CA2240392C - Multimer forms of interleukin-16 (il-16), process for the preparation and use thereof - Google Patents

Abstract

A multimeric form of IL-16 which optionally contains metal ions in a defined amount, is more active than the known IL-16.

Description

pei 4166 Multimeric forms of IL-16, processes for their production and their use The invention concerns multimeric forms of polypeptides with IL-16 activity, processes for their production and their use.
IL-16 (interleukin-16) is a lymphokine which is also referred to as lymphocyte chemoattracting factor (LCF) or immunodeficiency virus suppressing lymphokine (ISL).
ISL and its properties are described in WO 94/28134, W096/31607 and by Cruikshank, W.W., et al., Proc. Natl.
Acad. Sci. USA 91 (1994) 5109-5113 and by Baier, M., et al., Nature 378 (1995) 563. The recombinant production of IL-16 is also described in these references.
According to these monomeric IL-16 is a protein with a molecular mass of 13,385 D. Cruikshank also found that ISL eluted in a molecular sieve chromatography as a multimeric form with a molecular weight of 50-60, and 55-60 kD. The chemoattractant activity has been attributed to this multimeric form. A homodimeric form of IL-16 with a molecular weight of 28 kD is described by Baier. However, the chemoattractant activity described by Cruikshank et al. in J. Immunol. 146 (1991) 2928-2934 and the activity of recombinant human IL-16 described by Baier are very small.
The object of the present invention is to improve the activity of IL-16 and to reproducibly provide IL-16 forms which are advantageously suitable for a therapeutic application.
The object of the invention is achieved by a biologically active multimeric form of IL-16 subunits with a molecular weight of at least ca. 70 kD (measured with gel filtration HPLC) and/or with a defined amount of metal ions.
It has surprisingly turned out that a multimeric form of IL-16 subunits which contains more than four subunits has considerably more IL-16 activity than monomeric, dimeric or tetrameric forms. The molecular weight of these multimers differs significantly from the forms of IL-16 described by Baier et al and Cruikshank et al.
It was surprisingly found that a dimeric form of IL-16 with a molecular weight of 28 kD which was described by Baier et al. as being inactive can be converted by an incubation with metal ions into a higher molecular active form with a molecular weight of 45 ~ 4 kD which is referred to as the tetramer in the following to distinguish it from the dimers.
The production of this tetrameric form of IL-16 by addition of metal ions to solutions containing IL-16 as well as the tetrameric form of IL-16 with a molecular weight of at least 45 ~ 4 kD (depending on the molecular weight of the subunit) obtainable in this manner are a further subject matter of this invention.
Furthermore it was found that the tetrameric form of IL-16 can be converted into a polymeric of IL-16 with a molecular weight of at least ca. 70 kD which is also a subject matter of the invention.

A multimeric form of IL-16 with a molecular weight of at least ca. 70 kD preferably contains a defined number of subunits wherein the number of subunits in such a multimeric form is more than six, preferably between 6 and 32, and particularly preferably between 8 and 16.
Multimeric forms of IL-16 with 8 or 16 subunits and defined mixtures thereof are especially preferred.
It has also turned out that the activity of IL-16 can be further increased by the presence of metal ions. In this conrnection it is preferred that a preparation of an active monomeric or multimeric form of IL-16 subunits contains metal ions at a molar concentration of at least 50 % of the molar concentration of the IL-16 subunits present in the solution. The proportion of metal ions per subunit is preferably between 0.5 and 2, and particularly preferably between 0.5 and 1. Also in this case it is preferred that the IL-16 form according to the invention has a defined content of metal ions in the said ranges.
Preferred preparations for this are:
- IL-16 subunit with a molecular weight of ca.
13-35 kD containing one metal ion per subunit, - dimer of IL-16 subunits containing one or two metal ions, - tetramer of IL-16 subunits containing two or four metal ions.
A preparation of IL-16 within the sense of the invention is for example an aqueous, preferably buffered, solution or a lyophilisate. The preparation is preferably suitable for a therapeutic application or for the production of a pharmaceutical agent. In this case the concentration of IL-16 is in a therapeutically effective range. The preparation can additionally contain auxiliary substances and in particular pharmaceutical auxiliary substances such as solubilizers, fillers etc..
A biological activity of IL-16 is understood as its property of binding to T cells via the CD4 receptor and preferably to suppress the replication of HIV and SIV as described in the international Application WO 96/31607 ~'whi~h for this purpose is a subject matter of the disclosure of this present invention.
The term IL-16 is understood within the sense of the invention as a polypeptide with the activity of IL-16.
In a preferred embodiment IL-16 has an immunomodulating activity as described in WO 94/28134 and which for this purpose is a subject matter of the present invention.
The immunomodulating property can be determined via stimulation of cell division with IL-16, with a growth factor such as IL-2 or with anti-CD3 antibodies. Such a method is described in WO 94/28134.
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.
An IL-16 subunit (IL-16 momomer) is understood as a polypeptide which has IL-16 activity after multimerization and a) is coded by a DNA sequence according to SEQ ID NO:1 or a complementary sequence, b) is coded by DNA sequences which hybridize with SEQ ID NO:1 under stringent conditions.
The polypeptide preferably exhibits the stated action in the test procedure described in the international Application WO 96/31607 or stimulates cell division according to WO 94/28134.
The sequence of an IL-16 subunit can differ to a certain extent from protein sequences coded by such DNA
sequences. Such sequence variations may be amino acid substitutions, deletions or additions. However, the amino acid sequence of the IL-16 subunit is preferably at least 75 % and particularly preferably at least 90 identical to SEQ ID NO:1 and the active region of IL-16 contained therein. This region is N-terminally and/or C-terminally truncated compared to SEQ ID NO:1. The molecular weight of a subunit is preferably ca. 13-35 kD.
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 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 selected 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.
Numerous metal ions are suitable as metal ions within the sense of the invention. It has turned out that alkaline earth metals as well as elements of the side groups are suitable. Alkaline earth metals, cobalt, zinc, selenium, manganese, nickel, copper, iron, magnesium, calcium, molybdenum and silver are particularly suitable. The ions may be monovalent, divalent, trivalent or tetravalent. Divalent ions are particularly preferred, especially Cu(II) ions. The ions are preferably added as solutions of MgCl2, CaCl2, MnCl2, BaCl2, LiCl2, Sr(N03)2, Na2Mo04, AgCl2, Cu(II) acetate IL-16 is preferably produced recombinantly in prokaryotic or eukaryotic host cells. Such production processes are described for example in WO 94/28134 and 7 which are also for this purpose a subject matter of the disclosure of the present invention.
However, in order to obtain the forms according to the invention of IL-16 by recombinant production in a defined and reproducible manner, additional measures have to be taken beyond the processes for recombinant production familiar to a person skilled in the art.

The invention therefore concerns a process for the production of a biologically active multimeric form of IL-16 by expression of a nucleic acid coding for IL-16 in a prokaryotic or eukaryotic host cell and isolation of the said multimeric form characterized in that during the production or purification at least 0.25 metal ions, preferably at least 0.5 metal ions per IL-16 subunit are present and catalyse the defined oligomerisation of IL-_ 16. In the production process a mixture of several multimeric IL-16 forms may also be formed. This mixture can for example be used therapeutically, preferably after purification, either when separated into its individual constituents or as a mixture. The process according to the invention surprisingly enables polypeptides with IL-16 activity to be multimerized in a defined and reproducible manner. In this process IL-16 is obtained in a defined degree of oligomerisation depending on the reaction conditions (e. g. temperature, pH value etc.).
The addition of metal ions can take place during the fermentation or during the purification. It has turned out that IL-16 forms according to the invention are obtained when about 0.1 ~mol/1 to 10 mmol/1 metal ions are present during the fermentation per gram of recombinant IL-16 being formed. In this connection the upper limit of the metal ion concentration is not critical and merely depends on the ability of the microorganisms or cell lines to tolerate these metal ions and on the solubility of the metal compound or salt used. A metal ion concentration of 0.5 ~mol/1 to mmol/1 is preferably used. An increase in the yield of active metalloproteins by the addition of metal ions to the fermentation medium is described for example by Hoffman et al., in Protein Expression & Purific. 6 -(1995) 646-654.
The addition of metal ions or metal compounds from which metal ions can be detached preferably takes place during lysis of the fermentation preparation, during the purification or at the stage of purified IL-16. It is expedient to use the metal ions before or during a dialysis or a chromatographic step in an application or elution buffer.
A preparation of a multimeric form of IL-16 subunits is obtained in the recombinant production of the IL-16 forms according to the invention in which the number of subunits is preferably 6 to 32 and which is substantially free of mammalian cell proteins as a product of a recombinant production in prokaryotes or is substantially free of natural human proteins as a product of a recombinant production in eukaryotes.
A further embodiment of the invention concerns a process for the production of multimeric IL-16 subunits by incubating monomeric IL-16 subunits or IL-16 dimers with metal ions. Such a process can be used regardless of whether the IL-16 subunits have been isolated in a recombinant manner or by other means (e. g. synthetically or from natural sources).
The invention furthermore concerns a process for increasing the degree of multimerization of a monomeric or dimeric form of IL-16 subunits. In such a process the monomeric or dimeric form is incubated with metal ions by which means one or several higher multimerized forms of IL-16 subunits are formed.

The multimerization is preferably carried out in a subalkaline, neutral or acidic range preferably between pH 3 and 9 particularly preferably in a range between pH 3 and 8. The yield of multimeric forms and the duration of the multimerization can be improved by adding denaturing agents such as guanidine hydrochloride or urea. It is expedient to add such denaturing agents at concentrations of 0 to 8 mol/1. In the incubation with metal ions the concentration of denaturing agent can be reduced preferably to a non-denaturing concentration (fir guanidine hydrochloride e.g, ca. 0 to 2 mol/1) by dilution or dialysis.
The metal ion-dependent multimerization can be inhibited by chelating agents such as e.g. EDTA or by free binding sites for metal ions on the metal affinity chelate Sepharose used for the purification.
A particularly efficient tetramerization is achieved in a short time when metal ions are incubated with IL-16 in an approximately equimolar concentration or in an excess with respect to IL-16, the reaction rate depending on the concentration of the reactants. Therefore when the metal ion concentration is low it must be incubated for a correspondingly longer time. The tetramerization is carried out in a broad pH range, in particular in a range of pH 2.5 to 10. Following the tetramerization the further multimerization can be preferably carried out between pH 5 to 7 or 7.5 even without addition of further metal ions.
IL-16 multimers that elute as a discrete peak from an analytical molecular sieve HPLC column with an apparent molecular weight of ca. above 70 kD, preferably 90 to 150 kD, are particularly preferred. The polymer formation can be induced by a prior metal ion-dependent tetramerization. In addition the degree of multimerization is influenced by the respective buffer composition (type of buffer, e.g. imidazole, MES, acetate, glycine, citrate, pH value, GdmCl cone ).
A further preferred embodiment of biologically active IL-16 has a molecular weight of at least 45 + 4 kD and is obtainable by incubation of monomers or dimers with metal ions.
Such IL-16 preparations are particularly suitable for therapeutic use in therapeutic compositions. Such compositions advantageously also contain the usual fillers, auxiliary substances and additives.
The following examples and publications, the sequence protocol and the figures further elucidate the invention the scope of which results from the patent claims. The described processes are to be understood as examples which still describe the subject matter of the invention even after modification.
Fig.i: SDS-PAGE of IL-16 under reducing conditions (sample with 0.1 M DTE) 1) before cleavage with thrombin, 2) after cleavage with thrombin and 3)-5) after cleavage and purification by means of Q-Sepharose~(various fractions).
Fig.2: RP-HPLC elution diagram of IL-16 after cleavage with thrombin and purification by means of Q-Sepharose~
Fig.3A, Fig.3B: Molecular sieve HPLC elution diagram *Trademark A) of the IL-16 fusion protein before cleavage with thrombin, B) of IL-16 after cleavage with thrombin and purification by means of Q-Sepharose.
Fig.4: Molecular sieve HPLC elution diagram of dimeric (RT = 11.4 min) and tetrameric (RT = 10.06 min) IL-16 after renaturation of denatured IL-16 in 0.1 M Tris/HC1, 250 ~M Cu(II)acetate, pH 8.5 (see example 4).
Fig. S: Calibration run of the HPLC sieve column using BSA (MW 66kD; RT 9..17 min), ovalbumin (MW 43kD;
RT 9.98 min), chymotrypsinogen (MW 25kD, RT
11.89 min) and ribonuclease A (MW 13.7 kD;
RT 12.87 min).
Fig.6: Molecular sieve HPLC elution diagram of tetrameric IL-16 after incubation of 137 ;CM
IL-16 with 250 ACM Cu(II) acetate (see example 10).
Fig.7: Molecular sieve HPLC elution diagram of dimers (3 %; RT 11.66 min), tetramers (32 %; RT 10.30 min) and polymers (65 %; RT 7.93 min) after incubation of IL-16 for 16 hours in 50 mM MES, 250 ACM Cu(II)acetate, pH 5.5.
SEQ ID NO:1 shows the sequence of human IL-16.
SEQ ID N0:2 shows the amino acid sequence according to SEQ ID NO:1.
SEQ ID N0:3-8 show primer sequences.
SEQ ID N0:9-li show peptide sequences.

Example 1 Cloning and expression of IL-16 1.1 RNA isolation x 10~ PBMC (from humans or monkeys) were cultured for 48 hours with 10 ug/ml concanavalin A and 180 U/ml IL-2.
In order to prepare the RNA, the cells were washed once with PBS and subsequently lysed with 5 ml denaturation solution (RNA isolation kit, Stratagene)~ The lysate was kept on ice for 15 min after addition of 1 ml Na acetate, 5 ml phenol and 1 ml chloroform/isoamyl alcohol (24:1). The aqueous phase was subsequently mixed with 6 ml isopropanol in order to precipitate the RNA and stored for 2 hours at -20°C. The precipitate was finally washed once with pure ethanol and dissolved in 150 ~1 H2o. The yield was determined photometrically and was 120 ~lg.
1.2 cDNA synthesis The mixture for the cDNA synthesis contained 10 ~,g RNA, 0'.2 ~Cg oligo-dT, 13 mM DTT and 5 ~C1 bulk first strand reaction mix (first strand cDNA synthesis kit, Pharmacia) in an amount of 15 ~1. The mixture was incubated for 1 hour at 37°C and subsequently stored at -20°C for later use.
1.3 Amplification and cloning of IL-16 cDNA
The following oligonucleotides were synthesized for the amplification of IL-16 cDNA by means of PCR and for the subsequent cloning:
Primer 1: GCTGCCTCTCATATGGACCTCAACTCCTCCACTGACTCT (SEQ ID N0:3) *Trademark Primer 2: GATGGACAGGGATCCCTAGGAGTCTCCAGCAGCTGTGG (SEQ ID N0:4) The primers introduce additional NdeI or BamHI cleavage sites.
The PCR mixtures (100 ~C1 reaction volumes) each contained 1 ~1 cDNA (from the synthesis in section 3), 50 pmol primer 1 and 2, 12.5 ~mol dNTPs, 10 ~,1 lOxTAQ
buffer and 2.5 units Taq polymerase (Perkin-Elmer).
The cycle conditions were 30 sec., 94°C, l min, 53°C and 1 min, 72°C. 35 cycles were carried out.
1.4 Preparation of an expression clone with thrombin cleavage sites The PCR products were purified and digested for 16 hours at 37°C with NdeI and BamHI. For the cloning preparation the vector pETl5b (Novagen) was also cleaved with NdeI
and BamHI and subsequently purified on Agarose gel.
The ligations were carried out for 2 hours at room temperature in 20 ~C1 mixtures that contained 100 ng vector, 25 ng PCR product (insert), 2 ~1 10 x ligase buffer and 0.2 ~,1 ligase (New England Biolabs). After transformation by electroporation at 2.5 kV, 25 ~ Farad, 200 ohm (BIO-RAD electroporator) into E. coli, the cells were placed on ampicillin-resistant plates. Suitable E. coli, e.g. DH5, are known to a person skilled in the art.
Recombinant clones were identified by restriction analysis of plasmid preparations (pMISLB) and transformed into an E. coli strain for the desired protein expression. It was possible to additionally confirm the cloning of IL-16 cDNA by determining the nucleotide sequences. The sequences found correspond to the. published LCF sequence (Cruikshank, W.W., et al., Proc. Natl. Acad. Sci. USA 91 (1994) 5109-5113) except for a discrepancy at codon 96. In contrast to the published sequence codon 96 does not consist of the base sequence TTG but of the sequence TTT. Sequencing of further IL-16 clones, which were obtained from independent PCR amplifications, clearly showed that the authentic IL-l6 sequence in codon 96 is in reality represented by the sequence TTT.
1.5 Production of expression clones for shortened IL-16 Cloning example IL-16:
The following oligonucleotides were synthesized for the amplification of IL-16 cDNA and for subsequent expression cloning:
Primer ISL1: (SEQ ID N0:5) ccc gaa ttc tat gca tca cca cca cca cca cga tga cga cga caa acc cga cct caa ctc ctc cac t Primer ISL2: (SEQ ID N0:6) ccc gaa ttc tat gcc cga cct caa ctc ctc c Primer ISL3: (SEQ ID N0:7) ccc gaa ttc tat gca tca cca cca cca cca cga tga cga cga caa aat gcc cga cct caa ctc ctc c Primer ISL4: (SEQ ID N0:8) gcg gat cca agc tta gga gtc tcc agc agc tgt After PCR on the IL-16 gene primer ISL1 inserts an EcoRI
cleavage site as well as a "t" to produce lacZ fusions in pUC, 6 histidine codons and the codons for an enterokinase cleavage site (DDDDK; SEQ ID N0:9).
After PCR on the IL-16 gene primer ISL2 inserts an EcoRI
cleavage site as well as a "t" to produce lacZ fusions in pUC.
Primer ISL3 inserts the same properties as primer ISL1 and additionally a further ATG after the AAA (Lys) codon.
Primer ISL4 is the counter-primer to ISL1 to ISL3, it inserts a BamHI as well as a HindIII cleavage site at the 3' end of the IL-16 gene.
It is possible to express various types of IL-16 in E. coli by suitable combination of the above primers and cloning the PCR products into suitable vectors:
A combination of ISL1 and ISL4, results for example after PCR, recleaving the product with EcoRI and HindIII
and cloning behind for example a lac promoter with expression, in an IL-16 which when expressed contains 6 N-terminal histidines and an enterokinase cleavage site and yields mature IL-16 without an N-terminal Met after processing and cleaving with enterokinase (N-terminus PDLNS; SEQ ID NO:10).
A combination of ISL2 and ISL4 directly,yields a mature IL-16 after expression which starts with the sequence MPDLNS,.(SEQ ID NO:11) after PCR, recleaving the product with EcoRI and HindIII and cloning behind for example a lac promoter.
A combination of ISL3 and ISL4 yields a mature IL-16 after expression and cleavage with enterokinase which starts with the N-terminal sequence MPDLNS,.(SEQ ID
NO:11) after PCR, recleaving the product with EcoRI and HindIII and cloning behind for example a lac promoter.
A lacZ fusion can be formed depending on the plasmid used (e. g. when cloning in pUC plasmids).
It is obvious that any promoter that functions well in E. coli can be used in addition to the lac promoter.
Examples would be e.g. the tac promoter or even the mgl promoter. Low-copy as well as high-copy plasmids come into consideration as plasmids.
Example 2 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 1-10 vol. % in each case. Ampicillin (50-100 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 extract/C sources dosage. The fermentation temperature is 25-37°C. The dissolved partial oxygen pressure (p02) is kept 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 ca. 10 hours the biomass is harvested by centrifugation at OD standstill. The biomass is taken up in 50 mM sodium phosphate, 5 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 further.
Example 3 Purification of recombinant IL-16 550 ml lysis supernatant in 50 mM sodium phosphate, 5 mM
EDTA, 100 mM NaCl, pH 7.2 was admixed with 55 ml 5 M
NaCl, 60 mM MgCl2, pH 8.0, stirred for 30 minutes and subsequently centrifuged for 30 minutes at 20,000 g.
400 ml of the supernatant was taken up on a nickel-chelate Sepharose column (V = 6o ml; Pharmacia), which had previously been loaded with 30 ~Mol NiCl2/ml gel and equilibrated with 50 mM sodium phosphate, 0.2 M NaCl, pH 8Ø The column was subsequently washed with 300 ml 50 mM sodium phosphate, 0.5 M NaCl, pH 7.0 and the IL-16 fusion protein was then eluted with a gradient of 0 M to 300 mM imidazole, pH 7.0 in 50 mM sodium phosphate, 0.1 M NaCl, pH 7.0 (2* 0.5 1 gradient volumes).
Fractions containing IL-16 were identified by means of SDS-PAGE and pooled.
300 mg of the fusion protein obtained in this way was dialysed at 4°C against 20 1 20 mM imidazole, pH 5.5 and subsequently centrifuged for 30.minutes at 20,000 g in order to remove turbidities. The supernatant of the centrifugation was subsequently adjusted to pH 8.5 with NaOH, admixed with 0.3 g thrombin (Boehringer Mannheim GmbH) and incubated for 4 hours at 37°C. Subsequently the cleavage mixture was adjusted to pH 6.5 with HC1 and the conductivity was set to 1.7 mS by dilution with H20.
The sample was applied to a Q-Sepharose FF column (45 ml; Pharmacia) which had previously been equilibrated with 20 mM imidazole, pH 6.5. IL-16 was eluted using a gradient of 0 to 0.3 N NaCl in 20 mM
imidazole, pH 6.5. Fractions containing IL-16 were identified by means of SDS-PAGE and pooled. The identity of IL-16 was confirmed by means of mass analysis (molecular weight 13,566 ~ 3 D) and automated N-terminal sequence analysis. In order to determine the concentration the W absorbance of IL-16 was determined at 280 nm and a calculated molar extinction coefficient of 5540 M-1 cm'1 at this wave-length (Mack et al. (1992) Analyt.~Biochem. 200, 74-80) and a molecular weight of 13,566 D was used.
The IL-16 obtained in this manner had a purity of more than 95 % in SDS-PAGE under reducing conditions.
IL-16 eluted from an analytical HPLC gel filtration column (SuperdeX HR 75; Pharmacia) which had been calibrated with BSA, ovalbumin, chymotrypsinogen and ribonuclease A, with an apparent molecular weight of ca.
27,000 D (see Fig. 3) which is why this molecular *Trademark species is denoted "dimer" in the following.
The analytical Superdex 75 FPLC column (Pharmacia) was eluted with 25 mM Na phosphate, 0.5 M NaCl, 10 %
glycerol, pH 7.0 and at a flow rate of 1 ml/min. The amount of protein applied in a volume of 100 to 150 ~cl was 1.5 to 15 ~g protein. Detection was at 220 nm.
A Vydac Protein & Peptide C18, 4x180 mm column was used to analyse purity by means of RP-HPLC. It was eluted with a linear gradient of 0 % to 80 % B (solvent B: 90 %
acetonitrile in 0.1 % TFA; solvent A: 0.1 % TFA in HZO) within 30 minutes at a flow rate of 1 ml/min. Detection was at 220 nm.
Since Cruikshank W.W. et al., in Proc. Natl, Acad. Sci.
USA 91 (1994) 5109-5113 described a polymeric form of IL-16 with an apparent molecular weight of 50-60 kD as an active molecular species, it was attempted to convert the dimeric form into a polymeric form by denaturation and renaturation experiments using the highly pure IL-16 isolated in this way.
Example 4 Denaturation and renaturation of LL-16 (a) Influence of the pH value, EDTA and copper(II) acetate on the renaturation/tetramerization of IL-16 37 mg IL-16 was dialysed against 20 mM sodium phosphate, pH 7.0 and subsequently concentrated to a concentration of 7.1 mg/ml. IL-16 was denatured by adding 1 ml 8 M
GdmCl, 0.1 M glycine/HC1, 2 mM EDTA, 1 mM DTE, pH 1.8 to *Trademark 0.5 ml of the IL-16 concentrate. After incubating for 1 hour 100 ~1 aliquots of this solution were diluted in 2.2 ml of the renaturation buffers described in Table 1 at room temperature (23+/-3°C). The formation of polymeric IL-16 was analysed after an incubation of at least 2 hours by means of molecular sieve HPLC.
As can be seen from Table 1 no higher molecular IL-16 species were formed independent of the pH value when the renaturation was carried out in buffers which had previously been gassed with N2 and contained EDTA.
Higher molecular IL-16 associates were surprisingly only formed in buffers without EDTA and without nitrogen gassing and in particular in the presence of copper acetate. The higher molecular form elutes as a homogeneous peak from an analytical molecular sieve HPLC
column (see Fig. 4) and has an apparent molecular weight of ca. 45 + 4 kD. In order to differentiate it from the "dimeric" form this molecular species is referred to as "tetramer" in the following.
Since copper ions are also used for the oxidative production of disulfide bridges, the influence of redox systems on tetramerization were examined in more detail in the following.

Table 1 Influence of the pH value, EDTA and copper(II)acetate on the renaturation of IL-16 No. Renaturation buffer Dimer Tetramer L ~l L ~l 1 20 mM Tris/HC1, pH 8.0, 1 mM EDTA 100 0 2 20 mM Na phosphate, pH 7, 1 mM EDTA 100 0 3 20 mM Na phosphate, pH 7.0, 1 mM EDTA, 100 0 150 mM NaCl 4 20 mM Na phosphate, pH 6.0, 1 mM EDTA 100 0 150 mM NaCl 20 mM Na phosphate, pH 5.0, 1 mM EDTA 100 0 150 mM NaCl 6 20 mM Na phosphate, pH 4.0, 1 mM EDTA 100 0 150 mM NaCl 7 0.1 M Tris/HC1, pH 8.5 88 12 8 0.1 M Tris/HC1, pH 8.5, 64 36 250 ~M Cu(II) acetate (b) Influence of denaturation, of redox systems and of metal ions on the "tetramerization" of IL-16 400 ~C1 of the concentrate described in example 1 at a concentration of 7.1 mg/ml was diluted in 1) 800 ~,1 8.0 M GdmCl, 0.1 M glycine/HC1, 1 mM DTE, pH 1.8 (=denatured IL-16) or 2) 800 ~C1 20 mM sodium phosphate, pH 7.0 (=native IL-16) and incubated for 1 hour. 100 ~C1 of each of these dilutions was subsequently diluted in 2.2 ml of an aqueous solution of 0.1 M Tris/HC1, pH 8.5 and the additives described in Table 2, and the buffers in which the "native IL-16" from 2) were diluted additionally contained 0.23 M GdmCl so that all renaturation solutions had identical GdmCl concentrations.
As shown in Table 2, redox reactions do not seem to have any influence on the tetramerization since the usual redox systems for proteins comprising GSH and GSSG do not have any effect. Moreover metal ions seem to be essential for stabilizing the tetramer since magnesium and calcium ions can also induce tetramerization in addition to copper ions. Denaturation of IL-16 by high concentrations of denaturing agent is obviously not necessary for tetramerization since this also occurs without previous denaturation of IL-16.
As would be expected in an adjustment of equilibrium and also in an association reaction of the second order, the yield of tetrameric IL-16 increases further as the concentration of metal ions increases as can be seen for Cu acetate in Table 2.

Table 2 Influence of denaturation, redox systems and metal ions on the tetramerization of IL-16 No. IL-16 Dilution buffer % Tetramer 1 denatl~ 0.1 M Tris/HC1, pH 8.5 without 0 additives 2 native2> 0.1 M Tris/HC1, pH 8.5 without 0 additives 3 denat. 0.1 M Tris/HC1, pH 8.5, 42 250 uM Cu(II) acetate 4 native 0.1 M Tris/HC1, pH 8.5, 45 2 5 0 ACM Cu ( I I ) acetate denat. 0.1 M Tris/HC1, pH 8.5, 56 750 ~M Cu(II) acetate 6 native 0.1 M Tris/HC1, pH 8.5, 60 750 ~M Cu(II) acetate 7 denat. 0.1 M Tris/HC1, pH 9.5, 49 2 5 0 ACM Cu ( I I ) acetate 8 native 0.1 M Tris/HC1, pH 9.5, 60 250 ~M Cu(II) acetate 9 denat. 20 mM Na phosphate, pH 6.0, 65 250 ~M Cu(II) acetate native 20 mM Na phosphate, pH 6.0, 56 250 ~M Cu(II) acetate 11 denat. 0.1 M Tris/HC1, pH 8.5, 750 uM 34 CaCl2 12 native 0.1 M Tris/HC1, pH 8.5, 750 ~M 38 CaCl2 13 denat. 0.1 M Tris/HC1, pH 8.5, 750 ~,M 38 MgCl2 14 native 0.1 M Tris/HC1, pH 8.5, 750 ACM 42 MgC 12 No. IL-16 Dilution buffer % Tetramer 15 denat. 0.1 M Tris/HC1, pH 8.5 0 0.5 mM GSH, 1 mEDTA

16 native 0.1 M Tris/HCl, pH 8.5 0 0.5 mM GSH, 1 mM EDTA

17 denat. 0.1 M Tris/HC1, pH 8.5 0 0.5 mM GSH, 5.0 mM GSSG

18 native 0.1 M Tris/HC1, pH 8.5 p 0.5 mM GSH, 5.0 mM GSSG

19 denat. 0.1 M Tris/HC1, pH 8.5 0 5.0 mM GSSG, 1 mM EDTA

20 native 0.1 M Tris/HC1, pH 8.5 p 5.0 mM GSSG, 1 mM EDTA

The intermediate dilution of the IL-16 stock solution was in 8 M GdmCl.
2> The intermediate dilution of the IL-16 stock solution was in 20 mM sodium phosphate, pH 7.0 Example 5 Influence of GdmCl on tetramerization Since a complete denaturation of IL-16 is apparently not necessary for the tetramerization, the influence of low GdmCl concentrations was examined. For this the IL-16 concentrate described in example 4a was diluted 71-fold in 0.1 M Tris/HC1, pH 8.5 that additionally contained the additives described in Table 3.
As can be seen in Table 3, the tetramerization can be supported at low IL-16 concentrations by addition of non-denaturing concentrations of denaturation agents for various metal ions.
Table 3 Influence of GdmCl on the tetramerization of IL-16 by metal ions No. Buffer M Tris/HC1, pH 8.5 % Tetramers additive to 0.1 1 without addition 0 2 0.23M GdmCl 0 3 250 ~M MgCl2 0 4 250 ~M MgCl2 0.23 GdmCl 10 + M

250 ACMCaCl2 0 6 250 ~M CaCl2 0.23 GdmCl 34 + M

7 2 ACMMnC 12 0 8 250 ~M MnCl2 0.23 GdmCl 10 + M

9 250 ~M Cu(II) acetate 48 250 ACMCu(II) acetate + 0.125 M GdmCl ~2 11 250 ~,MCu(II) acetate + 0.25 M GdmCl 83 12 250 ~M Cu(II) acetate + 0.50 M GdmCl 92 13 250 ~M Cu(II) acetate + 1.0 M GdmCl 94 14 250 ACMCu(II) acetate + 2.0 M GdmCl 92 Example 6 Kinetics of the tetramerization of IL-16 with Cu(II) acetate Since the tetramerization of IL-16 is a reaction of at least a second or higher order, the rate of the tetramerization in the presence of Cu(II) acetate was examined.
For this 20 ~1 of an IL-16 concentrate described in example 4a was diluted in 800 ~1 0.1 M Tris/HC1, 250 ~,M
Cu(II) acetate, 0.25 M GdmCl, pH 8.5 and samples were examined for their content of tetramers after various incubation periods by means of an analytical molecular sieve HPLC column. As can be seen in Table 4 tetramerization is a relatively slow reaction the rate of which, however, depends on the concentration of the reactants.
Table 4 Kinetics of the tetramerization of IL-16 with Cu(II) acetate Incubation period (min) Tetramers (%) min 12 60 min 75 150 min 81 ' Example 7 Influence of EDTA on the tetramerization of IL-16 by Cu(II) acetate EDTA forms a high affinity complex with divalent metal ions. The inhibition of tetramerization by EDTA should therefore prove that this is induced by divalent metal ions.
For this an IL-16 concentrate described in example 4a - 2? -was diluted 40-fold in buffers which were composed of 0.1 M Tris/HC1, 0.25 M GdmCl, 250 ACM Cu(II) acetate, pH
8.5 and increasing concentrations of EDTA. The content of tetramers was again determined by means of a molecular sieve HPLC after an incubation period of at least one hour.
As the results in Table 5 show, equimolar concentrations of EDTA relative to Cu(II) acetate are really able to inhibit the tetramerization of IL-16.
Table 5 Inhibition of the tetramerization of IL-16 by EDTA
EDTA concentration [ACM] Tetramers (%) Example 8 a) Influence of the concentration of Cu(II) acetate on the tetramerization of IL-16 with a short incubation period If IL-16 is a metalloprotein, then it would be expected that stoichiometric amounts with respect to the monomer or dimer would be necessary to stabilize the tetrameric form. In contrast smaller amounts should be sufficient for a catalytic function of Cu2+ ions e.g. in the case of an oxidation of IL-16 under optimal conditions.
An IL-16 concentrate described in example 4a was diluted to a concentration of 15 ~cM in buffer which contained 50 mM MES, 250 mM GdmCl, pH 6.5 and in addition increasing concentrations of Cu(II) acetate. Samples of these dilutions were examined for their content of tetramers after at least 9 hours incubation by means of molecular sieve HPLC.
As can be seen in Table 5a catalytic amounts of Cu(II) acetate are not sufficient under these conditions for a tetramerization.
Table 5a Cu(II) acetate concentration [~M] Tetramers 2.5 2 5.0 10.0 5 15.0 17 20.0 93 40.0 g7 80.0 g7 250.0 gg b) Influence of the concentration of Cu(II) acetate on the tetramerization of IL-16 with a long incubation period The IL-16 concentrate was diluted to a concentration of ~,M (0.14 mg/ml) in buffer which contained 50 mM
HEPES, 250 mM GdmCl, pH 7.5 and in addition increasing concentrations of Cu(II) acetate. Samples of these dilutions were examined for their content of tetramers after a ca. 100 hours incubation period by means of molecular sieve HPLC.
As can be seen in the following Table 5b catalytic amounts of Cu(II) acetate are adequate under these conditions for a tetramerization since the lowest concentration of Cu acetate (2.5.~CM) used in this case is much smaller than the IL-16 concentration (10 ACM) and nevertheless ca. 80% of the molecules are tetramerized.
Table Sb Cu(II) acetate concentration [~M] Tetramers (%) 2.5 83 5.0 84 7.5 88 12.5 85 15.0 g5 20.0 g7 Example 9 Influence of various metal ions on the tetramerization of IL-16 In order to get an impression of the specificity of the metal binding of IL-16 and of its affinity for various metal ions, an IL-16 concentrate described in example 4a was diluted to a concentration of 0.2 mg/ml in a buffer (0.1 M Tris/HC1, 0.23 M GdmCl, 1 mM EDTA, pH 8.5) which contained different metal salts at a concentration of 500 ACM in each case. The content of tetramers in the samples was examined by means of molecular sieve HPLC
ca. 3 hours after the dilution and after subsequent dialyses against buffer that contained no GdmCl (1st dialysis) or no metal salt (2nd dialysis).
As can be seen in Table 6 various metal ions are in principle able to induce the tetramerization of IL-16.
The lower yields of tetramers in comparison to Cu(II) acetate may be caused by the specific unphysiological buffer conditions and may be substantially higher under other conditions.

Table 6 Influence of various metal ions on the tetramerization of IL-16 Buffer additive Tetramer before Tetramer after dialysis [%] dialyses [%]

without addition 0 4 1 mM EDTA 0 0 MnCl2 4 11 CdCl2 0 0 BaCl2 10 22 LiCl2 10 15 Sr(N03)2 22 29 Na2Mo04 13 14 Cu ( I I ) acetate 85 72 CuCl2 87 not determined-Example 10 Tetramerization of IL-16 at high protein concentrations with and without GdmCl and stability of the complex For the preparative production of tetrameric IL-16, an IL-16 solution containing 1.85 mg IL-16/ml (137 ~M IL-16) in 20 mM imidazole, 150 mM NaCl, pH 6.5 is admixed with 250 ~M Cu(II) acetate in the presence and absence of 0.25 M GdmCl and the amount of tetramer is determined after 90 min by means of molecular sieve HPLC. As can be seen in Table 7 the tetramerization is almost complete under these conditions independent of the GdmCl concentration.
In a subsequent dialysis of these samples containing mainly tetramers against 20 mM Na phosphate, 150 mM
NaCl, pH 7.0 no significant decrease of tetramers could be observed.
Table 7 Tetramerization of IL-16 at high protein concentrations with aid without GdmCl Buffer additives Tetramers [%]

0.25 GdmCl + 250 ~M Cu(II) acetate 96 M

250 Cu(II) acetate (without GdmCl) 92 ACM

0.25 GdmCl (without Cu acetate) 0 M

Example li Dependency of the tetramerization and polymerization of IL-16 on the pH value in the presence of 0.25 GdmCl An IL-16 stock solution described in example 4a was diluted to a concentration of 0.2 mg/ml in buffers at various pH values which each contained 250 ~,M Cu(II) acetate and 0.25 M GdmCl.
The amount of tetramers was analysed by means of molecular sieve HPLC. In this process a new polymeric species of IL-16 was detected which is formed in the acidic pH range, elutes with an apparent molecular weight of ca. 100 kD and is referred to as "polymer" in the following in order to distinguish it from the dimeric and tetrameric form of IL-16. IL-16 can apparently be present in three discrete association states (dimer, tetramer and polymer) independent of the buffer conditions.
The tetramerization is essentially dependent on metal ions and has a broad pH optimum whereas the polymerization takes place in a relatively narrow pH
range.
Table 8 Dependency of the tetramerization and polymerization of IL-16 on the pH value in the presence of 0.25 GdmCl Buffer and pH value Tetramers [%] Polymers [%]

50 mM CAPS, pH 10.5 8 /

50 mM Tris/HC1, pH 9.5 85 /

50 mM Tris/HC1, pH 8.5 85 /

50 mM HEPES, pH 7.5 97 /

50 mM MES, pH 6.5 96 2 50 mM MES, pH 5.5 96 2 50 mM acetic acid, pH 4.0 55 40 IL50mM glycine/HC1, pH 2.0 I 8 ~ 62 Example 12 Dependency of the tetramerization and polymerization of IL-16 on the pH value in the absence of GdmCl Since it is apparent from the above examples that a tetramerization can also take place in the absence of GdmCl, the pH value-dependency of this reaction was investigated analogously to example 10 also in the absence of GdmCl.
As shown in Table 9 the pH range of the tetramerization is narrower under these conditions and above all polymers are formed almost solely at pH 5.5.
An increased yield of polymeric IL-16 of 65 % was achieved when the incubation of IL-16 was carried out in 50 mM MES, pH 5.5 at a protein concentration of 0.4 instead of the 0.2 mg/ml IL-16 used in Table 9 and when the incubation period before the analysis was increased by ca. 3 hours to 16 hours. Thus by optimizing the conditions polymeric IL-16 can be probably produced almost quantitatively.
Table 9 Dependency of the tetramerization and polymerization of IL-16 on the pH value in the absence of GdmCl Buffer and pH value Tetramers [%] Polymers [%]

50 mM CAPS, pH 10.5 0 50 mM Tris/HC1, 9.5 56 /
pH

50 mM Tris/HC1, 8.5 60 /
pH

50 mM HEPES, pH 91 /
7.5 50 mM imidazole, 6.5 83 /
pH

50 mM MES, pH 6.5 91 /

50 mM MES, pH 5.5 45 51 50 mM acetic acid, pH 4.0 92 /

L50mM glycine/HC1, pH 2.0 I 86 I /

List of references Baier M., et al., Nature 378 (1995) 563 Cruikshank, W.W., et al., J. Immunol. 146 (1991) 2928-2934 Cruikshank, W.W., et al., Proc. Natl. Acad. Sci. USA 91 (1994) Hoffman et al. in Protein Expression & Purific. 6 (1995) 646-654 Mack et al., Analyt. Biochem. 200 (1992) 74-80 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

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Met Lys Ser Leu Leu Cys Leu Pro Ser Ser Ile Ser Cys Ala Gln Thr ACA

ProCysIlePro LysGluGlyAla SerProThr SerSerSer AsnGlu AspSerAlaAla AsnGlySerAla GluThrSer AlaLeuAsp ThrGly PheSerLeuAsn LeuSerGluLeu ArgGluTyr ThrGluGly LeuThr GluAlaLysGlu AspAspAspGly AspHisSer SerLeuGln SerGly GlnSerValIle SerLeuLeuSer SerGluGlu LeuLysLys LeuIle GluGluValLys ValLeuAspGlu AlaThrLeu LysGlnLeu AspGly Ile His Val Thr Ile Leu His Lys Glu Glu Gly Ala Gly Leu Gly Phe SerLeu AlaGlyGly AlaAspLeu GluAsnLys ValIleThr ValHis ArgVal PheProAsn GlyLeuAla SerGlnGlu GlyThrIle GlnLys GlyAsn GluValLeu SerIleAsn GlyLysSer LeuLysGly ThrThr HisHis AspAlaLeu AlaIleLeu ArgGlnAla ArgGluPro ArgGln Ala Val Ile Val Thr Arg Lys Leu Thr Pro Glu Ala Met Pro Asp Leu Asn Ser Ser Thr Asp Ser Ala Ala Ser Ala Ser Ala Ala Ser Asp Val Ser Val Glu 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 Ile Ile Lys Ala Leu Pro Asp Gly Pro Val Thr Ile Val Ile Arg Arg Lys Ser Leu Gln Ser Lys Glu Thr Thr Ala Ala Gly Asp Ser (2) INFORMATION FOR SEQ ID NO: 2:

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Met Lys Ser Leu Leu Cys Leu Pro Ser Ser Ile Ser Cys Ala Gln Thr Pro Cys Ile Pro Lys Glu Gly Ala Ser Pro Thr Ser Ser Ser Asn Glu Asp Ser Ala Ala Asn Gly Ser Ala Glu Thr Ser Ala Leu Asp Thr Gly Phe Ser Leu Asn Leu Ser Glu Leu Arg Glu Tyr Thr Glu Gly Leu Thr Glu Ala Lys Glu Asp Asp Asp Gly Asp His Ser Ser Leu Gln Ser Gly Gln Ser Val Ile Ser Leu Leu Ser Ser Glu Glu Leu Lys Lys Leu Ile Glu Glu Val Lys Val Leu Asp Glu Ala Thr Leu Lys Gln Leu Asp Gly Ile His Val Thr Ile Leu His Lys Glu Glu Gly Ala Gly Leu Gly Phe Ser Leu Ala Gly Gly Ala Asp Leu Glu Asn Lys Val Ile Thr Val His Arg Val Phe Pro Asn Gly Leu Ala Ser Gln Glu Gly Thr Ile Gln Lys Gly Asn Glu Val Leu Ser Ile Asn Gly Lys Ser Leu Lys Gly Thr Thr His His Asp Ala Leu Ala Ile Leu Arg Gln Ala Arg Glu Pro Arg Gln Ala Val Ile Val Thr Arg Lys Leu Thr Pro Glu Ala Met Pro Asp Leu Asn Ser Ser Thr Asp Ser Ala Ala Ser Ala Ser Ala Ala Ser Asp Val Ser Val Glu 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 LeuGly Gly Ala MetGlnGly ThrArg PheGlu Ala Trp Thr Leu Asn IleIle Lys Leu ProAspGly ValThr IleVal Ile Arg Ala Pro Arg LysSer Leu Ser LysGluThr AlaAla GlyAsp Ser Gln Thr (2)INFORMATION FOR SEQID
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Met Pro Asp Leu Asn Ser

Claims (8)

Claims

1. Biologically active multimeric IL-16 polypeptide consisting of a defined number of IL-16 subunits, wherein the number of subunits is between 6 and 32

2. The multimeric IL-16 polypeptide of claim 1, wherein the number of subunits is between 8 and 16.

3. Process for the production of a biologically active multimeric form of IL-16 subunits, wherein IL-16 subunits in at least one of a monomeric form and a dimeric form are incubated with metal ions and the multimeric form of IL-16 subunits formed in this process is isolate d either individually or as a mixture.

4. Process for increasing the degree of multimerization of a monomeric and/or multimeric form of IL-16 subunits (initial form), wherein the monomeric or multimeric form is incubated with metal ions during which one or several higher multimerized form o r forms of IL-16 subunits are formed and the higher multimerized form or forms are separated from the initial forms.

5. Process as claimed in claim 3 or 4, wherein a denaturing agent is additionally added.

6. The process of claim 5, wherein t he denaturing agent is guanidine hydrochloride or urea.

7. Process as claimed in any one of claims 3 to 5, wherein the incubation is carried out at a pH 3-9.

8. The process of claim 7, wherein the incubation is carried out at pH 5-7.5.