Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1997-09-09
2001-06-12
Romeo, David S. (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S300000, C514S012200, C435S069100
Reexamination Certificate
active
06245889
ABSTRACT:
The present invention relates to a novel family of purified proteins designated BMP-2 and BMP-4 proteins and processes for obtaining and producing them. These proteins may be used to induce bone and/or cartilage formation and in wound healing and tissue repair.
BMP-2 and BMP-4 proteins have previously been referred to collectively as BMP-2 proteins (BMP-2 previously referred to as BMP-2A or BMP-2 Class I and BMP-4 as BMP-2B or BMP-2 Class II).
Human BMP-2 proteins are characterized by an amino acid sequence comprising amino acid #299 (His, Pro, Leu . . . )—#396 (Arg) of
FIG. 2
(SEQ ID NO: 4). Human BMP-2 proteins are further characterized as diners of BMP-2 subunits. Mature BMP-2 is characterized by comprising amino acid #283 (Gln, Ala, Lys . . . )—#396 (Arg) of FIG.
2
. Mature BMP-2 is further characterized as a disulfide linked dimer wherein each subunit comprises amino acids #283-#396 of
FIG. 2
(SEQ ID NO: 4).
Human BMP-2 may be produced by culturing a cell transformed with a DNA sequence comprising the nucleotide coding sequence from nucleotide #356 to #1543 as shown in
FIG. 2
(SEQ ID NO: 3) and recovering and purifying from the culture medium a protein comprising amino acid #299 to #396 as shown in
FIG. 2
(SEQ ID NO: 4), substantially free from other proteinaceous materials with which it is co-produced. Human BMP-2 is characterized by the ability to induce bone formation. Human BMP-2 is further characterized by the ability to induce cartilage formation. Human BMP-2 may be further characterized by the ability to demonstrate cartilage and/or bone formation activity in the rat bone formation assay described below. In preferred embodiments, the proteins of the invention demonstrate activity in this assay at a concentration of 10 &mgr;g-500 &mgr;g/gram of bone. BMP-2 proteins may be characterized by the ability of 1 &mgr;g of the protein to score at least +2 in the rat bone formation assay of Example III using the modified scoring method described in Example VII.
The bovine BMP-2 protein is a member of the family of BMP-2 proteins of the invention. Bovine BMP-2 proteins comprise the amino acid sequence represented by amino acid #32 to amino acid #129 of
FIG. 1
(SEQ ID NO: 2). These proteins are capable of inducing the formation of cartilage and/or bone. Bovine BMP-2 may be further characterized by the ability to demonstrate cartilage and/or bone formation activity in the rat bone formation assay described below. In preferred embodiments, the proteins of the invention demonstrate activity in this assay at a concentration of 10 &mgr;g-500 &mgr;g/gram of bone. These proteins may be characterized by the ability of 1 &mgr;g of the protein to score at least +2 in the rat bone formation assay described in Example III using the modified scoring method as described in Example VII.
Human BMP-4 proteins are characterized by an amino acid sequence comprising amino acids #311 (His, Ser, Leu . . . )—#408 (Arg) as shown in
FIG. 3
(SEQ ID NO: 6). Mature BMP-4 comprises amino acids #293 (Ser, Pro, Lys . . . )—#408 (Arg) of FIG.
3
. BMP-4 proteins are further characterized as dimers of BMP-4 subunits. Mature BMP-4 is further characterized as a disulfide linked dimer wherein each subunit comprises amino acids #293-#408 of
FIG. 3
(SEQ ID NO: 6).
BMP-4 may be produced by culturing a cell transformed with a DNA sequence comprising the nucleotide coding sequence from nucleotide #403 to nucleotide #1626 substantially as shown in
FIG. 3
(SEQ ID NO: 5) and recovering and purifying from the culture medium a protein containing the amino acid sequence from amino acid #311 to #408 as shown in
FIG. 3
(SEQ ID NO: 6) substantially free from other proteinaceous materials with which it is co-produced. BMP-4 proteins are capable of inducing the formation of bone. BMP-4 proteins are capable of inducing formation of cartilage. BMP-4 proteins are further characterized by the ability to demonstrate cartilage and/or bone formation activity in the rat bone formation assay described below. In preferred embodiments, the proteins of the invention demonstrate activity in this assay at a concentration of 10 &mgr;g-500 &mgr;g/gram of bone. These proteins may be characterized by the ability of 1 &mgr;g of the protein to score at least +2 in the rat bone formation assay of Example III using the modified scoring method described in Example VII.
Another aspect of the invention provides pharmaceutical compositions containing a therapeutically effective amount of a BMP-2 or BMP-4 protein in a pharmaceutically acceptable vehicle or carrier. These compositions of the invention may be utilized in the formation of cartilage. These compositions may further be utilized in the formation of bone. They may also be used for wound healing and tissue repair. In further embodiments the compositions of the invention may be utilized for neuronal survival.
Further compositions of the invention may comprise a therapeutically effective amount of BMP-2 and BMP-4 in a pharmaceutically acceptable vehicle. Compositions of the invention may further include, in addition to a BMP-2 or BMP-4 protein, at least one other therapeutically useful agent such as the BMP proteins BMP-1, BMP-3, BMP-5, BMP-6, BMP-7, and BMP-8 disclosed respectively in co-owned U.S. patent application Ser. Nos. 655,578, 179,197 withdrawn in favor of U.S. Ser. No. 07/692,827 now, U.S. Pat. No. 5,111,738, Ser. No. 370,547, now U.S. Pat. No. 5,106,798, Ser. No. 370,544, abandoned in favor of U.S. Ser. No. 490,033 now U.S. Pat. No. 5,187,076 Ser. No. 370,549, abandoned in favor of U.S. Ser. No. 438,919 now U.S. Pat. No. 5,141,905 and Ser. No. 525,357 abandoned in favor of U.S. Ser. No. 641,204 abandoned in favor of U.S. Ser. No. 800,364 now U.S. Pat. No. 5,688,678. The compositions of the invention may comprise, in addition to a BMP-2 or BMP-4 protein, other therapeutically useful agents including growth factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF), and transforming growth factor (TGF-&agr; and TGF-&bgr;). The compositions may also include an appropriate matrix for instance, for supporting the composition and providing a surface for bone and/or cartilage growth. The matrix may provide slow release of the BMP protein and/or the appropriate environment for presentation of the BMP protein.
The BMP-2 and BMP-4 compositions may be employed in methods for treating a number of bone and/or cartilage defects, periodontal disease and various types of wounds. These methods, according to the invention, entail administering to a patient needing such bone and/or cartilage formation wound healing or tissue repair, an effective amount of a BMP-2 or BMP-4 protein. These methods may also entail the administration of a protein of the invention in conjunction with at least one of the novel BMP proteins disclosed in the co-owned applications described above. In addition, these methods may also include the administration of a BMP-2 or BMP-4 protein with other growth factors.
Still a further aspect of the invention are DNA sequences encoding a BMP-2 or BMP-4 protein. Such sequences include the sequence of nucleotides in a 5′ to 3′ direction illustrated in
FIGS. 1 through 3
(SEQ ID NO: 1,3, and 5) or DNA sequences which hybridize under stringent conditions with the DNA sequences of
FIGS. 1-3
and encode a protein having the ability to induce the formation of cartilage and/or bone. Finally, allelic or other variations of the sequences of
FIGS. 1 through 3
, 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 entails a vector comprising a DNA sequence as described above in operative association with an expression control sequence therefor. Such vector may be employed in a novel process for producing a BMP-2 or BMP-4 protein of the invention in which a cell line transformed with a DNA sequence encoding a BMP-2 or BMP-4 protein in operati
Rosen Vicki A.
Wang Elizabeth A.
Wozney John M.
Genetics Institute Inc.
Kapinos Ellen J.
Romeo David S.
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