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 defmed 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 (hGM-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 (α) chain exhibiting low affinity binding to GM-CSF (Gearing et al, 1989) and a second (β) 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 α chain (Hayashide et al, 1990).

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 (O ₂ ^" ) 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 eosinophils 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 non- haemopoietic 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

evidence 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 α-helix wherein one or more exposed amino acids in said first α-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, IL-10, 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 α-helix, the amino acid sequence of the HGF may

, be the same as the naturally occurring molecule (i.e. 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 α-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 α-helix in other haemopoietic growth factors. The position may also be determined by analogy to GM-CSF structure.

Reference to "unglycosylated form" herein means that the molecule is completely unglycosylated such as when expressed in recombinant form in a prokaryotic organism (e.g. E. colϊ). 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 α-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:

(i) being in unglycosylated form;

(ii) comprising a sequence of amino acids within the first α-helix; (iii) one or more exposed amino acids in said α-helix which have acidic or acidic-like properties being substituted by a basic amino acid residue;

(iv) being in recombinant or synthetic form;

(v) 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 ²¹ " are unable to bind to high affinity receptors but are still able to fully bind the low affinity α chain of the GM-CSF receptor. Importantly, the non-glycosylated GM-CSF Arg ²¹ and GM-CSF Lys ²¹ act as antagonists, preventing the stimulatory effect of native GM-CSF. For convenience, the numbering of amino acid

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 (i.e. Xaa ⁿ , 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 lie 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

The present invention is exemplified using GM-CSF and in particular hGM-CSF Arg 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 IL-5 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 acidic- like amino acid residues in the first α-helix of the molecule. The location of the N- terminal 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 α-helix of said HGF selected from the group consisting of:

i) His Val Asn Ala lie 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 He Xaa Xaa He He Thr His Leu (SEQ ID No. 3) ; iv) Leu Leu Leu Xaa Leu Gin Met He Leu (SEQ ID No. 4) ; v) He Thr Leu Gin Xaa He He Lys Thr Leu (SEQ ID No. 5) ; vi) Arg Tyr He Leu Xaa Gly He Ser Ala Leu Arg Lys

(SEQ ID No. 6) ; vii) Gly Asp Gin Tyr Xaa Ser Val Leu Met Val Ser He (SEQ ID No. 7) ;

viii) Ala Gly He Leu Xaa He Asn Phe Leu He 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 He

(SEQ ID No. 10) ; and xi) Tyr Leu Leu Glu Ala Lys Xaa Ala Glu Asn He 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 α-helix.

The HGF antagonists of the present invention and in particular GM-CSF Arg ²¹ 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 or be 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 defmed and in particular hGM-CSF Arg ²¹ 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 ²¹ and hGM-CSF Lys ²¹ .

TABLE 2 Cytokines related to GM-CSF exhibit a conserved acidic residue analogous to E21 in GM-CSF

CYTOKINE ² HELIX ¹ AMINO ACID SEQUENCE SEQ ID (Amino Acid No. Residue No.)

hIL-10 21-31 Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser 20 hG-CSF 13-24 Phe Leu Leu Lys Cys Leu Glu Gin Val Arg Lys He 21 hEPO 4-28 Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn He Thr Thr Gly 22

Only the pertinent portion of each helix is shown.

Predicted helices are from: for IL-2, (Brandhuber 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, 1990a); for hG-CSF, (Parry et al, 1988) ; for hEPO, (Bazan, 1990) ; for hGM-CSF the first helix was determined from the crystal structure (Karplus, 1991) . The location of the N-terminal helix in the other cytokines was based on comparable motifs from these secondary structure predictions.

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 0 ₂ ^" production in human neutrophils (D) and to antagonise the enhancement of 0 ₂ ^" by wild type GM-CSF tested at 1.0 ng/ml (Δ) and at 0.1 ng/ml (closed triangle).

Figure 2 is a graphical representation showing failure of E. cø/ -derived GM-CSF Arg ²¹ to antagonise the enhancement of 0 ₂ ^" production in human neutrophils stimulated with rumour necrosis factor-α (TNF-α).

Figure 3 is a graphical representation showing competitive inhibition of ¹²⁵ I-GM-CSF binding to COS cells transfected with the GM-CSF receptor α chain alone (top) or α and β chains (bottom) by GM-CSF Arg ²¹ .

Figure 4 is a graphical representation showing titration of GM-CSF Arg ²¹ for its ability to antagonise GM-CSF (A) in contrast to no effect on IL-3 (B)-mediated proliferation of TF-1 cells.

Figure 5 is a graphical representation showing the titration of GM-CSF Arg for its ability to antagonise TF-1 proliferation stimulated by either E. co/t ^' -derived GM-CSF (A), yeast-derived GM-CSF (B) or CHO-derived GM-CSF (C).

Figure 6 is a graphical representation showing the titration of GM-CSF .Arg for its ability to antagonise three primary human myeloid leukaemia (A, B & C) ex vivo.

Figure 7 is a graphical representation showing that E21R antagonises both GM-CSF but not TNF-α-mediated stimulation of human neutrophils (A), and both E21R and E21K also antagonise neurtrophil stimulation by CHO cell-derived GM-CSF (B). In panel A, titrations of E. co/ -derived wild type GM-CSF (•), TNF- ( ♦ ) and E21R (■) are shown. In antagonistic experiments, E21R was titrated against 1 ng/ml of E. coli-deήxeά GM-CSF (D) or 3ng/ml TNF-α (0). In panel B, titrations of CHO cell-derived wild type GM-CSF (O), E21R (■) and E21K (A) are shown. In antagonistic experiments, E21R (D) or E21K (Δ) 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. co// 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^→Arg ²¹ substitution and was derived from the pSGM-CSF parental plasmid. GME21K was expressed from the plasmid pSGM21.4 containing Glu ²¹ → Lys ²¹ 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 pSGMVl. A 64 bp Nco 1/SacII fragment was then excised from the pSGMVl plasmid and replaced by double- stranded 64bp oligonucleotides containing the appropriate mutation in the DNA sequence.

pSGM21.4 was generated by excising an 88 bp Bgll l/SacII fragment from pSGFl 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 β-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 10mm 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 estimated 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 anti- sheep 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.

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 TF- 1 cells 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 5 Radioreceptor assay

(a) Radioiodination of GM-CSF.

Yeast derived human GM-CSF or E. co/t-derived human GM-CSF was radioiodinated by the IC1 method (Contreras et al, 1983). Iodinated protein was separated from free ¹²⁵ I by chromatography on a Sephadex G-25 PD10 column (Phaπnacia, 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°C for up to 5 days. The radiolabelled GM-CSF retained >90% biological activity as judged from titration curves using non-iodinated GM-CSF as controls.

(b) Competition binding assays.

Competition for binding to high affinity and low affinity receptors used stably transfected CHO cell lines expressing either the α and β chains, or the α chain alone. The cells were suspended in binding medium consisting of RPMI-1640 supplemented with 20mmol/l HEPES and 0.5% w/v bovine serum albumin (BSA) and 0.1% w/v sodium azide. Typically, equal volumes (50μl) of 4 x 10 ⁴ CHO cells, iodinated GM-CSF and different

concentrations of GM-CSF and GM-CSF Arg ²¹ 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°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 ²¹ could not be expressed in MCI 061. Of twenty strains tested for GM-CSF Arg ²¹ expression, BL21 was the highest producer and used for subsequent studies.

To obtain purified GM-CSF .Arg ²¹ 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 ²¹ was then purified by reverse-phase HPLC and quantitated by HPLC before being analysed for biological and binding activities.

It was found that E. co/i ^' -derived GM-CSF Arg ²¹ was unable to enhance neutrophil 0 ₂ _ production up to a concentration of 3,000 ng/ml (Figure 1). This is different to the inventors' previous results with CHO-derived (i.e. glycosylated) GM-CSF Arg ²¹ which was able to enhance neutrophil function at approximately 30 ng/ml which represents a 300 fold reduced potency compared to wild type GM-CSF (Lopez et al, 1992).

Despite its inability to activate neutrophils, E. cø/t-derived GM-CSF Arg ²¹ was able to bind as well as wild type GM-CSF to the α-chain of the GM-CSF receptor (Figure 3). In contrast, GM-CSF Arg ²¹ exhibited an approximate 100-fold reduction in binding to the αβ GM-CSF receptor complex (Figure 3) indicating that the influence of the β 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 α-chain as well as being devoid of agonistic activity. This indicates that whilst binding to the α chain is necessary for GM-CSF activity it is not sufficient, and that GM-CSF binding to the β chain is required for GM-CSF-mediated activation.

Since E. coli-deήved GM-CSF Arg ²¹ was not able to stimulate neutrophils yet fully bound the GM-CSF receptor α 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. cø/t ^' -derived wild type GM-CSF (Figure 1). 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 0 ₂ ^" production (Figure 2).

The antagonistic effect of E. coli-deήved 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 5C). In keeping with the differences in binding affinity between heavily glycosylated CHO GM-CSF (lower affinity), partially glycosylated yeast GM-CSF (intermediate affinity) and unglycosylated E. coli GM-CSF (higher affinity), E. cø//-derived GM-CSF Arg ²¹ antagonised better the CHO wild type GM-CSF (Figure 5).

The antagonism of E. cø//-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. cø/z-derived GM-CSF Arg ²¹ antagonised the proliferative effect of wild type E. coli GM-CSF with an ΕC50 that varied with each leukaemia (Figure 6).

These results show that an unglycosylated GM-CSF molecule with a mutated Glu for an Arg in position 21 of the first α-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 (e.g. Asp) are present in related growth factors (see Table 2), this invention extends to antagonistic molecules for these growth factors constructed by incorporating the analogous charge reversal mutation. In particular, given that the GM- CSF, IL-3 and IL-5 receptor share the β chain, the Glu ²² in IL-3 and the Glu ¹³ in IL-5 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 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 α-helix carrying the substitution (see Table 2).

EXAMPLE 7 hGM-CSF Lys ²¹

His Val Asn Ala He Gin Lys Ala Arg Arg Leu Leu Asn Leu (SEQ ID NO. 23)

EXAMPLE 8 hlL-5 Lys ¹³ Ala Leu Val Lys Lys Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu (SEQ ID NO.24)

EXAMPLE 9 hlL-5 Arg ¹³

Ala Leu Val Lys Arg Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu (SEQ ID NO. 25)

EXAMPLE 10 hlL-3 Variants Asn Met He Asp Lys lie He Thr His Leu hIL-3 Lys ²² (SEQ ID NO. 26)

Asn Met He Lys Glu He He Thr His Leu hIL-3 Lys ²¹ (SEQ ID NO. 27) Asn Met He Asp Arg He He Thr His Leu hIL-3 Arg ²² (SEQ ID NO. 28)

Asn Met He Arg Glu He He Thr His Leu hIL-3 Arg ²¹ (SEQ ID NO. 29)

Asn Met He Lys Lys He He Thr His Leu hIL-3 Lys ²¹ Lys ²² (SEQ ID NO. 30)

Asn Met He Arg Arg He He Thr His Leu hIL-3 Arg ²¹ Arg ²² (SEQ ID NO. 31)

EXAMPLE 11 hH -2 Lys ²⁰ Leu Leu Leu Lys Leu Gin Met He Leu (SEQ ID NO. 32)

EXAMPLE 12 hIL-2 Arg ²⁰ Leu Leu Leu Arg Leu Gin Met He Leu (SEQ ID NO. 33)

EXAMPLE 13 hIL-4 Lys ¹² He Thr Leu Gin Lys He He Lys Thr Leu (SEQ ID NO. 34)

EXAMPLE 14 hIL-4 Arg ¹² He Thr Leu Gin Arg He He Lys Thr Leu (SEQ ID NO. 35)

EXAMPLE 15 hIL-6 Lys ²²

Arg Tyr He Leu Lys Gly He Ser Ala Leu Arg Lys (SEQ ID NO. 36)

EXAMPLE 16 hIL-6 Arg ²² Arg Tyr He Leu Arg Gly He Ser Ala Leu Arg Lys (SEQ ID NO. 37)

EXAMPLE 17 hIL-7 Lys ¹³

Gly Asp Gin Tyr Lys Ser Val Leu Met Val Ser He (SEQ ID NO. 38)

EXAMPLE 18 hIL-7 Arg ¹³

Gly Asp Gin Tyr Arg Ser Val Leu Met Val Ser He (SEQ ID NO. 39)

EXAMPLE 19 hIL-9 Lys ¹¹

Ala Gly He Leu Lys He Asn Phe Leu He Asn Lys Met Gin Glu Asp (SEQ ID NO. 40)

EXAMPLE 20 hIL-9 Arg ¹¹

Ala Gly He Leu Arg He Asn Phe Leu He Asn Lys Met Gin Glu Asp (SEQ ID NO. 41)

EXAMPLE 21 hIL-10 Lys ²⁵

Asn Met Leu Arg Lys Leu Arg Asp Ala Phe Ser (SEQ ID NO. 42)

EXAMPLE 22 hIL-10 Arg ²⁵ Asn Met Leu Arg Arg Leu Arg Asp Ala Phe Ser (SEQ ID NO. 43)

EXAMPLE 23 hG-CSF Lys ¹⁹ Phe Leu Leu Lys Cys Leu Lys Gin Val Arg Lys He (SEQ ID NO. 44)

EXAMPLE 24 hG-CSF Arg ¹⁹

Phe Leu Leu Lys Cys Leu Arg Gin Val Arg Lys He (SEQ ID NO. 45)

EXAMPLE 25 hEPO Lys ¹⁰ Tyr Leu Leu Glu Ala Lys Lys Ala Glu Asn He Thr Thr Gly (SEQ ID NO. 46)

EXAMPLE 26 hEPO Arg ¹⁰ Tyr Leu Leu Glu Ala Lys Arg Ala Glu Asn He 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.

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SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT

(other than the US): MEDVET SCIENCE PTY LTD

APPLICANT/INVENTORS

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

(ii) TITLE OF INVENTION: "Haemopoietic Growth Factor Antagonists"

(iii) NUMBER OF SEQUENCES: 47

(iv) CORRESPONDENCE ADDRESS:

(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: floppy disc

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: International PCT App.

(B) FILING DATE: 28-JUL-1994

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: Provisional AU PM0186

(B) FILING DATE: 28-JUL-1993

(C) APPLICATION NUMBER: Provisional AU PM4772

(D) FILING DATE: 30-MAR-1993

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: HUGHES, DR E JOHN L

(C) REFERENCE/DOCKET NUMBER: EJH/EK

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: +61 3 254 2777

(B) TELEFAX: +61 3 254 2770

(2) INFORMATION FOR SEQ ID NO. 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acid residues

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

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

His Val Asn Ala lie Gin Xaa Ala Arg Arg Leu Leu Asn Leu -

(2) INFORMATION FOR SEQ ID NO. 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) 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

(2) INFORMATION FOR SEQ ID NO. 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acid residues

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

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

Asn Met lie Xaa Xaa lie lie Thr His Leu

(2) INFORMATION FOR SEQ ID NO. 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

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

Leu Leu Leu Xaa Leu Gin Met lie Leu

(2) INFORMATION FOR SEQ ID NO. 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 5:

lie Thr Leu Gin Xaa lie lie Lys Thr Leu

(2) INFORMATION FOR SEQ ID NO. 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 6

Arg Tyr lie Leu Xaa Gly lie Ser Ala Leu Arg Lys

(2) INFORMATION FOR SEQ ID NO. 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 7:

Gly Asp Gin Tyr Xaa Ser Val Leu Met Val Ser lie

(2) INFORMATION FOR SEQ ID NO. 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO. 8:

Ala Gly lie Leu Xaa lie Asn Phe Leu lie Asn Lys Met Gin Glu Asp

(2) INFORMATION FOR SEQ ID NO. 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 9

Asn Met Leu Arg Xaa Leu Arg Asp Ala Phe Ser

(2) INFORMATION FOR SEQ ID NO. 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 10:

Phe Leu Leu Lys Cys Leu Xaa Gin Val .Arg Lys lie

(2) INFORMATION FOR SEQ ID NO. 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 11:

Tyr Leu Leu Glu Ala Lys Xaa Ala Glu Asn lie Thr Thr Gly

(2) INFORMATION FOR SEQ ID NO. 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 12:

is Val Asn Ala lie Gin Glu Ala Arg Arg Leu Leu Asn Leu

(2) INFORMATION FOR SEQ ID NO. 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) 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

(2) INFORMATION FOR SEQ ID NO. 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 14:

Asn Met lie Asp Glu lie lie Thr His Leu

(2) INFORMATION FOR SEQ ID NO. 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 15:

Leu Leu Leu Asp Leu Gin Met lie Leu

(2) INFORMATION FOR SEQ ID NO. 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 16:

lie Thr Leu Gin Asp lie lie Lys Thr Leu

(2) INFORMATION FOR SEQ ID NO. 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 17:

Arg Tyr lie Leu Asp Gly lie Ser Ala Leu Arg Lys

(2) INFORMATION FOR SEQ ID NO. 1 ^' 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO. 18:

Gly Asp Gin Tyr Glu Ser Val Leu Met Val Ser lie

(2) INFORMATION FOR SEQ ID NO. 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 19:

Ala Gly lie Leu Asp lie Asn Phe Leu lie Asn Lys Met Gin Glu Asp

(2) INFORMATION FOR SEQ ID NO. 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 20:

Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

(2) INFORMATION FOR SEQ ID NO. 21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 21:

Phe Leu Leu Lys Cys Leu Glu Gin Val Arg Lys lie

(2) INFORMATION FOR SEQ ID NO. 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 22:

Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn lie Thr Thr Gly

(2) INFORMATION FOR SEQ ID NO. 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 23:

His Val Asn Ala lie Gin Lys Ala Arg Arg Leu Leu Asn Leu

(2) INFORMATION FOR SEQ ID NO. 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) 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

(2) INFORMATION FOR SEQ ID NO. 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 25:

Ala Leu Val Lys Arg Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu

(2) INFORMATION FOR SEQ ID NO. 26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 26:

Asn Met lie Asp Lys lie lie Thr His Leu

(2) INFORMATION FOR SEQ ID NO. 27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 27:

Asn Met lie Lys Glu lie lie Thr His Leu

(2) INFORMATION FOR SEQ ID NO. 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 28:

Asn Met lie Asp Arg lie lie Thr His Leu

(2) INFORMATION FOR SEQ ID NO. 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 29:

Asn Met lie Arg Glu lie lie Thr His Leu

(2) INFORMATION FOR SEQ ID NO. 30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 30:

Asn Met lie Lys Lys lie lie Thr His Leu

(2) INFORMATION FOR SEQ ID NO. 31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 31:

Asn Met lie Arg Arg lie lie Thr His Leu

(2) INFORMATION FOR SEQ ID NO. 32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 32:

Leu Leu Leu Lys Leu Gin Met lie Leu

(2) INFORMATION FOR SEQ ID NO. 33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 33:

Leu Leu Leu Arg Leu Gin Met lie Leu

(2) INFORMATION FOR SEQ ID NO. 34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 34:

lie Thr Leu Gin Lys lie lie Lys Thr Leu

(2) INFORMATION FOR SEQ ID NO. 35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 35:

lie Thr Leu Gin Arg lie lie Lys Thr Leu

(2) INFORMATION FOR SEQ ID NO. 36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 36:

Arg Tyr lie Leu Lys Gly lie Ser Ala Leu Arg Lys

(2) INFORMATION FOR SEQ ID NO. 37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 37:

Arg Tyr lie Leu Arg Gly lie Ser Ala Leu Arg Lys

(2) INFORMATION FOR SEQ ID NO. 38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 38:

Gly Asp Gin Tyr Lys Ser Val Leu Met Val Ser lie

(2) INFORMATION FOR SEQ ID NO. 39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 39:

Gly Asp Gin Tyr Arg Ser Val Leu Met Val Ser lie

(2) INFORMATION FOR SEQ ID NO. 40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 40:

Ala Gly lie Leu Lys lie Asn Phe Leu lie Asn Lys Met Gin Glu Asp

(2) INFORMATION FOR SEQ ID NO. 41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 41:

Ala Gly lie Leu Arg lie Asn Phe Leu lie Asn Lys Met Gin Glu Asp

(2) INFORMATION FOR SEQ ID NO. 42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 42:

Asn Met Leu Arg Lys Leu Arg Asp Ala Phe Ser

(2) INFORMATION FOR SEQ ID NO. 43:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 43:

Asn Met Leu Arg Arg Leu Arg Asp Ala Phe Ser

(2) INFORMATION FOR SEQ ID NO. 44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 44:

Phe Leu Leu Lys Cys Leu Lys Gin Val Arg Lys lie

(2) INFORMATION FOR SEQ ID NO. 45:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 45

Phe Leu Leu Lys Cys Leu Arg Gin Val Arg Lys lie

⁽

(2) INFORMATION FOR SEQ ID NO. 46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 46:

Tyr Leu Leu Glu Ala Lys Lys Ala Glu Asn lie Thr Thr Gly

(2) INFORMATION FOR SEQ ID. NO. 47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 47:

Tyr Leu Leu Glu Ala Lys Arg Ala Glu Asn lie Thr Thr Gly