Specific enzyme-linked immunosorbent assays for quantitation of antibody-cytokine fusion proteins.
(9/4412)
Preliminary testing has shown in vitro and in vivo that antitumor activity can be obtained with fusion proteins linking tumor-reactive monoclonal antibodies to cytokines, such as granulocyte-macrophage colony-stimulating factor or interleukin 2 (IL-2). Preclinical and clinical testing of these reagents requires their in vitro and in vivo quantitation and pharmacokinetic evaluation. We have focused on the detection of a fusion protein which links one human IL-2 molecule to the carboxy terminus of each heavy chain of the tumor-reactive human-mouse chimeric anti-GD2 antibody, ch14.18. We have developed enzyme-linked immunosorbent assays (ELISAs) to evaluate intact tumor-reactive fusion proteins. By these ELISAs we can reliably measure nanogram quantities of intact ch14.18-IL-2 fusion protein and distinguish the intact protein from its components (ch14.18 and IL-2) in buffer, mouse serum, and human serum with specificity and reproducibility. The measurement of intact ch14.18-IL-2 fusion protein is not confounded by free IL-2 or free ch14.18 when 100 ng or less of total immunoglobulin per ml is used during the assay procedure. Our results indicate that these ELISAs are suitable for preclinical and clinical testing and with slight modifications are applicable to the analysis of a variety of other fusion proteins. (+info)
Advances in the biological therapy and gene therapy of malignant disease.
(10/4412)
Biological and gene therapy of cancer have become important components of clinical cancer research. Advances in this area are based on evidence for the presence of tumor antigens, antitumor immune responses, evasion of host control by tumors, and the recognition of host defense failure in cancer patients. These mechanisms are being corrected or exploited in the development of biological and gene therapy. Over the last decade, 9 biological therapies have received Food and Drug Administration approval, and another 12 appear promising and will likely be approved in the next few years. Our approach to gene therapy has been to allogenize tumors by the direct intratumoral injection of HLA-B7/beta2-microglobulin genes as plasmid DNA in a cationic lipid into patients with malignant melanoma. In four Phase I studies, we found a 36% response by the local injected tumor and a 19% systemic antitumor response. In other cancers, gene transfer, expression, and an intratumoral T-cell response were seen, but no clinical response was seen. A variety of follow-up studies with HLA-B7 and other genes are planned. Evasion of host control is now a major target of gene therapy. Strategies to overcome this include up-regulation of MHC and introduction of cell adhesion molecules into tumor cells, suppression of transforming growth factor and interleukin 10 production by tumor cells, and blockade of the fas ligand-fas interaction between tumor cells and attacking lymphocytes. With these approaches, it seems likely that gene therapy may become the fifth major modality of cancer treatment in the next decade. (+info)
Comparison of interferon-gamma, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor for priming leukocyte-mediated hyphal damage of opportunistic fungal pathogens.
(11/4412)
Proinflammatory cytokines have been proposed as adjunctive therapeutic agents to enhance the host immune response during infections caused by opportunistic fungi. The study compared the differential in vitro priming effects of interferon-gamma (IFN-gamma), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) on hyphal damage of opportunistic fungi mediated by isolated neutrophils (polymorphonuclear leukocytes, PMNL) and buffy coat cells (polymorphonuclear leukocytes/peripheral blood mononuclear cells, PMNL/PBMC) from healthy donors. IFN-gamma (1000 U/mL) effectively primed both PMNL and PMNL/PBMC for enhanced hyphal damage of Aspergillus fumigatus, Fusarium solani, and Candida albicans. G-CSF (100 ng/mL) increased hyphal damage mediated by both PMNL and PMNL/PBMC against F. solani, and GM-CSF (100 ng/mL) augmented the antifungal activity of PMNL/PBMC against hyphal forms of both F. solani and C. albicans. IFN-gamma may be superior to G-CSF or GM-CSF for enhancing the microbicidal activity of PMNL and PMNL/PBMC against opportunistic fungi. (+info)
Cytokine-based tumor cell vaccine is equally effective against parental and isogenic multidrug-resistant myeloma cells: the role of cytotoxic T lymphocytes.
(12/4412)
Tumor cells that survive initial courses of chemotherapy may do so by acquiring a multidrug-resistant phenotype. This particular mechanism of drug resistance may also confer resistance to physiological effectors of apoptosis that could potentially reduce the efficacy of immune therapies that use these pathways of cell death. We have previously demonstrated high efficacy for a cytokine-based tumor cell vaccine in a murine MPC11 myeloma model. In the present study, the effects of this vaccination were compared in MPC11 cells and their isogenic sublines selected for mdr1/P-glycoprotein (Pgp)-mediated multidrug resistance (MDR). Immunization with MPC11 cells expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-12 (IL-12) led to long-lasting protection of mice against subcutaneous (sc) challenge with both parental cells or their MDR variants. Similarly, immunization with GM-CSF/IL-12-transfected MDR sublines caused rejection of transplantation of both parental cells and the MDR sublines. Whereas MPC11 cells and their MDR variants were resistant to APO-1/CD95/Fas ligand, the immunization generated potent granzyme B/perforin-secreting cytotoxic T lymphocytes (CTLs) that were similarly effective against both parental and isogenic MDR cells. We conclude that MDR mediated by mdr1/Pgp did not interfere with lysis by pore-forming CTLs. Immunotherapy based on pore-forming CTLs may be an attractive approach to the treatment of drug-resistant myeloma. (+info)
Inhibition of IL-6 and IL-8 induction from cultured rheumatoid synovial fibroblasts by treatment with aurothioglucose.
(13/4412)
Gold compounds have long been used in the treatment of rheumatoid arthritis (RA). However, their actions in RA have not been clarified. In this study, we examined the effect of one of the monovalent gold compounds, aurothioglucose (AuTG), on the IL-1-induced production of IL-6, IL-8 and granulocyte macrophage colony stimulating factor (GM-CSF) from rheumatoid synovial fibroblasts (RSF) isolated from three RA patients. IL-6 and IL-8 induction but not GM-CSF induction was inhibited in most of the RSF after pretreatment with AuTG. Since gene expression of these cytokines is known to be under the control of a common transcription factor, NF-kappaB, the effect of AuTG on the cellular localization of NF-kappaB (p65 subunit) and on NF-kappaB-DNA binding was examined. Although AuTG treatment did not prevent NF-kappaB nuclear translocation, AuTG blocked the DNA-binding activity of NF-kappaB when examined in vitro. Morphologically, both metal-specific cell staining using p-dimethylaminobenzylidene rhodamine and transmission electron microscopic examinations demonstrated the accumulation of metal gold in the cytoplama and some organella (mitochondria and lysosomes) of the AuTG-treated RSF. These results indicate that one of the anti-rheumatic actions of AuTG might be through its inhibitory action on NF-kappaB. (+info)
Heterogeneity in the ability of cytotoxic murine NK cell clones to enhance Ig secretion in vitro.
(14/4412)
We recently described a panel of cytotoxic murine NK cell clones that also enhanced Ig secretion by B cells activated in an in vitro model of T cell-independent type 2 (TI-2) responses. We employed dextran-conjugated anti-IgD (alphadelta-dex) as a model antigen. Here we study the mechanism of Ig induction by these clones. Addition of the various NK clones to sort-purified B cells stimulated with alphadelta-dex and IL-2 resulted in a markedly heterogeneous increase in Ig secretion, which varied from 3-fold, as mediated by clone PKO 56, to 15-fold, as induced by clone PKO 101. The other NK cells showed intermediate levels of Ig induction. Furthermore, while addition of as few as 0.04% of PKO 101 cells stimulated significant increases and 1% induced near maximum Ig production, a 3% addition of PKO 56 cells was required for significant enhancement of Ig secretion. Supernatant material collected from the NK clones mediated Ig production at levels that mirrored the induction by the corresponding cells. Cytokine analysis showed that while all members of the NK panel produced IFN-gamma only two secreted granulocyte macrophage colony stimulating factor and that the levels of Ig induction mediated by the NK clones correlated only with their levels of IFN-gamma secretion. Culture of B and NK cells in the presence of anti-IFN-gamma demonstrated that IFN-gamma was the critical cytokine in NK-induced Ig production. These findings establish heterogeneity in the ability of NK cells to increase Ig secretion in vitro and show that NK-produced IFN-gamma is an important factor in determining this heterogeneity. (+info)
Cellular and molecular characterization of the scurfy mouse mutant.
(15/4412)
Mice hemizygous (Xsf/Y) for the X-linked mutation scurfy (sf) develop a severe and rapidly fatal lymphoproliferative disease mediated by CD4+CD8- T lymphocytes. We have undertaken phenotypic and functional studies to more accurately identify the immunologic pathway(s) affected by this important mutation. Flow cytometric analyses of lymphoid cell populations reveal that scurfy syndrome is characterized by changes in several phenotypic parameters, including an increase in Mac-1+ cells and a decrease in B220+ cells, changes that may result from the production of extremely high levels of the cytokine granulocyte-macrophage CSF by scurfy T cells. Scurfy T cells also exhibit strong up-regulation of cell surface Ags indicative of in vivo activation, including CD69, CD25, CD80, and CD86. Both scurfy and normal T cells are responsive to two distinct signals provided by the TCR and by ligation of CD28; scurfy cells, however, are hyperresponsive to TCR ligation and exhibit a decreased requirement for costimulation through CD28 relative to normal controls. This hypersensitivity may result, in part, from increased costimulation through B7-1 and B7-2, whose expression is up-regulated on scurfy T cells. Although the specific defect leading to this hyperactivation has not been identified, we also demonstrate that scurfy T cells are less sensitive than normal controls to inhibitors of tyrosine kinases such as genistein and herbimycin A, and the immunosuppressant cyclosporin A. One interpretation of our data would suggest that the scurfy mutation results in a defect, which interferes with the normal down-regulation of T cell activation. (+info)
Preclinical development of human granulocyte-macrophage colony-stimulating factor-transfected melanoma cell vaccine using established canine cell lines and normal dogs.
(16/4412)
Tumor vaccines and gene therapy have received significant attention as means of increasing cellular and humoral immune responses to cancer. We conducted a pilot study of seven research dogs to determine whether intradermal injection of canine tumor cells transfected via the Accell particle-mediated gene transfer device with the cDNA for human granulocyte-macrophage colony-stimulating factor (hGM-CSF) would generate biologically relevant levels of protein and result in demonstrable histological changes at sites of vaccination. Tumor cell vaccines of 10(7) irradiated canine melanoma cells were nontoxic, safe, and well tolerated. No significant alterations in blood chemistry values or hematological profiles were detected. A histological review of control vaccine sites revealed inflammatory responses predominated by eosinophils, whereas vaccine sites with hGM-CSF-transfected tumor cells had an influx of neutrophils and macrophages. Enzyme-linked immunosorbent assays of skin biopsies from vaccine sites had local hGM-CSF production (8.68-16.82 ng/site of injection) at 24 hours after injection and detectable levels (0.014-0.081 ng/site) for < or =2 weeks following vaccination. Flow cytometric analysis of hGM-CSF-transfected cells demonstrated < or =25% transfection efficiency, and hGM-CSF levels obtained during time-course assays demonstrated biologically relevant levels for both irradiated and nonirradiated samples. These data demonstrate the in vivo biological activity of irradiated hGM-CSF-transfected canine tumor cells and help provide evidence for a valid translational research model of spontaneous tumors. (+info)