WO2008051526A2 - Bone morphogenetic proteins - Google Patents

Abstract

The present invention provides modified highly potent bone morphogenetic proteins. In particular, the present invention features a chimeric bone morphogenetic protein including a dimer with each monomer containing a type I receptor binding site derived from a member of the BMP subfamily and a type II receptor binding site derived from a member of the TGF-β subfamily. As a result, the chimeric bone morphogenetic protein has improved binding affinity to type II receptor relative to the naturally-occurring BMP subfamily member from which the type I receptor binding site is derived. Also provided are modified bone morphogenetic proteins having reduced affinity for Noggin. Other embodiments of modified BMPs, compositions and method of use are also included.

Description

BONE MORPHOGENETIC PROTEINS

FIELD OF THE INVENTION

[0001] The invention relates to designed or modified bone morphogenetic proteins with improved properties, in particular, with improved receptor-binding affinities and/or decreased inhibition by BMP antagonists, and to methods of making and using compositions utilizing the designed or modified bone morphogenetic proteins.

BACKGROUND OF THE INVENTION

[0002] Bone morphogenetic proteins (BMPs) belong to the superfamily of transforming growth factor β (TGF-β), and control a diverse set of cellular and developmental processes, such as embryonic pattern formation and tissue specification as well as promoting wound healing and repair processes in adult tissues. BMPs were initially isolated by their ability to induce bone and cartilage formation.

[0003] BMPs initiate signaling by binding to and bringing together the type I and type II receptor Ser/Thr kinases on the cell surface. This allows the type II receptors to phosphorylate the type I receptor kinases. The type I receptor kinases then phosphorylate members of the Smad family of transcription factors, and the Smads translocate to the nucleus and activate the expression of a number of genes. BMP signaling is inducible upon bone fracture and related tissue injury, leading to bone regeneration and repair. Certain cells, on the other hand, selectively secret BMP antagonists, such as Noggin and Chordin, in response to BMP signaling to allow them to escape from BMP signaling. Although antagonists may help to provide spatial regulation of the BMP signaling, their action may extend beyond the region where they are secreted and result in reduced BMP activity near or at the bone regeneration site since the antagonists are generally secreted into the extracellular compartment. BMP molecules which have increased affinity for their receptors and/or decreased affinity for their antagonists would have improved biological activity relative to the native proteins. Such BMPs with increased in vivo activity would have therapeutic utility in the field of tissue regeneration, providing potent activity at lower protein levels than the currently used therapeutic doses.

SUMMARY OF THE INVENTION

[0004] It is therefore an object of the present invention to provide designed highly potent bone morphogenetic proteins (BMPs) suitable for therapeutic uses.

[0005] It is an object of the present invention to provide designed BMPs with improved receptor binding affinities. Particularly, it is an object of the present invention to provide designed BMPs with high binding affinities to both type I and type II receptors.

[0006] It is also an object of the present invention to provide designed BMPs with reduced binding affinities to BMP antagonists.

[0007] It is a further object of the invention to provide nucleic acid sequences which encode the designed BMPs of the invention and methods of using such nucleic acid sequences for producing the designed BMPs of the invention.

[0008] Thus, in one aspect, the present invention features a chimeric bone morphogenetic protein including a dimer with each monomer containing a type I receptor binding site derived from a member of the BMP subfamily and a type II receptor binding site derived from a member of the TGF-β subfamily. As a result, the chimeric bone morphogenetic protein has improved binding affinity to type II receptor relative to the naturally-occurring BMP subfamily member from which the type I receptor binding site is derived and the chimeric bone morphogenetic protein is capable of inducing bone morphogenesis or tissue morphogenesis. For example, the chimeric bone morphogenetic protein of the present invention is capable of inducing formation of bone, cartilage, non-mineralized skeletal or connective tissue, kidney, liver, periodontal or nerve tissues. [0009] In one embodiment, the chimeric bone morphogenetic protein of the invention contains a type I receptor binding site derived from BMP-7. In another embodiment, the chimeric bone morphogenetic protein of the invention contains a type I receptor binding site derived from BMP-2. [0010] In some embodiments, the chimeric bone morphogenetic protein of the invention contains the type I receptor binding site including an amino acid consensus sequence Xaal Xaa2 Xaa3 Xaa4 Phe Xaa5 Xaa6 Xaa7 Xaa8 Trp Xaa9 XaalO Trp, wherein Xaal, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, Xaa9, or XaalO is any amino acid. Preferably, Xaal is Leu; Xaa2 is Tyr or Phe; Xaa3 is VaI; Xaa4 is Asp or Ser; Xaa5 is Ser, Asn, or Arg; Xaa6 is Asp; Xaa7 is VaI or Leu; Xaa8 is GIy; Xaa9 is Asn or GIn; and XaalO is Asp.

[0011] In some embodiments, the chimeric bone morphogenetic protein of the invention contains the type I receptor binding site including an amino acid consensus sequence Pro Xaal Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Asn, wherein Xaal, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, or Xaa8 is any amino acid.

Preferably, Xaal is Leu; Xaa2 is Ala or Asn; Xaa3 is Asp or Ser; Xaa4 is His, Tyr or Phe; Xaa5 is Leu or Met; Xaa6 is Asn; Xaa7 is Ser or Ala; and Xaa8 is Thr.

[0012] In some embodiments, the chimeric bone morphogenetic protein of the invention contains the type I receptor binding site including an amino acid sequence Xaal Xaa2 He Xaa3 Xaa4 Thr Leu VaI Xaa5 Xaa6 Xaa7, wherein Xaal , Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, or Xaa7 is any amino acid. Preferably, Xaal is His; Xaa2 is Ala; Xaa3 is VaI; Xaa4 is GIn; Xaa5 is Asn or His; Xaa6 is Ser or Phe; or Xaa7 is VaI or He.

[0013] In one embodiment, the chimeric bone morphogenetic protein of the invention contains the type II receptor binding site derived from TGF-βl.

[0014] In a preferred embodiment, the chimeric bone morphogenetic protein of the invention contains the type I receptor binding site derived from BMP-7 and the type II receptor binding site derived from TGF-βl.

[0015] In another aspect, the present invention provides a modified bone morphogenetic protein with improved binding affinity to a type II receptor relative to the corresponding naturally-occurring bone morphogenetic protein. The modified bone morphogenetic protein includes a replacement fragment derived from a member of the TGF-β subfamily. The fragment replaced comprises a sequence of native human BMP-7 selected from the group consisting of IAPE (157 to E60 of the amino acid sequence set forth in Figure IB),, GYA (G61 to A63 of the amino acid sequence set forth in Figure IB), ISVL (Il 12 to Ll 15 of the amino acid sequence set forth in Figure IB), and VILKKY (V123 to Y128 of the amino acid sequence set forth in Figure IB). The modified bone morphogenetic protein is capable of inducing bone morphogenesis or tissue morphogenesis. For example, the modified bone morphogenetic protein of the present invention is capable of inducing formation of bone, cartilage, non-mineralized skeletal or connective tissue, kidney, liver, periodontal or nerve tissues.

[0016] In one embodiment, the modified bone morphogenetic protein of the invention includes the replaced fragment of human BMP-7 containing the sequence IAPE, and wherein the replacement fragment includes a sequence selected from the group consisting of IAPS, HEPK and LIPE.

[0017] In one embodiment, the modified bone morphogenetic protein of the invention includes the replaced fragment of human BMP-7 containing the sequence GYA, and wherein the replacement fragment includes a sequence selected from the group consisting of GYH and TYQ. [0018] In one embodiment, the modified bone morphogenetic protein of the invention includes the replaced fragment of human BMP-7 containing the sequence ISVL, and wherein the replacement fragment includes a sequence selected from the group consisting of MSML, LPIV and GKLL.

[0019] In one embodiment, the modified bone morphogenetic protein of the invention includes the replaced fragment of human BMP-7 containing the sequence VILKKY, and wherein the replacement fragment includes a sequence selected from the group consisting of IIKKDI, KVEQL and ISAHHV.

[0020] In a preferred embodiment, the modified bone morphogenetic protein of the invention is a modified BMP-7. In a further embodiment, the modified BMP- 7 further comprises a second replacement fragment derived from BMP-2 including a sequence selected from the group consisting of NSVNSKI, and wherein the modified BMP-7 has improved binding affinity to a type I receptor relative to the naturally-occurring BMP-7.

[0021] In yet another aspect, the present invention provides a modified bone morphogenetic protein with reduced binding affinity to Noggin. The modified bone morphogenetic protein contains at least one amino acid mutation corresponding to a position selected from the group consisting of Arg48, Asp54, Ile57, Ilel 12, VaIl 14, Tyrl 16, Phel 17, Aspl 18, Aspl 19, Serl20, Serl21, Asnl22, lie 124, Leul25, Lysl26 and Argl29 of human BMP-7. The modified bone morphogenetic protein is capable of inducing bone morphogenesis or tissue morphogenesis. For example, the modified bone morphogenetic protein of the present invention is capable of inducing formation of bone, cartilage, non-mineralized skeletal or connective tissue, kidney, liver, periodontal or nerve tissues.

[0022] In one embodiment, the modified bone morphogenetic protein includes a mutation at a position corresponding to L 125 of human BMP-7. In a preferred embodiment, the modified bone morphogenetic protein includes a charged amino acid at the position corresponding to Arg48, Asp54, Ile57, Ilel 12, VaIl 14, Tyrl 16, Phel 17, Aspl 18, Aspl 19, Serl20, Serl21, Asnl22, Ilel24, Leul25, Lysl26 or Argl29 of human BMP-7. In preferred embodiments, the modified bone morphogenetic protein is a modified human BMP-7. [0023] The present invention further provides therapeutic compositions comprising the modified BMPs described in various embodiments above. The invention also contemplates methods for inducing tissue regeneration. For example, the compositions of the invention can be used to induce formation of bone, cartilage, non-mineralized skeletal or connective tissue, kidney tissue, liver tissue, nerve tissue in vitro or in vivo, or to inhibit the formation of fibrotic tissue.

[0024] In another embodiment, the invention provides nucleic acid sequences which encode the designed BMPs of the invention, vectors comprising such nucleic acid sequences and recombinant cells which comprise a heterologous DNA sequence comprising a nucleic acid sequence which encodes a designed protein of the invention. [0025] In yet another aspect, the present invention provides a method for redesigning a bone morphogenetic protein including the steps of: (a) generating a structural model including the bone morphogenetic protein complexed with a type I receptor and a type II receptor, wherein the transmembrane domains of the type I receptor and the type II receptor form a heterodimer; and (b) identifying a modification in the bone morphogenetic protein that facilitates the formation of the heterodimer.

[0026] Other features, objects, and advantages of the present invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments of the present invention, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description.

[0027] As used herein, "designed BMPs," "modified BMPs" and "variant BMPs" are used as equivalents unless stated otherwise. By "designed BMPs" or "modified BMPs" and grammatical equivalents thereof herein is meant non- naturally occurring BMPs which differ from a wild type or parent BMP by at least one amino acid insertion, deletion, or substitution. It should be noted that unless otherwise stated, all positional numbering of designed or modified BMPs is based on the sequences of the mature native BMPs. Designed BMPs are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the BMP sequence. BMP variants must retain at least 50% of wild type BMP activity in one or more cell types, as determined using an appropriate assay described below. Variants that retain at least 75%, 80%, 85%, 90% or 95% of wild type activity are more preferred, and variants that are more active than wild type are especially preferred. Alternatively, in some embodiments, designed BMPs may be engineered to have different activities than a wild type BMP. For example, competitive inhibitors may be designed. A designed or modified BMP may contain insertions, deletions, and/or substitutions at the N-terminus, C-terminus, or internally. In a preferred embodiment, designed or modified BMPs have at least 1 residue that differs from the most similar human BMP sequence, with at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different residues being more preferred. Designed or modified BMPs also include chimeric BMPs containing sequences derived from both TGF-β and BMP subfamily members. Designed or modified BMPs may contain further modifications, for instance mutations that alter additional protein properties such as stability or imniunogenicity or which enable or prevent posttranslational modifications such as PEGylation or glycosylation. Modified BMPs may be subjected to co- or posttranslational modifications, including but not limited to synthetic derivatization of one or more side chains or termini, glycosylation, PEGylation, circular permutation, cyclization, fusion to proteins or protein domains, and addition of peptide tags or labels. By "designed BMP nucleic acids," "modified BMP nucleic acids," "chimeric BMP nucleic acids," "variant BMP nucleic acids" and grammatical equivalents herein is meant nucleic acids that encode designed or modified BMPs. Due to the degeneracy of the genetic code, an extremely large number of nucleic acids may be made, all of which encode the designed or modified BMPs of the present invention, by simply modifying the sequence of one or more codons in a way that does not change the amino acid sequence of the designed BMP. In this application, the use of "or" means "and/or" unless stated otherwise.

[0028] The proteins disclosed herein are described by reference to substitutions of specific amino acid residues or amino acid sequences. The amino acid residues are referred to by their full name, by the three letter code or by the one letter code, all of which are known in the art. The amino acids and their corresponding three- and one-letter codes are as follows: (alanine, Ala, A); (aspartic acid, Asp, D), (arginine, Arg, R), (asparagine, Asn, N), (cysteine, Cys, C), (glutamic acid, GIu, E), (glutamine, GIn, Q), (glycine, GIy, G), (histidine, His, H), (isoleucine, He, I), (leucine, Leu, L), (lysine, Lys, K), (methionine, Met, M), (phenylalanine, Phe, F), (proline, Pro, P), (serine, Ser, S), (threonine, Thr, T), (tryptophan, Trp, W), (tyrosine, Tyr, Y), and (valine, VaI, V). BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The drawings are provided for illustration, not limitation.

[0030] Figure IA shows the amino acid sequence of a human BMP-7 pro- protein (GenBank accession number NM_001719.1). Amino acid 1 starts at the beginning of the mature BMP-7 protein.

[0031] Figure IB shows the amino acid sequence of a mature human BMP-7 protein (GenBank accession number NM_001719.1).

[0032] Figure 2A shows a nucleotide sequence encoding a human BMP-7 pro-protein (GenBank accession number NM_001719.1). [0033] Figure 2B shows a nucleotide sequence encoding a mature human

BMP-7 protein (GenBank accession number NM_001719.1).

[0034] Figure 3 A shows the amino acid sequence of a human BMP-2 pro- protein (GenBank accession number NM_001200.2). Amino acid 1 starts at the beginning of the mature BMP-2 protein. [0035] Figure 3B shows the amino acid sequence of a mature human BMP-2 protein (GenBank accession number NM_001200.2).

[0036] Figure 4A shows a nucleotide sequence encoding a human BMP-2 pro-protein (GenBank accession number NM_001200.2).

[0037] Figure 4B shows a nucleotide sequence encoding a mature human BMP-2 protein (GenBank accession number NM_001200.2).

[0038] Figure 5 depicts mechanisms of BMP and TGF-β signaling.

[0039] Figure 6 depicts a hypothetical model for heterodimerization of one type I and one type II receptors upon binding of the ligands.

[0040] Figure 7 depicts a hypothetical model of BMP-induced activation of type I receptor kinase.

[0041] Figure 8 depicts a BMP-7-Noggin structural complex. Noggin (green and cyan) binding site on BMP-7 (yellow and golden) span over both type I and type II receptor binding sites. [0042] Figure 9A is an alignment of protein sequences of mature BMP-2 and

BMP-7 homologs.

[0043] Figure 9B is an alignment of protein sequences of mature BMP-2,

BMP-7 homologs and exemplary TGF-β subfamily members. [0044] Figure 10 depicts a type I receptor binding site consensus sequence corresponding to L19-W31 of human BMP-2 and L43-W55 of human BMP-7.

[0045] Figure 11 depicts a type I receptor binding site consensus sequence corresponding to P50-N59 of human BMP-2 and P74-N83 of human BMP-7.

[0046] Figure 12 depicts a type I receptor binding site consensus sequence corresponding to H60-V70 of human BMP-2 and H84-I94 of human BMP-7.

[0047] Figure 13 depicts a simulated BMP-7-BR1A structural complex.

[0048] Figure 14A depicts the location of Noggin-disruption site L 125 in region G.

[0049] Figure 14B depicts hydrophobic residue L 125 in BMP-7 not directly in contact with the type II receptor at the interface.

[0050] Figure 14C depicts that a L125R mutation in BMP-7 may disrupt its interaction with Noggin.

[0051] Figure 15 depicts the interaction of Noggin Ile218 with the hydrophobic patch of BMP-7 (Ile57, Leul 15, Phel 17, VaI 123). Noggin is depicted as blue sticks relative to the surface of BMP-7.

[0052] Figure 16 depicts BMP-7 viewed from the "top," modeled with the type I receptor domain (orange) and the type II receptor domain (cyan), with a segment of Noggin (blue sticks) that links between the two sites.

DETAILED DESCRIPTION OF THE INVENTION

[0053] The present invention provides designed or modified BMPs with improved properties relating to BMP signaling. In one aspect, the present invention provides modified BMPs that can bind to both type I and type II receptors with high affinities. In another aspect, the present invention provides modified BMPs with reduced binding affinities to BMP inhibitors. In yet another aspect, the present invention provides therapeutic compositions comprising the modified BMPs of the invention. Thus, the present invention represents a significant advance in BMP therapeutics. [0054] Various aspects of the invention are described in further detail in the following subsections. The use of subsections is not meant to limit the invention. Each subsection may apply to any aspect of the invention.

Bone Morphogenetic Proteins

[0055] BMPs belong to the TGF-β superfamily. The TGF-β superfamily proteins are cytokines characterized by six-conserved cysteine residues (Lander et al, (2001) Nature. 409:860-921). The human genome contains about 42 open reading frames encoding TGF-β superfamily proteins. The TGF-β superfamily proteins can at least be divided into the BMP subfamily and the TGF-β subfamily based on sequence similarity and the specific signaling pathways that they activate. The BMP subfamily includes, but is not limited to, BMP-2, BMP-3 (osteogenin), BMP-3b (GDF- 10), BMP-4 (BMP-2b), BMP-5, BMP-6, BMP-7 (osteogenic protein- 1 or OP-I), BMP-8 (OP-2), BMP-8B (OP-3), BMP-9 (GDF-2), BMP-IO, BMP-I l (GDF-I l), BMP- 12 (GDF-7), BMP- 13 (GDF-6, CDMP-2), BMP- 15 (GDF-9), BMP-16, GDF-I, GDF-3, GDF-5 (CDMP-I), and GDF-8 (myostatin). BMPs are also present in other animal species. Furthermore, there is some allelic variation in BMP sequences among different members of the human population. As used herein, "BMP subfamily," "BMPs," "BMP ligands" and grammatical equivalents thereof refer to the BMP subfamily members, unless specifically indicated otherwise. [0056] The TGF-β subfamily includes, but is not limited to, TGFs {e.g. ,

TGF-β 1, TGF-β2, and TGF-β3), activins {e.g., activin A) and inhibins, macrophage inhibitory cytokine- 1 (MIC-I), Mullerian inhibiting substance, anti-Mullerian hormone, and glial cell line derived neurotrophic factor (GDNF). As used herein, "TGF-β subfamily," "TGF-βs," "TGF-β ligands" and grammatical equivalents thereof refer to the TGF-β subfamily members, unless specifically indicated otherwise. [0057] The TGF-β superfamily is in turn a subset of the cysteine knot cytokine superfamily. Additional members of the cysteine knot cytokine superfamily include, but are not limited to, platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), placenta growth factor (PIGF), Noggin, neurotrophins (BDNF, NT3, NT4, and βNGF), gonadotropin, follitropin, lutropin, interleukin-17, and coagulogen.

[0058] Structurally, BMPs are dimeric cysteine knot proteins. Each BMP monomer comprises multiple intramolecular disulfide bonds. An additional intermolecular disulfide bond mediates dimerization in most BMPs. BMPs may form homodimers. Some BMPs may form heterodimers.

[0059] BMPs are naturally expressed as pro-proteins comprising a long pro- domain, one or more cleavage sites, and a mature domain. This pro-protein is then processed by the cellular machinery to yield a dimeric mature BMP molecule. The pro-domain is believed to aid in the correct folding and processing of BMPs. Furthermore, in some but not all BMPs, the pro-domain may noncovalently bind the mature domain and may act as a chaperone, as well as an inhibitor (e.g., Thies et al. (2001) Growth Factors, 18:251-259). The amino acid sequence of the native pro- protein of human BMP-7 is shown in Figure IA. The amino acid sequence of the native mature human BMP-7 is shown in Figure IB. The cDNA sequence of the native pro-protein of human BMP-7 is shown in Figure 2A. The cDNA sequence of the native mature human BMP-7 is shown in Figure 2B. The amino acid sequence of the native pro-protein of human BMP-2 is shown in Figure 3 A. The amino acid sequence of the native mature human BMP-2 is shown in Figure 3B. The cDNA sequence of the native pro-protein of human BMP-2 is shown in Figure 4A. The cDNA sequence of the native mature human BMP-2 is shown in Figure 4B. Unless otherwise noted, identification of specific amino acid residues herein refers to the native human mature BMP-7 sequence, as set forth in Figure IB.

[0060] BMP signal transduction is initiated when a BMP dimer binds two type I and two type II serine/threonine kinase receptors. Type I receptors include, but are not limited to, ALK-I, ALK-2 (also called ActRla or ActRI), ALK-3 (also called BMPRIa), and ALK-6 (also called BMPRIb). Type II receptors include, but are not limited to, ActRIIa (also called ActRII), ActRIIb, and BMPRII. The human genome contains 12 members of the receptor serine/threonie kinase family, including 7 type I and 5 type II receptors, all of which are involved in TGF-β signaling (Manning et al, (2002) Science, 298:1912-1934, the disclosures of which are hereby incorporated by reference). Following BMP binding, the type II receptors phosphorylate the type I receptors, the type I receptors phosphorylate members of the Smad family of transcription factors, and the Smads translocate to the nucleus and activate the expression of a number of genes. Mechanisms of BMP and TGF-β signaling are further illustrated in Figure 5: upon binding of the ligants, specific pairs of the two types of Ser/Thr kinase receptors heterodimerize, one type I and the other type II. Type II kinase then phosphorylates the type I receptor kinase upon heterodimerization to allow type I receptor kinase to bind ATP and Smad protein substrates for signaling. The ligands can be divided into three groups according to signaling pathways (Avidin, TBF- β, and BMPs) or two groups according to sequence similarity and the mode of binding (TGF- β /Avidin, and BMPs).

[0061] BMPs also interact with inhibitors, soluble receptors, and decoy receptors, including, but not limited to, BAMBI (BMP and activin membrane bound inhibitor), BMPER (BMP-binding endothelial cell precursor-derived regulator), Cerberus, cordin, cordin-like, Dan, Dante, follistatin, follistatin-related protein (FSRP), ectodin, gremlin, Noggin, protein related to Dan and cerberus (PRDC), sclerostin, sclerostin-like, and uterine sensitization-associated gene-1 (USAG-I). Furthermore, BMPs may interact with co-receptors, for example BMP-2 and BMP -4 bind the co-receptor DRAGON (Samad et al. (2005) J. Biol. Chem., 280: 14122-9), and extracellular matrix components such as heparin sulfate and heparin (Irie et al. (2003) Biochem. Biophys. Res. Commun., 308:858-865).

Receptor Activation by BMP/TGF-β Subfamily Proteins

[0062] Typically, each type I or type II receptor Ser/Thr kinase includes about 500 amino acid residues. Each receptor protein includes an N-terminal extracellular ligand binding domain, a transmembrane (TM) helix domain, and a C- terminal cytoplasmic Ser/Thr kinase domain (Massague, (1998) Annu. Rev. Biochem., 67:753-791; Shi & Massague, (2003) Cell, 1 13:685-700). The overall structures of the extracellular ligand binding domains from both type I and type II receptors exhibit a similar finger toxin fold, with each finger formed by a pair of anti-parallel β strands. The extracellular ligand binding domains of type I and type II receptors bind at two adjacent, nonoverlapping sites on the surface of each monomer of the dimeric ligands. Hence an active receptor signaling complex includes both types of receptors bound to the ligand. A hypothetical model for heterodimerization of one type I and one type II receptors upon binding of the ligands is shown in Figure 6: the model was generated by superposition of dimeric ligands in the two existing crystal structures of BMP7-ActRII (in yellow, brown, and red) and BMP2-R1A (in cyan, green and magenta) complexes. Two receptors bind two different parts of the ligand and do not directly interact with each other. There are no-induced structural differences in the ligands between the two complexes. These data suggest that the structural basis for synergetic binding of the second receptor to the pre-assembly of one receptor with the ligand is at the transmembrane helices and their juxtamembrane regions.

[0063] The TM helix of the receptors is located between the N-terminal extracellular ligand-binding domain and the C-terminal cytoplasmic Ser/Thr kinase domain with a relatively short stretch of unstructured sequence of about 20 amino acids. There are about 10 or more cysteines at the extracellular ligand-binding domain. Three cysteines form a characteristic cluster near the TM domain. The spacing between the three cysteines in type I receptors appears to be more conserved than that in type II receptors. There is a Ser in the juxtamembrane region whose phosphorylation appears to selectively modulate the intensity of different responses. Without wishing to be bound by the theory, the TM helix and its flanking residues are likely to be involved in the heterodimerization of one type I receptor and one type II receptor.

[0064] Type I, but not type II, receptors contain a characteristic SGSGSG sequence, known as the GS domain, immediately N-terminal to the kinase domain. The activation of the type I receptor kinase involves the phosphorylation of its GS domain by the type II receptor upon ligand-induced heterodimerization. This is an important step in BMP/TGF-β signaling (Figure 7). Among many ligand-induced phosphorylation sites, it has been proposed that important phosphorylation targets include one Thr and three serines within the GS domain of the type I receptor (Massague, (1998) Annu. Rev. Biochem.. 67:753-791 ; Shi & Massague, (2003) Cell, 113:685-700). As illustrated in Figure 7, before activation, type I receptor kinase is in a closed conformation in which it cannot bind ATP. Activation requires phosphorylation of the GS domain so that the GS loop is displaced out and the kinase can bind ATP. In addition to the GS domain, there are other phosphorylation sites that further modulate the intensity of signaling. Such phosphorylation sites include those immediately next the TM helix as disclosed in Souchelnytsky et al, (1996) EMBO J.. 15:6231-6240, the disclosures of which are hereby incorporated by reference. Without wishing to be bound by the theory, phosphorylation may affect the heterodimerization of TM helices.

[0065] When two receptors each binds to a monomer of the dimeric ligand, the conformation can be represented as (R1LR2)-(R1LR2), where L stands for the ligand and Rl and R2 stand for type I and type II receptors, respectively. The ligand brings the two cytoplasmic kinase domains of the receptors to the proximity for direct phosphorylation of the GS loop. Such phosphorylation appears possible without needing of any higher oligomerization. Interestingly, in the inactive conformation of the kinase domain of type I receptors, the protein substrate-binding site is often blocked and occupied by various receptor-binding proteins, such as, for example, immunophilin FKBP12 (Huse et al, (1999) CeU, 96:425-436). These proteins stabilize the inactive conformation of the receptor kinase and serve to eliminate spurious signaling caused by receptor oligomerization in the absence of ligands. The FKBP12-binding site overlaps with the GS domain. Therefore, the phosphorylation of the GS domain by type II receptor kinase inhibits such binding. As a result, the phosphorylation of the GS domain causes an inhibitor-to-substrate binding switch, including the eviction of the bound FKBP 12 (Huse et al, (2001) Molecular Cell, 8:671-682). Upon removal of FKBP12, this site becomes a protein substrate-binding site, for example, for downstream Smad proteins. Distinct Binding Modes of BMP and TGF-β to the Receptors

[0066] The BMP subfamily ligands and the TGF-β subfamily ligands bind to the receptors in two distinct binding modes, a cooperative mode that is characteristic of the BMP subfamily members and a sequential mode that is characteristic of the TGF-β subfamily members (Massague, (1998) Annu. Rev. Biochem., 67:753-791). Specifically, the BMP subfamily members can bind both types of receptors independently, but exhibit very different affinities for each receptor. The BMP subfamily members often exhibit a high affinity for type I receptors and a low affinity for type II receptors. However, once a BMP ligand binds to a type-I receptor, the pre-assembled type I receptor-BMP complex exhibits a high affinity for type II receptors.

[0067] On the other hand, the TGF-β subfamily members display a high affinity for type II receptors and do not interact with the isolated type I receptors. In this case, a TGF-β ligand binds tightly to the extracellular binding domain of a type II receptor first. This binding allows the subsequent binding of a type I receptor. In either mode, the binding affinity increases for the second receptor to the pre- assembled first receptor-ligand complex. The increased affinity suggests cooperation of the heterodimerization of receptors, or synergetic interactions between the two receptors on the surface of the ligands. This synergy is absence without the ligand and is ligand-induced. However, the heterodimerization of one type I receptor with one type II receptor is highly specific, i.e., one given type II receptor heterodimerize only with one particular type I receptor (Massague, (1998) Annu. Rev. Biochem.. 67:753-791; Shi & Massague, (2003) CeU, 113:685-700).

Formation of Heterotetrameric Receptors with the Ligand [0068] Two crystal structures of receptor-ligand complexes have been reported. One includes a ligand complexed with a type I receptor ligand-binding domain and the other includes a ligand complexed with a type II receptor ligand- binding domain (Greenwald et al, (2003) Molecular Cell, 11 :605-617; Keller et al, (2004) Nat. Struct. MoI. Biol, 11(5):481-8). There is no crystal structure of heterotetramerization of two type I and two type II receptors complexed with one dimeric ligand. The present invention contemplates a hypothetical structure of heterotetramerization by superposition of the ligands in the two solved structures.

[0069] Such hypothetical heterotetramerization structural model does not reveal any receptor-induced large conformational changes in the ligands. This is not unexpected, because the ligands have seven (7) disulfide bonds with rigid framework. It is unlikely that the heterotetramerization will cause conformational changes in the receptors since the ligand-binding domains of the receptors also contain multiple disulfide bonds with rigid framework. Without structural changes in ligands or the receptors, it is also unlikely that attached carbohydrate moieties to either ligands or receptors are involved in some conformational switch. In summary, the formation of ligand-receptor complex appears not to cause any significant structural changes. Surprisingly, the proposed heterotetrameric structural model also reveals that the ligand-binding domains of the type I and type II receptors do not physically interact with each other. [0070] Furthermore, the structures of the cytoplasmic kinase domains in an inactive conformation or in a complex with receptor binding protein FKBP 12 have been determined (Huse et al, (1999) Cell, 96:425-436). Dimerization might be involved in the inactive kinase domains. However, this dimerization is ligand independent. [0071] This model failed to explain how the binding of one ligand-binding domain onto the ligand increases affinity for the second ligand-binding domain. This observation leads to an interesting, testable hypothesis: is heterodimerization of the TM helix domains from the two types of the receptors kinases involved in specificity and synergetic interaction? Potential Roles of Higher Oligomeric Receptor-BMP/TGF-β Complexes

[0072] It has been shown that the TGF-β receptor complex contains homodimers or higher oligomers of the type I TGF-β receptors (TβR-Is) as well as homooligomers of the type II TGF-β receptors (TβR-IIs). The experimental data for the existence of oligomers in vivo are reported by Chen, R.H. & Derynck, R. (1994) J. Biol. Chem.. 269:22868-22874; Henis et al., (1994) J. Cell Biol., 126: 139-154; Yamashita et al. J. Biol. Chem., 1994 269 20172-8; Luo & Lodish, (1996) EMBO J₁, 15:4485-4496; and Weis-Garcia & Massague, (1996) EMBO J., 15:276-289. However, whether higher oligomerization is essential for activation remains unknown. In one study, a kinase-defective TβR-I mutant can functionally complement an activation-defective TβR-I mutant, by rescuing its TβR-II dependent phosphorylation. See, Weis-Garcia & Massague, (1996) EMBO J.. 15:276-289. However, this study only suggests that oligomers are functional in these mutants, but it does not provide any direct evidence that such oligomerization is required for the wild type cells. In fact, the existing structural data suggest otherwise, as explained below. [0073] In the hypothetic heterotetrameric receptor-ligand complex model,

(R1LR2)-(R1LR2), the C-terminal end distance between the extracellular ligand- binding domains of one type I receptor and one type II receptor that bind to one same ligand is shorter than any distance between the two same type receptors in the heterotetrameric complex. The distance between the type I and II receptors is about 35 A. The distance between the two type II receptors is about 82 A and the distance between the two type I receptors is about 67 A. Without wishing to be bound by the theory, two type II receptors of inactive homodimeric receptor (R2R2), which was shown to exist (Chen, R.H. & Derynck, R. (1994) J. Biol. Chem., 269:22868- 22874), cannot simultaneously bind one dimeric ligand over 82 A apart. The dimeric receptors must either first dissociate into monomer or solicit for higher order of oligomerization upon cross-linking with other dimeric ligands. This may apply similarly to inactive homodimeric type I receptor (RlRl). Cross-linking multiple dimeric ligands with only one type of receptors without the second would greatly reduce the accessibility of the second receptor, thus reducing the effectiveness and potency of signaling. Moreover, a higher order of aggregation of receptor-ligand complexes often leads to their internalization and exocytosis.

[0074] On the other hand, a higher order of oligomerization of receptor kinases is likely involved in the BMP/TGF-β signaling. For example, a recombinant heterodimeric BMP-7/BMP-4 is more potent in bioassays than BMP-7 or BMP4 homodimers. Without wishing to be bound by the theory, while each homodimer can activate one pair of type I/type II receptor kinases, each monomer of the heterodimeric ligand can activate two different types of I receptor kinases at once. Therefore, the potency of the heterodimeric BMP-7/4 ligand is greater than equally mixed homodimeric BMP-7 and homodimeric BMP4, suggesting that there is a synergy between two monomers of the dimeric ligand.

[0075] Without wishing to be bound by the theory, one explanation for why heterodimeric ligands possess higher potency is that each monomeric ligand activates one type I receptor kinase by its paired type II receptor kinase upon heterotetramerization (RlaLlR2a)-(RlbL2R2b), where Ll and L2 stand for the two distinct ligands BMP-7 and BMP4 within the hybrid, RIa and RIb stand for two different type I receptor kinases, and R2a and R2b stand for two different type II receptor kinases, respectively. This heterotetrameric receptor complex could involve four different receptors, two type I and two type II, without any homodimerization. The two activated different type I receptor kinases then preferentially phosphorylate two different Smad proteins that are part of activated Smad heterooligomers. Therefore, the heterodimeric ligand may eliminate an extra step of formation of activated heteromeric Smad complex from two individually phosphorylated Smad proteins. In this situation, the formation of activated heteromeric Smad proteins is nearly the first order of reaction due to a local concentration effect. In contrast, the formation of such complex is the second order of the reaction in proportional to the product of two Smad proteins when the two Smad proteins are phosphorylated by two different type I receptor kinases that are not physically associated within a large receptor complex. Therefore, the heterodimeric ligands may better serve as the Smad anchor for receptor activation, and for transportation through nucleopores to nucleus. In summary, the heterodimeric ligand, when properly chosen, should possess a higher potency, due to the added concentration effects of phosphorylated Smad proteins for heteromeric assembly.

[0076] Furthermore, homo-oligomerization without ligands may serve as a store for receptors. Upon binding to the ligands, oligomeric receptors may remain inactive until the TM helices of correctly paired receptors are dimerized with specificity. In this process, homo-oligomeric receptors dissociate and re-associate into heteromers on the surface of the ligands. This process can be represented by the following equation: (R1R1)+LL+(R2R2) -> R1(R1L)-R2(R2L) -> R2(R1L)- R1(R2L) -> (R1LR2)-(R1LR2). In this equation, Rl(RlL) stands for one monomer of the homodimeric type I receptor Rl bound to one molecule of ligand L while the second monomer of the homodimer is tethered. Similar illustration is used for R2(R2L), R1(R2L), and R2(R1L). This process is also known as isomerization of receptors on the surface of the ligands.

Interactions between BMPs and Their Antagonists

[0077] BMP Antagonists like Noggin may be important to eliminate and restrict BMP signaling. They could provide a spatial regulation through a gradient. However, since these antagonists are secreted into the extracellular compartment, their action may extend beyond where they are secreted, which would undesirably decrease the potency of BMP signaling. The present invention contemplates increasing the potency of BMPs upon decreasing their interactions with BMP antagonists such as Noggin.

[0078] The Noggin-binding site on BMPs overlaps with both binding sites of type I and type II receptors. Therefore, Noggin binding will prevent any receptors from binding to the BMP proteins. For example, the x-ray crystal structure of BMP- 7-Noggin complex is illustrated in Figure 8 (Groppe et al. (2002), Nature 420, 636- 642; Groppe et al. (2003), J. Bone Joint Surg. Am., 85-A Suppl. 3, 52-58. Structural information such as that provided in Figure 8 can be used to deduce BMP-7 residues which interact with the antagonist. For example, as shown in Figures 15 and 16, which are based on the crystal structure, several potential sites of interaction between BMP-7 and Noggin can be determined, and appropriate substitution of one or more key residues of BMP-7 could reduce these interactions, thereby decreasing the extent of Noggin binding. For example, Figure 15 shows the interaction between Noggin and hydrophobic residues Ile57, Leu 115, Phe 117, VaI 123 of BMP-7. Figure 16 illustrates the interaction between Noggin and BMP-7 in a region of BMP-7 between the type I and type II receptor binding sites.

[0079] Unlike the TGF-β subfamily members, the BMP subfamily members can bind independently to both type I and type II receptors. When the BMP subfamily members bind to any one receptor, its affinity would increase for a second receptor, possibly through dimerization of the TM domains. Therefore, if a designed BMP has reduced affinity at either receptor-binding site for Noggin, the designed BMP would initiate binding to a first receptor more easily. The second receptor would then effectively compete with Noggin and evict it through the synergy resulted from the TM dimerization. Designing Modified BMPs

[0080] The present invention contemplates at least two possible routes for designing potent BMPs. The first route is to improve the binding efficiency of heterodimeric receptor kinases to each monomer of dimeric ligands. As discussed above, the BMP subfamily members often exhibit a high affinity for type I receptors and a low affinity for type II receptors, while the TGF-β subfamily members display a high affinity for type II receptors. Thus, in one embodiment, the present invention contemplates grafting a high-affinity type II receptor-binding site from a TGF-β subfamily protein onto a BMP subfamily protein to generate a potent modified BMP that retains high affinities for both type I and type II receptors. [0081] In one embodiment, the present invention contemplates a chimeric bone morphogenetic protein including a dimer with each monomer containing a type I receptor binding site derived from a member of the BMP subfamily and a type II receptor binding site derived from a member of the TGF-β subfamily. As a result, the chimeric bone morphogenetic protein has improved binding affinity to type II receptor relative to the naturally-occurring BMP subfamily member from which the type I receptor binding site is derived. The chimeric BMP of the invention may contain a type I receptor binding site derived from BMP-2, BMP-7 or other members of the BMP subfamily. The chimeric BMP of the invention may contain a type II receptor binding site derived from TGF-βl, activin, MIS, or other members of the TGF- β subfamily. The second route for designing potent BMPs involves altering interactions between the BMP and its protein antagonists. Thus, the present invention further contemplates a modified BMP with reduced or diminished antagonist-binding affinities.

Protein Designing Methods [0082] A number of methods can be used to design modifications (that is, insertion, deletion, or substitution mutations) that will generate a modified BMP with improved binding affinities. These methods include, but are not limited to, sequence profiling (Bowie and Eisenberg (1991) Science 253:164-170), rotamer library selections (Dahlyat and Mayo (1996) Protein ScL 5:895-903; Dahlyat and Mayo, Science (1997) 278:82-87; Desjarlais and Handel (1995) Prot. ScL (1995) 4:2006- 1 2018; Harbury et al. (1995) Prac. Nat. Acad. Sci. USA. 92:8408-8412; Kono et al, Proteins (1994) 19:244-255; Hellinga and Richards (1994) Proc. Nat. Acad. Sci. USA. 91 :5803- 5807); and residue pair potentials (Jones (1994) Prot. Sci.. 3: 567-574).

[0083] In some embodiments, one or more sequence alignments of BMPs and related proteins is analyzed to identify residues that are likely to be compatible with each position. For example, the PFAM or BLAST alignment algorithm is used to generate alignments of the BMP subfamily, the TGF-β subfamily, or the cysteine knot cytokine superfamily. For each variable position, suitable substitutions may be defined as those residues that are observed at the same position in homologous sequences. Especially preferred substitutions are those substitutions that are frequently observed in homologous sequences. In some embodiments, an Analogous Contact Environment (ACE) algorithm, U. S. Patent Application Publication No. 20050143929, filed December 8 2004, is used in conjunction with the sequence alignment information to identify alternate suitable residues that are located in structurally similar environments in other BMPs, TGF-βs or homologs. In other embodiments, rational design of improved BMP variants is achieved by using Protein Design Automations^® (PDA^®) technology; see U.S. Patent Nos. 6,188,965; 6, 269,312; 6,403,312; 6,708,120; W098/47089, or using the sequence prediction algorithm (SPA) (Raha et al. (2000) Protein Sci.. 9:1106-1119). Structural Analysis of BMPs and TGF-βs

[0084] PDA technology calculations require a template protein structure.

Structures of human BMPs suitable for the present invention can be obtained by x- ray crystallography or NMR. Exemplary structures of BMPs include, but are not limited to BMP-2 (PDB code 3BMP, Scheufler et al (1999) J. MoI. Biol.. 287:103), BMP-2 mutant L51P (PDB code IREU, Keller et al. (2004) Nat. Struct. MoI. Biol.. 11 :481) and wild type human BMP-7 (PDB code ILXI Griffith et al. (1996) Proc. Nat. Acad. Sci. USA 93:1 878-883). Exemplary structures of TGF-βs and other cysteine knot cytokine proteins include, but are not limited to, crystal structure of TGF-β2 (PDB code ITFG, Schlunegger and Grutter (1992) Nature, 358:430), NMR structures of cysteine knot cytokine proteins such as TFG-βl (PDB codes IKLA, IKLC, and IKLD; Hinck et al. (1996) Biochem., 35:5817). Preferably, the crystal structure is a co-crystal structure comprising a BMP and a BMP receptor, or a TGF- β and a receptor. High-resolution structures are available for BMP-7 in complex with the receptor ActRlla (PDB code 1 LX5, Greenwald et al. (2003) MoI. Cell, 11 :605-617), activin A bound to ActRllb (PDB codes INYS and INYU, Thompson et al. (2003) EMBO J., 22:1555-1566), BMP-2 bound to ALK-3 (PDB code 1 ES7, Kirsch et al. (2000) Nat. Struct Biol., 7:492; and PDB code 1 REW, Keller et al (2004) Nat. Struct. MoI. Biol.. 11 :481), and TGF-β3 complexed with TβR2 extodomain (Hart PJ. et al. (2002), Nature Struct. Biol., 9:203-208). In another preferred embodiment, the crystal structure is a co-crystal structure comprising BMP and a BMP inhibitor. A high resolution structure is available for BMP-7 bound to the soluble inhibitor Noggin (PDB code 1 M4U, Groppe et al. (2002) Nature, 420:636). The disclosures of the above-identified structures are hereby incorporated by reference. Structures of additional BMPs or TGF-βs alone and bound to one or more receptors or inhibitors may be built using NMR or x-ray crystal structures including but not limited to those described above in conjunction with homology modeling, structural alignment, and protein-protein docking methods known in the art.

Identifying Modifications

[0085] Based on the structural analysis and sequence alignments described above and shown in Figures 9A and 9B as discussed further below, the type I receptor binding site includes, but is not limited to, regions A, B, D, E, F and G.

The type II receptor binding site includes, but is not limited to, regions C, F and G.

In particular, in BMP-2, amino acids important for the type I receptor binding include, but are not limited to, Kl 5 in region A; F23, V26, W28 and W31 in region B; P50, A52, D53, H54 and N59 in region D; 162, T65, L66, S69 and V70 in region

E; Y91 in region F; and Y 103 in region G. As another example, in BMP-7, amino acids important for the type II receptor binding include, but are not limited to, 157, A58, P59, E60 and A63 in region C; Sl 13, Ll 15 and Fl 17 in region F; and L125 and K 127 in region G. Figure 9A was compiled by mapping the receptor-binding sites on primary sequences. Regions taken from BMP7/ActRII complex are shown in black boxes, and regions taken from BMP2/R1A complex are shown in red boxes. These regions are labeled as regions A to G. BMP7/ActII interface includes region

C, I57/A58/P59/E60/A63, region F, Sl 13/Ll 15/Fl 17, and region G, Ll 25/Kl 27. BMP2/R1A interface includes region A, K15, region B, F23/V26/W28/W31, region

D, P50/A52/D53/H54/N59, region E, I62/T65/L66/S69/V70, region F, Y91, and region G, Y 103. An insertion /deletion difference (depicted by a cyan underlie) is located at region E. Figure 9B further depicts three human sequences, Activin, TGF- βl, and MIS. These newly added proteins do not interact with the isolated type I receptor.

[0086] Thus, the present invention contemplates that the type I receptor binding site derived from the BMP subfamily may include an amino acid consensus sequence Xaal Xaa2 Xaa3 Xaa4 Phe Xaa5 Xaa6 Xaa7 Xaa8 Trp Xaa9 Xaal 0 Tip, wherein Xaal, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, Xaa9, or XaalO is any amino acid. As shown in Figure 10, this consensus sequence corresponds to the sequence Leu43-Trp55 in the native human BMP-7 sequence and L19-W31 in the native human BMP-2 sequence. In some embodiments, Xaal is Leu; Xaa2 is Tyr or Phe; Xaa3 is VaI; Xaa4 is Asp or Ser; Xaa5 is Ser, Asn, or Arg; Xaa6 is Asp; Xaa7 is VaI or Leu; Xaa8 is GIy; Xaa9 is Asn or GIn; and XaalO is Asp.

[0087] The present invention also contemplates that the type I receptor binding site derived from the BMP subfamily may include an amino acid consensus sequence Pro Xaal Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Asn, wherein Xaal, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, or Xaa8 is any amino acid. As shown in

Figure 11, this consensus sequence corresponds to the sequence Pro74-Asn83 in the native human BMP-7 sequence and Pro50-Asn59 in the native human BMP-2 sequence. In some embodiments, Xaal is Leu; Xaa2 is Ala or Asn; Xaa3 is Asp or Ser; Xaa4 is His, Tyr or Phe; Xaa5 is Leu or Met; Xaa6 is Asn; Xaa7 is Ser or Ala; and Xaa8 is Thr. [0088] The present invention further contemplates that the type I receptor binding site derived from the BMP subfamily may include an amino acid consensus sequence Xaal Xaa2 He Xaa3 Xaa4 Thr Leu VaI Xaa5 Xaa6 Xaa7, wherein Xaal, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, or Xaa7 is any amino acid. As shown in Figure 12, this consensus sequence corresponds to the sequence His84-Ile94 in the native human BMP-7 sequence and His60-Val70 in the native human BMP-2 sequence. In some embodiments, Xaal is His; Xaa2 is Ala; Xaa3 is VaI; Xaa4 is GIn; Xaa5 is Asn or His; Xaa6 is Ser or Phe; or Xaa7 is VaI or He.

[0089] Therefore, a modified BMP of the present invention may contain the type I receptor binding site having one or more or any combination of the consensus sequences described above.

[0090] The present invention further contemplates a designed BMP-7 with improved binding affinity to the type I receptor. Structural analysis has shown that, upon binding to the ligand, the type I receptor-BMP-2 interface buries 9 large hydrophobic residues (F23, V26, W28, W31, P50, 162, L66, Y91, Y103), plus buried hydrogen bonds from two residues (Kl 5, D53), while the BMP-7-type II receptor interface buries only 5 hydrophobic residues (157, P59, Ll 15, Fl 17, L125). Structural modeling also suggests that BMP-7 binds to type I receptors not as energetically as BMP-2 does near the insertion/deletion site as shown in Figure 13. [0091] Thus, based on the structural analysis and the sequence alignments shown in Figure 9A, a modified BMP-7 with improved binding affinity to the type I receptor may include one or more of the following designed features: (1) replacement of residue Lysl27 in the native human BMP-7 sequence with Leu or Phe; (2) replacement of the sequence His92-Phe93-Ile94-Asn95-Pro96-Glu97- Thr98-Val99 in BMP-7 deletion loop with the sequence Asn-Ser-Val-Asn-Ser-Lys- He, corresponding to the sequence Asn68 to Ile74 to in the native human BMP-2 sequence; (4) replacement of residue Ala58 in the native human BMP-7 sequence with VaI; (5) replacement of Ile57 with Ala, Asp, Pro or Arg; .

[0092] The sequences of representative members of the TGF-β subfamily, including human TGF-β 1, Activin, and MIS, are included in the alignment as shown in Figure 9B. The alignment shows that there is less conservation among the TGF-β subfamily members than the BMP subfamily members, possibly due to insertions, deletions or sequence divergence, in the regions B, D, and E. As discussed above, these regions form part of the type I receptor binding site. As shown in Figure 9B, the predicted type II receptor binding site of the TGF-β subfamily members also includes, but is not limited to, regions C, F and G. However, the sequences of the type II receptor binding site of the TGF-β subfamily members are significantly different from those of the BMP subfamily members. The sequences also differ from each other within the TGF-β subfamily.

[0093] Thus, a modified BMP-7 with improved binding affinity to the type II receptor may include one or more of the following designed features: (1) replacement of Ala63 of the native human BMP-7 sequence Gly61-Tyr62-Ala63 with His, as found in BMP-2, Activin, and TGF-β 1 to generate the sequence Glyόl- Tyr62-His63; (2) replacement of Lysl27 of the native human BMP-7 sequence with Asp, as found in Activin, or with GIn, as found in TGF-β 1, or with His, as found in MIS in region G; (3) replacement of the sequence Vall23-Ilel24-Leul25-Lysl26- Lysl27-Tyrl28 in the native human BMP-7 sequence with the sequence Ile-Ile-Lys- Lys-Asp-Ile found in Activin, corresponding to Ilel25-Ilel30 of the native human activin sequence; (4) replacement of VaI 123 in the native human BMP-7 sequence with Ala or Asp; (5) replacement of Asp54 in the native human BMP-7 sequence with GIu, Arg or Lys; and (6) the replacement of Arg48 in the native human BMP-7 sequence with Asp, Ala or GIn.

[0094] Based on the sequence alignment shown in Figure 9B, a modified bone morphogenetic protein with improved binding affinity to a type II receptor relative to the corresponding naturally-occurring bone morphogenetic protein may include a replacement sequence derived from a member of the TGF-β subfamily.

For example, the replacement sequence may substitute a sequence in the native bone morphogenetic protein that corresponds to a sequence of native human BMP-7 selected from the group consisting of Ile57-Ala58-Pro59-Glu60; Gly61-Tyr62- Ala63; Ilel l2-Serl l3-Vall l4-Leul l5 and Vall23-Ilel24-Leul25-Lysl26-Lysl27- Tyrl28. A suitable replacement fragment for Ile-Ala-Pro-Glu may include, but is not limited to, a sequence selected from the group consisting of Ile-Ala-Pro-Ser; His-Glu-Pro-Lys and Leu-Ile-Pro-Glu. A suitable replacement fragment for GIy- Tyr-Ala may include, but is not limited to, a sequence selected from the group consisting of Gly-Try-His and Thr-Tyr-Gln. A suitable replacement fragment for Ile-Ser-Val-Leu may include, but is not limited to, a sequence selected from the group consisting of Met-Ser-Met-Leu; Leu-Pro-Ile-Val and Gly-Lys-Leu-Leu. A suitable replacement fragment for Val-Ile-Leu-Lys-Lys-Tyr may include, but is not limited to, a sequence selected from the group consisting of Ile-Ile-Lys-Lys-Asp-Ile; Lys-Val-Glu-Gln-Leu and Ile-Ser-Ala-His-His-Val. Further, GluόO in the sequence IAPE can be substituted with Ly s.

[0095] As discussed above, the present invention further contemplates a modified bone morphogenetic protein with reduced or diminished antagonist- binding affinities. In particular, the present invention contemplates a modified bone morphogenetic protein with reduced binding affinity to Noggin. The modified bone morphogenetic protein may contain at least one amino acid mutation corresponding to a position selected from the group consisting of Del 12, VaIl 14, Tyrl 16, Phel 17, Aspl 18, Aspl 19, Serl20, Serl21, Asnl22, Vall23, Ilel24, Leul25, Lysl26, Lysl27 and Argl29 of native human BMP-7.

[0096] In particular, the position corresponding to Leul25 of native human

BMP-7 may be mutated to a charged amino acid, such as Arg or Lys, to prevent Noggin from binding to the BMP ligand. As shown in Figure 14A, Leul25 is located in region G of BMP-7. As shown in Figure 14B, Leul25 lies at the BMP-7- ActRII interface; however, it does not directly contact with the receptor. There are large spaces between them. As shown in Figure 14C, a mutation to Arg in BMP-7 at this position could potentially improve its interaction with E29 of the receptor, while preventing Noggin from binding. Other preferred substitutions of residues in native human BMP-7 involved in BMP-7/Noggin binding include, but are not limited to, substitution of Phel 17 by Lys, Ala, Asp, Thr, or Trp; substitution of Lys 127 by Ala, Asp, GIu, He, Asn, Phe, Tyr ,Trp or His; substitution of Lys 126 by Ala, Asp, GIu, His, Trp or Arg and substitution of Arg 129 by Ala, Asp, GIu, He, Asn, Phe, Tyr ,Trp or His . Other mutations in the Noggin-binding region include the substitution of Ilel 12 by Ala, Asp, His or Pro; the substitution of Leul25 by Ala, Arg or Asp; the substitution of He 124 by Ala or Asp; the substitution of VaI 123 sequence with Ala or Asp and the substitution of Arg 134 by GIu. [0097] The modified BMPs of the invention can also include combinations of the substitutions disclosed herein, such as 2, 3, 4, 5 or more substitutions. Preferred multiple mutants are based on the human BMP-7 native sequence set forth in Figure IB, and include the double mutants (K126E, K127E), (R48A, Fl 17W), (R48A, Il 12A). A preferred triple substitution includes (R48A, Il 12A, Fl 17W). The modified BMPs can further include any one or more of the substitutions disclosed herein, together with the triple substitution (Y65N, Y78H, Rl 34E). The (Y65N, Y78H, Rl 34E) mutation has been shown to enhance the yield of modified BMP-7 proteins in recombinant expression systems. Additional preferred multiple mutants contemplated herein include (D54R, Y65N, Y78H, Rl 34E); (I57A,Y65N, Y78H, R134E); (Y65N, Y78H, Il 12D, R134E); (Y65N, Y78H, Fl 17D, R134E); (Y65N, Y78H, F117T, R134E); (Y65N, Y78H, L125D, R134E); (Y65N, Y78H, K126E, R134E); (Y65N, Y78H, K127E, R134E) and (Y65N, Y78H, K126E, K127E, R134E). [0098] A summary of certain of the specific substitutions of the native human BMP-7 amino acid sequence (Figure IB) is provided in Tables 1 and 2.

[0099] The modified BMPs disclosed herein can further include modifications in the pro-peptide which are designed to enhance protein processing following expression. As discussed above, BMPs are initially expressed as pro- proteins comprising an N-terminal pro-peptide, which is then cleaved by cellular proteases to yield the mature protein. Replacement of the native cleavage recognition sequence in the pro-peptide with an enhanced site can increase protein yields. For example, in the pro-BMP-7 sequence set forth in Figure IA, the cleavage recognition sequence is RSIR, which immediately precedes Ser(l), the N- terminal residue of the mature peptide. Replacement of this recognition sequence with a sequence optimized for cleavage by a specific protease results in increased yields of mature BMP-7. In a preferred embodiment, the modified BMPs of the invention are initially expressed as pro-proteins in which the pro-peptide comprises a recognition sequence enhanced for the protease furin. In a preferred embodiment, the RSIR sequence of pro-BMP-7 or a modified form thereof, is replaced by the furin recognition sequence RSKR. In a particularly preferred embodiment, the pro- BMP or modification thereof is produced by co-expression in host cells with recombinant furin.

Table 1 : Amino acid substitutions in the native human BMP-7 amino acid sequence (Figure IB).

Table 2: Amino acid sequence substitutions in the native human BMP-7 sequence (Figure IB)

I57-A58-P59-E60: IAPS

HEPK

LIPE

G61-Y62-A63: TYQ

Il 12-Sl 13-Vl 14-Ll 15: MSML

LPIV

GKLL

V123-I124-L125-K126-K127-Y128: IIKKDI KEVQL ISAHHV

H92-F93-I94-N95-P96-E97-T98-V99: NSVNSKI Generating Modified BMPs

[00100] As described above, BMPs are naturally expressed as pro-proteins comprising a long pro-domain, one or more cleavage sites, and a mature domain. This pro-protein is then processed by the cellular machinery to yield a dimeric mature BMP molecule. In a preferred embodiment, the modified BMPs of the invention are produced in a similar manner. The pro-domain is believed to aid in the correct folding and processing of BMPs. Furthermore, in some but not all BMPs, the pro-domain may noncovalently bind the mature domain and may act as a chaperone, as well as an inhibitor {e.g., Thies et al. (2001) Growth Factors, 18:251- 259). Preferably, the modified BMPs of the invention are produced and/or administered therapeutically in this form. Alternatively, BMPs may be produced in other forms, including, but not limited to, mature domain produced directly or refolded from inclusion bodies, or full-length intact pro protein. The modified BMPs of the invention are expected to find use in these and other forms.

[00101] In a preferred embodiment, the modified bone morphogenetic protein of the invention is a modified BMP-7, such as a modified or mutant human BMP-7. The amino acid sequence of the native pro-protein of human BMP-7 is shown in Figure IA. The amino acid sequence of the native mature human BMP-7 is shown in Figure IB. It is to be understood that, although the amino acid sequence of a subunit of the mature dimeric form of human BMP-7 is set forth in Figure IB, each subunit can be independently full length or truncated at the N-terminus. For example, a subunit can have any or all of the residues 1 to 37 of the full length mature form as shown in Figure IB. The cDNA sequence of the native pro-protein of human BMP-7 is shown in Figure 2A. The cDNA sequence of the native mature human BMP-7 is shown in Figure 2B.

[00102] Modified BMP nucleic acids and proteins of the invention may be produced using a number of methods known in the art, as elaborated below.

Preparing Nucleic Acids Encoding Modified BMPs [00103] In a preferred embodiment, nucleic acids encoding modified BMPs are prepared by total gene synthesis, or by site-directed mutagenesis of a nucleic acid encoding wild type or modified BMPs. Methods including template-directed ligation, recursive PCR, cassette mutagenesis, site-directed mutagenesis or other techniques that are well known in the art may be utilized (see for example Strizhov et al. PNAS 93:15012-15017 (1996V Prodromou and Perl. Prot. Eng. 5: 827-829 (1992), Jayaraman and Puccini, Biotechniques 12: 392-398 (1992), and Chalmers et al. Biotechniques 30: 249-252 (2001)).

Expression Vectors

[00104] In a preferred embodiment, an expression vector that comprises the components described below and a gene encoding a modified BMP is prepared. Numerous types of appropriate expression vectors and suitable regulatory sequences for a variety of host cells are known in the art. The expression vectors may contain transcriptional and translational regulatory sequences including but not limited to promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, transcription terminator signals, polyadenylation signals, and enhancer or activator sequences. In a preferred embodiment, the regulatory sequences include a promoter and transcriptional start and stop sequences. In addition, the expression vector may comprise additional elements. For example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification. Furthermore, for integrating expression vectors, the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences, which flank the expression construct. The integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art. In addition, in a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used. The expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome. [00105] The expression vector may include a secretory leader sequence or signal peptide sequence that provides for secretion of the modified BMP from the host cell. Suitable secretory leader sequences that lead to the secretion of a protein are known in the art. The signal sequence typically encodes a signal peptide comprised of hydrophobic amino acids, which direct the secretion of the protein from the cell. The protein is either secreted into the growth media or, for prokaryotes, into the periplasmic space, located between the inner and outer membrane of the cell. For expression in bacteria, bacterial secretory leader sequences, operably linked to a variant BMP encoding nucleic acid, are usually preferred.

Trans fection/Transformation

[0100] The modified BMP nucleic acids are introduced into the cells either alone or in combination with an expression vector in a manner suitable for subsequent expression of the nucleic acid. The method of introduction is largely dictated by the targeted cell type. Exemplary methods include CaPO₄ precipitation, liposome fusion {e.g., using the reagent Lipofectin^® or FuGene), electroporation, viral infection (e.g., as outlined in PCT/US97/01019,), dextran-mediated transfection, polybrene mediated transfection, protoplast fusion, direct microinjection, etc. The modified BMP nucleic acids may stably integrate into the genome of the host cell or may exist either transiently or stably in the cytoplasm.

Hosts for Expression of Modified BMPs

[0101] Appropriate host cells for the expression of modified BMPs include yeast, bacteria, archaebacteria, fungi, and insect and animal cells, including mammalian cells. Of particular interest are fungi such as Saccharomyces cerevisiae and Pichia pastoris and mammalian cell lines including 293 (e.g., 293-T and 293- EBNA), BHK, CHO (e.g., CHOKl and DG44), COS, Jurkat, NIH3T3, etc. (see the ATCC cell line catalog). Modified BMPs can also be produced in more complex organisms, including but not limited to plants (such as corn, tobacco, and algae) and animals (such as chickens, goats, cows); see for example Dove, Nature Biotechnol., 20:777- 779 (2002). In one embodiment, the cells may be additionally genetically engineered, that is, contain exogenous nucleic acid other than the expression vector comprising the modified BMP nucleic acid.

Expression Methods

[0102] The modified BMPs of the present invention are produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding a modified BMP, under the appropriate conditions to induce or cause expression of the modified BMP. Either transient or stable transfection methods may be used. The conditions appropriate for modified BMP expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation.

Purification

[0103] In a preferred embodiment, the modified BMPs are purified or isolated after expression. Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusing. For example, a modified BMP may be purified using a standard anti-recombinant protein antibody column. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. For general guidance in suitable purification techniques, see Scopes, R., Protein Purification, Springer- Verlag, NY, 3d ed. (1994). The degree of purification necessary will vary depending on the desired use, and in some instances no purification will be necessary.

Posttranslational Modification and Derivatization

[0104] Once made, the modified BMPs may be covalently modified. Covalent and non-covalent modifications of the protein are thus included within the scope of the present invention. Such modifications may be introduced into a modified BMP polypeptide by reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Optimal sites for modification can be chosen using a variety of criteria, including but not limited to, visual inspection, structural analysis, sequence analysis and molecular simulation. Sites for modification may be located in the pro-domain or the mature domain.

[0105] In one embodiment, the modified BMPs of the invention are labeled with at least one element, isotope or chemical compound. In general, labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) immune labels, which may be antibodies or antigens, and c) colored or fluorescent dyes. The labels may be incorporated into the compound at any position. Labels include but are not limited to biotin, tag (e.g., FLAG, Myc) and; fluorescent labels (e.g., fluorescein). Derivatization with bifunctional agents is useful, for instance, for cross linking a modified BMP to a water-insoluble support matrix or surface for use in the method for purifying anti-modified BMP antibodies or screening assays, as is more fully described below. Commonly used cross linking agents include, e.g., 1,1- bis(diazoacetyl)-2- phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4- azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'- dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N- maleimido-l,8-octane and agents such as methyl-3- [(p-azidophenyl) dithio] propioimidate. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of praline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C- terminal carboxyl group. Such derivatization may improve the solubility, absorption, transport across the blood brain barrier, serum half-life, and the like. Modifications of modified BMP polypeptides may alternatively eliminate or attenuate any possible undesirable side effect of the protein. Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa. (1980). [0106] Another type of covalent modification of modified BMP comprises linking the modified BMP polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol ("PEG"), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301 , 144; 4,670,417; 4,791 , 192 or 4, 179,337. A variety of coupling chemistries may be used to achieve PEG attachment, as is well known in the art.

[0107] Another type of modification is chemical or enzymatic coupling of glycosides to the modified BMP. Such methods are described in the art, e.g., in WO 87/05330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0108] Alternatively, removal of carbohydrate moieties present on the modified BMP polypeptides may be accomplished chemically or enzymatically. Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al, Arch. Biochem. Biophvs., 259:52 (1987) and by Edge et al, Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo-and exo- glycosidases as described by Thotakura et al, Meth. Enzvmol.. 138:350 (1987). Biophysical and Biochemical Characterization of Designed BMPs

[0109] The designed BMPs of the invention can be characterized by their binding affinities for Type I and Type II receptors and for inhibitors such as Noggin, using methods known in the art, for example, Biacore methods. The modified BMPs can also be characterized using cell-based and in vivo assays routinely used in evaluating osteogenic and chondrogenic factors, for example, the alkaline phosphatase assay, the osteoblast proliferation assay, the bone nodule assay. The biophysical and biochemical characterization of modified BMPs are elaborated below.

Assaying the Activity of Modified BMPs [0110] In preferred embodiments, the activity of the wild-type and modified

BMPs are analyzed using in vitro receptor binding assays, cell-based activity assays, or in vivo activity assays.

Receptor Binding Assays

[0111] The affinity of the modified BMPs for one or more BMP receptors can be determined by receptor binding assays. For example, affinities for ALK-2, ALK-3, ALK-6, ActRII, ActRllb, or BMPRII can be determined. Suitable binding assays include, but are not limited to, ELISA, fluorescence anisotropy and intensity, scintilation proximity assays (SPA), Biacore (Pearce et al., Biochemistry 38:81-89 (1999)), DELFIA assays, and AlphaScreen™ (commercially available from PerkinElmer; Bosse R., Illy C, and Chelsky D (2002)).

[0112] In a preferred embodiment, Biacore or surface plasmon resonance assays are used. See, for example, McDonnell (2001) Curr. Opin. Chem. Biol. 5:572- 577. Typically, Biacore experiments may be performed, for example, by binding BMP receptor-Fc fusion proteins to a protein A derivitized chip or an NTA chip and testing each modified BMP as an analyte. It is also possible to bind an anti-BMP antibody to the chip, or to bind the modified BMP to the chip and test soluble receptor or Fc-receptor fusion proteins as analytes. Biacore experiments have been used previously to characterize binding of TGF-β isoforms to their receptors (De Crescenzo et al. (2001) J. Biol. Chem., 276: 29632-29643; De Crescenzo et al (2003) L_MoLJ3ioL, 328: 1173-1 183).

[0113] Alternatively, a plate-based Direct Binding Assay is used to determine the affinity of one or more modified BMPs for one or more BMP receptors. This method is a modified sandwich ELISA in which BMP is captured using an anti-BMP monoclonal antibody and then detected using a BMP receptor-Fc fusion protein.

[0114] AlphaScreen™ assays (Bosse R. et al. (2002) Principles of

AlphaScreen™, PerkinElmer Literaure Aplication Note Ref #4069. http://lifesciences.perkinelmer. com/Notes/S4069-0802.pdf) can be used to characterize receptor and inhibitor binding. AlphaScreen™ is a bead-based non- radioactive luminescent proximity assay where the donor beads are excited by a laser at 680 nm to release singlet oxygen. The singlet oxygen diffuses and reacts with the thioxene derivative on the surface of acceptor beads leading to fluorescence emission at600 nm. The fluorescence emission occurs only when the donor and acceptor beads are brought into close proximity by molecular interactions occurring when each is linked to ligand and receptor (or ligand and inhibitor) respectively. This interaction may be competed away by adding an appropriate amount of unlabeled modified BMP that binds the relevant receptor or inhibitor.

[0115] In one embodiment, AlphaScreen™ assays are performed using 1) native BMP labeled by a first suitable tag or label; 2) donor beads capable of binding the first tag or label; 3) a BMP receptor or inhibitor labeled by a second suitable tag or label; 4) acceptor beads capable of binding the second tag or label, and 5) varying amounts of an unlabeled modified BMP molecule (e.g., a modified BMP-7), which acts as a competitor. It is also possible to coat the donor or acceptor beads with antibodies that specifically recognize the native BMP or BMP receptor, or to bind the receptor to the donor beads and the ligand to the acceptor beads. In an alternate embodiment, AlphaScreen™ assays are performed using 1) a type I BMP receptor labeled by a first suitable tag or label; 2) donor beads capable of binding the first tag or label; 3) a type II BMP receptor labeled by a second suitable tag or label; 4) acceptor beads capable of binding the second tag or label; 5) native BMP, and 6) varying amounts of an unlabeled modified BMP molecule (e.g., a modified BMP-7), which acts as a competitor. It is also possible to bind the type I BMP receptor to the acceptor beads and the type II BMP receptor to the donor beads.

[0116] Fluorescence assays can also be used to characterize receptor and inhibitor binding. For example, either BMP-7 or a BMP-7 receptor or inhibitor may be labeled with a fluorescent dye (for examples of suitable dyes, see the Molecular Probes catalog). As is known in the art, the fluorescence intensity or anisotropy of a labeled molecule may change upon binding to another molecule. Fluorescence assays may be performed using 1) fluorescently labeled native BMP (e.g., BMP-7), 2) a BMP receptor or inhibitor, and 3) varying amounts of an unlabeled modified BMP (e.g., modified BMP-7), which acts as a competitor.

[0117] Additionally, scintillation proximity assays (SPA) can be used to determine receptor binding affinity. For example, BMP receptor-Fc fusions may be bound to protein A coated SPA beads or flash-plate and treated with S³⁵-labeled BMP; the binding event results in production of light. Cell-Based Activity Assays

[0118] BMPs promote the growth and differentiation of a number of types of cells. BMP activity may be monitored, for example, by measuring BMP-induced differentiation of MC3T3-E1 (an osteoblast-like cell derived from murine calvaria), C3H10T1/2 (a mouse mesenchymal stem cell line derived from embryonic connective tissue), ATDC5 (a mouse embryonal carcinoma cell), L-6 (a rat myoblast cell line) or C2C12 (a mouse myoblastic cell line) cells. Differentiation may be monitored using, for example, luminescence reporters for alkaline phosphatase or calorimetric reagents such as Alcian Blue or PNPP (Asahina et al. (1996) Exp. Cell Res,, 222:38-47; Inada et al. (1996) Biochem. Biophvs. Res. Commun.. 222:317- 322; Jortikka et al. (1998) Life ScL 62:2359-2368; Cheng et al. (2003) J. Bone Joint Surgery 95A:1544-1552).

[0119] The rat limb bud cartilage differentiation assay may also be used to monitor activity in primary cells. In alternative embodiments, reporter gene or kinase assays may be used. Since BMPs activate the JAK-STAT signal transduction pathway, a BMP responsive cell line containing a STAT-responsive reporter such as GFP or luciferase may be used (Kusanagi et al. (2000) MoI Biol. Cell., 11 :555-565). For example, BMP activity in kidney cells may be determined using cell-based assays; see for example Wang and Hirschberg (2004) J. Biol. Chem., 279:23200- 23206.

Animal Models of BMP Activity

[0120] Typically, BMP activities in an animal are measured by bone induction following subcutaneous injection. In a preferred embodiment, the activities of one or more modified BMPs are determined in an animal model of a BMP -responsive disease or condition. For example, nimal models of renal disease include, but are not limited to, the rat nephrotoxic serum nephritis model (Zeisberg et al. 2003)), the rat chronic cyclosporine A-induced nephropathy model (Li et al. (2004) Am. J. Physiol. Renal Physiol., 286:F46-57), the mouse unilateral uretreral obstruction model (Schanstra et al. (2003) Thromb. Haemost, 89:735-740), streptozotocin- induced diabetic nephropathy (Taneda et al. (2003) J. Am. Soc. Nephrol., 14:968-980), the anti-thy 1.1 mAb and Habu snake venom induced glomerulonephritis models (Dimmler et al. (2003) Diagn. MoI. Pathol, 12: 108-117), and the rat 5/6 remnant kidney model (Romero et al. (1999) Kidney Int., 55:945- 955). Animal models of liver disease include, but are not limited to, rat bile duct ligation/scission model (Park et al. (2000) Pharmacol. Toxicol., 87:261-268), CCI4 plus ethanol-induced liver damage (Hall et al. (1991) Hepatology, 12:815-819), dimethyinitrosamine-induced liver cirrhosis (Kondou et al. (2003) J. Hepatol., 39:742-748), and thioacetamide-induced liver damage (Muller et al. (1988) Exp. Pathol., 34:229-236). Animal models of lung disease include, but are not limited to, ovalbumin-induced airway fibrosis (Kenyon et al. (2003) Toxicol. Appl. Pharmacol., 186:90-100), bleomycin-induced lung fibrosis (Izbicki et al. (2002) Int. J. Exp. Pathol, 83:111-119), monocrotaline-induced pulmonary fibrosis (Hayashi et al (1995) Toxicol. Pathol, 23: 63-71), and selective irradiation (Pauluhn et al. (2001) Toxicology, 161 : 153-163). Animal models of neurological disease include, but are not limited to, animal models for Parkinson's disease such as the 6- hydroxydopamine (6-OHDA) hemilesioned rat model and MPTP-induced

Parkinson's disease, animal models of ALS such as rats or mice expressing mutant SODl (Shibata et al. (2002) Neuropathology, 22:337-349), and animal models of stroke induced by intracortical microinjection of endothelin or quinolinic acid (Gilmour et al. (2004) Behav. Brain Res., 150:171-183) or cerebral artery occlusion (Merchenthaler et al. (2003) Ann. NY Acad. ScI 1007: 89-100).

Formulation and A dministration

[0121] Designed BMPs of the present invention can be formulated for administration to a mammal, preferably a human in need thereof as part of a pharmaceutical composition. The composition can be administered by any suitable means, e.g., parenterally, orally or locally. Where the designed BMPs is to be administered locally, as by injection, to a desired tissue site, or systemically, such as by intravenous, subcutaneous, intramuscular, intraorbital, ophthalmic, intraventricular, intracranial, intracapsular, intraspinal, intracistemal, intraperitoneal, buccal, rectal, vaginal, intranasal or aerosol administration, the composition preferably comprises an aqueous solution. The solution preferably is physiologically acceptable, such that administration thereof to a mammal does not adversely affect the mammal's normal electrolyte and fluid volume balance. The aqueous solution thus can comprise, e.g., normal physiologic saline (0.9% NaCl, 0.15M), pH 7-7.4.

[0122] Useful solutions for oral or parenteral systemic administration can be prepared by any of the methods well known in the pharmaceutical arts, described, for example, in "Remington's Pharmaceutical Sciences" (Gennaro, A., ed., Mack Pub., 1990, the disclosure of which is incorporated herein by reference). Formulations can include, for example, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like. Formulations for direct administration, in particular, can include glycerol and other compositions of high viscosity. Biocompatible, preferably bioresorbable polymers, including, for example, hyaluronic acid, collagen, tricalcium phosphate, polybutyrate, polylactide, polyglycolide and lactide/glycolide copolymers, may be useful excipients to control the release of the designed BMPs in vivo.

[0123] Other potentially useful parenteral delivery systems for the present designed BMPs can include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation administration can contain as excipients, for example, lactose, or can be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate or deoxycholate, or oily solutions for administration in the form of nasal drops or as a gel to be applied intranasally.

[0124] Alternatively, the designed BMPs, including designed OP-I , identified as described herein may be administered orally. For example, liquid formulations of designed BMPs can be prepared according to standard practices such as those described in "Remington's Pharmaceutical Sciences" (supra). Such liquid formulations can then be added to a beverage or another food supplement for administration. Oral administration can also be achieved using aerosols of these liquid formulations. Alternatively, solid formulations prepared using art-recognized emulsifiers can be fabricated into tablets, capsules or lozenges suitable for oral administration. [0125] Optionally, the designed BMPs can be formulated in compositions comprising means for enhancing uptake of the analog by a desired tissue. For example, tetracycline and diphosphonates (bisphosphonates) are known to bind to bone mineral, particularly at zones of bone remodeling, when they are provided systemically in a mammal. Accordingly, such components can be used to enhance delivery of the present designed BMPs to bone tissue. Alternatively, an antibody or portion thereof that binds specifically to an accessible substance specifically associated with the desired target tissue, such as a cell surface antigen, also can be used. If desired, such specific targeting molecules can be covalently bound to the present analog, e.g., by chemical crosslinking or by using standard genetic engineering techniques to create, for example, an acid labile bond such as an Asp- Pro linkage. Useful targeting molecules can be designed, for example, according to the teachings of U.S. Pat. No. 5,091,513.

[0126] It is contemplated also that some of the designed BMPs may exhibit the highest levels of activity in vivo when combined with carrier matrices, i. e. , insoluble polymer matrices. See for example, U.S. Pat. No. 5,266,683 the disclosure of which is incorporated by reference herein. Currently preferred carrier matrices are xenogenic, allogenic or autogenic in nature. It is contemplated, however, that synthetic materials comprising polylactic acid, polyglycolic acid, polybutyric acid, derivatives and copolymers thereof may also be used to generate suitable carrier matrices. Preferred synthetic and naturally derived matrix materials, their preparation, methods for formulating them with the designed BMPs of the invention, and methods of administration are well known in the art and so are not discussed in detailed herein. See for example, U.S. Pat. No. 5,266,683. [0127] Still further, the present designed BMPs can be administered to the mammal in need thereof either alone or in combination with another substance known to have a beneficial effect on tissue morphogenesis. Examples of such substances (herein, cofactors) include substances that promote tissue repair and regeneration and/or inhibit inflammation. Examples of useful cofactors for stimulating bone tissue growth in osteoporotic individuals, for example, include but are not limited to, vitamin D3, calcitonin, prostaglandins, parathyroid hormone, dexamethasone, estrogen and IGF-I or IGF-II. Useful cofactors for nerve tissue repair and regeneration can include nerve growth factors. Other useful cofactors include symptom-alleviating cofactors, including antiseptics, antibiotics, antiviral and antifungal agents, analgesics and anesthetics.

[0128] Modified BMPs preferably are formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable, nontoxic excipients and carriers. As noted above, such compositions can be prepared for systemic, e.g., parenteral, administration, particularly in the form of liquid solutions or suspensions; for oral administration, particularly in the form of tablets or capsules; or intranasally, particularly in the form of powders, nasal drops or aerosols. Where adhesion to a tissue surface is desired, the composition can comprise a fibrinogen-thrombin dispersant or other bioadhesive such as is disclosed, for example, in PCT US91/09275, the disclosure of which is incorporated herein by reference. The composition then can be painted, sprayed or otherwise applied to the desired tissue surface. [0129] The compositions can be formulated for parenteral or oral administration to humans or other mammals in therapeutically effective amounts, e.g., amounts which provide appropriate concentrations of the designed BMPs to target tissue for a time sufficient to induce the desired effect. Preferably, the present compositions alleviate or mitigate the mammal's need for a morphogen-associated biological response, such as maintenance of tissue-specific function or restoration of tissue-specific phenotype to senescent tissues (e.g., osteopenic bone tissue).

[0130] As will be appreciated by those skilled in the art, the concentration of the compounds described in a therapeutic composition will vary depending upon a number of factors, including the dosage of the drug to be administered, the chemical characteristics (e.g., hydrophobicity) of the compounds employed, and the route of administration. The preferred dosage of drug to be administered also is likely to depend on such variables as the type and extent of a disease, tissue loss or defect, the overall health status of the particular patient, the relative biological efficacy of the compound selected, the formulation of the compound, the presence and types of excipients in the formulation, and the route of administration. In general terms, the compounds of this invention may be provided in an aqueous physiological buffer solution containing about 0.1 to 10% w/v compound for parenteral administration. Typical doses ranges are from about 10 ng/kg to about 1 g/kg of body weight per day; with a preferred dose range being from about 0.1 mg/kg to 100 mg/kg of body weight. Therapeutic Uses

[0131] The modified BMPs of this invention are capable of inducing the developmental cascade of bone morphogenesis and tissue morphogenesis for a variety of tissues in mammals different from bone or bone cartilage. This morphogenic activity includes the ability to induce proliferation and differentiation of progenitor cells, and the ability to support and maintain the differentiated phenotype through the progression of events that results in the formation of bone, cartilage, non-mineralized skeletal or connective tissues, and other adult tissues.

[0132] For example, the modified BMPs of the present invention may be used for treatment to prevent loss of and/or increase bone mass in metabolic bone diseases. General methods for treatment to prevent loss of and/or increase bone mass in metabolic bone diseases using osteogenic proteins are disclosed in U.S. Patent No. 5,674,844, the disclosures of which are hereby incorporated by reference. The modified BMPs of the present invention may be used for periodontal tissue regeneration. General methods for periodontal tissue regeneration using osteogenic proteins are disclosed in U.S. Patent No. 5,733,878, the disclosures of which are hereby incorporated by reference. The modified BMPs of the present invention may be used for liver regeneration. General methods for liver regeneration using osteogenic proteins are disclosed in U.S. Patent No. 5,849,686, the disclosures of which are hereby incorporated by reference. The modified BMPs of the present invention may be used for treatment of chronic renal failure. General methods for treatment of chronic renal failure using osteogenic proteins are disclosed in U.S. Patent No. 6,861,404, the disclosures of which are hereby incorporated by reference. The modified BMPs of the present invention may be used for enhancing functional recovery following central nervous system ischemia or trauma. General methods for enhancing functional recovery following central nervous system ischemia or trauma using osteogenic proteins are disclosed in U.S. Patent No. 6,407,060, the disclosures of which are hereby incorporated by reference. The modified BMPs of the present invention may be used for inducing dendritic growth. General methods for inducing dendritic growth using osteogenic proteins are disclosed in U.S. Patent No. 6,949,505, the disclosures of which are hereby incorporated by reference. The modified BMPs of the present invention may be used for inducing neural cell adhesion. General methods for inducing neural cell adhesion using osteogenic proteins are disclosed in U.S. Patent No. 6,800,603, the disclosures of which are hereby incorporated by reference. The modified BMPs of the present invention may be used for treatment and prevention of Parkinson's disease. General methods for treatment and prevention of Parkinson's disease using osteogenic proteins are disclosed in U.S. Patent No. 6,506,729, the disclosures of which are hereby incorporated by reference. It is within skills of an ordinary artisan to modify the general methods using the modified BMPs of the present invention for various therapeutic uses described above. Exemplary embodiments of therapeutic applications of the modified BMPs of the present invention are further described below.

Refieneration of Damaged or Diseased Tissue

[0133] The modified BMPs of this invention may be used to repair diseased or damaged mammalian tissue. The tissue to be repaired is preferably assessed, and excess necrotic or interfering scar tissue removed as needed, by surgical, chemical, ablating or other methods known in the medical arts. The modified BMPs then may be provided directly to the tissue locus as part of a sterile, biocompatible composition, either by surgical implantation or injection. Alternatively, a sterile, biocompatible composition containing modified BMP-stimulated progenitor cells may be provided to the tissue locus. The existing tissue at the locus, whether diseased or damaged, provides the appropriate matrix to allow the proliferation and tissue-specific differentiation of progenitor cells. In addition, a damaged or diseased tissue locus, particularly one that has been further assaulted by surgical means, provides a morphogenically permissive environment. For some tissues, it is envisioned that systemic provision of the modified BMPs will be sufficient. [0134] In some circumstances, particularly where tissue damage is extensive, the tissue may not be capable of providing a sufficient matrix for cell influx and proliferation. In these instances, it may be necessary to provide the modified BMPs or modified BMP-stimulated progenitor cells to the tissue locus in association with a suitable, biocompatible formulated matrix, prepared by any of the means described below. The matrix preferably is tissue-specific, in vivo biodegradable, and comprises particles having dimensions within the range of 70-850 μm, most preferably 150-420 μm.

[0135] The modified BMPs of this invention also may be used to prevent or substantially inhibit scar tissue formation following an injury. If a modified BMP is provided to a newly injured tissue locus, it can induce tissue morphogenesis at the locus, preventing the aggregation of migrating fibroblasts into non-differentiated connective tissue. The modified BMP preferably is provided as a sterile pharmaceutical preparation injected into the tissue locus within five hours of the injury.

[0136] For example, the modified BMPs may be used for protein-induced morphogenesis of substantially injured liver tissue following a partial hepatectomy. Variations on this general protocol may be used for other tissues. The general method involves excising an essentially nonregenerating portion of a tissue and providing the modified BMP, preferably as a soluble pharmaceutical preparation to the excised tissue locus, closing the wound and examining the site at a future date. Like bone, liver has a potential to regenerate upon injury during post-fetal life.

[0137] As another example, the modified BMPs of this invention can also be used to induce dentinogenesis. To date, the unpredictable response of dental pulp tissue to injury is a basic clinical problem in dentistry. Using standard dental surgical procedures, small areas (e.g., 2 mm) of dental pulps can be surgically exposed by removing the enamel and dentin immediately above the pulp (by drilling) of sample teeth, performing a partial amputation of the coronal pulp tissue, inducing hemostasis, application of the pulp treatment, and sealing and filling the cavity by standard procedures. [0138] As another example, the modified BMP-induced regenerative effects on central nervous system (CNS) repair may be assessed using a rat brain stab model. Briefly, male Long Evans rats are anesthesized and the head area prepared for surgery. The calvariae is exposed using standard surgical procedures and a hole drilled toward the center of each lobe using a 0.035K wire, just piercing the calvariae. 25 μl solutions containing either a modified BMP or PBS then is provided to each of the holes by Hamilton syringe. Solutions are delivered to a depth approximately 3 mm below the surface, into the underlying cortex, corpus callosum and hippocampus. The skin then is sutured and the animal allowed to recover. [0139] Three days post surgery, rats are sacrificed by decapitation and their brains processed for sectioning. Scar tissue formation is evaluated by immunofiuoresence staining for glial fibrillary acidic protein, a marker protein for glial scarring, to qualitatively determine the degree of scar formation.

Exemplification [0140] The following BMP-7 mutants were preparted and characterized using the methods set forth below: R48A; R48D; R48Q; Il 12A; Fl 17W; K126A; K126D; K127H; R134E; AND (R48A, Fl 17W).

[0141] Preparation of variant cDNA

Mutations in the mature region of the BMP-7 protein were introduced by site directed mutagenesis, using QuikChange protocol (Stratagene). The sequence of each variant was confirmed by DNA sequencing. DNA preparation of variants was performed using Qiagen Maxi kit.

Expression of variants Variants were expressed in HEK 293T cells (ATCC) and analyzed by

Western blot analysis and ELISA. 293T cells were seeded in 6-well tissue culture plates at 4 x 10⁵ cells/well in DMEM supplemented with 10% FBS without antibiotics. The next day, cells were transfected with the appropriate DNA constructs using the Fugene HD reagent as described by the manufacturer (Roche). 24 hr post transfection, the culture medium was changed to DMEM containing 0.5% FBS. Transfected cells were then incubated for an additional 48 to 72 hour in a humidified tissue-culture incubator with 5% CO₂ at 37° C. Conditioned medium was collected at end of this incubation period for BMP-7 expression and activity analysis.

Western Blot Analysis

Proteins in conditioned media were resolved by SDS PAGE using pre-cast gels (Invitrogen) following manufacturer's recommendations. Resolved proteins were then transferred to a nitrocellulose membrane using a semi-dry transfer apparatus (Bio-Rad). Membranes containing blotted proteins were subsequently blocked, incubated with a rabbit polyclonal anti-BMP-7 primary antibody solution, washed, and then incubated with a near-infra red (NIRF) dye labeled goat anti-rabbit secondary antibody. Antibody-antigen complexes were visualized upon exposure of the blotted membranes in a Licor NIRF scanner according to the manufacturer's recommendations .

Assessing BMP-7 Mutant Activity and Susceptibility to Noggin Inhibition

The activity of BMP-7 mutants can be assessed using A549-BRE cells. This cell line is stably transfected with a BMP-7 reporter construct consisting of a BMP response element (BRE) upstream of the Firefly luciferase gene. A549-BRE cells are seeded in 96-well plates at a density of 15 x 10³ cells/well in 1 % FBS containing DMEM, supplemented with 0.2% G418. Seeding medium is removed 24 hrs later and replaced with diluted conditioned medium from transfected 293 cells. BMP-7 induced luciferase expression is then quantified by performing a luciferase activity assay on A549 BRE cells 24 hour post exposure to 293 cell conditioned medium. In order to assess BMP-7 mutant susceptibility to Noggin inhibition, A549-BRE cells are exposed to 293 cell conditioned medium in the presence of increasing concentrations (0.1, 0.5 or 1 ug/ml) of recombinant mouse Noggin (R&D Systems). In this assay, the highest Noggin concentration tested (1 ug/ml) results in a 100 % inhibition of wild type BMP-7 activity. INCORPORATION BY REFERENCE

[0142] All sequence and structure access numbers, publications and patent documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if the contents of each individual publication or patent document was incorporated herein.

Claims

CLAIMS We claim:

1. A chimeric bone morphogenetic protein comprising a dimer, wherein each monomer comprises a type I receptor binding site derived from a member of the BMP subfamily and a type II receptor binding site derived from a member of the TGF-β subfamily, wherein (i) said chimeric bone morphogenetic protein has improved binding affinity to type II receptor relative to the naturally-occurring BMP subfamily member from which the type I receptor binding site is derived and (ii) the chimeric bone morphogenetic protein is capable of inducing bone morphogenesis and tissue morphogenesis.

2. The chimeric bone morphogenetic protein of claim 1, wherein the chimeric bone morphogenetic protein is capable of inducing formation of bone, cartilage, non- mineralized skeletal or connective tissue, kidney, liver, periodontal or nerve tissues.

3. The chimeric bone morphogenetic protein of claim 1, wherein the type I receptor binding site is derived from BMP-7.

4. The chimeric bone morphogenetic protein of claim 1, wherein the type I receptor binding site is derived from BMP-2.

5. The chimeric bone morphogenetic protein of claim 1, wherein the type I receptor binding site comprises an amino acid sequence Xaal Xaa2 Xaa3 Xaa4 Phe Xaa5 Xaa6 Xaa7 Xaa8 Trp Xaa9 XaalO Tip, wherein Xaal, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, Xaa9, or XaalO is any amino acid.

6. The chimeric bone morphogenetic protein of claim 5, wherein Xaal is Leu; Xaa2 is Tyr or Phe; Xaa3 is VaI; Xaa4 is Asp or Ser; Xaa5 is Ser, Asn, or Arg; Xaa6 is Asp; Xaa7 is VaI or Leu; Xaa8 is GIy; Xaa9 is Asn or GIn; and XaalO is Asp.

7. The chimeric bone morphogenetic protein of claim 1, wherein the type I receptor binding site comprises an amino acid sequence Pro Xaal Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Asn, wherein Xaal, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, or Xaa8 is any amino acid.

8. The chimeric bone morphogenetic protein of claim 7, wherein Xaal is Leu; Xaa2 is Ala or Asn; Xaa3 is Asp or Ser; Xaa4 is His, Tyr or Phe; Xaa5 is Leu or Met; Xaa6 is Asn; Xaa7 is Ser or Ala; and Xaa8 is Thr.

9. The chimeric bone morphogenetic protein of claim 1, wherein the type I receptor binding site comprises an amino acid sequence Xaal Xaa2 He Xaa3 Xaa4 Thr Leu

VaI Xaa5 Xaa6 Xaa7, wherein Xaal, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, or Xaa7 is any amino acid.

10. The chimeric bone morphogenetic protein of claim 9, wherein Xaal is His; Xaa2 is Ala; Xaa3 is VaI; Xaa4 is GIn; Xaa5 is Asn or His; Xaa6 is Ser or Phe; or Xaa7 is VaI or Ue.

11. The chimeric bone morphogenetic protein of claim 1 , wherein the type II receptor binding site is derived from TGF-βl.

12. The chimeric bone morphogenetic protein of claim 1, wherein the type I receptor binding site is derived from BMP-7 and the type II receptor binding site is derived from TGF-βl.

13. A modified bone morphogenetic protein with improved binding affinity to a type II receptor relative to the corresponding naturally-occurring bone morphogenetic protein, the modified bone morphogenetic protein comprising a replacement fragment derived from a member of the TGF-β subfamily, wherein (i) the replacement fragment replaces a fragment of human BMP-7 comprising a sequence selected from the group consisting of IAPE, GYA, ISVL and VILKKY, and (ii) the modified bone morphogenetic protein is capable of inducing bone morphogenesis or tissue morphogenesis.

14. The modified bone morphogenetic protein of claim 13, wherein the modified bone morphogenetic protein is capable of inducing formation of bone, cartilage, non-mineralized skeletal or connective tissue, kidney, liver, periodontal or nerve tissues.

15. The modified bone morphogenetic protein of claim 13, wherein the replaced fragment of human BMP-7 comprises the sequence IAPE, and wherein the replacement fragment comprises a sequence selected from the group consisting of IAPS, HEPK and LIPE.

16. The modified bone morphogenetic protein of claim 13, wherein the replaced fragment of human BMP-7 comprises the sequence GYA, and wherein the replacement fragment comprises a sequence selected from the group consisting of GYH and TYQ.

17. The modified bone morphogenetic protein of claim 13, wherein the replaced fragment of human BMP-7 comprises the sequence ISVL, and wherein the replacement fragment comprises a sequence selected from the group consisting of MSML, LPIV and GKLL.

18. The modified bone morphogenetic protein of claim 13, wherein the replaced fragment of human BMP-7 comprises the sequence VILKKY, and wherein the replacement fragment comprises a sequence selected from the group consisting of IIKKDI, KVEQL and ISAHHV.

19. The modified bone morphogenetic protein of claim 13, wherein the modified bone morphogenetic protein is a modified BMP-7.

20. The modified bone morphogenetic protein of claim 19, wherein the modified BMP-7 further comprises a second replacement fragment derived from BMP-2 comprising the sequence NSVNSKI, wherein the replacement fragment replaces the fragment H92-F93-I94-N95-P96-E97-T98-V99 in the native human BMP-7 sequence and wherein the modified BMP-7 has improved binding affinity to a type I receptor relative to the naturally-occurring BMP-7.

21. A modified BMP-7, wherein the amino acid sequence H92-F93-I94-N95-P96- E97-T98-V99 in the native human BMP-7 sequence is substituted with the sequence NSVNSKI.

22. A modified bone morphogenetic protein with reduced binding affinity to Noggin comprising at least one amino acid mutation corresponding to a position selected from the group consisting of Ilel 12, VaIl 14, Tyrl 16, Phel 17, Aspl 18, Aspl 19, Serl20, Serl21, Asnl22, Ilel24, Leul25, and Lysl26 of human BMP-7, wherein the modified bone morphogenetic protein is capable of inducing bone morphogenesis or tissue morphogenesis.

23. The modified bone morphogenetic protein of claim 22, wherein the modified bone morphogenetic protein is capable of inducing formation of bone, cartilage, non-mineralized skeletal or connective tissue, kidney, liver, periodontal or nerve tissues.

24. The modified bone morphogenetic protein of claim 22, wherein the modified bone morphogenetic protein is a modified BMP-7.

25. The modified bone morphogenetic protein of claim 22, wherein the modified bone morphogenetic protein comprises a mutation at a position corresponding to L 125 of human BMP-7.

26. The modified bone morphogenetic protein of claim 25, wherein the modified bone morphogenetic protein comprises a charged amino acid at the position corresponding to L 125 of human BMP-7.

27. The modified bone morphogenetic protein of claim 26, wherein the modified bone morphogenetic protein comprises Arg at the position corresponding to L 125 of human BMP-7.

28. A modified BMP-7 comprising at least one amino acid substitution selected from the group consisting of R48A, I57P, D54E, K126A, A63H, K127W, Fl 17T, K127Y, F117W, V123A, I112A, V123D, I112D, L125A, I112P L125D I124A L125R I124D K126H, Il 12H, R129A, R129D, R129E, R129H, R129I, R129N, R129W, R129Y, R129F.

29. A modified BMP-7 comprising a set of amino acid substitutions selected from the group consisting of (D54R, Y65N, Y78H, Rl 34E); (I57A,Y65N, Y78H,

Rl 34E); (Y65N, Y78H, Il 12D, Rl 34E); (Y65N, Y78H, Fl 17D, Rl 34E); (Y65N, Y78H, Fl 17T, R134E); (Y65N, Y78H, L125D, R134E); (Y65N, Y78H, K126E, R134E); (Y65N, Y78H, K127E, R134E) and (Y65N, Y78H, K126E, K127E, Rl 34E)

30. A modified BMP-7 comprising the amino acid substitutions K126E and K127E.

31. The modified BMP-7 of any of claims 15 to 30, further comprising the amino acid substitutions Y65N, Y78H and R134E.