Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1998-12-01
2001-11-13
Carlson, Karen Cochrane (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S012200, C530S300000, C530S324000, C530S325000, C530S326000, C530S327000, C530S328000, C530S329000, C530S330000, C530S350000, C530S387100, C530S387900
Reexamination Certificate
active
06316412
ABSTRACT:
DESCRIPTION
1. Technical Field
The present invention relates to methods and compositions for promoting cell attachment to substrates. The invention particularly relates to the use of newly identified binding sites of fibronectin for binding to integrin receptors on cells.
2. Background
Regulation of cell adhesive events has broad biomedical implications. For instance, inhibition of cell adhesion may be of benefit in the treatment of thrombotic disorders through inhibition of platelet aggregation, of inflammatory disorders through inhibition of leukocyte adhesion and transmigration, and in malignant disease through inhibition of tumor cell lodgement and metastasis. Conversely, promotion of cell adhesion is, in some cases, desirable. For example, in the seeding of endothelial cells onto vascular grafts, in the stability of medical prostheses, and in promotion of wound healing. Adhesive events are widely recognized to involve interactions of extracellular receptors, i.e., integrin receptors, with substances surrounding the cell, e.g., fibronectin. Integrins are a functionally and structurally related group of receptors that interact with a wide variety of ligands including extracellular matrix glycoproteins, complement and other cells. Integrins participate in cell-matrix and cell—cell adhesion in many physiologically important processes including embryological development, hemostasis, thrombosis, wound healing, immune and nonimmune defense mechanisms and oncogenic transformation. See Hynes,
Cell,
48:549-554 (1987). Several integrins that participate in dynamic cell adhesion bind a tripeptide, arginine-glycine-aspartic acid (RGD), present in their ligand. See Ruoslahti et al.,
Science,
238:491-497 (1987).
Fibronectin is an adhesive glycoprotein found in plasma and on cell surfaces and extracellular matrices. By binding other macromolecules as well as cells, fibronectin promotes anchorage of cells to substrata. Hynes, in
Cell Biology of the Extracellular Matrix,
Hay ed., Plenum Press, pages 295-334 (1982); Hynes et al.,
J. Cell Biol.,
95:369-77 (1982). Also, fibronectin is known to accumulate at sites of injury and inflammation in vivo [Pettersson et al.,
Clin. Immunol. Immunopath,
11:425-436 (1978); Grinnel et al.,
J. Invest. Derm.,
76:181-189 (1981); Repesh et al.,
J. Histochem. Cytochem.,
30(4):399-408 (1985); Carsons et al.,
Arth. Rheum,
24(10):1261-67 (1981)] and is produced by cells in blood vessel walls at these sites. Clark et al.,
J. Exp. Med.,
156:646-51 (1982); Clark et al.,
J. Immunol.,
126(2):787-93 (1981); Clark et al.,
J. Invest. Derm.,
79:269-76 (1982); Clark et al.,
J. Clin Invest.,
74:1011-16 (1984).
Fibronectin is composed of subunits of variable primary structure [average relative molecular mass of 250 kilodaltons (kDa)]. The subunits are disulfide-linked to form dimers or multimers derived from a pool of similar but nonidentical polypeptides. Hynes, in
Cell Biology of the Extracellular Matrix,
Hay ed., Plenum Press, pages 295-334 (1982); Hynes et al.,
Cell Biol.,
95:369-77 (1982); Schwarzbauer et al.,
Proc. Natl., Acad. Sci. USA,
82:1424-28; Kornblihtt et al.,
EMBO J.,
4(7): 1755-59 (1985). Thus, the term “fibronectin” refers to several species of glycoprotein, some of which are more fully characterized than others.
Two major fibronectin (Fn) classes are plasma fibronectin and cellular fibronectin. Plasma fibronectin (pFn) is secreted by hepatocytes, whereas cellular fibronectin (cFn) is secreted by a variety of cultured cells including endothelial cells and fibroblasts. Jaffe et al.,
J. Exp Med.,
147:1779-91 (1978); Birdwell et al.,
Biochem. Biophys. Res. Commun.,
97(2):574-8 (1980). Despite extensive physical and immunologic similarities, the two classes of fibronectin differ in electrophoretic behavior, solubility, and biologic activities. Tamkun et al.,
J. Biol. Chem.,
258 (7):4641-47 (1983); Yamada et al.,
J. Cell Biol.,
80:492-98 (1979); Yamada et al.,
Biochemistry,
16 (25):2552-59, (1977).
Primary structural differences between plasma and cellular fibronectins have been found by peptide mapping [Hayashi et al.,
J. Biol. Chem.,
256(21):11,292-11,300 (1981)], cDNA cloning [Xornblihtt et al.,
EMBO J.,
4:1755-1759 (1985)], and immunologic techniques [Atherton et al.,
Cell,
25:133-41 (1981)]. From these data, it has been determined that the primary structure of fibronectin monomer contains three different types of internal repeats known as homology Types I, II and III, having lengths of about 40, 60 and 90 amino acids residues, respectively [Kornblihtt et al.,
EMBO J.,
4:1755-1759 (1985)]. All of the various distinct Fn moieties are produced by a single gene, with differences in primary structure resulting from alternative splicing of the primary mRNA transcript in at least three regions. Kornblihtt et al.,
EMBO J.,
4(7):1755-59 (1985); Schwarzbauer et al.,
Proc. Natl. Acad. Sci. USA,
82:1424-28 (1985); Gutman et al.,
Proc. Natl. Acad. Sci. USA,
84:7179-82 (1987); Schwarzbauer et al.,
EMBO J.,
6(9):2573-80 (1987).
A site containing the Arg-Gly-Asp (RGD) sequence in the 10th Type III repeat of Fn is known to be involved in cell adhesive events. Peptides containing this sequence inhibit certain cell adhesive events, or alternatively, can be used to promote cell adhesion. See, e.g., U.S. Pat. Nos. 4,589,881; 4,661,111; 4,517,686; 4,683,291; 4,578,079; 4,614,517; and 4,792,525.
Recently, site-directed mutagenesis studies of fibronectin have implicated non-RGD sequences as participating in cell adhesion phenomena. [Obara, M. et al.
Cell,
53:649-57 (1988)]. The proposed second binding site was not defined by this study; however, activity loss data indicated that a second site was involved in adhesion, probably in a synergistic fashion with the RGD sequence. This result helps to explain why other RGD-containing proteins do not bind integrins as well as fibronectin.
In view of the importance of promoting cell adhesion or, conversely, for inhibiting adhesion, non-RGD containing polypeptides suitable for these purposes are desired. In the event such polypeptides are found to complement RGD amino acid residue sequences in cell binding processes, compositions including both RGD sequences and adhesive non-RGD compounds are also desired.
BRIEF SUMMARY OF THE INVENTION
The present invention is for polypeptides that bind to integrin receptors, particularly GPIIb-IIIa, which polypeptides comprise a binding region for the integrin that is independent of the well-known RGD sequence of fibronectin (Fn). The new binding site is located at least fifty amino acid residues upstream (toward the N-terminus) of the RGD sequence of human Fn. The amino acid residue sequence of human Fn is described in Kornblihtt, et al.,
EMBO J.,
4:1755 (1985), which description is incorporated herein by reference. Selected regions of human Fn are depicted in
FIGS. 1-3
and include the sequence of fibronectin described by Kornblihtt et al.
In one embodiment, the present invention contemplates a polypeptide having a length of no more than about 100 amino acid residues. The peptide binds GPIIb-IIIa and includes an amino acid residue sequence represented by the formula: -DRX
1
PHX
2
R-, where X
1
and X
2
are any amino acid residue (SEQ ID NO 1).
Preferably, the polypeptide includes an amino acid residue sequence represented by the formula: -DRX
1
PHX
2
RU-, wherein X
1
is V or A, X
2
is S or A, and U is a sequence of amino acids represented by formula selected from the group consisting of: -X
3
X
4
X
5
X
6
-, -X
3
SIT-, -NX
4
IT-, -NSX
5
T-, and -NSIX
6
-, respectively, SEQ ID NO 2 through 6, wherein X
3
is N or A, X
4
is S or A, X
5
is I or A, and X
6
is T or A.
A preferred embodiment contemplates a polypeptide shown in SEQ ID NOs 2-6 which have the respective amino acid residue sequences of DRX
1
PHX
2
RX
3
X
4
X
5
X
6
, DRX
1
PHX
2
RX
3
SIT, DRX
1
PHX
2
RNX
4
IT, DRX
1
PHX
2
RNSX
5
T and DRX
1
PHI
2
RNSIX
6
.
In a preferred embodiment of the invention the instant
Bowditch Ronald
Ginsberg Mark H.
Plow Edward F.
Carlson Karen Cochrane
Fitting Thomas
Holmes Emily
The Scripps Research Institute
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