Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1998-10-29
2001-08-21
Borin, Michael (Department: 1631)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S011400, C530S317000, C530S323000, C424S001690, C435S325000
Reexamination Certificate
active
06277818
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to cyclic peptides that bind to the cell surface receptor (uPAR) for urokinase plasminogen activator (uPA) and, thus, are capable of delivering therapeutic agents or diagnostic probes to the surfaces of cells expressing this receptor. The invention also relates to pharmaceutical compositions comprising these peptides and their use to inhibit the binding of uPA to its cell surface receptor. By targeting therapeutic agents to uPAR or by inhibiting the binding of uPA to uPAR, it is possible to achieve a number of biological effects that include cell death, the inhibition of cell movement and migration and the inhibition of angiogenesis.
The peptides of the invention are capable of carrying a suitable detectable or imageable label so that they can be used to quantitate uPAR levels in vitro and in vivo. Such compositions are therefore useful as diagnostic, prognostic and imaging tools in all diseases and conditions where this receptor plays a pathological or otherwise undesirable role.
The peptides of the invention can also be immobilized to a suitable matrix and can be used for research applications to identify and isolate cells expressing uPAR and to identify and isolate uPAR from biological samples.
2. Description of the Background Art
The urokinase -type plasminogen activator (uPA) system is strongly linked to pathological processes, such as cell invasion and metastasis in cancer (Dan&phgr; et al.,
Adv. Cancer Res.,
44:139-266 (1985)). Cells produce uPA in an inactive form, pro-uPA or single-chain uPA (scuPA), which then binds to its receptor, uPAR. This binding event is a prerequisite for the efficient activation of scuPA to two-chain uPA (tcuPA) in a cell milieu (Ellis et al.,
J Biol. Chem.,
264:2185-88 (1989)).
The amino acid sequence of the N-terminus of human pro-uPA [residues 1-44 of SEQ ID NO:1] is
Ser Asn Glu Leu His Gln Val Pro Ser Asn Cys Asp Cys Leu Asn Gly 1 10
Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser Asn Ile His Trp Cys Asn Cys 20 30
Pro Lys Lys Phe Gly Gly Gln His Cys Glu Ile 40
The structure of pro-uPA [SEQ ID NO:1] is shown in FIG.
1
.
uPA is a three-domain protein comprising (1) an N-terminal epidermal growth factor-like domain, (2) a kringle domain, and (3) a C-terminal serine protease domain. uPAR, the receptor for pro-uPA, is also a multi-domain protein anchored by a glycosylphosphatidylinositol anchor to the outer leaf of the cell membrane (Behrendt et al.,
Biol. Chem. Hoppe
-
Seyler,
376:269-279 (1995)). The binding of uPA to uPAR initiates two separate events: the first, extracellular proteolysis, is mediated through the activation of plasminogen to plasmin, a broad-spectrum protease which can itself activate matrix metalloprotease (MMP) zymogens (Mazzieri et al.,
EMBO J.,
16: 2319-32 (1997)), release latent growth factors such as TGF-&bgr;, IGF-I, and bFGF from their binding proteins or from their binding sites within the extracellular matrix (ECM) (Falcone et al.,
J Biol. Chem.,
268(16): 11951-11958 (1993); Lamarre et al.,
Biochem J,
302: 199-205 (1994); Remacle-Bonnet et al.,
Int. J Cancer
72:835-843 (1997)), and directly remodel certain ECM components such as fibronectin and vitronectin (Wachtfogel et al.,
J. Clin. Invest.,
81:1310-1316 (1988); Sordat et al.,
Invasion Metastasis
14: 223-33 (1994).
The second series of events, triggered by uPA binding to uPAR depends upon transmembrane signal transduction and leads to the stimulation of cell differentiation and motility in several cell types, most notably endothelial cells, epithelial cells and leukocytes (Nusrat et al.,
Fibrinolysis
6 (suppl 1):71-76 (1992); Fazioli et al.,
EMBO J.
16: 7279-86 (1997); Schnaper et al.,
J. Cell. Physiol.
165:107-118 (1994)). This second activity is independent of the proteolytic cascade described above. uPAR mediates these signaling events despite its lack of a transmembrane domain presumably through an adaptor protein(s) which couples extracellular binding to intracellular signaling cascades . The signaling mediated by uPAR probably involves multiple pathways, as with other cytokines. Jak/STAT and MAP-dependent pathways (which overlap with Jak/STAT) have been implicated (Koshelnick et al.,
J. Biol. Chem.
272:28563-28567 (1997); Tang et al.,
J. Biol. Chem.
273:18268-18272 (1998); Dumler et al,
J. Biol. Chem.
273:315-321 (1998)).
uPAR is not normally expressed at detectable levels on quiescent cells and must therefore be upregulated before it can initiate the activities of the uPA system. uPAR expression is stimulated in vitro by differentiating agents such as phorbol esters (Lund et al.,
J. Biol. Chem.
266:5177-5181 (1991)), by the transformation of epithelial cells, and by various growth factors and cytokines such as VEGF, bFGF, HGF, IL-1, TNF&agr;, (in endothelial cells) and GM-CSF (in macrophages) (Mignatti et al.,
J. Cell Biol.
113:1193-1201 (1991); Mandriota et al.,
J. Biol. Chem.
270:9709-9716; Yoshida et al.,
Inflammation
20:319-326 (1996)). This up-regulation has the functional consequence of increasing cell motility, invasion, and adhesion (Mandriota et al., supra). More importantly, uPAR appears to be up-regulated in vivo in most human carcinomas examined to date, specifically, in the tumor cells themselves, in tumor-associated endothelial cells undergoing angiogenesis and in macrophages (Pyke et al.,
Cancer Res.
53:1911-15 (1993) which may participate in the induction of tumor angiogenesis (Lewis et al.,
J. Leukoc. Biol.
57:747-751 (1995)). uPAR expression in cancer patients is present in advanced disease and has been correlated with a poor prognosis in numerous human carcinomas (Hofinann et al.,
Cancer
78:487-92 (1996); Heiss et al.,
Nature Med.
1:1035-39 (1995). Moreover, uPAR is not expressed uniformly throughout a tumor but tends to be associated with the invasive margin and is considered to represent a phenotypic marker of metastasis in human gastric cancer. The fact that uPAR expression is up-regulated only in pathological states involving ECM remodeling and cell motility such as cancer makes it an attractive marker for diagnosis as well as a selective target for therapy.
In order to design the peptides of the present invention, it was necessary first to identify the minimal binding epitope of uPA for uPAR. It had been shown earlier that the amino terminal fragment of uPA (residues 1-135) that lacked the serine protease domain, sufficed for high affinity (sub-nanomolar) binding. (Stoppelli et al.,
Proc. Natl. Acad. Sci. USA
82:4939-43 (1985). Subsequent work showed that the growth factor domain alone (residues 1-48) conferred this binding. (Robbiati et al.,
Fibrinolysis,
4:53-60 (1990); Stratton-Thomas et al.,
Protein Engineering
8:463-470 (1995.))
Dan&phgr; et al., WO 90/12091 (Oct. 18, 1990), disclosed that the binding of uPA to uPAR could be prevented by administering a substance comprising a sequence identical or substantially identical to a uPAR binding site of uPA amino residues 12-32. Rosenberg et al., WO 94/28145(Dec. 9, 1994) disclosed the preparation and use of non-fucosylated HuPA
1-48
that prevented uPA binding to uPAR.
Earlier studies with peptide fragments within the growth factor domain had shown that residues 20-30 conferred the specificity of binding, but that residues 13-19 were also needed if residues 20-30 were to attain the proper binding conformation. Specifically, the peptide [Ala
19
]uPA(12-32), which contains two cysteines (the third cysteine being replaced by Ala to avoid undesired disulfide bond formations) in its open chain form prevented uPA binding to uPAR with an IC
50
of 100 nM. In its oxidized cyclic form with an intrachain disulfide bond between Cys
13
and Cys
31
, the peptide prevented uPA binding with an IC
50
of 40 nM. The authors proposed that residues 13-19 might act indirectly to provide a scaffold that would help residues 20-30 attain the correct binding conformation (Appella et al.,
J. Biol. Chem.,
262:4437-4440 (1987).
These results were par
Jones Terence R.
Mazar Andrew P.
Angstrom Pharmaceuticals, Inc.
Borin Michael
Livnat Shmuel
Venable
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