Drug – bio-affecting and body treating compositions – Lymphokine
Reexamination Certificate
1998-12-22
2001-01-09
Mertz, Prema (Department: 1646)
Drug, bio-affecting and body treating compositions
Lymphokine
C514S002600, C514S008100, C514S012200, C514S885000, C435S069500, C435S071100, C435S325000, C435S252300, C435S320100, C435S471000, C536S023100, C536S023500, C536S024100, C536S024310
Reexamination Certificate
active
06171584
ABSTRACT:
DESCRIPTION
The present invention concerns a new growth/ differentiation factor of the TGF-&bgr; family and DNA sequences coding therefor.
The BMP-, TGF- and inhibin-related proteins are members of the TGF-&bgr; family of growth factors (Roberts and Sporn, Handbook of Experimental Pharmacology 95, 419-472 (1990)). They are relevant for a wide range of medical therapeutic methods and applications. These factors are suitable for methods relating to wound healing and tissue regeneration. Moreover several members of the TGF-&bgr; family induce tissue growth for example the growth of bones.
Wozney (Progress in Growth Factor Research 1 (1989), 267-280) and Vale et al. (Handbook of Experimental Pharmacology 95 (1990), 211-248) describe various growth factors for example those which are related to the BMP and the activin/inhibin group. The members to this group have significant structural similarities. The precursor to the protein is composed of an amino-terminal signal sequence, a propeptide sequence and a carboxy-terminal sequence of 110 to 140 amino acids which is cleaved from the precursor and represents the mature protein. Furthermore its members are defined by an amino acid sequence homology. The mature protein contains the sequences that are conserved most, in particular seven cysteine residues which are conserved among the family members. The TGF-&bgr;-like proteins are multifunctional, hormonally active growth factors. They also have related biological activities for example chemotactic attraction of cells, promotion of cell differentiation and tissue-inducing capabilities. EP 0 222 491 A1 discloses sequences of inhibin alpha and beta chains.
On the whole the proteins of the TGF-&bgr; family show differences in their structure which leads to considerable variations in their exact biological function. In addition they are found in a wide range of different types of tissues and stages of development. As a consequence they may be different with regard to their exact function e.g. the required cellular physiological environment, their life span, their target areas, their requirements for auxiliary factors and their resistance to degradation. Although numerous proteins that show tissue-inductive potential have been described, their natural functions in the organism and—even more importantly—their medical relevance still has to be researched in detail. It can in all probability be assumed that there are still unknown members of the TGF-&bgr; family which are of importance for the differentiation/induction of various types of tissue. However, a major difficulty in the isolation of these new TGF-&bgr;-like proteins is that their functions cannot yet be described precisely enough to develop a highly discriminating bioassay. On the other hand the expected nucleotide sequence homology to known members of the family is too small to enable screening by classical nucleic acid hybridization techniques. Nevertheless the further isolation and characterization of new TGF-&bgr;-like proteins is urgently required in order to provide further inducing and differentiation proteins which fulfil all medical requirements. These factors could be used medically in healing injuries and treating degenerative diseases of various tissues.
A nucleotide and amino acid sequence for the TGF-&bgr; protein MP121 is given in the patent application PCT/EP93/00350 in which a major part of the sequence corresponding to the mature protein is stated. The complete sequence of the propeptide MP121 is not disclosed.
The underlying object of the present invention is to provide DNA sequences which code for new members of the TGF-&bgr; protein family with mitogenic and/or differentiation-inductive potential. The object of the present invention is in particular to provide the complete DNA and amino acid sequence of the TGF protein MP121.
This object is achieved by a DNA molecule that codes for a protein of the TGF-&bgr; family and which comprises
(a) the part coding for the mature protein and if necessary further functional parts of the nucleotide sequence shown in SEQ ID NO. 1,
(b) a nucleotide sequence corresponding to the sequence from (a) within the scope of the degeneracy of the genetic code,
(c) a nucleotide sequence corresponding to an allelic derivative of one of the sequences from (a) and (b) or
(d) a sequence which differs from sequence (a) due to the fact that it originates from other vertebrates
(e) a sequence hybridizing with one of the sequences from (a), (b), (c) or (d)
provided that a DNA molecule according to (e) contains at least the part coding for a mature protein of the TGF-&bgr; family.
Further embodiments of the present invention concern the subject matter of claims
2
to
10
. Other features and advantages of the invention emerge from the description of the preferred embodiments. The sequence protocols and drawings are now briefly described.
SEQ ID NO. 1 shows the complete nucleotide sequence of the DNA coding for the human TGF-&bgr; protein MP121. The ATG start codon begins at nucleotide 128. The start of the complete mature protein particularly preferably begins at nucleotide 836.
SEQ ID NO. 2 shows the complete amino acid sequence of the preproprotein of the human TGF-&bgr; protein MP121 which was derived from the nucleotide sequence shown in SEQ ID NO. 1. The start of the mature protein is preferably in the region of amino acids 217-240, particularly preferably at amino acid 236 or 237 and most preferably at amino acid 237.
SEQ ID NO.3 shows the complete nucleocide sequence of the DNA coding for the TGF-&bgr; protein MP121 from the mouse. The coding region begins at the ATG start codon at nucleotide 131 and ends at the stop codon beginning at position 1187. The start of the mature protein preferably begins at nucleotide 839. A ca. 5.5 kb large intron is located in the genomic DNA between position 446 and 447.
SEQ ID NO. 4 shows the complete amino acid sequence of the preproprotein of the TGP-&bgr; protein MP121 from the mouse which has been derived from the nucleotide sequence shown in SEQ ID NO. 3. The mature protein begins in the region of amino acids 217-240 in analogy to the human MP121 of SEQ ID NO.2. Tt is most preferred when the mature protein starts at amino acid 237 so that the mature part consists of 116 amino acids as in the human MP121. Members of the TGF-&bgr; family are frequently cleaved behind a RXXR cleavage site in order to separate the mature part from the precursor (see {umlaut over (O)}zkaynak et al., J. Biol. Chem. 267, 25220-25227 (1992) and the literature cited therein). In the case of MP121 from the mouse it is also conceivable that the beginning of the mature protein is at least sometimes at amino acid 236.
SEQ ID NO.5 and 6 show the nucleotide sequence of the human MP121 gene at the exon/intron junctions.
REFERENCES:
patent: 4798885 (1989-01-01), Mason et al.
patent: 0 222 491 (1987-05-01), None
patent: 93/16099 (1993-08-01), None
patent: 95/04819 (1995-02-01), None
patent: PCT/EP/9502552 (1995-06-01), None
patent: 96/01316 (1996-01-01), None
Hötten et al., Cloning of a New Member of the TFG-&bgr; Family: A putative New Activin &bgr;cChain:, Biochem. & Biophys. Res. Comm. vol. 206, No. 2, 1995.
Forage et al. (1986) Proc. Natl. Acad. Sci. vol. 83, pp. 3091-3095.
Bowie et al. (1990) Science vol. 247, pp. 1306-1310.
Bechtold Rolf
Hotten Gertrud
Neidhardt Helge
Paulista Michael
Pohl Jens
Arent Fox Kintner & Plotkin & Kahn, PLLC
Biopharm Gesellschaft zur Biotechnologischen Entwicklung von Pha
Mertz Prema
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