Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1995-05-10
2001-06-19
Pak, Michael (Department: 1646)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S002600
Reexamination Certificate
active
06248715
ABSTRACT:
DESCRIPTION
1. Technical Field
This invention relates to the fields of cellular biology and protein expression. More particularly, the invention relates to peptide ligands of the urokinase plasminogen activator receptor, and methods for preparing the same.
2. Background of the Invention
Urokinase-type plasminogen activator (uPA) is a multidomain serine protease, having a catalytic “B” chain (amino acids 144-411), and an amino-terminal fragment (“ATF”, aa 1-143) consisting of a growth factor-like domain (4-43) and a kringle (aa 47-135). The uPA kringle appears to bind heparin, but not fibrin, lysine, or aminohex-anoic acid. The growth factor-like domain bears some similarity to the structure of epidermal growth factor (EGF), and is thus also referred to as an “EGF-like” domain. The single chain pro-uPA is activated by plasmin, cleaving the chain into the two chain active form, which is linked together by a disulfide bond.
uPA binds to its specific cell surface receptor (uPAR). The binding interaction is apparently mediated by the EGF-like domain (S. A. Rabbani et al.,
J Biol Chem
(1992) 267:14151-56). Cleavage of pro-uPA into active uPA is accelerated when pro-uPA and plasminogen are receptor-bound. Thus, plasmin activates pro-uPA, which in turn activates more plasmin by cleaving plasminogen. This positive feedback cycle is apparently limited to the receptor-based proteolysis on the cell surface, since a large excess of protease inhibitors is found in plasma, including &agr;
2
antiplasmin, PAI-1 and PAI-2.
Plasmin can activate or degrade extracellular proteins such as fibrinogen, fibronectin, and zymogens. Plasminogen activators thus can regulate extracellular proteolysis, fibrin clot lysis, tissue remodeling, developmental cell migration, inflammation, and metastasis. Accordingly, there is great interest in developing uPA inhibitors and uPA receptor antagonists. E. Appella et al.,
J Biol Chem
(1987) 262:4437-40 determined that receptor binding activity is localized in the EGF-like domain, and that residues 12-32 appear to be critical for binding. The critical domain alone (uPA
12-32
) bound uPAR with an affinity of 40 nM (about 100 fold less than intact ATF).
S. A. Rabbani et al., supra, disclosed that the EGF-like domain is fucosylated at Thr
18
, and reported that fucosylated EGF-like domain (uPA
4-43
, produced by cleavage from pro-uPA) was mitogenic for an osteosarcoma cell line, SaOS-2. In contrast, non-fucosylated EGF-like domain bound uPAR with an affinity equal to the fucosylated EGF-like domain, but exhibited no mitogenic activity. Non-fucosylated EGF-like domain competed for binding to uPAR with fucosylated EGF-like domain, and reduced the mitogenic activity observed. Neither EGF-like domain was mitogenic in U937 fibroblast cells.
Previously, it was suggested that an “epitope library” might be made by cloning synthetic DNA that encodes random peptides into filamentous phage vectors (Parmley and Smith,
Gene
(1988) 73:305). It was proposed that the synthetic DNA be cloned into the coat protein gene III because of the likelihood of the encoded peptide becoming part of pIII without significantly interfering with pIII's function. It is known that the amino terminal half of pIII binds to the F pilus during infection of the phage into
E. coli
. It was suggested that such phage that carry and express random peptides on their cell surface as part of pIII may provide a way of identifying the epitopes recognized by antibodies, particularly using antibody to affect the purification of phage from the library. Devlin, PCT WO91/18980 (incorporated herein by reference) described a method for producing a library consisting of random peptide sequences presented on filamentous phage. The library can be used for many purposes, including identifying and selecting peptides that have a particular bioactivity. An example of a ligand binding molecule would be a soluble or insoluble cellular receptor (i.e., a membrane bound receptor), but would extend to virtually any molecule, including enzymes, that have the sought after binding activity. Description of a similar library is found in Dower et al., WO91/19818. The present invention provides a method for screening such libraries (and other libraries of peptides) to determine bioactive peptides or compounds. Kang et al., WO92/18619 disclosed a phage library prepared by inserting into the pvIII gene.
However, both the pIII and pVIII proteins are expressed in multiple copies in filamentous bacteriophage. As a result, the phage are selected and amplified based on their avidity for the target, rather than their affinity. To overcome this problem, a method for monovalent (only one test peptide per phage) phage display has been developed (H. B. Lowman et al.,
Biochem
(1991) 30:10832-38). To obtain monovalent display, the bacterial host is coinfected with the phage library and a large excess of “helper” phage, which express only wild-type pIII (and/or pVIII) and are inefficiently packaged. By adjusting the ratio of display phage to helper phage, one can adjust the ratio of modified to wild-type display proteins so that most phage have only one modified protein. However, this results in a large amount of phage having only wild-type pIII (or pVIII), which significantly raises the background noise of the screening.
DISCLOSURE OF THE INVENTION
One aspect of the invention is a method for producing non-fucosylated uPA EGF-like domain, particularly uPA
1-48
.
Another aspect of the invention is non-fucosylated uPA
1-48
, which is useful for inhibiting the mitogenic activity of uPA in cancer cells.
Another aspect of the invention is a method for treating cancer and metastasis by administering an effective amount of a non-fucosylated uPA EGF-like domain, particularly uPA
1-48
.
Another aspect of the invention is a method treating a uPA-mediated disorder by administering a composition comprising an effective amount of a non-fucosylated polypeptide consisting of the EDF-like domain by instillation in the eye.
Another aspect of the invention is a method for pre-enriching a monovalent phage display mixture prior to screening for binding to a target, by providing a mixture of monovalent display phage and non-displaying phage, wherein the monovalent display phage display both a candidate peptide and a common peptide, the common peptide is identical for each monovalent display phage, and the candidate peptide is different for different monovalent display phage; and separating all phage displaying the common peptide from phage not displaying a common peptide.
MODES OF CARRYING OUT THE INVENTION
A. Definitions
The term “huPA” refers specifically to human urokinase-type plasminogen activator. The “EGF-like domain” is that portion of the huPA molecule responsible for mediating huPA binding to its receptor (uPAR). The EGF-like domain, sometimes called the growth factor-like domain (“GFD”), is located within the first 48 residues of huPA. The critical residues (essential for binding activity) have been localized to positions 12-32, although a peptide containing only those residues does not exhibit a binding affinity high enough to serve as a useful receptor antagonist.
The term “huPAR antagonist polypeptide” refers to a polypeptide having a sequence identical to the EGF-like domain of huPA (residues 1-48), or an active portion thereof. An “active portion” is one which lacks up to 10 amino acids, from the N-terminal or C-terminal ends, or a combination thereof, of the huPA
1-48
polypeptide, and exhibits a K
d
≦5 nM with huPAR. The term “active analog” refers to a polypeptide differing from the sequence of the EGF-like domain of huPA
1-48
or an active portion thereof by 1-7 amino acids, but which still exhibits a K
d
≦5 nM with huPAR. The differences are preferably conservative amino acid substitutions, in which an amino acid is replaced with another natually-occurring amino acid of similar character. For example, the following substitutions are considered “conservative”: Gly
Ala; Val
Ile
Leu; Asp
Glu; Lys
Arg; Asn
Gln; and Phe
Trp
Tyr. Nonconservative
Rosenberg Steven
Stratton-Thomas Jennifer R.
Blackburn Robert P.
Chiron Corporation
Lenti David P.
Pak Michael
Teskin Robin L.
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