Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-04-06
2001-08-28
Mertz, Prema (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C536S023100, C536S023500, C530S351000, C435S069520, C435S071100, C435S071200, C435S325000, C435S320100, C435S252300, C435S254110, C435S471000, C424S085100, C424S085200, C514S002600, C514S008100, C514S012200
Reexamination Certificate
active
06280975
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a new IL-6 mutein, a DNA sequence coding for it, its use in therapy as well as a pharmaceutical composition comprising it.
BACKGROUND OF THE INVENTION
Interleukin-6 is released into the plasma upon injury or infection by different cell typs. It is involved in a spectrum of activities like immune defense, hematopoiesis, maturation of megakaryocytes, platelet production and acute phase response (1).
Besides playing a central role in host defense, IL-6 is involved in the pathogenesis of a variety of diseases like plasmocytoma/myeloma, osteoporosis and neoplastic and autoimmune diseases (1).
The IL-6 receptor complex on target cells consists of two different subunits, an 80-kDa specific ligand binding subunit (IL-6Ra) and a 130-kDa signal-transducing protein (gp130) (2-4). IL-6 binds to the IL-6Ra and the complex of IL-6/IL-6Ra becomes associated with a dimer of gp130, thereby initiating the IL-6 signal. IL-6 by itself has no measurable affinity to gp130 (5,6).
Interleukin-6 is a protein characterised by N-terminal heterogeneity. It has been reported (7) as a 184 amino acids (this amino acids numbering will be followed in this patent application). Secondary structure predictions and protein modeling pointed out that IL-6 is a member of the hematopoietic cytokine family characterized by four antiparallel a-helices (A, B, C, and D) (8,9). LIF (leukemia ihibitory factor), CNTF (ciliary neurotrophic factor), IL-11, CT-1 (cardiotrophin-1) and OSM (oncostatin M) also belongs to this family. They all use the gp130 protein in their receptor complex, which explains their overlaping bioactivities (1, 10, 11).
Deletion studies of IL-6 showed that the N-terminal 28 amino acid residuces are dispensable for the biological activity of this molecule. Removal of more than 28 amino acids inactived the protein (12). Further studies predicted that the C-terminus and the end of the A-B loop/beginning of the B-helix (region 2c, residues G77-E95) are involved in the interaction with the IL-6R&agr; (9, 13-16). These results were corroborated by the recently published human IL-6 model (9) where these two regions were in close proximity.
At present, two interaction sites of IL-6 with gp130 are identified.
i. Epitope mapping of the IL-6 protein with neutralizing mAbs provided evidence that the residues Q152-T162 (beginning of the D-helix) are involved in gp130 interaction (17, 18). Analysis of chimeric human/mouse IL-6 proteins revealed the presence of an epitope within the beginning of the A-B loop of IL-6 which was involved in contacting and activating gp130 (9, 19). Recently, this result was confirmed by demonstrating that leucine 57 is invoved in this interaction (20). This region is in close proximity of the beginning of helix D leading to the assumption that these two regions together form a common interaction site with one gp130 (9, 19, 21).
ii A second interaction site with gp130 was definded in analogy to the GH (growth hormone)/GHR
2
complex, the structure of which was solved by X-ray analysis (22). It was speculated, that the parts of the GH important for the interaction with the second GHR are the same in the IL-6 protein important for interaction with one gp130 (23, 24). Indeed, the substitution of two amino acids in the A-helix (Y31D/G35F) and two amino acids in the C-helix (S118R/V121D) also lead to an IL-6 mutant protein with nearly normal affinity to the IL-6Ra, but no bioactivity. These four amino acids seem to be important for the interaction with a second gp130 protein (24, 25).
In view of the previously discussed IL-6 involvement in the pathogenesis of some diseases, the development of inhibitors of IL-6 activity has therefore been the subject of active research. For this purpose, different approaches have been pursued, including the use of antibodies against IL-6, gp130 or gp80; the use of soluble gp130; or the use of muteins for IL-6, or IL-6 Receptor.
The Applicant has investigated the possibility of synthesising new IL-6 muteins that can act as IL-6 receptor antagonists. With this aim, one scientific approach to follow is to synthesise muteins that retain the ability to bind IL-6R&agr;, but have lost the capacity to recruit gp130. Therefore, the optimal molecule should be the one that does not show IL-6 activity but shows a higher IL-6R&agr; binding than IL-6 and that contains as few mutations as possible with respect to IL-6, in order to reduce the risks of antigenicity.
DISCLOSURE OF THE INVENTION
The Applicant has now found that by combining point mutations at position 54 with two mutations F170L/S176R, which increase the affinity to the IL-6Ra, and two mutations Q159E/T162P, which decreased the IL-6Ra-dependent interaction with gp130, obtained human IL-6 muteins, which retained receptor binding but failed to activate gp130. In particular, the main object of the present invention is human IL-6 mutein, comprising the amino acid sequence reported in FIG.
2
and in SEQ ID NO:1 as well as fragments thereof. This molecule behaved as efficient IL-6 antagonist on the human IL-6-dependent myeloma cell line XG-1 and shows all the advantages described above.
Another object of the invention is a DNA molecule comprising the DNA sequence coding for the poylpeptide of SEQ ID NO:1, as well as its variants resulting from the degeneracy of the genetic code or point mutations coding for a polypeptide having the same activity as that of SEQ ID NO:1.
A further object of the present invention is a plasmid vector containing the nucleotide sequence of the invention.
In a further aspect, the present invention provides the use of the protein as a medicament. In particular, it relates to the use of the protein of the invention in the manufacture of a medicament for the treatment of diseases in which IL-6 has a pathogenetic action, such as, for example, plasmocytoma/myeloma, osteoporosis and neoplastic and auotoimmune diseases.
The medicament is preferably presented in the form of a pharmaceutical composition comprising the protein of the invention together with one or more pharmaceutically acceptable carriers and/or excipients. Such pharmaceutical compositions form yet a further aspect of the present invention.
One method for preparing the mutein of the invention is by way of PCR technology using synthetic oligonucleotides, which contain a mismatch at the base that one wants to mutate, as primers.
Expression of any of the recombinant proteins of the invention as mentioned herein can be effected in eukaryotic cells (e.g. yeasts, insect or mammalian cells) or prokaryotic cells, using the appropriate expression vectors. Any method known in the art can be employed.
For example, the DNA molecule coding for the polypeptide of the invention are inserted into appropriately constructed expression vectors by techniques well known in the art (see Sambrook et al, 1989). Double stranded cDNA is linked to plasmid vectors by homopolymeric tailing or by restriction linking involving the use of synthetic DNA linkers or blunt-ended ligation techniques: DNA ligases are used to ligate the DNA molecules and undesirable joining is avoided by treatment with alkaline phosphatase.
In order to be capable of expressing the desired protein, an expression vector should comprise also specific nucleotide sequences containing transcriptional and translational regulatory information linked to the DNA coding the desired protein in such a way as to permit gene expression and production of the protein. First, in order for the gene to be transcribed, it must be preceded by a promoter recognizable by RNA polymerase, to which the polymerase binds and thus initiates the transcription process. There are a variety of such promoters in use, which work with different efficiencies (strong and weak promoters).
For eukaryotic hosts, different transcriptional and translational regulatory sequences may be employed, depending on the nature of the host. They may be derived form viral sources, such as adenovirus, bovine papilloma virus, Simian virus or the like, where the regulatory signals are ass
Ehlers Marc
Grotzinger Joachim
Rose-John Stefan
Applied Research Systems ARS Holding N.V.
Browdy and Neimark
Mertz Prema
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