Drug – bio-affecting and body treating compositions – Lymphokine – Interleukin
Reexamination Certificate
1999-05-24
2002-09-03
Spector, Lorraine (Department: 1647)
Drug, bio-affecting and body treating compositions
Lymphokine
Interleukin
C435S069100, C435S325000, C435S252300, C435S320100, C536S023500, C530S351000
Reexamination Certificate
active
06444202
ABSTRACT:
The invention concerns polypeptides with IL-16 activity, processes for their production and their use. The invention describes processed IL-16 with a high activity.
IL-16 (interleukin-16) is a lymphokine which is also referred to as lymphocyte chemoattracting factor (LCF) or immunodeficiency virus suppressing lymphokine (ISL). IL-16 and its properties are described in WO 94/28134 and in WO 96/31607 and by Cruikshank, W. W., et al., Proc. Natl. Acad. Sci. USA 91 (1994) 5109-5113 and by Baier, M., et al., Nature 378 (1995) 563. The recombinant production of IL-16 is also described in these references. According to these IL-16 is a protein with a molecular mass of 13,385 D. Cruikshank also found that ISL elutes in a molecular sieve chromatography as a multimeric form with a molecular weight of 50-60 and 55-60 kD. The chemoattractant activity has been attributed to this multimeric form which is a cationic homotetramer (product information AMS Biotechnology Ltd., Europe, Cat. No. 11177186). A homodimeric form of IL-16 with a molecular weight of 28 kD is described by Baier. 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 small.
The object of the present invention is to improve the activity of IL-16 and to provide IL-16 forms which have a low immunogenicity and are advantageously suitable for a therapeutic application.
The object of the invention is achieved by a nucleic acid which can be used to express a polypeptide with interleukin-16 activity in a prokaryotic or eukaryotic host cell wherein the said nucleic acid codes for a polypeptide having the sequence SEQ ID NO:2 which is optionally N-terminally elongated by Ala-Ser-(GCCTCT).
In a further preferred embodiment the nucleic acid codes for a polypeptide with IL-16 activity which is shortened at the C-terminus by up to 8 amino acids.
Such a nucleic acid codes for a processed polypeptide with IL-16 activity with a very low antigenicity, particularly preferably natural IL-16 from primates such as human IL-16 or IL-16 of an ape species or of another mammal such as the mouse.
It has turned out that FIG. 2 of WO 94/28134 does not describe the correctly processed IL-16. The start codon “ATG” of the precursor form of the protein does not begin with nucleotide 783 but rather with nucleotide 54 or 174. This reading frame results when an A is inserted after nucleotide 156, a C is inserted after nucleotide 398 and a G is inserted after nucleotide 780. The sequence also shows further differences to FIG. 2 of WO 94/28134. These are for example nucleotide substitutions (313 G into A, 717 C into A). IL-16 is processed during the expression in eukaryotic cells. In this way a polypeptide according to SEQ ID NO:2 is formed and/or a polypeptide with a sequence which is N-terminally elongated by Ala-Ser in comparison to SEQ ID NO:2. Knowledge of the processed IL-16 enables the production of IL-16 and derivatives with high activity and low immunogenicity.
The sequence of IL-16 can differ to a certain extent from protein sequences coded by such DNA sequences. Such sequence variations may be amino acid substitutions, deletions or additions. However, the amino acid sequence of IL-16 is preferably at least 75% and particularly preferably at least 90% identical to the amino acid sequence of SEQ ID NO:2. Variants of parts of the amino and of the nucleic acid sequences SEQ ID NO:1/SEQ ID NO:2 are for example described in WO 96/31607 and the International Patent Applications PCT/EP96/05662 and PCT/EP96/05661. However, it is essential that the polypeptides have the N-terminus defined according to the invention. Thus proteins are preferred in which the first three to ten amino acids at the N-terminus are unchanged and therefore begin N-terminally with the amino acid sequences SEQ ID NO:7 to 10 or with analogous sequences which are N-terminally elongated by Ala-Ser. Proteins are also preferred which are shortened by up to 8 amino acids at the C-terminus.
Nucleic acids within the sense of the invention are understood for example 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 referred to as PNA and are described in WO 92/20702. Since PNA-DNA bonds are for example stronger than DNA-DNA bonds, the stringent conditions described below are not applicable to PNA-DNA hybridization. However, suitable hybridization conditions are described in WO 92/20703.
The term “EL-16” is understood within the sense of the invention as a polypeptide with the activity of IL-16. IL-16 preferably exhibits the stated action in the test procedure described in WO 96/31607 or 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.
In particular IL-16 exhibits one or several of the following properties:
binding to T cells via the CD4 receptor,
stimulation of the expression of the IL-2 receptor and/or HLA-DR antigen on CD4
+
lymphocytes,
stimulation of the proliferation of T helper cells in the presence of IL-2,
suppression of the proliferation of T helper cells stimulated with anti-CD3 antibodies,
suppression of the replication of viruses, preferably of HIV-1, HIV-2 or SIV.
Nucleic acids are preferred which hybridize under stringent conditions with nucleic acids which are complementary to nucleic acids of sequence SEQ ID NO:1 and code for a polypeptide of sequence SEQ ID NO:2 which is optionally N-terminally elongated by Ala-Ser-(GCCTCT). The term “hybridize under stringent conditions” means that two nucleic acid fragments hybridize with one another under standardized hybridization conditions as described for example 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. Such conditions are for example hybridization in 6.0×SSC at about 45° C. followed by a washing step with 2×SSC at 50° C. In order to select the stringency the salt concentration in the washing step can for example be chosen between 2.0×SSC at 50° C. for low stringency and 0.2×SSC at 50° C. for high stringency. In addition the temperature of the washing step can be varied between room temperature, ca. 22° C., for low stringency and 65° C. for high stringency.
IL-16 is preferably produced recombinantly in prokaryotic or eukaryotic host cells. Such production processes are described for example in WO 94/28134 and WO 96/31607 which are also for this purpose a subject matter of the disclosure of the present invention. However, in order to obtain the forms according to the invention of IL-16 by recombinant production in a defined and reproducible manner, additional measures have to be taken beyond the processes for recombinant production familiar to a person skilled in the art.
Recombinant IL-16 can be produced by methods familiar to a person skilled in the art as a heterologous expression or homologous expression (after homologous recombination of the IL-16 nucleic acid into the genome of the host organism). For this a DNA is firstly produced which is able to produce a protein which has the activity of IL-16. The DNA is cloned into a vector which can be transferred into a host cell and can be replicated there. Such a vector contains regulatory elements in addition to the IL-16 sequence which are necessary for the expression of the DNA sequence. This vector which contains the IL-16 sequence and the regulatory elements is transferred into a vector which is able to express the DNA of IL-16. The host cell is cultured under conditions which are suitable for the amplification of the vector and IL-16 is isolated. In this process suitable measures ensure that the protein can adopt an active tertiary structure in wh
Ambrosius Dorothee
Baier Michael
Kurth Reinhard
Lanzendörfer Martin
Werner Albrecht
Arent Fox Kintner & Plotkin & Kahn, PLLC
Bundesrepublic Deutschland, vertreten durch den Bundesminister f
Spector Lorraine
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