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Sodium stibogluconate (SSG), an inhibitor of SHP-1 that negatively regulates cytokine signaling and immunity, suppressed growth of murine Renca tumors in combination with interleukin-2 (IL-2) via a T-cell-dependent mechanism. The ability of SSG to interact with IL-2 in activating primary human immune cells was evaluated herein by assessing its induction of interferon (IFN)-gamma(+) TH1 cells in human peripheral blood in vitro. The significance of IFN-gamma(+) cells was also investigated by assessing SSG/IL-2 antitumor activity in wild-type and IFN-gamma(-/-) mice. IFN-gamma(+) cells but not IL-5(+) cells were induced markedly (9.1x) in healthy peripheral blood by SSG/IL-2 in contrast to the modest induction by SSG alone (2.1x) at its clinically achievable dose (20 microg/mL) or by IL-2 (3.1x) at its C(max) of low-dose schedule (30 IU/mL). SSG at a higher dose (100 microg/mL) was less effective alone (1.5x) or in combination with IL-2 (7.8x). Peripheral IFN-gamma(+) cells were induced after 4 or 16 h treatment with SSG/IL-2 within CD4(+) and CD8(+) lymphocytes coincided with heightened CD69 expression (approximately 3-4x). SSG/IL-2 was also more effective than the single agents in inducing IFN-gamma(+) cells in the peripheral blood of melanoma patients, whose basal IFN-gamma(+) cell levels were approximately 5% of healthy controls. Renca tumor growth was inhibited by SSG/IL-2 in wild-type but not IFN-gamma(-/-) mice. These results demonstrate SSG interactions with IL-2 in vitro to activate key antitumor immune cells in peripheral blood of healthy and melanoma donors, providing further evidence for proof of concept clinical trials for effecting augmentation of IL-2 through inhibiting negative regulatory protein tyrosine phosphatases.

IFNs were the first new therapeutic products resulting from recombinant DNA technology. IFNs were also the first human proteins effective in cancer treatment. There is however much to be discovered which will lead to new clinical applications. Areas which represent major research challenges for full understanding and application of the IFN system are: (i) the diversity of the IFN family; (ii) the role of induction; (iii) molecular mechanism of action; (iv) cellular modulatory effects; (v) advantages of combinations, and (vi) identification of new therapeutic indications. This review will emphasize the diversity of the IFN family and chemical modifications which will result in second-generation IFNs. Pre-clinical and clinical findings form the basis for new therapeutic directions in chronic myelogenous leukemia, lymphomas, myelomas, melanoma, urologic malignancies, primary brain tumors, and ovarian carcinoma.

Interferon-gamma (IFN-gamma) has the most potent immunomodulatory activity of all the interferons. This phase II-B study was performed to define time- and dose-dependent immunomodulatory effects mediated by IFN-gamma in a subset of patients with melanoma treated in the dose-seeking therapeutic trial conducted by the Eastern Cooperative Oncology Group E4687 (13). The effects of IFN-gamma (Genentech, San Francisco, CA) were evaluated for phenotype and function of peripheral blood lymphocytes obtained twice prestudy, and on days 2, 9, and 29 of IFN-gamma therapy for 50 patients. Early significant increases in CD4/CD8 ratio (p = 0.001) were noted, largely due to a rise in CD4+ and fall in CD8+ T-cell populations sustained through day 29 at only the lowest dosage. Increased natural killer cell (NK) activity (p = 0.001 on day 9; p = 0.01 on day 29) was accompanied by durable increases in circulating activated NK cells (CD56+DR+% p = 0.001, day 9; p = 0.001, day 29). After initial depression of CD56+ and CD16+ cells on day 2, the total percent of CD56+ and CD16+ cells increased significantly by day 29. Increases in NK cell activity were maximal at doses > or =0.1 mg. Monocyte CD14+ expression of DQ+ rose early (p = 0.011 and 0.001 on days 2 and 9), accompanied by elevation in CD14+DR+ cells that was less significant. Immunomodulatory effects of IFN-gamma reported in this trial have major implications for interpretation of past and current clinical trials, and the design of future trials. This is the first trial in which IFN-gamma has been shown to have significant effects on the T-cell compartment of the immune system.

ISG-15, a 15-kDa protein encoded by an interferon (IFN)-stimulated gene (ISG), was produced in human corneal cell cultures prepared from donor corneas in response to each of three major types of IFN, IFN-alpha, IFN-beta, and IFN-gamma. IFN-alpha and IFN-beta induced more ISG-15 in the first 24 h of treatment than did IFN-gamma. ISG-15 was detectable within the first 3 h of treatment with either type I IFN, and production peaked at 24 h, whereas IFN-gamma did not induce detectable ISG-15 until 16 h and did not induce peak production until 48 h. Conjugates of ISG-15 to cellular proteins were detectable by Western blot beginning at 9 h after IFN-alpha or IFN-beta treatment. ISG-15 persisted in IFN-treated cells for as long as 96 h. Free, unconjugated, ISG-15 was secreted by keratocytes into cell culture medium. The extent and kinetics of production, the conjugation of ISG-15 to cell proteins, and the secretion of ISG-15 in human fibroblast derived from corneas were IFN type dependent and dose dependent.

ISG15 is a 15-kDa protein of unique primary amino acid sequence, which is transcriptionally regulated by interferon (IFN) alpha and IFN-beta. Because it is synthesized in many cell types and secreted from human monocytes and lymphocytes, we postulated that ISG15 might act to modulate immune cell function. ISG15 stimulated B-depleted lymphocyte proliferation in a dose-dependent manner with significant proliferation induced by amounts of ISG15 as low as 1 ng/ml (58 pM). Maximal stimulation of [3H]thymidine incorporation by B-depleted lymphocytes occurred at 6-7 days. Immunophenotyping of ISG15-treated B-depleted lymphocyte cultures indicated a 26-fold expansion of natural killer (NK) cells (CD56+). In cytotoxicity assays, ISG15 was a potent inducer of cytolytic activity directed against both K562 (100 lytic units per 10(6) cells) and Daudi (80 lytic units per 10(6) cells) tumor cell targets, indicating that ISG15 enhanced lymphokine-activated killer-like activity. ISG15-induced NK cell proliferation required coculturing of T and NK cells, suggesting that soluble factor(s) were required. Measurement of ISG15-treated cell culture supernatants for cytokines indicated production of IFN-gamma (> 700 units/ml). No interleukin 2 or interleukin 12 was detected. IFN-gamma itself failed to stimulate lymphocyte proliferation and lymphokine-activated killer cell activation. Further, induced expression of IFN-gamma mRNA was detected by reverse transcription-PCR in T lymphocytes after ISG15 treatment but not in NK cells. Enhancement of NK cell proliferation, augmentation of non-major histocompatibility complex-restricted cytotoxicity, and induction of IFN-gamma from T cells identify ISG15 as a member of the cytokine cascade and suggest that it may be responsible for amplifying and directing some of the immunomodulatory effects of IFN-alpha or IFN-beta.

IFN-gamma is a potent immunomodulator, which has activity against melanoma in vitro and in murine models. However, preclinical data suggests that the optimal therapeutic and immunomodulatory dose may not be the maximally tolerated clinical dose. We conducted a Phase II/III trial in good prognosis patients with metastatic melanoma to determine whether a therapeutic and immunomodulatory dose-response curve of IFN-gamma could be identified, and whether the two could be correlated. Ninety-eight patients with metastatic melanoma were randomized to one of seven dose levels of IFN-gamma ranging from 0.01 to 0.90 mg/m2. All patients were required to have s.c., skin, soft tissue, or nodal disease, although visceral disease was also allowed, and no more than one prior chemotherapy regimen. Patients received IFN-gamma as a 1-h i.v. infusion three times per week for at least 8 weeks or until progressive disease. Ninety-five patients were eligible for toxicity evaluation; 81 were eligible for tumor response. Four patients responded to therapy (response rate, 5%) at three dose levels: two patients at 0.01 mg/m2 and one each at 0.5 and 0.9 mg/m2. The duration of response ranged from 5 to 58 weeks. Toxicities were typical of IFNs and included flu-like constitutional symptoms. No dose-response relationship was identified for efficacy. A dose-response relationship for toxicity was observed only for fever and chills (p = 0.035) and hepatic toxicity (p = 0.034). IFN-gamma has minimal activity in metastatic melanoma, and a therapeutic dose-response curve could not be identified. Although potent dose-dependent effects on immunomodulation were identified (J. M. Kirkwood, J. Bryant, J. H. Schiller, M. M. Oken, E. C. Borden, and T. L. Whiteside. Immunomodulatory function of interferon gamma in patients with metastatic melanoma: results of a phase IIB trial in subjects with metastatic melanoma: ECOG Study E4987, submitted for publication), this biological activity does not translate into therapeutic activity in the metastatic disease setting in this trial.

Interferons (IFNs) are known to have antiviral effects and have been shown to enhance the expression of tumor-associated antigens (TAA) on different target cells. In our current study, we investigated the potential of IFN-alpha or IFN-gamma to enhance the expression of the TAAs recognized by monoclonal antibodies (MAbs) 19-9, B72.3, 17-1A, and BR55-2 on pancreatic cancer cell lines and the potential of IFN-gamma to modulate the expression of a single TAA, BR55-2, on nonpancreatic cancer cell lines. Expression of these TAAs, percentage of positive cells and mean fluorescence intensity, was measured by flow cytometry. In these studies, we provide evidence that one prostate (DU 145) and two pancreatic (HPAF and BxPC-3) cancer cell lines that moderately express BR55-2 can be upregulated by IFN-gamma treatment, with optimal enhancement occurring between 48 and 72 h with 1,000 IU/ml. Cell lines that highly expressed BR55-2 could not be further upregulated by the doses of IFNs tested during the various periods used. IFN-alpha or IFN-gamma treatments did not significantly change the levels of TAA expression on pancreatic cancer cell lines that bound MAbs 17-1A or 19-9. Cell lines that did not bind MAbs 17-1A, 19-9, B72.3, or BR55-2 before IFN treatments could not be induced to express these antigens after treatment. Although antigen expression does not ensure detectable therapeutic benefit, increased antigen expression on tumor tissues may augment the efficacy of MAbs bearing radionuclides, toxins, or effector cells to the tumor site. In each of these situations, the use of IFNs to enhance TAA expression, particularly IFN-gamma, may merit consideration.

The effects of recombinant human interferon (IFN)-gamma on functions of human granulocytes (PMNL) were investigated. Incubation with IFN-gamma for up to 24 h enhanced the chemoluminescent (CL) response of polymorphonuclear leukocytes (PMNL) to Staphylococcus aureus, FMLP, opsonized zymosan, and PMA in a dose-dependent fashion for concentrations of 1-1000 units/mL. Short exposures to tumor necrosis factor (TNF)-alpha also enhanced the CL responses of PMNL, but the effects IFN-gamma and TNF alpha together, at the concentrations used, were less than additive. Incubation of PMNL for 8 h with IFN-gamma preserved their bactericidal capabilities relative to control PMNL incubated for the same length of time without IFN-gamma. Preservation of neutrophil function may be a subtle but important role for IFN-gamma.

Four isoforms of 2',5'-oligoadenylate (2-5A) synthetase have been identified (40, 44, 69, and 100 kD). The 40- and 44-kD enzymes are encoded by the same gene, probably different from the genes encoding the larger isoforms. In this study, induction of the 100- and 69-KD (p100, p69) isoforms in different individuals and in different cell types was investigated after treatment with recombinant human interferons (IFN): IFN-alpha 2, IFN-beta ser, or IFN-gamma. The p69 and p100 isoforms were quantitated in cell extracts on Western blots using specific polyclonal antibodies, or their activity was measured after purification of cell extracts on immunoaffinity columns. The p69 and p100 isoforms were differentially induced in Daudi, fibroblast, and colon adenocarcinoma cells treated with IFNs. Considerable individual variations in both basal and induced levels of p69 and p100 were observed in peripheral blood mononuclear cells from normal donors cultured with IFNs in vitro, and from cancer patients treated with IFN-alpha 2 or with IFN-beta ser. These results demonstrate that the p69 and p100 isoforms are present in vivo in peripheral blood mononuclear cells and their level is increased following IFN administration. Furthermore, both the in vivo and in vitro observations indicate that the expression of these enzymes is specific to each cell type and varies among individuals.

The formation of new blood vessels is critical to tumor growth and metastasis and since endothelial cells are the principal cell type comprising such blood vessels, we carried out experiments to test whether the growth of endothelial cells in vitro can be inhibited by low doses of combinations of cytokines. We show that growth of mouse aortic endothelial cells can be inhibited in vitro by combinations of cytokines that, administered singly even at ten times higher concentrations, have only marginal effects. In particular, interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma), acting synergistically at low, subthreshold concentrations, are able to completely inhibit the growth of endothelial cells in vitro.

Interferons can induce neopterin biosynthesis and tryptophan degradation in monocytic cells. Indoleamine 2,3-dioxygenase (IDO), an inducible cellular enzyme, metabolizes tryptophan to N-formyl-L-kynurenine. Tryptophan degradation has been linked to interferon-mediated inhibition of replication by intracellular pathogens and inhibition of cancer cell proliferation. We evaluated the ability of the recombinant human interferons beta ser and gamma to stimulate neopterin production and tryptophan degradation in vitro by alveolar macrophages (AM) obtained from normal volunteers by bronchoalveolar lavage. Additionally, because other biologic response modifiers such as lipopolysaccharide (LPS) can also stimulate monocytic cells to produce increased amounts of neopterin and degrade tryptophan, we evaluated the effects of LPS on interferon-induced neopterin production and tryptophan degradation by AM. Both interferon-gamma (IFN-gamma) and interferon-beta (IFN-beta) induced neopterin production and tryptophan degradation by AM with corresponding inhibition of intracellular replication by Chlamydia psittaci in AM, but IFN-gamma was a more potent inducer of these responses than IFN-beta. LPS enhanced neopterin production and tryptophan degradation by interferon-exposed cells. This effect was particularly evident at lower concentrations of interferon, and LPS synergy was more pronounced with IFN-beta than IFN-gamma. Concentrations of LPS that alone had no stimulatory effect on tryptophan degradation synergistically enhanced the induction of IDO activity by lower concentrations of interferon. These studies suggest that IFN-gamma stimulates human AM to produce neopterin and degrade tryptophan more potently than IFN-beta, and that low concentrations of LPS can synergistically enhance such effects of interferons on tissue macrophage metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

Purpose: The study was designed to determine whether in vivo interferon gamma (IFN-gamma) administration could enhance tumor antigen expression on the surface of human tumor cells., Materials and Methods: Eight patients (six with ovarian and two with gastrointestinal tumors) with a diagnosis of adenocarcinoma with secondary malignant ascites were given weekly escalating doses of IFN-gamma (ie, 0.1 to 100 MU) intraperitoneally (IP) each week for 8 weeks. Tumor cells were isolated from the patients' ascites samples, which were collected three times per week: before and 24 and 48 hours post-IFN-gamma administration. The level of expression of tumor-associated glycoprotein-72 (TAG-72) and carcinoembryonic antigen (CEA) was measured using flow cytometry and immunocytochemistry., Results: IFN-gamma administered IP dramatically increased TAG-72 (as measured by binding of anti-TAG-72 monoclonal antibodies [MoAbs] B72.3 and CC 49) and CEA (measured by MoAb COL-1) expression on the surface of the carcinoma cells. The ascites-derived tumor cells from seven of the eight patients constitutively expressed TAG-72, and the level of TAG-72 expression was increased by IFN-gamma in all seven patients, as evidenced by the enhanced binding of anti-TAG-72 MoAbs to the tumor-cell surface. In some cases, IFN-gamma treatment increased the percentage of MoAb B72.3-reactive tumor cells from 10% to greater than 90%, and those changes were further corroborated by similar increases in the MoAb staining intensity observed with immunoperoxidase analysis. In addition, ascites-derived tumor cells from two patients with gastrointestinal carcinoma also expressed enhanced CEA levels after IFN-gamma. The increased TAG-72 and CEA expression were observed at low IFN-gamma doses (ie, 0.1 to 1.0 MU), which were well tolerated by all patients., Conclusions: The ability of IFN-gamma given IP to increase TAG-72 and CEA expression on tumor cells in vivo provides additional argument for the use of the cytokine as an adjuvant to enhance MoAb binding to human carcinoma-cell populations.

A 15,000 molecular weight protein (15-kDa), induced and secreted by human PBMC after treatment with IFN-alpha or -beta, was assessed for its ability to modulate cellular function. Although it had no effect on growth or 2'5'-A synthetase activity in Daudi, U-937, or HL-60 cells, when incubated with fresh human PBMC, LPS-induced monocyte cytotoxicity against WEHI-164 target cells was augmented. This stimulation was inhibited by both an antibody against TNF-alpha and a rabbit polyclonal antiserum to the 15-kDa protein. Furthermore, when the 15-kDa protein was added to PBMC an increase in GTP cyclohydrolase I activity, as assessed by neopterin secretion, resulted. Neopterin secretion by PBMC in response to the 15-kDa was increased in a dose-responsive manner up to more than sixfold over baseline, with a 15-kDa concentration of less than 10 ng/ml effective. The 15-kDa protein also stimulated indoleamine 2,3-dioxygenase (IDO) activity in fresh, human PBMC. Induction of neopterin secretion and IDO activity was inhibited by a polyclonal antiserum to 15-kDa. LPS-induced cytotoxic activity was not augmented by 15-kDa pretreatment of purified monocytes, indicating the need for the presence of a second cell population and the indirect action of the 15-kDa on the induction of monocyte activities. When PBMC or purified CD3+ cells, but not purified CD14+ cells, were incubated with the 15-kDa protein, secretion of a factor was induced that resulted in the induction of IDO activity in PMA-differentiated THP-1 cells. An antibody to IFN-gamma, but not IFN-alpha, inhibited the induction of IDO activity by this secreted factor. In addition, antiserum to the 15-kDa blocked the secretion of IFN-gamma from the CD3+ cells. Thus, a 15-kDa product of IFN-alpha- and IFN-beta-treated monocytes and lymphocytes can stimulate secretion of IFN-gamma from CD3+ cells.

Biological agents such as the interferons (IFNs) or lipopolysaccharides (LPSs) can prime phagocytic cells to generate increased amounts of oxygen metabolites upon exposure to various stimuli. The priming of human peripheral blood monocytes and alveolar macrophages (AM) by recombinant IFN-beta ser (rIFN-beta ser) and rIFN-gamma for an enhanced respiratory burst was compared. Both rIFN-beta ser and rIFN-gamma increased phorbol myristate acetate-stimulated superoxide anion generation by AM in a dose-dependent fashion. rIFN-beta ser was capable of priming AM for an enhanced superoxide anion release nearly as well as rIFN-gamma. In contrast, rIFN-beta ser was much less effective as a priming agent for monocytes when compared to either its effect on AM or to the priming effect of rIFN-gamma on monocytes. The respiratory burst of IFN-exposed AM was not inhibited by co-incubation with low concentrations of LPS. However, the ability of IFN to augment superoxide anion release by cells simultaneously exposed to LPS in comparison to superoxide anion generation by cells exposed to LPS only was attenuated.

Sera were collected from 111 patients diagnosed with adenocarcinoma or nonadenocarcinoma malignancies who received different schedules of interferon (IFN)-gamma or IFN-beta ser alone or in combination. Serum carcinoembryonic antigen (CEA) and tumor-associated glycoprotein-72 (TAG-72) antigen levels were measured to determine whether interferon could enhance the tumor shedding and, thereby, the serum level of either tumor antigen. Less than 10% of the sera samples from patients diagnosed with nonadenocarcinoma malignancies (e.g., hairy cell leukemia, melanoma) had positive titers of TAG-72 or CEA, and interferon neither increased nor resulted in the appearance of either tumor antigen in those sera. In contrast, 59.2% and 75.4% of the patients with adenocarcinoma had positive serum levels of TAG-72 and CEA, respectively, prior to interferon. IFN-gamma and IFN-beta ser alone or in combination significantly increased serum TAG-72 or CEA in approximately 65% of those patients. The results suggest that interferon administration to patients with adenocarcinoma can result in increased serum levels of selected tumor-associated antigens used in the diagnosis of malignancy. These preliminary findings may be important in the development of new strategies to obtain more sensitive tumor antigen serum assays for the diagnosis and monitoring for disease progression of adenocarcinoma.

Monoclonal antibodies (MAbs) which recognize a human tumor antigen, termed tumor-associated glycoprotein-72 (TAG-72), have successfully been used to localize primary as well as metastatic colorectal tumor lesions in patients. The localization of the anti-TAG-72 MAbs has also been exploited intraoperatively using a hand-held gamma probe. That procedure, termed radioimmunoguided surgery (RIGS), has identified occult tumors which were not detected using standard external imaging techniques. In another clinical trial, interferon-gamma (IFN-gamma) was administered intraperitoneally to patients diagnosed with either gastrointestinal or ovarian carcinoma with secondary ascites. Analysis of the tumor cells isolated from the malignant ascites revealed a substantial increase in TAG-72 expression on the surface of tumor cells isolated from seven of eight patients. The results provide evidence that the combination of an anti-carcinoma MAb with the administration of a cytokine, such as IFN-gamma, may be an effective approach for the detection and subsequent treatment, of colorectal carcinoma.

To directly compare clinical side effects and biological response modification, IFN-beta ser, IFN-gamma, or the combination of IFN-beta ser plus IFN-gamma was administered to 21 cancer patients. Each IFN or the combination was given intravenously on days 1, 8, and 15 in varied order. Each IFN and the combination resulted in significant (P less than 0.05) modulation of IFN-induced proteins. IFN-beta ser was more effective than IFN-gamma in enhancing 2-5A synthetase activity (P = 0.001). IFN-gamma was more effective than IFN-beta ser in enhancing serum beta 2 microglobulin expression (P = 0.05) and indoleamine dioxygenase activity, as assessed by decreased serum tryptophan (P = 0.03). The combination enhanced tryptophan catabolism more effectively than IFN-beta ser in a dose-dependent manner (P less than 0.03). IFN-beta ser/IFN-gamma did not potentiate natural killer cells or antibody-dependent cellular toxicity (ADCC). IFN-beta ser/IFN-gamma enhanced monocyte guanylate cyclase activity, as assessed by serum neopterin, more effectively than IFN-gamma alone (P = 0.005). Both IFNs and the combination resulted in increases in HLA class II expression on monocytes. However, no significant difference in the level of induction of HLA DQ and HLA DR expression between IFN-beta ser/IFN-gamma and either IFN-beta ser or IFN-gamma was noted. Although frequency and servity of side effects of IFN-beta ser, IFN-gamma, or the combination were dose related, induction of induced proteins (with exception of influences on tryptophan catabolism) were not a function of dose administered over the 10-fold range. Continued treatment with the combination intravenously three times a week for 4 wk sustained but did not further potentiate, most of the changes in interferon-induced proteins. Thus, IFN-beta ser and IFN-gamma each resulted in effective and essentially equivalent patterns of induction of induced proteins. When combined, however, these IFNs did not result in potentiation of biological response modification in vivo.

A phase I trial involving continuous infusion of both beta- and gamma-interferon (IFN-beta and IFN-gamma) was conducted in 20 patients in order to determine whether combinations of high doses of IFN-beta and IFN-gamma were tolerable when administered under conditions which mimic conditions of in vitro antiproliferative studies. Patients received a 5-day continuous infusion of IFN-beta/IFN-gamma, followed by a 9-day rest period. Two cycles were administered. Doses of IFN-beta/IFN-gamma were escalated between 4 dose levels, with 5 patients per dose level. Dose-dependent side effects, consisting primarily of constitutional symptoms typical of those experienced with IFN, were observed. The maximally tolerated dose of continuous IFN-beta/IFN-gamma infusion was 3 x 10(6) units of IFN-beta and 200 micrograms of IFN-gamma. Dose-limiting side effects consisted of severe headache, fatigue, fever, and hepatic toxicity. No clinical responses were observed. Serum IFN was measurable only at the highest 3 dose levels. Only 5 patients (4 at the highest dose level) had total serum levels which exceeded 50 laboratory units/ml (55, 63, 800, 800, and 550 laboratory units/ml, respectively). In order to confirm the biological effectiveness of this schedule, we measured IFN-inducible proteins prior to therapy, 24 h after the initiation of the infusion, and at the completion of the 5-day infusion. 2'-5'-Oligoadenylate synthetase, serum beta 2-microglobulin, neopterin, and p78 levels all increased significantly, and serum tryptophan decreased significantly within 24 h after the initiation of treatment (P less than 0.0001). A dose-response effect was observed for serum beta 2-microglobulin, neopterin, and p78 (P less than 0.02). We retrospectively compared the results of this trial with those of another IFN-beta/IFN-gamma trial in which IFN-beta and IFN-gamma were administered by i.v. bolus. Within the limitations of a retrospective comparison, continuous infusion was less well tolerated than our previous schedule of bolus administration 3 times/week. However, the continuous infusion schedule appeared to be more effective in enhancing 2'-5'-oligoadenylate synthetase levels in mononuclear cells. We conclude that tolerable doses of IFN-beta and IFN-gamma do not result in serum IFN levels which produce significant synergistic antiproliferative responses in vitro. This study and other findings suggest that, unless higher doses can be achieved, combinations of IFN-beta and IFN-gamma are unlikely to have significant therapeutic activity.

2',5'-Oligoadenylate (2-5A) synthetase is an intracellular enzyme induced during viral diseases and after clinical administration of interferons (IFN). Because monocytes and T and B lymphocytes in peripheral blood are targets of viral infection, we compared the basal levels and induction of 2-5A synthetase activity in these three cell types. IFN-beta induced significant, three- to eightfold increases in 2-5A synthetase activity in monocytes and T and B lymphocytes, both in vitro and in vivo. IFN-gamma was less effective in inducing 2-5A synthetase than IFN-beta, yielding up to 2.6-fold increases in monocytes and slight increases in lymphocytes in vitro. IFN-beta also had greater 2-5A synthetase-inducing activity than IFN-gamma in HL-60 and U937 monocytic cell lines and in human peritoneal macrophages. Baseline levels of 2-5A synthetase in untreated peripheral blood monocytes separated by adherence or density gradients were three- to fivefold higher than in lymphocytes. 2-5A synthetase levels were twofold higher in B than in T lymphocytes separated by fluorescence-activated cell sorting. Tumor necrosis factor-alpha (TNF-alpha) did not induce this enzyme in peripheral blood mononuclear cells (PBMCs) or peritoneal macrophages. Thus, monocytes had greater basal and induced levels of 2-5A synthetase activity than did lymphocytes and contributed disproportionately to total 2-5A synthetase activity in peripheral blood.

Combination treatment of SKCO1 human colon carcinoma cells with beta ser-interferon (IFN-beta ser) and gamma-interferon (IFN-gamma) results in a synergistic antiproliferative effect. The role of IFN-beta ser and IFN-gamma receptor modulation was investigated as a possible mechanism for this response. IFN-gamma (0.05-50 ng/ml) pretreatment of SKCO1 cells for 24 h decreased specific binding of 125I-IFN-beta ser by 35-60%. Scatchard analysis of binding data obtained following 24-h treatment with 5 ng/ml IFN-gamma showed that this reduction in binding was due to a decreased receptor affinity (control cells, Kd = 46 +/- 1.6 pM; IFN-gamma-treated cells, Kd = 106 +/- 6 pM, n = 2) rather than a significant change in receptor number (receptor number/control cell = 1214 +/- 471, receptor number/IFN-gamma treated cell = 1118 +/- 153, n = 2). In contrast, pretreatment of SKCO1 cells with IFN-beta ser (5 ng/ml) resulted in slight (10-35%) increases in 125I-IFN-gamma-specific binding. Scatchard analysis of binding data obtained following 24-h treatment with 5 ng/ml IFN-beta ser showed a decrease in binding affinity (control cells, Kd = 28 +/- 7 pM; IFN-beta ser-treated cells, Kd = 38 +/- 7 pM, n = 2) and a 32% increase in IFN-gamma receptor sites (receptor number/control cell = 4257 +/- 464, receptor number/IFN-beta ser-treated cell = 5570 +/- 730; n = 2). 125I-IFN-gamma internalization studies performed at 37 degrees C confirmed the cell surface binding assays; IFN-beta ser-treated cells internalized 30-50% more labeled IFN-gamma than untreated cells. However, it is unlikely that differences in binding and internalization of this magnitude play a primary role in the synergistic antiproliferative effect of IFN-gamma with IFN-beta ser in SKCO1 cells. Biochemical modulation at sites distal to the ligand receptor interaction should be investigated.

Interferon-gamma (IFN-gamma) previously has been shown to inhibit the replication of Chlamydia psittaci in epithelial cells by inducing indoleamine 2,3-dioxygenase, the enzyme that decyclizes tryptophan to N-formylkynurenine. The role of indoleamine 2,3-dioxygenase in IFN-mediated inhibition of C. psittaci in human macrophages has now been examined. Peripheral blood monocytes from normal donors were isolated and cultivated 10-14 days to allow differentiation to macrophages. Cells were then treated with either IFN-gamma or IFN-beta for 48 h before infection with sufficient C. psittaci to infect approximately 30% of the cells. Infected cells were incubated 24 h, at which time coverslips were fixed, stained with Giemsa, and examined for development of C. psittaci inclusions by light microscopy. Complete inhibition of inclusion development was observed with IFN-gamma. In the absence of lipopolysaccharide, inhibition of C. psittaci by IFN-beta was variable; however, in the presence of lipopolysaccharide, IFN-beta also completely inhibited C. psittaci replication. The addition of excess tryptophan to the culture medium at the time of infection partially reversed the effect of IFN on the inhibition of C. psittaci growth in a concentration-dependent manner. Indoleamine 2,3-dioxygenase activity was determined by measurement of the concentrations of tryptophan and its metabolites in the culture medium after reversed-phase high-performance liquid chromatography. Significant indoleamine 2,3-dioxygenase activity was observed only in macrophages treated with IFN-gamma or combined IFN-beta plus lipopolysaccharide, and resulted in greater than 50% of available tryptophan being catabolized in a 4-h period.(ABSTRACT TRUNCATED AT 250 WORDS)

This study assessed biologic response modification at three different dose levels (0.15, 1.5, and 15 mg/m2) of interferon-gamma (IFN-gamma) administered by intravenous bolus three times weekly. A final total of 24 patients were evaluable. Dose-limiting toxicity occurred at the highest dose level (15 mg/m2) and included fatigue, leukopenia, and hepatotoxicity. Evaluation of biologic response modification included assessment of 2',5'-oligoadenylate (2-5A) synthetase activity in peripheral mononuclear cells, measurement of serum beta 2-microglobulin and expression of beta 2-microglobulin on monocytes, measurement of monocyte HLA Class II expression (HLA-DR, HLA-DQ), and measurement of hydrogen peroxide generation by monocytes 24 h after the first and fourth IFN-gamma treatments. Significant increases (p less than 0.05) from baseline were seen at 24 h with all parameters except H2O2 generation. Except for enhancement of HLA-DR, even the lowest dose (0.15 mg/m2) augmented synthesis of 2-5A synthetase and HLA proteins. A dose-response effect was noted for changes in serum and monocyte beta 2-microglobulin levels but not for 2-5A synthetase levels or HLA Class II antigen expression on monocytes. After 4 doses administered over 9 days, most parameters remained increased when compared to pretreatment, but were not further enhanced when compared with levels attained after the first dose. The results of this study document the efficacy of IFN-gamma for biological activation over a wide dose range and are consistent with the postulate that immunoregulatory effects of biological therapeutics can be obtained in man at doses substantially less than those that are maximally tolerated. Further documentation of biologic response parameters by IFN-gamma at low doses will be necessary to determine the importance of biologic activation in relation to antitumor activity.

Interferon-alpha, interferon-beta, and interferon-gamma differ in their antiproliferative effects for several cell lines. Interferons were thus assessed for their activity in inhibiting proliferation of three renal cell carcinoma cell lines. The malignant epithelial phenotype of each of these cell lines was confirmed by electron microscopy, histology, karyotype and tumorigenicity. When compared on an anti-viral unit basis, naturally produced interferon-beta was more effective than natural interferon-alpha for all cell lines and clones. Proliferation of each of the cell lines was inhibited by interferon-gamma. In all cases, removal of interferons from culture media resulted in resumption of the rate of cell growth after a variable delay of 6-10 days. If the antiproliferative effects of interferons predominate in mediating tumor regression, clinical response may depend upon the type of interferon to which the tumor is exposed.

HL-60, a promyelocytic cell line, was treated with both recombinant interferon-gamma (IFN-gamma) and 1,25-(OH)2 vitamin D3 (D3), and the effect on monocyte-specific markers was assessed. IFN-gamma and D3 modulated different stages in the monocytic differentiation with respect to interleukin-1 (IL-1) production and secretion. D3 induced production of an intracellular IL-1 activity that was not secreted after lipopolysaccharide stimulation. IFN-gamma did not induce intracellular IL-1 but differentiated HL-60 cells, which had been treated with D3, so that lipopolysaccharide stimulated IL-1 release. Both D3 and IFN-gamma individually enhanced expression of nonspecific esterase; in combination the two agents potentiated each other. Expression of cell surface Leu-M3 antigen was also enhanced by the combination of these two agents. Thus, IFN-gamma not only potentiated expression of D3-induced markers but also conferred phenotypic properties characteristic of monocytes. IFN-gamma may play a role in the differentiation of bone marrow cells to mature monocytes.

Based upon the in vitro synergistic activity of interferon-beta (IFN-beta) and interferon-gamma (IFN-gamma) observed in melanoma cells, we initiated a Phase II trial using the combination to determine the clinical antitumor efficacy in patients with advanced disease. Fifteen patients with metastatic malignant melanoma were given 2,000 micrograms of recombinant IFN-gamma (rIFN-gamma) (Biogen) intravenously (i.v.) over 10 min, followed by a 10 min i.v. injection of 30 million units of recombinant IFN-beta (rIFN-beta ser) (Triton) 3 x/week. Six patients had skin, soft tissue, nodal, or subcutaneous metastases, 6 had visceral disease only, and 3 had both. Seven patients had received prior treatment, including chemotherapy (6), radiotherapy (3), and/or immunotherapy (3). Side effects included typical IFN constitutional symptoms such as anorexia, fatigue, nausea, and myalgias, but were not dose limiting. The mean drop in the white blood cell count (WBC) following 1 month of therapy, compared to baseline, was 3.3 x 10(3)/mm2 (p = 0.002); the mean increase in SGOT was 24.1 U/l (p less than 0.001). One patient had a dose reduction for Grade III anorexia and fatigue which did not resolve with repeated treatment. One patient with liver metastases had radiographical and clinical stabilization of his disease for 1 year. No responses were seen. The median time to progression was 6 weeks. Two patients' tumors were evaluable in the human tumor colony forming assay (HTCFA) and were markedly sensitive to the antiproliferative effects of IFN combinations. Both patients, however, failed to respond clinically.(ABSTRACT TRUNCATED AT 250 WORDS)

Human recombinant gamma interferon (rHuIFN-gamma) was found to induce tryptophan degradation in vitro in human cell cultures and in vivo in participants in phase I clinical trials. When human lung fibroblasts were treated with various concentrations of rHuIFN-gamma, they degraded tryptophan in a dose- and time-dependent manner. No tryptophan degradation was observed when cells were incubated in growth medium alone or in medium supplemented with human recombinant beta-interferon (rHuIFN-beta ser). Similarly human bladder carcinoma cells were induced to catabolize tryptophan after incubation with rHuIFN-gamma, but no activity was observed in untreated cells or cells treated with either rHuIFN-beta ser or human naturally produced alpha-interferon (HuIFN-alpha). When tryptophan plasma levels were measured in cancer patients who had received i.v. bolus injections of rHuIFN-gamma as part of a phase I clinical trial, decreased tryptophan levels were observed when compared with pretreatment values or values obtained from individuals who had received i.v. injections of HuIFN-alpha. Urine analyses were suggestive that plasma tryptophan degradation occurred via the kynurenine catabolic pathway in individuals who received rHuIFN-gamma. We conclude that tryptophan degradation is an activity induced in vitro and in vivo in response to exogenous IFN-gamma but not to IFN-alpha or IFN-beta. Tryptophan degradation may play an important role in the mechanism of antiproliferative, immunologic, and clinical side effects of IFN-gamma.