Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-08-16
2004-01-13
Spector, Lorraine (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S399000, C435S440000, C435S445000, C435S069100, C536S023400, C514S002600
Reexamination Certificate
active
06677432
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to modified proteins, and DNAs encoding the same, which are biosynthetic, chemosynthetic or recombinant constructs derived from the TGF-&bgr; superfamily of structurally related proteins, including modified morphogenic proteins.
BACKGROUND OF THE INVENTION
The TGF-&bgr; superfamily includes five distinct forms of TGF-&bgr; (Sporn and Roberts (1990) in
Peptide Growth Factors and Their Receptors
, Sporn and Roberts, eds., Springer-Verlag: Berlin pp. 419-472), as well as the differentiation factors Vg-1 (Weeks and Melton (1987)
Cell
51: 861-867), DPP-C polypeptide (Padgett et al. (1987)
Nature
325: 81-84), the hormones activin and inhibin (Mason et al. (1985)
Nature
318: 659-663; Mason et al. (1987)
Growth Factors
1: 77-88), the Mullerian-inhibiting substance, MIS (Cate et al. (1986)
Cell
45:685-698), osteogenic and morphogenic proteins OP-1 (PCT/US90/05903), OP-2 (PCT/US91/07654), OP-3 (PCT/WO94/10202), the BMPs, (see U.S. Pat. Nos. 4,877,864; 5,141,905; 5,013,649; 5,116,738; 5,108,922; 5,106,748; and 5,155,058), the developmentally regulated protein Vgr-1 (Lyons et al. (1989)
Proc. Natl. Acad. Sci. USA
86: 4554-4558) and the growth/differentiation factors GDF-1, GDF-3, GDF-9 and dorsalin-1 (McPherron et al. (1993)
J. Biol. Chem.
268: 3444-3449; Basler et al. (1993)
Cell
73: 687-702), to name but a few.
The proteins of the TGF-&bgr; superfamily are disulfide-linked homo- or heterodimers that are expressed as large precursor polypeptide chains containing a hydrophobic signal sequence, a long and relatively poorly conserved N-terminal pro region sequence of several hundred amino acids, a cleavage site, a mature domain comprising an N-terminal region that varies among the family members and a highly conserved C-terminal region. This C-terminal region, present in the processed mature proteins of all known family members, contains approximately 100 amino acids with a characteristic cysteine motif having a conserved six or seven cysteine skeleton. Although the position of the cleavage site between the mature and pro regions varies among the family members, the cysteine pattern of the C-terminus of all of the proteins is in the identical format, ending in the sequence Cys-X-Cys-X (Sporn and Roberts (1990), supra).
A unifying feature of the biology of the proteins of the TGF-&bgr; superfamily is their ability to regulate developmental processes, including endochondral bone morphogenesis. These structurally related proteins have been identified as being involved in a variety of developmental events. For example, TGF-&bgr; and the polypeptides of the inhibin/activin group appear to play a role in the regulation of cell growth and differentiation. MIS causes regression of the Mullerian duct in development of the mammalian male embryo, and dpp, the gene product of the Drosophila decapentaplegic complex, is required for appropriate dorsal-ventral specification. Similarly, Vg-1 is involved in mesoderm induction in Xenopus, and Vgr-1 has been identified in a variety of developing murine tissues. Regarding bone formation, proteins in the TGF-&bgr; superfamily, for example OP-1 and a subset of other proteins identified as BMPs (bone morphogenic proteins) play the major role. OP-1 (BMP-7) and other osteogenic proteins have been produced using recombinant techniques (U.S. Pat. No. 5,011,691 and PCT Application No. US 90/05903) and shown to be able to induce formation of true endochondral bone in vivo. The osteogenic proteins generally are classified in the art as a subgroup of the TGF-&bgr; superfamily of growth factors (Hogan (1996),
Genes & Development
, 10:1580-1594).
Recently certain members of this same family of proteins have been recognized to be morphogenic, i.e., capable of inducing the developmental cascade of tissue morphogenesis in a mature mammal (See PCT Application No. US 92/01968). In particular, these morphogens are capable of inducing the proliferation of uncommitted progenitor cells, and inducing the differentiation of these stimulated progenitor cells in a tissue-specific manner under appropriate environmental conditions. In addition, the morphogens are capable of supporting the growth and maintenance of these differentiated cells. These morphogenic activities allow the proteins to initiate and maintain the developmental cascade of tissue morphogenesis in an appropriate, morphogenically permissive environment, stimulating stem cells to proliferate and differentiate in a tissue-specific manner, and inducing the progression of events that culminate in new tissue formation. These morphogenic activities also allow the proteins to induce the “redifferentation” of cells previously stimulated to stray from their differentiation path. Under appropriate environmental conditions it is anticipated that these morphogens also may stimulate the “redifferentiation” of committed cells.
Members of this morphogenic class of proteins include the mammalian osteogenic protein-1 (OP-1, also known as BMP-7, and the Drosophila homolog 60A), osteogenic protein-2 (OP-2, also known as BMP-8), osteogenic protein-3 (OP-3), BMP-2 (also known as BMP-2A or CBMP-2A, and the Drosophila homolog DPP), BMP-3, BMP-4 (also known as BMP-2B or CBMP-2B), BMP-5, BMP-6 and its murine homolog Vgr-1, BMP-9, BMP-10, BMP-11, BMP-12, GDF-3 (also known as Vgr2), GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, BMP-13, BMP-14, BMP-15, GDF-5 (also known as CDMP-1 or MP52), GDF-6 (also known as CDMP-2 or BMP-13), GDF-7 (also known as CDMP-3 or BMP-12), the Xenopus homolog Vg1 and NODAL, UNWIN, SCREW, ADMP, and NEURAL, to name but a few.
By way of illustration using exemplary family members, the tertiary and quaternary structure of both TGF-&bgr;2 and OP-1 have been determined. Although TGF-&bgr;2 and OP-1 exhibit only about 35% amino acid identity in their respective amino acid sequences the tertiary and quaternary structures of both molecules are strikingly similar. Both TGF-&bgr;2 and OP-1 are dimeric in nature and have a unique folding pattern involving six of the seven C-terminal cysteine residues. In each subunit four cysteines bond to generate an eight residue ring, and two additional cysteine residues form a disulfide bond that passes through the ring to form a knot-like structure. With a numbering scheme beginning with the most N-terminal cysteine of the 7 conserved cysteine residues assigned number 1, the 2nd and 6th cysteine residues bond to close one side of the eight residue ring while the 3rd and 7th cysteine residues close the other side. The 1st and 5th conserved cysteine residues bond through the center of the ring to form the core of the knot. The 4th cysteine forms an interchain disulfide bond with the corresponding residue in the other subunit.
The TGF-&bgr;2 and OP-1 monomer subunits comprise three major structural elements and an N-terminal region. The structural elements are made up of regions of contiguous polypeptide chain that possess over 50% secondary structure of the following types: (1) loop, (2) &agr;-helix and (3) &bgr;-sheet. Furthermore, in these regions the N-terminal and C-terminal strands are not more than 7 Å apart. The residues between the 1st and 2nd conserved cysteines form a structural region characterized by an anti-parallel &bgr;-sheet finger, referred to herein as the finger 1 region (F1). Similarly the residues between the 5th and 6th conserved cysteines also form an anti-parallel &bgr;-sheet finger, referred to herein as the finger 2 region or sub-domain (f2). A &bgr;-sheet finger is a single amino acid chain, comprising &bgr;-strand that folds back on itself by means of a &bgr;-turn or some larger loop so that the entering and exiting strands forth one or more anti-parallel &bgr;-sheet structures. The third major structural region, involving the residues between the 3rd and 4th conserved cysteines is characterized by a three turn &agr;-helix referred to herein as the heel region (H). In the dimeric forms of both TGF-&bgr;2 and OP-1, the subunits are oriented such that the heel region of one subunit contacts the finger regions
McCartney John
Oppermann Hermann
Tai Mei-Sheng
Fish & Neave
Haley Jr. James F.
Mangasarian Karen
Seharaseyon Jegatheesan
Spector Lorraine
LandOfFree
Mutations of the C-terminal portion of TGF-&bgr; superfamily... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Mutations of the C-terminal portion of TGF-&bgr; superfamily..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Mutations of the C-terminal portion of TGF-&bgr; superfamily... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3221436