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
1997-12-18
2002-01-15
Spector, Lorraine (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S325000, C435S252300, C435S252330, C435S320100, C536S023500
Reexamination Certificate
active
06338950
ABSTRACT:
This invention relates to a new leukocyte specific and immunoregulatory protein (LST-1) and to the corresponding protein produced by recombinant techniques as well as nucleic acids which code for proteins with LST-1 activity, methods of use and of production.
Cytokines are proteins with a molecular weight of less than 50 kD, which mediate the exchange of autocrine, paracrine or endocrine signals between the cellular components of tissues or between different tissues. The cytokines identified so far include growth factors, interleukins and interferons and act on cells in many systems of the body: the hematopoietic system, the immune system, the nervous system, the skeletal system, connective tissues, and probably most other tissues and organs of the body (for reference see A. Thompson ed. (1991), The Cytokine Handbook, London, Academic Press and A. Miyajima et al., Annual Reviews Immunol. 10 (1992) 295-331). Examples of cytokines are EGF, NGF, PDGF, FGF, IL-1 to IL-7, GM-CSF, G-CSF, MCSF, IFN, TNF-&agr;, TG-&agr; and -&bgr;.
More than 100 cytokines have been identified so far, but there is a need for further new cytokines which might be important for potential health care advances.
SUMMARY OF THE INVENTION
Accordingly it is an object of the present invention to provide a new cytokine showing new biological properties.
It is a further object of the present invention to provide the new protein using recombinant DNA molecules capable of expressing said protein in order that the binding protein will be more readily available. These and other objects of the invention have been accomplished by providing a purified protein according to the invention, selected from a group consisting of a protein which is at least 85% homologous to the amino acid sequence SEQ ID NO:3 and fragments thereof, wherein said protein is capable of binding to an antibody specific for said protein or its cell surface receptor on leukocytes.
DESCRIPTION OF SPECIFIC EMBODIMENTS
The invention comprises especially novel therapeutic compositions comprising recombinant proteins produced using nucleic acid sequences encoding proteins with LST-1 activity. The LST-1 cDNA which can be used for the production of the recombinant protein was initially isolated from U-937 cells (DSM ACC 5) stimulated with IFN-&ggr;. RT-PCR cloning of mMRA from these cells resulted in a cDNA clone designated pLST-1 having a length of 636 bp. Analysis of the LST-1 cDNA reveals that LST-1 consists of six exons. These exons are designated SEQ ID NO:1 exons 1A (bp 48-162), 1B (bp 544-652), 2 (bp 1044-1162), 3 (bp 1475-1567), 4 (bp 1775-1797) and 5 (bp-2325-2709). In SEQ ID NO:2 exon 5, after position 2345, there is an internal 5′ donor splice site. By alternative splicing, thus, two isoforms of the LST-1 protein can be formed, which accordingly have a length of either 104 or 97 amino acids (cf. SEQ ID NOS: 3 and 4). The human pLST-1 cDNA done contains an extended 5′ region encoding stop codons.
The invention is based on a new cytokine-like protein (denoted LST-1 protein, or LST-1, in the following) whose production is stimulated in U-937 cells by IFN-&ggr; by a factor of more than 100 preferably 1000, which binds to the surface of leukocytes and which
a) is coded by the DNA sequence shown in SEQ ID NO:2 for the mature protein or by the genomic sequence shown in SEQ ID NO:1,
b) is coded by DNA sequences which hybridize under stringent conditions with the DNA sequences shown in SEQ ID NO:1 or 2 or fragments of the DNA sequences in the DNA region which codes for the mature protein, or
c) is coded by DNA sequences which, if there was no degeneracy of the genetic code, would hybridize with the sequences defined in a) or b) and code for a polypeptide with amino acid sequence,
d) and the reading frame of said protein is defined by ATG starting at position 1144 of SEQ ID NO:1 following within no shift of the reading frame in the protein coding region following said ATG.
The protein can be defined by its DNA sequence (preferably by its cDNA sequence deduced from SEQ ID NO:1) and by the amino acid sequence derived therefrom. The LST-1 protein can occur in natural allelic variations which differ from individual to individual. Such variations of the amino acids are usually amino acid substitutions. However, they may also be deletions, insertions or additions of amino acids to the total sequence. The LST-1 protein according to the invention—depending, both in respect of the extent and type, on the cell and cell type in which it is expressed—can be in glycosylated or non-glycosylated form The LST-1 proteins according to SEQ ID NO:3 and SEQ ID NO:4 are preferred.
LST-1 mRNA is expressed constitutively in T cells, macrophages, U-937 and at low levels in human tonsilla, lung and liver, which may be due to the lymphocytes and macrophages present in these tissues the protein shows an immunoregulatory activity. Transcription can be strongly enhanced by IFN-&ggr; in U-937 cells. Upon stimulation of the Jurkat T cell line (ATCC TIB 152) with TPA (12-o-tetradecanoylphorbol-13-acetate) only a short induction of LST-1 mRNA was observed. An increase of mRNA expression starts after four hours with a peak after 8 hours of stimulation and further decrease after 24 hours of stimulation.
“Immunoregulatory activity” means that the protein modulates directly or non-directly the cooperation of T-cells with macrophages.
The binding to the surface of leucocytes is preferably estimated in vitro. Such methods are known in the state of the art.
The term “hybridize under stringent conditions” means that two nucleic acid fragments are capable of hybridization to one another under standard hybridization conditions described in Sambrook et al., “Expression of cloned genes in
E. coli
” in Molecular Cloning: A laboratory manual (1989) Cold Spring Harbor Laboratory Press, New York, USA, 9.47-9.62 and 11.45-11.61.
More specifically, stringent conditions as used herein refer to hybridization in 1 mol/l NaCl 1% SDS and 10% dextransulfate. This is followed by two washes of the filter at room temperature of 5 minutes in 2×SSC and one final wash for 30 minutes. This final wash may be at 0.5×SSC, 0.1% SDS, more preferably at 0.2×SSC, 0.1% SDS and most preferably at 0.1×SSC, 0.1% SDS, final wash taking place at 65° C. Those of ordinary skilled in the art will recognize to that other conditions will afford the same degrees of stringency and are encompassed by the phraseology “under stringent conditions” and are encompassed herein.
LST-1 is a protein which is active in its glycosylated or unglycosylated form. The unglycosylated form can be produced by recombinant technology in prokaryotic cells.
“Proteins with LST-1 activity” means also proteins with minor amino acid variations but with substantially the same LST1 activity. Substantially the same means that the activities are of the same biological properties and preferably at least 85% homology in amino acid sequence. More preferably, the amino acid sequences are at least 90% identical. LST-1 can be purified from U-937 cells by affinity chromatography using a monoclonal antibody against LST-1. It is also preferred to use other known protein purification techniques, including immunoprecipitation, gel filtration, ion exchange, chromatography, chromatofocussing, isoelectric focussing, selective precipitation, electrophoresis, and the like. Fraction isolated during purification procedures can be analyzed for the presence of LST-1 activity by using LST-1 specific antibodies.
The protein according to the invention can also be produced by recombinant means. Non-glycosylated LST-1 protein is obtained when it is produced recombinantly in prokaryotes. With the aid of the nucleic acid sequences provided by the invention it is possible to search for the LST-1 gene or its variants in genomes of any desired cells (e.g. apart from human cells, also in cells of other mammals), to identify these and to isolate the desired gene coding for the LST-1 protein Such processes and suitable hybridization conditions are known to a person
Dubberley F. Aaron
Johnston George W.
Roche Diagnostics GmbH
Spector Lorraine
Tramaloni Dennis P.
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