Whole tumor cell vaccine with irradiated S180 cells as adjuvant.

Whole tumor cell vaccines have been widely studied and remain promising cancer immunotherapies. In the present study, we discovered that vaccination with irradiated mouse sarcoma S180 tumor cells stimulated robust antitumor immunity to autologous tumor cells in both syngenic and allogenic mice. The antitumor activity requires both T and B cells, but not NK cells. When a mouse lung carcinoma (TC-1) whole tumor cell vaccine was combined with the S180 vaccine, the antitumor immunity against live TC-1 tumor cells is significantly enhanced compared to a TC-1 whole cell vaccine alone. This antitumor immunity not only prevents live tumor challenge but also eradicates existing tumor cells. A similar phenomenon was also observed when S180 vaccine was combined with LL2 Lewis lung carcinoma tumor cells. Therefore, S180 vaccine may serve as an adjuvant for other whole tumor cell vaccines.

Fermented Noni exudate (fNE): a mediator between immune system and anti-tumor activity.

The anti-tumor activity of Morinda citrifolia fruit juice (Noni) has been previously reported. However, the mechanism behind this activity remains unknown. In the present study, we studied the anti-tumor activity of fermented Noni exudate (fNE) and demonstrated that intraperitoneal injection of this material significantly increased the percentages of granulocytes and NK cells in the peripheral blood, peritoneum, and spleen. Furthermore, in preventive and treatment settings, fNE injection induced complete tumor rejection in normal C57BL/6J mice, partial tumor rejection in C57 nude mice lacking functional lymphocytes, and no tumor rejection in NK cell deficient beige mice. Over 85% of the C57BL/6J mice that received fNE survived the first tumor injection and rejected up to 5 x 10(6) tumor cells when re-challenged. The anti-tumor activity remains in the heat-inactivated and filtrated supernatant of fNE. These data demonstrate that fNE appears to be able to stimulate the innate immune system and the adaptive immune system to reject tumor cells. NK cells respond quickly and appear to be among the major players of the innate immune system, while the adaptive immune system reacts later with a retained memory.

Combined treatment of dendritoma vaccine and low-dose interleukin-2 in stage IV renal cell carcinoma patients induced clinical response: A pilot study.

Vaccination using dendritic/tumor cell hybrids represents a novel and promising cancer immunotherapy. We have developed a technology that can instantly purify the hybrids (dendritomas) from the fusion mixture of dendritic cells (DCs) and tumor cells. Our animal studies and a phase I study of stage IV melanoma patients demonstrated that dendritoma vaccination could be conducted without major toxicity and induced tumor cell-specific immunological and clinical responses. In this pilot study, ten stage IV renal cell carcinoma patients were studied. Dendritomas were made from autologous DCs and tumor cells and administered by subcutaneous injection. After initial vaccination, three escalating doses of IL-2 (3, 6, and 9 million units each) were followed within five days. This treatment regimen was tolerated well without severe adverse events directly related to the dendritoma vaccine. Most adverse events were related to IL-2 administration or pre-existing disease. Patient-specific immune responses were evaluated by flow cytometric measurement of interferon-gamma-producing T-cells before and after vaccination in response to stimulation with tumor antigens. Nine out of nine patients eligible for the analysis showed an increase of IFN-gamma-expressing CD4+ T cells after vaccination(s); while five out of eight patients eligible for the analysis showed an increase of IFN-gamma-expressing CD8+ T cells. Clinical responses were documented in 40% of the patients, three with stabilization of disease and one with a partial response documented by a reduction in tumor size. This pilot study demonstrated that dendritoma vaccines could be administered safely to patients with metastatic renal cell carcinoma, while producing both clinical and immunologic evidence of response.

Transfer of in vitro expanded T lymphocytes after activation with dendritomas prolonged survival of mice challenged with EL4 tumor cells.

Adoptive T cell transfer after in vitro expansion represents an attractive cancer immunotherapy. The majority of studies so far have been focusing on the expansion of tumor infiltrated lymphocytes (TIL) and some have shown very encouraging results. Recently, we have developed a unique tumor immune response activator, dendritomas, by fusion of dendritic cells and tumor cells. Animal studies and early clinical trials have shown that dendritomas are able to activate tumor specific immune responses. In this study, we hypothesized that naïve T cells can be primed with dendritomas and expanded in vitro to develop an adoptive transfer therapy for patients who do not have solid tumors, such as leukemia. T cells were isolated and purified from lymph nodes of mice. The cells were then incubated with dendritomas made from syngeneic DCs and tumor cells and expanded in vitro using Dynabeads mouse CD3/CD28 T cell expander for approximately three weeks. The in vitro primed and expanded T cells showed tumor cell specific CTL activity and increased secretion of IFN-gamma. Tumor bearing mice receiving the in vitro expanded T cells survived significantly longer than control mice. Furthermore, the depletion of regulator T cells enhanced the survival of the mice that received the adoptive transfer therapy.

Dendritoma vaccination combined with low dose interleukin-2 in metastatic melanoma patients induced immunological and clinical responses.

A pilot clinical trial using dendritomas, purified hybrids from the fusion of dendritic/tumor cells combined with a low dose of IL-2, in metastatic melanoma patients was conducted in order to determine its safety and potential immunological and clinical responses. Ten metastatic melanoma patients were enrolled into this study. Dendritoma vaccines were created by fusing dendritic cells stained with green fluorescent dye with irradiated autologous tumor cells stained with red fluorescent dye and purifying the hybrids using immediate fluorescent-activated cell sorting. Initial vaccine was given subcutaneously and followed by IL-2 in serially elevated doses from 3-9 million units/m2 for 5 days. Repeated vaccinations were administered without IL-2, at 3-month intervals for a maximum of 5 times. Immune reactions were measured by the increase of interferon-gamma (IFN-gamma) expressing T cells. Vaccine doses ranged from 250,000 to 1,000,000 dendritomas. There was no grade 2 or higher toxicity directly attributable to the vaccine. All patients experienced toxicity due to IL-2 administration (9-grade 2, 3-grade 3, 1-grade 4). Eight of nine evaluable patients demonstrated immunologic reactions by increased IFN-gamma expressing T cells. One patient developed partial response at 12 weeks after the first vaccine. Nine months later, this patient achieved a complete response. In addition, two patients had stable disease for 9 and 4 months, respectively; one patient had a mixed response. Our findings demonstrated that dendritoma vaccines with a low dose of IL-2 can be safely administered to patients with metastatic melanoma and induce immunological and clinical responses.

Inhibition or promotion of tumor growth by granulocyte-macrophage colony stimulating factor derived from engineered tumor cells is dose-dependent.

BACKGROUND: Granulocyte-macrophage colony stimulating factor (GM-CSF) has been widely investigated as an adjuvant factor for tumor immunotherapy. However, the results are controversial with antitumor effects in some studies and a tumor growth promotion effect in others. MATERIALS AND METHODS: In order to determine whether there is a dose-dependent effect of GM-CSF on tumor growth, murine GM-CSF-expressing vector was constructed and transfected into TC-1 tumor cells and various clones stably expressing different levels of GM-CSF were obtained. The growth of these clones in vivo was studied. RESULTS: Although these clones grow at a similar rate in vitro, their growth in vivo is dramatically different. Clones expressing high levels (>10,000 pg/ml) of GM-CSF grow significantly faster than the control (p <0.001); clones expressing low levels (<100 pg/ml) of GM-CSF grow significantly slower than the control (p<0.001); while clones expressing intermediate levels (1000-2000 pg/ml) of GM-SCF grow at a similar rate as the control (p >0. 05). The high levels of GM-CSF secreted by tumor cells induced granulocytosis and lymphopenia. The antitumor growth effect induced by low levels of GM-CSF is not due to the function of lymphocytes. CONCLUSION: The inhibition or promotion of tumor growth by GM-CSF secreted from tumor cells is dose-dependent.

Synergistic anti-tumor effect of glycosylphosphatidylinositol-anchored IL-2 and IL-12.

BACKGROUND: Preclinical and clinical studies have demonstrated that interleukin 2 (IL-2), interleukin 12 (IL-12), and some other cytokines, play important roles in activating host immune responses against tumor growth. However, severe side effects caused by systemic high-dose administration of these cytokines limit their clinical application. In our previous study, local high doses of IL-2 were achieved by a GPI-anchoring technology; therefore, it will be interesting to know if this technology works for other cytokines. METHODS: A fusion gene containing murine IL-12 and the glycosylphosphatidylinositol (GPI) anchor signal sequence was generated and transfected into the murine melanoma tumor cell line B16F0 either alone or together with a vector encoding GPI-anchored IL-2. The GPI-anchored cytokine expression of the selected stable clones was assayed in vitro by ELISA and their anti-tumor effects were analyzed in vivo by tumor lymphocyte infiltration and tumor growth studies. RESULTS: GPI-anchored IL-12 was successfully expressed on the cell surface as indicated by FACS analysis and IL-12 ELISA assay. The GPI-anchored IL-12 enhanced lymphocyte infiltration and significantly inhibited tumor growth. More importantly, when GPI-anchored IL-12 and GPI-anchored IL-2 were co-delivered, a synergistic anti-tumor effect was observed in both subcutaneous and intravenous tumor models. CONCLUSIONS: GPI anchorage of cytokines represents a new approach to locally deliver high doses of cytokines without the severe adverse effects normally accompanied with systematic high-dose administration of these cytokines.

Fusion protein from RGD peptide and Fc fragment of mouse immunoglobulin G inhibits angiogenesis in tumor.

Targeting tumor vasculature represents an interesting approach for the treatment of solid tumors. The alpha v beta 3 integrins have been found to be specifically associated with angiogenesis in tumors. By using bacteriophage display technology, Ruoslahti et al found that a group of peptides containing the RGD (Arg-Gly-Asp) motif have high-binding affinity to the alpha v beta 3 integrins in tumors. In this study, we designed a fusion protein containing the RGD sequence and the Fc fragment of mouse IgG in order to target the Fc portion of IgG to the tumor vasculature to elicit an antiangiogenesis immune response. In vivo angiogenesis and tumor studies demonstrated that the fusion protein (RGD/mFc) inhibited tumor angiogenesis and tumor growth and improved overall survival. This approach may generate new therapeutic agents for solid tumor treatment.

Targeting foreign major histocompatibility complex molecules to tumors by tumor cell specific single chain antibody (scFv).

Down-regulation of the major histocompatibility complex (MHC) is one of the major mechanisms that tumor cells adopted to escape immunosurveillance. Therefore, specifically coating tumor cells with foreign MHC may make tumor cells a better target for immune recognition and surveillance. In this study, we designed and generated a fusion protein, H2Kd/scPSMA, consisting of a single chain antibody against human prostate specific membrane antigen (PSMA) and the extracellular domain of mouse H-2Kd. The expression of this fusion protein in B16F0 mouse melanoma cells was confirmed by RT-PCR and fluorescent activated cell sorting (FACS). Our animal study showed that the expression of H2Kd/scPSMA in B16F0/PSMA5, a B16F0 cell line expressing human PSMA, significantly inhibited tumor growth as demonstrated in the pulmonary metastasis assay and tumor growth study and improved overall survival.

Glycoinositol phospholipid-anchored interleukin 2 but not secreted interleukin 2 inhibits melanoma tumor growth in mice.

Whereas cancer immunotherapy with interleukin (IL) 2 and/or other cytokines has proved effective in activating immune responses against tumor cells, the major obstacle with the use of these cytokines in cancer patients is their severe side effects when delivered systemically at high doses. In an effort to overcome this problem, in the present study, a fusion protein containing human IL-2 and a glycoinositol phospholipid (GPI) anchor sequence of decay accelerating factor was generated. When expressed by transfected cells, these fusion proteins were presented on the cell surface in the GPI-anchored form as demonstrated by fluorescence-activated cell sorter and ELISA analyses. This GPI-anchored IL-2 is highly functional as indicated by significantly increased T-cell infiltration in tumor masses. Immunohistochemical analysis of tumor cells isolated from experimental tumors indicated that a local high level of IL-2 was achieved by GPI-anchored IL-2. More importantly, when injected into mice i.v., the growth of these B16F0 melanoma cells that were engineered to express this fusion protein was significantly inhibited. In contrast, the inhibition of secreted IL-2 on tumor growth was not observable in this study. These studies may provide a novel approach to locally deliver high doses of cytokines for cancer immunotherapy.