IMPROVED OSTEOGENIC PROTEINS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of, and claims the benefit of priority under 35 U.S.C. §120 to, U .S. Patent Application No. 14/589,468 filed January 5, 2015 by Berasi ef a/. , which is a continuation of U.S. Patent Application No. 13/21 1 ,755 filed August 17, 201 1 now U .S. Patent 8,952, 131 , which in turn claims the benefit of priority under 35 U.S.C § 1 19(e) to U.S. Provisional Patent Application No. 61/375,636, filed August 20, 2010. Each of the foregoing applications are incorporated by reference herein in their entirety and for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCI I copy, created on April 3, 2015, is named 7362174001 and is 17,395 bytes in size.

FIELD OF THE INVENTION

This application relates to the field of osteogenic proteins, methods of making improved osteogenic proteins, and methods of treating patients with osteogenic proteins.

BACKGROUND OF THE INVENTION

The cystine knot cytokine superfamily is divided into subfamilies, which include, the transforming growth factor β (ΤΰΡβ) proteins, the glycoprotein hormones, the platelet-derived growth factor-like (PDGF-like) proteins, nerve growth factors (NGF), and the differential screening-selected gene aberrative in neuroblastoma (DAN) family (e.g., cerberus). In turn, the TGFfi superfamily comprises approximately 43 members, subdivided into three subfamilies: the ΤΰΡβε, the activins and the bone morphogenetic/growth differentiation factor proteins (BMP/GDF).

The TGF-β superfamily members contain the canonical cystine knot topology. That is, cystine knots are the result of an unusual arrangement of six cysteine residues. The knot consists of bonds between cysteines 1 -4, cysteines 2-5, and the intervening sequence forming a ring, through which the disulfide bond between cysteines 3-6 passes. The active forms of these proteins are homodimers or heterodimers. In each case the monomer topology is stabilized by the cysteine knot and additional cysteines contribute to additional intrachain bonds and/or mediate dimerization with another protein unit. See Kingsley, 1994, Genes Dev. 8: 133-146; Lander et al, 2001 , Nature 409:860-921 .

BMP/GDFs are the most numerous members of the TGF-β protein superfamily. The BMP/GDF subfamily includes, but is not limited to, BMP2, BMP3 (osteogenin), BMP3b (GDF-10), BMP4 (BMP2b), BMP5, BMP6, BMP7 (osteogenic protein-1 or OP1 ), BMP8 (OP2), BMP8B (OP3), BMP9 (GDF2), BMP10, BMP1 1 (GDF1 1 ), BMP12 (GDF7), BMP13 (GDF6, CDMP2), BMP15 (GDF9), BMP16, GDF1 , GDF3, GDF5 (CDMP1 ; MP52), and GDF8 (myostatin). BMPs are sometimes referred to as Osteogenic Protein (OPs),

Growth Differentiation Factors (GDFs), or Cartilage-Derived Morphogenetic Proteins (CDMPs). BMPs are also present in other animal species. Furthermore, there is some allelic variation in BMP sequences among different members of the human population.

BMPs are naturally expressed as pro-proteins comprising a long pro-domain, one or more cleavage sites, and a mature domain. This pro-protein is then processed by the cellular machinery to yield a dimeric mature BMP molecule. The pro-domain is believed to aid in the correct folding and processing of BMPs. Furthermore, in some but not all BMPs, the pro-domain may noncovalently bind the mature domain and may act as a chaperone, as well as an inhibitor (e.g., Thies et al., Growth Factors 18:251 -9 (2001 )).

BMP signal transduction is initiated when a BMP dimer binds two type I and two type I I serine/threonine kinase receptors. Type I receptors include, but are not limited to, ALK-1 (Activin receptor-Like Kinase 1 ), ALK-2 (also called ActRIa or ActRI), ALK-3 (also called BMPRIa), and ALK-6 (also called BMPRIb). Type II receptors include, but are not limited to, ActRlla (also called ActRII), ActRl lb, and BMPRI I. The human genome contains 12 members of the receptor serine/threonine kinase family, including 7 type I and 5 type I I receptors, all of which are involved in TGF-β signaling (Manning et al., Science 298: 1912-34 (2002)), the disclosures of which are hereby incorporated by reference). Thus, there are 12 receptors and 43 superfamily members, suggesting that at least some TGF-β superfamily members bind the same receptor(s). Following BMP binding, the type I I receptors phosphorylate the type I receptors, the type I receptors phosphorylate members of the Smad family of transcription factors, and the Smads translocate to the nucleus and activate the expression of a number of genes.

BMPs are among the most numerous members of TGF-β superfamily, and control a diverse set of cellular and developmental processes, such as embryonic pattern formation and tissue specification as well as promoting wound healing and repair processes in adult tissues in, among other places, the nervous system and the skeletal system. In fact, BMPs were initially isolated by their ability to induce bone and cartilage formation: BMP signaling is inducible upon bone fracture and related tissue injury, leading to bone regeneration and repair. Given their roles in development and normal wound healing , BMPs hold immense promise for the regeneration and repair of the skeletal system, nervous system, and other tissues where BMP receptors are expressed. This promise would be even greater for BMPs with altered affinity for their receptors and/or improved biological activity relative to the native proteins. The inventors have previously described designer BMPs with altered binding to BMP receptors and with increased activity in various in vitro and in vivo assays. Yet the universe of designer BMPs currently known remains relatively small, and there is an ongoing need in the field for the development of new and novel designer BMPs.

SUMMARY OF THE INVENTION

The present invention addresses this need by providing, in its various aspects, novel designer BMPs based on BMPs 2, 4, 6 and 7, compositions and methods relating to these designer BMPs, and methods of making and using the same. In one aspect, the invention relates to a designer BMP comprising , in various embodiments, an amino acid sequence selected from the group consisting of SEQ ID NOS: 8,

9, 10, 1 1 , 12, 13, 14 and 15. The designer BMP optionally includes one or more mutations not in the Type I, Type II A, or Type II B binding domains; there may be, variously, 1 , 2, 3, 4, 5, 6 7, 8, 9, 10 or more such mutations. Alternatively or additionally, the designer BMP exhibits a receptor binding profile which differs from a wild-type BMP, which binding profile includes, one, several or all of the following: binding to the ALK2 receptor with a KD not greater than about 2 nM; binding to the ALK3 receptor with a KD not greater than about 2 nM; binding to the ALK6 receptor with a KD not greater than about 1 nM; binding to the ActRMA receptor with a KD not greater than about 2 nM; binding to the ActRIIB receptor with a KD not greater than about 0.5 nM; and binding to the BMPRIIA receptor with a KD not greater than about 3.5 nM.

In another aspect, the invention relates to a designer BMP comprising the following mutations relative to SEQ ID NO: 1 : V33I, P36E, H39A, H44E, P48A, A52N, D53S, H54Y, L55M, S57A, N68H, S69F, V70I, S72P, K73E, insertion of a T following K73, I74V, A77P, V80A, S85N, M89V, L92F, E94D, N95S, E96S, K97N, V99I. In some embodiments, the designer BMP optionally includes one or more mutations not in the Type I, Type II A, or Type II B binding domains; there may be, variously, 1 , 2, 3, 4, 5, 6 7, 8, 9, 10 or more such mutations. Alternatively or additionally, the designer BMP exhibits a receptor binding profile which differs from a wild-type BMP, which binding profile includes, one, several or all of the following: binding to the ALK2 receptor with a KD not greater than about 2 nM; binding to the ALK3 receptor with a KD not greater than about 2 nM; binding to the ALK6 receptor with a KD not greater than about 1 nM; binding to the ActRMA receptor with a KD not greater than about 2 nM; binding to the ActRIIB receptor with a KD not greater than about 0.5 nM; and binding to the BMPRIIA receptor with a KD not greater than about 3.5 nM. The designer BMP may have an amino acid sequence comprising SEQ ID NO:8 in some instances, while in other instances, the designer BMP further comprises the following mutations relative to SEQ ID NO:8: E84K, N86R, A87P, I88M, V90M, F93Y, S96G, S97Q, V99I, L101 K, N103D, Y104I, D106N, V108I, G1 1 1 E, R1 15S .

In another aspect, the invention relates to a designer BMP comprising the following mutations relative to SEQ ID NO: 1 : V33I, P36E, H39A, H44E, P48A, A52N, D53S, H54Y, L55M, S57A, N68H, S69F, V70I, S72P, K73E, insertion of a T following K73, I74V, A77P, V80A, E83K, S85R, A86P, I87M, L92Y, E94D, N95G, E96Q, K97N, V98I, V99I,L100K, N102D, Y103I, D105N, V107I, G1 10E, R1 14S. In some embodiments, the designer BMP optionally includes one or more mutations not in the Type I, Type II A, or Type II B binding domains; there may be, variously, 1 , 2, 3, 4, 5, 6 7, 8, 9, 10 or more such mutations. Alternatively or additionally, the designer BMP exhibits a receptor binding profile which differs from a wild-type BMP, which binding profile includes, one, several or all of the following: binding to the ALK2 receptor with a KD not greater than about 2 nM; binding to the ALK3 receptor with a KD not greater than about 2 nM; binding to the ALK6 receptor with a KD not greater than about 1 nM; binding to the ActRMA receptor with a KD not greater than about 2 nM; binding to the ActRIIB receptor with a KD not greater than about 0.5 nM; and binding to the BMPRIIA receptor with a KD not greater than about 3.5 nM. The designer BMP may have an amino acid sequence comprising SEQ ID NO: 9 in some instances.

In yet another aspect, the invention relates to a designer BMP comprising the following mutations relative to SEQ ID NO:2: V35I, P38K, Q41A, H46D, D48E, P50S, A54N, D55A, L57M, S59A, N70H, S71 L, V72P, S74P, S75E, insertion of a Y after S75, I76V, A79P, V82A, S87N, M91 V, L94F, E96D, Y97N, D98S, K99N, V101 I. In some embodiments, the designer BMP optionally includes one or more mutations not in the Type I, Type II A, or Type II B binding domains; there may be, variously, 1 , 2, 3, 4, 5, 6 7, 8, 9, 10 or more such mutations. Alternatively or additionally, the designer BMP exhibits a receptor binding profile which differs from a wild-type BMP, which binding profile includes, one, several or all of the following: binding to the ALK2 receptor with a KD not greater than about 2 nM; binding to the ALK3 receptor with a KD not greater than about 2 nM; binding to the ALK6 receptor with a KD not greater than about 1 nM; binding to the ActRIIA receptor with a KD not greater than about 2 nM; binding to the ActRIIB receptor with a KD not greater than about 0.5 nM; and binding to the BMPRIIA receptor with a KD not greater than about 3.5 nM. The designer BMP may have an amino acid sequence comprising SEQ ID NO: 10 in some instances. In other cases, the designer BMP may further include a mutation of K38R relative to SEQ ID NO: 10, and/or may comprise the amino acid sequence of SEQ ID NO: 12.

In yet another aspect, the present invention relates to a designer BMP comprising the following mutations relative to SEQ ID NO:2: V35I, P38K, Q41A, H46D, D48E, P50S, A54N, D55A, L57M, S59A, N70H, S71 L, V71 M, S74P, S75E, insertion of a Y after S75, I76V, A79P, V82A, E85K, S87R, A88P, I89M, L94Y, E96D, Y97G, D98Q, K99N, V100I, V1010I, L102K, N104D, Y105I, E107N, V109I, G1 12E, R1 16S. In some embodiments, the designer BMP optionally includes one or more mutations not in the Type I, Type II A, or Type II B binding domains; there may be, variously, 1 , 2, 3, 4, 5, 6 7, 8, 9, 10 or more such mutations. Alternatively or additionally, the designer BMP exhibits a receptor binding profile which differs from a wild-type BMP, which binding profile includes, one, several or all of the following: binding to the ALK2 receptor with a KD not greater than about 2 nM; binding to the ALK3 receptor with a KD not greater than about 2 nM; binding to the ALK6 receptor with a KD not greater than about 1 nM; binding to the ActRIIA receptor with a KD not greater than about 2 nM; binding to the ActRIIB receptor with a KD not greater than about 0.5 nM; and binding to the BMPRIIA receptor with a KD not greater than about 3.5 nM. The designer BMP may have an amino acid sequence comprising SEQ ID NO: 1 1 in some instances.

In yet another aspect, the present invention relates to a designer BMP comprising the following mutations relative to SEQ ID NO:2: V35I, P38R, Q41A, H46D, D48E, P50S, A54N, D55A, L57M, S59A, N70H, S71 L, V72M, S74P, S75E, insertion of a Y after S75, I76V, A79P, V82A, E85K, S87R, A88P, I89M, L94Y, E96D, Y97G, D98Q, K99N, V100I, V101 I, L102K, N104D, Y105I, E107N, V109I, G1 12E, R1 16S. In some embodiments, the designer BMP optionally includes one or more mutations not in the Type I, Type II A, or Type II B binding domains; there may be, variously, 1 , 2, 3, 4, 5, 6 7, 8, 9, 10 or more such mutations. Alternatively or additionally, the designer BMP exhibits a receptor binding profile which differs from a wild-type BMP, which binding profile includes, one, several or all of the following: binding to the ALK2 receptor with a KD not greater than about 2 nM; binding to the ALK3 receptor with a KD not greater than about 2 nM; binding to the ALK6 receptor with a KD not greater than about 1 nM; binding to the ActRIIA receptor with a

KD not greater than about 2 nM; binding to the ActRIIB receptor with a KD not greater than about 0.5 nM; and binding to the BMPRIIA receptor with a KD not greater than about 3.5 nM. The designer BMP optionally has an amino acid sequence comprising SEQ ID NO: 13.

In yet another aspect, the present invention relates to a designer BMP comprising the following mutations relative to SEQ ID NO:2: V35I, P38E, Q41A, H46E, D48E, P50A, A54N, D55S, H56Y, L57M, S59A, N70H, S71 F, V72I, insertion of a P after N73, S74E, S75T, I76V, A79P, V82A, S87N, M91V, L94F, E96D, Y97S, D98S, K99N, V101 I. In some embodiments, the designer BMP optionally includes one or more mutations not in the Type I, Type II A, or Type II B binding domains; there may be, variously, 1 , 2, 3, 4, 5, 6 7, 8, 9, 10 or more such mutations. Alternatively or additionally, the designer BMP exhibits a receptor binding profile which differs from a wild-type BMP, which binding profile includes, one, several or all of the following: binding to the ALK2 receptor with a KD not greater than about 2 nM; binding to the ALK3 receptor with a KD not greater than about 2 nM; binding to the ALK6 receptor with a KD not greater than about 1 nM; binding to the ActRIIA receptor with a KD not greater than about 2 nM; binding to the ActRIIB receptor with a KD not greater than about 0.5 nM; and binding to the BMPRIIA receptor with a KD not greater than about 3.5 nM. The designer BMP may have an amino acid sequence comprising SEQ ID NO: 14.

And in yet another aspect, the present invention relates to a designer BMP comprising the following mutations relative to SEQ ID NO: 2: V35I, P38E, Q41A, H46E, D48E, P50A, A54N, D55S, H56Y, L57M, S59A, N70H, S71 F, V72I, insertion of a P after N73, S74E, S75T, I76V, A79P, V82A, E85K, S87R, A88P, I89M, L94Y, E96D, Y97G, D98Q, K99N, V100I, V101 I, L102K, N 104D, Y105I, E107N, V109I, G1 12E, R1 16S. In some embodiments, the designer BMP optionally includes one or more mutations not in the Type I, Type II A, or Type II B binding domains; there may be, variously, 1 , 2, 3, 4, 5, 6 7, 8, 9, 10 or more such mutations. Alternatively or additionally, the designer BMP exhibits a receptor binding profile which differs from a wild-type BMP, which binding profile includes, one, several or all of the following: binding to the ALK2 receptor with a KD not greater than about 2 nM; binding to the ALK3 receptor with a KD not greater than about 2 nM; binding to the ALK6 receptor with a KD not greater than about 1 nM; binding to the ActRIIA receptor with a KD not greater than about 2 nM; binding to the ActRIIB receptor with a KD not greater than about 0.5 nM; and binding to the BMPRIIA receptor with a KD not greater than about 3.5 nM. The designer BMP may have an amino acid sequence comprising SEQ ID NO: 15.

The invention further includes methods of producing a designer BMP proteins of the types described above. The methods generally include introducing a nucleic acid encoding the protein into a host cell, culturing the cell under conditions where the protein is produced, and purifying the protein.

In various aspects the invention relates to a nucleic acid that encodes an amino acid sequence selected from the group consisting of SEQ ID NOS: 8-15, or specifically encodes. SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:1 1 , SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, and/or SEQ ID NO: 15. The nucleic acid can be incorporated into any suitable expression vector, which in turn can be introduced into a cell for expression and harvesting, as discussed below.

The invention also includes methods of treating a bone disease associated with bone loss in a patient in need thereof. In one aspect, the method includes administering a therapeutically effective amount of a designer BMP protein as described above, thereby treating bone disease in the patient. In various embodiments, the designer BMP protein includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 8-15, or specifically SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and/or SEQ ID NO: 15.

The invention includes a method of treating fibrosis in a patient in need thereof. In one aspect, the invention relates to a method which includes administering a therapeutically effective amount of a designer BMP protein as described above, thereby treating fibrosis. In various embodiments, the designer BMP protein includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 8-15, or specifically SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and/or SEQ ID NO: 15.

Additionally, the invention includes a method of inducing bone formation in a tissue. In this aspect, the method comprises contacting the tissue with a designer BMP protein as described above, thereby inducing bone formation in said tissue. In various embodiments, the designer BMP protein is amino acid sequence selected from the group consisting of SEQ ID NOS: 8-15, or specifically SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and/or SEQ ID NO: 15.

Finally, one aspect of the invention relates to a kit for treating a patient, which includes a designer BMP as described above. In various embodiments, the designer BMP protein includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 8-15, or specifically SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and/or SEQ ID NO: 15. Kits of the present invention optionally or additionally include a fluid for diluting the designer BMP, thereby creating a designer BMP solution, and/or a medical instrument or an implant material for use with the designer BMP solution.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

Figure 1 is a diagram showing the alignment of various wild type and designer BMP amino acid sequences and indicating (by being within a box) the regions of these proteins potentially involved in type I and type II receptor interactions. Figure 1 shows the amino acid sequence alignment of wild type BMP2 (SEQ ID NO: 1 ), BMP4 (SEQ ID NO: 2), BMP5 (SEQ ID NO: 3), BMP6 (SEQ ID NO: 4), BMP7 (SEQ ID NO: 5), BMP8 (SEQ ID NO: 6) and BMP9 (SEQ ID NO: 7).

Figure 2 is an illustration of a structural model showing a wild type BMP2 homodimer binding to two type I and two type II BMP receptors.

Figure 3, comprising panels A and B, is an diagram of a structural model showing the position of the histidine doorstop (H54) in human BMP2 produced in Chinese Hamster Ovary (CHO) (Figure 3A) and E. coli cells (Figure 3B).

Figure 4, comprising panels A and B, is a diagram illustrating the location of the glycan tether and potential histidine (His) doorstop. Figure 4A shows the glycan tether (N-linked glycan at N56) and histidine 54, in the non-doorstop orientation, as well as the interaction of the glycan tether with R16 all in CHO-produced BMP2. Figure 4B shows the glycan tether (N-linked glycan at N80) and the histidine in the non-doorstop configuration at H78 in BMP6, as well as the R39 corresponding to R16 in BMP2.

Figure 5, comprising panels A-D, shows various steps in the process for purification of BMPs and designer BMPs. Figure 5A shows a chromatogram showing gradient elution of BMPs using a cellufine sulfate column. Figure 5B is an image of a Coomassie stained SDS-PAGE (non-reduced on the left and reduced on the right side) gel containing samples of fractions from the cellufine sulfate column step. Figure 5C shows a chromatogram showing the profile from preparative reversed phase purification step. Figure 5D is an image of a Coomassie stained SDS-PAGE (non-reduced on the left and reduced on the right) gel of BMP containing samples of the fractions obtained by the preparative reversed phase purification step.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates a "designer" bone morphogenetic protein, referred to herein as "designer BMP," "designer osteogenic protein" and "designer protein." The designer BMPs of the invention may correspond to the amino acid sequences of wild type unmodified BMP, such as, but not limited to, BMP2 (SEQ ID NO: 1 ), BMP4 (SEQ ID NO:2), BMP5 (SEQ ID NO:3), BMP6 (SEQ ID NO:4), BMP7 (SEQ ID NO:5), BMP8 (SEQ ID NO:6), and BMP9 (SEQ ID NO:7). In particular embodiments, the designer BMPs show altered binding to a type I and/or type II BMP receptor when compared to its corresponding wild type BMP. In further embodiments, the designer BMP may be modified to have altered half-life, immunogenicity, or any pharmacokinetic/pharmacodynamic (PK/PD) parameter when compared to its corresponding BMP. Definitions

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art.

The methods and techniques of the present invention are generally performed according to methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Such references include, e.g., Sambrook and Russell, Molecular Cloning, A Laboratory Approach , Cold Spring Harbor Press, Cold Spring Harbor, NY (2001 ), Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002), and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990), which are incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

As used herein, each of the following terms has the meaning associated with it in this section. The articles "a" and "an" are used herein to refer to one or to more than one (i.e. , to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

In this application, the use of "or" means "and/or" unless stated otherwise.

Conventional notation is used herein to portray polypeptide sequences: the left-hand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxyl-terminus. As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991 )), which is incorporated herein by reference. As used herein, amino acids are represented by the full name thereof, by the three letter code corresponding thereto, or by the one-letter code corresponding thereto, as indicated as follows:

Full Name Three-Letter Code One-Letter Code

Aspartic Acid Asp D

Glutamic Acid Glu E

Lysine Lys K

Arginine Arg R

Histidine His H

Tyrosine Tyr Y

Cysteine Cys C

Asparagine Asn N

Glutamine Gin Q

Serine Ser S

Threonine Thr T

Glycine Gly G

Alanine Ala A

Valine Val V

Leucine Leu L

Isoleucine lie I

Methionine Met M

Proline Pro P

Phenylalanine Phe F

Tryptophan Trp W

A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson, Methods Mol. Biol. 243:307-31 (1994).

Examples of groups of amino acids that have side chains with similar chemical properties include 1 ) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al., Science 256: 1443-1445 (1992), herein incorporated by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

Preferred amino acid substitutions are those which: (1 ) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, and (4) confer or modify other physicochemical or functional properties of such analogs. Analogs comprising substitutions, deletions, and/or insertions can include various muteins of a sequence other than the specified peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the specified sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts, e.g. , outside of the CDRs or the type I or type II receptor binding sites). A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991 )); and Thornton et al., Nature 354: 105 (1991 ), which are each incorporated herein by reference.

The terms "polynucleotide", "nucleotide sequence", "nucleic acid", "nucleic acid molecule", "nucleic acid sequence", and "oligonucleotide" refer to a series of nucleotide bases (also called "nucleotides") in DNA and RNA, and mean any chain of two or more nucleotides. The polynucleotides can be chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, its hybridization parameters, etc. A nucleotide sequence typically carries genetic information, including the information used by cellular machinery to make proteins and enzymes. These terms include double- or single-stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and antisense polynucleotides. This also includes nucleic acids containing modified bases, for example, thio-uracil, thio-guanine, and fluoro-uracil, or containing carbohydrate, or lipids.

In the context of a nucleotide sequence, the term "substantially identical" is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences having at least about 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96% , 97%, 98% or 99% identity to a reference sequence.

By "designer BMP nucleic acids," and grammatical equivalents herein is meant nucleic acids that encode designer BMPs.

The terms "protein" and "polypeptide" are used interchangeably herein. These terms refer to a sequential chain of amino acids linked together via peptide bonds. The terms include one or more proteins that function as a discrete unit. If a single polypeptide is the discrete functioning unit and does not require permanent or temporary physical association with other polypeptides in order to form the discrete functioning unit, the terms "polypeptide" and "protein" may be used interchangeably. If the discrete functional unit is comprised of multiple polypeptides that physically associate with one another, the term "protein" as used herein refers to the multiple polypeptides that are physically coupled and function together as the discrete unit. A protein to be expressed according to the present invention can be a protein therapeutic. A protein therapeutic is a protein that has a biological effect on a region in the body on which it acts or on a region of the body on which it remotely acts via intermediates. Examples of protein therapeutics are discussed in more detail below.

"Designer BMP," as the term is used herein, relates to a BMP protein comprising at least one amino acid mutation compared to a corresponding wild type BMP without the mutation, wherein the designer BMP has detectably altered binding for at least a type I receptor and/or at least one type II receptor compared with the binding of the corresponding wild type BMP for the type I and/or type II receptor.

By "corresponding wild type protein" it is meant the wild type version of the designer BMP prior to the introduction of any mutations. For example, if the designer BMP is a designer BMP2, the corresponding wild-type BMP is wild-type BMP2. Thus, in one embodiment, design of a designer BMP can, but need not, begin with a wild type BMP sequence wherein mutations (e.g. , amino acid substitutions, deletions and/or insertion) are introduced into the wild type sequence. Therefore, the designer BMP can correspond with a wild type BMP, and the locations of the mutations can be said, for instance, to correspond with, be relative to and/or be respective with the amino acid sequence of the wild type corresponding or "reference" BMP sequence.

The proteins of the present invention include fragments, derivatives, analogs, or variants of the polypeptides described herein, and any combination thereof. The terms "fragment," "variant," "derivative" and "analog" when referring to proteins of the present invention include any proteins which retain at least some of the functional properties of the protein from which it was derived.

By the term "fragment" as used herein refers to a polypeptide and is defined as any discrete portion of a given polypeptide that is unique to or characteristic of that polypeptide. The term as used herein also refers to any discrete portion of a given polypeptide that retains at least a fraction of the activity of the full-length polypeptide. In certain embodiments, the fraction of activity retained is at least 10% of the activity of the full-length polypeptide. In certain embodiments, the fraction of activity retained is at least 20%, 30%, 40%, 50% , 60%, 70%, 80% or 90% of the activity of the full-length polypeptide. In certain embodiments, the fraction of activity retained is at least 95% , 96%, 97%, 98% or 99% of the activity of the full-length polypeptide. In certain embodiments, the fraction of activity retained is 100% or more of the activity of the full-length polypeptide. Alternatively or additionally, the term as used herein also refers to any portion of a given polypeptide that includes at least an established sequence element found in the full-length polypeptide. In some embodiments, the sequence element spans at least about 4-5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of the full-length polypeptide. Fragments of proteins of the present invention include proteolytic fragments, as well as deletion fragments.

Variants of the proteins of the present invention include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants may occur naturally or be non-naturally occurring. Non-naturally occurring variants may be produced using art-known mutagenesis techniques. Variant proteins may comprise conservative or non-conservative amino acid substitutions, deletions or additions.

The proteins of the invention include proteins having one or more residues chemically derivatized by reaction of a functional side group. Also included as proteins of the invention are polypeptides that contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.

"Recombinantly expressed polypeptide" and "recombinant polypeptide" as used herein refer to a polypeptide expressed from a host cell that has been manipulated to express that polypeptide. In certain embodiments, the host cell is a mammalian cell. In certain embodiments, this manipulation may comprise

one or more genetic modifications. For example, the host cells may be genetically modified by the introduction of one or more heterologous genes encoding the polypeptide to be expressed. The heterologous recombinantly expressed polypeptide can be identical or similar to polypeptides that are normally expressed in the host cell. The heterologous recombinantly expressed polypeptide can also be foreign to the host cell, e.g. heterologous to polypeptides normally expressed in the host cell. In certain embodiments, the heterologous recombinantly expressed polypeptide is chimeric. For example, portions of a polypeptide may contain amino acid sequences that are identical or similar to polypeptides normally expressed in the host cell, while other portions contain amino acid sequences that are foreign to the host cell. Additionally or alternatively, a polypeptide may contain amino acid sequences from two or more different polypeptides that are both normally expressed in the host cell. Furthermore, a polypeptide may contain amino acid sequences from two or more polypeptides that are both foreign to the host cell. In some embodiments, the host cell is genetically modified by the activation or upregulation of one or more endogenous genes.

Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g. , gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a typical embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, at least 40% , at least 50% or 60%, or at least 70%, 80%, 90%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology").

To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g. , gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions X 100). The determination of percent homology between two sequences can be accomplished using a mathematical algorithm. A preferred , non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin et al. , Proc Natl Acad Sci U S A 87:2264-8 (1990), modified as in Karlin et al. , Proc Natl Acad Sci U S A 90:5873-7 (1993). Such an algorithm is incorporated into the N BLAST and XBLAST programs of Altschul et al. , J Mol Biol 215:403-10 (1990). BLAST nucleotide searches can be performed with the NBLAST program, score= 100, wordlength= 12.

BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res 25:3389-402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g. , XBLAST and NBLAST) can be used.

The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the percent identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman et al. , J Mol Biol 48:443-53 (1970)) which has been incorporated into the GAP program in the GCG software package (available on at gcg .com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. In yet another embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available on the internet at gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6. One typical set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Myers and W. Miller (Myers et al. , Comput Appl Biosci 4: 1 1 -7 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

"Instructional material," as that term is used herein, includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compound, combination, and/or composition of the invention in the kit for affecting, alleviating or treating the various diseases or disorders recited herein. Optionally, or alternately, the instructional material can describe one or more methods of alleviating the diseases or disorders in a cell, a tissue, or a mammal, including as disclosed elsewhere herein.

The instructional material of the kit may, for example, be affixed to a container that contains the compound and/or composition of the invention or be shipped together with a container which contains the compound and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively.

Except when noted, the terms "patient" or "subject" are used interchangeably and refer to mammals such as human patients and non-human primates, as well as veterinary subjects such as rabbits, rats, and mice, and other animals. Preferably, patient refers to a human.

"Effective amount", or "therapeutically effective amount," as the terms are used interchangeably herein, is an amount that when administered to a tissue or a mammal, preferably a human, mediates a detectable therapeutic response compared to the response detected in the absence of the compound . A therapeutic response, such as, but not limited to, inhibition of and/or decreased fibrosis, increased bone mass or bone density, and the like, can be readily assessed by a plethora of art-recognized methods, including , e.g. , such methods as disclosed herein.

The skilled artisan would understand that the effective amount of the compound or composition administered herein varies and can be readily determined based on a number of factors such as the disease or condition being treated, the stage of the disease, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound being administered, and the like

As used herein, to "treat" means reducing the frequency with which symptoms of a disease (e.g. , decreased bone density, fracture, fibrosis, and the like) are experienced by a patient. The term includes the administration of the compounds or agents of the present invention to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease, alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.

By the phrase "specifically binds," as used herein, is meant a compound, e.g., a protein, a nucleic acid , an antibody, and the like, which recognizes and binds a specific molecule, but does not substantially recognize or bind other molecules in a sample. For instance, an BMP protein, an antibody or a peptide inhibitor which recognizes and binds a cognate receptor (e.g. , a BMP type I or type I I receptor, an antibody that binds with its cognate antigen, and the like) in a sample, but does not substantially recognize or bind other molecules in the sample. Thus, under designated assay conditions, the specified binding moiety (e.g., a BMP or a receptor binding fragment thereof) binds preferentially to a particular target molecule and does not bind in a significant amount to other components present in a test sample. A variety of assay formats may be used to select an antibody that specifically binds a molecule of interest. For example, solid-phase ELISA immunoassay, immunoprecipitation, BIAcore, FACS, Octet, and Western blot analysis are among many assays that may be used to identify a BMP that specifically reacts with a BMP receptor. Typically, a specific or selective reaction will be at least twice background signal or noise, more preferably, at least fivefold greater than background signal or noise, and more typically, more than 10 times background , even more specifically, a BMP is said to "specifically bind" a BMP receptor when the equilibrium dissociation constant (KD) is < 100 μΜ, more preferably < 10 μΜ, even more preferably < 1 μΜ, yet more preferably < 100 nM and most preferably < 10 nM.

The term "KD" refers to the equilibrium dissociation constant of a particular ligand-receptor interaction.

"Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a binding site of a molecule (e.g. , a BMP ligand) and its binding partner (e.g ., a BMP type I or type I I receptor). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g. , BMP and its cognate receptor). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd).

Affinity can be measured by common methods known in the art, including those described herein. Low-affinity BMPs generally bind a receptor slowly and tend to dissociate readily, whereas high-affinity BMPs generally bind a receptor faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative embodiments are described elsewhere herein.

The term "k0n", as used herein is intended to refer to the association or on rate constant, or specific reaction rate, of the forward , or complex-forming, reaction, measured in units: M sec-1.

The term "ko/r", as used herein, is intended to refer to the dissociation or off rate constant, or specific reaction rate, for dissociation of an antibody from the antibody/antigen complex, measured in units: sec-1.

The term "Kd", as used herein, is intended to refer to the dissociation constant of a particular antibody-antigen interaction. It is calculated by the formula:

The term "altered binding" as used herein means the designer BMP comprises a different binding specificity for at least a type I receptor and/or a type I I receptor when compared with the binding of a corresponding wild type BMP to the same type I and/or type I I receptor. The designer BMP may bind with greater or lesser affinity with the receptor compared to the binding of the wild type BMP to that receptor. For instance, if the wild type BMP bound a certain type I receptor with a certain binding affinity, the corresponding designer BMP binds that receptor with greater or lesser affinity compared with the wild type BMP. It may even be that the designer BMP will specifically bind a receptor that the wild type BMP did not detectably bind and vice-a-versa where the designer BMP will no longer detectably bind a receptor that the wild type BMP binds. Thus, altered binding encompasses any detectable change in binding by a designer BMP to a type I or type I I receptor compared with the binding of that receptor by the corresponding wild type BMP. It may be that the designer BMP has a greater or lesser kon value compared with the kon value for a corresponding wild type BMP and/or the designer BMP has a greater or lesser kon value compared with the k0ff value of the corresponding wild type BMP such that the Kd of the designer BMP is greater or lesser than the Kd of a corresponding wild type BMP for the same BMP receptor. Thus, any difference in a binding characteristic and/or affinity value between a designer BMP and a corresponding wild type BMP are encompassed by the term "altered binding" as used herein.

The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden, and Piscataway, N.J.). For further descriptions, see, e.g. , Johnsson, et al. , Ann. Biol.

Clin. 51 : 19-26 (1993); Johnsson, et al. , Biotechniques 1 1 : 620-627 (1991 ); Johnsson, et al. , J. Mol. Recognit. 8: 125-131 (1995); and Johnnson, et al. , Anal. Biochem. 198: 268-277 (1991 ).

As used herein, "substantially pure" means an object species is the predominant species present (i.e. , on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species (e.g. , a designer BMP) comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95% , and 99%. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.

Description

Bone Morphoqenetic Proteins (BMPs)

As stated above, BMPs are members of the TGF-β protein superfamily all of which are characterized by six-conserved cysteine residues (Lander et al, (2001 ) Nature, 409:860-921 . The BMP/GDF subfamily includes, but is not limited to, BMP2 , BMP3 (osteogenin) (see, e.g. , US Patent No. 6, 177,406), BMP3b (GDF-10) (see, e.g. , US Patent No. 6,204,047), BMP4 (BMP2b) (see, e.g. , US Patent No. 6,245,889), BMP5 (see, e.g. , US Patent No. 5,543,394), BMP6 (see, e.g ., US Patent No. 6,613,744), BMP7 (osteogenic protein-1 or OP1 ) (see, e.g ., US Patent No. 5, 141 ,905), BMP8 (OP2) (see, e.g. , US Patent No. 5,688,678), BMP8B (OP3) (see, e.g., US Patent No. 5,854,071 ), BMP9 (GDF2) (see, e.g., US Patent No. 6,287,816), BMP10 (see, e.g ., US Patent No. 5,703,043), BMP1 1 (GDF1 1 ) (see, e.g., US Patent No. 6,437, 1 1 1 ), BMP12 (GDF7) (see, e.g. , US Patent No. 6,027,919), BMP13 (GDF6, CDMP2) (see, e.g., US Patent No. 6,027,919), BMP15 (GDF9) (see, e.g., US Patent No. 6,034,229), BMP16 (see, e.g., US Patent No. 6,331 ,612), GDF1 (see, e.g., US Application No. 2004/0039162), GDF3 (see, e.g ., US Patent No. 6,025,475), GDF5 (CDMP1 ; MP52) (see, e.g ., US Patent No. 5,994,094), and GDF8 (myostatin) (see, e.g. , US Patent No. 5,827,733).

BMPs specifically bind their cognate receptors, which include Type I receptors: ALK-I , ALK-2 (also called ActRIa or ActRI), ALK-3 (also called BMPRIa), and ALK-6 (also called BMPRIb); and Type II receptors: ActRl la (also called ActRII), ActRl lb, and BMPRII . The BMP-receptor binding interactions have been studied extensively, and the binding specificities of each wild type BMP for each type I and/or type II receptor is generally known in the art and are shown in Table 1 . See, e.g. , Nickel et al. , Cytokine Growth Factor Rev 20:367-77 (2009); Heinecke et al. , BMC Biol 7:59 (2009).

TABLE 1

Designer Bone Morphoqenetic Proteins with Improved Osteogenic Activity

The present invention is based in part on the understanding that each BMP dimer binds to four BMP receptors: two type I receptors and two type I I receptors. The specificities of each BMP for each receptor are known in the art as shown above in Table 1 . Also, the receptor binding regions of various BMPs that mediate binding of the BMP for each receptor have been mapped and are shown in Table 2. For instance, it is well established that wild type BMP2 and BMP4 bind type I BMP receptors Alk-3 and ALK-6 with high affinity and bind type II BMP receptors with lower affinity. On the other hand, wild type BMP6 and BMP7 are known to have bind type I I receptors Actrl lA, Actrl lB, and BMPRI I with high affinity but bind type I receptors with lower affinity than they do to type II . It is believed that the differing cellular responses from the approximately forty-three TGFfi superfamily members signaling through interaction with approximately twelve receptors is believed to be due to each ligand utilizing a specific repertoire of receptors with which it binds with differing affinities. The type I and I I binding domains are described in Table 2.

TABLE 2

BMP9 (SEQ ID NO:7) 25-39 42-71 78-96

Rational amino acid substitution to alter receptor binding of designer BMPs

It is well known in the art that wild type BMP2 shows a relatively high affinity for type I receptors, while wild type BMP6 shows a high affinity for type II receptors. It is further known in the art that heterodimers of wild type BMP2 and BMP6 bind to both type I and type II receptors with relatively high affinity each BMP apparently providing the higher affinity binding site for each receptor. See Table 3, below. The BMP2/6 heterodimers are known to be more active that BMP2 or BMP6 alone or as homodimers, in both in vitro and in vivo bone formation assays. Table 3 shows an example of BMP2 and BMP6 binding affinities to type I and II receptors.

TABLE 3

Accordingly, it is an object of the invention to provide designer BMPs with improved binding to type I and/or type II receptors, including (without limitation) binding that mimics, and preferably improves upon, the binding of BMP heterodimers. As shown in Figure 1 and Table 2, each BMP comprises three binding sites that contribute to receptor binding. From N- to C-terminus, each BMP comprises a type II receptor binding site A, a type I receptor binding site, and a second type II receptor binding site B. Although an exemplary alignment of wild type BMP2, BMP4, BMP5, BMP6, BMP7, BMP8, and BMP9 is illustrated in Figure 1 , the skilled artisan will appreciate that there are well-known alignments providing the relative positioning of various amino acids among the members of the ΤΘΒβ superfamily. Such alignments are provided, among others, in International Publication Nos. WO 2009/086131 (e.g., Figures 15-17, Figure 31A), WO 2008/051526 (Figures 9-12), WO 2005/1 18636 (Figure 6), WO 2005/1 18635, WO 2005/1 13585 (Figure 3), WO 2001/92298 (Figure 6A-6C), Kirsch et al., EMBO J. 19 :3314-3324 (2000) (Figure 1 ), US Patent Application Publication No., 2007/0293425 (Figure 6), Groppe et al., Nature 420 :636-642 (2002), Nickel et al., J. Bone Joint Surg. Am. 83:7-14 (2001 ), and Weber et al., BMC Structural Biol. 7 :6 (2007). Thus, using protein seguence alignment algorithms and tools well-known in the art, including the alignments of the amino acid seguences of the various TGFfi superfamily members, as well as the disclosure provided herein, the corresponding amino acid in one BMP/GDF protein relative to the amino

acid at any position in another BMP/GDF protein can be determined. In one embodiment, the corresponding amino acid residues in BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8 and BMP-9 are shown (see, e.g. , Figure 1 ).

Designer BMPs according to the present invention generally include mutations in the type I binding domain and/or the type II binding domain, wherein the mutations confer altered binding to a type I or type I I BMP receptor. In some embodiments, the designer BMP comprises one or more mutations in both a type I binding domain and a first (binding domain A) and/or second (binding domain B) type II binding domain. In other embodiments, the designer BMP comprises one or more mutations in both type II binding domains. In other embodiments, the designer BMP comprises one or more mutations in the first type II binding domain, in the second type II binding domain, and in the type I binding domain. In some embodiments, the designer BMP comprises one or more mutations in the type I binding domain.

The mutations described above are, in preferred embodiments, generated by one or more "swaps" between corresponding domains of different TGFfi superfamily members, creating chimeric designer BMPs. For instance, in Berasi ef a/. , swaps between BMP-2 and BMP-6 are used to create various designer BMPs. In the present disclosure, the teachings of Berasi ef a/, are expanded to include swaps between BMP-2, 4, 6 and 7, though the skilled artisan will appreciate that other swaps are possible among these and other TGFfi superfamily members.

As non-limiting examples, BMP-GE27 (SEQ ID NO:8) includes swaps of the Type I and Type I I A and B domains of BMP-7 into BMP-2, resulting in a BMP-2/7 chimera. Similarly, BMP-GE46 (SEQ ID NO: 10) and BMP-GE47 (SEQ ID NO: 14)include Type I and Type II A and B domains of BMPs 6 and 7 (respectively) into BMP-4.

In addition to the mutation strategies above, the inventors discovered (and disclosed Berasi ef a/.) that a separate swap of the C-terminal region of activin-A into a designer BMPs results in a designer BMP with extremely high affinity for the ActRIIB receptor, such that the resulting molecules are functionally indifferent to Noggin, binding BMP receptors even in the presence of high concentrations of Noggin. Accordingly, the invention includes, in certain embodiments, dBMPs which incorporate one or more C-terminal mutations point mutations and/or "swaps" such as BMP-GE27-NR (SEQ ID NO. 9), BMP-GE46-NR (SEQ ID NO: 1 1 ), BMP-GER46-NR (SEQ ID NO: 13) or BMP-GE47-NR (SEQ ID NO: 15).

In some embodiments, the mutations improve binding to a type I receptor. In other embodiments, the mutations improve binding to a type II receptor. In other embodiments, the mutations decrease binding to a type I or type I I receptors. In some embodiments, the mutations create or destroy a glycan tether as more fully set forth below. In some embodiments, the mutations create or destroy a His doorstop as more fully set forth below.

Because BMPs are so well characterized and understood in the art, it would be understood , once provided with the disclosure provided herein, the location of possible mutations that can be made that do not further affect the activity of the designer BMPs would be understood. Accordingly, the designer BMPs of the invention encompass variant BMPs which differ from a corresponding wild type or designer BMP in that it contains additional insertions, deletions, or substitutions which do not affect the receptor binding affinity of the variant BMPs. In some non-limiting embodiments, those of skill in the art would understand that the cysteines involved in cysteine knot formation and amino acids involved in receptor interactions should not be mutated or should be changed with conservative substitutions, while other amino acids may be more freely substituted, inserted, or deleted without adversely affecting biological activity of the designer BMP.

It should be noted that unless otherwise stated, all positional numbering of designed or modified BMPs is based on the sequences of the mature native BMPs. Designer BMPs are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the BMP sequence. Variants of designer BMPs must retain at least 50% of the activity of the corresponding wild type or designer BMP activity in one or more cell types, as determined using an appropriate assay described below. Variants that retain at least 75%, 80%, 85%, 90% or 95% of wild type activity are more preferred, and variants that are more active than wild type are especially preferred. A designer BMP may contain insertions, deletions, and/or substitutions at the N- terminus, C-terminus, or internally. In a preferred embodiment, designed or modified BMPs have at least 1 residue that differs from the most similar human BMP sequence, with at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different residues being more preferred.

Designer BMPs of the invention maintain at least 80%, at least 81 %, at least 82%, at least 83%, at least 84% , at least 85%, at least 86%, at least 87%, at least 88%, at least 89% , at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , at least 97%, at least 98%, or at least 99% identity with the corresponding wild-type BMP protein sequence.

Designer BMPs of the invention may maintain at least 80%, at least 81 % , at least 82% , at least 83%, at least 84% , at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 % , at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , at least 97%, at least 98%, or at least 99% identity with the conserved cysteine domain of the C-terminal region of the corresponding wild-type BMP protein sequence.

Designer BMPs may contain further modifications, for instance mutations that alter additional protein properties such as stability or immunogenicity or which enable or prevent posttranslational modifications such as PEGylation or glycosylation. Designer BMPs may be subjected to co- or post-translational modifications, including but not limited to synthetic derivatization of one or more side chains or termini, glycosylation, PEGylation, circular permutation, cyclization, fusion to proteins or protein domains, and addition of peptide tags or labels.

Due to the degeneracy of the genetic code, an extremely large number of nucleic acids may be made, all of which encode the designer BMPs of the present invention, by simply modifying the sequence of one or more codons in a way that does not change the amino acid sequence of the designer BMP. The designer BMPs of the invention do not comprise these sequences set forth in WO2008/051526 or WO2009/086131 .

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

In particular embodiments, the designer BMP of the invention comprises a backbone BMP, i.e. , the wild type BMP, to which the designer BMP corresponds. In particular embodiments, this backbone BMP may be a wild type BMP2, BMP4, BMP5, BMP6, BMP7, BMP8, or BMP9 backbone.

In some embodiments of the invention, the designer BMP comprises at least one mutation in a type I binding domain and/or a type II binding domain, wherein the mutation confers altered binding to a type I or type I I BMP receptor compared with the binding of a corresponding wild type BMP not comprising the mutation. In some embodiments, the designer BMP comprises at least one mutation in both a type I binding domain and at least one mutation in a type II binding domain. In other embodiments, the designer BMP comprises at least one mutation within the type I I binding domain A and the type I I binding domain B. In other embodiments, the designer BMP comprises at least one mutation in type II binding domain A, type II binding domain B, and a type I binding domain.

In certain embodiments, the mutation may comprise an amino or nucleic acid substitution, deletion and/or insertion. In a preferred embodiment, the mutation comprises an amino acid substitution.

In some embodiments, the backbone BMP is a wild type BMP and the mutations are one or more of the mutations listed in Tables 4 to 6. The designer BMP may contain any combination and any number of mutations listed in these tables.

In some embodiments, the backbone BMP is a wild type BMP and the mutations are one or more of the mutations listed in Tables 4 to 6. The designer BMP may contain a permutation and any and all of the mutations listed in these tables or disclosed elsewhere herein.

ims:

1. A designer BMP protein comprising the amino acid sequence of any one of SEQ ID NOs: 8, 9, 10, 1 1 , 12, 13, 14 or 15.

2. The designer BMP protein of claim 1 , further comprising at least one mutation, the mutation not being within a Type I, Type MA, or Type MB binding domain of the designer BMP protein.

3. The designer BMP protein of claim 2, wherein the at least one mutation is not more than 10

mutations.

4. The designer BMP protein of claim 1 , having a binding profile which differs from a binding profile of a corresponding wild-type BMP.

5. The designer BMP protein of claim 4, wherein the binding profile of the designer BMP protein includes at least one of the following: binding to the ALK2 receptor with a KD not greater than about 2 nM; binding to the ALK3 receptor with a KD not greater than about 2 nM; binding to the ALK6 receptor with a KD not greater than about 1 nM; binding to the ActRIIA receptor with a KD not greater than about 2 nM; binding to the ActRIIB receptor with a KD not greater than about 0.5 nM; and binding to the BMPRIIA receptor with a KD not greater than about 3.5 nM.

6. A nucleic acid encoding the designer BMP protein of claim 1 .

7. A method of producing a designer BMP protein comprising the steps of introducing an expression vector comprising the nucleic acid of claim 6 into a cell.

8. A method of treating a patient, comprising the step of contacting the patient with a designer BMP protein according to claim 1.

9. The method of claim 8, wherein treating a patient comprises treating a bone disease associated with bone loss, and wherein the step of contacting a patient with a designer BMP includes administering to the patient a dose of the designer BMP effective to treat the bone disease.

10. The method of claim 8, wherein treating a patient comprises treating fibrosis, and wherein the step of contacting a patient with a designer BMP includes administering to the patient a dose of the designer BMP effective to treat fibrosis.

1 1. A designer BMP protein comprising the following mutations relative to SEQ ID NO:1 : V33I, P36E, H39A, H44E, P48A, A52N, D53S, H54Y, L55M, S57A, N68H, S69F, V70I, S72P, K73E, insertion of a T following K73, I74V, A77P, V80A, S85N, M89V, L92F, E94D, N95S, E96S, K97N, V99I.

12. The designer BMP protein of claim 1 1 , further comprising at least one mutation, the mutation not being within a Type I, Type MA, or Type MB binding domain of the designer BMP protein.

13. The designer BMP protein of claim 1 1 , comprising the amino acid sequence of SEQ ID NO: 8.

14. The designer BMP protein of claim 1 1 , having a binding profile which differs from a binding profile of a corresponding wild-type BMP.

15. The designer BMP protein of claim 14, wherein the binding profile of the designer BMP protein includes at least one of the following: binding to the ALK2 receptor with a KD not greater than

about 2 nM; binding to the ALK3 receptor with a KD not greater than about 2 nM; binding to the ALK6 receptor with a KD not greater than about 1 nM; binding to the ActRMA receptor with a KD not greater than about 2 nM; binding to the ActRIIB receptor with a KD not greater than about 0.5 nM; and binding to the BMPRIIA receptor with a KD not greater than about 3.5 nM.