Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1998-05-11
2000-04-04
Fitzgerald, David L.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
536 2352, 530351, 424 855, 435325, 4352523, 43525233, 4353201, C12P 2104, C07K 2104, C07K 100, A61K 3821
Patent
active
060460342
DESCRIPTION:
BRIEF SUMMARY
The invention relates to variants of the human recombinant interferon-.gamma. with increased thermal stability.
Interferon-.gamma. is suitable as an antiviral, antiproliferative, immunomodulatory human therapeutic agent, particularly for the treatment of kidney tumours and chronic granulomatosis.
Like the interleukins, interferons belong to the class of the cytokines and are listed in various classes: interferon-.alpha., interferon-.beta., interferon-.gamma., interferon-.omega. and interferon-.tau.. Interferon-.gamma. is a glycoprotein, whose amino acid sequence has been known since 1982 (Nucl. Acid Res. 10, pages 2487 and following (1982)). In the mature condition the interferon-.gamma. has 143 amino acids and a molecular weight of 63 to 73 kilodaltons. The amino acid sequence of interferon-.gamma. is shown in FIG. 1. It will be noticed that the sequence contains no cystein.
The tertiary and quaternary structure of the non-glycosylised protein was clarified in 1991 (Science 252, page 698 and following (1991)). According to this, interferon-.gamma. exists as a homodimer, the monomers being orientated in contrary directions in such a way that the C-terminal end of one monomer is located in the vicinity of the N-terminal end of the other monomer. Each of these monomers in all has six .alpha.-helices.
Interferon-.gamma. is also called immunointerferon, as it has non-specific antiviral, antiproliferative and in particular immunomodulatory effects. Its production in T-helper-lymphocytes is stimulated by mitogens and antigens. The effect of the expressed interferon-.gamma. has not yet been precisely clarified, but is subject to intensive research. In particular, interferon-.gamma. leads to the activation of macrophages and to the synthesis of histocompatability antigens of the class 2. In vitro, the activity of interferon-.gamma. is normally determined as a reduction in the virus-induced cytopathic effect, which arises from treatment with interferon-.gamma.. Due to its antigen-non-specific antiviral, antiproliferative and immunomodulatory activity it is suitable as a human therapeutic agent, for example of kidney tumours and chronic granulomatosis. Clinical studies of various tumours are being carried out at present. Great expectations are set also on the interferon-.gamma. therapy of neurodermitis. Furthermore, interferon-.gamma. is used in the research field also as a fine chemical for example for the stimulation of cell cultures or for determining interferon levels.
Since 1982 it has been possible to express variants of the human interferon-.gamma. in bacteria such as escherichia coli (Nature 295, pages 503 and following (1982)). The monomer of these recombinant variants of the human interferon-.gamma. has, in addition to the 143 amino acids of the native human monomer, an additional N-terminal methionin as an additional amino acid. This recombinant human interferon-.gamma. (rhu-IFN-.gamma.) is not glycosylised. It can be obtained in large quantities and was therefore extremely accurately characterised.
The recombinant human interferon-.gamma. also exists as a dimer and has normal biological activity. It is acid-sensitive and temperature-sensitive with a melting point of 52.degree. C.
Of all the interferons, the thermal stability of human interferon-.gamma. is the lowest. This low thermal stability of interferon-.gamma. renders it difficult to use as a human therapeutic agent.
Tests were carried out at an early stage in order to generate variants with improved properties of the recombinant human interferon-.gamma. with the aid of molecular-biological methods. In EP 0 306870 A2, variants of recombinant human interferon-.gamma. were presented, whose activity was significantly increased by splitting off the C-terminal 7-11 amino acids. In addition, WO 92-08737 indicates a variant of recombinant human interferon-.gamma. (Interferon-.gamma. C-10L) which has an increased biological activity. However, in none of these variants of recombinant interferon-.gamma. was the thermal stability significantly increased. There
REFERENCES:
Modelling of Interhelical Contacts in Interferons-.beta., -.gamma., and Dric Interleukin-5, Biochemical and Biophysical Research Communications, vol. 201, No. 3, Jun. 30, 1994, pp. 1401-1405.
Expression of human immune interferon cDNA in E.coli and monkey cells, Nature, vol. 295, Feb. 11, 1982, pp. 503-508.
Molecular cloning of human immune interferon cDNA and its expression in eukaryotic cells, Nucleic Acids Research, vol. 10, No. 8, 1982, pp. 2487-2501.
Three-Dimensional Structure of Recombinant Human Interferon-.gamma., Science, vol. 252, May 3, 1991, pp. 698-702.
Li Volkhart
Otto Bernd
Waschutza Gero
Fitzgerald David L.
Fraunhofer-Gesellschaft zur Forderung der Angewandten Forschung
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