CA2320624A1 - Mutants of il-16, processes for their production, and their use - Google Patents
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Abstract
A nucleic acid, which can be used to express an IL-16 mutant in a prokaryotic or eukaryotic host cell, wherein the said nucleic acid codes for a polypeptide with an amino acid sequence beginning with amino acids 1-5 and ending with amino acids 93-107 of SEQ ID NO:2, is suitable for producing IL-16 mutants with improved activity.
Description
Mutants of IL-16, processes for their production, and their use The invention concerns new IL-I6 mutants with a high activity, processes for their production. and their use.
IL-16 (interleukin-I6) is a lymphokine which is also referred to as lymphocyte chemo-attracting 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 (I994) SI09-5113 and by Baier, M., et al., Nature 378 (1990 X63. 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 13,38 D.
Cruikshank also found that ISL elutes in a molecular sieve chromatography as a multimeric form with a molecular weight of 50-60 and S~-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 Ba.ier. 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 y mutants which have, in addition, 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 starting with amino acids 1-5 and ending with amino acids 93-107 of the sequence SEQ ID N0:2.
In a preferred embodiment of the invention, the IL-16 mutant consists of amino acids 1-100, ~-107, or, most preferably, of amino acids 5-93 of of SEQ ID N0:2.
_7-Such a nucleic acid codes for an IL-16 mutant with improved activity. Its sequence is preferably based on the sequence of 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.
NMR and proteolytic data indicated that the N-terminal residues up to Glu 4 and the C-terminal residues starting from Lys 94 were disordered in solution and that these residues did not belong to the proper IL-16 structure. For example, these residues lacked long- and medium-range NOES and showed low values of heteronuclear I SN NOEs for backbone amides; features characteristic of highly flexible residues. Two shorter fragments; starting as S 1 to E 100 and from A~ to S 107, show full chemoattractant activity and no change in structural properties of the 89 residue folded part in the four constructs.
Therefore, the shortest fully folded and cytokine active IL-16 has a size of 89 amino acids;
this corresponds to residues A~ - R93 of the IL-16 amino acid sequence SEQ ID N0:2.
The structure of IL-16 was primarily determined from the 3D 1H-13C NOESY-HSQC
spectra acquired on a double 13C/15N labelled IL-16. 1H, 1~N and 13C chemical shift assignments were obtained using standard triple resonance NMR techniques using uniformly 15N~ 13C/15N labelled and selectively 15N-Ala, 15N-Gly/Ser, 15N-Leu labelled samples of IL-161-130. The tertiary structure of IL-16 was calculated by simulated annealing calculations from 689 experimental constraints, containing 588 interresidue distance constraints, 68 backbone dihedral angles and 21 x 1 side chain angles for the 89 residue folded part of the protein.
The structure of IL-16 consists of a central up-and-down 13-sandwich (formed by 13-strands 131-f35) which is flanked by an a-helix (amino acids R7I-A81). The N-terminal !3-strand (l31, T6-E13) makes an antiparallel 13-sheet with the C-terminal 13-strand a5 (V87-R93) and fortes, together with the short f3-strand 134 (I59-L62), one side of the f3-barrel.
The second antiparallel !3-sheet Ii2-(33, which is formed by the residues F21-24 and F37-I41, contains a I3-bulge in !3-strand f33 at residue I38. The two 13-sheets, which build the 13-sandwich, are packed against each othcr in a parallel manner, with a rotation of 39° degrees between a central axis of 13-sheet 132 Ii3 and a central axis of f3-sheet al 135. The main body core residues in IL-I6 are all hydrophobic (I38, F21, F73, W76, I89,179, L23). Poorly defined regions in structures are localized at loops K14 to A17, G28 to D32 and G44 to T52, where no NOES to the core of the protein were found. This was also in agreement with 1 SN relaxation measurements which showed low values of the heteronuclear 1 SN NOEs for these regions indicating increased flexibility of these fragments. Not all loops are flexible, however, the connecting loops WO 99/37781 - ~ - PCT/EP99/00428 between the a-helix and 13-strand f35, (3-strand f34 and the a-helix are well defined in the IL-16 structure.
PDZ domains are intracellular protein modules that mediate clustering of ion channels, receptors and other membrane proteins and connect them to their appropriate signal transduction complexes (Ponting and Phillips, Trends in Biochem. Sci. 20 (1990 102-103).
PDZ domains are identified by the presence of a conserved GLGF sequence that is responsible for binding of defined peptide consensus sequences. For example, PDZ domains were found to bind carboxyl termini of several membrane proteins that possess a consensus sequence (Ser/Thr)Xaa-Val(COOH) (Songyang et al., Science 275 (1997) 73-77).
sequence also contains the GLGF motif. IL-16 might belong to the family of PDZ
domains where the peptide binding capability is important for autoaggregation of tetrameric IL-16 or for clustering of receptors on the CD4''' surface (Rumsaeng et al., J.
Immunol. 1 ~9 (1997) 2904-2910).
The sequence of IL-16 can differ to a certain extent from protein sequences coded by such DNA sequences. Such sequence variations may preferably be amino acid substitutions.
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 N0:2.
Variants of parts of the amino and of the nucleic acid sequences SEQ iD N0:1/SEQ ID N0:2 are for example described in WO 96/31607 and the International Patent Applications PCT/EP96/05662 and PCT/EP96/05661. Proteins are also preferred which are shortened by up to 14 amino acids at the C-terminus of SEQ ID N0:2.
,,~~
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 "IL-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. The activity of IL-16 is measured suitably as the ratio of CD4+/CD?5+ cells which describes the inhibition of T cell stimulation.
IL-16 binds to CD4+ lymphocytes and can suppress the replication of viruses such as for example HIV-l, 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 an IL-I6 mutant according to the invention. 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 x SSC at about ATE
45°C followed by a washing step with 2 x 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 x SSC at 50°C
for low stringency and 0.2 x 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-I6 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 IL-16 mutants according to the invention ~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, which is essentially free of other human proteins, 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 which it exhibits IL-16 properties.
The IL-16 mutant according to the invention can occur in monomeric or oligomeric (e.g., tetrameric) form. However, a monomeric IL-16 polypeptide which cannot be cleaved into further subunits is preferred.
The nucleic acid sequence of the protein can also be modified. Such modifications are for example:
- , modification of the nucleic acid in order to introduce various recognition sequences of restriction enzymes to facilitate the steps of ligation, cloning and mutagenesis - modification of the nucleic acid to incorporate preferred codons for the host cell - extension of the nucleic acid by additional operator elements in order to optimize expression in the host cell.
The protein is preferably expressed in microorganisms in particular in prokaryotes and in this case in E. coli. The expression in prokaryotes yields an unglycosylated polypeptide.
The expression vectors must contain a promoter which allows expression~of the protein in the host organism. Such promoters are known to a person skilled in the art and are for example the lac promoter (Chang et al., Nature 198 (1977) 106), trp promoter (Goeddel et al., Nuc.
Acids Res. 8 ( i 980) 4057), ~.pL promoter (Shimatake et al., Nature 292 ( 1981 ) 128) and TS
promoter (US Patent No. 4,689,406). Synthetic promoters such as for example the tac promoter (US Patent No. 4,551,433) are also suitable. Coupled promoter systems are equally suitable such as for example the T7-RNA polymerase/ promoter system (Studier et al., J.
Mol. Biol. 189 (1986) 113). Hybrid promoters composed of a bacteriophage promoter and the operator region of the microorganism (EP-A 0 267 851 ) are also suitable. An effective ribosome binding site is necessary in addition to the promoter. In the case of E. coli this ribosome binding site is referred to as the Shine-Dalgarno (SD) sequence (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).
In order to improve expression it is possible to express the protein as a fusion protein. In this case a DNA sequence which codes for the I~r-terminal part of an endogenous bacterial protein or another stable protein is usually fused to the 5' end of the sequence coding for IL-16.
Examples of this are for example lacZ (Phillips and Silhavy, Nature 344 (1990) 882-884), trpE (Yansura, Meth. Enzymol. 185 (1990) 161-166).
Afrer expression of the vector which is preferably a biologically functional plasmid or a viral vector, the fusion proteins are preferably cleaved with enzymes (e.g.
enterokinase or factor Xa) (Nagai et al., Nature 309 ( 1984) 810). Further examples of cleavage sites are the IgA
protease cleavage site (WO .91/11520, EP-A 0 495 398) and the ubiquitin cleavage site (Miller et al., Bio/Technology 7 {1989) 698).
The proteins expressed in this manner in bacteria are obtained in the usual manner by disrupting the bacteria and isolating the protein.
In a further embodiment it is possible to secrete the proteins from the microorganisms as active proteins. For this a fusion product is preferably used which is composed of the signal sequence that is suitable for secretion of proteins in the host organisms used and of the nucleic acid that codes for the protein. In this case the protein is either secreted into the medium (in gram-positive bacteria) or into the periplasmatic space (in gram-negative bacteria). It is expedient to insert a cleavage site between the signal sequence and the sequence coding for IL-16 which allows cleavage of the protein either during processing or in an additional step. Such signal sequences are derived for example from ompA
(Ghrayeb et al., EMBO J. 3 (1984) 2437), phoA (Oka et al., Proc. Natl. Acad. Sci. USA 82 (1985) 7212).
The vectors additionally contain terminators. Terminators are DNA sequences that signal the end of a transcription process. They are usually characterized by two structural features: a 7_ reversely repetitive G/C-rich region which can form a double helix intramolecularly as well as a number of U.(or T) residues. Examples are the main terminator in the DNA
of the phages fd (Beck and Zink, Gene 16 (1981) 35-38) and rmB (Brosius et al., J.
viol. Biol. 148 (1981) 107-127).
In addition the expression vectors usually contain a selectable marker in order to select transformed cells. Such selectable markers are for example the resistance genes for ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracyclin {Davies et al., Ann. Rev. Microbiol. 32 (1978) 469). Selectable markers which are equally suitable are the genes for essential substances for the biosynthesis of substances necessary for the cell ,;._, such as e.g. histidine, tryptophan and leucine.
Numerous suitable bacterial vectors are known. Vectors have for example been described for the following bacteria: Bacillus subtilis (Palva et al., Proc. Natl. Acad.
Sci. USA 79 (1982) X582), E. coli {Arran et al., Gene 40.(1985) 183; Studier et al., J. Mol.
Biol. 189 (1986) 113), Streptococcus cremoris (Powell et al., Appl. Environ. Microbiol. 54 (1988) 65~), Streptococcus lividans and Streptomyces lividans (US Patent No. 4,747,056).
Further genetic engineering methods for the production and expression of suitable vectors are 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, USA.
,r In addition to prokaryotic microorganisms it is also possible to express recombinant IL-16 in ~-'' eukaryotes (such as for example CHO cells; yeast or insect cells). The yeast system or insect cells are preferred as a eukaryotic expression system. Expression in yeast can be achieved by means of three types of yeast vectors: (integrating YIp (yeast integrating plasmids) vectors, replicating YRp (yeast replicon plasmids) vectors and episomal YEp (yeast episomal plasmids) vectors. More details of this are described for example in S. M.
Kingsman et al.
Tibtech 5 (1987) 53-57. In a particular embodiment of the invention it is possible to use a nucleic acid which represents a precursor form of the processed IL-16 polypeptide according to the invention. Sueh precursor forms are N-terminally elongated by further amino acids of the sequence of the IL-16 mutant according to the invention. According to the invention it is then necessary to ensure the desired processing (cleavage) in the recombinant production.
This can be done for example by treating the expression product (before or after separation from the fermentation broth) with an endonuclease which cleaves at the desired N-terminus of the IL-16 mutant.
-g_ The invention in addition concerns a prokaryotic or eukaryotic host cell which is transformed or transfected with a nucleic acid which codes for an IL-16 mutant according to the invention in such a way that the host cell expresses the said polypeptide. Such a host cell usually contains a biologically functional nucleic acid vector, preferably a DNA
vector, a plasmid DNA, which contains this nucleic acid.
The IL-16 mutant according to the invention can be produced by culturing a prokaryotic or eukaryotic host cell which has been transformed or transfected with a corresponding coding nucleic acid under suitable nutrient conditions and optionally isolating the desired polypeptide. If it is intended to produce the polypeptide in vivo in the context of a gene therapy treatment, the polypeptide is of course not isolated from the cell.
A further subject matter of the invention is a pharmaceutical composition which contains an IL-16 mutant according to the invention in an amount and/or specific activity which is sufficient for a therapeutic application as well as optionally a pharmaceutically suitable diluent, adjuvant and/or carrier.
The IL-16 mutants according to the invention are especially suitable for treating pathological states which are caused by viral replication, in particular retroviral replication, and for immunomodulation. Such therapeutic applications are also described in the WO
96/31607.
This also describes diagnostic test procedures.
The IL-16 mutants according to the invention can be preferably used for immunosuppression.
This immunosuppression is preferably achieved by an inhibition of the helper function of the THO and/or TH1 and TH2 cells. Hence the polypeptides according to the invention are of therapeutic value in all diseases in which an immunodysregulatory component is postulated in the pathogenesis and in particular a hyperimmunity. Diseases which can be treated by IL-16 in cardiology/angiology are for example myocarditis, endocarditis and pericarditis, in pulmonology for example bronchitis, asthma, in haematology autoimmune neuropenias and transplant rejections, in gastroenterology chronic gastritis, in endocrinology diabetes mellitus type I, in nephrology glomerulonephritis, rheumatic diseases, diseases in ophthalmology. in neurology such as multiple sclerosis and eczemas in dermatology. The polypeptides according to the invention can be used in particular for autoimmune diseases, allergies and to avoid transplant rejections.
The invention furthermore concerns the use of the nucleic acids according to the invention within the context.of gene therapy. Retroviral or non-viral vector systems are for example suitable vector systems for this.
The following examples and publications as well as the sequence listing further elucidate the invention the protective scope of which results from the patent claims. The processes described are to be understood as examples that still describe the object of the invention even after modifications.
Example 1 ~,:-~ Production of an IL-16 mutant (amino acids x-100 of SEQ ID N0:2) using an enterokinase cleavage site 1.1 Expression clone The amplification, cloning of IL-16 cDNA and production of an expression clone is carried out as described in WO 94/28134 or WO 96/31607 taking the modified sequences into consideration. An appropriate oligonucleotide is used as a forward primer which contains an EcoRI site, 6 His, an enterokinase cleavage site, and the ~'-end of the IL-16 mutant sequence (approximately 7 codons). An appropriate oligonucleotide with approximately 12 codons of the 3' end of the IL-16 mutant is used as the reverse primer. The two reverse primers contain BamHI and HindIII cleavage sites for the cloning. A sequence is obtained using IL-16 Rl .-;. which codes for a protein according to SEQ ID N0:2. The PCR reaction, cloning and cat' ~
production of the expression clone (fusion protein with N-terminal poly-His part for purification) is carried out according to standard conditions:
0.2 mM dNTP mix, 1 pmoUpl each forward and reverse primer, 1 x high fidelity buffer (Boehringer Mannheim GmbH, DE) 1.5 mM MgCl2, 2.6 U high fidelity enzyme mix (Boehringer Mannheim GmbH, DE).
20 ~l final volume Instrument: Perkin Elmer GeneAmp 9600 Reaction course:
3 min 94°C, 1 min ~6°C, 2 min 72°C, then 2~ cycles (20 sec 94°C, 20 sec 56°C, 1 min 72°C) I.2 Fermentation 1 fermentation of an E. coli expression clone for IL-16 and high pressure disruption Precultures are set up from stock cultures (plate smear or ampoules stored at -20°C) which are incubated at 37°C while shaking. The inoculation volume into the next higher dimension is i - I 0 vol. % in each case. AmpiciIlin (~0-100 mg/1) is used in the preculture and main culture to select against plasmid loss.
Enzymatically digested protein and/or yeast extract as a N- and C-source as well as glycerol and/or glucose as an additional C-source are used as nutrients. The medium is buffered to pH
7 and metal salts are added at physiologically tolerated concentrations to stabilize the fermentation process. The fermentation is carried out as a feed batch with a mixed yeast extractlC sources dosage. The fermentation temperature is 2~-37°C. The dissolved partial oxygen pressure (p02) is kept approximately at < 20 % by means of the aeration rate, r.p.m.
regulation and dosage rate. The growth is determined by determining the optical density (OD) at 528 nm. The expression of IL-16 is induced by means of IPTG. After a fermentation period of 10 to 20 hours the biomass is harvested by centrifugation at OD standstill.
The biomass is taken up in SO mM sodium phosphate, ~ mM EDTA, 100 mM sodium chloride, pH 7 and is disrupted at 1000 bar by means of a continuous high pressure press. The suspension obtained in this manner is centrifuged again and the supernatant which contains the dissolved IL-16 is processed fturther.
1.3 Purification and cleavage 700 ml lysis supernatant in 50 mM sodium phosphate, ~ mM EDTA, 100 mM NaCI, pH
7.2 was admixed with 70 ml 5 M NaCI, 60 mM MgCl2, pH 8.0, stirred for 30 min and subse-quently centrifuged for 30 min at 20,000 g. The centrifuged supernatant was applied to a nickel-chelate column (V = 200 ml, Pham~.acia) which had previously been loaded with a NiS04 solution (c = 10 mg/ml) and equilibrated with 50 mM sodium phosphate, 0.5 NaCI, pH 8Ø The column was subsequently washed with equilibration buffer until the base line (UV detection at 280 nm) was nearly reached. Afterwards the column was rinsed with 1 1 of 50 mM sodium phosphate, 0.~ M NaCI, pH 7.0 and with 1 I of 50 mM sodium phosphate, 0.1 M NaCI, pH 7Ø The fusion protein was eluted with a gradient of 0 - 300 mM
imidazole, pH
7.0 in 50 mM sodium phosphate, 0.1 M NaCI, pH 7.0 (2 x 1.6 l). Fractions containing IL-16 were identified by means of SDS-PAGE and pooled. This IL-16 pool was 'concentrated in a Provario (Filtron, .membrane omega ~ K) to a concentration of ~ mg protein/ml and dialysed against 50 mM Tris, pH 8Ø
An equivalent of the pool containing 100 mg fusion protein was diluted to a protein concentration of c = 1 mg/ml with 50 mM Tris, pH $.0 for the enterokinase cleavage. After adding 33 pg enterokinase (Boehringer Mannheim GmbH; 1:3000 w/w) the cleavage preparation was incubated overnight (14 h) at 37°C. Subsequently the pH
value was adjusted to pH 6.5 with HCI.
') Uncleaved IL-16 was removed by stirring in 20 ml nickel-chelate sepharose (prepared as above; binding time 2 h) and subsequent centrifugation ( 10,000 g) or filtering the supernatant over a putsch filter. The indentity of the cleaved IL-16 contained in the supernatant was co~rmed by N-terminal sequencing and mass analysis. The purity was checked by SDS-PAGE and RP-HPLC (Vydac, biphenyl, 4.6 x 150 mm, linear gradient from 20 % to 95 % B
within 45 minutes; solution A: 20 mM potassium phosphate in H20, pH 7.5;
solution B:
100 % acetonitrile).
Example 2 Determination of the activity of the IL-16 mutant 3 x 10~ peripheral blood monocytes (PBMC) were in each case placed in 200 pI
of medium (RPMI/10% FCS) and incubated with the IL-16 polypeptide for 1 h at 37°C. The IL-16 .~,;~.~
concentration was 1 ug/ml in each case. Then the samples were transferred to a 96-well microtiter plate which had been pretreated as follows.
100 ul of anti-CD3-antibodies (UCHT obtained from Pharmingen, San Diego, USA, order No. 30100D) at a concentration of 2.5 p.g/ml were placed in PBS. Then the plate was incubated for 90 min. at 37°C and washed twice with PBS prior to the addition of the cells.
After four days the cells were in each case labeled with 5 pl of an anti-CD4-antibody PE
conjugate (13B.$.2, order No. 0449, Coulter Coop., Miami, USA) and anti-CD?5-antibody FITC conjugate (B 1.49.9, order No. 0478. Coulter Coop., Miami, USA) and analyzed in a FACS (Becton Dickinson). The table below shows the percentage of CD4+/CD25+
double-positive cells.
15-03-2000 ~ EP 009900428 The expression of CD25 is a measure of T cell activation. Thus, the decrease in CD25 expression afrer incubation with IL-I6 is a measure of IL-16-induced inhibition of stimularion by the anti-CD3-antibody.
Table I
IL-16 type CD4+ICD25+
control (PBS}1) 46 (100%
_ IL-1621 39 84%) :.
IL-I6 xnutant3) 24 (52%) II,-16 muntant4) 20 (43 %) IL-16 mutant3) 13 28 %) > > only phosph$te buffered saline ~l recombinant IL-16 according to WO 94/28138 3~ IL-15 mutant (amino acids 1-100 of SEQ ID N0:2) 4> TF.-16 (amino acids 5-107 of SEQ ID N0:2) 5~ IL-16 (amino acids 5-93 of SEQ ID N0:2) The percentage of CD4iGD25-positive eGIIs decreases by about 20% far recombinant IL-162) and at~ut 50% for the IL-lb mutant according to the invention-'). Thus, the IL-16 mutant according to the invention exhibits a distinctly higher activity compared to IL-16 according to the state ef the art.
Lfat of references Arran et al., Gcne 40 (1985) 183 Baier, M., et eL, Nature 378 (1995) 563 Eeck and Zink, Gene 16 (1981) 35-S$
Hrosiits et al., J. Mol. Biol. 148 (1981) 107 - 127 Chang et at., I3ature 198 (1977) lOSb Cruikshank, W,W,, et al., J. Irnmunol. 146 (1991) 2928-2934 Cruikshank, W. W., et al., Proc. Nail, Acad. Sci. USA 91 (1994) 5109-51 I 3 Davies et al., Ann. Rev. Microhiol. 32 ( I 978) 469 AMENDED SHEET
. ..
Ghrayeb et al., EMB4 3. 3 (I984) 2437 Goeddel et al., I~'uc. Acids Res. 8 (1980) 4057 International Patent Application PCTlBP96/0566i International Patent Application PCT/EP9bl05662 Kingsman, S.M., et al, Tibtech 5 (1987) 53-57 Mack et al., Analyt. Biochem. 200 (1992) 74-80 Miller et al., l3iolTecluiology 7 (1959) 698 Nagai et aL, Nattue 309 (1984) 8t0 i ,,; Oka et al., Proc. Natl. Acad. Sci. USA 82 (1985) 7212 Palva of al., Pmc. ATatl. Acad. Sci. USA 79 (1982) 5582 Phillips and Silhavy, Nature 344 (1990) 882-884 Ponting and Phillips, Trends in Bioch~rn,. Sci. 20 (1995) 102-103 Powell et al., Appl. >rnviron. Microbiol. 54 (1988) 655 Rumsa,eng et al., J. Imrnnnol. 159 (1997) 2904-2910 Sambmok et al., "Expression of cloned gents in E. colt" in Molecular Cloning:
A laboratory manual (I989), Cold Spring ~-iarbor Laboratory Press, New York, USA
Shimatake et al., Nature 292 (1981) 128 Songyar~ e~k aL, Scicace 275 (i997) 73-77 Studier ct ai., J', lVtol. Biol. 189 (1986) 113 US-Patent No. 4,551,433 US-Patent No. 4,6$9,406 US-Patent No. 4,747,056 WO 9 t Il I 520 W O 92120?02 WO 94128 i 34 Yansura, Meth. Enzymol. I 8 5 ( 1990) I 61-16b AMENDED SHEET
_ 14_ SEQUENCE LISTT_NG
(1) GENERAL INFORMATION:
(i) APPLICANT.:
(A) NAME: Bundesrepublik De utschland (B) CITY: Bonn (C) COUNTRY: Germany (D) POSTAL CODE (ZIP): D-53108 (E) TELEPHONE: 0228/941-1530 (E) TELEFAX: 0228/941-4983 (ii) TITLE OF INVENTION: Mutants of IL-16, processes for their production and their use (iii) NUMBER OF SEQUENCES: 2 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release n1.0, Version #1.308 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 324 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..324 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 1:
Ser Thr Ala Glu Ala Thr Val Cys Thr Val Thr Leu Glu Lys Met Ser Ala Gly LeaGly Phe SerLeu Glu GlyGly Lys GlySer Leu HisGly GAC AAG CCTCTC ACC ATTAAC AGG ATTTTC A.rLAGGAGCA GCC TCAGAA 144 Asp Lys ProLeu Thr IleAsn Arg IlePhe Lys GlyAla Ala SerGlu Gln Ser GluTtirVal G1nPro Gly AspGlu I1e LeuGln Leu GlyGly Thr Ala MetGln Gly LeuThr Arg PheGlu Ala TrpAsn Ile IleLys C 'A
Ala Leu ProAsp Gly ProVal Thr IleVal Ile ArgArg Lys SerLeu Gln Ser LysGlu Thr ThrAla Ala GlyAsp Ser (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 108 amino acids (E) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Ser Thr Ala Glu Ala Thr Val Cys Thr Val Thr Leu Glu Lys Met Ser Ala Gly Leu Gly Phe Ser Leu Glu Gly Gly Lys Gly Ser Leu His.Gly Asp Lys Pro Leu Thr Ile Asn Arg Ile Phe Lys Gly Ala Ala Ser Glu Gln Ser Glu Thr Val Gln Pro Gly Asp Glu Ile Leu Gln Leu Gly Gly Thr Ala Met Gln Gly Leu Thr Arg Phe Glu Ala Trp Asn Iie Ile Lys A1a Leu Pro Asp G1y Pro Val Thr Ile Val Ile Arg Arg Lys Ser Leu 85 9~ 95 Gln Ser Lys Glu Thr Thr Ala Ala G1y Asp Ser '-~~ 105
IL-16 (interleukin-I6) is a lymphokine which is also referred to as lymphocyte chemo-attracting 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 (I994) SI09-5113 and by Baier, M., et al., Nature 378 (1990 X63. 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 13,38 D.
Cruikshank also found that ISL elutes in a molecular sieve chromatography as a multimeric form with a molecular weight of 50-60 and S~-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 Ba.ier. 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 y mutants which have, in addition, 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 starting with amino acids 1-5 and ending with amino acids 93-107 of the sequence SEQ ID N0:2.
In a preferred embodiment of the invention, the IL-16 mutant consists of amino acids 1-100, ~-107, or, most preferably, of amino acids 5-93 of of SEQ ID N0:2.
_7-Such a nucleic acid codes for an IL-16 mutant with improved activity. Its sequence is preferably based on the sequence of 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.
NMR and proteolytic data indicated that the N-terminal residues up to Glu 4 and the C-terminal residues starting from Lys 94 were disordered in solution and that these residues did not belong to the proper IL-16 structure. For example, these residues lacked long- and medium-range NOES and showed low values of heteronuclear I SN NOEs for backbone amides; features characteristic of highly flexible residues. Two shorter fragments; starting as S 1 to E 100 and from A~ to S 107, show full chemoattractant activity and no change in structural properties of the 89 residue folded part in the four constructs.
Therefore, the shortest fully folded and cytokine active IL-16 has a size of 89 amino acids;
this corresponds to residues A~ - R93 of the IL-16 amino acid sequence SEQ ID N0:2.
The structure of IL-16 was primarily determined from the 3D 1H-13C NOESY-HSQC
spectra acquired on a double 13C/15N labelled IL-16. 1H, 1~N and 13C chemical shift assignments were obtained using standard triple resonance NMR techniques using uniformly 15N~ 13C/15N labelled and selectively 15N-Ala, 15N-Gly/Ser, 15N-Leu labelled samples of IL-161-130. The tertiary structure of IL-16 was calculated by simulated annealing calculations from 689 experimental constraints, containing 588 interresidue distance constraints, 68 backbone dihedral angles and 21 x 1 side chain angles for the 89 residue folded part of the protein.
The structure of IL-16 consists of a central up-and-down 13-sandwich (formed by 13-strands 131-f35) which is flanked by an a-helix (amino acids R7I-A81). The N-terminal !3-strand (l31, T6-E13) makes an antiparallel 13-sheet with the C-terminal 13-strand a5 (V87-R93) and fortes, together with the short f3-strand 134 (I59-L62), one side of the f3-barrel.
The second antiparallel !3-sheet Ii2-(33, which is formed by the residues F21-24 and F37-I41, contains a I3-bulge in !3-strand f33 at residue I38. The two 13-sheets, which build the 13-sandwich, are packed against each othcr in a parallel manner, with a rotation of 39° degrees between a central axis of 13-sheet 132 Ii3 and a central axis of f3-sheet al 135. The main body core residues in IL-I6 are all hydrophobic (I38, F21, F73, W76, I89,179, L23). Poorly defined regions in structures are localized at loops K14 to A17, G28 to D32 and G44 to T52, where no NOES to the core of the protein were found. This was also in agreement with 1 SN relaxation measurements which showed low values of the heteronuclear 1 SN NOEs for these regions indicating increased flexibility of these fragments. Not all loops are flexible, however, the connecting loops WO 99/37781 - ~ - PCT/EP99/00428 between the a-helix and 13-strand f35, (3-strand f34 and the a-helix are well defined in the IL-16 structure.
PDZ domains are intracellular protein modules that mediate clustering of ion channels, receptors and other membrane proteins and connect them to their appropriate signal transduction complexes (Ponting and Phillips, Trends in Biochem. Sci. 20 (1990 102-103).
PDZ domains are identified by the presence of a conserved GLGF sequence that is responsible for binding of defined peptide consensus sequences. For example, PDZ domains were found to bind carboxyl termini of several membrane proteins that possess a consensus sequence (Ser/Thr)Xaa-Val(COOH) (Songyang et al., Science 275 (1997) 73-77).
sequence also contains the GLGF motif. IL-16 might belong to the family of PDZ
domains where the peptide binding capability is important for autoaggregation of tetrameric IL-16 or for clustering of receptors on the CD4''' surface (Rumsaeng et al., J.
Immunol. 1 ~9 (1997) 2904-2910).
The sequence of IL-16 can differ to a certain extent from protein sequences coded by such DNA sequences. Such sequence variations may preferably be amino acid substitutions.
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 N0:2.
Variants of parts of the amino and of the nucleic acid sequences SEQ iD N0:1/SEQ ID N0:2 are for example described in WO 96/31607 and the International Patent Applications PCT/EP96/05662 and PCT/EP96/05661. Proteins are also preferred which are shortened by up to 14 amino acids at the C-terminus of SEQ ID N0:2.
,,~~
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 "IL-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. The activity of IL-16 is measured suitably as the ratio of CD4+/CD?5+ cells which describes the inhibition of T cell stimulation.
IL-16 binds to CD4+ lymphocytes and can suppress the replication of viruses such as for example HIV-l, 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 an IL-I6 mutant according to the invention. 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 x SSC at about ATE
45°C followed by a washing step with 2 x 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 x SSC at 50°C
for low stringency and 0.2 x 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-I6 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 IL-16 mutants according to the invention ~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, which is essentially free of other human proteins, 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 which it exhibits IL-16 properties.
The IL-16 mutant according to the invention can occur in monomeric or oligomeric (e.g., tetrameric) form. However, a monomeric IL-16 polypeptide which cannot be cleaved into further subunits is preferred.
The nucleic acid sequence of the protein can also be modified. Such modifications are for example:
- , modification of the nucleic acid in order to introduce various recognition sequences of restriction enzymes to facilitate the steps of ligation, cloning and mutagenesis - modification of the nucleic acid to incorporate preferred codons for the host cell - extension of the nucleic acid by additional operator elements in order to optimize expression in the host cell.
The protein is preferably expressed in microorganisms in particular in prokaryotes and in this case in E. coli. The expression in prokaryotes yields an unglycosylated polypeptide.
The expression vectors must contain a promoter which allows expression~of the protein in the host organism. Such promoters are known to a person skilled in the art and are for example the lac promoter (Chang et al., Nature 198 (1977) 106), trp promoter (Goeddel et al., Nuc.
Acids Res. 8 ( i 980) 4057), ~.pL promoter (Shimatake et al., Nature 292 ( 1981 ) 128) and TS
promoter (US Patent No. 4,689,406). Synthetic promoters such as for example the tac promoter (US Patent No. 4,551,433) are also suitable. Coupled promoter systems are equally suitable such as for example the T7-RNA polymerase/ promoter system (Studier et al., J.
Mol. Biol. 189 (1986) 113). Hybrid promoters composed of a bacteriophage promoter and the operator region of the microorganism (EP-A 0 267 851 ) are also suitable. An effective ribosome binding site is necessary in addition to the promoter. In the case of E. coli this ribosome binding site is referred to as the Shine-Dalgarno (SD) sequence (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).
In order to improve expression it is possible to express the protein as a fusion protein. In this case a DNA sequence which codes for the I~r-terminal part of an endogenous bacterial protein or another stable protein is usually fused to the 5' end of the sequence coding for IL-16.
Examples of this are for example lacZ (Phillips and Silhavy, Nature 344 (1990) 882-884), trpE (Yansura, Meth. Enzymol. 185 (1990) 161-166).
Afrer expression of the vector which is preferably a biologically functional plasmid or a viral vector, the fusion proteins are preferably cleaved with enzymes (e.g.
enterokinase or factor Xa) (Nagai et al., Nature 309 ( 1984) 810). Further examples of cleavage sites are the IgA
protease cleavage site (WO .91/11520, EP-A 0 495 398) and the ubiquitin cleavage site (Miller et al., Bio/Technology 7 {1989) 698).
The proteins expressed in this manner in bacteria are obtained in the usual manner by disrupting the bacteria and isolating the protein.
In a further embodiment it is possible to secrete the proteins from the microorganisms as active proteins. For this a fusion product is preferably used which is composed of the signal sequence that is suitable for secretion of proteins in the host organisms used and of the nucleic acid that codes for the protein. In this case the protein is either secreted into the medium (in gram-positive bacteria) or into the periplasmatic space (in gram-negative bacteria). It is expedient to insert a cleavage site between the signal sequence and the sequence coding for IL-16 which allows cleavage of the protein either during processing or in an additional step. Such signal sequences are derived for example from ompA
(Ghrayeb et al., EMBO J. 3 (1984) 2437), phoA (Oka et al., Proc. Natl. Acad. Sci. USA 82 (1985) 7212).
The vectors additionally contain terminators. Terminators are DNA sequences that signal the end of a transcription process. They are usually characterized by two structural features: a 7_ reversely repetitive G/C-rich region which can form a double helix intramolecularly as well as a number of U.(or T) residues. Examples are the main terminator in the DNA
of the phages fd (Beck and Zink, Gene 16 (1981) 35-38) and rmB (Brosius et al., J.
viol. Biol. 148 (1981) 107-127).
In addition the expression vectors usually contain a selectable marker in order to select transformed cells. Such selectable markers are for example the resistance genes for ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracyclin {Davies et al., Ann. Rev. Microbiol. 32 (1978) 469). Selectable markers which are equally suitable are the genes for essential substances for the biosynthesis of substances necessary for the cell ,;._, such as e.g. histidine, tryptophan and leucine.
Numerous suitable bacterial vectors are known. Vectors have for example been described for the following bacteria: Bacillus subtilis (Palva et al., Proc. Natl. Acad.
Sci. USA 79 (1982) X582), E. coli {Arran et al., Gene 40.(1985) 183; Studier et al., J. Mol.
Biol. 189 (1986) 113), Streptococcus cremoris (Powell et al., Appl. Environ. Microbiol. 54 (1988) 65~), Streptococcus lividans and Streptomyces lividans (US Patent No. 4,747,056).
Further genetic engineering methods for the production and expression of suitable vectors are 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, USA.
,r In addition to prokaryotic microorganisms it is also possible to express recombinant IL-16 in ~-'' eukaryotes (such as for example CHO cells; yeast or insect cells). The yeast system or insect cells are preferred as a eukaryotic expression system. Expression in yeast can be achieved by means of three types of yeast vectors: (integrating YIp (yeast integrating plasmids) vectors, replicating YRp (yeast replicon plasmids) vectors and episomal YEp (yeast episomal plasmids) vectors. More details of this are described for example in S. M.
Kingsman et al.
Tibtech 5 (1987) 53-57. In a particular embodiment of the invention it is possible to use a nucleic acid which represents a precursor form of the processed IL-16 polypeptide according to the invention. Sueh precursor forms are N-terminally elongated by further amino acids of the sequence of the IL-16 mutant according to the invention. According to the invention it is then necessary to ensure the desired processing (cleavage) in the recombinant production.
This can be done for example by treating the expression product (before or after separation from the fermentation broth) with an endonuclease which cleaves at the desired N-terminus of the IL-16 mutant.
-g_ The invention in addition concerns a prokaryotic or eukaryotic host cell which is transformed or transfected with a nucleic acid which codes for an IL-16 mutant according to the invention in such a way that the host cell expresses the said polypeptide. Such a host cell usually contains a biologically functional nucleic acid vector, preferably a DNA
vector, a plasmid DNA, which contains this nucleic acid.
The IL-16 mutant according to the invention can be produced by culturing a prokaryotic or eukaryotic host cell which has been transformed or transfected with a corresponding coding nucleic acid under suitable nutrient conditions and optionally isolating the desired polypeptide. If it is intended to produce the polypeptide in vivo in the context of a gene therapy treatment, the polypeptide is of course not isolated from the cell.
A further subject matter of the invention is a pharmaceutical composition which contains an IL-16 mutant according to the invention in an amount and/or specific activity which is sufficient for a therapeutic application as well as optionally a pharmaceutically suitable diluent, adjuvant and/or carrier.
The IL-16 mutants according to the invention are especially suitable for treating pathological states which are caused by viral replication, in particular retroviral replication, and for immunomodulation. Such therapeutic applications are also described in the WO
96/31607.
This also describes diagnostic test procedures.
The IL-16 mutants according to the invention can be preferably used for immunosuppression.
This immunosuppression is preferably achieved by an inhibition of the helper function of the THO and/or TH1 and TH2 cells. Hence the polypeptides according to the invention are of therapeutic value in all diseases in which an immunodysregulatory component is postulated in the pathogenesis and in particular a hyperimmunity. Diseases which can be treated by IL-16 in cardiology/angiology are for example myocarditis, endocarditis and pericarditis, in pulmonology for example bronchitis, asthma, in haematology autoimmune neuropenias and transplant rejections, in gastroenterology chronic gastritis, in endocrinology diabetes mellitus type I, in nephrology glomerulonephritis, rheumatic diseases, diseases in ophthalmology. in neurology such as multiple sclerosis and eczemas in dermatology. The polypeptides according to the invention can be used in particular for autoimmune diseases, allergies and to avoid transplant rejections.
The invention furthermore concerns the use of the nucleic acids according to the invention within the context.of gene therapy. Retroviral or non-viral vector systems are for example suitable vector systems for this.
The following examples and publications as well as the sequence listing further elucidate the invention the protective scope of which results from the patent claims. The processes described are to be understood as examples that still describe the object of the invention even after modifications.
Example 1 ~,:-~ Production of an IL-16 mutant (amino acids x-100 of SEQ ID N0:2) using an enterokinase cleavage site 1.1 Expression clone The amplification, cloning of IL-16 cDNA and production of an expression clone is carried out as described in WO 94/28134 or WO 96/31607 taking the modified sequences into consideration. An appropriate oligonucleotide is used as a forward primer which contains an EcoRI site, 6 His, an enterokinase cleavage site, and the ~'-end of the IL-16 mutant sequence (approximately 7 codons). An appropriate oligonucleotide with approximately 12 codons of the 3' end of the IL-16 mutant is used as the reverse primer. The two reverse primers contain BamHI and HindIII cleavage sites for the cloning. A sequence is obtained using IL-16 Rl .-;. which codes for a protein according to SEQ ID N0:2. The PCR reaction, cloning and cat' ~
production of the expression clone (fusion protein with N-terminal poly-His part for purification) is carried out according to standard conditions:
0.2 mM dNTP mix, 1 pmoUpl each forward and reverse primer, 1 x high fidelity buffer (Boehringer Mannheim GmbH, DE) 1.5 mM MgCl2, 2.6 U high fidelity enzyme mix (Boehringer Mannheim GmbH, DE).
20 ~l final volume Instrument: Perkin Elmer GeneAmp 9600 Reaction course:
3 min 94°C, 1 min ~6°C, 2 min 72°C, then 2~ cycles (20 sec 94°C, 20 sec 56°C, 1 min 72°C) I.2 Fermentation 1 fermentation of an E. coli expression clone for IL-16 and high pressure disruption Precultures are set up from stock cultures (plate smear or ampoules stored at -20°C) which are incubated at 37°C while shaking. The inoculation volume into the next higher dimension is i - I 0 vol. % in each case. AmpiciIlin (~0-100 mg/1) is used in the preculture and main culture to select against plasmid loss.
Enzymatically digested protein and/or yeast extract as a N- and C-source as well as glycerol and/or glucose as an additional C-source are used as nutrients. The medium is buffered to pH
7 and metal salts are added at physiologically tolerated concentrations to stabilize the fermentation process. The fermentation is carried out as a feed batch with a mixed yeast extractlC sources dosage. The fermentation temperature is 2~-37°C. The dissolved partial oxygen pressure (p02) is kept approximately at < 20 % by means of the aeration rate, r.p.m.
regulation and dosage rate. The growth is determined by determining the optical density (OD) at 528 nm. The expression of IL-16 is induced by means of IPTG. After a fermentation period of 10 to 20 hours the biomass is harvested by centrifugation at OD standstill.
The biomass is taken up in SO mM sodium phosphate, ~ mM EDTA, 100 mM sodium chloride, pH 7 and is disrupted at 1000 bar by means of a continuous high pressure press. The suspension obtained in this manner is centrifuged again and the supernatant which contains the dissolved IL-16 is processed fturther.
1.3 Purification and cleavage 700 ml lysis supernatant in 50 mM sodium phosphate, ~ mM EDTA, 100 mM NaCI, pH
7.2 was admixed with 70 ml 5 M NaCI, 60 mM MgCl2, pH 8.0, stirred for 30 min and subse-quently centrifuged for 30 min at 20,000 g. The centrifuged supernatant was applied to a nickel-chelate column (V = 200 ml, Pham~.acia) which had previously been loaded with a NiS04 solution (c = 10 mg/ml) and equilibrated with 50 mM sodium phosphate, 0.5 NaCI, pH 8Ø The column was subsequently washed with equilibration buffer until the base line (UV detection at 280 nm) was nearly reached. Afterwards the column was rinsed with 1 1 of 50 mM sodium phosphate, 0.~ M NaCI, pH 7.0 and with 1 I of 50 mM sodium phosphate, 0.1 M NaCI, pH 7Ø The fusion protein was eluted with a gradient of 0 - 300 mM
imidazole, pH
7.0 in 50 mM sodium phosphate, 0.1 M NaCI, pH 7.0 (2 x 1.6 l). Fractions containing IL-16 were identified by means of SDS-PAGE and pooled. This IL-16 pool was 'concentrated in a Provario (Filtron, .membrane omega ~ K) to a concentration of ~ mg protein/ml and dialysed against 50 mM Tris, pH 8Ø
An equivalent of the pool containing 100 mg fusion protein was diluted to a protein concentration of c = 1 mg/ml with 50 mM Tris, pH $.0 for the enterokinase cleavage. After adding 33 pg enterokinase (Boehringer Mannheim GmbH; 1:3000 w/w) the cleavage preparation was incubated overnight (14 h) at 37°C. Subsequently the pH
value was adjusted to pH 6.5 with HCI.
') Uncleaved IL-16 was removed by stirring in 20 ml nickel-chelate sepharose (prepared as above; binding time 2 h) and subsequent centrifugation ( 10,000 g) or filtering the supernatant over a putsch filter. The indentity of the cleaved IL-16 contained in the supernatant was co~rmed by N-terminal sequencing and mass analysis. The purity was checked by SDS-PAGE and RP-HPLC (Vydac, biphenyl, 4.6 x 150 mm, linear gradient from 20 % to 95 % B
within 45 minutes; solution A: 20 mM potassium phosphate in H20, pH 7.5;
solution B:
100 % acetonitrile).
Example 2 Determination of the activity of the IL-16 mutant 3 x 10~ peripheral blood monocytes (PBMC) were in each case placed in 200 pI
of medium (RPMI/10% FCS) and incubated with the IL-16 polypeptide for 1 h at 37°C. The IL-16 .~,;~.~
concentration was 1 ug/ml in each case. Then the samples were transferred to a 96-well microtiter plate which had been pretreated as follows.
100 ul of anti-CD3-antibodies (UCHT obtained from Pharmingen, San Diego, USA, order No. 30100D) at a concentration of 2.5 p.g/ml were placed in PBS. Then the plate was incubated for 90 min. at 37°C and washed twice with PBS prior to the addition of the cells.
After four days the cells were in each case labeled with 5 pl of an anti-CD4-antibody PE
conjugate (13B.$.2, order No. 0449, Coulter Coop., Miami, USA) and anti-CD?5-antibody FITC conjugate (B 1.49.9, order No. 0478. Coulter Coop., Miami, USA) and analyzed in a FACS (Becton Dickinson). The table below shows the percentage of CD4+/CD25+
double-positive cells.
15-03-2000 ~ EP 009900428 The expression of CD25 is a measure of T cell activation. Thus, the decrease in CD25 expression afrer incubation with IL-I6 is a measure of IL-16-induced inhibition of stimularion by the anti-CD3-antibody.
Table I
IL-16 type CD4+ICD25+
control (PBS}1) 46 (100%
_ IL-1621 39 84%) :.
IL-I6 xnutant3) 24 (52%) II,-16 muntant4) 20 (43 %) IL-16 mutant3) 13 28 %) > > only phosph$te buffered saline ~l recombinant IL-16 according to WO 94/28138 3~ IL-15 mutant (amino acids 1-100 of SEQ ID N0:2) 4> TF.-16 (amino acids 5-107 of SEQ ID N0:2) 5~ IL-16 (amino acids 5-93 of SEQ ID N0:2) The percentage of CD4iGD25-positive eGIIs decreases by about 20% far recombinant IL-162) and at~ut 50% for the IL-lb mutant according to the invention-'). Thus, the IL-16 mutant according to the invention exhibits a distinctly higher activity compared to IL-16 according to the state ef the art.
Lfat of references Arran et al., Gcne 40 (1985) 183 Baier, M., et eL, Nature 378 (1995) 563 Eeck and Zink, Gene 16 (1981) 35-S$
Hrosiits et al., J. Mol. Biol. 148 (1981) 107 - 127 Chang et at., I3ature 198 (1977) lOSb Cruikshank, W,W,, et al., J. Irnmunol. 146 (1991) 2928-2934 Cruikshank, W. W., et al., Proc. Nail, Acad. Sci. USA 91 (1994) 5109-51 I 3 Davies et al., Ann. Rev. Microhiol. 32 ( I 978) 469 AMENDED SHEET
. ..
Ghrayeb et al., EMB4 3. 3 (I984) 2437 Goeddel et al., I~'uc. Acids Res. 8 (1980) 4057 International Patent Application PCTlBP96/0566i International Patent Application PCT/EP9bl05662 Kingsman, S.M., et al, Tibtech 5 (1987) 53-57 Mack et al., Analyt. Biochem. 200 (1992) 74-80 Miller et al., l3iolTecluiology 7 (1959) 698 Nagai et aL, Nattue 309 (1984) 8t0 i ,,; Oka et al., Proc. Natl. Acad. Sci. USA 82 (1985) 7212 Palva of al., Pmc. ATatl. Acad. Sci. USA 79 (1982) 5582 Phillips and Silhavy, Nature 344 (1990) 882-884 Ponting and Phillips, Trends in Bioch~rn,. Sci. 20 (1995) 102-103 Powell et al., Appl. >rnviron. Microbiol. 54 (1988) 655 Rumsa,eng et al., J. Imrnnnol. 159 (1997) 2904-2910 Sambmok et al., "Expression of cloned gents in E. colt" in Molecular Cloning:
A laboratory manual (I989), Cold Spring ~-iarbor Laboratory Press, New York, USA
Shimatake et al., Nature 292 (1981) 128 Songyar~ e~k aL, Scicace 275 (i997) 73-77 Studier ct ai., J', lVtol. Biol. 189 (1986) 113 US-Patent No. 4,551,433 US-Patent No. 4,6$9,406 US-Patent No. 4,747,056 WO 9 t Il I 520 W O 92120?02 WO 94128 i 34 Yansura, Meth. Enzymol. I 8 5 ( 1990) I 61-16b AMENDED SHEET
_ 14_ SEQUENCE LISTT_NG
(1) GENERAL INFORMATION:
(i) APPLICANT.:
(A) NAME: Bundesrepublik De utschland (B) CITY: Bonn (C) COUNTRY: Germany (D) POSTAL CODE (ZIP): D-53108 (E) TELEPHONE: 0228/941-1530 (E) TELEFAX: 0228/941-4983 (ii) TITLE OF INVENTION: Mutants of IL-16, processes for their production and their use (iii) NUMBER OF SEQUENCES: 2 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release n1.0, Version #1.308 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 324 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..324 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 1:
Ser Thr Ala Glu Ala Thr Val Cys Thr Val Thr Leu Glu Lys Met Ser Ala Gly LeaGly Phe SerLeu Glu GlyGly Lys GlySer Leu HisGly GAC AAG CCTCTC ACC ATTAAC AGG ATTTTC A.rLAGGAGCA GCC TCAGAA 144 Asp Lys ProLeu Thr IleAsn Arg IlePhe Lys GlyAla Ala SerGlu Gln Ser GluTtirVal G1nPro Gly AspGlu I1e LeuGln Leu GlyGly Thr Ala MetGln Gly LeuThr Arg PheGlu Ala TrpAsn Ile IleLys C 'A
Ala Leu ProAsp Gly ProVal Thr IleVal Ile ArgArg Lys SerLeu Gln Ser LysGlu Thr ThrAla Ala GlyAsp Ser (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 108 amino acids (E) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Ser Thr Ala Glu Ala Thr Val Cys Thr Val Thr Leu Glu Lys Met Ser Ala Gly Leu Gly Phe Ser Leu Glu Gly Gly Lys Gly Ser Leu His.Gly Asp Lys Pro Leu Thr Ile Asn Arg Ile Phe Lys Gly Ala Ala Ser Glu Gln Ser Glu Thr Val Gln Pro Gly Asp Glu Ile Leu Gln Leu Gly Gly Thr Ala Met Gln Gly Leu Thr Arg Phe Glu Ala Trp Asn Iie Ile Lys A1a Leu Pro Asp G1y Pro Val Thr Ile Val Ile Arg Arg Lys Ser Leu 85 9~ 95 Gln Ser Lys Glu Thr Thr Ala Ala G1y Asp Ser '-~~ 105
Claims (9)
1. Nucleic acid which can be used to express an IL-16 mutant in a prokaryotic or eukaryotic host cell, wherein the said nucleic acid consisting of a sequence which codes for a polypeptide one of the group of peptides beginning with one of the amino acids 1-5 and ending with one of the amino acids 93-107 of the sequence SEQ ID
NO:2.
NO:2.
2. Nucleic acid as claimed in claim 1, which codes for IL-16 mutants of amino acids 1-100, 5-107 or 5-93 of SEQ ID NO:2.
3. Prokaryotic or eukaryotic host cell which is transformed or transfected with a nucleic acid as claimed in claim 1 or 2 in such a way that the host cell expresses the said IL-16 mutant.
4. Biologically functional nucleic acid vector that contains a nucleic acid as claimed in claim 1 or 2.
5. IL-16 mutant as it can be obtained as the product of a eukaryotic or prokaryotic expression of a nucleic acid as claimed in claim 1 or 2 which is essentially free of other human proteins.
6. IL-16 mutant as claimed in claim 5, consisting of amino acids 1-107 of SEQ
ID NO:2.
ID NO:2.
7. Process for the production of an IL-16 mutant as claimed in claim 5 or 6, wherein a prokaryotic or eukaryotic host cell which is transformed or transfected with a nucleic acid sequence as claimed in claim 1 or 2 is cultured under suitable nutrient conditions and the desired polypeptide is optionally isolated.
8. Pharmaceutical composition which contains an IL-16 mutant as claimed in claim 5 or 6 as well as a pharmaceutically suitable diluent, adjuvant and/or carrier.
9. Pharmaceutical composition containing an IL-16 mutant as claimed in claim 5 or 6 in an amount adequate for a therapeutic application.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP98101232.1 | 1998-01-24 | ||
| EP98101232 | 1998-01-24 | ||
| PCT/EP1999/000428 WO1999037781A1 (en) | 1998-01-24 | 1999-01-22 | Mutants of il-16, processes for their production, and their use |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2320624A1 true CA2320624A1 (en) | 1999-07-29 |
Family
ID=8231307
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002320624A Abandoned CA2320624A1 (en) | 1998-01-24 | 1999-01-22 | Mutants of il-16, processes for their production, and their use |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP1049782A1 (en) |
| JP (1) | JP2002500889A (en) |
| AU (1) | AU2424399A (en) |
| BR (1) | BR9907216A (en) |
| CA (1) | CA2320624A1 (en) |
| EA (1) | EA200000769A1 (en) |
| MX (1) | MXPA00006059A (en) |
| WO (1) | WO1999037781A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6699466B1 (en) | 1999-08-05 | 2004-03-02 | Research Corporation Technologies, Inc. | IL-16 antagonist peptides and DNA encoding the peptides |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ATE419356T1 (en) * | 1993-05-21 | 2009-01-15 | Univ Boston | CHEMOATTRACTION TRIGGERING FACTOR FROM LYMPHOCYTES AND ITS USE |
| AU1301797A (en) * | 1995-12-22 | 1997-07-17 | Bundesrepublik Deutschland Vertreten Durch Den Bundesminister Fur Gesundheit | Polypeptides with interleukin 16 activity, process for the preparation and use thereof |
| DE19617202A1 (en) * | 1996-04-30 | 1997-11-06 | Boehringer Mannheim Gmbh | Recombinant interleukin 16 |
| US6444202B1 (en) * | 1996-11-25 | 2002-09-03 | Bundesrepublic Deutschland, Vertreten Durch Den Bundesminister Fur Gesundheit | Processed polypeptides with IL-16 activity, processes for their production and their use |
-
1999
- 1999-01-22 BR BR9907216-5A patent/BR9907216A/en not_active IP Right Cessation
- 1999-01-22 CA CA002320624A patent/CA2320624A1/en not_active Abandoned
- 1999-01-22 AU AU24243/99A patent/AU2424399A/en not_active Abandoned
- 1999-01-22 WO PCT/EP1999/000428 patent/WO1999037781A1/en not_active Ceased
- 1999-01-22 EA EA200000769A patent/EA200000769A1/en unknown
- 1999-01-22 MX MXPA00006059A patent/MXPA00006059A/en unknown
- 1999-01-22 JP JP2000528688A patent/JP2002500889A/en not_active Ceased
- 1999-01-22 EP EP99903675A patent/EP1049782A1/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| MXPA00006059A (en) | 2003-08-01 |
| EP1049782A1 (en) | 2000-11-08 |
| JP2002500889A (en) | 2002-01-15 |
| WO1999037781A1 (en) | 1999-07-29 |
| BR9907216A (en) | 2000-10-24 |
| AU2424399A (en) | 1999-08-09 |
| EA200000769A1 (en) | 2001-02-26 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |