WO2010130830A2

WO2010130830A2 - Amino acid sequences directed against sclerostin and polypeptides comprising the same for the treatment of bone diseases and disorders

Info

Publication number: WO2010130830A2
Application number: PCT/EP2010/056657
Authority: WO
Inventors: Karen Cromie; Maria Gonzalez Pajuelo; Peter Vanlandschoot
Original assignee: Ablynx N.V.
Priority date: 2009-05-15
Filing date: 2010-05-14
Publication date: 2010-11-18
Also published as: WO2010130830A3

Abstract

Amino acid sequences are provided that are directed against sclerostin, as well as compounds or constructs, and in particular proteins and polypeptides, that comprise or essentially consist of one or more such amino acid sequences. The invention also relates to pharmaceutical compositions that comprise such amino acid sequences and to uses of such amino acid sequences or polypeptides for the prevention and/or treatment of bone diseases and disorders.

Description

Amino acid sequences directed against sclerostin and polypeptides comprising the same for the treatment of bone diseases and disorders

The present invention relates to amino acid sequences that are directed against (as defined herein) sclerostin (also known as SOST and as defined herein), as well as to compounds or constructs, and in particular proteins and polypeptides, that comprise or essentially consist of one or more such amino acid sequences (also referred to herein as ""amino acid sequences of the invention ', "compounds of the invention", and "polypeptides of the invention'^', respectively). The invention also relates to nucleic acids encoding such amino acid sequences and polypeptides (also referred Io herein as "nucleic acids of the invention" or "nucleotide sequences of the invention"); to methods for preparing such amino acid sequences and polypeptides; to host cells expressing or capable of expressing such amino acid sequences or polypeptides: to compositions, and in particular to pharmaceutical compositions, that comprise such amino acid sequences, polypeptides, nucleic acids and/or host cells; and to uses of such amino acid sequences or polypeptides, nucleic acids, host cells and/or compositions, in particular for prophylactic, therapeutic or diagnostic purposes, such as the prophylactic, therapeutic or diagnostic purposes mentioned herein.

Other aspects, embodiments, advantages and applications of the invention will become clear from the further description herein.

Sclerosteosis and Van Buchem disease are rare, high- bone-mass disorders that have been linked to deficiency in the SOST gene, encoding sclerostin. Sclerosteosis is attributed to premature termination mutations in the SOST gene, whereas Van Buchem disease has been associated with a 52 kb deletion downstream of the SOST gene that most likely inhibits

SOST gene transcription (Balemans and Van HuI J. Musculoskclet. Neuronal Interact. 2004; 4(2): 139-42; Van Bezooijen et al. J. Endocrinol. Invest. 2005; 28 (8 Suppl):15-7; Martin et al. Osteoporos Int. 2008; 19(8): 1 125-38). Over-expression of human sclerostin with use of a mouse osteocalcin promoter led to mice that exhibited low bone mass and decreased bone strength as a result of reduced osteoblast activity (Winkler el al. EMBO J. 2003: 22: 6267- 76). On the other hand, deletion of SOST gene in mice results in increased bone formation and bone strength (Li et al. J. Bone Min. Res. 2008 23: 860-869). It has been shown that sclerostin inhibits osteoblast proliferation and differentiation, suppresses mineralization of osteoblastic cells in vitro and stimulates osteoblast apoptosis (van Bezooijen et al. J. Exp. Med. 2004 199: 805-814; Winkler et ai. EMBO J. 2003; 22: 6267-76; Sutherland et al. Bone 2004; 35: 448-454). Accordingly, based on the observed phenotypes associated with sclerostin expression, scierostin was found to act as a negative regulator of bone formation.

The S¹OST gene encodes a 213 amino acid propeptide with a calculated molecular weight of 24 KDa including a signal sequence for secretion in the first 23 residues. The secreted protein (sclerostin) is characterized by a cysteine-knot motif, very homologous to that of the Bone Morphogenetic Protein (BMP) antagonists of the DAN/Cerberus family

(Balemans and Van HuI J. Musculoskelet. Neuronal Interact. 2004; 4(2):139-42; Balemans et al. Human Molecular Genetics 2001; 10(5): 537-543; Avsian-Kretchmer and Hsueh MoI Endocrinol. 2004; 18(1):I-12). Analysis of recombinant sclerostin by nonreducing SDS- polyacrylamide gel electrophoresis followed by Western blotting and gel filtration chromatography indicates that sclerostin behaves like a monomer (ten Dijke et al. J. Bone Joint Surg, Am. 2008; 90: 31 -35). At the amino acid level mouse sclerostin sequence is 88% identical to the human sequence (Brunkow et al. Am. J. Hum. Genet. 2001 ; 68: 577-89). Sclerostin belongs to the DAN family of glycoproteins, of which multiple family members have been shown to antagonize bone morphogenetic protein (BMP) and/or Writ activity. Scierostin is specifically expressed by osteocytes and inhibits BMP-induced osteoblast differentiation and ectopic bone formation. Considering sclerostin as a DAN family member, it was originally suggested that sclerostin inhibit bone formation by antagonizing BMP activity (Brunkow et al. Am. J. Hum. Genet. 2001; 68: 577-589; Kusu et al. J. Biol. Chem. 2003; 278: 241 13-24117; Winkler et al. EMBO J. 2003: 22: 6267-76). However, later results showed that sclerostin binds only weakly to BMPs and does not inhibit direct BMP-induced responses, suggesting a different mechanism of action in which sclerostin could antagonize another signaling pathway which cooperates with BMPs in stimulating bone formation (van Bezooijen et al. J. Exp. Med. 2004; 199: 805-814; van Bezooijen et al. Cytokine Growth Factor Rev. 2005; 16: 319-327; 28: 15-7; van Bezooijen et al. J, Bone Miner. Res. 2007; 22(l):19-28). Indeed, it was found that sclerostin acts as a ligand for LRP5 and LRP6, thereby antagonizing canonical Wnt signaling (Li et al. J. Biol. Chem. 2005; 280: 19883-19887; Semenov et al. J. Biol. Chem. 2005; 280: 26770-26775; Clevers, Cell 2006; 127: 469-480). Although sclerostin does not appear to compete with Wnt for binding LRP5 or LRP6, it has been shown that sclerostin antagonizes the Wnt-induced transcriptional reporter activity and alkaline phosphatase activity in different cell lines (van Bezooijen et al. J. Bone Miner. Res. 2007; 22(l):19-28; Li et al. J. Biol. Chem. 2005; 280: 19883-19887; Semenov et al. J. Biol. Chem. 2005; 280: 26770-26775). Thus, it has been suggested that high bone mass formation occurring in sclerosteosis and Van Buchem disease may result from increased Wnt signaling due to the absence of or insensitivity to sclerostin (ten Dijke et al. J. Bone Joint Surg. Am. 2008: 90: 31-35).

Based on the prior art above, it is clear that there is increasing evidence for an important biologica] role of sclerostin in various bone diseases and disorders. As a result, sclerostin has been proposed as a therapeutic target to treat patients with bone diseases and disorders, which are characterized by low bone mass, such as osteoporosis. Osteoporosis has become a major public health problem, which is caused by an imbalance between osteoblast- mediated bone formation and osteoclast-mediated bone resorption. This leads to bone fragility and increased fracture risk. Current therapies rely on anti-resorptive agents, such as bisphosphonates. oestrogens. calcitonin and selective oestrogen receptor modulators.

Bisphosphonates are most often used for the treatment of osteoporosis. Although this drug class is generally very safe, oral dosing is complex (Shoback, J. Clin. Endocrinol. Metab. 2007; 92: 747-753). Furthermore, these agents reduce but do not eliminate fracture risk and do not restore lost bone structure. UCB Celltech (formerly Celltech) is developing, in collaboration with Amgen, an injectable sclerostin-neutralizing monoclonal antibody (mAb), CDP-7851 (Sclerostin Ab, AMG-785), for the potential treatment of osteoporosis. Such full-size antibodies, however, face drawbacks such as high production costs, low stability, and large size, which e.g. impedes their access to certain hidden epitopes. Accordingly, there remains a need for alternative or improved active principles that can be used for the prevention or treatment of sclerostin-mediated bone diseases and disorders and disorders. The present invention solves this problem by providing alternative and improved amino acid sequences and polypeptides that are specific or selective for sclerostin. According to one aspect polypeptides or amino acid sequences are provided which comprise Nanobodies® or fragments thereof and compositions comprising such polypeptides or amino acid sequences directed to sclerostin. as well as nucleotide sequences encoding such polypeptides and amino acid sequences. According to one aspect, the polypeptides or amino acids and compositions of the present invention are used to modulate signalling pathways in which sclerostin is involved. In one embodiment, the polypeptides and compositions of the present invention are used to modulate, and in particular inhibit and/or prevent binding of sclerostin to low-density lipoprotein receptor-related proteins 5 and/or 6 (LRP5 and/or LRP6). As sclerostin antagonizes canonical Wnt signalling, the polypeptides and compositions of the present invention that inhibit binding of sclerostin to LRP 5 and/or LRP6 can generally be used to modulate, and in particular induce, stimulate, antagonize the inhibitory effect on, or agonize signalling that is mediated by the interaction of Wnt proteins with Frizzled (Fzd) receptors and LRP5/LRP6 coreceptors, and/or to modulate the biological pathways in which Wnt proteins. Frizzled (Fzd) receptors and/or LRP5/LRP6 coreceptors are involved, and/or to modulate the biological mechanisms, responses and effects associated with such signalling or these pathways. In addition, the polypeptides and compositions of the present invention that inhibit binding of sclerostin to LRP5 and/or LRP6 can be used to modulate and in particular induce, stimulate, antagonize the inhibitory effect on, or agonize other signalling pathways cooperating with Wnt proteins in stimulating bone formation. For instance, the polypeptides and compositions of the present invention that inhibit binding of sclerostin to LRP5 and/or LRP6 can also be used to modulate and in particular induce, stimulate, antagonize the inhibitory effect on, or agonize signalling that is mediated by BMPs, BMP type-I and typε-ll transmembrane serine -threonine kinase receptors and/or intracellular Smad effector proteins, to modulate the biological pathways in which BMP proteins, BMP type-I and type-II transmembrane serine-threonine kinase receptors and/or intracellular Smad effector proteins are involved, and/or to modulate the biological mechanisms, responses and effects associated with such signalling or these pathways. In the presence of an amino acid sequence. Nanobody® or polypeptide of the invention, the binding of sclerostin to LRP5 and/or LRP6 may be inhibited and/or prevented by at least 1%, preferably at least 5%, such as at least 10% or at least 25%. for example by at least 50%. at least 60%, at least 70%, at least 80%, or 90% or more, compared to the binding of sclerostin to LRP5 and/or LRP6 under the same conditions but without the presence of the amino acid sequence, Nanobody® or polypeptide of the invention. Also, in the presence of the amino acid sequence. Nanobody® or polypeptide of the invention, the signalling that is mediated by Wnt proteins, Frizzled (Fzd) receptors and LRP5/LRP6 coreceptors and/or BMP proteins, BMP type-I and type-II transmembrane serine-threonine kinase receptors and intracellular Smad effector proteins may be induced, stimulated, or agonized (or the inhibitory effect of sclerostin on this signaling may be antagonized) by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%. at least 80%, or 90% or more, compared to the signalling that is mediated by Wnt proteins, Frizzled (Fzd) receptors and LRP5/LRP6 coreceptors and/or BMP proteins, BMP type-I and type-II transmembrane serine-threonine kinase receptors and intracellular Smad effector proteins (or the inhibitory effect of sclerostin on this signaling) under the same conditions but without the presence of the amino acid sequence, Nanobody® or polypeptide of the invention. It is furthermore envisaged that the polypeptides and compositions of the present invention that inhibit binding of sclerostin to LRP5 and/or LRP6 may or may not additionally inhibit the binding of sclerostin to other proteins naturally interacting with sclerostin; for instance, polypeptides and compositions of the invention binding to sclerostin may or may not inhibit the binding of sclerostin to bone morphogenetic proteins (BMPs), including but not limited to BMP-2, -4, -6 and/or -7.

In particular embodiments of the present invention, the amino acid sequences, polypeptides and compositions of the invention can be used to induce and/or stimulate the differentiation and/or proliferation of osteoblasts. The differentiation and/or proliferation of osteoblasts may be increased, respectively, by at least 1 %, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to the differentiation and/or proliferation of osteoblasts under the same conditions but without the presence of the amino acid sequence, Nanobody® or polypeptide of the invention.

In particular embodiments of the present invention, the amino acid sequences, polypeptides and compositions of the invention can be used to modulate bone remodelling. Bone remodelling may be modulated at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to bone remodelling under the same conditions but without the presence of the amino acid sequence, Nanobody®, polypeptide or composition of the invention.

As such, the polypeptides, amino acid sequences and compositions of the present invention can be used for the prevention and treatment (as defined herein) of bone diseases and disorders. Generally, "bone diseases and disorders" can be defined as diseases and disorders that can be prevented and/or treated, respectively, by suitably administering to a subject in need thereof (i.e. having the disease or disorder or at least one symptom thereof and/or at risk of attracting or developing the disease or disorder) of either a polypeptide, amino acid sequence or composition of the invention (and in particular, of a pharmaceutically active amount thereof) and/or of a known active principle active against sclerostin or a biological pathway or mechanism in which sclerostin is involved (and in particular, of a pharmaceutically active amount thereof).

Bone diseases and disorders encompass diseases and disorders associated with the regulation of bone formation and resorption. Bone diseases and disorders characterized by bone resorption are also referred to as osteopenic disorders, including ostopenia. osteoporosis and osteolysis. The polypeptides, amino acid sequences and compositions of the present invention that modulate, and in particular inhibit and/or prevent, binding of sclerostin to LRP5 and/or LRP6 will generally be used for the prevention and treatment (as defined herein) of bone diseases and disorders characterized by bone resorption. Examples of such bone diseases and disorders will be clear to the skilled person based on the disclosure herein, and for example include the following diseases and disorders: osteoporosis, including, but not limited to, primary osteoporosis, endocrine osteoporosis (including, but not limited to. hyperthyroidism, hyperparathyroidism, Cushing's syndrome, and acromegaly), hereditary and congenital forms of osteoporosis (including, but not limited to. osteogenesis imperfecta, homocystinuria, Menkes' syndrome, Riley-Day syndrome), osteoporosis due to immobilization of extremities, glucocortico id-induced osteoporosis and post-menopausal osteoporosis, a bone related disorder selected from the group consisting of achondroplasia, cleidocranial dysostosis, enchondromatosis, fibrous dysplasia, Gaucher's Disease, hypophosphatemic rickets. Marfan's syndrome, multiple hereditary exotoses, neurofibromatosis, osteogenesis imperfecta, osteopetrosis, osteopoikilosis, sclerotic lesions, pseudoarthrosis, pyogenic osteomyelitis, periodontal disease, anti-epileptic drug induced bone loss, primary and secondary hyperparathyroidism, familial hyperparathyroidism syndromes, weightlessness induced bone loss, osteoporosis in men. postmenopausal bone loss, osteoarthritis, renal osteodystrophy, infiltrative disorders of bone, oral bone loss, osteonecrosis of the jaw, juvenile Paget's disease, melorheostosis. metabolic bone diseases, mastocytosis, sickle cell anemia/disease, organ transplant related bone loss, kidney transplant related bone loss, systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenile arthritides, thalassemia, mucopolysaccharidoses. Fabry Disease. Turner Syndrome, Down Syndrome, Klinefelter Syndrome, leprosy. Perthe's Disease, adolescent idiopathic scoliosis, infantile onset multi-system inflammatory disease, Winchester Syndrome, Menkes Disease, Wilson's Disease, ischemic bone disease (such as Legg-Calve-Perthes disease and regional migratory osteoporosis), anemic states, conditions caused by steroids, glucocorticoid-induced bone loss, heparm-induced bone loss, bone marrow disorders, scurvy, malnutrition, calcium deficiency, osteoporosis, osteopenia, alcoholism, chronic liver disease, postmenopausal state, chronic inflammatory conditions, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, inflammatory colitis, Crohn's disease, oligomenorrhea, amenorrhea, pregnancy, diabetes mellitus, hyperthyroidism, thyroid disorders, parathyroid disorders. Cushing's disease, acromegaly, hypogonadism, immobilization or disuse, reflex sympathetic dystrophy syndrome, regional osteoporosis, osteomalacia, bone loss associated with joint replacement, HIV associated bone loss, bone loss associated with loss of growth hormone, bone loss associated with cystic fibrosis, chemotherapy-associated bone loss, tumor- induced bone loss, cancer-related bone loss, hormone ablative bone loss, multiple myeloma, drug- induced bone loss, anorexia nervosa, disease-associated facial bone loss, disease-associated cranial bone loss, disease-associated bone loss of the jaw. disease-associated bone loss of the skull, bone loss associated with aging, facial bone loss associated with aging, cranial bone loss associated with aging, jaw bone loss associated with aging, skull bone loss associated with aging, and bone loss associated with space travel. In particular, the polypeptides, amino acid sequences and compositions of the present invention can be used for the prevention and treatment of bone diseases and disorders which are mediated by the pathway(s) in which sclerostin is involved. Examples of such bone diseases and disorders will again be clear to the skilled person based on the disclosure herein. In particular the particular the polypeptides, amino acid sequences and compositions of the present invention are used in the prevention and treatment, but more particularly the treatment of osteoporosis.

Thus, without being limited thereto, the amino acid sequences, polypeptides and compositions of the invention can for example be used to prevent and/or to treat all diseases and disorders that are currently being prevented or treated with active principles that can modulate the pathway (s) in which sclerostin is involved, such as those mentioned in the prior art cited above. It is also envisaged that the polypeptides of the invention can be used to prevent and/or to treat all diseases and disorders for which treatment with such active principles is currently being developed, has been proposed, or ^"will be proposed or developed in future. In addition, it is envisaged that, because of their favourable properties as further described herein, the polypeptides of the present invention may be used for the prevention and treatment of other diseases and disorders than those for which these known active principles are being used or will be proposed or developed; and/or that the polypeptides of the present invention may provide new methods and regimens for treating the diseases and disorders described herein.

Thus, without being limited thereto, the amino acid sequences, polypeptides and compositions of the invention can for example be used to prevent and/or to treat all diseases and disorders that are currently being prevented or treated with CDP-7851 (Sclerostin Ab, AMG-785).

Other applications and uses of the amino acid sequences, polypeptides and compositions of the invention will become clear to the skilled person from the further disclosure herein.

Generally, it is an object of the invention to provide pharmacologically active agents, as well as compositions comprising the same, that can be used in the diagnosis, prevention and/or treatment of bone diseases and disorders and of the further diseases and disorders mentioned herein; and to provide methods for the diagnosis, prevention and/or treatment of such diseases and disorders that involve the administration and/or use of such agents and compositions. In particular, it is an object of the invention to provide such pharmacologically active agents, compositions and/or methods that have certain advantages compared to the agents, compositions and/or methods that are currently used and/or known in the art. These advantages will become clear from the further description below.

More in particular, it is an object of the invention to provide therapeutic proteins that can be used as pharmacologically active agents, as well as compositions comprising the same, for the diagnosis, prevention and/or treatment of bone diseases and disorders and of the further diseases and disorders mentioned herein; and to provide methods for the diagnosis, prevention and/or treatment of such diseases and disorders that involve the administration and/or the use of such therapeutic proteins and compositions. Accordingly, it is a specific object of the present invention to provide amino acid sequences that are directed against (as defined herein) sclerostin, in particular against sclerostin from a warm-blooded animal, more in particular against sclerostin from a mammal. and especially against human sclerostin; and to provide proteins and polypeptides comprising or essentially consisting of at least one such amino acid sequence.

In particular, it is a specific object of the present invention to provide such amino acid sequences and such proteins and/or polypeptides that are suitable for prophylactic, therapeutic and/or diagnostic use in a warm-blooded animal, and in particular in a mammal, and more in particular in a human being.

More in particular, it is a specific object of the present invention to provide such amino acid sequences and such proteins and/or polypeptides that can be used for the prevention, treatment, alleviation and/or diagnosis of one or more diseases, disorders or conditions associated with sclerostin and/or mediated by sclerostin (such as the diseases, disorders and conditions mentioned herein) in a warm-blooded animal, in particular in a mammal, and more in particular in a human being.

It is also a specific object of the invention to provide such amino acid sequences and such proteins and/or polypeptides thai can be used in the preparation of pharmaceutical or veterinary compositions for the prevention and/or treatment of one or more diseases. disorders or conditions associated with and/or mediated by sclerostin (such as the diseases, disorders and conditions mentioned herein) in a warm-blooded animal, in particular in a mammal, and more in particular in a human being.

In the invention, generally, these objects are achieved by the use of the amino acid sequences, proteins, polypeptides and compositions that are described herein.

In general, the invention provides amino acid sequences that are directed against (as defined herein) and/or can specifically bind (as defined herein) to sclerostin; as well as compounds and constructs, and in particular proteins and polypeptides, that comprise at least one such amino acid sequence. More in particular, the invention provides amino acid sequences that can bind to sclerostin with an affinity (suitably measured and/or expressed as a Kp-value (actual or apparent), a K_A- value (actual or apparent), a k_on-rate and/or a k_ofrrate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein; as well as compounds and constructs, and in particular proteins and polypeptides, that comprise at least one such amino acid sequence.

In particular, amino acid sequences and polypeptides of the invention are preferably such that they: bind to scleiOstin with a dissociation constant (K_D) of 1 (T⁵ to 1 Cf¹² moles/liter or less. and preferably 10^"7 to 10^"12 moles/liter or less and more preferably 10^"8 to 10^~12 moles/liter (i.e. with an association constant (K_A) of 10^s to 10¹² liter/ moles or more, and preferably 10⁷ to 10¹² liter/moles or more and more preferably 10⁸ to 10^!2 liter /moles); and/or such that they: bind to sclerostin with a k_on-rate of between 10² M^-1S^"1 to about 10⁷ M^" V¹, preferably between 10³ M^'V¹ and 10⁷ JvT¹S^"1, more preferably between 10⁴ M^'V¹ and M)⁷ M^'ls^"\ such as between 10⁵ M^-1S^'1 and 10⁷ M^-1S^"1; and/or such that they: bind to sclerostin with a k_Off rate between i s^"! (t[_/2=0.69 s) and 10^"6 s^"3 (providing a near irreversible complex with a im of multiple days), preferably between 10^~2 s^"1 and K)^"6 s^" \ more preferably between 10^~J s^"! and 10^"6 s^"1, such as between 10^"4 s^"! and 10^"6 s^"1.

Preferably, a monovalent amino acid sequence of the invention (or a polypeptide that contains only one amino acid sequence of the invention) is preferably such that it will bind to sclerostin with an affinity less than 500 nM, preferably less than 200 nM. more preferably- less than 10 nM. such as less than 500 pM.

Some preferred IC50 values for binding of the amino acid sequences or polypeptides of the invention to sclerostin will become clear from the further description and examples herein.

For binding to sclerostin. an amino acid sequence of the invention will usually contain within its amino acid sequence one or more amino acid residues or one or more stretches of amino acid residues (i.e. with each "stretch" comprising two or amino acid residues that are adjacent to each other or in close proximity to each other, i.e. in the primary or tertiary structure of the amino acid sequence) via which the amino acid sequence of the invention can bind to sclerostin, which amino acid residues or stretches of amino acid residues thus form the ''site" for binding to sclerostin (also referred to herein as the ''antigen binding site'^'). The amino acid sequences provided by the invention are preferably in essentially isolated form (as defined herein), or form part of a protein or polypeptide of the invention (as defined herein), which may comprise or essentially consist of one or more amino acid sequences of the invention and which may optionally further comptise one or more further amino acid sequences (all optionally linked via one or more suitable linkers). For example, and without limitation, the one or more amino acid sequences of the invention may be used as a binding unit in such a protein or polypeptide, which may optionally contain one or more further amino acid sequences that can serve as a binding unit (i.e. against one or more other targets than sclerostin). so as to provide a monovalent, multivalent or multispecific polypeptide of the invention, respectively, all as described herein. Such a protein or polypeptide may also be in essentially isolated form (as defined herein).

The amino acid sequences and polypeptides of the invention as such preferably essentially consist of a single amino acid chain that is not linked via disulphide bridges to any other amino acid sequence or chain (but that may or may not contain one or more intramolecular disulphide bridges. For example, it is known that Nanobodies® - as described herein - may sometimes contain a disulphide bridge between CDR3 and CDRl or FR2).

However, it should be noted that one or more amino acid sequences of the invention may be linked to each other and/or to other amino acid sequences (e.g. via disulphide bridges) to provide peptide constructs that may also be useful in the invention (for example Fab' fragments, F(ab')i fragments, ScFv constructs, "diabodies'^" and other multispecific constructs. Reference is for example made to the review by Holliger and Hudson, Nat Biotechnol. 2005 Sep; 23 (9):1126-36).

Generally, when an amino acid sequence of the invention (or a compound, construct or polypeptide comprising the same) is intended for administration to a subject (for example for therapeutic and/or diagnostic purposes as described herein), it is preferably either an amino acid sequence that does not occur naturally in said subject; or, when it does occur naturally in said subject, in essentially isolated form (as defined herein).

It will also be clear to the skilled person that for pharmaceutical use, the amino acid sequences of the invention (as well as compounds, constructs and polypeptides comprising the same) are preferably directed against human sclerostin; whereas for veterinary purposes, the amino acid sequences and polypeptides of the invention are preferably directed against sclerostin from the species to be treated, or at at least cross-reactive with sclerostin from the species to be treated.

Furthermore, an amino acid sequence of the invention may optionally, and in addition to the at least one binding site for binding against sclerostin, contain one or more further binding sites for binding against other antigens, proteins or targets.

The affinity of the amino acid sequences and polypeptides of the invention for sclerostin can be tested using any suitable in vitro binding assay. The efficacy of the amino acid sequences and polypeptides of the invention, and of compositions comprising the same, can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and/or animal model known per se. or any combination thereof, depending on the specific disease or disorder involved. Suitable assays and animal models will be clear to the skilled person, and for example include and for example include ELISA: FACS binding assay; Biacore; competition binding assay (AlphaScreen®, Perkin Elmer. Massachusetts, USA; FMAT): TRAP assay (osteoclast differentiation assay; Rissanen el al. 2005, J. Bone Miner. Res. 20, Suppl. 1: S256). EC50 values for binding of the Nanobodies®. amino acid sequences or polypeptides of the invention to sclerostin in, for example ELISA or FACS are preferably 1 μM to 1 pM. more preferably 1 nM to 3 pM and more preferably 100 pM to IpM. IC50 values for binding of the Nanobodies©. amino acid sequences or polypeptides of the invention to sclerostin in, for example, AlphaScreen® or TRAP assay are preferably 1 μM to 1 pM, more preferably I nM to IpM and more preferably 100 pM to IpM. Suitable cell-based assays will be clear to the skilled person, and for example include but are not limited to alkaline phosphatase activity assay, such as the inhibition by human sclerostin of BMP- 2/4/6/7 induced ALP activity in mouse preosteoblastic KS483 cells (van Bezooijen et al. J. Exp. Med. 2004: 199: 805-814) as well as BMP-ό and Wnt-induced ALP activity in mouse mesenchymal C3H10T1/2 cells (Winkler et al. EMBO J. 2003; 22: 6267-76) and inhibition of BMP-6 induced ALP activity in Primary human mesenchymal cells (Sutherland et al. Bone 2004; 35: 448-454) or binding of sclerostin- AP to LRP-6 on HEK cells transfected with human LRP-6 (Li et al. J. Biol. Chem. 2005; 280: 19883-19887); Luciferase Reporter Assay for Monitoring Canonical Wnt signaling Cells transfected with a Wnt luciferase reporter construct in combination with other constructs to induce Wnt signaling (WO 06/015373. WO 07/084344; WO 08/097510; van Bezooijen RL et al. 2007. J. Bone Miner. Res. 22: 19-28); Mineralization assay for inhibition of BMP-2/4/6/7-induced mineralization by human sclerostin using the alizarin red staining method (van Bezooijen et al. J. Exp. Med. 2004: 199: 805-814); Cell proliferation assay for inhibition of proliferation of primary human mesenchymal cells treated with sclerostin pulsed with [methyl-³H]-thymidine (Sutherland et al. Bone 2004; 35: 448^154).

Suitable animal models will be clear to the skilled person, and for example include but are not limited to inhibition of BMP -induced bone formation by X-ray analysis of bone formation in calf muscle of mice using DNA electroporation of equal amounts of expression plasmids for BMP and sclerostin (van Bezooijen et al. J. Bone Miner. Res. 2007; 22(1):19- 28) and osteopenic sclerostin-transgenic mice (Winkler et al. EMBO J. 2003; 22: 6267-76). intact mice models in which BMD is measured (WO 06/015373), Synergeπeic mouse model (WO 07/084344), Oviarectomized mice, as well as the assays and animal models used in the experimentaϊ part below and in the prior ait cited herein.

Also, according to the invention, amino acid sequences and polypeptides that are directed against scleroslin from a first species of warm-blooded animal may or may not show cross-reactivity with sclerostin from one or more other species of warm-blooded animal. For example, amino acid sequences and polypeptides directed against human sclerostin may or may not show cross reactivity with sclerostin from one or more other species of primates (such as, without limitation, monkeys from the genus Macaca (such as, and in particular, cynomolgus monkeys {Macaco fascicularis) and/or rhesus monkeys {Macaca mulatto)) and baboon (Papio ur sinus)) and/or with sclerostin from one or more species of animals that are often used in animal models for diseases (for example mouse, rat, rabbit, pig or dog), and in particular in animal models for diseases and disorders associated with sclerostin (such as the species and animal models mentioned herein). In this respect, it will be clear to the skilled person that such cross-reactivity, when present, may have advantages from a drug development point of view, since it allows the amino acid sequences and polypeptides against human sclerostin to be tested in such disease models.

More generally, amino acid sequences and polypeptides of the invention that are cross-reactive with sclerostin from multiple species of mammal will usually be advantageous for use in veterinary applications, since it will allow the same amino acid sequence or polypeptide to be used across multiple species. Thus, it is also encompassed within the scope of the invention that amino acid sequences and polypeptides directed against sclerostin from one species of animal (such as amino acid sequences and polypeptides against human sclerostin) can be used in the treatment of another species of animal, as long as the use of the amino acid sequences and/or polypeptides provide the desired effects in the species to be treated. SEQ ID NO^"s 189 to 195 represent examples of amino acid sequences according to the present invention that are cross-reactive with human sclerostin and mouse sclerostin.

The present invention is in its broadest sense also not particularly limited to or defined by a specific antigenic determinant, epitope, part, domain, subunit or conformation (where applicable) of sclerostin against which the amino acid sequences and polypeptides of the invention are directed. For example, the amino acid sequences and polypeptides may or may not be directed against an "interaction site" (as defined herein). However, it is generally assumed and preferred that the amino acid sequences and polypeptides of the invention are preferably directed against an interaction site (as defined herein), and in particular against an interaction site (as defined herein), and in particular against the binding site on sclerostin for LRP5 and/or LRP6. . Thus, in one preferred, but non-limiting aspect, the amino acid sequences and polypeptides of the invention are directed against the LRP5 and/or LRP6 interaction site on sclerostin, and are as further defined herein.

Binding of the amino acid sequences and polypeptides of the invention to the LRP5 and/or LRP6 interaction site on sclerostin may inhibit and/or prevent binding of sclerostin to LRP5 and/or LRP6. and thus inhibit biological activity of sclerostin. As sclerostin antagonizes canonical Wnt signalling, the amino acid sequences and polypeptides of the present invention that inhibit binding of sclerostin to LRP5 and/or LRP6 act as agonists of Wnt signalling that is mediated by the interaction of Wnt proteins with Frizzled (Fzd) receptors and LRP5/LRP6 coreceptors., and/or act as agonists of the biological pathways in which Wnt proteins. Frizzled (Fzd) receptors and/or LRP5/LRP6 coreceptors are involved. In addition, the polypeptides and amino acid sequences of the present invention that inhibit binding of sclerostin to LRP5 and/or LRP6 act as agonists of BMP signalling that is mediated by the interaction of BMPs with BMP type-I and type-II transmembrane serine-threonine kinase receptors and/or intracellular Smad effector proteins.

In one specific embodiment, the amino acid sequences and polypeptides of the present invention that inhibit binding of sclerostin to LRP5 and/or LRP6 additionally inhibit the binding of sclerostin to bone morphogenetic proteins (BMPs). including but not limited to BMP-2. -4, -6 and/or -7. Alternatively, the amino acid sequences and polypeptides of the invention are directed against the LRP5 and/or LRP6 interaction site on sclerostin while not interfering with the sclerostin/BMP interaction.

In particular embodiments, the amino acid sequences and polypeptides of the present invention are preferably directed against an epitope on sclerostin that overlaps with the epitope of CDP-7851 (Sclerostin Ab, AMG-785). Binding of the amino acid sequences and polypeptides of the invention to an epitope on scierostin that overlaps with the epitope of CDP-7851 (Sclerostin Ab, AMG-785) may inhibit and/or prevent binding of CDP-7851 (Sclerostin Ab. AMG-785) to sclerostin. The amino acid sequences and polypeptides of the invention may therefore act as a competitive or as a non-competitive inhibitor of the binding of CDP-7851 (Sclerostin Ab. AMG-785) to sclerostin (e.g. in ELISA, in AlphaScreen® assay or in TRAP assay). As further described herein, a polypeptide of the invention may contain two or more amino acid sequences of the invention that are directed against sclerostin. Generally, such polypeptides will bind to sclerostin with increased avidity compared to a single amino acid sequence of the invention. Such a polypeptide may for example comprise two amino acid sequences of the invention that are directed against the same antigenic determinant, epitope, part, domain, subunit or conformation (where applicable) of sclerostin (which may or may not be an interaction site); or comprise at least one "first'^" amino acid sequence of the invention that is directed against a first same antigenic determinant, epitope, part, domain, subunit or conformation (where applicable) of sclerostin (which may or may not be an interaction site); and at least one '"second" amino acid sequence of the invention that is directed against a second antigenic determinant, epitope, part, domain, subunit or conformation (where applicable) different from the first (and which again may or may not be an interaction site). Preferably, in such "biparatopic" polypeptides of the invention, at least one amino acid sequence of the invention is directed against an interaction site (as defined herein), although the invention in its broadest sense is not limited thereto.

Thus, in particular embodiments, an amino acid sequence or polypeptide of the invention may comprise two or more amino acid sequences of the invention that are directed against the binding site for LRP5 and/or LRP6 on sclerostin; or comprise at least one "first" amino acid sequence of the invention that is directed against the binding site for site for LRP5 and/or LRP6 on sclerostin; and at least one "second" amino acid sequence of the invention that is directed against a second antigenic determinant, epitope, part, domain, subunit or conformation different from the first and which is not a binding site for site for LRP5 and/or LRP6 on sclerostin.

Also, when the target is part of a binding pair (for example, a receptor-ligand binding pair), the amino acid sequences and polypeptides may be such that they compete with the cognate binding partner (e.g. the ligand, receptor or other binding partner, as applicable) for binding to the target, and/or such that they (fully or partially) neutralize binding of the binding partner to the target

The amino acid sequences, Nanobodies®, polypeptides and compositions of the present invention are directed against sclerostin (also referred to as SOST herein). The SOST gene encodes a 213 amino acid propeptide with a calculated molecular weight of 24 KDa including a signal sequence for secretion in the first 23 residues. This secreted protein (sclerostin) is characterized by a cysteine-knot motif, very homologous to that of the Bone Morphogenetic Protein (BMP) antagonists of the DAN/Cerbems family. Analysis of recombinant sclerostin by nonreducing SDS-polyacrylamide gel electrophoresis followed by Western blotting and gel filtration chromatography indicates that sclerostin behaves like a monomer. At the amino acid level mouse sclerostin sequence is 88% identical to the human sequence.

Accordingly, the nucleotide sequences and amino acid sequences of sclerostin of a wide variety of species are generally kno wn in the art. For instance, the amino acid sequence and nucleotide sequence of human sclerostin are for example represented in the NCBI nucleotide database (see website at http://www.ncbi.nlm.nih.gov/) as Genbank Accession No. AF326736 and Genbank Accession No's. NPJJ79513, AAKl 3451 and AAKl 3454, respectively. Preferred sequences of sclerostin against which the amino acid sequences, Nanobodies®, polypeptides and compositions of the present invention are directed are represented herein as SEQ ID NO¹S: 198 and 199.

It is also within the scope of the invention that, where applicable, an amino acid sequence of the invention can bind to two or more antigenic determinants, epitopes, parts, domains, subunits or conformations of sclerostin. In such a case, the antigenic determinants, epitopes, parts, domains or subunits of sclerostin to which the amino acid sequences and/or polypeptides of the invention bind may be essentially the same (for example, if sclerostin contains repeated structural motifs or occurs in a multimeric form) or may be different (and in the latter case, the amino acid sequences and polypeptides of the invention may bind to such different antigenic determinants, epitopes, parts, domains, subunits of sclerostin with an affinity and/or specificity which may be the same or different). Also, for example, when sclerostin exists in an activated conformation and in an inactive conformation, the amino acid sequences and polypeptides of the invention may bind to either one of these conformation, or may bind to both these conformations (i.e. with an affinity and/or specificity which may be the same or different). Also, for example, the amino acid sequences and polypeptides of the invention may bind to a conformation of sclerostin in which it is bound to a pertinent ligand, may bind to a conformation of sclerostin in which it not bound to a pertinent ligand, or may bind to both such conformations (again with an affinity and/or specificity which may be the same or different).

It is also expected that the amino acid sequences and polypeptides of the invention will generally bind to all naturally occurring or synthetic analogs, variants, mutants, alleles, parts and fragments of sclerostin; or at least to those analogs, variants, mutants, alleles, parts and fragments of sclerostin that contain one or more antigenic determinants or epitopes that are essentially the same as the antigenic determinant(s) or epitope(s) to which the amino acid sequences and polypeptides of the invention bind in sclerostin (e.g. in wild-type sclerostin). Again, in such a case, the amino acid sequences and polypeptides of the invention may bind to such analogs, variants, mutants, alleles, parts and fragments with an affinity and/or specificity that are the same as, or that are different from (i.e. higher than or lower than), the affinity and specificity with which the amino acid sequences of the invention bind to (wild- type) sclerostin. It is also included within the scope of the invention that the amino acid sequences and polypeptides of the invention bind to some analogs, variants, mutants, alleles, parts and fragments of sclerostin, but not to others. When sclerostin exists in a monomeric form and in one or more muitimeric forms, it is within the scope of the invention that the amino acid sequences and polypeptides of the invention only bind to sclerostin in monomeric form, only bind to sclerostin in muitimeric form, or bind to both the monomeric and the muitimeric form. Again, in such a case, the amino acid sequences and polypeptides of the invention may bind to the monomeric form with an affinity and/or specificity that are the same as. or that are different from (i.e. higher than or lower than), the affinity and specificity with which the amino acid sequences of the invention bind to the muitimeric form.

Also, when sclerostin can associate with other proteins or polypeptides to form protein complexes (e.g. with multiple subunits), it is within the scope of the invention that the amino acid sequences and polypeptides of the invention bind to sclerostin in its non- associated state, bind to sclerostin in its associated state, or bind to both. In all these cases, the amino acid sequences and polypeptides of the invention may bind to such multimers or associated protein complexes with an affinity and/or specificity that may be the same as or different from (i.e. higher than or lower than) the affinity and/or specificity with which the amino acid sequences and polypeptides of the invention bind to sclerostin in its monomeric and non-associated state.

Also, as will be clear to the skilled person, proteins or polypeptides that contain two or more amino acid sequences directed against sclerostin may bind with higher avidity to sclerostin than the corresponding monomeric amino acid sequence(s). For example, and without limitation, proteins or polypeptides that contain two or more amino acid sequences directed against different epitopes of sclerostin may (and usually will) bind with higher avidity than each of the different monomers, and proteins or polypeptides that contain two or more amino acid sequences directed against sclerostin may (and usually will) bind also with higher avidity to a multimer of sclerostin. Generally, amino acid sequences and polypeptides of the invention will at least bind to those forms of sclerostin (including monomeric, muitimeric and associated forms) that are the most relevant from a biological and/or therapeutic point of view, as will be clear to the skilled person. It is also within the scope of the invention to use parts, fragments, analogs, mutants, variants, alleles and/or derivatives of the amino acid sequences and polypeptides of the invention, and/or to use proteins or polypeptides comprising or essentially consisting of one or more of such parts, fragments, analogs, mutants, variants, alleles and/or derivatives, as long as these are suitable for the uses envisaged herein. Such parts, fragments, analogs. mutants, variants, alleles and/or derivatives will usually contain (at least part of) a functional antigen-binding site for binding against sclerostin; and more preferably will be capable of specific binding to sclerostin, and even more preferably capable of binding to sclerostin with an affinity (suitably measured and/or expressed as a Kϋ-value (actual or apparent), a K_A- value (actual or apparent), a k_on-rate and/or a k_Off rate, or alternatively as an IC50 value, as further described herein) that is as defined herein. Some non-limiting examples of such parts, fragments, analogs, mutants, variants, alleles, derivatives, proteins and/or polypeptides will become clear from the further description herein. Additional fragments or polypeptides of the invention may also be provided by suitably combining (i.e. by linking or genetic fusion) one or more (smaller) parts or fragments as described herein.

In one specific, but non-limiting aspect of the invention, which will be further described herein, such analogs, mutants, variants, alleles, derivatives have an increased half- life in serum (as further described herein) compared to the amino acid sequence from which they have been derived. For example, an amino acid sequence of the invention may be linked (chemically or otherwise) to one or more groups or moieties that extend the half-life (such as PEG), so as to provide a derivative of an amino acid sequence of the invention with increased half-life.

In one specific, but non-limiting aspect, the amino acid sequence of the invention may be an amino acid sequence that comprises an immunoglobulin fold or may be an amino acid sequence that, under suitable conditions (such as physiological conditions) is capable of forming an immunoglobulin fold (i.e. by folding). Reference is inter alia made to the review by Halaby et al., J. (1999) Protein Eng. 12, 563-71. Preferably, when properly folded so as to form an immunoglobulin fold, such an amino acid sequence is capable of specific binding (as defined herein) to sclerostin; and more preferably capable of binding to sclerostin with an affinity (suitably measured and/or expressed as a K_D-value (actual or apparent), a K_A-value (actual or apparent), a Ic_0n -rate and/or a k_off-rate, or alternatively as an ΪC₅₀ value, as further described herein) that is as defined herein. Also, parts, fragments, analogs, mutants, variants, alleles and/or derivatives of such amino acid sequences are preferably such that they comprise an immunoglobulin fold or are capable for forming, under suitable conditions, an immunoglobulin fold.

In particular, but without limitation, the amino acid sequences of the invention may be amino acid sequences that essentially consist of 4 framework regions (FRl to FR4 respectively) and 3 complementarity determining regions (CDRl to CDR3 respectively); or any suitable fragment of such an amino acid sequence (which will then usually contain at least some of the amino acid residues that form at least one of the CDR^" s. as further described herein).

The amino acid sequences of the invention may in particular be an immunoglobulin sequence or a suitable fragment thereof, and more in particular be an immunoglobulin variable domain sequence or a suitable fragment thereof, such as light chain variable domain sequence (e.g. a V| -sequence) or a suitable fragment thereof: or a heavy chain variable domain sequence (e.g. a V_H-sequence) or a suitable fragment thereof. When the amino acid sequence of the invention is a heavy chain variable domain sequence, it may be a heavy chain variable domain sequence that is derived from a conventional four-chain antibody (such as. without limitation, a V_H sequence that is derived from a human antibody) or be a so-called Vi_4H-sequence (as defined herein) that is derived from a so-called "heavy chain antibody'" (as defined herein).

However, it should be noted that the invention is not limited as to the origin of the amino acid sequence of the invention (or of the nucleotide sequence of the invention used to express it), nor as to the way thai the amino acid sequence or nucleotide sequence of the invention is (or has been) generated or obtained. Thus, the amino acid sequences of the invention may be naturally occurring amino acid sequences (from any suitable species) or synthetic or semi-synthetic amino acid sequences. In a specific but non-limiting aspect of the invention, the amino acid sequence is a naturally occurring immunoglobulin sequence (from any suitable species) or a synthetic or semi -synthetic immunoglobulin sequence, including but not limited to "humanized" (as defined herein) immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized V_ΠH sequences or Nanobodies®). '"camelized" (as defined herein) immunoglobulin sequences, as well as immunoglobulin sequences that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing. Reference is for example made to the standard handbooks, as well as to the further description and prior art mentioned herein. Similarly, the nucleotide sequences of the invention may be naturally occurring nucleotide sequences or synthetic or semi-synthetic sequences, and may for example be sequences that are isolated by PCR from a suitable naturally occurring template (e.g. DNA or RNA isolated from a cell), nucleotide sequences that have been isolated from a library (and in particular, an expression library), nucleotide sequences that have been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR). nucleotide sequence that have been, prepared by PCR using overlapping primers, or nucleotide sequences that have been prepared using techniques for DNA synthesis known per se.

The amino acid sequence of the invention may in particular be a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody), a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a "dAb" (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody®™ (as defined herein, and including but not limited to a V_HH sequence); other single variable domains, or any suitable fragment of any one thereof. For a, general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684. For the term "dAb's". reference is for example made to Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), to Holt et al., Trends BiotechnoL, 2003, 21(1 1):484-490; as well as to for example WO 06/030220, WO 06/003388 and other published patent applications of Domantis Ltd. It should also be noted that, although less preferred in the context of the present invention because they are not of mammalian origin, single domain antibodies or single variable domains can be derived from certain species of shark (for example, the so- called "IgNAR domains", see for example WO 05/18629).

In particular, the amino acid sequence of the invention may be a Nanobody© (as defined herein) or a suitable fragment thereof. [Note: Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx N. V.J Such Nanobodies© directed against sclerostin will also be referred to herein as "Nanobodies® of the invention".

For a general description of Nanobodies®, reference is made to the further description below, as well as to the prior art cited herein. In this respect, it should however be noted that this description and the prior art mainly described Nanobodiεs® of the so-called "V_H3 class'¹ (i.e. Nanobodies® with a high degree of sequence homology to human germiine sequences of the V_H3 class such as DP-47, DP-51 or DP-29), which Nanobodies® form a preferred aspect of this invention. It should however be noted that the invention in its broadest sense generally covers any type of Nanobody® directed against sclerostin. and for example also covers the Nanobodies® belonging to the so-called "Vn4 class" (i.e. Nanobodies® with a high degree of sequence homology Io human germiine sequences of the V_H4 class such as DP-78), as for example described in WO 07/118670.

Generally, Nanobodies® (in particular V_HH sequences and partially humanized Nanobodies®) can in particular be characterized by the presence of one or more ''Hallmark residues^"' (as described herein) in one or more of the framework sequences (again as further described herein).

Thus, generally, a Nanobody® can be defined as an amino acid sequence with the (general) structure

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which one or more of the Hallmark residues are as further defined herein.

In particular, a Nanobody® can be an amino acid sequence with the (general) Structure

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FRl to FR4 refer to framework regions 1 to 4. respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 3 to 3, respectively, and in which the framework sequences are as further defined herein.

More in particular, a Nanobody® can be an amino acid sequence with the (general) structure

FRI - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: i) preferably one or more of the amino acid residues at positions 11, 37. 44. 45. 47. 83,

84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table B-2 below; and in which: ii) said amino acid sequence has at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO^"s: I to 22. in which for the purposes of determining the degree of amino acid identit), the amino acid residues that form the CDR sequences (indicated with X in the sequences of SEQ ID NO's: 1 to 22) are disregarded.

In these Nanobodies©, the CDR sequences are generally as further defined herein. Thus, the invention also relates to such Nanobodies® that can bind to (as defined herein) and/or are directed against sclerostin, to suitable fragments thereof, as well as to polypeptides that comprise or essentially consist of one or more of such Nanobodies® and/or suitable fragments.

SEQ ID NO's: 189 to 197 (see Table A-I) give the amino acid sequences of a number of V_Hji sequences that have been raised against sclerostin.

In particular, the invention in some specific embodiments provides: - amino acid sequences that are directed against (as defined herein) sclerostin and that have at least 80%, preferably at least 85%. such as 90% or 95% or more sequence identity with at least one of the amino acid sequences of SEQ ID NO^'s: 189 to 197 (see Table A-I). These amino acid sequences may further be such that they neutralize binding of LRP5 and/or LRP6to sclerostin: and/or compete with LRP5 and/or LRPόfor binding to sclerostin; and/or are directed against an interaction site (as defined herein) on sclerostin (such as the LRP5 binding site and/or the LRP6 binding site); - amino acid sequences that cross-block (as defined herein) the binding of at least one of the amino acid sequences of SEQ ID NO's: 189 to 197 (see Table A-I) to sclerostin and/or that compete with at least one of the amino acid sequences of SEQ ID NO's: 189 to 197 (see Table A-I) for binding to sclerostin. Again, these amino acid sequences maj further be such that they neutralize binding of LRP5 and/or LRPόto sclerostin; and/or compete with LRP5 and/or LRPβfor binding to sclerostin; and/or are directed against an interaction site (as defined herein) on sclerostin (such as the LRP5 binding site and/or the LRP6 binding site); which amino acid sequences may be as further described herein (and ma) for example be Nanobodies©); as well as polypeptides of the invention that comprise one or more of such amino acid sequences (which may be as further described herein, and may for example be bispecific and/or biparatopic polypeptides as described herein), and nucleic acid sequences that encode such amino acid sequences and polypeptides. Such amino acid sequences and polypeptides do not include any naturally occurring ligands.

Some particularly preferred Nanobodies® of the invention are Nanobodies® which can bind (as further defined herein) to and/or are directed against sclerostin and which: i) have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 189 to 197 (see Table A-I), in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded. In this respect, reference is also made to Table B-] , which lists the framework 1 sequences (SEQ ID NO^'s: 126 to 134). framework 2 sequences (SEQ ID

NO^'s: 144 to 152), framework 3 sequences (SEQ ID NO's: 162 to 170) and framework 4 sequences (SEQ ID NO's: 180 to 188) of the Nanobodies^ of SEQ ID NO^'s: 189 to 197 (see Table A-I) (with respect to the amino acid residues at positions 1 to 4 and 27 to 30 of the framework 1 sequences, reference is also made to the comments made below. Thus, for determining the degree of amino acid identity, these residues are preferably disregarded); and in which: ii) preferably one or more of the amino acid residues at positions 11, 37, 44. 45, 47, 83,

84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table B-2 below.

In these Nanobodies®. the CDR sequences are generally as further defined herein. Again, such Nanobodies© may be derived in any suitable manner and from any suitable source, and may for example be naturally occurring V_HH sequences (i.e. from a suitable species of Camelid) or synthetic or semi-synthetic amino acid sequences, including but not limited to "humanized^" (as defined herein) Nanobodies®. "'camelized" (as defined herein) immunoglobulin sequences (and in particular camelized heavy chain variable domain sequences), as well as Nanobodies® that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences). CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing as further described herein. Also, when a Nanobody® comprises a V_HH sequence, said Nanobody® may be suitably humanized, as further described herein, so as to provide one or more further (partially or fully) humanized Nanobodies® of the invention. Similarly, when a Nanobody© comprises a synthetic or semisynthetic sequence (such as a partially humanized sequence), said Nanobody® may optionally be further suitably humanized, again as described herein, again so as to provide one or more further (partially or fully) humanized Nanobodies® of the invention.

In particular, humanized Nanobodies® may be amino acid sequences that are as generally defined for Nanobodies® in the previous paragraphs, but in which at least one amino acid residue is present (and in particular, in at least one of the framework residues) that is and/or that corresponds to a humanizing substitution (as defined herein). Some preferred, but non-limiting humanizing substitutions (and suitable combinations thereof) will become clear to the skilled person based on the disclosure herein. In addition, or alternatively, other potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring V_HH sequence with the corresponding framework sequence of one or more closely related human V_H sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said V_HH sequence (in any manner known per se, as further described herein) and the resulting humanized V_HH sequences can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. In this way. by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person based on the disclosure herein. Also, based on the foregoing, (the framework regions of) a Nanobody® may be partially humanized or fully humanized.

Some particularly preferred humanized Nanobodies® of the invention are humanized variants of the Nanobodies® of SEQ ID NO's: 189 to 197 (see Table A-I). Thus, some other preferred Nanobodies® of the invention are Nanobodies® which can bind (as further defined herein) to sclerostin and which: i) are a humanized variant of one of the amino acid sequences of SEQ ID NO's: 189 to 197 (see Table A-I); and/or ii) have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ΪD NO's: 189 to 197 (see Table A-I) and/or humanized variants thereof, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded; and in which: i) preferably one or more of the amino acid residues at positions 11, 37, 44. 45. 47. 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table B-2 below.

According to another specific aspect of the invention, the invention provides a number of stretches of amino acid residues (i.e. small peptides) that are particularly suited for binding to sclerostin. These stretches of amino acid residues may be present in. and/or may be incorporated into, an amino acid sequence of the invention, in particular in such a way that they form (part of) the antigen binding site of an amino acid sequence of the invention. As these stretches of amino acid residues were first generated as CDR sequences of heavy chain antibodies or V_HH sequences that were raised against sclerostin (or may be based on and/or derived from such CDR sequences, as further described herein), they will also generally be referred to herein as ^UCDR sequences" (i.e. as CDRl sequences, CDR2 sequences and CDR3 sequences, respectively). It should however be noted that the invention in its broadest sense is not limited to a specific structural role or function that these stretches of amino acid residues may have in an amino acid sequence of the invention, as long as these stretches of amino acid residues allow the amino acid sequence of the invention to bind to sclerostin. Thus, generally, the invention in its broadest sense comprises any amino acid sequence that is capable of binding to sclerostin and that comprises one or more CDR sequences as described herein, and in particular a suitable combination of two or more such CDR sequences, that are suitably linked to each other via one or more further amino acid sequences, such that the entire amino acid sequence forms a binding domain and/or binding unit that is capable of binding to sclerostin. Il should however also be noted that the presence of only one such CDR sequence in an amino acid sequence of the invention may by itself already be sufficient to provide an amino acid sequence of the invention that is capable of binding to sclerostin; reference is for example again made to the so-called "Expedite fragments" described in WO 03/050531.

Thus, inparticular. but non-limiting embodiments, the amino acid sequences of the invention may be amino acid sequences that comprises at least one amino acid sequence that is chosen from the group consisting of the CDRl sequences, CDR2 sequences and CDR3 sequences that are described herein (or any suitable combination thereof). In particular, an amino acid sequence of the invention may be an amino acid sequence that comprises at least one antigen binding site, wherein said antigen binding site comprises at least one amino acid sequence that is chosen from the group consisting of the CDRl sequences, CDR2 sequences and CDR3 sequences that are described herein (or any suitable combination thereof).

Generally, in these embodiments of the invention, the amino acid sequence of the invention may be any amino acid sequence that comprises at least one stretch of amino acid residues, in which said stretch of amino acid residues has an amino acid sequence that corresponds to the sequence of at least one of the CDR sequences described herein. Such an amino acid sequence may or may not comprise an immunoglobulin fold. For example, and without limitation, such an amino acid sequence may be a suitable fragment of an immunoglobulin sequence that comprises at least one such CDR sequence, but that is not large enough to form a (complete) immunoglobulin fold (reference is for example again made to the "Expedite fragments" described in WO 03/050531). Alternatively, such an amino acid sequence may be a suitable "protein scaffold" that comprises least one stretch of amino acid residues that corresponds to such a CDR sequence (i.e. as part of its antigen binding site). Suitable scaffolds for presenting amino acid, sequences will be clear to the skilled person, and for example comprise, without limitation, to binding scaffolds based on or derived from immunoglobulins (i.e. other than the immunoglobulin sequences already described herein), protein scaffolds derived from protein A domains (such as Affibodies™), tendamistat fibronectin, lipocalin, CTLA-4, T-cell receptors, designed ankyrin repeats, avimers and PDZ domains (Binz et al, Nat. Biotech 2005, VoI 23: 1257), and binding moieties based on DNA or RNA including but not limited to DNA or RNA aptamers (Ulrich et al._^Comb Chem High Throughput Screen 2006 9(8):619-32). Again, any amino acid sequence of the invention that comprises one or more of these

CDR sequences is preferably such that it can specifically bind (as defined herein) to sclerostin, and more in particular such that it can bind to sclerostin with an affinity (suitably measured and/or expressed as a K_D- value (actual or apparent), a K_A-value (actual or apparent), a k_on-rate and/or a k_Off-rate, or alternatively as an IC₅₀ value, as further described herein), that is as defined herein.

In particular embodiments, the amino acid sequences of the invention may be any amino acid sequence that comprises at least one antigen binding site, wherein said antigen binding site comprises at least two amino acid sequences that are chosen from the group consisting of the CDRl sequences described herein, the CDR2 sequences described herein and the CDR3 sequences described herein, such that (i) when the first amino acid sequence is chosen from the CDRl sequences described herein, the second amino acid sequence is chosen from the CDR2 sequences described herein or the CDR3 sequences described herein; (ii) when the first amino acid sequence is chosen from the CDR2 sequences described herein, the second amino acid sequence is chosen from the CDRl sequences described herein or the CDR3 sequences described herein; or (iii) when the first amino acid sequence is chosen from the CDR3 sequences described herein, the second amino acid sequence is chosen from the CDRl sequences described herein or the CDR3 sequences described herein. Even more in particular, the amino acid sequences of the invention may be amino acid sequences that comprise at least one antigen binding site, wherein said antigen binding site comprises at least three amino acid sequences that are chosen from the group consisting of the CDRl sequences described herein, the CDR2 sequences described herein and the CDR3 sequences described herein, such that the first amino acid sequence is chosen from the CDRl sequences described herein, the second amino acid sequence is chosen from the CDR2 sequences described herein, and the third amino acid sequence is chosen from the CDR3 sequences described herein. Preferred combinations of CDRl, CDR2 and CDR3 sequences will become clear from the further description herein. As will be clear to the skilled person, such an amino acid sequence is preferably an immunoglobulin sequence (as further described herein), but it may for example also be any other amino acid sequence that comprises a suitable scaffold for presenting said CDR sequences.

Thus, in one specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against sclerostin, that comprises one or more stretches of amino acid residues chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO's: 135 to 143; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; d) the amino acid sequences of SEQ ID NO's: 153 to 161 ; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 153 to 161 ; f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 153 to 161 : g) the amino acid sequences of SEQ ID NO's: 171 to 179; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid, sequences of SEQ ID NO's: 171 to 179; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 171 to 179; or an)^f suitable combination thereof.

When an amino acid sequence of the invention contains one or more amino acid sequences according to b) and/or c): i) any amino acid substitution in such an amino acid sequence according to b) and/or c) is preferably, and compared to the corresponding amino acid sequence according to a), a conservative amino acid substitution, (as defined herein); and/or ii) the amino acid sequence according to b) and/or c) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding amino acid sequence according to a); and/or iii) the amino acid sequence according to b) and/or c) may be an amino acid sequence that is derived from an amino acid sequence according to a) by means of affinity maturation using one or more techniques of affinity maturation known per se.

Similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to e) and/or f): i) arty amino acid substitution in such an amino acid sequence according to e) and/or f) is preferably, and compared to the corresponding amino acid sequence according to d), a conservative amino acid substitution, (as defined herein): and/or ii) the amino acid sequence according to e) and/or f) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding amino acid sequence according to d); and/or iii) the amino acid sequence according to e) and/or f) may be an amino acid sequence that is derived from an amino acid sequence according to d) by means of affinity maturation using one or more techniques of affinity maturation known per se.

Also, similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to h) and/or i): i) any amino acid substitution in such an amino acid sequence according to h) and/or i) is preferably, and compared to the corresponding amino acid sequence according to g). a conservative amino acid substitution, (as defined herein); and/or ii) the amino acid sequence according to h) and/or i) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding amino acid sequence according to g); and/or iii) the amino acid sequence according to h) and/or i) may be an amino acid sequence that is derived from an amino acid sequence according to g) by means of affinity maturation using one or more techniques of affinity maturation known per se.

It should be understood that the last preceding paragraphs also general!}' apply to any amino acid sequences of the invention that comprise one or more amino acid sequences according to b), c), e), f), h) or i). respectively. In these embodiments, the amino acid sequence preferably comprises one or more stretches of amino acid residues chosen from the group consisting of: i) the amino acid sequences of SEQ ID NO^"s: 135 to 143; ii) the amino acid sequences of SEQ ID NO^'s: 153 to 161; and iii) the amino acid sequences of SEQ ID NO's: 171 to 179; or any suitable combination thereof.

Also, preferably, in such an amino acid sequence, at least one of said stretches of amino acid residues forms part of the antigen binding site for binding to sclerostin.

In more specific, but again non-limiting embodiments, the invention relates to an amino acid sequence directed against sclerostin that comprises two or more stretches of amino acid residues chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO^'s: 135 to 143; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; d) the amino acid sequences of SEQ ID NO's: 153 to 161 ; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 153 to 161; f) amino acid sequences that have 3, 2. or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 153 to 161; g) the amino acid sequences of SEQ ID NO's: 171 to 179; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 171 to 179; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 171 to 179; such that (i) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b) or c). the second stretch of amino acid residues corresponds to one of the amino acid sequences according to d), e), f), g), h) or i); (ii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to d), e) or f), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b), c), g), h) or i); or (iii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to g), h) or i), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b), c), d), e) or f).

In these specific embodiments, the amino acid sequence preferably comprises two or more stretches of amino acid residues chosen from the group consisting of: i) the amino acid sequences of SEQ ID NO's: 135 to 143; ii) the amino acid sequences of SEQ ID NO's: 153 to 161; and iii) the amino acid sequences of SEQ ID NO's: 171 to 179; such that, (i) when the first stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 135 to 143, the second stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 153 to 161 or of SEQ ID NO's: 171 to 179: (ii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 153 to 161, the second stretch of amino acid revsidues corresponds to one of the amino acid sequences of SEQ ID NO's: 135 to 143 or of SEQ ID NO's: 171 to 179; or (iii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO^'s: 171 to 179, the second stretch of amino acid- residues corresponds to one of the amino acid sequences of SEQ ID NO's: 135 to 143 or of SEQ ID NO's: 153 to 161.

Also, in such an amino acid sequence, the at least two stretches of amino acid residues again preferably form part of the antigen binding site for binding to sclerostin.

In even more specific, but non-limiting embodiments, the invention relates to an amino acid sequence directed against sclerostin, that comprises three or more stretches of amino acid residues, in which the first stretch of amino acid residues is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO^'s: 135 to 143; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; c) amino acid sequences that have 3, 2. or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; the second stretch of amino acid residues is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NO^'s: 153 to 161; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 153 to 161 ; f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 153 to 161 : and the third stretch of amino acid residues is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NO's: 171 to 179; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 171 to 179; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO^'s: 171 to 179.

Preferably, in this specific aspect, the first stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 135 to 143; the second stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO^'s: 153 to 161; and the third stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 171 to 179.

Again, preferably, in such an amino acid sequence, the at least three stretches of amino acid residues forms part of the antigen binding site for binding to sclerostin. Preferred combinations of such stretches of amino acid sequences will become clear from the further disclosure herein.

Preferably, in such amino acid sequences the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 189 to 197 (see Table A-I). This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 189 to 197 (see Table A-I), in which the amino acid residues that form the framework regions are disregarded. Also, such amino acid sequences of the invention can be as further described herein.

Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to sclerostin: and more in particular bind to sclerostin with an affinity (suitably measured and/or expressed as a K_D- value (actual or apparent), a K_A- value (actual or apparent), a kon-rale and/or a k_0fp-rate. or alternatively as an IC50 value, as further described herein) that is as defined herein.

When the amino acid sequence of the invention essentially consists of 4 framework regions (FRl to FR4. respectively) and 3 complementarity determining regions (CDRl to CDR3, respectively), the amino acid sequence of the invention is preferably such that: CDRl is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO's: 135 to 143; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; and/or

CDR2 is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NO's: 353 to 161; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 153 to 161 ; f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 153 to 161; and/or

CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NO's: 171 to 179; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO' s: 171 to 179: i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 171 to 179.

In particular, such an amino acid sequence of the invention may be such that CDRl is chosen from the group consisting of the amino acid sequences of SEQ ID NO^'s: 135 to 143; and/or CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 153 to 161; and/or CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 171 to 179.

In particular, when the amino acid sequence of the invention essentially consists of 4 framework regions (FRl to FR4, respectively) and 3 complementarity determining regions (CDRl to CDR3, respectively), the amino acid sequence of the invention is preferably such that:

CDRl is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO's: 135 to 143; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; c) amino acid sequences that have 3, 2, or I amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; and

CDR2 is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NO's: 153 to 161 ; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 153 to 161; f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 153 to 161 : and

CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NO's: 171 to 179; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO^'s: 171 to 379; i) amino acid sequences that have 3. 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO^'s: 171 to 179; or any suitable fragment of such an amino acid sequence.

In particular, such an amino acid sequence of the invention may be such that CDRl Is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 135 to 143; and CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 153 to 161 : and CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO^"s: 171 to 179.

Again, preferred combinations of CDR sequences will become clear from the further description herein.

Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to sclerostin: and more in particular bind to sclerostin with an affinity (suitably measured and/or expressed as a KD- value (actual or apparent), a KΛ-value (actual or apparent), a k_oπ-rate and/or a k_o*rrate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein.

In particular, but non-limiting embodiments, the invention relates to amino acid sequences that essentially consist of 4 framework regions (FRl to FR4, respectively) and 3 complementarity determining regions (CDRl to CDR3. respectively), in which the CDR sequences of said amino acid sequence have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 189 to 197 (see Table A-I). This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 189 to 197 (see Table A-I). in which the amino acid residues that form the framework regions are disregarded. Such amino acid sequences of the invention can be as further described herein. In such an amino acid sequence of the invention, the framework sequences may be any suitable framework sequences, and examples of suitable framework sequences will be clear to the skilled person, for example on the basis the standard handbooks and the further disclosure and prior art mentioned herein. The framework sequences are preferably (a suitable combination of) immunoglobulin framework sequences or framework sequences that have been derived from immunoglobulin framework sequences (for example, by humanization or camelization). For example, the framework sequences may be framework sequences derived from a light chain variable domain (e.g. a Vi_-sequence) and/or from a heavy chain variable domain (e.g. a V_H- sequence). In one particularly preferred aspect, the framework sequences are either framework sequences that have been derived from a Vpiπ-sequence (in which said framework sequences may optionally have been partially or fully humanized) or are conventional V_H sequences that have been camelized (as defined herein). The framework sequences are preferably such that the amino acid sequence of the invention is a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody): is a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody); is a "dAb" (or an amino acid sequence that is suitable for use as a dAb); or is a Nanobody® (including but not limited to V_HH sequence). Again, suitable framework sequences will be clear to the skilled person, for example on the basis the standard handbooks and the further disclosure and prior ait mentioned herein.

In particular, the framework sequences present in the amino acid sequences of the invention may contain one or more of Hallmark residues (as defined herein), such that the amino acid sequence of the invention is a Nanobody®. Some preferred, but non-limiting examples of (suitable combinations of) such framework sequences will become clear from the further disclosure herein.

Again, as generally described herein for the amino acid sequences of the invention, it is also possible to use suitable fragments (or combinations of fragments) of any of the foregoing, such as fragments that contain one or more CDR sequences, suitably flanked by and/or linked via one or more framework sequences (for example, in the same order as these CDR^'s and framework sequences may occur in the full-sized immunoglobulin sequence from which the fragment has been derived). Such fragments may also again be such that they comprise or can form an immunoglobulin fold, or alternatively be such that they do not comprise or cannot form an immunoglobulin fold. In specific embodiments, such fragments comprise a single CDR sequence as described herein (and in particular a CDR3 sequence), that is flanked on each side by (part of) a framework sequence (and in particular, part of the framework sequence(s) that, in the immunoglobulin sequence from which the fragment is derived, are adjacent to said CDR sequence. For example, a CDR3 sequence may be preceded by (part of) a FR3 sequence and followed by (part of) a FR4 sequence). Such a fragment may also contain a disulphide bridge, and in particular a disulphide bridge that links the two framework regions that precede and follow the CDR sequence, respectively (for the purpose of forming such a disulphide bridge, cysteine residues that naturally occur in said framework regions may be used, or alternatively cysteine residues may be synthetically added to or introduced into said framework regions). For a further description of these "Expedite fragments'^', reference is again made to WO 03/050531. as well as to the US provisional application of Ablynx N. V. entitled "Peptides capable of binding to serum proteins" of Ablynx N.V. (inventors: Revets, Hilde Adi Pierrette; Kolkman, Joost Alexander; and Hoogenboom, Hendricus Renerus Jacobus Mattheus) filed on December 5, 2006 (see also PCT/EP2007/063348).

In another aspect, the invention relates to a compound or construct, and in particular a protein or polypeptide (also referred to herein as a "compound of the invention" or "polypeptide of the invention", respectively) that comprises or essentially consists of one or more amino acid sequences of the invention (or suitable fragments thereof), and optionally further comprises one or more other groups, residues, moieties or binding units. As will become clear to the skilled person from the further disclosure herein, such further groups, residues, moieties, binding units or amino acid sequences may or may not provide further functionality to the amino acid sequence of the invention (and/or to the compound or construct in which it is present) and may or may not modify the properties of the amino acid sequence of the invention.

For example, such further groups,, residues, moieties or binding units may be one or more additional amino acid sequences, such that the compound or construct is a (fusion) protein or (fusion) polypeptide, In a preferred but non-limiting aspect, said one or more other groups, residues, moieties or binding units are immunoglobulin sequences. Even more preferably, said one or more other groups, residues, moieties or binding units are chosen from the group consisting of domain antibodies, amino acid sequences that are suitable for use as a domain antibody, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, "dAb" ^"s, amino acid sequences that are suitable for use as a dAb, or Nanobodies® .

Alternatively, such groups, residues, moieties or binding units may for example be chemical groups, residues, moieties, which may or may not by themselves be biologically and/or pharmacologically active. For example, and without limitation, such groups may be linked to the one or more amino acid sequences of the invention so as to provide a "derivative" of an amino acid sequence or polypeptide of the invention, as further described herein.

Also within the scope of the present invention are compounds or constructs, that comprises or essentially consists of one or more derivatives as described herein, and optionally further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more linkers. Preferably, said one or more other groups, residues, moieties or binding units are amino acid sequences.

In the compounds or constructs described above, the one or more amino acid sequences of the invention and the one or more groups, residues, moieties or binding units may be linked directly to each other and/or via one or more suitable linkers or spacers. For example, when the one or more groups, residues, moieties or binding units are amino acid sequences, the linkers may also be amino acid sequences, so that the resulting compound or construct is a fusion (protein) or fusion (polypeptide). As will be clear from the further description above and herein, this means that the amino acid sequences of the invention can be used as "building blocks" to form polypeptides of the invention, i.e. by suitably combining them with other groups, residues, moieties or binding units, in order to form compounds or constructs as described herein (such as. without limitations, the biparatopic. bi/multivalent and bi/multi specific polypeptides of the invention described herein) which combine within one molecule one or more desired properties or biological functions.

The compounds or polypeptides of the invention can generally be prepared by a method which comprises at least one step of suitably linking the one or more amino acid sequences of the invention to the one or more further groups, residues, moieties or binding units, optionally via the one or more suitable linkers, so as to provide the compound or polypeptide of the invention. Polypeptides of the invention can also be prepared by a method which generally comprises at least the steps of providing a nucleic acid that encodes a polypeptide of the invention, expressing said nucleic acid in a suitable manner, and recovering the expressed polypeptide of the invention. Such methods can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the methods and techniques further described herein.

The process of designing/selecting and/or preparing a compound or polypeptide of the invention, starting from an amino acid sequence of the invention, is also referred to herein as "formatting"^" said amino acid sequence of the invention; and an amino acid of the invention that is made part of a compound or polypeptide of the invention is said to be ""formatted^' or to be "in the format of said compound or polypeptide of the invention. Examples of ways in which an amino acid sequence of the invention can be formatted and examples of such formats will be clear to the skilled person based on the disclosure herein: and such formatted amino acid sequences form a further aspect of the invention.

In one specific aspect of the invention, a compound of the invention or a polypeptide of the invention may have an increased half-life, compared to the corresponding amino acid sequence of the invention. Some preferred, but non-limiting examples of such compounds and polypeptides will become clear to the skilled person based on the further disclosure herein, and for example comprise amino acid sequences or polypeptides of the invention that have been chemically modified to increase the half-life thereof (for example, by means of pegylation); amino acid sequences of the invention that comprise at least one additional binding site for binding to a serum protein (such as serum albumin); or polypeptides of the invention that comprise at least one amino acid sequence of the invention that is linked to at least one moiety (and in particular at least one amino acid sequence) that increases the half- life of the amino acid sequence of the invention. Examples of polypeptides of the invention that comprise such half-life extending moieties or amino acid sequences will become clear to the skilled person based on the further disclosure herein; and for example include, without limitation, polypeptides in which the one or more amino acid sequences of the invention are suitable linked to one or more serum proteins or fragments thereof (such as (human) serum albumin or suitable fragments thereof) or to one or more binding units that can bind to serum proteins (such as, for example, domain antibodies, amino acid sequences that are suitable for use as a domain antibody, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody. "dAb"^"s. amino acid sequences that are suitable for use as a dAb, or Nanobodies® that can bind to serum proteins such as serum albumin (such as human serum albumin), serum immunoglobulins such as IgG, or transferrine; reference is made to the further description and references mentioned herein); polypeptides in which an amino acid sequence of the invention is linked to an Fc portion (such as a human Fc) or a suitable part or fragment thereof; or polypeptides in which the one or more amino acid sequences of the invention are suitable linked to one or more small proteins or peptides that can bind to serum proteins (such as, without limitation, the proteins and peptides described in WO 91/01743, WO 01/45746, WO 02/076489 and to the US provisional application of Ablynx N. V. entitled "Peptides capable of binding to serum proteins" of Ablynx N. V. filed on December 5. 2006 (see also PCT/EP2007/063348).

Generally, the compounds or polypeptides of the invention with increased half-life preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding amino acid sequence of the invention per se. For example, the compounds or polypeptides of the invention with increased half-life may have a half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24. 48 or 72 hours, compared to the corresponding amino acid sequence of the invention per se.

In preferred, but non-limiting embodiments of the invention, such compounds or polypeptides of the invention have a serum half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24. 48 or 72 hours, compared to the corresponding amino acid sequence of the invention per se.

In particular, but non-limiting embodiments of the invention, such compounds or polypeptides of the invention exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more. For example, compounds or polypeptides of the invention may have a half- life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 1 1 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).

In a further aspect, the invention relates to a nucleic acid that encodes an amino acid sequence of the invention or a polypeptide of the invention (or a suitable fragment thereof). Such a nucleic acid will also be referred to herein as a "nucleic acid of the invention^'1' and may for example be in the form of a genetic construct, as further described herein.

In another aspect, the invention relates to a host or host cell that expresses (or that under suitable circumstances is capable of expressing) an amino acid sequence of the invention and/or a polypeptide of the invention; and/or that contains a nucleic acid of the invention. Some preferred but non-limiting examples of such hosts or host cells will become clear from the further description herein. The invention further relates to a product or composition containing or comprising at least one amino acid sequence of the invention, at least one polypeptide of the invention (or a suitable fragment thereof) and/or at least one nucleic acid of the invention, and optionally one or more further components of such compositions known per se. i.e. depending on the intended use of the composition. Such a product or composition may for example be a pharmaceutical composition (as described herein), a veterinary composition or a product or composition for diagnostic use (as also described herein). Some preferred but non-limiting examples of such products or compositions will become clear from the further description herein. Also, generally, amino acid sequences, polypeptides and compositions of the invention are preferably such that they are capable of inhibiting the activity of sclerostin. as measured by a suitable assay (such as suitable cell-based assays, for example including alkaline phosphatase activity assays (van Bezooijen et al. J. Exp. Med. 2004; 199: 805-814; Winkler et al. EMBO J. 2003: 22: 6267-76; Li et al. J. Biol. Chem. 2005; 280: 19883- 19887). luciferase reporter assay for monitoring canonical Wnt signaling cells transfected with a Wnt luciferase reporter construct in combination with other constructs to induce Wnt signaling (WO 06/015373. WO 07/084344; WO 08/097510; van Bezooijen RL et al. 2007, J. Bone Miner. Res. 22: 19-28), mineralization assay for inhibition of BMP-2/4/6/7-induced mineralization by human sclerostin using the alizarin red staining method (van Bezooijen et al. J. Exp. Med. 2004: 199: 805-814) and cell proliferation assay for inhibition of proliferation of primary human mesenchymal cells treated with sclerostin pulsed with [methyl-³H]-thymidine (Sutherland et al. Bone 2004; 35: 448-454)) under conditions usually applied for such an assay (as will be clear to the skilled person), by at least 1%, preferably at least 5%. more preferably at least 10%. such as at least 25% or even more than 50% and up to 75% or more, such as 90% or more, compared to the activity of sclerostin without the presence of the amino acid sequence or polypeptide of the invention.

Some other preferred amino acid sequences and polypeptides may be such that they are capable of competing for binding to sclerostin with LRP5 and/or LRP 6. Accordingly, the amino acid sequences, polypeptides and compositions of the present invention are generally such that they are capable of inhibiting binding of sclerostin to LRP5 and/or LRP6, as measured by a suitable assay (such as for instance ELlSA, Alpha Screen or TRAP assay) under conditions usually applied for such an assay (as will be clear to the skilled person), by at least 1%. preferably at least 5%. more preferably at least 10%. such as at least 25% or even more than 50% and up to 75% or more, such as 90% or more, compared to the binding of sclerostin to LRP5 and/or LRP6 without the presence of the amino acid sequence or polypeptide of the invention.

In addition, the amino acid sequences, polypeptides and compositions of the present invention are generally such that they are capable of inducing, stimulating, or agonizing Wnt signalling that is mediated by the interaction of Wnt proteins with Frizzled (Fzd) receptors and LRP5/LRP6 coreceptors (as measured by a suitable assay, known to the skilled person per se) under conditions usually applied for such an assay (as will be clear to the skilled person), by at least 1%, preferably at least 5%, more preferably at least 10%, such as at least 25% or even more than 50% and up to 75% or more, such as 90% or more, compared to the signalling that is mediated by Wnt proteins, Frizzled (Fzd) receptors and LRP5/LRP6 coreceptors under the same conditions but without the presence of the amino acid sequence. Nanobody® or polypeptide of the invention.

Furthermore, the polypeptides and compositions of the present invention that inhibit binding of sclerostin to LRP5 and/or LRPό may in addition be capable of modulating and in particular inducing, stimulating, or agonizing other signalling pathways cooperating with Wnt proteins in stimulating bone formation (as measured by a suitable assay, known to the skilled person per se) under conditions usually applied for such an assay (as will be clear to the skilled person). For instance, the polypeptides and compositions of the present invention that inhibit binding of sclerostin to LRP5 and/or LRP6 may be such that they are capable of inducing, stimulating, or agonizing signalling that is mediated by BMPs, BMP type-I and type-II transmembrane seπne-threonine kinase receptors and/or intracellular Smad effector proteins (as measured by a suitable assay, known to the skilled person per se) under conditions usually applied for such an assay (as will be clear to the skilled person), by at least 1%, preferably at least 5%, more preferably at least 10%, such as at least 25% or even more than 50% and up to 75% or more, such as 90% or more, compared to the signalling that is mediated by BMPs. BMP type-1 and type-II transmembrane serine-threonine kinase receptors and/or intracellular Smad effector proteins under the same conditions but without the presence of the amino acid sequence, Nanobody© or polypeptide of the invention. Also, the amino acid sequences, polypeptides and compositions of the invention are generally such that they are capable of inducing and/or stimulating the differentiation and/or proliferation of osteoblasts (as measured by a suitable assay, known to the skilled person per se) under conditions usually applied for such an assay (as will be clear to the skilled person). The differentiation and/or proliferation of osteoblasts may be increased, respectivel}', by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to the differentiation and/or proliferation of osteoblasts under the same conditions but without the presence of the amino acid sequence, Nanobody® or polypeptide of the invention.

In particular, the amino acid sequences, polypeptides and compositions of the invention are generally such that they are capable of modulating bone remodeling (as measured by a suitable assay, known to the skilled person per se) under conditions usually applied for such an assay (as will be clear to the skilled person). Bone remodelling may be modulated at least 1%. preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to bone remodelling under the same conditions but without the presence of the amino acid sequence, Nanobody© or polypeptide of the invention,

The invention also relates to the use of an amino acid sequence, Nanobody® or polypeptide of the invention, or of a composition comprising the same, in (methods or compositions for) modulating sclerostin, either in vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an a single cell or in a multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from a bone disease and/or disorder). The invention also relates to methods for modulating sclerostin, either in vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an a single cell or multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from a bone disease and/or disorder), which method comprises at least the step of contacting sclerostin with at least one amino acid sequence, Nanobody® or polypeptide of the invention, or with a composition comprising the same, in a manner and in an amount suitable to modulate sclerostin, with at least one amino acid sequence, Nanobody® or polypeptide of the invention.

The invention also relates to the use of an one amino acid sequence, Nanobody® or polypeptide of the invention in the preparation of a composition (such as, without limitation, a pharmaceutical composition or preparation as further described herein) for modulating sclerostin, either in vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an a single cell or multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from a bone disease and/or disorder).

In the context of the present invention, "modulating" or "to modulate" generally means either reducing or inhibiting the activity of, or alternatively increasing the activity of, sclerostin, as measured using a suitable in vitro, cellular or in vivo assay (such as those mentioned herein). In particular, "modulating"' or "to modulate" may mean either reducing or inhibiting the activity of, or alternatively increasing the activity of sclerostin, as measured using a suitable in vitro, cellular or in vivo assay (such as those mentioned herein), by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to activity of sclerostin in the same assay under the same conditions but without the presence of the amino acid sequence, Nanobody® or polypeptide of the invention.

As will be clear to the skilled person, "modulating" may also involve effecting a change (which may either be an increase or a decrease) in affinity, avidity, specificity and/or selectivity of sclerostin for one or more of its targets, ligands or substrates; and/or effecting a change (which may either be an increase or a decrease) in the sensitivity of sclerostin for one or more conditions in the medium or surroundings in which sclerostin is present (such as pH, ion strength, the presence of co -factors, etc.), compared to the same conditions but without the presence of the amino acid sequence, Nanobody® or polypeptide of the invention. As will be clear to the skilled person, this may again be determined in any suitable manner and/or using any suitable assay known per se, such as the assays described herein or in the prior art cited herein.

"Modulating" may also mean effecting a change (i.e. an activity as an agonist or as an antagonist, respectively) with respect to one or more biological or physiological mechanisms, effects, responses, functions, pathways or activities in which sclerostin (or in which its substrate(s), ϋgand(s) or pathway(s) are involved, such as its signalling pathway or metabolic pathway and their associated biological or physiological effects) is involved. Again, as will be clear to the skilled person, such an action as an agonist or an antagonist may be determined in any suitable manner and/or using any suitable (in vitro and usually cellular or in assay) assay known per se, such as the assays described herein or in the prior art cited herein. In particular, an action as an agonist or antagonist may be such that an intended biological or physiological activity is increased or decreased, respectively, by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to the biological or physiological activity in the same assay under the same conditions but without the presence of the amino acid sequence, Nanobody® or polypeptide of the invention.

Modulating may for example involve reducing or inhibiting the binding of sclerostin to a natural binding partner and/or competing with a natural binding partner for binding to sclerostin. Modulating may also involve activating sclerostin or the mechanism or pathway in which it is involved. Modulating may be reversible or irreversible, but for pharmaceutical and pharmacological purposes will usually be in a reversible manner.

The invention further relates to methods for preparing or generating the amino acid sequences, polypeptides, nucleic acids, host cells, products and compositions described herein. Some preferred but non-limiting examples of such methods will become clear from the further description herein.

Generally, these methods may comprise the steps of: a) providing a set, collection or library of amino acid sequences; and b) screening said set, collection or library of amino acid sequences for amino acid sequences that can bind to and/or have affinity for sclerostin; and c) isolating the amino acid sequence(s) that can bind to and/or have affinity for sclerostin.

In such a method, the set, collection or library of amino acid sequences may be any suitable set, collection or library of amino acid sequences. For example, the set, collection or library of amino acid sequences may be a set, collection or library of immunoglobulin sequences (as described herein), such as a naϊve set, collection or library of immunoglobulin sequences; a synthetic or semi-synthetic set, collection or library of immunoglobulin sequences; and/or a set. collection or library of immunoglobulin sequences that have been subjected to affinity maturation.

Also, in such a method, the set, collection or library of amino acid sequences may be a set, collection or library of heavy chain variable domains (such as V_H domains or V_HH domains) or of light chain variable domains. For example, the set. collection or library of amino acid sequences may be a set, collection or library of domain antibodies or single domain antibodies, or may be a set, collection or library of amino acid sequences that are capable of functioning as a domain antibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library of amino acid sequences may be an immune set. collection or library of immunoglobulin sequences, for example derived from a mammal that has been suitably immunized with sclerostin or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

In the above methods, the set, collection or library of amino acid sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set. collection or library of) amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology. 23. 9, 1 105-1 1 16 (2005),

In another aspect, the method for generating amino acid sequences comprises at least the steps of: a) providing a collection or sample of cells expressing amino acid sequences; b) screening said collection or sample of cells for cells that express an amino acid sequence that can bind to and/or have affinity for sclerostin; and c) either (i) isolating said amino acid sequence: or (ii) isolating from said cell a nucleic acid sequence that encodes said amino acid sequence, followed by expressing said amino acid sequence.

For example, when the desired amino acid sequence is an immunoglobulin sequence, the collection or sample of cells may for example be a collection or sample of B-cells. Also, in this method, the sample of cells may be derived from a mammal that has been suitably immunized with sclerostin or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

The above method may be performed in any suitable manner, as will be clear to the skilled person. Reference is for example made to EP 0 542 810. WO 05/19824, WO 04/051268 and WO 04/106377. The screening of step b) is preferably performed using a flow cytometry technique such as FACS. For this, reference is for example made to Lieby et al. Blood. Vol. 97, No. 12, 3820 (2001). In another aspect, the method for generating an amino acid sequence directed against sclerostin may comprise at least the steps of: a) providing a set, collection or library of nucleic acid sequences encoding amino acid sequences: b) screening said set, collection or library of nucleic acid sequences for nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for sclerostin; and c) isolating said nucleic acid sequence, followed by expressing said amino acid sequence. In such a method, the set, collection or library of nucleic acid sequences encoding amino acid sequences may for example be a set, collection or library of nucleic acid sequences encoding a naive set, collection or library of immunoglobulin sequences; a set. collection or library of nucleic acid sequences encoding a synthetic or semi- synthetic set, collection or library of immunoglobulin sequences: and/or a set. collection or library of nucleic acid sequences encoding a set, collection or library of immunoglobulin sequences that have been subjected to affinity maturation.

Also, in such a method, the set, collection or library of nucleic acid sequences may encode a set. collection or library of heavy chain variable domains (such as V_H domains or V_HII domains) or of light chain variable domains. For example, the set, collection or library of nucleic acid sequences may encode a set. collection or library of domain antibodies or single domain antibodies, or a set, collection or library of amino acid sequences that are capable of functioning as a domain antibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library of nucleic acid sequences may be an immune set, collection or library of nucleic acid sequences, for example derived from a mammal that has been suitably immunized with sclerostin or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s). The set, collection or library of nucleic acid sequences may for example encode an immune set. collection or library of heavy chain variable domains or of light chain variable domains. In one specific aspect, the set, collection or library of nucleotide sequences may encode a set. collection or library of V_HH sequences. In the above methods, the set. collection or library of nucleotide sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the art. for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1 105-1 116 (2005). in another aspect, the method for generating an amino acid sequence directed against sclerostin may comprise at least the steps of: a) providing a set, collection or library of nucleic acid sequences encoding amino acid sequences; b) screening said set, collection or library of nucleic acid sequences for nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for sclerostin and that is cross-blocked or is cross blocking a Nanobody® of the invention, e.g. SEQ ID NG: 189 to 197 (Table A-I); and c) isolating said nucleic acid sequence, followed by expressing said amino acid sequence. In particular embodiments, methods for generating an amino acid sequence directed against sclerostin may comprise at least the steps of: a) providing a set, collection or library of nucleic acid sequences encoding amino acid sequences; b) screening said set, collection or library of nucleic acid sequences for nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for sclerostin and that can induce, stimulate or agonize signalling mediated by the interaction of Wnt proteins with Frizzled (Fzd) receptors and LRP5/LRP6 coreceptors; and c) isolating said nucleic acid sequence, followed by expressing said amino acid sequence. In particular embodiments of the invention, methods for generating an amino acid sequence directed against sclerostin as provided according to the invention may comprise at least the steps of: a) providing a set, collection or library of nucleic acid sequences encoding amino acid sequences; b) screening said set, collection or library of nucleic acid sequences for nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for sclerostin and that can induce, stimulate or agonize signalling mediated by the interaction of BMP proteins and BMP type-I and type-Il transmembrane serine- threonine kinase receptors and/or intracellular Smad effector proteins; and c) isolating said nucleic acid sequence, followed by expressing said amino acid sequence. The invention also relates to amino acid sequences that are obtainable or obtained by the above methods, or alternatively by a method that comprises one of the above methods and in addition at least the steps of determining the nucleotide sequence or amino acid sequence of said immunoglobulin sequence; and of expressing or synthesizing said amino acid sequence in a manner known per se. such as by expression in a suitable host cell or host organism or by chemical synthesis.

Also, following the steps above, one or more amino acid sequences of the invention may be suitably humanized (or alternatively camelized); and/or the amino acid sequence(s) thus obtained may be linked to each other or to one or more other suitable amino acid sequences (optionally via one or more suitable linkers) so as to provide a polypeptide of the invention. Also, a nucleic acid sequence encoding an amino acid sequence of the invention may be suitably humanized (or alternatively camelized) and suitably expressed; and/or one or more nucleic acid sequences encoding an amino acid sequence of the invention may be linked to each other or to one or more nucleic acid sequences that encode other suitable amino acid sequences (optionally via nucleotide sequences that encode one or more suitable linkers), after which the nucleotide sequence thus obtained may be suitably expressed so as to provide a polypeptide of the invention.

The invention further relates to applications and uses of the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein, as well as to methods for the prevention and/or treatment for diseases and disorders associated with sclerostin. Some preferred but non-limiting applications and uses will become clear from the further description herein.

The invention also relates to the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein for use in therapy. In particular, the invention also relates to the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein for use in therapy of a disease or disorder that can be prevented or treated by administering, to a subject in need thereof, of (a pharmaceutically effective amount of) an amino acid sequence, compound, construct or polypeptide as described herein.

More in particular, the invention relates to the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein for use in therapy of bone diseases and disorders.

Other aspects, embodiments, advantages and applications of the invention will also become clear from the further description herein, in which the invention will be described and discussed in more detail with reference to the Nanobodies® of the invention and polypeptides of the invention comprising the same, which form some of the preferred aspects of the invention.

As will become clear from the further description herein, Nanobodies® generally offer certain advantages (outlined herein) compared to "dAb"s" or similar (single) domain antibodies or immunoglobulin sequences, which advantages are also provided by the Nanobodies® of the invention. However, it will be clear to the skilled person that the more genera] aspects of the teaching below can also be applied (either directly or analogously) to other amino acid sequences of the invention.

Detailed description of the invention

In the present description, examples and claims: a) Unless indicated or defined otherwise, all terms used have their usual meaning in the art. which will be clear to the skilled person. Reference is for example made to the standard handbooks mentioned in paragraph a) on page 46 of WO 08/020079. b) Unless indicated otherwise, the terms ''immunoglobulin sequence", "sequence", "nucleotide sequence" and "nucleic acid" are as described in paragraph b) on page 46 of WO 08/020079. c) Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to the standard handbooks and the general background art mentioned herein and to the further references cited therein: as well as to for example the following reviews Presta, Adv. Drug Deliv. Rev. 2006, 58 (5-6): 640-56; Levin and Weiss. MoI. Biosyst 2006, 2(1): 49-57: Irving et a!.. J. Immunol. Methods, 2001 , 248(1-2), 31-45: Schmitz et at., Placenta. 2000, 21 Suppl A, S 106-12, Gonzales et a!.. Tumour Biol.. 2005, 26(1), 31-43, which describe techniques for protein engineering, such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins. d) Amino acid residues will be indicated according to the standard three-letter or one- letter amino acid code. Reference is made to Table A-2 on page 48 of the International application WO 08/020079 of Ablynx N. V. entitled '^'Amino acid sequences directed against IL-6R and polypeptides comprising the same for the treatment of diseases and disorders associated with 11-6 mediated signalling". e) For the purposes of comparing two or more nucleotide sequences, the percentage of "sequence identity between a first nucleotide sequence and a second nucleotide sequence may be calculated or determined as described in paragraph e) on page 49 of WO 08/020079 (incorporated herein by reference), such as by dividing [the number of nucleotides in the first nucleotide sequence that are identical to the nucleotides at the corresponding positions in the second nucleotide sequence} by [the total number of nucleotides in the first nucleotide sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of a nucleotide in the second nucleotide sequence - compared to the first nucleotide sequence - is considered as a difference at a single nucleotide (position); or using a suitable computer algorithm or technique, again as described in paragraph e) on pages 49 of WO 08/020079 (incorporated herein by reference). f) For the purposes of comparing two or more amino acid sequences, the percentage of "'sequence identity" between a first amino acid sequence and a second amino acid sequence (also referred to herein as "amino acid identity") may be calculated or determined as described in paragraph f) on pages 49 and 50 of WO 08/020079 (incorporated herein by reference), such as by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence - compared to the first amino acid sequence - is considered as a difference at a single amino acid residue (position), i.e. as an "amino acid difference" as defined herein; or using a suitable computer algorithm or technique, again as described in paragraph f) on pages 49 and 50 of WO 08/020079 (incorporated herein by reference).

Also, in determining the degree of sequence identity between two amino acid sequences, the skilled person may take into account so-called "'conservative'^" amino acid substitutions, as described on page 50 of WO 08/020079.

Any amino acid substitutions applied to the polypeptides described herein may also be based on the analysis of the frequencies of amino acid variations between homologous proteins of different species developed by Schulz et aL Principles of Protein Structure. Springer- Ver lag, 1978, on the analyses of structure forming potentials developed by Chou and Fasman, Biochemistry 13 : 211. 1974 and Adv. Enzymol.. 47: 45-149, 1978, and on the analysis of hydrophobicity patterns in proteins developed by Eisenberg et aL, Proc. Natl. Acad Sci. USA 81 : 140-144, 1984; Kyte & Doolittle; J Molec. Biol. 157: 105-132. 198 1, and Goldman et aL, Ann. Rev. Biophys. Chem. 15: 321 -353, 1986, all incorporated herein in their entirety by reference. Information on the primary, secondary and tertiary structure of Nanobodies® is given in the description herein and in the general background art cited above. Also, for this purpose, the crystal structure of a V_ΠH domain from a llama is for example given by Desmyler et a!.. Nature Structural Biology, Vol. 3, 9, 803 (1996); Spinelli et a!.. Natural Structural Biology (1996); 3, 752-757; and Decanniere et aL. Structure. Vol. 7, 4, 361 (1999). Further information about some of the amino acid residues that in conventional Vj j domains form the V_H/V_L interface and potential camelizing substitutions on these positions can be found in the prior art cited above. g) Amino acid sequences and nucleic acid sequences are said to be "exactly the same" if they have 100% sequence identity (as defined herein) over their entire length. h) When comparing two amino acid sequences, the term ''amino acid difference" refers to an insertion, deletion or substitution of a single amino acid residue on a position of the first sequence, compared to the second sequence; it being understood that two amino acid sequences can contain one, two or more such amino acid differences, i) When a nucleotide sequence or amino acid sequence is said to "comprise'^" another nucleotide sequence or amino acid sequence, respectively, or to "essentially consist of another nucleotide sequence or amino acid sequence, this has the meaning given in paragraph i) on pages 51-52 of WO 08/020079. j) The term "in essentially isolated form" has the meaning given to it in paragraph j) on pages 52 and 53 of WO 08/020079. k) The terms "domain" and "binding domain" have the meanings given to it in paragraph k) on page 53 of WO 08/020079. 1) The terms ''antigenic determinant" and "epitope", which may also be used interchangeably herein, have the meanings given to it in paragraph I) on page 53 of WO 08/020079. m) As further described in paragraph m) on page 53 of WO 08/020079, an amino acid sequence (such as a Nanobody®, an antibody, a polypeptide of the invention, or generally an antigen binding protein or polypeptide or a fragment thereof) that can

(specifically) bind to, that has affinity for and/or that has specificity for a specific antigenic determinant, epitope, antigen or protein (or for at least one part, fragment or epitope thereof) is said to be "against" or "directed against said antigenic determinant, epitope, antigen or protein. n) The term "specificity^'' has the meaning given to it in paragraph n) on pages 53-56 of WO 08/020079; and as mentioned therein refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding molecule or antigen-binding protein (such as a Nanobody® or a polypeptide of the invention) molecule can bind. The specificity of an antigen-binding protein can be determined based on affinity and/or avidity, as described on pages 53-56 of WO 08/020079

(incorporated herein by reference), which also describes some preferred techniques for measuring binding between an antigen-binding molecule (such as a Nanobody® or polypeptide of the invention) and the pertinent antigen. Typically, antigen-binding proteins (such as the amino acid sequences, Nanobodies® and/or polypeptides of the invention) will bind to their antigen with a dissociation constant (K_D) of 10° to 10^"12

7 ϊ 7 moles/liter or less, and preferably 10^" to 10^' moles/liter or less and more preferably 10^"8 to 10^"12 moles/liter (i.e. with an association constant (K_AJ of 10^S to 10¹² liter/ moles or more, and preferably 10⁷ to 10¹² liter/moles or more and more preferably 10⁸ to 10¹² liter/moles). Any K_D value greater than 10⁴ mo I/liter (or any K_A value lower than 10⁴ M^'1) liters/mol is generally considered to indicate non-specific binding. Preferably, a monovalent immunoglobulin sequence of the invention will bind to the desired antigen with an affinity less than 500 nM, preferably less than 200 nM. more preferably less than 10 nM, such as less than 500 pM. Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se. including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA). enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned herein. As will be clear to the skilled person, and as described on pages 53-56 of WO 08/020079, the dissociation constant may be the actual or apparent dissociation constant. Methods for determining the dissociation constant will be clear to the skilled person, and for example include the techniques mentioned on pages 53-56 of WO 08/020079. o) The half-life of an amino acid sequence, compound or polypeptide of the invention can generally be defined as described in paragraph o) on page 57 of WO 08/020079 and as mentioned therein refers to the time taken for the serum concentration of the amino acid sequence, compound or polypeptide to be reduced by 50%. in vivo, for example due to degradation of the sequence or compound and/or clearance or sequestration of the sequence or compound by natural mechanisms. The in vivo half-life of an amino acid sequence, compound or polypeptide of the invention can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the ait, and may for example generally be as described in paragraph o) on page 57 of WO 08/020079. As also mentioned in paragraph o) on page 57 of WO 08/020079, the half-iife can be expressed using parameters such as the tl/2- alpha, tl/2-beta and the area under the curve (AUC). Reference is for example made to the Experimental Part below, as well as to the standard handbooks, such as Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al, Pharmacokinete analysis: A Practical Approach (1996). Reference is also made to "Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition (1982). The terms "increase in half-life" or "increased half-life" as also as defined in paragraph o) on page 57 of WO 08/020079 and in particular refer to an increase in the tl/2-beta, either with or without an increase in the tl/2-alpha and/or the AUC or both. p) In the context of the present invention,, "modulating" or "to modulate" generally means either reducing or inhibiting the activity of, or alternatively increasing the activity of, a target or antigen, as measured using a suitable in vitro, cellular or in vivo assay. In particular, "modulating'^" or "to modulate" may mean either reducing or inhibiting the activity of, or alternatively increasing a (relevant or intended) biological activity of, a target or antigen, as measured using a suitable in vitro, cellular or in vivo assay (which will usually depend on the target or antigen involved), by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to activity of the target or antigen in the same assay under the same conditions but without the presence of the construct of the invention.

As will be clear to the skilled person, "modulating" may also involve effecting a change (which may either be an increase or a decrease) in affinity, avidity, specificity and/or selectivity of a target or antigen for one or more of its ligands, binding partners, partners for association into a homomultimeric or heteromultimeric form, or substrates; and/or effecting a change (which may either be an increase or a decrease) in the sensitivity of the target or antigen for one or more conditions in the medium or surroundings in which the target or antigen is present (such as pH, ion strength, the presence of co-factors, etc.), compared to the same conditions but without the presence of the construct of the invention. As will be clear to the skilled person, this may again be determined in any suitable manner and/or using any suitable assay known per se, depending on the target or antigen involved. "Modulating" may also mean effecting a change (i.e. an activity as an agonist, as an antagonist or as a reverse agonist, respectively, depending on the target or antigen and the desired biological or physiological effect) with respect to one or more biological or physiological mechanisms, effects, responses, functions, pathways or activities in which the target or antigen (or in which its substrate(s), ligand(s) or pathway(s) are involved, such as its signalling pathway or metabolic pathway and their associated biological or physiological effects) is involved. Again, as will be clear to the skilled person, such an action as an agonist or an antagonist may be determined in any suitable manner and/or using any suitable (in vitro and usually cellular or in assay) assay known per se, depending on the target or antigen involved. In particular, an action as an agonist or antagonist may be such that an intended biological or physiological activity is increased or decreased, respectively, by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to the biological or physiological activity in the same assay under the same conditions but without the presence of the construct of the invention.

Modulating may for example also involve allosteric modulation of the target or antigen; and/or reducing or inhibiting the binding of the target or antigen to one of its substrates or iigands and/or competing with a natural ligand, substrate for binding to the target or antigen. Modulating may also involve activating the target or antigen or the mechanism or pathway in which it is involved. Modulating may for example also involve effecting a change in respect of the folding or conformation of the target or antigen, or in respect of the ability of the target or antigen to fold, to change its conformation (for example, upon binding of a ligand), to associate with other (sub)units, or to disassociate.

Modulating ma}' for example also involve effecting a change in the ability of the target or antigen to transport other compounds or to serve as a channel for other compounds (such as ions). Modulating may be reversible or irreversible, but for pharmaceutical and pharmacological purposes will usually be in a reversible manner. q) In respect of a target or antigen, the term "interaction site'^" on the target or antigen means a site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen that is a site for binding to a ligand, receptor or other binding partner, a catalytic site, a cleavage site, a site for allosteric interaction, a site involved in multimerisation (such as homomerizaiion or heterodimerization) of the target or antigen; or any other site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen that is involved in a biological action or mechanism of the target or antigen. More generally, an "interaction site'^" can be any site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen to which an amino acid sequence or polypeptide of the indention can bind such that the target or antigen (and/or any pathway, interaction, signalling, biological mechanism or biological effect in which the target or antigen is involved) is modulated (as defined herein), r) An amino acid sequence or polypeptide is said to be "''specific for^1' a first target or antigen compared to a second target or antigen when is binds to the first antigen with an affinity (as described above, and suitably expressed as a K_D value. K_Λ value, K_Off rate and/or K_0n rate) that is at least 10 times, such as at least 100 times, and preferably at least 1000 times, and up to 10.000 times or more better than the affinity with which said amino acid sequence or polypeptide binds to the second target or polypeptide. For example, the first antigen may bind to the target or antigen with a K_D value that is at least 10 times less, such as at least 100 times less, and preferably at least 1000 times less, such as 10.000 times less or even less than that, than the K_D with which said amino acid sequence or polypeptide binds to the second target or polypeptide. Preferably, when an amino acid sequence or polypeptide is "specific for" a first target or antigen compared to a second target or antigen, it is directed against (as defined herein) said first target or antigen, but not directed against said second target or antigen, The terms "cross-block", " 'cross-blocked" and " 'cross-blocking" are used interchangeably herein to mean the ability of an amino acid sequence or other binding agents (such as a Nanobody®. polypeptide or compound or construct of the invention) to interfere with the binding of other amino acid sequences or binding agents of the invention to a given target. The extent to which an amino acid sequence or other binding agents of the invention are able to interfere with the binding of another to sclerostin, and therefore whether it can be said to cross-block according to the invention, can be determined using competition binding assays. One particularly suitable quantitative cross-blocking assay uses a Biacore machine which can measure the extent of interactions using surface plasmon resonance technology. Another suitable quantitative cross-blocking assay uses an ELISA-based approach to measure competition between amino acid sequences or other binding agents in terms of their binding to the target.

The following generally describes a suitable Biacore assay for determining whether an amino acid sequence or other binding agent cross-blocks or is capable of cross-blocking according to the invention. It will be appreciated that the assay can be used with any of the amino acid sequences or other binding agents described herein. The Biacore machine (for example the Biacore 3000) is operated in line with the manufacturer's recommendations. Thus in one cross-blocking assay, the target protein is coupled to a CM5 Biacore chip using standard amine coupling chemistry to generate a surface that is coated with the target. Typically 200- 800 resonance units of the target would be coupled to the chip (an amount that gives easily measurable levels of binding but that is readily saturable by the concentrations of test reagent being used). Two test amino acid sequences (termed A* and B*) to be assessed for their ability to cross-block each other are mixed at a one to one molar ratio of binding sites in a suitable buffer to create the test mixture. When calculating the concentrations on a binding site basis the molecular weight of an amino acid sequence is assumed to be the total molecular weight of the amino acid sequence divided by the number of target binding sites on that amino acid sequence. The concentration of each amino acid sequence in the test mix should be high enough to readily saturate the binding sites for that amino acid sequence on the target molecules captured on the Biacore chip. The amino acid sequences in the mixture are at the same molar concentration (on a binding basis) and that concentration would typically be between 1.00 and 1.5 micromolar (on a binding site basis). Separate solutions containing A* alone and B* alone are also prepared. A* and B* in these solutions should be in the same buffer and at the same concentration as in the test mix.

The test mixture is passed over the target-coated Biacore chip and the total amount of binding recorded. The chip is then treated in such a way as to remove the bound amino acid sequences without damaging the chip-bound target. Typically this is done by treating the chip with 30 mM HCl for 60 seconds. The solution of A* alone is then passed over the target-coated surface and the amount of binding recorded. The chip is again treated to remove all of the bound amino acid sequences without damaging the chip-bound target. The solution of B* alone is then passed over the target-coated surface and the amount of binding recorded. The maximum theoretical binding of the mixture of A* and B* is next calculated, and is the sum of the binding of each amino acid sequence when passed over the target surface alone. If the actual recorded binding of the mixture is less than this theoretical maximum than the two amino acid sequences are cross-blocking each other. Thus, in general, a cross-blocking amino acid sequence or other binding agent according to the invention is one which will bind to the target in the above Biacore cross-blocking assay such that, during the assay and in the presence of a second amino acid sequence or other binding agent of the invention, the recorded binding is between 80% and 0.1% (e.g. 80% to 4%) of the maximum theoretical binding, specifically between 75% and 0.1% (e.g. 75% to 4%) of the maximum theoretical binding, and more specifically between 70% and 0.1% (e.g. 70% to 4%) of maximum theoretical binding (as just defined above) of the two amino acid sequences or binding agents in combination. The Biacore assay described above is a primary assay- used to determine if amino acid sequences or other binding agents cross-block each other according to the invention. On rare occasions particular amino acid sequences or other binding agents may not bind to target coupled via amine chemistry to a CMS Biacore chip (this usually occurs when the relevant binding site on target is masked or destroyed by the coupling to the chip). In such cases cross-blocking can be determined using a tagged version of the target, for example a N-terminal His-tagged version, ϊn this particular format, an anti-His amino acid sequence would be coupled to the Biacore chip and then the His-tagged target would be passed over the surface of the chip and captured by the anti-His amino acid sequence. The cross blocking analysis would be carried out essentially as described above, except that after each chip regeneration cycle, new His-tagged target would be loaded back onto the anti-His amino acid sequence coated surface, ϊn addition to the example given using N-terminal His-tagged target, C-terminal His-tagged target could alternatively be used. Furthermore, various other tags and tag binding protein combinations that are known in the art could be used for such a cross-blocking analysis (e.g. HA tag with anti-HA antibodies; FLAG tag with anti-FLAG antibodies; biotin tag with streptavidin). The following generally describes an ELISA assay for determining whether an amino acid sequence or other binding agent directed against a target cross-blocks or is capable of cross-blocking as defined herein. It will be appreciated that the assay can be used with any of the amino acid sequences (or other binding agents such as polypeptides of the invention) described herein. The general principal of the assay is to have an amino acid sequence or binding agent that is directed against the target coated onto the wells of an ELISA plate. An excess amount of a second, potentially cross-blocking, anti- target amino acid sequence is added in solution (i.e. not bound to the ELISA plate). A limited amount of the target is then added to the wells. The coated amino acid sequence and the amino acid sequence in solution compete for binding of the limited number of target molecules. The plate is washed to remove excess target that has not been bound by the coated amino acid sequence and to also remove the second, solution phase amino acid sequence as well as any complexes formed between the second, solution phase amino acid sequence and target. The amount of bound target is then measured using a reagent that is appropriate to detect the target. An amino acid sequence in solution that is able to cross-block the coated amino acid sequence will be able to cause a decrease in the number of target molecules that the coated amino acid sequence can bind relative to the number of target molecules that the coated amino acid sequence can bind in the absence of the second, solution phase, amino acid sequence. In the instance where the first amino acid sequence, e.g. an Ab-X. is chosen to be the immobilized amino acid sequence, it is coated onto the wells of the ELISA plate, after which the plates are blocked with a suitable blocking solution to minimize non-specific binding of reagents that are subsequently added. An excess amount of the second amino acid sequence, i.e. Ab-Y. is then added to the ELISA plate such that the moles of Ab-Y target binding sites per well are at least 10 fold higher than the moles of Ab-X target binding sites that were used, per well, during the coating of the ELISA plate. Target is then added such that the moles of target added per well are at least 25-fold lower than the moles of Ab-X target binding sites that were used for coating each well. Following a suitable incubation period the ELISA plate is washed and a reagent for detecting the target is added to measure the amount of target specifically bound by the coated anti [target amino acid sequence (in this case Ab-X). The background signal for the assay is defined as the signal obtained in wells with the coated amino acid sequence (in this case Ab-X), second solution phase amino acid sequence (in this case Ab-Y)₃ target buffer only (i.e. without target) and target detection reagents. The positive control signal for the assay is defined as the signal obtained in wells with the coated amino acid sequence (in this case

Ab-X), second solution phase amino acid sequence buffer only (i.e. without second solution phase amino acid sequence), target and target detection reagents. The ELISA assay may be run in such a manner so as to have the positive control signal be at least 6 times the background signal. To avoid any artefacts (e.g. significantly different affinities between Ab-X and Ab-Y for the target) resulting from the choice of which amino acid sequence to use as the coating amino acid sequence and which to use as the second (competitor) amino acid sequence, the cross-blocking assay may to be run in two formats: 1) format 1 is where Ab-X is the amino acid sequence that is coated onto the ELISA plate and Ab-Y is the competitor amino acid sequence that is in solution and 2) format 2 is where Ab-Y is the amino acid sequence that is coated onto the ELISA plate and Ab-X is the competitor amino acid sequence that is in solution. Ab-X and Ab- Y are defined as cross-blocking if, either in format 1 or in format 2, the solution phase anti -target amino acid sequence is able to cause a reduction of between 60% and 100%, specifically between 70% and 100%, and more specifically between 80% and 100%, of the target detection signal {i.e. the amount of target bound by the coated amino acid sequence) as compared to the target detection signal obtained in the absence of the solution phase anti- target amino acid sequence (i.e. the positive control wells). t) An amino acid sequence is said to be "cross-reactive^'" for two different antigens or antigenic determinants (such as serum albumin from two different species of mammal, such as human serum albumin and cyno serum albumin) if it is specific for (as defined herein) both these different antigens or antigenic determinants. u) By binding that is "essentially independent ofthepff is generally meant herein that the association constant (K_A) of the amino acid sequence with respect to the serum protein (such as serum albumin) at the pH valυe(s) that occur in a cell of an animal or human body (as further described herein) is at least 5%, such as at least 10%, preferably at least 25%, more preferably at least 50%. even more preferably at least 60%, such as even more preferably at least 70%, such as at least 80% or 90% or more (or even more than 100%, such as more than 110%. more than 120% or even 130% or more, or even more than 150%, or even more than 200%) of the association constant (K_A) of the amino acid sequence with respect to the same serum protein at the pH value(s) that occur outside said cell. Alternatively, by binding that is "essentially independent of the pW is generally meant herein that the k_Off rate (measured by Biacore) of the amino acid sequence with respect to the serum protein (such as serum albumin) at the pH value(s) that occur in a cell of an animal or human body (as e.g. further described herein, e.g. pH around 5.5, e.g. 5.3 to 5.7) is at least 5%, such as at least 10%, preferably at least 25%, more preferably at least 50%, even more preferably at least 60%, such as even more preferably at least 70%, such as at least 80% or 90% or more (or even more than

100%, such as more than 110%, more than 120% or even 130% or more, or even more than 150%, or even more than 200%) of the k_Off rate of the amino acid sequence with respect to the same serum protein at the pH value(s) that occur outside said cell, e.g. pH 7.2 to 7.4. By "the pH value (sj that occur in a cell of an animal or human body is meant the pH value(s) that may occur inside a cell, and in particular inside a cell that is involved in the recycling of the serum protein. In particular, by "the pH value (s) that occur in a cell of an animal or human body" is meant the pH value(s) that may occur inside a (sub)cellular compartment or vesicle that is involved in recycling of the serum protein (e.g. as a result of pinocytosis, endocytosis, transcytosis, exocytosis and phagocytosis or a similar mechanism of uptake or internalization into said cell), such as an endosome, lysosome or pinosome. v) As further described herein, the total number of amino acid residues in a Nanobody® can be in the region of 1 10-1.20, is preferably 112-115. and is most preferably 113. It should however be noted that parts, fragments, analogs or derivatives (as further described herein) of aNanobody® are not particularly limited as to their length and/or size, as long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are also preferably suitable for the purposes described herein; w) As further described in paragraph q) on pages 58 and 59 of WO 08/020079

(incorporated herein by reference), the amino acid residues of a Nanobody® are numbered according to the general numbering for V_H domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services. NIH Bethesda, MD, Publication No. 91), as applied to V_HH domains from Camelids in the article of Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun 23; 240 (1 -2): 185-195 (see for example Figure 2 of this publication), and accordingly FRl of a Nanobody® comprises the amino acid residues at positions 1-30, CDRl of a Nanobody® comprises the amino acid residues at positions 31 -35, FR2 of a Nanobody® comprises the amino acids at positions 36-49, CDR2 of a Nanobody® comprises the amino acid residues at positions 50-65, FR3 of aNanobody® comprises the amino acid residues at positions 66-94, CDR3 of a Nanobody® comprises the amino acid residues at positions 95-102, and FR4 of a Nanobody® comprises the amino acid residues at positions 103-113. x) The Figures, Sequence Listing and the Experimental Part/Examples are only given to further illustrate the invention and should not be interpreted or construed as limiting the scope of the invention and/or of the appended claims in any way, unless explicit!}' indicated otherwise herein.

For a general description of heavy chain antibodies and the variable domains thereof, reference is inter alia made to the prior art cited herein, as well as to the prior art mentioned on page 59 of WO 08/020079 and to the list of references mentioned on pages 41-43 of the International application WO 06/040153, which prior art and references are incorporated herein by reference.

In accordance with the terminology used in the art (see the above references), the variable domains present in naturally occurring heavy chain antibodies will also be referred to as "F_//// domains", in order to distinguish them from the heavy chain variable domains that are present in conventional 4-chain antibodies (which will be referred to hereinbelow as "F_# domains") and from the light chain variable domains that are present in conventional 4-chain antibodies (which will be referred to hereinbelow as ~Υ_L domains^'').

As mentioned in the prior ait referred to above, V_HH domains have a number of unique structural characteristics and functional properties which make isolated V_HH domains (as well as Nanobodies® based thereon, which share these structural characteristics and functional properties with the naturally occurring V_HH domains) and proteins containing the same highly advantageous for use as functional antigen-binding domains or proteins. In particular, and without being limited thereto, V_HH domains (which have been "designed^'' by nature to functionally bind to an antigen without the presence of, and without any interaction with, a light chain variable domain) and Nanobodies® can function as a single, relatively small, functional antigen-binding structural unit, domain or protein. This distinguishes the V_HH domains from the V^ and V_L domains of conventional 4-chain antibodies, which by themselves are generally not suited for practical application as single antigen-binding proteins or domains, but need to be combined in some form or another to provide a functional antigen-binding unit (as in for example conventional antibody fragments such as Fab fragments; in ScFv^" s fragments, which consist of a VH domain covalently linked to a V_L domain).

Because of these unique properties, the use of V_HH domains and Nanobodies® as single antigen -binding proteins or as antigen-binding domains (i.e. as part of a larger protein or polypeptide) offers a number of significant advantages over the use of conventional V_h and V_L domains, scFv's or conventional antibody fragments (such as Fab- or F(ab^*)2- fragments). including the advantages that are listed on pages 60 and 61 of WO 08/020079.

In a specific and preferred aspect, the invention provides Nanobodies® against sclerostin. and in particular Nanobodies® against sclerostin from a warm-blooded animal. and more in particular Nanobodies® against sclerostin from a mammal, and especially

Nanobodies® against human sclerostin; as well as proteins and/or polypeptides comprising at least one such Nanobody®.

In particular, the invention provides Nanobodies® against scierostin, and proteins and/or polypeptides comprising the same, that have improved therapeutic and/or pharmacological properties and/or other advantageous properties (such as, for example, improved ease of preparation and/or reduced costs of goods), compared to conventional antibodies against sclerostin or fragments thereof, compared to constructs that could be based on such conventional antibodies or antibody fragments (such as Fab' fragments, F(ab^")2 fragments, ScFv constructs, "diabodies" and other multispecific constructs (see for example the review by Holliger and Hudson, Nat BiotechnoL 2005 Sep;23(9): l 126-36)), and also compared to the so-called ''dAb's'" or similar (single) domain antibodies that may be derived from variable domains of conventional antibodies. These improved and advantageous properties will become clear from the further description herein, and for example include, without limitation, one or more of: increased affinity and/or avidity for sclerostin. either in a monovalent format, in a multivalent format (for example in a bivalent format) and/or in a multispecific format

(for example one of the multispecific formats described hereinbelow): - better suitability for formatting in a multivalent format (for example in a bivalent format); better suitability for formatting in a multispecific format (for example one of the multispecific foπnats described hereinbelow); improved suitability or susceptibility for ''humanizing'^' substitutions (as defined herein); less immunogenicity, either in a monovalent format- in a multivalent format (for example in a bivalent format) and/or in a multispecific format (for example one of the multispecific formats described hereinbelow): increased stability, either in a monovalent format, in a multivalent format (for example in a bivalent format) and/or in a multispecific format (for example one of the mult! specific formats described hereinbelow); increased specificity towards sclerostin, either in a monovalent format, in a multivalent format (for example in a bivalent format) and/or in a multispecific format (for example one of the multispecific formats described hereinbelow); - decreased or where desired increased cross-reactivity with sclerostin from different species; and/or one or more other improved properties desirable for pharmaceutical use (including prophylactic use and/or therapeutic use) and/or for diagnostic use (including but not limited to use for imaging purposes), either in a monovalent format, in a multivalent format (for example in a bivalent format) and/or in a multispecific format (for example one of the multispecific formats described hereinbelow). As generally described herein for the amino acid sequences of the invention, the Nanobodies® of the invention are preferably in essentially isolated form (as defined herein), or form part of a protein or polypeptide of the invention (as defined herein), which may comprise or essentially consist of one or more Nanobodies® of the invention and which may optionally further comprise one or more further amino acid sequences (all optionally linked via one or more suitable linkers). For example, and without limitation, the one or more amino acid sequences of the invention may be used as a binding unit in such a protein or polypeptide, which may optionally contain one or more further amino acid sequences that can serve as a binding unit (i.e. against one or more other targets than sclerostin), so as to provide a monovalent, multivalent or multispecifϊc polypeptide of the invention, respectively, all as described herein. In particular, such a protein or polypeptide may comprise or essentially consist of one or more Nanobodies® of the invention and optionally one or more (other) Nanobodies® (i.e. directed against other targets than sclerostin). all optionally linked via one or more suitable linkers, so as to provide a monovalent, multivalent or multi specific Nanobody® construct, respectively, as further described herein. Such proteins or polypeptides may also be in essentially isolated form (as defined herein).

In a Nanobody® of the invention, the binding site for binding against sclerostin is preferably formed by the CDR sequences. Optionally, a Nanobody® of the invention may also, and in addition to the at least one binding site for binding against sclerostin, contain one or more further binding sites for binding against other antigens, proteins or targets. For methods and positions for introducing such second binding sites, reference is for example made to Keck and Huston. Biophysical Journal. 71, October 1996, 2002-2011 : EP 0 640 130: and WO 06/07260.

As generally described herein for the amino acid sequences of the invention, when a Nanobody® of the invention (or a polypeptide of the invention comprising the same) is intended for administration Io a subject (for example for therapeutic and/or diagnostic purposes as described herein), it is preferably directed against human sclerostin: whereas for veterinary purposes, it is preferably directed against sclerostin from the species to be treated. Also, as with the amino acid sequences of the invention, a Nanobody® of the invention may or may not be cross-reactive (i.e. directed against sclerostin from two or more species of mammal, such as against human sclerostin and sclerostin from at least one of the species of mammal mentioned herein). Also, again as generally described herein for the amino acid sequences of the invention, the Nanobodies® of the invention may generally be directed against any antigenic determinant, epitope, part, domain, subunit or conformation (where applicable) of sclerostin. However, it is generally assumed and preferred that the Nanobodies® of the invention (and polypeptides comprising the same) are directed against the LRP5 and/or LRP6 interaction site on sclerostin, In particular embodiments of the present invention, the amino acid sequences and polypeptides of the invention are directed against an epitope of sclerostin that overlaps with the epitope of monoclonal antibody CDP-7851 (Sclerostin Ab, AMG-785).

As already described herein, the amino acid sequence and structure of a Nanobody® can be considered - without however being limited thereto - to be comprised of four framework regions or "FR's" (or sometimes also referred to as "FWs"), which are referred to in the art and herein as "Framework region 1" or "FRl"; as "Framework region 2" or "FR2"; as "Framework region 3" or ''FR3"; and as "Framework region 4" or "FR4", respectively; which framework regions are interrupted by three complementary determining regions or "CDR' s'\ which are referred to in the art as "Complementarity Determining Region I "or "CDRl "; as "Complementarity Determining Region 2" or "CDR2"; and as "Complementarity Determining Region 3" or "CDR3'^η, respectively. Some preferred framework sequences and CDR^' s (and combinations thereof) that are present in the Nanobodies® of the invention are as described herein. Other suitable CDR sequences can be obtained by the methods described herein.

According to a non-limiting but preferred aspect of the invention, (the CDR sequences present in) the Nanobodies® of the invention are such that: the Nanobodies® can bind to sclerostin with a dissociation constant (K_D) of 1 O^"5 to 10^" ¹² moles/liter or less, and preferably IG^"7 to 10^"12 moles/liter or less and more preferably IG^"8 to 10^"12 moles/liter (i.e. with an association constant (K_A) of IO⁵ to 10¹² liter/ moles or more, and preferably 10⁷ to 10¹² liter/moles or more and more preferably 10 to 10¹² liter/moles); and/or such that: the Nanobodies© can bind to sclerostin with a k_on-rate of between 10² M^-1S^"1 to about 10⁷ M^-1S^"3 , preferably between 10³ M^-1S^"1 and 10⁷ JvT¹S^"1, more preferably between 10⁴

M-'s^"1 and 10⁷ IvT¹S^'1 , such as between 10⁵ VF¹S^"1 and 10⁷ NfY¹; and/or such that they: the Nanobodies® can bind to sclerostin with a k_off rate between 1 s^'1 (ti_/2=0.69 s) and 10^~ό s^"1 (providing a near irreversible complex with a ty? of multiple days), preferably between 10^"2 s^"1 and 10^"6 s^"1, more preferably between 10^"3 s^"1 and 10^"6 s^"1, such as between 10^"4 S^"1 and 10^"6 s^"'. Preferably, (the CDR sequences present in) the Nanobodies® of the invention are such that: a monovalent Nanobody® of the invention (or a polypeptide that contains only one Nanobody® of the invention) is preferably such that it will bind to sclerostin with an affinity less than 500 liM, preferably less than 200 iiM. more preferably less than 10 nM, such as less than 500 pM. The affinity of the Nanobody® of the invention against sclerostin can be determined in a manner known per se, for example using the general techniques for measuring KD- K_A, k_off or k_oπ mentioned herein, as well as some of the specific assays described herein.

Some preferred ΪC50 values for binding of the Nanobodies® of the invention (and of polypeptides comprising the same) to sclerostin will become clear from the further description and examples herein.

In particular but non-limiting embodiments, the invention relates to a Nanobody® (as defined herein) against sclerostin, which consists of 4 framework regions (FRl to FR4 respectively) and 3 complementarity determining regions (CDRl to CDR3 respectively), in which: - CDRl is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO's: 135 to 143; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; and/or

CDR2 is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NO^!s: 153 to 161 : e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 153 to 161 ; f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 153 to 161 ; and/or CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NO's: 171 to 179: h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 171 to 179; i) amino acid sequences that have 3, 2. or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO^'s: 171 to 179; or any suitable fragment of such an amino acid sequence.

In particular, according to particular embodiments, the invention relates to a Nanobody® (as defined herein) against sclerostin, which consists of 4 framework regions (FRl to FR4 respectively) and 3 complementarity determining regions (CDRl to CDR3 respectively), in which:

CDRl is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO"s: 135 to 143: b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143 ; c) amino acid sequences that have 3, 2. or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO^'s: 135 to 143; and

CDR2 is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NO^'s: 153 to 161; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO^'s: 153 to 161; f) amino acid sequences that have 3. 2. or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO^'s; 153 to 161 ; and

CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NO^'s: 171 to 179; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 171 to 179; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO^'s: 171 to 179; or any suitable fragment of such an amino acid sequences. As generally mentioned herein for the amino acid sequences of the invention, when a Nanobody© of the invention contains one or more CDRl sequences according to b) and/or c): i) any amino acid substitution in such a CDR according to b) and/or c) is preferably, and compared to the corresponding CDR according to a), a conservative amino acid substitution (as defined herein); and/or ii) the CDR according to b) and/or c) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR according to a): and/or iii) the CDR according to b) and/or c) may be a CDR that is derived from a CDR according to a) by means of affinity maturation using one or more techniques of affinity maturation known per se. Similarly, when a Nanobody® of the invention contains one or more CDR2 sequences according to e) and/or f): i) any amino acid substitution in such a CDR according to e) and/or f) is preferably, and compared to the corresponding CDR according to d). a conservative amino acid substitution (as defined herein): and/or ii) the CDR according to e) and/or f) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR according to d); and/or iii) the CDR according to e) and/or f) may be a CDR that is derived from a CDR according to d) by means of affinity maturation using one or more techniques of affinity maturation known per sc.

Also, similarly, when a Nanobody® of the invention contains one or more CDR3 sequences according to h) and/or i): i) any amino acid substitution in such a CDR according to h) and/or i) is preferably, and compared to the corresponding CDR according to g). a conservative amino acid substitution (as defined herein); and/or ii) the CDR according to h) and/or i) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR according to g); and/or iii) the CDR according to h) and/or i) may be a CDR that is derived from a CDR according to g) by means of affinity maturation using one or more techniques of affinity maturation known per se.

It should be understood that the last three paragraphs generally apply to any Nanobody® of the invention that comprises one or more CDRl sequences, CDR2 sequences and/or CDR3 sequences according to b), c), e), f). h) or i), respectively.

Of the Nanobodies® of the invention, Nanobodies® comprising one or more of the CDR' s explicitly listed above are particularly preferred: Nanobodies® comprising two or more of the CDR' s explicitly listed above are more particularly preferred; and Nanobodies® comprising three of the CDR' s explicitly listed above are most particularly preferred. Some particularly preferred, but non-limiting combinations of CDR sequences, as well as preferred combinations of CDR sequences and framework sequences, are mentioned in Table B-I below, which lists the CDR sequences and framework sequences that are present in a number of preferred (but non-limiting) Nanobodies® of the invention. As will be clear to the skilled person, a combination of CDRl . CDR2 and CDR3 sequences that occur in the same clone (i.e. CDRl , CDR2 and CDR3 sequences that are mentioned on the same line in Table B-I ) will usually be preferred (although the invention in its broadest sense is not limited thereto, and also comprises other suitable combinations of the CDR sequences mentioned in Table B-I). Also, a combination of CDR sequences and framework sequences that occur in the same clone (i.e. CDR sequences and framework sequences that are mentioned on the same line in Table B-I) will usually be preferred (although the invention in its broadest sense is not limited thereto, and also comprises other suitable combinations of the CDR sequences and framework sequences mentioned in Table B-I. as well as combinations of such CDR sequences and other suitable framework sequences, e.g. as further described herein). Also, m the Nanobodies® of the invention that comprise the combinations of CDR's mentioned in Table B-I, each CDR can be replaced by a CDR chosen from the group consisting of amino acid sequences that have at least 80%. preferably at least 90%, more preferably at least 95%. even more preferably at least 99% sequence identity (as defined herein) with the mentioned CDR 's; in which: i) any amino acid substitution in such a CDR is preferably, and compared to the corresponding CDR sequence mentioned in Table B-I, a conservative amino acid substitution (as defined herein); and/or ii) any such CDR sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR sequence mentioned in Table B-I; and/or iii) any such CDR sequence is a CDR that is derived by means of a technique for affinity maturation known per se, and in particular starting from the corresponding CDR sequence mentioned in Table B-L

However, as will, be clear to the skilled person, the (combinations of) CDR sequences. as well as (the combinations of) CDR sequences and framework sequences mentioned in Table B-I will generally be preferred.

Thus, in particular embodiments . at least one of the CDRl, CDR2 and CDR3 sequences present in the Nanobodies® of the invention is suitably chosen from the group consisting of the CDRl , CDR2 and CDR3 sequences, respectively, listed in Table B-I : or from the group of CDRL CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%. more preferably at least 95%. even more preferably at least 99% "'sequence identity'^" (as defined herein) with at least one of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table B-I; and/or from the group consisting of the CDRl₅ CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 "'amino acid difference(s)" (as defined herein) with at least one of the CDRl, CDR2 and CDR3 sequences, respectively. listed in Table B-I .

In this context, by '"suitably chosen'^" is meant that, as applicable, a CDRl sequence is chosen from suitable CDRl sequences (i.e. as defined herein), a CDR2 sequence is chosen from suitable CDR2 sequences (i.e. as defined herein), and a CDR3 sequence is chosen from suitable CDR3 sequence (i.e. as defined herein), respectively. More in particular, the CDR sequences are preferably chosen such that the Nanobodies® of the invention bind to sclerostin with an affinity (suitably measured and/or expressed as a KQ- value (actual or apparent), a K._A-value (actual or apparent), a k_on-rate and/or a k_off-rate. or alternatively as an IC₅₀ value, as further described herein) that is as defined herein.

In particular, in the Nanobodies® of the invention, at least the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 sequences listed in Table B-I or from the group of CDR3 sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR3 sequences listed in Table B-I : and/or from the group consisting of the CDR3 sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDR3 sequences listed in Table B-I .

Preferably, in the Nanobodies® of the invention, at least two of the CDRl. CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table B-I or from the group consisting of CDRl, CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%. even more preferably at least 99% sequence identity with at least one of the CDRl , CDR2 and CDR3 sequences, respectively, listed in Table B-I ; and/or from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, that have 3. 2 or only 1 "amino acid difference(s)" with at least one of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table B-I .

In particular embodiments, in the Nanobodies® of the invention, at least the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 sequences listed in Table B-I or from the group of CDR3 sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR3 sequences listed in Table B-I, respectively; and at least one of the CDRl and CDR2 sequences present is suitably chosen, from the group consisting of the CDRl and CDR2 sequences, respectively, listed in Table B-I or from the group of CDRl and CDR2 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDRl and CDR2 sequences, respectively, listed in Table B-I ; and/or from the group consisting of the CDRl and CDR2 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDRl and CDR2 sequences, respectively, listed in Table B-L

Most preferably, in the Nanobodies® of the invention, all three CDRl, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDRl , CDR2 and CDR3 sequences, respectively, listed in Table B-I or from the group of CDRl, CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%. more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDRl. CDR2 and CDR3 sequences, respectively, listed in Table B-I ; and/or from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDRl , CDR2 and CDR3 sequences, respectively, listed in Table B-I . Even more preferably, in the Nanobodies® of the invention, at least one of the CDRl,

CDR2 and CDR3 sequences present is suitably chosen from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table B-I. Preferably, in this aspect, at least one or preferably both of the other two CDR sequences present are suitably chosen from CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the corresponding CDR sequences, respectively, listed in Table B-I; and/or from the group consisting of the CDR sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the corresponding sequences, respectively, listed in Table B-L In particular embodiments, in the Nanobodies® of the invention, at least the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 listed in Table B- 1. Preferably, in this aspect, at least one and preferably both of the CDRl and CDR2 sequences present are suitably chosen from the groups of CDRl and CDR2 sequences. respectively, that have at least 80%. preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the CDRl and CDR2 sequences, respectively, listed in Table B-L and/or from the group consisting of the CDRl and CDR2 sequences, respectively, that have 3. 2 or only 1 amino acid difference(s) with at least one of the CDRl and CDR2 sequences, respectively, listed in Table B-I . Even more preferably, in the Nanobodies© of the invention, at least two of the CDRL

CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDRl. CDR2 and CDR3 sequences, respectively, listed in Table B-I . Preferably, in this aspect, the remaining CDR sequence present is suitably chosen from the group of CDR sequences that have at least 80%. preferably at least 90%. more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the corresponding CDR sequences listed in Table B-I; and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the corresponding sequences listed in Table B-I.

In particular, in the Nanobodies® of the invention, at least the CDR3 sequence is suitably chosen from the group consisting of the CDR3 sequences listed in Table B-L and either the CDRl sequence or the CDR2 sequence is suitably chosen from the group consisting of the CDRl and CDR2 sequences, respectively, listed in Table B-I . Preferably, in this aspect, the remaining CDR sequence present is suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the corresponding CDR sequences listed in Table B-I; and/or from the group consisting of CDR sequences that have 3. 2 or only 1 amino acid difference(s) with the corresponding CDR sequences listed in Table B-L

Even more preferably, in the Nanobodies© of the invention, all three CDRl, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDRL CDR2 and CDR3 sequences, respectively, listed in Table B-L

Also, generally, the combinations of CDR^" s listed in Table B-I (i.e. those mentioned on the same line in Table B-I) are preferred. Thus, it is generally preferred that, when a CDR in a Nanobody® of the invention is a CDR sequence mentioned in Table B-I or is suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%. more preferably at least 95%, even more preferably at least 99% sequence identity with a CDR sequence listed in Table B-] ; and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with a CDR sequence listed in Table B-L that at least one and preferably both of the other CDR^rs are suitably chosen from the CDR sequences that belong to the same combination in Table B-I (i.e. mentioned on the same line in Table B-I) or are suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the CDR sequence(s) belonging to the same combination and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with the CDR sequence(s) belonging to the same combination. The other preferences indicated in the above paragraphs also apply to the combinations of CDR' s mentioned in Table B-I. Thus, by means of non-limiting examples, a Nanobody® of the invention can for example comprise a CDRl sequence that has more than 80 % sequence identity with one of the CDRl sequences mentioned in Table B-I , a CDR2 sequence that has 3, 2 or 1 amino acid difference with one of the CDR2 sequences mentioned in Table B-I (but belonging to a different combination), and a CDR3 sequence. Some preferred Nanobodies® of the invention may for example comprise: (1) a

CDRl sequence that has more than 80 % sequence identity with one of the CDRl sequences mentioned in Table B-I; a CDR2 sequence that has 3, 2 or 1 amino acid difference with one of the CDR2 sequences mentioned in Table B-I (but belonging to a different combination); and a CDR3 sequence that has more than 80 % sequence identity with one of the CDR3 sequences mentioned in Table B-I (but belonging to a different combination); or (2) a CDRl sequence that has more than 80 % sequence identity with one of the CDRl sequences mentioned in Table B-I ; a CDR2 sequence, and one of the CDR3 sequences listed in Table B-I; or (3) a CDRl sequence; a CDR2 sequence that has more than. 80% sequence identity with one of the CDR2 sequence listed in Table B-I; and a CDR3 sequence that has 3, 2 or 1 amino acid differences with the CDR3 sequence mentioned in Table B-I that belongs to the same combination as the CDR2 sequence.

Some particularly preferred Nanobodies® of the invention may for example comprise: (1) a CDRl sequence that has more than 80 % sequence identity with one of the CDRl sequences mentioned in Table B- 1 ; a CDR2 sequence that has 3, 2 or 1 amino acid difference with the CDR2 sequence mentioned in Table B-I that belongs to the same combination; and a CDR3 sequence that has more than 80 % sequence identity with the CDR3 sequence mentioned in Table B-I that belongs to the same combination; (2) a CDRl sequence: a CDR 2 listed in Table B-I and a CDR3 sequence listed in Table B-I (in which the CDR2 sequence and CDR3 sequence may belong to different combinations).

Some even more preferred Nanobodies® of the invention may for example comprise: (1) a CDRl sequence that has more than 80 % sequence identity with one of the CDRl sequences mentioned in Table B-I; the CDR2 sequence listed in Table B-I that belongs to the same combination: and a CDR3 sequence mentioned in Table B-I that belongs to a different combination; or (2) a CDRl sequence mentioned in Table B-I; a CDR2 sequence that has 3, 2 or 1 amino acid differences with the CDR2 sequence mentioned in Table B-I that belongs to the same combination; and a CDR3 sequence that has more than 80% sequence identity with the CDR3 sequence listed in Table B-I that belongs to the same or a different combination.

Particularly preferred Nanobodies® of the invention may for example comprise a CDRl sequence mentioned in Table B-I , a CDR2 sequence that has more than 80 % sequence identity with the CDR2 sequence mentioned in Table B-I that belongs to the same combination; and the CDR3 sequence mentioned in Table B- 1 that belongs to the same combination.

In the most preferred Nanobodies® of the invention, the CDRl, CDR2 and CDR3 sequences present are suitably chosen from one of the combinations of CDRl₅ CDR2 and CDR3 sequences, respectively, listed in Table B-I.

According to particular, but non-limiting embodiments of the invention (a) CDRl has a length of between 1 and 12 amino acid residues, and usually between 2 and 9 amino acid residues, such as 5, 6 or 7 amino acid residues; and/or (b) CDR2 has a length of between 13 and 24 amino acid residues, and usually between 15 and 21 amino acid residues, such as 16 and 17 amino acid residues; and/or (c) CDR3 has a length of between 2 and 35 amino acid residues, and usually between 3 and 30 amino acid residues, such as between 6 and 23 amino acid residues.

In particular, but non-limiting embodiments, the invention provides to a Nanobody© in which the CDR sequences (as defined herein) have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO^'s:

189 to 197 (see Table A-I).

Generally, Nanobodies® with the above CDR sequences may be as further described herein, and preferably have framework sequences that are also as further described herein. Thus, for example and as mentioned herein, such Nanobodies® may be naturally occurring

Nanobodies® (from any suitable species), naturally occurring V_HΠ sequences (i.e. from a suitable species of Camelid) or synthetic or semi-synthetic amino acid sequences or

Nanobodies®, including but not limited to partially humanized Nanobodies® or V_HH sequences, fully humanized Nanobodies® or V_HH sequences, camelized heavy chain variable domain sequences, as well as Nanobodies® that have been obtained by the techniques mentioned herein.

Thus, inparticular embodiments, the invention relates to a humanized Nanobody®, which consists of 4 framework regions (FRl to FR4 respectively) and 3 complementarity determining regions (CDRl to CDR3 respectively), in which CDRl to CDR3 are as defined herein and in which said humanized Nanobody® comprises at least one humanizing substitution (as defined herein), and in particular at least one humanizing substitution in at least one of its framework sequences (as defined herein).

Inparticular, but non-limiting embodiments, the invention relates to a Nanobody® in which the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 189 to 197 (see Table A-I).

This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said Nanobody® and one or more of the sequences of SEQ ID NO^'s: 189 to 197 (see Table A-I). in which the amino acid residues that form the framework regions are disregarded. Such Nanobodies® can be as further described herein.

Inparticular, but non-limiting embodiments, the invention relates to a Nanobody® with an amino acid sequence that is chosen from the group consisting of SEQ ID NO's: 189 to 197 (see Table A-I) or from the group consisting of from amino acid sequences that have more than 80%. preferably more than 90%, more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with at least one of the amino acid sequences of

SEQ ID NO's: 189 to 197 (see Table A-I). Particular, but non-limiting embodiments of the invention relate to humanized variants of the Nanobodies® of SEQ ID NO's: 189 to 197 (see Table A-I), that comprise, compared to the corresponding native V_HH sequence, at least one humanizing substitution (as defined herein), and in particular at least one humanizing substitution in at least one of its framework sequences (as defined herein).

It will be clear to the skilled person that the Nanobodies® that are mentioned herein as "preferred" (or "more preferred^", "even more preferred", etc.) are also preferred (or more preferred, or even more preferred, etc.) for use in the polypeptides described herein. Thus, polypeptides that comprise or essentially consist of one or more "preferred" Nanobodies® of the invention will generally be preferred, and polypeptides that comprise or essentially consist of one or more "more preferred" Nanobodies® of the invention will generally be more preferred, etc.

Generally, proteins or polypeptides that comprise or essentially consist of a single Nanobody® (such as a single Nanobody® of the invention) will be referred to herein as "monovalent" proteins or polypeptides or as "monovalent constructs". Proteins and polypeptides that comprise or essentially consist of two or more Nanobodies® (such as at least two Nanobodies® of the invention or at least one Nanobody® of the invention and at least one other Nanobody®) will be referred to herein as "multivalent" proteins or polypeptides or as "multivalent constructs", and these may provide certain advantages compared to the corresponding monovalent Nanobodies® of the invention. Some non- limiting examples of such multivalent constructs will become clear from the further description herein.

According to particular, but non-limiting embodiments, a polypeptide of the invention comprises or essentially consists of at least two Nanobodies® of the invention, such as two or three Nanobodies® of the invention. As further described herein, such multivalent constructs can provide certain advantages compared to a protein or polypeptide comprising or essentially consisting of a single Nanobody® of the invention, such as a much improved avidity for sclerostin. Such multivalent constructs will be clear to the skilled person based on the disclosure herein. According to particular, but non-limiting embodiments, a polypeptide of the invention comprises or essentially consists of at least one Nanobody® of the invention and at least one other binding unit (i.e. directed against another epitope, antigen, target, protein or polypeptide), which is preferably also a Nanobody®. Such proteins or polypeptides are also referred to herein as "multispecific" proteins or polypeptides or as 'multispecific constructs", and these may provide certain advantages compared to the corresponding monovalent Nanobodies® of the invention (as will become clear from the further discussion herein of some preferred, but-nonlimiting multispecific constructs). Such multispecific constructs will be clear to the skilled person based on the disclosure herein. According to particular, but non-limiting embodiments, a polypeptide of the invention comprises or essentially consists of at least one Nanobody® of the invention, optionally one or more further Nanobodies®, and at least one other amino acid sequence (such as a protein or polypeptide) that confers at least one desired property to the Nanobody® of the invention and/or to the resulting fusion protein. Again, such fusion proteins may provide certain advantages compared to the corresponding monovalent Nanobodies® of the invention. Some non-limiting examples of such amino acid sequences and of such fusion constructs will become clear from the further description herein.

It is also possible to combine two or more of the above embodiments, for example to provide a trivalent bispecific construct comprising two Nanobodies® of the invention and one other Nanobody®, and optionally one or more other amino acid sequences. Further non- limiting examples of such constructs, as well as some constructs that are particularly preferred within the context of the present invention, will become clear from the further description herein.

In the above constructs, the one or more Nanobodies® and/or other amino acid sequences may be directly linked to each other and/or suitably linked to each other via one or more linker sequences. Some suitable but non-limiting examples of such linkers will become clear from the further description herein.

In particular embodiments of the invention, a Nanobody® of the invention or a compound, construct or polypeptide of the invention comprising at least one Nanobody® of the invention may have an increased half-life, compared to the corresponding amino acid sequence of the invention. Some preferred, but non-limiting examples of such Nanobodies®, compounds and polypeptides will become clear to the skilled person based on the further disclosure herein, and for example comprise Nanobodies® sequences or polypeptides of the invention that have been chemically modified to increase the half-life thereof (for example, by means of pegylati on); amino acid sequences of the invention that comprise at least one additional binding site for binding to a serum protein (such as serum albumin, see for example EP 0 368 684 Bl, page 4); or polypeptides of the invention that comprise at least one Nanobody® of the invention that is linked to at least one moiety (and in particular at least one amino acid sequence) that increases the half- life of the Nanobody® of the invention. Examples of polypeptides of the invention that comprise such half-life extending moieties or amino acid sequences will become clear to the skilled person based on the further disclosure herein; and for example include, without limitation, polypeptides in which the one or more Nanobodies® of the invention are suitable linked to one or more serum proteins or fragments thereof (such as serum albumin or suitable fragments thereof) or to one or more binding units that can bind to serum proteins (such as, for example. Nanobodies® or (single) domain antibodies that can bind to serum proteins such as serum albumin, serum immunoglobulins such as IgG, or transferrine); polypeptides in which a Nanobody© of the invention is linked Io an Fc portion (such as a human Fc) or a suitable part or fragment thereof; or polypeptides in which the one or more Nanobodies® of the invention are suitable linked to one or more small proteins or peptides that can bind to serum proteins (such as, without limitation, the proteins and peptides described in WO 91/01743, WO 01/45746, WO 02/076489 and to the US provisional application of Ablynx N.V. entitled "Peptides capable of binding to serum proteins" of Ablynx N.V. filed on December 5, 2006 (see also PCT/EP/2007/063348).

Again, as will be clear to the skilled person, such Nanobodies®, compounds, constructs or polypeptides may contain one or more additional groups, residues, moieties or binding units, such as one or more further amino acid sequences and in particular one or more additional Nanobodies® (i.e. not directed against sclera stin). so as to provide a tri- of multispecific Nanobody® construct.

Generally, the Nanobodies® of the invention (or compounds, constructs or polypeptides comprising the same) with increased half-life preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding amino acid sequence of the invention per se. For example, the Nanobodies®. compounds, constructs or polypeptides of the invention with increased half-life may have a half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence of the invention per se. In particular, but non-limiting embodiments of the invention, such Nanobodies®. compound, constructs or polypeptides of the invention exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more. For example, compounds or polypeptides of the invention may have a half-life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 1 1 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).

In particular embodiments of the invention, a polypeptide of the invention comprises one or more (such as two or preferably one) Nanobodies® of the invention linked (optionally via one or more suitable linker sequences) to one or more (such as two and preferably one) amino acid sequences that allow the resulting polypeptide of the invention to cross the blood brain barrier. In particular, said one or more amino acid sequences that allow the resulting polypeptides of the invention to cross the blood brain barrier may be one or more (such as two and preferably one) Nanobodies®, such as the Nanobodies® described in WO 02/057445. of which FC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO 06/040154) are preferred examples. In particular, polypeptides comprising one or more Nanobodies® of the invention are preferably such that they: bind to sclerostin with a dissociation constant (K_D) of 10^"1 to 10^"i2 moles/liter or less, and preferably 10^~7 to 10^"12 moles/liter or less and more preferably 10^"8 to 10^"ϊ2 moles/liter (i.e. with an association constant (K_A) of 10 to 10° liter/ moles or more, and preferably 10' to 10¹² liter/moles or more and more preferably 10⁸ to 10¹² liter/moles); and/or such that they: bind to sclerostin with a k_αn-rate of between 10² M^"V to about 10⁷ IVfY¹, preferably between 10³ M^-1S^"1 and 10⁷ WT¹S^*1, more preferably between 10⁴ M^-1S^"1 and 10⁷ M^~V. such as between 10⁵ M^'V¹ and 10⁷ M^'Y¹ ; and/or such that they: bind to sclerostin with a k_off rate between 1 s^"1 (U _/2=0.69 s) and 10^~6 s^"1 (providing a near irreversible complex with a Xm of multiple days), preferably between 10^~2 s^"1 and 10^~6 s^" \ more preferably between 10^"3 s^"1 and 10^"6 s^"1, such as between 10^"4 s^"1 and 10^"6 s^"!. Preferably, a polypeptide that contains only one amino acid sequence of the invention is preferably such that it will bind to sclerostin with an affinity less than 500 nM, preferably less than 200 nM. more preferably less than 10 nM, such as less than 500 pM. In this respect, it will be clear to the skilled person that a polypeptide that contains two or more Nanobodies® of the invention may bind to sclerostin with an increased avidity, compared to a polypeptide that contains only one amino acid sequence of the invention.

Some preferred IC₃₀ values for binding of the amino acid sequences or polypeptides of the invention to sclerostin will become clear from the further description and examples herein.

Another aspect of this invention relates to nucleic acids that encode amino acid sequences of the invention (such as a Nanobody® of the invention) or polypeptides of the invention comprising the same. Again, as generally described herein for the nucleic acids of the invention, such a nucleic acid may be in the form of a genetic construct, as defined herein. In another aspect, the invention relates to host or host cells that express or that are capable of expressing an amino acid sequence (such as a Nanobody®) of the invention and/or a polypeptide of the invention comprising the same: and/or that contain a nucleic acid of the invention. Some preferred but non-limiting examples of such hosts or host cells will become clear from the further description herein. Another aspect of the invention relates to a product or composition containing or comprising at least one amino acid sequence of the invention, at least one polypeptide of the invention and/or at least one nucleic acid of the invention, and optionally one or more further components of such compositions known per se, i.e. depending on the intended use of the composition. Such a product or composition may for example be a pharmaceutical composition (as described herein), a veterinary composition or a product or composition for diagnostic use (as also described herein). Some preferred but non-limiting examples of such products or compositions will become clear from the further description herein.

The invention further relates to methods for preparing or generating the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein. Some preferred but non-limiting examples of such methods will become clear from the further description herein.

Other aspects, embodiments, advantages and applications of the invention will also become clear from the further description hereinbelow. Generally, it should be noted that the term Nanobody® as used herein in its broadest sense is not limited to a specific biological source or to a specific method of preparation. For example, as will be discussed in more detail below, the Nanobodies® of the invention can generally be obtained by any of the techniques (1) to (8) mentioned on pages 61 and 62 of WO 08/020079. or any other suitable technique known per se. One preferred class of

Nanobodies® corresponds to the V_HH domains of natural!} occurring heavy chain antibodies directed against sclerostin. As further described herein, such V_HH sequences can generally be generated or obtained by suitably immunizing a species of Camelid with sclerostin (i.e. so as to raise an immune response and/or heavy chain antibodies directed against sclerostin), by obtaining a suitable biological sample from said Camelid (such as a blood sample, serum sample or sample of B-cells), and by generating V_11H sequences directed against sclerostin. starting from said sample, using any suitable technique known per se. Such techniques will be clear to the skilled person and/or are further described herein.

Alternatively, such naturally occurring V_HH domains against sclerostin, can be obtained from naive libraries of Camelid V_HH sequences, for example by screening such a library using sclerostin. or at least one part, fragment, antigenic determinant or epitope thereof using one or more screening techniques known per se. Such libraries and techniques are for example described in WO 99/37681, WO 01/90190. WO 03/025020 and WO 03/035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naϊve V_HH libraries may be used, such as V_HH libraries obtained from naϊve Vnn libraries by techniques such as random mutagenesis and/or CDR shuffling, as for example described in WO 00/43507.

Thus, in another aspect, the invention relates to a method for generating Nanobodies® that are directed against sclerostin. In one aspect, said method at least comprises the steps of: a) providing a set, collection or library of Nanobody® sequences; and b) screening said set, collection or library of Nanobody® sequences for Nanobody® sequences that can bind to and/or have affinity for sclerostin; and c) isolating the Nanobody® or Nanobodies® that can bind to and/or have affinity for sclerostin.

In such a method, the set, collection or library of Nanobody® sequences may be a naϊve set. collection or library of Nanobody® sequences; a synthetic or semi -synthetic set, collection or library of Nanobody® sequences; and/or a set, collection or library of Nanobody® sequences that have been subjected to affinity maturation.

In particular embodiments of this method, the set, collection or library of Nanobody® sequences may be an immune set, collection or library of Nanobody® sequences, and in particular an immune set, collection or library of V_HH sequences, that have been derived from a species of Camelid that has been suitably immunized with sclerostin or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

In the above methods, the set, collection or library of Nanobody® or V_HH sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set. collection or library of) Nanobody© sequences will be clear to the person skilled in the art. for example on the basis of the further disclosure herein. Reference is also made to WO 03/054016 and to the review by Hoogenboom in Nature Biotechnology, 23, 9, 11054116 (2005).

In particular embodiments of the invention, the method for generating Nanobody® sequences comprises at least the steps of: a) providing a collection or sample of cells derived from a species of Camelid that express immunoglobulin sequences; b) screening said collection or sample of cells for (i) cells that express an immunoglobulin sequence that can bind to and/or have affinity for sclerostin; and (ii) cells that express heavy chain antibodies, in which substeps (i) and (ii) can be performed essentially as a single screening step or in any suitable order as two separate screening steps, so as to provide at least one cell that expresses a heavy chain antibody that can bind to and/or has affinity for sclerostin; and c) either (i) isolating from said cell the V_HH sequence present in said heavy chain antibody; or (ii) isolating from said cell a nucleic acid sequence that encodes the V_ilu sequence present in said heavy chain antibody, followed by expressing said V_HH domain. In the methods according to these embodiments of the invention, the collection or sample of cells may for example be a collection or sample of B-cells. Also, in this method, the sample of cells may be derived from a Camelid that has been suitably immunized with sclerostin or a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

The above method may be performed in any suitable manner, as will be clear to the skilled person. Reference is for example made to EP 0 542 810. WO 05/19824, WO 04/051268 and WO 04/106377. The screening of step b) is preferably performed using a flow cytometry technique such as FACS. For this, reference is for example made to Lieby et al., Blood, Vol. 97, No. 12. 3820. Particular reference is made to the so-called "Nanoclone™" technique described in International application WO 06/079372 by Ablynx N.V.

In particular embodiments, the method for generating an amino acid sequence directed against sclerostin may comprise at least the steps of: a) providing a set, collection or library of nucleic acid sequences encoding heavy chain antibodies or Nanobody® sequences: b) screening said set. collection or library of nucleic acid sequences for nucleic acid sequences that encode a heavy chain antibody or a Nanobody® sequence that can bind to and/or has affinity for sclerostin: and c) isolating said nucleic acid sequence, followed by expressing the V_HH sequence present in said heavy chain antibody or by expressing said Nanobody® sequence, respectively.

In such a method, the set. collection or library of nucleic acid sequences encoding heavy chain antibodies or Nanobody® sequences may for example be a set. collection or library of nucleic acid sequences encoding a naϊve set, collection or library of heavy chain antibodies or V_HH sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set. collection or library of Nanobody© sequences; and/or a set. collection or library of nucleic acid sequences encoding a set, collection or library of Nanobody® sequences that have been subjected to affinity maturation.

In particular embodiments of this method, the set, collection or library of nucleic acid sequences may be an immune set, collection or library of nucleic acid sequences encoding heavy chain antibodies or V_HH sequences derived from a Camelid that has been suitably immunized with sclerostin or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof, in one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s). In the above methods, the set, collection or library of nucleotide sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the ait, for example on the basis of the further disclosure herein. Reference is also made to WO 03/054016 and to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1 105-11 16 (2005).

As will be clear to the skilled person, the screening step of the methods described herein can also be performed as a selection step. Accordingly the term '"screening" as used in the present description can comprise selection, screening or any suitable combination of selection and/or screening techniques. Also, when a set, collection or library of sequences is used, it may contain any suitable number of sequences, such as 1. 2, 3 or about 5, 10, 50, 100, 500, 1000, 5000, 10⁴, 10⁵, 10⁶, 10⁷, 10^s or more sequences.

Also, one or more or all of the sequences in the above set, collection or library of amino acid sequences may be obtained or defined by rational or semi -empirical approaches such as computer modelling techniques or biostatics or datamining techniques.

Furthermore, such a set. collection or library can comprise one, two or more sequences that are variants from one another (e.g. with designed point mutations or with randomized positions), compromise multiple sequences derived from a diverse set of naturally diversified sequences (e.g. an immune library)), or any other source of diverse sequences (as described for example in Hoogenboom et al, Nat Biotechnol 23:1105, 2005 and Binz et al, Nat

Biotechnol 2005, 23:1247). Such set, collection or library of sequences can be displayed on the surface of a phage particle, a ribosome, a bacterium, a yeast cell, a mammalian cell, and linked to the nucleotide sequence encoding the amino acid sequence within these carriers. This makes such set, collection or library amenable to selection procedures to isolate the desired amino acid sequences of the invention. More generally, when a sequence is displayed on a suitable host or host cell, it is also possible (and customary) to first isolate from said host or host cell a nucleotide sequence that encodes the desired sequence, and then to obtain the desired sequence by suitably expressing said nucleotide sequence in a suitable host organism. Again, this can be performed in any suitable manner known per se, as will be clear to the skilled person.

Yet another technique for obtaining VH_H sequences or Nanobody® sequences directed against sclerostin involves suitably immunizing a transgenic mammal that is capable of expressing heavy chain antibodies (i.e. so as to raise an immune response and/or heavy chain antibodies directed against sclerostin), obtaining a suitable biological sample from said transgenic mammal that contains (nucleic acid sequences encoding) said V_HH sequences or Nanobody© sequences (such as a blood sample, serum sample or sample of B-cells). and then generating V_HH sequences directed against sclerostin, starting from said sample, using any suitable technique known per se (such as any of the methods described herein or a hybridoma technique). For example, for this purpose, the heavy chain antibody-expressing mice and the further methods and techniques described in WO 02/085945, WO 04/049794 and WO 06/008548 and Janssens et al.. Proc. Natl. Acad. Sci .USA. 2006 Oct 10; 103(41): 15130-5 can be used. For example, such heavy chain antibody expressing mice can express heavy chain antibodies with any suitable (single) variable domain, such as (single) variable domains from natural sources (e.g. human (single) variable domains, Camelid (single) variable domains or shark (single) variable domains), as well as for example synthetic or semi-synthetic (single) variable domains.

The invention also relates to the Vnπ sequences or Nanobody® sequences that are obtained by the above methods, or alternatively by a method that comprises the one of the above methods and in addition at least the steps of determining the nucleotide sequence or amino acid sequence of said V_HH sequence or Nanobody® sequence: and of expressing or synthesizing said V_ΠH sequence or Nanobody® sequence in a manner known per se, such as by expression in a suitable host cell or host organism or by chemical synthesis. As mentioned herein, a particularly preferred class of Nanobodies® of the invention comprises Nanobodies® with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring V_HH domain, but that has been "humanized'^*, i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring V_HH sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a Vn domain from a conventional 4-chain antibody from a human being (e.g. indicated above), as further described on, and using the techniques mentioned on, page 63 of WO 08/020079. Another particularly preferred class of Nanobodies® of the invention comprises Nanobodies® with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring V_H domain, but that has been "camelized", i.e. by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring V_H domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a V_HH domain of a heavy chain antibody, as further described on, and using the techniques mentioned on, page 63 of WO 08/020079.

Other suitable methods and techniques for obtaining the Nanobodies® of the invention and/or nucleic acids encoding the same, starting from naturally occurring V_H sequences or preferably V_HH sequences, will be clear from the skilled person, and may for example include the techniques that are mentioned on page 64 of WO 08/00279As mentioned herein, Nanobodies® may in particular be characterized by the presence of one or more "Hallmark residues" (as described herein) in one or more of the framework sequences. Thus, according to particular, but non-limiting embodiments of the invention, a Nanobody® in its broadest sense can be generally defined as a polypeptide comprising: a) an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 108 according to the Kabat numbering is Q; and/or: b) an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 45 according to the Kabat numbering is a charged amino acid (as defined herein) or a cysteine residue, and position 44 is preferably an E: and/or: c) an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting of R and S.

Thus, in particular, but non-limiting embodiments, a Nanobody® of the invention may have the structure

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which a) the amino acid residue at position 108 according to the Kabat numbering is Q; and/or in which: b) the amino acid residue at position 45 according to the Kabat numbering is a charged amino acid or a cysteine and the amino acid residue at position 44 according to the

Kabat numbering is preferably E; and/or in which: c) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S. and is in particular chosen from the group consisting of R and S; and in which: d) CDRl. CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the embodiments herein.

In particular, a Nanobody® in its broadest sense can be generally defined as a polypeptide comprising: a) an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 108 according Io the Kabat numbering is Q: and/or: b) an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 44 according to the Kabat numbering is E and in which the amino acid residue at position 45 according to the Kabat numbering is an R; and/or: c) an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P. R and S. and is in particular chosen from the group consisting of R and S.

Thus, according to particular, but non-limiting embodiments, a Nanobody® of the invention may have the structure FRI - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which a) the amino acid residue at position 108 according to the Kabat numbering is Q; and/or in which: b) the amino acid residue at position 44 according to the Kabat numbering is E and in which the amino acid residue at position 45 according to the Kabat numbering is an R; and/or in which: c) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting of R and S; and in which: d) CDRL CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the embodiments herein.

In particular, a Nanobody® against sclerostin according to the invention may have the structure:

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer Io the complementarity determining regions 1 to 3, respectively, and in which a) the amino acid residue at position 108 according to the Kabat numbering is Q; and/or in which: b) the amino acid residue at position 44 according to the Kabat numbering is E and in which the amino acid residue at position 45 according to the Kabat numbering is an R; and/or in which: c) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S. and is in particular chosen from the group consisting of R and S; and in which: d) CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the embodiments herein.

In particular, but non-limiting embodiments of the invention, a Nanobody® can generally be defined as a polypeptide comprising an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which; a-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A, G. E, D, G₅ Q, R, S, L; and is preferably chosen from the group consisting of G, E or Q; and a-2) the amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L, R or C; and is preferably chosen from the group consisting of L or R; and a-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of W, R or S; and is preferably W or R, and is most preferably W; a-4) the amino acid residue at position 108 according to the Kabat numbering is Q: or in which: b-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of E and Q; and b-2) the amino acid residue al position 45 according to the Kabat numbering is R; and b-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of W. R and S; and is preferably W; b-4) the amino acid residue at position 108 according to the Kabat numbering is chosen from the group consisting of Q and L; and is preferably Q; or in which: c-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A. G. E, D, Q, R, S and L; and is preferably chosen from the group consisting of G, E and Q; and c-2) the amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L, R and C; and is preferably chosen from the group consisting of L and R; and c-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S; and is in particular chosen from the group consisting of R and S; and c-4) the amino acid residue at position 108 according to the Kabat numbering is chosen from the group consisting of Q and L; is preferably Q; and in which d) CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the embodiments herein. Thus, particular, but non- limiting embodiments, a Nanobody® of the invention may have the structure

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: a-1 ) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A, G. E. D. G, Q, R, S, L; and is preferably chosen from the group consisting of G, E or Q; and in which: a-2) the amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L, R or C; and is preferably chosen from the group consisting of L or R; and in which: a-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of W, R or S; and is preferably W or R, and is most preferably W; and in which a-4) the amino acid residue at position 108 according to the Kabat numbering is Q; and in which: d) CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein. In particular, but non-limiting embodiments. aNanobody® of the invention may have the structure

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: b-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of E and Q; and in which: b-2) the amino acid residue at position 45 according to the Kabat numbering is R; and in which: b-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of W, R and S; and is preferably W; and in which: b-4) the amino acid residue at position 108 according to the Kabat numbering is chosen from the group consisting of Q and L; and is preferably Q: and in which: d) CDRl . CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the embodiments herein.

In particular, but non-limiting embodiments, a Nanobody® of the invention may have the structure

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3_f respectively, and in which: c-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A, G. E, D, Q₅ R. S and L; and is preferably chosen from the group consisting of G, E and Q; and in which: c-T) the amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L₅ R and C; and is preferably chosen from the group consisting of L and R; and in which: c- 3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S; and is in particular chosen from the group consisting of R and S; and in which: c-4) the amino acid residue at position 108 according to the Kabat numbering is chosen from the group consisting of Q and L; is preferably Q; and in which: d) CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the embodiments herein. Two particularly preferred, but non-limiting groups of the Nanobodies® of the invention are those according to a) above: according to (a-1) to (a-4) above: according to b) above; according to (b-1) to (b-4) above; according to (c) above: and/or according to (c-1) to (c-4) above, in which either: i) the amino acid residues at positions 44-47 according to the Kabat numbering form the sequence GLEW (or a GLEW -like sequence as described herein) and the amino acid residue at position 108 is Q; or in which: ii) the amino acid residues at positions 43-46 according to the Kabat numbering form the sequence KERE or KQRE (or a KERE-like sequence as described) and the amino acid residue at position 108 is Q or L. and is preferably Q.

Thus, in another preferred, but non-limiting aspect, a Nanobody® of the invention may have the structure

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FRl to FR4 refer to framework regions 1 to 4. respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3. respectively, and in which: i) the amino acid residues at positions 44-47 according to the Kabat numbering form the sequence GLEW (or a GLEW-like sequence as defined herein) and the amino acid residue at position 108 is Q; and in which: ii) CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the embodiments herein.

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: i) the amino acid residues at positions 43-46 according to the Kabat numbering form the sequence KERE or KQRE (or a KERE -like sequence) and the amino acid residue at position 108 is Q or L, and is preferably Q; and in which: ii) CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the embodiments herein.

In the Nanobodies® of the invention in which the amino acid residues at positions 43- 46 according to the Kabat numbering form the sequence KERE or KQRE, the amino acid residue at position 37 is most preferably F. In the Nanobodies® of the invention in which the amino acid residues at positions 44-47 according to the Kabat numbering form the sequence GLEW. the amino acid residue at position 37 is chosen from the group consisting of Y, H, I, L, V or F, and is most preferably V.

Thus, without being limited hereto in any way, on the basis of the amino acid residues present on the positions mentioned above, the Nanobodies® of the invention can generally be classified on the basis of the following three groups: i) The "GLE W- group^*': Nanobodies® with the amino acid sequence GLEW at positions 44-47 according to the Kabat numbering and Q at position 108 according to the Kabat numbering. As further described herein, Nanobodies® within this group usually have a V at position 37, and can have a W, P, R or S at position 103, and preferably have a W at position 103. The GLEW group also comprises some GLEW-like sequences such as those mentioned in Table B-2 below. More generally, and without limitation, Nanobodies® belonging to the GLEW-group can be defined as Nanobodies® with a G at position 44 and/or with a W at position 47, in which position 46 is usually E and in which preferably position 45 is not a charged amino acid residue and not cysteine; ii) The "KERE-group^'': Nanobodies® with the amino acid sequence KERE or KQRE (or another KERE -like sequence) at positions 43-46 according to the Kabat numbering and Q or L at position 108 according to the Kabat numbering. As further described herein. Nanobodies© within this group usually have a F at position 37. an L or F at position 47; and can have a W. P, R or S at position 103, and preferably have a W at position 103. More generally, and without limitation. Nanobodies® belonging to the KERE-group caii be defined as Nanobodies® with a K. Q or R at position 44 (usually K) in which position 45 is a charged amino acid residue or cysteine, and position 47 is as further defined herein; iii) The "103 P, R, S-group": Nanobodies® with a P, R or S at position 103. These Nanobodies® can have either the amino acid sequence GLEW at positions 44-47 according to the Kabat numbering or the amino acid sequence KERE or KQRE at positions 43-46 according to the Kabat numbering, the latter most preferably in combination with an F at position 37 and an L or an F at position 47 (as defined for the

KERE-group); and can have Q or L at position 108 according to the Kabat numbering, and preferably have Q.

Also, where appropriate, Nanobodies® may belong to (i.e. have characteristics of) two or more of these classes. For example, one specifically preferred group of Nanobodies® has GLEW or a GLEW-like sequence at positions 44-47; P.R or S (and in particular R) at position 103; and Q at position 108 (which may be humanized to L).

More generally, it should be noted that the definitions referred to above describe and apply to Nanobodies® in the form of a native (i.e. non-humanized) V_HH sequence, and that humanized variants of these Nanobodies® may contain other amino acid residues than those indicated above (i.e. one or more humanizing substitutions as defined herein). For example, and without limitation, in some humanized Nanobodies® of the GLEW-group or the 103 P, R. S-group. Q at position 108 may be humanized to 108L. As already mentioned herein, other humanizing substitutions (and suitable combinations thereof) will become clear to the skilled person based on the disclosure herein. In addition, or alternatively, other potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring V_HΠ sequence with the corresponding framework sequence of one or more closely related human V_H sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said V_HΠ sequence (in any manner known per se, as further described herein) and the resulting humanized V_HH sequences can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person based on the disclosure herein. Also, based on the foregoing, (the framework regions of) a Nanobody® may be partially humanized or fully humanized.

Thus, particular, but non-limiting embodiments, a Nanobody® of the invention may be a Nanobody® belonging to the GLEW-group (as defined herein), and in which CDRl , CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

In particular, but non-limiting embodiments, a Nanobody® of the invention may be a Nanobody® belonging to the KERE-group (as defined herein), and CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

Thus, particular, but non-limiting embodiments, a Nanobody® of the invention may be a Nanobody® belonging to the 103 P, R. S-group (as defined herein), and in which CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

Also, more generally and in addition to the 108Q, 43E/44R and 103 P.R.S residues mentioned above, the Nanobodies® of the invention can contain, at one or more positions that in a conventional V_H domain would form (part of) the V_H/V_L interface, one or more amino acid residues that are more highly charged than the amino acid residues that naturally occur at the same position(s) in the corresponding naturally occurring V_H sequence, and in particular one or more charged amino acid residues (as mentioned in Table A -2 on page 48 of the International application WO 08/020079). Such substitutions include, but are not limited to. the GLEW-like sequences mentioned in Table B-2 below; as well as the substitutions that are described in the International Application WO 00/29004 for so-called "'microbodies", e.g. so as to obtain a Nanobody® with Q at position 108 in combination with KLEW at positions 44-47. Other possible substitutions at these positions will be clear to the skilled person based upon the disclosure herein.

In particular embodiments of the Nanobodies® of the invention, the amino acid residue at position 83 is chosen from the group consisting of L. M₅ S. V and W; and is preferably L. Also, in particular embodiments of the Nanobodies® of the invention, the amino acid residue at position 83 is chosen from the group consisting of R, K. N, E. G, L T and Q; and is most preferably either K or E (for Nanobodies® corresponding to naturally occurring V_HH domains) or R (for '"humanized^"' Nanobodies®, as described herein). The amino acid residue at position 84 is chosen from the group consisting of P. A, R, S₅ D T, and V in one aspect, and is most preferably P (for Nanobodies® corresponding to naturally occurring VHH domains) or R (for "humanized^"' Nanobodies®. as described herein).

Furthermore, in particular embodiments of the Nanobodies® of the invention, the amino acid residue at position 104 is chosen from the group consisting of G and D; and is most preferably G. Collectively, the amino acid residues at positions 11, 37. 44, 45. 47, 83, 84. 103, 104 and 108, which in the Nanobodies® are as mentioned above, will also be referred to herein as the "Hallmark Residues". The Hallmark Residues and the amino acid residues at the corresponding positions of the most closely related human V_H domain. V_H3, are summarized in Table B-2. Some especially preferred but non-limiting combinations of these Hallmark Residues as occur in naturally occurring VH_H domains are mentioned in Table B-3. For comparison, the corresponding amino acid residues of the human V_H3 called DP-47 have been indicated in italics.

In the Nanobodies®, each amino acid residue at any other position than the Hallmark Residues can be any amino acid residue that naturally occurs at the corresponding position (according to the Kabat numbering) of a naturally occurring V_HH domain.

Such amino acid residues will be clear to the skilled person. Tables B-4 to B-7 mention some non-limiting residues that can be present at each position (according to the Kabat numbering) of the FRl, FR2. FR3 and FR4 of naturally occurring V_HH domains. For each position, the amino acid residue that most frequently occurs at each position of a naturally occurring V_HH domain (and which is the most preferred amino acid residue for said position in a Nanobody®) is indicated in bold; and other preferred amino acid residues for each position have been underlined (note: the number of amino acid residues that are found at positions 26-30 of naturally occurring V_HH domains supports the hypothesis underlying the numbering by Chothia (supra) that the residues at these positions already form part of CDR1 ).

In Tables B-4 - B-7, some of the non-limiting residues that can be present at each position of a human V[p domain are also represented. Again, for each position, the amino acid residue that most frequently occurs at each position of a naturally occurring human V_H3 domain is indicated in bold; and other preferred amino acid residues have been underlined.

For reference only. Tables B-4-B-7 also contain data on the V_HH entropy C V_HH Ent ") and V_HH variability C"V_HH Var.^"1) at each amino acid position for a representative sample of 7732 V_HH sequences (including a.o. data kindly provided by David Lutje HuI sing and Prof. Theo Verrips of Utrecht University). The values for the V_HH entropy and the V_HH variability provide a measure for the variability and degree of conservation of amino acid residues between the 7732 V_HH sequences analyzed: low values (i.e. <] , such as < 0.5) indicate that an amino acid residue is highly conserved between the V_HH sequences (i.e. little variability). For example, the G at position 9 and the W at position 36 have values for the V_HH entropy of 0.01 and 0 respectively, indicating that these residues are highly conserved and have little variability (and in case of position 36 is W in all 7732 sequences analysed), whereas for residues that form part of the CDR' s generally values of 1.5 or more are found (data not shown). Note that the data represented below support the hypothesis that the amino acid residues at positions 27-30 and maybe even also at positions 93 and 94 already form part of the CDR^* s (although the invention is not limited to any specific hypothesis or explanation, and as mentioned above, herein the numbering according to Kabat is used). For a general explanation of sequence entropy, sequence variability and the methodology for determining the same, see Oliveira et aL PROTEINS: Structure, Function and Genetics, 52: 544-552 (2003).

Thus, in particular, but not limiting embodiments, a Nanobody® of the invention can be defined as an amino acid sequence with the (general) structure

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: i) one or more of the amino acid residues at positions 1 L 37. 44. 45. 47, 83. 84. 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table B -2; and in which: ii) CDRl . CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

The above Nanobodies® may for example be Vim sequences or may be humanized Nanobodies®. When the above Nanobody® sequences are VHH sequences, they may be suitably humanized, as further described herein. When the Nanobodies® are partially humanized Nanobodies®, they may optionally be further suitably humanized, again as described herein.

In particular, a Nanobody® of the invention can be an amino acid sequence with the (general) structure

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl Io CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: i) (preferably) one or more of the amino acid residues at positions 11, 37, 44. 45, 47, 83. 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table B-2 (it being understood that VΠH sequences will contain one or more Hallmark residues; and that partially humanized Nanobodies® will usually, and preferably, [still] contain one or more Hallmark residues [although it is also within the scope of the invention to provide - where suitable in accordance with the invention - partially humanized Nanobodies® in which all Hallmark residues, but not one or more of the other amino acid residues, have been humanized]; and that in fully humanized Nanobodies®, where suitable in accordance with the invention, all amino acid residues at the positions of the Hallmark residues will be amino acid residues that occur in a human V_H3 sequence. As will be clear to the skilled person based on the disclosure herein that such V_HH sequences, such partially humanized Nanobodies® with at least one Hallmark residue, such partially humanized Nanobodies® without Hallmark residues and such fully humanized Nanobodies® all form aspects of this invention): and in which: ii) said amino acid sequence has at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 1 to 22, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences (indicated with X in the sequences of SEQ ID NO^'s: 1 to 22) are disregarded; and in which: iii) CDRL CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

The above Nanobodies® may for example be V_HH sequences or may be humanized Nanobodies®. When the above Nanobody® sequences are V_{1 ΪH} sequences, they may be suitably humanized, as further described herein. When the Nanobodies® are partially humanized Nanobodies®, they may optionally be further suitably humanized, again as described herein.

In particular, a Nanobody® of the invention of the KERE group can be an amino acid sequence with the (general) structure

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4 in which: i) the amino acid residue at position 45 according to the Kabat numbering is a charged amino acid (as defined herein) or a cysteine residue, and position 44 is preferably an E; and in which: ii) FRl is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:

and in which: iii) FR2 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:

and in which: iv) FR3 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:

and in which: v) FR4 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following ammo acid sequences:

and in which: vi) CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

In the above Nanobodies®, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V_HH sequences or partially humanized Nanobodies®).

Also, the above Nanobodies® may for example be V_HU sequences or may be humanized Nanobodies®. When the above Nanobody® sequences are V_HH sequences, they may be suitably humanized, as further described herein. When the Nanobodies® are partially humanized Nanobodies®, they may optionally be further suitably humanized, again as described herein.

With regard to framework I₅ it will be clear to the skilled person that, when an amino acid sequence as outlined above is generated by expression of a nucleotide sequence, the first four amino acid sequences (i.e. amino acid residues 1-4 according to the Kabat numbering) may often be determined by the primer(s) that have been used to generate said nucleic acid. Thus, for determining the degree of amino acid identity, the first four amino acid residues are preferably disregarded.

Also, with regard to framework 1, and although amino acid positions 27 to 30 are according to the Kabat numbering considered to be part of the framework regions (and not the CDR' s). it has been found by analysis of a database of more than 1000 V_HH sequences that the positions 27 to 30 have a variability (expressed in terms of V_HH entropy and V_HH variability - see Tables B-4 to B-7) that is much greater than the variability on positions 1 to 26. Because of this, for determining the degree of amino acid identity, the amino acid residues at positions 27 to 30 are preferably also disregarded. In view of this, a Nanobody® of the KERE class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which: i) the amino acid residue at position 45 according to the Kabat numbering is a charged amino acid (as defined herein) or a cysteine residue, and position 44 is preferably an E; and in which: ii) FRl is an amino acid sequence that, on positions 5 to 26 of the Kabat numbering, has at least 80% amino acid identity with at least one of the following amino acid sequences:

and in which: iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4 of Nanobodies® of the KERE-class; and in which: iv) CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

The above Nanobodies® may for example be V_HH sequences or may be humanized Nanobodies®. When the above Nanobody® sequences are V_HH sequences, they may be suitably humanized, as further described herein. When the Nanobodies® are partially humanized Nanobodies®, they may optionally be further suitably humanized, again as described herein.

A Naiiobody® of the GLEW class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which i) preferably, when the Nanobody© of the GLEW-class is a non-humanized Nanobody®, the amino acid residue in position 108 is Q; ii) FRl is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:

and in which: v) FR.4 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:

and in which: vi) CDRl , CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein,

In the above Nanobodies®, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V_Hfi sequences or partially humanized Nanobodies®). With regard to framework 1, it will again be clear to the skilled person that, for determining the degree of amino acid identity, the amino acid residues on positions 1 to 4 and 27 to 30 are preferably disregarded.

In view of this, a Nanobody® of the GLEW class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which: i) preferably, when the Nanobody® of the GLEW-class is a non-humanized Nanobody®, the amino acid residue in position 108 is Q: and in which: ii) FRl is an amino acid sequence that, on positions 5 to 26 of the Kabat numbering, has at least 80% amino acid identity with at least one of the following amino acid sequences:

and in which: iii) FR2. FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4 of Nanobodies© of the GLEW-class; and in which: iv) CDRi, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

The above Nanobodies® may for example be V_HH sequences or may be humanized Nanobodies®. When the above Nanobody® sequences are V_HH sequences, they may be suitably humanized, as further described herein. When the Nanobodies® are partially humanized Nanobodies®, they may optionally be further suitably humanized, again as described herein. In the above Nanobodies®, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V_HH sequences or partially humanized Nanobodies®). A Nanobody® of the P, R, S 103 class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which i) the amino acid residue at position ] 03 according to the Kabat numbering is different from W; and in which: ii) preferably the amino acid residue at position 103 according to the Kabat numbering is

P, R or S, and more preferably R; and in which: iii) FRl is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:

and in which iv) FR2 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:

and in which: v) FR3 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:

and in which: vi) FR4 is an amino acid sequence that has at least 80% amino acid identity with at least- one of the following amino acid sequences:

and in which: vii) CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

In the above Nanobodies®, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V_HH sequences or partially humanized Nanobodies®). With regard to framework 1, it will again be clear to the skilled person that, for determining the degree of amino acid identity, the amino acid residues on positions 1 to 4 and 27 to 30 are preferably disregarded.

In view of this, aNanobody® of the P₃R₅S 103 class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which: i) the amino acid residue at position 103 according to the Kabat numbering is different from W; and in which: ii) preferably the amino acid residue at position 103 according to the Kabat numbering is P, R or S, and more preferably R; and in which: iii) FRl is an amino acid sequence that, on positions 5 to 26 of the Kabat numbering, has at least 80% amino acid identity with at least one of the following amino acid sequences:

and in which: iv) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4 of Nanobodies® of the P,R,S 103 class; and in which: v) CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

In the above Nanobodies®, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V_HH sequences or partially humanized Nanobodies®). in particular, but non-limiting embodiments, the invention relates to a Nanobody® as described above, in which the CDR sequences have at least 70% amino acid identity. preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 189 to 197 (see Table A-I). This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said Nanobody® and one or more of the sequences of SEQ ID NO's: 189 to 197 (see Table A-I), in which the amino acid residues that form the framework regions are disregarded. Such Nanobodies® can be as further described herein.

As already mentioned herein, preferred but non-limiting embodiments of the invention relate to a Nanobody® with an amino acid sequence that is chosen from the group consisting of SEQ ID NO's: 189 to 197 (see Table A-I) or from the group consisting of from amino acid sequences that have more than 80%, preferably more than 90%, more preferably more than

95%, such as 99% or more sequence identity (as defined herein) with at least one of the amino acid sequences of SEQ ID NO^'s: 189 to 197 (see Table A-I). Also, in the above Nanobodies®: i) any amino acid substitution (when it is not a humanizing substitution as defined herein) is preferably, and compared to the corresponding amino acid sequence of SEQ ID NO^'s: 189 to 197 (see Table A-I). a conservative amino acid substitution, (as defined herein); and/or: ii) its amino acid sequence preferably contains either only amino acid substitutions, or otherwise preferably no more than 5, preferably no more than 3, and more preferably only 1 or 2 amino acid deletions or insertions, compared to the corresponding amino acid sequence of SEQ ID NO's: 189 to 197 (see Table A-I); and/or iii) the CDR's may be CDR's that are derived by means of affinity maturation, for example starting from the CDR^"s of to the corresponding amino acid sequence of SEQ ID NO's: 189 to 197 (see Table A-I). Preferably, the CDR sequences and FR sequences in the Nanobodies® of the invention are such that the Nanobodies® of the invention (and polypeptides of the invention comprising the same): bind to sclerostin with a dissociation constant (K_D) of 10^"5 to 10^"1" moles/liter or less, and preferably 10^" to 10^" moles/liter or less and more preferably 10^" Io 10^" moles/liter (i.e. with an association constant (K_A) of IO³ to 10ⁱ² liter/ moles or more, and preferably 10⁷ to 10¹² liter/moles or more and more preferably 10⁸ to 10¹² liter/moles); and/or such that they: bind to sclerostin with a k_On-rate of between 10² M^-1S^"1 to about 10⁷ M^-1S^"1, preferably between 10³ M^-1S^"1 and 10⁷ JVf¹S^"1. more preferably between 10⁴ Vf¹S^"1 and 10⁷ M^-1S^"1, such as between IO⁵ M⁴S^"1 and 10⁷ M^"!s⁴: and/or such that they: - bind to sclerostin with a k_off rate between 1 s^"1 (ti_/i=0.69 s) and 10^"6 s^'1 (providing a near irreversible complex with a i_\n of multiple days), preferably between 10^"2 s^"J and 10^"6 s^" \ more preferably between lO^"3 s^"1 and 10^"6 s^"1. such as between 10^"4 s^"] and 10^"6 s^"\ Preferably. CDR sequences and FR sequences present in the Nanobodies® of the invention are such that the Nanobodies® of the invention will bind to sclerostin with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 iiM, such as less than 500 pM.

According to particular embodiments of the invention, a Nanobody® may be as defined herein, but with the proviso that it has at least "one amino acid difference" (as defined herein) in at least one of the framework regions compared to the corresponding framework region of a naturally occurring human V_H domain, and in particular compared to the corresponding framework region of DP -47. More specifically, according to one non-limiting aspect of the invention, a Nanobody® may be as defined herein, but with the proviso that it has at least "one amino acid difference" (as defined herein) at at least one of the Hallmark residues (including those at positions 108, 103 and/or 45) compared to the corresponding framework region of a naturally occurring human V_H domain, and in particular compared to the corresponding framework region of DP-47. Usually, a Nanobody® will have at least one such amino acid difference with a naturally occurring V_H domain in at least one of FR2 and/or FR4, and in particular at at least one of the Hallmark residues in FR2 and/or FR4 (again, including those at positions 108, 103 and/or 45).

Also, a humanized Nanobody® of the invention may be as defined herein, but with the proviso that it has at least "one amino acid difference" (as defined herein) in at least one of the framework regions compared to the corresponding framework region of a naturally occurring V_HH domain. More specifically, according to one non-limiting aspect of the invention, a humanized Nanobody® may be as defined herein, but with the proviso that it has at least "one amino acid difference" (as defined herein) at at least one of the Hallmark residues (including those at positions 108, 103 and/or 45) compared to the corresponding framework region of a naturally occurring V_HH domain. Usually, a humanized Nanobody© will have at least one such amino acid difference with a naturally occurring V_HH domain in at least one of FR2 and/or FR4, and in particular at at least one of the Hallmark residues in FR2 and/or FR4 (again, including those at positions 108, 103 and/or 45).

As will be clear from the disclosure herein, it is also within the scope of the invention to use natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs (herein collectively referred to as "analogs^''^') of the Nanobodies® of the invention as defined herein, and in particular analogs of the Nanobodies® of SEQ ID NO's 189 to 197 (see Table A-I). Thus, according to particular embodiments of the invention, the term "Nanobody® of the invention" in its broadest sense also covers such analogs.

Generally, in such analogs, one or more amino acid residues may have been replaced, deleted and/or added, compared to the Nanobodies® of the invention as defined herein. Such substitutions, insertions or deletions may be made in one or more of the framework regions and/or in one or more of the CDR's. When such substitutions, insertions or deletions are made in one or more of the framework regions, they may be made at one or more of the Hallmark residues and/or at one or more of the other positions in the framework residues, although substitutions, insertions or deletions at the Hallmark residues are generally less preferred (unless these are suitable humanizing substitutions as described herein).

By means of non-limiting examples, a substitution may for example be a conservative substitution (as described herein) and/or an amino acid residue may be replaced by another amino acid residue that naturally occurs at the same position in another V_HH domain (see Tables B-4 to B-7 for some non-limiting examples of such substitutions), although the invention is generally not limited thereto. Thus, any one or more substitutions, deletions or insertions, or any combination thereof, that either improve the properties of the Nanobody® of the invention or that at least do not detract too much from the desired properties or from the balance or combination of desired properties of the Nanobody® of the invention (i.e. to the extent that the Nanobody® is no longer suited for its intended use) are included within the scope of the invention. A skilled person will generally be able to determine and select suitable substitutions, deletions or insertions, or suitable combinations of thereof, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible substitutions and determining their influence on the properties of the Nanobodies® thus obtained.

For example, and depending on the host organism used to express the Nanobody® or polypeptide of the invention, such deletions and/or substitutions may be designed in such a way that one or more sites for post-translational modification (such as one or more glycosylation sites) are removed, as will be within the ability of the person skilled in the art. Alternatively, substitutions or insertions may be designed so as to introduce one or more sites for attachment of functional groups (as described herein), for example to allow site-specific pegylation (again as described herein).

As can be seen from the data on the V_HH entropy and V_HH variability given in Tables B-4 to B-7 above, some amino acid residues in the framework regions are more conserved than others. Generally, although the invention in its broadest sense is not limited thereto, any substitutions, deletions or insertions are preferably made at positions that are less conserved. Also, generally, amino acid substitutions are preferred over amino acid deletions or insertions.

The analogs are preferably such that they can bind to sclerostin with an affinity (suitably measured and/or expressed as a Ko-value (actual or apparent), a KA-value (actual or apparent), a k_on-rate and/or a k_Off-rate, or alternatively as an IC₅0 value, as further described herein) that is as defined herein for the Nanobodies® of the invention.

The analogs are preferably also such that they retain the favourable properties the Nanobodies®, as described herein.

Also, according to particular embodiments, the analogs have a degree of sequence identity of at least 70%. preferably at least 80%, more preferably at least 90%. such as at least 95% or 99% or more: and/or preferably have at most 20, preferably at most 10. even more preferably at most 5, such as 4, 3. 2 or only 1 amino acid difference (as defined herein), with one of the Nanobodies® of SEQ ID NOs: 189 to 197 (see Table A-I). Also, the framework sequences and CDR' s of the analogs are preferably such that they are in accordance with the preferred embodiments defined herein. More generally, as described herein, the analogs will have (a) a Q at position 108; and/or (b) a charged amino acid or a cysteine residue at position 45 and preferably an E at position 44, and more preferably E at position 44 and R at position 45; and/or (c) P. R or S at position 103.

One preferred class of analogs of the Nanobodies® of the invention comprise Nanobodies® that have been humanized (i.e. compared to the sequence of a naturally occurring Nanobody® of the invention). As mentioned in the background ait cited herein, such humanization generally involves replacing one or more amino acid residues in the sequence of a naturally occurring V_HH with the amino acid residues that occur at the same position in a human Vu domain, such as a human V_H3 domain. Examples of possible humanizing substitutions or combinations of humanizing substitutions will be clear to the skilled person, for example from the Tables herein, from the possible humanizing substitutions mentioned in the background art cited herein, and/or from a comparison between the sequence of a Nanobody® and the sequence of a naturally occurring human V_H domain.

The humanizing substitutions should be chosen such that the resulting humanized Nanobodies® still retain the favourable properties of Nanobodies® as defined herein, and more preferably such that they are as described for analogs in the preceding paragraphs. A skilled person will generally be able to determine and select suitable humanizing substitutions or suitable combinations of humanizing substitutions, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible humanizing substitutions and determining their influence on the properties of the Nanobodies® thus obtained.

Generally, as a result of humanization, the Nanobodies® of the invention may become more "human-like'^', while still retaining the favorable properties of the Nanobodies® of the invention as described herein. As a result, such humanized Nanobodies® may have several advantages, such as a reduced immunogenicity, compared to the corresponding naturally occurring V_HN domains. Again, based on the disclosure herein and optionally after a limited degree of routine experimentation, the skilled person will be able to select humanizing substitutions or suitable combinations of humanizing substitutions which optimize or achieve a desired or suitable balance between the favourable properties provided by the humanizing substitutions on the one hand and the favourable properties of naturally occurring V_HH domains on the other hand.

The Nanobodies® of the invention may be suitably humanized at any framework residue(s), such as at one or more Hallmark residues (as defined herein) or at one or more other framework residues (i.e. non-Hallmark residues) or any suitable combination thereof. One preferred humanizing substitution for Nanobodies® of the ^uP,R.S-103 group" or the '"KERE group" is Ql 08 into Ll 08. Nanobodies© of the "GLEW class'^' may also be humanized by a Q 108 into L 108 substitution, provided at least one of the other Hallmark residues contains a camelid (camelizing) substitution (as defined herein). For example, as mentioned above, one particularly preferred class of humanized Nanobodies® has GLEW or a GLEW-like sequence at positions 44-47; P, R or S (and in particular R) at position 103, and an L at position 108.

The humanized and other analogs, and nucleic acid sequences encoding the same, can be provided in any manner known per se, for example using one or more of the techniques mentioned on pages 103 and 104 of WO 08/020079.

As mentioned there, it will be also be clear to the skilled person that the Nanobodies® of the invention (including their analogs) can be designed and/or prepared starting from human V_H sequences (i.e. amino acid sequences or the corresponding nucleotide sequences), such as for example from human V_H3 sequences such as DP-47. DP-51 or DP-29. i.e. by introducing one or more camelizing substitutions (i.e. changing one or more amino acid residues in the amino acid sequence of said human V_H domain into the amino acid residues that occur at the corresponding position in a V_HH domain), so as to provide the sequence of a Nanobody® of the invention and/or so as to confer the favourable properties of a Nanobody® to the sequence thus obtained. Again, this can generally be performed using the various methods and techniques referred to in the previous paragraph, using an amino acid sequence and/or nucleotide sequence for a human V_H domain as a starting point.

Some preferred, but non-limiting camelizing substitutions can be derived from Tables B-4 - B-7. It will also be clear that camelizing substitutions at one or more of the Hallmark residues will generally have a greater influence on the desired properties than substitutions at one or more of the other amino acid positions, although both and any suitable combination thereof are included within the scope of the invention. For example, it is possible to introduce one or more camelizing substitutions that already confer at least some the desired properties, and then to introduce further camelizing substitutions that either further improve said properties and/or confer additional favourable properties. Again, the skilled person will generally be able to determine and select suitable camelizing substitutions or suitable combinations of camelizing substitutions, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible camelizing substitutions and determining whether the favourable properties of Nanobodies® are obtained or improved (i.e. compared to the original VH domain).

Generally, however, such camelizing substitutions are preferably such that the resulting an amino acid sequence at least contains (a) a Q at position 108: and/or (b) a charged amino acid or a cysteine residue at position 45 and preferably also an E at position 44, and more preferably E at position 44 and R at position 45; and/or (c) P, R or S at position 103; and optionally one or more further camelizing substitutions. More preferably, the camelizing substitutions are such that they result in a Nanobody® of the invention and/or in an analog thereof (as defined herein), such as in a humanized analog and/or preferably in an analog that is as defined in the preceding paragraphs.

Nanobodies® can also be derived from V₁₁ domains by the incorporation of substitutions that are rare in nature, but nonetheless, structurally compatible with the VH domain fold. For example, but without being limiting, these substitutions may include on or more of the following: GIy at position 35, Ser, VaI or Thr at position 37, Ser, Thr. Arg. Lys, His, Asp or GIu at position 39, GIu or His at position 45, Trp, Leu, VaI. Ala, Thr, or GIu at position 47, S or R at position 50. (Barthelemy et al. J Bio] Chem. 2008 Feb 8;283(6):3639- 54. Epub 2007 Nov 28)

As will also be clear from the disclosure herein, it is also within the scope of the invention to use parts or fragments, or combinations of two or more parts or fragments, of the Nanobodies® of the invention as defined herein, and in particular parts or fragments of the Nanobodies® of SEQ ID NO's: 189 to 197 (see Table A-I ). Thus, according to particular embodiments of the invention, the term "Nanobody® of the invention" in its broadest sense also covers such parts or fragments. Generally, such parts or fragments of the Nanobodies® of the invention (including analogs thereof) have amino acid sequences in which, compared to the amino acid sequence of the corresponding full length Nanobody® of the invention (or analog thereof), one or more of the amino acid residues at the N-teπninal end, one or more amino acid residues at the C- terminal end, one or more contiguous internal amino acid residues, or any combination thereof, have been deleted and/or removed.

The parts or fragments are preferably such that they can bind to sclerostin with an affinity (suitably measured and/or expressed as a Ko-value (actual or apparent), a K__Λ-value (actual or apparent), a k_on-rate and/or a k_ofr-rate₅ or alternatively as an IC₅₀ value, as further described herein) that is as defined herein for the Nanobodies® of the invention.

Any part or fragment is preferably such that it comprises at least 10 contiguous amino acid residues, preferably at least 20 contiguous amino acid residues, more preferably at least 30 contiguous amino acid residues, such as at least 40 contiguous amino acid residues, of the amino acid sequence of the corresponding full length Nanobody® of the invention.

Also, any part or fragment is such preferably that it comprises at least one of CDRl. CDR2 and/or CDR3 or at least part thereof (and in particular at least CDR3 or at least part thereof). More preferably, any part or fragment is such that it comprises at least one of the CDR^" s (and preferably at least CDR3 or part thereof) and at least one other CDR (i.e. CDRl or CDR2) or at least part thereof, preferably connected by suitable framework sequence(s) or at least part thereof. More preferably, any part or fragment is such that it comprises at least one of the CDR^" s (and preferably at least CDR3 or part thereof) and at least part of the two remaining CDR" s, again preferably connected by suitable framework sequence(s) or at least part thereof.

According to particular, but non-limiting embodiments, such a part or fragment comprises at least CDR3, such as FR3. CDR3 and FR4 of the corresponding full length Nanobody® of the invention, i.e. as for example described in the International application WO 03/050531 (Lasters et al).

As already mentioned above, it is also possible to combine two or more of such parts or fragments (i.e. from the same or different Nanobodies® of the invention), i.e. to provide an analog (as defined herein) and/or to proλ'ide further parts or fragments (as defined herein) of a Nanobody® of the invention. It is for example also possible to combine one or more parts or fragments of a Nanobody® of the invention with one or more parts or fragments of a human Vu domain. According to particular embodiments, the parts or fragments have a degree of sequence identity of at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, such as at least 90%, 95% or 99% or more with one of the Nanobodies® of SEQ ID NOs 189 to 197 (see Table A-I).

The parts and fragments, and nucleic acid sequences encoding the same, can be provided and optionalry combined in any manner known per se. For example, such parts or fragments can be obtained by inserting a stop codon in a nucleic acid that encodes a full-sized Nanobody© of the invention, and then expressing the nucleic acid thus obtained in a manner known per se (e.g. as described herein). Alternatively, nucleic acids encoding such parts or fragments can be obtained by suitably restricting a nucleic acid that encodes a full-sized

Nanobody® of the invention or by synthesizing such a nucleic acid in a manner known per se. Parts or fragments may also be provided using techniques for peptide synthesis known per se

The invention in its broadest sense also comprises derivatives of the Nanobodies® of the invention. Such derivatives can generally be obtained by modification, and in particular by chemical and/or biological (e.g. enzymatical) modification, of the Nanobodies® of the invention and/or of one or more of the amino acid residues that form the Nanobodies© of the invention.

Examples of such modifications, as well as examples of amino acid residues within the Nanobody® sequence that can be modified in such a manner (i.e. either on the protein backbone but preferably on a side chain), methods and techniques that can be used to introduce such modifications and the potential uses and advantages of such modifications will be clear to the skilled person.

For example, such a modification may involve the introduction (e.g. by covalent linking or in an other suitable manner) of one or more functional groups, residues or moieties into or onto the Nanobody® of the invention, and in particular of one or more functional groups, residues or moieties that confer one or more desired properties or functionalities to the Nanobody® of the invention. Example of such functional groups will be clear to the skilled person.

For example, such modification may comprise the introduction (e.g. by covalent binding or in any other suitable manner) of one or more functional groups that increase the half-life, the solubility and/or the absorption of the Nanobody® of the invention, that reduce the immunogenicily and/or the toxicity of the Nanobody® of the invention, that eliminate or attenuate any undesirable side effects of the Nanobody® of the invention, and/or that confer other advantageous properties to and/or reduce the undesired properties of the Nanobodies® and/or polypeptides of the invention; or any combination of two or more of the foregoing. Examples of such functional groups and of techniques for introducing them will be clear to the skilled person, and can generally comprise all functional groups and techniques mentioned in the general background art cited hereinabove as well as the functional groups and techniques known per se for the modification of pharmaceutical proteins, and in particular for the modification of antibodies or antibody fragments (including ScFv' s and single domain antibodies), for which reference is for example made to Remington's Pharmaceutical Sciences, 16th ecL Mack Publishing Co., Eastern, PA (1980). Such functional groups may for example be linked directly (for example covalently) to a Nanobody© of the invention, or optionally via a suitable linker or spacer, as will again be clear to the skilled person.

One of the most widely used techniques for increasing the half-life and/or reducing the immunogenicity of pharmaceutical proteins comprises attachment of a suitable pharmacologically acceptable polymer, such as poly(ethyleneglycol) (PEG) or derivatives thereof (such as methoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form of pegylation can be used, such as the pegylation used in the art for antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv's); reference is made to for example Chapman. Nat. BiotechnoL 54, 531 -545 (2002): by Veronese and Harris. Adv. Drug Deliv. Rev. 54, 453-456 (2003). by Harris and Chess, Nat. Rev. Drug. Discov.. 2. (2003) and in WO 04/060965. Various reagents for pegylation of proteins are also commercially available, for example from Nektar Therapeutics. USA.

Preferably, site-directed pegylation is used, in particular via a cysteine -residue (see for example Yang et al.. Protein Engineering, 16, 10. 761-770 (2003). For example, for this purpose. PEG may be attached to a cysteine residue that naturally occurs in a Nanobody® of the invention, a Nanobody© of the invention may be modified so as to suitably introduce one or more cysteine residues for attachment of PEG, or an amino acid sequence comprising one or more cysteine residues for attachment of PEG may be fused to the N- and/or C-terminus of a Nanobody® of the invention, all using techniques of protein engineering known per se to the skilled person.

Preferably, for the Nanobodies® and proteins of the invention, a PEG is used with a molecular weight of more than 5000, such as more man 10,000 and less than 200,000, such as less than 100.000; for example in the range of 20.000-80.000.

Another, usually less preferred modification comprises N- linked or O-linked glycosylation, usually as part of co-translational and/or post-translational modification, depending on the host eel! used for expressing the Nanobody® or polypeptide of the invention.

Yet another modification may comprise the introduction of one or more detectable labels or other signal-generating groups or moieties, depending on the intended use of the labelled Nanobody®. Suitable labels and techniques for attaching, using and detecting them will be clear to the skilled person, and for example include, but are not limited to, the fluorescent labels, phosphorescent labels, chemiluminescent labels, bioluminescent labeis, radio-isotopes, metals, metal chelates, metallic cations, chromophores and enzymes, such as those mentioned on page 109 of WO 08/020079. Other suitable labels will be clear to the skilled person, and for example include moieties that can be detected using NMR or ESR spectroscopy.

Such labelled Nanobodies® and polypeptides of the invention may for example be used for in vitro, in vivo or in situ assays (including immunoassays known per se such as ELISA. RIA, EIA and other "'sandwich assays'^", etc.) as well as in vivo diagnostic and imaging purposes, depending on the choice of the specific label.

As will be clear to the skilled person, another modification may involve the introduction of a chelating group, for example to chelate one of the metals or metallic cations referred to above. Suitable chelating groups for example include, without limitation, diethyl- enetriaminepentaacetic acid (DTPA) or ethyl enediaminetetraacetic acid (EDTA). Yet another modification may comprise the introduction of a functional group that is one part of a specific binding pair, such as the biotin-( strep t)avidin binding pair. Such a functional group may be used to link the Nanobody® of the invention to another protein, polypeptide or chemical compound that is bound to the other half of the binding pair, i.e. through formation of the binding pair. For example, a Nanobody® of the invention may be conjugated to biotin, and linked to another protein, polypeptide, compound or carrier conjugated to avidin or streptavidin. For example, such a conjugated Nanobody® may be used as a reporter, for example in a diagnostic system where a detectable signal-producing agent is conjugated to avidin or streptavidin. Such binding pairs may for example also be used to bind the Nanobody® of the invention to a carrier, including carriers suitable for pharmaceutical purposes. One non-limiting example are the liposomal formulations described by Cao and Suresh, Journal of Drug Targetting, 8, 4, 257 (2000). Such binding pairs may also be used to link a therapeutically active agent to the Nanobody® of the invention.

For some applications, in particular for those applications in which it is intended to kill a cell that expresses the target against which the Nanobodies® of the invention are directed (e.g. in the treatment of cancer), or to reduce or slow the growth and/or proliferation such a cell, the Nanobodies® of the invention may also be linked to a toxin or to a toxic residue or moiety. Examples of toxic moieties, compounds or residues which can be linked to a Nanobody® of the invention to provide - for example - a cytotoxic compound will be clear to the skilled person and can for example be found in the prior art cited above and/or in the further description herein. One example is the so-called ADEPT™ technology described in WO 03/055527.

Other potential chemical and enzymatical modifications will be clear to the skilled person. Such modifications may also be introduced for research purposes (e.g. to study function-activity relationships). Reference is for example made to Lundblad and Bradshaw, Biotechnol. Appl. Biochem., 26, 143-151 (1997).

Preferably, the derivatives are such that they bind to sclerostin with an affinity (suitably measured and/or expressed as a Ko-value (actual or apparent), a K_A-value (actual or apparent), a k_on-rate and/or a k_off-rate, or alternatively as an IC_5O value, as further described herein) that is as defined herein for the Nanobodies® of the invention.

As mentioned above, the invention also relates to proteins or polypeptides that essentially consist of or comprise at least one Nanobody® of the invention. By "essentially consist of is meant that the amino acid sequence of the polypeptide of the invention either is exactly the same as the amino acid sequence of a Nanobody® of the invention or corresponds to the amino acid sequence of a Nanobody® of the invention which has a limited number of amino acid residues, such as 1 -20 amino acid residues, for example 1-10 amino acid residues and preferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, added at the amino terminal end, at the carboxy terminal end, or at both the amino terminal end and the carboxy terminal end of the amino acid sequence of the Nanobody®.

Said amino acid residues may or may not change, alter or otherwise influence the (biological) properties of the Nanobody® and may or may not add further functionality to the Nanobody®. For example, such amino acid residues: can comprise an N -terminal Met residue, for example as result of expression in a heterologous host cell or host organism. - may form a signal sequence or leader sequence that directs secretion of the Nanobody® from a host eel] upon synthesis. Suitable secretory leader peptides will be clear to the skilled person, and may be as further described herein. Usually, such a leader sequence will be linked to the N-temvinus of the Nanobody®, although the invention in its broadest sense is not limited thereto; may form a sequence or signal that allows the Nanobody® to be directed towards and/or to penetrate or enter into specific organs, tissues, cells, or parts or compartments of cells, and/or that allows the Nanobody® to penetrate or cross a biological barrier such as a cell membrane, a cell layer such as a layer of epithelial cells, a tumor including solid tumors, or the bϊood-brain-barrier. Examples of such amino acid sequences will be clear to the skilled person and include those mentioned in paragraph c) on page 112 of WO 08/020079. may form a "tag", for example an amino acid sequence or residue that allows or facilitates the purification of the Nanobody®, for example using affinity techniques directed against said sequence or residue. Thereafter, said sequence or residue may be removed (e.g. by chemical or enzymatica] cleavage) to provide the Nanobody® sequence (for this purpose, the tag may optionally be linked to the Nanobody® sequence via a cleavable linker sequence or contain a cleavable motif). Some preferred, but non-limiting examples of such, residues are multiple histidine residues, glutatione residues and a myc-tag (see for example SEQ ID NO: 31 of WO 06/12282). may be one or more amino acid residues that have been functionalized and/or that can serve as a site for attachment of functional groups. Suitable amino acid residues and functional groups will be clear to the skilled person and include, but are not limited to, the amino acid residues and functional groups mentioned herein for the derivatives of the Nanobodies® of the invention.

According to particular embodiments, a polypeptide of the invention comprises a Nanobody® of the invention, which is fused at its amino terminal end, at its carboxy terminal end, or both at its amino terminal end and at its carboxy terminal end to at least one further amino acid sequence, i.e. so as to provide a fusion protein comprising said Nanobody® of the invention and the one or more further amino acid sequences. Such a fusion will also be referred to herein as a "Nanobody® fusion".

The one or more further amino acid sequence may be any suitable and/or desired amino acid sequences. The further amino acid sequences may or may not change, alter or otherwise influence the (biological) properties of the Nanobody®. and may or may not add further functionality to the Nanobody® or the polypeptide of the invention. Preferably, the further amino acid sequence is such that it confers one or more desired properties or functionalities to the Nanobody® or the polypeptide of the invention.

For example, the further amino acid sequence may also provide a second binding site, which binding site may be directed against any desired protein, polypeptide, antigen, antigenic determinant or epitope (including but not limited to the same protein, polypeptide, antigen, antigenic determinant or epitope against which the Nanobody® of the invention is directed, or a different protein, polypeptide, antigen, antigenic determinant or epitope).

Example of such amino acid sequences will be clear to the skilled person, and may generally comprise all amino acid sequences that are used in peptide fusions based on conventional antibodies and fragments thereof (including but not limited to ScFv's and single domain antibodies). Reference is for example made to the review by Holliger and Hudson, Nature Biotechnology, 23, 9, 1126-1136 (2005).

For example, such an amino acid sequence may be an amino acid sequence that increases the half-life, the solubility, or the absorption, reduces the immunogenicity or the toxicity, eliminates or attenuates undesirable side effects, and/or confers other advantageous properties to and/or reduces the undesired properties of the polypeptides of the invention, compared to the Nanobody® of the invention per se. Some non-limiting examples of such amino acid sequences are serum proteins, such as human serum albumin (see for example WO 00/27435) or haptenic molecules (for example haptens that are recognized by circulating antibodies, see for example WO 98/22141 ).

In particular, it has been described in the art that linking fragments of immunoglobulins (such as V_H domains) to serum albumin or to fragments thereof can be used to increase the half-life. Reference is for made to WO 00/27435 and WO 01/077137). According to the invention, the Nanobody® of the invention is preferably either directly linked to serum albumin (or to a suitable fragment thereof) or via a suitable linker, and in particular via a suitable peptide linked so that the polypeptide of the invention can be expressed as a genetic fusion (protein). According to one specific aspect, the Nanobody® of the invention may be linked to a fragment of serum albumin that at least comprises the domain III of serum albumin or part thereof. Reference is for example made to WO 07/1 12940 of Ablynx N.V.

Alternatively, the further amino acid sequence may provide a second binding site or binding unit that is directed against a serum protein (such as, for example, human serum albumin or another serum protein such as IgG), so as to provide increased half-life in serum. Such amino acid sequences for example include the Nanobodies® described below, as well as the small peptides and binding proteins described in WO 91/01743, WO 01/45746 and WO 02/076489 and the dAb's described in WO 03/002609 and WO 04/003019. Reference is also made to Harmsen et al, Vaccine, 23 (41); 4926-42, 2005, as well as to EP 0 368 684, as well as to WO 08/028977, WO 08/043821, WO 08/043822 by Ablynx N.V. and US provisional application of Ablynx N.V. entitled "Peptides capable of binding to serum proteins" filed on December 5, 2006 (see also PCT/EP2007/063348).

Such amino acid sequences may in particular be directed against serum albumin (and more in particular human serum albumin) and/or against IgG (and more in particular human IgG). For example, such amino acid sequences may be amino acid sequences that are directed against (human) serum albumin and amino acid sequences that can bind to amino acid residues on (human) serum albumin that are not involved in binding of serum albumin to FcRn (see for example WO 06/0122787) and/or amino acid sequences that are capable of binding to amino acid residues on serum albumin that do not form part of domain III of serum albumin (see again for example WO 06/0122787); amino acid sequences that have or can provide an increased half-life (see for example WO 08/028977 by Ablynx N. V.); amino acid sequences against human serum albumin that are cross-reactive with serum albumin from at least one species of mammal, and in particular with at least one species of primate (such as, without limitation, monkeys from the genus Macaco, (such as, and in particular, cynomologus monkeys (Macaco fascicularis) and/or rhesus monkeys (Macaca mulatto)) and baboon (Papio vr sinus), reference is again made to WO 08/028977; amino acid sequences that can bind to serum albumin in a pH independent manner (see for example WO 08/043821by Ablynx N.V. entitled "Amino acid sequences that bind to serum proteins in a manner that is essentially independent of the pH, compounds comprising the same, and uses thereof") and/or amino acid sequences that are conditional binders (see for example WO 08/043822 by Ablynx N.V. entitled "Amino acid sequences thai bind to a desired molecule in a conditional manner").

According to particular embodiments, the one or more further amino acid sequences may comprise one or more parts, fragments or domains of conventional 4-chain antibodies (and in particular human antibodies) and/or of heavy chain antibodies. For example, although usually less preferred, a Nanobody® of the invention may be linked to a conventional (preferably human) V_H or V_L domain or to a natural or synthetic analog of a V_H or V₁, domain, again optionally via a linker sequence (including but not limited to other (single) domain antibodies, such as the dAb's described by Ward et al.).

The at least one Nanobody® may also be linked to one or more (preferably human) CH U C_H2 and/or C_H3 domains, optionally via a linker sequence. For instance, a Nanobody® linked to a suitable C_H I domain could for example be used - together with suitable light chains - to generate antibody fragments/structures analogous to conventional Fab fragments or F(ab')₂ fragments, but in which one or (in case of an F(ab')₂ fragment) one or both of the conventional V_H domains have been replaced by a Nanobody® of the invention. Also, two Nanobodies® could be linked to a C_H3 domain (optionally via a linker) to provide a construct with increased half-life in vivo.

According Io particular embodiments of the polypeptides of the invention, one or more Nanobodies® of the invention may be linked (optionally via a suitable linker or hinge region) to one or more constant domains (for example, 2 or 3 constant domains that can be used as pail of/to form an Fc portion), to an Fc portion and/or to one or more antibody parts, fragments or domains that confer one or more effector functions to the polypeptide of the invention and/or maj confer the ability to bind to one or more Fc receptors. For example, for this purpose, and without being limited thereto, the one or more further amino acid sequences may comprise one or more C_H2 and/or Qp domains of an antibody, such as from a heavy chain antibody (as described herein) and more preferably from a conventional human 4 -chain antibody; and/or may form (part of) and Fc region, for example from IgG (e.g. from IgGl, IgG2. IgG3 or IgG4). from IgE or from another human Ig such as IgA. IgD or IgM. For example. WO 94/04678 describes heavy chain antibodies comprising a Camelid V_HH domain or a humanized derivative thereof (i.e. a Nanobody®), in which the Camelidae C_R2 and/or C₁-_P domain have been replaced by human C_f [2 and C_H3 domains, so as to provide an immunoglobulin that consists of 2 heavy chains each comprising a Nanobody® and human C]j2 and C_H3 domains (but no QiI domain), which immunoglobulin has the effector function provided by the Q|2 and C_H3 domains and which immunoglobulin can function without the presence of any light chains. Other amino acid sequences that can be suitably linked to the Nanobodies® of the invention so as to provide an effector function will be clear to the skilled person, and may be chosen on the basis of the desired effector function(s). Reference is for example made to WO 04/058820. WO 99/42077. WO 02/056910 and WO 05/017148, as well as the review by Holliger and Hudson, supra; and to WO 09/068628. Coupling of a Nanobody® of the invention to an Fc portion may also lead to an increased half-life, compared to the corresponding Nanobody® of the invention. For some applications, the use of an Fc portion and/or of constant domains (i.e. C_H2 and/or Qp domains) that confer increased half-life without any biologically significant effector function may also be suitable or even preferred. Other suitable constructs comprising one or more Nanobodies® and one or more constant domains with increased half-life in vivo will be clear to the skilled person, and may for example comprise two Nanobodies® linked to a C_H3 domain, optionally via a linker sequence. Generally, any fusion protein or derivatives with increased half-life will preferably have a molecular weight of more than 50 kD, the cut-off value for renal absorption. In another particular, but non-limiting, embodiments of the invention, in order to form a polypeptide of the invention, one or more amino acid sequences of the invention may be linked (optionally via a suitable linker or hinge region) to naturally occurring, synthetic or semisynthetic constant domains (or analogs, variants, mutants, parts or fragments thereof) that have a reduced (or essentially no) tendency to self-associate into dimers (i.e. compared to constant domains that naturally occur in conventional 4-chain antibodies). Such monomer ic (i.e. not self-associating) Fc chain variants, or fragments thereof, will be clear to the skilled person. For example. Helm et aL J Biol Chem 1996 271 7494, describe monomelic Fcε chain variants that can be used in the polypeptide chains of the invention. Also, such monomeric Fc chain variants are preferably such that they are still capable of binding to the complement or the relevant Fc receptor(s) (depending on the Fc portion from which they are derived), and/or such that they still have some or all of the effector functions of the Fc portion from which they are derived (or at a reduced level still suitable for the intended use). Alternatively, in such a polypeptide chain of the invention, the monomeric Fc chain may be used to confer increased half-life upon the polypeptide chain, in which case the monomeric Fc chain may also have no or essentially no effector functions.

Bivalent/multivalent. bispecific/multispecific or biparatopic/multiparatopic polypeptides of the invention may also be linked to Fc portions, in order to provide polypeptide constructs of the type that is described in WO 09/068630A1. The further amino acid sequences may also form a signal sequence or leader sequence that directs secretion of the Nanobody® or the polypeptide of the invention from a host cell upon synthesis (for example to provide a pre-, pro- or prepro- form of the polypeptide of the invention, depending on the host cell used to express the polypeptide of the invention).

The further amino acid sequence may also form a sequence or signal that allows the Nanobody© or polypeptide of the invention to be directed towards and/or to penetrate or enter into specific organs, tissues, cells, or parts or compartments of cells, and/or that allows the Nanobody® or polypeptide of the invention to penetrate or cross a biological barrier such as a cell membrane, a cell layer such as a layer of epithelial cells, a tumor including solid tumors, or the blood-brain-barrier. Suitable examples of such amino acid sequences will be clear to the skilled person, and for example include, but are not limited to, those mentioned on page 1 18 of WO 08/020079. For some applications, in particular for those applications in which it is intended to kill a cell that expresses the target against which the Nanobodies© of the invention are directed (e.g. in the treatment of cancer), or to reduce or slow the growth and/or proliferation of such a cell, the Nanobodies® of the invention may also be linked to a (cyto)toxic protein or polypeptide. Examples of such toxic proteins and polypeptides which can be linked to a Nanobody® of the invention to provide - for example - a cytotoxic polypeptide of the invention will be clear to the skilled person and can for example be found in the prior art cited above and/or in the further description herein. One example is the so- called ADEPT™ technology described in WO 03/055527.

According to particular, but non-limiting embodiments of the invention, said one or more further amino acid sequences comprise at least one further Nanobody®, so as to provide a polypeptide of the invention that comprises at least two. such as three, four, five or more Nanobodies®, in which said Nanobodies® may optionally be linked via one or more linker sequences (as defined herein). As described on pages 119 and 120 of WO 08/020079, polypeptides of the invention that comprise two or more Nanobodies®, of which at least one is a Nanobody® of the invention, will also be referred to herein as "multivalent" polypeptides of the invention, and the Nanobodies® present in such polypeptides will also be referred to herein as being in a "multivalent format". For example, "bivalent" and "trivalent^" polypeptides of the invention may be as further described on pages 119 and 120 of WO 08/020079.

Polypeptides of the invention thai contain at least two Nanobodies©, in which at least one Nanobody® is directed against a first antigen (i.e. against sclerostin) and at least one Nanobody® is directed against a second antigen (i.e. different from sclerostin), will also be referred to as "multispecific" polypeptides of the invention, and the Nanobodies® present in such polypeptides will also be referred to herein as being in a "multispecific format^"'. Thus, for example, a "bispecific" polypeptide of the invention is a polypeptide that comprises at least one Nanobody® directed against a first antigen (i.e. sclerostin) and at least one further Nanobody® directed against a second antigen (i.e. different from sclerostin), whereas a "trispecific'' polypeptide of the invention is a polypeptide that comprises at least one Nanobody® directed against a first antigen (i.e. sclerostin), at least one further Nanobody® directed against a second antigen (i.e. different from sclerostin) and at least one further Nanobody® directed against a third antigen (i.e. different from both sclerostin, and the second antigen); etc. Accordingly, in its simplest form, a bispecific polypeptide of the invention is a bivalent polypeptide of the invention (as defined herein), comprising a first Nanobody® directed against sclerostin, and a second Nanobody® directed against a second antigen, in which said first and second Nanobody® may optionally be linked via a linker sequence (as defined herein); whereas a trispecific polypeptide of the invention in its simplest form is a trivalent polypeptide of the invention (as defined herein), comprising a first Nanobody® directed against sclerostin, a second Nanobody® directed against a second antigen and a third Nanobody® directed against a third antigen, in which said first, second and third Nanobody® may optionally be linked via one or more, and in particular one and more, in particular two, linker sequences.

However, as will be clear from the description hereinabove, the invention is not limited thereto, in the sense that a multispecifϊc polypeptide of the invention may comprise at least one Nanobody® against sclerostin and any number of Nanobodies® directed against one or more antigens different from sclerostin. Furthermore, although it is encompassed within the scope of the invention that the specific order or arrangement of the various Nanobodies® in the polypeptides of the invention may have some influence on the properties of the final polypeptide of the invention (including but not limited to the affinity, specificity or avidity for sclerostin, or against the one or more other antigens), said order or arrangement is usually not critical and may be suitably chosen by the skilled person, optionally after some limited routine experiments based on the disclosure herein. Thus, when reference is made to a specific multivalent or multispecific polypeptide of the invention, it should be noted that this encompasses any order or arrangements of the relevant Nanobodies®, unless explicitly indicated otherwise.

Finally, it is also within the scope of the invention that the polypeptides of the invention contain two or more Nanobodies® and one or more further amino acid sequences (as mentioned herein).

For multivalent and multispecific polypeptides containing one or more V_HH domains and their preparation, reference is also made to Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001; Muyldermans, Reviews in Molecular Biotechnology 74 (2001), 277-302; as well as to for example WO 96/34103 and WO 99/23221. Some other examples of some specific multispecific and/or multivalent polypeptide of the invention can be found in the applications by Ablynx N.V. referred to herein.

One preferred, but non-limiting example of a multispecific polypeptide of the invention comprises at least one Nanobody® of the invention and at least one Nanobody® that provides for an increased half-life. Such Nanobodies® may for example be Nanobodies® that are directed against a serum protein, and in particular a human serum protein, such as human serum albumin, thyroxine-binding protein, (human) transferrin, fibrinogen, an immunoglobulin such as IgG, IgE or IgM, or against one of the serum proteins listed in WO 04/003019. Of these. Nanobodies® that can bind to serum albumin (and in particular human serum albumin) or to IgG (and in particular human IgG, see for example Nanobody® VH-I described in the review by Muyldermans. supra) are particularly preferred (although for example- for experiments in mice or primates. Nanobodies® against or cross-reactive with mouse serum albumin (MSA) or serum albumin from said primate, respectively, can be used. However, for pharmaceutical use. Nanobodies® against human serum albumin or human IgG will usually be preferred). Nanobodies® that provide for increased half-life and that can be used in the polypeptides of the invention include the Nanobodies® directed against serum albumin that are described in WO 04/041865. in WO 06/122787 and in the further patent applications by Ablynx N.V., such as those mentioned above. For example, the some preferred Nanobodies® that provide for increased half-life for use in the present invention include Nanobodies® that can bind to amino acid residues on (human) serum albumin that are not involved in binding of serum albumin to FcRn (see for example WO 06/0122787): Nanobodies® that are capable of binding to amino acid residues on serum albumin that do not form part of domain III of serum albumin (see for example WO 06/0122787); Nanobodies® that have or can provide an increased half-life (see for example WO 08/028977 by Ablynx N.V mentioned herein); Nanobodies® against human serum albumin that are cross-reactive with serum albumin from at least one species of mammal, and in particular with at least one species of primate (such as, without limitation, monkeys from the genus Macaca (such as. and in particular, cynomologuε monkeys {Macaca fascicularis) and/or rhesus monkeys (Macaca mulatto)) and baboon (Papio ursinus)) (see for example WO 08/028977 by Ablynx N.V)); Nanobodies® that can bind to serum albumin in a pH independent manner (see for example WO 08/043821 by Ablynx N.V. mentioned herein) and/or Nanobodies® that are conditional binders (see for example WO 08/043822by Ablynx N.V.).

Some particularly preferred Nanobodies® that provide for increased half-life and that can be used in the polypeptides of the invention include the Nanobodies® ALB-I to ALB-IO disclosed in WO 06/122787 (see Tables II and III) of which ALB-8 (SEQ ID NO: 62 in WO 06/122787) is particularly preferred.

According to particular, but non-limiting embodiments of the invention, the polypeptides of the invention contain, besides the one or more Nanobodies® of the invention, at least one Nanobody® against human serum albumin.

Generally, any polypeptides of the invention with increased half-life that contain one or more Nanobodies® of the invention, and any derivatives of Nanobodies® of the invention or of such polypeptides that have an increased half-life, preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding Nanobody® of the invention per se. For example, such a derivative or polypeptides with increased half-life may have a half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding Nanobody® of the invention per se.

In particular, but non-limiting embodiments of the invention, such derivatives or polypeptides may exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more. For example, such derivatives or polypeptides may have a half-life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 1 1 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).

According to particular embodiments of the invention the polypeptides are capable of binding to one or more molecules which can increase the half-life of the polypeptide in vivo.

The polypeptides of the invention are stabilised in vivo and their half-life increased by binding to molecules which resist degradation and/or clearance or sequestration. Typically, such molecules are naturally occurring proteins which themselves have a long half-life in vivo.

Another preferred, but non-limiting example of a multispecifϊc polypeptide of the invention comprises at least one Nanobody® of the invention and at least one Nanobody® that directs the polypeptide of the invention towards, and/or that allows the polypeptide of the invention to penetrate or to enter into specific organs, tissues, cells, or parts or compartments of cells, and/or that allows the Nanobody® to penetrate or cross a biological barrier such as a cell membrane, a cell layer such as a layer of epithelial cells, a tumor including solid tumors, or the blood-brain-barrier. Examples of such Nanobodies® include Nanobodies® that are directed towards specific cell-surface proteins, markers or epitopes of the desired organ, tissue or cell (for example cell-surface markers associated with tumor cells), and the single-domain brain targeting antibody fragments described in WO 02/057445 and WO 06/040153, of which FC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO 06/040154) are preferred examples.

In the polypeptides of the invention, the one or more Nanobodies® and the one or more polypeptides may be directly linked to each other (as for example described in WO 99/23221) and/or may be linked to each other via one or more suitable spacers or linkers, or any combination thereof.

Suitable spacers or linkers for use in multivalent and multispecific polypeptides will be clear to the skilled person, and may generally be any linker or spacer used in the art to link amino acid sequences. Preferably, said linker or spacer is suitable for use in constructing proteins or polypeptides that are intended for pharmaceutical use.

Some particularly preferred spacers include the spacers and linkers that are used in the art to link antibody fragments or antibody domains. These include the linkers mentioned in the general background art cited above, as well as for example linkers that are used in the art to construct diabodies or ScFv fragments (in this respect, however, its should be noted that, whereas in diabodies and in ScFv fragments, the linker sequence used should have a length, a degree of flexibility and other properties that allow the pertinent V_H and V_L domains to come together to form the complete antigen-binding site, there is no particular limitation on the length or the flexibility of the linker used in the polypeptide of the invention, since each Nanobody® by itself forms a complete antigen-binding site).

For example, a linker may be a suitable amino acid sequence, and in particular amino acid sequences of between 1 and 50, preferably between 1 and 30, such as between 1 and 10 amino acid residues. Some preferred examples of such amino acid sequences include gly-ser linkers, for example of the type (gly^ser^z, such as (for example (g!y₄ser)₃ or (gly₃ser₂)₃. as described in WO 99/42077 and the GS30. GS 15, GS9 and GS7 linkers described in the applications by Ablynx mentioned herein (see for example WO 06/040153 and WO 06/122825), as well as hinge-like regions, such as the hinge regions of naturally occurring heavy chain antibodies or similar sequences (such as described in WO 94/04678 ).

Some other particularly preferred linkers are poly-alanine (such as AAA). as well as the linkers GS30 (SEQ ID NO: 85 in WO 06/122825) and GS9 (SEQ ID NO: 84 in WO 06/122825).

Other suitable linkers generally comprise organic compounds or polymers, in particular those suitable for use in proteins for pharmaceutical use. For instance, poly(ethyleneglycol) moieties have been used to link antibody domains, see for example WO 04/081026.

It is encompassed within the scope of the invention that the length, the degree of flexibility and/or other properties of the linker(s) used (although not critical, as it usually is for linkers used in ScFv fragments) may have some influence on the properties of the final polypeptide of the invention, including but not limited to the affinity, specificity or avidity for sclerostin, or for one or more of the other antigens. Based on the disclosure herein, the skilled person will be able to determine the optimal linker(s) for use in a specific polypeptide of the invention, optionally after some limited routine experiments.

For example, in multivalent polypeptides of the invention that comprise Nanobodies® directed against a multimeric antigen (such as a multimeric receptor or other protein), the length and flexibility of the linker are preferably such that it allows each Nanobody® of the invention present in the polypeptide to bind to the antigenic determinant on each of the subunits of the multimer. Similarl} , in a multispecific polypeptide of the invention that comprises Nanobodies® directed against two or more different antigenic determinants on the same antigen (for example against different epitopes of an antigen and/or against different subunits of a multimeric receptor, channel or protein), the length and flexibility of the linker are preferably such that it allows each Nanobody® to bind to its intended antigenic determinant. Again, based on the disclosure herein, the skilled person will be able to determine the optimal lmker(s) for use in a specific polypeptide of the invention, optionally after some limited routine experiments

It is also within the scope of the invention that the linker(s) used confer one or more other favourable properties or functionality to the polypeptides of the invention, and/or provide one or more sites for the formation of derivatives and/or for the attachment of functional groups (e.g. as described herein for the derivatives of the Nanobodies® of the invention). For example, linkers containing one or more charged amino acid residues (see Table A-2 on page 48 of the International application WO 08/020079) can provide improved hydrophilic properties, whereas linkers that form or contain small epitopes or tags can be used for the purposes of detection, identification and/or purification. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linkers for use in a specific polypeptide of the invention, optionally after some limited routine experiments.

Finally, when two or more linkers are used in the polypeptides of the invention, these linkers may be the same or different. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linkers for use in a specific polypeptide of the invention, optionally after some limited routine experiments. Usually, for easy of expression and production, a polypeptide of the invention will be a lineai- polypeptide. However, the invention in its broadest sense is not limited thereto. For example, when a polypeptide of the invention comprises three of more Nanobodies®, it is possible to link them by use of a linker with three or more "arms^"', which each "arm" being linked to a Nanobody®, so as to provide a "star-shaped'^' construct. It is also possible, although usually less preferred, to use circular constructs.

The invention also comprises derivatives of the polypeptides of the invention, which may be essentially analogous to the derivatives of the Nanobodies® of the invention, i.e. as described herein.

The invention also comprises proteins or polypeptides that "essentially consist'^" of a polypeptide of the invention (in which the wording "essentially consist of has essentially the same meaning as indicated hereinabove).

According to particular embodiments of the invention, the polypeptide of the invention is in essentially isolated from, as defined herein.

The amino acid sequences, Nanobodies®, polypeptides and nucleic acids of the invention can be prepared in a manner known per se. as will be clear to the skilled person from the further description herein. For example, the Nanobodies® and polypeptides of the invention can be prepared in any manner known per se for the preparation of antibodies and in particular for the preparation of antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments). Some preferred, but non-limiting methods for preparing the amino acid sequences, Nanobodies®, polypeptides and nucleic acids include the methods and techniques described herein.

As will be clear to the skilled person, one particularly useful method for preparing an amino acid sequence, Nanobody® and/or a polypeptide of the invention generally comprises the steps of: i) the expression, in a suitable host cell or host organism (also referred to herein as a "host of the invention") or in another suitable expression system of a nucleic acid that encodes said amino acid sequence, Nanobody® or polypeptide of the invention (also referred to herein as a "nucleic acid of the invention"), optionally followed by: ii) isolating and/or purifying the amino acid sequence, ^"Nanobody® or polypeptide of the invention thus obtained.

In particular, such a method may comprise the steps of: i) cultivating and/or maintaining a host of the invention under conditions that are such that said host of the invention expresses and/or produces at least one amino acid sequence.,

Nanobody® and/or polypeptide of the invention; optionally followed by: ii) isolating and/or purifying the amino acid sequence, Nanobody® or polypeptide of the invention thus obtained. A nucleic acid of the invention can be in the form of single or double stranded DNA or RNA. and is preferably in the form of double stranded DNA. For example, the nucleotide sequences of the invention may be genomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage that has been specifically adapted for expression in the intended host cell or host organism).

According to particular embodiments of the invention, the nucleic acid of the invention is in essentially isolated from, as defined herein.

The nucleic acid of the invention may also be in the form of, be present in and/or be part of a vector, such as for example a plasmid, cosmid or YAC, which again may be in essentially isolated form.

The nucleic acids of the invention can be prepared or obtained in a manner known per se, based on the information on the amino acid sequences for the polypeptides of the invention given herein, and/or can be isolated from a suitable natural source. To provide analogs, nucleotide sequences encoding naturally occurring V_{HI I} domains can for example be subjected to site-directed mutagenesis, so at to provide a nucleic acid of the invention encoding said analog. Also, as will be clear to the skilled person, to prepare a nucleic acid of the invention, also several nucleotide sequences, such as at least one nucleotide sequence encoding a Nanobody® and for example nucleic acids encoding one or more linkers can be linked together in a suitable manner. Techniques for generating the nucleic acids of the invention will be clear to the skilled person and may for instance include, but are not limited to. automated DNA synthesis; site- directed mutagenesis; combining two or more naturally occurring and/or synthetic sequences (or two or more parts thereof), introduction of mutations that lead to the expression of a truncated expression product; introduction of one or more restriction sites (e.g. to create cassettes and/or regions that may easily be digested and/or ligated using suitable restriction enzymes), and/or the introduction of mutations by means of a PCR reaction using one or more "mismatched" primers, using for example a sequence of a naturally occurring form of sclerostin as a template. These and other techniques will be clear to the skilled person, and reference is again made to the standard handbooks, such as Sambrook et al. and AusubeS et al., mentioned above, as well as the Examples below.

The nucleic acid of the invention may also be in the form of, be present in and/or be part of a genetic construct, as will be clear to the person skilled in the art and as described on pages 131-134 of WO 08/020079 (incorporated herein by reference). Such genetic constructs generally comprise at least one nucleic acid of the invention that is optionally United to one or more elements of genetic constructs known per se, such as for example one or more suitable regulatory elements (such as a suitable promoter(s), enhancer(s), terminator(s), etc.) and the further elements of genetic constructs referred to herein. Such genetic constructs comprising at least one nucleic acid of the invention will also be referred to herein as "genetic constructs of the invention".

The genetic constructs of the invention may be DNA or RNA, and are preferably double- stranded DNA. The genetic constructs of the invention may also be in a form suitable for transformation of the intended host cell or host organism, in a form suitable for integration into the genomic DNA of the intended host cell or in a form suitable for independent replication, maintenance and/or inheritance in the intended host organism. For instance, the genetic constructs of the invention may be in the form of a vector, such as for example a plasmid, cosmid, YAC, a viral vector or transposon. In particular, the vector may be an expression vector, i.e. a vector that can provide for expression in vitro and/or in vivo (e.g. in a suitable host cell, host organism and/or expression system).

In a preferred but non-limiting embodiment, a genetic construct of the invention comprises i) at least one nucleic acid of the invention; operably connected to ii) one or more regulatory elements, such as a promoter and optionally a suitable terminator; and optionally also iii) one or more further elements of genetic constructs known per se; in which the terms "operably connected" and "operably linked" have the meaning given on pages 131 -134 of WO 08/020079; and in which the "regulatory elements", "promoter", "terminator" and "'further elements" are as described on pages 131 -134 of WO 08/020079; and in which the genetic constructs may further be as described on pages 131-134 of WO 08/020079.

The nucleic acids of the invention and/or the genetic constructs of the invention may be used to transform a host cell or host organism, i.e. for expression and/or production of the amino acid sequence, Nanobody® or polypeptide of the invention. Suitable hosts or host cells will be clear to the skilled person, and may for example be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism, for example those described on pages 134 and 135 of WO 08/020079.; as well as all other hosts or host cells known per se for the expression and production of antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments), which will be clear to the skilled person. Reference is also made to the genera] background art cited hereinabove, as well as to for example WO 94/29457; WO 96/34103; WO 99/42077; Frenken et a!.. (1998), supra: Riechmann and Muyldermans, (1999). supra: van der Linden, (2000), supra; Thomassen et al., (2002), supra; Joosten et al., (2003). supra; Joosten et al., (2005), supra; and the further references cited herein.

The amino acid sequences. Nanobodies® and polypeptides of the invention can also be introduced and expressed in one or more cells, tissues or organs of a multicellular organism, for example for prophylactic and/or therapeutic purposes (e.g. as a gene therapy), as further described on pages 135 and 136 of in WO 08/020079and in the further references cited in WO 08/020079.

For expression of the Nanobodies® in a cell, they may also be expressed as so-called "intrabodies", as for example described in WO 94/02610, WO 95/22618 and US-A-7004940; WO 03/014960; in Caltaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Development and Applications. Landes and Springer- Verlag; and in Kontermann. Methods 34. (2004). 163- 170.

The amino acid sequences, Nanobodies® and polypeptides of the invention can for example also be produced in the milk of transgenic mammals, for example in the milk of rabbits, cows, goats or sheep (see for example US-A-6.741.957, US-A-6,304,489 and US-A- 6.849,992 for general techniques for introducing transgenes into mammals), in plants or parts of plants including but not limited to their leaves, flowers, fruits, seed, roots or turbers (for example in tobacco, maize, soybean or alfalfa) or in for example pupae of the silkworm Bombϊx mori.

Furthermore, the amino acid sequences. Nanobodies® and polypeptides of the invention can also be expressed and/or produced in cell-free expression systems, and suitable examples of such systems will be clear to the skilled person. Some preferred, but non-limiting examples include expression in the wheat germ system; in rabbit reticulocyte lysates: or in the E, coli Zubay system.

As mentioned above, one of the advantages of the use of Nanobodies® is that the polypeptides based thereon can be prepared through expression in a suitable bacterial system, and suitable bacterial expression systems, vectors, host cells, regulatory elements, etc., will be clear to the skilled person, for example from the references cited above. It should however be noted that the invention in its broadest sense is not limited to expression in bacterial systems.

Preferably, in the invention, an (in vivo or in vitro) expression system, such as a bacterial expression system, is used that provides the polypeptides of the invention in a form that is suitable for pharmaceutical use, and such expression systems will again be clear to the skilled person. As also will be clear to the skilled person, polypeptides of the invention suitable for pharmaceutical use can be prepared using techniques for peptide synthesis.

For production on industrial scale, preferred heterologous hosts for the (industrial) production of Nanobodies® or Nanobody®-containing protein therapeutics include strains of E. coli, Pichia pastoris, S. cerevfsiae that are suitable for large scale expression/production/fermentation, and in particular for large scale pharmaceutical (i.e. GMP grade) expression/production/fermentation. Suitable examples of such strains will be clear to the skilled person. Such strains and production/expression systems are also made available by companies such as Biovitrum (Uppsala, Sweden).

Alternatively, mammalian cell lines, in particular Chinese hamster ovary (CHO) cells, can be used for large scale expression/production/fermentation, and in particular for large scale pharmaceutical expression/production/fermentation. Again, such expression/production systems are also made available by some of the companies mentioned above.

The choice of the specific expression system would depend in part on the requirement for certain post-translational modifications, more specifically glycosylation. The production of a Nanobody®-containing recombinant protein for which glycosylation is desired or required would necessitate the use of mammalian expression hosts that have the ability to glycosylate the expressed protein. In this respect, it will be clear to the skilled person that the glycosylation pattern obtained (i.e. the kind, number and position of residues attached) will depend on the cell or cell line that is used for the expression. Preferably, either a human cell or cell line is used (i.e. leading to a protein that essentially has a human glycosylation pattern) or another mammalian cell line is used that can provide a glycosylation pattern that is essentially and/or functionally the same as human glycosylation or at least mimics human glycosylation. Generally, prokaryotic hosts such as E. coli do not have the ability to glycosylate proteins, and the use of lower eukaryotes such as yeast usually leads to a glycosylation pattern that differs from human glycosylation. Nevertheless, it should be understood that all the foregoing host cells and expression systems can be used in the invention, depending on the desired amino acid sequence, Nanobody® or polypeptide to be obtained.

Thus, according to particular embodiments of the invention, the amino acid sequence, Nanobody® or polypeptide of the invention is glycosylated. According to other non-limiting embodiments of the invention, the amino acid sequence, Nanobody® or polypeptide of the invention is non-glycosylated. According to a preferred, but non-limiting embodiment of the invention, the amino acid sequence, Nanobody® or polypeptide of the invention is produced in a bacterial cell, in particular a bacterial cell suitable for large scale pharmaceutical production, such as cells of the strains mentioned above.

According to a particular, but non-limiting embodiment of the invention, the amino acid sequence. Nanobody® or polypeptide of the invention is produced in a yeast cell, in particular a yeast cell suitable for large scale pharmaceutical production, such as cells of the species mentioned above.

According to a particular, but non-limiting embodiment of the invention, the amino acid sequence. Nanobody® or polypeptide of the invention is produced in a mammalian cell, in particular in a human cell or in a cell of a human cell line, and more in particular in a human cell or in a cell of a human cell line that is suitable for large scale pharmaceutical production, such as the cell lines mentioned hereinabove.

As further described on pages 138 and 139 of WO 08/020079, when expression in a host cell is used to produce the amino acid sequences. Nanobodies® and the polypeptides of the invention, the amino acid sequences, Nanobodies® and polypeptides of the invention can be produced either intracellullarly (e.g. in the cytosol, in the periplasms or in inclusion bodies) and then isolated from the host cells and optionally further purified; or can be produced extracellularly (e.g. in the medium in which the host cells are cultured) and then isolated from the culture medium and optionally further purified. Thus, according to one non- limiting aspect of the invention, the amino acid sequence, Nanobody® or polypeptide of the invention is an amino acid sequence, Nanobody® or polypeptide that has been produced intracellulaiiy and that has been isolated from the host cell, and in particular from a bacterial cell or from an inclusion body in a bacterial cell. According to another non-limiting aspect of the invention, the amino acid sequence, Nanobody® or polypeptide of the invention is an amino acid sequence. Nanobody® or polypeptide that has been produced extracellularly, and that has been isolated from the medium in which the host cell is cultivated.

Some preferred, but non-limiting promoters for use with these host cells include those mentioned on pages 139 and 140 of WO 08/020079. Some preferred, but non-limiting secretory sequences for use with these host cells include those mentioned on page 140 of WO 08/020079.

Suitable techniques for transforming a host or host cell of the invention will be clear to the skilled person and may depend on the intended host cell/host organism and the genetic construct to be used. Reference is again made to the handbooks and patent applications mentioned above.

After transformation, a step for detecting and selecting those host cells or host organisms that have been successfully transformed with the nucleotide sequence/genetic construct of the invention may be performed. This may for instance be a selection step based on a selectable marker present in the genetic construct of the invention or a step involving the detection of the amino acid sequence of the invention, e.g. using specific antibodies.

The transformed host cell (which may be in the form or a stable cell line) or host organisms (which may be in the form of a stable mutant line or strain) form further aspects of the present invention.

Preferably, these host cells or host organisms are such that they express, or are (at least) capable of expressing (e.g. under suitable conditions), an amino acid sequence. Nanobody© or polypeptide of the invention (and in case of a host organism: in at least one cell, part, tissue or organ thereof). The invention also includes further generations, progeny and/or offspring of the host cell or host organism of the invention, that may for instance be obtained by cell division or by sexual or asexual reproduction.

To produce/obtain expression of the amino acid sequences of the invention, the transformed host cell or transformed host organism may generally be kept, maintained and/or cultured under conditions such that the (desired) amino acid sequence, Nanobody® or polypeptide of the invention is expressed/produced. Suitable conditions will be clear to the skilled person and will usually depend upon the host cell/host organism used, as well as on the regulatory elements that control the expression of the (relevant) nucleotide sequence of the invention. Again, reference is made to the handbooks and patent applications mentioned above in the paragraphs on the genetic constructs of the invention.

Generally, suitable conditions may include the use of a suitable medium, the presence of a suitable source of food and/or suitable nutrients, the use of a suitable temperature, and optionally the presence of a suitable inducing factor or compound (e.g. when the nucleotide sequences of the invention are under the control of an inducible promoter); all of which may be selected by the skilled person. Again, under such conditions, the amino acid sequences of the invention may be expressed in a constitutive manner, in a transient manner, or only when suitably induced.

It will also be clear to the skilled person that the amino acid sequence, Nanobody® or polypeptide of the invention may (first) be generated in an immature form (as mentioned above), which may then be subjected to post-translational modification, depending on the host cell/host organism used. Also, the amino acid sequence. Nanobody® or polypeptide of the invention may be glycosylated, again depending on the host cell/host organism used.

The amino acid sequence, Nanobody® or polypeptide of the invention may then be isolated from the host cell/host organism and/or from the medium in which said host cell or host organism was cultivated, using protein isolation and/or purification techniques known per se, such as (preparative) chromatography and/or electrophoresis techniques, differential precipitation techniques, affinity techniques (e.g. using a specific, cleavable amino acid sequence fused with the amino acid sequence, Nanobody© or polypeptide of the invention) and/or preparative immunological techniques (i.e. using antibodies against the amino acid sequence to be isolated).

Generally, for pharmaceutical use, the polypeptides of the invention may be formulated as a pharmaceutical preparation or compositions comprising at least one polypeptide of the invention and at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more further pharmaceutically active polypeptides and/or compounds. By means of non- limiting examples, such a formulation may be in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), for topical administration, for administration by inhalation, by a skin patch, by an implant, by a suppository, etc.. Such suitable administration forms - which may be solid, semi-solid or liquid, depending on the manner of administration - as well as methods and earners for use in the preparation thereof, will be clear to the skilled person, and are further described herein.

Thus, in a further aspect, the invention relates to a pharmaceutical composition that contains at least one amino acid of the invention, at least one Nanobody® of the invention or at least one polypeptide of the invention and at least one suitable carrier, diluent or excipient (i.e. suitable for pharmaceutical use), and optionally one or more further active substances.

Generally, the amino acid sequences. Nanobodies® and polypeptides of the invention can be formulated and administered in any suitable manner known per se, for which reference is for example made to the general background art cited above (and in particular to WC) 04/041862, WO 04/041863, WO 04/041865. WO 04/041867 and WO 08/020079) as well as to the standard handbooks, such as Remington's Pharmaceutical Sciences, 18^th Ed., Mack Publishing Company, USA (1990). Remington, the Science and Practice of Pharmacy, 21th Edition, Lippincott Williams and Wilkins (2005); or the Handbook of Therapeutic Antibodies (S. Dubel, Ed.), Wiley. Weinheim, 2007 (see for example pages 252-255). For example, the amino acid sequences, Nanobodies® and polypeptides of the invention may be formulated and administered in any manner known per se for conventional antibodies and antibody fragments (including ScFv^' s and diabodies) and other pharmaceutically active proteins. Such formulations and methods for preparing the same will be clear to the skilled person, and for example include preparations suitable for parenteral administration (for example intravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal, intra-arterial or intrathecal administration) or for topical (i.e. transdermal or intradermal) administration .

Preparations for parenteral administration may for example be sterile solutions, suspensions, dispersions or emulsions that are suitable for infusion or injection. Suitable carriers or diluents for such preparations for example include, without limitation, those mentioned on page 143 of WO 08/020079. Usually, aqueous solutions or suspensions will be preferred.

The amino acid sequences, Nanobodies® and polypeptides of the invention can also be administered using gene therapy methods of delivery. See. e.g., U.S. Patent No. 5.399346. which is incorporated by reference in its entirety. Using a gene therapy method of delivery, primary cells transfected with the gene encoding an amino acid sequence, Nanobody® or polypeptide of the invention can additionally be transfected with tissue specific promoters to target specific organs, tissue, grafts, tumors, or cells and can additionally be transfected with signal and stabilization sequences for subcellular^ localized expression.

Thus, the amino acid sequences. Nanobodies® and polypeptides of the invention may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the amino acid sequences. Nanobodies® and polypeptides of the invention may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of the amino acid sequence. Nanobody® or polypeptide of the invention. Their percentage in the compositions and preparations may, of course, be varied and ma> conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of the amino acid sequence. Nanobody® or polypeptide of the invention in such therapeutically useful compositions is such that an effective dosage level will be obtained. The tablets, troches, pills, capsules, and the like ma)' also contain binders, excipients. disintegrating agents, lubricants and sweetening or flavouring agents, for example those mentioned on pages 143-144 of WO 08/020079. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the amino acid sequences. Nanobodies® and polypeptides of the invention, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the amino acid sequences, Nanobodies® and polypeptides of the invention may be incorporated into sustained-release preparations and devices.

Preparations and formulations for oral administration may also be provided with an enteric coating that will allow the constructs of the invention to resist the gastric environment and pass into the intestines. More generally, preparations and formulations for oral administration may be suitably formulated for delivery into any desired part of the gastrointestinal tract. In addition, suitable suppositories may be used for delivery into the gastrointestinal tract. The amino acid sequences, Nanobodies® and polypeptides of the invention may also be administered intravenously or intraperitoneally by infusion or injection, as further described on pages 144 and 145 of WO 08/020079.

For topical administration, the amino acid sequences, Nanobodies® and polypeptides of the invention may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid, as further described on page 145 of WO 08/020079.

Generally, the concentration of the amino acid sequences, Nanobodies® and polypeptides of the invention in a liquid composition, such as a lotion, will be from about 0.1 - 25 wt-%, preferably from about 0.5-10 wt-%. The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt~ %.

The amount of the amino acid sequences. Nanobodies® and polypeptides of the invention required for use in treatment will vary not only with the particular amino acid sequence, Nanobody® or polypeptide selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. Also the dosage of the amino acid sequences, Nanobodies® and polypeptides of the invention varies depending on the target cell, tumor, tissue, graft, or organ.

The desired dose may conveniently be presented in a single dose or as divided doses administered, at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

An administration regimen could include long-term, daily treatment. By "long-term" is meant at least two weeks and preferably, several weeks, months, or years of duration. Necessary modifications in this dosage range may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. See Remington's Pharmaceutical Sciences (Martin, E. W., ed. 4), Mack Publishing Co., Easton, PA. The dosage can also be adjusted by the individual physician in the event of any complication.

In another aspect, the invention relates to a method for the prevention and/or treatment of at least one bone disease and/or disorder, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody® of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same,

In the context of the present invention, the term "prevention and/or treatment" not only comprises preventing and/or treating the disease, but also generally comprises preventing the onset of the disease, slowing or reversing the progress of disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and/or alleviating one or more symptoms associated with the disease, reducing the severity and/or the duration of the disease and/or of any symptoms associated therewith and/or preventing a further increase in the severity of the disease and/or of any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease, and generally any pharmacological action that is beneficial to the patient being treated.

The subject to be treated may be any warm-blooded animal, but is in particular a mammal, and more in particular a human being. As will be clear to the skilled person, the subject to be treated will in particular be a person suffering from, or at risk of, the diseases and disorders mentioned herein. The invention relates to a method for the prevention and/or treatment of at least one disease or disorder that is associated with sclerostin, with its biological or pharmacological activity, and/or with the biological pathways or signalling in which sclerostin is involved, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of aNanobody® of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same. In particular, the invention relates to a method for the prevention and/or treatment of at least one disease or disorder that can be treated by modulating sclerostin. its biological or pharmacological activity, and/or the biological pathways or signalling in which sclerostin is involved, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody® of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same. In particular, said pharmaceutically effective amount may be an amount that is sufficient to modulate sclerostin, its biological or pharmacological activity, and/or the biological pathways or signalling in which sclerostin is involved; and/or an amount that provides a level of the amino acid sequence of the invention, of aNanobody® of the invention, of a polypeptide of the invention in the circulation that is sufficient to modulate sclerostin, its biological or pharmacological activity, and/or the biological pathways or signalling in which sclerostin is involved. The invention furthermore relates to a method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering an amino acid sequence of the invention, a Nanobody® of the invention or a polypeptide of the invention to a patient, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of aNanobody® of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same.

More in particular, the invention relates to a method for the prevention and/or treatment of at least one disease or disorder chosen from the group consisting of the diseases and disorders listed herein, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody® of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same.

In another aspect, the invention relates to a method for immunotherapy, and in particular for passive immunotherapy, which method comprises administering, to a subject suffering from or at risk of the diseases and disorders mentioned herein, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody® of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same,

In the above methods, the amino acid sequences. Nanobodies® and/or polypeptides of the invention and/or the compositions comprising the same can be administered in any suitable manner, depending on the specific pharmaceutical formulation or composition to be used. Thus, the amino acid sequences. Nanobodies® and/or polypeptides of the invention and/or the compositions comprising the same can for example be administered orally, intraperitoneally (e.g. intravenously, subcutaneously, intramuscularly, or via any other route of administration that circumvents the gastrointestinal tract), intranasally. transdermally. topically, by means of a suppository, by inhalation, again depending on the specific pharmaceutical formulation or composition to be used. The clinician will be able to select a suitable route of administration and a suitable pharmaceutical formulation or composition to be used in such administration, depending on the disease or disorder to be prevented or treated and other factors well known to the clinician.

The amino acid sequences. Nanobodies® and/or polypeptides of the invention and/or the compositions comprising the same are administered according to a regime of treatment that is suitable for preventing and/or treating the disease or disorder to be prevented or treated. The clinician will generally be able to determine a suitable treatment regimen, depending on factors such as the disease or disorder to be prevented or treated, the severity of the disease to be treated and/or the severity of the symptoms thereof, the specific amino acid sequence, Nanobody® or polypeptide of the invention to be used, the specific route of administration and pharmaceutical formulation or composition to be used, the age, gender, weight, diet, general condition of the patient, and similar factors well known to the clinician.

Generally, the treatment regimen will comprise the administration of one or more amino acid sequences, Nanobodies® and/or polypeptides of the invention, or of one or more compositions comprising the same, in one or more pharmaceutically effective amounts or doses The specific amount(s) or doses to be administered can be determined by the clinician, again based on the factors cited above.

Generally, for the prevention and/or treatment of the diseases and disorders mentioned herein and depending on the specific disease or disorder to be treated, the potency of the specific amino acid sequence, Nanobody® and polypeptide of the invention to be used, the specific route of administration and the specific pharmaceutical formulation or composition used, the amino acid sequences, Nanobodies® and polypeptides of the invention will generally be administered in an amount between 1 gram and 0.01 microgram per kg body weight per day, preferably between 0.1 gram and 0.1 microgram per kg body weight per day, such as about 1. 10. 100 or 1000 microgram per kg body weight per day. either continuously (e.g. by infusion), as a single daily dose or as multiple divided doses during the day. The clinician will generally be able to determine a suitable daily dose, depending on the factors mentioned herein. It will also be clear that in specific cases, the clinician may choose to deviate from these amounts, for example on the basis of the factors cited above and his expert judgment. Generally, some guidance on the amounts to be administered can be obtained from the amounts usually administered for comparable conventional antibodies or antibody fragments against the same target administered via essentially the same route, talcing into account however differences in affinity/avidity, efficacy, biodistribution, half-life and similar factors well known to the skilled person.

Usually, in the above method, a single amino acid sequence. Nanobody® or polypeptide of the invention will be used. It is however within the scope of the invention to use two or more amino acid sequences, Nanobodies® and/or polypeptides of the invention in combination.

The Nanobodies®. amino acid sequences and polypeptides of the invention may also be used in combination with one or more further pharmaceutically active compounds or principles, i.e. as a combined treatment regimen, which may or may not lead to a synergistic effect. Again, the clinician will be able to select such further compounds or principles, as well as a suitable combined treatment regimen, based on the factors cited above and his expert judgement.

In particular, the amino acid sequences. Nanobodies® and polypeptides of the invention may be used in combination with other pharmaceutically active compounds or principles that are or can be used for the prevention and/or treatment of the diseases and disorders cited herein, as a result of which a synergistic effect may or may not be obtained. Examples of such compounds and principles, as well as routes, methods and pharmaceutical formulations or compositions for administering them will be clear to the clinician. In particular, the pharmaceutical compositions of the invention may comprise one or more amino acid sequences, Nanobodies® and/or polypeptides of the invention and at least one additional therapeutic agent selected from monoclonal antibody (mAb). CDP-7851 (Sclerostin Ab, AMG-785). a bone morpho genie factor, transforming growth factor- β: (TGF- β). an interleukin-1 (IL-I) inhibitor. IL-lra, Kinεret™; a TNFα inhibitor, a soluble TNFα receptor, Enbrel™, an anti- TNFα antibody. Remicade™. a D2E7 antibody, a parathyroid hormone, an analog of a parathyroid hormone, a parathyroid hormone related protein, an analog of a parathyroid hormone related protein, a prostaglandin, a bisphosphonate, an alendronate, fluoride, calcium, a non-steroidal anti -inflammatory drug (NSAID). a COX-2 inhibitor, Celebrex™, Vioxx™, an immunosuppressant, methotrexate, leflunomide, a serine protease inhibitor, a secretory leukocyte protease inhibitor (SLPI), an IL-6 inhibitor, an antibody or Nanobody® against IL-6, an IL-8 inhibitor, an antibody or Nanobody® against 1L-8. an IL- 18 inhibitor, an IL-18 binding protein, an antibody or Nanobody® against IL-18. an Interleukin-1 converting enzyme (ICE) modulator, a fibroblast growth factor (FGF), an FGF modulator, a PAF antagonist, an oestrogen, a selective oestrogen receptor modulator, a keratinocyte growth factor (KGF), a KGF -related molecule, a KGF modulator, a matrix melalloproteinase (MMP) modulator, a nitric oxide synthase (NOS) modulator, a modulator of glucocorticoid receptor, a modulator of glutamate receptor, a modulator of lipopolysaccharide (LPS) levels, a noradrenaline, a noradrenaline mimetic, and a noradrenaline modulator as described, for example, in US 2004/00335353.

When two or more substances or principles are to be used as part of a combined treatment regimen, they can be administered via the same route of administration or via different routes of administration, at essentially the same time or at different times (e.g. essentially simultaneously, consecutively, or according to an alternating regime). When the substances or principles are to be administered simultaneously via the same route of administration, they may be administered as different pharmaceutical formulations or compositions or part of a combined pharmaceutical formulation or composition, as will be clear to the skilled person.

Also, when two or more active substances or principles are to be used as part of a combined treatment regimen, each of the substances or principles may be administered in the same amount and according to the same regimen as used when the compound or principle is used on its own, and such combined use may or may not lead to a synergistic effect. However, when the combined use of the two or more active substances or principles leads to a synergistic effect, it may also be possible to reduce the amount of one, more or all of the substances or principles to be administered, while still achieving the desired therapeutic action. This may for example be useful for avoiding, limiting or reducing any unwanted side- effects that are associated with the use of one or more of the substances or principles when they are used in their usual amounts, while still obtaining the desired pharmaceutical or therapeutic effect. The effectiveness of the treatment regimen used according to the invention may be determined and/or followed in any manner known per se for the disease or disorder involved, as will be clear to the clinician. The clinician will also be able, where appropriate and on a case-by-case basis, to change or modify a particular treatment regimen, so as to achieve the desired therapeutic effect, to avoid, limit or reduce unwanted side-effects, and/or to achieve an appropriate balance between achieving the desired therapeutic effect on the one hand and avoiding, limiting or reducing undesired side effects on the other hand.

Generally, the treatment regimen will be followed until the desired therapeutic effect is achieved and/or for as long as the desired therapeutic effect is to be maintained. Again, this can be determined by the clinician. in another aspect, the invention relates to the use of an amino acid sequence, Nanobody® or polypeptide of the invention in the preparation of a pharmaceutical composition for prevention and/or treatment of at least one bone disease and/or disorder; and/or for use in one or more of the methods of treatment mentioned herein.

The subject to be treated may be any warm-blooded animal, but is in particular a mammal, and more in particular a human being. As will be clear to the skilled person, the subject to be treated will in particular be a person suffering from, or at risk of. the diseases and disorders mentioned herein.

The invention also relates to the use of an amino acid sequence, Nanobody® or polypeptide of the invention in the preparation of a pharmaceutical composition for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering an amino acid sequence, Nanobody© or polypeptide of the invention to a patient.

More in particular, the invention relates to the use of an amino acid sequence. Nanobody® or polypeptide of the invention in the preparation of a pharmaceutical composition for the prevention and/or treatment of bone diseases and disorders, and in particular for the prevention and treatment of one or more of the diseases and disorders listed herein.

Again, in such a pharmaceutical composition, the one or more amino acid sequences. Nanobodies® or polypeptides of the invention may also be suitably combined with one or more other active principles, such as those mentioned herein.

Finally, although the use of the Nanobodies® of the invention (as defined herein) and of the polypeptides of the invention is much preferred, it will be clear that on the basis of the description herein, the skilled person will also be able to design and/or generate, in an analogous manner, other amino acid sequences and in particular (single) domain antibodies against sclerostin, as well as polypeptides comprising such (single) domain antibodies.

For example, it will also be clear to the skilled person that it may be possible to "graft" one or more of the CDR's mentioned above for the Nanobodies® of the invention onto such (single) domain antibodies or other protein scaffolds, including but not limited to human scaffolds or non-immunoglobulin scaffolds. Suitable scaffolds and techniques for such CDR grafting will be clear to the skilled person and are well known in the art, see for example those mentioned in WO 08/020079. For example, techniques known per se for grafting mouse or rat CDR's onto human frameworks and scaffolds can be used in an analogous manner to provide chimeric proteins comprising one or more of the CDR's of the Nanobodies® of the invention and one or more human framework regions or sequences.

It should also be noted that, when the Nanobodies® of the inventions contain one or more other CDR sequences than the preferred CDR sequences mentioned above, these CDR sequences can be obtained in any manner known per se, for example using one or more of the techniques described in WO 08/020079.

Further uses of the amino acid sequences, Nanobodies®, polypeptides, nucleic acids, genetic constructs and hosts and host cells of the invention will be clear to the skilled person based on the disclosure herein. For example, and without limitation, the amino acid sequences of the invention can be linked to a suitable carrier or solid support so as to provide a medium than can be used in a manner known per se to purify sclerostin from compositions and preparations comprising the same. Derivatives of the amino acid sequences of the invention that comprise a suitable detectable label can also be used as markers to determine (qualitatively or quantitatively) the presence of sclerostin in a composition or preparation or as a marker to selectively detect the presence of sclerostin on the surface of a cell or tissue (for example, in combination with suitable cell sorting techniques).

The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference, in particular for the teaching that is referenced hereinabove. The invention will now be further described by means of the following non-limiting preferred aspects, examples and figures:

Preferred Aspects: Aspect A-I : An amino acid sequence that is directed against and/or that can specifically bind to sclerostin. Aspect A-2: An amino acid sequence according to aspect A-I. that is in. essentially isolated form. Aspect A-3: An amino acid sequence according to aspect A-I or A-2, for administration to a subject, wherein said amino acid sequence does not naturally occur in said subject. Aspect A-4: An amino acid sequence that can specifically bind to sclerostin with a dissociation constant (K_D) of 10^"5 to 10^"12 moles/litre or less, and preferably ] 0^" ⁷ to 10^"12 moles/litre or less and more preferably 10^" to 10^"12 moles/litre. Such an amino acid sequence may in particular be an amino acid sequence according to any of the preceding aspects. Aspect A-5: An amino acid sequence thai can specifically bind to sclerostin with a rate of association (k_on-rate) of between 10² M^" V¹ to about 10⁷ M^-1S^"1. preferably between 10³ M^-1S^"1 and 10⁷ M^-1S^'1, more preferably between 10⁴ M^-1S^"1 and 10⁷

M^" V¹ , such as between 10^s M^-1S^"1 and 10⁷ IVfV¹. Such an amino acid sequence may in particular be an amino acid sequence according to any of the preceding aspects.

Aspect A-6: An amino acid sequence that can specifically bind Io sclerostin with a rate of dissociation (k_Off rate) between 1 s^'! and 10^"6 s^" , preferably between 10^"2 s^"! and

10^"6 s^"1. more preferably between 10^~J s^"' and 10^"6 s^"1, such as between 10^"4 s^'1 and 10^"6 s^"1. Such an amino acid sequence may in particular be an amino acid sequence according to any of the preceding aspects. Aspect A-7: An amino acid sequence that can specifically bind to sclerostin with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. Such an amino acid sequence may in particular be an amino acid sequence according to any of the preceding aspects. Aspect A-8: An amino acid sequence according to any of the preceding aspects that is a naturally occurring amino acid sequence (from any suitable species) or a synthetic or semi -synthetic amino acid sequence.

Aspect A-9: An amino acid sequence according to any of the preceding aspects that comprises an immunoglobulin fold or that under suitable conditions is capable of forming an immunoglobulin fold. Aspect A-IO: An amino acid sequence according to any of the preceding aspects, that essentially consists of 4 framework regions (FRl to FR4 respectively) and 3 complementarity determining regions (CDRl to CDR3 respectively).

Aspect A-I l : An amino acid sequence according to any of the preceding aspects that is an immunoglobulin sequence.

Aspect A-12: An amino acid sequence according to any of the preceding aspects that is a naturally occurring immunoglobulin sequence (from any suitable species) or a synthetic or semi-synthetic immunoglobulin sequence.

Aspect A-13: An amino acid sequence according to any of the preceding aspects that is a humanized immunoglobulin sequence, a camelized immunoglobulin sequence or an immunoglobulin sequence that has been obtained by techniques such as affinity maturation.

Aspect A-14: An amino acid sequence according to any of the preceding aspects, that essentially consists of a light chain variable domain sequence (e.g. a VL- sequence); or of a heavy chain variable domain sequence (e.g. a VH-sequence).

Aspect A-15: An amino acid sequence according to any of the preceding aspects, that essentially consists of a heavy chain variable domain sequence that is derived from a conventional four-chain antibody or that essentially consist of a heavy chain variable domain sequence thai is derived from heavy chain antibody. Aspect A- 16: An amino acid sequence according to any of the preceding aspects, that essentially consists of a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody), of a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), of a "dAb" (or an amino acid sequence that is suitable for use as a dAb) or of a Nanobody® (including but not limited to a VHH sequence).

Aspect A- 17: An amino acid sequence according to any of the preceding aspects, that essentially consists of a Nanobody®.

Aspect A-18: An amino acid sequence according to any of the preceding aspects, that essentially consists of a Nanobody® that i) has at least 80% amino acid identity with at least one of the An amino acid sequences of SEQ ID NO's: ] to 22, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded; and in which: ii) preferably one or more of the amino acid residues at positions 1 1 , 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table B -2. Aspect A-19: An amino acid sequence according to any of the preceding aspects, that essentially consists of a polypeptide that i) has at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO^'s: 189 to 197, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded; and in which: ii) preferably one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table B-2. Aspect A-20: An amino acid sequence according to any of the preceding aspects, that essentially consists of a Nanobody® that i) has at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 189 to 197, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded; and in which: ϋ) preferably one or more of the amino acid residues at positions 11. 37. 44. 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table B-2, Aspect A-21 : An amino acid sequence according to any of the preceding aspects, that essentially consists of a humanized Nanobody®.

Aspect A-22: An amino acid sequence according to any of the preceding aspects, that in addition to the at least one binding site for binding against sclerostin, contains one or more further binding sites for binding against other antigens, proteins or targets.

Aspect B-I : An amino acid sequence that is directed against and/or that can specifically bind sclerostin, and that comprises one or more stretches of amino acid residues chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO's: 135 to 143: b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; d) the amino acid sequences of SEQ ID NO's: 153 to 161 ; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 153 to 161 ; f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 153 to 161 ; g) the amino acid sequences of SEQ ID NO's: 171 to 179; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid, sequences of SEQ ID NO's: 171 to 179; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 171 to 179; or any suitable combination thereof.

Such an amino acid sequence may in particular be an amino acid sequence according to any of the aspects A-I to A-22. Aspect B-2: An amino acid sequence according to aspect B-L in which at least one of said stretches of amino acid residues forms part of the antigen binding site for binding against sclerostin.

Aspect B-3 : An amino acid sequence that is directed against and/or that can specifically bind sclerostin and that comprises two or more stretches of amino acid residues chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO's: 135 to 143; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; d) the amino acid sequences of SEQ ID NO's: 153 to 161; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 153 to 161 ; f) amino acid sequences that have 3, 2. or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 153 to 161; g) the amino acid sequences of SEQ ID NO's: 171 to 179; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 171 to 179; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 171 to 179; such that (i) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b) or c), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to d), e), f), g). h) or i): (ii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to d), e) or f), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b), c). g). h) or i); or (iii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to g). h) or i), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b), c), d), e) or f).

Such an amino acid sequence may in particular be an amino acid sequence according to any of the aspects A-I to A-22, B-I or B-2.

Aspect B-4: An amino acid sequence according to aspect B-3, in which the at least two stretches of amino acid residues forms part of the antigen binding site for binding against sclerostiii. Aspect B-5: An amino acid sequence that is directed against and/or that can specifically bind sclerostin and that comprises three or more stretches of amino acid residues, in which the first stretch of amino acid residues is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO's: 135 to 143; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO^'s: 135 to 143: c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; the second stretch of amino acid residues is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NO's: 153 to 161 : e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO^'s: 153 to 161: f) amino acid sequences that have 3. 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO^"'s: 153 to 161 : and the third stretch of amino acid residues is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NO^'s: 171 to 179; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 171 to 179; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 171 to 179. Such an amino acid sequence may in particular be an amino acid sequence according Io any of the aspects A-I to A-22 and/or B-I to B-4.

Aspect B-6: An amino acid sequence according to aspect B-5, in which the at least three stretches of amino acid residues forms part of the antigen binding site for binding against sclerostin.

Aspect B-7: An amino acid sequence that is directed against and/or that can specifically bind sclerostin in which the CDR sequences of said amino acid sequence have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 189 to

197. Such an amino acid sequence may in particular be an amino acid sequence according to any of the aspects A-I to A-22 and/or B-I to B-6.

Aspect C-I : An amino acid sequence that is directed against sclerostin and that cross-blocks the binding of at least one of the amino acid sequences of SEQ ID NO's: 189 to 197 to sclerostin. Such an amino acid sequence may in particular be an amino acid sequence according to any of the aspects A-I to A-22 and/or according to aspects B-I to B-7. Also, preferably, such an amino acid sequence is able to specifically bind to sclerostin.

Aspect C-2: An amino acid sequence that is directed against sclerostin and that is cross- blocked from binding to sclerostin by at least one of the amino acid sequences of SEQ ID NO's: 189 to 197. Such an amino acid sequence may in particular be an amino acid sequence according to any of the aspects A-I to A-22 and/or according to aspects B-] to B-7. Also, preferably, such an amino acid sequence is able to specifically bind to sclerostin. Aspect C-3: An amino acid sequence according to any of aspects C-I or C-2. wherein the ability of said amino acid sequence to cross-block or to be cross-blocked is detected in a Biacore assay.

Aspect C-4: An amino acid sequence according to any of aspects C-I to C-3 wherein the ability of said amino acid sequence to cross-block or to be cross-blocked is detected in an ELISA assay.

Aspect D-I: An amino acid sequence according to any of aspects B-I to B-7 or C-I to C-4. that is in essentially isolated form.

Aspect D-2: An amino acid sequence according to any of aspects B-I to B-7. C-I to C-4, and/or Dl for administration to a subject, wherein said amino acid sequence does not naturally occur in said subject.

Aspect D-3: An amino acid sequence according to any of aspects B- 1 to B-7. C-I to C-4. and/or Dl to D-2 that can specifically bind to sclerostin with a dissociation constant (K_D) of 10° to 10^"12 moles/litre or less, and preferably 10^"7 to 10^"12 moles/litre or less and more preferably 10^~8 to 3 O^"12 moles/litre.

Aspect D-4: An amino acid sequence according to any of aspects B-I to B-7, C-I to C-4, and/or D-I to D-3 that can specifically bind to sclerostin with a rate of association (k_on-rate) of between 10² M^"V to about 10⁷ Nf ¹S^'1, preferably between 10³ M-V¹ and 10⁷ M^" V, more preferably between 10⁴ M^"'s^"! and 10⁷ M⁴S^"1. such as between 10⁵ M^' V¹ and 10⁷ M^'Y¹.

Aspect D- 5: An amino acid sequence according to any of aspects B- 1 to B-7, C-I to C-4, and/or D-I to D-4 that can specifically bind to sclerostin with a rate of dissociation (karate) between 1 s^"1 and 10^"6 s^"1 preferably between 10^"2 s^"1 and 10^"6 s^"1, more preferably between 10^~3 s^"1 and 10^"6 s^"1, such as between 10^~4 s^"1 and 10^'6 S^"1.

Aspect D-6: An amino acid sequence according to any of aspects B-I to B-7, C-] to C-4. and/or D-I to D-5 that can specifically bind to sclerostin with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. The amino acid sequences according to aspects D-I to D-6 may in particular be an amino acid sequence according to any of the aspects A-I to A-22.

Aspect E-I : An amino acid sequence according to any of aspects B-I to B-7. C-I to C-4 and/or Dl to D-6 that is a naturally occurring amino acid sequence (from any suitable species) or a synthetic or semi-synthetic amino acid sequence. Aspect E-2: An amino acid sequence according Io any of aspects B-I to B-7, C-I to C-4. Dl to D-6, and/or E-I that comprises an immunoglobulin fold or that under suitable conditions is capable of forming an immunoglobulin fold.

Aspect E-3: An amino acid sequence according to any of aspects B-I to B-7, C-I to C-4. Dl to D-6, and/or D-I or D-2, that is an immunoglobulin sequence.

Aspect E-4: An amino acid sequence according to any of aspects B-I to B-7, C-I to C-4. Dl to D-6, and/or E- 1 to E-3. that is a naturally occurring immunoglobulin sequence (from any suitable species) or a synthetic or semi-synthetic immunoglobulin sequence. Aspect E-5: An amino acid sequence according to any of aspects B-I to B-7, C-I to C-4, D] to D-6, and/or E-I to E-4 that is a humanized immunoglobulin sequence, a camelized immunoglobulin sequence or an immunoglobulin sequence that has been obtained by techniques such as affinity maturation.

Aspect E-6: An amino acid sequence according to any of aspects B-I to B-7, C-I to C-4. Dl to D-6, and/or E-I to E-5 that essentially consists of a light chain variable domain sequence (e.g. a V_L-sequence); or of a heavy chain variable domain sequence (e.g. a V_H-sequence).

Aspect E-7: An amino acid sequence according to any of aspects B-I to B-7, C-I to C-4.

Dl to D-6.. and/or E-I to E-6. that essentially consists of a heavy chain variable domain sequence that is derived from a conventional four-chain antibody or that essentially consist of a heavy chain variable domain sequence that is derived from heavy chain antibody.

Aspect E-8: An amino acid sequence according to any of aspects B-I to B-7. C-I to C-4,

Dl to D-6. and/or E-I to E-7, that essentially consists of a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody), of a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), of a "dAb" (or an amino acid sequence that is suitable for use as a dAb) or of a Nanobody® (including but not limited to a V₁]_H sequence).

Aspect E-9: An amino acid sequence according to any of aspects B-I to B-7, C-I to C-4. Dl to D-6. and/or E-I to E-8 that essentially consists of a Nanobody®.

Aspect E-IO: An amino acid sequence according to any of aspects B-I to B-7. C-I to C-4, Dl to D-6. and/or E-I to E-9 that essentially consists of a Nanobody® that i) has at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO^'s: 1 to 22, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded; and in which: ii) preferably one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table B-2.

Aspect E-1 1 : An amino acid sequence according to any of aspects B-I to B-7, C-I to C-4, D 1 to D-6, and/or E- 1 to E- 10, that essentially consists of a Nanobody® that i) has at least 80% amino acid identity with at least one of the An amino acid sequences of SEQ ID NO's: 189 to 197. in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded; and in which: ii) preferably one or more of the amino acid residues at positions 1 1, 37. 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table B -2.

Aspect E-12: An amino acid sequence according to any of aspects B-I to B-7, C-I to C-4, D 1 to D-6, and/or E- 1 to E- 11. that essentially consists of a humanized

Nanobody®. Aspect E-13: An amino acid sequence according to any of the aspects B-I to B-7, C-I to C-

4, Dl to D-6, and/or E-I to E-12. that in addition to the at least one binding site for binding formed by the CDR sequences, contains one or more further binding sites for binding against other antigens, proteins or targets.

The amino acid sequences according to aspects E-I to E-13 may in particular be an amino acid sequence according to any of the aspects A-I to A-22.

Aspect F-I : An amino acid sequence that essentially consists of 4 framework regions (FRl to FR4. respectively) and 3 complementarity determining regions (CDRl to

CDR3, respectively), in which:

CDRl is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO^'s: 135 to 143: b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO^'s: 135 to 143; and/or

CDR2 is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NO's: 153 to 161; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO^"s: 153 to 161; f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 153 to 161 ; and/or

CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NO's: 171 to 179; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 171 to 179; i) amino acid sequences that have 3. 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO^'s: 171 to 179. Such an amino acid sequence is preferably directed against sclerostin and/or an amino acid sequence that can specifically bind to sclerostin. Also, such an amino acid sequence is preferably an amino acid sequence according to any of the aspects A-! to A-22, C-I to C-4, Dl to D-6 and/or E-I to E-13.

Aspect F-2: An amino acid sequence that essentially consists of 4 framework regions (FRl to FR4, respectively) and 3 complementarity determining regions (CDRl to CDR3, respectively), in which:

CDRl is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO's: 135 to 143; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; and

CDR2 is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NO's: 153 to 161 ; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 153 to 161; f) amino acid sequences that have 3, 2. or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 153 to 161; and

CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NO^'s: 171 to 179; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 171 to 179: i) amino acid sequences that have 3. 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO"s: 171 to 179. Such an amino acid sequence is preferably directed against sclerostin and/or an amino acid sequence that can specifically bind to sclerostin. Also, such an amino acid sequence is preferably an amino acid sequence according to any of the aspects A-I to A-22, C-I to C-4, Dl to D-6 and/or E-I to E-13.

Aspect F-3: An amino acid sequence according to any of aspects F-I and F-2, in which the CDR sequences of said amino acid sequence have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO^'s: 189 to 197.

Such an amino acid sequence is preferably directed against sclerostin and/or an amino acid sequence that can specifically bind to sclerostin. Also, such an amino acid sequence is preferably an amino acid sequence according to any of the aspects A-I to A-22, C-I to C-4. Dl to D-6 and/or E-I to E-13.

Aspect F -4: An amino acid sequence according to any of aspects F-I to F-3 that is directed against sclerostin and that cross-blocks the binding of at least one of the amino acid sequences according to any of aspects the amino acid sequences of SEQ ID NO's: 189 to 197. Aspect F-5: An amino acid sequence according to any of aspects F-I to F-3 that is directed against sclerostin and that is cross-blocked from binding to sclerostin by at least one of the amino acid sequences of SEQ ID NO^'s: 189 to 197. Aspect F-6: Amino acid sequence according to any of aspects F -4 or F-5 wherein the ability of said amino acid sequence to cross-block or to be cross-blocked is detected in a Biacore assay.

Aspect F-7: Amino acid sequence according to any of aspects F4 or F-5 wherein the ability of said amino acid sequence to cross-block or to be cross-blocked is detected in an ELISA assay.

Aspect F-8: An amino acid sequence according to any of aspects F-I to F-7, that is in essentially isolated form.

Aspect F-9: An amino acid sequence according to any of aspects F-I to F-8, for administration Io a subject, wherein said an amino acid sequence does not naturally occur in said subject.

Aspect F-10: An amino acid sequence according to any of aspects F-I to F-9. that can specifically bind to sclerostin with a dissociation constant (KQ) of 10^"' to 10^{" "} moles/litre or less, and preferably 10^"7 to 10^4~ moles/litre or less and more preferably 10^"8 to 10⁴² moles/litre.

Aspect F-I l: An amino acid sequence according to any of aspects F-I to F-IO, that can specifically bind to sclerostin with a rate of association (k_on-rate) of between 10² M^-1S^"1 to about 10⁷ IvT¹S^"1, preferably between 10³ M⁴S⁴ and 10⁷ M^-1S^"1, more preferably between 10⁴ M^" 's⁴ and 10⁷ M⁴S^"1. such as between 10⁵ M⁴S^"1 and 10⁷ M⁴S⁴.

Aspect F-12: An amino acid sequence according to any of aspects F-I to F-I l, that can specifically bind to sclerostin with a rate of dissociation (k_Off rate) between 1 s⁴ and 10^'6 s⁴ preferably between 10^"2 s⁴ and 10^"6 s⁴, more preferably between 10^"3 s⁴ and 10^"6 s⁴. such as between 10^"4 s⁴ and 10^"6 s⁴.

Aspect F-13: An amino acid sequence according to any of aspects F-I to F-12, that can specifically bind to sclerostin with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

Aspect F-14: An amino acid sequence according to any of aspects F-I to F-13 that is a naturally occurring amino acid sequence (from any suitable species) or a synthetic or semi-synthetic amino acid sequence.

Aspect F-15: An amino acid sequence according to any of aspects F-I to F-14 that comprises an immunoglobulin fold or that under suitable conditions is capable of forming an immunoglobulin fold. Aspect F-16: An amino acid sequence according to any of aspects F-I to F-15 that is an immunoglobulin sequence. Aspect F-17: An amino acid sequence according to any of aspects F-I to F- 16 that is a naturally occurring immunoglobulin sequence (from an}' suitable species) or a synthetic or semi-synthetic immunoglobulin sequence. Aspect F-18: An amino acid sequence according to any of aspects F-I to F- 17 that is a humanized immunoglobulin sequence, a camelized immunoglobulin sequence or an immunoglobulin sequence that has been obtained by techniques such as affinity maturation. Aspect F- 19: An amino acid sequence according to any of aspects F-I to F-19, that essentially consists of a light chain variable domain sequence (e.g. a VL- sequence); or of a heavy chain variable domain sequence (e.g. a Ve-sequence). Aspect F-20: An amino acid sequence according to any of aspects F-I to F- 19 that essentially consists of a heavy chain variable domain sequence that is derived from a conventional four-chain antibody or that essentially consist of a heavy chain variable domain sequence that is derived from heavy chain antibody. Aspect F -21 : An amino acid sequence according to any of aspects F-I to F-20 that essentially consists of a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody), of a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), of a

"dAb" (or an amino acid sequence that is suitable for use as a dAb) or of a Nanobody® (including but not limited to a V_HH sequence). Aspect F~22: An amino acid sequence according to any of aspects F-I to F-21 that essentially consists of a Nanobody®.

Aspect F-23: An amino acid sequence according to any of aspects F-I to F-22 that essentially consists of a Nanobody® that i) has at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO^'s: 1 to 22, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded; and in which: ii) preferably one or more of the amino acid residues at positions 11₅ 37, 44, 45, 47. 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table B-2. Aspect F-24: An amino acid sequence according to any of aspects F-I to F-23, that essentially consists of aNanobody© that i) has at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO^'s: ] 89 to 197, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded; and in which: ii) preferably one or more of the amino acid residues at positions IL 37, 44,

45. 47. 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table B -2.

Aspect F-25: An amino acid sequence according to any of aspects F-I to F-24. that essentially consists of a humanized Nanobody®.

Aspect G-I : An amino acid sequence according to any of the preceding aspects, that in addition to the at least one binding site for binding formed by the CDR sequences, contains one or more further binding sites for binding against another antigen, protein or target. Aspect FI-I : Nanobody® that is directed against and/or that can specifically bind to sclerostin.

Aspect H-2: Nanobody® according to aspect H-I, that is in essentially isolated form. Aspect H-3: Nanobody® according to any of aspects H-I to H-2, that can specifically bind to sclerostin with a dissociation constant (K_D) of 10^"5 to 10^"12 moles/litre or less, and preferably 10^"7 to 10^"12 moles/litre or less and more preferably 10^"8 to 10^"'² moles/litre.

Aspect H-4: Nanobody® according to any of aspects H-I to H-3, that can specifically bind to sclerostin with a rate of association (k._on-rale) of between 10² M^" V¹ to about 10⁷ Vf's^'1, preferably between 10^J M^" V¹ and 10⁷ M⁴S^"1. more preferably between 10⁴ M^"' s^"' and 10⁷ M^'V¹, such as between 10⁵ M^-1S^"1 and 10⁷ M^-1S^"1. Aspect H- 5: Nanobody® according to any of aspects H- 1 to H-4, that can specifically bind to sclerostin with a rate of dissociation (k_Off rate) between 1 s^"s and 10^"6 s^"1 preferably between 10^"2 s^"1 and 10^"6 s^"1, more preferably between 10^'3 s^"1 and

10^"6 s^"], such as between 10^"4 s^'1 and 10^"6 s^"1.

Aspect H-6: Nanobody® according to any of aspects H-I to H-5. that can specifically bind to sclerostin with an affinity less than 500 nM. preferably less than 200 nM, more preferably less than 10 iiM, such as less than 500 pM. Aspect H-7: Nanobody© according to any of aspects H-I to H-6. that is a naturally occurring Nanobody® (from any suitable species) or a synthetic or semisynthetic Nanobody®.

Aspect H-8: Nanobody® according to any of aspects to H-I to H-7, that is a V_HH sequence, a partially humanized V_HH sequence, a fully humanized V_HH sequence, a camelized heavy chain variable domain or a Nanobody® that has been obtained by techniques such as affinity maturation. Aspect H-9: Nanobody® according to any of aspects H- 1 to H-8, that i) has at least 80% amino acid identity with at least one of the An amino acid sequences of SEQ ID NO^'s: 1 to 22, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded; and in which: ii) preferably one or more of the amino acid residues at positions 11 , 37, 44. 45. 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table B-2. Aspect H- 10: Nanobody® according to any of aspects H-I to H-9, that i) has at least 80% amino acid identity with at least one of the An amino acid sequences of SEQ ID NO's: 189 to 197, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded: and in which: ii) preferably one or more of the amino acid residues at positions 1 L 37, 44. 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table B-2. Aspect H-I l: Nanobody® according to any of aspects H-I to H-IO, in which: CDRl is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO^'s: 135 to 143; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO^"s: 135 to 143; c) amino acid sequences that have 3. 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; and/or

CDR2 is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NO^' s: 153 to 161 : e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 153 to 161 ; f) amino acid sequences that have 3. 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 153 to 161 ; and/or

CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NO's: 171 to 179: h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 171 to 179; i) amino acid sequences that have 3. 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO^'s: 171 to 179. Aspect H- 12: Nanobody® according to any of aspects H-I to H-11, in which: CDRl is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO^'s: 135 to 143; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; c) amino acid sequences that have 3. 2. or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; and

CDR2 is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NO's: 153 to 161; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO^'s: 153 to 161 ; f) amino acid sequences that have 3. 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO^'s: 153 to 161; and

CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NO's: 171 to 179; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO^'s: 171 to 179: i) amino acid sequences that have 3. 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO^'s: 171 to 179. Aspect H-13: Nanobody® according to any of aspects H-I to H- 12, in which the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO^'s: 189 to 197.

Aspect H- 14: Nanobody® according to any of aspects H-I to H- 13, which is a partially humanized Nanobody®.

Aspect H-15: Nanobody® according to any of aspects H-I to H- 14, which is a fully humanized Nanobody®.

Aspect H-16: Nanobody® according to any of aspects H-I to H- 15, that is chosen from the group consisting of SEQ ID NO's: 189 to 197 or from the group consisting of from amino acid sequences that have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with at least one of the amino acid sequences of SEQ ID NO^'s: 189 to 197.

Aspect H-17: Nanobody® according to any of aspects H-I Io H-16. w^rhich is a humanized

Nanobody® that is chosen from the group consisting of humanized variants of SEQ ID NO^"s: 189 to 197 or from the group consisting of amino acid sequences that have more than 80%, preferably more than 90%. more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with at least one humanized variant of one of SEQ ID NO's: 189 to 197.

Aspect H- 18: Nanobody® according to any of aspects H-I to H-17 that is chosen from the group consisting of SEQ ID NO^'s: 189 to 197.

Aspect H-19: Nanobody® directed against sclerostin that cross-blocks the binding of at least one of the amino acid sequences of SEQ ID NO's: 189 to 197 to sclerostin.

Aspect H-20: Nanobody® directed against sclerostin that is cross-blocked from binding to sclerostin by at least one of the amino acid sequences of SEQ ID NO^'s: 189 to 197.

Aspect H-21 : Nanobody® according to any of aspects H-19 or H-20 wherein the ability of said Nanobody® to cross-block or to be cross-blocked is detected in a Biacore assa> . Aspect H-22: Nanobody® according to any of aspects H- 19 to H-21 wherein the ability of said Nanobody® to cross-block or to be cross-blocked is detected in an ELISA assay.

Aspect K-I : Polypeptide that comprises or essentially consists of one or more amino acid sequences according to any of aspects A-I to A-22, B-I to B-7, C-I to C-4, D- 1 to D-6, E-I to E- 13, F-I to F-25 or G-I and/or one or more Nanobodies® according to any of aspects H-I to H-22, and optionally further comprises one or more peptidic linkers.

Aspect K-2: Polypeptide according to aspect K-I, in which said one or more binding units are immunoglobulin sequences.

Aspect K-3: Polypeptide according to any of aspects K-I or K-2, in which said one or more other groups, residues, moieties or binding units are chosen from the group consisting of domain antibodies, amino acid sequences that are suitable for use as a domain antibody, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, "dAb"'s, amino acid sequences that are suitable for use as a dAb, or Nanobodies®.

Aspect K-4: Polypeptide according to any of aspects K-I to K-3, in which said one or more amino acid sequences of the invention are immunoglobulin sequences.

Aspect K-5: Polypeptide according to any of aspects K-I to K-4, in which said one or more amino acid sequences of the invention are chosen from the group consisting of domain antibodies, amino acid sequences that are suitable for use as a domain antibody, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, "dAb'"s, amino acid sequences that are suitable for use as a dAb, or Nanobodies®.

Aspect K-6: Polypeptide according to any of aspects K-I to K-5, that comprises or essentially consists of one or more Nanobodies® according to any of aspects H-I to H-22 and in which said one or more other binding units are Nanobodies®.

Aspect K-7: Polypeptide according to any of aspects K-I to K- 6, wherein at least one binding unit is a multivalent construct.

Aspect K-8: Polypeptide according to any of aspects K-I to K-8, wherein at least one binding unit is a multiparatopic construct.

Aspect K-9: Polypeptide according to any of aspects K-I to K-8, wherein at least one binding unit is a multispecific construct. Aspect K-IO: Polypeptide according to any of aspects K-I to K-9, which has an increased half-life, compared to the corresponding amino acid sequence according to any of aspects A-I to A-22, B-I to B-7, C-I to C-4. D-I to D-6, E-I to E-13, F-I to F-25 or G-I per se or Nanobody® according to any of aspects H-I to H-22 per se, respectively.

Aspect K-I l : Polypeptide according to aspect K-IO, in which said one or more other binding units provide the polypeptide with increased half-life, compared to the corresponding amino acid sequence according to any of aspects A-I to A-22, B-I to B-7, C-I to C-4, D-I to D-6, E-I to E-13, F-I to F-25 or G-I per se or Nanobody® according to any of aspects H-I to H-22 per se, respectively.

Aspect K- 12: Polypeptide according to aspect K-I O or K-I l. in which said one or more other binding units that provide the polypeptide with increased half-life is chosen from the group consisting of serum proteins or fragments thereof, binding units that can bind to serum proteins, an Fc portion, and small proteins or peptides that can bind to serum proteins.

Aspect K- 13 : Polypeptide according to any of aspects K- 10 to K- 12, in which said one or more other binding units that provide the polypeptide with increased half-life is chosen from the group consisting of human serum albumin or fragments thereof. Aspect K- 14 : Polypeptide according to any of aspect K- 10 to K- 13. in which said one or more other binding units that provides the polypeptide with increased half-life are chosen from the group consisting of binding units that can bind to serum albumin (such as human serum albumin) or a serum immunoglobulin (such as IgG).

Aspect K- 15 : Polypeptide according to any of aspects K- 10 to K- 14. in which said one or more other binding units that provides the polypeptide with increased half-life are chosen from the group consisting of domain antibodies, amino acid sequences that are suitable for use as a domain antibody, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, "dAb"'s , amino acid sequences that are suitable for use as a dAb, or

Nanobodies® that can bind to serum albumin (such as human serum albumin) or a serum immunoglobulin (such as IgG).

Aspect K-16: Polypeptide according to aspect K-IO to K-15, in which said one or more other binding units that provides the polypeptide with increased half-life is a Nanobody® that can bind to serum albumin (such as human serum albumin) or a serum immunoglobulin (such as IgG).

Aspect K- 17: Polypeptide according to any of aspects K-IO to K- 16. thai has a serum half- life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding amino acid sequence according to any of aspects A-I to A- 22. B-I to B-7, C-I to C-4. D-I to D-6, E-I to E-13, F-I to F-25 or G-I per se or Nanobody® according to any of aspects H-I to H-22 per se, respectively.

Aspect K-18: Polypeptide according to any of aspects K-I O to K-17. that has a serum half- [0 life that is increased with more than 1 hours, preferably more than 2 hours. more preferably more than 6 hours, such as more than 12 hours, or even more than 24. 48 or 72 hours, compared to the corresponding amino acid sequence according to any of aspects A-I to A-22, B-I to B-7, C-I to C-4. D-I to D-6, E-I to E-13, F-I to F-25 or G-I per se or Nanobody® according to any of aspects H-I to H-22 per se, respectively.

Aspect K-19: Polypeptide according to any of aspects K-I to K-18, that has a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more: for example, of at least 5 days (such as about 5 to 10 days), preferably at least 9 20 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).

Aspect L-I : Compound or construct, that comprises or essentially consists of one or more amino acid sequences according to any of aspects A-I to A-22. B-I to B-7. C-I to C-4, D-I to D-6, E-I to E-13, F-I to F-25 or G-I and/or one or more Nanobodies® according to any of aspects H-I to H-22, and optionally further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more linkers.

30 Aspect L-2: Compound or construct according to aspects L-I, in which said one or more other groups, residues, moieties or binding units are amino acid sequences.

Aspect L-3: Compound or construct according to aspect L-I or L-2, in which said one or more linkers, if present, are one or more amino acid sequences. Aspect L-4: Compound or construct according to any of aspects L-I to L-3, in which said one or more other groups, residues, moieties or binding units are immunoglobulin sequences.

Aspect L-5: Compound or construct according to any of aspects L-I to L-4, in which said one or more other groups, residues, moieties or binding units are chosen from the group consisting of domain antibodies, amino acid sequences that are suitable for use as a domain antibody, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, "dAb'"s. amino acid sequences that are suitable for use as a dAb, or Nanobodies®. Aspect L-6: Compound or construct according to any of aspects L-I to L-5, in which said one or more amino acid sequences of the invention are immunoglobulin sequences.

Aspect L-7: Compound or construct according to any of aspects L-I to L-6, in which said one or more amino acid sequences of the invention are chosen from the group consisting of domain antibodies, amino acid sequences that are suitable for use as a domain antibody, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, "dAb"'s, amino acid sequences that are suitable for use as a dAb. or Nanobodies®.

Aspect L-8: Compound or construct, that comprises or essentially consists of one or more Nanobodies® according to any of aspects H-I to H-22 and in which said one or more other groups, residues, moieties or binding units are Nanobodies®.

Aspect L-9: Compound or construct according to any of aspects L-I to L-9, which is a multivalent construct.

Aspect L- 10: Compound or construct according to any of aspects L- 1 to L-IO, which is a multispecific construct.

Aspect L-I l: Compound or construct according to any of aspects L-I to L- 10, which has an increased half-life, compared to the corresponding amino acid sequence according to any of aspects A-I to A-22, B-I to B-7, C-I to C-4, D-I to D-6, E-I to E- 13, F-I to F-25 or G-I per se or Nanobody® according to any of aspects H-I to H-22 per se_? respectively.

Aspect L-12: Compound or construct according to aspect L-I to L-11 , in which said one or more other groups, residues, moieties or binding units provide the compound or construct with increased half-life, compared to the corresponding amino acid sequence according to any of aspects A-I to A-22, B-I to B-7, C-I to C-4, D-I to D-6, E-I to E-13, F-I to F-25 or G-I per se or Nanobody® according to any of aspects H-I to H-22 per se, respectively.

Aspect L-13 : Compound or construct according to aspect L-12. in which said one or more other groups, residues, moieties or binding units that provide the compound or construct with increased half-life is chosen from the group consisting of serum proteins or fragments thereof, binding units that can bind to serum proteins, an Fc portion, and small proteins or peptides that can bind to serum proteins.

Aspect L- 14: Compound or construct according to aspect L- 12 or L- 13, in which said one or more other groups, residues, moieties or binding units that provide the compound or construct with increased half-life is chosen from the group consisting of human serum albumin or fragments thereof.

Aspect L-15: Compound or construct according to any of aspects L- 12 to L- 14. in which said one or more other groups, residues, moieties or binding units that provides the compound or construct with increased half-life are chosen from the group consisting of binding units that can bind to serum albumin (such as human serum albumin) or a serum immunoglobulin (such as IgG).

Aspect L-16: Compound or construct according to any of aspects L-12 to L-14, in which said one or more other groups, residues, moieties or binding units that provides the compound or construct with increased half-life are chosen from the group consisting of domain antibodies, amino acid sequences that are suitable for use as a domain antibody, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, "dAb'"s . amino acid sequences that are suitable for use as a dAb. or Nanobodies® that can bind to serum albumin (such as human serum albumin) or a serum immunoglobulin (such as IgG).

Aspect L-17: Compound or construct according to any of aspects L-12 to L-14. in which said one or more other groups, residues, moieties or binding units that provides the compound or construct with increased half- life is a Nanobody® that can bind to serum albumin (such as human serum albumin) or a serum immunoglobulin (such as IgG).

Aspect L-18: Compound or construct according to any of aspects L-12 to L- 17, that has a serum half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding amino acid sequence according to any of aspects A-I to A-22, B-I to B-7, C-I to C-4, D-I to D-ό, E-I to E-13, F-I to F- 25 or G-I per se or Nanobody© according to any of aspects H-I to H-22 per se. respectively.

Aspect L-19: Compound or construct according to any of aspects L-12 to L-] 8, that has a serum half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence according to any of aspects A-I to A-22, B-I to B-7, C-I to C-4, D-I to D-6. E-I to E-13. F-I to F-25 or G-I per se or Nanobody® according to any of aspects H-I to H-22 per se, respectively.

Aspect L-20: Compound or construct according to any of aspects L-12 to L-19, that has a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more; for example, of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).

Aspect L-2Ϊ : Monovalent construct, comprising or essentially consisting of one amino acid sequence according to any of aspects A-I to A-22. B-I to B-7, C-I to C-4. D-I to D-6. E-I to E-13, F-I to F-25 or G-I and/or one Nanobody® according to any of aspects H-I to H-22.

Aspect L-22: Monovalent construct according to aspect L-21, in which said amino acid sequence of the invention is chosen from the group consisting of domain antibodies, amino acid sequences that are suitable for use as a domain antibody, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, "dAb"^'s, amino acid sequences that are suitable for use as a dAb. or Nanobodies®.

Aspect L-23: Monovalent construct, comprising or essentially consisting of one Nanobody® according to any of aspects H- ϊ to H-22. Aspect M-I : Nucleic acid or nucleotide sequence, that encodes an amino acid sequence according to any of aspects A-I to A-22. B-I to B-7, C-I to C-4, D-I to D-6, E-I to E-13. F-I to F-25 or G-I, a Nanobody® according to any of aspects H-I to H-22, a polypeptide according to any of aspects K-I to K-219, or a compound or construct according to any of aspects L-I to L-21 that is such that it can be obtained by expression of a nucleic acid or nucleotide sequence encoding the same, or a monovalent construct according to any of aspects L-22 or L-23.

Aspect M-2: Nucleic acid or nucleotide sequence according to aspect M-I, that is in the form of a genetic construct.

Aspect N-I : Host or host cell that expresses, or that under suitable circumstances is capable of expressing, an amino acid sequence according to any of aspects A-I to A- 22, B-I to B-7, C-I to C-4, D-I to D-6, E-I to E-13, F-I to F-25 or G-L a

Nanobody® according to any of aspects H-I to H-22, a polypeptide according to any of aspects K-I to K- 19, a compound or construct according to any of aspects L-I to L-21 that is such that it can be obtained by expression of a nucleic acid or nucleotide sequence encoding the same, or a monovalent construct according to any of aspects L-22 or L-23; and/or that comprises a nucleic acid or nucleotide sequence according to aspect M-I or a genetic construct according to aspect M-2.

Aspect O-l : Composition comprising at least one amino acid sequence according to any of aspects A-I to A-22, B-I to B-7, C-I to C-4, D-I to D-6, E-I to E-13, F-I to F- 25 or G-I, Nanobody® according to any of aspects H-I to H-22, polypeptide according to any of aspects K-I to K- 19, compound or construct according to any of aspects L-I to L-21, monovalent construct according to any of aspects L-22 or L-23, or nucleic acid or nucleotide sequence according to aspects M-] or M-2.

Aspect O-2: Composition according to aspect 0-1, which is a pharmaceutical composition.

Aspect 0-3 : Composition according to aspect 0-2, which is a pharmaceutical composition, that further comprises at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and that optionally comprises one or more further pharmaceutical! y active polypeptides and/or compounds. Aspect P-I : Method for producing an amino acid sequence according to any of aspects A-I to A-22, B-I to B-7, C-I to C-4, D-I to D-6, E-I to E-13, F-I to F-25 or G-I, a Nanobody® according to any of aspects H-I to H-22, a polypeptide according to any of aspects K-I to K-19, a compound or construct according to any of aspects L-I to L-21 that is such that it can be obtained by expression of a nucleic acid or nucleotide sequence encoding the same, or a monovalent construct according to any of aspects L-22 or L-23, said method at least comprising the steps of: a) expressing, in a suitable host cell or host organism or in another suitable expression system, a nucleic acid or nucleotide sequence according to aspect M-I, or a genetic construct according to aspect M-2; optionally followed by: b) isolating and/or purifying the amino acid sequence according to any of aspects A-I to A-22, B-I to B-7, C-I to C-4, D-I to D-6, E-I to E-13, F-I to F-25 or G-I , the Nanobody® according to any of aspects H-I to H-22, the polypeptide according to any of aspects K-I to K-19, the compound or construct according to any of aspects L-I to L-21, or the monovalent construct according to any of aspects L-22 or L-23 thus obtained.

Aspect P -2: Method for producing an amino acid sequence according to any of aspects A-I to A-22, B-I to B-7, C-I to C-4, D-I to D-6, E-I to E-13, F-I to F-25 or G-I, a Nanobody® according to any of aspects H-I to H-22, a polypeptide according to any of aspects K-I to K- 19, a compound or construct according to any of aspects L-I to L-21 that is such that it can be obtained by expression of a nucleic acid or nucleotide sequence encoding the same, or a monovalent construct according to any of aspects L-22 or L-23, said method at least comprising the steps of: a) cultivating and/or maintaining a host or host cell according to aspect N-I under conditions that are such that said host or host cell expresses and/or produces at least one amino acid sequence according to any of aspects A- 1 to A-22₅ B-I to B-7, C-I to C-4, D-I to D-6, E-I to E-13, F-I to F-25 or G-I, Nanobody® according to any of aspects H-I to H-22, polypeptide according to any of aspects K-I to K- 19, compound or construct according to any of aspects L-I to L-21, or monovalent construct according to any of aspects L-22 or L~23; optionally followed by: b) isolating and/or purifying the amino acid sequence according to any of aspects A-I to A-22, B-I to B-7, C-I to C-4, D-I to D-6, E-I to E-13, F-I to F-25 or G-I, Nanobody® according to any of aspects H-I to H-22, polypeptide according to any of aspects K-I to K- 19, compound or construct according to any of aspects L-I to L-21, or monovalent construct according to any of aspects L-22 or L-23 thus obtained.

Aspect Q-I: Method for screening amino acid sequences directed against sclerostin that comprises at least the steps of: a) providing a set, collection or library of nucleic acid sequences encoding amino acid sequences; b) screening said set collection or library of nucleic acid sequences for nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for sclerostin and that is cross-blocked or is cross blocking a Nanobody® of the invention, e.g. SEQ ID NO: 189 to 191

(Table- 1); and c) isolating said nucleic acid sequence, followed by expressing said amino acid sequence.

Aspect R-I : Method for the prevention and/or treatment of at least one bone disease and/or disorder, said method comprising administering to a subject in need thereof a pharmaceutically active amount of at least one amino acid sequence according to any of aspects A-I to A-22, B-I to B-7, C-I to C-4, D-I to D-6, E-I to E-13, F-I to F-25 or G-I, Nanobody® according to any of aspects H-I to H-22, polypeptide according to any of aspects K-I to K- 19, compound or construct according to any of aspects L-I to L-21, monovalent construct according to any of aspects L-22 or L-23; or composition according to aspect O-2 or 0-3. Aspect R-2: Method for the prevention and/or treatment of at least one disease or disorder that is associated with sclerostin, with its biological or pharmacological activity, and/or with the biological pathways or signalling in which sclerostin is involved, said method comprising administering to a subject in need thereof a pharmaceutically active amount of at least one amino acid sequence according to any of aspects A-I to A-22, B-I to B-7, C-I to C-4, D-I to D-6, E-I to E-13, F-I to F-25 or G-I, Nanobody® according to any of aspects H-I to H-22, polypeptide according to any of aspects K-I to K- 19, compound or construct according to any of aspects L-I to L-21, monovalent construct according to any of aspects L-22 or L-23; or composition according to aspect 0-2 or 0-3. Aspect R- 3 : Method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering to a subject in need thereof at least one amino acid sequence according to any of aspects A-I to A- 22. B-I to B-7. C-I to C-4, D-I to D-ό, E-I to E-13, F-I to F-25 or G-I. Nanobody® according to any of aspects H-I to H- 22, polypeptide according to any of aspects K-I to K- 19, compound or construct according to any of aspects L- 1 to L-21. monovalent construct according to any of aspects L-22 or L-23; or composition according to aspect O-2 or 0-3, said method comprising administering to a subject in need thereof a pharmaceutically active amount of at least one at least one amino acid sequence according to any of aspects A-I to A-22, B-I to B-7, C-I to C-4, D-I to D-6, E-I to E-13, F-I to F-25 or G-I, Nanobody® according to any of aspects H-I to H- 22, polypeptide according to any of aspects K-I to K- 19, compound or construct according to any of aspects

L-I to L-21, monovalent construct according to any of aspects L-22 or L-23; or composition according to aspect 0-2 or 0-3.

Aspect R.-4: Method for immunotherapy, said method comprising administering to a subject in need thereof a pharmaceutically active amount of at least one amino acid sequence according to any of aspects A-I to A-22, B-I to B-7. C-I to C-4, D-I to D-6, E-I to E-13, F-I to F-25 or G-L Nanobody® according to any of aspects H-I to H-22. polypeptide according to any of aspects K-I to K- 19, compound or construct according to any of aspects L-I to L-21, monovalent construct according to any of aspects L-22 or L-23; or composition according to aspect 0-2 or 0-3.

Aspect R-5: Use of an amino acid sequence according to any of aspects A-I to A-22, B-I to B-7, C-I to C-4, D-I to D-6. E-I to E-13, F-I to F-25 or G-I, a Nanobody® according to any of aspects H-I to H-22. a polypeptide according to any of aspects K-I to K-19, a compound or construct according to any of aspects L-I to L-21, or a monovalent construct according to any of aspects L-22 or L-23 in the preparation of a pharmaceutical composition for prevention and/or treatment of at least one bone disease and/or disorder: and/or for use in one or more of the methods according to aspects R-I to R-4. Aspect R-6: Amino acid sequence according to any of aspects A-I to A-22, B-I to B-7, C-I to C-4. D-I to D-6, E-I to E-13, F-I to F-25 or G-I, Nanobody® according to any of aspects H-I to H-22, polypeptide according to any of aspects K-I to K- 19. compound or construct according to any of aspects L-I to L-21, monovalent construct according to any of aspects L-22 or L-23, or composition according to aspect 0-2 or 0-3 for the prevention and/or treatment of at least one bone disease and/or disorder,

Aspect S-I : Part or fragment of an amino acid sequence according to any of aspects A- 1 to A-22, B-I to B-7, C-I to C-4, D-I to D-6, E-I to E-13, F-I to F-25 or G-L or of a Nanobody® according to any of aspects H-I to H-22.

Aspect S-2: Part or fragment according to aspect S-I, that can specifically bind to sclero stin. Aspect S-3: Part of fragment according to any of aspects S-I or S-2, that can specifically bind to sclerostin with a dissociation constant (K_D) of 10^"5 to 10^'12 moles/litre or less, and preferably 10^~7 to 10^'12 moles/litre or less and more preferably 10^'8 to 10^"12 moles/litre. Aspect S-4: Part or fragment according to any of aspects S-I to S-3, that can specifically bind to sclerostin with a rate of association (Ic_0n -rate) of between 10² M^"'s^~l to about 10⁷ M^"Y\ preferably between W M^-1S^"1 and 10⁷ M^-1S^"1. more preferably between 10⁴ IVT' S^"1 and 10⁷ M^"V\ such as between 10⁵ IvT¹S^"1 and 10⁷ M^-1S^"1. Aspect S-5: Part or fragment according to any of aspects S-I to S-4, that can specifically bind to sclerostin with a rate of dissociation (karate) between 1 s^"! and 10^"6 s^"! preferably between K)^"2 s^"1 and 10^~6 s^"1. more preferably between 10^""1 s^"! and

10^"6 s^'1. such as between 10^"4 s^"! and 1(T⁶ s^"!. Aspect S-6: Compound or construct, that comprises or essentially consists of one or more parts or fragments according to any of aspects S-] to S-4. and optionally further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more linkers. Aspect S -7: Compound or construct according to aspect S-6. in which said one or more other groups, residues, moieties or binding units are amino acid sequences. Aspect S-8: Compound or construct according to aspect S-6 or S-7, in which said one or more linkers, if present, are one or more amino acid sequences. Aspect S-9: Nucleic acid or nucleotide sequence, that encodes a part or fragment according to any of aspects S-I to S-7 or a compound or construct according to aspect S- 8.

Aspect S-10: Composition, comprising at least one part or fragment according to any of aspects S-I to S-7, compound or construct according to any of aspects S-6 to S- 8, or nucleic acid or nucleotide sequence according to aspect S-9. Aspect T-I : Derivative of an amino acid sequence according to any of aspects A-I to A-22, B-I to B-7, C-I to C-4, D-I to D~6, E-I to E-13, F-I to F-25 or G-I. or of a Nanobody® according to any of aspects H-I to H- 22.

Aspect T-2: Derivative according to aspect T-L that can specifically bind to sclerostin.

Aspect T-3 : Derivative according to any of aspects T-I or T-2, that can specifically bind to sclerostin with a dissociation constant (Kj)) of 10^"5 to 10^~12 moles/litre or less, and preferably 10^"7 to 10^~12 moles/litre or less and more preferably 10^~s to IG^'12 moles/litre.

Aspect T-4: Derivative according to any of aspects T-I to T-3, that can specifically bind to scierostin with a rate of association (k_on-rate) of between 10² NfV¹ to about 10⁷ MV, preferably between IG³ M^'V¹ and 10⁷ M^-1S⁴, more preferably between IG⁴ M^-1S^'1 and 10⁷ M^-1S^"1. such as between IQ⁵ M^"'s^"' and 10⁷ M^'V¹.

Aspect T-5: Derivative according to any of aspects T-I to T-4, that can specifically bind to sclerostin with a rate of dissociation (k_off rate) between 1 s^"1 and 10^"6 s^"1 preferably between 10 v^"2" „ s-^"1 and 10 i-^"6 „ s-^" . more preferably between 10 ,-^"3^J s -^"1 and 10^"6 s^"1, such as between 10^"4 s^"1 and 10^"6 s^"1.

Aspect T-6: Derivative of a polypeptide according to any of aspects K-I to K-19 or compound or construct according to any of aspects L-I to L-23.

Aspect T-7: Derivative according to aspect T-6, that can specifically bind to sclerostin. Aspect T- 8: Derivative according to any of aspects T-6 or T-7. that can specifically bind to sclerostin with a dissociation constant (K_D) of 10^" to 10^"12 moles/liter or less, and preferably 10^'7 to 10^"12 moles/liter or less and more preferably 10^"8 to 10^"12 moles/liter.

Aspect T-9: Derivative according to any of aspects T-6 to T-8, that can specifically bind to sclerostin with a rate of association (k_on-rate) of between 10² NfV¹ to about 10⁷ M^-1S^"1, preferably between 3 O³ IVT¹S^"1 and 10⁷ M^'V, more preferably between 10⁴ M^"'s^"' and 10⁷ NT's^"1, such as between 10⁵ MV¹ and 10⁷ M^"V^].

Aspect T-10: Derivative according to any of aspects T-6 to T-9, that can specifically bind to sclerostin with a rate of dissociation (k_Off rate) between 1 s^{" 1} and 10^"6 s^"1 preferably between 10^"2 s^"! and 10^" s^"1, more preferably between 10^"3 s^"1 and

10 i-^"6° „ S-^"1¹, such as between I (T -4 s . -^"1¹ and 10 -i-^"6° s -1. Aspect T-I l : Derivative according to any of aspects T-I to T-IO₅ that has a serum half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding amino acid sequence according to any of aspects A-I to A-22, B-I to B-7, C-I to C-4, D-I to D-6. E-I to E-13, F-I to F-25 or G-I per se, Nanobody® according to any of aspects H-I to H-22 per se, polypeptide according to any of aspects K-I to K-19 or compound or construct according to any of aspects L-I to L-23 per se.

Aspect T-12: Derivative according to any of aspects T-I to T-1 1 , that has a serum half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence according to any of aspects A-I to A-22, B-I to B-7. C-I to C-4, D-I to D-6, E-I to E-13, F-I to F-25 or G-I per se, Nanobody® according to any of aspects H-I to H-23 per se, polypeptide according to any of aspects K-I to K-19 or compound or construct according to any of aspects L-I to L-23 per se, respectively.

Aspect T-13: Derivative according to any of aspects T-I to T-12, that has a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more; for example, at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 1 1 days (such as about 1 1 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).

Aspect T-14: Derivative according to any of aspects T-I to T-13, that is a pegylated derivative.

Aspect T-15 : Compound or construct, that comprises or essentially consists of one or more derivatives according to any of aspects T-I to T-14, and optionally further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more linkers. Aspect T-16: Compound or construct according to aspect T-15, in which said one or more other groups, residues, moieties or binding units are amino acid sequences.

Aspect T-17: Compound or construct according to aspect T-16. in which said one or more linkers, if present, are one or more amino acid sequences.

Aspect T-18: Nucleic acid encoding a compound or construct according to aspect T-16 or T- 17.

Aspect T-19: Composition, comprising at least one derivative to any of aspects T-I to T-14. compound or construct according to any of aspects T- 15 to T-17. or nucleic acid or nucleotide sequence according to aspect T- 18,

Figure Legends:

Figure 1. Human sclera stin phage binding assay for a selection of clones. Negative controls are addition of irrelevant phage selected against a viral antigen (GPA2E6), no phage addition and non coated protein.

Figure 2. Human sclera stin P. E. binding assay for a selection of clones. Negative controls are addition of irrelevant P.E. selected against a viral antigen (GPA2E6), no P.E. addition and non coated protein.

Figure 3. Binding of purified Nanobodies® to human sclerostin. Negative control is an irrelevant Nanobody® selected against a viral antigen (GPA2E6).

Figure 4. Binding of 40 iiM of purified Nanobodies® to human and mouse sclerostin. Negative control is an irrelevant Nanobody® selected against a viral antigen (GPA2E6), no Nanobody® addition and non coated protein.

Figure 5. Purified Nanobodies® binding to mouse sclerostin. Negative control are an irrelevant Nanobody® selected against a viral antigen (GPA2E6). no Nanobody® added (Bl) and non-coated protein.

Examples:

A. Identification of Nanobodies® binding sclerostin

Example 1: Animal immunizations Four llamas (294, 295. 296 and 297) were immunized, according Io standard protocols, with 4 boosts of a cocktail containing recombinant human sclerostin (R&D Systems Cat No 1406-ST/CF, Lot HMH120804A). This recombinant protein was obtained from a DNA sequence encoding the mature human SOST (Sclerostin). Amino acid residues 24-213 of sclerostin were fused to the signal peptide of human CD33 and a seven-histidine tag at the N- terminus and expressed in a mouse myeloma cell line. NSO (see SEQ ID NO: 199 herein and Balemans, W. et al. 2001, Hum. MoI. Genet. 10(5):537-543; Brunkow, M.E. et al, 2001, Am. J. Hum. Genet. 68(3):577-589). Blood was collected from these animals 4 and 8 days after boost 4.

Example 2: Library construction

Peripheral blood mononuclear cells were prepared from blood samples using Ficoll- Hypaque according to the manufacturer's instructions. Next, total RNA was extracted from these cells and used as starting material for RT-PCR to amplify Nanobody® encoding gene fragments. These fragments were cloned into an expression vector derived from pUC119 which contained the LacZ promoter, a coliphage pill protein coding sequence, a resistance gene for ampicillin or carbenicillin. a multicloning site and the gen3 leader sequence. In frame with the Nanobody® coding sequence, the vector coded for a C-terminal c-myc tag and a (His)6 tag.. Phages were prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein).

Example 3: Selections of phage displaying human sclerostin binding Nanobodies®

Phage libraries 294, 295, 296 and 297 were used for selections on recombinant human sclerostin (R&D Systems Cat No 1406-ST/CF. Lot HMH 120804 A).

Sclerostin was immobilized directly on Maxisorp 96 well microliter plates (Nunc) at 5 ug/ml, 0.5 μg/ml and 0 μg/mJ (control). Following incubation with the phage libraries and extensive washing, bound phages were totally eluted with trypsin. The eluted phage were amplified and applied in a second round of selection on 2 μg/ml, 0.2 μg/ml, 0.02 μg/ml and 0 μg/ml (control) immobilized sclerostin. Individual colonies of E. coli TGl infected with the obtained eluted phage pools were grown and i) induced for new phage production and ii) induced with IPTG for Nanobody® expression and extraction (periplasmic extracts) according to standard methods (see for example the prior art and applications filed by applicant cited herein). Example 4: Screening for human selerostiπ binding Nanobodies®

In order to determine binding specificity to human sclerostin. the clones were tested in an ELISA binding assay setup, using the monoclonal phage pools or periplasmic extracts (P.E.). Phage binding to recombinant human sclerostin (R&D Systems Cat No 1406-ST/CF. Lot HMH120804A) were tested. Briefly, 1 μg/ml of protein was immobilized on Maxisorp ELISA plates (Nunc) and free binding sites were blocked using 4% Marvel skimmed milk in PBS. Next, 10 ul of supernatant from the monoclonal phage inductions of the different clones in 100 ul 2% Marvel PBS were allowed to bind to the immobilized antigen. After incubation and a wash step, phage binding was revealed using a HRP-conjugated monoclonal -anti-M 13 antibody (Gentaur Cat# 27942101 ). Figure 1 shows a selection of phage binding to human sclerostin. Binding specificity was determined based on OD values compared Io controls having received irrelevant (GPA2E6) or no phage.

In the case of P.E. a mouse anti myc antibody (Roche. Cat 1 1667149001) and an anti mouse immunoglobulins-AP antibody (SIGMA A2429) was used to detect binding. Binding specificity was determined based on OD values compared to controls contacted with irrelevant or no phage or P.E. or Nanobody®. Figure 2 shows a selection of P.E. binding to human sclerostin.

Example S: Determination! of binding potency of purified Nanobodies® to human scSerostin

Nanobodies® were produced and purified as follows: 250 ml exponential growth cultures were induced with IPTG for four hours. The bacteria were pelleted and Iysed by freeze-thawing. The pellet was resuspended in PBS and centrifuged. The Nanobodies® were purified from the supernatant containing the periplasmic fraction by using affinity chromatography columns (His Trap™ FF crude (GE Healthcare, 1 1-0004-58) and a desalting column (HiPrep™ 26/10. Code no 17-508701) in the AKTAXpress system (GE Healthcare). Purity and concentration of the purified Nanobodies® were determined by SDS-PAGE analysis. In order to determine the potency of binding, the purified Nanobodies® were tested at different concentration for binding Io immbolized human sclerostin. Briefly, 1 μg/ml of protein was immobilized on Maxisorp ELISA plates (Nunc) and free binding sites were blocked using 4% Marvel skimmed milk in PBS. Next, different amounts of purified Nanobodies® in 100 ul 2% Marvel PBS were allowed to bind to the immobilized antigen. After incubation and some wash steps, bound Nanobodies® were revealed using a mouse anti myc antibody (Roche, Cat 11667149001), and a rabbit anti mouse immunoglobulins-AP antibody (SIGMA A2429). Binding specificity was determined based on OD values compared to the irrelevant controJ (GPA2E6). According to the results shown in Figure 3 some Nanobodies® can be classified as the highest affinity ones (3-C8, 3-D7 and 3-Bl), with roughly estimated binding 1C50 values of around 3-6 iiM; moderate affinity Nanobodies® (2- F6. 6-E12 and 4-H2) with binding IC50 values up to 20 nM and the lowest affinity Nanobodies® (1-Fl, 3-Fl and 7-G5). with binding IC50 values over 20 nM.

Example 6: Screening for Nanobodies® binding mouse scierostin

To test if the Nanobodies® binding human sclerostin are also recognizing mouse sclerostin an ELISA was performed as follows: in parallel 1 μg/ml of human scierostin (R&D Systems Cat No 1406-ST/CF. Lot HMH120804A) and 1 μg/ml of mouse sclerostin (R&D Systems Cat No 1589-ST/CF, Lot IUL0407041) were immobilized on Maxisorp ELISA plates (Nunc) and free binding sites were blocked using 4% Marvel skimmed milk in PBS. Next. 40 nM of purified Nanobodies® in 100 μl 2% Marvel PBS were allowed to bind to the immobilized antigens. After incubation and some wash steps, bound Nanobodies® were revealed using a mouse anti myc antibody (Roche. Cat 11667149001), and an anti mouse immunoglobulins-AP antibody (SIGMA A2429). Binding specificity was determined based on OD values compared to controls having received irrelevant or no Nanobody®. Figure 4 shows the obtained results. Nanobodies® 3-D7, 3-Bl, 3-Fl and 6-E12 clearly bind both human and mouse SOST. Nanobodies® 4-H2. 1 -Fl and 7-G5, although cross reactive, seem to bind better the human than the mouse protein. Human SOST Nanobodies® 3-C8 and 2-Fό are not mouse cross- reactive.

Nanobodies® against human sclerostin that showed mouse cross reactivity were used in an ELISA to determine the potency of binding to the mouse protein. 1 μg/ml of mouse sclerostin (R&D Systems Cat No 1589-ST/CF, Lot IUL0407041) was immobilized on Maxisorp ELISA plates (Nunc) and free binding sites were blocked using 4% Marvel skimmed milk in PBS. Next, different amounts of purified Nanobodies® in 100 μl 2% Marvel PBS were allowed to bind to the immobilized antigens. After incubation and some wash steps, bound Nanobodies® were revealed using a mouse anti myc antibody, and a rabbit anti mouse immunoglobulins-HRP antibody. Binding specificity was determined based on OD values compared to controls having received irrelevant or no Nanobody®. SOST Nanobodies® 3~D7, 3-Bl, 3-Fl and 6-E12 are clearly human-mouse cross reactive and present binding IC50s values between 5-1 OnM for mouse sclerostin (mSOST). Nanobodies® 4-H2. 1-Fl and 7-G5 are cross reactive between the two proteins but in much less extent, presenting lC50s much higher in what refers to mSOST (note that althought testing a concentration of 10OnM it is not possible to determine an approximate IC 50 value for these Nanobodies®. Nanobodies® 3-C8 and 2-F6 are not cross-reactive with the mouse protein as observed from the results presented in Figure 5.

Claims

1. An amino acid sequence that is directed against and/or that can specifically bind to sclerostin comprising one or more stretches of amino acid residues chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO's: 135 to 143; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 135 to 143; c) amino acid sequences that have 3, 2. or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO^'s: 135 to 143; d) the amino acid sequences of SEQ ID NO's: 153 to 161 ; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 153 to 161; f) amino acid sequences that have 3. 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 153 to 161 ; g) the amino acid sequences of SEQ ID NO^'s: 171 to 179: h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO^"s: 171 to 179: i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 171 to 179; or any suitable combination thereof.

2. The amino acid sequence according to claim 1 , in which said one or more stretches of amino acid residues have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentiall} 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 189 to 197,

3. The amino acid sequence according to claim 1 or 2 mat cross-blocks the binding of at least one of the amino acid sequences of SEQ ID NO^'s: 1 89 to 197 to sclerostin.

4. The amino acid sequence according to any of claims 1 to 3 that is cross-blocked from binding to sclerostin by at least one of the amino acid sequences of SEQ ID NO^'s: 189 to 197.

5. An amino acid sequence according to any of claims 1 to 4, that essentially consists of a Nanobody® that i) has at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 1 to 22 and 189 to 197. in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded; and in which: ii) preferably one or more of the amino acid residues at positions 11. 37, 44, 45, 47, 83, 84, 103. 104 and 108 according to the Kabat numbering are chosen from the

Hallmark residues mentioned in Table B-2.

6. Nanobody® according claim 5. which is a partially or fully humanized Nanobody®.

7. Nanobody® according to claim 6, that is chosen from the group consisting of SEQ ID NO^'s: 189 to 197 or from the group consisting of from amino acid sequences that have more than 80%, preferably more than 90%. more preferably more than 95%. such as 99% or more sequence identity (as defined herein) with at least one of the amino acid sequences of SEQ ID NO^'s: 189 to 197.

8. Polypeptide that comprises or essentially consists of one or more amino acid sequences according to any of claims 1 to 5 and/or one or more Nanobodies® according to any of claims 6 or 7, and optionally further comprises one or more peptidic linkers, other groups or residues and/or other binding units.

9. Polypeptide according to claim 8, which has an increased half-life, compared to the corresponding amino acid sequence according to any of claims 1 Io 5 per se or Nanobody® according to any of claims 6 or 7 per se, respectively.

10. Nucleic acid or nucleotide sequence, that encodes an amino acid sequence according to any of claims 1 to 5, a Nanobody® according to any of claims 6 or 7, or a polypeptide according to any of claims 8 or 9, optionally in the forma of a genetic construct.

11. Host or host cell that expresses, or that under suitable circumstances is capable of expressing, an amino acid sequence according to any of claims 1 to 5, aNanobody® according to any of claims 6 or 7. a polypeptide according to any of claims 8 or 9; and/or that comprises a nucleic acid or nucleotide sequence according to claim 10.

12. A composition comprising at least one amino acid sequence according to any of claims 1 to 5, at least one Nanobody® according to any of claims 6 or 7 and/or a polypeptide according to any of claims 8 or 9.

13. The composition according to claim 10. which is a pharmaceutical composition, which further comprises at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally comprises one or more further pharmaceutically active polypeptides and/or compounds.

14. Method for producing an amino acid sequence according to any of claims 1 to 5, a Nanobody® according to any of claims 6 or 7. a polypeptide according to any of claims 8 or 9. said method at least comprising the steps of: a) expressing, in a suitable host cell or host organism or in another suitable expression system, a nucleic acid or nucleotide sequence or a genetic construct according to claim 10; or cultivating and/or maintaining a host or host cell according to claim 1 1 under conditions that are such that said host or host cell expresses and/or produces at least one amino acid sequence according to any of claims 1 to 5. Nanobody® according to any of claims 6 or 7, polypeptide according to any of claims 8 or 9; optionally followed by: b) isolating and/or purifying the amino acid sequence according to any of claims 1 to 5, the Nanobody® according to any of claims 6 or 7, the polypeptide according to any of claims 8 or 9 thus obtained.

15. Method for the prevention and/or treatment of at least one bone disease and/or disorder, said method comprising administering to a subject in need thereof a pharmaceutically active amount of at least one amino acid sequence according to any of claims 1 to 5, Nanobody® according Io any of claims 6 or 7, polypeptide according to any of claims 8 or 9, or composition according to any of claims 12 or 13.

16. Use of an amino acid sequence according to any of claims 1 to 5, a Nanobody® according to any of claims 6 or 7, or a polypeptide according to any of claims 8 or 9 in the preparation of a pharmaceutical composition for prevention and/or treatment of at least one bone disease and/or disorder; and/or for use in the method according to claim 15.

17. Amino acid sequence according to any of claims 1 to 5. Nanobody® according to any of claims 6 or 7, polypeptide according to any of claims 8 or 9, or composition according to any of claims 12 or 13 for the prevention and/or treatment of at least one bone disease and/or disorder.