Recombinant human recombinant human interleukin-1&agr;

Details Recombinant human recombinant human interleukin-1&agr; Recombinant human recombinant human interleukin-1&agr;

1999-04-13

2001-07-31

Martinell, James (Department: 1632)

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, C536S023100, C536S023500

Reexamination Certificate

active

06268180

ABSTRACT:

BACKGROUND OF THE INVENTION
Interleukin-1 (IL-1) is a protein synthesized and secreted by activated macrophages. As part of the body's defense mechanism against infection and other forms of injury, this polypeptide hormone stimulates the proliferation and/or differentiation of a broad spectrum of cell types (including T and B lymphocytes, liver cells, bone marrow cells, connective tissue elements, skeletal muscle, brain cells, etc.). Through its actions on these diverse cell populations, IL-1 modulates immune function, fever, liver cell function (increased synthesis and secretion of acute phase reactants: increased uptake of amino acids, iron and zinc), production and release of neutrophils from the bone marrow, skeletal muscle proteolysis, changes in connective tissue, etc. IL-1 has also been described in the prior art as lymphocyte activating factor (LAF), leukocyte endogeneous mediator (LEM), endogeneous pyrogen (EP), and mononuclear cell factor (MCF). Until recently all studies of IL-1 were conducted with partially purified protein preparations, therefore, it has not been certain whether all the activities associated with IL-1 are contained within one molecule, or whether fragments of IL-1 or other macrophage proteins are responsible for certain of the functions outlined above.
Since it has been difficult to prepare sufficient amounts of human IL-1 for structural and activity studies, the biochemical nature of this molecule is poorly understood. IL-1 preparations show evidence of size and charge heterogeneity. IL-1 activity is associated with single polypeptide chains with molecular weights anywhere in the range between 12,000 and 19,000.
Recently, the gene coding for mouse IL-1 was cloned, sequenced, and expressed in
Escherichia Coli
. See in this regard Lomedico et al., Nature 312, pp. 458-462 (Nov. 29, 1984). In conjunction with the sequencing studies on purified “natural” mouse IL-1, it is possible to understand how this hormone is synthesized to yield the population of molecules possessing size and charge heterogeneity. When purified natural mouse IL-1 is electrophoresed on SDS-polyacrylamide gels, one finds multiple polypeptides with molecular weights between 12,000 and 19,000, all of which are biologically active. These polypeptides have different amino-terminal sequences and demonstrate charge heterogeneity on Tris-glycinate polyacrylamide gels. Sequencing of the cloned mouse IL-1 cDNA and in vitro translation experiments proved that IL-1 is initially synthesized as a precursor polypeptide of 270 amino acids. Biologically active IL-1 can be obtained from
E. coli
by expressing the carboxy-terminal 156 amino acids of this precursor. Hence, IL-1 activity is proteolytically released from the carboxy-terminus of the 270 amino acid precursor protein. Multiple points of protease attack will generate a population of molecules with “ragged” amino-termini, thus providing an explanation of the size and charge heterogeneity observed in purified “natural” IL-1.
The cloning of a putative gene for human IL-1 was described by Auron et al., Proc. Natl. Acad. Sci. USA, 81, 7907 (1984) which was published in February 1985. The DNA and protein sequences described therein are only partially homologous to the sequences described below.
The purification of natural human IL-1 to homogeneity has been reported by Lachman, Fed. Proc. 42, No. 3. 2639-2645 (June 1983). The method used molecular weight fractionation, isoelectric focusing and preparative polyacrylamide gel electrophoresis. Due to the use of sodium dodecyl sulfate in the last step, the product was denatured and exhibited only a trace of its original biological activity. See also Schmidt, J. Exp. Med. 160, 772-787 (September 1984) for a purification scheme using HPLC methods to produce a single charged species of human IL-1 and Kronheim et al., J. Exp. Med. 161, 490-502 (March 1985).
It has also been known in the art to produce antibodies directed against murine IL-1. See Mizel et al. J. Immun. 131, 1834 (1983). These antibodies, which were raised in goat, were utilized to develop an assay for IL-1 and also in the production of an anti-IL-1 immunoabsorbent column which in turn is useful for further purification of either natural or recombinant murine IL-1. The anti murine IL-1 antibody crossreacts poorly with human IL-1.
SUMMARY OF THE INVENTION
The present invention relates to the cloning of the human IL-1 gene, its engineering into suitable expression vectors, transformation of host organisms with such expression vectors and production of biologically active recombinant human IL-1 by culture of such transformed cells. Additionally, the present invention relates to the isolation and use of the resulting recombinant human IL-1 polypeptide.
Thus, the present invention utilizes recombinant DNA technology as the means to discover the DNA sequence and the deduced amino acid sequence for human interleukin-1 and to its production and to its use.
More particularly, the present invention relates to the isolation and identification of DNA sequences coding for biologically active forms of human interleukin-1. This was accomplished by employing a mouse IL-1 cDNA clone to isolate a partial human IL-1 genomic clone. This genomic clone was used in turn to isolate a human IL-1 cDNA clone. The sequence of this cDNA revealed the structure of the human IL-1 precursor protein. Expression of the carboxy-terminal 154 amino acids of this precursor in
E. coli
resulted in the production of biologically active IL-1 protein.
Thus, more particularly, the present invention relates to the isolation and identification of DNA sequences encoding the human IL-1 precursor and biologically active molecules contained therein, and to the construction of recombinant DNA expression vehicles containing such DNA sequences operatively linked to expression—effecting promoter sequences and to the expression vehicles so constructed. In another aspect, the present invention relates to host culture systems, such as various microorganism and vertebrate cell cultures transformed with such expression vehicles and thus directed in the expression of the DNA sequences referred to above. In other aspects, this invention relates to the means and methods of converting the end products of such expression to novel entities, such as pharmaceutical compositions, useful for the prophylactic or therapeutic treatment of humans or in diagnostic assay systems. In preferred embodiments, this invention provides particular expression vehicles that are constructed properly such that human interleukin-1 is produced in the host cell in mature form.


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