A novel reporter gene assay for interferons based on CHO-K1 cells.

Interferons (IFNs) are cytokines playing an important role in the immune response and defence against viruses. They are widely used as biopharmaceuticals. Currently, the anti-viral assay (AVA) is the most commonly used bioassay for determining interferon potency. In the search for rapid and robust but reliable methods, reporter gene assays (RGA) appear to be the most promising approach, therefore we have designed a new reporter cell line, CHO-ISRE-SEAP, suitable for determination of type I interferon potency. Chinese hamster ovary (CHO-K1) cells were stably transfected with secretory alkaline phosphatase (SEAP) gene under the control of interferon stimulated response element (ISRE) promoter. The amount of SEAP in the cell culture medium can be easily measured colorimetrically and has been found to correlate with the amount of IFN added. The new assay is widely applicable for determination of type I IFNs, such as IFN-alpha, IFN-beta and IFN-omega, in research, development of IFN biopharmaceuticals, in batch release, etc. Interestingly, in this assay, IFN-beta shows approximately 6 times higher response than IFN-alpha, which makes it especially appropriate for measuring low levels of IFN-beta. Compared to other known RGAs, the novel CHO-ISRE-SEAP cell line-based RGA appears to have certain advantages with respect to cost and performance.

Chemometric approach in quantification of structural identity/similarity of proteins in biopharmaceuticals.

We present a chemometrics study in which we show the identity or degree of similarity of 3D protein structures of various G-CSF (Granulocyte Colony-Stimulating Factor) isolates. The G-CSF isolates share the same amino acid sequence, but the preparation was carried out by somehow diverse technologies. The comparison of 3D structures was made on the basis of 2D NMR NOESY (Nuclear Overhauser Enhancement Spectroscopy) spectra of proteins. In searching for the most appropriate criteria to determine the identity or degree of similarity of selected spectral regions of different isolates, two methods for quantitative evaluation of identity/similarity were used. The first method compares all peaks in the two investigated protein spectral regions; the extent of peaks that overlap is determined. The second method includes spectral invariants originating from graph theory. The criteria of identity/similarity were calculated from graphs, derived from a collection of up to 200 peaks of investigated 2D NMR spectral region. The peaks were linked into a graph according to the sequential nearest neighborhoods. According to the first method all peaks were relevant, considering that spectral noise was previously removed; the largest similarity was found between the protein of a commercially available G-CSF drug and one of the three new isolates produced in the laboratory. The second method indicated that the pairwise similarity of the three new isolates is larger than the similarity of any of the new isolates with the commercially available drug. This is an expected result taking into account that the new isolates are produced by the same technology, while the commercial product has additives for long-term storage that could not be completely compensated. The proposed measure of similarity may help the developers of biosimilar products to optimize the controllable parameters of the production technology and eventually to argue the identity of the new isolate in comparison with the originator commercial product.

Characterisation of metal-chelate methacrylate monoliths.

Monoliths are attractive stationary phases for purification of large biomolecules like proteins because of their flow-unaffected properties. Isolation of histidine containing proteins to high purity can be efficiently performed using metal-chelate interactions within a single chromatographic step. In this work, we investigated properties of commercial metal-chelate methacrylate monoliths-Convective Interaction Media (CIM). Analytical CIM disk monolithic columns and CIM 8 ml monolithic columns were used for purification of tumor necrosis factor-alpha (TNF-alpha) analog LK-801 and green fluorescence protein with 6 histidine tag (GFP-6His). In both cases, purity over 90% was achieved. Dynamic binding capacity at 10% of breakthrough was around 17-18 mg/ml for LK-801 and around 30 mg/ml for GFP-6His. Adsorption isotherm revealed that the maximal capacity is achieved at protein concentration above 60 microg/ml. Dynamic binding capacity and resolution were found to be flow unaffected.

Perspectives of immobilized-metal affinity chromatography.

Immobilized Metal-Affinity Chromatography (IMAC) represents a relatively new separation technique that is primarily appropriate for the purification of proteins with natural surface-exposed histidine residues and for recombinant proteins with engineered histidine tags or histidine clusters. Because the method has gained broad popularity in recent years, the main recent developments in the field of new sorbents, techniques and possible applications are discussed in this article. Advantages of the method and new prospects are described as well as the problems and concerns that appear when the method is to be used for production of pharmaceutical-grade proteins.

Construction of an expression cassette with hTNF-alpha gene for transient expression of the gene in mammalian cells.

BACKGROUND: Tumor vaccines, which are created by the insertion of cDNA encoding different cytokines into the tumor cells, are capable of inducing a very complex immune reaction including activation of CD8 T cells, granulocytes, macrophages, the triggering of cytokine cascades and antibody production. Aiming to create genetically modified tumor cells which could produce and secrete Human Tumor Necrosis Factor-alpha (hTNF-alpha), we constructed the expression cassette containing hTNF-alpha gene in pcDNA3 plasmid vector. MATERIALS AND METHODS: The successful ligation of cDNA encoding for hTNF-alpha into pcDNA3 plasmid vector was confirmed by PCR, restriction mapping and sequence determination. The constructed expression cassette in pcDNA3 vector was than transferred in vitro into malignant melanoma B16 tumor cells by the method of Receptor Mediated Gene Transfer (RMGT). RESULTS: Measurable amounts of hTNF-alpha protein detected in the medium of transfected cells proved that tumor cells modified in this manner became producers of hTNF-alpha protein. CONCLUSION: The expression of the transferred gene was transient and the produced protein was biologically active. Furthermore, the production of hTNF-alpha protein was also observed in sub-lethally irradiated tumor cells, showing that the expression cassette was preserved during the irradiation and that the cells were potentially applicable as a tumor vaccine.

Soluble tumor necrosis factor receptor I (sTNFRI) as a prognostic factor in melanoma patients in Slovene population.

Tumor necrosis factor alpha (TNF-alpha) and its receptors (TNFRI and TNFRII) which exist in soluble form as a product of cleavage of the extracellular domain of membrane integrated receptors, still rise debate about their importance. It was reported that TNF-alpha has numerous actions in diseases such as inflammation, autoimmunity, infectious diseases, septic shock and many types of cancer [1, 2]. Several authors have reported the significance of sTNFRI level in serum of cancer patients [3, 4]. This study was performed in collaboration with the Institute of Oncology of Slovenia. At least two different mouse monoclonal antibodies (MAbs) against human sTNFRI have been prepared to obtain a sensitive and reliable sandwich ELISA. It was compared with commercially available R&D and Endogen ELISAs for the determination of sTNFRI. Groups of patients with different stages of melanoma and epithelial ovarian carcinoma were tested and their clinical records were reexamined. Levels of sTNFRI were measured and compared with the normal serum levels of sTNFRI.

Early events in TNFa-p55 receptor interations--experiments with TNF dimers.

The first essential step in TNF signal transduction is believed to be clustering of the membrane bound receptors around the trimeric TNF molecule. To check if one receptor binding site would be enough to trigger the signal, we tried to prepare several types of TNF dimer. For this purpose, two TNF analogs bearing different cysteine mutations at the inner subunit binding surfaces were designed, expressed in E. coli and prepared in pure form. By mixing equimolar quantities of these analogs under appropriate conditions, two different types of dimer were prepared. The first, Dim/S2, proved to be composed mainly of a disulfide-linked dimer, which was still capable of trapping the third subunit of either of the precursor analogs, thus showing relatively high residual cytotoxicity. To avoid trimeric structures, Dim/S2 was further transformed into Dim/Iaa2 by alkylation of -SH groups of the newly introduced cysteines, allowing binding of only two TNF subunits through native contact surfaces. These dimers showed substantially reduced cytotoxicity on the L929 cell line. In addition, it appears that Dim/Iaa2 is able to competitively inhibit cytotoxicity of native TNF, as assessed on the L-M cell line.

Histidines in affinity tags and surface clusters for immobilized metal-ion affinity chromatography of trimeric tumor necrosis factor alpha.

In order to achieve efficient IMAC (immobilized metal-ion affinity chromatography) purification of tumor necrosis factor alpha (TNF-alpha) and its analogs by a common chromatographic procedure, we tested four histidine-rich affinity tags attached to the N-termini of the trimeric TNF-alpha molecule. Using low cultivation temperature and appropriate protease deficient E. coli strains, it was possible to obtain intact, full-length proteins with NHis2Xa and HisArg tags, which could be purified to over 95% purity in a single step. However, in comparison to model proteins bearing a surface histidine cluster, accumulation of the histidine-tagged proteins in E. coli was significantly reduced, even in protease deficient strains. In addition, the histidine tagged TNF-alpha proteins never displayed good chromatographic behavior, which was otherwise easily achieved with model proteins. Although the most popular hexa-histidine tag is generally recognized as very convenient for single step isolation of monomeric proteins, our results with trimeric TNF-alpha indicate that oligomeric proteins may require further optimization of the tag, with respect to its length, composition, and location. Histidines, relatively rigidly inserted in the structure, as in our model proteins, display superior chromatographic characteristics vis a vis flexible tags with the same total number of histidines.

New TNF-alpha analogues: a powerful but less toxic biological tool against tumours.

Our approach to the modification of recombinant human tumour necrosis factor alpha (rhTNF-alpha) comprised changes in flexible loop regions on the surface of the TNF molecule. Using this approach, two different rhTNF-alpha analogues LK 801 and LK 805 were synthesized and tested for their ability to affect the growth of Sa-1 tumour cells. Results obtained in vitro indicate that neither rhTNF-alpha nor its analogues have a direct cytotoxic effect. In vivo experiments were performed on subcutaneous Sa-1 tumours in A/J mice, where the antitumour effect and the toxic side effects of the cytokines were followed. There was no significant difference between growth delay of tumours in animals treated with native rhTNF-alpha and in animals treated with one of the analogues. On the contrary, the LD50 for rhTNF-alpha was 29.1 microg, for LK 801 59.3 microg, and for LK 805 even 66.1 microg, indicating that LK 801 and especially LK 805 were significantly better tolerated. The results confirm that the rhTNF-alpha molecule has been successfully modified resulting in two new analogues with a potent antitumour activity and much lower systemic toxicity. A particularly low systemic toxicity and a strong antitumour effect were observed after treatment with LK 805 suggesting that this analogue merits further investigation in pre-clinical and clinical trials.

Anti-tumor effectiveness of electrochemotherapy with bleomycin is increased by TNF-alpha on SA-1 tumors in mice.

With the aim to increase anti-tumor effectiveness of electrochemotherapy, adjuvant immunotherapy with tumor necrosis factor-alpha (TNF-alpha) was tested on tumors in mice. Increased anti-tumor effectiveness on SA-1 tumors was observed after combining TNF-alpha, injected either intratumorally or peritumorally, with electrochemotherapy using suboptimal dose of bleomycin (BLM). The increased anti-tumor effectiveness was neither the result of potentiated anti-tumor effectiveness of TNF-alpha due to exposure of tumors to electric pulses, nor due to interaction with BLM. Therefore, the effect of adjuvant TNF-alpha treatment might be immunomodulatory, augmenting the anti-tumor activity of electrochemotherapy, and possibly adding a systemic component to the localized electrochemotherapy treatment.

Searching for new TNF-alpha analogs having potential application in cancer therapy.

Two new TNF-alpha analogs were prepared and tested for their anti-tumor activity on fibrosarcoma SA-1 tumor model in vivo. In analog LK-801 two histidines (His107His108) were introduced into the surface loop thus enabling efficient purification by metal-affinity chromatography. This analog showed less side effects and can serve as a lead compound to look for other useful mutations. Another analog LK-802 was designed by introduction of additional pair of mutations (Cys95Cys148) into LK-801 in order to prepare disulfide linked TNF trimers. Cytotoxicity on mouse cell line L929 was comparable to TNF-alpha, but effect on tumor growth was quite reduced. Pharmacokinetic study revealed that serum levels of LK-802 were quite low in comparison to native TNF-alpha. This at least partially explains why anti-tumor activity of LK-802 is reduced and also illustrates the problems in designing the analogs with desired in vivo biological properties.

Purification of human tumour necrosis factor by membrane chromatography.

The recombinant human tumour necrosis factor alpha from an extract of Escherichia coli was enriched to homogeneity according to specific activity and sodium dodecyl sulphate-polyacrylamide gel electrophoresis by purification using anion-exchange HPLC and hydrophobic interaction HPLC. Parallel experiments with the same separation methods, but carried out with membrane chromatography on compact discs, gave similar results in terms of yield and purity of the product. The active form of the protein is a trimer. The second isolation step, hydrophobic interaction chromatography, causes dissociation of the trimer into monomers and a partial loss of the biological activity of the protein. The phenomenon occurs on both the column and the disc. This in turn indicates strongly that the dissociation of the protein is a consequence of interaction between the samples and the hydrophobic ligand, and is not caused by non-specific interaction with the matrix.