Nucleic acid ligands to integrins

Details Nucleic acid ligands to integrins Nucleic acid ligands to integrins

1999-07-29

2001-12-18

Zitomer, Stephanie (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S023100, C536S025400

Reexamination Certificate

active

06331394

ABSTRACT:

FIELD OF THE INVENTION
This invention is directed towards nucleic acid ligands of integrins isolated using the SELEX process. SELEX is an acronym for Systematic Evolution of Ligands by EXponential Enrichment. This invention relates to integrin proteins, and methods and compositions for treating and diagnosing diseases involving integrins.
BACKGROUND OF THE INVENTION
The integrins are a class of heterodimeric integral membrane proteins, one or homologous alpha subunits and 8 homologous beta subunits associate in various combinations to yield an extensive family of receptors. Each integrin heterodimer has a large extracellular domain that mediates binding to specific ligands. These ligands may include plasma proteins, proteins expressed on the surface of adjacent cells, or components of the extracellular matrix. Several of the integrins show affinity for more than one ligand and many have overlapping specificities (Hynes (1992) Cell 69:11-25). Both the &agr; and &bgr; subunits contribute to a small intracellular domain that contacts components of the actin cytoskeleton, thus forming a physical link between proteins outside and inside the cell. Integrins play an important role in cellular adhesion and migration, and these properties are controlled by the cell, in part, by modulation of integrin affinity for its ligands (so-called “inside-out” signaling). Conversely, the presence or absence of integrin ligation provides specific information about the cellular microenvironment, and in many instances integrins serve as a conduit for signal transduction. Ligand binding by an integrin may promote its incorporation into focal adhesions, the assembly of cytoskeletal and intracellular signaling molecules into supra-molecular complexes, and the initiation of a cascade of downstream signaling events including protein phosphorylation, calcium release, and an increase in intracellular pH (reviewed by Schwartz et al. (1995) Ann. Rev. Cell Dev. Biol. 11:549-99). Such “outside-in” signaling ties into pathways controlling cell proliferation, migration and apoptosis (Stromblad et al. (1996) J. Clin. Invest. 98:426-33; Eliceiri et al. (1998) J. Cell. Biol. 140:1255-63). Integrins have been shown to play a role in such diverse physiological settings as embryonic development, wound healing, angiogenesis, clot formation, leukocyte extravasation, bone resorption and tumor metastasis.
The &bgr;
3
-containing integrins are among the best studied of the receptor superfamily. The &bgr;
3
subunit forms heterodimers with either &agr;
v
(&agr;
v
&bgr;
3
) or &agr;
IIb
(&agr;
IIb
&bgr;
3
). While these integrins show substantial overlap in ligand specificity, they play very different roles in normal physiology and in disease.
&agr;
v
&bgr;
3
is expressed by activated endothelial cells, smooth muscle cells, osteoclasts, and, at a very low level, by platelets. It is also expressed by a variety of tumor cell types. The integrin binds to a number of plasma proteins or proteins of the extracellular matrix, many of which are associated with sites of inflammation or wound healing (Albelda (1991) Am. J. Resp. Cell Mol. Biol. 4:195-203). These include vitronectin, fibronectin, osteopontin, von Willebrand factor, thrombospondin, fibrinogen, and denatured collagen Type I (Hynes (1992) Cell 69:11-25). Each of these proteins share a common sequence motif, arginine-glycine-aspartic acid (RGD), that forms the core of the integrin binding site.
&agr;
v
&bgr;
3
has been most intensely studied in the context of new blood vessel formation (angiogenesis) where it mediates the adhesion and migration of endothelial cells through the extracellular matrix. Angiogenesis in adults is normally associated with the cyclical development of the corpus luteum and endometrium and with the formation of granulation tissue during wound repair. In the latter case, microvascular endothelial cells form vascular sprouts that penetrate into the temporary matrix within a wound. These cells transiently express &agr;
v
&bgr;
3
and inhibition of the ligand binding function of the integrin temporarily inhibits the formation of granulation tissue (Clark et al. (1996) Am. J. Pathol. 148:1407-21). In cytokine-stimulated or unstimulated angiogenesis on the chick chorioallantoic membrane, blockade of &agr;
v
&bgr;
3
with a heterodimer-specific antibody prevents new vessel formation without affecting the pre-existing vasculature (Brooks et al. (1994) Science 264:569-71). Furthermore, the loss of adhesive contacts by endothelial cells activated for angiogenesis induces a phenotype characteristic of apoptotic cells (Brooks et al. (1994) Cell 79:1157-64); that is, ligand binding by &agr;
v
&bgr;
3
appears to transmit a survival signal to the cell. Thus, adhesion and/or signaling mediated by &agr;
v
&bgr;
3
is essential for the formation of new blood vessels.
Solid tumors are unable to grow to significant size without an independent blood supply. It is currently hypothesized that the acquisition of an angiogenic phenotype is one of the limiting steps in the growth of primary tumors and of tumors at secondary sites (Folkman (1995) Nat. Med. 1:27-31). In addition, while the vasculature that penetrates a tumor mass provides a source of oxygen and nutrients, it also serves as a conduit for metastatic cells to leave the primary tumor and migrate throughout the body. Thus, inhibition of angiogenesis may limit both the growth and metastasis of cancerous lesions. In experimental settings of tumor-induced angiogenesis, inhibition of ligand-binding by endothelial &agr;
v
&bgr;
3
prevented the formation of new blood vessels (Brooks et al. (1994) Cell 79:1157-64; Brooks et al. (1995) J. Clin. Invest. 96:1815-22), and inhibitors of &agr;
v
&bgr;
3
were shown to reduce the growth of experimental tumors in vivo (Brooks et al. (1995) J. Clin. Invest. 96:1815-22; Carron et al. (1998) Canc. Res. 58:1930-5).
&agr;
v
&bgr;
3
is not only expressed by the microvasculature within tumors, but in some cases, is also found on the surface of tumor cells themselves. In particular, expression of &agr;
v
&bgr;
3
integrin has been detected in tissue sections from tumors of melanocytic and astroglial origin (Albelda et al. (1990) Canc. Res. 50:6757-64; Gladson and Cheresh (1991) J. Clin. Invest. 88:1924-32), and the level of integrin expression has been correlated with the stage or metastatic potential of the tumor (Albeldaet al. (1990) Canc. Res. 50:6757-64; Gladson et al. (1996) Am. J. Pathol. 148:1423-34; Hieken et al. (1996) J. Surg. Res. 63:169-73). Furthermore, melanoma cells grown in vitro in a three-dimensional matrix of denatured collagen undergo apoptosis upon &agr;
v
&bgr;
3
blockade.
Data such as these have driven an interest in inhibitors of &agr;
v
&bgr;
3
for the treatment of cancer. At present, two such inhibitors are in or near clinical trial: Vitaxin is a chimeric Fab fragment derived from the &agr;
v
&bgr;
3
-specific monoclonal antibody, LM609 (Wu et al. (1998) Proc. Nat. Acad. Sci. 95:6037-42). A phase I trial in late-stage cancer patients has been completed and no significant treatment-associated toxicities were observed (Gutheil et al. (1998) Am. Soc. Clin. Onc.). EMD121974 is a cyclic pentapeptide inhibitor of &agr;
v
&bgr;
3
. A Phase I study of this compound in Kaposi's sarcoma, brain tumors and solid tumors is scheduled to begin in 1999.
Angiogenesis (and &agr;
v
&bgr;
3
) are implicated in the pathology of several other diseases, including psoriasis (Creamer et al. (1995) Am. J. Pathol. 147:1661-7), rheumatoid arthritis (Walsh et al. (1998) Am. J. Pathol. 152:691-702; Storgard et al. (1999) J. Clin. Invest. 103:47-54), endometriosis (Healy et al. (1998) Hum. Reprod. Update 4:736-40), and several proliferative diseases of the eye (Casaroli Marano et al. (1995) Exp. Eye Res. 60:5-17; Friedlander et al. (1996) Proc. Nat. Acad. Sci. 93:9764-9; Hammes et al. (1996) Nat. Med. 2:529-33). Inhibition of integrin ligand binding in each of these contexts may provide significant therapeutic benefit.
Atheromatous plaque and restenosis following angioplasty are

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