Activin / bone morphogenetic protein 7 chimeras: superactive SAB704 and SAB715 and their noggin-sensitized variants NAB704, NAB715 and NAB204

Abstract

The present invention relates to chimeric polypeptides having transforming growth factor (TGF)-β activity, nucleic acids encoding the polypeptides, and host cells for producing polypeptides having improved or new biological and therapeutic properties.

Description

Technical Field

[0001] This application claims priority to U.S. Patent Application No. 62 / 767,707, filed on November 15, 2018, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the manipulation and design of biomolecules and the manipulated proteins and nucleic acids. Background Art

[0003] Activins and bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily. Their pervasiveness in various developmental and cell signaling processes makes TGF-β ligands candidates for therapeutic use. The discovery presented in the present invention is that such biological properties can be specifically modified, enhanced, and altered by processing TGF-β ligands. Summary of the invention

[0004] Technical issues

[0005] According to one embodiment of the present invention, the present invention provides a recombinant polypeptide comprising two or more peptide segments respectively derived from different polypeptides, wherein the first segment of the above polypeptide comprises a sequence having more than 90% identity with a first transforming growth factor-β family protein, and the second peptide segment comprises a sequence having more than 90% identity with a second transforming growth factor-β family protein, and the above two segments are operably connected, and have at least two activities of the first parental transforming growth factor-β family protein or the second parental transforming growth factor-β family protein (parental TGF-beta family protein), or have new in vivo signal transduction activity and cell-specific activity, and the two or more polypeptide segments comprise 6 peptide segments operably connected from the N-terminus to the C-terminus, wherein the above polypeptide regulates the SMAD pathway.

[0006] In one example of the present invention, the present invention provides a recombinant polypeptide comprising a total of 6 segments. Two or more peptide segments comprise a sequence having a 90% or more identity with a first transforming growth factor-β family parent protein, and another peptide segment comprises a sequence having a 90% or more identity with a second transforming growth factor-β family parent protein, wherein these 6 segments are operably linked in the order of segment 1, segment 2, segment 3, segment 4, segment 5, and segment 6, and the resulting polypeptide chimera enhances the activity of one or more or all two of the transforming growth factor-β family proteins.

[0007] In one example of the present invention, the polypeptide comprises the N-terminal segment 1 derived from bone morphogenetic protein-2, and the two or more polypeptide segments comprise six peptide segments operably linked from the N-terminus to the C-terminus.

[0008] In one example of the present invention, the first transforming growth factor-β family protein is bone morphogenetic protein-2, the second transforming growth factor-β family protein is activin or other family members, each segment corresponds to the structural motif of the common segment of the two transforming growth factor-β family proteins, and the polypeptide regulates the SMAD pathway.

[0009] In one example of the present invention, the polypeptide comprises the N-terminal segment 1 derived from bone morphogenetic protein-7, and the two or more polypeptide segments comprise six peptide segments operably linked from the N-terminus to the C-terminus.

[0010] In one example of the present invention, the first transforming growth factor-β family protein is bone morphogenetic protein-7, and the second transforming growth factor-β family protein is activin or other family members, wherein each segment corresponds to the structural motif of the common segment of the above two transforming growth factor-β family proteins, and the above polypeptide regulates the SMAD pathway.

[0011] In one example of the present invention, in the above polypeptide, segments 1, 3 and 4 derived from bone morphogenetic protein-7 are operably linked to segments 2, 5 and 6 derived from activin to prepare SAB704, and the above polypeptide regulates the SMAD pathway.

[0012] In one example of the present invention, in the above polypeptide, segments 1, 3, 4 and 5 derived from bone morphogenetic protein-7 and segments 2 and 6 derived from activin are operably linked to prepare SAB715, and the above polypeptide regulates the SMAD pathway.

[0013] In a more preferred embodiment, the present invention provides design-augmented (DA) TGF-beta ligands, which can be synthesized by screening different sequence segments that bind to growth factor-beta superfamily ligands and joining them to construct new ligands ("design-augmented chimeras"). These new ligands have a completely new protein sequence library that is different from the naturally occurring parent ligand. This approach mainly comes from identifying structural commonality between natural TGF-beta superfamily ligands. All ~40 TGF-beta superfamily ligands share the same overall architecture with generic features for various regions of the above proteins. The framework of the TGF-beta ligand can be divided into (usually) 6 subdomains (also called sequence segments; in Figure 1 6 different colors in the figure).

[0014] According to another embodiment of the present invention, the present invention also provides a design principle for a chimeric transforming growth factor-β family polypeptide comprising a first transforming growth factor-β family protein segment (e.g., a protein represented by "A") operably linked to a second different transforming growth factor-β family protein segment (e.g., a protein represented by "B"), wherein the above-mentioned polypeptide chimera has the order of segment 1-segment 2-segment 3-segment 4-segment 5-segment 6, and each segment of the chimera is derived from the corresponding numbered segment of A or B, as described in the present invention, different SMAD-regulatory activities are provided by generating different chimeras (Gray & Choe, 2019, "Design-augmented (DA) biologics: BMP Chimeras for bone and cartilage regeneration, Osteoarthritis and Cartilage, in press, DOI: doi.org / 10.1016 / j.joca.2019.09.004).

[0015] The sequence boundaries of these chimeras are from U.S. Provisional Application No. 61 / 155,066 filed on February 24, 2009. The activin and bone morphogenetic protein-2 chimeras (comprising AB204 and AB215) have super active signaling capabilities compared to their parent proteins (see Figure 2 ).

[0016] According to another embodiment of the present invention, the present invention provides a chimeric transforming growth factor-β family polypeptide, comprising a first transforming growth factor-β family protein segment operably linked to a second transforming growth factor-β family protein segment to provide a chimeric polypeptide with SMAD-regulatory activity, wherein each segment corresponds to a structural motif of a common segment of the above-mentioned two transforming growth factor-β family proteins.

[0017] According to another embodiment of the present invention, the present invention provides sequence variants, which are the sequence variants described in embodiments 1, 2, 3, 4, 5, 6, 7 or 8. In order to prepare NAB204 using AB204, prepare NAB704 using SAB704 or prepare NAB715 using SAB715 by regulating the noggin binding ability, a noggin sensitizer sequence of 6 amino acids replaces the structurally equivalent region of the above-mentioned polypeptide.

[0018] According to another embodiment of the present invention, the present invention provides sequence variants of polypeptides of the transforming growth factor-β superfamily, using a noggin-sensitive sequence of 6 amino acids in length derived from a related subfamily sequence to introduce a noggin-sensitive (NS-)-sequence. As an example of such a chimera, a noggin-sensitive form such as noggin-sensitive-activin (derived from activin) can be generated, or a noggin-insensitive (NIS-)-sequence derived from a related subfamily sequence is used to replace the same 6 amino acids in length noggin-sensitive sequence to remove the noggin-sensitive-sequence. As an example of such a chimera, bone morphogenetic protein 2 can be used to generate noggin-insensitive-bone morphogenetic protein 2.

[0019] Specifically, the present invention provides three non-naturally occurring chimeras, each of which comprises a protein derived from a parent protein, activin and bone morphogenetic protein 7 (BMP-7, SEQ ID NO: 6, Figure 2 ) protein sequence. Two chimeras named SAB704 and SAB715 are operably linked from the N-terminus to the C-terminus in the order of segment 1, segment 2, segment 3, segment 4, segment 5, and segment 6, thereby regulating the interaction with activin and bone morphogenetic protein-7 (SEQ ID NO: 9 and SEQ ID NO: 11, respectively, Figure 2 ) naturally associated SMAD pathway.

[0020] Named NAB704 and NAB715 (SEQ ID NO: 10 and SEQ ID NO: 12, respectively, Figure 2) are sequence variants of SAB704 and SAB715, for which less than 5 amino acid residues are changed in the sequence of segment 6 of SAB704 and SAB715, respectively. As a result, NAB704 and NAB715 are Noggin-sensitive. Specifically, Noggin-sensitivity means that Noggin can be physically bound to and react with Noggin in vivo through a design for biological function. SAB704 and SAB715 are Noggin-insensitive and are designed to have super activity in cell signaling ability. Additionally, the present invention also provides a third design-enhanced chimera sequence (identifies) as Noggin-sensitive, which is AB204 (derived from U.S. Provisional Application No. 61 / 155066). Figure 2 7), is a similar variant of NAB204 ( Figure 2 SEQ ID NO:8).

[0021] Basic design of SAB704, SAB715, NAB204, NAB704 and NAB715

[0022] In one example of the present invention, the polypeptide chimera SAB704 comprises segments 2, 5 and 6 of three segments derived from activin A ("A"), and segments 1, 3 and 4 of three segments derived from bone morphogenetic protein-7 ("B"), such as "1B2A3B4B5A6A", and these segments are operably linked from the N-terminus to the C-terminus in the order of segment 1, segment 2, segment 3, segment 4, segment 5 and segment 6. The sequence boundaries of SAB704 are derived from U.S. Provisional Application No. 61 / 155066 filed on February 24, 2009 ( Figure 5 ).

[0023] In another example of the present invention, the polypeptide chimera SAB715 comprises two segments derived from activin A ("A"), segments 2 and 6, and four segments derived from bone morphogenetic protein-7 ("B"), segments 1, 3, 4, and 5, such as "1B2A3B4B5B6A", which are operably linked from the N-terminus to the C-terminus in the order of segment 1, segment 2, segment 3, segment 4, segment 5, and segment 6. The sequence boundaries of SAB715 are derived from U.S. Provisional Application No. 61 / 155066 filed on February 24, 2009.

[0024] In another embodiment of the present invention, the polypeptide chimera NAB204 has 5 new amino acids replacing 6 residues in segment 6 of AB204 (replaced by VVLKKY in IIKKDI). IIKKDI is derived from activin A (the underlined portion of SEQ ID NO: 4, Figure 2 ), whereas VVLKKY is derived from bone morphogenetic protein 2 (the underlined portion of SEQ ID NO: 5, Figure 2 In the present application, these 6 residues in segment 6 are defined as "Noggin-sensitizer", whose protein sequence is similar to all Noggin-binding bone morphogenetic proteins including bone morphogenetic protein-2 and bone morphogenetic protein-7 (VILKKY in bone morphogenetic protein 7, the underlined part in SEQ ID NO: 7, Figure 2 ). These amino acid changes introduced into AB204 alter the biological properties of AB204 to make NAB204 (SEQ ID NO: 8, Figure 2 ) binds to Noggin ( Figure 4b ), and thus, is functionally sensitive to the antagonistic effects of Noggin in vivo, and is defined in this application as "Noggin-sensitized." The protein sequence of AB204 is derived from U.S. Provisional Application No. 61 / 155,066, filed on February 24, 2009.

[0025] In another embodiment of the present invention, the polypeptide chimera NAB704 has 4 new amino acids (replaced by VILKKY in IIKKDI) that replace the noggin-sensitive sequence of SAB704. These amino acid changes alter the biological properties of SAB704 to make NB704 bind to noggin, thereby making it functionally sensitive to the antagonistic effect of noggin in vivo ( Figure 4a ).

[0026] In another embodiment of the present invention, the polypeptide chimera NAB715 has 4 new amino acids (replaced by VILKKY in IIKKDI) that replace the noggin-sensitive sequence of SAB715. These amino acid changes alter the biological properties of SAB715 to make NB715 bind to noggin, thereby making it functionally sensitive to the antagonistic effect of noggin in vivo ( Figure 4a ).

[0027] Due to the sequence similarity, the noggin-sensitive sequence may contain no more than 5 amino acid substitutions that are identical or conservatively similar to VVLKKY of bone morphogenetic protein-2 or VILKKY of bone morphogenetic protein-7 within the noggin-sensitive sequence of the activin / bone morphogenetic protein chimera, thereby converting the noggin-sensitive sequence of the activin / bone morphogenetic protein chimera into noggin-sensitive or noggin-insensitive.

[0028] According to another embodiment of the present invention, the present invention provides a polynucleotide encoding the above polypeptide.

[0029] The present invention also provides polynucleotides encoding the polypeptides of the present invention. However, in this example, the present invention provides an identical polypeptide sequence that is at least 90%, 95%, 98%, 99% or more identical to the sequence consisting of the segment assembly of the activin / bone morphogenetic protein chimera, regardless of the presence of noggin-sensitivity.

[0030] In one example of the present invention, the polynucleotide comprises sequences derived from a plurality of transforming growth factor-β family polynucleotides operably linked to encode a functional chimeric polypeptide having SMAD-regulated activity including bone and cartilage regeneration through a combinatorial time-dependent mix of the ligands, thereby regulating cellular activity in vivo.

[0031] According to another embodiment of the present invention, the present invention provides a vector comprising the above-mentioned polynucleotide.

[0032] According to another embodiment of the present invention, the present invention provides a host cell comprising the above-mentioned vector.

[0033] According to another embodiment of the present invention, the present invention provides a host cell comprising the above polynucleotide.

[0034] According to another embodiment of the present invention, the present invention provides a method for producing a chimeric transforming growth factor-β polypeptide, comprising: step (a), aligning two or more transforming growth factor-β family member protein sequences; step (b), identifying six structurally related segments of the two family proteins; step (c), identifying the cross-over points of the two transforming growth factor-β proteins containing segments at the ends that are structurally related to more than 90% sequence identity among more than 5 consecutive amino acids; and step (d), producing a chimeric transforming growth factor-β polypeptide comprising at least segment 1 from a bone morphogenetic protein to a second transforming growth factor-β family member protein, wherein the above segments are sequentially connected at the cross-over points, and as an example of such a chimera, 1B2-bone morphogenetic protein 7 is produced.

[0035] According to another embodiment of the present invention, the present invention provides a chimeric polypeptide produced by the above method for producing the polypeptide.

[0036] According to another embodiment of the present invention, the present invention provides a method for regulating cell proliferation or activity associated with the SMAD pathway, comprising the step of contacting the above polypeptide with cells.

[0037] According to another embodiment of the present invention, the present invention provides a method for treating a disease or condition associated with bone, cartilage, nerve tissue, cardiac tissue, skeletal muscle or endocrine tissue, comprising the step of contacting the above polypeptide with the tissue.

[0038] According to another embodiment of the present invention, the present invention provides a method for treating a cell proliferative disease or disorder, comprising the step of contacting the above polypeptide with cells having the cell proliferative disease or disorder.

[0039] According to another embodiment of the present invention, the present invention provides a pharmaceutical composition for preventing or treating a disease associated with bone, cartilage, nerve tissue, cardiac tissue, skeletal muscle or endocrine tissue, comprising the above polypeptide.

[0040] According to another embodiment of the present invention, the present invention provides a pharmaceutical composition for preventing or treating a cell proliferative disorder, comprising the above polypeptide.

[0041] In an additional embodiment, the present invention provides additional basis for providing new ligands for other members of the transforming growth factor-β superfamily in U.S. Provisional Application No. 61 / 155,066 filed on February 24, 2009. The ligands are chimeric proteins having six segments of the same sequence arrangement from different members of the transforming growth factor-β superfamily. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1A ribbon diagram showing six segments of a single subunit of a transforming growth factor-β superfamily ligand scaffold. A single transforming growth factor-β superfamily ligand consists of two subunits covalently bound. The two subunits are twice rotationally symmetric with an axis that is perpendicular (light blue arrow) in this view. Therefore, the left and right panels are rotated with a conventional rotational symmetry axis, thus showing the opposite directions of the subunits. The six segments are sequentially shown in red (segment 1), blue (segment 2), yellow (segment 3), green (segment 4), brown (segment 5) and purple (segment 6) from the N-terminus to the C-terminus.

[0043] Figure 2 The structure / sequence alignment of the member sequences of the transforming growth factor-β family and various chimera sequences described in the present invention is shown. SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 are nucleotide sequences synthesized and designed for expressing activin A, bone morphogenetic protein 2 and 1B2-bone morphogenetic protein 7 in bacteria in the present invention, respectively. 1B2-bone morphogenetic protein 7 refers to the sequence of a chimera of segment 1 derived from bone morphogenetic protein 2 combined with segments 2 to 6 derived from bone morphogenetic protein 2. The chimera is easily expressed in bacteria and has the same function as bone morphogenetic protein 7. Due to their nature, the nucleotide sequence can be changed while the amino acid sequence described in SEQ ID NO: 4 to SEQ ID NO: 13 remains unchanged.

[0044] SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 are the amino acid sequences of the mature regions of human activin A (black), human bone morphogenetic protein 2 (red), and human bone morphogenetic protein 7 (blue) for bacterial expression. For this reason, in these three parent molecules, methionine is used as the first amino acid to start. In order to indicate their origin, all relevant segments of these chimeras are distinguished in red, blue, and black. For illustrative purposes only, all 6 segments are distinguished by "-". SEQ ID NO:7 and SEQ ID NO:8 are the amino acid sequences of AB204 and NAB204 as designed-enhanced chimeras. NAB204 contains a 6-amino acid-long noggin sensitivity derived from bone morphogenetic protein 2 (underlined in red). SEQ ID NO:9 and SEQ ID NO:10 are the amino acid sequences of SAB704 and NAB704 as designed-enhanced chimeras. NAB704 contains a 6-amino acid-long noggin sensitivity derived from bone morphogenetic protein 7 (underlined in blue). SEQ ID NO: 11 and SEQ ID NO: 12 are the amino acid sequences of SAB715 and NAB715 as designed-enhanced chimeras. NAB715 contains a 6-amino acid length of noggin sensitivity derived from bone morphogenetic protein 7 (underlined in blue). SEQ ID NO: 13 is the amino acid sequence of 1B2-bone morphogenetic protein 7 as a designed-enhanced chimera. 1B2-bone morphogenetic protein 7 contains segment 1 (red) derived from bone morphogenetic protein 2 and segment 2 (blue) derived from bone morphogenetic protein 7. For bacterial expression and refolding, 1B2-bone morphogenetic protein 7 replaces bone morphogenetic protein 7.

[0045] Figure 3a The cell signaling activity of BMP-7 of SAB704 and SAB715 as designer-enhanced chimeras and their parent molecules as control molecules is shown ( Figure 3a ). Figure 3a It was shown that SAB704 showed approximately 30-fold increased signaling activity in C2C12 cells, particularly through the SAMD1-mediated signaling pathway (Allendorph et al., 2011 Designer TGF-beta Superfamily Ligands with Diversified Functionality, PLOS One, https: / / doi.org / 10.1371 / journal.pone.0026402, (2011)).

[0046] Figure 3bThe relative levels of RNA (RNA) induction by 4 assimilation genes (ACAN, Aggrecan) are shown, which are known to be associated with cartilage formation and cartilage regeneration. The background level of RNA expression associated with each panel is shown by a red vertical line standardized at 1.0. After processing with these design-enhanced chimeras and bone morphogenetic protein 7, Smad-1 luciferase signaling activity was measured by the same method on the 4th, 8th and 11th days.

[0047] Figure 3c The relative levels of RNA induction by 4 assimilation genes (COL2A1, Collagen 2A1) are shown, which are known to be related to cartilage formation and cartilage regeneration. The background level of RNA expression related to each panel is shown by the red vertical line standardized by 1.0. After utilizing these design-enhanced chimeras and bone morphogenetic protein 7 to process, Smad-1 luciferase signaling activity was measured by the same method on the 4th, 8th and 11th days.

[0048] Figure 3d The relative levels of RNA induction by 4 assimilation genes (SOX9) are shown, which are known to be associated with cartilage formation and cartilage regeneration. The background level of RNA expression associated with each panel is shown by a red vertical line standardized at 1.0. After processing with these design-enhanced chimeras and bone morphogenetic protein 7, Smad-1 luciferase signaling activity was measured by the same method on the 4th, 8th and 11th days.

[0049] Figure 3e The relative levels of RNA induction by 4 assimilation genes (SOX6) are shown, which are known to be associated with cartilage formation and cartilage regeneration. The background level of RNA expression associated with each panel is shown by a red vertical line standardized at 1.0. After processing with these design-enhanced chimeras and bone morphogenetic protein 7, Smad-1 luciferase signaling activity was measured by the same method on the 4th, 8th and 11th days.

[0050] Figure 3f Relative levels of RNA induction by 4 anabolic genes (MMP-13) are shown, which are known to be associated with cartilage formation and cartilage regeneration. The background level of RNA expression associated with each panel is shown by a red vertical line standardized at 1.0. After processing with these design-enhanced chimeras and bone morphogenetic protein 7, Smad-1 luciferase signaling activity was measured by the same method on the 4th, 8th and 11th days.

[0051] Figure 3gThe relative level of RNA induction by 4 assimilation genes (ADAMTS5) is shown, and the above-mentioned gene is related to cartilage formation and cartilage regeneration and is known. The background level of RNA expression related to each panel is shown by the red vertical line standardized by 1.0. After utilizing these designs-enhanced chimeras and bone morphogenetic protein 7 to process, Smad-1 luciferase signaling activity was measured by the same method at the 4th day, the 8th day, the 11th day.

[0052] Figure 3h The relative level of RNA induction by 4 assimilation genes (COLX, Collagen X) is shown, and the above-mentioned genes are related to cartilage formation and cartilage regeneration and are well known. The background level of RNA expression related to each panel is shown by the red vertical line standardized by 1.0. After utilizing these design-enhanced chimeras and bone morphogenetic protein 7 to process, Smad-1 luciferase signaling activity was measured by the same method on the 4th day, the 8th day, and the 11th day.

[0053] Figure 3i The relative levels of RNA induction by 4 assimilation genes (RUNX2) are shown, which are known to be related to cartilage formation and cartilage regeneration. The background level of RNA expression associated with each panel is shown by a red vertical line standardized at 1.0. After utilizing these design-enhanced chimeras and bone morphogenetic protein 7 to process, Smad-1 luciferase signaling activity was measured by the same method on the 4th, 8th and 11th days.

[0054] Figure 4a Shown are the noggin sensitivities of BMP2, BMP7, and AB204 as a designer-enhanced chimera, compared for each ligand in the absence or presence of noggin. Smad-1 luciferase signaling activity was measured in the absence (white) and presence (dark grey) of noggin, where noggin and chimera were treated at a 1:1 molar ratio.

[0055] Figure 4bThe physical binding between NAB204 and noggin as a noggin-sensitizing chimera in the presence of noggin (red) is shown, and the AB204 mixture as a noggin-non-sensitive chimera in the presence of noggin is distinguished from not showing binding to noggin. As a result, in the size exclusion chromatography analysis, the elution time of the noggin-NAB204 complex is shown to the right. Noggin alone is shown as a reference (blue), and noggin elutes slower than the NAB204-noggin complex. As a result, similar to the results of comparing bone morphogenetic protein 2 with AB204, noggin binding for NAB204, NAB714 and NAB715 in the presence of noggin results in reduced signaling activity.

[0056] Figure 5 As a reference for the sequences of the present invention, the sequence alignment between the transforming growth factor-β superfamily ligands is shown, and they are divided into 6 segments to illustrate the similarity of sequence and structure, thereby demonstrating the similar design basis for the design-enhanced chimeras of this superfamily. DETAILED DESCRIPTION

[0057] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Therefore, the terms used throughout this specification may have the following meanings.

[0058] The transforming growth factor-β superfamily member used in the present application refers to the transforming growth factor-β superfamily (including bone morphogenetic factor) gene or protein of any species, in particular, mammal species including cattle, sheep, pigs, mice, horses and humans but not limited thereto. "Transforming growth factor-β superfamily polypeptide" refers to the amino acid sequence of a purified transforming growth factor-β superfamily protein obtained from any species, in particular, mammal species including cattle, sheep, pigs, mice, horses and humans, and regardless of natural, synthetic, semi-synthetic or recombinant, obtained from any source.

[0059] "Peptide" refers to a portion or fragment of a larger polypeptide or a portion or fragment of a protein. Peptides need not themselves have functional activity, although in some cases they correspond to domains of a polypeptide that have their own biological activity. Stability-associated peptides are peptides found in polypeptides that enhance stability, function, or folding compared to the associated polypeptide without such peptide.

[0060] "Fused", "operably linked" and "operably associated" are used as synonyms in this application and, in all other aspects, broadly refer to the chemical or physical association of two different domains, wherein each domain has a separate biological function. Therefore, in the present invention, mutually fused transforming growth factor-β (chicken house, bone morphogenetic protein or activin) domains are proposed, which function as polypeptides with improved or altered transforming growth factor-β family activity or ligand specificity of polypeptides of the transforming growth factor-β family.

[0061] "Chimera" or "chimeric protein" or "chimeric polypeptide" refers to a combination of two or more segments of two or more different parent proteins. As will be understood by those of ordinary skill, the segments need not actually be derived from the parents, respectively, as in the case of the specific sequences involved, and need not themselves be physical nucleic acids. For example, a bone morphogenetic protein chimera possesses two different parent bone morphogenetic proteins; or a bone morphogenetic protein and other members of the transforming growth factor-β superfamily, or as an alternative, possesses at least two segments from unrelated proteins. A chimeric protein may also be an "interspecies", "intergenic", etc. fusion of protein structures (same or different member proteins) expressed by different species of organisms. In a preferred embodiment, two segments are connected in order to calculate a new chimeric protein. In other words, if a protein possesses the same sequence as one side of the full-length parent protein, it is not a chimera. A chimeric protein may contain two or more segments from two different parent proteins. For example, to calculate the final chimera or library of chimeras, there may be 2, 3, 4, 5, 6 or 10 to 20 or more parents from which these domains are derived. The segments of each parent protein may be very short or very long, and these segments may vary in length from one to about the entire length of the above-mentioned protein. In one example, the minimum length is 5 amino acids. Typically, such a segment or subdomain is one of the 6 subdomains (see Figure 1 and Figure 2 ).

[0062] The "six segments" of the transforming growth factor-β superfamily members are identified based on the structural system of the primary amino acid sequence of the member protein and / or other homologous member proteins (including proteins of other species). As identified, in general, in order to minimize the distortion of the intrinsic transforming growth factor-β member sequence in the chimera operating device, or, as an alternative, to maximize the distortion, the member protein is divided into 6 different parts (as an alternative, 5 different parts) based on the derived segments. Generally, Figure 1 and Figure 2The relative positions of different segments repeated on the arranged sequences of each of the multiple transforming growth factor-β superfamily members are shown. Although relative, these segments may contain substitutions, insertions, additional amino acids or specific amino acid sequences or original amino acid sequences that conform to other deformations at one or both ends of the original sequence. "Conformed" means that the structural integrity of each segment is maintained as compared to the structural domain of the original sequence. For example, in the segments of the transforming growth factor-β superfamily members described in the present application, such as in order to confirm that the segments are adjacent to each other, 5, 3, 2 or 1 amino acids may be moved (shift) at one or both ends of the segment, or preferably, only 1 amino acid may be moved.

[0063] In one example, the chimeric protein of the present invention comprises a fusion of one or more segments derived from a transforming growth factor-β member and a second segment derived from a second transforming growth factor-β member, wherein the first segment is unrelated to the second transforming growth factor-β member. In a single subunit of a transforming growth factor-β superfamily ligand, 5 to 6 subdomains (segments) are used as a scaffold skeleton, and the new (designed-enhanced chimera) sequence is recombinantly linked by mixing segments derived from different transforming growth factor-β ligands in the same order in which they appear in nature. This combination is similar to a segment in a variety of different target sequences, but the new sequence is calculated to be clearly different from any naturally occurring sequence.

[0064] As will be appreciated by those of ordinary skill, there are not only exact sequences, but also "variants" of chimeras. In other words, conservative amino acid substitutions (e.g., about 1 to 10 conservative amino acid substitutions) can be generated within a chimera. Thus, in the case of a variant chimera, each segment is 100% absent from the final chimera. Based on the definition of the term variant, the amount that can be altered by the addition of residues or the removal or alteration of residues is defined. Of course, as will be appreciated by those of ordinary skill, the above discussion applies not only to amino acids, but also to nucleic acids encoding these amino acids.

[0065] "Conservative" amino acid substitutions refer to the interchangeability of residues having similar side chains and, therefore, are generally accompanied by substitutions of amino acids that are defined as identical or similar classes of amino acids within a polypeptide. By way of example, and without limitation, amino acids with aliphatic side chains may be substituted with other aliphatic amino acids, such as alanine, valine, leucine, isoleucine, and methionine; amino acids with hydroxyl side chains may be substituted with other amino acids with hydroxyl side chains, such as serine and threonine; amino acids with aromatic side chains may be substituted with other amino acids with aromatic side chains, such as phenylalanine, tyrosine, tryptophan, and histidine; amino acids with basic side chains may be substituted with other amino acids with basic side chains, such as lysine, arginine, and histidine; amino acids with acidic side chains may be substituted with other amino acids with acidic side chains, such as aspartic acid or glutamic acid; and hydrophobic or hydrophilic amino acids may be substituted with other hydrophobic or hydrophilic amino acids, respectively.

[0066] "Non-conservative" substitutions refer to substitutions with amino acids that have significantly different side chain properties within the polypeptide. Non-conservative substitutions utilize amino acids between the defined groups compared to those within the defined groups and affect (a) the structure of the peptide backbone at the site of the substitution (e.g., substitution of glycine for proline), (b) the charge or hydrophobicity, or (c) the size of the side chain. As examples, and without limitation, exemplary non-conservative substitutions may be substitutions of acidic amino acids with basic or aliphatic amino acids; substitutions of aromatic amino acids with small amino acids; and substitutions of hydrophilic amino acids with hydrophobic amino acids.

[0067] "Isolated polypeptide" refers to a polypeptide separated from other naturally associated contaminants, such as proteins, lipids and polynucleotides. The above term includes (embrace) naturally occurring polypeptides or polypeptides removed or purified using an expression system (e.g., host cells or in vitro synthesis).

[0068] A "substantially pure" polypeptide refers to a composition that is primarily composed of the above-mentioned polypeptide species (i.e., when on a molar or weight basis, the polypeptide species is more abundant than any other individual macromolecular species in the composition), and, generally, when the target species accounts for more than about 50% of the macromolecular species on a molar or weight percentage basis, the composition is substantially purified. Generally, a substantially pure polypeptide composition accounts for about 90% or more, about 95% or more, about 98% or more of all macromolecular species present in the composition on a molar or weight percentage basis. In some examples, the target species is purified substantially uniformly (i.e., contaminating species cannot be detected from the composition by conventional detection methods), wherein the above-mentioned composition is essentially composed of a single macromolecular species. Solvent species, small molecules (<500 Daltons) and elemental ion species are not considered macromolecular species.

[0069] "Reference sequence" refers to a sequence used as a basis for sequence comparison. A reference sequence can be a subset of a larger sequence, such as a segment of a full-length gene or polypeptide sequence. Typically, a reference sequence can be at least 20 nucleotides or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, or a full-length nucleic acid or full-length polypeptide. Two polynucleotides or polypeptides respectively (1) contain a sequence that is similar to the two sequences (i.e., a portion of the complete sequence), and (2) also contain a sequence that is different from the two sequences. Therefore, typically, sequence comparison between two (or more than two) polynucleotides or polypeptides is performed by comparing the sequences of the two polynucleotides or polypeptides on a "comparison window", thereby confirming and comparing local regions of sequence similarity.

[0070] Table 1 shows the Figure 5 The sequence of the mature form of the transforming growth factor-β superfamily ligand is referenced. Figure 1 SEQ ID NO:5 (bone morphogenetic protein 2), SEQ ID NO:6 (bone morphogenetic protein 7) and SEQ ID NO:4 (activin) described in part (b) in which Met is located in front as the first amino acid, and SEQ ID NO:14 (bone morphogenetic protein 2), SEQ ID NO:19 (bone morphogenetic protein 7) and SEQ ID NO:35 (activin A) in Table 1 are reproduced in the form of recombinant expression.

[0071]

[0072]

[0073]

[0074] "Sequence identity" refers to two amino acid sequences that are substantially identical over a comparison window (i.e., on an amino acid-amino acid basis). The term "sequence similarity" refers to similar amino acids that share the same biophysical properties. The term "ratio of sequence identity" or "ratio of sequence similarity" is calculated by comparing two optimally aligned sequences over a comparison window, calculating the number of identical positions by determining the number of positions generated by identical residues (or similar residues) in the polypeptide sequences on both sides, dividing the number of identical positions by the total number of positions (i.e., the window size) within the comparison window, and multiplying the obtained result by 100 to calculate the ratio of sequence identity (or ratio of sequence similarity). With respect to polynucleotide sequences, the terms sequence identity and sequence similarity have meanings similar to those described for protein sequences, and the term "ratio of sequence identity" refers to two polynucleotide sequences that are identical over a comparison window (on a nucleotide-nucleotide basis). Therefore, the ratio of polynucleotide sequence identity (or, based on an analysis algorithm, for example, the ratio of polynucleotide sequence similarity for silent substitutions or other substitutions) can also be calculated. Maximum correspondence can be determined using one of the sequence algorithms described in this application (or otherwise available to one of ordinary skill) or by visual inspection.

[0075] When applied to polypeptides, the term substantial identity or substantial similarity refers to the sequence identity or sequence similarity shared by two peptide sequences when they are optimally aligned by the programs BLAST, GAP or BESTFIT using the default gap weights, or by visual inspection. Similarly, when applied to the context of two nucleic acids, the term substantial identity or substantial similarity refers to the sequence identity or sequence similarity shared by two nucleic acid sequences when they are optimally aligned by the programs BLAST, GAP or BESTFIT using the default gap weights (described in detail below), or by visual inspection.

[0076] "Functional" refers to a polypeptide that has one of the inherent biological activities of a naturally-occurring protein, or any specific desired activity, depending on the type of polypeptide, as well as the ability to bind to a ligand or cognate molecule.

[0077] The "Transforming Growth Factor-β (TGF-beta)" superfamily of proteins consists of extracellular cytokines observed in most human cells. The less than 40 TGF-β superfamily ligands can be divided into smaller families including TGF-β, bone morphogenetic proteins (BMPs), activins and inhibins, Growth and Differentiation Factors (GDFs), Nodal, Mullerian Inhibiting Substance (MIS), and glial cell line-derived neurotrophic factor (GDNF). TGF-β superfamily members are observed in a wide range of cell types and play a role in fundamental cellular phenomena including dorsal / ventral patterning and left / right axis determination for bone formation and tissue repair. Recently, various TGF-β ligands have been found to be involved in the maintenance of stem cells or directly involved in the differentiation of stem cells. Their pervasiveness makes modulation of transforming growth factor-β ligand signaling applicable to the treatment of a wide variety of conditions ranging from skeletal and muscle disorders to various neoplastic events. Exemplary sequences associated with various members of this family or protein are disclosed in this application, and one of ordinary skill can readily identify homologs and variants using publicly available databases by word search or sequence BLAST search.

[0078] Transforming growth factor-β ligands are collectively referred to as inactive precursor molecules, which are composed of an N-terminal prodomain and a C-terminal mature domain connected by a protease cleavage site. Generally, for activation, the mature domain is cleaved from the prodomain by a convertase, such as furin. Members of the transforming growth factor-β superfamily are classified together due to the conserved structural system observed in their mature domains. Generally, each mature ligand monomer contains 7 cysteines, of which 6 form 3 intra-disulfide bonds arranged in a "cystine knot" motif. Four β chains extend from the "cystine knot" to form two curved fingers. The last remaining cysteine ​​forms an external disulfide bond with the second ligand monomer to generate a covalently linked dimer. In this dimer, the "cystine knot" is the theme, and the finger shape is extended like a food, and the overall shape is a butterfly. The functional subunits of the transforming growth factor-β superfamily are dimers as described above, and they exist as homodimers and heterodimers in vivo. Although some family members, such as growth and differentiation factor-9 and bone morphogenetic protein-15, do not have the cysteine ​​required for the formation of external disulfide bonds, they can still form stable dimers.

[0079] In order to disclose the signaling process, the transforming growth factor-β dimer needs to have two groups of receptors named type I and type II. These receptors are serine / threonine kinases with an extracellular domain (ECD) arranged in a 3-finger toxin fold, a single transmembrane domain, and a large intracellular kinase domain. The transforming growth factor-β ligands show a preference in their affinity for different receptor types. Activin and node show high affinity for type II receptors, while bone morphogenetic protein-2 and growth and differentiation factor-5 have higher affinity for type I receptors. After the transforming growth factor-β ligand binds to the two high-affinity receptors, two low-affinity receptors bind to the above complex. If all four receptors bind to the transforming growth factor-β ligand, a 6-member ternary complex is formed, and the downstream signaling cascade is disclosed. In essence, the active type II receptor phosphorylates the type I receptor, after which the type I receptor binds and phosphorylates the intracellular signaling molecule called Smad. Smad molecules can then translocate to the nucleus and interact directly with transcriptional regulators. At multiple steps in this cascade, a variety of mechanisms are used to tightly regulate TGF-β signaling: extracellular antagonists including noggin, follistatin, and inhibin; pseudoreceptors similar to BAMBI that lack an intracellular kinase domain; or intracellular molecules such as inhibitory Smads.

[0080] The transforming growth factor-β superfamily ligands show a high degree of promiscuity for their ligands through the receptors. There are more than 40 transforming growth factor-β ligands, in contrast, there are only 12 receptors (7 type I and 5 type II). Therefore, the receptors can interact with multiple different ligands. For example, ActRII is known to bind to activin and bone morphogenetic protein-7 with high affinity, but binds to bone morphogenetic protein-2 with lower affinity. It is understood how the structural details of affinity and specificity for ligand: receptor are regulated and what significance they have in the overall signaling process (fate). In growth and differentiation factor-5, a single amino acid was found to determine its type I receptor preference, and in bone morphogenetic protein-3, a single point mutation was found to alter type II receptor affinity and confer function to the above ligands. The present invention has new receptor binding properties, and thus provides methods for preparing modified transforming growth factor-β ligands having various transforming growth factor-β ligand functions and compositions having such activity.

[0081] The present invention provides novel chimeric transforming growth factor-β ligands that have the ability to be expressed and refolded using E. coli or mammalian expression systems and methods for preparing these ligands. The chimeras mimic the signaling characteristics of specific transforming growth factor-β ligands or show unique signaling properties that are not observed in nature. In one example, in the present invention, in order to calculate the activin / bone morphogenetic protein-2 chimera having the refolding efficiency of bone morphogenetic protein-2 and the signaling properties of activin-βA, activin-βA and bone morphogenetic protein-2 are used as templates, but it is understood that any other transforming growth factor-β protein family members can be used.

[0082] The chimera design scheme of the present invention computes additional TGF-β member chimeras (e.g., activin / BMP-2) ligands with non-native signaling characteristics and biological activity. Furthermore, the present invention demonstrates that a targeted chimera can be incorporated into a small Noggin-sensitive portion of BMP-2 or BMP-7 in order to generate a "Noggin-sensitized" ligand. This chimeric TGF-β family polypeptide invention can expand the library of TGF-β ligands that can be used for functional studies and facilitate the development of new TGF-β ligands as therapeutic agents.

[0083] The nucleic acid sequence and polypeptide sequence of bone morphogenetic protein-2 and naturally occurring variants are known. A wild-type bone morphogenetic protein-2 nucleic acid sequence (SEQ ID NO: 2) is provided. In the N-terminal position of residue 1, Met (Q) is caused by the translation of the bacterial start codon (ATG). In addition, activin is also known in the art (SEQ ID NO: 1). The present invention provides a method for generating a chimeric bacterium by a codon-optimized polynucleotide sequence in order to express the same polypeptide sequence of the chimera. The present invention also provides two or more segments (segment 2 and segment 6) derived from one or more N-terminal segments 1 of bone morphogenetic protein-2 or bone morphogenetic protein-7 after Met together with the start codon of ATG, and the chimeric polypeptides generated here exhibit high activity (display) different from their parent proteins.

[0084] In one embodiment of the present invention, two factors are considered when designing chimeric segments. The first is a structural consideration. The entire transforming growth factor-β monomer fold is naturally divided into six parts: β strands 1 and β strands 2, pre-helix loops, α helix 1, and β strands 3 and β strands 4. The second consideration is to minimize changes to the chimera within such segment boundaries, to minimize duplication of the DNA sequence in the region for the polymerase chain reaction (PCR) strategy, and to minimize all changes to the natural sequence. Figure 2 The sequences and structures of these contemplated entities are shown. The segment boundaries of the chimeras are derived from US Provisional Application No. 61 / 155,066.

[0085] For example, the chimeric polypeptide comprising the algorithm 1B2B3B4B5B6A indicates 6 segments 1 to 6, with the text indicating the parent chain of each segment by "A" or "B". Thus, in the embodiment "1B2B3B4B5B6A", segment 1 is derived from the parent chain "B" for bone morphogenetic protein-2 or bone morphogenetic protein-7 corresponding to the AB215 or SAB715 chimera, respectively, segment 2 is derived from the parent chain "A" for activin, segment 3 is derived from the parent chain "B", segment 4 is derived from the parent chain "B", segment 5 is derived from the parent chain "B", and segment 6 is derived from the parent chain "A".

[0086] It has been found that when these recombinant functional chimeric transforming growth factor-β family polypeptides are prepared, their ligand specificity can be improved, or biological activity can be altered or increased, as disclosed in this specification, compared to the non-recombinant parent polypeptide. Due to the differences in activity / ligand profiles, these manipulated chimeric transforming growth factor-β family polypeptides are screened for active substances for ligand specific activation and inhibition, and provide a unique basis for providing new therapeutic polypeptides and research reagents.

[0087] In another embodiment of the present invention, the present invention provides a chimeric transforming growth factor-β polypeptide, which comprises segment 1 derived from bone morphogenetic protein 2, followed by segments 2 to 6 derived from bone morphogenetic protein-7 (e.g., 1B2-bone morphogenetic protein-7 polypeptide, see SEQ ID NO: 3). These chimeric 1B2-bone morphogenetic protein-7 exhibit the transforming growth factor-β protein activity of bone morphogenetic protein-7.

[0088] In another example of the present invention, the segment of the chimeric polypeptide may be 100% identical to the parent chain derived from the segment. In one example of the present invention, the segment may comprise an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% or more identical to the corresponding segment in the parent chain. For example, the segment may comprise more than one conservative amino acid substitution (assuming that 1 to 5 conservative amino acid substitutions).

[0089] In one embodiment of the present invention, the biological activity of the chimera of the present invention can be determined using a variety of assays for transforming growth factor-β activity recognized in the art. Such assays include, but are not limited to: BIAcore (surface plasmon resonance); C2C12 luciferase assay; Smad 1 / 5 reporter system; HEK293 luciferase assay; Smad 2 / 3 reporter system; FSH (follicle stimulating hormone) release assay; BRE (bone morphogenetic protein response factor) luciferase assay in rat pituitary cells; Smad 1 / 5 reporter HEK 293 cells; Cripto binding assay; luciferase response measured in the presence / absence of Cripto; human stem cell assay; cartilage regeneration assay; in vitro cartilage particle culture; NMR binding studies; bone and cartilage formation assays in rats and other animals; visualization of ligand: receptor complexes; size exclusion chromatography (SEC); speed rice seedling ultracentrifugation for visualization of ligand: receptor complex formation; and Seldi gravimetric analysis for accurate ligand size determination.

[0090] Also, for example, modified chimeras can be prepared by amino acid substitutions, deletions or additions. For example, it is reasonable to expect that an isolated replacement of leucine with isoleucine or valine, an isolated replacement of aspartate with glutamate, an isolated replacement of threonine with serine, or similar replacement of amino acids with structurally related amino acids (assuming, conservative mutations) will have no other effect on the biological activity of the resulting molecule. Conservative alternations occur within families of amino acids related in side chains.

[0091] In particular instances, the present invention contemplates mutations in a manner that reduces the sensitivity of the proteolytic cleavage site of the chimeric sequence to proteolytic cleavage. Computer analysis (using commercially available software such as MacVector, Omega, PCGene, Molecular Simulation, Inc.) can be used to confirm the proteolytic cleavage site. As will be appreciated by those of ordinary skill, most of these described mutations, variants, or modifications can be prepared at the nucleic acid level (or, in some cases, by post-translational deformation or chemical synthesis). These descriptions are well known in the art.

[0092] In a specific example, the present invention contemplates specific mutations of the chimera sequence in order to alter the glycosylation of the chimera. Such mutations may be selected to introduce or remove one or more glycosylation sites, such as O-linked or N-linked glycosylation sites. Asparagine-linked glycosylation recognition sites typically comprise a tripeptide sequence, asparagine-X-threonine (where "X" is any amino acid), which is specifically recognized by appropriate cellular glycosylation enzymes. Modifications may also be prepared by appending one or more serine or threonine residues to the sequence of the wild-type polypeptide or by substitution of one or more serine or threonine residues (in the case of O-linked glycosylation sites). In the first or third amino acid position of the glycosylation recognition site, various amino acid substitutions or deletions on one or both sides (and / or amino acid deletions in the second position) result in non-glycosylation of the modified tripeptide sequence. Other methods for increasing the number of carbohydrate moieties are chemical or enzymatic coupling of polypeptides to glycosides.

[0093] The present invention also provides a polynucleotide encoding a portion of a chimeric polypeptide disclosed in the present application. The polynucleotide can be codon-optimized and can be operably linked to one or more heterologous regulatory sequences or control sequences that control gene expression in order to generate a recombinant polynucleotide that can express the polypeptide in a specific expression host. An expression construct comprising a polynucleotide encoding a chimeric polypeptide can be introduced into an appropriate host cell to express the chimeric polypeptide.

[0094] Given the knowledge of the specific sequences of proteins of the transforming growth factor-β family and the specific descriptions of the chimeric polypeptides in the present application (e.g., the segment structure of the chimeric domain), the skilled artisan can clearly understand the chimeric nucleic acid sequences to be manipulated. The knowledge of the amino acid sequences of these polypeptides and the knowledge of the codons corresponding to the various amino acids enables the skilled artisan to prepare different polynucleotides encoding the polypeptides of the present invention. Therefore, the present invention contemplates every possible variation of polynucleotides that can be prepared by selecting combinations based on codon usage as much as possible, and all of these variations are deemed to be explicitly disclosed for any polypeptide described in this application.

[0095] An example of an expression system in question is the E. coli expression system, and these proteins can be easily cloned into and expressed by a variety of expression systems. Advantages include, but are not limited to, post-translational modifications as observed in the organism from which the protein is derived (in the present case, H. sapiens), expression of ligands without the initial methionine required for bacterial expression, and ease of incorporation of unnatural amino acids or additional chemical modifications. Suitable prokaryotes include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as E. coli. Various strains of E. coli are publicly available, such as E. coli K12 strain MM294 (ATCC 31446), E. coli X1776 (ATCC 31537), E. coli strain W3110 (ATCC 27325), and K5 772 (ATCC 53635). In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeast, are also suitable as cloning or expression hosts for VEGF-E-encoding vectors. Saccharomyces cerevisiae is a commonly utilized lower eukaryotic host microorganism.

[0096] Host cells suitable for expressing the chimeras of the present invention are derived from unicellular and multicellular organisms. Examples of invertebrates include insect cells, such as Drosophila S2 and Spodoptera Sf9, and plant cells. Plant expression systems have also been successfully used to express modified proteins. Examples of useful mammalian host cell strains include Chinese hamster ovary (CHO) and COS cells. More specific examples include monkey kidney CV1 cell line transformed by SV40 (COS-7, ATCC CRL 1651), human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)), Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)), mouse testicular supporting cells (Sertoli cells) (TM4, Mather, Biol. Reprod., 23:243-251 (1980)), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065) ​​and mouse breast tumor (MMT 060562, ATCC CCL51). The selection of an appropriate host cell is within the skill of the art.

[0097] Alternative protein expression systems include but are not limited to human embryonic kidney (HEK) 293 cells, insect cell strains (S. frugiperda) utilizing baculovirus expression systems, yeast expression systems of Pichia pastoris and S. cerevisiae, and, include a variety of microalgae strains. To express precisely modified proteins, transformed animals may be utilized. In essence, these animals may be "bioreactors" that can express large quantities of the desired protein in readily harvested fluids or tissues, such as milk obtained from cows. Cell-free in vitro expression systems utilizing bacterial or wheat germ cell lysates may be used. Cell-free expression systems allow for the insertion of a wide range of non-natural amino acids or epitope tags with greater efficiency and specificity.

[0098] In a specific embodiment, the present invention provides methods and agents for promoting cartilage and bone formation and for increasing the amount thereof. Thus, in chimeric proteins of the present invention that are predicted to affect the bone-related functions of transforming growth factor-β superfamily proteins, such as bone morphogenetic protein-2, bone morphogenetic protein-3, growth and differentiation factor-10, bone morphogenetic protein-4, bone morphogenetic protein-7 or bone morphogenetic protein-8, in order to confirm the ability to modulate bone or cartilage growth, they can be tested in vivo or in vitro in intact cells or tissues. Various methods known in the art can be used for this purpose. Therefore, the effect of the chimeric protein of interest on bone or cartilage growth, preferably a chimeric protein comprising a fragment derived from bone morphogenetic protein-2 or bone morphogenetic protein-3, can be confirmed by the effect on bone formation and chondrogenesis activity, as an example, in a cell-based assay, there is measurement of positive or negative differentiation marker genes for chondrogenesis including ACAN (positive), Col2A1 (positive), MMP13 (negative), ADAMTS-5 (negative) and ColX (negative), or induction of Msx2 (see also, e.g., Daluiski et al., Nat. Genet. 2001, 27(1):84-8; Hino et al., Front Biosci. 2004, 9:1520-9).

[0099] It should be understood that the screening assays of the present invention are applicable not only to the subject chimeric proteins and their variants, but also to any test compound including active substances (agonists) and antagonists (antagonists) of their chimeric proteins or their variants. Moreover, these screening assays are useful for drug target validation and quality management purposes.

[0100] The chimeras of the present invention can be prepared for use in various biological systems in vitro and in vivo. In order to administer the chimeras alone or with other active agents, any known method can be used. For example, they can be gradually injected into the tissue area according to time or administered non-orally by injection. The above-mentioned agents can be administered by methods such as oral, rectal, buccal (presumably, sublingual), guided, non-oral (for example, subcutaneous, intramuscular, intradermal, intravenous, intraperitoneal, including skeletal muscle, cardiac muscle, diaphragm muscle and smooth muscle), topical (i.e., mucosal surfaces including skin, i.e., airway surfaces), intranasal, transdermal, intraarticular, intrathecal, intracavity, inhalation administration, administration to the gang through intraportal delivery, and direct organ injection (for example, injection into the liver, injection into the brain to deliver to the central nervous system, injection into the pancreas). In any case, the most suitable route is affected by the nature and severity of the disorder to be treated and the nature of the specific compound to be utilized.

[0101] The present invention also provides a pharmaceutical composition comprising a chimeric protein of interest and a pharmaceutically acceptable carrier. The pharmaceutical composition can be used to enhance the growth of a tissue in an object, preferably a human, or to reduce or prevent the loss of a tissue. For example, the targeted tissue can be bone, cartilage, skeletal muscle, myocardium, and / or neural tissue.

[0102] In another embodiment, the chimera can be prepared together with other agents (e.g., emulsions, creams, sprays, gels or ointments) for administration alone or together. In order to reduce toxicity or increase the efficiency of biological utilization, it can be prepared as a liposome. As another delivery method, oral methods with microspheres or proteinoid encapsulation (encapsulation), aerosol delivery (e.g., to the lungs), or transdermal delivery (e.g., by iontophoresis or transdermal electroporation) are included. Ordinary technicians can clearly grasp other methods of administration.

[0103] Example

[0104] Example 1

[0105] Preparation of transforming growth factor-β chimeras. For extensive use as a design principle, this is also part of the design to maintain two structural segments, part 3 and part 4, and a part of one side of the parent gene can be used for processing (called part 3*4). This strategy is implemented in the following manner, that is, activin-βA is used as a target ligand, and bone morphogenetic protein-2 is used as a backbone ligand, thereby preparing activin / bone morphogenetic protein-2 chimeras. This invention has been proposed and authorized for publication in U.S. Provisional Application No. 61 / 155066 filed on February 24, 2009.

[0106] In the case of the activin / bone morphogenetic protein-2 chimera, the mature domains of human bone morphogenetic protein-2 and human activin-βA were each pre-divided into 6 segments, and primers were designed for each segment. In the case of bone morphogenetic protein-2, primers encoding the following 6 segments were designed. The 6-residue-length noggin-sensitive sequence in segment 6 is indicated by underlining.

[0107] Segment 1, QAKHKQRKRLKSSCKRHPLYVDFSDVGWND;

[0108] Segment 2, WIVAPPGYHAFYCHGECP;

[0109] Segment 3, FPLADHLNSTNHAIVQTLVN;

[0110] Segment 4, SVNSKIPKACCVP;

[0111] Segment 5, TELSAISMLYYD;

[0112] Segment 6, ENEK VVL QDMVVEGCGCR.

[0113] In the case of activin-βA, primers were designed encoding the following 6 segments. The 6-residue-length Noggin-sensitive sequence in segment 6 is indicated by underlining.

[0114] Segment 1, RGLECDGKVNICCKKQFFVSFKDIGWNDW;

[0115] Segment 2, WIIAPSGYHANYCEGECP;

[0116] Segment 3, SHIAGTSGSSLSFHSTLVN;

[0117] Segment 4, HYRMRGHSPFANLKSCCVP;

[0118] Segment 5, TKLRPMSMLYYD;

[0119] Segment 6, DGQNI IKKDI QNMI VEECGCS.

[0120] An overlapping PCR strategy was used whereby the full-length chimera was calculated by mixing the various segments of BMP-2 and activin [beta]A operably linked in the order of 1, 2, 3, 4, 5 and 6 after the start codon ATG for Met.

[0121] In the case of the activin / bone morphogenetic protein-7 chimera, the mature domains of human bone morphogenetic protein-7 and human activin-βA were pre-divided into 6 segments, respectively, and primers were designed for each segment. In the case of bone morphogenetic protein-7, primers encoding the following 6 protein sequences were designed. The 6-residue-length noggin-sensitive sequence in segment 6 is indicated by underlining.

[0122] Segment 1, MSTGSKQRSQNRSKTPKNQEALRMANVAENSSSDQRQACKKHELYVSFR DLGWQD;

[0123] Segment 2, WIIAPEGYAAYYCEGECA;

[0124] Section 3, GPLNSYMNATNHAIVQTLVH;

[0125] Segment 4, FINPETVPKPCCAP;

[0126] Segment 5, TQLNAISVLYFD;

[0127] Segment 6, DSSN VILKKY RNMVVRACGCH.

[0128] In the case of activin-βA, primers were designed encoding the following 6 segments. The 6-residue-length Noggin sensitizer sequence in segment 6 is indicated by underlining.

[0129] Segment 1, RGLECDGKVNICCKKQFFVSFKDIGWNDW;

[0130] Segment 2, WIIAPSGYHANYCEGECP;

[0131] Segment 3, SHIAGTSGSSLSFHSTLVN;

[0132] Segment 4, HYRMRGHSPFANLKSCCVP;

[0133] Segment 5, TKLRPMSMLYYD;

[0134] Segment 6, DGQN IIKKDI QNMI VEECGCS.

[0135] An iterative polymerase chain reaction strategy was used whereby the full-length chimera was calculated by mixing the various segments of BMP-7 and activin [beta]A operably linked in the order of 1, 2, 3, 4, 5 and 6 after the start codon ATG for Met.

[0136] To construct the 1B2 chimera of BMP-7, 1B2-BMP-7, amino acids II (underlined) QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDII were inserted into the target gene after BMP-2 segment 1 using two oligonucleotides. The two amino acids II were added to create convenient restriction enzyme sites. For cloning into the pET21a expression vector, external primers for all constructs were prepared in such a way that a 5'NdeI site and a 3'XhoI site were incorporated. Figure 2 In the description of 1B2-BMP-7, the N-terminus of segment 1B2 was used to set the basis for various chimeras and variants thereof, including 1B2-NAB704, 1B2-AB704 and 1B2-NAB715. However, these two amino acids inserted at the C-terminus of segment 1 are not necessary for the preparation of 1B2-BMP-7. Both sequences operate identically.

[0137] Segment 1, QAKHKQRKRLKSSCKRHPLYVDFSDVGWND II ;(bone morphogenetic protein-2)

[0138] Segment 2, WIIAPEGYAAYYCEGECA; (bone morphogenetic protein-7)

[0139] Segment 3, GPLNSYMNATNHAIVQTLVH; (bone morphogenetic protein-7)

[0140] Segment 4, FINPETVPKPCCAP; (bone morphogenetic protein-7)

[0141] Segment 5, TQLNAISVLYFD; (bone morphogenetic protein-7)

[0142] Segment 6, DSSNVILKKYRNMVVRACGCH. (bone morphogenetic protein-7)

[0143] Protein expression and purification. Activin / bone morphogenetic protein-2 chimeras, activin / bone morphogenetic protein-7 chimeras, activin-βA, 1B chimeras and their noggin-sensitized variants were expressed using a conventional E. coli expression system, and chimeras were observed in inclusion body fractions. The expressed inclusion bodies were isolated, purified and refolded. The refolded ligands were purified using Hi-trap heparin columns (GE Healthcare) and reversed phase chromatography (reversed phase chromatography) (GraceVydac). These ligands were freeze-dried and, in the case of all cell-based analysis evaluations, resuspended in 4mM HCl, pH 1, or in the case of all biophysical analysis evaluations, resuspended in 10mM sodium acetate (Na acetate), pH 4. Among them, a portion was expressed in a stably transformed CHO cell line and purified by techniques known in the art. Noggin was expressed and purified based on the scheme described above.

[0144] Smad-1 luciferase assay was evaluated in C2C12 cells. Smad1-dependent luciferase assay was performed by techniques known in the art. In brief, C2C12 myoblasts were cultured in Dulbecco's minimum essential medium (DMEM) + 5% fetal bovine serum (FBS) supplemented with L-glutamine and antibiotics. To analyze and evaluate the luciferase reporter gene, the cells were trypsinized, washed twice with phosphate buffered saline, and smeared in 48-well plates containing DMEM + 0.1% fetal bovine serum. Luciferase activity was measured 24 hours after ligand stimulation, and these values ​​were standardized for conversion efficiency by using β-galactosidase activity. The activity of the luciferase reporter gene was expressed as fold-induction relative to the control value obtained using -927Id1-luciferase (Id1-Luc mut) without the Smad binding domain. To investigate the ability of Noggin to attenuate Smad1 signaling of these ligands, the luciferase assay was repeated as described above, and a defined volume of Noggin was included in the assay. FIG3 shows that SAB704 has a signaling ability approximately 30-fold higher than native BMP-7, with ED50 values ​​of 15.6 nM and 423.1 nM, respectively ( Figure 3a ).exist Figure 3b to Figure 3iAs shown in , the expression of hypertrophic genes including ADAMTS5, COLX and MMP13, or the expression of chondrogenic genes including aggrecan and COL2A1, was reduced, and thus, these activities were associated with the enhanced RNA-induced activity of SAB704.

[0145] Evaluation of Smad-2 luciferase assay in HEK293 cells. HEK293T cells were seeded at a density of 150,000 cells / well in 24-well plates coated with poly-lysine. After 24 h, cells were transformed overnight into a mixture of A3 Lux (25 ng) and β-galactosidase (25 ng) reporter plasmids, transcription factor FAST2 (50 ng) and empty pCDNA3 vector (400 ng) using Perfectin transformation reagent (GenLantis) according to the manufacturer's recommendations. Afterwards, these cells were treated with increasing doses of activin-βA or activin / bone morphogenetic protein-2 chimeras for 16 to 24 h. The cells were harvested from an ice-cold lysis buffer (1% Triton X-100 in 25 mM dithiothreitol, 4 nM ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA), 15 mM MgSO4) and luciferase and β-galactosidase activity was assessed using standard methods. To assess the ability of chimeras to bind to known transforming growth factor-β co-receptors, HEK293T cells were treated with increasing doses of activin-βA or activin / bone morphogenetic protein-7 chimeras. Activity was determined by the methods described above.

[0146] AB204, SAB704 and SAB715 are noggin-insensitive. Noggin directly binds to ligands and inhibits signal transduction activity, making the above ligands unable to bind to their own receptors for signal transduction. Compared with bone morphogenetic protein-2, which is blocked to background levels in the presence of noggin, the high signal transduction of AB204, SAB704 and SAB715 is not inhibited by noggin. Figure 4 shows the cell activity of noggin-sensitive bone morphogenetic protein 2, noggin-sensitive bone morphogenetic protein 7, noggin-insensitive AB204 or noggin-insensitive SAB704 or SAB8715. Figure 4bThe size exclusion chromatography results of NAB204 are shown to illustrate NAB204 physically bound to Noggin compared to AB204 not bound to Noggin. This physical binding provides a direct demonstration of the Noggin-insensitivity of the signaling activities of AB204, SAB704, and SAB715. This property may be particularly effective in modulating cell signaling capabilities in vivo through time-dependent combinatorial mixing between them, including bone and cartilage regeneration. <110> OSR Holdings Ltd <120> Activin / BMP7 chimeras: superactive SAB704 and SAB715 and their noggin-sensitized variants NAB704, NAB715 and NAB204 <130> PP190068 <150> US 62 / 767,707 <151> 2018-11-15 <160> 42 <170> KoPatentIn 3.0 <210> 1 <211> 345 <212> DNA <213> Artificial sequence <220> <223> Activin A <400> 1 atgcaagcca aacacaaaca gcggaagcgt cttaagtcca gctgcaaaag gcaccctttg 60 tatgtggact tcagtgatgt ggggtggaat gactggatca ttgctccctc tggctatcat 120 gccaactact gcgacggaga atgccctttt cctctggctg atcatctgaa ctccactaat 180 catgccattg ttcagacgtt ggtcaactct gttaactcta agattcctaa ggcatgctgt 240 gtcccgacca agctgagacc catgtccatg ttgtactatg atgatggtca aaacatcatc 300 aaaaaggaca ttcagaacat gatcgtggag gagtgtgggt gctca 345 <210> 2 <211> 345 <212> DNA <213> Artificial Sequence <220> <223> Bone Morphogenetic Protein 2 <400> 2 atgcaagcca aacacaaaca gcggaagcgt cttaagtcca gctgcaaaag gcaccctttg 60 tatgtggact tcagtgatgt ggggtggaat gactggatca ttgctccctc tggctatcat 120 gccaactact gcgacggaga atgccctttt cctctggctg atcatctgaa ctccactaat 180 catgccattg ttcagacgtt ggtcaactct gttaactcta agattcctaa ggcatgctgt 240 gtcccgacca agctgagacc catgtccatg ttgtactatg atgatggtca aaacatcatc 300 aaaaaggaca ttcagaacat gatcgtggag gagtgtgggt gctca 345 <210> 3 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> 1B2 - Bone Morphogenetic Protein - 7 <400> 3 atgcaagcca aacacaaaca gcggaaacgc cttaagtcca gctgtaagag acaccctttg 60 tacgtggact tcagtgacgt ggggtggaat gactggatta tcgcgcctga aggctacgcc 120 gcctactact gtgaggggga gtgtgccttc cctctgaact cctacatgaa cgccaccaac 180 cacgccatcg tgcagacgct ggtccacttc atcaacccgg aaacggtgcc caagccctgc 240 tgtgcgccca cgcagctcaa tgccatctcc gtcctctact tcgatgacag ctccaacgtc 300 atcctgaaga aatacagaaa catggtggtc cgggcctgtg gctgccac 348 <210> 4 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Activin A <400> 4 Met Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys Cys Lys Lys Gln 1 5 10 15 Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp Ile Ile Ala 20 25 30 Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys Pro Ser His 35 40 45 Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser Thr Val Ile 50 55 60 Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn Leu Lys Ser 65 70 75 80 Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu Tyr Tyr Asp 85 90 95 Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn Met Ile Val Glu 100 105 110 Glu Cys Gly Cys Ser 115 <210> 5 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Bone Morphogenetic Protein 2 <400> 5 Met Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30 Ile Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys 35 40 45 Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile Val 50 55 60 Gln Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala Cys Cys 65 70 75 80 Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu Asn 85 90 95 Glu Lys Val Val Leu Lys Asn Tyr Gln Asp Met Val Val Glu Gly Cys 100 105 110 Gly Cys Arg 115 <210> 6 <211> 140 <212> PRT <213> Artificial Sequence <220> <223> Bone Morphogenetic Protein 7 <400> 6 Met Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro 1 5 10 15 Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser 20 25 30 Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe 35 40 45 Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala 50 55 60 Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met 65 70 75 80 Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Phe Ile Asn 85 90 95 Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala 100 105 110 Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys 115 120 125 Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130 135 140 <210> 7 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> AB204 <400> 7 Met Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30 Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Asp Gly Glu Cys 35 40 45 Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile Val 50 55 60 Gln Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala Cys Cys 65 70 75 80 Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu Tyr Tyr Asp Asp Gly 85 90 95 Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn Met Ile Val Glu Glu Cys 100 105 110 Gly Cys Ser 115 <210> 8 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> NAB204 <400> 8 Met Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30 Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Asp Gly Glu Cys 35 40 45 Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile Val 50 55 60 Gln Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala Cys Cys 65 70 75 80 Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu Tyr Tyr Asp Asp Gly 85 90 95 Gln Asn Val Val Leu Lys Asn Tyr Gln Asn Met Ile Val Glu Glu Cys 100 105 110 Gly Cys Ser 115 <210> 9 <211> 140 <212> PRT <213> Artificial sequence <220> <223> SAB704 <400> 9 Met Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro 1 5 10 15 Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser 20 25 30 Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe 35 40 45 Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Ser Gly Tyr His 50 55 60 Ala Asn Tyr Cys Glu Gly Glu Cys Pro Phe Pro Leu Asn Ser Tyr Met 65 70 75 80 Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Phe Ile Asn 85 90 95 Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Lys Leu Arg Pro 100 105 110 Met Ser Met Leu Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp 115 120 125 Ile Gln Asn Met Ile Val Glu Glu Cys Gly Cys Ser 130 135 140 <210> 10 <211> 140 <212> PRT <213> Artificial sequence <220> <223> NAB704 <400> 10 Met Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro 1 5 10 15 Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser 20 25 30 Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe 35 40 45 Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Ser Gly Tyr His 50 55 60 Ala Asn Tyr Cys Glu Gly Glu Cys Pro Phe Pro Leu Asn Ser Tyr Met 65 70 75 80 Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Phe Ile Asn 85 90 95 Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Lys Leu Arg Pro 100 105 110 Met Ser Met Leu Tyr Tyr Asp Asp Gly Gln Asn Val Ile Leu Lys Lys 115 120 125 Tyr Gln Asn Met Ile Val Glu Glu Cys Gly Cys Ser 130 135 140 <210> 11 <211> 140 <212> PRT <213> Artificial Sequence <220> <223> SAB715 <400> 11 Met Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro 1 5 10 15 Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser 20 25 30 Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe 35 40 45 Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Ser Gly Tyr His 50 55 60 Ala Asn Tyr Cys Glu Gly Glu Cys Pro Phe Pro Leu Asn Ser Tyr Met 65 70 75 80 Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Phe Ile Asn 85 90 95 Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala 100 105 110 Ile Ser Val Leu Tyr Phe Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp 115 120 125 Ile Gln Asn Met Ile Val Glu Glu Cys Gly Cys Ser 130 135 140 <210> 12 <211> 140 <212> PRT <213> Artificial sequence <220> <223> NAB715 <400> 12 Met Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro 1 5 10 15 Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser 20 25 30 Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe 35 40 45 Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Ser Gly Tyr His 50 55 60 Ala Asn Tyr Cys Glu Gly Glu Cys Pro Phe Pro Leu Asn Ser Tyr Met 65 70 75 80 Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Phe Ile Asn 85 90 95 Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala 100 105 110 Ile Ser Val Leu Tyr Phe Asp Asp Gly Gln Asn Val Ile Leu Lys Asn 115 120 125 Tyr Gln Asn Met Ile Val Glu Glu Cys Gly Cys Ser 130 135 140 <210> 13 <211> 116 <212> PRT <213> Artificial sequence <220> <223> 1B2 bone morphogenetic protein 7 <400> 13 Met Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30 Ile Ile Ala Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys 35 40 45 Ala Phe Pro Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala Ile Val 50 55 60 Gln Thr Leu Val His Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys 65 70 75 80 Cys Ala Pro Thr Gln Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp 85 90 95 Ser Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala 100 105 110 Cys Gly Cys His 115 <210> 14 <211> 114 <212> PRT <213> Artificial sequence <220> <223> Bone morphogenetic protein-2 <400> 14 Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys Arg 1 5 10 15 His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp Ile 20 25 30 Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys Pro 35 40 45 Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile Val Gln 50 55 60 Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala Cys Cys Val 65 70 75 80 Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu Asn Glu 85 90 95 Lys Val Val Leu Lys Asn Tyr Gln Asp Met Val Val Glu Gly Cys Gly 100 105 110 Cys Arg <210> 15 <211> 110 <212> PRT <213> Artificial sequence <220> <223> Bone morphogenetic protein-3 (osteogenin) <400> 15 Gln Trp Ile Glu Pro Arg Asn Cys Ala Arg Arg Tyr Leu Lys Val Asp 1 5 10 15 Phe Ala Asp Ile Gly Trp Ser Glu Trp Ile Ile Ser Pro Lys Ser Phe 20 25 30 Asp Ala Tyr Tyr Cys Ser Gly Ala Cys Gln Phe Pro Met Pro Lys Ser 35 40 45 Leu Lys Pro Ser Asn His Ala Thr Ile Gln Ser Ile Val Arg Ala Val 50 55 60 Gly Val Val Pro Gly Ile Pro Glu Pro Cys Cys Val Pro Glu Lys Met 65 70 75 80 Ser Ser Leu Ser Ile Leu Phe Phe Asp Glu Asn Lys Asn Val Val Leu 85 90 95 Lys Val Tyr Pro Asn Met Thr Val Glu Ser Cys Ala Cys Arg 100 105 110 <210> 16 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Bone Morphogenetic Protein-4 (Bone Morphogenetic Protein-2b) <400> 16 Ser Pro Lys His His Ser Gln Arg Ala Arg Lys Lys Asn Lys Asn Cys 1 5 10 15 Arg Arg His Ser Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp 20 25 30 Trp Ile Val Ala Pro Pro Gly Tyr Gln Ala Phe Tyr Cys His Gly Asp 35 40 45 Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile 50 55 60 Val Gln Thr Leu Val Asn Ser Val Asn Ser Ser Ile Pro Lys Ala Cys 65 70 75 80 Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu 85 90 95 Tyr Asp Lys Val Val Leu Lys Asn Tyr Gln Glu Met Val Val Glu Gly 100 105 110 Cys Gly Cys Arg 115 <210> 17 <211> 134 <212> PRT <213> Artificial Sequence <220> <223> Bone Morphogenetic Protein-5 <400> 17 Ala Ala Asn Gln Asn Arg Asn Lys Ser Ser Ser His Gln Asp Ser Ser 1 5 10 15 Arg Met Ser Ser Val Gly Asp Tyr Asn Thr Ser Glu Gln Lys Gln Ala 20 25 30 Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln 35 40 45 Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala Phe Tyr Cys Asp Gly 50 55 60 Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His Ala 65 70 75 80 Ile Val Gln Thr Leu Val His Leu Met Phe Pro Asp His Val Pro Lys 85 90 95 Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Ile Ser Val Leu Tyr Phe 100 105 110 Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val 115 120 125 Arg Ser Cys Gly Cys His 130 <210> 18 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> Bone Morphogenetic Protein-6 (Vgr-1) <400> 18 Gln Gln Ser Arg Asn Arg Ser Thr Gln Ser Gln Asp Val Ala Arg Val 1 5 10 15 Ser Ser Ala Ser Asp Tyr Asn Ser Ser Glu Leu Lys Thr Ala Cys Arg 20 25 30 Lys His Glu Leu Tyr Val Ser Phe Gln Asp Leu Gly Trp Gln Asp Trp 35 40 45 Ile Ile Ala Pro Lys Gly Tyr Ala Ala Asn Tyr Cys Asp Gly Glu Cys 50 55 60 Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His Ala Ile Val 65 70 75 80 Gln Thr Leu Val His Leu Met Asn Pro Glu Tyr Val Pro Lys Pro Cys 85 90 95 Cys Ala Pro Thr Lys Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp 100 105 110 Asn Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala 115 120 125 Cys Gly Cys His 130 <210> 19 <211> 139 <212> PRT <213> Artificial Sequence <220> <223> Osteogenic Protein-7 (OP-1) <400> 19 Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser Ser 20 25 30 Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg 35 40 45 Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala 50 55 60 Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn 65 70 75 80 Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Phe Ile Asn Pro 85 90 95 Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala Ile 100 105 110 Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr 115 120 125 Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130 135 <210> 20 <211> 139 <212> PRT <213> Artificial Sequence <220> <223> Osteogenic Protein-8 (OP-2) <400> 20 Ala Val Arg Pro Leu Arg Arg Arg Gln Pro Lys Lys Ser Asn Glu Leu 1 5 10 15 Pro Gln Ala Asn Arg Leu Pro Gly Ile Phe Asp Asp Val His Gly Ser 20 25 30 His Gly Arg Gln Val Cys Arg Arg His Glu Leu Tyr Val Ser Phe Gln 35 40 45 Asp Leu Gly Trp Leu Asp Trp Val Ile Ala Pro Gln Gly Tyr Ser Ala 50 55 60 Tyr Tyr Cys Glu Gly Glu Cys Ser Phe Pro Leu Asp Ser Cys Met Asn 65 70 75 80 Ala Thr Asn His Ala Ile Leu Gln Ser Leu Val His Leu Met Lys Pro 85 90 95 Asn Ala Val Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr 100 105 110 Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val Ile Leu Arg Lys His 115 120 125 Arg Asn Met Val Val Lys Ala Cys Gly Cys His 130 135 <210> twenty one <211> 110 <212> PRT <213> Artificial sequence <220> <223> Bone Morphogenetic Protein-9 (Growth and Differentiation Factor-2) <400> twenty one Ser Ala Gly Ala Gly Ser His Cys Gln Lys Thr Ser Leu Arg Val Asn 1 5 10 15 Phe Glu Asp Ile Gly Trp Asp Ser Trp Ile Ile Ala Pro Lys Glu Tyr 20 25 30 Glu Ala Tyr Glu Cys Lys Gly Gly Cys Phe Phe Pro Leu Ala Asp Asp 35 40 45 Val Thr Pro Thr Lys His Ala Ile Val Gln Thr Leu Val His Leu Lys 50 55 60 Phe Pro Thr Lys Val Gly Lys Ala Cys Cys Val Pro Thr Lys Leu Ser 65 70 75 80 Pro Ile Ser Val Leu Tyr Lys Asp Asp Met Gly Val Pro Thr Leu Lys 85 90 95 Tyr His Tyr Glu Gly Met Ser Val Ala Glu Cys Gly Cys Arg 100 105 110 <210> 22 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Bone Morphogenetic Protein-10 <400> 22 Asn Ala Lys Gly Asn Tyr Cys Lys Arg Thr Pro Leu Tyr Ile Asp Phe 1 5 10 15 Lys Glu Ile Gly Trp Asp Ser Trp Ile Ile Ala Pro Pro Gly Tyr Glu 20 25 30 Ala Tyr Glu Cys Arg Gly Val Cys Asn Tyr Pro Leu Ala Glu His Leu 35 40 45 Thr Pro Thr Lys His Ala Ile Ile Gln Ala Leu Val His Leu Lys Asn 50 55 60 Ser Gln Lys Ala Ser Lys Ala Cys Cys Val Pro Thr Lys Leu Glu Pro 65 70 75 80 Ile Ser Ile Leu Tyr Leu Asp Lys Gly Val Val Thr Tyr Lys Phe Lys 85 90 95 Tyr Glu Gly Met Ala Val Ser Glu Cys Gly Cys Arg 100 105 <210> twenty three <211> 125 <212> PRT <213> Artificial sequence <220> <223> Bone Morphogenetic Protein-15 (Growth and Differentiation Factor-9b) <400> twenty three Gln Ala Asp Gly Ile Ser Ala Glu Val Thr Ala Ser Ser Ser Lys His 1 5 10 15 Ser Gly Pro Glu Asn Asn Gln Cys Ser Leu His Pro Phe Gln Ile Ser 20 25 30 Phe Arg Gln Leu Gly Trp Asp His Trp Ile Ile Ala Pro Pro Phe Tyr 35 40 45 Thr Pro Asn Tyr Cys Lys Gly Thr Cys Leu Arg Val Leu Arg Asp Gly 50 55 60 Leu Asn Ser Pro Asn His Ala Ile Ile Gln Asn Leu Ile Asn Gln Leu 65 70 75 80 Val Asp Gln Ser Val Pro Arg Pro Ser Cys Val Pro Tyr Lys Tyr Val 85 90 95 Pro Ile Ser Val Leu Met Ile Glu Ala Asn Gly Ser Ile Leu Tyr Lys 100 105 110 Glu Tyr Glu Gly Met Ile Ala Glu Ser Cys Thr Cys Arg 115 120 125 <210> 24 <211> 118 <212> PRT <213> artificial sequence <220> <223> Growth factor-1 <400> 24 Asp Ala Glu Pro Val Leu Gly Gly Gly Pro Gly Gly Ala Cys Arg Ala 1 5 10 15 Arg Arg Leu Tyr Val Ser Phe Arg Glu Val Gly Trp His Arg Trp Val 20 25 30 Ile Ala Pro Arg Gly Phe Leu Ala Asn Tyr Cys Gln Gly Gln Cys Ala 35 40 45 Leu Pro Val Ala Leu Ser Gly Ser Gly Gly Pro Pro Ala Leu Asn His 50 55 60 Ala Val Leu Arg Ala Leu Met His Ala Ala Ala Pro Gly Ala Ala Asp 65 70 75 80 Leu Pro Cys Cys Val Pro Ala Arg Leu Ser Pro Ile Ser Val Leu Phe 85 90 95 Phe Asp Asn Ser Asp Asn Val Val Leu Arg Gln Tyr Glu Asp Met Val 100 105 110 Val Asp Glu Cys Gly Cys 115 <210> 25 <211> 114 <212> PRT <213> artificial sequence <220> <223> Growth factor-3(Vgr-2) <400> 25 Ala Ala Ile Pro Val Pro Lys Leu Ser Cys Lys Asn Leu Cys His Arg 1 5 10 15 His Gln Leu Phe Ile Asn Phe Arg Asp Leu Gly Trp His Lys Trp Ile 20 25 30 Ile Ala Pro Lys Gly Phe Met Ala Asn Tyr Cys His Gly Glu Cys Pro 35 40 45 Phe Ser Leu Thr Ile Ser Leu Asn Ser Ser Asn Tyr Ala Phe Met Gln 50 55 60 Ala Leu Met His Ala Val Asp Pro Glu Ile Pro Gln Ala Val Cys Ile 65 70 75 80 Pro Thr Lys Leu Ser Pro Ile Ser Met Leu Tyr Gln Asp Asn Asn Asp 85 90 95 Asn Val Ile Leu Arg His Tyr Glu Asp Met Val Val Asp Glu Cys Gly 100 105 110 CysGly <210> 26 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Growth and differentiation factor-5 (bone morphogenetic protein-14) <400> 26 Ala Pro Leu Ala Thr Arg Gln Gly Lys Arg Pro Ser Lys Asn Leu Lys 1 5 10 15 Ala Arg Cys Ser Arg Lys Ala Leu His Val Asn Phe Lys Asp Met Gly 20 25 30 Trp Asp Asp Trp Ile Ile Ala Pro Leu Glu Tyr Glu Ala Phe His Cys 35 40 45 Glu Gly Leu Cys Glu Phe Pro Leu Arg Ser His Leu Glu Pro Thr Asn 50 55 60 His Ala Val Ile Gln Thr Leu Met Asn Ser Met Asp Pro Glu Ser Thr 65 70 75 80 Pro Pro Thr Cys Cys Val Pro Thr Arg Leu Ser Pro Ile Ser Ile Leu 85 90 95 Phe Ile Asp Ser Ala Asn Asn Val Val Tyr Lys Gln Tyr Glu Asp Met 100 105 110 Val Val Glu Ser Cys Gly Cys Arg 115 120 <210> 27 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Growth and differentiation factor-6 (bone morphogenetic protein-13) <400> 27 Thr Ala Phe Ala Ser Arg His Gly Lys Arg His Gly Lys Lys Ser Arg 1 5 10 15 Leu Arg Cys Ser Lys Lys Pro Leu His Val Asn Phe Lys Glu Leu Gly 20 25 30 Trp Asp Asp Trp Ile Ile Ala Pro Leu Glu Tyr Glu Ala Tyr His Cys 35 40 45 Glu Gly Val Cys Asp Phe Pro Leu Arg Ser His Leu Glu Pro Thr Asn 50 55 60 His Ala Ile Ile Gln Thr Leu Met Asn Ser Met Asp Pro Gly Ser Thr 65 70 75 80 Pro Pro Ser Cys Cys Val Pro Thr Lys Leu Thr Pro Ile Ser Ile Leu 85 90 95 Tyr Ile Asp Ala Gly Asn Asn Val Val Tyr Lys Gln Tyr Glu Asp Met 100 105 110 Val Val Glu Ser Cys Gly Cys Arg 115 120 <210> 28 <211> 129 <212> PRT <213> Artificial sequence <220> <223> Growth and differentiation factor-7 (bone morphogenetic protein-12) <400> 28 Thr Ala Leu Ala Gly Thr Arg Thr Ser Gln Gly Ser Gly Gly Gly Ala 1 5 10 15 Gly Arg Gly His Gly Arg Arg Gly Arg Ser Arg Cys Ser Arg Lys Pro 20 25 30 Leu His Val Asp Phe Lys Glu Leu Gly Trp Asp Asp Trp Ile Ile Ala 35 40 45 Pro Leu Asp Tyr Glu Ala Tyr His Cys Glu Gly Leu Cys Asp Phe Pro 50 55 60 Leu Arg Ser His Leu Glu Pro Thr Asn His Ala Ile Ile Gln Thr Leu 65 70 75 80 Leu Asn Ser Met Ala Pro Asp Ala Ala Pro Ala Ser Cys Cys Val Pro 85 90 95 Ala Arg Leu Ser Pro Ile Ser Ile Leu Tyr Ile Asp Ala Ala Asn Asn 100 105 110 Val Val Tyr Lys Gln Tyr Glu Asp Met Val Val Glu Ala Cys Gly Cys 115 120 125 Arg <210> 29 <211> 109 <212> PRT <213> Artificial sequence <220> <223> Growth and differentiation factor-8 (myostatin) <400> 29 Asp Phe Gly Leu Asp Cys Asp Glu His Ser Thr Glu Ser Arg Cys Cys 1 5 10 15 Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile 20 25 30 Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu 35 40 45 Phe Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala 50 55 60 Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser 65 70 75 80 Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly 85 90 95 Lys Ile Pro Ala Met Val Val Asp Arg Cys Gly Cys Ser 100 105 <210> 30 <211> 135 <212> PRT <213> Artificial sequence <220> <223> Growth and differentiation factor-9 <400> 30 Gly Gln Glu Thr Val Ser Ser Glu Leu Lys Lys Pro Leu Gly Pro Ala 1 5 10 15 Ser Phe Asn Leu Ser Glu Tyr Phe Arg Gln Phe Leu Leu Pro Gln Asn 20 25 30 Glu Cys Glu Leu His Asp Phe Arg Leu Ser Phe Ser Gln Leu Lys Trp 35 40 45 Asp Asn Trp Ile Val Ala Pro His Arg Tyr Asn Pro Arg Tyr Cys Lys 50 55 60 Gly Asp Cys Pro Arg Ala Val Gly His Arg Tyr Gly Ser Pro Val His 65 70 75 80 Thr Met Val Gln Asn Ile Ile Tyr Glu Lys Leu Asp Ser Ser Val Pro 85 90 95 Arg Pro Ser Cys Val Pro Ala Lys Tyr Ser Pro Leu Ser Val Leu Thr 100 105 110 Ile Glu Pro Asp Gly Ser Ile Ala Tyr Lys Glu Tyr Glu Asp Met Ile 115 120 125 Ala Thr Lys Cys Thr Cys Arg 130 135 <210> 31 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Growth and differentiation factor-10 (bone morphogenetic protein-3b) <400> 31 Lys Thr Met Gln Lys Ala Arg Arg Lys Gln Trp Asp Glu Pro Arg Val 1 5 10 15 Cys Ser Arg Arg Tyr Leu Lys Val Asp Phe Ala Asp Ile Gly Trp Asn 20 25 30 Glu Trp Ile Ile Ser Pro Lys Ser Phe Asp Ala Tyr Tyr Cys Ala Gly 35 40 45 Ala Cys Glu Phe Pro Met Pro Lys Ile Val Arg Pro Ser Asn His Ala 50 55 60 Thr Ile Gln Ser Ile Val Arg Ala Val Gly Ile Ile Pro Gly Ile Pro 65 70 75 80 Glu Pro Cys Cys Val Pro Asp Lys Met Asn Ser Leu Gly Val Leu Phe 85 90 95 Leu Asp Glu Asn Arg Asn Val Val Leu Lys Val Tyr Pro Asn Met Ser 100 105 110 Val Asp Thr Cys Ala Cys Arg 115 <210> 32 <211> 109 <212> PRT <213> Artificial sequence <220> <223> Growth and differentiation factor-11 (bone morphogenetic protein-11) <400> 32 Asn Leu Gly Leu Asp Cys Asp Glu His Ser Ser Glu Ser Arg Cys Cys 1 5 10 15 Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile 20 25 30 Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Gln Cys Glu 35 40 45 Tyr Met Phe Met Gln Lys Tyr Pro His Thr His Leu Val Gln Gln Ala 50 55 60 Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser 65 70 75 80 Pro Ile Asn Met Leu Tyr Phe Asn Asp Lys Gln Gln Ile Ile Tyr Gly 85 90 95 Lys Ile Pro Gly Met Val Val Asp Arg Cys Gly Cys Ser 100 105 <210> 33 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Growth and Differentiation Factor-15 <400> 33 Ala Arg Asn Gly Asp Asp Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg 1 5 10 15 Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp 20 25 30 Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys 35 40 45 Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser 50 55 60 Leu His Arg Leu Lys Pro Asp Thr Glu Pro Ala Pro Cys Cys Val Pro 65 70 75 80 Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val 85 90 95 Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile 100 105 110 <210> 34 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Node <400> 34 His His Leu Pro Asp Arg Ser Gln Leu Cys Arg Lys Val Lys Phe Gln 1 5 10 15 Val Asp Phe Asn Leu Ile Gly Trp Gly Ser Trp Ile Ile Tyr Pro Lys 20 25 30 Gln Tyr Asn Ala Tyr Arg Cys Glu Gly Glu Cys Pro Asn Pro Val Gly 35 40 45 Glu Glu Phe His Pro Thr Asn His Ala Tyr Ile Gln Ser Leu Leu Lys 50 55 60 Arg Tyr Gln Pro His Arg Val Pro Ser Thr Cys Cys Ala Pro Val Lys 65 70 75 80 Thr Lys Pro Leu Ser Met Leu Tyr Val Asp Asn Gly Arg Val Leu Leu 85 90 95 Asp His His Lys Asp Met Ile Val Glu Glu Cys Gly Cys Leu 100 105 110 <210> 35 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Activin - bA <400> 35 Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys Cys Lys Lys Gln Phe 1 5 10 15 Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp Ile Ile Ala Pro 20 25 30 Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys Pro Ser His Ile 35 40 45 Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser Thr Val Ile Asn 50 55 60 His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn Leu Lys Ser Cys 65 70 75 80 Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu Tyr Tyr Asp Asp 85 90 95 Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn Met Ile Val Glu Glu 100 105 110 Cys Gly Cys Ser 115 <210> 36 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Activin - bB <400> 36 Gly Leu Glu Cys Asp Gly Arg Thr Asn Leu Cys Cys Arg Gln Gln Phe 1 5 10 15 Phe Ile Asp Phe Arg Leu Ile Gly Trp Asn Asp Trp Ile Ile Ala Pro 20 25 30 Thr Gly Tyr Tyr Gly Asn Tyr Cys Glu Gly Ser Cys Pro Ala Tyr Leu 35 40 45 Ala Gly Val Pro Gly Ser Ala Ser Ser Phe His Thr Ala Val Val Asn 50 55 60 Gln Tyr Arg Met Arg Gly Leu Asn Pro Gly Thr Val Asn Ser Cys Cys 65 70 75 80 Ile Pro Thr Lys Leu Ser Thr Met Ser Met Leu Tyr Phe Asp Asp Glu 85 90 95 Tyr Asn Ile Val Lys Arg Asp Val Pro Asn Met Ile Val Glu Glu Cys 100 105 110 Gly Cys Ala 115 <210> 37 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Activin - bC <400> 37 Gly Ile Asp Cys Gln Gly Gly Ser Arg Met Cys Cys Arg Gln Glu Phe 1 5 10 15 Phe Val Asp Phe Arg Glu Ile Gly Trp His Asp Trp Ile Ile Gln Pro 20 25 30 Glu Gly Tyr Ala Met Asn Phe Cys Ile Gly Gln Cys Pro Leu His Ile 35 40 45 Ala Gly Met Pro Gly Ile Ala Ala Ser Phe His Thr Ala Val Leu Asn 50 55 60 Leu Leu Lys Ala Asn Thr Ala Ala Gly Thr Thr Gly Gly Gly Ser Cys 65 70 75 80 Cys Val Pro Thr Ala Arg Arg Pro Leu Ser Leu Leu Tyr Tyr Asp Arg 85 90 95 Asp Ser Asn Ile Val Lys Thr Asp Ile Pro Asp Met Val Val Glu Ala 100 105 110 Cys Gly Cys Ser 115 <210> 38 <211> 114 <212> PRT <213> Artificial sequence <220> <223> Activin-bE <400> 38 Thr Pro Thr Cys Glu Pro Ala Thr Pro Leu Cys Cys Arg Arg Asp His 1 5 10 15 Tyr Val Asp Phe Gln Glu Leu Gly Trp Arg Asp Trp Ile Leu Gln Pro 20 25 30 Glu Gly Tyr Gln Leu Asn Tyr Cys Ser Gly Gln Cys Pro Pro His Leu 35 40 45 Ala Gly Ser Pro Gly Ile Ala Ala Ser Phe His Ser Ala Val Phe Ser 50 55 60 Leu Leu Lys Ala Asn Asn Pro Trp Pro Ala Ser Thr Ser Cys Cys Val 65 70 75 80 Pro Thr Ala Arg Arg Pro Leu Ser Leu Leu Tyr Leu Asp His Asn Gly 85 90 95 Asn Val Val Lys Thr Asp Val Pro Asp Met Val Val Glu Ala Cys Gly 100 105 110 Cys Ser <210> 39 <211> 134 <212> PRT <213> Artificial sequence <220> <223> Inhibin-a <400> 39 Ser Thr Pro Leu Met Ser Trp Pro Trp Ser Pro Ser Ala Leu Arg Leu 1 5 10 15 Leu Gln Arg Pro Pro Glu Glu Pro Ala Ala His Ala Asn Cys His Arg 20 25 30 Val Ala Leu Asn Ile Ser Phe Gln Glu Leu Gly Trp Glu Arg Trp Ile 35 40 45 Val Tyr Pro Pro Ser Phe Ile Phe His Tyr Cys His Gly Gly Cys Gly 50 55 60 Leu His Ile Pro Pro Asn Leu Ser Leu Pro Val Pro Gly Ala Pro Pro 65 70 75 80 Thr Pro Ala Gln Pro Tyr Ser Leu Leu Pro Gly Ala Gln Pro Cys Cys 85 90 95 Ala Ala Leu Pro Gly Thr Met Arg Pro Leu His Val Arg Thr Thr Ser 100 105 110 Asp Gly Gly Tyr Ser Phe Lys Tyr Glu Thr Val Pro Asn Leu Leu Thr 115 120 125 Gln His Cys Ala Cys Ile 130 <210> 40 <211> 112 <212> PRT <213> artificial sequence <220> <223> transforming growth factor-b1 <400> 40 Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser Thr Glu Lys Asn Cys Cys 1 5 10 15 Val Arg Gln Leu Tyr Ile Asp Phe Arg Lys Asp Leu Gly Trp Lys Trp 20 25 30 Ile His Glu Pro Lys Gly Tyr His Ala Asn Phe Cys Leu Gly Pro Cys 35 40 45 Pro Tyr Ile Trp Ser Leu Asp Thr Gln Tyr Ser Lys Val Leu Ala Leu 50 55 60 Tyr Asn Gln His Asn Pro Gly Ala Ser Ala Ala Pro Cys Cys Val Pro 65 70 75 80 Gln Ala Leu Glu Pro Leu Pro Ile Val Tyr Tyr Val Gly Arg Lys Pro 85 90 95 Lys Val Glu Gln Leu Ser Asn Met Ile Val Arg Ser Cys Lys Cys Ser 100 105 110 <210> 41 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Transforming Growth Factor - b2 <400> 41 Ala Leu Asp Ala Ala Tyr Cys Phe Arg Asn Val Gln Asp Asn Cys Cys 1 5 10 15 Leu Arg Pro Leu Tyr Ile Asp Phe Lys Arg Asp Leu Gly Trp Lys Trp 20 25 30 Ile His Glu Pro Lys Gly Tyr Asn Ala Asn Phe Cys Ala Gly Ala Cys 35 40 45 Pro Tyr Leu Trp Ser Ser Asp Thr Gln His Ser Arg Val Leu Ser Leu 50 55 60 Tyr Asn Thr Ile Asn Pro Glu Ala Ser Ala Ser Pro Cys Cys Val Ser 65 70 75 80 Gln Asp Leu Glu Pro Leu Thr Ile Leu Tyr Tyr Ile Gly Lys Thr Pro 85 90 95 Lys Ile Glu Gln Leu Ser Asn Met Ile Val Lys Ser Cys Lys Cys Ser 100 105 110 <210> 42 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Transforming Growth Factor - b3 <400> 42 Ala Leu Asp Thr Asn Tyr Cys Phe Arg Asn Leu Glu Glu Asn Cys Cys 1 5 10 15 Val Arg Pro Leu Tyr Ile Asp Phe Arg Gln Asp Leu Gly Trp Lys Trp 20 25 30 Val His Glu Pro Lys Gly Tyr Tyr Ala Asn Phe Cys Ser Gly Pro Cys 35 40 45 Pro Tyr Leu Arg Ser Ala Asp Thr Thr His Ser Thr Val Leu Gly Leu 50 55 60 Tyr Asn Thr Leu Asn Pro Glu Ala Ser Ala Ser Pro Cys Cys Val Pro 65 70 75 80 Gln Asp Leu Glu Pro Leu Thr Ile Leu Tyr Tyr Val Gly Arg Thr Pro 85 90 95 Lys Val Glu Gln Leu Ser Asn Met Val Val Lys Ser Cys Lys Cys Ser 100 105 110

Claims

1. A recombinant chimeric polypeptide comprising peptide segments each derived from a transforming growth factor-β family protein consisting of bone morphogenetic protein-7, bone morphogenetic protein-2 and activin, wherein the recombinant chimeric polypeptide consists of the following amino acid sequence: The amino acid sequence shown in SEQ ID NO: 8, The amino acid sequence shown in SEQ ID NO: 9, or The amino acid sequence shown in SEQ ID NO:

11.

2. A polynucleotide encoding the chimeric polypeptide of claim 1. A vector comprising the polynucleotide according to claim 2. A host cell comprising the vector according to claim 3.

Images