FI118736B - Bone morphogenetic protein - Google Patents

Description

118736

BACKGROUND OF THE INVENTION This invention relates to bone formation inducing proteins called bone morphogenetic proteins (BMPs), particularly BMP-3c, nucleic acid molecules encoding said proteins, vectors containing said proteins, and vectors containing said proteins. said protein. The present invention also relates to the use of these bone morphogenetic pro-10 proteins in the treatment of disorders such as those related to bone and cartilage formation. The present invention further relates to osteogenic devices and pharmaceutical compositions containing said proteins.

BACKGROUND OF THE INVENTION

In 1945, Lancroix discovered a phenomenon related to bone formation when he showed that acidic alcohol-bone extract induces heterotropic bone formation at ectopic sites. Twenty years later, Urist and his colleagues decalcified the bone matrix, and when implanted intramuscularly, he noticed new cartilage and bone forming at the implant site. These findings led to the isolation and purification of a bone inducing agent, designated BMP, from bone matrices of various animal species and, years later, to the cloning and characterization of the cDNA of these novel proteins. Biological activity of BMP has been determined by bioassay in which BMP is implanted in rat or mouse muscle mass, or in mammalian cultures measuring alkaline phosphatase (ALP).

Earlier studies since 1965 have shown that BMPs are a member of the TGF-β superfamily, and like other members of this gene family, φ * ·: They have multiple effects on cell motility, growth and differentiation: ··· * 30 especially during bone formation and tissue repair processes, but also on embryogenesis and cancer. They are small, hydrophobic glyco- * · * * proteins of molecular size that are soluble in chaotropic substances such as urea and guanidine hydrochloride, but are resistant to several proteases, such as collagenases. effect.

The BMPs are produced as large precursor molecules that are processed into proteolytically mature peptides after translation. Like other members of the TGF-β superfamily ·: · *: BMPs contain a sequence of seven cysteine residues in the mature C-2 118736 terminal portion. In mature BMP monomers, between these cysteines, there are three disulfide bonds and one disulfide bond that combines the two monomers to form a biologically active dimer.

The effect of BMP is mediated through specific receptors on the cell membrane of target cells. BMP receptors are serine-threonine kinases that are similar to TGF-? Receptors and are divided into two subgroups: type I and type II receptors. BMPs can only bind tightly to the receptor that forms the heterotetrameric complex. The formation of such complex 10 is a prerequisite for BMP signal transduction. Within the target cell, BMP signals are transferred to the nucleus using specific molecules specialized in signal transduction called Smadit, which are also capable of attenuating BMP signals.

To date, 16 different BMP molecules have been characterized, seven of which (BMPs 2-7 and 9) have been shown to be capable of inducing bone formation when implanted into ectopic sites. Based on the amino acid sequence of the mature portion, the BMPs are divided into two subgroups. BMPs 2 and 4 are 86% identical to each other and BMPs 5, 6 and 7 are 78% identical. The identity between these two subgroups is only about 56%. The amino acid sequence of BMP-3 is about 45% similar to that of BMP-2 and BMP-4 and BMP-9 is 50-55% identical. BMPs 2, 4, 5, 6 and 7. Due to the high homology and low level:! ·. differences in molecular size, separation of BMPs, purification, and identification at the protein level is quite difficult, very time consuming and expensive. For this reason, most BMPs are nowadays produced using molecular biology techniques. Various recombinant protein techniques have been tried and both * * * eukaryotic and prokaryotic systems have been used.

• · • · φ · ·

Most of the research has been directed to recombinant human BMP, but the 30 Cervidae are also of interest because they undergo effective bone induction in the horns. Horns are bone-containing skull structures. which are typical of the Ce / v / dae family and which differ from the Bovidae horns in their manner of growth. The horns grow from the tip of the horn and are dropped by males * ·: * 'once a year. It has been argued that horns are the fastest-growing structures in m:: 35 min, and these structures are known to be the only ones that ·: ··: completely renew each year. A certain type of endochondral ossification occurs during the formation of horns, where the process proceeds through the presence of abundant vascular cartilage, which eventually becomes bone upon calcification. The horns form an interesting model of mineralizing tissue regeneration in an adult animal, and bone regeneration continues until the horns are dropped. Although the root cause of the astounding growth rate of horns has not been elucidated, several hormone BMPs are known to exist in horns. Deer antler-5 has been shown to produce BMP-2 and BMP-4 (Feng et al. 1997 Biochim Biophys Acta 1350: 47-52; Feng et al. 1995 Biochim Biophys Acta 1263: 163-168). In addition, BMP-3b is expressed in reindeer horns (Kapanen et al. 2002 J Biomed Mat Res 59: 78-83). However, it is also possible that the horns have one or more unknown factors that cause rapid growth of the horn.

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Because of their osteoinductive properties, both BMPs extracted from demineralized bone matrix and BMPs produced using recombinant technology are very interesting and highly potential alternatives to bone graft. Various BMP proteins have been used in several experimental and clinical studies.

BMP-3 and 3b are important regulatory molecules for bone induction and osteogenesis.

BMP-3 has been shown to be an important component of osteogenin with osteogenic activity, but there is still conflicting information regarding the biological activity of recombinant human BMP-3. Some studies suggest that it has osteogenic activity, but some studies claim the exact opposite, demonstrating its inhibitory function on bone:! ·. in the formation process, being a negative factor in the regulation of bone density.

• · · | For example, WO 02/43759 discloses methods and compositions for the treatment of defects and diseases such as osteoporosis or osteopenic conditions. In the i * V method in question, a composition comprising a BMP-3 inhibitor or antagonist is applied to the osteoporotic or osteopenic state.

* * • * «·· To date, BMP-3b is the only BMP that is characterized by a reindeer antler *. · *? 30 (Kapanen et al. 2002).

• · · • · • »• · ·. US 6245889 discloses purified BMP-2 and BMP-4 proteins and their production methods. Also described is a pharmaceutical composition containing said BMP-4.

• · **: * 'As is commonly known in the art, these proteins and compositions can be used to: V: 35 treat bone and cartilage defects, heal wounds, and repair similar *: * ·· other tissue damage. In addition, the above pharmaceutical composition may include a matrix capable of delivering said BMP protein to a site of bone and / or cartilage injury, providing a structure for developing bone and cartilage, and also capable of being optimally resorbed into the body. Such matrices can be formed from materials currently in use in other medical implant applications.

US 5,399,677 discloses DNA molecules encoding mutant forms of bone morphogenetic proteins. Mutant forms of BMP can be produced in bacteria and folded into the biologically active form either as a homodimer or heterodimer of BMP. The method by which said mutant BMP is made is also disclosed. These mutant forms are useful because, when produced in a bacterial host, they are folding correctly.

WO 98/51354 discloses osteogenic devices and methods of using them to repair bone and cartilage defects. A method for growing new bone at a damaged bone site in a mammal includes the step of implanting a calcium phosphate matrix containing at least one osteogenic protein into the lesion site. These osteogenic proteins comprise a number of morphogens, such as bone morphogenetic proteins.

EP 1131087 discloses the additional use of morphogenetic proteins such as BMP. It has been shown that contact of cancer cells with morphogens inhibits the growth of cancer cells and causes their differentiation away from the cancer cell phenotype. . ·. Injecting. The use of morphogens can affect the cellular fate of cancer cells and help to reduce the symptoms of cancer.

Although some applications of some BMP proteins as bone and cartilage inducers, as well as in the alleviation of the symptoms of cancer, are known, there is a need for: however, to obtain even better methods for these proteins. and · ··, especially to obtain better morphogenetic proteins, such as those that are more effective as bone inducers or more soluble than before. Such proteins would be of use in better therapeutic methods and applications.

Surprisingly, the novel BMP-3-type protein of the present invention, hereinafter referred to as the BMP-3c, is known as: BMP-3c. , was found in a reindeer. Although it has a high sequence homology to other already known BMP-3 proteins, BMP-3c has very useful properties related to bone and cartilage formation. These properties are significantly better than those of the already known BMP proteins. The bone morphogenetic proteins of the present invention and their homologs are useful as bone and cartilage inducers in a variety of applications, such as therapeutic applications.

The reindeer BMP-3c protein of this invention is 95% homologous to the already known reindeer BMP-3b and 93% homologous to human BMP-3b. Because BMP-3b was already known in the reindeer, the novel protein of the present invention was named BMP-3c.

One aspect of the present invention pertains to an isolated morphogenetic protein comprising the BMP-3c consensus sequence.

Another aspect of this invention relates to an isolated DNA molecule encoding the morphogenetic protein of interest.

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Yet another aspect of the present invention pertains to a nucleic acid vector containing said isolated DNA molecule.

Yet another aspect of the present invention pertains to a recombinant host cell containing the DNA molecule or nucleic acid vector mentioned above.

Yet another aspect of the present invention pertains to a bone morphogenetic protein produced by growing said host cell so as to express: said bone morphogenetic protein and said bone morphogenetic protein is recovered from said host cell.

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Yet another aspect of the present invention pertains to a recombinant host cell, which expresses said bone morphogenetic protein.

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Yet another aspect of the present invention relates to a pharmaceutical composition comprising said bone morphogenetic protein.

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Still another aspect of the present invention relates to said isolated bone morphogenetic protein for use as a medicament.

A further aspect of the present invention relates to the use of said isolated bone morphogenetic protein in the manufacture of a medicament for use in the treatment of bone and cartilage disorders in which regeneration, repair, or growth is desirable, or other diseases such as cancers.

Yet another aspect of the present invention pertains to an osteogenic device for treating said disorders, which device comprises a morphogenetic protein of the bone in question.

Yet another aspect of the present invention pertains to a method of inducing cartilage and / or bone formation by treating said cartilage and / or bone 10 with said isolated bone morphogenetic protein.

Yet another aspect of the present invention pertains to a method of treating such bone and cartilage disorders where regeneration, repair, or growth is desirable, or other diseases such as cancer by administering said isolated bone morphogenetic protein to a patient suffering from said disorder. .

BRIEF SUMMARY OF THE FIGURES Figure 1 shows plasmids containing rdBMP-3c inserts in the PCR vector pGEM-T® (Promega).

Figure 2 shows plasmids containing rdBMP-3c inserts in the expression vector. pTrcHis2A (Invitrogen).

··:. ·. Figure 3 shows plasmids containing rdBMP-3c inserts in the expression vector pET-22b (+) (Novagen).

Figure 4 shows the mature: ···: 30 parts of the amino acid and nucleotide sequences of reindeer BMP-3c expressed from pTrcrd3c. The mature portion of the reindeer BMP-3c protein is flanked and the cysteines typical of the TGF-3 superfamily are marked *; '··· in bold.

• · · • * • * • · ·

Figure 5 shows the amino acid and nucleotide sequences of the mature portion of reindeer BMP-3c expressed from pETrd3c. The mature portion of reindeer BMP-3c * ·: ** is flanked and the cysteines typical of the TGF-β superfamily are marked: ***: in bold.

·· # ♦ ♦ ··· 7 118736

Figure 6 shows part of the amino acid and nucleotide sequence of reindeer BMP-3c. The mature portion is flanked and the cysteines typical of the TGF-β superfamily are highlighted in bold.

Figure 7 shows X-rays of the hind foot muscles of the mouse. A) a control and B) an implanted implant containing the BMP-3c protein of the present invention.

Figure 8 shows an SDS-PAGE gel stained with Coomassie Blue dye with fractions eluted from a HiTrap column (Example 3C). The zones represent 10 1) starting material; 2) material flowing through the column; 3) first wash; 4) second wash; 5) pH gradient elution, pH 8.0; 6) pH gradient elution, pH 6.2; 7) pH gradient elution pH 5.3; 8) pH gradient elution, pH 4.0 and 9) standard.

Detailed Description of the Invention 15

There is a high degree of homology between the mature portions of the previously known BMP-3 proteins in mammals. Cloning and characterization of the mature portion of reindeer BMP-3c showed that at the amino acid level the highest homology was with reindeer BMP-3b (95%) and human BMP-3b (93%).

The nucleotide (Table 1) and amino acid (Table 2) homologies of BMP-3b and BMP-3 proteins between different mammals are shown in Tables 1 and 2. Human, mouse, and rat BMP-3b have been previously characterized. The homology comparison at nucleotide level varies between 87-92% and at amino acid level 94-97%. Previously ···:. In addition to the organisms mentioned above, BMP-3b has also been cloned from a reindeer. Various mammals: The amino acid homology of the derived BMP-3b varies from 94% to 97% and the nucleic acid homology of nucleic acid from 87% to 92%. The nucleotide sequence of the reindeer BMP-3c partial cDNA and the corresponding amino acid sequence are shown in Figure 6. In general, "I.5, BMP-3c also has homology with other BMP types.

• · • 1 ·· 1 ·· • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · to · Close «Back * · 8 118736

Human Human Mouse Rat Poro rdBMP-3c ah nt ah nt ah nt ah nt ah nt

Human 100 100 92 88 92 87 89 88 93 89

Mouse 92 88 100 100 97 96 87 86 91 87

Rat 92 87 97 96 100 100 87 85 91 86

Poro 89 88 87 86 87 85 100 100 95 96 rdBMP-3c 93 89 91 87 91 86 95 96 100 100

Table 1. Homology of mature portions of different mammalian BMP-3b at nucleotide and amino acid levels (%) (nt = nucleotides; ah = amino acids)

Human Human Mouse Rat rdBMP-3c ah nt ah nt ah nt ah nt

Human 100 100 94__88_ 97_ 87__81_J> 9_

Mouse__94 88 100 100 97 92 73 71_

Rat 97_ 87 97 92 100 100 80 71. rdBMP-3c 81 69 73 71 80 71 100 100 • · · * · · · ** · “(^ - ^ * • t · • · ·» M *

Table 2. Homology of mature portions of different mammalian BMP-3 at nucleotide and amino acid levels (%) (nt = nucleotides / ah = amino acids).

»M *, _. . 10 • · *

The alignment below shows the amino acid sequence of mature human and reindeer BMP-3b proteins and the sequence of reindeer BMP-3c of this invention (comparison made using ClustalX 1.8 (Thompson, JD, Higgins, DG and Gibson) , TJ! *** (1994) Nucleic Acids Research, 22: 4673-680), rdBMP-3c = reindeer BMP-3c, ...: 15 rdBMP-3b = reindeer BMP-3b, hBMP-3b = human BMP-3b, star indicates identical amino acids).

• · • · ··· * ··· * · · · · · · 9 118736

rdBMP-3c RKKGQDVFMASSQVLDFDEKTMQKARKKQWDEPRVCSRRYLKVDFADIGWNEWIISPKSF

rdBMP-3b RKKGQDVFMAS SQVLDFDEKTMQKA-KKQVGEPRVCSRRYLKVDFADIGWNEWI X SPKSF

hBMP-3b RKKGQEVFMAASQVLDFDEKTMQKARRKQWDEPRVCSRRYLKVDFADIGWNEWIISPKSF

*** ★ **** ★ * · * ★ ************ - ** ******* ★ ** ★ · ***** ♦ **** ★ ******* 5

rdBMP-3 c DAYYCSGACEFPMPKMVRPSNHATIQSIVRAVGIVPGIPEPCCVPDKMSSLGVLFLDENR

rdBMP-3b DAYYCSGACEFPMPRWVRPSNHATIQSIVRAVGIVPGIPEPCCAPDKMSSLGVLFLDENR

hBMP-3b DAYYCAGACEFPMPKIVRPSNHATIQSIVRAVGIIPGIPEPCCVPDKMNSLGVLFLDENR

*****. ********. ******************. ******** * * * * *********** 10

rdBMP-3 c NWLKVYPNMSVETCACQ

rdBMP-3b NWLKVYPNMSVETCACQ

hBMP-3b NWLKVYPNMSVDTCACR

************ · * * * * · 15 "BMP-3 protein of the invention" or "bone morphogenetic protein of the invention" refer to a protein that has bone morphogenetic activity (or both, words can be used as synonyms), such as the reindeer-derived BMP-3c protein described herein (SEQ ID NO: 1 or 20 rdBMP-3c in the above alignment). A feature of the BMP of the present invention, for example, as set forth in the above alignment, are those amino acids that are different from other known BMP-3 proteins, such as those different from human BMP-3c and reindeer BMP-3c. between. Preferably, the regions in which these amino acids are located are conserved M · *. *. 25 in the BMP of the present invention. Amino acids 26 and 104 define the region where the amino acids differ when compared to reindeer BMP-3b and amino acids 6 and 138 define the region where the amino acids differ when compared to human BMP-3b.

On the other hand, additions, deletions, and substitutions far from said characteristic region are unlikely to result in a substantial change in the BMP of the invention. protein function, potency, or folding. For example, homologs with deletions, such as deletion of a few amino acids, preferably 1-10 amino acids, more preferably 1-5 amino acids, most preferably 1-3 amino acids, at either the 35 carboxyl or amino terminus, wherein the polypeptide is short in size. are within the scope of this invention so long as such deletions do not affect the amino acids characteristic of M *! ...: the BMP of the invention. It is preferred that the homologues in question have the beneficial properties of the original reindeer BMP-4, associated with said characteristic amino acids Pro6 and / or Pro21 and / or their region. Such homologues may have amino acid substitutions that do not substantially affect the function and potency of said protein. For example, an amino acid not located at or near the active site 5, may be replaced by another having similar structure and / or chemical properties (e.g., hydrophobicity or hydrophilicity), this means conservative amino acid exchange as long as the substitution in question does not substantially alter the function or folding of the mature protein. Such substitutions are well known and understood in the art. Examples of these 10 amino acid properties divided into different groups are hydrophobic (Met, Ala, Val, Leu, Ile), neutral hydrophilic (Cys, Ser, Thr), acidic (Asp, Glu), basic (Asn, Gln, His, Lys, Arg) ), amino acids that affect chain orientation (Gly, Pro), and aromatic (Trp, Tyr, Phe) amino acids. Substitutions within the groups in question are unlikely to result in significant changes in the backbone structure of the poly-15 peptide chain (for example, pleated or helical conformation), charge, or hydrophobicity of the molecule or side chain size.

Compared to BMP-3c with known reindeer BMP-3b, BMP-3c has one additional amino acid R26 and five substituted amino acids W30, D31, K75, M76

and V104, calculated from the mature portion of the BMP-3c protein as set forth in SEQ ID NO:

NO: 1 ° C. When compared to the known human BMP-3b protein, BMP-3c contains nine substituted amino acids D6, S11, K27, S66: M76, V95, S109, E133 and Q138 as defined by the mature portion of BMP-3c ··· · 25 as shown in SEQ ID NO: 1. These amino acids are characteristic of the BMP protein of this invention. However, since E133 and Q138 are located at the carboxy terminus of the mature portion of the protein, they are not likely to play a major role in the function of the · · * | * Y protein.

The BMP-3c protein in question contains the amino acids D6, S11, K27, S66, M76, V95 and S109 at their respective numbering positions, as defined by the matrix of the BMP-3c protein. - ··: * · * of this portion as shown in SEQ ID NO: 1. Such sites may be determined by aligning similar homologues, derivatives or fragments of those homologues with the sequence of SEQ ID NO: 1.

• i ·. ···. 35 • ·

The BMP-3 protein of the invention contains the sequence of the BMP-3 family shown below, derived from the ClustalX lineage, where several species of different species are linearized. sequences corresponding to amino acids 26-104 of SEQ ID NO: 1.

r KLK-Q-W-D-E-P-R-V / N-C-S / A-R-R-Y-L-D-V-D-F-A-D-I-g-w-n / p-e-w-i-i-p-p-D-p-F-D-A

5 Y-Y-C-S-G-A-C-E / Q-F-P-M-P-K-M-V / L-R / K-P-S-N-H-A-T-I-Q-S-I-V-R-A-V-G-I / V-V-F / S-G-I-P-E-P-C-C-V

Other consensus sequences may also be defined, such as those comprising the amino acid residues 6-104, 6-109, or 6-138 of SEQ ID NO: 10, or the like, which differ in length, for example, 1-20 amino acids. Such consensus sequences can be defined from the alignment below, or from similar alignments using different BMP proteins related to each other. The aligned BMP sequences are derived from reindeer, human, rat and African nail frog (Xenopus Lae-15 vis).

♦ 4 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·• · ··· • * • • • • • • • • • • • • • • • • * * ··· ·······················································································• · • · * · ··· 12 118736

BMP3C_REINDEER RKKGQDVFMASSQVLDFDEKTMQKARKKQWDEPRVCSRRYLKVDFADIGWNEWIISPKSF

BMP3_HUMAN KKKQRKGPHRKSQTLQFDEQTLKKARRKQWIEPRNCARRYLKVDFADIGWSEWIISPKSF

BMP3_RAT KKKQRKGPHQKGQTLQFDEQTLKKARRKQWIEPRNCARRYLKVDFADIGWSEWIISPKSF

BMP3_XEN0PUS KKKLRKGSRQKSQTLQFDEQTLKKARRKQWNEPRNCARRYLKVDFADIGWSEWIISPKSF

5 BMP3B REINDEER RKKGQDVFMASSQVLDFDEKTMQKA-KKQVGEPRVCSRRYLKVDFADIGWNEWIISPKSF

BMP3 B_HUMAN RKKGQEVFMAASQVLDFDEKTMQKARRKQWDEPRVCSRRYLKVDFADIGWNEWIISPKSF

, * · * · * * 1 ★ * - ★ ·· ** · ** * * * ★ - ♦ ★ ♦ ********** i *********

BMP3C_REINDEER DAYYCSGACEFFMPKMVRPSNHATIQSIVRAVGIVPGIPEPCCVPDKMSSLGVLFLDENR

10 BMP3_HUMAN DAYYCSGACQFFMPKSLKPSNHATIQSIVRAVGWPGIPEPCCVPEKMSSLSILFFDENK

BMP3_RAT DAYYCSGACQFPMPKSLKPSNHATIQSIVRAVGWSGIPEPCCVPEKMSSLSILFFDENK

BMP3_XENOPUS DAYYCSGACQFPMPKSLKPSNHATIQSIVRAVGWPGIPEPCCVPEKMSSLSILFLDENK

BMP3B_REINDEER DAYYCSGACEFPMPRWVRPSNHATIQSIVRAVGIVPGIPEPCCAPDKMSSLGVLFLDENR

BMP3B_HUMAN DAYYCAGACEFPMPKIVRPSNHATIQSIVRAVGIIPGIPEPCCVPDKMNSLGVLFLDENR

*****. 1. * * ♦♦. .. .. *************** ********. ****. **. 1.

BMP3C_REINDEER nwlkvypnmsvetcacq BMP3_HUMAN nwlkvypnmtvescacr

BMP3_RAT NWLKVYPNMTVDSCACR

20 BMP3_XENOPUS nwlkvypnmtvescacr

BMP3B_REINDEER NWLKVYPNMSVETCACQ

BMP3B_HUMAN nwlkvypnmsvdtcacr ********** * In one embodiment of the present invention, the BMP in question is any BMP-3 comprising SEQ ID NO: 1. amino acids 26-104. These amino- • · *; the position numbers of the acids have been calculated for any generic BMP-3 protein • • «·:. part of the amino terminus, such as the BMP-3c-! · * ··] protein of SEQ ID NO: 1, wherein the amino terminal sequence begins with RKKGQ, such as in a reindeer (see above for SEQ ID NO. : 1), or .. the corresponding areas. If there are additions or deletions in the amino acid sequence of the protein in question that affect numbering, this should be taken into account when defining the positions of the essential amino acids, e.g., by aligning the sequences as described, and then defining the amino acid sequences of said amino acids. 35 places for boys. However, any of the BMPs in question should, in essence, exhibit the function and efficacy disclosed herein. However, since all known BMP-3 proteins are highly conserved, the positioning of these essential amino acids is unambiguous, as is the case with human BMP-3b. For example, rdBMP-3b has a single amino acid deletion compared to rdBMP-3c, as observed in the above alignment, but nonetheless, the corresponding amino acids in the sequences involved in the comparison can be readily determined. These sites 5 can also be readily determined from other BMP proteins (see alignment above). In general, their degree of homology at the amino acid level may be at least 70%, preferably 80%, more preferably 93% and most preferably 95%.

In one embodiment of the present invention, BMP is any BMP-3 containing amino acids 6-104 of SEQ ID NO: 1. In one embodiment of the present invention, BMP is any BMP-3 containing amino acids 6-109 of SEQ ID NO: 1. In one embodiment of the present invention, BMP is any BMP-3 containing amino acids 6-138 of SEQ ID NO: 1. In yet another embodiment of the present invention, BMP is a BMP-3 protein comprising the amino acid sequence of SEQ ID NO: 15.

One embodiment of the present invention provides a nucleic acid molecule, such as a DNA or RNA molecule encoding said BMP of this invention, with or without a heparin binding site or propeptide. Due to the degeneracy of the genetic code, there are several different nucleic acid sequences encoding the BMP of the invention, the heparin-binding site or the propeptide. All such nucleic acid variants are within the scope of the present invention. Preferably, the nucleic acid molecule is a DNA molecule. Examples of such DNA sequences are shown in Figures 4-6.

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One embodiment of the present invention provides a replicable vector comprising a nucleotide acid molecule as described above operably linked to such a nucleic acid molecule. a sequence that controls its expression. Such vectors can be used to produce recombinant BMPs of the invention in a suitable host system.

The nucleic acid encoding the BMP of the present invention can be inserted into that · ***** replicable vector for cloning or expression. There are several vectors: T: publicly available. The vector may be, for example, in the form of a plasmid, cosmid, viral particle: '1:35 or phage. The appropriate nucleic acid sequence can be inserted into the vector using a variety of procedures. In general, DNA is ligated to a suitable restriction endonuclease cleavage site using procedures well known in the art. The vector may be, for example, one or more sequences of the signal sequence, the origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of such suitable vectors containing one or more of these components utilizes standard ligation techniques well known to those skilled in the art.

In general, the BMP in question can be produced by gene expression by expression in any suitable host cell, such as a bacterial cell. Such methods are well known in the art and can be found in the literature. It is essential that the protein folds into the correct form during expression and that it contains all necessary post-translational modifications.

It is not always possible to produce and purify certain proteins properly, for example due to solubility and refolding problems. Generally, E coli cannot make the post-translational modifications that are typical of mammalian cell systems. However, the present inventors have produced the mature portion of reindeer BMP-3c as a recombinant protein in E. coli and, after purification and refolding, have shown that it is in a biologically active form.

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One embodiment of the present invention provides a host cell comprising a nucleotide molecule or nucleotide vector described above. Useful cells are all prokaryotic and eukaryotic cells capable of expressing this * »; ' protein of the invention. Such host cells are well known in the art and one of ordinary skill in the art can readily select a suitable host cell. One embodiment provides? · V *: BMP produced by growing said cells to express said protein and recovering said produced protein from said host cell. Any useful method may be used to provide protein or • * a ·: * · *; tea purification and purification and such methods are well known in the art.

* The BMP of the present invention may be used in the treatment of disorders affecting **: * bone, cartilage, tendon or tooth defects or diseases, or the like. : where their regeneration, repair, or growth is desirable, or for other 35 diseases. The protein of the present invention may also be used for wound healing. such as burns, surgical ulcers and stomach ulcers and similar tissue repair and also cancer treatment as disclosed in EP1131087. Because the BMP protein generally lacks species specificity, the patient suffering from the defect in question may be any suitable animal, preferably a mammal such as a human, and the BMP protein used in the treatment may be derived from any suitable source. The use of similar BMPs in many therapeutic applications is well known in the art (see, for example, US 5,645,889 and WO 98/51354).

"Disorders Related to Bone, Cartilage, Tendon or Tooth Defects" as used herein generally refers to any disorder in which the healing or rebuilding, i.e. regeneration, of bone, cartilage, tendon or teeth is desirable. Non-limiting examples of treatment measures for disorders or diseases of bone, cartilage, tendon or teeth, or the like, include regeneration, repair, and growth of bone and dental tissue; bone regeneration, repair and growth in mammals such as man; treatment measures to correct abnormalities in bone formation or regeneration; wound healing, ec-15 topical bone induction, and healing of segmental bone damage in vertebrates; treatment measures to correct skeletal disorders and deformities; repair of major bone damage due to trauma, tumor removal, or congenital malformations, reconstruction of bone stores that have been reduced due to implanted internal prosthesis in repair operations, or retarded bone fractures or unrelated bone fractures; repair of bone and cartilage defects such as "critical size defects", "non-critical size defects", unrelated fractures, segmental unrelated fractures; acute fractures, cartilage defects, bone-cartilage defects, cartilage bone defects, local bone and cartilage formation; defects caused by degenerative diseases; dental applications for repairing dental tissue, alveolar bone, dental cement, tooth root membrane, filling tooth root-j: *: channel content, developing and improving dental implant attachment. More examples of these disorders can be found in Ann Rheum Dis, Volume 62, • · · *. ·· *. 2003, 73-78: Reddy AH: Cartilage Morphogenic Proteins: Role in Joint Develop- • · * 30 ment, homeostasis and regeneration.

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Other diseases in which the BMP of this invention is useful include, for example, cancer, fibromyalgia, psoriasis and other dermatological disorders, and rheumatic disorders: T and the like. Examples of such cancers and methods and compositions for treating them are provided. in EP1131087.

· * «• ·

One embodiment of the invention provides a BMP, such as BMP-3, for use in any of the embodiments described herein with one or more other morphological proteins, such as another type of BMP or the like. Generally, this achieves synergy benefits, as is known in the art. Examples of other suitable BMP proteins include, but are not limited to, BMP-1, BMP-2, other BMP-3, BMP-4, BMP-5, BMP-6, BMP-7 and BMP-8. Other therapeutically useful agents may also be used, such as epidermal growth factor, fibroplastic growth factor and transforming growth factor (US 6,245,889). In one embodiment of the invention, the complementary morphogenetic protein in question is derived from a reindeer, like any other reindeer BMP protein. In one embodiment of the invention, BMP is provided for use as a dimer, homodimer or heterodimer in combination with another BMP protein as described above. In another embodiment, the BMP is provided for use in dimeric form or in combination with another agent or protein, as described above, for the manufacture of a medicament for the treatment of disorders described in the specification.

15

One embodiment of the invention provides an osteogenic device, such as an implant, containing the BMP of the present invention. The osteogenic device may include a biocompatible matrix such as calcium phosphate, carboxymethylcellulose, collagen matrix, or combinations thereof. In one embodiment, the calcium phosphate matrix is a hydroxyapatite matrix. Such a matrix may allow slow release of the BMP protein and / or a suitable environment for release of the BMP protein. The osteogenic device may also include a metallic implant surrounded by said biocompatible matrix. One example of this metal is titanium. Some examples of such osteogenic devices are disclosed in WO 98/51354.

• ♦ • • • • • •: Non-limiting examples of various supports, carriers, or supports that

Iti 1 17 118736 Mineralized bone, bio-glass, any biodegradable material and combinations thereof; binding agents selected from the group consisting of mannitol, dextrans, white petrolatum, alkyl and methylcelluloses, wetting agents such as sodium salts, phobrin glue, mammalian fibrinogen and thrombin, and combinations and mixtures thereof. The osteogenic device may be, for example, a structurally stable, three-dimensional implant in the shape of a cube, cylinder or block or anatomically shaped or injectable. Examples of osteogenic tools, useful materials and techniques are disclosed in Skeletal reconstruction and bioimplantation (T. Sam Lindholm, 1997, Sprin-10 ger-Verlag, Heidelberg, Germany).

One embodiment of the invention provides a pharmaceutical composition comprising a therapeutically effective amount of the BMP of this invention in a pharmaceutically acceptable carrier or carrier. Such pharmaceutical compositions may be used to treat disorders associated with bone, cartilage, tendon or tooth damage or other diseases, wounds and other tissue damage, or any of the disorders described herein.

One embodiment of the invention provides a method of inducing bone, cartilage, tendon, tooth or the like, wherein said bone, cartilage, tendon, tooth, or the like is treated with the BMP of the present invention or combinations thereof as described above, in vitro or in vitro. vivo. Yet another embodiment of the invention provides a method of treating the disorders described in the specification, comprising administering an isolated BMP of the invention to a patient suffering from such disorders. The BMP in question may be administered as a pharmaceutical composition or as an osteogenic device as described above. Complementary morphogenetic proteins or other useful substances may be co-administered with the · · *: BMP of the present invention as described above, · ··· ·. ** ·. therapeutic effect.

30 ... The following description and examples describe how the mature portion of the reindeer *. /. * Nant BMP-3c, as disclosed in one aspect of the present invention, is produced in E. coli. After purification and folding, osteoinductive T activity was confirmed by bioassay in the mouse hindlimb muscle pocket. This in vivo bioassay has been a standard method for determining BMP activity since the discovery of the y protein. It involves implanting BMP in the hind foot of a mouse ***. assessment of intramuscular and heterotropic new bone induction radiologically and histologically 10 to 21 days after implantation.

18 118756

Osteoinductivity was observed in all four groups studied (1mg, 3mg, 5mg, and 8mg reindeer recombinant BMP-3c) and the response increased with dose (Table 3).

5 BMP-3C138

Doses 1 mg 3 mg 5 mg 8 mg

Pore _____ + __- ++ _ (+) + __ + __ + _ (+) __ BMP-3cno 10 __ ^ __

Dosage 1 mg 3 mg 5 mg 10 mg

The porous l | l | | l - I + I (+) I + I ++ ++ + + + ++ ++ • ♦ · / "1 Table 3. Reindeer BMP-3c138 and BMP-3c110 Osteoinductive Anti-1 · '· · '· 1 · tea comparison at different doses.

The results of the in vivo bioassay are shown in Figure 7. Figure 7A is a control and 7B is a · ·: 15 sample implanted with the BMP-3c protein of the present invention. Bioassay was performed as described in Marshall R. Urist, J.J. Chang, A. Lietze, Y.K.

Huo, A.G. Brownell and J. DeLange (1987): Preparation and Bioassay of Bone Morphogenetic Protein and Polypeptide Fragments, Methods Enzymol 146: 294-: ·. 312.

t ·· 20 I · * · 1 Examples

Ml III • · ·

Cloning and Homology Analysis of Reindeer BMP-3c cDNA ···

• I

• ·

M1 · 19118736 PCR product of about 400 base pairs was isolated and cloned into pGEM-T® vector. The nucleotide sequence obtained from the ABI Prism reactions was analyzed by computer and compared with already known human BMP sequences. In homology comparisons, the new cloned cDNA was found to be more homologous to human BMP-3b (nucleotide homology 89% and amino acid homology 93%) than to human BMP-3 (nucleotide homology 69% and amino acid homology 81%). When comparing the new cloned cDNA to all known BMPs, homology was highest with reindeer BMP-3b (nucleotide homology 96% and amino acid homology 95%). Therefore, the cDNA of the present invention was designated as reindeer BMP-10 3c.

Expression of mature portion of reindeer BMP-3c

In previous studies, human BMP-3 protein has been produced in CHO cells, 15 E. coli cells and the retroviral system. In addition, human BMP-3 and rat BMP-3b proteins have been produced as recombinant proteins in the adenoviral system. However, no member of the deer BMP-3 gene family has been produced as a recombinant protein.

A nucleotide molecule of 414 nucleotides in length corresponding to the mature portion of the 138 amino acid reindeer BMP-3c was subcloned from the pGEM-T® vector into the TrcHis2A vector and transformed into E. coli TOP10 cells (Invitrogen) to give the vector pTrc3. / 138. Expression of the recombinant protein was induced by: i.i .: IPTG. The production of recombinant protein was confirmed by SDS-PAGE but by induction • ·! : 25 It was not observed. To induce expression of the protein, pET22b (+) having the His6 tag and the pelB leader region was selected as an expression vector and a new vector constructed: designated pETrd3c / 138. E. coli strains of Rosetta (DE3) (Novagen) and Origami B (DE3) (Nova: *: gen) were used for expression. Results of recombinant rdBMP-3c ···. IPTG induction was confirmed by SDS-PAGE, but the expression level was lower than the expression levels of rdBMP-4 and rdBMP-6 (Unpublished data).

• «• ·: .. 7 Expression of Reindeer BMP-3c / 110 • · • · ·· *

Another shorter construct was made from rdBMP-3c. Nucleotide mole: :) 35 kilos of 330 nucleotides corresponding to 110 amino acids of the reindeer BMP-3c protein were subcloned from pGEM-T® to TrcHis2A vector **** '. and transformed into E. coli TOP 10 cells to yield the pTrcrd3c / 110 vector. Re-expression of the recombinant protein was induced by IPTG. The production of the recombinant protein was confirmed by SDS-PAGE, but no induction was observed. Therefore, pET22b (+) with the His6 tag and the pelB leader region was selected as the expression vector and the pETrd3c / 110 vector was constructed. Rosetta (DE3) and Origami B (DE3j E. coli strains were used for expression. Successful IPTG induction was confirmed by SDS-PAGE.

5

Biological Activity of Reindeer BMP-3c / 138 and BMP-3c / 110 Recombinant Proteins

Recombinant human BMP-3 protein has demonstrated osteogenic activity in bioassay when produced in CHO cells or in E. coli, but when retroviral or adenoviral systems are used as human recombinant BMP-3 production systems, no osteogenic activity is observed. When BMP-3b was produced using the adenoviral system, it was found to affect bone formation synergistically with BMP-2, but was unable to induce bone induction on its own. However, BMP-3 from deer (Cervidae-15) has not even been cloned and the biological activity of BMP-3b, which has been cloned previously, has not been tested.

In this study, it has been shown that a new member of the BMP-3 gene family, recombinant reindeer BMP-3c produced in E. coli, can induce bone formation when implanted with collagen felt in mouse thigh pockets.

* • i · • «« * «·« 1 • · «« 1 ♦ · · «« 1 • 1 I ·· · · «·· • • • 1 ** 1 • ·« «• ·· · 2 • · • 1 * · 1 ··· • · # · · · · · · · 1

M

• · ♦ f 2 • tt • • »21 118736

Example 1: Cloning and Sequencing the 3'-End of Reindeer BMP-3c cDNA A. Isolation of RNA 5-year-old male reindeer horns were dissected and frozen in liquid nitrogen immediately after slaughter. The frozen horns were cut into 0.5 cm pieces and stored at -70 ° C. Reindeer horn mRNA was isolated using the QuickPrep® Micro mRNA Purification Kit (Pharmacia Biotech). A portion of the reindeer horn piece was cut into small pieces (about 1 mm 3) and 0.1 g of this tissue was added to 0.6 in 10 ml of Extraction buffer containing guanidine thiocyanate and N-lauroyl sarcosine. The tissue was homogenized on ice with Ultra Turras three times for 10 seconds and cooled between homogenization runs. 1.2 ml of Elution buffer was added and the suspension was homogenized once more for 10 seconds. The result was a uniform suspension.

15

The reindeer horn homogenate and the Oligo (dT) cellulose were centrifuged at full speed [14,000 rpm, room temperature, Centrifuge 5415 C (Eppendorf)] for one minute. The buffer on top of the oligo (dT) cellulose pellet was removed and the clear tissue homogenate was pipetted in its place. The tube was inverted to obtain a suspension of Oli-20 go (dT) cellulose. The suspension was gently stirred for five minutes and centrifuged at full speed [14,000 rpm, room temperature. . ·. Centrifuge 5415 C (Eppendorf)] for ten seconds. The supernatant was discarded.

• · · • M «• · • · ·

* 7 25 Oligo (dT) cellulose was resuspended in High-Salt Buffer [10 mM

• · *

Tris-HCl (pH 7.5), 1 mM EDTA, 0.5 M NaCl], and the suspension was centrifuged at full speed [14,000 rpm, room temperature, Centrifuge 5415 C (Eppendorf)] for ten seconds. Washes with High-Salt Buffer were repeated five times

and twice more with Low Salt Buffer [10 mM Tris-HCl (pH 7.5), 1 mM

i.:i: 30 EDTA, 0.1 M NaCl]. 3 ml of Low Salt Buffer buffer was added and the suspension transferred to: ***: MicroSpin column. The MicroSpin column was placed in an Eppendorf tube and centrifuged. was run at full speed for five seconds. Column Oligo (dT) cellulose: r, was rinsed three times with Low Salt Buffer.

Y: 35 Reindeer Horn mRNA was eluted from the MicroSpin column to a clean Eppendorf tube

·: * ·: Add 0.2 ml of 65 ° C Elution buffer (QuickPrep® Micro mRNA) to the column

Purification Kit, Pharmacia Biotech) and centrifugation at full speed [14,000 rpm, room temperature, Centrifuge 5415 C (Eppendorf)] for 5 seconds.

22 118736

The elution step was repeated twice. The isolated mRNA was mixed by adding 5 μΙ of glycogen solution (5-10 mg / ml in DEPC-treated H2O), 1/10 volume of K-acetate solution (2.5 M potassium acetate, pH 5.0) and 0.5 ml of absolute ethanol to each eluant. (cooled to -20 ° C). Precipitation was allowed to proceed at -20 ° C for at least 30 minutes and the mRNA was centrifuged at full speed [14,000 rpm, 4 ° C, Centrifuge 5415 ° C (Eppendorf)] for five minutes. The precipitated mRNA was stored at -70 ° C until cDNA synthesis was performed.

B. Synthesis of cDNA 10

Reverse transcription of reindeer mRNA was performed by modifying the instructions of the Time Saver ™ cDNA Synthesis Kit (Pharmacia Biotech). 3 pg of mRNA was heat denatured at 65 ° C for ten minutes and cooled on ice. 0.2 pmol DTT, 0.5 µg Oligo (dT) 12-8 primer and heat denatured mRNA 15 were added to the First Strand reaction mixture containing FPLCpure ™ Cloned Murine Reverse Transcriptase, RNAguard ™, RNase / DNase-Free BSA, dNTPs (dATP, dCTP, dGTP and dTTP) in an aqueous buffer (Time Saver ™ cDNA Synthesis Kit, Pharmacia Biotech). The mixed solution was incubated at 37 ° C for one hour. After incubation, the First Strand reaction mixture was added to the Second Strand 20 reaction mixture containing E. coli RNase H and E. coli DNA polymerase! And dNTPs in an aqueous buffer (Time Saver ™ cDNA Synthesis Kit, Pharma.) . here Biotech). The solution was gently mixed and incubated at room temperature for 30 minutes. The synthesized cDNA was stored at + 4 ° C.

• · · ··· * • t • · · /! * '25 C. Mapping Reindeer Horn cDNA »· t • *» ··· · t ·

Part of the reindeer BMP-3c cDNA was amplified by PCR (Polymerase chain reaction) using degenerate primers (51-CGCAA (A / G) GACCGCA (A / G) GAAGAA (A / G) GGC-3 ') and s. .: V 30 (3′-TC (T / C) GT (A / G) GAGACCTGTGCCTG (T / C) CAA-5 ′), designed for the BMP-3b genes of various known mammalian species (human, rat and mouse) homology. based. The PCR reaction mixture (50 µl) contained 100 ng of reindeer hIII / cDNA, 40 pmol of each primer, 0.4 mM dNTP (PCR Core Kit, Roche) and 0.7 µL Expand High Fidelity Enzyme Mixture (Thermostable Taq Polymerase + Proofol: V: 35 Spring Polymerase, Roche) in Expand High Fidelity Buffer containing MgCl 2 ·: ··; (Expand High Fidelity PCR System, Roche). The reaction was carried out using a Mastercycler personal apparatus (Eppendorf) according to the following program: initial denaturation 94 ° C, 4 minutes, and the following 25 cycles: denaturation 94 ° C, 12 23 118736 minutes, priming 55 ° C, 1 minute, DNA extension of the chains at 72 ° C for 2 minutes. The final extension was performed at 72 ° C for 10 minutes.

D. Cloning into pGEM-T® Vector 5 The PCR product was purified using the Wizard® PCR Preps DNA Purification System kit (Promega) and ligated into the pGEM-T® vector (Figure 1) using T4 DNA ligase (pGEM-T® Vector). System I; Promega). 0.3 µg of purified PCR product and 2.3 µg / ml pGEM-T® vector were added to the ligation buffer containing 18 mM Tris-10 HCl (pH 7.8), 6 mM MgCl2, 6 mM DTT, 0.3 mM mM ATP, 3% polyethylene glycol and 0.14 U / μΙ T4 DNA ligase at a total volume of 66 µl. The reaction was allowed to proceed in a +16 ° C water bath which cooled to +4 ° C overnight. The resulting new plasmid was called pGEMrd3c / 142 (Figure 1).

15 E. Preparation of competent Escherichia coli TOP 10 F 'cells

Competent Escherichia coli TOP 10 F 'cells were prepared by the calcium chloride / magnesium chloride method. 2 ml of LB broth were inoculated with E. coli TOP 10 F 'cells and grown overnight at 37 ° C with shaking (225 rpm). The next 20 mornings, 100 ml of fresh LB broth was added to 1 ml of overnight culture and grown with shaking (225 rpm) at 37 ° C until. . *. ODeoo = 0.5-0.6. Cultured cells were harvested by centrifugation (2.500 x g, 5 minutes), suspended in 10 ml of 0.1 M MgCl 2 solution, and harvested by centrifugation (2.500 x g, 5 minutes). After treatment with MgCl2, the cells were suspended in 10 ml of 0.1 M CaCl2 solution, incubated in an ice bath for 30 minutes and collected again by centrifugation at 2500 x g for 5 minutes. The CaCl 2 treatment was repeated, however, with the addition of 3.5 ml of CaCl 2 solution a second time and the cells incubated for one hour. Glycerol (final concentration 14% (v / v)) was added to the suspension and the suspension was aliquoted in 200 μΙ. Competent E. coli TOP 10 F 'cells were frozen in nes-30 tetra nitrogen and stored at -70 ° C.

·· # • t • · ···. F. Transformation of Competent Escherichia coli TOP 10 F 'Cells and Selection of Clones Containing Reindeer BMP-3c: V: 35 Competent Escherichia coli TOP 10 F' Cells were thawed in an ice bath 15 minutes

·:··: I drive. 20 μΙ of the ligation mixture (described above) was added to 100 µl of TCM (10 mM

Tris-HCl, 10 mM CaCl2, 10 mM MgCl2, pH 7.0) and mixed with 200 μΙ of competent E. coli cells. The mixture was incubated on ice for 30 minutes before warming up to 1118366 poke shocks (43 ° C, 3 minutes). After heat treatment, 800 µl of LB broth was added and the cells were allowed to recover at 37 ° C for 30 minutes. Transformed cells were harvested by centrifugation at full speed for 2 minutes and resuspended in 30 µl culture broth. The cell suspension was plated on two LB plates containing si-5 containing 25 pg / ml ampicillin, 1 mmol IPTG (isopropyl-pD-thiogalactopyranoside) and 2.4 nmol of X-gal (5-bromo-4-chloro-3-indolyl) - D-galactoside) and cells were grown on plates overnight at 37 ° C. Positive clones were found to be white in color due to α-complementation of the IacZ gene. The method is described in detail in Sambrook and Russel (2001) Molecular Cloning, Cold 10 Spring Harbor Laboratory Press, New York.

Isolation of G. pGEMrd3c Plasmids and Sequencing of cDNA Inserts

Plasmids were isolated using the Wizard® Plus Minipreps DNA Purification System 15 (Promega) and further purified with ethanol precipitated. The identity of the cDNA was confirmed by sequencing on an ABI Prism (Perkin-Elmer Corporation). The sequencing reaction was performed using a DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech) and a Mastercycler Personel PCR (Eppendorf). The PCR primers used for sequencing were (5'-20 TAATACGACTCACTATAGGGCGA-3 ') and (3'-ATTTAGGTGACACTATAGAATAC-5') and the program used: 25 cycles of denaturation, .1, thio. 94 ° C, 30 seconds, primer attachment 50 °. C, 15 seconds, chain extension · 1, 60 ° C. Amplified PCR products were ethanol precipitated. To 10 µl of reaction mixture, 1 µl of 1.5 M Na acetate-250 mM EDTA buffer and 95-100% ethanol was added to give a final concentration of 75% ethanol. Precipitation was allowed; After stirring in an ice bath for 7 minutes and then centrifugation for 20 minutes, the supernatant was discarded and the pellet was washed at room temperature with 125 silicon 70% ethanol. The mixture was centrifuged rapidly and the ethanol used for washing was removed as efficiently as possible. for a few minutes 30 until all the ethanol had evaporated. The ABI Prism equipment was at the University of Oulu, Department of Medical Biochemistry and Molecular Biology, and the actual sequencing was carried out there Figure 6 shows a portion of the reindeer BMP-3c cDNA nucleotide and amino acid sequence.

• · • 1 ··· • · «« · ♦ · · «· · 25 118736

Example 2. Expression of the mature portion of reindeer's recombinant BMP-3c in Escherichia coli TOP 10 F ', Origami B (DE3) and Rosetta (DE3) cells A. Amplification of the mature portion of reindeer BMP-3c for the expression vector

The mature portion of reindeer BMP-3c was amplified from pGEMrd3c / 142 by PCR using homologous primers (5'-GGATCCGAGGAAGAAGGGCCAGGATGTTTTC-3 ') and (3'-AAGCTTTTGGCAGGCACAGGTCTCCAC-5') (see Example B). The 5 'end of the first primer of En-10 had the recognition site for the Bam HI restriction enzyme and the 3' end of the second primer had the recognition site for Hind III.

The 50 μΙ: η reaction mixture contained, in addition to plasmid pGEMrd3c / 142 (0.05 pg) and both primers (40 pmol) 0.4 mM dNTPs (PCR Core Kit, Roche) and 0.7 U / μΙ Ex- 15 pandas High Fidelity Enzyme Mixture (Thermostable Taq Polymerase + Proofreading Polymerase, Roche) and Expand High Fidelity Buffer containing MgCl2 (Roche). The PCR reaction was performed on a Mastercycler Personel (Eppendorf) according to the following program: 94 ° C initial, 4 minutes, and 25 cycles: 94 ° C denaturation, 1 minute, primer 55 ° C, 1 minute, DNA strands Extension at 72 ° C for 2 minutes. The final extension was performed at 72 ° C for 10 minutes. The PCR product was purified from the PCR reaction mixture using Wizard® PCR Preps. . ·. DNA Purification System kit (Promega) and ligated into pGEM-T1 vector.

The resulting new plasmid containing the mature portion of the reindeer BMP-3c was called i as pGEMrd3c / 138 (Figure 1).

'' 25: B. Subcision of the mature portion of reindeer BMP-3c from pGEM-T® vector into the expression vector pTrcHis 2A (Invitrogen) and transformation into competent Escherichia coli TOP 10 F 'cells 30 Reindeer BMP-3c mature subcloning of the portion from the pGEMrd3c / 138 vector into the expression vector pTrcHis 2A (Figure 2) was accomplished by removing the mature portion ···. \ pGEMrd3c / 138 with Bam HI and Hind III restriction enzymes and ligation

insert in pTrcHis 2A vector digested with the same enzymes. ! »· * Digestion of pGEMrd3c / 138 and pTrcHis 2A vectors (1 pg) with Bam HI (Roche) and Hind III

Y: 35 (Roche) enzymes were performed in 10 µl of 10 mM Tris-HCl, 10 mM NaCl, 5 mM

·: ··: MgCl 2, 1 mM 2-mercaptoethanol, pH 8.0 (SuRE / Cut B, Roche) using 1 U / μΙ coat restriction enzyme. The reaction was allowed to proceed for 1.5 hours at 37 ° C, after which the restriction enzymes were inactivated at 65 ° C for 20 minutes and frozen at 26118736 at -20 ° C. The ligation (0.1 U / μg ligase concentration) was performed in 2X Rapid Ligation Buffer (supplied with the pGEM-T® vector, Promega) in a +16 ° C water bath which was allowed to cool slowly to + 4 ° C overnight.

The new construct was verified by sequencing (the method is described in Example 1, section G) using primers: (5'-AGAGGTATATATTAATGTATCG -3 ') and (3'-ATGGTCGACGGCGCTATTCAG-5'). An expression vector containing the mature cDNA of pTrcHis 2A and the mature portion of reindeer BMP-3c was called pTrcrd3c (Figure 2). pTrcrd3c was transformed into competent Escherichia coli TOP 10 F 'cells as described in Example 1, Part F.

C. Extraction of the mature portion of reindeer BMP-3c into pET22b (+) (Novagen) expression vector and transformation into competent Escherichia coli Origami B (DE3) and Rosetta (DE3) cells

Subcloning of the mature portion of reindeer BMP-3c into the expression vector pET22b (+) (Novagen) (Figure 3) was performed as described above (see Example 2, Part B).

The resulting new plasmid containing the cDNA of the mature portion of the pET22b (+) vector and the reindeer BMP-3c ligated therein was verified by sequencing using primers 20 (5'-GGATCCGAGGAAGAAGGGCCAGGATGTTTTC-3 ') and (3'-CGCAGCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG -5 ') (see method in: described in Example 1, Part G) and the plasmid was called pETrd3c (Figure 3).

: Competent Origami B (DE3) and Rosetta (DE3) cells were transformed according to the instructions of the manufacturer (Novagen).

. * D. Expression of the mature portion of the recombinant protein of Poron BMP-3c in Escherichia coli cultures and cell harvesting.

Mf E. coli cells [TOP 10, Origami B (DE3) or Rosetta (DE3)] containing either 30 pTrcrd3c or pETrd3c plasmids were grown overnight in 50 ml SOB broth containing ampicillin (100 pg / ml) and Rosetta (DE3) cells also:. chloramphenicol (34 pg / ml), shaking at +37 ° C (225 rpm). The following morning, 24 ml of overnight culture was added to 1200 ml of SOB medium containing the above antibiotics and cultured at + 37 ° C. I (225 rpm) until ODeoo was 0.6, whereby the cells are in the middle of the logarithmic growth phase m. At this point, the induction of the recombinant protein was accomplished by adding IPTG to a final concentration of 1 mM. After induction, cells were grown for four to five hours, after which they were harvested by centrifugation. · - · · il .--. · · 118736 27 la. The amino acid sequences of the recombinant proteins derived from the nucleotide sequences are shown in Figure 4 (pTrcrd3c) and Figure 5 (pETrd3c).

Example 3: Purification and folding of the mature portion of reindeer BMP-3c mature protein A. Washing of inclusion bodies

The harvested cells were suspended by shaking in 50 mM Na-phosphate buffer (pH 7.0; 220 g 10 cells / liter buffer). The suspension was centrifuged at 5500 x g for 45 minutes at + 4 ° C.

Washing with Na-phosphate solution was repeated once. The cell pellet was weighed and stored at -70 ° C overnight. The frozen pellet with partially disrupted cells was thawed and suspended (25 mg / ml) in 20 mM Tris-HCl buffer, pH 8.5 containing 0.5 mM EDTA, shaking for 2 minutes. The suspension was centrifuged at 26,000 x g for 40 minutes at + 4 ° C and the Tris-HCl-EDTA wash was repeated once. The resulting cell pellet was weighed. After the final wash, the cell pellet was suspended (35 mg / ml) with shaking (200 rpm / minute, overnight, at room temperature) in lysis buffer (6 M GuHCl - 20 mM Na phosphate - 0.5 M NaCl, pH 8.0), leaving all the rest whole E. coli cells are disrupted and the inclusion bodies are soluble. The suspension is centrifuged (26,000 x g, 20 45 minutes, at room temperature), the pellet is discarded, leaving the recombinant protein soluble in the supernatant. Finally, the supernatant is filtered through 45 µm salt; · *; through the datum to make sure you get rid of all cell debris.

B. Deposition at the isoelectric point (pl) · · · 25 TV T reel BMP-3c recombinant protein expressed from pETrd3c plasmid Es- · V .: cherichia coli in Origami B (DE3) or Rosetta (DE3) cells, were precipitated at isoelectric point pH 9.54 (pETrd3c / 138) and pH 6.35 (pETrd3c / 110). The isoelectric point was determined by computer calculation from the amino acid sequence of recombinant reindeer BMP-3c (Figure 5). The precipitate was collected by centrifugation (12,000 x g, 30 × 2 × · * min, room temperature) and suspended in lysis buffer (6 M GuHCl - 20 mM Na · · · Phosphate – 0.5 M NaCl, pH 8.0).

• Immobilized Metal Affinity Chromatography (IMAC) \ 0 35

·; **: Pre-packaged HiTrap Chelating HP was used in the IMAC method

affinity columns (Amersham Pharmacia Biotech). The column matrices were charged with Co2 + -,

Cu2 + or Ni2 + ions according to the manufacturer's instructions. The filtered, post-wash 28118736 supernatant was applied to the column after loading the column matrix. Most of the impurities were removed by washing the column with lysis buffer (6 M GuHCl -20 mM Na-phosphate - 0.5 M NaCl, pH 8.0) in a 5-10 fold column volume. A second wash, 5-10 times the column volume, was performed with lysis-5 buffer with 6M urea instead of 6M GuHCl. The reindeer recombinant BMP-3c was eluted from a HiTrap column using a pH gradient from pH 7.0 to pH 4.0 (6M urea - 20mM Na-phosphate - 0.5M NaCl). Fractions were analyzed by SDS-PAGE and the purest fractions containing the rdBMP-3c recombinant were pooled for folding.

D. Folding of the mature portion of recombinant rdBMP-3c BMP-3c fractions analyzed by SDS-PAGE were pooled and dialyzed against water. Protein precipitated by dialysis was collected by centrifugation and suspended for 15 hours at 25 ° C in 8 M urea - 0.1 M Tris / HCl, pH 8, additionally containing 100 mM DTT, 1 mM EDTA. The pH was lowered to pH 3-4 by adding dropwise

1M HCl solution. The DTT was completely removed by dialysis for two hours at 25 ° C at 6 M

urea against -10 mM HCl. Dialysis was continued at + 4 ° C overnight against 6 M urea. Folding of recombinant rdBMP-3c was performed in a biphasic fashion

20 dialysis. The first dialysis solution was 20 mM Tris-HCl - 150 mM NaCl - 3 M

urea (pH 7.5). The dialysis buffer was changed at least six times over two or three days. In a second step, all salts were removed with a thorough aqueous *. by dialysis. The desalted sample was centrifuged and the pellet dried by lyophilization. At this point, BMP-3c had a purity of 75% and folded at 50-25% as determined by non-reducing SDS-PAGE. The amount of the folded dimer of reindeer recombinant! '*. * BMP-3c was quantitated by a Coomassie • · · ** densitometer; I,: Brilliant Blue stained gels.

• · • · ·· *

Example 4. Testing for biological activity of the recombinant protein of mature portion of reindeer BMP-3c ··· • v • · · *:! ·. The biological activity of the lyophilized reindeer BMP-3c recombinant protein was tested by implanting 1 mg, 3 mg or 5 mg of recombinant protein in a mouse thigh pocket absorbed into a Lyostrypt® collagen blanket or weighed in a gelatin capsule.

• · v .: 35 BSA served as control. The hind legs were x-rayed and the implant sites were dissected and *: · *: fixed in 10% neutral formalin solution. Fixed implants were cut into 7 µm sections and stained with hematoxylin-eosin solution. The sections were examined under a light microscope. New bone formation was evaluated by X-ray imaging to determine surface area and optical density. X-rays were transferred to a computer using an optical scanner (HP Scan Jet, Hewett-Packard, USA). The formation of ectopic and orthotopic new bone was assessed by the area of calcified tissue seen in X-rays (mm 2) using the Scion Image Beta 4.02 (Scion Corp., 5 USA) software. The average optical density (mmAl) of a given region was measured with the same device. The optical density calibration was performed using an aluminum plate (Al) with 0.25mmAl steps and a calibrated density giving a range up to 4mmAl.

10 Results

Cloning of the partial cDNA of reindeer BMP-3c

The nucleotide sequence obtained from ABI Prism sequencing reactions was analyzed by computer and compared to known BMP sequences. Based on homology comparisons, the new cloned cDNA was found to be most homologous to reindeer BMP-3b (nucleotide homology 96% and amino acid homology 95%) and human BMP-3b (amino acid homology 93%), Table 1. Homology of BMP-3c to other nis -3 proteins had the highest 20 human BMP-3 (91% amino acid homology), Table 2.

: Expression of the mature portion of reindeer BMP-3c First, two mature portions of reindeer BMP-3c were inserted into the * ·· 25 pTrcHis2A vector and E. coHTOP 10 cells were transformed pTrcrd3c / 138-1 * Y and pTrcrd3c / 110 vectors obtained. Recombinant protein production was induced by IPTG.

The production of the recombinant protein was confirmed by SDS-PAGE, but neither of the vectors was induced. This was thought to be due to many of the rdBMP-3c codons rarely used in E. coli. It is also possible that the in-30 non-showers may be due to some degree to the high GC content of both: ** '. * En at the beginning of the coding regions for rdBMP-3c (BMP-3c / 138 ·) ·. The GC content of the 10 codons is 53% and the GC of the first 10 codons of BMP-3C / 110 is 63%).

• * ·· »• 0 .: 35 For these reasons, it was decided to try a different vector system and a different E.

*: ··: coli strain. pET22b (+) (Novagen) with His6 tag and pelB leader was selected as the new expression vector and Rosetta (DE3) and Origami B (DE3) as new E. coli strains. Both reindeer BMP-3c cDNAs were cloned into p112236 (+) vector and the new plasmids were named pETrd3c / 138 and pETrd3c / 110, and both Rosetta (DE3) and Origami B (DE3) cells were transformed with these vectors. Analyzes by SDS-PAGE showed the expression of rdBMP-3c (Figure 8). Based on the expression experiments, mainly Rosetta (DE3) -5 cells containing pETrd3c vectors were used to produce rdBMP-3c proteins.

Purification of rdBMP-3c Proteins of reindeer rdBMP-3c were produced in E coli. After washing, isoelectric point precipitation, and solubilization of the inclusion bodies, the content of the reindeer rdBMP-3c was 85%.

The next purification step was immobilized metal affinity chromatography (IMAC). The sample was eluted from the column using a pH gradient and the rdBMP-3c protein was at least 75% pure as determined by SDS-PAGE (Figure 8). The mature portion of the isolated rdBMP-3c / 138 protein had a molecular weight of 20,400 Da and 17,100 Da of rdBMP-3c / 110 as determined by electrophoretic mobility by SDS-PAGE under reducing conditions.

Folding and activity assay of rdBMP-3c / 138 and rdBMP-3c / 110. In vitro folding of denatured rdBMP-3c proteins was quantitated by measuring densities of Coomassie Brilliant Blue stained gels by densitometry. Protein folding was determined from non-reducing SDS-PAGE gels at 50%.

The osteoinductive activity of rdBMP-3c increased with dose size up to 5 mg (Table 3). Comparing the biological activity of rdBMP-3c / 138 and rdBMP-3c / 110, it appears that rdBMP-). 3c / 110 was a more potent inducer of bone formation.

The present invention has been described with reference to some preferred embodiments and embodiments. However, it will be apparent to those skilled in the art that variations of the embodiments disclosed may be made and practiced, and that the invention may be practiced within the scope of the following claims other than as specifically set forth herein.

31 118736

SEQUENCE LISTING

<110> BBS-Bioactive Bone Substitutes Oy 5 <120> Bone Morphogenetic Proteins <130> OP100919 <160> 1 10 <170> Patent Version 3.1 <210> 1 <211> 138

15 <212> PRT

<213> Rangifer tarar tarar <400> 1 20 Arg Lys Lys Gly Gin Asp Val Phe Met Ala Ser Ser Gin Val Leu Asp 15 10 15

Phe Asp Glu Lys Thr Met Gin Lys Ala Arg Lys Lys Gin Trp Asp Glu 20 25 30 25

Pro Arg Val Cys Ser Arg Tyr Leu Lys Val Asp Phe Ala Asp lie 35 40 45

Gly Trp Asn Glu Trp lie lie Ser Pro Lys Ser Phe Asp Ala Tyr Tyr 30 50 55 60

Cys Ser Gly Ala Cys Glu Phe Pro Met Pro Lys Met Val Arg Pro Ser 65 70 75 80 35 Asn His Ala Thr lie Gin Ser lie Val Arg Ala Val Gly lie Val Pro 85 90 95 •! ί ί Gly Ile Pro Glu Pro Cys Cys Val Pro Asp Lys Met Ser Ser Leu Gly. ** 100 105 110:. :: 40

Val Leu Phe Leu Asp Glu Asn Arg Asn Val Val Leu Lys Val Tyr Pro! .1.i 115 120 125 • · • ϊ: Asn Met Ser Val Glu Thr Cys Ala Cys Gin. 45 130 135

• · I

• 9 · · 1 · 9 ··· • 9 • · • 9 9 • · · 9 · · 1 · ··· · · * 99 • • 9 • ♦ 9 • · · * · «· * 99 • · • · • 9 · · * 1 · • 9 · • «9 9 9

Claims (19)

118736 1. The isolated bone morphogenetic protein 3 (BMP-3), characterized in that it contains the consensus sequence: 5-R-K-K-Q-W-D-E-P-R-V / N-C-S / A-R-R-Y-L-D-V-D-F-A-D-I-g-w-n / the w-i-i-p-p-D-p-F-D-A-Y-Y-C-S-G-A-C-E / Q-F-P-M-P-K-M-V / L-R / K-P-S-N-H-A-T-I-Q-S-I-V-R-A-V-G-I / V-V-P / S-G-I-P-E-P-C-C-W. Bone morphogenetic protein 3 according to claim 1, characterized in that it contains amino acids 6-104 of SEQ ID NO: 1. Bone morphogenetic protein 3 according to claim 1, characterized in that it contains amino acids 26-104 of SEQ ID NO: 1. Bone morphogenetic protein 3 according to claim 1, characterized in that it contains amino acids 6-109 of SEQ ID NO: 1. 5. Isolated bone morphogenetic protein 3, characterized in that it contains the amino acid sequence of SEQ ID NO: 1. 20 6. An isolated DNA molecule, characterized in that it encodes one of the preceding pa. . ·. A bone morphogenetic protein according to claim 1. ·· ♦ • · · · · ♦ · Nucleotide vector, characterized in that it contains the isolated DNA molecule of claim 6. A recombinant host cell, characterized in that it contains the nucleotide vector of claim 7. Pharmaceutical composition, characterized in that it contains a bone morphogenetic protein according to any one of claims 1 to 5. m • · ♦ · · M * Pharmaceutical composition according to claim 9, characterized in that it contains the bone morphogenetic protein in question as a homodimer, or as a hetero-T: 35 rodimer with another bone morphogenetic protein. • · • · · · 118736 Pharmaceutical composition according to claim 9 or 10, characterized in that it further contains a bone morphogenetic protein, an epidermal growth factor, a fibroblastic growth factor or a transforming growth factor. Use of a bone morphogenetic protein according to any one of claims 1 to 5 in the manufacture of a medicament for the treatment of bone, cartilage, tendon or dental disorders where regeneration, repair or growth is desirable. Use of a bone morphogenetic protein according to any one of claims 1 to 5 in the manufacture of a medicament for the treatment of cancer, fibromyalgia, psoriasis and other dermatological disorders, and rheumatic disorders. An osteogenic device, characterized in that it contains a bone morphogenetic protein according to any one of claims 1-5. An osteogenic device according to claim 14, characterized in that it contains said bone morphogenetic protein as a homodimer, or as a heterodimer with another bone morphogenetic protein. 20 An osteogenic device according to claim 14 or 15, characterized in that:. that it also contains another bone morphogenetic protein, an epidermal growth factor, a fibroblastic growth factor, or a transforming growth factor. • · · ·· · *** · j // 25 An osteogenic device according to any one of claims 14 to 16, characterized in that it contains a biocompatible matrix. • * · • · · ··· · · · An osteogenic device according to claim 17, characterized in that said biocompatible matrix contains calcium phosphate, carboxymethylcellulose or collagen or combinations thereof. ··· • «• · Λ 19. A method of inducing bone, cartilage, tendon or tooth bone formation in vitro, characterized in that the bone * ··· * morphogenetic protein of any one of claims 1 to 5 is used to treat said bone, cartilage, tendon or tooth. of view. • · • * ♦ * ·· • · 118736