Leukocyte Interferon Research Articles

Update on the Diagnosis and Treatment of Rare Neuroendocrine Tumors

A growing knowledge and awareness of bizarre clinical presentations, the physiology of new peptides, and the more frequent use of radioimmunoassay techniques has led to the identification of more patients with glucagonomas, somatostatinomas, vipomas, and ectopic tumors. Definite clinical syndromes, though sometimes seemingly "silent," occur as a result of hypersecretion of newly identified hormones and neurotransmitters that act in endocrine, neurocrine, or paracrine fashion to alter normal metabolism of carbohydrates and electrolytes. Metabolically and clinically, glucagonomas are catabolic, somatostatinomas are inhibitory, and vipomas are diarrheogenic. The clinical syndromes can be differentiated from other, more common, endocrinopathies; the measurement of the plasma concentrations of the specific peptides is not only diagnostic, but prognostic. Specific pathologic confirmation of the functional potential of these tumors by immunocytochemical techniques is now possible. The goal of diagnosis is detection and tumor localization before metastases occur so that surgical excision may be curative. Objective clinical and humoral responses to chemotherapy for nonresectable or metastatic lesions can be expected in about 50 per cent of patients; specifically, dacarbazine (DTIC) is the agent of choice for glucagonomas, streptozotocin for somatostatinomas, and leukocyte interferon for vipomas.

Interferon-alpha down-regulates insulin receptors in lymphoblastoid (Daudi) cells. Relationship to inhibition of cell proliferation.

The Daudi line of human lymphoblastoid cells requires insulin and transferrin for growth in serum-free medium and is highly sensitive to the inhibitory effect of human leukocyte interferon (IFN-alpha) on cell proliferation. A variant subline of Daudi cells, which is resistant to the antiproliferative action of IFN-alpha, also has been grown in serum-free medium containing insulin and transferrin. The proliferation of IFN-sensitive and -resistant Daudi cells is dependent on the occupancy of insulin receptors, with optimal cell proliferation observed at high receptor occupancy (nearly 100%). No evidence was found for receptors for insulin-like growth factor I on Daudi cells. IFN treatment of IFN-sensitive cells decreased the capacity of the cells to bind 125I-insulin. The altered binding capacity was due to diminished specific, lower affinity insulin binding, as detected at high 125I-insulin concentrations. Higher affinity insulin binding was not altered by IFN. Insulin binding was also reduced in detergent-solubilized extracts from IFN-treated sensitive Daudi cells and the magnitude of the effect was comparable to that observed in intact cells. This indicates that the total number of insulin binding sites (surface + internal) is decreased in IFN-treated sensitive cells. Insulin binding to IFN-sensitive cells decreased linearly with time between 6 and 48 h from the addition of IFN. The effect on lower affinity insulin binding developed more rapidly than the inhibitory effect of IFN on cell proliferation. The insulin-binding capacity of Daudi cells resistant to the antiproliferative effect of IFN was unaffected by IFN, despite the fact that these cells contain as many cell surface IFN receptors as sensitive cells. These observations raise the possibility that lower affinity insulin binding is important in the growth-promoting actions of insulin.

Chemical characterization of recombinant human leukocyte interferon A using fast atom bombardment mass spectrometry.

Proteolytic digests of biologically active fractions of recombinant human leukocyte interferon A expressed in large quantities in Escherichia coli were analyzed by fast atom bombardment mass spectrometry and high-performance liquid chromatography. The values observed in the mass spectra of digests of the major fraction of recombinant human leukocyte interferon A with trypsin and Staphylococcus aureus protease V8 accounted for 93% of the amino acid sequences of human leukocyte interferon A predicted from the nucleotide sequence of the gene encoding the protein, indicating that the major fraction of recombinant human leukocyte interferon A was expressed with the same amino acid sequence as that translated from the nucleotide sequence of the gene encoding the protein. Mass spectrometry of proteolytic digests of two minor fractions of recombinant human leukocyte interferon A and mass and amino acid analyses of their high-performance liquid chromatography fractions showed that the amino group of the N-terminal amino acid residue of interferon was in part acetylated, and the Cys-1 and Cys-98 residues were oxidized to cysteic acid or linked to glutathione. These findings suggest that amino acid residues in recombinant proteins prepared in large quantities in E. coli are modified post-translationally.

Recombinant interferon enhances monoclonal antibody-targeting of carcinoma lesions in vivo.

Heterogeneity in the expression of tumor-associated antigens, as defined by the binding of monoclonal antibodies, is a characteristic common to most, if not all, human carcinoma cell populations. Antigen-negative cells within the population can escape detection and therapy by their failure to bind the appropriate antibody. Therefore, the extent of antigenic heterogeneity is an important consideration when designing protocols for the management of cancer by administration of monoclonal antibodies. One approach to counteracting the effect of antigenic heterogeneity is the use of clone A of recombinant human leukocyte interferon (Hu-IFN-alpha A). Administration of Hu-IFN-alpha A in vivo effectively increased the amount of tumor antigen expressed by a human colon xenograft in situ and augmented the localization of a radiolabeled monoclonal antibody to the tumor site. Concomitant administration of Hu-IFN-alpha A and monoclonal antibody may thus be effective in overcoming the antigenic heterogeneity of carcinoma cell populations and in enhancing the efficacy of monoclonal antibodies in the detection and treatment of carcinoma lesions.

Immunochemical and functional analysis of HLA class II antigens induced by recombinant immune interferon on normal epidermal melanocytes.

The effect of recombinant immune interferon (IFN-gamma) on the expression and shedding of HLA antigens and of melanoma-associated antigens (MAA) by epidermal melanocytes was investigated by using serologic and immunochemical techniques. IFN-gamma enhances the expression and/or shedding of HLA class I antigens and of the cytoplasmic MAA defined by monoclonal antibody (MoAb) 465.12S and induces a slight reduction in the expression of the high m.w. melanoma-associated antigen (HMW-MAA). In agreement with the data in the literature, melanocytes incubated with IFN-gamma acquire HLA-DR, -DQ, and -DP antigens. Contrary to previous information in the literature, the effect is not restricted to HLA class II antigens, since IFN-gamma also induces the expression of the 96-kDa MAA recognized by MoAb CL203. The effect of IFN-gamma on HLA class II antigens and 96-kDa MAA is dose and time dependent and is specific, because recombinant leukocyte interferon affects the expression of neither type of antigen. In spite of the expression of HLA class II antigens, IFN-gamma-treated melanocytes do not acquire the ability to stimulate the proliferation of allogeneic lymphocytes. HLA-DR antigens are more susceptible to induction by IFN-gamma than HLA-DQ and -DP antigens, since the percentage of melanocytes acquiring HLA-DQ and -DP antigens is lower than that acquiring HLA-DR antigens. Furthermore, the dose of IFN-gamma is higher and the time of incubation is longer to induce HLA-DQ and -DP antigens than to induce HLA-DR antigens. The differential susceptibility of HLA-DR, -DQ, and -DP antigens as well as of melanocytes from various donors to the modulating effect of IFN-gamma may provide an explanation for the more frequent detection of HLA-DR than of HLA-DQ and -DP antigens in melanoma lesions and for the expression of HLA class II antigens by some, but not all, melanoma lesions.

Improvement of proteinuria in a case of hepatitis B-associated glomerulonephritis after treatment with interferon

Disappearance of proteinuria was observed in a patient with hepatitis B-associated chronic glomerulonephritis after treatment with leukocyte interferon. The decrease of proteinuria was preceded by the disappearance of both deoxyribonucleic acid polymerase activity and hepatitis B e antigen from the patient's sera.

2',5'-Oligoadenylate synthetase activity in peripheral blood lymphocytes as a clinical marker in interferon therapy for chronic hepatitis B.

Treatment of chronic hepatitis B (CHB) with human leukocyte interferon (IFN-alpha) was studied in terms of increase of 2',5'-oligoadenylate (2-5A) synthetase activity in peripheral blood lymphocytes (PBL) after IFN-alpha administration. The 2-5A synthetase activity in PBL increased to a maximum 16-24 h after IFN-alpha injection and then gradually decreased, while serum HB virus DNA polymerase (HBV-DNAP) activity, which corresponds to the amount of HBV virions in the serum, decreased to a minimum after about 48 h. Increase in 2-5A synthetase activity was followed about 1 day later by decrease in DNAP activity. Furthermore, of 9 patients treated with IFN for at least 22 weeks and observed for over 1 year after IFN treatment, the effective 5 cases in which HBeAg disappeared during IFN therapy and did not reappear showed rather high increases in 2-5A synthetase activity, whereas the ineffective 4 cases in which HBeAg remained positive at the end of IFN therapy showed little increases in enzyme activity. The present study suggests that prescreening by 2-5A synthetase assay before IFN therapy should be useful for obtaining better results in IFN treatment of CHB patients.

Purification of human leukocyte interferon A derived from Escherichia coli: An aging process to prevent the formation of its oligomers

Recombinant human leukocyte interferon A (rIFN-αA) was purified from Escherichia coli cells by monoclonal antibody affinity chromatography (Ab) and CM-cellulose column chromatography (CM). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the CM eluate was found to contain a large amount of rIFN-αA oligomers, in addition to the monomer, linked by intermolecular disulfide bonds. In contrast, the Ab eluate was found to contain a large amount of rIFN-αA monomers. These results show that the oligomers were formed at the CM step. Free SH groups were detected at a significant level in the Ab eluate, but were detected barely in the CM eluate, indicating that the Ab eluate contained some amount of the unstable monomer with free SH groups. To prevent the formation of the oligomers we established a simple and easily scaled-up process, called an aging process, during which the Ab eluate was incubated at pH 3.3 at 37°C for several days. This process is effective for changing the unstable monomer to the stable monomer and thus preventing the formation of the oligomers.

Anomalous behavior of human leukocyte interferon subtypes on polyacrylamide gel electrophoresis in the presence of dodecyl sulfate

35S-labeled human leukocyte interferon (IFN) subtypes produced in a cell-free system derived from Escherichia coli were analyzed by polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE). Some IFN subtypes anomalously showed lower electrophoretic mobilities than those expected from their formula molecular masses. The results with hybrid IFNs and esterification suggest that this anomaly of IFN subtypes on SDS-PAGE is due to the introduction of one or two negative charges in the middle of the molecule.

3] Design, chemical synthesis, and molecular cloning of a gene for human epidermal growth factor

Since the first successful construction and genetic expression of a chemically synthesized gene coding for somatostatin, many total human gene syntheses have been reported, including growth hormone, insulin, leukocyte interferon, insulin-like growth factors I and II, and lymphotoxin. Given the amino acid sequence of a peptide hormone or growth factor, it is easier to synthesize the gene than to isolate it by cDNA cloning if it contains less than 350 amino acids. Chemical synthesis permits gene design. Codon usage and restriction sites can be tailored for a specific expression system. This chapter discusses the methods used for chemical synthesis, enzymatic assembly, and expression in the yeast of human epidermal growth factor (hEGF). The chapter presents a simple manual protocol for rapid oligonucleotide synthesis by a solid supported phosphoramidite method using commercially available reagents for the people who do not have access to automated synthesizers. There is also a description of the design of synthetic genes for facile subcloning into expression systems. By using hEGF as an example, the chapter presents a general method for the one-step enzymatic ligation and subsequent gel purification of a full-length double-stranded gene constructed from several single-stranded synthetic oligomers. The techniques discussed in the chapter are used to construct several genes and gene derivatives.

4678751 Hybrid human leukocyte interferons: David Goeddel assigned to Genentech Inc

4678751 Hybrid human leukocyte interferons: David Goeddel assigned to Genentech Inc

Interferons in the treatment of multiple myeloma

A review of the clinical studies in which interferons have been involved has shown that they may have a role in the treatment of multiple myeloma. Twelve studies, each of which involved at least 10 evaluable cases (352 in total) with various dose schedules involving leukocyte, lymphoblastoid and recombinant alpha-IFNs, reported 8-33% objective responses. The response duration was rather short but, in a few cases, it lasted for more than a year. In addition to a decrease in the levels of M-protein and/or urine Bence-Jones protein, a decrease in the number of plasma cells in the bone marrow, disappearance of bone pain, healing of bone lesions, increase of hemoglobin and/or restoration of normal immunoglobulins were observed. Higher doses of recombinant alpha-interferons seemed to exert a stronger effect. No clear difference in response rate was observed between myeloma which had been previously treated and that which was not treated. At least clinically, therefore, there seems to be no cross-resistance between alpha-interferons and conventional anti-tumor drugs. A randomized study comparing low-dose leukocyte interferon with intermittent high-dose melphalan-prednisone has given a lower response rate for interferon. Beta- and gamma-interferons have not yet been extensively studied. They have been used at low doses producing an objective response in 7% of 68 and 2% of 45 evaluable cases, respectively. Since the myelosuppression of interferons is transient and, after discontinuation of interferon therapy, peripheral blood cells usually recover within a week, it may be possible to use interferon in combination with agents that have strong myelosuppressive effects provided there is no synergism.

The effect of cultivation and interferon treatment on the surface morphology of hairy cell leukemia cells.

The ability of interferon to induce alterations in the surface morphology of malignant B cells from 4 patients with hairy cell leukemia (HCL) was investigated under scanning electron microscopy. Peripheral blood hairy cells showed the same surface features as those isolated from spleens involved with HCL, and constantly exhibited both ruffles and microvilli. Cultured hairy cells displayed extreme polarization of their surface microprojections, and very active surfaces with elongated microvilli and broad-based ruffles were evident. All 4 cases of HCL were treated with recombinant human leukocyte interferon in vitro, and one-third of the HCs from 3 cases displayed deformed surfaces with "bubbling" membrane and altered microprojections. Most of these IFN-treated hairy cells appeared to be larger in size compared to the untreated control cells, and frequently showed a villous bud-like formation at their extremity. The possibility that these unique surface alterations reflect IFN-induced changes in the cytoskeletal proteins and/or membrane components is discussed in light of the clinical efficacy of in vivo IFN treatment in HCL.

4678751 Hybrid human leukocyte interferons: David Goeddel assigned to Genentech Inc

4678751 Hybrid human leukocyte interferons: David Goeddel assigned to Genentech Inc

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.

TREATMENT OF MALIGNANT ENDOCRINE PANCREATIC TUMOURS WITH HUMAN LEUCOCYTE INTERFERON

22 patients with advanced malignant endocrine pancreatic tumours were treated with human leucocyte interferon 3-6 × 10 6 IU per day. Objective responses (more than 50% reduction in tumour markers or tumour size) were seen in 7/7 with the watery diarrhoea/hypokalaemia/achlorhydria syndrome, 3/4 with the Zollinger-Ellison syndrome, 6/9 with "non-functioning" tumours, and 1 with a mixed tumour mainly producing somatostatin. The median duration of response was 8.5 months, and all responders improved clinically. Adverse effects seemed more tolerable than those of cytotoxic treatment.

A monoclonal antibody with broad reactivity to human interferon-alpha subtypes useful for purification of leukocyte-derived interferon.

A monoclonal antibody to human interferon-alpha, termed HT-1 antibody, with a broad reactivity to various subtypes of interferon-alpha was prepared. It bound and neutralized all of the four subtypes of E. coli-derived human recombinant interferon-alpha (alpha 1, alpha 2, alpha 4, and alpha 6) tested; it also neutralized human natural leukocyte interferon but only partially. Human interferon-beta and -gamma were not bound. The antibody conjugated to Sepharose beads retained over 90% of human leukocyte interferon induced by Sendai virus. The bound interferon was recovered by acid elution in good yields and in almost pure form (specific activity was about 2 X 10(8) international units/mg protein). The purified interferon showed, in SDS-polyacrylamide gel electrophoresis, an activity profile with major peaks in a mol. wt. range of 17,000-22,000, which completely agreed with the profile shown by polyclonal antibody-purified interferon. Such purified leukocyte interferon-alpha preparations containing most of the naturally occurring subtypes can be useful for clinical and other purposes.

Phase II study of alpha interferon on renal cell carcinoma. Summary of three collaborative trials.

A cooperative study was carried out in 44 institutions in Japan to evaluate the clinical efficacy of two kinds of recombinant human leukocyte interferon (Ro 22-8181 and Sch 30500) and a human lymphoblastoid interferon (MOR-22) on metastatic lesions of renal cell carcinoma. Of 226 evaluable cases, efficacy was observed in 40 cases; complete response (CR) in 4 cases, and partial response (PR) in 36 cases, indicating an overall response rate of 17.7%. Although there were no statistically significant differences in age range, sex distribution, and performance status between responders and nonresponders, the incidence of poor response was significantly higher among patients in whom primary renal cell carcinoma had remained or in those who had prior chemotherapy and/or radiation therapy. When response was assessed with regard to individual metastatic sites of renal cell carcinoma, pulmonary metastasis was significantly susceptible to interferon treatment, compared to other metastatic sites. The median period to achieve CR or PR was 45 days (range, 22-211 days) and the median length to maintain CR or PR was 79 days (range, 29-278 days). The median of pulmonary metastatic area that was measured by the sum of the products of the largest perpendicular diameters was 7.2 cm2 (0.5-107.8 cm2), which was reduced to 1.1 cm2 (0-48.8 cm2) by alpha interferons. Cumulative survival of all responder patients was significantly longer than that of other patients with poor response.

Phase II study of alpha interferon on renal cell carcinoma. Summary of three collaborative trials.

A cooperative study was carried out in 44 institutions in Japan to evaluate the clinical efficacy of two kinds of recombinant human leukocyte interferon (Ro 22-8181 and Sch 30500) and a human lymphoblastoid interferon (MOR-22) on metastatic lesions of renal cell carcinoma. Of 226 evaluable cases, efficacy was observed in 40 cases; complete response (CR) in 4 cases, and partial response (PR) in 36 cases, indicating an overall response rate of 17.7%. Although there were no statistically significant differences in age range, sex distribution, and performance status between responders and nonresponders, the incidence of poor response was significantly higher among patients in whom primary renal cell carcinoma had remained or in those who had prior chemotherapy and/or radiation therapy. When response was assessed with regard to individual metastatic sites of renal cell carcinoma, pulmonary metastasis was significantly susceptible to interferon treatment, compared to other metastatic sites. The median period to achieve CR or PR was 45 days (range, 22-211 days) and the median length to maintain CR or PR was 79 days (range, 29-278 days). The median of pulmonary metastatic area that was measured by the sum of the products of the largest perpendicular diameters was 7.2 cm2 (0.5-107.8 cm2), which was reduced to 1.1 cm2 (0-48.8 cm2) by alpha interferons. Cumulative survival of all responder patients was significantly longer than that of other patients with poor response.

Chromatographic methods for purification of leukocyte interferon

A chromatographic method has been developed for obtaining interferon products of three types: (a) with a specific activity of 10 5 I.U./mg of protein (on the basis of leukocyte interferon); (b) with a specific activity of 10 6 I.U./mg of protein (on the basis of leukocyte interferon); (c) a near-homogeneous preparation with an activity of 2 · 10 8—4 · 10 8 I.U./mg of protein (for leukocyte and recombinant interferons). In the first two cases, only gel permeation chromatography (GPC) is used, which yields interferon preparations containing the oligomeric forms of interferon and also such biologically active proteins as factor D and interleukin-1. In the last case, GPC and immunoaffinity chromatography may be conveniently supplemented by interferon purification by ion exchange to remove immunoglobulins and the products formed by their hydrolytic or proteolytic decomposition.