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  Bmp-2 variants with improved propertiesUSPTO Application #: 20060235204Title: Bmp-2 variants with improved properties Abstract: The invention relates to variants of BMP-2 with improved properties and methods for their use. (end of abstract) Agent: Dorsey & Whitney LLP - San Francisco, CA, US Inventors: John R. Desjarlais, Shannon Alicia Marshall, Jonathan Zalevsky USPTO Applicaton #: 20060235204 - Class: 530350000 (USPTO) Related Patent Categories: Chemistry: Natural Resins Or Derivatives; Peptides Or Proteins; Lignins Or Reaction Products Thereof, Proteins, I.e., More Than 100 Amino Acid Residues The Patent Description & Claims data below is from USPTO Patent Application 20060235204. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application claims benefit under 35 U.S.C. .sctn.119(e) to U.S. Ser. No. 60/558,189 filed on Mar. 31, 2004, entitled "Cysteine Knot Cytokine Variants with Improved Properties"; U.S. Ser. No. 60/570,520 filed on May 11, 2004 entitled "Cysteine Knot Cytokine Variants with Improved Properties"; U.S. Ser. No. 60/578,432 filed on Jun. 9, 2004 entitled "Cysteine Knot Cytokine Variants with Improved Properties"; and U.S. Ser. No. 60/587,464, filed on Jul. 13, 2004 entitled "Cysteine Knot Cytokine Variants with Improved Properties", all of which are expressly incorporated by reference in their entirety. FIELD OF THE INVENTION [0002] The invention relates to variants of bone morphogenetic proteins and other cysteine knot growth factors with improved properties, and to methods of making and using compositions utilizing these variants. BACKGROUND OF THE INVENTION [0003] Bone morphogenetic proteins (BMPs) are a well-known family of growth factors that contribute to developmental processes such as pattern formation and tissue specification as well as promoting wound healing and repair processes in adult tissues. BMPs were initially isolated by their ability to induce bone and cartilage formation and are now known to regulate cell proliferation, migration, differentiation, and apoptosis in a number of tissues and organs. [0004] BMPs include a number of related human proteins, such as BMP-2, BMP-3 (osteogenin), BMP-3b (GDF-10), BMP-4 (BMP-2b), BMP-5, BMP-6, BMP-7 (osteogenic protein-1 or OP-1), BMP-8 (OP-2), BMP-8B (OP-3), BMP-9 (GDF-2), BMP-10, BMP-11 (GDF-11), BMP-12 (GDF-7), BMP-13 (GDF-6, CDMP-2), BMP-15 (GDF-9), BMP-16, GDF-1, GDF-3, GDF-5 (CDMP-1), and GDF-8 (myostatin). BMPs may be grouped into subfamilies. For example, BMP-2 and BMP-4 are closely related, as are BMP-5, BMP-6, BMP-7, BMP-8, and BMP-8B. BMP-13, BMP-14, and BMP-12 also constitute a subfamily. BMPs are also present in other animal species. Furthermore, there is some allelic variation in BMP sequences among different members of the human population. [0005] BMPs are a subset of the transforming growth factor-.beta. (TGF-.beta.) family, which also includes TGFs (TGF-.beta.1, TGF-.beta.2, and TGF-.beta.3), activins (activin A) and inhibins, macrophage inhibitory cytokine-1 (MIC-1), Mullerian inhibiting substance, anti-Mullerian hormone, and glial cell line derived neurotrophic factor (GDNF). The TGF-.beta. family is in turn a subset of the cysteine knot cytokine superfamily. Additional members of the cysteine knot cytokine superfamily include, but are not limited to, platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), placenta growth factor (PIGF), noggin, neurotrophins (BDNF, NT3, NT4, and .beta.NGF), gonadotropin, follitropin, lutropin, interleukin-17, and coagulogen. [0006] BMPs have demonstrated utility in the treatment of a variety of conditions and diseases. BMP-2 and BMP-7 have been used to promote bone formation, bone fracture healing, and spinal fusion. BMP-4 (Rundle et. al. (2003) Bone 32: 591-601), BMP-5 (Arosarena and Collins (2003) Arch. Otolaryngol. Head Neck Surg. 129: 1125-1130), BMP-6 (Helm (2003) Gene Ther. 10: 1735-1743), and BMP-9 (Li et. al. (2003) J. Gene Med. 5: 748-756), have also been demonstrated to promote bone healing in animal models. Animal studies indicate that BMP-7 may be used to treat renal fibrosis and renal failure (Wang et. al. (2001) J. Am. Soc. Nephrol. 12: 2392-2399; Wang and Hirshberg (2003) Am. J. Physiol. Renal Physiol. 284: 1006-1013; Zeisberg et. al. (2003) Nat. Med. 9: 964-968; and Zeisberg et. al. (2003) Am. J. Physiol. Renal Physiol. 285: F1060-F1067), ischemic stroke (Chang et. al. (2003) Stroke 34: 558-564 and Harvey et. al. Pharmacol. Ther. (2005) 105: 113-125) and inflammatory bowel diseases (Maric et. al. (2003) J. Cell Physiol. 196: 258-264). [0007] Structurally, BMPs are dimeric cysteine knot proteins. Each BMP monomer comprises multiple intramolecular disulfide bonds. An additional intermolecular disulfide bond mediates dimerization in most BMPs. BMPs may form homodimers; furthermore some BMPs may form heterodimers. BMPs are expressed as pro-proteins comprising a long pro-domain, one or more cleavage sites, and a mature domain. 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 an inhibitor (eg. Thies et. al. (2001) Growth Factors 18: 251-259). [0008] BMP signal transduction is initiated when a BMP dimer binds two type I and two type 11 serine/threonine kinase receptors. Type I receptors include but are not limited to ALK-1, ALK-2 (also called ActRIa or ActRI), ALK-3 (also called BMPRIa), and ALK-6 (also called BMPRIb) and type II receptors include but are not limited to ActRIIa (also called ActRII), ActRIIb, and BMPRII. Following BMP binding, the type II receptors phosphorylate the type I receptors, the type I receptors phosphorylate members of the Smad family of transcription factors, and the Smads translocate to the nucleus and activate the expression of a number of genes. [0009] BMPs also interact with inhibitors, soluble receptors, and decoy receptors, including BAMBI (BMP and activin membrane bound inhibitor), BMPER (BMP-binding endothelial cell precursor-derived regulator), Cerberus, cordin, cordin-like, Dan, Dante, follistatin, follistatin-related protein (FSRP), ectodin, gremlin, noggin, protein related to Dan and cerberus (PRDC), sclerostin, sclerostin-like, and uterine sensitization-associated gene-1 (USAG-1). Furthermore, BMPs may interact with co-receptors, for example BMP-2 and BMP-4 bind the co-receptor DRAGON (Samad et. al. (2005) J. Biol. Chem.), and extracellular matrix components such as heparin sulfate and heparin (Irie et. al. (2003) Biochem. Biophys. Res. Commun. 308: 858-865) [0010] For further background on the BMP family, see Balemans and Hul (2002) Dev. Biol. 250: 231-250; Bubnoff and Cho (2001) Dev. Biol. 239: 1-14; Celeste et. al. (1990) Proc. Nat. Acad. Sci. USA 87: 9843-9847; and Cheng et. al. (2003) J. Bone Joint Surgery 85A: 1544-1552 [0011] A number of unfavorable properties of naturally occurring BMPs limit the development and use of BMP therapeutics. BMP expression yields are typically poor and suitable expression hosts are limited, hindering development and production. BMPs often possess multiple biological effects, including unwanted side effects. Many BMPs are poorly soluble, reducing storage stability and bioavailability. Finally, BMPs may induce unwanted immune responses. [0012] Earlier studies have identified BMP variants with a number of interesting properties. BMP variants with improved yield in the context of E. coli expression and subsequent refolding from inclusion bodies have been disclosed (U.S. Pat. No. 5,399,677; U.S. Pat. No. 5,804,416; and U.S. Pat. No. 6,677,432). Consensus BMP variants with BMP-like activity have also been described (U.S. Pat. No. 5,011,691; U.S. Pat. No. 6,395,883; U.S. Pat. No. 6,531,445). A BMP-2 point mutant, L51P, has been described that does not bind type I receptors but binds type II receptors normally (Keller et. al. (2004) Nat. Struct. Mol. Biol. 11: 481-488). Deletion mutants of BMP-4 that act as competitive inhibitors of BMP signaling have been disclosed (Weber et. al. (2003) J. Bone Miner. Res. 18: 2142-2151). Mutagenesis experiments have also been performed on BMP-2 to identify residues important for receptor binding; some of these variants were found to act as antagonists (Kirsch et. al. (2000) EMBO J. 19: 3314-3324 and Nickel et. al. (2001) J. Bone Joint Surg. Am. 83-A: S7-S14). In addition, methods for identifying analogs of morphogenetic proteins have been claimed (U.S. Pat. No. 6,273,598). Furthermore, several point mutants of ActA with reduced ALK-4 binding have been identified: S60P, 163P, M91 E, 1105E, and M108A (Harrison et. al. (2004) J. Biol. Chem. 279: 28036-28044). SUMMARY OF THE INVENTION [0013] The present invention is related to variants of human bone morphogenetic proteins and other cysteine knot cytokine proteins with improved properties, including increased expression yield, expression in the absence of a pro-domain, increased solubility, increased specific activity, altered receptor, co-receptor, and inhibitor specificity, and decreased immunogenicity. [0014] In one aspect, the invention provides variant BMP-7 protein comprising the sequence: Fx(1-20)-Vb(21)-Fx(22-38)-Vb(39)-Fx(40-64)-Vb(65)-Fx(66-71)-Vb(72)-Fx(73-- 77)-Vb(78)-Fx(79-92)-Vb(93)-Fx(94-119)-Vb(120)-Fx(121-134)-Vb(135) [0015] wherein [0016] Fx1(1-20) corresponds to amino acid residues 1-20 of human BMP-7 (SEQ ID NO:5); [0017] Vb(21) is selected from the group consisting of L and G; [0018] Fx(22-38) corresponds to amino acid residues 22-38 of human BMP-7 (SEQ ID NO:5); [0019] Vb(39) is selected from the group consisting of K, A and S; [0020] Fx(40-64) corresponds to amino acid residues 40-64 of human BMP-7 (SEQ ID NO:5); [0021] Vb(65) is selected from the group consisting of Y and N; [0022] Fx(66-71) corresponds to amino acid residues 66-71 of human BMP-7 (SEQ ID NO:5); [0023] Vb(72) is selected from the group consisting of A and D; [0024] Fx(73-77) corresponds to amino acid residues 73-77 of human BMP-7 (SEQ ID NO:5); [0025] Vb(78) is selected from the group consisting of Y and H; [0026] Fx(79-92) corresponds to amino acid residues 79-92 of human BMP-7 (SEQ ID NO:5); [0027] Vb(93) is selected from the group consisting of F, H, S and T; [0028] Fx(94-119) corresponds to amino acid residues 94-119 of human BMP-7 (SEQ ID NO:5); [0029] Vb(120) is selected from the group consisting of S and D; [0030] Fx(121-134) corresponds to amino acid residues 121-134 of human BMP-7 (SEQ ID NO:5); [0031] Vb(135) is selected from the group consisting of A and E; [0032] wherein the variant comprises an amino acid substitution as compared to human BMP-7 (SEQ ID NO:5). [0033] In a further aspect, the invention provides variant BMP-7 proteins comprising a substitution as compared to human BMP-7 (SEQ ID NO:5) selected from the group consisting of: L21G, K39A, K39S, Y65N, A72D, Y78H, F93H, F93S, F93T, SI 20D and A135E. In both cases, particular variants with single substitutions include, L21G, K39A, K39S, Y65N, A72D, Y78H, F93H, F93S, F93T, S120D and A135E. Particular sets of substitutions include, K39S-F93S; K39S-S120D; K39S-S120D-Y65N; K39S-S120D-A72D; K39S-S120D-Y78H; K39S-S120D-F93H; K39S-S120D-F93S; Y65N-L21G; Y65N-L21R; Y65N-K39S; Y65N-Y78H; Y65N-S120D; Y78H-A72D; Y78H-F93H-Y65N; Y78H-F93H-A72D; Y78H-F93H-S120D; Y78H-S120D and F93H-K39S. [0034] In an additional aspect, the invention provides variant BMP-7 proteins having altered receptor binding affinity compared to wild-type BMP-7 (SEQ ID NO:5), the variant BMP-7 protein comprising one or more substitutions selected from the group consisting of: M23N, Q53G, Q53H and I86D. BRIEF DESCRIPTION OF THE DRAWINGS [0035] FIG. 1 shows sequence alignments of the mature domains of human BMP and GDF proteins (Residues 14-114 of SEQ ID NO:1, residues 16-116 of SEQ ID NO:2, residues 37-138 of SEQ ID NO:3, residues 38-139 of SEQ ID NO:4, residues 38-139 of SEQ ID NO:5, residues 38-139 of SEQ ID NO:6 and SEQ ID NOS:71-83). The consensus BMP sequence and the MIC-1 sequence are shown for reference. The N-terminal most residues of the mature domain, which are significantly less well aligned, are not shown. [0036] FIG. 2 shows the hexameric structure comprising BMP-7 dimer bound to two type I receptors and two type II receptors. The structure was generated by superimposing BMP-2 (white) bound to ALK-3 (gray, upper right and lower left) and BMP-7 (black) bound to ActRIIa (gray, upper left and lower right). [0037] FIG. 3 shows alignments of human type I BMP receptors and human type II BMP receptors used for homology modeling (SEQ ID NOS:7-12). Continue reading... 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