Drug – bio-affecting and body treating compositions – Lymphokine – Interleukin
Reexamination Certificate
1999-05-11
2002-01-15
Mertz, Prema (Department: 1646)
Drug, bio-affecting and body treating compositions
Lymphokine
Interleukin
C536S023100, C536S023500, C536S024300, C536S024310, C435S069500, C435S071100, C435S071200, C435S325000, C514S002600, C514S008100, C514S012200
Reexamination Certificate
active
06338844
ABSTRACT:
The invention concerns regulatory elements of the expression of IL-16, genomic nucleic acids, cDNA and mRNA that code for polypeptides with IL-16 activity, processes for the production thereof and their use.
IL-16 (interleukin-16) is a lymphokine that is also denoted “lymphocyte chemoattracting factor” (LCF) or “immunodeficiency virus suppressing lymphokine” (ISL). IL-16 and its properties are described in WO 94/28134, the International Application PCT/EP96/01486 as well as by Cruikshank, W. W., et al., Proc. Natl. Acad. Sci. U.S.A. 91 (1994) 5109-5113 and Baier, M., et al., Nature 378 (1995) 563. The recombinant production of IL-16 is also described therein. According to this IL-16 is a protein with a molecular mass of 13,385 D. Cruikshank also found that ISL elutes as a multimeric form with a molecular weight of 50-60 or 55-60 kD in molecular sieve chromatography. The chemoattractant activity is attributed to this multimeric form which is a cationic homotetramer (product information AMS Biotechnology Ltd., Europe, Cat. No. 11177186). Baier describes a homodimeric form of IL-16 with a molecular weight of 28 kD. However, the chemoattractant activity described by Cruikshank et al., in J. Immunol. 146 (1991) 2928-2934 and the activity of recombinant human IL-16 described by Baier are very low.
The object of the present invention is to provide regulatory elements of IL-16 expression, to improve the activity of IL-16 and to provide forms of IL-16 which exhibit low immunogenicity and are advantageously suitable for therapeutic use.
The object of the invention is achieved by a nucleic acid with which expression of a polypeptide having interleukin-16 activity can be achieved or regulated in a eukaryotic host cell, wherein the said nucleic acid
a) corresponds to the DNA sequence SEQ ID NO:1 or its complementary strand;
b) hybridizes under stringent conditions with the DNA of sequence SEQ ID NO:1, preferably with nucleotides 1-6297 of SEQ ID NO:1;
c) or is a nucleic acid sequence which, if there was no degeneracy of the genetic code, would hybridize under stringent conditions with the nucleic acid sequences defined by a) or b),
d) and, if it codes for a polypeptide having IL-16 activity, has a length of at least 1179 coding nucleotides.
A preferred sequence is the cDNA sequence shown in SEQ ID NO:6, the complementary strand thereof or a sequence which under stringent conditions hybridizes with the sequence SEQ ID NO:6. SEQ ID NO:5 and the plasmid pCI/IL16 PROM also describe the genomic DNA of IL-16 and contain the introns and exons each parity or completely.
Such a nucleic acid preferably codes a polypeptide with IL-16 activity, particularly preferably the natural IL-16 of primates such as human IL-16 or IL-16 of a species of monkey or another mammal such as e.g. mouse.
It surprisingly turned out the FIG. 2 of WO 94/28134 does not describe the complete sequence of IL-16. The start codon “ATG” of the precursor form of the protein does not begin with nucleotide 783. The sequence has yet more differences to FIG. 2 of WO 94/28134. These are for example nucleotide substitutions (313 G by A, 717 C by A, 1070 G by T).
The sequence of IL-16 can differ to a certain extent from protein sequences coded by such DNA sequences. Such sequence variations can be amino acid substitutions, deletions or additions. However, the amino acid sequence of IL-16 is preferably at least 75% especially preferably at least 90% identical to the amino acid sequence of IL-16. Variants of parts of the amino acid sequence or nucleic acid sequence are for example described in the International Patent Application Nos. PCT/EP96/01486, PCT/EP96/05662 and PCT/EP96/05661.
Nucleic acids within the sense of the invention are for example understood as DNA, RNA and nucleic acid derivatives and analogues. Preferred nucleic acid analogues are those compounds in which the sugar phosphate backbone is replaced by other units such as e.g. amino acids. Such compounds are denoted PNA and are described in WO 92/20702. Since for example PNA-DNA bonds are stronger the DNA-DNA bonds the stringent conditions for PNA-DNA hybridization described in the following are not applicable. Suitable hybridization conditions are, however, described in WO 92/20703.
SEQ ID NO:6 describes the cDNA derived from the mRNA. The cDNA is suitable, for instance, for the determination of the corresponding RNA in tissue fluids and body fluids of mammals and humans. The cDNA is preferably used, however, for the expression of full length IL-16 in prokaryotes, preferably in
E.coli.
For that purpose the cDNA is inserted into an appropriate vector, transformed into a prokaryotic host cell, said host cell is cultivated, and, after cultivation, IL-16 is isolated. This can be done according to the methods known to one skilled in the art. If the protein is not secreted but obtained within the cell as a denatured insoluble protein (inclusion bodies), solubilisation and naturation must be carried out thereafter. These methods are also known to one skilled in the art.
SEQ ID NO:7 describes the amino acid sequence of IL-16 in its precursor form, which is also a subject matter of the invention.
The term “IL-16” within the sense of the invention is understood as a polypeptide with the activity of IL-16. IL-16 preferably exhibits the effect stated in the International Patent Application No. PCT/EP96/01486 or it stimulates cell division according to WO 94/28134.
IL-16 binds to CD4
+
lymphocytes and can suppress the replication of viruses such as for example HIV-1, HIV-2 and SIV. The function of IL-16 is not limited by its presentation in the MHC complex.
IL-16 in particular exhibits one or several of the following properties:
binding to T cells via the CD4 receptor,
stimulating the expression of the IL-2 receptor and/or HLA-DR antigen on CD4
+
lymphocytes,
stimulating the proliferation of T helper cells in the presence of IL-2,
suppressing the proliferation of T helper cells stimulated with anti-CD3 antibodies,
suppressing the replication of viruses preferably HIV-1, HIV-2 or SIV.
The term “hybridizing under stringent conditions” means that two nucleic acid fragments hybridize with one another under standardized hybridization conditions as for example 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, U.S.A. Such conditions are for example hybridization in 6.0×SSC at about 45° C. followed by a washing step at 2×SSC at 50° C. To select the stringency, the salt concentration in the washing step can be selected for example between 2.0×SSC at 50° C. for low stringency and 0.2×SSC at 50° C. for high stringency. In addition the temperature in the washing step can be varied between room temperature ca. 22° C. for low stringency and 65° C. for high stringency.
A “regulatory element” is understood as a DNA sequence which regulates the expression of genes (e.g. promoter, attenuator, enhancer). A promoter is understood as a cis-acting DNA sequence which is usually 80-120 base pairs long and is located 5′ upstream of the initiation site of the gene to be expressed. A promoter is in addition characterized in that RNA polymerase can bind to it and can initiate the correct transcription. A preferred DNA fragment with promoter activity spans nucleotides 2053-3195 of SEQ ID NO:1.
An enhancer is usually understood as a cis-acting DNA sequence of ca. 50-100 bp in length which is of paramount importance for an efficient transcription. Enhancer sequences work independently of orientation and position.
An intron is understood as a nucleotide sequence which is present in eukaryotic genes and is transcribed into pre-mRNA and is removed from the mRNA in a further step (splicing). The IL-16 gene contains several introns and exons which are described in SEQ ID NO:1, pCI/IL16 PROM and/or SEQ ID NO:5.
Plasmid pCI/IL-16 PROM contains a sequence upstream of SEQ ID NO:5. SEQ ID NO:5 describes the 3′ terminal part of the genomic DNA whereas the plasm
Baier Michael
Bannert Norbert
Kurth Reinhard
Metzner Karin
Werner Albrecht
Arent Fox Plotkin Kintner Kahn
Bundesrepublik Deutschland vertreten durch de Bundesminister für
Mertz Prema
Prasad Sarada C
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