AU690128B2 - Haemopoietic growth factor antagonists - Google Patents

Description

I WO 95/046075 PCT/AU94/00432 -1- HAEMOPOIETIC GROWTH FACTOR ANTAGONISTS The present invention relates to modified and variant forms of haemopoietic growth factors capable of acting as antagonists to the corresponding native haemopoietic growth factors and their use in ameliorating aberrant effects caused by the native molecules.

Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description. Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined following the bibliography.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one member of a family of haemopoietic growth factors (HGFs) which have a similar predicted tertiary configuration (Parry et al, 1988) and whose receptors also belong to a common family (Gearing et al, 1989, Bazan, 1990). This family of haemopoietic growth factors includes, for example, in addition to GM-CSF, the cytokines IL-2, IL-3, IL-5, IL-6 and IL-10. A distinct subfamily comprising GM-CSF, IL-3 and IL-5 can be discerned based on structural similarities (Goodall et al, 1993) and on their ability to interact with a common receptor component (Lopez et al, 1992).

Human GM-CSF (bGM-CSF) comprises 127 amino acids and is available in recombinant form (rhGM-CSF). The hGM-CSF receptor has also been cloned and shown to comprise a binding chain exhibiting low affinity binding to GM-CSF (Gearing et al, 1989) and a second chain which does not measurably bind GM-CSF by itself but it allows the formation of a high affinity receptor when co-expressed with the a chain (Hayashide et al, 1990).

-9 ~b' WO 95/04075 PCT/AU94/00432 -2- GM-CSF exhibits a range of activities extending over neutrophil, eosinophil and monocyte lineages. Specifically, GM-CSF stimulates the progenitors of these cells to proliferate and differentiate to become mature cells. In addition, it stimulates mature cells to greater function. The stimulation of mature cells results in greater capacity to phagocytose and kill micro-organisms, kill antibody-coated tumour cells and parasites and generate superoxide anion in response to various stimuli. The purpose of this activation is presumed to enable the mature cells to become better effector cells in inflammatory reactions.

Therapeutically, the HGFs form an important group of molecules for their actual or potential properties. For example, the main indications for GM-CSF are for its effects on progenitor cells or mature cells. Using its effects on progenitor cells, GM-CSF is used in the treatment of bone marrow failure as seen in aplastic anaemia or chemotherapy or AIDS-induced marrow suppression. In the treatment of infections, the capacity to stimulate mature cells is especially relevant. The capacity of GM-CSF-activated neutrophils and eosincphils to kill tumour cells that have bound antibody is especially remarkable and could be used in tumour therapy.

However, despite the actual and potential benefits of HGFs, they can exhibit some detrimental side effects. For example, GM-CSF can exhibit toxicity due to stimulation of mature cells causing blood vessel damage or thrombosis. The eosinophilia caused by GM-CSF appears especially damaging in this regard. The molecule can also have detrimental effects by stimulating growth of leukaemia cells and tumour cells of nonhaemopoietic origin and stimulating production of inflammatory mediators.

International Patent Application No. PCT/AU89/00177 and an article by Lopez et al.

(1992) disclose amino acid variants of GM-CSF which have exhibited reduced potency.

These variants were investigated further for their potential as GM-CSF antagonists.

However, the variants cause classical stimulation at concentrations 100 fold greater compared to the native GM-CSF molecule. Furthermore, attempts to find antagonistic properties failed since mixing large concentrations of one of the variants with suboptimal concentrations of native GM-CSF resulted in stronger GM-CSF stimulation with no r I1 M7 L_ L L_ I ~I_ WO 95/04075 PCT/AU94/00432 -3evidence of inhibition being observed.

There is a need, therefore, to develop antagonists to HGFs and in particular GM-CSF which are capable of ameliorating the aberrant effects of the corresponding native molecules. There is also a need for such antagonists not to exhibit agonist properties in respect of the corresponding HGFs.

Accordingly, one aspect of the present invention provides a haemopoietic growth factor characterised by being in unglycosylated form and comprising a sequence of amino acids within a first a-helix wherein one or more exposed amino acids in said first a-helix having acidic or acidic-like properties are substituted with a basic amino acid residue.

In accordance with the present invention, it is proposed that the modified HGFs defined above act as antagonists of the native form of the corresponding HGF but not other HGFs. The term "modified" is considered herein synonymous with terms such as "variant", "derivative" and "mutant".

The HGFs are preferably GM-CSF, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, granulocyte colony-stimulating factor (G-CSF) and erythropoietin (EPO) modified in accordance with the present invention. Most preferably, the HGF is GM-CSF. The HGFs are preferably in recombinant or synthetic form and, with the exception of the amino acid substitution(s) in the first a-helix, the amino acid sequence of the HGF may be the same as the naturally occurring molecule native molecule) or may carry single or multiple amino acid substitutions, deletions and/or additions to the native amino acid sequence. The HGF sequences are preferably of mammalian origin such as from humans, livestock animals, companion animals or laboratory test animals. Most preferably, the HGFs are of human origin or of a mammalian origin capable of functioning in humans.

The first a-helix of GM-CSF has been determined at 2.4 angstrom resolution by X-ray crystallography and encompasses amino acid residues 13 to 28. Similar procedures may be adopted to determine the first a-helix in other haemopoietic growth factors. The position may also be determined by analogy to GM-CSF structure.

I WO 95/04075 PCTIAU94/00432 -4- Reference to "unglycosylated form" herein means that the molecule is completely unglycosylated such as when expressed in recombinant form in a prokaryotic organism E. coli). Alternatively, a glycosylation-deficient mammalian cell may be used or complete deglycosylation may occur in vitro using appropriate enzymes. Accordingly, the present invention extends to chemically synthesised GM-CSF which is in unglycosylated form.

An "exposed" amino acid is taken herein to refer to an amino acid on an exposed or outer portion of an a-helix compared to those amino acids orientated towards the inside of the molecule.

An acidic amino acid includes, for example, Glu and Asp. Preferred basic amino acids are Arg and Lys.

According to another aspect of the present invention, there is provided a haemopoietic growth factor characterised by: being in unglycosylated form; (ii) comprising a sequence of amino acids within the first a-helix; (iii) one or more exposed amino acids in said a-helix which have acidic or acidic-like properties being substituted by a basic amino acid residue; (iv) being in recombinant or synthetic form; being capable of acting as an antagonist for at least one property of the corresponding native HGF.

This aspect of the present invention is predicated in part on the surprising discovery that a mutation in amino acid 21 (Glu) of hGM-CSF to Arg or Lys together with the variant in GM-CSF being in unglycosylated form results in the hGM-CSF variant being unable to detectably exhibit classical GM-CSF function. The variants, referred to herein "GM- CSF Arg 21 and "GM-CSF Lys 2 1 are unable to bind to high affinity receptors but are still able to fully bind the low affinity a chain of the GM-CSF receptor. Importantly, the non-glycosylated GM-CSF Arg 2 1 and GM-CSF Lys 2 1 act as antagonists, preventing the stimulatory effect of native GM-CSF. For convenience, the numbering of amino acid

'I-

WO 95/04075 IPCT/AU94/00432 residues in hGM-CSF is taken from Wong et al. (1985).

By way of a shorthand notation the following three letter abbreviations for amino acid residues are used in the specification as defined in Table 1.

Where a specific amino residue is referred to by its position in the polypeptide of an HGF, the amino acid abbreviation is used with the residue number given in superscript Xaa", wherein Xaa is the amino acid residue).

Table 1 Amino acid Three-letter Corresponding abbreviation single-letter abbreviation Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gin Q Glutamic acid Glu E Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V -c -I I P _I _I C _I WO 95/04075 PCT/AU94/00432 -6- The present invention is exemplified using GM-CSF and in particular hGM-CSF Arg 2 1 and hGM-CSF Lys 21 This is done, however, with the understanding that the present invention extends to all HGFs as hereinbefore described.

For example, given that there is a Glu at position 22 of IL-3 and position 13 in which in the three dimensional structure (in the case of IL-5) occupies an equivalent position to Glu 21 of GM-CSF, then the present invention provides the basis for the creation of antagonists in IL-3 and IL-5. The substitution of Glu at these positions in IL- 3 and IL-5 may be the sole mutation or it may be in combination with other amino acid mutations (including substitutions, deletions and/or additions) for the development of effective antagonists. This similarly applies to other HGFs based on the acidic or acidiclike amino acid residues in the first a-helix of the molecule. The location of the Nterminal helix can in each case be readily determined on comparable motifs from predicted helices. Such HGFs are listed in Table 2 showing the acidic or acidic-like amino acid residue in bold in the equivalent position to Glu 21 of hGM-CSF. The acidic or acidic-like amino acid residues are readily substituted by, for example, recombinant DNA technology.

According to another aspect of the present invention there is provided a modified variant including HGF comprising an amino acid sequence in the first a-helix of said HGF selected from the group consisting of: i) His Val Asn Ala Ile Gin Xaa Ala Arg Arg Leu Leu Asn Leu (SEQ ID No. 1); ii) Ala Leu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu (SEQ ID No. 2); iii) Asn Met Ile Xaa Xaa Ile Ile Thr His Leu (SEQ ID No. 3); iv) Leu Leu Leu Xaa Leu Gin Met Ile Leu (SEQ ID No. 4); v) Ile Thr Leu Gin Xaa Ile Ile Lys Thr Leu (SEQ ID No. vi) Arg Tyr Ile Leu Xaa Gly Ile Ser Ala Leu Arg Lys (SEQ ID No. 6); vii) Gly Asp Gin Tyr Xaa Ser Val Leu Met Val Ser Ile (SEQ ID No. 7); s I -II- WO 95/04075 PCT/AU94/00432 -7viii) Ala Gly Ile Leu Xaa Ile Asn Phe Leu Ile Asn Lys Met Gin Glu Asp (SEQ ID No. 8); ix) Asn Met Leu Arg Xaa Leu Arg Asp Ala Phe Ser (SEQ ID No. 9); x) Phe Leu Leu Lys Cys Leu Xaa Gin Val Arg Lys Ile (SEQ ID No. 10); and xi) Tyr Leu Leu Glu Ala Lys Xaa Ala Glu Asn Ile Thr Thr Gly (SEQ ID No. 11) wherein Xaa is a basic amino acid, preferably selected from the group consisting of Arg and Lys, and wherein said variant HGF is in unglycosylated form and acts as an antagonist for at least one property of the corresponding native HGF. Preferably, the haemopoietic growth factor is hGM-CSF and Xaa is Arg or Lys at position 21 of the first c-helix.

The HGF antagonists of the present invention and in particular GM-CSF Arg 2 1 and GM- CSF Lys 21 are useful inter alia in the treatment of myeloid and lymphocyte leukaemias, some tumours of non-haemopoietic origins and acute and chronic inflammation such as asthma and rheumatoid arthritis. These and other conditions are considered herein to result from o' 1: facilitated by the aberrant effects of an endogenous HGF such as GM- CSF. hGM-CSF Arg 21 and hGM-CSF Lys 21 will also be useful in mobilising stem cells and progenitor cells into the circulation without the risk of activating neutrophils and monocytes. Other related molecules may have different useful properties.

The present invention, therefore, contemplates a method of treatment comprising the administration to a mammal of an effective amount of a modified HGF as hereinbefore defined and in particular hGM-CSF Arg 2 1 or hGM-CSF Lys 21 or both for a time and under conditions sufficient for effecting said treatment. Generally, the mammal is a human, livestock animal, companion animal or laboratory test animal. Most preferably, the mammal is a human. A single modified HGF may be administered or a combination of variants of the same HGF. For example, a range of hGM-CSF variants could be used such as a combination of hGM-CSF Arg 2 1 and hGM-CSF Lys 21 u 0 TABLE 2 Cytokines related to GM-CSF exhibit a conserved acidic residue analogous to E21 in GM-CSF

CYTOKINE

2 HELIX' AMINO ACID SEQUENCE SEQ ID (Amino Acid No.

Residue No.) hGM-CSF 15-28 His Val Asn Ala Ile Gin Giu Ala Arg Arg Leu Leu Asn Leu 12 9-24 Ala Leu Val Lys Giu Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu 13 hIL-3 18-27 Asn Met Ile Asp Glu Ile Ile Thr His Leu 14 hIL-2 17-25 Leu Leu Leu Asp Leu Gin Met Ile Leu hIL-4 8-17 Ilie Thr Leu Gin Asp Ile Ile Lys Thr Leu 16 hIL-6 18-43 Arg Tyr Ile Leu Asp Gly Ile Ser Ala Leu Arg Lys 17 hIL-7 9-20 Gly Asp Gin Tyr Giu Ser Val Leu Met Val Ser Ile 18 hIL-9 7-22 Ala Gly Ile Leu Asp Ile Asn Phe Leu Ile Asn Lys Met Gin Giu Asp 19 hIL-lO 21-31 Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser hG-CSF 13-24 Phe Leu Leu Lys Cys Leu Giu Gin Vai Arg Lys Ile 21 hEPO 4-28 Tyr Leu Leu Glu Ala Lys Giu Ala Giu Asn Ile Thr Thr Giy 22 1 oniy the pertinent portion of each helix is shown.

2 Predicted helices are from: for IL-2, (Braudhuber et al, 1987; Zurawski and Zurawski, 1989) for hIL-3 (Parry et al, 1988); for mIL-5, (Parry et al, 1988); for hIL-6 (Bazan, i990a); for hG-CSF, (Parry et al, 1988); for hEPO, (Bazan, 1990); for hGM-CSF the first helix was determined from the crystal structure (Karpius, 1991).- The iocation of the N-terminal helix in the> other cytokines was based on comparable motifs from these secondary structure predictions. C WO 95/04075 PCT/AU94/00432 -9- The present invention also provides a pharmaceutical composition comprising the variant HGFs as hereinbefore defined or combinations thereof. Most particularly, the pharmaceutical composition comprises hGM-CSF Arg 21 or hGM-CSF Lys 21 or both.

Methods for preparing pharmaceutical compositions are well known in the art and reference can conveniently be made to Remington's Pharmaceutical Sciences, Mack Publishing Company, Eaton, Pennsylvania, USA and may also include one or more pharmaceutical acceptable carriers and/or diluents.

The present invention is further described by reference to the following non-limiting Examples and/or Figures.

In the Figures: Figure 1 is a graphical representation showing titration of E. coli derived GM-CSF Arg 21 for its ability to affect 02" production in human neutrophils and to antagonise the enhancement of 02' by wild type GM-CSF tested at 1.0 ng/ml and at 0.1 ng/ml (closed triangle).

Figure 2 is a graphical representation showing failure of E. coli-derived GM-CSF Arg 2 1 to antagonise the enhancement of 02" production in human neutrophils stimulated with tumour necrosis factor-a (TNF-a).

Figure 3 is a graphical representation showing competitive inhibition of 1 25

I-GM-CSF

binding to COS cells transfected with the GM-CSF receptor a chain alone (top) or a and p chains (bottom) by GM-CSF Arg 21 Figure 4 is a graphical representation showing titration of GM-CSF Arg 2 1 for its ability to antagonise GM-CSF in contrast to no effect on IL-3 (B)-mediated proliferation of TF-1 cells.

WO 95/04075 PCT/AU94/00432 10 Figure 5 is a graphical representation showing the titration of GM-CSF Arg 2 1 for its ability to antagonise TF-1 proliferation stimulated by either E. coli-derived GM-CSF yeast-derived GM-CSF or CHO-derived GM-CSF Figure 6 is a graphical representation showing the titration of GM-CSF Arg 21 for its ability to antagonise three primary human myeloid leukaemia B C) ex vivo.

Figure 7 is a graphical representation showing that E21R antagonises both GM-CSF but not TNF-a-mediated stimulation of human neutrophils and both E21R and E21K also antagonise neurtrophil stimulation by CHO cell-derived GM-CSF In panel A, titrations of E. coli-derived wild type GM-CSF TNF-c and E21R are shown. In antagonistic experiments, E21R was titrated against Ing/ml of E. coli-derived GM-CSF or 3ng/ml TNF-a In panel B, titrations of CHO cell-derived wild type GM-CSF E21R and E21K are shown. In antagonistic experiments, E21R or E21K were titrated against 3ng/ml CHO cell-derived GM-CSF. Each value represents the mean of triplicate determinations and error bars represent the SEM.

EXAMPLE 1 Expression of wild type and GM-CSF Arg 21 and GM-CSF Lys 21 in an E. coli expressed system Wild type GM-CSF was expressed in E. coli using a plasmid (designated pshGM-CSF) containing a synthetic human GM-CSF cDNA cloned into the E. coli expression vector pIN-III-OmpH3, a derivative of the vector pIN-III-OmpA2 (Ghrayeb et al, 1984).

GME21R was expressed from the plasmid pSGM21.1 containing Glu 21 -Arg 2 1 substitution and was derived from the pSGM-CSF parental plasmid. GME21K was expressed from the plasmid pSGM21.4 containing Glu 21 Lys 2 1 substitution and was derived from the pSGM-CSF parental plasmid.

pSGM21 was generated by initially eliminating a SacII site from the wild type GM-CSF using oligonucleotide cassette mutagenesis to generate plasmid pSGMV1. A 64 bp Nco 1!SacII fragment was then excised from the pSGMV1 plasmid and replaced by doublestranded 64bp oligonucleotides containing the appropriate mutation in the DNA sequence.

WO 95/04075 PCT/AU94/00432 11 pSGM21.4 was generated by excising an 88 bp Bgl11/SacII fragment from pSGFI and replacing it with 88bp oligonucleotides containing the appropriate mutation site directed mutagenesis (Zoller Smith, 1984).

Protein was expressed in either MC1061, for wild type GM-CSF or BL21 for GME21R or GME21K, after induction by isopropyl p-D-thiogalactoside and recovered from the periplasmic space by osmotic shock (Koshland and Botstein, 1980).

GM-CSF protein was purified using a monoclonal antibody 4A12 generated in the laboratory coupled to Sepharose beads. Further purification was achieved by reversed phase HPLC using a BioRad controller and a Brownlee Aquaport C8 100 x column. GM-CSF was eluted using a 30-50% gradient of acetonitrile in 0.1% trifluoroacetic acid.

Resultant purified GM-CSF was lyophilised and resuspended in 1 x PBS before being quantitated by HPLC gel filtration. Samples were fractionated on a Beckman Ultraspherogel SEC3000 7.5 x 300mm using a 0.1M Na Phosphate pH 7.0/01M Na 2

SO

4 mobile phase. Purity was 3stimated at >95% and area under peaks corresponding to GM- CSF integrated as the extinction coefficient of 0.95 absorbance units.ml.mg 1 EXAMPLE 2 Visualisation of mutant GM-CSF protein GM-CSF unpurified or purified from E. coli was size-fractionated by NaDodSo 4 /12.5% w/v polycarylamide gel electrophoresis (Laemmli, 1970). For Western blot analysis, protein was transferred to nitrocellulose as described (Towbin et al, 1979). Filters were probed with a sheep anti-GM-CSF followed by a second layer of biotinylated-rabbit antisheep IgG. After a further incubation with an avidin-biotinylated-horseradish peroxidase conjugate, the complex was visualised using a diaminobenzidine substrate solution. For silver staining, the method of Morrissey (1981) was used.

'I 'I WO 95/04075 PCT/AU94/00432 12- EXAMPLE 3 Stimulation of haemopoietic cell proliferation The human erythroleukaemia cell line TF-1 (and myeloid leukaemia) cells were used to measure the proliferative function of GM-CSF and GM-CSF Arg 21 Proliferation of TFcells were measured by the ability to incorporate 3 H]-thymidine in response to increasing doses of GM-CSF. This assay was performed as described by Lopez et al (1988).

EXAMPLE 4 Functional activation of human granulocytes and monocytes The superoxide anion production assay was carried out as previously described (Lopez et al, 1986).

EXAMPLE Radioreceptor assay Radioiodination of GM-CSF.

Yeast derived human GM-CSF or E. coli-derived human GM-CSF was radioiodinatd by the IC method (Contreras et al, 1983). Iodinated protein was separated from free 1251 by chromatography on a Sephadex G-25 PD10 column (Pharmacia, Uppsala, Sweden), equilibrated in phosphate buffered saline (PBS) containing 0.02% w/v Tween 20, and stored at 4°C for up to 4 weeks. Before use, the iodinated protein was purified from Tween and non-protein-associated radioactivity by cation exchange chromatography on a 0.3ml CM-Sepharose CL-6B column (Pharmacia) and stored at 4 0 C for up to 5 days.

The radiolabeled GM-CSF retained >90% biological activity as judged from titration curves using non-iodinated GM-CSF as controls.

Competition binding assays.

Competition for binding to high affinity and low affinity receptors used stably transfected CHO cell lines expressing either the a and B chains, or the a chahi alone. The cells were suspended in binding medium consisting of RPMI-1640 supplemented with HEPES and 0.5% w/v bovine serum albumin (BSA) and 0.1% w/v sodium azide.

Typically, equal volumes (50Wl) of 4 x 104 CHO cells, iodinated GM-CSF and different

Y

WO 95/04075 PCT/AU94/00432 13 concentrations of GM-C3F and GM-CSF Arg 2 1 were mixed in siliconised glass tubes for 3 hr at 4°C. At the end of the incubation period, cell suspensions were overlaid on 0.2ml foetal calf serum (FCS) at 4 0 C, centrifuged in a Beckman Microfuge 12, and the tip of each tube containing the visible cell pellet cut off and counted in a gamma counter.

Specific counts were determined by first subtracting the counts, obtained in the presence of excess wild type GM-CSF.

EXAMPLE 6 Generation of hGM-CSF Variants By way of example only, the generation of GM-CSF Arg 21 is hereinafter described in detail. A human GM-CSF cDNA was subjected to mutagenesis to introduce the amino acid Arg for Glu at position 21. Two mutants were obtained, one containing the Glu 21 -+Arg mutation and a second one containing a double mutation X 10 -Ile and Glu 21 -*Arg. These mutants were cloned into the expression system piN OMPIII and expressed in E. coli. Wild type (WT) GM-CSF was expressed in MC1061. GM-CSF Arg 2 1 could not be expressed in MC1061. Of twenty strains tested for GM-CSF Arg 2 1 expression, BL21 was the highest producer and used for subsequent studies.

To obtain purified GM-CSF Arg 2 1 in high yields a two-step purification procedure was devised. In the first step, GM-CSF Arg 21 was purified by an affinity column constructed with a monoclonal antibody (4A12) that binds to GM-CSF in solution and with high affinity. Affinity purified GM-CSF Arg 2 1 was then purified by reverse-phase HPLC and quantitated by HPLC before being analysed for biological and binding activities.

It was found that E. coli-derived GM-CSF Arg 21 was unable to enhance neutrophil 02.

production up to a cont ntration of 3,000 ng/ml (Figure This is different to the inventors' previous results with CHO-derived glycosylated) GM-CSF Arg 2 1 which was able to enhance neutrophil function at approximately 30 ng/ml which represents a 300 fold reduced potency compared to wild type GM-C"F (Lopez et al, 1992).

WO 95/04075 PCT/AU94/00432 14- Despite its inability to activate neutrophils, E. coli-derived GM-CSF Arg 2 1 was able to bind as well as wild type GM-CSF to the a-chain of the GM-CSF receptor (Figure 3).

In contrast, GM-CSF Arg 21 exhibited an approximate 100-fold reduction in binding to thn ap GM-CSF receptor complex (Figure 3) indicating that the influence of the P chain has been selectively lost.

This is the first time that a GM-CSF mutant is shown to have unaltered binding to the GM-CSF receptor a-chain as well as being devoid of agonistic activity. This indicates that whilst binding to the a chain is necessary for GM-CSF activity it is not sufficient, and that GM-CSF binding to the p chain is required for GM-CSF-niediated activation.

Since E. coli-derived GM-CSF Arg 21 was not able to stimulate neutrophils yet fully bound the GM-CSF receptor a chain, it was tested for antagonistic activity. The inventors found that this mutant fully inhibited the effect of wild type GM-CSF with an approximately 300-fold excess required to induce 50% inhibition of E. coli-derived wild type GM-CSF (Figure This antagonistic effect was specific for GM-CSF as judged by the lack of antagonistic effect of GM-CSF Arg 21 on TNF enhancement of neutrophil 02" production (Figure 2).

The antagonistic effect of E. coli-derived GM-CSF Arg 21 was present whether wild type GM-CSF was expressed in E. coli (Figure 5A), yeast (Figure 5B) or CHO cells (Figure In keeping with the differences in binding affinity between heavily glycosylated CHO GM-CSF (lower affi-ity), partially glycosylated yeast GM-CSF (intermediate affinity) and unglycosylated E. coli GM-CSF (higher affinity), E. coli-derived GM-CSF Arg 2 1 antagonised better the CHO wild type GM-CSF (Figure The antagonism of E. coli-derived GM.CSF Arg 21 was not restricted to proliferation of the established TF-1 cell line but was also seen in primary myeloid leukaemias. In three different leukaemias, E. coli-derived GM-CSF Arg 21 antagonised the proliferative effect of wild type E. coli GM-CSF with an EC50 that varied with each leukaemia (Figure 6).

L WO 95/04075 PCT/AU94/00432 15 These results show that an unglycosylated GM-CSF molecule with a mutated Glu for an Arg in position 21 of the first a-helix is able to antagonise native GM-CSF.

Since the mutated Glu in GM-CSF is in a position where the same acidic residue or similar acidic residues Asp) are present in related growth factors (see Table this invention extends to antagonistic molecules for these growth factors constricted by incorporating the analogous charge reversal mutation. In particular, given that the GM- CSF, IL-3 and IL-5 receptor share the P chain, the Glu 22 in IL-3 and the Glu 1 3 in are predicted to play a similar role. Other variant HGFs are shown in Examples 3 to 22 in which the equivalent or similar amino acid residue to Glu 2 1 of hGM-CSF is replaced by either Arg or Lys. In these Examples, the amino acid sequences are provided for the relevant portion of the first a-helix carrying the substitution (see Table 2).

EXAMPLE 7 hGM-CSF Lys 21 His Val Asn Ala Ile Gin Lys Ala Arg Arg Leu Leu Asn Leu (SEQ ID NO. 23) EXAMPLE 8 Lys 13 Ala Leu Val Lys Lys Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu (SEQ ID NO.24) EXAMPLE 9 Arg 13 Ala Leu Val Lys Arg Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu (SEQ ID NO. EXAMPLE hlL-3 Variants Asn Met Ile Asp Lys Ile Ile Thr His Leu hIL-3 Lys 22 (SEQ ID NO. 26) Asn Met Ile Lys Glu Ile Ile Thr His Leu hIL-3 Lys 21 (SEQ ID NO. 27) Asn Met Ile Asp Arg Ile Ile Thr His Leu hIL-3 Arg 22 (SEQ ID NO. 28) Asn Met Ile Arg Glu Ile Ile Thr His Leu hIL-3 Arg 2 1 (SEQ ID NO. 29) Asn Met Ile Lys Lys Ile Ile Thr His Leu hIL-3 Lys 2 1 Lys 22 (SEQ ID NO. WO 95/04075 WO 9504075PCT/AU94/00432 16 Asn Met Ile Arg Arg le Ile Thr His Leu hIL-3 Arg 21 EXAMPLE 11 hIL-2 Lys 20 Leu Leu Leu Lys Leu Gin Met Ile Leu (SEQ ID NO. 32) EXAMPLE 12 hL0L-2 Arg 2 Leu Leu Leu Arg Leu Gin Met le Leu (SEQ ID NO. 33) EXAMPLE 13 hIL-4 Lys 12 le Tbr Leu Gin Lys le le Lys Thr Leu (SEQ ID NO. 34) EXAMPLE 14 hIL.-4 Arg 12 Ile Thr Leu Gin Arg le le Lys Thr Leu (SEQ ID NO. EXAMPLE hlL-6 LyS 22 Arg Tyr Ile Leu Lys Gly le Ser Ala Leu Arg Lys (SEQ ID EXAMPLE 16 h[L-.6 Arg 22 Arg Tyr Ile Leu Arg Giy Ile Ser Ala Leu Arg Lys (SEQ ID EXAMPLE 17 hIL-7 Lys" 3 Gly Asp Gin Tyr Lys Ser Vai Leu Met Vai Ser Ile (SEQ II Arg 22 (SEQ ID NO. 3 1) NO. 36) NO. 37) NO. 38)

I

WO 95/04075 PCT/AU94/00432 17 EXAMPLE 18 hIL,-7 Arg' 3 Giy Asp Gin Tyr Arg Ser Val Leu Met Val Ser Ile (SEQ ID NO. 39) EXAMPLE 19 hIL-9 Lys" 1 Ala Gly Ile Leu Lys Ile Asn Phe Leu Ile Asn Lys Met Gin Glu Asp (SEQ ID NO. EXAMPLE hIL-9 Arg 1 Ala Gly le Leu Arg Ile Asn Phe Leu le Asn Lys Met Gin Glu Asp (SEQ ID NO. 41) EXAMPLE 21 hIL-lO Lys 2 Asn Met Leu Arg Lys Leu Arg Asp Ala Phe Ser (SEQ ID NO. 42) EXAMPLE 22 hil-iD1 Arg 2 Asn Met Leu Arg Arg Leu Arg Asp Ala Phe Ser (SEQ ID NO. 43) EXAMPLE 23 hG-CSF Lys" 9 Phe Leu Leu Lys Cys Leu Lys Gin Val Arg Lys Ile (SEQ ID NO. 44) EXAMPLE 24 hG-CSF Arg 1 9 Phe Leu Lee~ Lys Cys Leu Arg Gin Val Arg Lys le (SEQ ID NO. m WO 95/04075 PCT/AU94/00432 18 EXAMPLE hEPO Lys 1 0 Tyr Leu Leu Glu Ala Lys Lys Ala Glu Asn Ile Thr Thr Gly (SEQ ID NO. 46) EXAMPLE 26 hEPO Arg 1 0 Tyr Leu Leu Glu Ala Lys Arg Ala Glu Asn Ile Thr Thr Gly (SEQ ID NO. 47) 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.

WO 95/04075 PTA9/03 IICT/AU94/00432 19

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I T~- WO 95/04075 PCT/AU94/00432 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT

(other than the US): MEDVET SCIENCE PTY LTD

APPLICANT/INVENTORS

(US only): LOPEZ, SHANNON, M.F. and VADAS, M.A.

(ii) TITLE OF INVENTION: "Haemopoietic Growth Factor Antagonists" (iii) NUMBER OF SEQUENCES: 47 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Davies Collison Cave STREET: 1 Little Collins Street CITY: Melbourne STATE: Victoria COUNTRY: Australia ZIP: 3000 COMPUTER READABLE FORM: MEDIUM TYPE: floppy disc COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: Patentln Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: International PCT App.

FILING DATE: 28-JUL-1994 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: Provisional AU PM0186 FILING DATE: 28-JUL-1993 APPLICATION NUMBER: Provisional AU PM4772 FILING DATE: 30-MAR-1993 (viii) ATTORNEY/AGENT INFORMATION: NAME: HUGHES, DR E JOHN L REFERENCE/DOCKET NUMBER: EJH/EK (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: +61 3 254 2777 TELEFAX: +61 3 254 2770 P~ 9 I_ WO 95/04075 PCT/AU94/00432 21 INFORMATION FOR SEQ ID NO. 1: SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acid residues TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 1: Hiq Val Asn Ala Ile Gln Xaa Ala Arg Arg Leu Leu Asn Leu INFORMATION FOR SEQ ID NO. 2: SEQUENCE CHARACTERISTICS: LENGTH: 16 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO, 2: Ala Leu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu INFORMATION FOR SEQ ID NO. 3: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acid residues TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 3: Asn Met Ile Xaa Xaa Ile Ile Thr -is Leu WO 95/04075 PCT/AU94/00432 -22- INFORMATION FOR SEQ ID NO. 4: SEQUENCE CHARACTERISTICS: LENGTH: 9 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 4: Leu Leu Leu Xaa Leu Gln Met Ile Leu INFORMATION FOR SEQ ID NO. SEQUENCE CHARACTERISTICS: LENGTH: TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. Ile Thr Leu Gin Xaa Ile Ile Lys Thr Leu INFORMATION FOR SEQ ID NO, 6: SEQUENCE CHARACTERISTICS: LENGTH: 12 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 6: Arg Tyr Ile Leu Xaa Gly Ile Ser Ala Leu Arg Lvs WO 95/04075 I'CT/AU94/00432 23 INFORMATION FOR SEQ ID NO. 7: SEQUENCE CHARACTERISTICS: LENGTH: 12 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: pclypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 7: Gly Asp Gin Tyr Xaa Ser Val Leu Met Val Ser Ile INFORMATION FOR SEQ ID NO. 8: SEQUENCE CHARACTERISTICS: LENGTH: 16 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 8: Ala Gly Ile Leu Xaa Ile Asn Phe Leu Ile Asn Lys Met Gin Glu Asp INFORMATION FOR SEQ ID NO. 9: SEQUENCE CHARACTERISTICS: LENGTH: 11 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 9: Asn Met Leu Arg Xaa Leu Arg Asp Ala Phe Ser PCT/AU94/00432 WO 95/04075 -24- INFORMATION FOR SEQ ID NO. SEQUENCE CHARACTERISTICS: LENGTH: 12 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. Phe Leu Leu Lys Cys Leu Xaa Gin Val Arg Lys Ile INFORMATION FOR SEQ ID NO. 11: SEQUENCE CHARACTERISTICS: LENGTH: 14 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 11: Tyr Leu Leu Glu Ala Lys Xaa Ala Glu Asn Ile Thr Thr Gly INFORMATION FOR SEQ ID NO. 12: SEQUENCE CHARACTERISTICS: LENGTH: 14 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 12: His Val Asn Ala Ile Gin Glu Ala Arg Arg Leu Leu Asn Leu WO 95/04075 P'CT/AU94/00432 25 INFORMATION FOR SEQ ID NO. 13: SEQUENCE CHARACTERISTICS: LENGTH: 16 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 13: Ala Leu Val Lys Glu Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu INFORMATION FOR SEQ ID NO. 14: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO, 14: Asn Met Ile Asp Glu Ile Ile Thr His Leu INFORMATION FOR SEQ ID NO. SEQUENCE CHARACTERISTICS: LENGTH: 9 TYPE: amino acid STRANDEDN': 1: single TOPOLOGY: .inear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. Leu Leu Leu Asp Leu Gln Met Ile Leu u I~I WO 95/04075 0PCT1AU94/00432 26- INFORMATION FOR SEQ ID NO. 16: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 16: Ile Thr Leu Gin Asp Ile i. Lys Thr Leu INFORMATION FOR SEQ ID NO. 17: SEQUENCE CHARACTERISTICS: LENGTH: 12 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 17: Arg Tyr Ile Leu Asp Gly Ile Ser Ala Leu Arg Lys INFORMATION FOR SEQ ID NO. 18: SEQUENCE CHARACTERISTCS: LENGTH: 12 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 18: Gly Asp Gin Tyr Glu Ser Val Leu Met Val Ser Ile I -r SWO 95/04075 -27- INFORMATION FOR SEQ ID NO. 19: PCT/AU94/00432 SEQUENCE CHARACTERISTICS: LENGTH: 16 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 19: Ala Gly Ile Leu Asp Ile Asn Phe Leu Ile Asn Lys Met Gin Glu Asp INFORMATION FOR SEQ ID NO. SEQUENCE CHARACTERISTICS: LENGTH: 11 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser INFORMATION FOR SEQ ID NO. 21: SEQUENCE CHARACTERISTICS: LENGTH: 12 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 21: Phe Leu Leu Lys Cys Leu Glu Gin Val Arg Lys Ile -~pM C1- WO 95/04075 28 INFORMATION FOR SEQ ID NO. 22: I'CT/AU94/00432 SEQUENCE CHARACTERISTICS: LENGTH: 14 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 22: Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly INFORMATION FOR SEQ ID NO. 23: SEQUENCE CHARACTERISTICS: LENGTH: 14 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 23: His Val Asn Ala Ile Gln Lys Ala Arg Arg Leu Leu Asn Leu INFORMATION FOR SEQ ID NO. 24: SEQUENCE CHARACTERISTICS: LENGTH: 16 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 24: Ala Leu Val Lys Lys Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu WO 95/04075 PCT/AU94/00432 29- INFORMATION FOR SEQ ID NO. SEQUENCE CHARACTERISTICS: LENGTH- 16 TYPE: anino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. Ala Leu Val Lys Arg Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu INFORMATION FOR SEQ ID NO. 26: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 26: Asn Met Ile Asp Lys Ile Ile Thr His Leu INFORMATION FOR SEQ ID NO. 27: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 27: Asn Met Ile Lys Glu Ile Ile Thr His Leu r I -I SWO 95/04075 PCT/AU94/00432 INFORMATION FOR SEQ ID NO. 28: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 28: Asn Met Ile Asp Arg Ile Ile Thr His Leu INFORMATION FOR SEQ ID NO. 29: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: amino acid STRANL.SDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 29: Asn Met Ile Arg Glu Ile Ile Thr His Leu INFORMATION FOR SEQ ID NO. SEQUENCE CHARACTERISTICS: LENGTH: TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. Asn Met Ile Lys Lys Ile Ile Thr His Leu

I

eb~r. WO 95/04075 -31 INFORMATION FOR SEQ ID NO. 31: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 31: Asn Met Ile Arg Arg Ile Ile Thr His Leu INFORMATION FOR SEQ ID NO. 32: SEQUENCE CHARACTERISTICS: LENGTH: 9 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 32: Leu Leu Leu Lys Leu Gin Met Ile Leu INFORMATION FOR SEQ ID NO, 33: SEQUENCE CHARACTERISTICS: LENGTH: 9 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 33: PCI/AU94/00432 Leu Leu Leu Arg Leu Gin Met Ile Leu 41 WO 95/04075 32 INFORMATION FOR SEQ ID NO. 34: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 34: Ile Thr Leu Gin Lys Ile Ile Lys Thr Leu INFORMATION FOR SEQ ID NO. SEQUENCE CHARACTERISTICS: LENGTH: TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. Ile Thr Leu Gln Arg Ile Ile Lys Thr Leu INFORMATION FOR SEQ ID NO. 36: SEQUENCE CHARACTERISTICS: LENGTH: 12 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 36: PCT/AU94/00432 Arg Tyr Ile Leu Lys Gly Ile Ser Ala Leu Arg Lys F 1- I WO 95/04075 'CT/AU94/00432 -33 INFORMATION FOR SEQ ID NO. 37: SEQUENCE CHARACTERISTICS: LENGTH: 12 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 37: Arg Tyr Ile Leu Arg Gly Ile Ser Ala Leu Arg Lys INFORMATION FOR SEQ ID NO. 38: SEQUENCE CHARACTERISTICS: LENGTH: 12 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 38: Gly Asp Gln Tyr Lys Ser Val Leu Met Val Ser Ile INFORMATION FOR SEQ ID 1O. 39: SEQUENCE CHARACTERISTICS: LENGTH: 12 TYPE: amino acie STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 39: Gly Asp Gln Tyr Arg Ser Val Leu Met Val Ser Ile _I C_ I 1~_1~ WO 95/04075 PCT/AU94/00432 -34 INFORMATION FOR SEQ ID NO. SEQUENCE CHARACTERISTICS: LENGTH: 16 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. Ala Gly Ile Leu Lys Ile Asn Phe Leu Ile Asn Lys Met Gin Glu Asp INFORMATION FOR SEQ ID NO. 41: SEQUENCE CHARACTERISTICS: LENGTH: 16 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 41: Ala Gly Ile Leu Arg Ile Asn Phe Leu Ile Asn Lys Met Gin Glu Asp INFORMATION FOR SEQ ID NO. 42: SEQUENCE CHARACTERISTICS: LENGTH: 11 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 42: Asn Met Leu Arg Lys Leu Arg Asp Ala Phe Ser WO 95/04075 PCT/AU94/00432 INFORMATION FOR SEQ ID NO. 43: SEQUENCE CHARACTERISTICS: LENGTH: 11 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 43: Asn Met Leu Arg Arg Leu Arg Asp Ala Phe Ser INFORMATION FOR SEQ ID NO. 44: SEQUENCE CHARACTERISTICS: LENGTH: 12 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 44: Phe Leu Leu Lys Cys Leu Lys Gln Val Arg Lys Ile INFORMATION FOR SEQ ID NO. SEQUENCE CHARACTERISTICS: LENGTH: 12 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. Phe Leu Leu Lys Cys Leu Arg Gln Val Arg Lys Ile WO 95/04075 PCT/AU94/00432 -36- INFORMATION FOR SEQ ID NO, 46: SEQUENCE CHARACTERISTICS: LENGTH: 14 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 46: Tyr Leu Leu Glu Ala Lys Lys Ala Glu Asn Ile Thr Thr Gly INFORMATION FOR SEQ ID. NO. 47: SEQUENCE CHARACTERISTICS: LENGTH: 14 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 47: Tyr Leu Leu Glu Ala Ly Arg Ala Glu Asn Ile Thr Thi Gly cl

Claims (12)

1. A modified haemopoietic growth factor (HGF) characterised by being in unglycosylated form and comprising a sequence of amino acids within a first a-helix wherein one or more exposed amino acids in said first a-helix having acidic or acidic-like properties are substituted with a basic amino acid residue.

2. A modified HGF according to claim 1, wherein said HGF is a modified form of an HGF s, :ted from granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukir IL-3, IL-4, IL-5, IL-6, 11-7, IL-9, IL-10, G-CSF, erythropoietin (EPO).

3. A modified HGF according to claim 2 wherein said HGF is a modified form of GM-CSF.

4. A modified HGF according to claim I or 2 or 3 wherein said HGF is of human, livestock animal, companion animal or laboratory test animal origin. A modified HGF according to claim 4 wherein said HGF is of human origin.

6. A modified HGF according to any one of the preceding claims wherein the acidic amino acid residue on the first a-helix is Glu and/or Asp and the basic amino acid residue substituted therefor is Arg and/or Lys.

7. A modified HGF comprising an amino acid sequence in the first a-helix of said HGF selected from the group consisting of: i) His Val Asn Ala Ile Gin Xaa Ala Arg Arg Leu Leu Asn Leu; ii) Ala Leu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu; iii) Asn Met Ile Xaa Xaa Ile Ile Thr His Leu; iv) Leu Leu Leu Xaa Leu Gin Met Ile Leu; v) Ile Thr Leu Gin Xaa Ile Ile Lys Thr Leu; WO 95/04075 P'CT/AU94/00432 38 vi) Arg Tyr Ile Leu Xaa Gly Ile Ser Ala Leu Arg Lys; vii) Gly Asp Gin Tyr Xaa Ser Val Leu Met Val Ser Ile; viii) Ala Gly Ile Leu Xaa Ile Asn Phe Leu Ile Asn Lys Met Gin Glu Asp; ix) Asn Met Leu Arg Xaa Leu Arg Asp Ala Phe Ser; x) Phe Leu Leu Lys Cys Leu Xaa Gin Val Arg Lys Ile; xi) Tyr Leu Leu Glu Ala Lys Xaa Ala Glu Asn Ile Thr Thr Gly; wherein Xaa is a basic amino acid, selected from the group consisting of Arg and Lys and wherein said modified HGF is in unglycosylated form and acts as an antagonist for at least one property of the corresponding native HGF.

8. A modified HGF according to claim 7 wherein the HGF is a modified form of an HGF selected from the list consisting of GM-CSF, IL-2, IL-3, IL-4, IL-5, IL-6, 11-7, IL-9, G-CSF, EPO.

9. A modified HGF according to claim 8, wherein the variant HGF is a modified form of GM-CSF. A modified HGF according to claim 7 or 8 wherein Xaa is Arg.

11. A modified human GM-CSF in unglycosylated form having an amino acid sequence in a first a-helix comprising His Val Asn Ala Ile Gin Arg Ala Arg Arg Leu Leu Asn Leu.

12. A modified human GM-CSF in unglycosylated form having an amino acid sequence in a first a-helix comprising His Val Asn Ala Ile Gin Lys Ala Arg Arg Leu Leu Asn Leu.

13. A modified human IL-5 in unglycosylated form having an amino acid sequence in a first a-helix comprising Ala Leu Val Lys Lys Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu. L WO 95/04075 W CT/AU94/00432

39- 14. A modified human IL-5 in unglycosylated form having an amino acid sequence in a first a-helix comprising Ala Leu Val Lys Arg Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu. A modified human IL-3 in unglycosylated form having an amino acid sequence in a first a-helix selected from the list consisting of: Asn Met Ile Asp Lys Ile Ile Thr His Leu; Asn Met Ile Lys Glu Ile Ile Thr His Leu; Asn Met Ile Asp Arg Ile Ile Thr His Leu; Asn Met Ile Arg Glu Ile Ile Thr His Leu; Asn Met Ile Lys Lys Ile Ile Thr His Leu; and Asn Met Ile Arg Arg Ile Ile Thr His Leu. 16. A modified human IL-2 in unglycosylated form having an amino acid sequence in a first a-helix comprising Leu Leu Leu Lys Leu Gin Met Ile Leu. 17. A modified human IL-2 in unglycosylated form having an amino acid se,''enc, in a first a-helix comprising Leu Leu Leu Arg Leu Gin Met Ile Leu. 18. A modified human IL-4 in unglycosylated form having an amino acid sequence in a first a-helix comprising Ile Thr Leu Gin Lys Ile Ile Lys Thr Leu. 19. A modified human IL-4 in unglycosylated form having an amino acid sequence in a first a-helix comprising lie Thr Leu Gin Arg Ile Ile Lys Thr Leu. A modified human IL-4 in unglycosylated form having an amino acid sequence in a first a-helix comprising Arg Tyr Ile Leu Lys Gly Ile Ser Ala Leu Arg Lys. 21. A modified human IL 4 in unglycosylated form having an amino acid sequence in a first a-helix comprising Arg Tyr Ile Leu Arg Gly Ile Ser Ala Leu Arg Lys. 22. A modified human IL-4 in unglycosylated form having an amino acid sequence in a first a-helix comprising *t Gly Asp Gin Tyr Lys Ser Val Leu Met Val Ser lie. 23. A modified human IL-4 in unglycosylated form having an amino acid sequence in a first a-helix comprising SGly Asp Gin Tyr Arg Ser Val Leu Met Val Ser Ile, 24. A modified human IL-4 in unglycosylated form having an amino acid sequence in a first a-helix comprising Ala Gly Ile Leu Lys Ile Asn Phe Leu Ile Asn Lys Met Gin Glu Asp. 0oO 25. A moditied human IL-4 in unglycosylated form having an amino acid sequence in a first c-helix comprising Ala Gly Ile Leu Arg Ile Asn Phe Leu Ile Asn Lys Met Gin Glu Asp. 26. A modified human IL-4 in unglycosylated form having an amino acid sequence in a first a-helix comprising Asn Met Leu Arg Lys Leu Arg Asp Ala Phe Ser. 27. A modified human IL-4 in unglycosylated form having an amino acid sequence in a first a-helix comprising s=Asn Met Leu Arg Arg Leu Arg Asp Ala Phe Ser. l-t WO 95/0407! PCT/AU94/00432 41 28. A modified human IL-4 in unglycosylated form having an amino acid sequence in a first a-helix comprising Phe Leu Leu Lys Cys Leu Lys Gin Val Arg Lys Ile. 29. A modified human IL-4 in unglycosylated form having an amino acid sequence in a first a-helix comprising Phe Leu Leu Lys Cys Leu Arg Gin Val Arg Lys IlI, A modified human IL-4 in unglycosylated form having an amino acid sequence in a first a-helix comprising Tyr Leu Leu Glu Ala Lys Lys Ala Glu Asn Ile Thr Thr Gly. 31. A modified human IL-4 in unglycosylated form having an amino acid sequence in a first a-helix comprising Tyr Leu Leu Glu Ala Lys Arg Ala Glu Asn Ile Thr Thr Gly. 32. A method of ameliorating the aberrant effects of an endogenous HGF in a mammal, said method comprising administering to said mammal an effective amount of a modified HGF characterised by being in unglycosylated form and comprising a sequence of amino acids within a first a-heliz wherein one or more exposed amino acids in said first a-helix having acidic or acidic-like properties are substituted with a basic amino acid residue. 33. A method according to claim 32 wherein the modified HGF is selected from the group consisting of: i) His Val Asn Ala Ile Gin Xaa Ala Arg Arg Leu Leu Asn Leu; ii) Ala Leu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu; iii) Asn Met Ile Xaa Xaa Ile Ile Thr His Leu; iv) Leu Leu Leu Xaa Leu Gin Met Ile Leu; v) Ile Thr Leu Gin Xaa Ile Ile Lys Thr Leu; vi) Arg Tyr Ile Leu Xaa Gly Ile Ser Ala Leu Arg Lys; vii) Gly Asp Gin Tyr Xaa Ser Val Leu Met Val Ser Ile; WO 95/04075S PCT/AU94/00432 -42- viii) Ala Gly Ile Leu Xaa Ile Asn Phe Leu Ile Asn Lys Met Gln Glu Asp; ix) Asn Met Leu Arg Xaa Leu Arg Asp Ala Phe Ser; x) Phe Leu Leu Lys Cys Leu Xaa Gin Val Arg Lys Ile; and xi) Tyr Leu Leu Glu Ala Lys Xaa Ala Glu Asn Ile Thr Thr Gly; wherein Xaa is a basic amino acid selected from the group consisting of Arg and Lys and wherein said variant HGF is in unglycosylated form and acts as an antagonist for at least one property of the corresponding native HGF. 34. A method according to claim 32 or 33 wherein the HGF is a modified form of an HGF selected from GM-CSF, IL-2, IL-3, IL-4, IL-5, IL-6, 11-7, IL-9, IL-10, G-CSF, EPO. A method according to claim 32 or 33 wherein the modified HGF is a modified human GM-CSF having an amino acid sequence in a first a-helix comprising His Val Asn Ala Ile Gin Arg Ala Arg Arg Leu Leu Asn Leu. 36. A method according to claim 32 or 33 wherein the modified HGF is a modified human GM-CSF having an amino acid sequence in a first a-helix comprising His Val Asn Ala Ile Gin Lys Ala Arg Arg Leu Leu Asn Leu. 37. Use of one or more modified HGFs each as defined in claim 1 or 7 in the manufacture of a medicamen' for the treatment of the affects of an aberrant endogenous HGF. 38. An agent comprising one or more modified HGFs each as defined in claim 1 or 7 for treating the affects of an aberrant endogenous HGF. 39. A pharmaceutical composition comprising one or more modified HGFs each as defined in claim 1 or 7 together with one or more pharmaceutically acceptable carriers and/or diluents. I