The present invention is in the field of medicine, particularly in the field of therapeutic proteins. Specifically, the present invention relates to variants of human bone morphogenic protein-7 (BMP7) useful for the treatment of cancer, cartilage damage and degeneration, pain associated with osteoarthritis or bone healing.

Bone morphogenic proteins are a well-known family of growth factors that regulate cell proliferation, migration, differentiation and apoptosis in a number of tissues and organs. Human BMP7 is a secreted signaling molecule of the TGF-beta superfamily and was originally identified for its ability to induce bone formation. It is now recognized as a multifunctional cytokine which mediates growth and differentiation of many different cell types.

BMP7 has been used to promote bone formation, bone fracture healing and spinal fusion. Nevertheless, its use as a therapeutic has been limited primarily to topical administration to the surface of the tissue because of its poor solubility/bioavailability and the propensity to cause ectopic bone formation (EBF) or a rapid formation of new bone in soft tissue as a result of precipitation at the injection site.

BMP7 protein variants with improved properties such as increased expression yield, increased solubility, increased specific activity and decreased immunogenicity have been reported in WO2005/097825. However, there is still a need for human BMP7 protein variants with improved solubility/bioavailability, increased specific activity, decreased binding to endogenous circulating inhibitors, and reduced EBF activity that could be useful as a therapeutic.

Cancer stem-like cells (CSC) in solid tumors are purported to contribute to cancer development and poor treatment outcome. The abilities to self-renew, differentiate, and resist anticancer therapies are hallmarks of these rare cells, and steering them into lineage commitment may be one strategy to curb cancer development or progression. However, despite the acknowledged potential of this approach, there still remains a need for effective cancer therapies which induce tumor cell transdifferentiation and revert CSCs into lineages that are not resistant or less resistant to killing by conventional chemo- and radiotherapy (see, for example, Li, R., et al., Oncotarget.2016 Oct 18; 7(42): 68360–68370).

The present invention provides alternative human BMP7 protein variants. Particularly, the present invention provides variants of human BMP7 protein with increased specific activity, improved solubility/bioavailability characteristics, decreased binding to endogenous circulating inhibitors, and/or reduced EBF activity as compared to the corresponding wild type human BMP7 protein. In addition, a method of treating cancer, cartilage damage and degeneration, pain associated with osteoarthritis or bone fracture healing comprising the administration of a human BMP7 protein variant of the present invention is described.

The present invention provides a variant of human BMP7 protein wherein the mature domain of the human BMP7 protein comprises a polypeptide comprising the amino acid sequence of:

STGSKQRSQNRSKTPKNQEALRMANVAENSSSXaa33QRQXaa37CKKHELYVSFRD LGWQDWIIAPXaa60GYAAXaa65YCEGECAFPLNSYMNATNHAXaa86Xaa87QXaa89L Xaa91HXaa93Xaa94NPETVPKPCCAPTQLXaa110AISXaa114LYFDDXaa120SNVILKKXaa128RN MXaa132VXaa134ACGCH (SEQ ID NO: 3), wherein

Xaa33 is D or M; Xaa37 is A or P; Xaa60 is E or Q; Xaa65 is Y, S, or G; Xaa86 is I, V, or L; Xaa87 is V or L; Xaa89 is T, S, or A; Xaa91 is V or M; Xaa93 is F or V; Xaa94 is I, F or M; Xaa110 is G; Xaa114 is V or M; Xaa120 is S or Q; Xaa128 is Y, F or W; Xaa132 is V, Q, or S; and, Xaa134 is R or K.

The invention also provides a variant of human BMP7 wherein the mature domain of the human BMP7 protein comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 wherein: (a) Xaa33 is D; (b) Xaa37 is A; (c) Xaa60 is E; (d) Xaa65 is Y, S, or G; (e) Xaa86 is I, V, or L; (f) Xaa87 is V; (g) Xaa89 is T or A; (h) Xaa91 is V; (i) Xaa93 is F or V; (j) Xaa94 is I; (k) Xaa110 is G; (l) Xaa114 is V or M; (m) Xaa120 is S; (n) Xaa128 is Y, F, or W; (o) Xaa132 is V; and (p) Xaa134 is R.

The invention also provides a variant of human BMP7 protein wherein the mature domain of the BMP7 protein comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 wherein: (a) Xaa33 is D; (b) Xaa37 is A; (c) Xaa60 is E; (d) Xaa65 is Y or G; (e) Xaa86 is I or L; (f) Xaa87 is V; (g) Xaa89 is T or A; (h) Xaa91 is V; (i) Xaa93 is F or V; (j) Xaa94 is I; (k) Xaa110 is G; (l) Xaa114 is V or M; (m) Xaa120 is S; (n) Xaa128 is Y, F, or W; (o) Xaa132 is V; and (p) Xaa134 is R.

The invention also provides a variant of human BMP7 protein wherein the mature domain of the BMP7 comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 3

wherein: (a) Xaa33 is D; (b) Xaa37 is A; (c) Xaa60 is E; (d) Xaa65 is G; (e) Xaa86 is L; (f) Xaa87 is V; (g) Xaa89 is T or A; (h) Xaa91 is V; (i) Xaa93 is V; (j) Xaa94 is I; (k) Xaa110 is G; (l) Xaa114 is V or M; (m) Xaa120 is S; (n) Xaa128 is F or W; (o) Xaa132 is V; and (p) Xaa134 is R.

Preferably, Xaa114 is V and Xaa128 is W.

The invention further provides a variant of human BMP7 protein wherein the mature domain of the BMP7 protein comprises a F93V/N110G variant as shown in SEQ ID NO:4; a Y65G/I86L/T89A/N110G variant as shown in SEQ ID NO: 5; a Y65G/I86L/N110G/Y128F variant as shown in SEQ ID NO: 6; a Y65G/I86L/N110G/Y128W variant as shown in SEQ ID NO: 7; a Y65G/I86L/F93V/N110G/Y128W variant as shown in SEQ ID NO: 8; a

Y65G/T89A/N110G/Y128F variant as shown in SEQ ID NO: 9; a Y65G/I86L/N110G variant as shown in SEQ ID NO: 10; or a Y65G/V114M variant as shown in SEQ ID NO: 11. More preferably, variants of human BMP7 protein of the present invention comprise a

Y65G/I86L/N110G/Y128W variant of the human mature BMP7 protein as shown in SEQ ID NO: 7 or a Y65G/I86L/F93V/N110G/Y128W variant of the human mature BMP7 protein as shown in SEQ ID NO: 8 (hereinafter, referred to as human mature BMP7 protein variant F9 or human mature BMP7 protein variant F9). Even more preferably, the human mature BMP7 protein variant of the present invention comprises human mature BMP7 protein variant F9, which is a Y65G/I86L/F93V/N110G/Y128W variant of the human mature BMP7 (see, SEQ ID NO: 8).

The invention also provides a variant of the human BMP7 protein wherein the mature domain of the BMP7 protein variant comprises the amino acid sequence of SEQ ID NO: 3 wherein:

Xaa33 is D or M; Xaa37 is A or P; Xaa60 is E or Q; Xaa65 is Y, S, or G; Xaa86 is I, V, or L; Xaa87 is V or L; Xaa89 is T, S, or A; Xaa91 is V or M; Xaa93 is F or V; Xaa94 is I, F or M; Xaa110 is G; Xaa114 is V or M; Xaa120 is S or Q; Xaa128 is Y, F or W; Xaa132 is V, Q, or S; and, Xaa134 is R or K, and wherein the N-terminus of the variant protein is covalently fused to the C-terminus of the human BMP7 pro-domain sequence of SEQ ID NO: 18.

The invention further provides a variant of human pro-BMP7 protein wherein said protein comprises a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; and SEQ ID NO: 16.

The invention further provides a variant of human mature BMP7 protein wherein said

variant comprises a polypeptide having the amino acid sequence as shown in SEQ ID NO: 17. The invention further provides human BMP7 protein variants that have increased specific activity, improved solubility characteristics, improved bioavailability, decreased binding to endogenous circulating inhibitors, and/or reduced EBF activity as compared to the corresponding wild type human BMP7 protein.

Another aspect of the present invention is a pharmaceutical composition comprising a human BMP7 protein variant of the present invention together with a pharmaceutically acceptable carrier, diluents, or excipient and optionally one or more other therapeutic ingredients.

Another aspect of the invention provides methods of treating cancer comprising administering to a patient in need thereof in simultaneous, separate, or sequential combination of an effective amount of a human BMP7 protein variant of the present invention with an effective amount of one or more chemotherapeutic agents or ionizing radiation. Preferably, the methods of treating cancer are methods for treating lung cancer, including, but not limited to, non-small cell lung cancer (NSCLC), brain cancer, cervical cancer, skin cancer, head and neck cancer, glioblastoma, neuroblastoma, or colorectal cancer. Preferably, the human BMP7 protein variant of the present invention is administered to the patient prior to the administration of one or more chemotherapeutic agent(s) and/or ionizing radiation.

An aspect of the invention is the use of a human BMP7 protein variant of the present invention in combination with all-trans retinoic acid (ATRA) for the treatment of cancers in which the retinoid receptors RAR alpha and RAR gamma are known to be important including, but not limited to, lung cancer, brain cancer, cervical cancer, skin cancer, head and neck cancer, glioblastoma, neuroblastoma, oral leukoplakia, oral squamous cell carcinoma, non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, T-cell lymphoma, soft tissue sarcoma, pancreatic cancer, colorectal cancer, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

An aspect of the invention is the use of a human BMP7 protein variant comprising a polypeptide having the amino acid sequence of SEQ ID NO: 7 or 8 in combination with ATRA for the treatment of cancers in which the retinoid receptors RAR alpha and RAR gamma are known to be important including, but not limited to, lung cancer, brain cancer, cervical cancer, skin cancer, head and neck cancer, glioblastoma, neuroblastoma, oral leukoplakia, oral squamous cell carcinoma, non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, T-cell lymphoma, soft tissue sarcoma, pancreatic cancer, colorectal cancer, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

The human BMP7 protein variants of the present invention may be achieved through generating appropriate gene sequences, i.e. by arranging the appropriate nucleotide sequences and expressing them in a suitable cell line. The desired nucleotide sequences can be produced using a method such as codon-based mutagenesis. Such procedures permit the production of any and all frequencies of amino acid residues at any desired codon positions within an oligonucleotide.

The invention provides a method for treating cancer, cartilage damage and degeneration, pain associated with osteoarthritis, or bone fracture healing comprising administering a therapeutically effective amount of a variant of human BMP7 of the present invention to a human patient in need thereof. Preferably, the invention also provides a method for treating cancer comprising administering a therapeutically effective amount of a variant of human BMP7 of the present invention to a human patient in need thereof wherein said cancer is selected from the group consisting of lung cancer, including, but not limited to, non-small cell lung cancer (NSCLC), brain cancer, cervical cancer, skin cancer, head and neck cancer, glioblastoma, neuroblastoma, and colorectal cancer.

The invention provides a method for treating cancer comprising administering a therapeutically effective amount of a variant of human BMP7 protein of the present invention in combination with all- trans retinoic acid to a patient in need thereof.

The invention provides a variant of human BMP7 protein of the present invention in combination with ATRA for use as a medicament.

The invention provides a variant of human BMP7 protein in combination with ATRA for use in the treatment of cancer said cancer is selected from the group consisting of lung cancer, brain cancer, cervical cancer, skin cancer, head and neck cancer, glioblastoma, neuroblastoma, oral leukoplakia, oral squamous cell carcinoma, non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, T-cell lymphoma, soft tissue sarcoma, pancreatic cancer, colorectal cancer, Hodgkin's lymphoma, and non-Hodgkin's lymphoma.

The retinoic acid receptor (RAR) is a type of nuclear receptor which is activated by both ATRA and 9-cis retinoic acid. There are three RARs: RAR-alpha, RAR-beta, and RAR-gamma. Activation of the RARs results in a stimulation of alkaline phosphatase activity, which can be measured essentially as described in Example 3.

The invention further provides the use of a variant of human BMP7 protein in combination with ATRA for the manufacture of a medicament for the treatment of cancer.

Another aspect of the present invention provides variants of human BMP7 protein in

combination with ATRA for use as a medicament.

Another aspect of the invention embodies a variant of human BMP7 protein in combination with ATRA according to the present invention for use in the treatment of cancer.

The invention provides variants of the human BMP7 protein for use as a medicament. The invention provides a variant of the human BMP7 protein for use in the treatment of cancer, cartilage damage and degeneration, pain associated with osteoarthritis or bone fracture healing.

The invention further provides the use of a variant of human BMP7 protein of the present invention for the manufacture of a medicament for the treatment of cancer, cartilage damage and degeneration, pain associated with osteoarthritis or bone fracture healing.

Other embodiments of the invention are drawn to polynucleotides encoding the variants of human BMP7 protein of the present invention. Another embodiment is a vector containing said polynucleotide(s) and a host cell carrying said vector. Another embodiment is drawn to processes for producing a human BMP7 protein variant of the present invention by culturing host cells carrying said vector containing DNA encoding said protein, expressing said protein from the host cells and recovering the protein from the culture media.

For purposes of the present invention, as described and claimed herein, terms are defined as follows:

The term“about” means up to a 10% variance of the value such term would be given depending on the number of significant figures. For example,‘about 200’ encompasses from 180 to 220 and‘about 1’ encompasses from 0.9 to 1.1.

The term“administering” refers to an act to transfer a pharmaceutical composition of the present invention into the body of a mammal, preferably a human, in need thereof.

Administration may be via any route known to be effective by the physician of ordinary skill. Parenteral administration is one form of administration commonly understood in the medical literature as the injection of a dosage form into the body by a sterile syringe or some other mechanical device such as an infusion pump. The parenteral administration may include intravenous injection, subcutaneous injection, muscular injection, intraperitoneal injection, endothelial administration, local administration, intranasal administration, intrapulmonary administration, and rectal administration. Administration at the time of surgery or through the use of x-ray imaging (fluoroscopy) are additional forms.

All-trans retinoic acid (ATRA) is a ligand for both the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). RARs and RXRs function as ligand inducible transcription factors that regulate the growth and differentiation of both normal and malignant cells (Zhou et al., Phil Trans. R. Soc. B 362: 959-971, 2007).

The term“bone healing” and“bone fracture healing” are used interchangeably herein and are intended to refer to bone repair associated with delayed union and non-union fractures of femoral and tibial bone, fractures of toe and metatarsal bone, proximal humerus bone fractures, other bone fractures, alveolar bone defects associated with dental implant fixtures, intervertebral disc degeneration, spinal fusions, and bone repair associated with cranio-maxillofacial surgeries or enhanced osseous integration to stabilize fixation of implants (screws, plates, prosthesis, dental implants).

Cartilage damage and degeneration refers to cartilage injury resulting from joint injuries associated with trauma, sports, falls, or collisions such as post-traumatic knee cartilage injury from joint dislocation, ligament tear, meniscus tear, post-traumatic shoulder cartilage injury, post-traumatic cartilage injury of the hip, post-traumatic cartilage injury of the elbow, or other cartilage damage such as osteoarthritis.

Pain associated with osteoarthritis refers to pain associated with post-traumatic osteoarthritis, knee osteoarthritic pain, osteochondral defect or related disorders.

Pharmaceutically acceptable excipient refers to a pharmaceutically acceptable formulation carrier, solution, or additive to enhance the formulation characteristics. Such excipients must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof and are well known to the skilled artisan, see, e.g., Remingtons

Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, 1995.

A stable formulation is one in which the protein remains soluble for an extended period of time under the conditions of storage.

Sulfated polysaccharides are compounds consisting of two or more saccharide units containing one or more sulfation sites per saccharide unit. Exemplary sulfated polysaccharides include heparin, heparin sulfate, dextran sulfate, sucrose octasulfate, sulfated β-cyclodextrin, myo-inositol hexasulfate, polypentosan sulfate, fucoidan, chonroitin sulfate A, chonroitin sulfate B, chonroitin sulfate C, and derivatives thereof.

Potency or specific activity is a measurement of the relative activity of variants of human BMP7 protein including those of the present invention and may be measured in the MFc7 cell assay described hereinbelow in Example 5, for example. Generally, the relative activity is

compared to the wild type human mature BMP7 to yield a relative potency for the human BMP7 protein variant.

Soluble or solubility refers to the reduction or relative absence of aggregated protein that is determined by an aggregation assay such as the one described herein in Example 6. Solubility is also a measure of the physical stability of the BMP7 protein variant which may be measured in a thermal unfolding assay essentially as described in Example 7.

The terms“subject”,“patient”, or“individual”, are used interchangeably herein, and all of them refer to a mammal, including, but not limited to, murines, simians, humans, mammalian farm animals (i.e., sheep, etc.), mammalian sport animals (i.e., horses), and mammalian pets (i.e., dogs or cats); preferably, the term refers to humans. In a certain embodiment, the subject, preferably a human, is further characterized with a disease or disorder or condition that would benefit from treatment with a variant of human BMP7 protein of the present invention.

Treating as used herein describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a pharmaceutical composition of the present invention to alleviate the symptoms or complications, or eliminate the disease, condition, or disorder.

The phrase“therapeutically effective amount” refers to the amount of an active agent necessary to impart a therapeutic benefit to a patient.

A therapeutically effective amount is the amount of an active agent necessary to impart a therapeutic benefit to a patient. For example, a therapeutically effective amount administered to a human patient in need of treatment for cartilage damage and degeneration, pain associated with osteoarthritis, or bone healing is such an amount which induces, ameliorates, or otherwise causes an improvement in the pathological symptoms, disease progression, or physiological conditions associated with cartilage damage and degeneration, pain associated with osteoarthritis, or bone repair/healing. Furthermore, a therapeutically effective amount of a human BMP7 protein variant of the present invention is an amount administered to a human patient in need of treatment for cancer is an amount which in mammals, preferably, humans, reduces the number of cancer cells; reduces the tumor size; inhibits (i.e., slow to some extent or stop) cancer cell infiltration into peripheral tissues organs; inhibit (i.e., slow to some extent or stop) tumor metastasis; inhibits, to some extent, tumor growth; and/or relieves to some extent one or more of the symptoms associated with the cancer. An effective amount of a human BMP7 protein variant of the invention may be administered in a single dose or in multiple doses. Furthermore, an effective

amount of a human BMP7 protein variant of the invention may be administered in multiple doses of amounts that would be less than an effective amount if not administered more than once.

As is well known in the medical arts, dosages for any one subject depends upon many factors, including the patient’s size, body surface area, age, the particular compound to be administered, gender, time and route of administration, general health, and other drugs being administered concurrently. Dose may further vary depending on the type and severity of the disease. A typical dose of a human BMP7 protein variant of the present invention can be, for example, in the range of about 10 mg to about 1000 mg; preferably, about 50 mg to about 500 mg; more preferably, about 200 mg to about 500 mg; even more preferably, about 200 mg to about 400 mg, even more preferably, about 200 mg to about 300 mg; even more preferably, about 225 mg to about 275 mg; even more preferably, about 250 mg to about 275 mg; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. A daily parenteral dosage regimen can be from about 250 µg/kg to about 10 mg/kg. Progress may be monitored by periodic assessment, and the dose adjusted accordingly.

In some embodiments of the present invention, a single dose of a human BMP7 protein of the present invention may be administered intravenously for treating a cancer in an adult patient. A typical single dose for intravenous administration of a variant of human BMP7 protein of the present invention can be, for example, in the range of about 10 mg to about 1000 mg; preferably, about 10 mg to about 500 mg; more preferably, about 10 mg to about 500 mg; more preferably, about 10 mg to about 400 mg; more preferably, about 10 mg to about 350 mg; more preferably, about 10 mg to about 300 mg; even more preferably, about 10 mg to about 275 mg; even more preferably, about 10 mg to about 250 mg; even more preferably, about 10 mg to about 200 mg; even more preferably, about 10 mg to about 175 mg; even more preferably, about 10 mg to about 150 mg; or most preferably, about 10 mg to about 125 mg; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.

Alternatively, a typical single dose for intravenous administration of a variant of human BMP7 protein of the present invention can be, for example, from about 0.2 mg/kg to about 15 mg/kg body weight; more preferably, about 0.2 mg/kg to about 10 mg/kg; even more preferably, about 0.2 mg/kg to about 7.5 mg/kg; even more preferably, about 0.2 mg/kg to about 5 mg/kg; even more preferably, about 0.2 mg/kg to about 4 mg/kg; even more preferably, about 0.2 mg/kg to about 3 mg/kg; even more preferably about 0.2 mg/kg to about 2.5 mg/kg; or most preferably, about 0.2 mg/kg to about 2 mg/kg. Such doses can be administered intravenously once every

week, once every two weeks, once every three weeks, or once every month, for example.

Progress may be monitored by periodic assessment, and the dose adjusted accordingly.

These suggested amounts of a variant of human BMP7 protein of the present invention are subject to a great deal of therapeutic discretion. The key factor in selecting an appropriate dose and scheduling is the result obtained. Factors for consideration in this context include the particular disorder being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the protein, the particular type of form of the protein (e.g., pro-BMP7 protein variant or mature BMP7 protein variant) the method of administration, the scheduling of administration, and other factors known to medical practitioners.

Human BMP7 protein is a secreted signaling molecule of the TGF-beta superfamily and was originally identified for its ability to induce bone formation but later became recognized as a multifunctional cytokine which mediates growth and differentiation of many different cell types. Human BMP7 protein is expressed in cells as a 292 amino acid precursor protein and the mature, biologically active BMP7 is generated by proteolytic removal of the signal peptide and pro-peptide. The wild type human BMP7 protein amino acid sequence containing the signal peptide (the first 29 amino acids), pro-domain, and mature peptide (underlined) is indicated as SEQ ID NO: 1: MHVRSLRAAAPHSFVALWAPLFLLRSALADFSLDNEVHSSFIHRRLRSQERREMQ REILSILGLPHRPRPHLQGKHNSAPMFMLDLYNAMAVEEGGGPGGQGFSYPYKA VFSTQGPPLASLQDSHFLTDADMVMSFVNLVEHDKEFFHPRYHHREFRFDLSKIP EGEAVTAAEFRIYKDYIRERFDNETFRISVYQVLQEHLGRESDLFLLDSRTLWASE EGWLVFDITATSNHWVVNPRHNLGLQLSVETLDGQSINPKLAGLIGRHGPQNKQ PFMVAFFKATEVHFRSIRSTGSKQRSQNRSKTPKNQEALRMANVAENSSSDQRQ ACKKHELYVSFRDLGWQDWIIAPEGYAAYYCEGECAFPLNSYMNATNHAIVQTL VHFINPETVPKPCCAPTQLNAISVLYFDDSSNVILKKYRNMVVRACGCH (SEQ ID NO: 1). It is understood by the skilled artisan that the signal peptide may be removed by proteolytic cleavage resulting in an intact pro-domain/mature peptide that is designated as pro-BMP7.

Wild type human mature BMP7 is a dimer of two glycosylated, 139 amino acid disulfide-linked, homodimeric proteins of about 35 kDa. Each homodimeric protein has the amino acid sequence as shown in SEQ ID NO: 2:

STGSKQRSQNRSKTPKNQEALRMANVAENSSSDQRQACKKHELYVSFRDLGWQ DWIIAPEGYAAYYCEGECAFPLNSYMNATNHAIVQTLVHFINPETVPKPCCAPTQ LNAISVLYFDDSSNVILKKYRNMVVRACGCH (SEQ ID NO: 2).

The variants of human BMP7 protein of the present invention include variants of human mature BMP7 of SEQ ID NO: 2, with the specific amino acid positions changed indicated in the consensus sequence as shown in SEQ ID NO: 3. Particular variants of human mature BMP7 protein of the present invention have increased specific activity, improved solubility

characteristics, improved bioavailability, decreased binding to endogenous circulating inhibitors, and/or reduced EBF activity compared to the wild type mature human BMP7 protein.

Preferred variants of human BMP7 protein are selected from the group consisting of F93V/N110G SEQ ID NO: 4; Y65G/I86L/T89A/N110G SEQ ID NO: 5;

Y65G/I86L/N110G/Y128F SEQ ID NO: 6; Y65G/I86L/N110G/Y128W SEQ ID NO:7;

Y65G/I86L/F93V/N110G/Y128W SEQ ID NO: 8, Y65G/T89A/N110G/Y128F SEQ ID NO: 9; Y65G/I86L/N110G SEQ ID NO: 10; and, Y65G/V114M SEQ ID NO: 11. The most preferred variants of BMP7 of the present invention are selected from the group consisting of

Y65G/I86L/N110G/Y128W (SEQ ID NO: 7) and Y65G/I86L/F93V/N110G/Y128W (SEQ ID NO: 8).

The present invention also provides variants of human pre-BMP7 (i.e., SEQ ID NO: 1) as well as variants of human pro-BMP7 (i.e., SEQ ID NO: 21). Preferred variants of human pro-BMP7 of the present invention that contain the pro-domain fused to the N-terminus of the human mature BMP7 protein variant are selected from the group consisting of SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO:14; SEQ ID NO: 15; and SEQ ID NO: 16.

The variants of human BMP7 protein of the present invention may be synthesized by recombinant organisms engineered using methods well known in the art, or alternatively, by chemical synthesis. Accordingly, other embodiments of the invention are directed to

polynucleotides encoding the variants of human BMP7 protein. Another embodiment is a vector containing said polynucleotide and a host cell carrying said vector. Another embodiment is directed to processes for producing a protein by culturing host cells carrying said vector containing DNA encoding said protein, expressing said protein from the host cells and recovering the protein from the culture media.

The polynucleotides that encode for a variant of human BMP7 of the present invention may include the following: only the coding sequence for the human mature BMP7 protein variant, the coding sequence for the variant and additional coding sequence such as a functional protein, or a signal or secretory sequence or a pro-domain sequence; the coding sequence for the human mature BMP7 protein variant and non-coding sequence, such as introns or non-coding

sequence 5’ and/or 3’ of the coding sequence for the variant. Thus, the term“polynucleotide encoding a variant” encompasses a polynucleotide that may include not only the coding sequence for the human mature BMP7 protein variant but also a polynucleotide which includes additional coding and/or non-coding sequence. It is known in the art that a polynucleotide sequence that is optimized for a specific host cell/expression system can readily be obtained from the amino acid sequence of the desired protein. Examples of polynucleotides of the present invention are SEQ ID NO: 19 and SEQ ID NO: 20 which are DNA sequences that encode pre-BMP7 forms of the human mature BMP7 protein variants as shown in SEQ ID NO: 7 and SEQ ID NO: 8 respectively.

The polynucleotides of the present invention are expressed in a host cell after the sequences have been operably linked to an expression control sequence. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers, e.g., tetracycline, neomycin, and/or dihydrofolate reductase, to permit detection of those cells transformed with the desired DNA sequences. The vectors containing the polynucleotide sequences of interest (e.g., the variants of BMP7 protein and expression control sequences) are transferred into a host cell by well-known methods, which vary depending on the type of cellular host.

The variants of human BMP7 protein of the present invention may readily be produced in mammalian cells such as CHO, NS0, HEK293 or COS cells, in bacterial cells such as E. coli or Pseudomonas flourescence or in fungal or yeast cells. Preferably, the host cell is a mammalian cell. The preferred mammalian cell is a CHO cell. The host cells are cultured using techniques well known in the art.

In one embodiment the present invention relates to a vector containing and expressing in a host a pre-BMP7 gene, a pro-BMP7 gene or a mature BMP7 gene. The BMP7 gene encoding the pre-BMP7 protein, the pro-BMP7 protein or the mature BMP7 protein may originate from a mammal. In a preferred embodiment, the expression vector may comprise a polynucleotide that encodes a variant of a human pre-BMP7, a variant of a human pro-BMP7 or a variant of a human mature BMP7 protein. The polynucleotide encoding these human BMP7 protein variants may be operatively linked to a promoter and optionally to an enhancer.

In some embodiments, the invention relates to a mammalian host cell harboring a vector comprising a polynucleotide encoding a human pre-BMP7 protein variant, a human pro-BMP7

protein variant, or a human mature BMP7 protein variant, wherein the pro-BMP7 protein variant is deleted of the“pre” or“signal” peptide at the N-terminus. Preferably, the mammalian host cell harbors a vector that comprises a polynucleotide that encodes for the expression of a human mature BMP7 protein variant having the amino acid sequence of any one of SEQ ID NO: 3-11, or a pre- or pro- form thereof. More preferably, the mammalian host cell harbors a vector that comprises a polynucleotide that encodes for the expression of a human pro-BMP7 protein variant having the amino acid sequence of any one of SEQ ID NO: 12-16, or a pre- form thereof. In some embodiments a“pre” or“signal” peptide sequence from a different origin is fused to the N-terminus of the human pro-BMP7 protein variant. In some embodiments of the present invention, the“pre” or“signal” peptide sequence may be the insulin-like growth factor 1 (IGF-1) or the tissue plasminogen activator (tPA) peptide signal sequence, for example.

Various methods of protein purification may be employed and such methods are known in the art and described, for example, in Protein Purification: Principals, High Resolution Methods, and Applications, 2nd Edition, Wiley-VCH Inc. (Germany, 1998).

Pharmaceutical compositions of the present invention can be prepared using conventional dissolution and mixing procedures. One of ordinary skill in the formulation sciences will recognize that the order of addition of a medically useful protein, osmolyte, and hydrophobic preservative, can be varied without compromising the effectiveness of the formulation. To further improve the bioavailability of the pharmaceutical compositions of the present invention, targeting strategies may be employed for the retention of the human BMP7 protein variants in the joint space. This involves the formulation in lactose buffer or encapsulation techniques to physically entrap molecules in the joint space (e.g. hydrogel, nanoparticles, liposomes) or targeting to proteins in the extracellular space that include but are not limited to collagens (type I and II) or integrins. For bone healing, the human BMP7 protein variant of the present invention may be mixed with carriers such as collagen type 1.

Hydrophobic preservative refers to a hydrophobic compound that may be added to a pharmaceutical formulation to act as an anti-microbial agent. Examples of hydrophobic preservatives acceptable in parenteral formulations are alkylparabens, phenolic preservatives i.e. phenol and cresol, benzyl alcohol, chlorobutanol, benzoic acid, and various mixtures thereof.

Reference to a wild type human BMP7 protein or variant thereof herein, including by reference to a SEQ ID NO: or a unique reference code (for example,“F9”) refers to a homodimer thereof. As a non-limiting example,“human mature BMP7 protein variant F9 (SEQ ID NO: 8)”

or interchangeably“BMP7 protein variant F9” or the like, as used herein, refers to a homodimer wherein each monomeric subunit has the sequence as shown in SEQ ID NO: 8 and the subunits are linked via disulfide bond(s).

For the functional assays described hereinbelow, treatment with or administration of a particular pro-BMP7 protein or a variant thereof to, refers to treatment with or administration of homodimers of the particular human mature BMP7, i.e., either wild type or a variant thereof, which are generally in a non-covalent complex with wild type human pro-domain.

Cresol refers to meta-cresol, ortho-cresol, para-cresol, chloro-cresol, or mixtures thereof. Isotonicity agent refers to a compound that is tolerated physiologically and imparts a suitable tonicity to a formulation to prevent the net flow of water across cell membranes.

Examples of isotonicity agents are glycerin, salts e.g. NaCl, KCl, and sugars, e.g. dextrose, mannitol, and sucrose.

Osmolytes are compounds that have the ability to stabilize proteins against denaturation and aggregation. Exemplary osmolytes include amino acids, polyols (e.g. sorbitol, mannitol, xylitol, and glycerol), sugars, sugar alcohols, sugar acids and the like. Preferred osmolytes include histidine, salts of histidine, glycine, salts of aspartic acid, salts of glutamic acid, salts of lysine, salts of arginine, serine, proline and alanine. The preferred osmolyte is arginine.

Preferably, the concentration of arginine will be about 100 mM to 1 M; more preferably about 125 mM to about 800 mM; still more preferably about 200 mM to about 500 mM; and, most preferably about 250 mM.

The preferred hydrophobic preservative is selected from the group consisting of phenol, m-cresol, methylparaben, propylparaben, benzyl alcohol, chlorocresol, and mixtures thereof.

The pharmaceutical compositions of the present invention optionally may contain other compounds in addition to the medically useful protein, hydrophobic preservative, and osmolyte. For example, pharmaceutically acceptable surfactants like Tween 20 (polyoxyethelene (20) sorbitan monolaurate), Tween 40 (polyoxyethelene (20) sorbitan monopalmitate), Tween 80 (polyoxyethelene (20) sorbitan monooleate), Pluronic F68 (polyoxyethelene polyoxypropylene glycol), and PEG (polyethylene glycol) may optionally be added to the formulation to reduce aggregation. These additives are particularly useful if a pump or plastic container is used to administer the formulation. A pharmaceutically acceptable surfactant may further reduce protein aggregation.

The pharmaceutical composition of the present invention may contain a sulfated polysaccharide. The preferred sulfated polysaccharide is selected from the group consisting of heparin, heparin sulfate, and dextran sulfate.

The pharmaceutical composition of the present invention may contain an aqueous buffer. The buffers suitable for the present invention are those having pH buffering capacity in the range of from about pH 6 to about pH 8 and are compatible with the dried protein. The pH of the formulation solution is about 6.5 to about 7.5. More preferably, 6.8 to about 7.5. Still more preferably, a pH between about 7.0 and about 7.4.

Representative buffer systems to maintain effective pH control include Tris-acetate, sodium citrate, potassium citrate, citrate-glycine and sodium phosphate. More preferred buffer systems include sodium citrate and sodium phosphate. The most preferred buffer is sodium citrate. The preferred concentration of the buffer system is about 1 mM to about 50 mM. A more preferred concentration is about 5 mM to about 30 mM. The most preferred concentration is about 10 mM. The skilled artisan will recognize that many other buffer systems are available which also can be used to maintain the pH in the preferred range.

In addition, an isotonicity agent, preferably NaCl or KCl, may be optionally added to the soluble, pharmaceutical composition/formulation. Most preferably the isotonicity agent is NaCl. The concentration of the isotonicity agent is in the range known in the art for parenteral formulations, preferably about 100 mM to about 250 mM, more preferably about 125 mM to about 200 mM and still more preferably about 150 mM.

The pharmaceutical compositions of the human BMP7 protein variants of the present invention may be administered by any means known in the art that achieve the generally intended purpose of treatment for cancer, cartilage damage and degeneration, pain associated with osteoarthritis, or bone healing.

We Claim:

1. A protein comprising a polypeptide comprising the amino acid sequence of:

STGSKQRSQNRSKTPKNQEALRMANVAENSSSXaa33QRQXaa37CKKHELYVSFR DLGWQDWIIAPXaa60GYAAXaa65YCEGECAFPLNSYMNATNHAXaa86Xaa87QXaa 89LXaa91HXaa93Xaa94NPETVPKPCCAPTQLXaa110AISXaa114LYFDDXaa120SNVILK KXaa128RNMXaa132VXaa134ACGCH (SEQ ID NO: 3),

wherein:

Xaa33 is D or M; Xaa37 is A or P; Xaa60 is E or Q; Xaa65 is Y, S, or G; Xaa86 is I, V, or L; Xaa87 is V or L; Xaa89 is T, S, or A; Xaa91 is V or M; Xaa93 is F or V; Xaa94 is I, F or M; Xaa110 is G; Xaa114 is V or M; Xaa120 is S or Q; Xaa128 is Y, F or W; Xaa132 is V, Q, or S; and, Xaa134 is R or K.

2. The protein of Claim 1 wherein Xaa33 is D; Xaa37 is A; Xaa60 is E; Xaa87 is V; Xaa89 is T or A; Xaa91 is V; Xaa94 is I; Xaa120 is S; Xaa132 is V; and, Xaa134 is R.

3. The protein of Claim 2 wherein Xaa65 is Y or G; Xaa86 is I or L.

4. The protein of Claim 3 wherein Xaa65 is G; Xaa86 L; Xaa89 is T; Xaa93 is V; and

Xaa128 is F or W.

5. The protein of Claim 4 wherein Xaa114 is V.

6. The protein of Claim 5 wherein the amino acid sequence of the polypeptide is SEQ ID NO: 8.

7. The protein of Claim 1 wherein the amino acid sequence of the polypeptide is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 10.

8. The protein of any one Claims 1-7 wherein the N-terminus of the polypeptide is

covalently fused to the C-terminus of a human BMP7 pro-domain polypeptide comprising an amino acid sequence of SEQ ID NO: 18.

9. The protein of any one of Claims 1-7 wherein the amino acid sequence of the polypeptide is selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

10. A pharmaceutical composition comprising a protein of any one of Claims 1-7 and one or more pharmaceutically acceptable excipients, diluents, or carriers.

11. The pharmaceutical composition of Claim 10 wherein the protein is a disulfide linked homodimer.

12. The pharmaceutical composition of Claim 11 wherein the composition further comprises a polypeptide having the amino acid sequence of SEQ ID NO: 18.

13. A method of treatment for cancer, cartilage damage and degeneration, pain associated with osteoarthritis, or bone healing comprising administering to a human patient in need thereof an effective amount of the pharmaceutical composition of any one of Claims 11-12.

14. The method of Claim 13 wherein the cancer is lung cancer, non-small cell lung cancer (NSCLC), brain cancer, cervical cancer, skin cancer, head and neck cancer, glioblastoma, neuroblastoma, or colorectal cancer.

15. The protein of any one of Claims 1-7, for use in therapy.

16. The protein of any one of Claims 1-7 for use in the treatment of c a n c e r , cartilage damage and degeneration, pain associated with osteoarthritis, or bone healing.

17. The protein for use of Claim 16, wherein the cancer is lung cancer, non-small cell lung cancer (NSCLC), brain cancer, cervical cancer, skin cancer, head and neck cancer, glioblastoma, neuroblastoma, or colorectal cancer.

18. The protein for use of any one of Claims 15-17, wherein the antibody is administered in simultaneous, separate, or sequential combination with ionizing radiation.

19. The protein for use of any one of Claims 15-17, wherein the antibody is administered in simultaneous, separate, or sequential combination with one or more chemotherapeutic agents.

20. A method of treating cancer comprising administering to a patient in need thereof in simultaneous, separate, or sequential combination of an effective amount of a protein of any one of Claims 1-7, with an effective amount of one or more chemotherapeutic agents or ionizing radiation.

21. The method of Claim 20, wherein the cancer is lung cancer, non-small cell lung cancer (NSCLC), brain cancer, cervical cancer, skin cancer, head and neck cancer, glioblastoma, neuroblastoma, or colorectal cancer.

22. A kit for the treatment of cancer, the kit comprising a first pharmaceutical composition comprising a protein of any one of Claims 1-7 and an acceptable carrier, diluent, or excipient and a second pharmaceutical composition comprising one or more chemotherapeutic agents an acceptable carrier, diluent, or excipient.

23. The kit of claim 22 wherein the first pharmaceutical composition further comprises a polypeptide having the amino acid sequence of SEQ ID NO: 18.

24. A combination medicine comprising a protein of any one of Claims 1-7, and one or more chemotherapeutic agents for simultaneous, separate, or sequential use in the treatment of cancer.

25. The combination medicine for use of Claim 24, wherein the cancer is lung cancer, non- small cell lung cancer (NSCLC), brain cancer, cervical cancer, skin cancer, head and neck cancer, glioblastoma, neuroblastoma, or colorectal cancer.

26. The combination medicine for use of any one of Claims 24-25, wherein the protein is administered in simultaneous, separate, or sequential combination with ionizing radiation.

27. The combination medicine for use of any one of Claims 24-26, wherein the protein is administered in simultaneous, separate, or sequential combination with one or more chemotherapeutic agents.

28. The use of a protein of any one of Claims 1-7 for the manufacture of a medicament for the treatment of cancer, c artilage damage and degeneration, pain associated with osteoarthritis, or bone healing.

29. The protein for use of Claim 28 wherein the cancer is lung cancer, non-small cell lung cancer (NSCLC), brain cancer, cervical cancer, skin cancer, head and neck cancer, glioblastoma, neuroblastoma, or colorectal cancer.

30. The protein for use of any one of Claims 28-29, wherein the protein is administered in simultaneous, separate, or sequential combination with ionizing radiation.

31. The protein for use of any one of Claims 28-30, wherein the protein is administered in simultaneous, separate, or sequential combination with one or more chemotherapeutic agents.

32. A pharmaceutical composition comprising a protein of any one of Claims 1-7 for use in simultaneous, separate, or sequential combination with a pharmaceutical composition comprising a chemotherapeutic agent in the treatment of cancer.

33. The pharmaceutical composition for use of Claim 32, wherein either pharmaceutical composition is administered in simultaneous, separate, or sequential combination with ionizing radiation.

34. A pharmaceutical composition comprising a protein of any one of Claims 1-7 for use in simultaneous, separate, or sequential combination with ionizing radiation in the treatment of cancer.

35. The pharmaceutical composition for use of any one of Claims 32-34, wherein the

cancer is lung cancer, non-small cell lung cancer (NSCLC), brain cancer, cervical cancer, skin cancer, head and neck cancer, glioblastoma, neuroblastoma, or colorectal cancer.