JP2002500889A - Mutants of IL-16, methods for their production and uses thereof - Google Patents

Abstract

  (57) [Summary] A nucleic acid that may be used to express an IL-16 variant in a prokaryotic or eukaryotic host cell, where the nucleic acid starts with amino acids 1-5 of SEQ ID NO: 2, and begins with amino acids 93-93. It encodes a polypeptide having an amino acid sequence ending in 107 and is suitable for producing an IL-16 variant having improved activity.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel IL-16 variant having high activity, a method for producing the same, and use thereof.

[0002] IL-16 (interleukin-16) relates to lymphocyte affinity factor (LCF) or immunodeficiency suppressive lymphokine (ISL). IL-16 and its properties are described in WO94 / 28134 and WO96 / 31607 and in Cluckshank, W.C. W. (Cruikshank, WW) et al., Proc. Natl. Aca
d. Sci. USA 91 (1994) pp. 5109-5113 and in Nature 378 (1995) 563 by Bayer, M. et al. In addition, production by recombination of IL-16 is described in these documents. According to these documents, IL-16 is a protein having a molecular mass of 13,385D. In addition, Brookshank found that the ISL eluted in molecular sieve chromatography in the form of multimers with a molecular weight of 50-60 and 55-60 kD. Affinity activity was determined by cationic homotetramer (product is AMS Biotechnology Ltd. Europe, C
at number 11177186). 28
A homodimer of IL-16 with a molecular weight of KD has been described by Bayer. However, Cluckshank et al. Immunol. 14
6 (1991) pp. 2928-2934 and the activity of recombinant human IL-16 described by Bayer is extremely low.

[0003] It is an object of the present invention to provide IL-16 variants which have improved IL-16 activity, have even lower immunogenicity and are advantageously suitable for therapeutic use.

[0004] The object of the invention is achieved by a nucleic acid which may be used to express a polypeptide having the activity of interleukin 16 in a prokaryotic or eukaryotic host cell, wherein the nucleic acid Encodes a polypeptide starting at amino acids 1-5 of SEQ ID NO: 2 and ending at amino acids 93-107.

[0005] In a preferred embodiment of the invention, the IL-16 variant comprises the amino acid 1 of SEQ ID NO: 2.
-100, 5-107 or most preferably 5-93.

[0006] Such nucleic acids encode IL-16 variants with improved activity.
Preferably, this sequence is based on the sequence of a primate-specific IL-16, eg, human IL-16, or an ape or other mammal, eg, mouse IL-16.

[0007] NMR and proteolysis data show that the N-terminal residues up to Glu4 and the C-terminal residues starting from Lys94 are impaired in solution and these residues are in the native IL-16 structure. It does not belong. For example, these residues
Heteronuclear ¹⁵ N NOEs to amide backbones that lack long range and medium range NOEs and are characterized by highly adaptable residue properties
s showed a low value. The two shorter fragments of S1 to E100 and A5 to S107 show that they do not alter the activity of the full affinity and the structural properties of the 89 residue fold in the four constructs. Thus, the shortest folded and cytokine active IL-16 has a size of 89 amino acids; this corresponds to the IL-16 amino acid residues A5-R93 of SEQ ID NO: 2.

[0008] The structure of IL-6 was mainly determined from the 3D ¹ H- ¹³ C NOESY-HSOC spectrum acquired on IL-16 double-labeled with ¹³ C / ¹⁵ N. ¹ H
, ¹⁵ N and ¹³ C chemical shift designations are labeled with uniform ¹⁵ N, ¹³ C / ¹⁵ N and labeled with ¹⁵ N-Ala, ¹⁵ N-Gly / Ser, ¹⁵ N-Leu.
-16 Obtained by standard triple resonance NMR techniques using a sample selectivity of ^1-130 . The three-dimensional structure of IL-16 shows a constraint of 588 residues distance, a 68 backbone dihedral angle (backbone) for the 89-residue fold of the protein.
Dihedral angles) and 21 _chi 689 experimental constraints with ₁ side chain angle (Experimenta
l constraints), the simulated annealing calculation method (simulated anneal
aling calculations).

[0009] The structure of IL-16 is a β-sandwich structure (β-strands β1 to β1) having an α-helix (amino acids R71 to A81) structure at the side and arranged in the center and having a central shape.
5). N-terminal β strand (β1, T6-E13
) Yields an antiparallel β-sheet with a C-terminal β-strand β5 (V87-R93) and together with a short β-strand β4 (I59-L62) forms one end of a β-barrel structure. In this case, the second antiparallel β sheet β2 to β3 is F21
-24 and formed from residues F37-I41, with a β ridge in β strand β3 at residue I38. The two β sheets forming the β-sandwich structure are fixed in parallel with each other at a rotation angle of 39 ° between the central axes of the β sheets β2β3 and β1β5. The major body core residues in IL-16 are all hydrophobic (I38, F21, F73, W76, I89, I7
9, L23). Regions that are not well defined in the structure are K14-A17, G2
Localized in the 8-D32 and G44-T52 loops, where no NOEs were found for the protein core. Also, this indicates that for those regions that show the increased flexibility of these fragments, low heteronuclear ¹⁵ N
It is consistent with that of the ¹⁵ N relaxation measurement, which indicates the value of NOEs. Not all loops are flexible, however, the loop connecting α-helix to β-strand β5, β-strand β4 to α-helix,
Well defined in the IL-16 structure.

[0010] The PDZ domain is an intercellular protein module that indirectly clusters ion channels, receptors and other membrane proteins, and these are suitable for signal transduction complexes (Ponting and Phill
ips, Trend in Biochem. Sci. 20 (1995) 102-103). PDZ domains are identified by the presence of a conserved GLGF sequence that is important for the binding of the defined peptide consensus sequence. For example, the PDZ domain has a consensus sequence (Ser / Thr) Xaa-Val (COOH) (Songyang et al., Science
275 (1997) 73-77) was found to bind to the carboxy terminus of some membrane proteins. Also, the IL-16 sequence has a GLGF motif. IL-
16 may belong to the family of PDZ domains, in which case this peptide avidity is important for IL-16 tetramer self-aggregation or clustering of receptors on the CD4 ⁺ surface. (Rumsaeng et al., J. Immunol. 159 (1
997) 2904-2910).

The sequence of IL-16 may differ to some extent from the protein sequence encoded by such a DNA sequence. Such sequence diversity may preferably be due to amino acid substitutions. However, the amino acid sequence of IL-16 is preferably at least 75%, relative to the amino acid sequence of SEQ ID NO: 2.
Particularly preferably, it has at least 90% homology. Partial variants of the amino acid sequence and the nucleic acid sequence of SEQ ID NO: 1 / SEQ ID NO: 2 are described, for example, in WO96 / 3160.
7 and International Patent Applications PCT / EP96 / 05662 and PCT / EP96 /
05661. Also, the protein is 14-terminal at the C-terminal of SEQ ID NO: 2.
It is preferably shortened by up to a number of amino acids.

[0012] Nucleic acids are to be understood within the scope of the present invention, for example, as DNA, RNA and nucleic acid derivatives and analogs. Preferred nucleic acid analogs are those compounds whose sugar phosphate backbone has been replaced with another unit, for example, an amino acid. Such compounds are called PNAs and are described in WO 92/20702. PNA-D
The stringent conditions described below are not suitable for PNA-DNA hybridization because NA binding is stronger than, for example, DNA-DNA binding.
However, suitable hybridization conditions are described in WO 92/20703.

[0013] Within the scope of the present invention, the term IL-16 refers to a polypeptide having IL-16 activity.
Is interpreted as IL-16 is preferably described in WO 96/31607.
Show the effect defined by the test method described or according to WO 94/28134
Stimulates cell division. The activity of IL-16 is CD4 showing suppression of T cell stimulation ⁺ / CD25⁺Is appropriately measured by the cell ratio of

[0014] IL-16 can bind to CD4 ⁺ lymphocytes and inhibit the growth of viruses such as HIV-1, HIV-2 and SIV. The function of IL-16 is
It is not limited by its expression in the MHC complex.

In particular, IL-16 exhibits one or several of the following properties: binds to T cells via the CD4 receptor, and binds to the IL-2 receptor on CD4 ⁺ lymphocytes and / or Stimulates the expression of HLA-DR antigen, stimulates the proliferation of helper T cells in the presence of IL-2, suppresses the proliferation of helper T cells stimulated with anti-CD3 antibody, viruses such as HIV-1, HIV -2 or inhibit the growth of SIV.

Preferably, the nucleic acid is complementary to the nucleic acid sequence of SEQ ID NO: 1 under stringent conditions and hybridizes with a nucleic acid encoding an IL-16 variant according to the invention. The term "hybridizes under stringent conditions" is described, for example, by Sambrook et al. In "Expression of c.
loned genes in E. coli ”in Molecular Cloning: A laboratory manual (1989
), Means that two nucleic acid fragments hybridize to each other under standardized hybridization conditions as described in Cold Spring Harbor Laboratory Press, New York, USA. Such a condition is, for example, 6.0XSS
Hybridization at about 45 ° C. in C followed by 50 ° C. in 2 × SSC
To perform the washing step. In order to select the stringency of the salt concentration during the washing step, for example, between a low stringency at 50 ° C. with 2.0 × SSC and a high stringency at 50 ° C. with 0.2 × SSC. You can choose. Furthermore, the temperature of the washing step is room temperature, low stringency of about 22 ° C. and 6 ° C.
It can vary between high stringency of 5 ° C.

[0017] IL-16 is preferably produced recombinantly in prokaryotic or eukaryotic host cells. Such production methods are described, for example, in WO 94/28134 and WO 96/31607, and are the subject disclosed in the present invention for this purpose. However, in order to obtain an IL-16 variant according to the invention by recombinant production in a defined and reproducible manner, additional measures must be taken which go beyond the recombinant production steps known to the person skilled in the art. Must.

Recombinant DNA, essentially free of other human proteins, has been produced by methods known to those skilled in the art for heterologous or homologous expression (after homologous recombination of the IL-16 nucleic acid into the genome of the host organism). Is also good. To this end, first, IL-16
A DNA capable of producing a protein having activity is produced. The DNA may be cloned in a vector, which may be transferred to a host cell, and replicated therein. Such vectors contain regulatory elements in addition to the IL-16 sequence necessary for the expression of the DNA sequence. This IL-16
The vector containing the sequence and the regulatory elements is transferred into a vector capable of expressing IL-16 DNA. The host cell is cultured under conditions suitable for amplifying the vector, and IL-16 is isolated. Suitable measurements in this way reveal that the protein can adopt an activated three-dimensional structure exhibiting IL-16 properties.

An IL-16 variant according to the invention may occur in the form of a monomer or an oligomer. However, it is preferred that the IL-16 polypeptide monomer is not attached in another subunit in this case.

Further, the nucleic acid sequence of the protein may be modified. Such modifications include, for example: modification of the nucleic acid to introduce different recognition sequences for restriction enzymes, to facilitate the ligation, cloning and mutagenesis steps, modification of the nucleic acid to incorporate preferred codons into the host cell, and Extension of nucleic acids by addition of operator elements to optimize expression in host cells.

The proteins are preferably in microorganisms, especially prokaryotes, and in this case E. coli.
Coli. Expressed in Prokaryotic expression results in an unglycosylated polypeptide.

[0022] The expression vector must contain a promoter that causes the protein to be expressed in the host organism. Such promoters are known to those skilled in the art and include, for example, the lac promoter (Chang et al., Nature 198 (1977) 1056), the trp promoter (Goeddel et al., Nuc. Acids Res. 8 (1980) 4057). , Λ _PL promoter (Shimatake et al., Nature 292 (1981) 128) and the T5 promoter (US Pat. No. 4,689,406). Also suitable are synthetic promoters, for example the tac promoter (US Pat. No. 4,551,433). Coupled promoter systems, such as the T7-RNA polymerase / promoter system (St
udier et al., J. Mol. Biol. 189 (1986) 113) are likewise suitable. Also suitable are composite promoters consisting of bacteriophage promoters and operator regions of microorganisms (EP-A-0267851). An effective ribosome binding site is required in addition to the promoter. E. FIG. Col
In the case of i, the ribosome binding site is a Shine-Dalgarno (SD) sequence (Sambrook et al., “Expression of Cloned Gene in E. Coli”).
n Molecular Cloning: A laboratory manual (1989) Cold Spring Habor Labora
tory Press, New York, USA).

[0023] To improve expression, it is possible to express the protein as a fusion protein. In this case, the DNA sequence encoding the N-terminal portion of the bacterial endogenous protein or other stable protein is usually fused to the 5 'end of the sequence encoding IL-16. This example is, for example, lacZ (Phillips and S
ilhavy, Nature 344 (1990) 882-884), trpE (Yansura, Meth. Enzymol. 1)
85 (1990) 161-166).

After expression of the vector, which is preferably a biofunctional plasmid or a viral vector, the fusion protein is preferably cleaved with an enzyme (eg, enteraloxinase or factor Xa) (Nagai et al., Nature 309 (1984) 81
0). Other examples of cleavage sites are the IgA protease cleavage site (WO 91/11520, EP 0 495 398) and the ubiquitin cleavage site (Miller et al., Bio / Technology 7 (1989) 698).

The protein expressed in bacteria in this way can be obtained in the usual way by disruption of the bacterium and isolation of the protein.

In another embodiment, the protein can be secreted from the microorganism as an active protein. To this end, the fusion product preferably comprises a single sequence suitable for secretion of the protein in the host organism used and a nucleic acid encoding the protein. In this case, the protein is secreted into the medium (Gram-positive bacteria) or into the periplasmic space (Gram-negative bacteria).
It is advantageous to insert a cleavage site between the signal sequence and the sequence encoding IL-16, which will cause the protein to be cleaved during processing or in an additional step. Such a signal sequence is, for example, ompA (Ghrayeb et al.,
EMBO J. 3 (1984) 2437), PhoA (Oka et al., Proc. Natl. Acad. Sci. US
A 82 (1985) 7212).

The vector additionally contains a terminator. The terminator is the DNA sequence of the last signal in the transcription process. They are usually characterized by two structural features: a G / C-rich region that repeats in the opposite direction, which can form a double helix in the molecule, as well as many U (or T). Residue. Examples are the major terminators in the DNA of phage fd (Beck and Zink, Gene 16 (1981) 35-38) and phage rrnB (Brosius et al., J. Mol. Biol. 148 (1981) 107-127). It is.

Furthermore, the expression vector usually has a selectable marker for selecting a transformed cell. Such selectable markers are, for example, genes resistant to ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline (Davies et al., Ann. Rev. Microbiol. 32 (1978).
) 469). Similarly suitable selection markers are genes for substances necessary for the cell, for example substances essential for the biosynthesis of histidine, tryptophan and leucine.

[0029] Many suitable bacterial vectors are known. Vectors include, for example, the following bacteria: Bacillus subtilis (Palva et al., Proc. N
atl. Acad. Sci. USA 79 (1982) 5582); Coli (Aman et al., Gene 40)
(1985) 183; Studier et al., J. Mol. Biol. 189 (1986) 113), Streptococcus cremoris (Powell et al., Appl. Envir.
on. Microbiol. 54 (1988) 655), Streptococcus lividan.
s lividans) and Streptomyces lividans (U.S. Pat. No. 4,747,056).

Other genetic engineering methods for producing and expressing suitable vectors are described in Sambrook et al., “Expression of Cloned Genes in E. coli” in Molecular Cloning: A laaboratory manual (1989) Cold Spring
Harbor Laboratory Press, New York, USA.

In addition to prokaryotic microorganisms, it is also possible to express recombinant IL-16 in eukaryotes (eg, CHO cells, yeast or insect cells). Yeast or insect cells are preferred as eukaryotic expression systems. Expression in yeast is 3
One type of yeast vector may be achieved: an integrated YIp (yeast integrated plasmid) vector, a replicative YRp (yeast replicon plasmid) vector and an episomal YEp (yeast episomal plasmid) vector. More specifically, for example, M. Kingsman et al. (SM Kingsman et al.
) Tibtech 5 (1987) 53-57. In a particular embodiment of the invention, it is possible to use a nucleic acid which expresses a precursor of an IL-16 polypeptide which has been treated according to the invention. Such precursors are those whose N-terminus has been extended by another amino acid of the sequence of the IL-16 variant according to the invention. Thereafter, according to the invention, it is later necessary to identify the preferred processing (cutting) during recombinant production. This may be performed, for example, by treating the expressed product with an endonuclease that cuts at the preferred N-terminus of the IL-16 variant (before or after separation from the fermentation broth).

Furthermore, the present invention relates to a method for expressing a polypeptide as described above, wherein a host cell is transformed with a nucleic acid encoding an IL-16 variant according to the present invention.
Alternatively, it relates to transfected prokaryotic or eukaryotic host cells.
Such host cells usually include a biologically functional nucleic acid vector containing the nucleic acid, preferably a DNA vector, a plasmid vector.

The IL-16 variant according to the invention can be used for culturing prokaryotic or eukaryotic host cells transformed or transfected with the corresponding encoding nucleic acids under suitable nutritional conditions, and May be produced by isolating the preferred polypeptide. When producing a polypeptide in vivo at the site of gene therapy, the polypeptide is of course not isolated from cells.

Another subject of the invention is that the IL-16 variant according to the invention contains a quantity and / or a specific activity sufficient for therapeutic use, and optionally a pharmaceutically acceptable dilution. Pharmaceutical products containing agents, auxiliaries and / or carriers.

The IL-16 variants according to the invention are suitable for the treatment and immunomodulation of pathological conditions caused by the propagation of viruses, in particular of retroviruses.
Such therapeutic uses are also described in WO 96/31607. In addition, it describes a diagnostic test.

[0036] The IL-16 variant according to the invention may preferably be used for immunosuppression. This immunosuppression is achieved preferably by TH ₀ and / or TH ₁ and TH ₂ inhibition of cell helper function. Thus, the polypeptides according to the invention are of therapeutic value in all diseases in which the etiology is suspected of causing immune dysregulation, in particular in hyperimmunity. Diseases that can be treated by IL-16 in cardiology / angiology are, for example, myocarditis, endocarditis and pericarditis, in pulmonology, for example, bronchitis, asthma, and in hematology, For example, autoimmune neuropathy (autoimmu
ne neuropenias) and transplant rejection, chronic gastritis in gastroenterology, diabetes mellitus type I in endocrinology, glomerulonephritis, rheumatic disease, ophthalmological disease in nephrology, and multiple sclerosis in neurology Dermatosis and eczema in dermatology. The polypeptide according to the invention can be used in particular for autoimmune diseases, allergies and to avoid transplant rejection.

The present invention furthermore relates to the use of the nucleic acids according to the invention in the context of gene therapy.
Retroviral or non-viral vector systems are, for example, suitable vector systems.

The following examples and publications and other described sequences further define the scope of protection resulting from the claims of the present invention. The described method is to be understood, even after modification, as an example which further describes the subject of the invention.

Example 1 IL-16 Mutant Using Enterokinase Cleavage Site (SEQ ID NO: 2 SEQ ID NO: 1
1.1 Expression Clones Amplification, cloning of IL-16 cDNA and production of expression clones are described in WO
Modified sequences are also considered and implemented as described in 94/28134 or WO 96/31607. Suitable oligonucleotides are used as forward primers containing an EcoRI site, a 6His, an enterokinase cleavage site and the 5 'end of the sequence of the IL-16 variant (about 7 codons). Suitable oligonucleotides having about 12 codons at the 3 'end of the IL-16 variant are:
Used as a reverse primer. The two reverse primers have BamHI and HindIII cleavage sites for cloning. Sequence is obtained using the IL-16R ₁ that encodes a protein with SEQ ID NO: 2. PCR reactions, cloning and production of expression clones (fusion protein with N-terminal poly-His site for purification) are performed under standard conditions: 0.2 mM dNTPmix, 1 pmol / μl forward primer and Reverse primer, 1X high fidelity buffer (high fidelity buffer) (
Boehringer Mannheim GmbH, Germany)
5 mM MgCl ₂ , 2.6 U highly compatible enzyme mix (Boehringer Mannheim, Germany).

Final liquid volume 20 μl Instrument: Perkin Elmer GeneAmp 9600 Reaction steps: 94 ° C. for 3 minutes, 56 ° C. for 1 minute, 72 ° C. for 2 minutes, followed by 25 cycles (94 ° C. for 20 seconds,
56 ° C for 20 seconds, 72 ° C for 1 minute).

1.2 Fermentation E. coli for IL-16 10 l fermentation broth and high pressure disruption of the E. coli expression clone Pre-culture is started from a stock culture (smeared plates or ampoules stored at -20 ° C) grown at 37 ° C with shaking. The inoculation dose on the next larger scale is in each case from 1 to 10% by weight. Ampicillin (50
１００100 mg / l) is used for selection for plasmid loss in preculture and main culture.

[0042] Enzymatically digested protein and / or yeast extract as N and C sources and glycerol and / or glucose as additional C sources may be used as nutrients. The medium is buffered to pH 7 and metal salts are added at pharmaceutically acceptable concentrations to stabilize the fermentation process. The fermentation is carried out using a feed of the mixed yeast extract / C source as a feed batch.
Fermentation temperature is 25-37 ° C. Aeration speed, r.p. p. m. Depending on regulation and feed rate, the dissolved oxygen partial pressure (pO ₂ ) is maintained at about 20%. Growth is measured by optical density (OD) at 528 nm. IL-16 expression is induced by IPTG. After a fermentation time of 10 to 20 hours, the biomass is recovered by centrifugation during the OD stationary phase.

The biomass is placed in 50 mM sodium phosphate, 5 mM EDTA, 100 mM sodium chloride, pH 7, and broken by continuous high pressure compression at 1000 bar. The suspension thus obtained is again centrifuged and the dissolved I
The supernatant containing L-16 is worked up.

1.3 Purification and cleavage 50 mM sodium phosphate, 5 mM EDTA, 100 mM NaCl, pH
700 ml of the dissolved supernatant in 7.2 are mixed with 70 ml of 5M NaCl, 60 mM of MgCl ₂ , pH 8.0, stirred for 30 minutes and then 20,000
g and centrifuged for 30 minutes. The supernatant liquid obtained by centrifugation is previously used for NiSo4
Solution (concentration 10 mg / ml) and 50 mM sodium phosphate, 0.
5 Nickel-chelate column (volume 20) equilibrated with NaCl, pH 8.0
0 ml, pharmacia). The column was then washed with the equilibration buffer until almost baseline (UV detection at 280 nm) was reached. Thereafter, the column was washed with 1 liter of 50 mM sodium phosphate, 0.5 M NaCl, pH7.
. Rinse with 0 and 1 l of 50 mM sodium phosphate, 0.1 M NaCl, p
Rinse with H7.0. The fusion protein was prepared using 50 mM sodium phosphate, 0.1 M
0-300 mM imidazole in NaCl, pH 7.0 (2 x 1.61), p
Eluted with a concentration gradient of H7.0. The fraction containing IL-16 was subjected to SDS-PAGE.
Identified and collected. This collected IL-16 was collected in Provatio (Filtro
n, membrane omega 5K) to a concentration of 5 mg / l protein and 50
Dialysis was performed against mM Tris, pH 8.0.

One equivalent of the harvest containing 100 mg of the fusion protein was diluted to a protein concentration of 1 mg / ml using 50 mM Tris, pH 8.0 for cleavage by enterokinase. 33 μg of enterokinase (Boehringer Mannheim; 1
: 3000 w / w), the cut preparation was incubated at 37 ° C. overnight (14 hours). Thereafter, the pH value was adjusted to pH 6.5 using HCl.

The uncleaved IL-16 is stirred in 20 ml of nickel-chelate sepharose (adjusted as above; binding time 2 hours), after which the supernatant is centrifuged (10000 g) or It was removed by filtration on a nutsch filter. Identification of the cleaved IL-16 contained in the supernatant,
Confirmed by N-terminal sequence and mass spectrometry. The purity, SDS-PAGE and RP-HPLC (Vydac, diphenyl, 4.6 × 150mm, 45 linear gradient 20% to 95% within minutes B; solution A: H ₂ O in the potassium 20mM phosphate, p H7.5 Solution B: 100% acetonitrile).

Example 2 Measurement of IL-16 Mutant Activity Peripheral blood mononuclear cells (PBMC) (3 × 10 ⁵ ) were each placed in 200 μl of medium (RPM I / 10% FCS), and IL-16 polypeptide was used. 1 at 37 ° C
The temperature was maintained over time. IL-16 concentrations were each 1 μg / ml.
Thereafter, the sample was placed in a 96-well microtiter plate (microtiter plate) pretreated as follows.
plate).

100 μl of the anti-CD3 antibody (obtained at UCHT, Pharmingen, San Diego, USA, order no. 30100D) was placed in PBS at a concentration of 2.5 μg / ml.
Thereafter, the plate was incubated at 37 ° C. for 90 minutes and washed twice with PBS before adding cells. Four days later, the cells were treated with 5 μl each of anti-CD4 antibody PE conjugate (13B.8.2, order no. 0449, Coulter Coop., Miami, USA) and 5 μl anti-CD25 antibody FITC conjugate (B1.49.9, order no. , Coulter Coop., Miami, USA) and FACS (Becto
n Dickinson). The table below shows the percentage of CD4 ⁺ / CD25 ⁺ double positive cells.

[0049] CD25 expression is a measure of T cell activity. Thus, a decrease in CD25 expression after culturing with IL-16 is a measure of the IL-16 induced suppression of anti-CD3 antibody stimulation.

[0050]

[Table 1]

The percentage of CD4 / CD25 positive cells is about 2 with recombinant IL-162 ^).
0% and the IL-16 variant according to the invention ³⁾ reduced by about 50%. Therefore,
The IL-16 variants according to the invention show a significantly higher activity compared to the prior art IL-16.

[0052]

[Outside 1]

[0053]

[Outside 2]

Sequence Listing (1) General Information: (i) Applicant: (A) Name :: Federal Republic of Germany (B) Town: Bonn (C) Country: Germany (D) Postal Code (ZIP): D- 53108 (E) Telephone: 0028 / 941-1530 (F) Telefax: 0028 / 941-4983 (ii) Title of invention: IL-16 variant, method for its production and its use (iii) Total number of sequences: 2 ( iv) Computer readable format: (A) Medium type: floppy disk (B) Computer: IBM PC compatible (C) Operating system: PC-DOS / MS-DOS (D) Software: Patent in Release # 1.0, Version # 1
. 30B (EPO) (2) Information on SEQ ID NO: 1 (i) Sequence characteristics: (A) Sequence length: 324 base pairs (B) Sequence type: Nucleic acid (C) Number of strands: Double strand (D ) Topology: linear (ii) Sequence type: cDNA (ix) Sequence characteristics (A) Name / symbol: CDS (B) Location: 1. . 324 (xi) Sequence description: SEQ ID NO: 1

[0055]

[Outside 3]

[0056]

[Outside 4]

(2) Information on SEQ ID NO: 2 (i) Sequence characteristics (A) Sequence length: 108 amino acids (B) Sequence type: amino acids (D) Topology: linear (ii) Sequence type: Description of the protein (xi) sequence: SEQ ID NO: 2

[0058]

[Outside 5]

[0059]

[Outside 6]

[Sequence list]

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] March 15, 2000 (2000.3.15)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0050

[Correction method] Change

[Correction contents]

[0050]

[Table 1]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 31/12 A61P 37/06 4C087 37/02 37/08 4H045 37/06 C07K 14/54 37/08 C12N 1/15 C07K 14/54 1/19 C12N 1/15 1/21 1/19 C12P 21/02 K 1/21 C12N 15/00 ZNAA C12P 21/02 A61K 37/02 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG) KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV , MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Drote Ambrosius Germany Minoir Strasse 2 (72) Inventor Kurt Lang Germany Penzberg Langor Strasse 10 (72) Inventor Martin Lanzen Delfer Germany Munich Walde Thrust 4 F-term (reference) 4B024 AA01 AA11 BA26 CA04 CA07 DA02 DA05 DA11 EA04 GA11 HA01 HA03 4B064 AG03 CA19 CC24 DA01 DA13 4B065 AA01X AA57X AA87X AA90Y AB01 BA02 4 NA14 ZA022 ZA332 ZA362 ZA592 ZA662 ZA812 ZB072 ZB112 ZB132 ZB152 ZC352 4C086 AA01 AA02 AA03 AA04 EA16 MA01 MA04 NA14 ZA02 ZA33 ZA36 ZA59 ZA66 ZA81 ZB07 ZB11 ZB13 ZB15 ZC15 4A087 4H045 AA10 BA10 CA40 DA02 EA20 EA50 FA72 FA74 GA26

Claims (9)

[Claims] 1. A nucleic acid that may be used to express an IL-16 variant in a prokaryotic or eukaryotic host cell, wherein the nucleic acid starts with amino acids 1-5 of SEQ ID NO: 2, and A nucleic acid that may be used to express an IL-16 variant in a prokaryotic or eukaryotic host cell, characterized in that it encodes a polypeptide ending with amino acids 93-107. 2. Amino acids 1 to 100, 5 to 107 or 5 to 9 of SEQ ID NO: 2.
3. The nucleic acid of claim 1, which encodes three IL-16 variants. 3. A prokaryotic or eukaryotic host cell transformed or transfected with a nucleic acid according to claim 1 or 2 so that the host cell expresses said IL-16 variant. 4. A biologically functional nucleic acid vector containing the nucleic acid according to claim 1 or 2. 5. An IL-16 variant obtainable as a product of eukaryotic or prokaryotic expression of a nucleic acid according to claim 1 or 2 which is essentially free of other human proteins. 6. The IL-16 variant of claim 5, consisting of amino acids 1-107 of SEQ ID NO: 2. 7. A method for producing an IL-16 variant according to claim 5 or 6, wherein the eukaryotic or prokaryotic host cell is transformed or transfected with the nucleic acid sequence according to claim 1 or 2. Is cultured under suitable nutrient conditions, and optionally the desired polypeptide is isolated.
The method for producing an IL-16 mutant according to the above. 8. A medicament comprising the IL-16 variant according to claim 5 or 6, and a pharmaceutically acceptable diluent, auxiliary and / or carrier. 9. A pharmaceutical comprising the IL-16 variant according to claim 5 or 6 in an amount suitable for therapeutic use.