WO2014187807A1

WO2014187807A1 - Follistatin derivatives

Info

Publication number: WO2014187807A1
Application number: PCT/EP2014/060312
Authority: WO
Inventors: Gert De Wilde; Hermanus Johannes Marco Eijken
Original assignee: Arcarios B.V.
Priority date: 2013-05-21
Filing date: 2014-05-20
Publication date: 2014-11-27

Abstract

The present invention relates to a recombinant human follistatin polypeptide in which the heparin binding sequence (HBS) in follistatin domain FSD1 is replaced by a corresponding non-heparin binding motif from follistatin domain FSD2for the treatment of bone related disorders. The polypeptide of the invention originates from the longer 315 residue containing splice variant of human follistatin that, in contrast to the shorter 288 residue containing splice variant, is able to stimulate bone formation in vivo upon parenteral, intravenous, intraperitoneal or intracapsular administration.

Description

FIELD OF THE INVENTION

The present invention relates to the field of medicine. In particular it relates to the field of bone-related disorders such as osteoporosis and the treatment thereof. More in particular the invention relates to derivatives of follistatin and pharmaceutical compositions comprising such derivatives in the treatment of bone-related disorders. BACKGROUND OF THE INVENTION

Osteoporosis is a disorder in which the density and quality of bone is reduced. This leads to weakness of the skeleton and increases the risk of fractures. Such fractures particularly occur in the spine, wrist, hip, pelvis and upper arm. Osteoporosis associated fractures are an important cause of mortality and morbidity. Bone quality is an important determinant of osteoporosis and proper osteoblast

differentiation plays an important role in the control and maintenance of bone quality. Osteoblasts have a mesenchymal origin and the differentiation of mesenchymal stem cells to an osteoblastic lineage is regulated by many endocrine, paracrine and autocrine factors .

During bone formation osteoblasts produce an organic extracellular matrix (ECM) or osteoid (the immature matrix before mineralizing) , which is primarily composed of type I collagen and non-collagenous proteins. This ECM then

mineralizes by the deposition thereon of calcium phosphates to form bone spicules. Initiation of bone mineralization, or ossification, requires the precipitation and attachment of calcium phosphate crystals, in particular hydroxyapatite to the ECM. Bone mineralization and in particular its regulation is a complicated process controlled by many factors including serum calcium and phosphate concentrations, hormones, enzymes and the structure of the ECM. The macromolecular organization of type I collagen is a factor facilitating bone

mineralization. Initially calcium phosphate is deposited in the holes of the collagen fibrils and later fills in the pores and the remainder of the area within the collagen fibrils.

Transforming growth factor-β (ΤΘΕβ) and bone morphogenic protein (BMP) are well known regulators of bone formation. Activins belong to the ΤΘΕβ superfamily; their structure is closely related to that of ΤΘΕβ and activins act via similar intracellular signaling molecules. Activins and their

relatives, inhibins, were initially purified from gonadal fluids and characterized on the basis of their ability to modulate FSH secretion from pituitary gonadotropes . Besides this classical role of activins, these proteins can affect the function of other cell types and tissues such as the adrenal gland, liver, neurons and pancreas. Activins and inhibins are composed of the inhibitory subunits , βΑ and βΒ.

Heterodimerization of the a and βΑ or βΒ subunit forms inhibin A or inhibin B. Homo- and heterodimerization of the βΑ and βΒ subunits result in formation of activin-A, activin-AB, or activin-B. Activins need type I and type II activin receptors for signal transduction. Activins bind to the activin type IIA or type IIB receptors, leading to recruitment and

phosphorylation of the activin type IB receptor. The

phosphorylated type I receptor, in turn, phosphorylates intracellular signaling proteins known as Smads .

Whereas activin-A was initially believed to enhance bone formation (Gaddy-Kurten et al . 2002 Endocrinology 143:74-83; Sakai et al . 2000 Bone 27(1): 91-6) it has now been clearly shown that inhibition of activin stimulates bone formation both in vitro as in vivo (WO 2008/060156; Eijken M et al . 2007. The activin A-follistatin system: potent regulator of human extracellular matrix mineralization . FASEB J 21:2949-60; Pearsall et al . 2008 PNAS 105 (19) : 7082-7) . Follistatin is a soluble protein that functions as an activin binding protein preventing activin from interacting with its receptor

(Nakamura T et al . 1990. Activin-binding protein from rat ovary is follistatin . Science 247:836-8) . The inventors of the present invention have found previously that activin signaling in osteoblasts prevents maturation/mineralisation of the ECM and that this could be effectively reversed by follistatin (WO 2008/060156; Eijken M et al . 2007. FASEB J 21:2949-60).

Although follistatin appeared to be a very useful protein in methods for stimulating bone healing, bone formation and for the treatment of osteoporosis, it also became clear that only relatively low plasma levels of follistatin could be achieved upon intravenous (iv) , intraperitoneal (ip) or subcutaneous (sc) injection during pharmacokinetic studies. Such low circulating levels could potentially compromise the effectiveness of the protein to interfere with bone metabolism in vivo. Clearly, it appeared that altered (and/or mutant) versions of follistatin with improved pharmacokinetic

properties over the wild type follistatin protein could potentially also have improved properties in respect of bone formation . SUMMARY OF THE INVENTION

The present invention relates to a recombinant 315-amino acid containing splice variant of the human follistatin polypeptide, wherein said splice variant comprises a mutation in the heparin binding sequence (HBS) rendering the

polypeptide unable to bind to heparin, for use in a medical treatment of a subject suffering from a condition wherein enhancing bone formation, bone strength, mineralization and/or osteoblast activity is beneficial to the physiology of said subject. Preferred examples of such conditions are

osteoporosis, secondary osteoporosis, osteopenia,

osteomalacia, osteodystrophy, osteomyeloma, bone fracture, Paget ' s disease, osteogenesis imperfecta, (bone-related) trauma, orthopedy, tumoral cavities, Ear Nose & Throat

disease, maxilla-facial surgery, periodontal surgery,

fractures with bone defects, pseudarthrosis with or without bone defects, vertebral arthrodesis (spinal fusion) and tibial osteotomy. Especially preferred are polypeptides for use in the treatment of osteoporosis. In a further preferred aspect said subject to be treated is a human subject. In yet a further preferred embodiment, said mutation of the heparin binding sequence comprises a replacement of the heparin binding sequence (as shown in SEQ ID NO: 6) present in FSD1, preferably wherein said heparin binding sequence is replaced by a corresponding region (SEQ ID NO: 7) from FSD2. In yet another preferred embodiment of the invention the recombinant human follistatin polypeptide according to the invention is linked to an Fc domain of human IgGl (SEQ ID NO:2), which adds to PK properties of the recombinant protein.

The present invention relates to a recombinant polypeptide comprising the amino acid sequence of SEQ ID NO: 23 (315 amino acids with a mutated HBS domain) for use in a medical

treatment of a subject suffering from a condition selected from the group consisting of: osteoporosis, secondary

osteoporosis, osteopenia, osteomalacia, osteodystrophy, osteomyeloma, bone fracture, Paget ' s disease, osteogenesis imperfecta, (bone-related) trauma, orthopedy, tumoral

cavities, Ear Nose & Throat disease, maxilla-facial surgery, periodontal surgery, fractures with bone defects,

pseudarthrosis with or without bone defects, vertebral

arthrodesis (spinal fusion) and tibial osteotomy. Preferably, the polypeptide as disclosed herein is used in the treatment of osteoporosis. In another preferred embodiment, said recombinant polypeptide for use according to the invention is linked to an Fc domain of human IgGl (SEQ ID NO: 2) .

The invention also relates to a pharmaceutical composition comprising a recombinant human follistatin polypeptide

according to the present invention, or a nucleic acid encoding said polypeptide according to the present invention, said composition further comprising a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers for purified recombinant proteins are well-known in the art. The invention further relates to the use of a recombinant polypeptide comprising the amino acid sequence of SEQ ID NO: 23 in the preparation of a medicament for use in a subject suffering from a condition selected from the group consisting of: osteoporosis, secondary osteoporosis, osteopenia,

disease, maxilla-facial surgery, periodontal surgery,

fractures with bone defects, pseudarthrosis with or without bone defects, vertebral arthrodesis (spinal fusion) and tibial osteotomy. Preferably, said condition to be treated is

osteoporosis .

In yet another aspect, the invention relates to a method of treating a subject suffering from a condition selected from the group consisting of: osteoporosis, secondary osteoporosis, osteopenia, osteomalacia, osteodystrophy, osteomyeloma, bone fracture, Paget ' s disease, osteogenesis imperfecta, (bone- related) trauma, orthopedy, tumoral cavities, Ear Nose &

Throat disease, maxilla-facial surgery, periodontal surgery, fractures with bone defects, pseudarthrosis with or without bone defects, vertebral arthrodesis (spinal fusion) and tibial osteotomy, comprising the step of administering a therapeutically effective amount of a recombinant polypeptide comprising the amino acid sequence of SEQ ID NO: 23.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1, panel (A) shows the primary amino acid sequence of human follistatin (SEQ ID NO:l) . Panel (B) shows the same sequence (SEQ ID NO:l) and the separate domains within the follistatin polypeptide: The signal peptide that is cleaved off upon secretion is indicated by SP; the C-terminal domain present in the 315 amino acid splice variant is indicated by CD. The N-terminal domain of the mature protein (FSDN; aa 1- 63) is followed by three successive so-called FSD regions (FSD1, FSD2 and FSD3), each containing ten cysteine residues (underlined) at regular intervals. The Heparin binding site in FSD1 (bold; SEQ ID NO: 6) was replaced by the non-heparin binding site at the similar position within FSD2 (also in bold; SEQ ID NO: 7) .

Figure 2 shows the schematic representation of the FST315 and FST315-AHBS proteins without the signal peptide at the N terminus. The Heparin binding site (HBS) in FSD1 (aa 75-86 in Figure IB; SEQ ID NO: 6) is replaced by a similar (but non- heparin binding) sequence that is found at positions 148-157 in FSD2 (SEQ ID NO : 7 ) . The amino acid sequence of FST315-AHBS (without the signal peptide and Fc domain) is provided in SEQ ID NO: 23. The amino acid sequence of wild type FST315 with the signal peptide and human Fc domain is provided in SEQ ID NO: 3. The amino acid sequence of the FST315-FC-AHBS protein with the cleaved-off signal peptide and human Fc domain is provided in SEQ ID NO: 4. Figure 3 displays the experimental setup of the

pharmacokinetics (PK) experiment. Mice (n=3 per group) were injected with vehicle (control), FST315-FC or FST315-FC-AHBS at t=0. Blood was drawn at the indicated times (black triangles) and mice were sacrificed at day 7 or day 14 as indicated (red/final triangles) . The numbers (#) relate to the identification numbers of the specific mouse in each group.

Figure 4 shows the serum levels of FST315-FC and FST315- FC-AHBS at different time points after iv injection of the respective proteins.

Figure 5 shows the results of an activin binding assay in which the ability of FST315, FST315-FC and FST315-FC-AHBS to bind activin were determined. Although the capacity of wild type follistatin to bind activin was somewhat higher than the mutant versions, there appeared to be no significant

difference between FST315-FC and FST315-FC-AHBS recombinant polypeptides .

Figure 6 shows the results of an experiment in which the amount of trabecular bone (percentage bone volume divided by the tissue volume; % BV/TV) was measured in mice that were treated with a single injection of 10 mg/kg FST315-mFC-AHBS in a 2 week study (panel A and B) , and in a 5-6 weeks study with twice weekly administrations (panel D) . Panel (C) shows the results of a comparison between FST288-FC-AHBS and FST315-mFC- AHBS in a 2 week experiment with thrice weekly

administrations. A similar experiment was performed in a 5-6 weeks study with twice weekly administrations in Black6 mice (panel E) with FST315-mFC-AHBS and NOD.SCID mice (panel F) treated with FST315-hFC-AHBS .

Figure 7 shows the results of the same experiments as shown in Figure 6, now in respect of the trabecular number parameter. The amount of trabeculae per volume unit is given.

Figure 8 shows the results of the same experiments as shown in Figures 6 and 7, now in respect of the trabecular pattern factor parameter which is a measurement for connectivity. The lower the factor the better the trabeculae are connected.

DETAILED DESCRIPTION

The present invention relates to the use of follistatin- derived polypeptides that have improved properties in

comparison to wild type human follistatin. The inventors of the present invention showed earlier that follistatin can be used in medical treatments because the protein appeared to stimulate bone healing, bone formation and prevent/inhibit osteoporosis (WO 2008/060156). However, pharmacokinetic (PK) studies in mice demonstrated that only low peak serum levels of human follistatin could be achieved through iv-, ip- or sc administration. Of the total amount injected only about 1-5 % was found back in serum shortly thereafter. It is known that follistatin can interact with cell surfaces through its heparin binding sequence (HBS, see Figure 1) in FSD1 (Sidis Y et al . Heparin and activin-binding determinants in follistatin and FSTL3. 2005. Endocrinology 146:130-6; Keutmann HT et al . The role of follistatin domains in follistatin biological action. 2004. Mol Endocrinol 18:228-40). It was hypothesized that the HBS domain might be responsible for the observed low peak serum levels because the majority of follistatin could be bound to cell surfaces in vivo. To address this question, the inventors generated a number of follistatin variants in which the HBS was replaced by a corresponding (but non-heparin binding) sequence found in FSD2 (see Figure 1 and 2; Sidis et al . 2005; Datta-Mannan A et al . An engineered human

follistatin variant : insights into the pharmacokinetic and pharmacodynamic relationships of a novel molecule with broad therapeutic potential . 2013. J Pharmacol Exp Ther 344:616-

623) . Notably, Sidis et al . and Datta-Mannan et al . replaced residues 75-86 for residues 148-159, whereas the inventors of the present invention replaced residues 75-84 (SEQ ID NO: 6) for residues 148-157 (SEQ ID NO:7). The follistatin polypeptides were linked to the Fc domain of human IgGl in order to further increase the half-life in circulation after administration. It should be noted that the presence of the Fc domain was not an essential property of the polypeptide of the present invention because the contribution to the PK

properties was there, but relatively minor as compared to the replacement of the HBS domain, as shown in Figure 4 (see examples below) . The follistatin variants generated as disclosed herein were first analyzed for their heparin binding competence. It appeared that whereas the wild type FST315-FC polypeptide was still able to bind heparin efficiently, the FST315-FC-AHBS lost this ability. Nevertheless, the activin-binding capacity of the FST315-FC-AHBS mutant was not lost. It was already known from literature that mutants of the follistatin splice variant that terminated at the 288 residue and that contained a similar HBS swap lost their heparin-binding capacity while retaining their ability to bind activin (Sidis et al . 2005) . Based on the prior art it was hypothesized that the removal of the HBS domain would, because heparin could no longer bind, result in improved pharmacokinetic properties. This indeed appeared to be the case: the FST315-FC (wild type follistatin fused to the FC domain of IgGl) disappeared from the serum in the first few hours after administration with relatively low peak serum levels (0.5 μg/ml) , whereas the FST315-FC-AHBS variant remained detectable for several days with a peak serum level that was much higher (30 μg/ml) . However, although the HBS mutant protein was likely unable to bind heparin (or perhaps displayed increased plasma exposure) this did not automatically mean that the biological effects on bone

formation would be improved. Despite the fact that the

activity to interact with activin was unchanged in the AHBS derivative, it could be that the domain was required for a proper bioavailability in bone that could be lost because the protein distributed widely throughout the body, without being present locally in the bone (in higher concentrations than the wild type follistatin protein that still contained the HBS domain) to act. That a removal of the HBS domain did not automatically result in an improved effect on bone formation was shown by the fact that the FST288-FC-AHBS variant that was similar to the AHBS variant known from the art (see Sidis et al . 2005), although being able to interact with activin, did not give rise to increased trabecular bone volume. In contrast however, the variant of the present invention (FST315-FC-AHBS) was able to increase trabecular bone volume as shown clearly in the data provided herein. This is even more surprising in view of publications that had shown that the follistatin splice variant that ends at residue 288 has an improved potency to inhibit activin and has an increased

transcriptional activity in comparison to the splice variant terminating at residue 315 (see Hashimoto 0 et al . Difference between follistatin isoforms in the inhibition of activin signaling: activin neutralizing activity of follistatin isoforms is dependent on their affinity for activin. 2000. Cell Signal 12:565-71) . The invention as disclosed herein now clearly shows that such increased activin binding capacity does not automatically translate into an improved effect on bone formation. It should be noted that the AHBS mutant disclosed by Datta-Mannan et al . (2013) was not used in bone forming experiments but was suggested to be useful in diseases related to skeletal muscle.

On top of the positive effects related to bone formation, as disclosed for the first time herein, the FST315-FC-AHBS also has other beneficial properties: 1) lack of heparin binding might lead to lower immunogenicity, because heparin binding might lead to binding to antigen presenting cells (Leonetti M et al . Cell surface heparan sulfate proteoglycans influence MHC class II-restricted antigen presentation. 2010. J Immunol 185:3847-56); 2) less heparin (or heparin-like) molecules will stick during the recombinant production process which leads to improved compatibility (less aggregation) with different culture media that contain heparin-like molecules; and 3) it is likely that lower cell clumping of producing cells will occur during recombinant production and growth of the cells.

The present invention relates to a recombinant human follistatin polypeptide comprising a mutated heparin binding sequence rendering said polypeptide unable to bind to heparin, for use in a medical treatment of a human subject suffering from a condition wherein enhancing bone formation, bone strength, mineralization and osteoblast activity is beneficial to the physiology of said human subject, and/or in a medical treatment for stimulating bone healing, bone formation and/or in a medical treatment of osteoporosis, wherein said

follistatin polypeptide is the full length splice variant. Many conditions exist wherein enhancing bone formation, bone strength, mineralization and osteoblast activity are

beneficial to the physiology of a human subject. Examples of such conditions are those relating to osteoporosis, secondary osteoporosis, osteopenia, osteomalacia, osteodystrophy, osteomyeloma, bone fracture, Paget ' s disease, osteogenesis imperfecta, (bone-related) trauma, orthopedy, tumoral

pseudarthrosis with or without bone defects, vertebral

arthrodesis (spinal fusion) and tibial osteotomy. The present invention also relates to a use of a

recombinant human follistatin polypeptide comprising a mutated heparin binding sequence rendering said polypeptide unable to bind to heparin, wherein said follistatin polypeptide is the full length splice variant, in the preparation of a medicament for use in a human subject suffering from a condition wherein enhancing bone formation, bone strength, mineralization and/or osteoblast activity is beneficial to the physiology of said human subject, and/or in a medical treatment for stimulating bone healing, bone formation and/or in a medical treatment of osteoporosis. Moreover, the present invention relates to a method of treating a human subject suffering from a condition wherein enhancing bone formation, bone strength,

mineralization and/or osteoblast activity is beneficial to the physiology of said human subject, and/or for the treatment for stimulating bone healing, bone formation and/or for the medical treatment of osteoporosis in said human subject, comprising the step of administering a therapeutically

effective amount of a recombinant human follistatin

polypeptide comprising a mutated heparin binding sequence rendering said polypeptide unable to bind to heparin, wherein said follistatin polypeptide is the full length splice

variant . In a highly preferred embodiment, said human follistatin polypeptide splice variant is based on the 315 amino acid- containing human follistatin protein, and contains the wild type sequences of that splice variant except for the

replacement of the HBS domain as disclosed herein. The person skilled in the art is aware of the wide variety of mutations that can be introduced in recombinant proteins. Examples of such mutations are (partial- or full) deletions, inversions, replacements and (point) mutations. In a preferred aspect the mutation of the heparin binding sequence comprises a

replacement of the heparin binding sequence (HBS; SEQ ID NO: 6) in FSD1, see Figure IB and Figure 2. More preferably, said heparin binding sequence is replaced by a corresponding region (SEQ ID NO: 7) from FSD2, see Figure IB and Figure 2, which leaves the remainder of the 315 amino acid-containing polypeptide intact. In yet another preferred embodiment, the recombinant human follistatin polypeptide according to the invention is fused (linked) at the C-terminus of the

polypeptide with an Fc domain of mouse IgG2A (for in vivo experiments in mice) or human IgGl (SEQ ID NO: 2) for the use in human subjects. Such Fc domains are often used in the art of protein expression to generate more stable proteins with improved pharmacokinetic properties and to simplify

purification procedures. Hence, the addition of the Fc domain contributes further to the already significant improved PK properties of the polypeptides of the present invention.

Nevertheless, as shown herein, only the addition of the Fc domain is insufficient to significantly increase the PK of the polypeptides . The present invention also relates to a nucleic acid encoding a human follistatin polypeptide according to the invention, and to an expression vector comprising the nucleic acid encoding a human follistatin polypeptide according to the invention. The present invention also relates to a

pharmaceutical composition comprising a recombinant human follistatin polypeptide or a nucleic acid according to the invention, said pharmaceutical composition further comprising a pharmaceutically acceptable carrier. The person skilled in the art is aware of the existence of a wide variety of

pharmaceutical acceptable carriers (sometimes referred to as pharmaceutically acceptable ^xexcipients' ) that may

appropriately be used in different settings for different therapeutic applications. The use or medical treatment method according to the present invention may be performed via parenteral, intravenous, intraperitoneal or intracapsular administration .

The recombinant human follistatin polypeptide of the present invention may be produced on different systems such as in vitro cultured cells (e.g. genetically engineered CHO, HEK- 293 or PER.C6 cells) or for instance in animal systems such as transgenic female mice, rats, goats, rabbits, bovines, pigs or camels that produce the recombinant protein in their milk. Amounts and regimens for the administration of follistatin can be determined readily by those with ordinary skill in the clinical art of treating bone-related disorders and bone formation defects. Generally, the dosage of treatment will vary depending upon considerations such as age, health, conditions being treated, kind of concurrent treatment (if any) , frequency of treatment and the nature of the effect desired, extent of tissue damage, gender, duration of the symptoms, counter indication (if any) and other variables to be adjusted by the individual physician. Dosage of follistatin can be administered in one or more applications to obtain the desired results. The dosage administered should preferably be chosen such that local concentrations are between about 5 and about 500 ng/ml follistatin. The dosage of follistatin

derivatives may suitably be from about 0.1 to about 30 mg/kg. The recombinant human follistatin derivatives of the present invention can be administered in any appropriate pharmalogical carrier for administration. They can be

administered in any form effective in prophylactic,

palliative, preventative, or curing conditions of bone-related disorders and bone formation defects. Preparations of the follistatin derivatives for parenteral administration include sterile aqueous or non-aqueous solvents, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, fish oil and injectable organic esters. Aqueous carriers include water, water-alcohol solutions, emulsions or suspensions, including saline and buffered medical parenteral vehicles including sodium chloride solution, Ringer' s dextrose solution, dextrose plus sodium chloride solution, Ringer' s solution containing lactose, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers , electrolyte replenishers , such as those based upon Ringer's dextrose and the like. The follistatin derivatives of the present invention may also be administered by means of pumps, or in sustained- release form, especially when the defect is prolonged or delayed rather than acute. The recombinant human follistatin polypeptides of the present invention may also be delivered to specific organs in high concentration by means of suitable inserted catheters, or by providing the polypeptides as a part of a chimeric molecule (or complex) , which is designed to target specific places in the body. Administration in a sustained-release form is more convenient for those subjects that require repeated injections over prolonged periods of time. Sustained-release forms using polymer-based nano- and microparticles are well-known in the art. It is desirable to administer the follistatin-derived polypeptides of the present invention in a sustained release form when the methods of the invention are being used to treat e genetic or chronic disease based upon a mineralization-related disorder so as to maximize the comfort to the human subject being treated.

The polypeptides of the present invention can be employed in dosage forms such as tablets, capsules, powder packets, liquid solutions for parenteral injection into the body, or liquid solutions for enteral (oral) administration.

Pharmaceutical compositions for use in aspects of the present invention can be obtained by mixing the recombinant human follistatin polypeptides with a pharmaceutically acceptable carrier or excipient, by means that are widely known in the art, such as by conventional mixing, granulating, dragee- making, dissolving, lyophilizing or similar processes. A very suitable application of a method of the present invention is the provision of tissue regeneration scaffolds that rapidly mineralize, and wherein said mineralization may occur either in vivo or in vitro. In yet another suitable application, the recombinant human follistatin polypeptides of the present invention are impregnated onto implants that subsequently are introduced into the human subject at the position where ECM mineralization is required, to have a direct effect at the targeted site. The invention is now further illustrated by the following non-limiting examples.

EXAMPLES

Example 1. Generation of follistatin variant polypeptides

Cloning procedures

Two splice variants of follistatin were chosen for further study: one that terminates at residue 288, herein referred to as FST288 and one that terminates at residue 315, herein referred to as FST315. Both variants were fused to a mouse IgG2A Fc domain (for experiments in mice) and to a human IgGl Fc domain (for use in humans) . SEQ ID NO:5 shows the FST288- Fc-AHBS protein sequence with the signal peptide at the N- terminus . The following cloning procedures were performed:

FST-Fc plasmid constructs were cloned by a fusion PCR reaction between hFST (template from Open Biosystems, pOTB7-H sapiens Follistatin accession number BC004107.2) and the Fc part of human IgGl (SEQ ID NO : 2 ; template derived from InVivoGen, pFUSE-hlgGl-Fc) or murine IgG2A. The DNA sequence for murine IgG2A (SEQ ID NO : 8 ) was ordered at GeneArt/Invitrogen in pMA-T plasmid .

To generate the various FST-FC variants FST and FC

fragments were amplified by PCR using the primers listed in Table I. Subsequently the two PCR fragments were fused by another PCR reaction. Table IIA lists the different PCR strategies used for the construction of the various FST-FC variants .

Table I. Primer sequences

Table IIA. PCR strategy

Table IIB. PCR strategy HBS variants

Fusion PCR products were cloned using TOPO cloning into pENTR/D-TOPO (Invitrogen art. K2400-20) . Subsequently product was subcloned into a pLenti-6.3/V5-DEST expression vector (Invitrogen, art. V533-06) using Gateway LR Clonase II enzyme (Invitrogen, art. 11791-020). From these FST-Fc construct the corresponding AHBS constructs were cloned. For PCR strategy see table IIB. First, a PCR reaction was performed to replace the HBS sequence by the corresponding amino acid sequence of FSD2 (generated PCR fragment: from start codon to end of HBS sequence) . Next this fragment and the 3' end of the FST-FC gene were used in a fusion PCR according to the PCR reaction described in table IIB (PCR 2,3 and fusion PCR) . This lead to a FST-FC AHBS PCR fragment that was used for subsequent cloning as described above.

Expression and purification

The pLenti6.3 expression plasmids were used in combination with ViraPower Lentiviral Packaging Mix (Invitrogen art . # K4975-00) for the production of lentivirus particles in

HEK293FT cells, following the protocols provided by the manufacturer. For expression of the preferred polypeptides encoded by the lentiviruses , CHO-S cells (Invitrogen, art . # 11619-012) were transduced with the respective lentiviruses and after 1 week of culture cells further selected on 2 yg/ml blasticidin (Invitrogen cat . # R210-01). Protein production was performed in CD CHO medium (Invitrogen, cat . # 10743-029) in 1 L spinner flasks (Corning, cat . # 3651) at 37°C, 115 rpm and 5% CO₂ for 72 h. The follistatin variant polypeptides were

purified from conditioned medium (cleared by centrifugation at 3000g and 0.2 ym filtration) using protein A based

chromatography (HiTrap MabSelect Xtra 5 mL, GE Healthcare art.# 28-4082-60), desalted using a HiPrep 26/10 Desalting column (GE Healthcare, art. 17-5087-01) into 50 mM KP0₄, 160 mM sucrose and 0.01% Tween-20, and stored at -80°C.

Example 2. Heparin- and activin-binding properties of the follistatin variant polypeptides

The generated follistatin variants FST315-FC and FST315- FC-AHBS were studied for their binding to heparin. This was analyzed by affinity chromatography (AKTAprime plus) . In total 200 yg purified protein was loaded on a 1 ml HiTrap Heparin HP column (GE, cat . # 17-0406-01) in PBS. Protein was washed with PBS and eluted with a 0.14-2M NaCl gradient in PBS. FST315-FC protein showed strong binding and was eluted at 62 mS/cm. De binding of FST315-FC-AHBS appeared much weaker and was eluted at 24 mS/cm. This shows that the heparin binding of protein was indeed significantly reduced by replacing the HBS domain in FSD1 by the corresponding non-heparin binding domain from FSD2. This is in agreement with the findings of Sidis and colleagues who showed that a FST mutant (splice variant terminated at residue 288) with a similar swap of the HBS domain had significantly lower cell surface binding capacity (Sidis Y et al . 2005. Endocrinology 146:130-6) .

Subsequently, the ability to bind activin A was determined in an activin A signaling bioassay. This assay was based on an activin A sensitive luciferase reporter assay in HEK293 cells (CAGAi₂-luciferase ; Dennler S et al . Direct binding of Smao!3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. 1998. EMBO J 17:3091-100) . Cells were stimulated with 1 ng/ml activin A (R&D systems) in the presence of various

concentrations protein. After 24 hours luciferase signal was measured. The results given in Figure 5 show that activin A binding capacities were similar between wild type follistatin (FST315), FST315-FC and FST315-FC-AHBS . It was concluded that the removal of the HBS domain from FSD1 and the replacement thereof of the corresponding region from FSD2 (that does not bind to heparin) does not influence the activity to bind activin A, which is in concert with the findings of Sidis et al . (2005) that show that the FST variant terminated at 288 and that contains a similar HBS swap did still bind labeled activin .

Example 3. Pharmacokinetic properties of the follistatin variant polypeptides

Figure 3 displays an overview of the experimental setup of the investigation on the pharmacokinetic properties of the follistation derivatives that were generated according to example 1. The delivery vehicle (50 mM KPO₄, 160 mM sucrose, 0.01% Tween, pH 7) was taken along as negative control.

FST315-FC and FST315-FC-AHBS (both in an amount of 2.0 mg/kg body weight) were injected iv in two sets of three 8-week old male Balb/c mice, as indicated. The injected volume ranged between 80 to 100 μΐ . Subsequently, serum samples were

collected at three time points (= 2 blood withdrawals at either lh, 3h, 24h or 3 days as indicated, and 1 cardiac puncture at sacrifice at either 7 days or 14 days, as

indicated) . Each time point gave 3 samples, from 3 mice each. Samples were stored at -20°C before use.

Next, serum was analyzed for protein concentrations using a human follistatin ELI SA (Peprotech) . The FST315-FC values were calculated using a FST315-FC standard curve and FST315- FC-AHBS values were calculated using a FST315-FC-AHBS standard curve. The results are given in Figure 4. Whereas the wild type protein FST315-FC had already disappeared from the serum at 24 h after administration, very high levels could still be detected of the FST315-FC-AHBS variant that only reached almost zero levels after 7 days. Peak serum levels at 1 h were about 20-40

for FST315-FC-AHBS and only 0.5 for FST315-FC. Exposure (area under the curve) was increased almost 200 fold. Values found with the vehicle administrations were all less than 0.01 μg/ml. The relevant pharmacokinetic parameters are summarized in Table III. With the exception of the total body clearance, all calculations are based on the terminal phases assuming a simple exponential model.

Table III. Pharmacokinetic parameters (in gram units) . AUC = area under the curve.

From these experiments it was concluded that the

replacement of the HBS domain in human follistatin not only influenced the ability of the follistatin protein to bind heparin, but also that the protein could remain in the system for a significant longer period after iv administration than the wild type follistatin protein.

Example 4. Effect of follistatin derivatives on bone formation

The results of example 3 show that the removal of the heparin binding sequence from the follistatin protein and replacement of that sequence by a corresponding domain from elsewhere in the protein results in a dramatic increase of pharmacokinetic properties. The protein appeared to remain in the circulation for a prolonged period of time. This however, does not directly mean that the protein should also have improved stimulating bone-forming activity. The heparin binding property of follistatin could act either positively: the HBS makes that follistatin is bound to the bone matrix locally resulting in efficient activin binding to the bone environment, or negatively: the low serum levels caused by the HBS result in low bioavailability in the bone because the majority of the follistatin derivative is present at other places in the body.

In a first study to investigate the bone-forming activity of the HBS deleted follistatin derivatives, the FST315-mFC- AHBS variant was taken and compared to a negative control (vehicle only) . For this 10 mg/kg protein (FST315-mFC-AHBS) was injected (ip) twice a week in 12 week old female C57B1/6 mice. At the end of the studies bones were collected and the following parameters were investigated in the proximal part of the tibiae using micro computed tomography (yCT) (Skyscan 7210) :

The results in respect of the amount of trabecular bone (first parameter) are shown in Figure 6A, 6B (two-week

studies) and Figure 6D (5-6 weeks study) . Notably, the amount of trabecular bone (%BV/TV) appeared to be significantly increased when FST315-mFC-AHBS was administered. Results in respect of trabecular number (same experiments) are shown in Figure 7A, B and D. There appeared to be no effect of any of the administered compounds on the averaged trabecular

thickness (data not shown) . Results in respect of the

trabecular pattern factor for these same experiments are shown in Figure 8A, B and D respectively. The overall conclusion from these experiments is that FST315-mFC-AHBS, while being unable to bind to heparin, is still capable of enhancing the amount of trabecular bone when administered ip over a period of several weeks.

To study the effect of the follistatin derivatives lacking the HBS domain on bone formation further, the two naturally occurring splice variants of follistatin were compared:

FST288-FC-AHBS (SEQ ID NO:5) and FST315-FC-AHBS (SEQ ID NO : 4 ) . Healthy female 12-week old Black6 mice were treated ip with 10 mg/kg body weight purified protein (FST288-mFC-AHBS and

FST315-mFC-AHBS) twice a week during a period of 2 weeks. At the end of the studies bones were collected and the following parameters were investigated in the proximal part of the tibiae using micro computed tomography (yCT) (Skyscan 7210) . The results are given in panels C of Figures 6-8. Similarly, experiments were performed using 10 mg/kg FST288-mFC-AHBS and FST315-mFC-AHBS in an experiment that lasted 6 weeks with twice weekly administrations. Results are shown in panels E and F of Figures 6-8. Panels E and F differed only in the type of mouse (panel E: Black6; panel F: NOD.SCID) . Unexpectedly, there appeared to be a striking difference between the

biological activities in respect of bone formation between the two natural splice variants: while the FST315-mFC-AHBS variant performed as in the previous experiments in that bone

formation was significantly induced over a number of weeks with regular administration intervals, the FST288-FC-AHBS variant appeared inactive, although it was previously shown that this variant could still interact with activin. The conclusion form these different experiments is that the longer 315 residue splice variant with a deleted HBS domain (in contrast to the known 288 residue splice variant with a similar HBS deletion) is preferably applied for bone forming therapy .

Claims

1. A recombinant 315-amino acid splice variant of the human follistatin polypeptide comprising a mutation in the heparin binding sequence of said splice variant rendering the polypeptide unable to bind to heparin, for use in a medical treatment of a subject suffering from a condition selected from the group consisting of: osteoporosis, secondary

pseudarthrosis with or without bone defects, vertebral

arthrodesis (spinal fusion) and tibial osteotomy.

2. The polypeptide for use according to claim 1, wherein said mutation comprises a replacement of the heparin binding sequence (SEQ ID NO: 6) in FSD1 by a corresponding region (SEQ ID NO: 7) from FSD2.

3. The polypeptide for use according to claim 1 or 2, wherein the polypeptide is linked to an Fc domain of human IgGl (SEQ ID NO : 2 ) .

4. A recombinant polypeptide comprising the amino acid sequence of SEQ ID NO: 23 for use in a medical treatment of a subject suffering from a condition selected from the group consisting of: osteoporosis, secondary osteoporosis,

osteopenia, osteomalacia, osteodystrophy, osteomyeloma, bone fracture, Paget ' s disease, osteogenesis imperfecta, (bone- related) trauma, orthopedy, tumoral cavities, Ear Nose &

Throat disease, maxilla-facial surgery, periodontal surgery, fractures with bone defects, pseudarthrosis with or without bone defects, vertebral arthrodesis (spinal fusion) and tibial osteotomy .

5. A recombinant polypeptide for use according to claim 4, wherein the polypeptide is linked to an Fc domain of human IgGl (SEQ ID NO : 2 ) .

6. A nucleic acid encoding the polypeptide of any one of claims 1-5.

7. An expression vector comprising the nucleic acid according to claim 6.

8. A pharmaceutical composition comprising a polypeptide of any one of claims 1-5, or a nucleic acid according to claim 6, said composition further comprising a pharmaceutically acceptable carrier.

9. Use of a recombinant polypeptide comprising the amino acid sequence of SEQ ID NO: 23 in the preparation of a

medicament for use in a subject suffering from a condition selected from the group consisting of: osteoporosis, secondary osteoporosis, osteopenia, osteomalacia, osteodystrophy, osteomyeloma, bone fracture, Paget ' s disease, osteogenesis imperfecta, (bone-related) trauma, orthopedy, tumoral

pseudarthrosis with or without bone defects, vertebral

arthrodesis (spinal fusion) and tibial osteotomy.

10. Use according to claim 9, wherein said polypeptide is linked to an Fc domain of human IgGl (SEQ ID NO: 2) .

11. A method of treating a subject suffering from a condition selected from the group consisting of: osteoporosis, secondary osteoporosis, osteopenia, osteomalacia, osteodystrophy, osteomyeloma, bone fracture, Paget ' s disease, osteogenesis imperfecta, (bone-related) trauma, orthopedy, tumoral cavities, Ear Nose & Throat disease, maxilla-facial surgery, periodontal surgery, fractures with bone defects, pseudarthrosis with or without bone defects, vertebral arthrodesis (spinal fusion) and tibial osteotomy, comprising the step of administering a therapeutically effective amount of a recombinant polypeptide comprising the amino acid sequence of SEQ ID NO:23.

12. Method according to claim 11, wherein said

polypeptide is linked to an Fc domain of human IgGl (SEQ ID NO: 2) .