoxynucleotides (CpG) to mediate antitumor effects by activating myeloid cells, such as macrophages in tumor-bearing mice. Separate teams have shown that chemotherapy with gemcitabine (GEM) or 5-fluorouracil (5-FU) can reduce tumor-induced myeloid-derived suppressor cells (MDSC) in mice. In this study we asked if the same chemotherapy regimens with GEM or 5-FU will enhance the antitumor effect of anti-CD40 and CpG. Using the model of B16 melanoma growing intraperitoneally in syngeneic C57BL/6 mice, we show that these GEM or 5-FU treatment regimens reduced MDSC in the peritoneal cavity of tumor-bearing mice. Treatment of mice with GEM or 5-FU did not significantly affect the antitumor function of macrophages as assessed in vitro. In vivo, treatment with these GEM or 5-FU regimens followed by anti-CD40/CpG resulted in antitumor effects similar to those of anti-CD40/CpG in the absence of GEM or 5-FU. Likewise, reduction of MDSC by in vivo anti-Gr-1 mAb treatment did not significantly affect anti-CD40/CpG antitumor responses. Together, the results show that the GEM or 5-FU chemotherapy regimens did not substantially affect the antitumor effects induced by anti-CD40/CpG immunotherapy.
doi:10.1016/j.intimp.2013.10.019
PMCID: PMC3873769  PMID: 24201083
anti-CD40; CpG; gemcitabine; 5-fluorouracil; immunotherapy
3.  Phase I Trial of Fenretinide Delivered Orally in a Novel Organized Lipid Complex in Patients with Relapsed/Refractory Neuroblastoma: A Report from the New Approaches to Neuroblastoma Therapy (NANT) Consortium 
Pediatric blood & cancer  2013;60(11):1801-1808.
Background
A phase I study was conducted to determine the maximum-tolerated dose, dose-limiting toxicities (DLTs), and pharmacokinetics of fenretinide (4-HPR) delivered in an oral powderized lipid complex (LXS) in patients with relapsed/refractory neuroblastoma.
Procedure
4-HPR/LXS powder (352 - 2210 mg/m2/day) was administered on Days 0 – 6, in 21-day courses, by standard 3+3 design.
Results
Thirty-two patients (median age = 8 years, range 3 – 27 years) enrolled with thirty evaluable for dose escalation. Prior therapies included stem cell transplantation/support (n = 26), 13-cis-retinoic acid (n = 22), 125/131I-MIBG (n = 13), and anti-GD2 antibody (n = 6). 170+ courses were delivered. Course 1 DLTs were a Grade 3 (n = 1) alkaline phosphatase at 352 mg/m2/day. Other major toxicities were Grade 4 (n = 1) alkaline phosphatases on Courses 5 and 6 at 774 mg/m2/day, and Grade 3 (n = 1) ALT/AST elevation on Course 2 at 1700 mg/m2/day. Of twenty-nine response-evaluable patients, six had stable disease (SD)(4 – 26 courses); four with marrow- or bone disease-only had complete responses (CR)(10 - 46 courses). 4-HPR plasma levels were several fold higher (P<0.05) than previously reported using capsular fenretinide. The Day 6 mean peak 4-HPR plasma level at 1700 mg/m2/day was 21 μM. An MTD was not reached.
Conclusions
4-HPR/LXS oral powder obtained higher plasma levels, with minimal toxicity and evidence of anti-tumor activity, than a previous capsule formulation. A recommended phase II schedule of 4-HPR/LXS powder is 1500 mg/m2/day, TID, on Days 0 – 6, of a 21-day course.
doi:10.1002/pbc.24643
PMCID: PMC4066886  PMID: 23813912
fenretinide; neuroblastoma; pediatric; powder; Lym-X-Sorb™
4.  Suppression of T-Cell Expansion by Melanoma is Exerted on Resting Cells 
Annals of surgical oncology  2011;18(13):3848-3857.
Background
Immunotherapeutic cancer protocols often rely on the ability to promote proliferative expansion of tumor-specific T-cell, but the influence of cancer on in vivo T-cell expansion remains largely undefined.
Methods
The ability of control and B16F10 melanoma-bearing C57BL/6 mice to expand lymphocytic choriomeningitis virus antigen-specific T-cell populations in response to acute viral infection was compared by using flow cytometric assays of splenocytes.
Results
The ability to expand virus-specific CD8+ and CD4+ T-cells was globally and markedly suppressed in tumor-bearing mice. Expanded cytotoxic T lymphocytes (CTLs) retained in vivo and in vitro functionality, suggesting that melanoma growth did not induce T-cell anergy. The magnitude of suppressed proliferative expansion was proportional to the extent of tumor burden. Melanoma-induced suppression of CTL expansion was correlated with upregulated apoptotic activity and hampered the induction of memory precursor effector cells. Adoptive transfer of resting LCMV antigen-specific T-cells before or after tumor establishment demonstrated that a critical period of in vivo exposure of resting T-cells to growing melanoma was responsible for the induction of suppressed expansion. This suppression was durable; surgical resection of melanoma after in vivo exposure to resting T-cells but before antigenic stimulation did not restore full expansion.
Conclusions
These data suggest that growing melanoma tumors exert a global, antigen-independent influence on resting T-cells that fundamentally reprograms their ability to undergo proliferative expansion in response to subsequent antigenic stimulation. This finding may have direct implications for T-cell-based immunotherapeutic strategies.
doi:10.1245/s10434-011-1667-6
PMCID: PMC4167760  PMID: 21465311
5.  Ab-IL2 fusion proteins mediate NK cell immune synapse formation by polarizing CD25 to the target cell-effector cell interface 
Cancer immunology, immunotherapy : CII  2011;60(12):1789-1800.
The huKS-IL2 immunocytokine (IC) consists of IL2 fused to a mAb against EpCAM, while the hu14.18-IL2 IC recognizes the GD2 disialoganglioside. They are under evaluation for treatment of EpCAM+ (ovarian) and GD2+ (neuroblastoma and melanoma) malignancies because of their proven ability to enhance tumor cell killing by antibody-dependent cell-mediated cytotoxicity (ADCC) and by antitumor cytotoxic T cells. Here, we demonstrate that huKS-IL2 and hu14.18-IL2 bind to tumor cells via their antibody components and increase adhesion and activating immune synapse (AIS) formation with NK cells by engaging the immune cells’ IL-2 receptors (IL2R). The NK leukemia cell line, NKL (which expresses high affinity IL2Rs), shows fivefold increase in binding to tumor targets when treated with IC compared to matching controls. This increase in binding is effectively inhibited by blocking antibodies against CD25, the α-chain of the IL2R. NK cells isolated from the peritoneal environment of ovarian cancer patients, known to be impaired in mediating ADCC, bind to huKS-IL2 via CD25. The increased binding between tumor and effector cells via ICs is due to the formation of AIS that are characterized by the simultaneous polarization of LFA-1, CD2 and F-actin at the cellular interface. AIS formation of peritoneal NK and NKL cells is inhibited by anti-CD25 blocking antibody and is 50–200% higher with IC versus the parent antibody. These findings demonstrate that the IL-2 component of the IC allows IL2Rs to function not only as receptors for this cytokine but also as facilitators of peritoneal NK cell binding to IC-coated tumor cells.
doi:10.1007/s00262-011-1072-9
PMCID: PMC4153733  PMID: 21792658
Immunocytokines; Natural killer; Cancer; Immunotherapy; Immune synapse
6.  Intratumoral hu14.18-IL2 (IC) Induces Local and Systemic Antitumor Effects that Involve Both Activated T- and NK cells as well as Enhanced IC Retention 
Hu14.18-IL2 is an immunocytokine (IC) consisting of human IL2 linked to hu14.18 mAb, which recognizes the GD2 disialoganglioside. Phase 2 clinical trials of intravenous hu14.18-IL2 (IV-IC) in neuroblastoma and melanoma are underway, and have already demonstrated activity in neuroblastoma. We have previously shown that intratumoral hu14.18-IL2 (IT-IC) compared to IV-IC results in enhanced antitumor activity in mouse models. Studies presented here were designed to determine the mechanisms involved in this enhanced activity and to support the future clinical testing of IT administration of immunocytokines. Improved survival and inhibition of growth of both local and distant tumors were observed in A/J mice bearing subcutaneous NXS2 neuroblastomas treated with IT-IC versus IV-IC or control mice. The local and systemic antitumor effects of IT-IC were inhibited by depletion of NK cells or T cells. IT-IC resulted in increased NKG2D receptors on intratumoral NKG2A/C/E+ NKp46+ NK cells and NKG2A/C/E+ CD8+ T cells when compared to control or IV-IC mice. NKG2D levels were augmented more in tumorinfiltrating lymphocytes (TILs) compared to splenocytes, supporting the localized nature of the intratumoral changes induced by IT-IC treatment. Prolonged retention of IC at the tumor site was seen with IT-IC compared to IV-IC. Overall, IT-IC compared to IV-IC resulted in increased numbers of activated T- and NK cells within tumors, better IC retention in the tumor, enhanced inhibition of tumor growth, and improved survival.
doi:10.4049/jimmunol.1200934
PMCID: PMC3424361  PMID: 22844125
7.  Current and Potential Uses of Immunocytokines as Cancer Immunotherapy 
Antibodies  2012;1(2):149-171.
Immunocytokines (ICs) are a class of molecules created by linking tumor-reactive monoclonal antibodies to cytokines that are able to activate immune cells. Tumor selective localization is provided by the ability of the mAb component to bind to molecules found on the tumor cell surface or molecules found selectively in the tumor microenvronment. In this way the cytokine component of the immunocytokine is selectively localized to sites of tumor and can activate immune cells with appropriate receptors for the cytokine. Immunocytokines have been made and tested by us, and others, using a variety of tumor-reactive mAbs linked to distinct cytokines. To date, the majority of clinical progress has been made with ICs that have linked human interleukin-2 (IL2) to a select number of tumor reactive mAbs that had already been in prior clinical testing as non-modified mAbs (Figure 1). Here we briefly review the background for the creation of ICs, summarize current clinical progress, emphasize mechanisms of action for ICs that are distinct from those of their constituent components, and present some directions for future development and testing.
doi:10.3390/antib1020149
PMCID: PMC3954573  PMID: 24634778
immunocytokine; ADCC; cancer; immunotherapy
8.  Intratumoral delivery of low doses of anti-CD40 mAb combined with monophosphoryl lipid A induces local and systemic antitumor effects in immunocompetent and T cell-deficient mice 
In this study, an agonistic anti-CD40 monoclonal antibody was combined with monophosphoryl lipid A (MPL), a nontoxic derivative of LPS and agonist of toll-like receptor 4, to assess the immunomodulatory and antitumor synergy between the two agents in mice. Anti-CD40 was capable of priming macrophages to subsequent ex vivo activation by MPL in immunocompetent and T cell-depleted mice. Intraperitoneal injections of anti-CD40+MPL induced additive to synergistic suppression of poorly immunogenic B16-F10 melanoma growing subcutaneously in syngeneic mice. When anti-CD40+MPL were injected directly into the subcutaneous tumor, the combination treatment was more effective, even with a 25-fold reduction in dose. Low-dose intratumoral treatment also slowed the growth of a secondary tumor growing simultaneously at a distant, untreated site. Antitumor effects were also induced in immunodeficient SCID mice and in T cell-depleted C57BL/6 mice. Taken together, our results show that the antitumor effects of anti-CD40 are enhanced by subsequent treatment with MPL, even in T cell-deficient hosts. These preclinical data suggest that an anti-CD40+MPL combined regimen is appropriate for clinical testing in human patients, including cancer patients that may be immunosuppressed from prior chemotherapy.
doi:10.1097/CJI.0b013e3182780f61
PMCID: PMC3521848  PMID: 23211623
immunotherapy; anti-CD40; monophosphoryl lipid A; macrophages; intratumoral
9.  Intratumoral hu14.18–IL-2 (IC) Induces Local and Systemic Antitumor Effects That Involve Both Activated T and NK Cells As Well As Enhanced IC Retention 
hu14.18–IL-2 (IC) is an immunocytokine consisting of human IL-2 linked to hu14.18 mAb, which recognizes the GD2 disialoganglioside. Phase 2 clinical trials of i.v. hu14.18–IL-2 (i.v.-IC) in neuroblastoma and melanoma are underway and have already demonstrated activity in neuroblastoma. We showed previously that intratumoral hu14.18–IL-2 (IT-IC) results in enhanced antitumor activity in mouse models compared with i.v.-IC. The studies presented in this article were designed to determine the mechanisms involved in this enhanced activity and to support the future clinical testing of intratumoral administration of immunocytokines. Improved survival and inhibition of growth of both local and distant tumors were observed in A/J mice bearing s.c. NXS2 neuroblastomas treated with IT-IC compared with those treated with i.v.-IC or control mice. The local and systemic antitumor effects of IT-IC were inhibited by depletion of NK cells or T cells. IT-IC resulted in increased NKG2D receptors on intratumoral NKG2A/C/E+ NKp46+ NK cells and NKG2A/C/E+ CD8+ T cells compared with control mice or mice treated with i.v.-IC. NKG2D levels were augmented more in tumor-infiltrating lymphocytes compared with splenocytes, supporting the localized nature of the intratumoral changes induced by IT-IC treatment. Prolonged retention of IC at the tumor site was seen with IT-IC compared with i.v.-IC. Overall, IT-IC resulted in increased numbers of activated T and NK cells within tumors, better IC retention in the tumor, enhanced inhibition of tumor growth, and improved survival compared with i.v.-IC.
doi:10.4049/jimmunol.1200934
PMCID: PMC3424361  PMID: 22844125
10.  Increasing the Clinical Efficacy of NK and Antibody-Mediated Cancer Immunotherapy: Potential Predictors of Successful Clinical Outcome Based on Observations in High-Risk Neuroblastoma 
Disease recurrence is frequent in high-risk neuroblastoma (NBL) patients even after multi-modality aggressive treatment [a combination of chemotherapy, surgical resection, local radiation therapy, autologous stem cell transplantation, and cis-retinoic acid (CRA)]. Recent clinical studies have explored the use of monoclonal antibodies (mAbs) that bind to disialoganglioside (GD2), highly expressed in NBL, as a means to enable immune effector cells to destroy NBL cells via antibody-dependent cell-mediated cytotoxicity (ADCC). Preclinical data indicate that ADCC can be more effective when appropriate effector cells are activated by cytokines. Clinical studies have pursued this by administering anti-GD2 mAb in combination with ADCC-enhancing cytokines (IL2 and GM-CSF), a regimen that has demonstrated improved cancer-free survival. More recently, early clinical studies have used a fusion protein that consists of the anti-GD2 mAb directly linked to IL2, and anti-tumor responses were seen in the Phase II setting. Analyses of genes that code for receptors that influence ADCC activity and natural killer (NK) cell function [Fc receptor (FcR), killer immunoglublin-like receptor (KIR), and KIR-ligand (KIR-L)] suggest patients with anti-tumor activity are more likely to have certain genotype profiles. Further analyses will need to be conducted to determine whether these genotypes can be used as predictive markers for favorable therapeutic outcome. In this review, we discuss factors that affect response to mAb-based tumor therapies such as hu14.18-IL2. Many of our observations have been made in the context of NBL; however, we will also include some observations made with mAbs targeting other tumor types that are consistent with results in NBL. Therefore, we hypothesize that the NBL observations discussed here may also be relevant to mAb therapy for other cancers, in which ADCC is known to play a role.
doi:10.3389/fphar.2012.00091
PMCID: PMC3353262  PMID: 22623917
ADCC; KIR; FcR; neuroblastoma; immunocytokine; mAb; IL2
11.  T-cell-independent Antitumor Effects of CD40 Ligation 
CD40 ligation has been shown to induce antitumor effects in mice and cancer patients. Most of the studies have focused on the ability of an agonistic anti-CD40 mAb to either directly kill CD40-positive tumor cells or activate T-cell immune responses. In this review the authors focus on the ability of CD40 ligation to activate antitumor effector mechanisms of the cells of innate immunity such as macrophages and NK cells.
doi:10.3109/08830185.2012.698337
PMCID: PMC3537496  PMID: 22804571
anti-CD40; macrophages; NK cells
12.  Antitumor Activity of Hu14.18-IL2 in Patients With Relapsed/Refractory Neuroblastoma: A Children's Oncology Group (COG) Phase II Study 
Journal of Clinical Oncology  2010;28(33):4969-4975.
Purpose
The hu14.18-IL2 fusion protein consists of interleukin-2 molecularly linked to a humanized monoclonal antibody that recognizes the GD2 disialoganglioside expressed on neuroblastoma cells. This phase II study assessed the antitumor activity of hu14.18-IL2 in two strata of patients with recurrent or refractory neuroblastoma.
Patients and Methods
Hu14.18-IL2 was given intravenously (12 mg/m2/daily) for 3 days every 4 weeks for patients with disease measurable by standard radiographic criteria (stratum 1) and for patients with disease evaluable only by [123I]metaiodobenzylguanidine (MIBG) scintigraphy and/or bone marrow (BM) histology (stratum 2). Response was established by independent radiology review as well as BM histology and immunocytology, and durability was assessed by repeat evaluation after more than 3 weeks.
Results
Thirty-nine patients were enrolled (36 evaluable). No responses were seen in stratum 1 (n = 13). Of 23 evaluable patients in stratum 2, five patients (21.7%) responded; all had a complete response (CR) of 9, 13, 20, 30, and 35+ months duration. Grade 3 and 4 nonhematologic toxicities included capillary leak, hypoxia, pain, rash, allergic reaction, elevated transaminases, and hyperbilirubinemia. Two patients required dopamine for hypotension, and one patient required ventilatory support for hypoxia. Most toxicities were reversible within a few days of completing a treatment course and were expected based on phase I results.
Conclusion
Patients with disease evaluable only by MIBG and/or BM histology had a 21.7% CR rate to hu14.8-IL2, whereas patients with bulky disease did not respond. Hu14.18-IL2 warrants further testing in children with nonbulky high-risk neuroblastoma.
doi:10.1200/JCO.2009.27.8861
PMCID: PMC3020698  PMID: 20921469
13.  Anti-tumor and immunomodulatory activity of resveratrol in vitro and its potential for combining with cancer immunotherapy 
International immunopharmacology  2011;11(11):1877-1886.
We evaluated the anti-tumor effect of Resveratrol (RV) on M21 and NXS2 tumor cell lines and its immunosuppressive activity on human and murine immune cells to determine the potential for combining RV and immunotherapy. In vitro, concentrations of RV ≥ 25mcM, inhibited cell proliferation, blocked DNA synthesis and induced G1 phase arrest in tumor and immune cells. RV at 12–50mcM inhibited antibody dependent cell mediated cytotoxicity (ADCC) of tumor cells facilitated by the hu14.18-IL2 immunocytokine (IC). The in vivo anti-tumor and immunomodulating activity of RV given systemically were assessed in mice. Results showed that this RV regimen inhibited the growth of NXS2 tumors in vivo but did not appear to interfere with blood cell count, splenocyte or macrophage function. Thus, RV may be a candidate for combining with immunotherapy.
doi:10.1016/j.intimp.2011.07.019
PMCID: PMC3204008  PMID: 21854876
Resveratrol; Immunocytokine; anti-GD2 antibody; Neuroblastoma; hu14.18-IL2
14.  Clinical Cancer Therapy by NK Cells via Antibody-Dependent Cell-Mediated Cytotoxicity 
Natural killer (NK) cells are powerful effector cells that can be directed to eliminate tumor cells through tumor-targeted monoclonal antibodies (mAbs). Some tumor-targeted mAbs have been successfully applied in the clinic and are included in the standard of care for certain malignancies. Strategies to augment the antitumor response by NK cells have led to an increased understanding of how to improve their effector responses. Next-generation reagents, such as molecularly modified mAbs and mAb-cytokine fusion proteins (immunocytokines, ICs) designed to augment NK-mediated killing, are showing promise in preclinical and some clinical settings. Continued research into the antitumor effects induced by NK cells and tumor-targeted mAbs suggests that additional intrinsic and extrinsic factors may influence the antitumor response. Therefore more research is needed that focuses on evaluating which NK cell and tumor criteria are best predictive of a clinical response and which combination immunotherapy regimens to pursue for distinct clinical settings.
doi:10.1155/2011/379123
PMCID: PMC3110303  PMID: 21660134
15.  The anti-tumor effect of resveratrol alone or in combination with immunotherapy in a neuroblastoma model 
We investigated the anti-tumor effect of peritumoral resveratrol in combination with immunotherapy in vivo in neuroblastoma-bearing mice. Subcutaneous NXS2 tumors were induced in A/J mice. On day 10 some mice received 15mcg of intravenous immunocytokine for 5 days, mice received of 20mg of peritumoral resveratrol twice a week (starting on day 12) for a total of 5 injections, and a separate group received a combination of both regimens. Tumor progression and survival were assessed every 3–4 days. Blood and primary tumor tissue samples were collected on day 20 for Complete Blood Count and CD45 immunohistochemistry and histology, respectively. The primary tumor regressed in all mice receiving peritumoral resveratrol. Most of these mice receiving peritumoral resveratrol alone developed metastatic tumors and recurrence of the primary tumor after cessation of therapy. When resveratrol and immunocytokine regimens were combined, 61% of the mice receiving this combination therapy resolved their primary tumors and survived without developing metastatic tumors, compared to 15% and 13% receiving resveratrol or immunocytokine alone, respectively. None of the therapeutic regimes prevented lymphocyte infiltration or affected the Complete Blood Count. Greater necrosis was observed microscopically in tumors from mice receiving the combination therapy. These results demonstrate that the combination therapy of peritumoral resveratrol plus intravenous immunocytokine provides better anti-tumor effects in this model than either therapy alone.
doi:10.1007/s00262-011-0971-0
PMCID: PMC3094716  PMID: 21340652
Resveratrol; Immunocytokine; anti-GD2 antibody; Neuroblastoma; hu14.18-IL2
16.  Radiofrequency Ablation Combined with KS-IL2 Immunocytokine (EMD 273066) Results in an Enhanced Antitumor Effect Against Murine Colon Adenocarcinoma 
Purpose
Radiofrequency ablation (RFA) is a common treatment modality for surgically unresectable tumors. However, there is a high rate of both local and systemic recurrence.
Experimental Design
In this pre-clinical study, we sought to enhance the antitumor effect of RFA by combining it with huKS-IL2 immunocytokine (tumor-specific monoclonal antibody fused to interleukin 2) in mice bearing CT26-KS colon adenocarcinoma. Mice were treated with RFA, huKS-IL2 via intratumoral injection, or combination therapy.
Results
Treatment of mice bearing subcutaneous tumors with RFA and huKS-IL2 resulted in significantly greater tumor growth suppression and enhanced survival, compared to mice treated with RFA or huKS-IL2 alone. When subtherapeutic regimens of RFA or huKS-IL2 were used, tumors progressed in all treated mice. In contrast, the combination of RFA and immunocytokine resulted in complete tumor resolution in 50% of mice. Treatment of a tumor with RFA and intratumoral huKS-IL2 also demonstrated antitumor effects against a distant untreated tumor. Tumor-free mice following treatment with RFA and huKS-IL2 demonstrated immunological memory based on their ability to reject subsequent challenges of CT26-KS and the more aggressive parental CT26 tumors. Flow cytometry analysis of tumor-reactive T-cells from mice with complete tumor resolution demonstrated that treatment with RFA and huKS-IL2 resulted in a greater proportion of cytokine-producing CD4 T-cells and CD8 T-cells compared to mice treated with RFA or huKS-IL2 alone.
Conclusions
These results show that the addition of huKS-IL2 to RFA significantly enhances the antitumor response in this murine model, resulting in complete tumor resolution and induction of immunological memory.
doi:10.1158/1078-0432.CCR-09-0110
PMCID: PMC2735865  PMID: 19638464
radiofrequency ablation; immunocytokine; colon cancer; IL2
17.  Tumor-associated myeloid cells can be activated in vitro and in vivo to mediate antitumor effects 
Cancer immunology, immunotherapy : CII  2012;61(10):1683-1697.
Tumor growth is often accompanied by the accumulation of myeloid cells in the tumors and lymphoid organs. These cells can suppress T cell immunity, thereby posing an obstacle to T cell-targeted cancer immunotherapy. In this study, we tested the possibility of activating tumor-associated myeloid cells to mediate antitumor effects. Using the peritoneal model of B16 melanoma, we show that peritoneal cells (PEC) in tumor-bearing mice (TBM) had reduced ability to secrete nitric oxide (NO) following in vitro stimulation with interferon gamma and lipopolysaccharide, as compared to PEC from control mice. This reduced function of PEC was accompanied by the influx of CD11b+ Gr-1+ myeloid cells to the peritoneal cavity. Nonadherent PEC were responsible for most of the NO production in TBM, whereas in naïve mice NO was mainly secreted by adherent CD11b+ F4/80+ macrophages. Sorted CD11b+ Gr-1− monocytic and CD11b+ Gr-1+ granulocytic PEC from TBM had a reduced ability to secrete NO following in vitro stimulation (compared to naïve PEC), but effectively suppressed proliferation of tumor cells in vitro. In vivo, treatment of mice bearing established peritoneal B16 tumors with anti-CD40 and CpG resulted in activation of tumor-associated PEC, reduction in local tumor burden and prolongation of mouse survival. Inhibition of NO did not abrogate the antitumor effects of stimulated myeloid cells. Taken together, the results indicate that in tumor-bearing hosts, tumor-associated myeloid cells can be activated to mediate antitumor effects.
doi:10.1007/s00262-012-1236-2
PMCID: PMC3572779  PMID: 22392192
Myeloid cells; Anti-CD40; CpG; Immunotherapy
18.  Naïve mouse macrophages become activated following recognition of L5178Y lymphoma cells via concurrent ligation of CD40, NKG2D, and CD18 molecules1 
Under different circumstances, tumors can inhibit or activate macrophage (Mϕ) effector functions. We studied the mechanisms of tumor-Mϕ interactions leading to Mϕ activation. The results show that L5178Y mouse T-cell lymphoma cells can prime naïve mouse Mϕ to subsequent LPS stimulation, resulting in increased NO production and anti-lymphoma effects in vitro. L5178Y cells, but not naïve splenocytes, primed Mϕ respond to ligation of TLR4 but not TLR9. L5178Y-primed Mϕ incubated with LPS showed down-regulation of CD40 and up-regulation of NKG2D expression. While L5178Y T cell lymphoma cells prime naïve mouse Mϕ, mouse A20 B cell lymphoma, B16 melanoma, or NIH-3T3 fibroblasts, and human Jurkat T cell lymphoma, Daudi B cell lymphoma, or M21 melanoma tumor cells lines all failed to prime mouse Mϕ. Neither L5178Y-conditioned supernatants nor co-culture of Mϕ and L5178Y cells in transwells resulted in priming, indicating that direct L5178Y cell-Mϕ contact was needed. Several receptor-ligand pairs are reciprocally expressed on Mϕ and L5178Y cell membranes and can be potentially involved in Mϕ priming. Of these, the CD40-CD154 pair played the most important role, as blocking the interaction of these molecules substantially reduced in vitro Mϕ priming. Furthermore, simultaneous blocking of interactions between CD40–CD154, NKG2D–H60, and CD18–ICAM-1/-2 led to complete abrogation of Mϕ-mediated NO secretion and complete inhibition of Mϕ-mediated tumor cell cytostasis. The priming of Mϕ to LPS with L5178Y cells was also observed in vivo. These results suggest that contact with certain tumor cells via CD40, NKG2D, and CD18 molecules on the Mϕ may facilitate Mϕ-mediated anti-tumor immune surveillance.
doi:10.4049/jimmunol.0800443
PMCID: PMC2757113  PMID: 19201847
Monocytes/macrophages; Innate immunity; Tumor recognition
19.  Phase II trial of hul4.18-IL2 for patients with metastatic melanoma 
Cancer immunology, immunotherapy : CII  2012;61(12):2261-2271.
Phase I testing of the hu14.18-IL2 immunocytokine in melanoma patients (pts) showed immune activation, reversible toxicities, and a maximal tolerated dose of 7.5 mg/m2/day. In this phase II study, fourteen pts with measurable metastatic melanoma were scheduled to receive hu14.18-IL2 at 6 mg/m2/day as 4-hour intravenous infusions on days 1, 2 and 3 of each 28 day cycle. Pts with stable disease (SD) or regression following cycle 2 could receive 2 additional treatment cycles. The primary objective was to evaluate anti-tumor activity and response duration. Secondary objectives evaluated adverse events and immunologic activation. All pts received 2 cycles of treatment. One pt had a partial response (PR) [1 PR of 14 pts = response rate of 7.1%; confidence interval 0.2%−33.9%] and 4 pts had SD and received cycles 3 & 4. The PR and SD responses lasted 3–4 months. All toxicities were reversible and those resulting in dose reduction included grade 3 hypotension (2 pts) and grade 2 renal insufficiency with oliguria (1 pt). Pts had a peripheral blood lymphocytosis on day 8 and increased C-reactive protein. While one PR in 14 pts met protocol criteria to proceed to stage 2 and enter 16 additional pts, we suspended stage 2 due to limited availability of hul 4.18-IL2 at that time and the brief duration of PR and SD. We conclude that subsequent testing of hu14.18-IL2 should involve melanoma patients with minimal residual disease based on compelling preclinical data and the confirmed immune activation with some antitumor activity in this study.
doi:10.1007/s00262-012-1286-5
PMCID: PMC3502633  PMID: 22678096
Immunocytokine; Advanced Melanoma; Immunotherapy; Interleukin-2; anti-ganglioside antibody; phase II clinical trial
20.  Phase II study of oral capsular 4-hydroxyphenylretinamide (4-HPR/fenretinide) in pediatric patients with refractory or recurrent neuroblastoma: A report from the Children’s Oncology Group NSC #374551; IND# 40294 
Purpose
To determine the response rate to oral capsular fenretinide in children with recurrent or biopsy proven refractory high-risk neuroblastoma.
Experimental Design
Patients received 7 days of fenretinide: 2475 mg/m2/day divided TID (<18 years) or 1800 mg/m2/day divided BID (≥18 years) every 21 days for a maximum of 30 courses. Patients with stable or responding disease after course 30 could request additional compassionate courses. Best response by course 8 was evaluated in Stratum 1 (measurable disease on CT/MRI +/− bone marrow and/or MIBG avid sites) and Stratum 2 (bone marrow and/or MIBG avid sites only).
Results
Sixty-two eligible patients, median age 5 years (range 0.6–19.9), were treated in Stratum 1 (n=38) and Stratum 2 (n=24). One partial response (PR) was seen in Stratum 2 (n=24 evaluable). No responses were seen in Stratum 1 (n=35 evaluable). Prolonged stable disease (SD) was seen in 7 patients in Stratum 1 and 6 patients in Stratum 2 for 4–45+ (median 15) courses. Median time to progression was 40 days (range 17–506) for Stratum 1 and 48 days (range 17–892) for Stratum 2. Mean 4-HPR steady state trough plasma concentrations were 7.25 µM (coefficient of variation 40–56%) at day 7 course 1. Toxicities were mild and reversible.
Conclusions
Although neither stratum met protocol criteria for efficacy, 1 PR + 13 prolonged SD occurred in 14/59 (24%) of evaluable patients. Low bioavailability may have limited fenretinide activity. Novel fenretinide formulations with improved bioavailability are currently in pediatric Phase I studies.
doi:10.1158/1078-0432.CCR-11-0995
PMCID: PMC3207022  PMID: 21908574
fenretinide; neuroblastoma; Phase II; ANBL0321
21.  In Situ Conversion of Melanoma Lesions into Autologous Vaccine by Intratumoral Injections of α-gal Glycolipids 
Cancers  2010;2(2):773-793.
Autologous melanoma associated antigens (MAA) on murine melanoma cells can elicit a protective anti-tumor immune response following a variety of vaccine strategies. Most require effective uptake by antigen presenting cells (APC). APC transport and process internalized MAA for activation of anti-tumor T cells. One potential problem with clinical melanoma vaccines against autologous tumors may be that often tumor cells do not express surface markers that label them for uptake by APC. Effective uptake of melanoma cells by APC might be achieved by exploiting the natural anti-Gal antibody which constitutes ~1% of immunoglobulins in humans. This approach has been developed in a syngeneic mouse model using mice capable of producing anti-Gal. Anti-Gal binds specifically to α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R). Injection of glycolipids carrying α-gal epitopes (α-gal glycolipids) into melanoma lesions results in glycolipid insertion into melanoma cell membranes, expression of α-gal epitopes on the tumor cells and binding of anti-Gal to these epitopes. Interaction between the Fc portions of bound anti-Gal and Fcγ receptors on APC induces effective uptake of tumor cells by APC. The resulting anti-MAA immune response can be potent enough to destroy distant micrometastases. A clinical trial is now open testing effects of intratumoral α-gal glycolipid injections in melanoma patients.
doi:10.3390/cancers2020773
PMCID: PMC3475649  PMID: 23087817
Melanoma immunotherapy; anti-Gal antibody; α-gal glycolipids; α1,3-galactosyltransferase; Fcγ receptors; antigen presenting cells
22.  Intratumoral Immunocytokine Treatment Results in Enhanced Antitumor Effects 
Cancer immunology, immunotherapy : CII  2008;57(12):1891-1902.
Immunocytokines (IC), consisting of tumor-specific monoclonal antibodies fused to the immunostimulatory cytokine interleukin 2 (IL2), exert significant antitumor effects in several murine tumor models. We investigated whether intratumoral (IT) administration of IC provided enhanced antitumor effects against subcutaneous tumors. Three unique ICs (huKS-IL2, hu14.18-IL2, and GcT84.66-IL2) were administered systemically or IT to evaluate their antitumor effects against tumors expressing the appropriate IC-targeted tumor antigens. The effect of IT injection of the primary tumor on a distant tumor was also evaluated. Here, we show that IT injection of IC resulted in enhanced antitumor effects against B16-KSA melanoma, NXS2 neuroblastoma, and human M21 melanoma xenografts when compared to intravenous (IV) IC injection. Resolution of both primary and distant subcutaneous tumors, and a tumor-specific memory response were demonstrated following IT treatment in immunocompetent mice bearing NXS2 tumors. The IT effect of huKS-IL2 IC was antigen-specific, enhanced compared to IL2 alone, and dose-dependent. Hu14.18-IL2 also showed greater IT effects than IL2 alone. The antitumor effect of IT IC did not always require T cells since IT IC induced antitumor effects against tumors in both SCID and nude mice. Localization studies using radiolabeled 111In-GcT84.66-IL2 IC confirmed that IT injection resulted in a higher concentration of IC at the tumor site than IV administration. In conclusion, we suggest that IT IC is more effective than IV administration against palpable tumors. Further testing is required to determine how to potentially incorporate IT administration of IC into an antitumor regimen that optimizes local and systemic anticancer therapy.
doi:10.1007/s00262-008-0519-0
PMCID: PMC2574976  PMID: 18438664
intratumoral; immunocytokine; hu14.18-IL2; huKS-IL2; melanoma; neuroblastoma
23.  A Phase I Clinical Trial of the hu14.18-IL2 (EMD 273063) as a Treatment for Children with Refractory or Recurrent Neuroblastoma and Melanoma: a Study of the Children’s Oncology Group 
Purpose
Evaluate the clinical safety, toxicity, immune activation/modulation, and maximal tolerated dose of hu14.18-IL2 (EMD 273063) in pediatric patients with recurrent/refractory neuroblastoma and other GD2-positive solid tumors.
Experimental Design
Twenty-seven pediatric patients with recurrent/refractory neuroblastoma and one with melanoma were treated with a humanized anti-GD2 monoclonal antibody linked to human interleukin 2 (IL-2). Cohorts of patients received hu14.18-IL2, administered i.v. over 4 hours for three consecutive days, at varying doses. Patients with stable disease, partial, or complete responses were eligible to receive up to three additional courses of therapy.
Results
Most of the clinical toxicities were anticipated and similar to those reported with IL-2 and anti-GD2 monoclonal antibody therapy and to those noted in the initial phase I study of hu14.18-IL2 in adults with metastatic melanoma. The maximal tolerated dose was determined to be 12 mg/m2/d, with agent-related dose-limiting toxicities of hypotension, allergic reaction, blurred vision, neutropenia, thrombocytopenia, and leukopenia. Three patients developed dose-limiting toxicity during course 1; seven patients in courses 2 to 4. Two patients required dopamine for hypotension. There were no treatment-related deaths, and all toxicity was reversible. Treatment with hu14.18-IL2 led to immune activation/modulation as evidenced by elevated serum levels of soluble IL-2 receptor α (sIL2Rα) and lymphocytosis. The median half-life of hu14.18-IL2 was 3.1 hours. There were no measurable complete or partial responses to hu14.18-IL2 in this study; however, three patients did show evidence of antitumor activity.
Conclusion
Hu14.18-IL2 (EMD 273063) can be administered safely with reversible toxicities in pediatric patients at doses that induce immune activation. A phase II clinical trial of hu14.18-IL2, administered at a dose of 12 mg/m2/d × 3 days repeated every 28 days, will be done in pediatric patients with recurrent/refractory neuroblastoma.
doi:10.1158/1078-0432.CCR-05-2000
PMCID: PMC2587020  PMID: 16551859
24.  Anti-GD2 Antibody with GM-CSF, Interleukin-2, and Isotretinoin for Neuroblastoma 
The New England journal of medicine  2010;363(14):1324-1334.
BACKGROUND
Preclinical and preliminary clinical data indicate that ch14.18, a monoclonal antibody against the tumor-associated disialoganglioside GD2, has activity against neuroblastoma and that such activity is enhanced when ch14.18 is combined with granulocyte–macrophage colony-stimulating factor (GM-CSF) or interleukin-2. We conducted a study to determine whether adding ch14.18, GM-CSF, and interleukin-2 to standard isotretinoin therapy after intensive multimodal therapy would improve outcomes in high-risk neuroblastoma.
METHODS
Patients with high-risk neuroblastoma who had a response to induction therapy and stem-cell transplantation were randomly assigned, in a 1:1 ratio, to receive standard therapy (six cycles of isotretinoin) or immunotherapy (six cycles of isotretinoin and five concomitant cycles of ch14.18 in combination with alternating GM-CSF and interleukin-2). Event-free survival and overall survival were compared between the immunotherapy group and the standard-therapy group, on an intention-to-treat basis.
RESULTS
A total of 226 eligible patients were randomly assigned to a treatment group. In the immunotherapy group, a total of 52% of patients had pain of grade 3, 4, or 5, and 23% and 25% of patients had capillary leak syndrome and hypersensitivity reactions, respectively. With 61% of the number of expected events observed, the study met the criteria for early stopping owing to efficacy. The median duration of follow-up was 2.1 years. Immunotherapy was superior to standard therapy with regard to rates of event-free survival (66±5% vs. 46±5% at 2 years, P = 0.01) and overall survival (86±4% vs. 75±5% at 2 years, P = 0.02 without adjustment for interim analyses).
CONCLUSIONS
Immunotherapy with ch14.18, GM-CSF, and interleukin-2 was associated with a significantly improved outcome as compared with standard therapy in patients with high-risk neuroblastoma.
doi:10.1056/NEJMoa0911123
PMCID: PMC3086629  PMID: 20879881
25.  Soluble Interleukin-2 Receptor α Activation in a Children’s Oncology Group Randomized Trial of Interleukin-2 Therapy for Pediatric Acute Myeloid Leukemia 
Pediatric blood & cancer  2011;57(3):398-405.
Purpose
To assess associations of soluble IL-2 receptor alpha (sIL-2rα) concentration with outcomes in pediatric acute myeloid leukemia in a phase 3 trial of IL-2 therapy.
Procedures
We randomized 289 children with AML in first remission after intensive chemotherapy to receive IL-2 infused on days 0-3 and 8-17 (IL-2 group) or no further therapy (AML control group). We measured sequential serum sIL-2rα concentrations in both groups before, during and after therapy in both groups and in reference controls without AML.
Results
Before treatment, mean sIL-2rα concentrations were similar in the IL-2 group and AML controls, but significantly higher than in reference controls. Both AML groups experienced reduction in sIL-2rα concentration after chemotherapy. Thereafter in the IL-2 group, mean sIL-2rα concentration increased from 2669 pg/ml before IL-2 to 15,534 pg/ml on day 4 (p<0.001) and 10,585 pg/ml on day 18 (p<0.001). In the control group sIL-2rα concentration did not change after 28 days of follow-up. Five-year disease-free survival (DFS) was 51% in the IL-2 group and 58% in the controls (p=0.489) and overall survival was 70% and 73% respectively (p=0.727).
Conclusion
SIL-2r α concentration was elevated in AML at diagnosis and tended to normalize after chemotherapy. IL-2 infusion significantly increased sIL-2rα concentration, but did not improve DFS or survival in pediatric AML. Furthermore, sIL-2rα concentration was not predictive of outcome before, during or after treatment for AML.
doi:10.1002/pbc.22966
PMCID: PMC3172052  PMID: 21681921
acute myeloid leukemia; interleukin-2; soluble interleukin-2 receptor alpha

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