HK1198768B - Gdf-5 mutant for inducing cartilage formation - Google Patents

Description

GDF-5 mutants for inducing chondrogenesis

Description of the invention

The present invention relates to GDF-5 related proteins having improved ability to induce cartilage formation and reduced ability to induce bone formation. The novel proteins are particularly useful in the treatment of cartilage defects where the formation of bone tissue is undesirable.

Synovial joints are essential for the biomechanical function of the bone. Abnormal function as observed in arthritic disease directly leads to a severe reduction in quality of life. Therefore, joint biology has been focused on a wide range of studies over the years, allowing us to understand joint anatomy and histology, as well as the biomechanical properties and roles of articular cartilage and other components in joint function and maintenance.

GDF-5(Et al 1994, biochem. biophysis Res. Commun.204,646-652) are morphogens that have been shown to promote cell proliferation, differentiation and/or tissue formation in a variety of tissues. The protein is also known as morphogenetic protein MP52, bone morphogenetic protein-14 (BMP-14) or cartilage derived morphogenetic protein-1 (CDMP-1). GDF-5 shows chondrogenic activity and congenital GDF-5 mutations lead to finger or toe (digit), wrist and ankle defects in mice and humans (Storm et al, 1994; Thomas et al, 1997). GDF-5 expression is most significantly restricted to the area where joints will develop and is one of the earliest hallmarks of joint formation (Storm and Kingsley, 1999). BMP receptor signaling is required for postnatal maintenance of articular cartilage (Rountree,2004, PLoS biol.2004November,2 (11)).

GDF-5 is closely related to GDF-6 and GDF-7, these three proteins form a unique subset of the TGF- β superfamily, showing comparable biological properties and an ultrahigh degree of amino acid sequence identity (see Wolfman et al 1997, j. clin. invest.100, 321-330.) all family members were initially synthesized as larger precursor proteins which subsequently undergo proteolytic cleavage at a set of basic residues about 110-140 amino acids from the C-terminus, releasing the C-terminal mature protein moiety from the N-terminal prodomain.

Members of the GDF-5/-6/-7 subgroup have been repeatedly demonstrated to be important inducers and regulators of bone and cartilage in the first place (Cheng et al 2003, J.bone & Joint Surg.85A, 1544-1552; Settle et al 2003, development. biol.254, 116-130). GDF-5 and related proteins bind to and oligomerize two types of membrane-bound serine-threonine kinase receptors (referred to as type I and type II). Upon ligand binding, these complexes transduce signals by phosphorylating members of the SMAD family of transcription factors that, when activated, enter the nucleus and regulate transcription of responsive genes (Massague, 1996). Recent experiments have involved two different type I receptors in bone type, BMPR-IA and BMPR-IB. Both receptors are expressed in a dynamic pattern during normal development. Overlapping expression of BMPR-IA and BMPR-IB is observed in several limb structures, e.g., in the interarticular zone and perichondrium (Mishina et al, 1995; Zou et al, 1997; Baur et al, 2000). For BMPR-IA and BMPR-IB expression patterns, GDF-5 signaling should be achieved by interaction with both BMPR-IA and BMPR-IB (Chang et al, 1994; Zou et al, 1997). Null mutations in the bmpr-1b gene produce viable mice defective in bone and joint formation, very close to those seen in GDF-5 deficient mice (Storm and Kingsley, 1996; Yi et al, 2000), whereas bmpr-la/mice are known to die early in embryogenesis (Mishina et al, 1995). However, conditional knockdown of BMPR-IA under control of the GDF-5-Cre driver circumvented embryonic death and produced surviving mice with normally formed joints. But after birth, articular cartilage in the joints wears out in a process similar to osteoarthritis, indicating the importance of this receptor in cartilage homeostasis and repair (roundree et al, 2004).

The activity of wild-type proteins of the GDF-5 related protein family often leads to cartilage and bone formation. However, there are different medical conditions in which cartilage formation is desired, however, bone tissue formation is not desired. For example, it is evident that in the case of joint defects, cartilage formation is desired and ossification should be avoided.

Therefore, it is an object of the present invention to specifically use the chondrogenic and bone formation inducing effects of GDF-5 related proteins and to shut down the bone formation inducing effects. Surprisingly, it was found possible to provide variants of GDF-5 related proteins with improved capacity to induce cartilage formation and reduced capacity to induce bone formation. This may be achieved by modifying the GDF-5 related protein such that the GDF-5 related protein has an increased affinity for BMPR-IB and/or a decreased affinity for BMPR-IA.

Wild-type GDF-5 binds BMPR-IB in vitro with affinity to BMPR-IA (K)_D1-1.1nM) is 40 to 120 times higher (K) than_D8-27 pM). It was found that cartilage formation is promoted and bone formation is reduced by modifying the binding affinity of the GDF-5 related protein such that the affinity for BMPR-IB is increased and the affinity for BMPR-IA is decreased. This may be achieved by specific substitution of one or more amino acid residues associated with the BMPR-IB and/or BMPR-IA binding site in the amino acid sequence of the GDF-5 related protein.

The binding affinity of the GDF-5 related protein with the specific substitution is compared with the binding affinity of a human wild-type GDF-5 related protein, in particular with the binding affinity of human wild-type GDF-5.

To avoid misunderstandings and ambiguities, some commonly used terms herein are defined and exemplified as follows:

the term "cystine knot domain" as used herein refers to the well-known and conserved cysteine-rich amino acid region that is present in the mature portion of a TGF- β superfamily protein, such as human GDF-5, and forms a three-dimensional protein structure known as a cystine knot, in which the cysteine residues are in relative position to each other and only slightly changed so as not to lose biological activity, the cystine knot domain alone has been shown to be sufficient for the biological function of the protein (Schreuder et al (2005), Biochem Biophys Res Commun.329, 1076-86). the consensus sequence of the cystine knot domain is well known in the art.

The term "GDF-5 related protein" as used herein refers to any naturally occurring or artificially created protein that is very closely related to human growth/differentiation factor 5 (hdgf-5). A common feature of all GFD-5 related proteins is that the emerging cystine-knot domain has at least 60% amino acid identity with the 102 amino acid cystine-knot domain of human GDF-5 (amino acids 400-501 of SEQ ID NO:2), which is sufficient for the biological function of the protein. The term "GDF-5 related protein" includes proteins belonging to the group of GDF-5, GDF-6 and GDF-7 proteins from vertebrate or mammalian species, as well as recombinant variants thereof, provided that these proteins exhibit the above-mentioned percentage of identity with the cystine-knot domain of human GDF-5. A limit of 60% is well suited for separating members of the GDF-5/-6/-7 group of proteins and variants thereof from other proteins such as more distantly related GDFs and BMPs. Comparison of the 102 amino acid cystine knot domains of human GDF-5, human GDF-6 and human GDF-7 (see FIG. 2) shows a high degree of amino acid identity between these proteins. Human GDF-6 shares 87 (85%) identical residues with the cystine-knot domain of human GDF-5, and human GDF-7 shares 83 (81%) identical residues with the cystine-knot domain of human GDF-5. The corresponding domains of GDF-5/-6/-7 molecules from other vertebrate and mammalian species, as determined so far, also show a very high percentage of identity of at least 75% (79% to 99%) when compared to human GDF-5. In contrast, GDF and BMP that do not belong to the GDF-5/-6/-7 subgroup show much lower identity below 60%.

The determination of the corresponding amino acid positions in the relevant amino acid sequences and the calculation of the percent identity can be readily carried out by means of well-known alignment algorithms and optionally computer programs using these algorithms. For example, amino acid identity in this patent application (i.e., FIG. 2) has been calculated by aligning sequences using the free program ClustalX (version 1.81) with default parameters and performing subsequent identical residue counts manually. Default settings for the two-sequence alignment (slow-exact) are: vacancy opening parameters: 10.00; a vacancy-extension parameter of 0.10; protein weight matrix: gonnet 250. ClustalX programs are described in detail in Thompson, J.D., Gibson, T.J., Plewniak.F., Jeanmougin.F., and Higgins.D.G. (1997): the ClustalX windows interface: flexible protocols for multiple sequence alignment aided by quality analysis tools, nucleic Acids Research24: 4876-4882. ClustalX is a windows interface to the ClustalW multiple sequence alignment program and is available from a variety of sources, namely by anonymous ftp from ftp-igbmc.u-strasbg.fr, ftp.embl-heidelberg.de, ftp.ebi.ac.uk or by download from the following web pages: http:// www-igbmc.u-strasbg.fr/Biolnfo/. ClustalW programs and algorithms are also described in detail in Thompson, J.D., Higgins, D.G., and Gibson, T.J, (1994): CLUSTALW: optimizing the sensitivity of progressive multiple sequence alignment through sequence alignment, positioning-specific gap polarities and weight matrix gradient, nucleic acids Research22: 4673-4680. Particularly preferred GDF-5 related proteins exhibit at least 70%, 80%, 90% or 95% amino acid identity with the 102 amino acid cystine knot domain of human GDF-5.

Non-limiting examples of vertebrate and mammalian GDF-5-related proteins are the precursor and mature proteins of human GDF-5 (disclosed as MP52 in WO95/04819 and as human GDF-5 in Hotten et al 1994, biochem. Biophys Res. Commun.204,646-652), recombinant human (rh) GDF-5/MP52(W096/33215), MP52Arg (WO 97/06254); HMW human MP52(WO97/04095), CDMP-1(WO96/14335), mouse (mus musculus) GDF-5(US5,801,014), rabbit (Oryctolagus cuniculus) GDF-5(Sanyal et al 2000, Mol Biotechnol.16,203-210), chicken (Gallus (Gallus garlus)) GDF-5(NCBI accession number NP-989669), Xenopus laevis (Xenopus laevis)) GDF-5(NCBI accession number AAT99303), monomeric GDF-5(WO01/11041 and WO99/61611), human GDF-6/BMP-13 (U.S. Pat. No. 5,658,882), mouse GDF-6(NCBI accession No. NP-038554), GDF-6/CDMP-2(WO96/14335), human GDF-7/BMP-12 (U.S. Pat. No. 5,658,882), mouse GDF-7(NCBI accession No. AAP97721), GDF-7/CDMP-3(WO 96/143335). Also encompassed by the present invention are GDF-5 related proteins having additional mutations, such as substitutions, additions and deletions, provided that these additional mutations do not completely disrupt the activity of the biological protein.

The invention is based on the findings of the inventors: it is possible to alter proteins in such a way that they have an improved ability to induce cartilage formation and a reduced ability to induce bone formation, by specific modifications in the region of the amino acid sequence involved in GDF-5 related proteins binding to BMPR-IB and/or BMPR-IA.

Proteins with increased affinity for BMPR-IB and/or proteins with reduced affinity for BMPR-IA have been found to be more useful for inducing cartilage formation while reducing bone formation. These properties are particularly evident in proteins that exhibit both increased affinity for BMPR-IB and decreased affinity for BMPR-IA.

The GDF-5 related proteins of the present invention may be obtained by chemical modification or genetic engineering techniques, preferably recombinant proteins. The protein may be obtained by replacing at least one amino acid residue in the amino acid sequence of the GDF-5 related protein which is involved in the binding site for BMPR-IB and/or BMPR-IA. In particular, it is preferred to replace one, two, three or more amino acid residues associated with the BMPR-IB binding site and/or the BMPR-IA binding site in the amino acid sequence of the GDF-5 related protein.

The above modifications may be introduced into any known GDF-5 related protein as defined above. For the therapeutic use of the protein, it is preferred to derive the protein from a human GDF-5 related protein, for example from a human wild-type GDF-5 related protein such as GDF-5, GDF-6 or GDF-7 derived protein. However, the proteins of the invention may also be derived from GDF-5 related proteins with additional mutations, such as substitutions, additions or deletions, as long as these additional mutations do not completely destroy the biological protein activity.

A GDF-5 related protein as defined herein comprises a cystine-knot domain having at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 90% and most preferably at least 95% amino acid identity with the 102 amino acid cystine-knot domain of human GDF-5.

The GDF-5 related proteins of the invention preferably comprise one or more amino acid substitutions in the region involved in binding to BMPR-IB and/or in the region involved in binding to BMPR-IA compared to the wild type. The regions involved in GDF-5 related proteins that bind to BMPR-IA and/or BMPR-IB are well known in the art or can be readily determined using methods within the common general knowledge.

With reference to the full-length amino acid sequence of GDF-5 wild type, it is particularly preferred to replace one or more of the following amino acids (in single letter code) with any different amino acid:

R399；

any one of F409 to W417, preferably M412, G413, W414 and/or W417;

any one of E434 to M456, preferably F435, P436, L437, R438, S439, H440, P443, N445, V448, I449, L452, M453, S455 and/or M456;

S475；

I476；

F478；

any one of K488 to M493, preferably K488, Y490 and/or D492.

Preferably, amino acid R399 is replaced with V, L, I, M, F, Y, W, E or D.

Preferably, amino acid M412 is replaced with V, L, I, F, Y, W, H, K or R.

Preferably, amino acid W414 is replaced with R, K, F, Y, H, E or D.

Preferably, amino acid W417 is replaced with R, K, F, Y, H, E or D.

Preferably, amino acid F435 is replaced with V, L, I, M, P, Y, W, H, K or R.

Preferably, amino acid P436 is replaced with V, L, I, M, F, Y or W.

Preferably, amino acid L437 is replaced with D or E.

Preferably, amino acid R438 is replaced with K, D, H, N, M, E, Q, S, T, Y or W.

Preferably, amino acid S439 is replaced with K, D, E, H, R, M, T, N, Q, Y or W.

Preferably, amino acid H440 is replaced with V, I, M, F, Y, W, E or D.

Preferably, amino acid P443 is replaced with V, L, I, M, F, Y, W, A or S.

Preferably, amino acid N445 is replaced with D, Q, H, F, L, R, K, M, S, Y or W.

Preferably, amino acid V448 is replaced by F, L, I, M, P, Y or W.

Preferably, amino acid I449 is replaced by F, L, V, M, P, Y or W.

Preferably, amino acid L452 is replaced with F, I, V, M, P, Y or W.

Preferably, amino acid M456 is replaced with F, I, L, P, Y, W, S, T, N, Q, K or D.

Preferably, amino acid S475 is replaced with M, T, N, Q, Y or W.

Preferably, amino acid K488 is replaced with R, M, S, T, N, Q, Y or W.

Preferably, amino acid Y490 is replaced by E, H, K, R, Q, F, T, M, S, N, Q or W.

Preferably, amino acid D492 is replaced with G, E, M, S, T, N, Q, Y, W, H, K or R.

Preferably, amino acid I476 is replaced by G, A, V, L, M, F, Y or W.

Preferably, amino acid F478 is replaced with G, A, V, L, I, Y or W.

The corresponding positions in the amino acid sequences of different GDF-5 related proteins can be readily derived from the above information regarding wild-type GDF-5.

According to a first embodiment, at least one hydrophobic amino acid in the BMPR-IB and/or BMPR-IA binding site of a GDF-5 related protein is replaced by a hydrophilic or polar amino acid. Examples of hydrophilic or polar amino acid residues are aspartic acid, glutamic acid, lysine, arginine, histidine, serine and threonine.

According to a second embodiment, at least one hydrophilic or polar amino acid in the BMPR-IB and/or BMPR-IA binding site of a GDF-5 related protein is replaced by a hydrophobic amino acid. Examples of hydrophobic amino acids are alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine.

According to another preferred embodiment, the protein of the invention comprises a conservative substitution of at least one amino acid in the BMPR-IB and/or BMPR-IA binding site of a GDF-5 related protein. This means that the properties of the amino acids originally present are retained. Thus, one hydrophilic or polar amino acid is replaced by another hydrophilic or polar amino acid or one hydrophobic amino acid is replaced by another hydrophobic amino acid.

Preferably, conservative substitutions are selected in such a way that one amino acid is exchanged for another with a different spatial requirement. According to this aspect of the invention, hydrophobic amino acids may be replaced with smaller or larger hydrophobic amino acids or hydrophilic or polar amino acids may be replaced with smaller or larger hydrophilic or polar amino acids.

Amino acid substitutions in GDF-5 related proteins can be grouped into 4 groups according to amino acid properties:

I. replacement of basic amino acid residue (R, K, H) with

a) Hydrophobic (V, L, I, M, P, F, Y, W)

b) Acidity (E, D)

c) A basic amino acid residue other than I (R, K, H)

d) Polarity (S, T, N, Q).

Replacement of an acidic amino acid residue (D) by

a) Hydrophobic (M, Y, W, G)

b) Acidity (E)

c) Basic (R, K, H)

d) Polarity (S, T, N, Q)

Replacement of hydrophobic amino acid residue (M, V, L, I, P, F, Y, W, A) by

a) A hydrophobic amino acid residue other than III (M, V, L, I, P, F, Y, W, G, A)

b) Acidity (E, D)

c) Basic (R, K, H)

d) Polarity (S, T, N, Q)

d) Small (A).

Substitution of polar amino acid residue (S, T, N)

a) Hydrophobic (M, V, L, I, P, F, Y, W)

b) Acidity (E, D)

c) Basic (R, K, H)

d) A polar amino acid residue other than IV (S, T, N, Q)

In a preferred embodiment, the GDF-5 related protein of the invention comprises a sequence matching one of the following amino acid sequences:

a)

ZCX₁X₂KX₃LHVX₄ZZZZZZZZZX₇IAPLX₈YEAX₉HCX₁₀GX₁₁CZZZZZZZZZZZZZ

ZZZZZZZZZZX₁₃PX₁₄X₁₅X₁₆PX₁₇X₁₈CCVPX₁₉X₂₀LX₂₁PIZILX₂₂X₂₃DX₂₄X₂₅NNW

YZZZZZZWEX₂₇CGCR or

b)

ZCX₁X₂KX₃LHVX₄FX₅X₆ZZZDDZX₇IAPLX₈YEAX₉HCX₁₀GX₁₁CX₁₂ZZZZZZLEZ

TZHAZZQTZZNZZX₁₃PX₁₄X₁₅X₁₆PX₁₇X₁₈CCVPX₁₉X₂₀LX₂₁PIZILX₂₂X₂₃DX₂₄X₂₅N

NVVYZX₂₆ZZZMWEX2₇CGCR

And wherein

Each X represents an arbitrary amino acid, and each X represents,

each Z represents any amino acid.

These universal sequences are compiled by comparison of the cystine knot domains of vertebrate GDF-5, GDF-6 and GDF-7 sequences. Positions that are not conserved in the aligned proteins are denoted as X in the universal sequence. The position of the mutation according to the invention is denoted as Z.

In a more preferred embodiment, the GDF-5 related protein of the present invention comprises a sequence matching one of the above-mentioned universal amino acid sequences, and wherein

X₁Represents asparagine (N) or serine(s)

X₂Represents arginine (R) or lysine (K)

X₃Represents alanine (A), glutamine (Q), proline (P) or serine (S)

X₄Represents arginine (R) or lysine (K)

X₅Represents aspartic acid (D) or glutamic acid (E)

X₆Represents leucine (L) or methionine (M)

X₇Represents isoleucine (I) or valine (V)

X₈Represents aspartic acid (D) or glutamic acid (E)

X₉Represents histidine (H), phenylalanine (F) or tyrosine (Y)

X₁₀Represents aspartic acid (D) or glutamic acid (E)

X₁₁Represents leucine (L)1 methionine (M) or valine (V)

X₁₂Represents aspartic acid (D) or glutamic acid (E)

X₁₃Represents alanine (A), asparagine (N) or aspartic acid (D)

X₁₄Represents arginine (R), asparagine (N), aspartic acid (D), glutamic acid (E), glycine (G) or serine (S)

X₁₅Represents alanine (A), asparagine (N), serine (S) or threonine (T)

X₁₆Represents alanine (A), methionine (M) or threonine (T)

X₁₇Represents alanine (A) or proline (P)

X₁₈Represents serine (S) or threonine (T)

X₁₉Represents alanine (A), serine (S) or threonine (T)

X₂₀Represents arginine (R) or lysine (K)

X₂₁Represents serine (S) or threonine (T)

X₂₂Represents phenylalanine (F) or tyrosine (Y)

X₂₃Represents isoleucine (I) or threonine (T)

X₂₄Represents alanine (A) or serine (S)

X₂₅Represents alanine (A) or glycine (G)

X₂₆Represents glutamic acid (E) or glutamine (Q)

X₂₇Represents alanine (A), glutamine (Q), serine (S) or threonine (T) and

each Z represents any amino acid.

In a particular embodiment, the GDF-5 related protein of the invention is derived from wild-type GDF-5. According to this particular aspect, the GDF-5 related protein comprises a sequence matching one of the following general amino acid sequences

a)

ZCSRKALHVNZZZZZZZZZIIAPLEYEAFHCEGLCZZZZZZZZZZZZZZZZZZZ

ZZZZDPESTPPTCCVPTRLSPIZILFIDSANNVVYZZZZZZWESCGCR

b)

ZCSRKALHVNFKDZZZDDZIIAPLEYEAFHCEGLCEZZZZZZLEZTZHAZZQT

ZZNZZDPESTPPTCCVPTRLSPIZILFIDSANNWYZQZZZMWESCGCR，

Wherein each Z represents any amino acid.

An example of a protein as described above is a variant of human GDF-5 whereby the tryptophan residue at position 414 is exchanged for arginine (W414R). Reference is made to the mature sequence of GDF-5(SEQ ID NO:4), which corresponds to the substitution at position 33. Surprisingly, it was found that this protein variant has a greatly reduced affinity for BMPR-IA. In contrast, the affinity for BMPR-IB was almost unaffected. Also preferred are other variants of the GDF-5 related protein comprising an amino acid substitution different from W414R.

An example of such a variant of a GDF-5 related protein is a variant of human GDF-5, whereby the isoleucine residue at position 449 is exchanged for valine (I449V). Reference is made to the mature sequence of GDF-5(SEQ ID NO:4), which corresponds to the substitution at position 68. The protein variants have reduced affinity for BMPR-IA and increased affinity for BMPR-IB.

Additional exemplary variants of a GDF-5 related protein include the amino acid substitution R399E. Reference is made to the mature sequence of GDF-5(SEQ ID NO:4), which corresponds to a substitution at position 18. The protein variants have reduced affinity for BMPR-IA.

Yet another exemplary variant of human GDF-5 is a variant whereby the serine residue at position 439 is exchanged for glutamic acid (S439E). Reference is made to the mature sequence of GDF-5(SEQ ID NO:4), which corresponds to the substitution at position 58. The protein variants also have reduced affinity for BMPR-IA.

Another exemplary variant of human GDF-5 is a variant whereby the arginine residue at position 399 is exchanged for methionine (R399M). Reference is made to the mature sequence of GDF-5(SEQ ID NO:4), which corresponds to a substitution at position 18. The protein variants have greatly increased affinity for BMPR-IB.

Preferably, the GDF-5 related protein of the invention is present as an "isolated" protein. This means that the proteins of the invention are substantially separated from other protein and peptide molecules present in the natural source of the isolated protein (e.g., other polypeptides of the protein of natural origin). For example, a recombinantly expressed peptide is considered isolated. According to a preferred embodiment of the invention, the GDF-5 related protein is a recombinant protein. In addition, a peptide is also considered isolated if it has been altered by human intervention or expressed by an organism that is not the natural source of the peptide. Furthermore, an "isolated" protein is free of some other cellular material or cell culture medium with which it is naturally associated when prepared by recombinant techniques, or free of chemical precursors or other chemicals when chemically synthesized. Unpurified mixtures or compositions are specifically not included in the definition of "isolated" proteins.

According to another embodiment, the invention relates to a nucleic acid encoding a protein according to the invention. The nucleic acid has a sequence such that substitution of one or more amino acid residues associated with the BMPR-IB and/or BMPR-IA binding site of the corresponding wild-type GDF-5 associated protein is effected. The base triplets and the degeneracy of the genetic code (degeneracy) which code for these amino acids are generally known. The nucleic acid may be a DNA sequence and/or an RNA sequence, as long as the protein according to the invention is obtainable from the nucleic acid when expressed in a suitable system. The nucleic acids of the invention may be wholly or partially synthetic. Nucleic acids include single-stranded and/or fully or partially double-stranded polynucleotide sequences. Nucleic acids may be prepared by any means, including genomic preparation, cDNA preparation, in vitro synthesis, PCR, RT-PCR, and/or in vitro or in vivo transcription.

Particularly preferred are "isolated" nucleic acids that are substantially separated from nucleic acid molecules (e.g., sequences encoding other polypeptides) that are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5 'and 3' ends of the nucleic acid) in at least some of its naturally occurring replicons. For example, cloned nucleic acids are considered isolated. A nucleic acid is also considered isolated if it has been altered or placed by human intervention at a locus or location that is not its natural side or if it is introduced into a cell. In addition, an isolated nucleic acid may be free of some other cellular material or culture medium with which it is naturally associated when prepared by recombinant techniques, or free of chemical precursors or other chemicals when chemically synthesized.

In a preferred manner, the nucleic acids of the invention can be prepared by whole gene synthesis or by site-directed mutagenesis (site-directed mutagenesis) of nucleic acids encoding wild-type or modified GDF-5-related proteins. Methods that may be utilized include template directed ligation, circular PCR, cassette mutagenesis, site directed mutagenesis, or other techniques well known in the art.

The nucleic acids of the invention may comprise additional nucleic acid sequences that may add additional functionality to the isolated nucleic acids of the invention. For example, such additional nucleic acid sequences may include nucleic acid sequences that allow for proper expression of the proteins of the invention, and may include promoter sequences, regulatory sequences, termination signals, origins of replication, and the like. The skilled person is well aware of such functional nucleic acid sequences and of how to arrange them to obtain nucleic acid molecules with the desired properties.

Expression vectors are a further subject of the invention, in which the nucleic acid is inserted into a suitable vector system, which is selected according to the desired protein expression. The vector system may be a eukaryotic vector system but preferably a prokaryotic vector system, by means of which the protein can be prepared in a particularly easy and simple manner. Suitable expression vectors are shown, for example, in WO 96/33215. The expression vector may also be a viral vector, which may be used, for example, in gene therapy methods.

Host cells and transgenic organisms are also subjects of the present invention. The host cells and transgenic organisms are characterized in that they contain a nucleic acid or an expression vector according to the invention and in that they are able to use the information present in the nucleic acid and in the expression vector, respectively, for expressing a protein according to the invention. The invention therefore relates to transgenic organisms or cells which are transiently or stably transformed or transfected with at least one nucleic acid or at least one vector encoding a protein according to the invention, or to the progeny of such transgenic organisms or cells. Furthermore, the invention relates to parts of the cells, cell cultures, tissues and/or transgenic organisms of the invention. It is understood that for the purposes of the present invention, the term "transgenic organism" not only includes organisms which have transiently or stably introduced the nucleic acids of the invention, but also refers to the progeny of such organisms, irrespective of the age, as long as they still comprise the nucleic acids of the invention and express the proteins of the invention.

Preferably, the transgenic organism or cell is of prokaryotic or eukaryotic origin. Preferably, the transgenic organism is a microorganism. Preferred microorganisms are bacteria, yeasts, algae or fungi. Suitable host cells are preferably prokaryotic cells, in particular strains of escherichia coli (e. Particularly useful host cells are E.coli W3110 defensins (safedants), as shown, for example, in WO 96/33215. In a preferred embodiment, host cells, especially of human origin, may also be useful for transplantation into a patient in need thereof.

The preparation of a transformed organism or a transformed cell requires the introduction of the appropriate DNA into an appropriate host organism or cell. A variety of methods can be used for this process, which is referred to as transformation. Thus, by way of example, DNA may be introduced directly by microinjection or by bombardment with DNA-coated micro-or nanoparticles. The cells may also be permeabilized chemically, for example using polyethylene glycol, so that the DNA can enter the cells by diffusion. DNA can also be transformed by protoplast fusion with other DNA-containing units such as minicells, cells, lysosomes, or liposomes. Another suitable method for introducing DNA is electroporation, in which cells are reversibly permeabilized by an electrical pulse.

Another subject of the present invention is a process for the preparation of a protein having an improved capacity to induce cartilage formation and a reduced capacity to induce bone formation, comprising the steps of:

(i) randomizing at least one amino acid position in a GDF-5 related protein region involved in the binding site of BMPR-IB and/or BMPR-IA to obtain a protein variant,

(ii) (ii) the protein variant obtained in (i) is analysed with respect to its affinity for BMPR-IB and/or BMPR-IA,

(iii) selecting those protein variants that provide enhanced affinity for BMPR-IB and/or reduced affinity for BMPR-IA.

Regions involved in GDF-5 related proteins binding to BMPR-IA or BMPR-IB are known in the art. In step (i), at least one amino acid position in one or both of these regions is randomized. Preferably at least two, three or more amino acid positions are randomized. Amino acids present in the wild-type sequence of a GDF-5 related protein are replaced by other amino acids by chemical modification or preferably by genetic engineering techniques. The method for preparing the randomized protein variant of step (i) comprises synthetic de novo synthesis of the protein and/or expression of the protein from the nucleic acid encoding it. In a particularly preferred manner, the protein variant of step (i) is prepared by expression using the corresponding nucleic acid.

Preferably, protein variants are obtained for all other possible amino acids at the relevant positions. However, it is also possible to replace only one or more amino acids specifically by other amino acids. For example, hydrophilic amino acids may be replaced with hydrophobic amino acids. Alternatively, hydrophobic amino acids may be replaced with hydrophilic amino acids. Conservative substitutions are also possible in which hydrophilic or hydrophobic properties are retained. By substitution, exchange of amino acids with another spatial requirement is preferably effected.

Subsequently, the plurality of protein variants obtained in step (i) is analyzed with respect to their affinity for BMPR-IB and/or for BMPR-IA. This can be done in a manner known and common in the art. Methods for assessing protein-receptor interactions are common practice.

In step (iii), those protein variants are selected which provide an increased affinity for BMPR-IB and/or a decreased affinity for BMPR-IA. It has surprisingly been found that these specific proteins have an improved capacity to induce cartilage formation and a reduced capacity to induce bone formation.

Another subject matter of the invention relates to antibodies directed against the GDF-5 related proteins of the invention. These antibodies are specific for the claimed recombinant GDF-5 related proteins. Preferably, the antibody is specific for a region of the GDF-5 related protein that contains one or more amino acid substitutions as described herein. Preferably, the antibody is specific for a region of a recombinant protein derived from a GDF-5 related protein associated with a BMPR-IB and/or BMPR-IA binding site. These antibodies according to the present invention can be produced by using antibodies produced by a known methodSuch as those described above, are produced as immunogens. The antibody may be monoclonal or polyclonal and it may be of any isotype. Also included are antibody fragments such as Fab fragments or Fab₂And (3) fragment. The antibody may be a humanized antibody, a genetically engineered antibody, or the like.

The antibodies of the invention are particularly suitable for use as analytical tools. The antibodies can be used to study the absorption and distribution of the proteins according to the invention in vivo. Furthermore, the above antibodies are suitable for studying release kinetics.

A further subject of the present application is a pharmaceutical composition comprising a recombinant GDF-5 related protein or nucleic acid or vector or host cell according to the invention. In principle, any of the disclosed pharmaceutical compositions with a GDF-5 related protein are suitable. The expression vector or host cell may be considered advantageous as an active substance in a pharmaceutical composition. Combinations of the protein according to the invention with other proteins may also be used in preferred pharmaceutical compositions. Of course, the invention also encompasses pharmaceutical compositions containing additional substances, such as pharmacologically acceptable additives or carriers. The formulations may include antioxidants, preservatives, coloring, flavoring and emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffering agents, delivery vehicles, excipients, and/or pharmaceutical adjuvants. For example, a suitable carrier or vehicle can be water for injection, physiological saline solution, or saline solution mixed with a suitable carrier protein such as serum albumin. A preferred antioxidant for use in preparing the compositions of the present invention is ascorbic acid.

The solvent or diluent of the pharmaceutical composition may be aqueous or non-aqueous, and may contain other pharmaceutically acceptable excipients capable of altering and/or maintaining the pH, osmolarity, viscosity, clarity, ratio (scale), sterility, stability, dissolution rate, or odor of the formulation. Similarly, other components may be included in the pharmaceutical compositions of the present invention to alter and/or maintain the release rate of the pharmaceutically effective agent. Such modifying components are those commonly employed in the art to formulate dosages for parenteral administration in unit dosage form or in multiple dosage form.

The finally formulated pharmaceutical compositions prepared according to the present invention may be stored in sterile vials in the form of solutions, suspensions, gels, emulsions, solids or dehydrated or lyophilized powders. These formulations may be stored in a ready-to-use form or in a form that requires reconstitution prior to administration (e.g., in the case of a lyophilized powder). The above and additional suitable Pharmaceutical formulations are known in the art and are described, for example, in Gus Remington's Pharmaceutical Sciences (18th ed., Mack Publishing co., Eastern, Pa.,1990, 1435-1712). Such formulations may affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the pharmaceutically effective component.

Other effective forms of administration include parenteral slow release (i.e. delayed) formulations, inhalation mist or orally active formulations. For example, sustained release formulations may include proteins conjugated to or incorporated into microparticulate formulations of polymers (e.g., polylactic acid, polyglycolic acid, etc.) or liposomes.

The pharmaceutical compositions according to the invention may also be formulated for parenteral administration, for example by infusion or injection, and may also include sustained release or sustained circulation formulations. Such parenterally administered therapeutic compositions are typically in the form of pyrogen-free parenterally acceptable aqueous solutions comprising the pharmaceutically effective component(s) in a pharmaceutically acceptable carrier and/or diluent.

The pharmaceutical composition may comprise a matrix material, for example in the case where cartilage is intended to be regenerated. It is advantageous for proteins, nucleic acids, expression vectors or host cells when applied in and/or on a biocompatible matrix material. Matrix material as used herein refers to a carrier or matrix that serves as a scaffold for cell recruitment, attachment, proliferation and differentiation and/or serves as a potential delivery and storage device for recombinant GDF-5 related proteins of the invention. In contrast to solid matrices, carriers are composed of amorphous materials that do not have a defined surface and lack a specific shape, i.e., alkylcelluloses, pluronics, gelatin, polyethylene glycols, dextrins, vegetable oils, sugars, and other liquid and viscous substances.

Exemplary matrix materials are described, for example, in WO 98/21972. These matrix materials are equally suitable for the proteins according to the invention. The matrix material may be surgically implanted into a patient, for example, where the protein or DNA encoding the protein may be slowly released from the matrix material and subsequently available for a longer period of time. All types of matrix materials according to the invention are useful as long as they are biocompatible and selected for the intended area of use or indication. The matrix material may be a natural material, a modified natural material, and a synthetic material. Including all known matrices for morphogenic proteins. Extracellular matrices include, for example, various collagens, such as types I, II, V, IX, X, XI, and XIII; additional proteoglycans and glycosaminoglycans, such as chondroitin sulfate, biglycan, decorin and/or hyaluronic acid; or non-collagenous proteins such as osteopontin, laminin, fibronectin, vitronectin, and cartilage matrix protein. All mentioned natural materials can also be used in artificially modified form. For non-limiting examples of useful carriers and matrices, see further Kirker-Head,2000, Advanced Drug Delivery43, 65-92.

A further subject of the invention relates to a liposomal formulation comprising a recombinant GDF-5 related protein according to the invention. Liposomes for use in the formulations are well known to those skilled in the art. In particular, preferred liposome formulations are disclosed in WO 2008/049588. More preferred liposome formulations are described on pages 9 to 13 of WO 2008/049588.

In addition, the GDF-5 related proteins of the present invention may be administered in combination with other pharmaceutically active substances. The pharmaceutically active substance may be, for example, an analgesic, such as a locally effective analgesic; or other substances having a positive effect on diseases in which cartilage formation is desired, such as protease inhibitors. These are only examples of possible additives and a worker skilled in the art can readily add other excipients for pharmaceutical formulations or generally recognized as safe.

Due to their improved ability to induce chondrogenesis, the recombinant GDF-5-related proteins of the invention are particularly useful in the treatment of diseases where chondrogenesis is desired but bone formation is not. Thus, another aspect of the invention is the use of a protein, nucleic acid, vector or host cell of the invention in the treatment of these diseases. In particular, the protein, nucleic acid, vector or host cell of the invention is used for the treatment of cartilage defects or for the treatment of traumatic rupture or detachment of cartilage, in particular age-related cartilage defects, such as age-related cartilage defects due to wear, osteoarthritis, rheumatoid arthritis, sports-related injuries; diseases that can affect cartilage, such as cartilage dystrophy; diseases characterized by interference with cartilage growth and subsequent ossification; achondroplasia; costal chondritis; disc herniation and disc repair; recurrent polychondritis; repair of cartilage defects associated with benign or malignant tumors, such as chondroma or chondrosarcoma.

Another embodiment of the invention is a method for the treatment of a disease wherein cartilage formation is desired but bone formation is not desired, comprising the step of administering to a patient in need of such treatment a protein, nucleic acid, vector or host cell according to the invention.

As used herein, the term "treating" or "treatment" refers to reversing, alleviating or inhibiting the progression of a disease, disorder or condition or one or more symptoms of such a disease, disorder or condition to which the term applies. As used herein, treating can also refer to reducing the likelihood or incidence of the occurrence of a disease, disorder or condition in a mammal as compared to an untreated control population or as compared to the same mammal prior to treatment. For example, as used herein, treating may refer to preventing a disease, disorder, or condition, and may include delaying or preventing the onset of a disease, disorder, or condition, or delaying or preventing symptoms associated with a disease, disorder, or condition. As used herein, treating may also refer to reducing the severity of a disease, disorder or condition, or symptoms associated with such a disease, disorder or condition, prior to the onset of such a disease, disorder or condition in a mammal. Such prevention or reduction of the severity of a disease, disorder or condition prior to a disease involves administering to a subject who is not suffering from the disease, disorder or condition at the time of administration a composition of the invention as described herein. As used herein, treatment may also refer to prevention of recurrence of a disease, disorder, or condition, or one or more symptoms associated with such a disease, disorder, or condition.

The following examples together with the figures and sequence schemes are intended to further illustrate the invention.

SEQ ID NO1 shows the DNA sequence of the human GDF-5 precursor, while SEQ ID NO 2 shows the protein sequence of the human GDF-5 precursor.

SEQ ID NO 3 shows the DNA sequence of human mature monomer GDF-5, while SEQ ID NO 4 shows the protein sequence of human mature monomer GDF-5.

Drawings

FIG. 1 shows additional features of the human GDF-5 precursor protein according to SEQ ID NO: 2:

amino acid 001-381 prepro domain (pre-prodomain) (bold letter)

Amino acid 001-027 Signal peptide (bold and underlined)

Amino acids 382-501 mature protein portion

Amino acid 400-501 cystine knot domain (underlined)

FIG. 2 shows a comparison of the 102 amino acid cystine knot domains of human GDF-5(SEQ ID NO:2), human GDF-6 (sequence from U.S. Pat. No. 5,658,882) and human GDF-7 (sequence 2 from U.S. Pat. No. 5,658,882). Amino acid residues that are identical in all three molecules are highlighted by the border.

FIG. 3 shows the results of alkaline phosphatase Assay (ALP) using recombinant human GDF-5 mutant W414R (as described in example 2).

FIG. 4 shows the results of alkaline phosphatase Assay (ALP) of recombinant human GDF-5 mutant I449V (as described in example 3).

FIG. 5 shows the results of alkaline phosphatase Assay (ALP) of recombinant human GDF-5 mutant R399E (as described in example 3).

FIG. 6 shows the results of alkaline phosphatase Assay (ALP) of recombinant human GDF-5 mutant S439E (as described in example 3).

FIG. 7 shows the results of alkaline phosphatase Assay (ALP) of recombinant human GDF-5 mutant R399M (as described in example 3).

FIG. 8 shows the results of alkaline phosphatase Assay (ALP) of recombinant human GDF-5 mutant W414R (as described in example 3).

Example 1: production, expression and purification of GDF-related proteins

The DNA encoding the mature portions of human GDF-5, human GDF-6 and human GDF-7 proteins was isolated from human ROB-C26 bone precursor cells by RT-PCR (Yamaguchi et al 1991, Calcif. tissue int.49,221-225) and subsequently ligated into prokaryotic plasmid vectors. To determine the functionally important amino acid residues of the mature part of GDF-5, -6 and-7, various single mutations were introduced into these sequences by site-directed mutagenesis.

All individual mutations were generated using the QuickChange site-directed mutagenesis kit according to the manufacturer's instructions, with PfuTurboTm DNA polymerase and DPN1 endonuclease from Stratagene.

Proteins were expressed in inclusion bodies using the plasmid transformed and IPTG induced bacterial strain W3110 BP. These inclusion bodies were separated using homogenization buffer (25mM Tris HCI pH7.3,10mM EDTA NaOH pH8,8M Urea) and wash buffer (1M Urea, 20mM Tris HCI, pH8.3,10mM EDTA NaOH pH8.0) according to standard procedures. On a reverse phase column Aquapore Octyl (Applied Biosys, (CV ═ 7,8ml)100 × 10,20 μ, No186470) over 104 minutes (flow rate: 3ml/min) a gradient was used from 100% eluent a (0.1% TFA, HPLC H₂O) eluent B (0.1% TFA, 90% CH) to 100%₃N，HPLC H₂O) for further purification. After western blot control, fractions containing the mutant proteins were pooled and lyophilized.

The mutein was dissolved in lysis buffer (6M guanidine hydrochloride, 50M Tris,150mM NaCl,3mM DTT, pH8.0), the protein concentration was adjusted to exactly 2.6mg/ml and the pH was adjusted to 8 to 9. After incubation at room temperature for 2 hours, renaturation (refolding) buffer (1M NaCl,50mM Tris,5mM EDTA,1mM GSSG,2mM gsh,33mM Chaps, pH 9.5) was added with gentle stirring to reach a final concentration of 0.16 mg/ml.

The solution was then incubated at 22 ℃ for 48h and renaturation was stopped by changing the pH to 3-4 by addition of 18% HCl. After centrifugation, the non-renatured monomer was separated from the dimeric form by performing a second RP-HPLC under the same conditions. The fractions containing the dimerized protein were pooled, lyophilized and stored at-70 ℃.

Example 2: measurement of biological activity of different variants of GDF-related proteins in vitro by ALP analysis

2.0x10⁵C2C12-lb cells (cell line stably overexpressing BMPR-IB receptor) and C2C12 cells at 37 deg.C and 5% CO₂、H₂incubate for 3-4 days in 20ml cell culture medium (α -MEM, penicillin/streptomycin, 2mM L-glutamine, 10% FCS) at O-saturation the cells were then washed with PBS (phosphate buffered saline), trypsinized and resuspended in medium to 3X10⁴Density of individual cells/ml. Transfer 150. mu.l to each well of a 96-well plate and incubate at 37 ℃ with 5% CO₂、H₂Incubate 24h under O-saturation. After washing with media, wells were filled with 120 μ l of fresh media. Mu.l of different dilutions of the mutein or wild-type protein (dissolved in 10mM HCl and diluted at least 250-fold with medium) were added, followed by 5% CO at 37 ℃₂、H₂Another incubation step was performed for 72h at O-saturation. After washing with PBS, 150. mu.l of lysis buffer (0.2% NonidetP40, 0.2g MgCl) was added₂x6H₂O, adjusted to 1000ml with water), followed by 5% CO at 37 ℃₂、H₂Incubate for 15-18h under O-saturation. Thereafter, 50. mu.l of each well was transferred to a new 96-well plate. Then 50. mu.l of substrate solution (2.5 Xconcentrated diethanolamine substrate buffer +148g/l PNPP (sodium p-nitrophenyl phosphate)) was added to each well and 5% CO at 37 ℃₂、H₂Plates were incubated for 4min at O-saturation. The ALP reaction was then stopped using 100. mu.l of 30g/l NaOH and finally the optical density was measured at 405nm using an automatic microplate reader taking into account blank value subtraction.

As an example, the results (average of 2 independent experiments) for the hGDF-5 mutant W414R for C2C12-lb cells are shown in FIG. 3. Five different protein concentrations (14ng/mL, 44.5ng/mL, 133.2ng/mL, 400ng/mL and 1200ng/mL) were used in this assay. The mutein W414R showed biological activity in cells overexpressing the BMPR-IB receptor (C2C12-lb cells), indicating that the BMPR-IB binding site of W414R is functionally active. The wild type protein (rhGDF-5) served as a control in the assay system.

Additional results of the biological activity of additional hdgf-5 mutants of cell lines C2C12 and C2C12-lb are shown in table 1.

Example 3: measurement of biological activity of different variants of GDF-related proteins in vitro by ALP analysis

5x10⁵ATDC-5 cells and 5x10⁵MCHT1/26 cells at 37 ℃ with 5% CO₂、H₂incubation in 20ml cell culture medium (α -MEM,2mM L-glutamine, 10% FCS for MCHT 1/26; DMEM/F12(1:1), 5% FCS) at O-saturation for 3-4 days, followed by washing the cells with PBS (phosphate buffered saline), trypsinization and resuspension in the medium to 3X10⁴Density of individual cells/ml. Transfer 150. mu.l to each well of a 96-well plate and incubate at 37 ℃ with 5% CO₂、H₂Incubate 24h under O-saturation. After washing with media, wells were filled with 120. mu.l of fresh media for MCHT1/26 and 120. mu.l for ATDC-5Medium (DMEM/F12(1:1), 0.5% FCS). Mu.l of different dilutions of the mutein or wild-type protein (dissolved in 10mM HCl and diluted at least 250-fold with medium) were added, followed by 5% CO at 37 ℃₂、H₂Another incubation step was performed for 72h at O-saturation. After washing with PBS, 150. mu.l of lysis buffer (MCHT1/26 lysis buffer: 0.2% Nonidet P40,1mM MgCI) were added₂(ii) a ATDC-5 lysate: 100mM sodium glycinate, 1% Nonidet P40,1mM MgCl₂) Followed by 5% CO at 37 ℃₂、H₂Incubation with ATDC-5 for 1h and MCHT1/26 for 15-18h at O-saturation. Subsequently 50 μ l of each well was transferred to a new 96-well plate. Then 50. mu.l of substrate solution (2.5 Xconcentrated diethanolamine substrate buffer +148g/l PNPP (sodium p-nitrophenyl phosphate)) was added to each well and 5% CO at 37 ℃₂、H₂Plates were incubated for a further 60min under O-saturation. The ALP reaction was then stopped using 100. mu.l of 30g/l NaOH and finally the optical density was measured at 405nm using an automatic microplate reader taking into account blank value subtraction.

Exemplary results (average of 2 independent experiments) for hdf-5 mutant I449V, R399E, S439E, R399M, W414R are shown in fig. 4-8, respectively. Five different protein concentrations (14.8ng/ml, 44.5ng/ml, 133.2ng/ml, 400ng/ml, 1200ng/ml) were used in the assay.

The muteins showed higher biological activity in this assay system on ATDC-5 cells than on MCHT1/26 cells compared to wild-type GDF-5.

Example 4: biacore affinity measurements of GDF-5 related proteins

The Biacore t100 system (Biacore, GE Healthcare, Chalfont st. giles, GB) was used for all biosensor experiments. Approximately 200 Resonance Units (RU) of the extracellular domain of the Fc-fusion protein receptor of BMPR-IB, BMPR-IA or BMPR-II were immobilized on a protein G CM5 biosensor chip. The interaction sensorgrams were recorded at a flow rate of 60. mu.l/min in 10mM HEPES (pH7.4), 300mM NaCl, 3.4mM EDTA, 0.005% Tween 20 at 30 ℃. Experiments were performed in duplicate using ligand concentrations of 0.05 to 100 nM. All apparent binding affinities were obtained using BIAevaluation v.2.2.4(Biacore, ge healthcare, Chalfont st.giles, GB). The affinity of ligand type I receptor interactions was deduced by fitting kinetic data into a 1:1Langmuir binding model (KD (kin)). Since the binding kinetics are too fast (over 106M-1s-1 (for kon) and 10-2s-1 (for koff)), for the ligand: the apparent binding affinity of the BMPR-11 interaction was determined by the dose dependence of equilibrium binding (KD (eq)).

The results of Biacore affinity measurements for different variants of human GDF-5 are shown in table 1.

TABLE 1

Results of affinity measurement 1 for GDF-5 wild-type, affinity for BMPR-IA: 1nM, affinity for BMPR-IB: 8pM

Results of affinity measurements 2 for GDF-5 wild-type, affinity for BMPR-IA: 1.1nM, affinity for BMPR-IB: 27pM

0-no ALP Activity

+ to ++++ ═ ALP Activity, the number + represents the intensity of ALP Activity

n.d. not measured.

Claims (13)

1. A GDF-5 related protein, wherein one of the following amino acids in the protein is replaced with a defined amino acid, with reference to the full-length amino acid sequence of the GDF-5 wild-type related protein:

replacement of amino acid R399 with E or M;

substitution of amino acid W414 with R;

replacement of amino acid W417 with R or F;

substitution of amino acid S439 with E; or

Amino acid I449 was replaced by V.

2. The protein of claim 1, wherein the protein is derived from a human wild-type GDF-5 related protein.

3. The protein of claim 1, wherein the protein is derived from human GDF-5, GDF-6, or GDF-7.

4. A nucleic acid encoding the protein of any one of claims 1 to 3.

5. Use of a protein according to any one of claims 1 to 3 or a nucleic acid according to claim 4 in the preparation of a medicament for the treatment of a disease in which cartilage formation is desired and bone formation is not desired.

6. The use according to claim 5, wherein the protein or nucleic acid is for the treatment of a cartilage defect or for the treatment of traumatic rupture or detachment of cartilage; diseases that can affect cartilage; diseases characterized by interference with cartilage growth and subsequent ossification; achondroplasia; costal chondritis; disc herniation and disc repair; recurrent polychondritis; cartilage defect repair associated with benign or malignant tumors.

7. The use according to claim 6, wherein the traumatic rupture or loss of cartilage is an age-related cartilage defect.

8. The use according to claim 6, wherein the traumatic rupture or detachment of cartilage is an age-related cartilage defect due to abrasion, osteoarthritis, rheumatoid arthritis, sports-related injuries.

9. Use according to claim 6, wherein the disease that can affect cartilage is cartilage dystrophy.

10. The use of claim 6, wherein the tumor is chondroma or chondrosarcoma.

11. A pharmaceutical composition comprising: the protein of any one of claims 1 to 3, the nucleic acid of claim 4, a vector comprising the nucleic acid of claim 4, or a host cell comprising the nucleic acid of claim 4 as an active agent.

12. The pharmaceutical composition of claim 11, wherein the protein, nucleic acid, vector or host cell is combined with a pharmaceutically acceptable additive or carrier.

13. An antibody directed against the protein of any one of claims 1 to 3, wherein the antibody specifically binds to the protein.