Drug – bio-affecting and body treating compositions – Lymphokine – Interleukin
Reexamination Certificate
1998-08-27
2001-03-27
Spector, Lorraine (Department: 1646)
Drug, bio-affecting and body treating compositions
Lymphokine
Interleukin
C435S383000, C435S069520, C435S069700, C435S325000, C435S252300, C530S351000, C536S023500
Reexamination Certificate
active
06207144
ABSTRACT:
The invention concerns polypeptides with IL-16 activity, processes for their production and their use.
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, 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 eluted 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 in the region coding for the said polypeptide
a) corresponds to the DNA sequence of nucleotides 54-1175 from SEQ ID NO:1 or to its complementary strand
b) hybridizes with the DNA of nucleotide sequence 54-785 from SEQ ID NO:1 under stringent conditions,
c) hybridizes with the DNA of nucleotide sequence 786-1175 from SEQ ID NO:1 under stringent conditions and at the 5′ end begins with nucleotides 756-785 from SEQ ID NO:1 or a part thereof which codes for part of the sequence of amino acids 235-244 from SEQ ID NO:1 or
d) it is a nucleic acid sequence which would hybridize under stringent conditions with the nucleic acid sequences defined by a) or b) without the degeneracy of the genetic code.
In a preferred embodiment the nucleic acid which is defined by b) also additionally hybridizes with the DNA of nucleotides 786-1175 from SEQ ID NO:1.
Such a nucleic acid preferably codes for a polypeptide with improved IL-16 activity, particularly preferably improved 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 surprisingly turned out that IL-16 forms with an N-terminally extended sequence compared to the IL-16 described in WO 94/28134 are still active. Such IL-16 forms even have an increased activity in vivo due to an increased half life.
It has surprisingly turned out that FIG. 2 of WO 94/28134 does not describe the correct sequence of IL-16. The start codon “ATG” does not begin with nucleotide 783 but rather with nucleotide 54. 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 correct reading frame is shown in SEQ ID NO:1. This sequence also shows further differences to FIG. 2 of WO 94/28134. However, these are only nucleotide substitutions (e.g. 313 G into A, 717 C into A). The nucleic acid according to the invention also codes for a polypeptide which can be processed during its production. Such a polypeptide which is thus extended compared to the known IL-16 from WO 94/28134 exhibits an improved activity. Monomeric IL-16 with improved activity has a molecular weight of 13.9-39 kD, preferably ca. 15-35 kD and is thus about three times as large as the C-terminal IL-16 fragment described in WO 94/28134.
The sequence of IL-16 can differ to a certain extent from protein sequences coded by such DNA sequences. Such sequence variations of IL-16 muteins 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:1. 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 the International Application WO 96/31607 and the German Patent Application 195 47 933.5.
Particularly preferred IL-16 muteins with improved activity begin at the N-terminus with the amino acids 235-244 from SEQ ID NO:1/2 or a part thereof. Particularly preferred N-termini are described in SEQ ID NO:3-12.
In a preferred embodiment the IL muteins with improved activity can be truncated at the C-terminus by preferably 1-20 amino acids.
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 above are not applicable to PNA-DNA hybridization. However, suitable hybridization conditions are described in Wo 92/20703.
The term IL-16 is understood within the sense of the invention as a polypeptide with the activity of IL-16 or preferably with an improved activity. IL-16 preferably exhibits the stated action in the test procedure described in the International Application 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.
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 the 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. 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 in a vector which can be transferred into a host cell and can be replicated there. Such a vector contains operator elements in addition to the IL-16 sequence which are necessa
Baier Michael
Bannert Norbert
Kurth Reinhard
Lang Kurt
Metzner Karin
Arent Fox Kintner Plotkin & Kahn
Roche Diagnostics GmbH
Spector Lorraine
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