WO1994003492A1 - Interleukin-6 variants and uses therefor - Google Patents

Abstract

The present invention relates to Interleukin-6 (IL-6) variants and in particular variants capable of stimulating thrombocytopoiesis while exhibiting a substantially low hybridoma growth factor activity compared to a non-variant IL-6.

Description

INTERLEUKIN-6 VARIANTS AND USES THEREFOR

The present invention is directed to variants of Interleukin-6 and the therapeutic and diagnostic use of same.

Interleukin-6 (IL-6) is a pleiotropic cytokine which can act on a wide variety of tissues. Depending on the nature of the target cell, IL-6 can exert growth promotion, growth inhibition, differentiation and induce specific gene expression. More specifically, IL-6 has been shown to be an effective stimulator of megakaryocytopoesis and platelet production (see Williams et al The Role of Interleukin-6 in megakaryocyte formation, megakaryocyte development and platelet production. In: Polyfunctional cytokines T -6 and TF. Wiley, Chichester (CIBA Foundation, ppl60-173, 1992).

Megakaryocytopoiesis is the formation of new platelets in megakaryocytes in haemopoietic tissue, predominantly in bone marrow. A number of conditions and disease states can result in a depletion in circulating platelets, a condition known as thrombocytopema. Although IL-6 is capable of stimulating thrombocytopoiesis, its pleiotrophic activities comprises its clinical use.

In work leading up to the present invention, the inventors investigated organ related factors stimulating megakaryocytopoesis and have surprisingly discovered variants of IL-6 with biological activity restricted to the megakaryocytic lineage.

Accordingly, one aspect of the present invention is directed to an isolated amino acid variant of a interleukin-6 (IL-6), said variant capable of stimulating thrombocytopoiesis while exhibiting a low hybridoma growth factor activity compared to a non-variant IL-6. In a related aspect of the present invention, there is provided an IL-6 variant molecule having an altered or otherwise modified C-terminal and/or N-terminal region such that the IL-6 variant molecule is capable of stimulating, enhancing or otherwise facilitating thrombocytopoiesis while having a low hybridoma growth factor activity compared to a non-variant IL-6.

In a preferred embodiment, the present invention contemplates an IL-6 variant polypeptide carrying a single or multiple amino acid substitution, deletion and /or addition to a region comprising at least 5 amino acids from the C-terminal end of IL-6 and/or at least 22 amino acids from the N-terminal end such that said IL-6 variant is capable of stimulating, enhancing or otherwise facilitating thrombocytopoiesis while having a low hybridoma growth factor activity.

The present invention is predicated, in part, on the discovery that the biological activities of IL-6 are ascribable to certain regions of the polypeptide. In accordance with the present invention, it has been surprisingly discovered tliat the first approximately 5 amino acids from the C-terminal end or the first approximately 22 amino acids from the N-terminal end are required for hybridoma growth factor activity but not thrombocytopoietic activity. Accordingly, by masking the activity associated with one or both of these two regions in an IL- 6 moelcule, a variant is produced capable of inducing thrombocytopoiesis without the side effects of other IL-6 activities. Hybridoma growth factor activity is a convenient measure of general IL-6 "activity" and the measure of this activity is described in Example 1. By "low hybridoma growth factor activity" is meant an activity of less than 60%, preferably less than 50%, more preferably less than 40%, even more preferably less than 30%, still more preferably less than 20% of the activity exhibited by a non-variant IL-6. In a most preferred embodiment, the IL-6 variant exhibits an activity of less than 10% (e.g. 0.1 - 5%) compared to a non-variant IL-6. The present invention is directed to an "isolated" IL-6 variant. By "isolated" is meant a composition comprising the IL-6 variant with substantially no other protein or polypeptide from the source from which the variant is isolated. Accordingly, "isolated" means that the variant is biologically pure, forming approximately 55%, preferably at least 65%, even more preferably at least 70% and even more preferably 75-95% or more of the composition by weight.

The IL-6 variant of the present invention is from an IL-6 of animal origin including birds and mammals. Preferably, the IL-6 is of mammalian origin such as from humans, livestock animals, companion animals or laboratory test animals (e.g. mice, rats, guinea pigs or rabbits). Most preferably, the IL-6 is of human or mouse origin. The numbering of the amino acids herein is principally taken from the human IL-6 molecule. One skilled in the art will immediately understand which amino acids correspond in other IL-6 molecules such as from mice. It is noted, for example, that mouse IL-6 is slightly shorter in length compared to human IL-6. Reference to a "non-variant" IL-6 is taken herein to include a molecule having the same amino acid sequence as naturally occurring IL-6 or which exhibits the pleiotrophic activities attributed to naturally occurring IL-6. The non-variant IL-6 may, therefore, be a naturally occurring or a recombinant o a synthetic molecule. From a functional viewpoint, a "non-variant IL-6" encompasses any IL-6 molecule exhibiting substantially normal hybridoma growth factor activity. In a preferred aspect of the present invention, a non-variant is considered a full length IL-6 molecule and a variant is a non-full length molecule.

The IL-6 from which the IL-6 variants are derivable may comprise the naturally occurring amino acid sequence or may contain single or multiple amino acid substitutions, deletions and/or additions to a region spanning from approximately amino acid 22 to approximately amino acid 175, provided that a molecule having an altered amino acid sequence in this region retains thrombocytopoetic activity. The IL-6 molecule may have its naturally occurring glycosylation pattern or association with carbohydrate moieties or may possess an altered pattern. Again, such altered molecules are constrained by the need to still possess thrombocytopoietic activity.

The IL-6 variants of the present invention preferably have the relevant C-terminal and/ or N-terminal amino acids deleted. One skilled in the art, however, will immediately appreciate that the relevant amino acids may be substituted for "non¬ functional" amino acids, i.e. amino acids the inclusion of which result in a loss of or a reduction in hybridoma growth factor activity. Alternatively, the end region(s) of the IL-6 molecule may be masked by, for example, specific antibodies or binding proteins or may be part of a fusion molecule such as with IL-3 or active parts thereof. All such IL-6 variants are encompassed by the present invention notwithstanding that the following description relates to the truncated IL-6.

Accordingly, in a preferred embodiment, there is provided a truncated (i.e. non- full length) IL-6 molecule comprising a polypeptide having an amino acid sequence with at least approximately 5 but not more than approximately 59 contiguous amino acids deleted from the C-terminal end and/or not more than approximately 22 contiguous amino acids from the N-terminal end wherein said truncated IL-6 molecule stimulates, enhances or otherwise facilitates thrombocytopoiesis but has a low hybridoma growth factor activity compared to a non-variant IL-6 molecule.

As stated above, the amino acid sequence of the variant may be the same as the corresponding region of the naturally occurring IL-6 molecule or may contain single or multiple amino acid substitutions, deletions and/or additions.

Amino acid insertional derivatives of the IL-6 variant of the present invention include amino and /or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Typical substitutions are those made in accordance with the following Table 1:

TABLE 1 Suitable residues for amino acid substitutions

Where the IL-6 variant is derivatised by amino acid substitution, the amino acids are generally replaced by other amino acids having like properties, such as hydrophobicity, hydrophilicity, electronegativity, bulky side chains and the like. Amino acid substitutions are typically of single residues. Amino acid insertions will usually be in the order of about 1-10 amino acid residues and deletions will range from about 1-20 residues. Preferably, deletions or insertions are made in adjacent pairs, i.e. a deletion of two residues or insertion of two residues.

The amino acid variants referred to above may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis (Merrifield, J. Am. Chem. Soc. 85: 2149, 1964) and the like, or by recombinant DNA manipulations. Techniques for making substitution mutations at predetermined sites in DNA having known or partially known sequence are well known and include, for example, M13 mutagenesis. The manipulation of DNA sequence to produce variant proteins which manifest as substitutional, insertional or deletional variants are conveniently described, for example, in Sambrook et aL, (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989).

Other examples of recombinant or synthetic mutants and derivatives of the IL-6 variants of the present invention include single or multiple substitutions, deletions and/ or additions of any molecule associated with the molecule such as carbohydrates, lipids and/or proteins or polypeptides.

IL-6 variants also extend to functional chemical equivalents or analogues thereof.

Analogues of the IL-6 variants contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/ or derivatising the molecule and the use of crosslinkers and other methods which impose conformational constraints on the molecule. Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH^ amidation with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthali anhydride; and pyridoxylation of lysine with pyridoxal-5'-phosphate followed by reduction with NaBH

The guanidino group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3- butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation yia O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4- chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4- nitrophenol and other mercurials; carbomoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hyci_roxy-5-nit_robenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative. Other modifications include iodination of tyrosine and biotinylation of lysine.

Modification of the imidaxole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy 6- methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.

Crosslinkers can be used, for example, to stabilise 3D conformations, using homo- bifunctional crosslinkers such as the bifunctional imido esters having (CH2)_n spacer groups with n = 1 to n= 6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group-specific reactive moiety such as maleimido or dithio moiety or carbodiimide. In addition, peptides could be conformationally constrained by, for example, incorporation of C_β and N_β- methylamino acids, introduction of double bonds between C_β and C_p atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.

The IL-6 variants of the present invention are conveniently prepared by recombinant techniques. Accordingly, the present invention extends to a nucleic acid molecule comprising a nucleotide sequence encoding, or complementary to a sequence encoding, an IL-6 variant as hereinbefore described and in particular an IL-6 variant comprising a polypeptide having an amino acid sequence with at least approximately 5 amino acids but not more than approximately 59 contiguous amino acids deleted from the C-terminal portion and/or not more than approximately 22 contiguous amino acids deleted from the N-terminal portion wherein said IL-6 variant stimulates, enhances or otherwise facilitates thrombopoiesis but has a comparatively low hybridoma growth factor activity. Where the nucleic acid molecule comprises a nucleotide sequence complementary to a sequence encoding the IL-6 variant, then such a nucleic acid molecule will be useful as a probe to screen for similar or related genetic sequences in other animals or for use as an antisense molecule to regulate IL-6 expression or for diagnostic purposes such as for use in a PCR. If the latter is the case, then it is preferable to have smaller oligonucleotides directed to specific parts of the IL-6 variant and the present invention extends to these.

The nucleic acid molecule may be RNA or DNA but is preferably cDNA The nucleotide sequence may correspond to the naturally occurring nucleotide sequence of the corresponding coding part of the gene in the cell's chromosome or corresponding to the appropriate portion on an IL-6 mRNA transcript or may contain single or multiple nucleotide substitutions, deletions and/or additions thereto. Preferably, where there is an alteration to the sequence, this corresponds to the derivatives of the IL-6 variants contemplated above.

The nucleotide acid molecule may be part of a larger molecule such as a vector or part of a fusion sequence. Preferably, the vector is an expression vector and is capable of replication and/or expression in one or more of a prokaryotic cell (e.g. IΞ cΩli, Pseudomonas sp or Bacillus sp cells) and/or a eukaryotic cell (e.g. yeast, fungal, insect, animal or mammalian, cells). Most preferably, the vector is pUC9.

The nucleic acid molecule may encode just the IL-6 variant of the present invention or may encode the full length IL-6 molecule but engineered to give rise to variants upon expression of the DNA sequence. Alternatively, the variants may be generated from a full length or near full length IL-6 encoding sequence by standard in ήlrα and/or in vivo mutagenic techniques. The present invention extends to an isolated nucleic acid molecule which (i) encodes a non-full length D -6 molecule of animal origin, said non-full length molecule capable of stimulating thrombocytopoiesis while exhibiting a low hybridoma growth factor activity compared to a full length IL-6 molecule of same animal origin; and (ii) hybridises under conditions of low stringency to a strand of a double stranded DNA molecule which DNA molecule encodes a full length IL-6 molecule of animal origin, preferably of murine and/or human origin.

The nucleic acid molecule may be RNA or DNA single stranded or double stranded, in linear or covalently closed circular form. For the purposes of defining the level of stringency, reference can conveniently be made to Sambrook et al., Supra at pp 387-389 which is herein incorporated by reference where the washing step at paragraph 11 is considered high stringency. A low stringency is defined herein as being in 0.1-0.5% w/v SDS at 37-45 °C for 2-3 hours. Depending on the source and concentration of nucleic acid involved in the hybridisation, alternative conditions of stringency may be employed such as medium stringent conditions which are considered herein to be 0.25%-0.5% w/v SDS at > 45 °C for 2-3 hours or high stringent conditions as disclosed by Sambrook et al., Supra.

The nucleotide sequences for murine and human B -6 are published in Van Snick et al (I) Eur. J. Immunol. 18: 193-197, 1988 and Van Snick et al (II) Proc. Nad. Acad. Sci. USA 83: 9679-9683, 1986, respectively, which are herein incorporated by reference. The nucleotide sequences are also set forth herein in SEQ ID No. 2 and SEQ ID No. 4, respectively.

The present invention extends to IL-6 variants encoded by nucleic acid molecules which hybridise to the murine and/or human DNAs referred to above. The IL-6 variants in the form of non-full length molecules are also definable in terms of amino acid sequence similarity to known full length molecules. In a most preferred embodiment, the present invention provides a non-full length IL- molecule capable of stimulating thrombocytopoiesis while exhibiting a low hybridoma growth factor activity compared to a full length IL-6 molecule wherei said non-full length molecule comprises an amino acid sequence having at least 35%, preferably at least 45%, more preferably at least 55-60%, still more preferably at least 70-80% and even more preferably greater than 90% similarit to the amino acid sequence of the corresponding region in a murine or human I 6 molecule. The amino acid sequences for murine and human IL-6 are set forth in Van Snick et al (I) and Van Snick et al (II), respectively and SEQ ID No. 1 and SEQ ID No. 3, respectively.

The recombinant or synthetic EL-6 variants of the present invention are contemplated to be particularly useful in inducing thrombopoiesis in thrombocytopenic patients or in potentially thrombocytopenic patients which might arise following surgery, chemotherapy, radiation therapy and following bo marrow transplantation amongst other conditions.

Accordingly, another aspect of the present invention is directed to a method for stimulating, enhancing or otherwise facilitating thrombocytopoiesis in an animal comprising administering to said animal a thrombocytopoietic effective amount o an IL-6 variant, said variant capable of stimulating thrombocytopoiesis while exhibiting substantially reduced hybridoma growth factor activity compared to a non-variant IL-6, for a time and under conditions sufficient to stimulate megakaryocytes.

In addition, the IL-6 variants of the present invention may be useful in cancer therapy where the fragments express anti-neoplastic activity. Such a method of use of the IL-6 variants is encompassed by the present invention. Preferably, the animal is a mammal and more preferably is a human.

The IL-6 variant may be administered in a single dose or as multiple doses over time. Generally, a human IL-6 variant will be used in the treatment of humans but there may be occasions where a heterologous IL-6 variant is appropriate (e.g. mouse IL-6 variant for use in humans).

The present invention further extends to the administration of the IL-6 variant in combination with other cytokines, thrombopoietic molecules or anti-cancer agents. Preferably, the IL-6 variant is administered with IL-3 or active fragments thereof.

The present invention, therefore, contemplates a pharmaceutical composition comprising an effective amount of an IL-6 variant as hereinbefore defined and one or more pharmaceutically acceptable carriers and/or diluents. The composition may also contain one or more other molecules including but not limited to IL-3 or other variants of IL-6. The active ingredients of a pharmaceutical composition comprising an IL-6 variant are contemplated to exhibit excellent therapeutic activity, for example, in stimulating, enhancing or otherwise facilitating thrombocytopoiesis in an animal and preferably a mammal and most preferably a human when administered in amount which depends on the particular case. For example, from about 0.5 ug to about 20 mg, preferably 0.5 ug to about lOmg, and more preferably 1.0 ug to about 5 mg per kilogram of body weight per day may be administered. Dosage regima may be adjusted to provide the optimum therapeutic response, For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation, The active compound may be administered in a convenient manner such as by the oral, intraveneous (where water soluble), intramuscular, subcutaneous, intranasal, intradermal or suppository routes, implanting (eg using slow release molecules) or using a nasal spray. Preferably, however, the IL-6 variant is administered sub-cutaneously. Depending on the route of administration, the active ingredients which comprise an IL-6 variant may be required to be coated in a material to protect said ingredients from the action of enzymes, acids and other natural conditions which may inactivate said ingredients. For example, the low lipophilicity of the IL-6 variants will allow it to be destroyed in the gastrointestinal tract by enzymes capable of cleaving peptide bonds and in the stomach by acid hydrolysis. In order to administer an IL-6 variant by other than parenteral administration, it will be coated by, or administered with, a material to prevent its inactivation. For example, an IL-6 variant may be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes. Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon. Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes.

The active compounds may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the temporaneous preparation of sterile injectable solutions or dispersion. In all cases the preparation must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene gloycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The The prevention of ths action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization, generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

When an IL-6 variant is suitably protected as described above, the active, compound may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 10 ug and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergree, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations.

As used herein "pharmaceutically acceptable carrier and/or diluent" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.

The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 ug to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 ug to about 2000 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

The present invention further relates to antibodies specific to the IL-6 variants contemplated herein and to thrombocytopoietic specific receptor(s) for the 11-6 variants. Such receptors may be particularly useful in treating cancers such as multiple myelomas which could be induced, stimulated or enhanced by naturally occurring IL-6 variants.

The present invention is further described by the following non-limiting Figure and Examples. In the Figure:

Figure 1 comprises graphical representations showing the responsiveness of immature megakaryocytes to titrated doses (ng/ml) of IL-6 and variants of IL-6. The results are the mean ± SD of triplicate cultures. The drawn line indicates the background activity in the cultures; the line joined by (•) points represents the level of responsiveness. (A), Alkylated mIL-6; (B), C^"16; (C), mIL-6; (D) C⁵ mIL-6.

EXAMPLE 1 Determination of Activity of IL-6 Variants

A range of variant IL-6 molecules were assayed for megakaryocyte activity and hybridoma growth activity and the results are shown in Table 2.

The megakaryocyte assay was determined on an immature megakaryocyte population obtained from mouse bone marrow. Single suspensions of bone marrow cells were centrifuged in a Percoll gradient, and the 1.07-1.085 g/cm³ fraction (400g, 4°C, 20 min) was used. The cells (10⁵ cells/ml) were cultured for 5 days at 37°C in a humidified incubator in the presence of titrated doses of the growth factor preparations and 5% (v/v) fetal calf serum (FCS) in DMEM. The FCS was titrated and used at a dose that gave maximum background stimulation. The 3% agar cultures were dried, stained for acetylcholinesterase (Williams and Jackson Cell Tissue Kinet. 15; 483-494, 1982) and the number of single positive- staining cells counted by light microscopy. Cell size (error ± 2%) was determined as area (μm²) for all acetylcholinesterase-positive cells in a culture dish by computer-drive image analysis. All assays were performed in triplicate and results were tested statistically using a Student's t-test (Williams et al Experimental HematQlogy 1& 69-72, 1990). The hybridoma growth factor assay was as previously described (Van Snick et al Proc. Natl. Acad. Sci. I IS A 83: 9679-9683, 1986). Briefly, IL-6-dependent 7TD1 mouse-mouse hybridoma cells (2000 cells /microwell) were incubated with serial dilutions of the test fractions in a final volume of 0.2 ml. Cell growth was determined after 4 days at 37°C by colorimetric measurement of the hexosaminidase levels (Landegren, U., J. Immunol. Methods 67: 379-388, 1984). A titre of 1 U/ml was arbitarily assigned to material that produced half maximal growth of the hybridoma 7TD1 cells.

TABLE 2 Growth of Immature Megakaryocytes by IL-6 Mutants

(a) Protein concentration was measured by amino acid analysis.

(b) Recombinant murine interleukin-6 (mIL-6) was expressed in E coli as a β- galactosidase fusion protein using the lac operon inducible plasmid pUC9. The first 8 amino acids of the expressed protein are from the N-terminus of bacterial β-galactosidase while the remaining 176 amino acids are residues 12 to 187 of nature muine IL-6. mIL-6 was purified as described in Example 2.

(c) C^'16IL-6 and C^'^IL-ό refer to truncated forms lacking 16 to 64 amino acids from the C-terminus of mIL-6, respectively. These forms were isolated as side products during purification of mIL-6 in Example 2. (d) C^"5IL-6 and N^'^IL-ό are lacking 5 amino acids from the C-terminus and 22 amino acids from N-terminus of IL-6, respectively. Both forms were generated by the polymerase chain reaction using the pUC9 plasmid containing mIL-6 as template. Both constructs were sequenced prior to expression and purification as described for mIL-6 in Example 2. Purity of the genetically engineered clones was confirmed by assessment of molecular mass (20,682 compared to a theoretical value of 20,680 daltons for C^'5 IL-6) DNA sequence analysis.

(e) IAA-IL-6 was generated by reducing and alkylating mD -6 with iodoacetic acid. All 4 cysteine residues of mIL-6 were modified and confirmed by amino acid analysis (and moles S-carboxymethyl cysteine/mole EL-6).

Megakaryocyte growth activity was determined by the appearance of detectable single megakaryocytes in cell culture. Variants of IL-6 (IL-6, IAA-IL6, N^"22 IL-6) also increased cell size (664 ± 45 μm²) significantly above background levels (439 ± 45 μm²). The effect of titrated doses of variants of IL-6 is shown in Figure 1. All growth factor preparations were maximally active in the 50-100 ng range.

EXAMPLE 2 Purification and Characterisation of Recombinant mlL-6 Variant

1. Materials and Methods

Materials

Trifluoroacetic acid (Sequanal grade), Tween-20, 2 mg/ml bovine serum albumin, and bioinchoninic acid were obtained from Pierce (Rockford, IL, USA). Dithiothreitol, isopropyl- β-D-thiogalactopyranoside, p-nitrophenyl-N-acetyl- β-D- glucosaminide, anti-(rat IgG)-alkaline-phosphatase conjugate, Tris base, 3(cyclohexylamino)-l -propane sulfonic acid (Caps), phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor and α- lactalbumin were purchased from Sigma (St. Lous, MO, USA). Guanidine hydrochloride (Gu/HCl), Nonidet P-40 and urea were from Fluka (Buchs, Switzerland). Coomassie brilliant blue R-250 and sodium dodecyl sulphate were purchased from LKB (Bromma, Sweden). Nitrocellulose membrane was obtained from Schleicher & Schull (Dassel, FRG). Triton X-100 and Staphylococcus aurens. strain V8 protease were from Miles (Naperville, IL, USA), DNase I, lysozyme and Asp-N endoproteinase were products of Boehringer Mannheim. HPLC-grade organic solvents were purchased from Mallinckrodt (Melbourne, Australia). All buffers were prepared with deionized water purified by a tandem Milli-RO and Milli-Q system (Millipore, Bedford, MA, USA).

Construction of murine D -6 expression vector

Murine IL-6 cDNA (Van Snick et al Eur. J. Tmmunol. 1& 193-197, 1988) was cloned into the EcoRl site of pUC9 located within the encoded lac. gene and transformed into E___coli (strain JM101) as previously described (Simpson et al Biochem. Biophys. Res. Commun. 152; 364-372, 1988). pUC9-transfoπned E. coli colonies were screened for IL-6 activity using the hybridoma growth factor assay (Example 1). One active clone, p9HPlB5B12, was found to encode a fusion protein comprising the eight N-terminal amino acids of bacterial β-galactosidase fused to residue 12 of mature mIL-6. Thus, the amino acid numbering throughout this example is based on the sequence of the rmIL-6.

Purification of recombinant mIL-6 a) Growth of E. coli and preparation of cell pellet

Escherichi coli containing the plasmid p9HPlB5B12 was grown overnight at 37°C in Luria broth (100 ml) and used to inoculate 51 medium containing ampicillin (40μg/ml) in shaker plasks. The expression of mIL-6 was induced, at a cell density corresponding to A₆₀₀ = 1.0, by the addition of isopropyl β-d- thiogalactopyranoside to a final concentration of 0.1 mM. After induction (3.5h), the E___coli cells (37 g wet mass) were harvested by centrifugation (4000 x g, 10 min, 4°C) and washed with phosphate-buffered saline pH 7.2 and then resuspended in 370 ml 50 mM Tris/HCl pH 7.5 containing 25% (w/v) sucrose and 0.1% (w/v) lysozyme. After stirring for 1 h at 4°C, the suspension was supplemented with Nonidet P-40 (0.5% v/v), MgCl₂ (5mM) and DNase I (40 μg/ml) and stirring was continued for a further 1 h. Insoluble aggregates (inclusion bodies) of mIL-6 were pelleted by centrifugation at 23000 x g for 30 min. The pellet (* 18g wet mass) was resuspended in 200 ml 100 ml Tris/HCl pH 8.0 containing 4.0 M urea, 1% (v/v) Triton X-100, strirred at 25°C for 1 h and then recentrifuged at 23000 x g for 30 min. This procedure was repeated twice and then the pellet (= 2g wet mass) was dissolved in 15ml 8 M Gu/HCl, 50 mM Tris/HCl pH 7.5 and gentyl stirred for 16-20 h at 4°C prior to gel filtration.

b) Gel-permeation chromatography

The Gu/HCl-solubilised protein (15ml) was chromatographed on a Fractogel TSK HW-55(S) column (60 x 5.0 cm) equihbrated in 6M Gu/HCl, 50 mM Tris/HCl pH 7.5. The flow rate was 120 ml/h and 4 ml fractions were collected.

Fractions from the gel-filtration step were monitored for rmIL-6 content by analytical reversed-phase (RP) HPLC and SDS /PAGE. Immunological reactivity was assessed by Western blotting.

c) Reversed-phase high-performance liquid chromatography (RP-HPLC) πnIL-6-containing fractions (120-150 ml as 4 ml fractions in 6 M Gu/HCl, 50 mM Tris/HCl pH 7.5) recovered from the gel-permeation column were pooled and diluted five-fold with aqueous 0.1% (v/v) trifluoroacetic acid and pumped directly at 10 ml/min, using an Altex model 110A pump, onto a Vydac C₄ column (22.5 mm internal diameter x 100 mm) which had been previously equilibrated with aqueous 0.1% (v/v) trifluoroacetic acid.

The column was developed with a 60-min linear gradient of 0-100% B, where solvent A was aqueous 0.1% (v/v) trifluoroacetic acid and solvent B was 60% acetonitrile/40% water containing 0.1% (v/v) trifluoroacetic acid. Flow rate was 20 ml/min and 10 ml fractions were collected automatically using a FRAC 100 (Pharmacia) fraction collector. Fractions containing pure rmIL-6, as assessed by analytical RP-HPLC and SDS/PAGE in 15% (w/v) acrylamide gels, were pooled and lyophilised.

Biological Assay rmIL-6 titers were determined in a hybridoma growth factor assay, as described in Example 1.

Electrophoretic Techniques a) Denaturing gels

SDS/PAGE was performed according to Laemmli (Nature.222; 6680-6685, 1970). For Gu/HCl-containing sample, Gu/HCl was removed by selective precipitation of protein with deoxycholate and trictchloroacetic acid. Proteins were visualised in the gels by Coomassie brilliant blue R-250 staining. Phosphorylase b, bovine serum albumin, ovalbumin were used as apparent molecular mass markers.

Isoelectric Focusing Gel

Isoelectric focusing on thin-layer polyacrylmide gel was performed on a Pharmacia PhastSystem and proteins were stained with Coomassie brilliant blue R-250 according to the manufacturer's instructions.

a) Urea-gradient electrophoresis

Urea-induced unfolding of rmIL-6 was monitored by ureagradient gel electrophoresis, essentially as described by Creighton Protein Structure: A Practical Approach IRL Press Ltd. Oxford, 199Q. Briefly, slab gels (6.0 cm x 8.8 cm x 0.1 cm) were prepared containing a transverse gradient of 0-8 M urea with an inverse gradient of 15-11% (w/v) acrylamide in order to compensate for the electrophoretic effects of the urea. Unfolding of murine IL-6 was monitored in 50 mM acetate/Tris pH 4.0. Electrophoresis of rmIL-6 (40μg in 150μl) was performed on a Bio-Rad Mini-protean™II apparatus at 25°C for 3 h at 6mA constant current (100 V). Pre-electrophoresis was performed for 1 h at 6 mA prior to sample loading. Protein was stained with Coomassie brilliant blue R-250. b) Western blot analysis

Proteins resolved by SDS /PAGE were electro transferred onto a nitrocellulose membrane using a Bio-Rad mino two dimensional gel system. The first probe was a rat anti-(mIL-6) monoclonal antibody (6B4); the second probe was a goat anti-(rat IgG) antibody conjugated with alkaline phosphatase (Sigma, St. Louis, MO, USA)

Protein Estimation

The concentration of protein in the early stages of the purification protocol was determined using the bicinchoninic acid procedure Brown et al Anal. Biochem. ISO; 136-139, 1989. The concentration of purified protein was determined by amino acid analysis using a Beckman model 6300 amino acid analyzer.

High Performance Liquid Chromatography a) Instrumentation

Gel-permeation chromatography was performed using a Pharmacia FPLC system. Preparative RP-HPLC was performed on a Beckman liquid chromatograph fitted with model HOB pumps, a model 421 A controller and a model 163 variable- wavelength detector. Analytical RP-HPLC and peptide mapping were performed on a Hewlett-Packard 1090A liquid chromatograph, fitted with a model 1040A diode-array detector.

b) Column supports

The following columns were used for the large-scale purification of rmIL-6; (a) Fractogel TSK HW 55(S) (Toyo Soda, Japan) packed into a glass-lined, low- pressure column (50 mm intenal diameter x 600 mm, Pharmacia, Sweden); (b) Vydac C₄ (30 nm pore size, 10-15 μm particle diameter butylsilica; the Separations Group, hesperia, CA USA). A slurry of this support (11.5 g/ 600ml n-propanol) was prepared by sonication (30 min). Using a Shandon column packer (Shandon Southern, Runcorn, UK), the slurry was rapidly packed at 55 MPa into a clean, highly polished stainless stell column [22.5 mm internal diameter x 100 mm tubing assembled with Parker Haniffin Co. (Huntsville, AL, USA) column end fittings] that had been previously filled with neat n-propanol. The column was consolidated at 55 MPa with 1000 ml aqueous 50% (v/v) methanol before dismantling and applying the final end-fitting. The column was further conditioned with 500 ml 60% (w/v) acetonitrile/40% water containing 0.1% (v/v) trifluoroacetic acid.

Analytical RP-HPLC and peptide mapping were performed using a Brownlee RP- 300 cartridge (either 2.1 mm internal diameter x 100 mm or 4.6 mm internal diameter x 100 mm, Applied Biosystems, Foster City, CA, USA).

Titration of Sulfhydryl Groups

The Ellman assay was performed by titration of rmIL-6 solution in 0.1 phosphate pH 7.3, 1 mM EDTA, containing 6 M Gu/HCl with 3 mM 5,5'-dithiobis (2- nitrobenzoic acid) according to the procedure of Riddles et al Methods Enzymol. 21; 49, 1983.

Analysis of Disulfide Bonding

Disulfide bond formation in rmIL-6 during its purification was monitored by one- dimensional SDS/PAGE under non-reducing conditions. Using this approach, reduced rmIL-6 and oxidised (disulfide-bonded) rmIL-6 can be discriminated by a difference in molecular mass on 12.5% (w/v) acrylamide gels. Reduced πnIL-6 migrates at a molecular mass of 22-23 kDa on SDS/PAGE relative to 20-21 kDa for oxidized rmIL-6. rmIL-6 samples taken from various steps in the purification protocol were treated with 50 mM iodoacetamide to arrest further disulfide bond formation. The pH of the reaction mixtures was adjusted to about 8.3 by adding concentrated Tris/HCl buffer. The reaction was allowed to proceed for 1 h at room temperature in the dark in *0.1 M Tris/HCl pH 8.3 containing 6 M Gu/HCl. Prior to performing SDS/PAGE under nonreducing conditions, salts were removed by selective precipitation of protein with deoxycholate and trichloroacetic acid. Kinetics of Disulfide Bond Formation in 8 M Gu/HCl

Pure πnIL-6(l mg/ml) in 6 M Gu/HCl, 50 mM Tris/HCl pH 7.5 was treated wit 100 mM dithiothreitol at 37°C for 4 h. Fully reduced rmIL-6 was recovered by reversed phase HPLC (Brownlee RP-300, 4.6mm internal diamter x 100 mm cartridge) using a linear gradient of acetonitrile in 0.1% (v/v) trifluoroacetic acid and then quickly dried (60 min) by centrifugal lyophilisation (Savant, Hicksville, NY, USA). Lyophilised reduced rmIL-6 (1 mg/ml) was reconstituted in 8 M Gu/HCl, 50 mM Tris/HCl pH 7.5 and cooled to 6°C. At various times indicated, an aliquot (20 μl) was automatically withdrawn and directly injected onto the same column used above. The solvents were aqueous 20 mM HC1 (solvent A) and 60% (w/v) acetonitrile/40% water containing 20 mM HC1 (solvent B). The HPLC configuration used in this study was a Hewlett-Packard HP 1090 series M liquid chromatography fitted with a temperature-controlled precolumn sample preparation system. Cooling of the autoinjection system was provided by a Thermomix

U liquid cooling system (B. Braun, Australia Pty. Ltd.).

Location of Disulfide Bonds Disulfide bond analysis of πnIL-6 was performed by S. aureus V8 protease peptide mapping.

Peptide Mapping with Asp-N Endoproteinase

Recombinant mIL-6 and variant forms of rmIL-6 (5 μg) were digested with Asp-N endoproteinase, at an enzyme /substrate mass ratio of 1:10, in 250 μl 0.05 M sodium phosphate pH 8.0 containing 0.02% (v/v) Tween-20 for 16 h at 37°C.

Circular Dichroism

Far-ultraviolet CD spectra were measured using an Aviv model 62DS circular dichroism spectrometer. The data were collected from 185 nm to 250 nm using a step size of 0.2 nm and a band width of 0.8 nm. Spectra were the average of 3-5 scans with the base line subtracted. Measurements were made with 88 μg/ml rmIL-6 in 10 mM sodiumphosphate pH 7.4 at 25°C using a 0.1 cm pathlength cell. The reported spectrum is expressed as mean residue ellipticity pfθ _j- and was calculated using a mean residue mass of 115.5 Da for rmIL-6. Analysis of the spectrum for contributions of particular secondary structural elements were performed using the CD reference spectra of Yang et al Methods Enzymol. 13Q; 225, 1986, and a multilinear regression programme (Aviv Plot, version 3.3m) supplied by Aviv (Lakewood, NJ, USA). All solutions were filtered before used with 0.22 μm pore size filters (Millipore).

Electrospray Mass Spectrometric Analysis

Recombinant mIL-6 was initially dissolved in 1% (v/v) aqueous formic acid. An aliquot was then taken and a four-fold dilution was made in methanol/water (50:50) with an overall concentration of 1.25% (v/v) formic acid. The final concentration of rmIL-6 was 0.25 μg/1. The sample was analysed by infusing a constant flow (0.5 μl/min) of the protein solution into an electrospry mass spectrometer (Finnigan, model TSQ700). Data were acquired by summing several scans to obtain the final spectrum.

2. Results

Murine interleukin-6 (IL-6), when expressed in E. coli using the pUC9 vector, accumulated as insoluble aggregates or "inclusion bodies". After selective urea washing of the inclusion bodies, to remove extraneous proteins, murine IL-6 was solubilized with 8 M guanidine hydrochloride and then rapidly purified to homogeneity by size-exclusion chromatography followed by reversed-phase HPLC. It was demonstrated that complete disulfide bond formation in murine IL-6 occurred during the early urea washing/ guanidine hydrochloride extraction steps, so no refolding step was required. When fully reduced murine EL-6 was dissolved in 8 M guanidine hydrochloride and allowed to air-oxidize, complete disulfide bond formation, monitored by analytical reversed-phase HPLC, was shown to occur within 13 h at 6 ^βC. About 25 mg pure protein was obtained from 37 g wet cells. This recombinant murine IL-6 had a specific activity in the hybridoma growth factor assay of 2 x 10 U/mg, which is equivalent to that of native murine IL-6.

During the purification procedure, a number of variant forms of murine IL-6 wer isolated and partially characterised. Two of these forms, Tl and T3, were C- terminal deletants of murine IL-6 lacking about 60 and 20 amino acids from the N-terminus. None of these variant forms of murine IL-6 bound to the murine IL 6 receptor and, consequently, all were inactive in the hybridoma growth factor assay.

EXAMPLE 3 In vivo Effects of an IL-6 Variant

The in vivo effect on platelet production of an IL-6 variant was shown in mice. Female C57B1/6 mice (18-20 grams: 5 mice per group) were treated with 2.5 μg of either recombinant mouse IL-6 or C⁵ variant IL-6 (see Example 1) every 8 hours for 56 hours and the mice sacrificed at 72 hours. The effect of IL-6 or variant IL-6 on peripheral blood values, including platelet numbers is shown in Table 3. The timing schedule used was that known to be optimal for full length recombinant human IL-6.

The results clearly shown that the IL-6 variant maintains its thrombocytopoietic activity as shown by the level of platelets per millilitre relative to the untreated mice. The results show that this effect is not due to a change in blood volume (i.e. no signficant change in hematocrit percentage). Nor was there any adverse effect on marrow cellularity. The megakaryocyte progenitor cell pool was higher in IL-6 variant-treated mice relative treatment with the full length molecule or in the control indicating that at the dose and timing used, the megakaryocytic processes may be delayed in IL-6 variant treated mice. TABLE 3 Effect of IL-6 Variant on Peripheral Blood Values in Mice¹

Results are the mean and S.E.M. from 5 mice

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT (US): Neil Thomas WILLIAMS

Richard John SIMPSON (OTHER THAN US): The University of Melbourne

Ludwig institute for Cancer Research

(ii) TITLE OF INVENTION: "lnterleukin-6 variants and uses therefor

(Hi) NUMBER OFSEQUENCES: 4

(iv) CORRESPONDENCEADDRESS:

(A) ADDRESSEE: Davies Collison Cave

(B) STREET: 1 Little Collins Street

(C) CITY: Melbourne

(D) STATE: Victoria

(E) COUNTRY: Australia

(F) ZIP: 3000

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 5.25" floppy disc

(B) COMPUTER: IBM compatible

(C) OPERATING SYSTEM: MS-DOS 5.0

(D) SOFTWARE: WordPerfect 5.1

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: International application

(B) FILING DATE: 1 July, 1993

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: Aust. Patent App. No. PL3983/92

(B) FILING DATE: 6 August, 1992

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: John M. Slattery

(B) REGISTRATION NUMBER:

(C) REFERENCE/DOCKET NUMBER: EJH/JMS/EK

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (03) 254 2777

(B) TELEFAX: (03) 254 2770 (2) INFORMATION FOR SEQ ID NO. 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 211 amino acid residues

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULETYPE: polypeptide

(Hi) SEQUENCE DESCRIPTION: SEQ ID NO.1:

Met Lys Phe Leu Ser Ala Arg Asp Phe His -15

Pro Val Ala Phe Leu Gly Leu Met Leu Val -5

Thr Thr Thr Ala Phe Pro Thr Ser Gin Val 6

Arg Arg Gly Asp Phe Thr Glu Asp Thr Thr 16

Pro Asn Arg Pro Val Tyr Thr Thr Ser Gin 26

Val Gly Gly Leu lie Thr His Val Leu Trp 36

Glu lie Val Glu Met Arg Lys Glu Leu Cys 46

Asn Gly Asn Ser Asp Cys Met Asn Asn Asp 56

Asp Ala Leu Ala Glu Asn Asn Leu Lys Leu 66

Pro Glu lie Gin Arg Asn Asp Gly Cys Tyr 76

Gin Thr Gly Tyr Asn Gin Glu lie Cys Leu 86

Leu Lys lie Ser Ser Gly Leu Leu Glu Tyr 96

His Ser Tyr Leu Glu Tyr Met Lys Asn Asn 106

Leu Lys Asp Asn Lys Lys Asp Lys Ala Arg 116

Val Leu Gin Arg Asp Thr Glu Thr Leu lie 126

His lie Phe Asn Gin Glu Val Lys Asp Leu 136

His Lys lie Val Leu Pro Thr Pro lie Ser 146

Asn Ala Leu Leu Thr Asp Lys Leu Glu Ser 156

Gin lLs Glu Trp Leu Arg Thr Lys Thr lie 166

Gin Phe lie Leu Lys Ser Leu Glu Glu Phe 176

Leu Lys Val Thr Leu Arg Ser Thr Arg Gin 186 Thr (3) INFORMATION FOR SEQ ID NO. 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 627

(B) TYPE: DNA

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

SEQUENCE DESCRIPTION: SEQ ID NO. 2:

ATG AAG TTC CTC TCT GCA AGA GAC TTC CAT CCA GTT GCC 39

TTC TTG GGA CTG ATG CTG GTG ACA ACC ACG GCC TTC CCT 78

ACT TCA CAA GTC CGG AGA GGA GAC TTC ACA GAG GAT ACC 117

ACT CCC AAC AGA CCT GTC TAT ACC ACT TCA CAA GTC GGA 156

GGC TTA ATT ACA CAT GTT CTC TGG GAA ATC GTG GAA ATG 195

AGA AAA GAG TTG TGC AAT GGC AAT TCT GAT TGT ATG AAC 234

AAC GAT GAT GCA CTT GCA GAA AAC AAT CTG AAA CTT CCA 273

GAG ATA CAA AGA AAT GAT GGA TGC TAC CAA ACT GGA TAT 312

AAT CAG GAA ATT TGC CTA TTG AAA ATT TCC TCT GGT CTT 351

CTG GAG TAC CAT AGC TAC CTG GAG TAC ATG AAG AAC AAC 390

TTA AAA GAT AAC AAG AAA GAC AAA GCC AGA GTC CTT CAG 429

AGA GAT ACA GAA ACT CTA ATT CAT ATC TTC AAC CAA GAG 468

GTA AAA GAT TTA CAT AAA ATA GTC CTT CCT ACC CCA ATT 507

TCC AAT GCT CTC CTA ACA GAT AAG CTG GAG TCA CAG AAG 546

GAG TGG CTA AGG ACC AAG ACC ATC CAA TTC ATC TTG AAA 585

TCA CTT GAA GAA TTT CTA AAA GTC ACT TTG AGA TCT ACT 624 CGG CAA ACC

(4) INFORMATION FOR SEQ ID NO. 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 212 amino acid residues

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULETYPE: polypeptide

(iii) SEQUENCE DESCRIPTION: SEQ ID NO.3:

Met Asn Ser Phe Ser Thr Ser Ala Phe Gly -19

Pro Val Ala Phe Ser Leu Gly Leu Leu Leu -9

Val Leu Pro Ala Ala Phe Pro Ala Pro Val 2

Pro Pro Gly Glu Asp Ser Lys Asp Val Ala 12

Ala Pro His Arg Gin Pro Leu Thr Ser Ser 22

Glu Arg lie Asp Lys Gin lie Arg Tyr lie 32

Leu Asp Gly lie Ser Ala Leu Arg Lys Glu 42

Thr Cys Asn Lys Ser Asn Met Cys Glu Per 52

Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu 62

Asn Leu Pro Lys Met Ala Glu Lys Asp Gly 72

Cys Phe Gin Ser Gly Phe Asn Glu Glu Thr 82

Cys Leu Val Lys lie lie Thr Gly Leu Leu 92

Glu Phe Glu Val Tyr Leu Glu Tyr Leu Gin 102

Asn Arg Phe Glu Ser Ser Glu Glu Gin Ala 112

Arg Ala Val Gin Met Ser Thr Lys Val Leu 122 lie Gin Phe Leu Gin Lys Lys Ala Lys Asn 132

Leu Asp Ala lie Thr Thr Pro Asp Pro Thr 142

Thr Asn Ala Ser Leu Leu Thr Lys Leu Gin 152

Ala Gin Asn Gin Trp Leu Gin Asp Met Thr 162

Thr His Leu lie Leu Arg Ser Phe Lys Glu 172

Phe Leu Gin Ser Ser Leu Arg Ala Leu Arg 182 Gin Met (5) INFORMATION FOR SEQ ID NO. 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 628

(B) TYPE: DNA

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(iii) SEQUENCE DESCRIPTION: SEQ ID NO. 4:

ATG AAC TCC TTC TCC ACA AGC GCC TTC GGT CCA GTT GCC 39

TTC TCC CTG GGG CTG CTC CTG GTG TTG CCT GCT GCC TTC 78

CCT GCC CCA TGA CCC CCA GGA GAA GAT TCC AAA GAT GTA 117

GCC GCC CCA CAC AGA CAG CCA CTC ACC TCT TCA GAA CGA 156

ATT GAC AAA CAA ATT CGG TAC ATC CTC GAC GGC ATC TCA 195

GCC CTG AGA AAG GAG ACA TGT AAC AAG AGT AAC ATG TGT 234

GAA AGC AGC AAA GAG GCA CTG GCA GAA AAC AAC CTG AAC 273

CTT CCA AAG ATG GCT GAA AAA GAT GGA TGC TTC CAA TCT 312

GGA TTC AAT GAG GAG ACT TGC CTG GTG AAA ATC ATC ACT 351

GCT CTT TTG GAG TTT GAG GTA TAC CTA GAG TAC CTC CAG 390

AAC AGA TTT GAG AGT AGT GAG GAA CAA GCC AGA GCT GTG 429

CAG ATG AGT ACA AAA GTC CTG ATC CAG TTC CTG CAG AAA 468

AAG GCA AAG AAT CTA GAT GCA ATA ACC ACC CCT GAC CCA 507

ACC ACA AAT GCC AGC CTG CTG ACG AAG CTG CAG GCA CAG 546

AAC CAG TGG CTG CAG GAC ATG ACA ACT CAT CTC ATT CTG 585

CGC AGC TTT AAG GAG TTC CTG CAG TCC AGC CTG AGG GCT 624 CTT CGG CAA ATG

Claims

CLAIMS:

1. An isolated amino acid variant of a interleukin-6 (IL-6), said variant capable of stimulating thrombocytopoiesis while exhibiting a low hybridoma growth factor activity compared to a non-variant EL-6.

2. An isolated amino acid variant of IL-6 according to claim 1 wherein said variant exhibits a hybridoma growth factor activity of less than 30% compared to a non-variant D -6.

3. An isolated amino acid variant of IL-6 according to claim 1 wherein said variant exhibits a hybridoma growth factor activity of less than 10% compared to a non-variant IL-6.

4. An isolated amino acid variant of IL-6 according to claim 1 wherein said IL-6 variant is of human or mouse origin.

5. An isolated amino acid variant of IL-6 according to claim 1 or 4 in recombinant form.

6. An isolated amino acid variant of IL-6 according to claim 1 wherein said variant comprises an N-terminal and/or C-terminal amino acid portion altered or otherwise modified relative to a non-variant EL-6.

7. An isolated amino acid variant of IL-6 according to claim 6 wherein said variant carries a single or multiple amino acid substitution, deletion and/or addition to a region comprising at least five amino acids from the C-terminal end and/ or to a region comprising at least 22 amino acids from the N-terminal end of a non-variant IL-6.

8. An isolated amino acid variant of IL-6 according to claim 7 wherein said variant has at least five but not more than 59 contiguous amino acids deleted from the C-terminal end and/or not more than approximately 22 contiguous amino acids from the N-terminal end of a non-variant D -6.

9. An isolated amino acid variant of IL-6 according to claim 8 lacking five contiguous amino acid residues from the C-terminal end of a non-variant IL-6.

10. An isolated amino acid variant of IL-6 according to claim 8 lacking 22 contiguous amino acid residues from the N-terminal end of non-variant IL-6.

11. An isolated amino acid variant of IL-6 according to claim 8 lacking 16 contiguous amino acid residues from the C-terminal end of non-variant IL-6.

12. An isolated amino acid variant of IL-6 according to claim 8 lacking 64 contiguous amino acid residues from the C-terminal end of non-variant IL-6.

13. An isolated amino acid variant of IL-6 according to claim 6 generated by reducing and alkylating a non-variant IL-6.

14. An isolated amino acid variant of IL-6 encoded by a first nucleic acid molecule having less than 100% nucleotide similarity to a second nucleic acid molecule encoding murine or human IL-6 said first nucleic acid molecule capable of hybridising under conditions of low stringency to said second nucleic acid molecule.

15. A nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an IL-6 variant, said variant capable of stimulating thrombocytopoiesis while exhibiting a low hybridoma growth factor activity compared to a non-variant IL-6.

16. A nucleic acid molecule according to claim 15 wherein the IL-6 variant is of human or mouse origin.

17. A nucleic acid molecule according to claim 15 wherein said IL-6 variant carries a single or multiple amino acid substitution, deletion and/or addition to a region comprising at least five amino acid residues from the C-terminal end and/or to a region comprising at least 22 amino acid residues from the N- terminal end of a non-variant IL-6.

18. A nucleic acid molecule according to claim 17 wherein the IL-6 variant has at least five but not more than 59 contiguous amino acids deleted from the C- terminal portion and/or not more than approximately 22 contiguous amino acids from the N-terminal portion of a non-variant DL-6.

19. A nucleic acid molecule according to claim 18 wherein the IL-6 variant lacks five contiguous amino acid residues from the C-terminal end of non-variant IL-6.

20. A nucleic acid molecule according to claim 18 wherein the IL-6 variant lacks 22 contiguous amino acid residues from the N-terminal end of non-variant IL-6.

21. A nucleic acid molecule according to claim 18 wherein the IL-6 variant lacks 16 contiguous amino acid residues from the C-terminal end of non-variant IL-6.

22. A nucleic acid molecule according to claim 18 wherein the IL-6 variant lacks 64 contiguous amino acid residues from the C-terminal end of non-variant IL-6.

23. An isolated nucleic acid molecule which:

(i) encodes a non-full length IL-6 molecule of animal origin, said non- full length molecule capable of stimulating thrombocytopoiesis while exhibiting a low hybridoma growth factor activity compared to a full length IL-6 molecule of same animal origin; and

(ii) hybridises under conditions of low stringency to one strand of a double stranded DNA molecule encoding a full length IL-6 molecule.

24. An isolated nucleic acid molecule according to claim 23 wherein the non- full length IL-6 molecule is of murine origin.

25. An isolated nucleic acid molecule according to claim 23 wherein the non- full length IL-6 molecule is of human origin.

26. A vector comprising the nucleic acid molecule according to any one of claims 15 or 18 to 25 operably linked to a promoter.

27. A vector according to claim 26 wherein the vector is pUC9.

28. A method of stimulating thrombocytopoiesis in an animal said method comprising administering to said animal a thrombocytopoietic stimulating effective amount of an IL-6 variant, said variant capable of stimulating thromboiesis while exhibiting a low hybridoma growth factor activity compared to a non-variant IL-6.

29. A method according to claim 28 wherein the IL-6 variant exhibits a hybridoma growth factor activity of less than 30% compared to a non-variant IL-6.

30. A method according to claim 28 wherein the EL-6 variant exhibits a hybridoma growth factor activity of less than 10% compared to a non-variant IL-6.

31. A method according to claim 28 wherein the variant IL-6 is in recombinan form.

32. A method according to claim 28 wherein the variant IL-6 comprises an N- terminal and /or C-terminal portion altered or otherwise modified relative to a non-variant IL-6.

33. A method according to claim 32 wherein the IL-6 variant carries a single or multiple amino acid substitution, deletion and/or addition to a region comprising at least five amino acids from the C-terminal end and/or to a region comprising at least 22 amino acids from the N-terminal end of a non-variant JL-6.

34. A method according to claim 33 wherein the IL-6 variant has at least five but not more than 59 contiguous amino acids deleted from the C-terminal end and/or not more than approximately 22 contiguous amino acids from the N- terminal end of a non-variant IL-6.

35. A method according to claim 33 wherein the IL-6 variant lacks five contiguous amino acid residues from the C-terminal end of a non-variant IL-6.

36. An isolated amino acid variant of IL-6 according to claim 34 lacking 22 contiguous amino acid residues from the N-terminal portion of non-variant IL-6.

37. An isolated amino acid variant of IL-6 according to claim 34 lacking 16 contiguous amino acid residues from the C-terminal portion of non-variant IL-6.

38. An isolated amino acid variant of IL-6 according to claim 34 lacking 64 contiguous amino acid residues from the C-terminal portion of non-variant EL-6.

39. An isolated amino acid variant of EL-6 according to claim 32 generated by reducing and alkylating a non-variant IL-6.

40. A pharmaceutical composition comprising an IL-6 variant according to claim 1 or 8 or 14 and one or more pharmaceutically acceptable carriers and/or diluents.