Human cytokines, tumor necrosis factor, and interferons: gene cloning, animal studies, and clinical trials.

Presented is a comprehensive program designed to isolate human cytokine genes and investigate their relative induction, and to analyze cytokine activities in cell culture, animal tumor models, and human clinical trials. Human cytokine cDNAs have been isolated from a cDNA library made from normal human peripheral blood leukocytes (PBLs) treated with Sendai virus and the relative induction of tumor necrosis factor (TNF), alpha and gamma interferons (IFN-alpha, IFN-gamma), and interleukin-1 beta IL-1 beta) genes has been analyzed. In the Sendai virus-induced PBL system, IL-1 beta mRNA was shown to be approximately twofold higher than TNF or IFN-alpha mRNA whereas IFN-gamma mRNA was 50-100-fold lower than TNF or IFN-alpha mRNA. The cytotoxic activity of TNF was analyzed on several cell lines and IFN-alpha and IFN-gamma were shown to potentiate TNF cytotoxicity about 2-200-fold depending on cell lines. The LD50 for recombinant TNF in BALB/c mice was determined to be 6 X 10(7) U/kg and the therapeutic dose of recombinant TNF in sarcoma 180 bearing BALB/c mice was 3 X 10(5) U/kg, indicating a wide therapetic index. Phase I clinical trials of recombinant TNF given I.V. indicated a tolerated dose of 150,000 U/kg with biphasic half-life (T-1/2) of 2 and 31 min following TNF injection. Phase II trials of TNF and trials of TNF combined with IFN-alpha are in progress. These studies indicate that cytokines such as TNF and IFN-alpha are subject to similar induction systems, potentiate each other's activities, and can be tolerated at specific doses for potential therapeutic use.

Antiproliferative assay of human leucocyte interferon.

An in vitro microtitration system of the antiproliferative effect of human leucocyte interferon (IFN-alpha(Le)) was investigated. The preliminary experiments suggested that the antiproliferative effect of IFN-alpha(Le) was increased by prolonging the incubation period, by reducing the target cell concentration, and might be prescribed by the total IFN amounts in the challenge medium. By employing a system consisted of 1.5 X 10(4) Daudi cells in 0.2 ml of medium containing IFN dilutions and an incubation period of 3 days at 37 degrees C, the antiproliferative effects of twenty-one lots of partially purified IFN-alpha(Le) (PIF-alpha(Le)) preparations were titrated. The interassay variations in the antiproliferative titers of three PIF-alpha(Le) established in the present system were found to be in the same range as those in the antiviral titers estimated by a standard macroplaque reduction assay. The each titration curve was parallel, and the antiproliferative titers, assessed by the reciprocals of the IFN dilutions which suppress the cell growth in 50%, were significantly (p less than 0.01) correlated to the antiviral international units of them.

Antitumor effect of human necrosis factor on human hepatoma cells PLC/PRF/5.

The antitumor activity of natural human tumor necrosis factor (TNF) on a human hepatoma cell line PLC/PRF/5 was studied in vitro. TNF produced by the LuKII human lymphoblastoid cell line showed a cytostatic effect on the hepatoma cells, whereas the growth of non-tumorigenic Chang liver cells was little affected. The combined effects of TNF and interferon-gamma (IFN-gamma) were additive on the PLC/PRF/5 cells as shown by statistical analyses. The same combination showed synergistic effects on a human breast cancer cell line BT-20, which was highly sensitive to TNF. These data may provide some informations concerning the use of TNF in the treatment of hepatoma.

Comparison of the antitumor effects of human natural and recombinant interferons (alpha and beta) on human cancer cell lines.

The antitumor effects of 5 different subtypes of human type I interferons (IFN-alpha(Le), rIFN-alpha A, rIFN-alpha D, IFN-beta, and rIFN-beta) were studied on 3 human cancer cell lines (Daudi lymphoma, PLC/PRF/' hepatoma, and G361 melanoma). IFN-alpha(Le) was the most potent against Daudi cells. The effects of IFNs were cytostatic. On the other hand, 500 IU/ml of IFN-alpha(Le), IFN-beta and 5000 IU/ml of rIFN-beta showed cytocidal effect against PLC/PRF/5 cells. The G361 cells were the least sensitive to the IFNs tested. This is the first report on the antitumor effect of rIFN-beta isolated from insect cells. The effect of this rIFN-beta was similar to that of IFN-beta.

Effects of cimetidine on various biological activities of human leucocyte interferon and on interferon production in lymphocytes.

In vitro effects of cimetidine on the antiviral, the antiproliferative and the natural killer (NK) cell stimulating activities of human leucocyte interferon (IFN-alpha (Le], and on the Sendai virus induced interferon production in lymphocytes were investigated. The antiviral activity of IFN-alpha (Le) was dosedependently enhanced by cimetidine, which alone did not demonstrate any antiviral action. The observed titer of the same IFN preparation titrated with 60 micrograms/ml of cimetidine was significantly higher (p less than 0.05) than that without cimetidine. Cimetidine slightly but dosedependently inhibited the growth of Daudi or G-361 cells. The combination effects of these two agents were revealed to be statistically additive or synergistic. The NK cell activity of peripheral blood lymphocytes (PBL) was suppressed after a 16 hr pretreatment with cimetidine. Furthermore, the interferon production in normal lymphocyte cultures was suppressed dosedependently by cimetidine. On the other hand, the augmentation of the NK cell activity by interferon was further potentiated by cimetidine.

Cimetidine enhances interferon induced augmentation of NK cell activity and suppresses interferon production.

Effects of cimetidine on the augmentation of natural killer (NK) cell activity by human leukocyte interferon (Hu IFN-alpha (Le] and on the IFN production from donor leukocytes were investigated in vitro. NK cell activity of PBL was suppressed after 16 hours pretreatment with 0.3, 3.0, and 30.0 micrograms/ml of cimetidine. On the other hand, there was dose dependent suppression of Hu IFN-alpha (Le) production in PBL by cimetidine. The NK cell activity of PBL augmented by Hu IFN-alpha (Le) was further enhanced when the effector cells were pretreated with cimetidine or when cimetidine was added together with IFN. K562, G361, and PLC/PRF/5 cells were used as target cells. Our results suggest that a 16 hour treatment with cimetidine alone may suppress NK cell activity of PBL, probably due to reduced IFN production by leukocytes. On the other hand, cimetidine may enhance the IFN induced NK augmentation in PBL in vitro.

Antiviral and antiproliferative activities of human leukocyte interferon potentiated by cimetidine in vitro.

The influence of cimetidine on antiviral activity of leukocyte interferon (IFN-alpha (Le] was studied in plaque-reduction assays using Utrecht (U) amnion cells challenged with vesicular stomatitis virus (VSV) and in CPE inhibition assays using A549 cells challenged with encephalomyocarditis (EMC) virus and WISH cells challenged with VSV. The IFN-alpha (Le)-induced antiviral activity was slightly enhanced in cells treated with cimetidine, whereas cimetidine treatment alone did not show any antiviral effect. The observed titer (OT) was significantly higher (p less than 0.05) in cells treated with cimetidine together with IFN-alpha (Le) compared with the control without cimetidine. The effect of cimetidine on IFN-alpha (Le)-induced cell growth inhibition was studied on Daudi (a Burkitt's lymphoma cell line) and on G361 (a melanoma cell line) cells. The growth of these cells was slightly suppressed by cimetidine alone. When cells were treated with IFN-alpha (Le)/cimetidine, the cell growth inhibition rates were significantly higher (p less than 0.02) than the rates obtained with IFN-alpha (Le) or cimetidine alone. These results indicate that cimetidine can enhance the antiviral as well as the antiproliferative activities of IFN-alpha (Le) in "in vitro" studies.

Monoclonal antibody (WI-2) reactive against human transformed and leukemic cells.

A monoclonal antibody (WI-2) was produced against HL-60 cells. This antibody was IgM Kappa and reacted with human cell lines derived from fibroblasts and from hemopoetic, lymphoid, and neoplastic tissues. It also reacted with lymphocytes transformed by mitogens. WI-2 lacked reactivity against normal human RBC's, mature granulocytes, and T and B lymphocytes. The target antigen for WI-2 had a molecular weight of 43,000 daltons. Specimens from patients with leukemia were tested with WI-2. The number of immature cells was compared with the number of WI-2 reactive cells in the peripheral blood and bone marrow and subjected to linear regression analysis. There was a highly significant (p less than 0.001) correlation between the two parameters. The antibody may be useful in monitoring the progress of the patients and in detecting early relapse in leukemia.

Temperature influences on different human alpha interferon activities.

Influences of different incubation temperatures within the physiological range on three different biological activities of human leukocyte-derived alpha interferon (IFN) (the antiviral effect, the antiproliferative activity, and the augmentation of natural killer cell (NK) activity) were investigated in vitro. Using the plaque-reduction assay (U cells challenged with VSV), the antiviral activity by IFN was found to be lower at 35 degrees C and higher at 38 degrees C and 39 degrees C than at 37 degrees C. Using the CPE (cytopathogenic effect) inhibition technique (Vero cells challenged with VSV), the antiviral activity was slightly enhanced at 38 degrees C, 39 degrees C, and 40 degrees C, respectively, when compared with 37 degrees C. The antiproliferative activity on Daudi cells and G-361 melanoma cells was enhanced at elevated temperatures. On the other hand, the antiproliferative activity on PLC/PRF/5 hepatoma cells was lower at 38 degrees C and 39 degrees C than at 37 degrees C, but higher at 40 degrees C. NK activity of PBL after 2 h incubation at 41 degrees C was remarkably lower than that at 37 degrees C, while it was not affected by 2 h incubation at 35 degrees C and 39 degrees C, respectively. When PBL was treated with IFN for 2 h at the temperatures described above, NK activity was equally augmented at all temperatures tested. Our results suggest that elevated incubation temperature potentiates the antiviral and the antiproliferative activities, but does not affect the NK augmenting activity of HuIFN-alpha (Le).

Mouse monoclonal antibody (WI-MN-1) against malignant melanoma.

A mouse monoclonal antibody, WI-MN-1, was raised against G-361 melanoma cell line. Reactivity of this antibody was characterized by indirect immunofluorescence against 22 cell lines and normal and neoplastic human tissues. Positive reactions were seen against three melanoma cell lines (G-361, HT-144, and MeWo). It also reacted against an epidermoid carcinoma cell line (Hep-2) and amnion cells (WISH). The antibody failed to react against cell lines derived from granulocytic leukemias, lymphocytic leukemias, Burkitt's lymphoma, carcinoma of the lung, carcinoma of the cervix, carcinoma of the colon, carcinoma of the breast, astrocytoma, and a human monocytoid cell line. Mouse melanoma and mouse fibroblast cell lines were also nonreactive. Similarly, there was no reaction against human peripheral blood, bone marrow, spleen, lymph node, thymus, breast, lung, stomach, heart, brain, kidney, liver, skin, or testis. The antibody was also tested by indirect immunofluorescence against 17 samples of metastatic malignant melanoma which were removed from 13 patients. WI-MN-1 gave positive reaction against 16 of these specimens, and it reacted with the cryostat section in the immunoperoxidase test, delineating neoplastic melanoma from the normal brain tissue. The antibody precipitated Mr 105,000 and 38,000 antigens from biosynthetically labeled G-361 cells. It was suggested that WI-MN-1 was a useful addition to the panel of monoclonal antibodies against melanoma-associated antigens.

Monoclonal antibody against myeloid leukemia cell line (KG-1).

A murine monoclonal antibody (WI-5) was produced against a myeloid leukemia cell line (KG-1). The antibody was immunoglobulin G3K. It reacted only against KG-1 cells and failed to react against 33 other cell lines representing fibroblasts, solid tumors, and cells of myeloid and lymphatic origin. It also showed no reaction against normal red blood cells, granulocytes, platelets, monocytes, and T- and B-lymphocytes. Similarly, there was no reaction against lymphocytes transformed by mitogens. Peripheral blood and bone marrow samples from acute granulocytic and acute lymphocytic leukemia, chronic granulocytic leukemia, chronic granulocytic leukemia in blastic crisis, and chronic lymphocytic leukemia failed to react with WI-5. It was suggested that WI-5 detected a unique antigen on KG-1 cells.

Monoclonal antibody against a unique antigen on human acute promyelocytic leukemia cell line (HL-60).

An IgM kappa monoclonal antibody (WI-1) reacted against HL-60 cells in indirect immunofluorescence and microcytotoxicity tests. It failed to react against 19 other cell lines representing acute myelogenous leukemia, chronic myelogenous leukemia, lymphocytic leukemias, multiple myeloma, Burkitt's lymphoma, monocytoid cells and virus induced lymphoid cell lines. Normal peripheral blood nucleated cells and bone marrow cells derived from acute granulocytic leukemia, chronic granulocytic leukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia also failed to react with WI-1. Normal peripheral blood lymphocytes, transferred with mitogens, showed no reaction against the antibody. There was some decrease in the reactivity of the cells, against WI-1, following maturation with dimethyl sulfoxide. However, a large percentage of the cells remained positive after maturation. WI-1 reacted specifically against an antigen on the HL-60 cells which had a molecular weight of about 42,000 dalton. Peripheral blood cells from one patient with acute promyelocytic leukemia failed to react with the antibody. It is possible that acute promyelocytic leukemia is an antigenically heterogeneous disease. A large population of acute promyelocytic leukemia patients needs to be tested to see if the specific antigen on HL-60 cells, detected by WI-1, is demonstrable in other patients with acute promyelocytic leukemia.

Transfer factor in the treatment of herpes simplex types 1 and 2.

Transfer factor potentiates cellular immunity and induces interferon. It was because of these properties that transfer factor was tried in 17 patients with recurrent herpes simplex types 1 and 2. Transfer factor was administered in doses ranging from 5 to 10 U/m2 i. m. The interval between injections varied from 1 week to 3 months. 16 patients could be evaluated clinically in whom the recurrence rate decreased from 10.7 +/- 6.1 to 2.1 +/- 2.5 (mean SD). The reduction was statistically significant. 8 patients were completely free of disease while the other 8 had reduced number of episodes during the period of observation, 7 patients had abnormal T cell function as reflected by the low number of T cells or low lymphocyte transformation. Statistically significant improvement in the T cell function was observed. Delayed hypersensitivity skin test reactions also improved significantly.

Immunocompetence and transfer factor therapy in uveitis.

A study of the clinical course of 20 patients with uveitis treated with transfer factor is reported. Twelve (60%) of the patients were initially immunoincompetent on screening. Eight of the 12 changed to an immunocompetent status after treatment and could either decrease or discontinue their anti-inflammatory drugs. Five had a statistically significant improvement in visual acuity. One of the 8 initially immunocompetent patients had a statistically significant visual improvement, and 2 decreased or discontinued all drugs, while 3 increased their drugs.