Method of treating a urokinase-type plasminogen activator-mediated disorder

A method of treating an uPA-mediated disorder is disclosed, which comprises providing and administering an effective amount of polypeptide consisting essentially of the EGF-like domain of human uPA or active analog thereof.

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 (u). 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 .alpha..sub.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.sub.12-32) bound u 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.sub.18, and reported that fucosylated EGF-like domain
 (uPA.sub.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 u with an affinity equal to the fucosylated EGF-like
 domain, but exhibited no mitogenic activity. Non-fucosylated EGF-like
 domain competed for binding to u 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.sub.1-48.
 Another aspect of the invention is non-fucosylated uPA.sub.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.sub.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 (u). 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 "hu 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.sub.1-48 polypeptide, and exhibits a K.sub.d.ltoreq.5
 nM with hu. The term "active analog" refers to a polypeptide differing
 from the sequence of the EGF-like domain of huPA.sub.1-48 or an active
 portion thereof by 1-7 amino acids, but which still exhibits a
 K.sub.d.ltoreq.5 nM with hu. 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{character pullout}Ala; Val{character pullout}Ile{character
 pullout}Leu; Asp{character pullout}Glu; Lys{character pullout}Arg;
 Asn{character pullout}Gln; and Phe{character pullout}Trp{character
 pullout}Tyr. Nonconservative changes are generally substitutions of one of
 the above amino acids with an amino acid from a different group (e.g.,
 substituting Asn for Glu), or substituting Cys, Met, His, or Pro for any
 of the above amino acids. The hu antagonist polypeptides of the
 invention should be substantially free of peptides derived from other
 portions of the huPA protein. For example, a hu antagonist composition
 should contain less than 20 wt % uPA B domain (dry weight, absent
 excipients), preferably less than 10 wt % uPA-B, more preferably less than
 5 wt % uPA-B, most preferably no detectable amount. The hu antagonist
 polypeptides also preferably exclude the kringle region of uPA.
 The term "expression vector" refers to an oligonucleotide which encodes the
 hu antagonist polypeptide of the invention and provides the sequences
 necessary for its expression in the selected host cell. Expression vectors
 will generally include a transcriptional promoter and terminator, or will
 provide for incorporation adjacent to an endogenous promoter. Expression
 vectors will usually be plasmids, further comprising an origin of
 replication and one or more selectable markers. However, expression
 vectors may alternatively be viral recombinants designed to infect the
 host, or integrating vectors designed to integrate at a preferred site
 within the host's genome. Expression vectors may further comprise an
 oligonucleotide encoding a signal leader polypeptide. When "operatively
 connected", the hu antagonist is expressed downstream and in frame with
 the signal leader, which then provides for secretion of the hu
 antagonist polypeptide by the host to the extracellular medium. Presently
 preferred signal leaders are the Saccharomyces cerevisiae .alpha.-factor
 leader (particularly when modified to delete extraneous Glu-Ala
 sequences), and the ubiquitin leader (for intracellular expression).
 The term "transcriptional promoter" refers to an oligonucleotide sequence
 which provides for regulation of the DNA.fwdarw.mRNA transcription
 process, typically based on temperature, or the presence or absence of
 metabolites, inhibitors, or inducers. Transcriptional promoters may be
 regulated (inducible/repressible) or constitutive. Yeast glycolytic enzyme
 promoters are capable of driving the transcription and expression of
 heterologous proteins to high levels, and are particularly preferred. The
 presently preferred promoter is the hybrid ADH2/GAP promoter described in
 Tekamp-Olson et al., U.S. Pat. No. 4,876,197 (incorporated herein by
 reference), comprising the S. cerevisiae glyceralde-hyde-3-phosphate
 dehydrogenase promoter in combination with the S. cerevisiae alcohol
 dehydrogenase II upstream activation site.
 The term "host" refers to a yeast cell suitable for expressing heterologous
 polypeptides. There are a variety of suitable genera, such as
 Saccharomyces, Schizosaccharomyces, Kluveromyces, Pichia, Hansenula, and
 the like. Presently preferred are yeast of the Saccharomyces genus,
 particularly Saccharomyces cerevisiae.
 The term "uPA-mediated disorder" refers to a disease state or malady which
 is caused or exacerbated by a biological activity of uPA. The primnary
 biological activity exhibited is plasminogen activation. Disorders
 mediated by plasminogen activation include, without limitation,
 inappropriate angiogenesis (e.g., diabetic retinopathy, corneal
 angiogenesis, Kaposi's sarcoma, and the like), metastasis and invasion by
 tumor cells, and chronic inflammation (e.g., rheumatoid arthritis,
 emphysema, and the like). Fucosylaied ATF is also mitogenic for some tumor
 cells (e.g., SaOS-2 osteosar-coma cells), which sometimes self-activate in
 an autocrine mechanism. Accordingly, the hu antagonist of the invention
 is effective in inhibiting the proliferation of uPA-activated tumor cells.
 The term "effective amount" refers to an amount of hu antagonist
 polypeptide sufficient to exhibit a detectable therapeutic effect. The
 therapeutic effect may include, for example, without limitation,
 inhibiting the growth of undesired tissue or malignant cells, inhibiting
 inappropriate angiogenesis, limiting tissue damage caused by chronic
 inflammation, and the like. The precise effective amount for a subject
 will depend upon the subject's size and health, the nature and severity of
 the condition to be treated, and the like. Thus, it is not possible to
 specify an exact effective amount in advance. However, the effective
 amount for a given situation can be determined by routine experimentation
 based on the information provided herein.
 The term "pharmaceutically acceptable" refers to compounds and compositions
 which may be administered to mammals without undue toxicity. Exemplary
 pharmaceutically acceptable salts include mineral acid salts such as
 hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the
 salts of organic acids such as acetates, propionates, malonates,
 benzoates, and the like.
 The term "pre-enriching" refers to increasing the concentration of
 candidate phage in a monovalent phage display mixture by removing phage
 which do not have a candidate peptide. A "monovalent phage display
 mixture" is a mixture of phage containing recombinant phage and helper
 phage in a ratio such that most phage display at most one recombinant
 surface protein.
 The term "common peptide" refers to a distinctive heterologous (not
 wild-type) peptide sequence which is displayed identically by all
 recombinant members of a phage (or other host) library. The common peptide
 is preferably an epitope recognized by a high-affinity antibody, which is
 not cross-reactive with any epitopes naturally occurring in the wild-type
 phage. The common peptide permits one to select all recombinant phage
 (having a common peptide and a random candidate peptide) as a set, and
 purify them away from non-recombinant (wild-type) phage. The presently
 preferred common peptide is Glu-Tyr-Met-Pro-Met-Glu.
 B. General Method
 The present invention relies on the fact that yeast do not fucosylate
 proteins upon expression, but are able to express properly folded, active
 uPA and fragments. One may employ other eukaryotic hosts in the practice
 of the invention as long as the host is incapable of fucosylating
 proteins, whether naturally or due to manipulation (e.g., genetic mutation
 or antibiotic treatment). Presently preferred hosts are yeasts,
 particularly Saccharomyces, Schizosaccharomyces, Kluveromyces, Pichia,
 Hansenula, and the like, especially S. cerevisiae. Strains AB110 and MB2-1
 are presently preferred.
 The expression vector is constructed according to known methods, and
 typically comprises a plasmid functional in the selected host. The uPA
 sequence used may be cloned following the method described in Example 1
 below. Variations thereof (i.e., active fragments and active analogs) may
 be generated by site-specific mutagenesis, imperfect PCR, and other
 methods known in the art. Stable plasmids generally require an origin of
 replication (such as the yeast 2.mu. ori), and one or more selectable
 markers (such as antibiotic resistance) which can be used to screen for
 transformants and force retention of the plasmid. The vector should
 provide a promoter which is functional in the selected host cell,
 preferably a promoter derived from yeast glycolytic enzyme promoters such
 as GAPDH, GAL, and ADH2. These promoters are highly efficient, and can be
 used to drive expression of heterologous proteins up to about 10% of the
 host cell weight. The presently preferred promoter is a hybrid ADH2/GAP
 promoter comprising the S. cerevisiae glyceraldehyde-3-phosphate
 dehydrogenase promoter in combination with the S. cerevisiae alcohol
 dehydrogenase II upstream activation site.
 The expression vector should ideally provide a signal leader sequence
 between the promoter and the hu antagonist polypeptide sequence. The
 signal leader sequence provides for translocation of the hu antagonist
 polypeptide through the endoplasmic reticulum and export from the cell
 into the extracellular medium, where it may be easily harvested. There are
 a number of signal leader sequences known that are functional in yeast.
 The yeast .alpha.-factor leader is presently preferred (see U.S. Pat. No.
 4,751,180, incorporated herein by reference).
 Alternatively, the vector may provide for integration into the host genome,
 as is described by Shuster, PCT WO92/01800, incorporated herein by
 reference.
 Transformations into yeast can be carried out according to the method of A.
 Hinnen et al., Proc Natl Acad Sci USA (1978) 75:1929-33, or H. Ito et al.,
 J Bacteriol (1983) 153:163-68. After DNA is taken up by the host cell, the
 vector integrates into the yeast genome at one or more sites homologous to
 its targeting sequence. It is presently preferred to linearize the vector
 by cleaving it within the targeting sequence using a restriction
 endonuclease, as this procedure increases the efficiency of integration.
 Following successful transformations, the number of integrated sequences
 may be increased by classical genetic techniques. As the individual cell
 clones can carry integrated vectors at different locations, a genetic
 cross between two appropriate strains followed by sporulation and recovery
 of segregants can result in a new yeast strain having the integrated
 sequences of both original parent strains. Continued cycles of this method
 with other integratively transformed strains can be used to further
 increase the copies of integrated plasmids in a yeast host strain. One may
 also amplify the integrated sequences by standard techniques, for example
 by treating the cells with increasing concentrations of copper ions (where
 a gene for copper resistance has been included in the integrating vector).
 Correct ligations for plasmid construction may be confirmed by first
 transforming E. coli strain MM294 obtained from E. coli Genetic Stock
 Center, CGSC #6135, or other suitable host with the ligation mixture.
 Successful transformants are selected by ampicillin, tetracycline or other
 antibiotic resistance or using other markers depending on the plasmid
 construction, as is understood in the art. Plasmids from the transformants
 are then prepared according to the method of D. B. Clewell et al., Proc
 Natl Acad Sci USA (1969) 62:1159, optionally following chloramphenicol
 amplification (D. B. Clewell, J Bacteriol (1972) 110:667). Isolated DNA is
 analyzed by restriction mapping and/or sequenced by the dideoxy method of
 F. Sanger et al., Proc Natl Acad Sci USA (1977) 74:5463 as further
 described by Messing et al., Nucl Acids Res (1981) 9:309, or by the method
 of Maxam and Gilbert, Meth Enzymol (1980) 65:499.
 hu antagonist polypeptides may be assayed for activity by methods known
 in the art. For example, one may assay competition of the antagonist
 against native uPA for cell surface receptor binding. Competition for the
 receptor correlates with inhibition of uPA biological activity. One may
 assay hu antagonist polypeptides for anti-mitogenic activity on
 appropriate tumor cell lines, such as the osteosarcoma cell line SaOS-2
 described in the art. Inhibition of mitogenic activity may be determined
 by comparing the uptake of .sup.3 H-T in osteosarcoma cells treated with
 the antagonist against controls. One may also assay hu antagonists for
 anti-invasive activity on appropriate tumor cell lines, such as HOC-1 and
 HCT116 (W. Schlechte et al., Cancer Comm (1990) 2:173-79; H. Kobayashi et
 al., Brit J Cancer (1993) 67:537-44).
 hu antagonists are administered orally, topically, or by parenteral
 means, including subcutaneous and intramuscular injection, implantation of
 sustained release depots, intravenous injection, intranasal
 administration, and the like. When used to treat tumors, it may be
 advantageous to apply the hu antagonist directly to the site, e.g.,
 during surgery to remove the bulk of the tumor. Accordingly, hu
 antagonist may be administered as a pharmaceutical composition comprising
 hu antagonist in combination with a pharmaceutically acceptable
 excipient. Such compositions may be aqueous solutions, emulsions, creams,
 ointments, suspensions, gels, liposomal suspensions, and the like.
 Suitable excipients include water, saline, Ringer's solution, dextrose
 solution, and solutions of ethanol, glucose, sucrose, dextran, mannose,
 mannitol, sorbitol, polyethylene glycol (PEG), phosphate, acetate,
 gelatin, collagen, Carbopol.RTM., vegetable oils, and the like. One may
 additionally include suitable preservatives, stabilizers, antioxidants,
 antimicrobials, and buffering agents, for example, BHA, BHT, citric acid,
 ascorbic acid, tetracycline, and the like. Cream or ointment bases useful
 in formulation include lanolin, Silvadene.RTM. (Marion), Aquaphor.RTM.
 (Duke Laboratories), and the like. Other topical formulations include
 aerosols, bandages, and other wound dressings. Alternatively, one may
 incorporate or encapsulate the hu antagonist in a suitable polymer
 matrix or membrane, thus providing a sustained-release delivery device
 suitable for implantation near the site to be treated locally. Other
 devices include indwelling catheters and devices such as the Alzet.RTM.
 minipump. Ophthalmic preparations may be formulated using commercially
 available vehicles such as Sorbi-care.RTM. (Allergan), Neodecadron.RTM.
 (Merck, Sharp & Dohme), Lacrilube.RTM., and the like, or may employ
 topical preparations such as that described in U.S. Pat. No. 5,124,155,
 incorporated herein by reference. Further, one may provide a hu
 antagonist in solid form, especially as a lyophilized powder. Lyophilized
 formulations typically contain stabilizing and bulking agents, for example
 human serum albumin, sucrose, mannitol, and the like. A thorough
 discussion of pharmaceutically acceptable excipients is available in
 Remington's Pharmaceutical Sciences (Mack Pub. Co.).
 The amount of hu antagonist required to treat any particular disorder
 will of course vary depending upon the nature and severity of the
 disorder, the age and condition of the subject, and other factors readily
 determined by one of ordinary skill in the art. The appropriate dosage may
 be determined by one of ordinary skill by following the methods set forth
 below in the examples. As a general guide, about 0.01 mg/Kg to about 50
 mg/Kg hu antagonist administered i.v. or subcutaneously is effective
 for inhibiting tissue damage due to chronic inflammation. For treating
 corneal angiogenesis, hu antagonist may be administered locally in a
 gel or matrix at a concentration of about 0.001 mg/Kg to about 5 mg/Kg.

C. EXAMPLES
 The examples presented below are provided as a further guide to the
 practitioner of ordinary skill in the art, and are not to be construed as
 limiting the invention in any way.
 Example 1
 (Cloning and Expression of huPA.sub.1-48)
 DNA encoding residues 1-48 of mature human uPA (huPA) was cloned into a
 yeast expression vector as a fusion with the yeast alpha-factor leader
 (.alpha.Fl), under transcriptional control of a hybrid ADH2-GAP promoter.
 The .alpha.Fl is described in Brake, U.S. Pat. No. 4,870,008, incorporated
 herein by reference. The hybrid ADH2-GAP promoter is described in
 Tekamp-Olson et al., U.S. Pat. No. 4,876,197, and Tekamp-Olson et al.,
 U.S. Pat. No. 4,880,734, both incorporated herein by reference.
 The gene encoding huPA was obtained by PCR using the following sense and
 nonsense primers:
 5'-ATGCTAGATCTAATGAACTTCATCAGGTACCATCG-3' (SEQ ID NO:1), and
 5'-CGATAGATCTTTATTTTGACTTATCTATITCACAG-3' (SEQ ID NO:2).
 Each of the above primers introduces a BglII site at the ends for cloning
 into the expression vector. Additionally, the sense strand primer
 introduces a KpnI site 14 nucleotides downstream from the signal peptide
 cleavage site, and the nonsense strand primer introduces a stop codon
 after Lys at position 48. The template DNA used was a clone of full length
 mature huPA in a yeast expression vector, as an alpha-factor fusion
 (pAB24UK300, consisting of the yeast shuttle vector pAB24 having a
 cassette inserted at the BamHI site, the cassette containing the ADH2-GAP
 hybrid promoter, the yeast ai-factor leader, the coding sequence for
 mature human uPA, and the GAP terminator, obtained from P. Valenzuela,
 Chiron Corporation) derived from a human kidney cDNA library (M. A. Truett
 et al., DNA (1985) 4:333-49). Polymerase chain reactions were carried out
 in 100 .mu.L volumes with the following components: 10 mM Tris-HCl, pH
 8.3, 50 mM KCl, 1.5 mM MgCl.sub.2, 0.2 mM each dATP, dCTP, dGTP, and dTTP,
 1 .mu.M each primer, 9 ng template plasmid, and 2.5 U Taq DNA polymerase.
 The reaction conditions were 94.degree. C. for 1 min, followed by
 37.degree. C. for 2 min, then 72.degree. C. for 3 min, for 30 cycles. Both
 the PCR fragment and a subcloning vector (pCBR, described by Frederik et
 al., J Biol Chem (1990) 265:3793) containing the yeast expression cassette
 were digested with BglII and then ligated together, after treatment of the
 pCBR vector with alkaline phosphatase. Once the subclone was obtained
 (pCBRuPA.alpha.13), the expression cassette was isolated via BamHI
 digestion and ligated into the yeast shuttle vector (pAB24) to yield
 pAB24.alpha.13u-48.
 The expression plasmid was transformed into Saccharomyces cerevisiae AB110
 (MAT.alpha. leu2-3-112 ura3-52 pep4-3 [cir].degree.) using the lithium
 acetate method (Ito et al., J Bacteriol (1983) 153:163), and selected for
 uracil prototrophy. The plasmid copy number was then amplified by growth
 on minimal media without leucine, containing 8% glucose to keep ADH2-GAP
 promoter-mediated expression repressed. High level expression of secreted
 huPA.sub.1-48 was obtained with pAB24.alpha.13u-48 transformants of
 AB110 grown in leu medium and inoculating at 1:10 into YEP 4% glucose
 medium. All yeast cultures were grown at 30.degree. C., 275 rpm, for 96
 hours.
 Example 2
 (Purification of huPA.sub.1-48)
 One liter of yeast supernatant was harvested by centrifuging the cells at
 2600.times.g. Protein was concentrated from the supernatant by adding 70%
 ammonium sulfate, incubating for 1 hr at 4.degree. C., and separating the
 protein precipitate by centrifuging at 11,000.times.g for 1 hr at
 4.degree. C. The protein pellets were resuspended in buffer containing 20
 mM potassium phosphate, pH 7.0, 50 mM NaCl, and 1 mM EDTA. The suspension
 was dialyzed against the same buffer, with two changes of 4 L, overnight
 at 4.degree. C. The entire dialysate was loaded onto a 1.8 L SEPHADEX.RTM.
 G-50 column at room temperature. Fractions were collected and monitored
 with UV at 254 nm, then pooled based on 16% Tris-Tricine SDS-PAGE (Novex)
 under non-reducing conditions. The peak fractions, containing monomeric
 huPA.sub.1-48, were then loaded onto a 22 mm C18 reverse phase HPLC column
 (Vydac) and the protein eluted with a 0.6% gradient of acetonitrile
 containing 1% TFA. The major peak eluting at 33.5 minutes was collected
 and lyophilized. The purification yield is summarized in Table 1:
 TABLE 1
 Purification of huPA.sub.1-48
 Sample Total Protein Total Units.sup.b Yield
 Crude supernatant .about.200 mg.sup.a 3.3 .times. 10.sup.6 --
 Ammonium sulfate 160 mg 2.0 .times. 10.sup.6 60%
 G50 Column 103 mg 1.3 .times. 10.sup.6 42%
 HPLC Purified 8.4 mg 7.4 .times. 10.sup.5 22%
 .sup.a Estimated protein concentration due to interference with BCA assay
 .sup.b Unit = volume of crude sample required to inhibit binding of
 .sup.125 I-ATF 50% in competition with biotinylated su.
 Example 3
 (Characterization of huPA.sub.1-48)
 Purified huPA.sub.1-48 was subjected to amino acid analysis and N-terminal
 sequencing, yielding the expected composition and sequence. The Edman
 degradation was performed through residue 20. A stoichiometric amount of
 threonine was observed at cycle 18, indicating that this residue was not
 modified by fucosylation, as is found for uPA purified from eukaryotic
 cells. The absence of post translational modification was later confirmed
 by electrospray mass spectrometry. The binding activity of the recombinant
 huPA.sub.1-48 was determined using a radio-receptor binding assay.
 Baculovirus-derived recombinant human urokinase receptor was expressed as a
 truncated, soluble molecule as described previously for mouse L-cells
 (Masucci et al., J Biol Chem (1991) 266:8655). The purified receptor was
 biotinylated with NHS-biotin, and immobilized at 1 .mu.g/mL in PBS/0.1%
 BSA on streptavidin coated 96-well plates. Human uPA ATF (residues 1-135,
 obtained from M. Shuman, University of California, San Francisco) was
 iodinated using the lodogen method (Pierce), and used as tracer. The
 .sup.125 I-ATF was incubated at 100-500 pM with increasing amounts of
 huPA.sub.1-48 in triplicate (100 pM-1 .mu.M) for 2 hours at room
 temperature in 0.1% BSA/PBS in a total volume of 200 .mu.L. The plates
 were then washed 3 times with PBS/BSA, and the remaining bound
 radioactivity determined. The apparent K.sub.d observed for huPA.sub.1-48
 was 0.3 nM, comparable to that reported for ATF and intact uPA.
 Example 4
 (Construction of huPA.sub.1-48 Muteins)
 In order to efficiently analyze the features of huPA.sub.1-48, we performed
 a series of mutagenesis experiments utilizing phage display. Attempts to
 employ the system described by Scott and Smith, Science (1990) 249:386-90,
 were not successful. However, the use of monovalent phage display, using a
 phagemid and helper phage as described by Lowman et al., Biochem (1991)
 30:10832-38, did result in functional display of the protein domain.
 Finally, we employed an affinity epitope "tag" to reduce the fraction of
 phage bearing only wild-type pIII protein, reducing the background in
 panning experiments.
 A.) Construction of Phagemids:
 The starting materials were a phagemid construct (pGMEGF) comprising a
 human epidermal growth factor (hEGF) gene linked to the lac promoter,
 using pBLUESCRIPT (Stratagene) as the backbone. The polylinker region of
 the vector contained within a PvuII fragment was replaced by a cassette
 comprising a leader sequence from the photo-bacterial superoxide dismutase
 fused to a synthetic gene for hEGF, in turn fused to residues 198-406 of
 the M13 pIII gene. The sequence of the insert is shown in SEQ ID NO:3. A
 synthetic gene encoding human urokinase residues 1-48 was obtained from J.
 Stratton-Thomas, Chiron Corporation.
 Fusion proteins were generated using PCR. A first set of primers EUKMPCR1
 and EUKGPCR1 were used with primer EUKPCR2 to add epitope tags to
 huPA.sub.1-48 at the N-terminus, and to add an amber codon (TAG) and a
 BamHI site within residues 249-254 of the pIII protein at the C-terminus.
 EUKMPCR1: CTCATCAAGCTTTAGCGGACTACAAAGACGATGACGATAAGAGC-AATGAACTTCATCAAG
 (SEQ ID NO:5);
 EUKGPCR1: CTCATCAAGCTTTAGCCGAATACATGCCAATGGAAAGCAATGAAC-TTCATCAAG (SEQ ID
 NO:6);
 EUKPCR2: CACCGGAACCGGATCCACCCTATTTTGACTTATC (SEQ ID NO:7).
 The PCR reactions yielded primary products of the expected sizes, 204 and
 197 bp.
 A second set of primers, SRO1 and EUKCPCR1, were used with the
 EGF-containing phagemid construct as template. These primers added a BamI
 site at pIII residues 250-251 and amplified a fragment ending at the
 unique Cla1 site at residues 297-299 of pIII.
 SRO1: GAAATAGATAAGTCAAAATAGGGTGGATCCGGTTCCGGTGATTTTGATT-ATG (SEQ ID NO:8);
 and
 EUKCPCR1: GAAACCATCGATAGCAGCACCG (SEQ ID NO:9).
 This PCR reaction yielded a primary product of approximately 180 bp. The
 PCR reaction products were separated from unreacted primers by size
 exclusion chromatography (Chromaspin-100, Clontech), digested with
 restriction enzymes Hd3 and BamHI (set 1) or BamHI and Cla1 (set 2), and
 isolated from a 2.5% agarose gel, using the MERMAID procedure (Bio-101).
 Each of the set 1 fragments were ligated with the C-terminal reaction 2
 fragment, the ligations digested with Hd3 and Cla1, and the resulting
 fragments ligated into pGMEGF (digested with Hd3 and Cla1,
 dephosphorylated with alkaline phosphatase). The ligations were
 transformed into E. coli JS5 (Biorad) by electroporation. Strain JS5
 overproduces lac repressor, and is sup0, preventing expression of the
 uPA.sub.1-48 -pIII fusion protein due to the amber stop codon between the
 uPA.sub.1-48 and pIII genes. Correct clones were identified by restriction
 analysis and confirmed by DNA sequencing. These steps yielded phagemids
 pHM1a (M1Flag-uPA.sub.1-48) and pHM3a (Glutag-uPA.sub.1-48). The DNA
 sequences of the fusion proteins in these phagemids are shown in SEQ ID
 NO: 10 and SEQ ID NO:12.
 The phagemid containing a synthetic gene for uPA.sub.1-48 was constructed
 in the same vector by the following steps. The sequence of the synthetic
 gene is shown in SEQ ID NO: 14. Plasmid pCBRuPA (16 .mu.g), a derivative
 of pCBR (Frederick et al., J Biol Chem (1990) 265:3793) containing this
 synthetic gene for uPA.sub.1-48, inserted between the yeast .alpha.-factor
 leader and GAPDH terminator as a BglII fragment, was digested with Sac1
 and Cla1, and adapted for phagemid expression using the following set of
 synthetic oligonucleotides:
 SRO35: AGCTTTAGCGGAATACATGCCAATGGAAAGCAATGAGCT (SEQ ID NO: 16);
 SRO36: CATTGCTTTCCATTGGCATGTATTCCGCTAA (SEQ ID NO: 17);
 SRO37: CGATAAGTCAAAATAGGGTG (SEQ ID NO: 18); and
 SRO38: GATCCACCCTATTTTGACTTAT (SEQ ID NO: 19).
 Oligonucleotides SRO36 and SRO37 (250 pmol) were phosphorylated with
 polynucleotide kinase and annealed with equimolar amounts of oligos SRO35
 and SRO38, respectively. The two annealed duplexes (125 pmol) were ligated
 overnight with the digested plasmid DNA, the ligase heat inactivated, and
 the ends phosphorylated with polynucleotide kinase. The DNA was run on a
 6% polyacrylamide gel and the correct sized band (ca. 200 bp) was excised
 and isolated. The insert was ligated with plasmid pHM1a (digested with Hd3
 and BamHI) and phosphatased, and the ligations transformed into E. coli
 JS5. The correct recombinants were identified by restriction analysis, and
 confirmed by DNA sequencing, yielding phagemid pHM3-3.
 B.) Production and Panning of Phagemids:
 To produce phagemid particles, DNAs were transformed into E. coli strain
 XL1-BLUE (Stratagene) by electroporation. This strain was used because it
 is supE44 (TAG codon encodes Gln), laciQ (overproduces lac repressor), and
 makes phage (F'+). Overnight cultures were grown in 2.times.YT broth
 containing 50 .mu.g/mL amnpicillin and 10 .mu.g/mL tetracycline (to
 maintain the F'). Cells were diluted 1:50 or 1:100 into the same media,
 grown for 20 minutes as 37.degree. C. for 10 minutes at 225 rpm to enhance
 phage attachment, and then grown with normal agitation at 325 rpm
 overnight. Phage particles were then purified and concentrated by two
 successive precipitations with polyethylene glycol. The concentrations of
 phage present were determined by infection of E. coli XL1-blue and plating
 on L broth plates containing 50 .mu.g/mL ampicillin.
 To pan for binding phage particles, small tissue culture plates were coated
 either with anti-Glu antibody (R. Clark, Onyx Corporation) or streptavidin
 at 10 .mu.g/mL in PBS overnight. Plates were then blocked with PBS
 containing 0.1% BSA. To the streptavidin plates was then added 1 .mu.g/mL
 of biotinylated secreted human urokinase receptor obtained by recombinant
 baculovirus infection of A. califormica Sf9 cells. After 2 hours at room
 temperature, the plates were again blocked with BSA, and phage (10.sup.6
 -10.sup.10 cfu) were added in 1 mL of PBS/BSA. After incubation for 1
 hour, non-specifically adhered phage were removed by washing (7.times.1 mL
 PBS/BSA), and the remaining phage eluted with 1 mL of 0.1 M glycine, pH
 2.2, for 30 minutes. The eluted phage were immediately neutralized with 1
 M Tris, pH 9.4, and stored at 4.degree. C. overnight. The number of phage
 eluted was determined by titering on E. coli XL1-blue on ampiciflin
 plates. The procedure, where phage are first bound and eluted from the
 Glu-Ab plates and then panned against receptor plates, reduces the high
 background that would otherwise result from the large number of phage
 containing only wild type pIII: only phage containing an insert in pIII
 display an epitope tag and are selected on anti-Glu MAbs plates.
 Table 2 shows that phagemids displaying uPA.sub.1-48 are specifically bound
 and eluted from immobilized urokinase receptor. Table 3 demonstrates that
 the phagemid which displays a Glu tag-uPA.sub.1-48 fusion is specifically
 retained by immobilized Glu Ab. Finally, Table 4 shows that a population
 of the Glu-uPA.sub.1-48 phagemid which has been specifically eluted from
 the Glu Ab plates, is retained with a much higher yield on urokinase
 receptor plates, than is the unenriched phagemid population.
 TABLE 2
 Panning on Immobilized Receptor
 % Yield
 Sample Phage/phagemid Input.sup.e -u +u
 1.sup.a 1a 9.4 .times. 10.sup.9 0.0018 0.094
 2.sup.b 3a 1.4 .times. 10.sup.10 0.0014 0.08
 3.sup.c pGMEGF 1.3 .times. 10.sup.10 0.0015 0.0012
 4.sup.d LP67 (control) 1.4 .times. 10.sup.9 -- 0.0099
 .sup.a M1-FLAG-UPAELD-short pIII (pHM1a)
 .sup.b Glu-tag-UPAELD-short pIII (pHM3a)
 .sup.c M1-FLAG-EGF-medium long pIII (pGMEGF)
 .sup.d LP67-control phage (Amp.sup..gamma. M13)
 .sup.e ampicillin resistant colonies, in cfu
 TABLE 2
 Panning on Immobilized Receptor
 % Yield
 Sample Phage/phagemid Input.sup.e -u +u
 1.sup.a 1a 9.4 .times. 10.sup.9 0.0018 0.094
 2.sup.b 3a 1.4 .times. 10.sup.10 0.0014 0.08
 3.sup.c pGMEGF 1.3 .times. 10.sup.10 0.0015 0.0012
 4.sup.d LP67 (control) 1.4 .times. 10.sup.9 -- 0.0099
 .sup.a M1-FLAG-UPAELD-short pIII (pHM1a)
 .sup.b Glu-tag-UPAELD-short pIII (pHM3a)
 .sup.c M1-FLAG-EGF-medium long pIII (pGMEGF)
 .sup.d LP67-control phage (Amp.sup..gamma. M13)
 .sup.e ampicillin resistant colonies, in cfu
 TABLE 2
 Panning on Immobilized Receptor
 % Yield
 Sample Phage/phagemid Input.sup.e -u +u
 1.sup.a 1a 9.4 .times. 10.sup.9 0.0018 0.094
 2.sup.b 3a 1.4 .times. 10.sup.10 0.0014 0.08
 3.sup.c pGMEGF 1.3 .times. 10.sup.10 0.0015 0.0012
 4.sup.d LP67 (control) 1.4 .times. 10.sup.9 -- 0.0099
 .sup.a M1-FLAG-UPAELD-short pIII (pHM1a)
 .sup.b Glu-tag-UPAELD-short pIII (pHM3a)
 .sup.c M1-FLAG-EGF-medium long pIII (pGMEGF)
 .sup.d LP67-control phage (Amp.sup..gamma. M13)
 .sup.e ampicillin resistant colonies, in cfu
 These enriched phagemid pools are used for multiple mutagenesis strategies
 in order to identify improved uPA.sub.1-48 ligands with altered
 specificity or improved affinity. For example the region between residues
 13 and 32 of human uPA has been implicated in receptor binding (E. Appella
 et al., J Biol Chem (1987) 262:4437-40). Key residues in the region from
 19-30 can be easily mutated by replacing the region between the unique
 restriction sites Kpnl and Munl.
 In order to rapidly and quantitatively assess the binding affinities of the
 resulting uPA.sub.1-48 variants, relatively large quantities of properly
 folded proteins are required. Although this could be done by bacterial
 expression, using the phagemid constructs in a sup0 strain and inducing
 with IPTG, such a strategy yields relatively small amounts of protein in
 the periplasm. A second strategy is to express the variants in yeast, as
 described above for the wild type protein. To accomplish this we have
 constructed a yeast expression vector which enables us to move fragments
 encoding residues 4-48 of uPA.sub.1-48 in a single step from the phagemid
 vectors. This was accomplished as follows: Plasmid pAG.alpha.G, identical
 to pCBR except for a small deletion of an Xba fragment in the ADH2-GAPDH
 promoter, was digested with Sac1, which cleaves once within the promoter,
 and then treated with Mung Bean nuclease which destroys the site.
 Subsequent religation yielded plasmid pAG.alpha.G-Sac. Digestion with
 BglII and treatment with alkaline phosphatase yielded a vector into which
 was ligated the BglII fragment corresponding to the synthetic gene for
 uPA.sub.1-48. Transformation of E. coli strain HB101 to ampicillin
 resistance and restriction analysis yielded the correct clone. The 2.4 kB
 BamHI fragment from this plasmid (pAG.alpha.G-Sac1-48synth), containing
 the expression cassette, was isolated and ligated into pAB24, which had
 been treated with BamHI and alkaline phosphatase. The resulting plasmid
 has unique Sac1 and Xhol sites which can be used for transfer of the
 phagemid 1-48 genes. This is accomplished by digesting the phagemid with
 BamHI, treating with Mung Bean Nuclease, digesting with Sac1 and isolating
 the 145 bp fragment. The vector is digested with Xhol, treated with Mung
 Bean Nuclease, digested with Sac1, and treated with alkaline phosphatase.
 Ligation then yields the correct recombinants in a single step in the
 yeast expression vector. Transformation of yeast strain AB110 then yields
 high levels of secreted 1-48 variants for analysis.
 Using this construct, one can express a library of uPA variations for
 screening. Variations may be constructed by a variety of methods,
 including low-fidelity PCR (which introduces a large number of random
 point mutations), site-specific mutation, primer-based mutagenesis, and
 ligation of the uPA.sub.1-48 sequence (or portions thereof) to a random
 oligonucleotide sequence (e.g., by attaching (NNS).sub.x to the
 uPA.sub.1-48 coding sequence, or substituting NNS for one or more
 uPA.sub.1-48 codons). Generation of random oligonucleotide sequences is
 detailed in Devlin, WO91/18980, incorporated herein by reference. Phage
 displaying uPA.sub.1-48 variants (having one or more amino acid
 substitutions) are screened according to the protocol described above
 (using, e.g., pHM3a as a positive control) and selected for improved
 binding.
 Example 5
 (Formulation of huPA.sub.1-48)
 huPA.sub.1-48 formulations suitable for use in chemotherapy are prepared as
 follows: