Construction of a recombinant human GM-CSF/MCAF fusion protein and study on its in vitro and in vivo antitumor effects

A novel cytokine fusion protein was constructed by fusing granulocyte macrophage colony stimulating factor (GM-CSF) with monocyte chemotactic activating factor (MCAF), which acts as a factor directing effector cells (monocytes) to a target site. The recombinant human GM-CSF/MCAF fusion protein could sustain the growth of GMCSF-dependent cell line TF1 and was chemotactic for monocytes. Thein vitro antitumor effect showed that rhGM-CSF/MCAF could activate monocytes to inhibit the growth of several human tumor cell lines, including a promyelocyte leukemia cell line HL-60, a lung adenocarcinoma cell line A549, a hepatoma cell line SMMC-7721 and a melanoma cell line Bowes. Furthermore, the cytotoxicity of monocytes activated by rhGM-CSF/MCAF against HL-60 and A549 was greater than that activated by GM-CSF or MCAF alone, even greater than that activated by a combination of GM-CSF and MCAF, suggesting that the fusion protein has synergistic or enhanced effects. Thein vivo antitumor effect indicated that rhGM-CSF/MCAF had marked antitumor effect against A549 tumor in nude mice and even completely suppressed tumor formation. rhGM-CSF/MCAF was significantly more effective in inhibiting tumor growth than rhGM-CSF. Histological analysis showed that tumor site injected with rhGM-CSF/MCAF was infiltrated by a large number of monocytes while a sparse infiltration of monocytes was observed at the tumor site injected with rhGM-CSF or normal saline, suggesting that the antitumor effect of rhGM-CSF/MCAF was mediated by the recruitment of a large number of monocytes to the tumor site.     

Construction of a recombinant human GM-CSF/MCAF fusion protein and study on its in vitro and in vivo antitumor effects

A novel cytokine fusion protein was constructed by fusing granulocyte macrophage colony stimulat-ing factor (GM-CSF) with monocyte chemotactic activating factor (MCAF), which acts as a factor directing effector cells (monocytes) to a target site. The recombinant human GM-CSF/MCAF fusion protein could sustain the growth of GM-CSF-dependent cell line TF1 and was chemotactic for monocytes. The in vitro antitumor effect showed that rhGM-CSF/MCAF could activate monocytes to inhibit the growth of several human tumor cell lines, including a promyelocyte leukemia cell line HL-60, a lung adenocarcinoma cell line A549, a hepatoma cell line SMMC-7721 and a melanoma cell line Bowes. Furthermore, the cytotoxicity of monocytes activated by rhGM-CSF/MCAF against HL-60 and A549 was greater than that activated by GM-CSF or MCAF alone, even greater than that activated by a combina-tion of GM-CSF and MCAF, suggesting that the fusion protein has synergistic or enhanced effects. The in vivo anti-tumor effect indicated that     

In vitro andin vivo antineoplastic effects of ortrovanadate

In the present study we have demonstrated that orthovanadate at concentrations of 5–10 uM is cytotoxic to proliferating cells including primary cultures and tumour cell lines. However, concentrations of up to 50 uM did not affect the viability of non-proliferating cells. The cytotoxicity appears to be dependent on the vanadium concentration rather than on the oxidation state of vanadium or the vanadium compound. Furthermore, tumour cell lines with different proliferative rates were equally sensitive to orthovanadate cytotoxicity. Although the mechanisms responsible for the cytotoxicity are not known, addition of H₂O₂ potentiated orthovanadate cytotoxicity suggesting that hydroxyl or vanadium radicals may be involved.In vivo subcutaneous injections of orthovanadate into mice containing MDAY-D2 tumours resulted in the inhibition of tumour growth by 85–100%. These data indicated that orthovanadate at concentrations greater than 5 uM has antineoplastic properties and may be useful as a chemotherapeutic agent.     

Dysfunctional monocytes from a patient with disseminatedMycobacterium kansasii infection are activatedin vitro andin vivo by GM-CSF

A 27 year-old woman presented with disseminated infection due toMycobacterium kansasii. Signs and symptoms of disseminated infection persisted despite the administration of multiple antimycobacterial agents to which her organism was sensitive for 15 months. She was seronegative for HIV-1 and functional studies of T and B lymphocytes and granulocytes failed to demonstrate any abnormality. Peripheral blood monocytes proved abnormally permissive to the intracellular growth ofMycobacterium avium andM. kansasii, and expressed normal number of receptors to interferon-gamma, but reduced numbers of receptors to granulocyte monocyte colony stimulating factor and tumor necrosis factor. These defects were partially reversed within vitro exposure of her cells to recombinant GM-CSF. In addition, administration of recombinant human GM-CSFin vivo (250 mg/M² per day) for 10 days armed her circulating monocytes as evidenced by increased production of O₂ ^– in response to phorbol esther and, when infectedex vivo withM. kansasii, enhanced inhibition of intracellular growth compared with pre-therapy monocytes. These defects reappeared with discontinuation of GM-CSF and resolved with its re-administration. While a salutary clinical and microbiologic effect was difficult to assess, administration of GM-CSFin vivo was associated within vitro activation of monocytes and enhanced mycobactericidal activity in this patient with a defect in monocyte function.     

A recombinant human G-CSF/GM-CSF fusion protein from E. coli showing colony stimulating activity on human bone marrow cells

Granulocyte-Macrophage colony stimulating factor (GM-CSF) and Granulocyte colony stimulating factor (G-CSF) are cytokines involved in the differentiation of bone marrow progenitor cells into myeloid cells. They also activate mature myeloid cells to mediate a variety of antimicrobial activities and inflammatory responses. Recombinant GM-CSF and G-CSF proteins have been used to treat various diseases including cancer and hematopoietic diseases and to isolate peripheral blood progenitor cells for bone marrow transplantation. A plasmid construct expressing recombinant human G-CSF/GM-CSF fusion protein has now been prepared by linking the human G-CSF and GM-CSF coding regions and the recombinant fusion protein has been successfully expressed in E. coli. The recombinant human G-CSF/GM-CSF fusion protein was extracted and purified from the cellular inclusion and refolded into the biologically active form to show colony stimulating activity. The recombinant fusion protein exhibited colony stimulating activity on human bone marrow cell cultures, indicating that the linkage of GM-CSF and G-CSF by a linker peptide may not interrupt activities of the cytokines in the fusion protein. The colony forming unit of the fusion protein was also higher than those of the cultures treated with the same molar numbers of the recombinant human GM-CSF and G-CSF separately, which suggests that the fusion protein presumably retains both G-CSF and GM-CSF activities.     

Construction and characterization of a recombinant human beta defensin 2 fusion protein targeting the epidermal growth factor receptor: in vitro study

The HER2/neu proto-oncogene encodes a 185-kDa trans-membrane glycoprotein kinase with extensive homology to the epidermal growth factor receptor and plays a key role in the transformation and growth of malignant tumors. To date, two antibody drugs targeting HER2/neu have been developed successfully. In order to reduce the cost and the time of clinical treatment, we produced a fusion protein composed of human beta defensin 2 (hBD2) and anti-HER2/neu single-chain variable fragment (scFv 4D5), which is capable of specifically targeting, significantly inhibiting, and promptly killing HER2/neu-positive cancer cells. The recombinant protein was expressed in Escherichia coli using the small ubiquitin-related modifier (SUMO) as the molecular chaperone, and the optimal expression level reached to 40.2 % of the total supernatant protein. After purifying by Ni-NTA affinity chromatography, the fusion protein was cleaved with a SUMO-specific protease to obtain hBD2–4D5, which was further purified by Ni-NTA affinity chromatography. The purity of hBD2–4D5 was higher than 95 %, and the yield was 19?±?2 mg/L in flask fermentation. The cell number count and flow cytometry results showed that hBD2–4D5 exerted cytotoxic and anti-proliferative effects on HER2/neu-positive breast cancer cell line, SKBR-3. The results of scanning electron microscope and transmission electron microscope observation indicated that hBD2–4D5 could induce intracellular ultrastructure changes and cell necrosis by disrupting the cell membrane. Immunofluorescence analysis showed that hBD2–4D5 could bind to SKBR-3 cells and further be internalized into the cytoplasm. Moreover, hBD2–4D5 could also mediate apoptosis of SKBR-3 cells by up-regulating the ratio of Bax to Bcl-2.     

重组人血小板生成素融合蛋白工程菌发酵工艺的研究

对表达重组人血小板生成素融合蛋白（rhTPO/GST)工程菌的发酵条件进行了较详细的研究,优化了影响工程菌发酵的条件,探讨了发酵条件对工程菌表达外源蛋白量的影响。结果发现采用复合培养基分阶段流加有机营养物,用异丙基硫代-B-D-半乳糖苷（IPTG）进行诱导,使rhTPO/GST融合蛋白的湿菌重达25g/L以上,表达水平约占菌体总蛋白的30%左右。在此基础上,建立了中试发酵生产工艺流程。     

A recombinant human monoclonal antibody to human metapneumovirus fusion protein that neutralizes virus in vitro and is effective therapeutically in vivo

Human metapneumovirus (hMPV) is a recently discovered paramyxovirus that is a major cause of lower-respiratory-tract disease. hMPV is associated with more severe disease in infants and persons with underlying medical conditions. Animal studies have shown that the hMPV fusion (F) protein alone is capable of inducing protective immunity. Here, we report the use of phage display technology to generate a fully human monoclonal antibody fragment (Fab) with biological activity against hMPV. Phage antibody libraries prepared from human donor tissues were selected against recombinant hMPV F protein with multiple rounds of panning. Recombinant Fabs then were expressed in bacteria, and supernatants were screened by enzyme-linked immunosorbent assay and immunofluorescent assays. A number of Fabs that bound to hMPV F were isolated, and several of these exhibited neutralizing activity in vitro. Fab DS7 neutralized the parent strain of hMPV with a 60% plaque reduction activity of 1.1 mug/ml and bound to hMPV F with an affinity of 9.8 x10(-10) M, as measured by surface plasmon resonance. To test the in vivo activity of Fab DS7, groups of cotton rats were infected with hMPV and given Fab intranasally 3 days after infection. Nasal turbinates and lungs were harvested on day 4 postinfection and virus titers determined. Animals treated with Fab DS7 exhibited a >1,500-fold reduction in viral titer in the lungs, with a modest 4-fold reduction in the nasal tissues. There was a dose-response relationship between the dose of DS7 and virus titer. Human Fab DS7 may have prophylactic or therapeutic potential against severe hMPV infection.     

In vivo effects of recombinant human lymphotoxin on human medulloblastoma xenograft: Enhancement of antitumor activity of etoposide

The authors investigated the antitumor activities of rHuLT alone and in combination with etoposide on human medulloblastoma xenografts growing subcutaneously in nude mice. Intravenous administration of rHuLT (1.0×10⁵U/kg, 5.0×10⁵U/kg, 2.5×10⁶U/kg, three times a week for three weeks) suppressed medulloblastoma growth depending on the dose. However, the highest dosage caused serious side effects. Combining rHuLT (intravenously, 5.0×10⁵U/kg, three times a week for three weeks) with etoposide (intraperitoneally, 20mg/kg, once a week for three weeks) increased the antitumor activity without causing serious toxicity. Microscopically, tumor specimen showed thrombosed tumor vessels and massive necrosis 3 weeks after rHuLT treatment. Ultrastructural examination revealed that 120 minutes after the administration of rHuLT alone, disruption of interendothelial junctions was evident, and that the endothelial cells were destroyed at 240 minutes.Concentration of etoposide in tumor tissue peaked 30 minutes after intraperitoneal administration, and then decreased with time. When etoposide was administered in combination with rHuLT, the concentration of etoposide in tumor tissue after 60 to 240 minutes was significantly higher than when etoposide was given alone, and the area under the concentrationversus time curve was also greater for the tumors of mice with combination treatment.The findings suggest that the proper combination of rHuLT and etoposide may have synergistic antitumor activities. Histological changes suggest that increased concentrations of etoposide within the tumor after combination therapy may occur due to increased vascular permeability and/or decreased etoposide clearance which is the result of blood stasis in the tumor vasculature.Abbreviations AUC area under the concentration versus time curve - LT lymphotoxin - rHuLT recombinant human lymphotoxin - rHuTNF recombinant human tumor necrosis factor - TNF tumor necrosis factor     

In vitro and in vivo action of recombinant human GM-CSF (rhGM-CSF) in patients with myelodysplastic syndromes

The availability of rhGM-CSF has allowed the in vivo treatment of patients with cytopenia. Therefore, a phase I-II trial was initiated to study the effect of rhGM-CSF in patients with myelodysplastic syndromes who were not eligible for other kinds of therapy. rhGM-CSF has been tested in 10 patients in doses from 15 micrograms/m2 to 150 micrograms/m2 given intravenously over 8 hours for a cycle of 7 days followed by an interval of 14 days and a second 7-day treatment course. A dose-dependent increase in leukocyte count was observed in 9 of 10 patients. No change in reticulocyte numbers was seen and only one patient experienced an increase in platelet count. Toxicity mainly consisted of mild phlebitis at the site of infusion and sternal pain after bolus injection. An increase in blast cell counts in some patients necessitated the start of low-dose Ara-C therapy.     

Thein vitro andin vivo effects of carrageenin on humoral and cellular factors of natural resistance

The inhibitory effect of carrageenin, a sulfated algal polygalactose, on humoral factors of natural resistance is discussed. In dependence on dose and time, the influence of non-specific and specific bacteriolysis, on thein vitro andin vivo opsonic activity and on the course of infection was studied.     

The expression of monocyte chemotactic protein (MCP-1) in human vascular endotheliumin vitro andin vivo

Total 6-phosphofructo-1-kinase (PFK) activity, amounts of each type of PFK subunit, and levels of fructose-2,6-P₂ in the cerebral cortex, midbrain, pons-medulla, and cerebellum of 3, 12, and 25 month rats were measured. Further, the role of fructose-2,6-P₂ in the regulation of brain PFK activity was examined. A positive correlation was found to exist between the reported losses of glucose utilization as measured by 2-deoxy-D-glucose uptake and PFK activity in each region. That is, both parameters decreased to their lowest level by 12 months of age and remained decreased and fairly constant thereafter. Fructose-2,6-P₂ levels did not appear to directly correlate with regional changes in glucose utilization. Also, region-specific and age-related alterations of the PFK subunits were found although these changes apparently did not correlate with decreased glucose utilization. Brain PFK is apparently saturated with fructose-2,6-P₂ due to the high endogenous levels, and it contains a large proportion of the C-type subunit which dampens catalytic efficiency. Consequently, brain PFK could exist in a conformational state such that it can readily consume fructose-6-P rather than in an inhibited state requiring activation. This may explain, in part, the ability of brain to efficiently but conservatively utilize available glucose in energy production.Abbreviations fructose-2,6-P₂ D-fructose 2,6-bisphosphate - fructose-6-P D-fructose 6-phosphate - PAGE Polyacrylamide Gel Electrophoresis - PFK 6-phosphofructo-1-kinase - PP_i-PFK Pyrophosphate-dependent Phosphofructokinase, ribose-1,5-P₂, ribose-1,5-bisphosphate - SDS Sodium Dodecyl Sulfate     

Effects of UV- and X-rays on histonesin vivo andin vitro

Summary Histones were isolated by acid extraction from Chinese hamster ovarian tissue culture cells and investigated by means of an amino acid analyzer. Irradiation was performed with a low pressure mercury lamp (2537 A) or with 30 kV-X-ray Bremsstrahlung either of the tissue culture cells before extraction or of the isolated histones in aqueous solution. Both types of radiation affected mainly the aminoacids Tyrosine, Phenylalanine and Histidine in thein vitro experiments. After X-irradiationin vivo comparable doses were without effect. UV-irradiationin vivo led to a decrease of Tyr and Phe like in thein vitro experiments, but in contrast to his finding to an increase of His-concentration. It is tried to give a hypothetic explanation of these results.Paper read at the 6th Annual Meeting of the European Society for Radiobiology, Interlaken, 5.–8. June, 1968. Round Table: Radiation Effectsin vitro andin vivo. Correlations and Discrepancies.     

Combined effect of protein fusion and signal sequence greatly enhances the production of recombinant human GM-CSF in Escherichia coli

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor, that has been used as a therapeutic agent in facilitating bone marrow and stem cell transplantation and in other clinical cases like neutropenia. Although biologically active recombinant GM-CSF has been successfully produced in Escherichia coli, the reported levels are extremely poor. In this study we looked into the possible reasons for poor expression and found that protein toxicity coupled with protease-based degradation was the principal reason for low productivity. To overcome this problem we attached a signal sequence, as well as an amino-terminal His-tag fusion to the GM-CSF gene. This combination had a dramatic effect on expression levels, which increased from 0.8 microg/mL in the control to 40 microg/mL. When a larger fusion partner, such as the Maltose-binding protein (MBP-tag), was used the expression levels increased further to 69.5 microg/mL, which along with the MBP-tag represented approx 12% of the total cellular protein.     

In vivo andin vitro studies of vanadate in human and rodent diabetes mellitus

In vivo vanadate and vanadyl have been shown to mimic the action of insulin and to be effective treatment for animal models of both Type I and Type II diabetes. The molecular mechanism of action of the vanadium salts on insulin sensitivity remains uncertain, and several potential sites proposed for the insulin-like effects are reviewed. In human trials, insulin sensitivity improved in patients with NIDDM, as well as in some patients with IDDM after two weeks of treatment with sodium metavanadate. This increase in insulin sensitivity was primarily due to an increase in non-oxidative glucose disposal, whereas oxidative glucose disposal and both basal and insulin stimulated suppression of hepatic glucose output (HGP) were unchanged. Clinically, oral vanadate was associated with a small decrease in insulin requirements in IDDM subjects. Of additional benefit, there was a decrease in total cholesterol levels in both IDDM and NIDDM subjects. Furthermore, there was an increase in the basal activities of MAP and S6 kinases to levels similar to the insulin-stimulated levels in controls, but there was little or no further stimulation with insulin was seen. Further understanding of the mechanism of vanadium action may ultimately be useful in the design of drugs that improve glucose tolerance.     

Combined effect of protein fusion and signal sequence greatly enhances the production of recombinant human GM-CSF in Escherichia coli

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor, that has been used as a therapeutic agent in facilitating bone marrow and stem cell transplantation and in other clinical cases like neutropenia. Although biologically active recombinant GM-CSF has been successfully produced in Escherichia coli, the reported levels are extremely poor. In this study we looked into the possible reasons for poor expression and found that protein toxicity coupled with protease-based degradation was the principal reason for low productivity. To overcome this problem we attached a signal sequence, as well as an amino-terminal His-tag fusion to the GM-CSF gene. This combination had a dramatic effect on expression levels, which increased from 0.8 μg/mL in the control to 40 μg/mL. When a larger fusion partner, such as the Maltose-binding protein (MBP-tag), was used the expression levels increased further to 69.5 μg/mL, which along with the MBP-tag represented approx 12% of the total cellular protein.     

Tumor necrosis therapy antibody interleukin-2 fusion protein elicits prolonged and targeted antitumor effects in vivo

Interleukin-2 (IL-2) is one of the most successful cytokines applied in tumor immunotherapy because of its ability to stimulate potent cellular immune response. The life-threatening toxicity of vascular leak syndrome (VLS) associated with the high-dose IL-2 treatment regimen has limited its use in tumor immunotherapy. To reverse this situation, a tumor-targeted fusion protein, recombinant human TNT-IL2 (rhTNT-IL2), was generated with both the cytokine activity of IL-2 and the tumor-targeting ability of TNT antibody. TNT is a human tumor necrosis therapy monoclonal antibody capable of binding intracellular antigens which are accessible and abundant in necrotic regions of tumors. The immunotherapeutic potential of this fusion protein was tested in murine melanoma and lung cancer models, and tumor-bearing mice showed satisfied tumor regressions after rhTNT-IL2 immunotherapy. Immunohistochemical study showed a distinct penetration of IL-2 in tumors in mice treated with rhTNT-IL2, indicating its evident tumor-targeting activity. Moreover, the rhTNT-IL2 was well tolerated in cynomolgus monkeys in a 12-week long-term repeated toxicity study. These studies indicate that the targeting of IL-2 to necrotic areas of tumors might be a new approach for the immunotherapy of solid tumors.     

重组人GM—CSF／MCAF融合蛋白的变性,复性及纯化研究

人粒细胞巨噬细胞集落刺激因子(GM-CSF)和单核细胞趋化激活因子(MCAF)融合蛋白在大肠杆菌中高效表达后,表达产物以包涵体形式存在。包涵体经分离和洗涤后,探索了rhGM-CSF/MCAF变性和复性的合适条件。复性后的样品经Sephadex G-75凝胶过滤和CM-Sepharose FF离子交换两步层析,得到了具有生物学活性的SDS-PAGE纯的rhGM-CSF/MCAF。Western blot检测表明,纯化的rhGM-CSF/MCAF能分别与GM-CSF和MCAF抗体发生特异反应。     

Growth and development of tomato fruitsin vivo andin vitro

Tomato (Lycopersicon esculentum Mill.) fruits of the male sterile cultivar Pearson (MS35 BC4, 61) were transferred toin vitro culture during the cell division period. Fruits grownin vivo andin vitro were compared throughout their development according to various growth parameters: fresh and dry weight, cell number, cell diameter, and DNA and total protein content. In all cases, the values pertaining to fruits grownin vitro were significantly lower than theirin vivo counterparts. The final fresh weight of fruits transferred to culture 2, 5, or 10 days after pollination was only 0.7, 1.2, and 3.4%, respectively, of that of plant-grown fruits. The results indicate that the reduced fruit sizein vitro is related to the reduction in both cell number and cell size. It is interesting to note that the DNA content per cell increased 15-fold during the growth of the plant-grown fruits while this accumulation was only between 2-and 3-fold in all the cultured fruits. The time to first colour appearance of fruits cultured 2, 5, or 10 days after pollination was 196, 132 and 85%, respectively, of that of plant-grown fruits.