WO2006001770A1 - Method for identifying modulators of cytokine class i receptor - Google Patents

Abstract

The present invention relates to methods for the identification of modulators of cytokine class I receptors, in particular the growth hormone receptor, said method comprising identifying a molecule binding to a site at the receptor different from the binding site of the natural ligand of the said receptor; and using the said molecule in a competitive binding assay for identifying modulators of the said cytokine class I receptor.

Description

Methods for identifying modulators of cytokine class I receptors.

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority from Swedish Patent Application No. 0401666- 3, filed June 28, 2004, and U.S. Provisional Patent Application No. 60/609,593, filed September 14, 2004. The prior applications are incorporated herein by reference in their entirety.

TECHNICAL FIELD The present invention relates to methods for the identification of modulators of cytokine class I receptors, in particular the growth hormone receptor. The modulators are useful as pharmaceutical compounds, e.g., in the treatment of acromegaly. The invention also provides variants of the growth hormone receptor, said variants essentially lacking capacity to bind human growth hormone.

BACKGROUND The Growth Hormone Receptor (GHR) belongs to the cytokine class I receptor family, together with, e.g., the prolactin receptor (PRLR), the erythropoietin receptor (EPOR), the thrombopoietin receptor (TPOR) [I]. GHR is expressed in several tissues, e.g., liver, pancreas, muscles, adipose tissue, bone and brain, where it is activated by its natural ligand, growth hormone (GH). Activation of the receptor triggers multiple signal transduction pathways, resulting in tissue / organ dependent responses known to be induced by GH [2]. The cDNA for human growth hormone receptor (GHR) encodes a 620-aa protein of about 70 kDa (positions 19-638 of SEQ ID NO:2), consisting of an extracellular ligand binding domain, a membrane spanning region, and a cytoplasmic part, involved in signaling [3, 4]. In addition to the full-length receptor, growth hormone binding protein, GHbp comprising the extracellular part of the receptor, can be isolated from plasma [5]. GHbp is generated by alternative splicing in rodents and by proteolytic cleavage of the extracellular part of receptor in humans [6]. Signaling is initiated by a two-step dimerization process. In the initial step a high affinity binding of a GH molecule to one GHR is taking place, followed by the recruitment of a second GHR molecule to this complex, thus forming a signaling- productive ternary complex [7]. The dimerization of GHR also occurs spontaneously, independently of GH molecule, as described recently by Waters et al. [8]. According to this hypothesis, the GHR occurs as already predimerized form on the cellular surface. Binding of GH to these predimerized receptors introduces the conformational change in the intracellular part of receptors proximal to the cellular membrane [8]. According to Waters, it is this conformational change and not dimerization process per se, which causes initiation of cellular signaling. Dimerization of the receptors independently in the presence of the natural ligand has previously been described for the erythropoietin receptor [9], which belongs to the same family of receptors. Regardless the exact mechanism of dimerization, the binding of a ligand to the ternary complex is essential for inducing intracellular signaling [7, 10], leading to the transcriptional activation of various genes [H]. The region required for GH binding has been identified as a hot spot [12]: a central functional epitope, comprising of 11 amino acids side chains [13]. Two tryptophan residues (Trp 104 and Tip 169) within this epitope are contributing most to the binding affinity between GH and its receptor. Replacing any of these tryptophans with alanine results in a receptor molecule incapable of GH binding. In addition to receptor activation involving hormone binding, an alternative mechanism for receptor activation has been suggested [14]. An algorithm, allowing for the identification of peptide sequences within the receptor sequence, was described. The peptides were capable of receptor activation, independently of the natural ligand, when added to cellular systems expressing a given receptor. Studies of the erythropoietin receptor showed that such a peptide binds to the site of its origin on the receptor molecule [15]. Receptor-specific peptides have been identified for several of the receptors in the class I cytokine family, including GHR [16, 17]. To this date there have been no low molecular weight (LMW) ligands identified in binding assays using GH as a competitive ligand, which are also capable of agonistic or antagonistic functions upon GHR. Other class I cytokine receptors are also known as difficult targets for such types of screenings. A LMW agonist of the granulocyte colony stimulating factor (GCMSF) has been described, but this compound was identified in a functional screen and was specific only to the mouse receptor; it was not active towards the human receptor [18]. The only example of a synthetic mimetic of a cytokine class I receptor is the thrombopoietin mimetic described by Kimura et al. (1998) [19]. The difficulties in identifying functional ligands via the hormone interacting site are connected with the size of molecular surface involved in the interaction between the hormone and its receptor. For GHR and GH, this size has been estimated to 3000 A. Consequently, the possibility of identifying LMW inhibitors via screening to another functional site on the receptor, offers the possibility of a new approach for identification of potential agonists or antagonists of said receptors.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a mass spectrogram depicting interactions between GH and GHbp_32-237 (upper panel), and between GH and GHbp_32-237wi₀4A (lower panel). Fig. 2 is a mass spectrogram depicting interactions between GH and GHbp_32-237 (upper panel), and between GH, GHbP_32-237, and a GHR-derived peptide (SEQ ID NO: 6) (lower panel). Fig. 3 is a mass spectrogram depicting interaction between GH, GHbp_32-237, and compound BVT.3693. Fig. 4 is a mass spectrogram depicting interaction between GHbpwi_04Aand compound BVT.3693. Fig. 5 is a mass spectrogram depicting interaction between GHbp₁₃o_-237 ([M], left panel) or GHbp_32-237 ([M], right panel), compound BVT.3693 [L], and a competing ligand BVT.39221 [R].

DETAILED DISCLOSURE The present invention provides a method for identification of a modulator of a growth hormone receptor, the method comprising: (i) identifying or providing a molecule that binds to a site at the growth hormone receptor different from the growth hormone-binding site of the growth hormone receptor; and (ii) using the molecule in an assay for identifying a modulator of the growth hormone receptor. Preferably, the said assay in step (ii) is a competitive binding assay comprising the steps: (a) contacting the growth hormone receptor or a variant thereof with a test compound under conditions that permit binding to the growth hormone receptor or variant thereof; and (b) determining whether the test compound binds to the growth hormone receptor or variant thereof by detecting the presence or absence of a signal generated from the interaction of the test compound with the growth hormone receptor or variant thereof, in the presence of the molecule provided in step (i). In the Examples of the present invention, binding of test compounds to the GH receptor was determined using Electrospray-Ionization Mass Spectrometry (ESI-MS) technology. However, the skilled person will understand that there are various alternative ways to carry out the assay mentioned in step (ii) above. For instance, the molecule provided in step (i) could be labeled and its displacement from the receptor by a competing compound can be monitored by known methods such as SPA (Scintillation Proximity Assay) technology. Alternatively, if the molecule identified or provided in step (i) is a peptide, a FRET assay can be employed. The said competitive binding assay can preferably be carried out using a variant of the said growth hormone receptor, said variant lacking capacity to bind growth hormone. The said variant could, e.g., have a point mutation inhibiting its capacity to bind growth hormone. Such a variant is exemplified by the polypeptide GHbp_32-237wio_4A (SEQ ID NO:4) which is a variant of the polypeptide having the amino acid sequence shown as SEQ ID NO:3 and which carries the point mutation W104A. Other suitable variants could carry other point mutations, for instance W 169 A. Alternatively, the growth hormone receptor variant lacking capacity to bind growth hormone could be a variant lacking the growth hormone binding region. Such a variant is exemplified by the polypeptide GHbpi₃o-₂₃₇ (SEQ ID NO:5) which is a variant of the polypeptide having the amino acid sequence shown as SEQ ID NO:3, which variant is lacking the amino acids shown as positions 1-98 in SEQ ID NO:3. The above-mentioned variants are derived from the extracellular polypeptide GHbp_32-237 (SEQ ID NO:3) corresponding to positions 50-255 of SEQ ID NO:2. However, it will be understood that variants can be obtained from other extracellular polypeptides (GH binding proteins) derived from GHR shown as SEQ ID NO:2. Examples of such extracellular polypeptides include polypeptides referred to as GHbpi. ₂₃₇ (positions 19-255 of SEQ ID NO:2); GHbp_1-246 (positions 19-264 of SEQ ID NO:2); and GHbp_32-246 (positions 50-264 of SEQ ID NO:2). The said molecule identified or provided in step (i) above could, e.g., be a peptide, as exemplified by the peptide having the amino acid sequence of SEQ ID NO:6. Alternatively, the molecule is a low-molecular weight molecule such as the compound designated BVT.3693 (N-[5-(aminosulfonyl)-2-methylphenyl]-5-bromo-2- furamide). Compounds identified in the methods described above will be of use in therapy. Accordingly, the present invention provides a compound identified as an agonist or an antagonist of GHR for use in therapy, in particular for treating acromegaly, cancer, diabetes, diabetic nephropathy, diabetic retinopathy and neuropathy, and other diseases with pathologically increased IGF-I levels, as well as for treatment of children with growth hormone deficiency, Prader- Willis syndrome, Turners syndrome, children with retarded growth due to chronic renal failure, substitution of adults with growth hormone deficiency, frail elderly, and wasting syndrome in AIDS. In another aspect, the invention provides a method for the identification of a modulator of a cytokine class I receptor, comprising: (i) identifying or providing a molecule that binds to a site at the cytokine class I receptor different from the binding site of the natural ligand of the cytokine class I receptor; (ii) using the molecule in a competitive binding assay for identifying a modulator to the cytokine class I receptor, wherein the competitive binding assay is carried out using a variant of the cytokine class I receptor that does not bind to the natural ligand of the cytokine class I receptor. The term "cytokine class I receptor" is well known in the art. The family of cytokine class I receptors comprises, e.g., the growth hormone receptor (GHR), the prolactin receptor (PRLR), the erythropoietin receptor (EPOR), and the thromobopoietin receptor (TPOR) [I]. In another aspect, the invention provides a polypeptide which is a variant of the polypeptide having the amino acid sequence shown as SEQ ID NO:3, but essentially lacking capacity to bind growth hormone. As described above, the said variant could, e.g., have a point mutation inhibiting its capacity to bind growth hormone, such as the variant GHbp_32-237wi04A (SEQ ID NO:4), or lack the growth hormone binding region, such as the variant GHbpi_30-237 (SEQ ID NO:5). The said variant should preferably retain the ability to interact in the extracellular stem region of GHR with a second GHR molecule in a dimerization process; and should preferably have an intact domain 2 of the extracellular part of the receptor. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Suitable methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. The invention will now be further illustrated through the description of examples of its practice. The examples are not intended as limiting in any way of the scope of the invention.

EXAMPLES

EXAMPLE 1 : Preparation of GH binding proteins Growth hormone binding protein (GHbp₃₂.₂₃₇; SEQ ID NO:3) comprising amino acids 32-237 of the extracellular part of the receptor has been described previously [20]. GHbp_32-237 was produced in E. coli as described and purified to homogeneity in a four- step purification process, which included osmotic chock of bacterial cells, followed by affinity purification on GH-sepharose, desalting on Superdex 30 XKl 6/60, and gel filtration on Superdex 75 XKl 6/60. The amount and the purity of the binding protein were verified by SDS-PAGE, N-terminal sequencing, amino acid analysis and MALDI- MS. A variant GH binding protein (GHbp_32-2₃₇wi04A; SEQ ID NO:4) was unable to bind GH due to a point mutation at position 104. A circular DNA, consisting of 5061 base pairs and coding for the same binding protein, but with TrplO4 replaced with Ala, and with the addition of a Hisg-tag sequence at the C-terminus, was prepared. The W 104 A mutation was inserted using oligonucleotides containing the new codon in a two-step Polymerase Chain Reaction method. Expression of the encoded protein was thus performed in E. coli strain BL21 in a 5 L bench-top fermenter. The protein, which could not be purified by a traditional GH-affϊnity chromatography, was purified to homogeneity in a three-step purification process comprising (i) osmotic chock; (ii) immobilized metal-ion affinity chromatography (IMAC) utilizing the His₆-tag; and (iii) gel filtration. The two chromatographic steps were performed on an Akta Explorer System (Amersham Biosciences) using a TALON column (Clontech) and a HiLoad X16/60 Superdex 75 (Amersham Biosciences). The amount and the purity of the produced protein were analyzed by SDS-PAGE, N-terminal sequencing, amino acid analysis and MALDI-MS. A further GHbp variant, comprising domain 2 (amino acids 130-137) of the GH receptor, was prepared (GHbp _I30-237; SEQ ID NO: 5). In this variant, all residues from domain I, which is involved in the interaction with growth hormone, have been removed. The protein, which could not be purified by a traditional GH-affinity chromatography, was purified to homogeneity in a three-step purification process comprising (i) Ammonium sulphate precipitation; (ii) gel filtration; and (iii) ion exchange chromatography. The two chromatographic steps were performed on an Akta Explorer System (Amersham Biosciences) using a HiLoad X26/60 Superdex 75 (Amersham Biosciences) and a Mono Q HR 10/10 IEX column (Amersham Biosciences). The amount and the purity of the produced protein were analyzed by SDS-PAGE, C- and N-terminal sequencing, amino acid analysis and MALDI-MS.

EXAMPLE 2: ESI-MS determination of ligand-receptor binding Binding of test compounds to the GH receptor was determined using Electrospray-Ionization Mass Spectrometry (ESI-MS) technology. Non-denaturing ESI- MS allows the transfer of non-covalently bound complexes from a solution into the gas phase. The identification of non-covalent host-guest interactions by mass spectrometry has been known since 1991 [21, 22,23]. In the ESI-MS process, the sample in solution is sprayed directly by a strong electric field gradient into the gas phase [24,25]. A new ESI-MS based procedure to determine the binding affinity between GHbp and low molecule mass inhibitors, that will solely reflect the binding conditions in the solution phase, was developed [26]. For this purpose, a reference ligand with a known dissociation constant was used. Normally, the compound N-[5-(aminosulfonyl)- 2-methylphenyl]-5-bromo-2-furamide (BVT.3693) was used as reference ligand, but any ligand with established K_j can be used as a reference ligand. No labeling of the reference ligand was necessary as the complexes were identified by their molecular masses. The peak intensity of the protein-reference ligand complex was measured before and after the addition of a further ligand, competing for the same binding site on the protein. Similar procedures for determination of binding affinities has previously been used by Kempen et al. [27] for measuring binding constants of crown ethers:alkali metal complexes, and by Oliv [28] for the complexes between model proteins and known low molecular mass binders (300-500 Da). The proteins were dialyzed offline against 10 mM ammonium acetate by means of a Slide-a-lyzer prior to analysis (MWCO 10000, Pierce, Rockford, IL). The ESI mass spectra were recorded on a Q-TOF-I instrument (Micromass, Manchester, UK). The instrument conditions for the ESI interface were as follows: Capillary voltage 3000 V, cone voltage 80 or 90 V, source block temperature 6O⁰C, desolvation temperature 80°C. In order to preserve the protein-ligand complex in the gas phase, an elevated instrument pressure in the source region was maintained by reducing analyzer pumping and introducing gas into the hexapole collision cell to achieve an analyzer pressure of approximately 4xlO'⁵ mbar. The mass spectra were recorded between 1000 and 5000 m/z, with data accumulation of 2 s per spectrum and an inter scan time delay of 0.1 s. 65 scans/experiment were performed. The mobile phase used was ammonium acetate (10 mM; pH 6.5), and the sample flow rate was 5 μl/min. A volume of 5 μl was injected by means of an autosampler (FAMOS, LC packings, Amsterdam, NL).

EXAMPLE 3: GHbpi?.??7 WIΓUA does not form a complex with growth hormone Human GH was obtained from Pharmacia, (Genotropin®, batch 25690-51). GHbp_32-237 (6 μM) was incubated with human GH (5.5 μM) for 15 minutes prior to MS analysis. For the experiments with the W104A mutant, 10 μM of GHbp_32-237 wio4A was preincubated with 13 μM of hGH. ESI-MS was then carried out as described in Example 2. As shown in Fig. 1 (upper panel) GHbP_32-237 formed a complex with human GH, whereas the mutant GHbp₃₂.₂₃₇ wi_O4_A was unable to form such a complex (Fig. 1, lower panel). The peaks A in the mass spectra represent free hGH (MW 22.1 kD); peak B in the upper panel represents hGHbp_32-237 in a 1 : 1 complex with hGH (MW 46 kD); peak C in the upper panel represents hGHbp₃₂.₂₃₇ in a 1 :2 complex with hGH (MW = 70.2 kD); and peak D in the lower panel represents free GHbp_32-237 wi04A (MW = 24.9 kD). These results, which were obtained with a technology different from previously published methods [12, 13], confirm the importance of Tip 104 in GHR for GH binding.

binding site It was confirmed by ESI MS assay, carried out as described in Example 2 that the GHR-derived peptide shown as SEQ ID NO:6 (previously published as SEQ ID NO:9 in US 6,333,031 [17]) binds to GHbp_32-237 at a site different from the GH binding site, since GH and the peptide, when added to GHbp_32-237 concomitantly, could form 1 : 1 :1 and 1:2:1 complexes with GHbp_32-237 (Fig. 2). The concentrations of GH, GHbP_32-237 and the GHR-derived peptide were 13, 10 and 33 μM, respectively. The 1 :1 and 1:2 complexes between GH and GHbp_32-237 are indicated as (A) and (B). Complexes formed in the absence of peptide are shown in the upper panel, and complexes formed in the presence of peptide in the lower panel. Additional peaks were detected, corresponding to the multiple charges 15 and 16 of the complex 1 :1 :1 at 49.6 kDa and to the multiple charges 18 and 19 of the complex 1 :2:1 at 73.6 kDa. This indicated that 1 :1 :1 as well as 1 :2:1 complexes were formed between GH-GHbp-peptide. In addition, the results indicate that the peptide binds to a site different from the GH-binding site on the receptor, since the peptide does not displace GH in the complex.

EXAMPLE 5: Screening for LMW compounds A homogenous screening assay, employing the GHR-derived peptide (SEQ ID NO: 6) as a competitive ligand, was designed based on Fluorescence Energy Transfer (FRET) principle [29]. Briefly, biotine labeled peptide and GHR specific monoclonal antibody labeled with Europium are bound to the receptor. The signaling molecule - allophycocyanin bound to streptavidin is then added to the reaction mixture, whereupon streptavidin forms a complex with a peptide-bound biotine. This interaction brings Eu and allophycocyanin in proximity, allowing for energy transfer between europium and allophycocyanin, resulting in emission signal of fluorescence at 665 run wavelength. If the peptide is displaced from the receptor by a competing ligand, a diminished signal is measured.

binding site Similar complexes as in Example 4 were formed when a low molecular weight (LMW) GHR ligand (BVT.3693; N-[5-(aminosulfonyl)-2-methylphenyl]-5-bromo-2- furamide), identified in the screening procedure according to Example 5, was incubated with GHbp_32-237 and GH (Fig. 3). The 1 :1 :1 complex GH-GHbp-BVT.3693 has a molecular weight of 46.5 kDa. Peaks corresponding to the multiple charges 15, 14, and 13 of the complex ("A+BVT compound"; indicated with arrows in Fig. 3). The 1 :2: 1 complex could not be clearly identified due to the technical limitations of the method (peak broadening at higher m/z). EXAMPLE 7: LMW compound binds to GHbpwKUA ESI-MS analysis indicated that BVT.3693 binds to the GHbp variant GHbp_Wi_04A (Fig. 4). The mutant protein and the compound formed 1 :1 complex. In contrast, the natural GHR ligand, growth hormone, did not show any complex formation with GHbpwi04A BVT.3693 was used at 25 μM concentration and the protein was 5 μM. Since the protein was purified with the aid of metal chelation chromatography, the presence of an adduct ion was detected. The 1 : 1 complex between the substance and the protein is indicated. The excess of the free protein shown in Fig. 4 is due to dissociation of the complex when the sample enters the gas phase inside the mass spectrometer. In solution, the 1 : 1 complex is probably more abundant.

EXAMPLE 8: Low-molecular weight compounds binds to GHbpnn-rv? Another protein lacking GH-binding domain used in the studies was GHbp₁₃o- ₂₃₇. In this construct the whole domain involved in GH-interactions was deleted. Despite this, the truncated protein was able to form a 1 :1 complex with BVT.3693. A competition study was done using BVT.3693 at a fixed concentration of 30 μM. A competing ligand, 5-bromo-N-[2,4,6-trimethyl-3-(morpholin-4- ylsulfonyl)phenyl]-2-furamide (BVT.39221), was added at increasing concentrations (0-300 μM). The proteins GHbPi_30-237 (left panel), or as a control GHbp_32-237 (right panel), were used at 6 μM concentrations. The results (Fig. 5) indicate that both LMW compounds could form complexes with GHbpπo_-237 to the same extent they form complexes with GHbp₃₂._237j which has an intact GH binding region. It could also be seen that both ligands compete for the same binding site on the proteins. These results indicate that GHbp variants, which lack the GH binding region, can still be used in screening assays to identify modulating molecules binding to a site at the GHR different from the GH binding site.

REFERENCES

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Claims

1. A method for identification of a modulator of a growth hormone receptor, the method comprising: (i) providing a molecule that binds to a site at the growth hormone receptor different from the growth hormone-binding site of the growth hormone receptor; and (ii) using the molecule in an assay for identifying a modulator of the growth hormone receptor.

2. The method according to claim 1, wherein the assay is a competitive binding assay comprising: (a) contacting the growth hormone receptor or a variant thereof with a test compound under conditions that permit binding to the growth hormone receptor or variant thereof; and (b) determining whether the test compound binds to the growth hormone receptor or variant thereof by detecting the presence or absence of a signal generated from the interaction of the test compound with the growth hormone receptor or variant thereof, in the presence of the molecule provided in step (i).

3. The method according to claim 2, wherein the competitive binding assay is carried out using a variant of the growth hormone receptor that does not bind to growth hormone.

4. The method according to claim 3, wherein the growth hormone receptor variant is a polypeptide having a point mutation that abrogates binding to growth hormone.

5. The method according to claim 4, wherein the growth hormone receptor variant is a variant of the polypeptide having the amino acid sequence shown as SEQ ID NO:3.

6. The method according to claim 5, wherein the point mutation is W104A.

7. The method according to claim 6, wherein me growxn normone receptor variant is GHbp_32-237Wio4A (SEQ ID NO:4).

8. The method according to claim 3, wherein the growth hormone receptor variant is a polypeptide lacking the growth hormone-binding region.

9. The method according to claim 8, wherein the growth hormone receptor variant is a variant of the polypeptide having the amino acid sequence shown as SEQ ID NO:3, essentially lacking the amino acids shown as positions 1-98 in SEQ ID NO:3.

10. The method according to claim 9, wherein the growth hormone receptor variant is GHbpi_30-237 (SEQ ID NO:5).

11. The method according to any one of claims 1 to 10, wherein the molecule provided in step (i) is a peptide.

12. The method according to claim 11, wherein the peptide has the amino acid sequence of SEQ ID NO:6.

13. The method according to any one of claims 1 to 10, wherein the molecule provided in step (i) is the compound N-[5-(aminosulfonyl)-2-methylphenyl]-5-bromo-2- furamide.

14. The method according to any one of claims 2 to 13, wherein the competitive binding assay is an ESI-MS assay.

15. A method for identification of a modulator of a cytokine class I receptor, the method comprising: (i) providing a molecule that binds to a site at the cytokine class I receptor different from the binding site of the natural ligand of the cytokine class I receptor; (ii) using the molecule in a competitive binding assay for identifying a modulator to the cytokine class I receptor, wherein the competitive binding assay is carrieα oui using a variant oi me cytokine class I receptor that does not bind to the natural ligand of the cytokine class I receptor.

16. A polypeptide comprising a variant of the amino acid sequence shown as SEQ ID NO:3, wherein the variant does not bind to growth hormone.

17. The polypeptide according to claim 16, wherein the variant has a point mutation that abrogates binding to growth hormone.

18. The polypeptide according to claim 17, wherein the point mutation is W104A.

19. The polypeptide according to claim 18, wherein the variant is GHbp_{32- 237}wio_4A (SEQ ID NO:4).

20. The polypeptide according to claim 16, wherein the variant lacks the growth hormone-binding region.

21. The polypeptide according to claim 20, wherein the growth hormone- binding region is positions 1-98 of SEQ ID NO:3.

22. The polypeptide according to claim 21, wherein the variant is GHbpi₃₀.₂₃₇ (SEQ ID NO:5).