Allelic variant of human STAT3

Details Allelic variant of human STAT3 Allelic variant of human STAT3

2002-04-08

2003-12-09

Gambel, Phillip (Department: 1644)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023100, C435S069100, C435S252300, C435S471000, C435S320100

Reexamination Certificate

active

06660848

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a human STAT
3
allelic variant, the cDNA sequence encoding it, its use in therapy and/or in diagnosis of autoimmune and/or inflammatory diseases, as well as pharmaceutical compositions comprising it.
BACKGROUND OF THE INVENTION
Signal Transducer and Activator of Transcription (STAT) proteins are a new class of intracellular transcription factors which play an essential function in the cellular responses to cytokines (Stahl et at., 1994; Gouilleux et at., 1995; Azam et al., 1995; Tian et at., 1994; May et al., 1996; and Iwatsuki et al., 1997).
Most of these proteins have been well characterized by sequencing, and their structure as well as the mechanism of their actions has been extensively analyzed and well documented (Wegenka et al., 1993; Akira et al., 1994; Wegenka et al., 1994; Quelle et al., 1995 and Silva et al., 1996).
These proteins contain SH
2
and SH
3
domains as well as a phosphorylation site at their carboxy-terminal region (Kapetein et al., 1996; and Herman et al., 1996). After cytokine receptor activation through ligand binding, the intracellular portion of the receptor becomes phosphorylated by an associated kinase of the JAK family. STAT proteins then bind to the phosphorylated receptor, through their SH
2
domain, and are in turn phosphorylated by JAKs (Stahl et al., 1995). Phosphorylated STAT proteins then dimerize and translocate to the nucleus, where they are able to recognize specific DNA responsive elements (Seidel et al., 1995; and Harroch et al., 1994).
STAT
3
has been identified as an important mediator of the signal imparted by the IL-6 family of cytokines, as well as by EGF and by a number of other interleukins and growth factors.
STAT
3
has been shown to play a central role in the upregulation of hepatic acute-phase proteins (Wegenka et al., 1993; and Zhang et al., 1996) in the growth arrest of monocytic cells (Yamanaka et al., 1996; and Minami et al., 1996) as well as in the survival of myeloma cells (Harroch et al., 1994).
DESCRIPTION OF THE INVENTION
During experiments on the analysis of STAT
3
interactions, we have amplified by RT-PCR from HepG2 cells a cDNA fragment corresponding to the SH
2
domain of human STAT
3
. We have found by DNA sequencing that the SH
2
domain we have isolated shows a divergence of 13 residues over the corresponding sequence of the original published human STAT
3
gene (Akira et al., 1994).
In order to determine the nature of this sequence variant, we have designed three pairs of primers with 3′ ends corresponding to nucleotide positions at variance between the two human cDNA sequences.
Upon such investigations it resulted that the new variant corresponds to at least the most frequent allele of human STAT
3
.
Therefore, the main object of this invention is the above-mentioned allelic variant of human STAT
3
protein. In particular, the object of the invention is a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or a functionally equivalent salt, or a functionally equivalent derivative, or an active fraction, or a fusion protein.
The definition “salt” as used herein refers both to salts of the carboxyl-groups and to the salts of the amino functions of the compound obtainable through known methods. The salts of the carboxyl-groups comprise inorganic salts as, for example, sodium, potassium, calcium salts and salts with organic bases as those formed with an amine as triethanolamine, arginine or lisine. The salts of the amino groups comprise for example salts with inorganic acids as hydrochloric acid and with organic acids as acetic acid.
The definition “derivative” as herein used refers to derivatives which can be prepared from the functional groups present on the lateral chains of the amino acid moieties or on the terminal N- or C-groups according to known methods and are comprised in the invention when they are pharmaceutically acceptable i.e. when they do not destroy the protein activity or do not impart toxicity to the pharmaceutical compositions containing them. Such derivatives include for example esters or aliphatic amides of the carboxyl-groups and N-acyl derivatives of free amino groups or O-acyl derivatives of free hydroxyl-groups and are formed with acyl-groups as for example alcanoyl- or aroyl-groups.
As “active fraction” of the protein the present invention refers to any fragment or precursor of the polypeptidic chain of the compound itself, alone or in combination with related molecules or residues bound to it, for example residues of sugars or phosphates, or aggregates of the polypeptide molecule when such fragments or precursors show the same activity of the protein of the invention, as medicament.
The definition “fusion protein” as herein used refers to polypeptides comprising the polypeptide of the invention above specified fused with another protein and having a longer lasting half-life in body fluids. It can for example be fused with another protein such as, for example, an immunoglobulin.
Another object of the invention is the DNA molecule comprising the DNA sequence coding for the allelic variant of the invention, including nucleotide sequences substantially the same.
“Nucleotide sequences substantially the same” includes all other nucleic acid sequences which, by virtue of the degeneracy of the genetic code, also code for the given amino acid sequences. In particular, the present invention refers to the nucleotide sequence comprising the SEQ ID NO:1.
The present invention also refers to recombinant DNA molecules which hybridize with the DNA sequence coding for the above-mentioned allelic variant of hSTAT
3
and whose nucleotide sequences show at least the same 13 differences in the SH
2
domain (with respect to the human STAT
3
sequence in Akira et al., 1994), as shown in FIG.
1
. The gene can contain, or not, the natural introns and can be obtained for example by extraction from appropriate cells and purification with known methods.
Furthermore, the present invention also includes recombinant DNA molecules which hybridize under stringent conditions with a probe having a nucleotide sequence selected between SEQ ID NO:16 and SEQ ID NO:17.
The term “stringent conditions” refers to hybridization and subsequent washing conditions which those of ordinary skill in the art conventionally refer to as “stringent”. See Ausubel et al.,
Current Protocols in Molecular Biologic supra. Interscience
. N.Y. pare. 6.3 and 6.4 (1987, 1992), and Sambrook et al, 1989. Without limitation, examples of stringent conditions include washing conditions 12-20° C. below the calculated Tm of the hybrid under study in, e.g. 2×SSC and 0.5% SDS for 5 minutes, 2×SSC and 0.1% SDS for 15 minutes; 0.1×SSC and 0.5% SDS at 37° C. for 30-60 minutes and then a 0.1×SSC and 0.5% SDS at 68° C. for 30-60 minutes. Those of ordinary skill in this art understand that stringency conditions also depend on the length of the DNA sequences, oligonucleotide probes (such as 10-40 bases) or mixed oligonucleotide probes. If mixed probes are used, it is preferable to use tetramethyl ammonium chloride (TMAC) instead of SSC. See
Ausubel
. supra.
The invention also includes expression vectors which comprise the above DNAs, host-cells transformed with such vectors and a process of preparation of such allelic variant of hSTAT
3
, its active fragments or fusion proteins, through the culture in appropriate culture media of said transformed cells.
The DNA sequence coding for the protein of the invention can be inserted and ligated into a suitable plasmid. Once formed, the expression vector is introduced into a suitable host cell, which then expresses the vector(s) to yield the desired protein.
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 molecules coding for the proteins obtained by any of the above methods are in

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