Granulocyte-macrophage Colony-stimulating Factor Secreting Research Articles

Anti–Programmed Death-1 Synergizes with Granulocyte Macrophage Colony-Stimulating Factor–Secreting Tumor Cell Immunotherapy Providing Therapeutic Benefit to Mice with Established Tumors

The purpose of the present study was to evaluate granulocyte macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell immunotherapy, which is known to stimulate potent and long-lasting antigen-specific immune responses, in combination with PD-1 blockade, which has been shown to augment cellular immune responses. Survival studies were done in the B16 melanoma and CT26 colon carcinoma tumor models. Immune monitoring studies were done in the B16 model. GM-CSF-secreting tumor cell immunotherapy was administered s.c. and the anti-PD-1 antibody was administered i.p. The studies reported here show that combining PD-1 blockade with GM-CSF-secreting tumor cell immunotherapy prolonged the survival of tumor-bearing animals compared with animals treated with either therapy alone. Prolonged survival correlated with strong antigen-specific T-cell responses detected by tetramer staining and an in vivo CTL assay, higher secretion levels of proinflammatory cytokines by splenocytes, and the persistence of functional CD8+ T cells in the tumor microenvironment. Furthermore, in the biweekly multiple treatment setting, repeated antigen-specific T-cell expansion was only observed following administration of the cellular immunotherapy with the PD-1 blockade and not when the cellular immunotherapy or PD-1 blockade was used as monotherapy. The combination of PD-1 blockade with GM-CSF-secreting tumor cell immunotherapy leads to significantly improved antitumor responses by augmenting the tumor-reactive T-cell responses induced by the cellular immunotherapy. Readministration of the cellular immunotherapy with the anti-PD-1 antibody in subsequent immunotherapy cycles was required to reactivate these T-cell responses.

Protein disulfide isomerases are antibody targets during immune-mediated tumor destruction

AbstractThe identification of cancer antigens that contribute to transformation and are linked with immune-mediated tumor destruction is an important goal for immunotherapy. Toward this end, we screened a murine renal cell carcinoma cDNA expression library with sera from mice vaccinated with irradiated tumor cells engineered to secrete granulocyte macrophage colony-stimulating factor (GM-CSF). Multiple nonmutated, overexpressed proteins that function in tumor cell migration, protein/nucleic acid homeostasis, metabolism, and stress responses were detected. Among these, the most frequently recognized clone was protein disulfide isomerase (PDI). High titer antibodies to human PDI were similarly induced in an acute myeloid leukemia patient who achieved a complete response after vac-cination with irradiated, autologous GM-CSF–secreting tumor cells in the setting of nonmyeloablative allogeneic bone marrow transplantation. Moreover, ERp5, a closely related disulfide isomerase involved in major histocompatibility complex (MHC) class I chain-related protein A (MICA) shedding, also evoked potent humoral reactions in diverse solid and hematologic malignancy patients who responded to GM-CSF–secreting tumor cell vaccines or antibody blockade of cytotoxic T lymphocyte–associated antigen 4 (CTLA-4). Together, these findings reveal the unexpected immunogenicity of PDIs and raise the possibility that these gene products might serve as targets for therapeutic monoclonal antibodies.

Treatment of Biochemical Recurrence of Prostate Cancer With Granulocyte-Macrophage Colony-Stimulating Factor Secreting, Allogeneic, Cellular Immunotherapy

This phase I-II study evaluated the safety, clinical activity and immunogenicity of an immunotherapy developed from human prostate cancer cell lines (PC-3 and LNCaP) modified to secrete granulocyte-macrophage colony-stimulating factor. Patients with noncastrate prostate cancer (19) with biochemical (prostate specific antigen) recurrence following prostatectomy or radiation therapy and no radiological evidence of metastasis were enrolled in the study. Patients were injected with an initial dose of 5 x 10(8) cells followed by 12 biweekly administrations of 1 x 10(8) cells. The adverse event profile, prostate specific antigen response, changes in prostate specific antigen kinetics and immunogenicity were assessed. Immunotherapy was well tolerated with no serious treatment related adverse events and no autoimmune reactions. A negative deflection in prostate specific antigen slope was observed in 84% of patients after treatment with a significant increase in median prostate specific antigen doubling time from 28.7 weeks before treatment to 57.1 weeks after treatment (p = 0.0095). Median time to prostate specific antigen progression was 9.7 months. Immunoblot analysis of patient serum demonstrated new or enhanced production of PC-3 or LNCaP reactive antibodies in 15 of 19 (79%) patients after immunotherapy. Induction of antibody responses reactive against PC-3 in general, and to the PC-3 associated filamin-B protein specifically, were positively associated with treatment associated changes in prostate specific antigen kinetics. Granulocyte-macrophage colony-stimulating factor secreting cellular immunotherapy has a favorable toxicity profile with signals of clinical and immunological activity against hormone naïve prostate cancer. An association between immune response and prostate specific antigen changes was observed. Phase 3 trials in patients with advanced, metastatic, hormone refractory prostate cancer are under way.

Concerted Potent Humoral Immune Responses to Autoantigens Are Associated with Tumor Destruction and Favorable Clinical Outcomes without Autoimmunity

The therapeutic importance of immune responses against single versus multiple antigens is poorly understood. There also remains insufficient understanding whether responses to one subset of antigens are more significant than another. Autoantibodies are frequent in cancer patients. They can pose no biological significance or lead to debilitating paraneoplastic syndromes. Autoreactivity has been associated with clinical benefits, but the magnitude necessary for meaningful results is unknown. Autologous tumor cells engineered to secrete granulocyte macrophage colony-stimulating factor generate immune infiltrates in preexisting metastases with associated tumor destruction. We sought to identify targets of responses from this vaccination strategy. Postvaccination sera used in screening a cDNA expression library prepared from a densely infiltrated metastasis of a long-term surviving melanoma patient identified several autoantigens. Additional autoantigens were identified through similar screenings in non-small cell lung cancer and murine models, and proteins implicated in cancer propagation. ELISAs for several targets were established using recombinant proteins, whereas others were evaluated by petit serologies. Eleven gene products were identified through serologic screening from two patients showing highly favorable clinical outcomes. A subset of antigens revealed significant changes in antibody titers compared with weak responses to other proteins. Time course analyses showed coordinated enhanced titers against several targets as a function of vaccination in responding patients. This study shows the range of biologically significant antigens resulting from a whole-cell vaccine. Targets include autoantigens that are components of cell cycle regulation. Potent antibody responses against multiple autoantigens are associated with effective tumor destruction without clinical autoimmunity.

Lymphocyte Activation Gene-3 Fusion Protein Increases the Potency of a Granulocyte Macrophage Colony-Stimulating Factor–Secreting Tumor Cell Immunotherapy

The purpose of the present study was to evaluate granulocyte macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell immunotherapy, which is known to stimulate a potent and long-lasting antigen-specific immune response in combination with lymphocyte activation gene-3 fusion protein (LAG-3Ig), which has been shown to act as an adjuvant for priming T helper type 1 and cytotoxic T-cell responses. Survival and immune monitoring studies were done in the B16 melanoma model. GM-CSF-secreting tumor cell immunotherapy was administered as a single s.c. injection and LAG-3Ig was administered s.c. at the immunotherapy site. The studies reported here show that combining LAG-3Ig with GM-CSF-secreting tumor cell immunotherapy prolonged the survival of tumor-bearing animals compared with animals treated with either therapy alone. Prolonged survival correlated with increased numbers of systemic IFN gamma-secreting CD8+ T cells and a significantly increased infiltration of activated effector CD8+ T cells into the tumor. Moreover, an increase in antigen-specific IgG1 humoral responses was detected in serum of animals injected with the combination therapy compared with animals injected with either therapy alone. LAG-3Ig combined with a GM-CSF-secreting tumor cell immunotherapy stimulated both cellular and humoral antitumor immune responses that correlated with prolonged survival in tumor-bearing animals.

Secretion of Biologically Active Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor by Transduced Gastric Cancer Cells

PurposeGastric cancer has the highest incidence rate among cancers in Asia. The advanced type of signet ring cell carcinoma has poor prognosis compared to other types of gastric cancer. The immuno-gene therapy with cytokine-based tumor vaccines has not yet been investigated for gastric cancer. The granulocyte macrophage colony-stimulating factor (GM-CSF)-based tumor vaccine has been demonstrated as the most potent stimulator for specific and long-lasting systemic tumor immunity.Materials and MethodsIn the present study, KATO III cells, the human signet ring cell gastric carcinoma cell line, were genetically modified by the transduction with the human GM-CSF cDNA or the modified hGM-CSF in replication-deficient retroviruses. The genomic integrations and mRNA expressions of the transgenes were determined by Southern and Northern blot analyses.ResultsWild type (wt) or modified hGM-CSF was integrated into the genome of KATO III cells. The modified hGM-CSF mRNA was more stable than that of wt. The KATO III cells with the modified hGM-CSF produced higher level of hGM-CSF (12.4 - 19 ng/106 cells/48 hrs) than that with wt hGM-CSF, when determined by enzyme-linked immunosorbent assay (ELISA). The secreted recombinant hGM-CSF could support the proliferation of the GM-CSF-dependent cell line, indicating that the hGM-CSF secreted by the transduced KATO III cells has biological activities. Irradiated, transduced KATO III cells continued to secret hGM-CSF without proliferation.ConclusionOur results suggest that GM-CSF secreting KATO III cells could be tested for the treatment of gastric cancer as an allogeneic tumor vaccine as a part of immunotherapeutic treatment.

In vivo responses to vaccination with Mage-b, GM-CSF and thioglycollate in a highly metastatic mouse breast tumor model, 4T1.

Metastatic breast cancer is an important contributor to morbidity and mortality. Hence, new therapies are needed that target breast cancer metastases. Here, we focus on Mage-b as a possible vaccine target to prevent the development of breast cancer metastases, through activation of Mage-b-specific cytotoxic T lymphocytes (CTL). The syngeneic cell line 4T1, highly expressing Mage-b, was used as a pre-clinical metastatic mouse breast tumor model. BALB/c mice received three preventive intraperitoneal immunizations with Mage-b DNA vaccine mixed with plasmid DNA, secreting granulocyte-macrophage colony stimulating factor (GM-CSF). In addition, antigen-presenting cells were more efficiently recruited to the peritoneal cavity by the injection of thioglycollate broth (TGB), prior to each immunization. Immunization with Mage-b/GM-CSF/TGB significantly reduced the number of metastases by 67% compared to the saline/GM-CSF/TGB and by 69% compared to the vector control/GM-CSF/TGB. Also, tumor growth was significantly reduced by 45% in mice vaccinated with Mage-b/GM-CSF/TGB compared to the saline/ GM-CSF/TGB and by 47% compared to the control vector/ GM-CSF/TGB group. In vivo, the number of CD8 T cells significantly increased in the primary tumors and metastases of mice vaccinated with Mage-b/GM-CSF/TGB compared to the saline/GM-CSF/TGB and the control vector/ GM-CSF/TGB group, while the number of CD4 T cells significantly decreased. The combination of Mage-b, GM-CSF and TGB did not only induce significantly higher levels of IFNgamma in the lymph nodes of vaccinated compared to control mice, but also induced significantly higher expression levels of Fas-ligand (FasL) in the primary tumors (expressing Fas protein constitutively), compared to the control mice. Whether the interaction between Fas and FasL may have contributed to the smaller tumors needs to be further analyzed.

Vaccine Impedes the Development of Reflux-Induced Esophageal Cancer in a Surgical Rat Model: Efficacy of the Vaccine in a Pre-Barrett’s Esophagus Setting

Background & AimsWe developed a granulocyte-macrophage-colony-stimulating factor (GM-CSF) tumor vaccine for esophageal cancer. We evaluated the effectiveness of the vaccine as a prevention option in a surgical reflux rat model of esophageal cancer. MethodsA surgical model involving a jejuno-esophagostomy was used to create Barrett’s esophagus and esophageal cancer in rats. No carcinogen exposure was utilized. Cell lines derived from these tumors were stably passaged in vitro. GM-CSF-secreting tumor cells were generated using stable transfection. All rats underwent a total gastrectomy, followed by a jejuno-esophagostomy. The surgery promoted the reflux of duodenal contents into the esophagus. All animals were administered either a GM-CSF secreting whole cell vaccine or a phosphate-buffered saline (PBS) placebo injection 4, 6, 14, and 16 weeks post-surgery. ResultsWhile 15 of 16 animals in the non-vaccinated placebo group developed esophageal cancer, 94% (15 of 16), animals in the vaccine group had an incidence of cancer of 25% (4 of 16) (p < 0.05). Barrett’s esophagus was seen in 100% (16 of 16) of the controls and 83% (13 of 16) of the vaccinated animals. ConclusionsA GM-CSF-secreting whole cell tumor vaccine impeded esophageal tumor growth, but not the development of Barrett’s esophagus, in a clinically relevant surgical reflux model.

MFG-E8–mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF

Granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances protection against tumors and infections, but GM-CSF-deficient mice develop inflammatory disease. Here we show that GM-CSF is required for the expression of milk fat globule EGF 8 (MFG-E8) in antigen-presenting cells, and that MFG-E8-mediated uptake of apoptotic cells is a key determinant of GM-CSF-triggered tolerance and immunity. Upon exposure to apoptotic cells, GM-CSF-deficient antigen-presenting cells (APCs) produce an altered cytokine profile that results in decreased Tregs and increased Th1 cells, whereas concurrent ablation of IFN-gamma promotes Th17 cells. In wild-type mice, MFG-E8 attenuates the vaccination activity of GM-CSF-secreting tumor cells through Treg induction, whereas a dominant-negative MFG-E8 mutant potentiates GM-CSF-stimulated tumor destruction through Treg inhibition. These findings clarify the immunoregulatory effects of apoptotic cells and suggest new therapeutic strategies to modulate CD4(+) T cell subsets in cancer and autoimmunity.

Granulocyte Macrophage Colony-Stimulating Factor–Secreting Allogeneic Cellular Immunotherapy for Hormone-Refractory Prostate Cancer

This trial evaluated the safety, clinical activity, and immunogenicity of an allogeneic cellular immunotherapy in 55 chemotherapy-naïve patients with hormone-refractory prostate cancer (HRPC). The immunotherapy, based on the GVAX platform, is a combination of two prostate carcinoma cell lines modified with the granulocyte macrophage colony-stimulating factor (GM-CSF) gene. HRPC patients with radiologic metastases (n = 34) or rising prostate-specific antigen (PSA) only (n = 21) received a prime dose of 500 million cells and 12 boost doses of either 100 million cells (low dose) or 300 million cells (high dose) biweekly for 6 months. End points were changes in PSA, time to progression, and survival. Median survival was 26.2 months (95% confidence interval, 17, 36) in the radiologic group: 34.9 months (8, 57) after treatment with the high dose (n = 10) of immunotherapy and 24.0 months (11, 35) with the low dose (n = 24). The median time to bone scan progression in the radiologic group was 5.0 months (2.6, 11.6) with the high dose and 2.8 months (2.8, 5.7) with the low dose. In the rising-PSA group (n = 21) receiving the low dose, the median time to bone scan progression was 5.9 months (5.6, not reached), and median survival was 37.5 months (29, 56). No dose-limiting or autoimmune toxicities were seen; the most common adverse events were injection site reaction and fatigue. These results suggest that this GM-CSF-secreting, allogeneic cellular immunotherapy is well tolerated and may have clinical activity in patients with metastatic HRPC. Phase 3 trials to confirm these results are under way.

Use of an oncolytic virus secreting GM-CSF as combined oncolytic and immunotherapy for treatment of colorectal and hepatic adenocarcinomas

Oncolytic cancer therapy using herpes simplex viruses (HSV) that have direct tumoricidal effects and cancer immunotherapy using the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) have each been effective in preclinical testing. NV1034 is a multimutated oncolytic HSV carrying the gene for murine GM-CSF that attempts to combine these 2 anticancer strategies. The purpose of this study was to compare NV1034 to NV1023, the parent HSV mutants lacking GM-CSF, to determine if such combined oncolytic and immunotherapy using a single vector has advantages over oncolytic therapy alone. Expression GM-CSF in vitro did not alter the infectivity, cytotoxicity, or replication of NV1034 compared to the noncytokine-secreting control. Tumors infected with NV1034 produced GM-CSF in picogram quantities. In vivo efficacy of the viruses against murine colorectal carcinoma CT26 and murine hepatoma Hepa l-6 was then tested in subcutaneous tumors in syngeneic Balb/c and C57 L/J mice, respectively. In these immune-competent models, NV1034 and NV1023 each demonstrated potent antitumor activity. Treatment with NV1034 had significantly better antitumor effect compared to treatment with NV1023. Furthermore, there was no difference in the antitumor efficacy of these viruses in mice depleted of CD4+ and CD8+ T lymphocytes. Viral vectors combining oncolytic and immunotherapy are promising agents in treatment of colorectal carcinoma and hepatoma.

Vascular Endothelial Growth Factor Blockade Reduces Intratumoral Regulatory T Cells and Enhances the Efficacy of a GM-CSF–Secreting Cancer Immunotherapy

The purpose of the present study was to evaluate granulocyte macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell immunotherapy in combination with vascular endothelial growth factor (VEGF) blockage in preclinical models. Survival and immune response were monitored in the B16 melanoma and the CT26 colon carcinoma models. VEGF blockade was achieved by using a recombinant adeno-associated virus vector expressing a soluble VEGF receptor consisting of selected domains of the VEGF receptors 1 and 2 (termed sVEGFR1/R2). Dendritic cell and tumor infiltrating lymphocyte activation status and numbers were evaluated by fluorescence-activated cell sorting analysis. Regulatory T cells were quantified by their CD4+CD25hi and CD4+FoxP3+ phenotype. The present study established that GM-CSF-secreting tumor cell immunotherapy with VEGF blockade significantly prolonged the survival of tumor-bearing mice. Enhanced anti-tumor protection correlated with an increased number of activated CD4+ and CD8+ tumor-infiltrating T cells and a pronounced decrease in the number of suppressive regulatory T cells residing in the tumor. Conversely, overexpression of VEGF from tumors resulted in elevated numbers of regulatory T cells in the tumor, suggesting a novel mechanism of VEGF-mediated immune suppression at the tumor site. GM-CSF-secreting cancer immunotherapy and VEGF blockade increases the i.t. ratio of effector to regulatory T cells to provide enhanced antitumor responses. This therapeutic combination may prove to be an effective strategy for the treatment of patients with cancer.

Surgical excision combined with autologous whole tumor cell vaccination is an effective therapy for murine neuroblastoma

Background Although a whole tumor cell vaccine strategy based on the synergistic action of granulocyte macrophage–colony-stimulating factor (GM-CSF) transduced tumor cells and CpG oligodeoxynucleotides induces potent tumor-specific immunity, such therapy is not curative in the face of large established tumors. Purpose The primary goal of this study was to determine if combining surgical resection of the tumor with whole tumor cell vaccination is an effective therapeutic strategy for established neuroblastoma. We also wished to determine if the phenotype of the immune response generated by this vaccine strategy was altered by the presence of bulky established tumor. Methods The murine neuroblastoma model was used in which syngeneic Neuro-2a was grown subcutaneously in A/J mouse. The whole tumor cell vaccine consisted of irradiated Neuro-2a as the vehicle for tumor antigens admixed with GM-CSF and CpG oligonucleotides (100 μg). Results In the presence of large tumors, dendritic cells were effectively activated by the vaccine, but secretion of intereferon- γ from vaccinated splenocytes in response to antigen stimulation was suppressed. The tumor's inhibitory effect on interferon- γ production from vaccinated splenocytes was reversed after resection of the primary tumor. The use of prepared GM-CSF–secreting bystander cells simplified production of an autologous whole tumor cell vaccine that was remarkably effective in curing large tumors when combined with tumor excision. Conclusions These findings suggest that at least part of the immunosuppressive effects of the cancer can be reversed after surgical excision of the primary tumor. Thus, in the context of minimal residual disease, this dendritic cell-targeted immunotherapeutic approach may prove effective for the treatment of neuroblastoma.

Therapy-induced antibodies to MHC class I chain-related protein A antagonize immune suppression and stimulate antitumor cytotoxicity.

The activation of NKG2D on innate and adaptive cytotoxic lymphocytes contributes to immune-mediated tumor destruction. Nonetheless, tumor cell shedding of NKG2D ligands, such as MHC class I chain-related protein A (MICA), results in immune suppression through down-regulation of NKG2D surface expression. Here we show that some patients who respond to antibody-blockade of cytotoxic T lymphocyte-associated antigen 4 or vaccination with lethally irradiated, autologous tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor generate high titer antibodies against MICA. These humoral reactions are associated with a reduction of circulating soluble MICA (sMICA) and an augmentation of natural killer (NK) cell and CD8(+) T lymphocyte cytotoxicity. The immunotherapy-induced anti-MICA antibodies efficiently opsonize cancer cells for dendritic cell cross-presentation, which is correlated with a diversification of tumor antigen recognition. The anti-MICA antibodies also accomplish tumor cell lysis through complement fixation. Together, these findings establish a key role for the NKG2D pathway in the clinical activity of cytotoxic T lymphocyte-associated antigen 4 antibody blockade and granulocyte-macrophage colony-stimulating factor secreting tumor cell vaccines. Moreover, these results highlight the therapeutic potential of anti-MICA antibodies to overcome immune suppression and effectuate tumor destruction in patients.

Highly Successful Therapeutic Vaccinations Combining Dendritic Cells and Tumor Cells Secreting Granulocyte Macrophage Colony-stimulating Factor

In an attempt to induce potent immune antitumor activities, we investigated, within the rat 9L gliosarcoma model, distal therapeutic vaccinations associating three therapies: dendritic cell vaccination, intratumoral granulocyte macrophage colony-stimulating factor (GM-CSF) gene transfer, and tumor apoptosis induction. Vaccines of dendritic cells coinjected with processed GM-CSF secreting 9L cells induced systemic responses, resulting in the complete regression of distant preimplanted 9L tumor masses in, with the best strategy, 94% of male rats. All of the cured rats developed a long-term resistance to a rechallenge with parental cells. The curative responses were correlated with the detection of elevated specific cytotoxic activities and a CD4+, CD8+ T cell-, and natural killer (NK) cell-mediated IFN-gamma production. The survival rate of the rat seemed more directly linked to the amount of GM-CSF secreted by the transduced tumor cells, which in turn depended on the toxicity of the apoptosis-inducing treatment, than to the level of apoptosis induced. Unexpectedly, alive GM-CSF secreting 9L cells became apoptotic when injected in vivo. Thus we documented the positive role of apoptosis in the induction of therapeutic antitumor responses by comparing, at equal GM-CSF exogenous supply, the effects of dendritic cells coinjected with apoptotic or necrotic 9L cells. The data showed the superior therapeutic efficiency of combined vaccines containing apoptotic tumor cells. In conclusion, vaccinations with dendritic cells associated with apoptotic tumor cells secreting GM-CSF show a very high therapeutic potency that should show promise for the treatment of human cancer.

CD1d-restricted T cells regulate dendritic cell function and antitumor immunity in a granulocyte-macrophage colony-stimulating factor-dependent fashion.

CD1d-restricted T cells contribute to tumor protection, but their precise roles remain unclear. Here we show that tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor induce the expansion of CD1d-restricted T cells through a mechanism that involves CD1d and macrophage inflammatory protein 2 expression by CD8 alpha-, CD11c+ dendritic cells (DCs). The antitumor immunity stimulated by vaccination with irradiated, granulocyte-macrophage colony-stimulating factor-secreting tumor cells was abrogated in CD1d- and J alpha 281-deficient mice, revealing a critical role for CD1d-restricted T cells in this response. The loss of antitumor immunity was associated with impaired tumor-induced T helper 2 cytokine production, although IFN-gamma secretion and cytotoxicity were preserved. DCs from immunized CD1d-deficient mice showed compromised maturation and function. Together, these results delineate a role for CD1d-restricted T cell-DC cross talk in the shaping of antitumor immunity.

Establishment and characterization of a rectal cancer model in mice: application to cytokine gene therapy.

We established an orthotopic animal model of rectal cancer in mice and applied this model to the study of the antitumor effects of cytokine-assisted tumor vaccine. The CT-26 murine colon adenocarcinoma cells were inoculated into the submucosa of the rectum of the mice to induce the rectal tumor. The tumor growth rate and the survival time of the mice were observed. The cDNA of granulocyte-macrophage colony-stimulating factor (GM-CSF) was transduced to the CT-26 cell line via a retroviral vector, and the therapeutic effects of irradiated GM-CSF secreting tumor vaccine on the rectal tumor were investigated. All the mice implanted with the wild-type tumor cells had tumor growth in the rectum and died. The mean survival time of the mice was 28.9 days. Two doses of irradiated GM-CSF secreting tumor vaccine administered on days 0 and 3 after tumor cell implantation significantly prolonged the survival of the mice with rectal tumor compared with that of the control groups ( P<0.0001). In contrast, no antitumor effect was observed when the treatment with GM-CSF secreting tumor vaccine was delayed to 3 days after tumor cell implantation ( P>0.17). The results suggest that cytokine gene therapy exerts an antitumor effect on small tumors and may be considered as an adjuvant immunotherapy of rectal cancers and prevention of reimplantation of tumor cells disseminated during or following surgery. The orthotopic animal model of the rectal cancer in mice could be applied to the in vivo experimental studies of rectal cancer.

Antitumor Effect on Murine Renal Cell Carcinoma by Autologous Tumor Vaccines Genetically Modified with Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-6 Cells

SUMMARY: The authors evaluted the efficacy of vaccination with murine renal cell carcinoma (Renca) secreting the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene and interleukin-6 (IL-6) gene for the treatment of Renca tumor. Murine GM-CSF and murine IL-6 genes were introduced and expressed in Renca cells (Renca-GM-CSF and Renca-IL-6). For a prevaccination study, wild-type Renca cells were injected subcutaneously into Balb/c mice that had been vaccinated three times with inactivated wild-type Renca, Renca-GM-CSF, Renca-IL-6, or a mixture of Renca-GM-CSF and Renca-IL-6 cells 7, 14, and 21 days before this tumor inoculation. For vaccination experiments, Renca tumor-bearing (8 to 10 mm) mice were injected subcutaneously weekly for 3 weeks with inactivated wild-type Renca cells, or either one or a combination of Renca-GM-CSF and Renca-IL-6. A nonvaccinated control was included in all experiments. The animals were monitored for survival and tumor development for 8 weeks. Mice inoculated with wild-type Renca alone died from the tumor within 35 days. Renca-IL-6 grew slower than wild-type Renca (p < 0.05). No tumor was produced by Renca-GM-CSF. Prevaccination with the combination of Renca-GM-CSF and Renca-IL-6 prevented subsequently inoculated wild-type Renca from forming tumors, and prevaccination with either one of them, compared with prevaccination with wild-type Renca, retarded tumor growth and prolonged survival time. Tumor-bearing mice vaccinated with wild-type Renca died within 42 days. Vaccination with Renca-GM-CSF or Renca-IL-6 alone prolonged the survival time, but only Renca-GM-CSF drastically reduced the tumor size. Vaccination with the combination of them achieved complete remission. Neither of the cytokine-secreting cells enhanced the expression of MHC class I or II molecules. Autologous tumor cell vaccine secreting GM-CSF is effective in preventing and treating established tumors. Its efficacy is enhanced by the cosecretion of IL-6.

CD40 Ligand Immunotherapy in Cancer: An Efficient Approach

Cancer cells do not elicit a clinically sufficient anti-tumor immune response that results in tumor rejection. Recently, many investigators have been trying to enhance anti-tumor immunity and encouraging results have been reported. This review will discuss current anti-cancer immunotherapy; interleukin-2 therapy, tumor vaccine secreting Granulocyte macrophage-colony stimulating factor, dendritic cells fused with tumor cells, and CD40 ligand immunotherapy. Moreover, we introduce our two kinds of CD40 ligand immuno-genetherapy; (1) oral CD40 ligand gene therapy against lymphoma using attenuated Salmonella typhimurium (published in BLOOD 2000), (2) cancer vaccine transfected with CD40 ligand ex vivo for neuroblastoma (unpublished). Both approaches resulted in a high degree of protection against the tumor progression and they are simple and safe in the murine system.

Efficient induction of an antigen-specific, T helper type 1 immune response by interleukin-12-secreting fibroblasts.

To determine whether the paracrine secretion of interleukin (IL)-12 can efficiently convert immune responses characterized by high levels of synthesis of IL-4 and immunoglobulin E (IgE) into T helper 1 (Th1)-dominated responses, 3T3 fibroblasts were stably transfected to secrete IL-12 (480 units/10(6) cells/48 hr). Their effects on the T helper cell-mediated immune response were investigated in ovalbumin (OVA)-primed mice. Free mouse recombinant IL-12 was included as a control group. IL-12-secreting fibroblasts (3T3/IL-12) were more effective than free recombinant IL-12 at increasing OVA-specific interferon-gamma (IFN-gamma) production and decreasing OVA-specific IL-4 production in CD4+ T cells. In addition, injection with 3T3/IL-12 cells significantly increased anti-OVA immunoglobulin G2a (IgG2a) levels and decreased anti-OVA IgE levels in OVA-primed mice. This work suggests that IL-12-secreting fibroblasts can efficiently induce an antigen-specific Th1 response and may be beneficial in the treatment of diseases caused by undesirable T helper 2 (Th2)-dominated responses, including allergic diseases.