AU624940B2 - Osteoinductive compositions - Google Patents

Description

WO90/11366 PCT/US90/01630 OSTEOINDUCTIVE COMPOSITIONS The present invention relates to proteins having utility in the formation of hone i' 5 and/or cartilage. In particular the invention relates to a number of families of purified proteins, termed BMP-5, BMP-6 and BMP-7 protein families (wherein BMP is Bone Morphogenic Protein) and processes for obtaining them. These proteins may exhibit the ability to induce cartilage and/o: bone formation. They may be used to induce bone and/or cartilage formation and in wo';nd healing and S'.ssue repair.

The invention provides a family of proteins. Purified human BMP-5 proteins are substantially free from other proteins with which they are co-produced, and characterized by an amino acid sequence comprising From amino acid #323 to amino acid #454 set forth in Table III. This amino acid sequence #323 to #454 is encoded by the DNA sequence comprising nucleotide #1665 to nucleotide #2060 of Table III. BMP-5 proteins may be further characterized by an apparent molecular weight of 1 28,000-30,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis i (SDS-PAGE). Under reducing conditions in SDS-PAGE the protein electrophoreses with a molecular weight of approximately 14,000 20,000 daltons. It .s contemplated that these proteins are capable cf stimulating, promoting, or otherwise inducing cartilage and/or bone formation.

The invention further provides bovine proteins comprising amino acid #9 to amino acid #140 set forth in Table I. Ths amino acid sequence ~$"~CBa~llrsl~R~ICIPr~ WO 90/11366 PC/US90/01 630 2 1 from #9 to #140 is encoded by the DNA sequence comprising nucleotide #32 to #427 of Table I.

i These proteins may be further characterized by an apparent molecular weight of 28,000 30,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Under reducing conditions in SDS-PAGE the protein electrophoreses with a nolecular weight of approximately 14,000-20,ouL daltons. It is contemplated that these proteins are capable of inducing cartilage and/or bone formation.

Human BMP-5 proteins of the invention may be produced by culturing a cell transformed with a DNA sequence containing the nucleotide sequence the same or substantially the same as the nucleotide sequence shown in Table III comprising nucleotide #699 to nucleotide #2060. BMP-5 proteins comprising the amino acid sequence the same or substantially the same as shown in Table III from amino acid 323 to amino acid 454 are recovered, isolated and purified from the culture medium.

Bovine BMP-5 proteins may be produced by culturing a cell transformed with a DNA sequence containing the nucleotide sequence the same or substantially the same as that shown in Table I comprising nucleotide #8 through nucleotide #427 and Lecovering and purifying from the culture medium a protein containing the amino acid sequence or a portion thereof as shown in Table I comprising amino acid #9 to amino acid #140.

The invention provides a family of BMP-6 proteins. Purified human BMP-6 proteins, substantially free from other proteins with which they are co-produced and are characterized by an amino acid sequence comprising acid #382 to amino WO 90/11366 PCT/US90/01630 16 the human gene ir fragments thereof. Sequences thus identifinrd nav i=C i WO 90/11366 PCT/US90/01630 acid #513 set forth in Table IV. The amino acid sequence from amino acid #382 to #513 is enOdded by the DNA sequence of Table IV from nucleotide #1303 to nucleotide #1698. These proteins may be further characterized by an apparent molecular weight of 28,000-30,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Under reducing conditions in SDS-PAGE the protein electrophoreses with a molecular weight i 10 of approximately 14,000 20,000 daltons. It is i contemplated that these proteins are capable of stimulating promoting, or otherwise inducing cartilage and/or bone formation.

The invention further provides bovine BMP-6 i 15 proteins characterized by the amino acid sequence comprising amino acid #121 to amino acid #222 set forth in Table II. The amino acid sequence from #121 to #222 is encoded by the DNA sequence of Table II from nucleotide #361 to #666 of Table II.

These proteins may be further characterized by an apparent molecular weight of 28,000 30,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Under reducing conditions in SDS-PAGE the protein electrophoreses with a molecu:lar weight of approximately 14,000-20,000 daltons. It is contemplated that these proteins are capable of inducing cartilage and/or bone formation.

A Human BMP-6 proteins oZ the invention are produced by culturing a cell transformed with a DNA sequence comprising nucleotide #160 to nucleotide #1698 as shown in Table III or a substantially similar sequence. BMP-6 proteins comprising amino acid #382 to amino acid #513 or a substantially similar sequence are recovered, isolated and -1 WO 90/11366 PCT/US90/01630 4 purified from the culture medium.

Bovine BMP-6 proteins may be produced by culturing a cell transformed with a DNA comprising i nucleotide #361 through nucleotide #666 as set forth in Table II or a substantially similar 1 sequence and recovering and purifying from the culture medium a protein comprising amino acid #121 i to amino acid #222 as set forth in Table II.

The invention provides a family of BMP-7 proteins. Which includes purified human BMP-7 proteins, substantially free from other proteins with which they are co-produced. Human BMP-7 j proteins are characterized by an amino acid isequence comprising amino acid #300 to amino acid #431 set forth in Table V. This amino acid sequence #300 to #431 is encoded by the DNA I sequence of Table V from nucleotide #994 to #1389.

BMP-7 proteins may be further characterized by an apparent molecular weight of 28,000-30,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Under reducing conditions in SDS-PAGE the protein electrophoreses with a molecular weight of approximately 14,000 20,000 daltons. It is j 25 contemplated that these proteins are capable of stimulating, promoting, or otherwise inducing cartilage and/or bone formation.

Human BMP-7 proteins of the invention may be produced by culturing a cell transformed with a DNA sequence containing the nucleotide sequence the same or substantially the same as the nucleotide sequence shown in Table V comprising nucleotide 97 to nucleotide #1389. BMP-7 proteins comprising the amino acid sequence the same or substantially the same as shown in Table V from amino acid #300 WO 90/11366 P(T/UFc3/01630 to amino acid #431 are recovered, isolated and purified from the culture medium.

The invention further provides a method wherein the proteins described above are utilized for obtaining related human protein/s or other mammalian cartilage and/or bone formation protein/s. Such methods are known to those skilled in the art of genetic engineering. One method for obtaining such proteins involves utilizing the human BMP-5, BMP-6 and BMP-7 coding sequences or portions thereof to design probes for screening human genomic and/or cDNA libraries to isolate human genomic and/or cDNA sequences. Additional methods within the art may employ the bovine and human BMP proteins of the invention to obtain other mammalian BMP cartilage and/or bone formation proteins.

Having identified the nucleotide sequences, the proteins are produced by culturing a cell transformed with the nucleotide sequence. This sequence or portions thereof hybridizes under stringent conditions to the nucleotide sequence of either BMP-5, BMP-6 or BMP-7 proteins and encodes a protein exhibiting cartilage and/or bone formation activity. The expressed protein is recovered and purified from the culture medium.

The purified BMP proteins are substantially free from other proteinaceous materials with which they are co-produced, as well as from other contaminants.

BMP-6 and BMP-7 proteins may be characterized by the ability to promote, stimulate or otherwise induce the formation of cartilage and/or bone formation. It is further contemplated that the ability of these proteins to induce the

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WO 90/11366 PCT/US90/01630 6 formation of cartilage and/or bone may be exhibited by the ability to demonstrate cartilage and/or bone pi formation activity in the rat bone formation assay described below. It is further contemplated that the proteins of the invention demonstrate activity in this rat bone formation assay at a concentration of 10pg 500g/gram of bone formed.

More particularly, it is contemplated these proteins may be characterized by the ability of lpg of the protein to score at least +2 in the rat bone formation assay described below using either the original or modified scoring method.

Another aspect of the invention provides pharmaceutical compositions containing a therapeutically effective amount of a BMP-5, BMP-6 or BMP-7 protein in a pharmaceutically acceptable vehicle or carrier. Further compositions comprise at least one BMP-5, BMP-6 or BMP-7 protein. It is therefore contemplated that the compositions may contain more than one of the BMP proteins of the present invention as BMP-5, BMP-6 and BMP-7 proteins may act in concert with or perhaps synergistically with one another. The compositions of the invention are used to induce bone and/or cartilage formation. These compositions may also be used for wound healing and tissue repair.

Further compositions of the invention may include in addition to a BMP-5, BMP-6 or BMP-7 protein of the present invention at least one other therapeutically useful agent such as the proteins designated BMP-1, BMP-2 (also having been designated in the past as BMP-2A, BMP-2 Class I), BMP-3 and BMP-4 (also having been designated in the past as BMP-2B and BMP-2 Class II) disclosed in coowned International Publication W088/00205

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T Ii j I 90/11366 PCT/US90/01630 published 14 January 1988 and International Publication W089/10409 published 2 November 1989.

Other i,,erapeutically useful agents include growth factors such as epidermal growth factor (EGF) fibroblast growth factor (FGF), transforming growtfactors (TGF-a and TGF-) and platelet derived growth factor (PDGF).

The compositions of the invention may also include an appropriate matrix, for instance, for delivery and/or support of the composition and/or providing a surface for bone and/or cartilage formation. The matrix may proide solw release of the BMP protein and/or the appropriate environment for presentation of the BMP protein of the invention.

The compositions of the invention may be employed in methods for treating a number of bone and/or cartilage defects, and periodontal disease.

They may also be employed in methods for treating various types of wounds and in tissue repair.

These methods, according to the invention, entail administering a composition of the invention to a patient needing such bone and/or cartilage formation, wound healing or tissue repair. The method therefore involves administration of a therapeutically effective amount of a protein of the invention. These methods may also entail the administration of a protein of the invention in conjunction with at least one of the "BMP" proteins disclosed in the co-owned applications described above. In addition, these methods may also include the administration of a protein of the invention with other growth factors including EGF, FGF, TGFa, TGF-3, and PDGF.

Still a further aspect of the invention are

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WO 90/11366 PCT/US90/01630 DNA sequences coding for expression of a protein of the invention. Such sequences include the sequence of nucleotides in a 5' to 3' direction illustrated in Tables I V or DNA sequences which hybridize under stringent conditionE with the DNA sequences of Tables I V and encode a protein demonstrating ability to induce cartilage and/or bone formation.

Such cartilage and/or bone formation may be demonstrated in the rat bone formation assay described below. It :Is contemplated that these proteins may demonstrate activity in this assay at a concentration of 10 ug 500 ug/gram of bone formed. More particularly, it is contemplated that these proteins demonstrate the ability of lug of the protein to score at least +2 in the rat bone formation assay. Finally, allelic or other variations of the sequences of Tables I V whether such nucleotide changes result in changes in the peptide sequence or not, are also included in the present invention.

A further aspect of the invention provides vectors containing a DNA sequence as described above in operative association with an expression control sequence therefor. These vectors may be employed in a novel process for producing a protein i of the invention in which a cell line transformed with a DNA sequence directing expression of a protein of the invention in operative association with an expression control sequence therefor, is cultured in a suitable culture medium and a protein of the invention is recovered and purified therefrom. This claimed process may employ a number of known cells, both prokaryotic and eukaryotic, as host cells for expression of the polypeptide. The revovered BMP proteins are c -i t WO 90/11366 PCT/US90/01630 9 purified by isolating them from other proteinaceous Smaterials with which they are co-produced as well as from other contaminants.

Other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description and preferred embodiments thereof.

Detailed Description of the Invention Purified human BMP-5 proteins may be produced by culturing a host cell transformed with the DNA sequence of Table III. The expressed proteins are isolated and purified from the culture medium. Purified human BMP-5 proteins are expected to be characterized an amino acid sequence comprising amino acid #323 to #454 as shown in Table III. Purified BMP-5 human cartilage/bone proteins of the present invention are therefore produced by culturing a host cell transformed with a NA sequence comprising nucleotide #699 to nucleotide #2060 as shown in Table III or substantially homologous sequences operatively linked to a heterologous regulatory control sequence and recovering and purifying from the culture medium a protein comprising the amino acid sequence as shown in Table III from amino acid #323 to amino acid #454 or a substantially homologous sequence.

In further embodiments the DNA sequence Jcomprises the nucleotides encoding amino acids #323- #454. BMP-5 protrins may therefore be produced by culturing a host cell transformed with a DNA sequence comprising nucleotide #1665 to nucleotide #2060 as shown in Table III or substantially homologous sequences operatively linked to a c

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WO 90/11366 PCT/US90/01630 heterologous regulatory control sequence and recov. ring and purifying from the culture medium a protein comprising amino acid #323 to amino acid #454 as shown in Table III or a substantially homologous sequence. The purified human proteins are substantially free from other proteinaceous materials with which they are coproduced, as well as from other contaminants.

Purified BMP-5 bovine cartilage/bone proteins of the present invention are produced by culturing a host cell transformed with a DNA sequence comprising the DNA sequence as shown in Table I from nucleotide 8 to nucleotide 578 or substantially homologous sequences and recovering and purifying from the culture medium a protein comprising the amino acid sequence as shown in Table I from amino acid 9 to amino acid 140 or a substantially homologous sequence. The purified bovine proteins as well as all of the BMP proteins of the invention, are substantially free from other protcinaceous materials with which they are co-produced, as well as from other contaminants.

Purified human BMP-6 proteins may be produced by culturing a host cell transformed with the DNA sequence of Table IV. The expressed proteins are isolated and purified from the culuture medium.

Purified human BMP-6 proteins of the invention are expected to be characterized by an amino acid sequence comprising amino acid #382 to #513 as set forth in Table IV. These purified BMP-6 human cartilage/bone proteins of the present invention are therefore produced by culturing a host cell transformed with a DNA sequence comprising nucleotide #160 to nucleotide #1698 as set forth WO 90/11366 PCT/US90/01630 11 in Table IV or substantially homologous sequence operatively linked to a hetero], jus regulatory control sequence and recovering, isolating and purifying from the culture medium a protein comprising amino acid #382 to amino acid #513 as i. set forth in Table IV or a substantially homologous sequence.

Further embodiments may utilize the DNA sequence comrising the nucleotides encoding amino acids #382 #513. Purified human BMP-6 proteins may therefore be produced by culturing a host cell transformed with the DNA sequence comprising nucleotide #1303 to #1698 as set forth in Table IV or substantially homologous sequences operatively linked to a heterologous regulatory control sequence and recovering and purifying from the culture medium a protein comprising amino acid #382 to #513 as set forth in Table IV or a substantially homologous sequence. The purified human BMP-6 proteins are substantially free from other proteinaceous materials with which they are coproduced, as well as from other contaminants.

Purified BMP-6 bovine cartilage/bone protein of the present invention are produced by culturing a host cell transfc-med with a DNA seqieon' comprising nucleotide #361 to nucleotide #666 a& i set forth in Table II or substantially homologous sequences and recovering from the culture medium a protein comprising amino acid #121 to amino acid #222 as set forth in Table II or a substantially homologous sequence. In another embodiment the bovine protein is produced by culturing a host cell transformed with a sequence comprising nucleotide #289 to #666 of Table II and rcovering and purifying a protein comprising amino acid #97 to c WO 90/11366 PCT/US90/01630 12 j! !ami n acid #222. The purified BMP-6 Dcvine proteins are substantially free from other proteinaceous materials with which they are coproduced, as well as from other contaminants.

Purified human BMP-7 proteins may be produced by culturing a host cell transformed with the DNA sequence of Table V. The expressed proteins are isolated and purified from the culture medium.

Purified human BMP-7 proteins are expec 4 id to be characterized by an amino acid sequence comprising amino acid #300-#431 as shown in Table V. These purified BMP-7 human cartilage/bone proteins of the present invention are therefore produced by culturing a host cell transformed with a DNA sequence comprising nucleotide #97 to nucleotide #1389 as shown in Table V or Rubstantially homologous sequences operatively linked to a heterologous regulatory control sequence and recovering, isolating ai.d purifying ftcm the culture medium a protein comprising the amino acid sequence as shown in Table V from amino acid #300 to anin, acid #431 or a substantially homologous sequence.

Further emodiments may utilize the DNA sequence comprising the nucleotides encoding amino acids #300 #431. Purified BMP-7 proteins may be produced by culturing a host cell transformed with a DNA comprising the DNA sequence as shown in Table V from nucleotide #994 #1389 or substantially homologous sequences operatively linked to a heterologous rcgualtory control sequence and recovering, and purifying from the culture medium a protein comprising the amino acid sequence as showr in Table V from amino aCid #300 to amino acid #431 or a substantially homologous sequence, The

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i ,S i 'i i! WO 90/11366 PCT/US90/01630 13 purified human BMP-7 proteins are substantially free from other proteinaceous materials from which they are co-produced, as wel' s from other contaminants.

BMP-5, BMP-6 and BMP-7 proteins 'may be further characterized by the ability to demonstrate cartilage and/or bone formation activity. This activity may be demonstrated, for example, in the rat bone formation assay as described in Example III. It is further contemplated that these proteins demonstrate activity in the assay at a concentration of 10 pg 500 Ig/gram of bone formed. The proteins may be further characterized by the ability of lpg to score at least +2 in this assay using either the uriginal or modified scoring method descirbed further herein below.

BMP-6 and BMP-7 proteins may be further characterized by an apparent molecular weight of 28,000-30,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Under reducing cond 4 .tions in SDS-PAGE the protein electrophoresis witn a molecular weight of approximately 14,000-20,000 dalton, The proteins provided herein also include factors encoded by the sequences similar to those of Tables I V but into which modifications are naturally provided ke.g. allelic variations in the nucleotide sequence which may result in amino acid changes in the polypeptide) or deliberately engineered. Similarly, synthetic polypeptides which wholly or partially duplicate continuous sequences of the amino acid residues of Tables I- V are encompassed by the invention. These sequences, by virtue of sharing primary, secondary, or tertiary structural and conformational WO 90/11366 PCT/US90/01630 14 characteristics with other cartilage/bone proteins of the invention may possess bone and/or cartilage growth factor biological properties in common therewith. Thus, they may be employed as biologically active substitutes for naturallyoccurring proteins in therapeutic processes.

Other specific mutations of the sequences of the proteins of the invention described herein involv,! modifications of a glycosylation site.

These modification may involve O-linked or N-linked glycosylation sites. For instance, the absence of glycosylation or only partial glycosylation results from amino acid subst .tution or deletion at the asparagine-llnked glycosylation recognition sites present in the sequences of the proteins of the invention, as shown in Table I V. The asparagine-linked glycosylation recognition sites comprise tripeptide sequences which are specifically recognized by appropriate cellular glycosylation enzymes. These tripeptide sequences are either asparagine-X-threonine or asparagine-Xserine, where X is usually any amino acid. A variety of amino acid substitutions or deletions at one or both of the first or third amino acid positions of a glycosylation recognition site (and/or amino acid deletion at the second position) results in ,.on-glycosylation at the modified tripeptide sequence. Expression of such altered i nucleotide sequences produces variants which are not glycosylated at that site.

The present invention also encompasses the novel DNA sequences, free of association with DNA sequences encoding other proteinaceous materials, anid coding on expression for the proteins of the invention. These DNA sequences include those WO 90/11366 PC'/US90/01630 depicted in Tables I V in a 5' to 3' direction.

Further included are those sequences which hybridize under stringent hybridization conditions [see, T. Maniatis et al, Molecular Cloning 'A Laboratory Manual), Cold Spring Harbor Laboratory (1982), pages 387 to 389] to the DNA sequence of Tables I V and demonstrate cartilage and/or bone formation activity in the rat bone formation assay.

An example of one such stringent hybridization condition is hybridization at[6~ 4 x SSC at 65 0

C,

followed by a washing in 0.1 x SCC at 65 0 C for an hour. Alternatively, an exemplary stringent hybridization condition is in 50% formamide, 4 x SCC at 42 0

C.

Similarly, DNA sequences which encode proteins similar to the protein encoded by the sequences of Tables I V, but which differ in codon sequence due :o the degeneracies of the genetic code or allelic variations (naturally-occurring base changes in the species population which may or may not -esult in an amino acid change) also encode the proteins of the invention described herein.

Variations in the DNA sequences of Tables I V which are caused by point mutations or by induced modifications (including insertion, deletion, and substitution) to enhance the activity, half-life or production of the polypeptides encoded thereby are also encompassed in the invention.

In a further aspect, the invention provides a method for obtaining related human proteins or other mammalian BMP-5, BMP-6 and BMP-7 proteins.

One method for obtaining such proteins entails, for instance, utilizing the human BMP-5, BMP-6 and BMP- 7 coding sequence disclosed herein to probe a human genomic library using standard techniques for WO 90/11366 PCT/US90/01630 16 the human gene or fragments thereof. Sequences thus identified may also be used as probes to identify a human cell line or tissue which synthesizes the analogous cartilage/bone protein.

A cDNA library is synthesized and screened with probes derived from the human or bovine coding sequences. The human sequence thus identified is transformed into a host cell, the host cell is cultured and the protein recovered, isolated and purified from the culture medium. The purified protein is predicted to exhibit cartilage and/or bone formation activity in the rat bone formation assay of Example III.

Another aspect of the present invention provides a novel method for producing the BMP-6 and BMP-7 proteins of the invention. The method of the present invention involves culturing a suitable cell or cell line, which has been transformed with a DNA sequence as described above coding for expression of a protein of the invention, under the control of known regulatory sequences. Regulatory sequences include promoter fragments, terminator fragments and other suitable i sequences which direct the expression of the 25 protein in an appropriate host cell. Methods for culturing suitable cell lines are within the skill i of the art. The transformed cells are cultured and the BMP proteins expressed thereby are recovered, i isolated and purified from the culture medium U 30 using purification techniques known to those skilled in the art. The purified BMP proteins are substantially free from other proteinaceous materials with which they are co-produced, as well as other contaminants. Purified BMP proteins of the invention are substantially free from Na -WOM out ri il WO 90/11366 PCT/US90/01630 17 materials with which the proteins of the invention exist in nature.

Suitable cells or cell lines may be mammalian cells, such as chinese hamster ovary cells (CHO).

5 The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, Gethinq and Sambrook, Nature, 293:620-625 (1981), or alternativ,-.y, Kaufman et al, Mol. Cell. Biol., .5(7):1750-1759 (1985) or Howley et al, U.S. Patent 4,419,446. Other suitable mammaliar, cell lines include but are not limited to the monkey COS-1 cell line and the CV-1 cell line.

Bacterial cells may also be suitable hosts.

For example, the various strains of E. coli HB101, MC1061) are well-known as host cells in the field of biotechnology. Various strains of B. subtilis, Pseudomonas, other bacilli and the like may also be employed in this method.

Many strains of yeast cells known to those skilled in the art may also be available as host cells for expression of the polypeptides of the pre3er' invention. Additionally, where desired, insect cells may be utilized as host cells in the method of the present invention. See, e.g. Miller et al, Genetic Engineering, 8:277-298 (Plenum Press 1986) and references cited therein.

Another aspect of the present invention provides vectors for use in the method of expression of the proteins cf the invention. The vectors contain the novel DNA sequences which code for the BMP-5, BMP-6 and BMP-7 proteins of the invention. Additionally, the vectors also contain appropriate expression control sequences permitting WO 90/11366 PCT/US90/01630 18 expression of the protein sequences.

Alternatively, vectors incorporating truncated or modified sequences as described abovu are also embodiments of the present invention and useful in the production of the proteins of the invention.

The vectors may be employed in the method of transforming cell lines and contain selected regulatory sequences in operative association with the DNA coding sequences of the invention which are capable of directing the replication and expression thereof in selected host cells. Useful regulatory sequences for such vectors are known to those skilled in the art and may be selected depending upon the selected host cells. Such selection is routine and does not form part of the present invention. Host cells transformed with such vectors and progeny thereof for use in producing BMP-5, BMP-6 and BMP-7 proteins are also provided by the invention.

One skilled in the art can construct mammalian expression vectors by employing the DNA sequences of the invention and known vectors, such as pCD [Okayama et al., Mol. Cell Biol., 2:161-170 (1982)] and pJL3, pJL4 [Gough et al., EMBO 4:645-653 (1985)]. Similarly, one skilled in the art could manipulate the sequences of the invention by eliminating or replacing the mammalian regulatory sequences flanking the coding sequence with bacterial sequences to create bacterial vectors for intracellular or extracellular expression by bacterial cells. For example, the coding sequences could be further manipulated (e.g.

ligated to other known linkers or modified by deleting non-coding sequences there-from or altering nucleotides therein by other known WO 90/11366 PCT/US90/01630 19 techniques). The modified coding sequence could then be inserted into a known bacterial vector using procedures such as described in T. Taniguchi et al., Proc. Natl Acad. Sci. USA, 77:5230-5233 (1980). This exemplary bacterial vector could then be transformed into bacterial host cells and a Iprotein of the invention expressed thereby. For a j strategy for producing extracellular expression of a cartilage and/or bone protein of the invention in bacterial cells., see, e.g. European patent application EPA 177,343.

Similar manipulations can be performed for the construction of an insect vector [See, e.g.

procedures described in published European patent application 155,476] for expression in insect cells. A yeast vector could also be constructed employing yeast regulatory sequences for intracellular or extracellular expression of the factors of the present invention by yeast cells.

[See, procedures described in published PCT application W086/00639 and European patent application EPA 123,289].

A method for producing high levels of a protein of the invention from mammalian cells involves the construction of cells containing multiple copies of the heterologous gene encoding proteins of the invention. The heterologous gene may be linked to an amplifiable marker, e.g. the dihydrofolate reductase (DHFR) gene for which cells containing increased gene copies can be selected for propagation in increasing concentrations of methotrexate (MTX) according to the procedures of Kaufman and Sharp, J. Mol. Biol., 159:601-629 (1982). This approach can be employed with a number of different cell types.

WO 90/11366 PCT/US00/01630 For instance, a plasmid containing a DNA sequence for a protein of the invention in operative association with other plasmid sequences enabling expression thereof and the DHFR expression plasmid pAdA26SV(A)3 [Kaufman and Sharp, Mol. Cell. Biol., 2:1304 (1982)] may be cointroduced into DHFR-deficient CHO cells, DUKX-BII, by calcium phosphate coprecipitation and transfection, electroperation or protoplast fusion.

DHFR expressing transformants are selected for growth in alpha media with dialyzed fetal calf serum, and subsequently selected for amplification by growth in increasing concentrations of MTX (sequential steps in 0.02, 0.2, 1.0 and 5uM MTX) as described in Kaufman et al., Mol Cell Biol., 5:1750 (1983). Protein expression should increase with increasing levels of MTX resistance.

Transformants are cloned, and the proteins of the invention are recovered, isolated, and purified from the culture medium. Characterization of expressed proteins may be carried out using stnadard techniques. For instance, characterization may include pulse labeling with

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methionine or cysteine, or polyacrylamide gel electrphoresis. Biologically active protein expression is monitored by the Rosen-modified Sampath Reddi rat bone formation assay described above in Example III. Similar procedures can be followed to produce other related proteins.

A protein of the present invention, which induces cartilage and/or bone formation in circumstances where bone and/or cartilage is not normally formed, has application in the healing of bone fractures and cartilage defects in humans and other animals. A preparation employing a protein a WO 90/11766 PCT/UJS90/01630 21 of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by 'n osteogenic agent contributes to the repair of ngenital, trauma I. induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery. A protein of the invention may be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells. A variety of osteogenic, cartilage-inducing and bone inducing factors have been described. See, e.g. European Patent Applications 148,155 and 169,016 for discussions thereof.

The proteins of the invention may also be used in wound healing and related tissue repair. The types of wounds include, but are not limited to burns, incisions and ulcers. See, e.g. PCT Publication W084/01106 for discussion of wound healing and related tissue repair.

A further aspect of the invention includes therapeutic methods and composition for repairing fractures and other conditions related to bone and/or cartilage defects or periodontal diseases.

In addition, the invention comprises therapeutic methods and compositions for wound healing and tissue repair. Such compositions comprise a therapeutically effective amount of at least one of the BMP proteins BMP-6 and BMP-7 of the invention in admixture with a pharmaceutically acceptable vehicle, carrier or

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WO 90/11366 matrix.

PCT/US90/01630 It is expected that the proteins of the invention may act in concert with or perhaps synergistically with one another or with nther related proteins and growth factors. Therapeutic methods and compositions of the invention therefore comprise one or more of the proteins of the present invention. Further therapeutic methods and compositions of the invention therefore comprise a therapeutic amount of at least one protein of the invention with a therapeutic amount of at least one of the other "BMP" proteins BMP-1, BMP-2, BMP-3 and BMP-4 disclosed in co-owned Published International Applications W088/00205 and W089/10409 as mentioned above. Such methods and compositions of the invention may comprise proteins of the invention or portions thereof in combination with the above-mentioned "BMP" proteins or portions thereof.

Such combination may comprise individual separate molecules of the proteins or heteromolecules such as heterodimers formed by portions of the respective proteins. For example, a method and composition of the invention may comprise a BMP protein of the present invention or a portion thereof linked with a portion of another "BMP" protein to form a heteromolecule.

Further therapeutic methods and compositions of the invention comprise the proteins of the invention or portions thereof in combination with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet derived r W090/11366 ~d~l- PCT/US90/01630 ji growth factor (PDGF), transforming growth factors (TGF-a and TGF-) K-fibroblast growth factor (kFGF), parathyroid hormone (PTH), leukemia inhibitory factor (LIF/HILDA, DIA) and insulin-like growth factor (IGF-I and IGF-II). Portions of these agents may also be used in compositions of the invention.

The preparation and formulation of such physiologically acceptable protein compositions, having due regard to pH, isotonicity, stability and the like, is within the skill of the art. The therapeutic compositions are also presently valuable for veterinary applications due to the apparent lack of species specificity in cartilage and bone growth factor proteins. Domestic animals and thoroughbred horses in addition to humans are desired patients for such treatment wi*h the proteins of the present invention.

The therapeutic method includes administering the composition topically, systemically, or locally as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of cartilage and/or bone or tissue damage. Topical administration may be suitable for wound healing and tissue repair.

Preferably for bone and/or cartilage formation, the composition would include a matrix capable of delivering the BMP proteins of the invention to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartiJage and optimally capable of being resorbed into the body. The matrix may provide

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WO 90/11366 PCT/US90/01630 24 slow release of the BMP proteins or other factors comprising the composition. Such matrices may be formed of materials presently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions of the invention will define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid and polyanhydrides. Other potential materials are biodegradable and biologically well defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are nionbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as Spolylactic acid and hydroxyapatite or collagen and tricalciumphosphate. The bioceramics may be altered in composition, such as in calciumaluminate-phosphate and processing to alter pore Ii size, particle size, particle shape, and i bi degradability.

The dosage regimen will be determined by the attending physician considering various factors which modify the action of the proteins of the invention. Factors which may modify the action of the proteins of the invention include the amount of bone weight desired to be forriied, the site of bone c

I

4I WO 90/11366 PCT/US90/01630 damage, the condition of the damaged bone, the size of a wound, type of damaged tissue, the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors.

The dosage may vary with the type of matrix used in the reconstitution and the type or types of bone i and/or cartilage proteins present in the composition. The addition of other known growth factors, such as EGF, PDGF, TGF-a, TGF-9, and IGF-I and IGF-II to the final composition, may also effect the dosage.

Progress can be monitored by periodic assessment of cartilage and/or bone growth and/or repair. The progress can be monitored, for example, using x-rays, histomorphometric determinations and tetracycline labeling.

The following examples illustrate practice of the present invention in recovering and characterizing bovine cartilage and/or bone proteins of the invention and employing these proteins to recover the corresponding human protein or proteins and in expressing the proteins via recombinant techniques.

EXAMPLE I Isolation of Bovine Cartilage/Bone Inductive Protein Ground bovine bone powder (20-120 mesh, Helitrex) is prepared according to the procedures of M. R. Urist et al., Proc. Natl Acad. Sci USA, 70:3511 (1973) with elimination of some extraction steps as identified below. Ten kgs of the ground powder is deminerali7,ed in successive changes of 0.6N HC1 at 41C over a 43 hour period with vigorous W091111366PCT/US90/01630 26 stirring. The resulting suspensAi on is extracted for 16 hours at 44C with 50 liters of 2M4 CaC1 2 and ethyl enediamine-tetraacptic acid [EDTA], and followed by -extraction for 4 hours in 50 liters of O.B14 EDTA. Th1 e residue is washed three t'imes with dist.Llled ioater before its resuspension in liters of~ 414 guanidine 'ydrochloride [GuCl] Tris (pH 1mM N-ethylmaleimide,~ 1mM iodoacetamide, 1mM phenylmethylsulfonyl fluorine as desct.ibed in Clin. OrthoD. Rsl-.e. 171: 213 (1982) After 16 to 20 hours the supernatant is removed and replaced with aro-t-her 10 liters of GuCl bu~fer. The resi"due is extractec. f or another 24A hours.

lfi The crude GuCi extracts are. combined, concentrated approximately 2-0 times on a Pellicon apparatus with a 10,000 molecular weight cut-off membrane, and then dialyzed in 50mM Tris, 0.lM NaCl, 614 urea (p147.2), tb~a starting buffer for the first column. After Pxcensive dialysis the protein is loaded on a 4 liter DEAE cellulose column and the unbound fractions are collected.

The unbound fractions are concentrated and dialyzed against 50mM NaAc, 50mM NaCl (pH 4.6) in 6M urea. 'The unbound fractions are ipplied to a carboxymethyl cellulose column. Protein not bound to the column is removed by extensive washing with starting buffer, and the material containing protein having bone and/or cartilarje formnation activity as measured by the Rosen-modified Sam}~ath- Rd d fromy thescri~~bed i0n Exaple III5m Naio Red a described ino the~ colum bye0MN~,02m alw 6M urea(pH 4 ).Tepoenfo hase Ilto is cnetae 0 o4-flte diluted 5 times with 80MM KPO 4 614 urea A .Mi UMMMUMONOWANUM 4 1 I- -M WO 90/11366 WO 90/ 1366PCT/US90/0I 630 S WO 90/i 1366 PCT/US90/01630 27 The pH of the solution is adjusted to 6.0 with 500mM K 2 HP0 4 The sample is applied to an hydroxylapatite column (LKB) equilibrated in KP0 4 6M urea (pH6.0) and all unbound protein is removed by washing the column with the same buffer. Protein having bone and/or cartilage formation activity is eluted with 100mM KP04 (pH7.4) and 6M urea.

The protein is concentrated approximately times, and solid NaCl added to a final concentration of 0.15M. This material is applied to a heparin Sepharose column equilibrated in KP0 4 150nmM NaCI, SM urea (pH7.4). After extensive washing of the column with scarting buffer, a protein with b;ie and/or cartilage inductive activity is eluted by 50mM KPO4, 700mM NaCI, 6M urea (pH7.4). This fraction is concentrated to a minimum volume, and 0.4ml aliquots are applied to Superose 6 and Superose 12 columns connected in series, equilibrated with 4M GuCl, Tris (pH7.2) and thu columns develope- at a flow rate of 0.25ml/min. The protein demonstrating bone and/or cartilage inductive activity corre.sponds to an approximate 30,000 dalton protein.

The above fractions from the supe::ose columns are pooled, dialyzed against 50mM NaAc, 6M urea (pH4.6), and applied to a Pharmacia MonoS HR column. TSQ cclumn is developed with a gradient to 1.OM NaCl, 50iM NaAc, 6M urea (pH4.6). Act.ve bone and/or cartilage formation fractions are pooled.

The material is applied to a 0.46 x 25cm Vydac C4 column in 0.1% TFA and the column developed with a grad' .c to 90% acetonitrile, 0.1% TFA (31.5% acetonitrile, 0.1% TFA to 49.5% acetonitrile, 0.1% I 35 TFA in 60 minutes at lml per minute). Active

A

WO 90/11366 PCT/US90/01630 28 material is eluted at approximately 40-44% acetonitrile. Fractions were assayed for cartilage and/or bone formation activity.The active material is further fractionated on a MonoQ column. The protein is dialyzed against 6M urea, diethanolamine, pH 8.6 and then applied to a 0.5 by cm MonoQ column (Pharmacia) which is developed with a gradient of 6M urea, 25mM diethanolamine, pH 8.6 and 0.5 M NaCl, 6M urea, 25mM diethanolamine, pH 8.6. Fractions are brought to pH3.0 with trifluoroacetic acid (TFA). Aliquots of the appropriate fractions are iodinated by one of the fol2.-wing methods: P. J. McConahey et al, Int. Arch. Allergy, 29:185-189 (1966); A. E. Bolton et al, Biochem 133:529 (1973); and D. F.

Bowen-Pope, J. Biol. Chem., 237:5161 (1982). The iodinated proteins present in these fractions are analyzed by SDS gel electrophoresis.

EXAMPLE II Characterization of Bovine Carti.ace/Bone Inductive Factor A. Molecular Weight Approximately 5g protein from Example I in 6M urea, 25mM die,"anolamine, pH 8.6, approximately 0.3 M NaCl is made 0.1% with respect to SDS and dialyzed against 50 mM tris/HC1 0.1% SDS pH 7.5 for 16 hrs. The dialyzed material is then i electrophorectically concentrated against a dialysis membrane [Hunkapillar et al Meth. Enzymol.

91: 227-236 (1983)] with a small amount of I 125 labelled counterpart. This material (volume approximately 1001) is loaded onto a 12% polyacrylamide gel and subjected to SDS-PAGE [Laemmli, U.K. Nature, 227:680-685 (1970)] without

WO_

II

0/1I a I1366 PC/US90/01 630 reducing the sample with dithiothreitol. The molecular weight is determined relative to prestained molecular weight standards (Bethesda Research Labs). Following autoradiography of the unfixed gel the approximate 28,000-30,000 dalton band is excised and the protein electrophoretically eluted from the gel (Hunkapillar et al supra) Based on similar purified bone fractions as described in the co-pending "BMP" applications described above wherein bone and/or cartilage activity is found in the 28,000-30,000 region, it is inferred that this band comprises bone and/or cartilage inductive fractions.

B. Subunit Characterization The subunit composition of the isolated bovine bone protein is also determined. The eluted protein described above is fully reduced and alkylated in 2% SDS using iodoacetate and standard procedures and reconcentrated by electrophoretic packing. The fully reduced and alkylated sample is then further submitted to SDS-PAGE on a 12% gel and the resulting approximate 14,000-20,000 dalton region having a doublet appearance located by autoradiography of the unfixed gel. A faint band remains at the 28,000-30,000 region. Thus the 28,000-30,000 dalton protein yields a broad region of 14,000-20,000 which may otherwise also be interpreted and described as comprising two broad bands of approximately 14,000-16,000 and 16,000- 20,000 daltons.

EXAMPLE III Rosen Modified Sampath-Reddi Assay A modified version of the rat bone i t WO 90/11366 PCT/US90/01630 formation assay described in Sampath and Reddi, Proc. Natl. Acad. Sci. 80:6591-6595 (1983) is used to evaluate bone and/or cartilage activity of the proteins of the invention. This modified assay is herein called the Rosen-modified Sampath- S Reddi assay. The ethanol precipitation step of the Sampath-Reddi procedure is replaced by dialyzing (if the composition is a solution) or diafiltering (if the composition is a suspension) the fraction to be assayed against water. The solution or suspension is then redissolved in 0.1 TFA, and the resulting solution added to 20mg of rat matrix.

A mock rat matrix sample not treated with the protein serves as a control. This material is frozen and lyophilized and the resulting powder enclosed in #5 gelatin apsules. The capsules are i implanted subcutaneously in the abdominal thoracic area of 21 49 day old male Long Evans rats. The implants are removed after 7 14 days. Half of each implant is used for alkaline phosphatase analysis [See, A. H. Reddi et al., Proc. Natl Acad Sci., 69:1601 (1972)].

The other half of each implant is fixed and processed for histological analysis.

Glycolmethacrylate sections (lpm) are stained with Von Kossa and acid fuschin or toluidine blue to score the amount of induced bone and cartilage formation present in each implant. The terms +1 through +5 represent the area of each histological A section of an implant occupied by new bone and/or cartilage cells and newly formed bone and matrix.

Two scoring methods are herein described. In the first scoring method a score of +5 indicates that greater than 50% of the implant is new bone and/or cartilage produced as a direct result of protein in WO9 90/11B366 PCT/US90/01630 i j i i i j :i

I

the implant. A score of +2 and +1 would indicate that greater than 40%, 30%, 20% and respectively of the implant contains new cartilage and/or bone. The second scoring method 5 (which hereinafter may be referred to as the modified scoring method) is as follows: three nonadjacent sections are evaluated from each implant and averaged. indicates tentative identification of cartilage or bone; indicates >10% of each section being new cartilage or bone; The scores of the individual implants are Labulated to indicate assay variability.

It is contemplated that the dose response nature of the cartilage and/or bone inductive protein containing samples of the matrix samples will demonstrate that the amount of bone and/or cartilage formed increases with the amount of cartilage/bone inductive protein in the sample. It is contemplated that the control samples will not result in any bone and/or cartilage formation.

As with other cartilage and/or bone inductive proteins such as the above-mentioned "BMP" proteins, the bone and/or cartilage formed is expected to be physically confined to the space occupied by the matrix. Samples are also analyzed by SDS gel electrophoresis and isoelectric focusing followed by autoradiography. The activity is correlated with the protein bands and pl. To estimate the purity of the rotein in a particular fraction an extinction coefftcient of 1 OD/i,t-cm is used as an estimate for protein and the protein is run on SDS-PAGE followed by silver staining or radioiodination and autoradiography.

WO 90/11366 PCT/US90/01630 32 r-XAMPLE IV A. Bovine Protein Composition The gel slice of the approximate 14,000l 20,000 dalton region described in Example IIB is fixed with methanol-acetic acid-water using standard procedures, briefly rinsed with water, then neutralized with 0.1M ammonium bicarbonate.

Following dicing the gel slice with a razor blade, the protein is digested from the gel matrix by adding 0.2 pg of TPCK-treated trypsin (Worthington) and incubating the gel for 16 hr. at 37 degrees centigrade. The resultant digest is then subjected to RPHPLC using a C4 Vydac RPHPLC column and 0.1% TFA-water 0.1% TFA water-acetonitrile gradient.

The resultant peptide peaks were monitored by UV absorbance at 214 and 280 nm and subjected to direct amino terminal amino acid sequence analysis using an Applied Biosystems gas phase reguenator (Model 470A). One tryptic fragment is isolated by standard procedures having the following amino acid sequence as represented by the amino acid standard three-letter symbols and where "Xaa" indicates an unknown amino acid the amino acid in parentheses indicates uncertainty in the sequence: Xaa-His-Glu-Leu-Tyr-Val-Ser-Phe-(Ser) The following four oligonucleotide probes are designed on the basis of the amino acid sequence of the above-identified tryptic fragment and synthesized on an automated DNA synthesizer.

PROBE GTRCTYGANATRCANTC PROBE GTRCTYGANATRCANAG r _~1~111 WO 90/11366 PCT/US90/01630 33 PROBE GTRCTYAAYATRCANTC PROBE GTRCTYAAYATRCANAG i i The standard nucleotide symbols in the above identified probes are as follows: A,adenosine; C,cytosine; G,guanine; T,thymine; N, adenosine or cytosine or guanine or thymine; R,adenosine or guanine; and Y,cytosine or thymine.

Each of the probes consists of pools of oligonucleotides. Because the genetic code is degenerate (more than one codon can code for the same amino acid), a mixture of oligonucleotides is synthesized that contains all possible nucleotide sequences encoding the amino acid sequence of the tryptic. These probes are radioactively labeled and employed to screen a bovine cDNA library as described below.

B. Bovine Poly(A) containing RNA is isolated by oligo(dT) celluL.ose chromatography from total RNA isolated from fetal bovine bone cells by the method of Gehron-Robey et al in current Advances in Skeletogenesis, lisviar Science Publishers (1985).

The total RNA was obtained from Dr. Marion Young, National Institute of Dental Research, National Institutes of Health. A cDNA library is made in lambda gtlO (Toole et al supra) and plated on plates at 8000 recombinants per plate. These recombinants (400,000) are screened on duplicate nitrocellulose filters with a combination of Probes 1, 2, 3, and 4 using the Tetramethylammonium chloride (TMAC) hybridization procedure [see Wozney et al Science, 242: 1528-1534 (1988)]. Twenty-

C-

WO 90/11366 PC/US90/01 630 eight positives are obtained and are replated for secondaries. Duplicate nitrocellulose replicas again are made. One set of filters are screened with Probes #1 and the other with Probes #3 and Six positives are obtained on the former, 21 Spositives with the latter. One of the six, called is plague purified, a phage plate stock made, and bacteriophage DNA isolated. This DNA is digested with EcoRI and subcloned into M13 and pSP65 (Promega Biotec, Madison, Wisconsin) [Melton, et al. Nucl. Acids Res. 12: 7035-7056 (1984)]. The DNA sequence and derived amino acid sequence of this fragment is shown in Table I.

DNA sequence analysis of this fragment in M13 indicates that it encodes the desired tryptic peptide sequence set forth above, and this derived amino acid sequence is preceded by a basic residue (Lys) as predicted by the specificity of trypsin.

The underlined portion of the sequence in Table I from amino acid #42 to #48 corresponds to the tryptic fragment identified above from which the oligonucleotide probes are designed. The derived amino acid sequence Ser-Gly-Ser-His-Gln-Asp-Ser- Ser-Arg as set forth in Table I from amino acid to #23 is noted to be similar to a tryptic fragment sequence Ser-Thr-Pro-Ala-Gln-Asp-Val-Ser-Arg found in the 28,000 30,000 dalton purified bone preparation as described in the "BMP'" Publications 1 W088/00205 and W089/10409 mentioned above. This fragment set forth in Table I is a portion of the DNA sequence which encodes a bovine BMP-5 protein.

The DNA sequence shown in Table I indicates an open reading frame from the 5' end of the clone of 420 base pairs, encoding a partial peptide of 140 amino acid residues (the first 7 nucleotides are of the !L L -L WO090/11366 PCI] US9O/0 1630 adaptors used in the cloning procedure). An inframne stop codon (TAA) indicates that this clone encodes the carboxy-terminal part of bovine 1 WO 90/11366 PC'r/US90/01 630 36 TABLE I 1 TCTAGAGGTGAGAGCAGCCLA~kAGAGAAAAAATCAAAACCGCAATAAATCCGGCTCTCAT 61 LeuGluValArgAlaA] -,.AsnLysArgLysAsnGlnAsnArgAsnLysSerGlVSerHis 62 CAGGACTCCTCTAGAATGTCCAGTGTTGGAGATTATAACACCAGTGAACAAAAACAAGCC 1 GlnAspSerSerArcgMetSerSerValGlyAspTyrAsnTlirSerGluGlnLysGlnAla (23) 122 TGTAAAAAG CATGAACTCTATGTGAGTTTC CGGGATCTGGGATGGCAGGACTGGATTATA 1 CysLysLysHi sGluLeuTyrValS erPheArgAspLeuGlyTrpGlnAspTrpl lelle (42) (48) 182 GCACCAGAAGGATATGCTGCATTTTATTGTGATGGAGAATGTTCTTTTCCACTCAATGCC 2 Al aProGluGlyTyrAlaAlaPheTyrCysAspGlyGluCysSerPheProLeuAsnAl a 242 CATATGAATGCCACCAATCATGCCATAGT'XCAGACTCTGGTTCACCTGATGTTTCCTGAC 3 HisMetAsnAl aThrAsnl-isAlalleValGlnThrLeuValHisLeuMetPheProAsp 302 CACGTACCAAAGCCTTGCTGCGCGACAAACAAACTAAATGCCATCTCTGTGTTGTACTTT 3 HisValProLysProCysCysAlaThrAsnLysLeuAsnAlal leSerValLeuTyrPhe 362 GATGACAGCTCCAATGTCATTTTGAAAAAGTACAGAAATATGGTCGTGCGTTCGTGTGGT 4 AspAspSerSerAsnVa.IleLeuLysLysTyrArgAsn~etValVa lArgSerCyscly 422 TG CCACTAATAGTG CATAATkATGGTAATAAGAAAAAAGATCTGTATGGAGGTTTATGA 4 CysHisEnd (140) 481 CTACAATAAAAAATATCTTTCGGATAAAAGGGGAATTTAATAAAATTACTCTGGCTCATT 541 TCATCTCTGTAACCTATGTACAAGAGCATGTATATAGT 578 l-1. t WO 90/11366 PCT/US90/01630 37 1 C. Bovine BMP-6 The remaining positive clones (the second set containing 21 positives) isolated with Probes #1, and #4 described above are screened with HEL5 and a further clone is identified that hybridizes under reduced hybridization conditions SSC, 0.1% SDS, 5X Denhardt's, 100 pg/ml salmon sperm DNA standard hybridization buffer (SHB) at 0 C, wash in 2XSSC 0.1% SDS at 65 0 This clone is plaque purified, a phage plate stock made and bacteriophage DNA isolated. The DNA sequence and derived amino acid sequence of a portion of this clone is shown in Table II.. This sequence represents a portion of the DNA sequence encoding a bovine BMP-6 cartilage/bone protein of the invention.

The first underlined portion of the sequence in Table II from amino acid #97 amino acid #105 corresponds to the tryptic fragment found in the 28,000-30,000 dalton purified bovine bone preparation (and its reduced form at approximately 18,000-20,000 dalton reduced form) as described in the "BMP" Publications W088/00205 and W089/10409 mentioned above. The second underlined sequence in Table II from amino acid #124 amino acid #130 corresponds to the tryptic fragment identified above from which the oligonucleotide probes are designed.

The DNA sequence of Table II indicates an open reading frame of 666 base pairs starting from the end of the sequence of Table II, encoding a partial peptide of 222 amino acid residues. An inframe stop codon (TGA) indicates that this clone encodes the carboxy-terminal part of a bovine BMP-6 C WO90/11366 PCT/US90/01630 38 j protein. Based on knowledge of other BMP proteins and other proteins in the TGF-p family, it is predicted that the precursor polypeptide would be cleaved at the three basic residues (ArgArgArg) to yield a mature peptide beginning with residue 90 or 91 of the sequence of Table II.

c 1 WO 90/11366PC!USO013 PCr/US90/01630 39 TABLE II 9 CIG GIG GGC Leu Leu Gly (1) ACG OT CT GIG CX "Q (GAj CG CGC 'IGG GIG GAG T=T GAO Thr Arg Ala 17-7 Tirp Ala .)er Giu Ala Gly Trp Lau Giu Phie Asp ATC AM CO ACM AGO AAC C= TGG =I ACT= CCGAG =A AAC A!IG GCG GI le Thr Ala U=r Ser Asn Teu Trp Val beau Thr Pro Gin His Asn MMT Gly teu 153 GAG MI AGO =I =T AMO =O GAT =C =I AGO A=O AGO OCT =GGGCCC COG =G Gin Leu Ser Val Vai Thx Arg Asp Gly 1-nu Ser le Ser Pro Gly Ala Ala Gly 2 C'7 =I GI =C AGG GAC C= 00 TMC GAC AAG CAS 00 TI GI CC TI TI Leu Val Gly Arg Asp Gly Pro Tyr Asp Lys Gixr Pro Phe IMT Val Ala Pne P'ne 225 261 AAG CO AGI GAG GM' =A =I =O AGI' CO CC= TCZ CGCCCC C OG Lys Ala Ser Giu Val His Vai Arg Ser Ala Arg Ser Ala Pro Gly Arg Arg Arg 279 315 GAG CAG GC W2C AAO G Gin Gin Ala Arg Asn Arg

TOOC

Ser (97) AGCC CG G AG GAO GIG T)G 11h.r Pro Ala Gin Aso Val Sar CGG GCO TOO AGO ~Ala Ser Ser (105) 369 378 CO TCA GAG Ala Ser Asp 387 TAO AAC AGO AGO GAG Tyr Asn Ser Ser Ciu 396 GIG AAG ACCG 'IC GO COC ILeu Lys n=r Ala Gys Arg (121) MAG OAT GAG GIC Lys H.LS Ciu Leg (1241 TMO GI AGOC G AG GAO GG Ty~r W 7 ai Ser Phe Gin Asp Lau Gly (130) =G GAG GAO AM MT CO =0 AAG C= Trp Gin Asp Thp le le Ala Pro Lys Gly MOCO CO A TA MCG =GAC GGA GAA =T rC TI OC MI AA GOA =A MG Tyr Ala Ala Asn Tryr Gys Asp Gly Ciu Gys Ser Phe Pro Le-u Asn Ala His MET 495 504 5i3 522 531 540 MACG C AGO AAC CAT C MTC GTG GAG AGO GIG GIT GAC CIC MTG AAG 000 GAG Asn Ala Thr Asn His Ala le Val Gin Thr Leu Val His Leu MET Asn Pro Glu WO 90/11366 PCr/US90/01630 TABLE II (page 2 of 2) 549 558 567 576 585 594 TAC GTC CCC AAA COG TGC WGC GCX CCC AM~ AA CIG MAC GCC ATC TOO GIG CTC Tyr Val Pro Lys Pro Cys Cys, Ala Pro M=r Lys Lu Asri Ala le Ser Val Lau 603 612 621 630 639 648 TfAC TIC GAC GhC MAC TCC AAT CIC AI'C Cjfl AAG A7C OGG AAC AG GTC GI!A Tyr Phe Asp. Asp Asn Ser Asn Val le Le, Lys Lys Ty~r Arg Asn MET~ Val. Val.

657 666 676 686 696 706 716 CGA GCG 'IGT GGG 'IG CAC TGACPCCGGG 'IG.AGIGC G(GACXCI GOACACACIG CCIGGAC=C Argi Ala Cys Gly Cys His (222) 72u 736 746 756 7G6 776 786 TGGATCACGT C03CUC]IAAG CCCACAGAGG CCCCOCGGC AC.-.GGAGGAG ACCCC2AGGC CACCI 1 0'GC 796 806 816 826 836 846 856

'IGCCI

T

IG CITITCC= AAQr-CAGACC CAAGGGACC CI7C=C CI'IGCICA CCGTGA=T 866 876 886 ,.GTGAGTAGC CA~TCGG=T TAWAGMGCAG CACTCGAG WO90/11366 PCT/US90/01630 41 EXAMPLE V A. Human Protein Composition i Human cell lines which synthesize BMP-5 and/or BMP-6 mRNAs are identified in the following manner.

RNA is isolated from a variety of human cell lines, selected for poly(A)-containing RNA by chromatography on oligo(dT) cellulose, electrophoresed on a formaldehyde-agarose gel, and transferred to nitrocellulose. A nitrocellulose replica of the gel is hybridized to a single stranded M13 3 2 P-labeled probe corresponding to the above mentioned BMP-5 EcoRI-BglII fragment containing nucleotides 1-465 of the sequence of Table I. A strongly hybridizing band is detected in the lane corresponding to the human osteosarcoma cell line U-20S RNA. Another nitrocellulose replica is hybridized to a single stranded M13 32plabeled probe containing the PstI-Smal fragment of bovine BMP-6 (correspondi'g to nucleotides 106- 261 of Table II). "t is found that several RNA species in the lane corresponding to U-20S RNA hybridize to this probe.

A cDNA Library is made in the vector lambda ZAP (Stratagene) from U-20S poly(A)-containing RNA using established techniques (Toole et al.).

750,000 recombinants of this library are plated and duplicate nitrocellulose replicas made. The SmaI fragment of bovine BMP-6 corresponding to nucleotides 259-751 of Table II is labeled by nicktranslation and hybridized to both sets of filters in SHB at 65'r. One set of filters is washed under stringent conditions (0.2X SSC, 0.1% SDS at the other under reduced stringency conditions (IX SSC, 0.1% SDS at 65'r) iany WO 90/11366 PCT/US90/01630 42 duplicate hybridizing recombinants (approximately 162) are noted. 24 are picked and replated for secondaries. Three nitrocellulose replicas are I made of each plate. One is hybridized to the BMP-6 SmaI probe, one to a nick-translated BMP-6 PstI- SacI fragment (nucleotides 106-378 of Table II), and the third to the nick-translated BMP-E XbaI fragments (nucleotides 1-76 of Table I).

Hybridization and washes are carried out under stringent conditions.

B. Human BMP-5 Proteins 17 clones that hybridize to the third probe more strongly than to the second probe are plaque purified. DNA sequence analysis of one of these, U2-16, indicates that it encodes human BMP-5. U2- 16 was deposited with the Ameri-an Type Culture Collection (ATCC), Rockville, Maryland on June 22, 1989 under accession number ATCC~ 4 6f9. This deposit as well as the other deposits described herein are made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the Regulations thereunder S(Budapest Treaty). U2-16 contains an insert of approximately 2.1 Kb. The DNA sequence and derived amino acid sequence of U2-16 is shown below in i Table III. This clone is expected to contain all i of the nucleotide sequence necessary to encode i human BMP-5 proteins. The cDNA sequence of Table III contains an open reading frame of 1362 bp, encoding a protein of 454 amino acids, preceded by a 5' untranslated region of 700 bp with stop codons in all frames, and contains a 3' untranslated region of 90 bp following the in frame stop codon

(TAA).

i i I i i I j ii I:i 1\ WO 90/11366 PCT/US90/01630 This protein of 454 amino acids has molecular weight of approximately 52,000 daltons as predicted by its amino acid sequence, and is contemplated to represent the primary translation product. Based on knc.:.edge of other BMP proteins and other proteins within the TGF-p family, it is predicted that the precursor polypeptide would be cleaved at the tribasic peptide Lys Arg Lys yielding a 132 amino acid mature peptide beginning with amino acid #323 "Asn". The processing of BMPinto the mature form is expected to involve dimerization and removal of the N-terminal region in a manner analogous to the processing of the related protein TGF-- Gentry, et al., IHolec.

Cell. Biol. 8:4162 (1988); R. Dernyck, et al., Nature 316:701 (1985)].

It is contemplated therefore that the mature active species of BMP-5 comprises a homodimer of 2 polypeptide subunits each subunit comprising amino acid #323 #454 with predicted molecular weight of approximately 15,000 daltons. Further active species are contemplated, for example, proprotein dimers or proprotein subunits linked to mature subunits. Additional active species may comprise amino acid #329 #454 such species including homologous the tryptic sequences found in the purified bovine material. Also contemplated are BMP-5 proteins comprising amino acids #353- #454 thereby including the first conserved cysteine residue.

The underlined sequence of Table III from amino acid #329 to #337 Ser-Ser-Ser-His-Gln-Asp- Ser-Ser-Arg shares homology with the bovine sequence of Table I from amino acid #15 to #23 as discussed above in Example IV. Each of these WO 90/11366 PCT/US90/01630 44 sequences shares homology with a tryptic fragment sequence Ser-Thr-Pro-Ala-Gln-Asp-Val-Ser-Arg found in the 28,000 30,000 dalton purified bone preparation (and its reduced form at approximately 18,000 20,000 daltons) as described in the "BMP" published applications W088/00205 and W089/10409 mentioned above.

The underlined sequence of Table III from amino acid #356 to #362 His-Glu-Leu-Tyr-Val-Ser-Phe corresponds to the tryptic fragment identified in the bovine bone preparation described above from which the oligonucleotide probes are designed.

H

WO 90/11366 PCT/US90/01 630 TABLE III 20 CTGGTATATT TGTGCCTGCT 70 GGGATTGAAT GGACTTACAG 110 120 ATTTACTTGA ATAGTACAAC 160 170 AAAAGATGTT AAAGTTATCA 210 220 ACCAAGGTGC AGATCAGCAT 260 270 TTGGAAAGAG CTCAAGGGTT 310 320 TTTGGGAACT ACAGTTTATC 360 370 AAAGGCCTGA TTATCATAAA 410 420 AAATAATATT AGCCGTC-TTC 460 470 AACTGTGGAT AATTGGAAAT 510 520 TCTTGACATA TTCCAAAATA 560 570 TGTTGTGCTC AGAAATGTCA 610 620 TCAGCTACTG GGAAACTGTA 660 670 AAGAGGACAA GAAGGACTAA 30 40 GGAGGTCGAA TTAACAGTAA. GAAGGAGAAA 80 90 100 GAAGGATTTC AAGTAAATTC AGGGAAACAC 130 140 150 CTAGAGTATT ATTTTACACT AAGACGACAC 180 190 200 CCAAGCTGCC GGACAGATAT ATATTCCkAC 230 240 250 AGATCTGTGA TTCAGAAATC AGGATTTGTT 280 290 300 GAGAAGAACT CAAAAGCAAG TGAAGATTAC 330 340 350 AGAAGATCAA CTTTTGCTAA TTCAAATACC 380 390 400 TTCATATAGG AATGCATAGG TCATCTGATC 430 440 450 TGCTACATCA ATGCAGCAAA AACT(. rAAC 480 490 500 CTGAGTTTCA GCTTTCTTAG AAATAACTAC 530 540 550 TTTAAAATAG GACAGGAAAA TCGGTGAGGA 580 590 600 CTGTCATGAA AAATAGGTAA ATTTGTTTTT 630 640 650 CCTCCTAGAA CCTTACGTTT TTTTTTTTTT 680 690 700 AAATATCAAC TTTTGCTTTT GGACAAAA WO90/11366 PCT/US90/01630 46 TABLE III (page 2 Of 4) 701 710 719 728 737 ATG CAT CTG ACT GTA TTT TTA CTT AAG GGT ATT GTG GGT TTC CTC MET His Leu Thr Val Phe Leu Leu Lys Gly Ile Val Gly Phe Leu (1) 746 755 764 773 782 TGG AGC TGC TGG GTT CTA GTG GGT TAT GCA AAA GGA GGT TTG GGA Trp Ser Cys Trp Val Leu Val Gly Tyr Ala Lys Gly Gly Leu Gly 791 800 809 818 827 GAC AAT CAT GTT CAC TCC AGT TTT ATT TAT AGA AGA CTA CGG AAC Asp Asn His Val His Ser Ser Phe Ile Tyr Arg Arg Leu Arg Asn 836 845 854 863 872 CAC GAA AGA CGG GAA ATA CAA AGG GAA ATT CTC TCT ATC TTG GGT His Glu Arg Arg Glu Ile Gin Arg Glu Ile Lev Ser Ile Leu Gly 881 890 899 908 917 TTG CCT CAC AGA CCC AGA CCA TTT TCA CCT GGA AAA ATG ACC AAT Leu Pro His Arg Pro Arg Pro Phe Ser Pro Gly Lys Gin Ala Ser 926 935 944 953 962 CAA GCG TCC TCT GCA CCT CTC TTJ ATC CTG GAT CTC TAC AAT GCC Ser Ala Pro Leu Phe MET Leu Asp Leu Tyr Asn Ala MET Thr Asn 971 980 989 998 1007 GAA GAA AAT CCT GAA GAG TCG GAG TAC TCA GTA AGG GCA TCC TTG Glu Glu Asn Pro Glu Glu Ser Glu Tyr Ser Val Arg Ala Ser Leu 1016 1025 1034 1043 1052 GCA GAA GAG ACC AGA GGG GCA AGA AAG GGA TAC CCA GCC TCT CCC Ala Glu Glu Thr Arg Gly Ala Arg Lys Gly Tyr Pro Ala Ser Pro 1061 1070 1079 1088 1097 AAT GGG TAT CCT CGT CGC ATA CAG TTA TCT CGG ACG ACT CCT CTG Asn Gly Tyr Pro Arg Arg Ile G1n Leu Ser Arg Thr Thr Pro Leu 1106 1115 1124 1133 1142 ACC ACC CAG AGT CCT CCT CTA GCC AGC CTC CAT GAT ACC AAC TTT Thr Thr Gin Ser Pro Pro Leu Ala Ser Leu His Asp Thr Asn Phe 1151 1160 1169 1178 1187 CTG AAT GAT GCT GAC ATG GTC ATG AGC TTT GTC AAC TTA GTT GAA Leu Asn Asp Ala Asp AET Val MET Ser Phe Val Asn Leu Val Glu 1196 1205 1.214 1223 1232 AGA GAC AAG GAT TTT TCT CAC CAG CGA AGG CAT TAC AAA GAA TTT Arg Asp Lys Asp Phe Ser His Gin Arg Arg His Tyr Lys Glu Phe

-L

i i i i ii/ 1: i 1

I

WO 90/11366 PCr/US9P/01 630 47 TABLE III (page 3 of 4) 1241 1250 1259 1268 1277 CGA TTT GAT CTT ACC CAA ATT CCT CAT GGA GAG GCA GTG ACA GCA Arg Phe Asp Leu Thr Gin Ile Pro His Gly Glu Ala Val Thr Ala 1286 1295 1304 1313 1322 GCT GAA TTC CGG ATA TAC AAG GAC CGG AGC AAC AAC CGA TTT GAA Ala Glu Phe Arg Ile Tyr Lys Asp Arg Ser Asn Asn Arg Phe Glu 1331 1340 1349 1358 1367 AAT GAA ACA ATT AAG ATT AGC ATA TAT CAA ATC ATC AAG GAA TAC Asn Glu Thr Ile Lys Ile Ser Ile Tyr Gln Ile Ile Lys Glu Tyr 1376 1385 1394 1402 1412 ACA AAT AGG GAT GCA GAT CTG TTC TTG TTA GAC ACA AGA AAG GCC Thr Asn Arg Asp Ala Asp Leu Phe Leu Leu Asp Thr Arg Lys Ala 1421 1430 1439 1448 1457 CAA GCT TTA GAT GTG GGT TGG CTT GTC TTT GAT ATC ACT GTG ACC Gin Ala Leu Asp Val Gly Trp Leu Val Phe Asp Ile Thr Val Thr 1466 1475 1484 1493 1502 AGC AAT CAT TGG GTG ATT AAT CCC CAG AAT AAT TTG GGC TTA CAG Ser Asn His Trp Val Ile Asn Pro Gin Asn Asn Leu Gly Leu Gin 1511 -520 1529 1538 1547 CTC TGT GCA GAA ACA GGG GAT GGA CGC AGT ATC AAC GTA AAA TCT Leu Cys Ala Glu Thr Gly Asp Gly Arg Ser Ile Asn Val Lys Ser 1556 1565 1574 1583 1592 GCT GGT CTT GTG GGA AGA CAG GGA CCT CAG TCA AAA CAA GCA TTC Ala Gly Leu Val Gly Arg Gln Gly Pro Gin Ser Lys Gli Pro Phe 1601 1610 1619 1628 1637 ATG GTG GCC TTC TTC AAG GCG AGT GAG GTA CTT CTT CGA TCC GTG MET Val Ala Phe Phe Lys Ala Ser Glu Val Leu Leu Arg Ser Val 1646 1655 1664 1673 1682 AGA GCA GCC AAC AAA CGA AAA AAT CAA AAC CGC AAT AAA TCC AGC Arg Ala Ala Asn Lys Arg Lys Asn Gin Asn Arg Asn Lys Ser Ser (323) (329) 1691 1700 1709 1718 1727 TCT CAT CAG GAC TCC TCC AGA ATG TCC AGT GTT GGA GAT TAT AAC Ser His Gin Asp Ser Ser Arq MET Ser Ser Val Gly Asp Tyr Asn (337) r WO 90/11366 PCT/US90/01630 TABLE III (page 4 of 4) 1736 1745 1754 1763 1772 ACA AGT GAG CAA AAA CAA GCC TGT AAG AAG CAC GAA CTC TAT GTG Thr Ser Glu Gin Lys Gin Ala Cys Lys Lys His Glu Leu Tyr Val (356) 1781 1790 1799 1808 1817 AGC TTC CGG GAT CTG GGA TGG CAG GAC TGG ATT ATA GCA CCA GAA Ser Phe Arg Asp Leu Gly Trp Gin Asp Trp Ile Ile Ala Pro Glu (362) 1826 1835 1844 1853 1862 GGA TAC GCT GCA TTT TAT TGT GAT GGA GAA TGT TCT TTT CCA CTT Gly Tyr Ala Ala Phe Tyr Cys Asp Gly Glu Cys Ser Phe Pro Leu 1871 1880 1889 1898 1907 AAC GCC CAT ATG AAT GCC ACC AAC CAC GCT ATA GTT CAG ACT CTG Asn Ala His MET A'n Ala Thr Asn His Ala Ile Val Gin Thr Leu 1916 1925 1934 1943 1952 GTT CAT CTG ATG TTT CCT GAC CAC GTA CCA AAG CCT TGT TGT GCT Val His Leu MET Phe Pro Asp His Val Pro Lys Pro Cys Cys Ala 1961 1970 1979 1988 1997 CCA ACC AAA TTA AAT GCC ATC TCT GTT CTG TAC TTT GAT GAC AGC Pro Thr Lys Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser 2006 2015 2024 2033 2042 TCC AAT GTC ATT TTG AAA AAA TAT AGA AAT ATG GTA GTA CGC TCA Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn MET Val Val Arg Ser (450) 2051 2060 2070 2080 2090 2100 TGT GGC TGC CAC TAATATTAAA TAATATTGAT AATAACAAAA AGATCTGTAT Cys Gly Cys His (454) 2110 2120 2130 2140 2150 TAAGGTTTAT GGCTGCAATA AAAAGCATAC TTTCAGACAA ACAGAAAAAA AAA WO 90/11366 PCT/US90/01630 49 The tryptic sequence His-Glu-Leu-Tyr-Val-Ser- Phe-(Ser) described above is noted to be similar to the sequence His-Pro-Leu-Tyr-Val-Asp-Phe-Ser found in the bovine and human cartilage/bone protein BMP- 2A sequence, for instance as described in Publication WO 88/00205. Human BMP-5 shares homology with other BMP molecules as well as other members of the TGF-p superfamily of molecules. The cysteine-rich carboxy-terminal 102 amino acid residues of human BMP-5 shares the following homologies with BMP proteins disclosed herein and in Publications WO 88/00205 and WO 89/10409 described above: 61% identity with BMP-2; 43% identity with BMP-3, 59% identity with BMP-4; 91% identity with BMP-6; and 88% identity with BMP-7.

Human BMP-5 further shares the following homologies: 38% identity with TGF-P3; 37% identity with TGF-f2; 36% identity with TGF-Il; 25% identity with Mullerian Inhibiting Substance (MIS), a testicular glycoprotein that causes regression of the Mullerian duct during development of the male embryo; 25% identity with inhibin a; 38% identity with inhibin fB; 45% identity with inhibin fA; 56% identity with Vgl, a Xenopus factor which may be involved in mesoderm induction in early embryogenesis (Weeks and Melton, Cell 51:861-867 (1987)]; and 57% identity with Dpp the product of the Drosophila decapentaplegic locus which is required for dorsal-ventral specification in early embryogenesis and is involved in various other developmental processes at later stages of development [Padgett, et al., Nature 325:81-84 (1987)].

C. Human BMP-6 Proteins WO 90/11366 PCT/US90/01630 Six clones which hybridize to the second probe described in Example V.A. more strongly than to the third are picked and transformed into plasmids.

j Restriction mapping, Southern blot analysis, and DNA sequence analysis of these plasmids indicate that there are two classes of clones. Clones U2-7 and U2-10 contain human BMP-6 coding sequence based on their stronger hybridization to the second probe and closer DNA homology to the bovine BMP-6 sequence of Table II than the other 4 clones. DNA sequence data derived from these clones indicates that they encode a partial polypeptide of 132 amino acids comprising the carboxy-terminus of the human BMP-6 protein. U2-7 was deposited with the American Type Culture Collection (ATCC), Rockville, Maryland on June 23, 1989 under accession number 68021 under the provisions of the Budapest Treaty.

A primer extended cDNA library is made from U- 2 OS mRNA using the oligonucleotide GGAATCCAAGGCAGAATGTG, the sequence being based on the 3' untranslated sequence of the human BMP-6 derived from the clone U2-10. This library is screened with an oligonucleotide of the sequence CAGAGTCGTAATCGC, derived from the BMP-6 coding sequence of U2-7 and U2-10. Hybridization is in standard hybridization buffer (SHB) at 42 degrees centigrade, with wash conditions of 42 degrees centigrade, 5X SSC, 0.1% SDS. Positively hybridizing clones are isolated. The DNA insert of one of these clones, PEH6-2, indicates that it extends further in a 5' direction than either U2-7 or U2-10. A primer extended cDNA library constructed from U-20S mRNA as above is screened with an oligonucleotide of the sequence GCCTCTCCCCCTCCGACGCCCCGTCCTCGT, derived from the WO 90/11366 PCT/US90/01630 51 sequence near the 5' end of PEH6-2. Hybridization is at 65 degrees centigrade in SHB, with washing at I 65 degrees centigrade in 2X SSC, 0.1% SDS.

Positively hybridizing recombinants are isolated and analyzed by restriction mapping and DNA sequence analysis.

The 5' sequence of the insert of one of the positively hybridizing recombinants, PE5834#7, is used to design an oligonucleotide of the sequence CTGCTGCTCCTCCTGCTGCCGGAGCGC. A random primed cDNA library [synthesized as for an oligo (dT) primed library except that (dN) 6 is used as the primer] is screened with this oligonucleotide by hybridization at 65 degrees centigrade in SHB with washing at 65 degrees centigrade in IX SSC, 0.1% SDS. A positively hybridizing clone, RP10, is identified, isolated, and the DNA sequence sequence from the 5' end of its insert is determined. This sequence is used to design an oligonucletide of the sequence

TCGGGCTTCCTGTACCGGCGGCTCAAGACGCAGGAGAAGCGGGAGATGCA.

A human placenta cDNA library (Stratagene catalog #936203) is screened with this oligonucleotide by hybridization in SHB at 65 degrees centigrade, and washing at 65 degrees centigrade with 0.2 X SSC, 0.1% SDS. A positively hybridizing recombinant designated BMP6C35 is isolated. DNA sequence analysis of the insert of this recombinant indicates that it encodes the complete human BMP-6 protein. BMP6C35 was deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland USA on March 1, 1990 under Accession Number 68245 under the provisions of the Budapest Treaty.

The DNA and derived amino acid sequence of the II WO 90/11366 PCT/US90/01630 52 majority of the insert of BMP6C35 is given in Table IV. This DNA sequence contains an open reading frame of 1539 base pairs which encodes the 513 amino acid human BMP-6 protein precursor. The presumed initiator methionine codon is preceded by a 5'untranslated sequence of 159 base pairs with stop codons in all three reading frames. The stop codon at nucleotides 1699-1701 is followed by at least 1222 base pairs of 3'untranslated sequence.

It is noted that U2-7 has a C residue at the position corresponding to the T residue at position 1221 of BMP6C35; U2-7 also has a C residue at the position corresponding to the G residue at position 1253 of BMP6C35. These do not cause amino acid differences in the encoded proteins, and presumably represent allelic variations.

T h e o 1 i g o n u c 1 e o t i d e

TCGGGCTTCCTGTACCGGCGGCTCAAGACGCAGGAGAAGCGGGAGATGCA

is used to screen a human genomic library (Toole et al supra), by hybridizing nitrocellulose replicas of 1 X 106 recombinants with the oligonucleotide in SHB at 65 degrees centigrade, and washing at degrees centigrade with 0.2 X SSC, 0.1% SDS.

Positively hybridizing clones are purified. The oligonucleotide hybridizing region is localized to an approximately 1.5 kb Pst I fragment. DNA sequence analysis of this fragment confirms the sequence indicated in Table IV.

The first underlined portion of the sequence in Table IV from amino acid #388 to #396, Ser-Thr- Gln-Ser-Gln-Asp-Val-Ala-Arg, corresponds to the similar sequence Ser-Thr-Pro-Alg-Gln-Asp-Val-Ser- Arg of the bovine sequence described above and set forth in Table II. The second underlined sequence WO 90n/11366 PC/US90/01630 53 Sin Table IV from amino acid #415 through #421 Hisj Glu-Leu-Tyr-Val-Ser-Phe, corresponds to the tryptic fragment identified above from which the oligonucleotide probes are designed. The tryptic sequence His-Glu-Leu-Tyr-Val-Ser-Phe-(Ser) is noted to be similar to a sequence found in other BMP proteins for example the sequence His-Pro-Leu- Tyr-Val-Asp-Phe-Ser found in the bovine and human cartilage/bone protein BMP-2 sequence as described in Publication WO 88/00205. BMP-6 therefore represents a new member of the BMP subfamily of TGF-8 like molecules which includes the molecules BMP-2, BMP-3, BMP-4 described in Publications WO 88/00205 and WO 89/10409, as well as BMP-5 and BMP- 7 described herein.

Based on knowledge of other BMP proteins, as well as other proteins in the TGF-9 family, BMP-6 is predicted to be synthesized as a precursor molecule and the precursor polypeptide would be cleaved between amino acid #381 and amino acid #382 yielding a 132 amino acid mature polypeptide with a calculated molecular weight of approximately The mature form of BMP-6 contains three potential N-linked glycosylation sites per polypeptide chain as does The processing of BMP-6 into the mature form is expected to involve dimerization and removal of the N-terminal region in a manner analogous to the processing of the related protein TGF-9 [L.E.

Gentry, et al., (1988); R. Dernyck, et al., (1985) sura] It is contemplated that the active BMP-6 protein molecule is a dimer. It is further contemplated that the mature active species of BMPcomprises protein molecule is a homodimer comprised of two polypeptide subunits each subunit WO 90/11366 PCT/' S90/01630 54 comprising amino acid #382 #513 as set forth in Table IV. Further active species of BMP-5 are contemplated such as phoprotein dimers or a p-oprotein subunit and a mature subunit.

Additional active BMP-5 proteins may comprise amino acid #388 #513 thereby including the tryptic fragments found in the purified bovine material.

Another BMP-5 protein of the invention comprises amino acid #412 #513 reby including the first conserved cystine resier

I

PCI, US90/0 1630 WO 90/11366 TAPA~ IV 20 30 40 CGACCATGAG AGATAAGGAC TGLGGGC.CAG GAAGGGGAAG CGAGCCCGCC "1,0 80 90 100 GAGAGGTGGC GGGGACTCCT CACGCCAAGG GCCXCAG CGG CCGCGCTCCG 110 120 130 140 150 GCCTCGCTCC GCCGCTCCAC GCCTCGCGGG ATCCGCGGGG GCAGCCCGGC 159 168 177 186 195 CGGGCGGGG ATG CCG GGG CTG GGG CCC AOG GCG CAG TGG CTG TGC MET :'ro Gly Leu Gly Arg Arg Ala Gin Trp Leu Cys (1)j 204 TGG TCG TGG Trp Trp Trp 249 CGG CCG CCC Arg Pro Pro 294 CTG CTG GGG Leu Leu Gly 213 222 GGG CTC CTG TGC AGC TGC Gly Leu Leu Cys Ser Cys 258 267 TTC CCC CCT GCC CC GCC Leu Pro Ala Ala Ala Ala 303 312 GAC CCC CGG AGC CCC GGC Asp Gly Gly Ser Pro Gly 231 240 TGC GGG CCC CCG CCG CTG Cys Gly Pro Pro Pro Leu 276 CCC CCC GCC CCC CCC Ala Ala Ala Gly Gly 285

CAC

Gin 339 348 357 CCC TCC CCC CAG TCC TCC TCC CCC TTC Pro Ser Pro Cln Ser Ser Ser Gly Phe 321 330 CCC ACG GAG CAG CCG CCG Arg Thr Clu Gin Pro Pro 366 17 CTC TAC CCC CGG CTC AAG LJcZ u Tyr Arg Arg Leu Lys 411 420 GAG ATC TTC TCG GTG CTG Glu Ile Leu Ser Val. Leu 384 ACC CAG GAG Thr Cln Glu 429 CCC CTC CCC Gly Leu Pro 393 11 A.AG CGC GAG ATG CAG I Lys Arg Clu MET Gin t>j 438 447 CAC CCC CCC CCC CCC CTC Flis Arg Pro Arg Pro Leu 456 CAC CCC CTC CAA CAG His Gly Leu Gin Gin 465

CCC

Pro I WO 90/11366 PC'!IS90/0 1630 56 Table IV (page 2 ofr 6) 474 CAG CCC CCG GCG Gin Pro Pro Ala 519 CAG C2G CCT CGC Gin Leu Pro Arg 564 CCC CTC TTC ATG Pro Leu Phe MET 609 GAC GAG GAC G G.G Asp Glu Asp Gly 654 CAC GAA OCA 0CC His Glu Ala Ala 699 GCC GCO CAC CCO Pro Pro Gly Ala 744 GGC AGC GGC GGC Gly Ser Gly G -1y 789 TTC CTC AAC GAC Phe Leu Asn Asp 483 CTC CGG CAG Leu Arg Gln 528 OGA GAG CCC Oly Glu Pro 573 CTG GAT CTG Leu Asp Leu 618 OCO TCG GAG Ala Ser Glu 663 AGC TCG TCC Ser Ser Ser 708 CTC AAC CGC Ala His Pro 753 GCG TCC CCA Ala Ser Pro 798 GCG GAC ATG Ala Asp MET 492 CAG GAO Gln Glu 537 CCT CCC Pro Pro 501 510 GAG CAG CAG CAG CAG CAG Glu Gin Gin Gin Gin Gin 546 555 000 CGA CTG AAO TCC OCG Gly Arg Leu Lys Ser Ala 582 TAC AAC CCC CTO Tyr Asn Ala Leu 627 000 GAO AGO CAG Oly Glu Arg Oln 672 CAG COT CGG CAG 0Th Arg Arg Gin 591 600 TCC 0CC GAC AAC Ser Ala Asp Asn 636 645 CAG TCC TOO CCC Gin Ser Trp Pro 681 6590 CCO CCC CC1 OGC Pro Pro Gly Ser 717 AAG AGC Leu Asn 762 CTG ACC Leu Thr 807 GTC ATO Val MET 852 CCT CGT Pro Arg 897 ATT CCT Ile Pro 726 735 CTT CTG 0CC CCC GOA TCT Arg Lys Ser Leu Leu Ala 771 780 A0C 0CC CAG GAC AOC GCC Ser Ala Gln Asp Ser Ala 816 825 AGC TTT GTG AAC CTG GTG Ser Phe Val Asn Leu Val 861 870 CAG CGA CAC CAC AAA GAO Gln Arg His His Lys Olu 906 915 GAG GOT GAG GTO GTG ACG Glu Gly Glu Val Val Thr 834 GAG TAC GAC AAG GAG Giu Tyr Asp Lys Glu 843 TTC TCC Phe Ser 879 TTC AAG TTC MAC Phe Lys 1?he Asn 888 TTA TCC CAG Leu Ser Oln e WO 90/11366 924 GCT GCA GAA TTC CGC Phe Arg Ile Tyr Lys i Q9

"U-~D~SB~

PCT/US90/01630 57 Table IV (page 3 of 6) 933 942 951 960 ATC TAC AAG GAC TGT GTT ATG GGG AGT TTT Asp Cys Val MET Ala Ala Glu Gly Ser Phe 978 987 996 1005 CTT ATC AGC ATT TAT CAA GTC TTA CAG GAG Leu Ile Ser Ile Tyr Gin Val Leu Gin Glu AAA AAC CAA Lys Asn Gin ACT TTT Thr Phe 1014 1023 1032 1041 1050 CAT CAG CAC AGA GAC TCT GAC CTG TTT TTG TTG GAC ACC CGT GTA His Gin His Arg Asp Ser Asp Leu Phe Leu Leu Asp Thr Arg Val 1059 1068 1077 1086 1095 GTA TGG GCC TCA GAA GAA GGC TGG CTG GAA TTT GAC ATC ACG GCC Val Trp Ala Ser Glu Glu Gly Trp Leu Glu Phe Asp Ile Thr Ala 1104 1113 1122 1131 1140 ACT AGC AAT CTG TGG GTT GTG ACT CCA CAG CAT AAC ATG GGG CTT Thr Ser Asn Leu Trp Val Val Thr Pro Gin His Asn MET Gly Leu 1149 1158 1167 1176 1185 CAG CTG AGC GTG GTG ACA AGG GAT GGA GTC CAC GTC CAC CCC CGA Gin Leu Ser Val Val Thr Arg Asp Gly Val His Val His Pro Arg 1194 1203 1212 1221 1230 GCC GCA GGC CTG GTG GGC AGA GAC GGC CCT TAC GAT AAG CAG CCC Ala Ala Gly Leu Val Gly Arg Asp Gly Pro Tyr Asp Lys Gin Pro 1239 1248 1257 1266 1275 TTC ATG GTG GCT TTC TTC AAA GTG AGT GAG GTC CAC GTG CGC ACC Phe MET Val Ala Phe Phe Lys Val Ser Glu Val His Val Arg Thr 1284 1293 1302 1311 1320 ACC AGG TCA GCC TCC AGC CGG CGC CGA CAA CAG AGT CGT AAT CGC Thr Arg Ser Ala Ser Ser Arg Arg Arg Gin Gin Ser Arg Asn Arg (382) 1329 1338 i347 1356 1365 TCT ACC CAG TCC CAG GAC GTG GCG COG GTC TCC AGT GCT TCA GAT Ser Thr Gin Ser Gin Asp Val Ala Arq Val Ser Ser Ala Ser Asp (388) I .4 iI

I

i j WO 90/11366 PCT/US90/01630 58 Table IV (page 4 of 6) 1374 TAC AAC AGC AGT Tyr Asn Ser Ser 1419 TAT GTG AGT TTC Tyr Val Ser Phe 1464 CCC AAG GGC TAT Pro Lys Gly Tyr 1509 CCA CTC AAC GCA Pro Leu Asn Ala 1383 GAA TTG Glu Leu 1428 CAA GAC Gln Asp 1473 GCT GCC Ala Ala 1518 CAC ATG His MET 1392 1401 1410 AAA ACA GCC TGC AGG AAG CAT GAG CTG Lys Thr Ala Cys Arg Lys His Glu Leu (412) 1437 1446 1455 CTG GGA TGG CAG GAC TGG ATC ATT GCA Leu Gly Trp Gln Asp Trp Ile Ile Ala 1482 1491 1500 AAT TAC TGT GAT GGA GAA TGC TCC TTC Asn Tyr Cys Asp Gly Glu Cys Ser Phe 1527 1536 1545 AAT GCA ACC AAC CAC GCG ATT GTG CAG Asn Ala Thr Asn His Ala Ile Val Gln 1554 1563 1572 1581 1590 ACC TTG GTT CAC CTT ATG AAC CCC GAG TAT GTC CCC AAA CCG TGC Thr Leu Val His Leu MET Asn Pro Glu Tyr Val Pro Lys Pro Cys 1599 1608 1617 1626 1635 TGT GCG CCA ACT AAG CTA AAT GCC ATC TCG GTT CTT TAC TTT GAT Cys Ala Pro Thr Lys Len Asn Ala Ile Ser Val Leu Tyr Phe Asp 1644 1653 1662 1671 1680 GAC AAC TCC AAT GTC ATT CTG AAA AAA TAC AGG AAT ATG GTT GTA Asp Asn Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn MET Val Val 1689 1698 1708 1718 1728 AGA GCT TGT GGA TGC CAC TAACTCGAAA CCAGATGCTG GGGACACACA Arg Ala Cys Gly Cys His (513) 1738 1748 1758 1/68 1778 TTCTGCCTTG GATTCCTAGA TTACATCTGC CTTAAAAAAA CACGGAAGCA 1788 1798 1808 1818 1828 CAGTTGGAGG TGGGACGATG AGACTTTGAA ACTATCTCAT GCCAGTGCCT 1838 1848 1858 1868 1878 WO 90/11366PT/S/063 PCr/US90/01630 Table IV (page 5 of 6) TATTACCCAG GAAGATTTTA AAGGACCTCA TTAA"AATTT GCTCACTTGG 1888 TAAkATGACGT 1938

GTAGCATAAG

1988

CCCTCCTCCC

20Z8

TATTTGGGGT

2088

ATGTATTCTT

2138

AGATTTTACA

2188 AG TTCATTC C 2238

CTCCACGGGG

2288

AGTTTTGTTG

2338

TACACCTCAA

2388

CTGATTACAC

2438 1898

GAGTAGTTGT

1948

GTCTGGTAAC

1998

CCAAAAACCC

2048

GTTTGTTAGT

2098

GGCTAAAGGA

2148

GAGAACAGAA

2198

CAGAAGTCCA

2248

CGCCCTTGTC

2298

GTGTGAAAAT

2348

TCCTCCATTT

2398

TGAGGTGAGG

2448 1908

TGGTCTGTAG

1958

TGCAGAAACA

2008

ACCA.AAATTA

2058

AAATAGGGAA

210q

TCAGUTGGTT

2158

ATCGGGGA.AG

2208

CAGGACGCAC

2258

TCAGTCATTG

2308

ACACTTATTT

2358 G CT GTA CT CT 2408

CTACAAGGGG

2458 1918

CAAGCTGAGT

1968

TAACCGTGAA

2018

GTTTTAGCTG

2068

AATAATCTCA

CAGTACTGTC

2168

TGGGGGGAAC

2218

AGCCCAGGCC

2268

CTGTTGTATG

2318

CAGCCAAAAC

2368

TTGCTAGTAC

2418

TGTGTAACCG

2468 1928

TTGGATGTCT

1978

GCTCTTCCTA

2028 TA GATCAAGC 2078

AAGGAGTTAA

2128

TATCAAAGGT

2178

GCCTCTGTTC

2228

ACAGCCAGGG

2278

TTCGTGCTGG

2328

ATACCATTTC

2378

CAAAAGTAGA

2428

TGTAACACGT

2478 GAAGGCAGTG CTCACCTCTT CTTTACCAGA ACGGTTCTTT GACCAGOACA WO 90/11366 PCU/US90/01 630 Table IV (page 6 of 6) 2488 2498 2508 2518 2528 TTAACTTCTG GACTGCCGGC TCTAGTACCT TTTCAGTAAA GTGGTTCTCT 2538

GCCTTTTTAC

2588

AAATAAAATG

2638

ATGCTGTTTA

268'8

GATTAAATTT

2.738

GGGAAGGCAA

2788

AACTGTTTGC

2838

TCTATTTTAT

2888

GGGGGGGGGG

2548

TATACAGCAT

2598

AGGGTGCCCA

2648

TGAACGGAAA

2698

TAGAATATTT

274,8

TTTCATACTA

2798

ACTTACAGCT

2848

ATCTGTTTTG

2898

GTTTGTTTGG

2 -58

ACCACGCCAC

2608

GCTTATAAGA

2658

'.CATGATTTC

2708

TCTAAATGTC

2758

AACTGATTAA

2808

TTTTTTGTAA

2858

CTGTGGCGTT

2908

GGGGTGTCGT

2568 2578 AGGGTTAGAA CCAACGAAGA 2618 2628 ATGGTGTTAG GGGGATGAGC 2668 2678 CCTGTAGAAA CTGAGGCTCA 2718 2728 TTTTTCACAA TCATGTGACT 2768 2778 ATAATACATT TATAATCTAC 2818 2828 ATATAAACTA TAATTTATTG 2868 2878 GGGGGGGGGG CCGGGCTTTT 2918 GGTGTGGGCG GGCGG WO 90/11366 PCT/US90/01630 61 Comparision of the sequence of murine Vgr-1 [Lyons, et al., PNAS 86:4554 (1989)] to human BMP-6 reveals I. a degree of amino acid sequence identity greater than 92% The murine Vgr-1 is likely the murine homologue of BMP-6. Human BMP-6 shares homology with other BMP molecules as well as other members of the TGF-p superfamily of molecules. The cysteine-rich carboxy-terminal 102 amino acid residues of human BMP-6 shares the following homologies with BMP proteins disclosed herein and in Publications WO 88/00205 and WO 89/10409: 61% identity with BMP-2; 44% identity with BMP-3, identity with BMP-4; 91% identity with BMP-5; and 87% identity with BMP-7. Human BMP-6 further shares the following homologies: 41% identity with TGF-P3; 39% identity with TGF-32; 37% identity with TGF-fl; 26% identity with Mullerian Inhibiting Substance (MIS), a testicular glycoprotein that causes regression of the Mullerian duct during development of the male embryo; 25% identity with inhibin a; 43% identity with inhibin 8 B; 49% identity with inhibin PA; 58% identity with Vgl, a Xenopus factor which may be involved in mesoderm induction in early embryogenesis (Weeks and Melton, (1987) Supra]; and 59% identity with Dpp the product of the Drosophila decapentaplegic locus which is required for dorsal-ventral specification in early embryogenesis and it involved in various S 30 other developmental processes at later stages of development [Padgett, et al., (1987) supra].

D. Human BMP-7 Proteins The other four clones of Example V.C. above which appear to represent a second class of clones WO 90/11366 PCT/US90/01630 encode a novel polypeptide which we designate as BMP-7. One of these clones, U2-5, was deposited with the ATCC on June 22, 1989 under accession number ATCC 68020 under the provisions of the Budapest Treaty. This clone was determined not to contain the entire coding sequence for BMP-7. An oligo of the squence GCGAGCAATGGAGGATCCAG (designed on the basis of the 3' noncoding sequence of was used to make a primer-extended cDNA library from U-2 OS mRNA (Toole, et 500,000 recombinants of this library were screened with the loigonucleotide GATCTCGCGCTGCAT (designed on the basis of the BMP-7 coding sequence) by hybridization in SHB at 420 and washing in 5X SSC, 0.1% SDS at 420. Several hybridizing clones were obtained. DNA sequence analysis and derived amino acid sequence of one of these clones, PEH7-9, is given in Table V. PEH7-9 was deposited with the American Type Culture Collection (ATCC), Rockville, Maryland on November 17, 1989 under accession number ATCC 68182 under the provisions of the Budapest Treaty. PEH7-9 contains an insert of 1448 base pairs. This clone, PEH7-9, is expected to contain all of the nucleotide sequence necessary to encode BMP-7 proteins. The cDNA sequence of Table V contains an open reading frame of 1292 base pairs, encoding a protein of 431 amino acids, preceded by a 5' untranslated region of 96 base pairs with stop codons in all frames, and contains a 3' untranslated region of 60 base pairs following the in frame stop codon TAG.

This protein of 431 amino acids has a molecular weight of 49,000 daltons as predicted by its amino acid sequence and is contemplated to represent the primary translation product. Based WO 90/11366 PCT/US90/01630 63 on knowledge of other BMP proteins as well as other proteins within the TGF-p family, it is predicted that the precursor polypeptide would be cleaved between amino acid #299 and #300, yielding a 132 amino acid mature peptide.

It is contemplated that processing of BMP-7 to the mature form involves dimerization of th proprotein and removal of the N-terminal re ion in a manner analogous to the processing of the related protein TGF-B Gentry, et al., (1988) Supra and; R. Dernyck, et al., (1985) Supra]. It is comtemplated therefore that the mature active species of BMP-7 comprises a homodimer of 2 polypeptide subunits each subunit cmprising amino acid #300 #431 as shown in Table V with a calculated weight of 15,000 daltons. Other active BMP-7 species are contemplated, for example, protein dimers or proprotein subunits linked to mature subunits. Additional active species may comprise amino acids #309 #431 of Table V such species including the tryptic sequences found in the purified bovine material. Also contemplated are BMP-7 proteins comprising amino acids #330- #431 thereby including the first conserved cysteine residue.

The underlined sequence of Table V from amino acid #309 #314 Asn-Gln-Glu-Ala-Leu-Arg is the same sequence as that of tryptic fragment #5 found in the 28,000 30,000 dalton purified bone preparation as described in the "BMP" Publications WO 88/00205 and WO 89/10409 mentioned above. The underlined sequence of Table V from amino acid #333-#339 His-Glu-Leu-Tyr-Val-Ser-Phe corresponds to the tryptic fragment identified in the bovine bone preparation described above from which the WO 90/1 1366 PCT/US90/01 630 oligonucleotide probes are d:esigned.I n

U

WO 90/11366 PCT/US90/01 630 TABLE V GIGAC~rAGC CAC GIG a.

His Val. Aa 00TGG C Leu Trp A) 20 30 40 GCCr-rCC; GC03CV= CC CTICA COIGG-GGCOG 70 80 90 99 GAGCC3GAG YC-vGTAGC GCGIAGAGCC GGCGCG ATG (1) 8 3 a 117 TWA CIG CGA Ser I.eu Arg 162 ccc cmG TMC Pro Leu Phe 126 CC GM C Ala Ala Ala 171 CIG CIG CC Lau Leu Arg 1 1Z COG CAC A( C Pro His Ser 180 TCO GCC CIG Ser Ala Lisu 225 TTC NTC CAC The le His 144 TIC =GIGC The Val. Ala 189 GCC GAC =I Al1a Asp The 234 OGG cxC CIc Arg Arg Leu 198 AGC CIG GAC Ser Leu Asp 243 CGC AGO CAG Arg Ser Gin 288 TIC GGC TIG Lau Gly Teu 333 AAC TOG GCA Asn Ser Ala 378 GMG GAG =A Val Glu Glu 423 TAC AAG CC Tyr Lys Ala 468 CMA CAT AGO Gin Asp Ser 207 AAO GAG GMG CAC Asn Glu Val His 216 TM XGO Ser Ser 252 GAG CCC COG Glu Arg Arg 261 2 0 GAG A2T3 CAG CXMC GAG NEC C:TC Glu METr Gin krg Giiu Ile Lou 279 ICC A!IT Ser le 297 306 CCCAC CGC 00m =C CCC A Pro HIs Arg Pro Arg Pro His 342 351 CCC AMS TIC AdG CIG GAC CIG Pro MET The M~r Leu Asp L-eu 315 C7C CAG 'Lau Gln 360 TMC MO 7 r Asn 405 CCC 'ITC Gly The 387 OXC CCC GGC CCC C Gly Gly Gly PrO Gly 396 CCC CAG Gly G~n 324 CCC AAG CAC Cly Lys His 369 CCC ATG GCC Ala, NLT Ala 414 'ICC TAO CCC V4r Ty~r Pro 4; ~9 CCC AGC CTG Ala Ser Leu AIG AGC TIC MT Ser The 432 GTC TIC AGT ACC Val Tne Set, Thr 4.i CAG GGC Gin Gly 450 000 O0T CIG Pro Pro Leu 477 CAT TIC CIC ACC CAC His The Ieu Thr Asp 486 GCC GAO A.'a Asp 495 AIrG GIC MET Val 513 522 531 540 54!: GIC AAC =CMr GIG CAT GAO MG GMA TIC IC C CA CGC TAC Val. Asn Leau Val. Glu His Asp Lys C1u Phe The His Pro Arg Ty~r WO 90/11256 Table V (page 2 of 3) 558 367 576 CAC CAT CGA GAG TM CGG TIT GAT CIT His His Arg Giu Phe Arg IPne Asp Leu PCT/US90/01 630 66 585 594 TCC AAG ATC CCA, GAA 1GG Ser Lys Ile Pro Glu Gly 603 GAA GOT GTC Glu Ala Val 648 OCG GAA CGC Arg Glu Arg 693 1=1 ci' GAG Val Leu Gin 738 GAC AGC CGT As Ser Arg 783 GAC AIC ACA Asp Ile Thr 828 CIG (&GC Asn Leu Gly 612 ACG GCA GCC Thr Ala Ala 657 T1C GAC AAT Phe Asp Asn 702 GAG CAC TIG Glu His Leu 621 GAA TIC OG Glu !'ne Arg 666 GAG ACG TIC Glu Thr Phe 630 ATC TAC AAG Ile Tyr Lys 675 COG ATC AGO Arg Ile Ser 711 G'C AGC GAA TOO GAT Gly Arg Giu Ser Asp 720

CIC

Lu 747 756 AC CC T GCC TOG GAG Thr Leu Trp Ala Ser Glu 792 GCC ACC AC AAC Ala Thr Ser Asn 801 CAC TG His Trp 837 GG CP GC Teu Gin Leu 873 ATC AA CCC AAG Ile Asn Pro Lys 882 TIG GC ILeu Ala 918 AAC AAG GAG Asn Lys Gin 927 CCC TO :ATG Pro Phe MET 846 TC GIG GAG Ser Val Glu 891 Cly Iu Ile 936 GTG GOT TIC Val Ala Phe 981 TCC ACC G(G Ser Thr Gly 1026 AAG MG GAG Lys Asn Gln (309) 1071 AGO AGO GAG Ser Ser Asp 765 GAG GGC IGG Gili Gly Trp 810 GIG GIC AAT Vda. Val Asn 855 ACS CIG GAT Thr Leu Asp 900 GGG COG CAC Gly !Xg His 945 TYC AAG GCC 1Pie Lys Ala 639 GAC TAG ATG Asp Tyr Ile 684 GIT TAT GAG Val yrr Gin 729 TIC GIG OTO Phe Leu Leu 774 GIG GIG TIT Leu Val Phe 819 CCG CG GAG Pr Arg His 864 3GG CAG AGO Galy Gin Ser 909 GGG CCC GAG Gly Pro Gin 954 ACO GAG GIC Thr Glu Val 963 CAC TIC COC AGO His Phe Arg Ser 972 ATC MGC Il Arg 990 999 AG AAA GAG CGC AGC CAG Ser Lys Gin Arg Ser Gin (300) 1035 1044 GAA GCC CIG CGG ATG GCC Glu Ala Teu M r T Ala 1008 1017 AAC CGC TG AAG ACG CCC Asn Axg Ser Lys Thr Pro 1053 AAC GIG GCA Asr. Val. Ala ?062 GAG AAC AGO Glu Asn Ser 1080 'AG AGG CAG Gin Arg Gin 1089 GCC MT AAG Ala -ys Lys (330) L I ~a tI'i WO 90/11366 PCT/US90/01 630 Table V (page 3 of 3)

AAG

Lys 1093 CAC GAG His r1iu 1107 CIG TAT GTC AGO 1pu Tvr Val Rer 1116 'C Oak The Arg 1143 TG ATC AIC GCG Trp Ile Ile Ala 1152 OCT GAA Pro Glu 1188 GAG 'IW GCC Glu Cys Ala 1233 GC' ATC GIG Ala Ile Val 1278 CCC AAG OCC Pro Lys Pro 1323 CIC TAC TTC Leu Tyr Phe 1157 TW CCT CIG Phe Pro tsu 1242 CAG A OG CIG Gin 'Th Leu 1287 TGC TGT GCG Cys Cys Ala 1161 GGC TAO GCC Gly Tyr Ala 1206 AAC TC TAC Asn Ser Tyr 1251 GIC CAC TC Va1 His The 1296 CCC ACG CAG Pro Thr Gin 1125 GAO CIG (3CC Asp Ieu Gly 1170 GCC TAC TA C Ala Tyr Tyr 1215 AG AAC GCC MT Asn Ala 1260 ATO AAC CCG Ile Asn Pro 1134 IGG GAG GAO Trp Gin Asp 1179 TGT GAG GGG Cys Glu Gly 1224 ACC AAC CAC Thr Asn His 1269 GAA ACG GTr, Ile Ser Val 1305 MC AAT GCC ATC Leu Asn Ala Ile 1314 TCC GTC Ser Val 1332 1341 1350 1359 GAT GAC AGC TOO MC GC CI AAG AM TAO AGA Asp Asp Sei Ser Asn Val Ile Lu Lys Lys Tyr Arg 1368 1377 1386 1399 AAC AIG GM GIIC COG GCC RIT GGC TGC CAC TAGCICCtC= Asri MET Val Val Arg Ala Cys Gly Cys His (431) 1409 1419 1429 1439 1448 GAGAUTCAG ACOCITICGG GCGAAGTI

T

TCTGrG1ACCT CCA=TGCTC WO90/11366 PCT/US90/01630 68 Like BMP-5 and BMP-6, human BMP-7 shares i homology with other BMP molecules as well as other 'i members of the TGF-P superfamily of molecules. The i 5 cysteine-rich carboxy-terminal 102 amino acids residues of human BMP-7 shares the following Shomologies with BMP proteins herein and in Publications WO 88/00205 and WO 89/10409 described 1above: 60% identity with BMP-2; 43% identity with BMP-3, 58% identity with BMP-4, 87% identity with !I BIP-6; and 88% identity with BMP-5. Human BMP-7 further shares the following homologies: ,i identity with TGF-93; 40% identity with TGF-p2; 36% identity with TGF-l; 29% identity with Mullerian Inhibiting Substance (MIS), a testicular glycoprotein that causes regression of the Mullerian duct during development of the male embryo; 25% identity with inhibin-a; 44% identity i with inhibin.-PB; 45% identity with jnhibin-PA; 57% identity with Vgl, a Xenopus factor which may be involved in mesoderm induction in early embryogenesis [Weeks adn Melton, (1987) Supra.]; and 58% identity with Dpp the product of the i Drosophila decapentaplegic locus which is required S 25 for dorsal-ventral specification in early embryogenesis and is involved in various other developmental processes at later stages of development [Padgett, et al., (1987) Supra.].

The invention encompasses the genomic sequences of BMP-5, BMP-6 and BMP-7. To obtain these sequences the cDNA sequences described herein are utilized as probes to screen genomic libraries using techniques known to those skilled in the art.

The prccedures described above and additional 1 r

L--L

WO 90/11366 PCT/US90/01630 69 methods known to those skilled in the art may be employed to isolate other related proteins of interest by utilizing the bovine or human proteins as a probe source. Such other proteins may find 3i similar utility in, inter alia, fracture repair, i wound healing and tissue repair.

SEXAMPLE VI Expression of BMP Proteins S 10 In order to produce bovine, human or other mammalian BMP-5, BMP-6 or BMP-7 proteins of the invention, the DNA encoding it is transfected into i an appropriate expression vector and introduced into mammalian cells or other preferred eukaryotic or prokaryotic hosts by conventional genetic engineering techniques. It is contemplated that the preferred expression system for biologically active recombinant human proteins of the invention will be stably transformed mammalian cells. For transient expression, the cell line of choice is transformed African green monkey kidney COS-1 or COS-7 which typically produce moderate amounts of the protein encoded within the plasmid for a period of 1-4 days. For stable high level expression of BMP-5, BMP-6 or BMP-7 the preferred cell line is Cinese hamster Ovary (CHO). It is therefore contemplated that the preferred mammalian cells will be CHO cells.

i The transformed host cells are cultured and the BMP proteins of the invention expressed thereby are recovered, isolated and purified.

Characterization of expressed proteins is carried out using standard techiques. For example, characterization may include pulse labeling with 3 5S] methionine or cysteine and analysis by L WO 90/11366 PcT/US9O/bi 0 polyacrylamide electrphoresis. The recombinantly expressed BMP proteins are fre- of proteinaceous materials with which they are co-produced and with which they ordinarily are associated in nature, as well as from other contaminants, such as materials found in the culture media.

A. Vector Construction As described above, numerous expression vectors known in the art may be utilized in the expression of BMP protnins of the invention. The vector utilized in the followi'g examples is pMT21, a derivitive of pMT 2 though other vectors may be suitable in practice of the invention.

pMT 2 is derived from pMT2-VWF, which has been deposited with the American Type Culture Collection (ATCC), Rockville, MD (USA) under accession number ATCC 67122 under the provisions of the Budapest on S9 Mci.y Icfc Treaty EcoRI digestion excises the cDNA insert present in pMT-VWF, yielding pMT2 in linear form which can be ligated and used to transform E. Coli HB 101 or DH-5 to ampicillin resistance. Plasmid pMT2 DNA can be prepared by conventional methods.

pMT21 is then constructed using loopout/in mutagenesis [Morinaga, et al., Biotechnology _J4:636 (1984)]. This removes bases 1075 to 1170 (inclusive). In addition it inserts the following Ssequence: 5' TCGA This sequence complates a new restrictio-i site, Xhol. This plasmid now S 30 contains 3 unique cloning sites PstI, EcoRI, an' Xhol.

In addition, pMT21 is digested with EcoRV and Xhol, treating the digested DNA with Klenow fragment of DNA polymerase I and ligating Clal linkers (NEBio Labs, CATCGATG). This removes bases WO 90/11366 PCT/US90/01 630 71 2171 to 2420 starting from the HindIII site near the SV40 origin of replication and enhancer sequences of pMT2 and introduces a unique Cla I site, but leaves the adenovirus VAI gene intact.

B. BMP-5 Vector Construction A derivative of the BMP-5 cDNA sequence set forth in Table III comprising the the nucleotide sequence from nucleotide #699 to #2070 is specifically amplified. The oligonucleotides CGACCTGCAGCCACCATGCATCTGACTGTA and TGCCTGCAGTTTAATATTAGTGGCAGC are utilized as primers to allow the amplification of nucleotide sequence #699 to #2070 of Table III from the insert of clone U2-16 described above in Example V. This procedure introduces the nucleotide sequence CGACCTGCAGCCACC immediately preceeding nucleotide #699 and the nucleotide sequence CTGCAGGCA immediately following nucleotide #2070. The addition of these sequences results in the creation of PstI restriction endonuclease recognition sites at both ends of the amplified DNA fragment. The resulting amplified DNA product of this procedure is digested with the restriction endonuclease PstI and subcloned into the PstI site of the pMT2 derivative pMT21 described above. The resulting clone is designated The insert of H5/5/pMT is excised by PstI digestion and subcloned into the plasmid vector pSP65 at the PstI site resulting in BMP5/SP6.

BMP5/SP6 and U2-16 are digested with the restriction endonucleases Nsil and Ndel to excise the portion of their inserts corresponding to nucleotides #704 to #1876 of Table III. The resulting 1173 nucleotide NsiI-Ndei fragment of WO 90/11366 PCT/US90/01630 72 clone U2-16 is ligated into the NsiI-NdeI site of BMP5/SP6 from which the corresponding 1173 nucleotide NsiI-NdeI fragment had been removed.

The resulting clone is designated BMP5mix/SP64.

Direct DNA sequence analysis of BMP5mix/SP64 is performed to confirm identity of the nucleotide sequences produced by the amplification to those set forth in Table III. The clone BMP5mix/SP64 is digested with the restriction endonuclease PstI resulting in the excision of an insert comprising the nucleotides #699 to #2070 of Table III and the additional sequences containing the PstI recognition sites as described above. The resulting 1382 nucleotide PstI fragment is subcloned into the PstI site of the pMT2 derivative pMT21. This clone is designated BMP5mix/pMT21#2.

C. BMP-6 Vector Construction A derivative of the BMP-6 cDNA sequence set forth in Table IV comprising the nucleotide sequence from nucleotide #160 to #1706 is produced by a series of techniques known to those skilled in the art. The clone BMP6C35 described above in i Example V is digested with the restriction endonucleases Apal and TaqI, resulting in the excision of a 1476 nucleotide portion of the insert comprising nucleotide #231 to #1703 of the sequence set forth in Table IV. Synthetic olignucloetides with Sall restriction endonuclease site converters are designed to replace those nucleotides corresponding to #160 to #230 and #1704 to #1706 which are not contained in the 1476 ApaI-TaqI fragment of the BMP-6 cDNA sequence.

Oligonucleotide/SalI converters conceived to r e p a c e t h e m i s s i n g 5 WO 90/11366 PCr/UlS90/01 630 73 (TCGA .CCACCATGCCGGGGCTGGGGCGGAGGGCGCAGTGGCTGTG CTGGTGGT GGGGGCTGTGCTGCAGCTGCTGCGGGCC and

CGCAGCAGCTGCACAGCAGCCCCCACCACCAGCACAGCCACTGCGCC

CTCCGCCCCAG CCCCGGCATGGTGGG)and 3' (TCGACTGGTTT and CGAAACCAG) sequences are annealed to each other independently. The annealed 5' and 3' converters are then ligated to the 1476 nucleotide ApaI-TaqI described above, creating a 1563 nucleotide fragment comprising the nucleotide sequence from #160 to #1706 of Table IV and the additional sequences contrived to create Sall restriction endonuclease sites at both ends. The resulting 1563 nucleotide fragment is subcloned into the Sall site of pSP64. This clone is designated BMP6/SP64#15.

DNA sequence analysis of BMP6/SP64#15 is performed to confirm identity of the 5' and 3' sequences replaced by the converters to the sequence set forth in Table IV. The insert of BMP6/SP64#15 is excised by digestion with the restriction endonuclease SalI. The resulting 1563 nucleotide SalI fragment is subcloned into the XhoI restriction endonuclease site of the pMT2 derivative pMT21 and designated herein as BMP6/pMT21.

D. BMP-7 Vector Construction A derivative of the BMP-7 sequence set forth in Table V comprising the nucleotide sequence from nucleotide #97 to #1402 is specifically amplified.

The oligonucleotides CAGGTCGACCCACCATGCACGTGCGCTCA and TCTGTCGACCTCGGAGGAGCTAGTGGC are utilized as primers to allow the amplification of nucleotide sequence #97 to #1402 of Table V from the insert of clone PEH7-9 described above. This procedure WO 90/11366 PCT/US90/01630 74 generates the insertion of the nucleotide sequence CAGGTCGACCCACC immediately preceeding nucleotide #97 and the irn, rtion of the nucleotide sequence GTCGACAGA immediately following nucleotide #1402.

The addition of these sequences results in the creation of a Sail restriction endonuclease recognition site at each end of the amplified DNA fragment. The resulting amplified DNA product of this procedure is digested with the restriction endonuclease SalI end subcloned into the SalI site of the plasmid vector pSP64 resulting in BMP7/SP6#2.

The clones BMP7/SP6#2 and PEH7-9 are digested with the restriction endonucleases NcoI And StuI to excise the portion of their inserts corresponding to nucleotides #363 to #1081 of Table V. The resulting 719 nucleotide NcoI-StuI fragment of clone PEH7-9 is ligated into the NcoI-StuI site of BMP7/SP6#2 from which the corresponding 719 nucleotide fragment is removed. The resulting clone is designated BMP7mix/SP6.

Direct DNA sequence analysis of BMP7mix/SP6 confirmed identity of the 3' region to the nucleotide sequence from #1082 to #1402 of Table V, however the 5' region contained one nucleotide misincorporation.

Amplification of the nucleotide sequence (#97 1 to #1402 of Table V) utilizing PEH7-9 as a template is repeated as described above. The resulting amplified DNA product of this procedure is digested wi-h the restriction endonucleases SalI and PstI.

This digestion results in the excision of a 747 nucleotide fragment comprising nucleotide #97 to #833 of Table V plus the additional sequences of the 5' priming oligonucleotide used to create the WO 90/11366 PCT/US90/01630 Sail restriction endonuclease recognition site described earlier. This 747 SalI-PstI fragment is subcloned into a SalI-PstI digested pSP65 vector resulting in 5'BMP7/SP65. DNA sequence analysis demonstrates that the insert of the 5'BMP7/SP65#1 comprises a sequence identical to nucleotide #97 to #362 of Table V.

The clones BMP7mix/SP6 and 5'BMP7/SP65 are digested with the restriction endnucleases SalI and NcoI. The resulting 3' NcoI-SalI fragment of BMP7mix/SP6 comprising nucleotides #363 to #1402 of Table V and 5' SalI-NcoI fragment of 5'BMP7/SP65 comprising nucleotides #97 to #362 of Table V are ligated together at the NcoI restriction sites to produce a 1317 nucleotide fragment comprising nucleotides #97 to #1402 of Table V plus the additional sequences derived from the 5' and 3' oligonucleotide primers which allows the creation of Sail restriction sites at both ends of this fragment. This 1317 nucleotide Sail fragment is ligated into the Sall site of the pMT2 derivative pMT2Cla-2. This clone is designated BMP7/pMT2.

The insert of BMP7/pMT2 is excised by digestion with the restriction endonuclease Sail.

The resulting 1317 nucleotide Sall fragment is subcloned into the Sall restriction site of the vector pSP64. This clone is designated BMP7/SP64#2d. The insert of BMP7/SP64#2d is excised by digestion with Sail and the resulting Sall fragment comprising nucleotides #97 to #1402 of Table V is subcloned into the Xhol restriction endonuclease site of the pMT2 derivative pIr21 described above.

Example VII i_ I -~mLb~g~ WO 90/11366 PCT/US90/01630 76 Transient COS Ceil Expression To obtain transient expression of BMP-5, BMP- 6, and BMP-7 proteins one of the vectors containing the cDNA for BMP-5, BMP-6 or BMP-7, BMP5mix/pMT21#2, BMP6/pMT21#2, or BMP7/pMT21 respectively, are transfected into COS-1 cells using the electroporation method. Othur suitable transfection methods include DEAE-dextran, and lipofection. Approximately 48 hours later, cells are analysed for expression of both intracellular and secreted BMP-5, BMP-6 or BMP-7 protein by metabolic labelling with 3 5 S] methionine and polyacrylamide gel electrophoresis. Intracellular BMP is analyzed in cells which are treated with tunicamycin, an inhibitor of N-linked glycosylation. In tunicamycin-treated cells, the nonglycosylated primary translation product migrates as a homogeneous band of predictable size and is often easier to discern in polyacrylamide gels than the glycosylated form of the protein. In each case, intracelluar protein in tunicamycintreated cells is compared to a duplicate plate of transfected, but untreated COS-1 cells.

A. BMP-5 COS Expression The results demonstrate that intracellular i forms of BMP-5 of approximately 52 Kd and 57 Kd are made by COS cells. The 52 Kd protein is the size i predicted by the primary sequence of the the cDNA clone. Following treatment of the cells with tunicamycin, only the 52 Kd form of BMP-5 is made, suggesting that the 57 Kd protein is a glycosylated derivative of the 52 Kd primary translation product. The 57 Kd protein is secreted into the conditioned medium and is apparently not -111 UII T.Y *-31"-EI WO 90/11366 PCT/US90/01630 77 efficiently processed by COS-1 cells into the pro and mature pepti, 's.

B. BMP-6 COS Expression Intracellular BMP-6 exists as a doublet of approximately 61 Kd and 65 Kd in untreated COS-1 cells. In the presence of tunicamycin, only the 61 Kd protein is observed, indicating that the 65 Kd protein is the glycosylated derivative of the 61 Kd primary translation product. This is similar to the molecular weight predicted by the cDNA clone for BMP-6. In the absence of tunicamycin, the predominant protein secreted from COS-1 cells is the 65 Kd glycosylated, unprocessed clipped form of BMP-6. There are also peptides of 46 Kd and 20 Kd present at lower abundance than the 65 Kd that likely represent the processed pro and mature peptides, respectively.

C. BMP-7 COS Expression Intracellular BMP--7 protein in tunicamycintreated COS-1 cells is detected as a doublet of 44 Kd and, 46 Kd. In the absence of tunicamycin, proteins of 46 Kd and perhaps 48 Kd are synthesized. These likely represent glycosylated derivatives of the BMP-7 primary translation products. The 48 Kd protein is the major BMP species secreted from COS-1 cells, again suggesting inefficient cleavage of BMP-7 at the propeptide i dibasic cleavage site.

S Example VIII CHO Cell Expression DHFR deficient CHO cells (DUKX B11) are transfected by electroporation with one of the BMP- 5, BMP-6 or BMP-7 expression vectors described WO 90/11366 PCT/US90/01630 78 above, and selected for expression of DHFR by growth in nucleoside-free media. Other metnt. of I transfection, including but not limited to CaPO 4 precipitation, protoplast fusion, microinjection, and lipofection, may also be employed. In order to obtain higher levels of expression more expediently, cells may be selected in nucleosidefree media supplemented with 5 nM, 20 nM or 100 nM MTX. Since the DHFR selectable marker is physically linked to the BMP cDNA as the second gene of a bicistronic coding region, cells which express DHFR should also express the BMP encoded within the upstream cistron. Either single clones, or pools of combined clones, are expanded and analyzed for expression of BMP protein. Cells are selected in stepwise increasing concentrations of MTX (5 nM, 20 nM, 100 nM, 500 nM, 2 uM, 10 uM, and 100 uM) in order to obtain cell lines which contain multiple copies of the expression vector DNA by virtue of gene amplification, and hence secrete large amounts of BMr protein.

Using standard techniques cell lines are screened for expression of BMP RNA, protein or activity, and high expressing cell lines are cloned or recloned at the appropriate level of selection to obtain a more homogeneous population of cells.

The resultant cell line is then further characterized for BMP DNA sequences, and expression of BMP RNA and protein. Suitable cell lines can then be used for producing recombinant BMP protein.

A. CHO Expression of The BMP-5 vector BMP5mix/pMT21#2 described above is transfected into CHO cells by electroporation, and cells are selected for

I

WO 90/11366 PCT/US90/01630 79 expression of DHFR. Clonal cell lines are obtained from individual colcnies selected stepwise for resistence to MTX, and analyzed for secretion of BMP-5 proteins. In some cases cell lines may be maintained as pools and cloned at later stages of MTX selection.

As described in Example V.B. the cDNA for BMPencodes for a protein of approximately 52 Kd.

Following processing within the cell that includes, but may not be limited to, propeptidc cleavage, glycosylation, and dimer or multimer formation, multiple BMP-5 peptides are produced. There are at least 4 candidate peptides for processed forms of the BMP-5 protein discernable following SDS PAGE under reducing conditions; a 65 Kd peptide, a 35 Kd peptide, and a doublet of approximately 22 Kd moleular weight. Other less abundant peptides may also be present. By comparison to thp processing of other related BMP molecules and the related protein TGF-beta, the 65 Kd protein likely represents unprocer. ed BMP-5, the 35 Kd species represents the propeptide, and the 22 Kd doublet *preents the mature peptide.

Material from a BMP-5 cell line is analyzed in a 2-dimensional gel system. In the first dimension, proteins are electrophoresed under nonreducing condicions. The material is then reduced, and electrophoresed in a second polyacrylamice gel. Proteins that form disulfidebonded dimers or multimers will run below a diagonal across the second reduced gel. Results from analysis of BMP-5 protein indicates that a significant amount of the mature BMP-5 peptides can form homodimers of approximately 30-35 Kd that reduce to the 22 Kd doublet observed in one WO 90/11366 PCT/US90/01630 dimensional reduced gels. A fraction of the mature peptides are apparently in a disulfide-bondeC complex with the pro peptide. The amount of this complex is minor relative to the mature homodimer.

In addition, some of the unprocessed protein can apparantly form homodimers or homomultimers.

B. CHO Expression of BMP-6 The BMP-6 expression vector BMP6/pMT21 described above is transfered into CHO cells and selected for stable transfornants via DHFR expression in a manner as described above in part A with relation to BMP-5. The mature active species of BMP-6 is contemplated to comprise amino acid #382 #513 of Table IV. It is contemplated that secreted BMP-6 protein will be processed in a manner similar to that described above for other related BMP molecules and analogous to the processing of the related protein TGF-P [Gentry, At al.; Dernyck, et al., Supra].

C. CHO Expression of BMP-7 The BMP-7 expression vector BMP7/pMT..

described above is transfected into CHO cells and selected for stable transformants via DHFR expression in a manner as described above in relation to BMP-5. The mature active species of SBMP-7 is contemplated to comprise amino acid #300- #431 of Table V. It is contemplated that secreted BMP-7 protein will processed in a manner similar to that described above for BMP-5, other related BMP molecules and analogous to the processing of the related protein TGF-9 [Gentry, at al.; Dernyck, et al., Supra.].

WO 90/11366 PCT/US90/01630 81 EXAMPLE IX Biological Activity of Expressed BMP Proteins To measure the biological activity of the expressed BMP-5, BMP-6 and BMP-7 proteins obtained in Example VII and VIII above, the BMP proteins are recovered from the culture media and purified by isolating the BMP proteins from other proteinaceous materials with which they are coproduced, as well as from other contaminants. The proteins may be partially purified on a Heparin Sepharose column and further purified using standard purification techniqueF known to those skilled in the art.

For instance, post transfection conditioned medium supernatant collected from the cultures is concentrated approximately 10 fold by ultrafiltration on a YM 10 membrane and then dialyzed against 20mM Tris, 0.15 M NaC1, pH 7.4 (starting buffer). This material is then applied to a Heparin Sepharose column in starting buffer.

Unbound proteins are removed by a wash of starting buffer, and bound proteinz. including proteins of the invention, are desorbed by a wash of 20 mM Tris, 2.0 M NaC1, pH 7.4. The proteins bound by the Heparin column are concentrated approximately on, for example, a Centricon 10 and the salt reduced by diafiltration with, for example, 0.1% trifluoroacetic acid. The appropriate amount of the resultant solution is nixeu with 20 mg of rat matrix and then assayed for in vtvo bone and/or cartilage formation activity by the Rosenmodified Sampath Reddi assay. A mock transfect.on supernatant fractionation is used as a control.

Further purification may be achieved by INTERNATIONAL SEARCH REPORT International Applicatnon No PCT/US 90/01630 CLASSIFICATION OF SUBJECT MATTER (if several classilication symbols apply, indicate all)I According to International Patent Classification (IPC) or to both National Classification and IPC 11 /In r. 1 7 V 1'3 If%^ ~i~BReQE--b3181LPBII~BgIIB~B~ WO 90/11366 PCT/US90/01630 82 preparative NaDodSO 4 /PAGE [:aemmli, Nature 227:680- 685 (1970)]. for instance, approximately 300 pg of protein is applied to a 1.5-mm-thick 12.5% gel: recovery is be estimated by adding L- 3 5 Sjmethionine-labeled BMP protein purified over heparin-Sepharose as described above. Protein may be visualized by copper staining of an adjacent lane [Lee, et al., Anal. Biochem. 166:308-312 (1987)]. Appropriate bands are excised and extracted in 0.1% NaDodSO 4 /20 mM Tris, pH 8.0. The supernatant may be acidified with 10% CF 3 COOH to pH 3 and the proteins are desalted on 5.0 x 0.46 cm Vydac C 4 column (The Separations Group, Hesperia, CA) developed with a gradient of 0.1% CF 3 COOH to 90% acetonitrile/0.1% CF 3

COOH.

The implants containing rat matrix to which specific amounts of human BMP-5, BMP-6 or BMP-7 proteins of the invention have been added are removed from rats after approximately seven days and processed for histological evaluation.

Representative sections from eacl implant are stained for the presence of new bone mineral with von Kossa and acid fuschin, and for the presence of cartilage-specific matrix formation using toluidine blue. The types of cells present within the section, as well as the extent to which these cells display phenotype are evaluated and scored as described in Example III.

Levels of activity may also be tested for host cell extracts. Purification is accomplished in a similar manner as described above except that 6 M urea is included in all the buffers.

The foregoing descriptions detail presently preferred embodiments of the present invention. Numerous WO 90/11366 PCT/US90/01630 83 modifications and variations in practice thereof are expected to occur to thcse skilled in the art upon consideration of these descriptions. Those modifications and variations are believed to be encompassed within the claims appended hereto.

Claims (31)

1. A purified human BMP protein selected from the group consisting of: BMP-5 characterized by the aminc acid sequence comprising amino acid #"23 to #454 of Table III; BMP-6 characterized by the amino acid sequence comprising amino acid #382 to #513 of Table IV; and BMP-7 characterized by the amino acid sequence comprising amino acid #300 to #431 of Table V.

2. A purified human BMP protein selected from the group consisting of BMP-5 protein produced by the steps of culturing a cell transformed with a DNA sequence comprising nucleotide #1665 to #2060 of Table III or a sequence substantially homologous thereto; and (ii) recovering, isolating and purifiying from said culture medium a protein comprising amino acid #323 to #454 as shown in Table III or a sequence substantially homologous thereto; BMP-6 produced by the steps of culturing a cell transformed with a DNA sequence comprising nucleotide #1303 to #1698 of Table IV or a sequence substantially homologous thereto; and (ii) recovering, isolating and purifying WO 90/11366 PCT/US90/01630 from said culture medium a protein comprising amino acid #382 -o #513 as sho~n in Table IV or a sequence substantially homologous thereto; and BMP-7 protein produced by the steps of culturing a cell transformed with a DNA sequence comprising nucleotide #994 to #1389 of Table V or a sequence substantially homologous thereto; and (ii) recovering, isolating and purifying from said culture medium a protein comprising the amino acid #300 to amino acid #431 as shown in Table V or a sequence substantially homologous thereto.

3. A purified human BMP protein selected from the group consisting of BMP-5 produced by the steps of culturing a cell transformed with a DNA sequence comprising nucleotide #699 to #2060 of Table III or a sequence substantially homologous thereto; and (ii) recovering, isolating and purifying from said culture medium said protein; BMP-6 produced by the steps of culturing a cell transformed with a DNA sequence comprising nucleotide #160 to #1698 of Table IV or a sequence substantially homologous thereto; and WO 90/11366 PCT/US90/01630 86 (ii) recovering, isolating and purifying from said culture medium said BMP-6 protein; and BMP-7 produced by the steps of culturing a cell transformed with a DNA sequence comprising nucleotide #97 to #1389 of Table V or a sequence substantially homologous thereto; and (ii) recovering, isolating and purifying from said culture medium said BMP-7 protein.

4. A purified BMP protein produced by the steps of: culturing a cell transformed with a DNA sequence comprising a sequence which hybridizes to the DNA sequence selected from the DNA sequences of Table III comprising nucleotide #1665 #2060, Table IV comprising nucleotide #1303- #1698 or Table V comprising nucleotide #994 #1389 under stringent hybridization conditions; and recovering, isolating and purifying from said culture medium a protein characterized by the ability to induce cartilage and/or bone formation. A protein of claim 1 further characterized by the ability to demonstrate the induction of cartilage and/or bone formation.

6. A protein of claim 2 further characterized by the ability to demonstrate the induction of L- WO 90/11366 PCT/US90/01630 87 cartilage and/or bone formation.

7. A protein of claim 3 further characterized by the ability to demonstrate the ind -tion of cartilage and/or bone formation.

8. A DNA sequence encoding a protein of claim 1.

9. A DNA sequence encoding a BMP protein said DNA sequence selected from the group consisting of a DNA sequence encoding BMP-5 comprising the nucleotide #1665 to #2060 of Table III and sequences which hybridize thereto under stringent hybridization conditions and encode a protein characterized by the ability to induce the formation of cartilage and/or bone; a DNA sequence encoding BMP-6 comrising nucleotide #1303 #1698 of Table IV and sequences which hybridize thereto under stringent hybridization conditions and encode a protein characterized by the ability to induce the formation of cartilage and/or bone; a DNA sequence encoding BMP-7 comprising nucleotide #994 #1389 of Table V and sequences which hybridize thereto under stringent hybridization conditions and encode a protein characterized by the ability to induce the formation of cartilage and/or bone; A DNA sequence encoding a BMP protein selected from the group consisting of c, 1 WO 90/11366 PCT/US90/01630 88 a DNA sequence encoding BMP-5 comprising the nucleotide #669 to #2060 of Table III i and sequences which hybridize thereto I under stringent hybridization conditions and encode a protein characterized by the ability to induce the formation of cartilage and/or bone; a DNA sequence encoding BMP-6 comrising nucleotide #160 #1698 of Table IV and sequences which hybridize thereto under stringent hybridization conditions and encode a protein characterized by the ability to induce the formation of cartilage and/or bone; a DNA sequence encoding BMP-7 mprising nucleotide #97 #1389 of Table V and sequences which hybridize thereto under stringent hybridization conditions and encode a protein characterized by the ability to induce the formation of cartilage and/or bone;

11. A vector comprising a DNA sequence of claim 8 in operative association with an expression control sequence therefor.

12. A vector comprising a DNA sequence of claim 9 in operative association with an expression contol sequence therefor. S13. A vector comprising a DNA sequence of claim in operative association with an expression control sequence therefor.

14. A host cell transformed with a vector of claim I- WO 90/11366 PCT/US90/01630 89 11. A host cell transformed with a vector of claim 12.

16. A host cell transformed with a vector of claim 13.

17. A method for producing a purified BMP protein said method comprising the steps of culturing in a suitable culture medium a transformed host cell of claim 14; and recovering, isolating and purifying said protein from said culture medium.

18. A method for producing a purified BMP protein said method comprising the steps of culturing in a suitable culture medium a transformed host cell of claim 15; and recovering, isolating and purifying said protein from sa4i culture medium.

19. A method for producing a purified BMP protein said method comprising the steps of culturing in a suitable culture medium a transformed host cell of claim 16; and recovering, isolating and purifying said protein from said culture medium. A pharmaceutical composition comprising an effective amount of a BMP-5, BMP-6 or BMP-7 protein in admixture with a pharmaceutically acceptable vehicle.

21. A pharmaceutical composition comprising an C L L_ L~i^ ~,~Dls~B d~"LY WO 90/11366 PCT/US90/01630 effective amount admixture with a vehicle. of a protein of claim 1 in pharmaceutically acceptable

22. A pharmaceutical effective amount admixture with a vehicle.

23. A pharmaceutical effective amount admixture with a vehicle.

24. A composition of pharmaceutically A composition of pharmaceutically

26. A composition of pharmaceutically

27. A composition of pharmaceutically

28. The composition matrix comprises group c-nsisting composition comprising an of a protein of claim 2 in pharmaceutically acceptable composition comprising an of a protein of claim 3 in pharmaceutically acceptable claim 20 further comprising a acceptable matrix. claim 21 further comprising a acceptable matrix. claim 22 further comprising a acceptable matrix. claim 23 further comprising a acceptable matrix. of claim 20 wherein said a material selected from the of hydroxyapatite, collagen, polylactic acid and tricalcium phosphate.

29. The composition of claim 21 wherein said matrix comprises a material selected from the group consisting of hydroxyapatite, collagen, polylactic acid and tiicalcium phospnate. WO 90/11366 PCT/US90/01630 The composition of claim 22 wherein said matrix comprises a material selected from the group consisting of hydroxyapatite, collagen, polylactic acid and tricalcium phosphate.

31. The composition of claim 23 wherein said matrix comprises a material selected from the group consisting of hydroxyapatite, collagen, polylactic acid and tricalcium phosphate.

32. Use of the composition treatment of a patient and/or bone formation.

33. Use of the composition treatment of a patient and/h.: bone formation.

34. Use of the composition treatment of a patient and/or bone formation. Use of the composition treatment of a patient and/or bone formation. of claim 20 for the in need of cartilage of claim 21 for the in need of cartilage of claim 22 for the in need of cartilage of claim 23 for the in need of cartilage

36. A pharmaceutical composition for wound healing and tissue repair said composition comprising an effective amount of a BMP-5, BMP-6 or BMP-7 protein in a pharmaceutically acceptable vehicle.

37. A pharmaceutical composition for wound healing and tissue repair said composition comprising I WO 94i 11366 PC1'/US9O/01630 an effective amount of the protein of claim 1 in a pharmaceutically acceptab~le vehicle.

38. A pharmadeutical coniposition for wound h -aling and tissue repair said composition comprising an effective amount of the protein of claim 2 in a pharmaceutically acceptabl: vehicle.

39. A pharmaceutical coc-nosition for wound healing P.nd tissue repair sa.id composition comprising an effective' amourt of~ the protein of claim 3 in a pharmaceuticoilly acceptable vehic'2.:.