CA2162855A1 - Expression of urokinase plasminogen activator inhibitors - Google Patents

Abstract

A method for preparing a urokinase-type plasminogen activator inhibitor by expressing HuPA1-48 from yeast is disclosed.

Description

~p 94128145 ~ PCT/US94/05669 Expression of Urokinase Pl~inogen Activator Inhibitors Description Technical Field This invention relates to the fields of cellular biology and protein eA~lGssion. More particularly, the invention relates to peptide ligands of the 10 urokinase p1~minogen activator lcc~lor, and methods for preparing the same.

Back~lvund of the Invention Urokinase-type pl~minogen activator (uPA) is a multidomain serine pro-tease, having a catalytic "B" chain (amino acids 144-411), and an amino-~,-,-inal fr~gmçnt ("ATF", aa 1-143) cO~ p of a growth factor-like domain (4-43) and a kringle (aa 47-135). The uPA kringle a~ to bind heparin, but not fibrin, lysine, or aminoheA~oic acid. The growth factor-like domain bears some similarity to the structure of epi~lenn~l growth factor (EGF), and is thus also referred to as an "EGF-like" domain. The single chain pro-uPA is activated by 20 pl~min, cleaving the chain into the two chain active form, which is linked together by a r~ de bond.
uPA binds to its spcecific cell surface lcceplor (uPAR). The binding int~,~ction iS a~arcnlly m~Ai~t~d by the EGF-like domain (S.A. Rabbani et al., JBiol Chem (1992) 267: 14151-56). Cleavage of pro-uPA into active uPA is 25 accelerated when pro-uPA and pl~minogen are receptor-bound. Thus, plasmin activates pro-uPA, which in turn activates more plasmin by cleaving pl~minogen.
This positive feeAb~k cycle is app~t..lly limited to the receptor-based proteolysis on the cell surface, since a large excess of protease inhibitors is found in plasma, including (X2 antiplasmin, PAI-1 and PAI-2.

WO 94/28145 PCT/I~S94/05669 ~
~IS2$~S

Plasmin can activate or degr~.1t extracellular proteins such as fibrinogen, fibronectin, and zymogens. pl~cminogen activators thus can regulate extracellular proteolysis, fibrin clot Iysis, tissue remodeling, developmental cell migr~tiQn,infl~mm~tion, and m.-,t~ct~cic. Accordingly, there is great interest in developing uPA inhibitors and uPA receptor antagonists. E. Appella et al., J Biol Chem (1987) ~ 4437-40 determined that receptor binding activity is ioc~li7ed in the EGF-like domain, and that residues 12-32 appear to be critical for binding. The critical domain alone (uPA12 32) bound uPAR with an aff~ity of 40 nM (about 100 fold less than intact AIP).
0 S.A. Rabbani et al., supra, disclosed that the EGF-like domain is fuco-sylated at Thrl8, and reported that fucosylated EGF-like domain (uPA~ 43, produced by cleavage from pro-uPA) was mitogenic for an osteosaf-;ol,la cell line, SaOS-2. In cQIltr~ct~ non-fuco~ylaled EGF-like domain bound uPAR with an affinity equal to the fucosylated EGF-like domain, but exhibited no mitogenic activity. Non-fucosylated EGF-lilce domain col-lpc~led for binding to uPAR with fuco~ylaled EGF-like domain, and reduced the mitogenic activity observed.
Neither EGF-l~e domain was mitogenic in U937 fibroblast cells.
Previously, it was suggested that an "epitope library" might be made by cloning ~ylllh~lic DNA that encodes random peptides into f~mentous phage vectors (Parmley and Smith, Gene (1988) 73:305). It was ~ posed that the synthetic DNA be cloned into the coat protein gene m because of the likelihood of the encoded peptide beco..~;ng part of pm without signifiç~ntly inlelreling with pm's function. It is known that the amino terminal half of pm binds to the F
pilus during infection of the phage into E. coli. It was suggested that such phage 25 that carry and express random peptides on their cell surface as part of pm may provide a way of idelllirying the ~il~es recognized by antibodies, particularly using antibody to affect the pnrifiç~tinn of phage from the library. Devlin, PCTWO91/18980 (incol~oldled herein by reference) described a method for produc-ing a library con~i~tin3~ of random peptide sequences pl~,s~,nled on filamentous ~ 94/28145 21 6 2 8 5 ~ PCT~S94/05669 phage. The library can be used for many purposes, including identifying and selo~ing peptides that have a particular bioactivity. An example of a ligand binding molecule would be a soluble or insoluble cellular l~ce~ulor (i.e., a mem-brane 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 co~ Jo-lnds. Kang et al., WO92/18619 disclosed a phage library p,~a~ed by inserting into the pvm gene.
0 However, both the pm and pvm prol~;ins are e,.~,~,3se~ in multiple copies in fil~m~ntous ba;l~li~hage. As a result, the phage are selec~d and amplified based on their avidity for the target, rather than their affinity. To ovel~o,ne 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 pm (and/or pVm) and are inefficiently p~ck~ged. By adjusting the ratio of display phage to helper phage, one can adjust the ratio of modified to wild-typedisplay ploteih-s so that most phage have only one modified protein. However,

2 o this results in a large amount of phage having only wild-type pm (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 uPAl 48.
Another aspect of the invention is non-fucosylated uPAI ~8, which is useful for inhibiting the mitogenic activity of uPA in cancer cells.

~7~5 Another aspect of the invention is a method for treating cancer and meta-stasis by ~lmini~t-p~ring an effective amount of a non-fucosylated uPA EGF-like domain, particularly uPAI 48.
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 ç~nrlitl~t~, peptide and a common peptide, the common peptide is identi~l for each monovalent display phage, and the c~ntli~tP peptide is dirÇerG.Il for dirrGIGIII monovalent displayphage; and s~.,.l;.~g 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 specific~lly to human urokinase-type pl~minogen activator. The "EGF-like domain" is that portion of the huPA
molecule l~s~,unsible for mPAi~ting huPA bill-ling to its lGc~lor (uPAR). The EGF-like domain, somPtimes called the growth factor-like domain ("GFD"), is located within the first 48 residues of huPA. The critical residues (es~nti~l for binding activity) have been loc~li7ed to positions 12-32, although a peptide co~ ing only those residues does not exhibit a l)indil~g affimity high enough toserve as a useful ~xe~tor antagonist.
The term "huPAR antagonist polypeptide" refers to a polypeptide having a seqllen~e identi~l to the EGF-like domain of huPA (residues 1-48), or an active portion thereof. An "active portion" is one which lacks up to lO amino acids, from the N-terminal or C-~e~ ",ii~l ends, or a combination thereof, of the huPAI ~8 polypeptide, and exhibits a Kd ~ 5 nM with huPAR. The term "active analog" refers to a polypeptide diffenng from the se~en~e of the EGF-like domain of huPAI 48 or an active portion thereof by 1-7 amino acids, but which ~ 94128145 PCT/US94/05669 ~ 2 8 5 5 still exhibits a Kd ~ 5 nM with huPAR. The dir~çe"ces are preferably conserv-ative amino acid substitutions, in which an amino acid is replaced with another n~h~ ly-occurring amino acid of similar character. For example, the following substitutions are considered "conservative": Gly ~ Ala; Val ~ Ile ~ Leu; Asp 5 ~ Glu; Lys ~ Arg; Asn ~ Gln; and Phe ~ Trp ~ Tyr. Nonconservative changes are generally substitutions of one of the above amino acids with an amino acid from a dirr~ènl group (e.g., substitutin~ Asn for Glu), or substinlting Cys, Met, His, or Pro for any of the above amino acids. The huPAR antagonist polypepti-~es of the invention should be sul~ lly free of peptides derived from 10 other portions of the huPA protein. For example, a huPAR antagonist composition should contain less than 20 wt% uPA B domain (dry weight, absent exci~ie"~), preferably less than lO wt% uPA-B, more preferably less than S wt%
uPA-B, most preferably no detect~hle ~monnt The huPAR antagonist polypep-tides also ~lGÇ~lal)ly ex~ le the kAngle region of uPA.
The term "t;A~ssion vector" refers to an oligonucleotide which encodes the huPAR antagonist polypeptide of the invention and provides the se~uen~es n~.ces~ / for its eA~les~ion in the select~l host cell. Expression vectors will generally include a tr~n~-rirtional promoter and l~ or, or will provide for incol~o.alion ~lja~Pnt to an endogenous promoter. EA~ ssion vectors will 20 usually be pl~mitl~, further compAsing an origin of replication and one or more select~hle l~ h~. However, eAlJlessioll vectors may alternatively be viral recombinants designPA to infect the host, or inleglalil~g vectors de~ignPd to il.legl~le at a ~rerelred site within the host's genome. Expression vectors may further compAse an oligonucleotide encoding a signal leader polypeptide. When 25 "operatively connected", the huPAR antagonist is c;AIJlGssed downstream and in frame with the signal leader, which then provides for secretion of the huPAR
antagonist polypeptide by the host to the extr~Plh-l~r mP~inm. Presently efelled signal leaders are the Saccharomyces cerevisiae a-factor leader (partic-WO 94/28145 . PCT/~S94/05669 ~ ~2~5~
-- 6ularly when modified to delete extraneous Glu-Ala sequences), and the ubiquitin leader (for intr~.ellnl~r expression).
The term "llalls.;~ ional promoter" refers to an oligonucleotide seq~-ence which provides for regulation of the DNA ~ mRNA transcription 5 process, typically based on temperature, or the presence or absence of metabolites, inhibitors, or inducers. TrAn~criptional promoters may be regulated(inducible/~pr~ssible) or consli~uli~/e. Yeast glycolytic enzyme promoters are capable of driving the transcription and e~.y,Gssion of heterologous ~r~teh~s tohigh levels, and are particularly "~rellGd. The plGselllly plG~l~d promoter is 10 the hybrid ADH2/GAP promoter described in Tekamp-Olson et al., US
4,876,197 (incol~,olaled herein by f~rclGnce), comprising the S. cerevisiae glycer aldehyde-3-phosphate dehydl~genase promoter in combination with the S.
cerevisiae alcohol dehydrogenase ~ ulJsll~ull activation site.
The term "host" refers to a yeast cell suitable for t;~ ,sing 15 heterologous polypeptides. There are a variety of suitable genera, such as Sacch~omyces, Schiz~7s~1~charomyces, Rluveromyces, Pichia, Nansenula, and the like. I~esel~lly p~cr~ ,d are yeast of the Saccharomyces genus, particularly Saccharomyces cerevisiae.
The term "uPA-mP~i~t~l disorder" refers to a disease state or malady 2 0 which is caused or exacGll,aled by a biologi~ l activity of uPA. The primarybiological activity exhibited is pl~cminogen activation. Disorders meAi~t~ by pl~cminogP.n activation include, without limit~tic)n, hla~l,liale ~ngiogen~cic (e.g., diabetic lGlih~opallly, corneal angiogenesis, Kaposi's sarcoma, and the like), m~.t~ct~cic and invasion by tumor cells, and chronic ;..rl~...--.,.li~ n (e.g., 25 rh~.um~toid arthritis, emphysema, and the like). ~ucosylated ATF is also mitogenic for some tumor cells (e.g., SaOS-2 osteos~o,l,a cells), which sometimes self-activate in an autocrine mechanism. Accoldil~gly, the huPAR
antagonist of the invention is effective in inhibiting the proliferation of uPA-activated tumor cells.

~l 94/28145 2 ~ j PCT/US94/05669 The term "effective amount" refers to an amount of huPAR antagonist polypeptide sufficient to exhibit a detect~hle therapeutic effect. The therapeutic O effect may include, for example, without limit~tion, inhibiting the growth of undesired tissue or m~lign~nt cells, inhibiting hla~ç~liate angiogenesis, limiting 5 tissue damage caused by chronic infl~mm~tion, 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 l~e. Thus, it is notpossible to specify an exact effective amount in advance. However, the effectiveamount for a given situation can be ietP~rmin~ by routine t;~)c.;lllpnt~tion based 10 on the il~on"~lion provided herein.
The term "~hal...~eutic~lly ~ecept~hle" refers to co~ ounds and compo-sitions which may be ~lminictered to m~mm~l~ wilhoul undue toxicity.
Exemplary ~h~....P~ceuti~lly ~cept~hle salts include miner~l acid salts such as hydrochlorides, hydlubr~,.l.ides, phosphales, slllf~tes, and the like; and the salts 15 of organic acids such as ~c~t~tes~ pl~iol~dles, malonates, bçn?o~les, and the like.
The term "pre-e.nri~hing" refers to increasing the conce.-l,ation of ç~n-lirl~te phage in a monovalent phage display ,-,i~lu,~ by removing phage which do not have a c~nr~ tp peptide. A "monovalent phage display mixture" is a Illi~lulc of phage co~ g recombinant phage and helper phage in a ratio such 2 0 that most phage display at most one recombinant surface protein.
The terrn "common peptide" refers to a ~ e heterologous (not wild-type) peptide se~ ence which is di~,layed identi~lly by all recombinant mP.mbers of a phage (or other host) library. The common peptide is preferably an epitope recogni~ed by a high-affinity antibody, which is not cross-reactive with 25 any epitopes naturally occurring in the wild-type phage. The common peptide el"-ils one to select all recombinant phage (having a common peptide and a random r~ntlitl~te peptide) as a set, and purify them away from non-recombinant (wild-t-ype) phage. The p,cse"lly ~ e"cd common peptide is Glu-Tyr-Met-Pro-Met-Glu.

Wo 94/28145 PcT~Sg4/05669 8 ~ 3 B. General Method The present invention relies on the fact that yeast do not fucosylate pl~teills upon e~ Gssion, but are able to express P1U~JGIIY folded, active uPA and frA~mPnt~. One may employ other eukaryotic hosts in the practice of the 5 inventiûn as long as the hûst is incapable of fucosylating proteins, whether naturally or due to manipulation (e.g., genetic mutation or antibiotic treatment).
I~GSGIII1Y l)rGrGll~d hosts are yeasts, particularly Saccharomyces, Schizosaccharo-myces, Kluveromyces, Pichia, Hansenula, and the l~e, especially S. cerevisiae.
Strains ABllO and MB2-1 are presently l"Gre.l.,d.
The eAplG~ion vector is constructed according to known metho-lc, and typically comprises a plasmid functional in the SPlPct-pcl host. The uPA sequence used may be cloned following the method described in Example 1 below.
Variations thereof (i. e., active frAgmPnt~ and active analogs) may be gÇ~51dtP~ by site-specific mutAgPnPC~ "~,Çect PCR, and other methor1s known in the art.
15 Stable pl~mills generally require an origin of replication (such as the yeast 2~u ori), and one or more se!ectAhle Illalh~l~ (such as antibiotic l~ e) which can be used to screen for tran~rul",a~ and force l~te.l~iGn of the plasmid. The vector should provide a promoter which is functional in the SPIçct~pcl host cell, preferably a promoter derived from yeast glycolytic enzyme promoters such as 20 GAPDH, GAL, and ADH2. These promoters are highly efficient, and can be used to drive e,~ ssion of heterologous ~n)l~ls up to about 10% of the host cellweight. The pl~se.llly ~,~,fe,l~,d promoter is a hybrid ADH2/GAP promoter comprising the S. cerevisiae glycer~klehyde-3-pho.~l.hAIe del.yd~ enase promoterin combination with the S. cerevisiae alcohol dehydlu~el~ase II u~
25 activation site.
The expression vector should ideally provide a signal leader se~uçnre bt;Lween the promoter and the huPAR antagonist poly-peptide sequence. The signal leader se~uen~e provides for translocation ûf the huPAR antagonist polypeptide through the endoplasmic reticulum and export from the cell into the 94/28145 ~16 2 8 5 5 PCT/US94/05669 extracellular mP~illm~ where it may be easily harvested. There are a number of signal leader selluen~es known that are functional in yeast. The yeast ~-factor leader is presently plGr~llGd (see U.S. 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 I C;rGI GllCe.
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., JBacteriol (1983) 153:163-68. After DNA is taken up by the host cell, the vector e~ P,s into the yeast genome at one or more sites homologous to its lar~,eling seq~Pn-e. It is ~JlGsGIllly prGrellGd to lillGa~i~G the vector by cleaving it within the t~,eLil~g sequ~Pn~e using a r~Pstrictic-n endonn~ e, as this procedure increasesthe efficiency of ;~IP,g~lion~
Following successful lla l,rull~.alions, the number of i~lP,g.~
sequ~Pnces may be illcl~,ased by cl~c;c~l genetic techniq~les. As the individualcell clones can carry i~lPg~led vectors at dirr~Gnl locations, a genetic cross belween two a~f~pliale strains followed by sporulation and recovery of seg~ega ll., can result in a new yeast strain having the i..le~laled seq~enre~ of both 20 original parent strains. Co..linued cycles of this mtothod with other ;,-~ rely llalnsrol...ed strains can be used to further increase the copies of inte.grat~dpl~cmi~l~ in a yeast host strain. One may also amplify the i"l~ A se~uences by standard techniques, for example by ll~aling the cells with increasing con~entr~tions of copper ions (where a gene for copper resi.~t~nl e has been 25 in-hlded in the inlegldlu~g vector).
Correct ligations for plasmid construction may be confirmed by first tran~rolllli,~g E. coli strain MM294 obtained from E. coli Genetic Stock Center,CGSC #6135, or other suitable host with the ligation llliXIUIG. Successful trans-follll~lls are selected by ampicillin, tetracycline or other ~llibiOIic re~i~t~nce or WO 94t28145 PCT/US94/05669 using other markers depending on the plasmid construction, as is understood in the art. Plasmids from the tran~ro,lllallLs are then prepared according to the method of D.B. Clewell el al., Proc Natl Acad Sci USA (1969) 62:1159, optionally following chloramphenicol amplification (D.B. Clewell, J Bacteriol t1972) 110:667). Isolated DNA is analyzed by restriction mapping and/or sequence~l by the dideoxy method of F. Sanger et al., Proc Natl Acad Sci USA
(1977) 74:5463 as further described by l\~escing el al., Nucl Acids Res (1981) 2:309, or by the method of Maxam and Gilbert, Meth Enzymol (1980) 65:499.
huPAR antagonist polypeptides may be assayed for activity by methods known in the art. For example, one may assay cc,lllpt;lilion of the antagonist against native uPA for cell surface l~c~lor binding. Co..-pc;l;linn for the receptor correlates with inhibition of uPA biological activity. One may assay huPAR
antagonist polypeptides for anti-lllilogenic activity on a~lu~"iale tumor cell lines, such as the osteos~;ol.la cell line SaOS-2 described in the art. Inhibition of 15 ~nilogenic activity may be d~ ed by co...pz.;,-g the uptake of 3H-T in o~eos~o..la cells treated with the antagonist against controls. One may also assay huPAR antagonists for anti-invasive activity on a~p,;ale tumor cell lines,such as HOC-l and HCT116 (W. Schlp~chte et al., Cancer Comm (1990) 2:173-79; H. Kobayashi et al., Brit J Cancer (1993) 67:537-44).
2 o huPAR antagonists are ~(~minictered orally, topically, or by p~ dl means, in~ ling ~ul)~;ul~euus and ;.~ l--scul~- injection, impl~lalion of sus-tained release depots, intravenous injection, intr~n~c~l a~lminictration, and the like. When used to treat tumors, it may be advantageous to apply the huPAR
antagonist directly to the site, e.g., during surgery to remove the bu~c ûf the tumor. Accordingly, huPAR antagonist may be ~lminictered as a pharm~ e~lti~
composition co-,-plisi.lg huPAR antagonist in combination with a pharm~- e~ltit~lly acceptable excipient. Such compositions may be aqueous solutions, emlllci~ ns, creams, ointm~ tc~ suspensions, gels, liposomal suspe..~ions, and the like.
Suitable excipients include water, saline, Ringer's solution, dextrose solution, and ~0 94/28145 PCT/US94/05669 2162~

solutions of ethanol, glucose, sucrose, dextran, mannose, mAnnhol, sorbitol, poly-ethylene glycol (PEG), phosphate, acetate, gelatin, collagen, Carbopol~, vegetable oils, and the like. One may additionally include suitable preservatives, stabilizers, antioxidants, ~ntimicrobials, and burr~ g agents, for example, BHA,5 BHT, citric acid, ascorbic acid, tetracycline, and the like. Cream or ointmentbases useful in formulation include lAnolin, Silvadene~ (Marion), A~lua~)ho~D
(Duke Laholato.ics), and the like. Other topical formulations include aerosols, bandages, and other wound dressings. Alternatively, one may incorporate or encapsulate the huPAR antagonist in a suitable polymer matrix or membrane, thus 10 providing a s~lstAin~A-release delivery device suitable for implantation near the site to be treated locally. Other devices include indwelling catheters and devices such as the Alzet~ m;n;l~un~l). OphthAlmic ~ dlions may be formnlAt~ using co~ llc.~ially available vehicles such as Sorbi-care~ (Allergan), Neodecadron0 (Merck, Sharp & Dohme), ~cril-~be~, and the like, or may employ topical p~ ~alions such as that llesc~be~l in US 5,124,155, incol~o,aled herein by reference. Further, one may provide a huPAR antagonist in solid form, especially as a Iyophilized powder. Lyophilized fonnnlAtions typically contain stabilizing and buLking agents, for example human serum albumin, sucrose, n~ ol, and the like. A thorough ~ cus~ion of phAnnA~uti~Ally acceptable 20 ex~ nl~ is available in ]~.n;.~ "'s PhAnn~ceutir,Al Scie1 ces ~Iack Pub. Co.).
The amount of huPAR antagonist required to treat any particular disorder will of course vary ~ie,pen~1ing upon the nature and severity of the disorder, the age and condiLioll of the subject, and other factors readily deter-mined by one of or~lin~ skill in the art. The ap~)lu~,l;ale dosage may be 25 deLGllllih~ed by one of or~ laly skill by following the methods set forth below in - the e~ .lcs. As a general guide, about 0.01 mg/Kg to about 50 mg/Kg huPAR
antagonist A-lmini~tered i.v. or suhc~tAn~usly is effective for inhibiting tissue damage due to chronic infiA.. AI;on. For treating corneal angiogenesis, huPAR

W O 94/28145 PCTrUS94/05669 ~

2~628~

antagonist may be ~lmini~tered 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 o~linaly skill in the art, and are not to be construed as limiting the invention in any way.

Example 1 0 (Cloning and Expression of huPAl~8) 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 (~Fl), under t~ncrriptional control of a hybrid ADH2-GAP promoter. The aFl is described in Brake, US 4,870,008,inco ~ ulaled herein by .~fel~,nce. The hybrid ADH2-GAP p,.,-l-olel is described in Tekamp-Olson et al., Us 4,876,197, and Tekamp-Olsûn et al., Us 4,880,734, both inco~llulaled herein by lGrcl~nce.
The gene encoding huPA was ol,~in~cl by PCR using the following sense and nonsense ~lilllcl~:
5 ' -ATGCTAGATCTAATGAACTTCATCAGGTACCATCG-3 ' (SEQ ID
2 0 NO: 1), and 5'-CGATA(3A~ -lAl-l-l-l~ACITATCTAl~TCACAG-3' (SEQ ID
NO:2).
Each of the above primers intrûduces a Bgl~ site at the ends fûr cloning into the e~ cssion vectûr. Additiûnally, the sense strand primer intrûduces a KpnI site 14 nucleotides downstream from the signal peptide cleavage site, and the nûn~en~e strand primer introduces a stop codon after Lys at position 48. The tt;lllpldle DNA uscd was a clone of full length mature huPA in a yeast e~ressionvector, as an alpha-factor fusion (pAB24UK300, con~i~ting of the yeast shuttle vector pAB24 having a cassette inserted at the BamH[ site, the C~ p~ttp~ Cont~ining 94/28145 ~ ~ ~ 2 8 ~ ~ PCT/US94/05669 the ADH2-GAP hybrid promoter, the yeast ~-factor leader, the coding sequence for mature human uPA, and the GAP lel...ilulor, obtained from P. V~le~7uela, Chiron Co~Jul~tion) derived from a human kidney cDNA library (M.A. Truett et al., DNA (1985) _:333-49). Polymerase chain reactions were carried out in 100 ~4L volumes with the following components: 10 mM Tris-HCI, pH 8.3t 50 mM
KCl, 1.5 mM MgCl2, 0.2 mM each dATP, dCTP, dGTP, and dlTP, 1 ,uM each primer, 9 ng template plasmid, and 2.5 U Taq DNA polymerase. The reaction conditions were 94C for 1 min, followed by 37C for 2 min, then 72C 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 e~lGssion cA~sette were digested with BglII and then ligated together, after l~,A~ .I of the pCBR vector with ~lk~line phosphatase. Once the subclone was obtAin~d (pCBRuPA~x13), the e,~ sion cA~sto,tte was isolated via BamHI
fli~estion and ligated into the yeast shuttle vector (pAB24) to yield pAB24~13uPA1-48.
The e~lGssion plasmid was transformed into Saccharomyces cerevisiae AB110 (MATcY leu2-3 -112 ura3 -52 pep4 -3 [cir3) using the lithium acetate method ~Ito et al., J Bacteriol (1983) 153:163), and selecte~l for uracil plvlollul~hy. The plasmid copy number was then amplified by growth on 20 ,;,.i."Al media without leucine, ~IIAi~ g 8% glucose to keep ADH2-GAP
promoter-...~fl;AI~ G,.~l~ssion l~ ,ssed. High level eA~ ,ssion of secreted huPAI ~8 was obtained with pAB24a13uPA1-48 tran~r~ ",.~,ls of AB110 grown in leu~ me~ m and inoclllAting at 1:10 into YEP 4% glucose m~illm. All yeast cultures were grown at 30C, 275 rpm, for 96 hours.
Example 2 (Purification of huPA~
One liter of yeast SUP~...AIA~I was harvested by ce,ll,iruging the cells at 2600 x g. Protein was conce..~l.AI~d from the ~e",_~,l by adding 70%

2~f~ i5 ammonium sulfate, inrl~b~ting for 1 hr at 4C, and separating the protein precipitate by ct;.~ uging at 11,000 x g for 1 hr at 4C. The protein pellets were resuspended in buffer cu.l~;~in;,lg 20 mM pot~ium phosphate, pH 7.0, 50 mM NaCl, and 1 mM FnTA. The suspension was dialyzed against the same 5 buffer, with two changes of 4 L, overnight at 4C. The entire dialysate was loaded onto a 1.8 L Sephadex0 G-50 column at room tt;lll,Util~ltUlC. Fractions were collected and monitored with W at 254 nm, then pooled based on 16%
Tris-Tricine SDS-PAGE (Novex) under non-re~ çing conditions. The peak fractions, co~ ;ning monomeric huPAl 48, were then loaded onto a 22 mm C18 10 reverse phase HPLC column (Vydac) and the protein eluted with a 0.6 % gradient of act;lol~illile co~ 1% TFA. The major peak eluting at 33.5 mimltes was collected and lyophilized. The purific~tion yield is ~ ed in Table 1:

TABLE 1: Purification of huPAI48 Sample Total Protein Total Unitsb Yield Crude ~u~ ~200 mg~ 3.3 x 106--Ammonium sulfate 160 mg 2.0 x 10660%
G50 Column 103 mg 1.3 x 10642 %
HPLC Purified 8.4 mg 7.4 x 10522 %
a) F! i protein - due to ~ , cc with BCA as~ay 25b) Unit = volume of crude ~ample required to inhibit binding of ~ ATF 50% in c . with b; .~
~uPAR.

Example 3 30(Char~ el;,illion of huPAl 48) Purified huPAl~8 was subjected to amino acid analysis and N-terminal se~uçn-~ing, yielding the expected composition and sequçnce. The Edman 94t28145 ~ ~ ~ 28 ~ ~ PCT/US94/05669 degradation was yt;lro~ ed through residue 20. A stoichiometric amount of Lhlc;onine was observed at cycle 18, in-lic~tin~ that this residue was not modified by fucosylation, as is found for uPA purified from eukaryotic cells. The absenceof post tr~n~l~tional mo(1ifiç~tion was later confirmed by elecln,~lay mass spectrometry. The binding activity of the recombinant huPAI 48 was dete~nined using a radio-receptor binding assay.
Baculovirus-derived recombinant human urokinase receptor was expressed as a tn-nr~ted; soluble molecule as described previously for mouse L-cells (~ CCi et al., J Biol Chem (1991) 266:8655). The purified receptor was biotinylated with NHS-biotin, and immobilized at 1 ~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 Fr~nri~co) was io-lin~t~l using the Iodogen method (Pierce), and used as tracer. The 12sI-ATF was inrub~t~ at 100-500 pM with increasing amounts of huPAI 48 in triplicate (100 pM - 1 ,uM) for 2 hours at room lelllyelalul~, in 0.1% BSA/PBS in a total volume of 200 ~L4L.
The plates were then washed 3 times with PBS/BSA, and the ,~,...~in;ng bound r~lio~tivity de~e, ...;ned. The apyal~nl Kd observed for huPA1 48 was 0.3 nM, co",ya,able to that reported for ATF and intact uPA.

2 o ~cample 4 (Construction of huPAI 48 ~ntt~in~) In order to efficiently analyze the fealurGs of huPAl 48, we ~lro,-"ed a series of mutagenesis G~)~ i utili7ing phage display. Attempts to employ the system described by Scott and Smith, Science (1990) ~L:386-90, were not succes~ful. However, the use of monovalent phage display, using a phagemid and helper phage as described by Lowman et a/., Biochem (1991) 30:10832-38, did result in filnr.tioll~l display of the protein dom~in Finally, we employed an affinity epitope "tag" to reduce the fraction of phage bearing only wild-type pmprotein, reducing the bacl~,ou,~d in p~nning t;~yclullents.

2~ ~8~5 A.) Construction of Phagemids:
The starting materials were a phagemid construct (p(3MFGF) 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 5 contained within a PvuII fragment was replaced by a cassette comprising a leader seque-nre from the photob~cteri~l su~ ide ~ mut~e fused to a synthetic gene for hEGF, in turn fused to residues 198-406 of the M13 pm gene. The se~uence 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 1 0 Corporation.
Fusion p~ eills were g~.n~ ~ using PCR. A first set of ~ lel~
EUl~PCRl and EUKGPCRl were used with primer EUKPCR2 to add epitope tags to huPAI 48 at the N-le..l.i,l.ls, and to add an amber codon (TAG) and a BamHI site within residues 249-254 of the pm protein at the C-termin--c.
15 EUKMPCRl: CTCATCAAGCTTTAGCGGACTACAAAGACGATGACGATAAGA-GCAATGAAC~CATCAAG (SEQ ID NO:5);
EUKGPCRl: CTCATCAAGCTTTAGCCGAATACATGCCAATGGAAAGCAATGA-ACITCATCAAG (SEQ ID NO:6);EUKPCR2: CACCGGAACCGGATCCACCCTA~ ~ACITATC (SEQ ID
2 0 NO:7).
The PCR reactions yielded IJ.ih..~ products of the expected sizes, 204 and 197 bp.
A second set of primers, SROl and EUKCPCRl, were used with the EGF-co-.l; ;n;ng phagemid construct as template. These primers added a BamHI
site at pm residues 250-251 and amplified a fragment ending at the unique Clal site at residues 297-299 of pm.
SROl: GAAATAGATAAGTCAAAATAGGGTGGATCCGGTTCCGGTGAlTITGA-TTATG (SEQ ID NO:8); and EUKCPCRl: GAAACCATCGATAGCAGCACCG (SEQ ID NO:9).

94/28145 ,~ PCT/US94/05669 This PCR reaction yielded a primary product of approximately 180 bp. The PCR
reaction products were srlJ~-AI~ from unreacted primers by size exclusion chromatography (Chromaspin-100, Clontech), digested with restriction enzymes Hd3 and BamHI (set 1) or BamHI and Clal (set 2), and isolated from a 2.5 %
5 agarose gel, using the Mermaid procedure (Bio-101). Each of the set 1 fragments were ligated with the C-te""h~al reaction 2 fr~mP.nt the ligations digested withHd3 and Clal, and the res~lting fr~gmPnt~ ligated into pGMEGF (digested with Hd3 and Clal, dephosphorylated with ~lk~line phosphatase). The ligations were transformed into E. coli JS5 (13iorad) by electroporation. Strain JS5 overproduces 10 lac ,el)fessor, and is supO, preventing e~lc;ssion of the uPAI 48-pm fusion pro-tein due to the amber stop codon belw~.~ the uPA1 A~8 and pm genes. Correct clones were identifi~P~ by restriction analysis and co-.ii....e~ by DNA se~uencing.
These steps yielded phagemids pHMla (MlFlag-uPAl ~8) and p~ (Glutag-uPAl 48). The DNA seqllences of the fusion proteins in these phagemids are 15 shown in SEQ ID NO:10 and SEQ ID NO:12.
The phagemid c~n~;nil-~ a synthetic gene for uPAl 48 was constructed in the same vector by the following steps. The se l~e-~ e of the synthetic gene is shown in SEQ ID NO:14. Plasmid pCBRuPA (16 ~g), a derivative of pCBR
delick et al., J Biol Chem (1990) 265:3793) co..~ this synthetic gene for 20 uPAl 48, inserted bel~n the yeast cY-factor leader and GAPDH te....;~ r as a BglII fr~gment was digested with Sacl and Clal, and adapted for phagemid ,ssion using the following set of ~ylllL~lic oligonucleotides:
SRO35: AGCl-l-lAGCGGAATACATGCCAATGGAAAGCAATGAGCT (SEQ ID
NO: 16);
25SRO36: CATTG~l~ CATTGGCATGTATTCCGCTAA (SEQ ID NO:17);
SRO37: CGATAAGTCAAAATAGGGTG (SEQ ~ NO:18); and SRO38: GATCCACCCTAl-l-l-l~ACTTAT(SEQ ~ NO: 19) .
Oligon-lclPotides SRO36 and SRO37 (250 pmol) were phosphorylated with polynucleotide kinase and ~nnP~l_d with equimolar amounts of oligos SRO35 and WO 94/28145 PCT/US94/05669 ~

~1~ 2 8 ~ S

SRO38, respectively. The two ~nn~ cl 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 5 isolated. The insert was ligated with plasmid pHMla (digested with Hd3 and BamHI) and phosph~t~ce~1, and the ligations transformed into E. coli JS5. The correct recombinants were identified by restriction analysis, and conrl,llled byDNA se~uencing, 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 elecllul)olation. This st~in was used because itis supE44 (TAG codon encodes Gln), laciQ (overproduces lac lG~U-GSsOl), and makes phage (F' +). Overnight cultures were grown in 2 x YT broth cont~ining 50 ~llg/mL ampicillin and 10 ~g/mL tetracycline (to ~ ;"~ the F'). Cells were diluted 1:50 or 1:100 into the same media, grown for 20 min-lte~ as 37C for 10 --"es at 225 rpm to enh~nce phage ~tt~-hment, and then grown with normal agitation at 325 Ipm overnight. Phage particles were then purified and conre~ ,.l~1 by two s~lccçc~ive ~ )i~lions with polyethylene glycol. The con~ntrAtions of phage present were dele~ d by infection of E. coli XLl-blue 2 0 and plating on L broth plates co,~ g 50 ,~g/mL ampicillin.
To pan for binding phage particles, small tissue culture plates were coated either with anti-Glu antibody (R. Clark, Onyx Co,~ola~ion) or streptavidin at 10 ,ug/mL in PBS overnight. Plates were then blocked with PBS co..~ g 0.1% BSA. To the streptavidin plates was then added l ,ug/mL of biotinylated 25 secreted human urokinase receptor obtained by recombin~nt baculovirus infection of A. californica Sf9 cells. After 2 hours at room te~ al~ , the plates were again blocked with BSA, and phage (106-101 cfu) were added in 1 mL of PBS/BSA. After in~ ub~tic)n for 1 hour, non-specifically adhered phage were removed by washing (7x 1 mL PBS/BSA), and the rem~ining phage eluted with ~ 94/28145 21 6 2 ~ 5 5 PCT/US91/05669 1 mL of 0.1 M glycine, pH 2.2, for 30 minutes. The eluted phage were immed-iately neutralized with 1 M Tris, pH 9.4, and stored at 4C overnight. The num-ber of phage eluted was determined by titering on E. coli XLl-blue on ampicillinplates. The procedure, where phage are first bound and eluted from the Glu-Ab 5 plates and then panned against receptor plates, reduces the high background that would otherwise result from the large number of phage cont~ining only wild type pm: only phage cont~inin~ an insert in pm display an epitope tag and are sel~ted on anti-Glu MAbs plates.
Table 2 shows that phagemids displaying uPAl 48 are specifically bound 10 and eluted from immobilized urokinase receptor. Table 3 demon~tr~tp~s that the phagemid which displays a Glu tag-uPA~ 48 fusion is specifically retained by immobilized Glu Ab. Finally, Table 4 shows that a population of the GlU-UPAl 48 phagemid which has been specifically eluted from the Glu Ab plates, is retained with a much higher yield on urokinase ,~ce~lor plates, than is the nP.nriehed phagemid population.

TABLE 2: Panning on ~nobilized ~ c e~to.
%Yield Sample Pha~e/phagemid Inpute -uPAR +uPAR
1~ la 9.4x109 0.0018 0.094 2b 3a 1.4x101 0.0014 0.08 3C P(~J~'C~F 1.3 x 101 0.0015 0.0012 4d LP67 (control) 1.4x109 - 0-0099 a Ml-FLAG-UPAELD-short pm (pHMla) b Glu-tag-UPAELD-short pIII (pHM3a) c M1-FLAG-EGF - ' Iong pm (pGMEGF)

3 0 d LP67 - control phage (Ampr M13) e a~npicillin resistant colonies, in cfu WO 94/28145 PCT/US94/05669 ~

2 ~

TAiBLE 3: Panning phage with Glu-Ab orsuPA~R
%Yield Sample Pha~e/pha~emid Inputa suPARI' GluAb 1 pHMla 1.5 x 101 0.55 % 0.003 %
2 p~M~?, 2.5x101 0.44% 0.048%
3 LP67 (control) 3.5xlOs 0.008%
a ampicillin resistant colonies, in cfu b soluble uPA receptor TAiBLE 4: Panning GluAb-~ r;JIed and enriched phage on suPA~R
%Yield Sample Phage/phagemid Inputa suPARh GluAb p~M~ 2.7 x 107 0.85 % 0.08 %
2 pHM3a (enriched) 6 X106 9.7% 3.3%
2 o 3 LP67 (control) 5.4 x 106 < 0.04 % < 0.02 %
~ ampicillin resistant colonies, in cfu b soluble uPA receptor These enrich~d phagemid pools are used for multiple mutagenesis str~te~ies in order to identify improved uPAl 48 ligands with altered specificity or improved affinity. For example the region bG~ween residues 13 and 32 of human uPA has been implicated in lGcel lol binding (E. Appella et al., J Biol Chem (1987) 262:4437-40). Key residues in the region from 19-30 can be easily ml~t~ted by replacing the region bGIW~n the unique restriction sites Kpnl and Munl.
3 0 In order to rapidly and 4u~ ely assess the binding ~ffiniti~s of theres~lltin~ uPAl~8 variants, relatively large qn~ntiti~s of l~lupGlly folded proteins are required. Although this could be done by b~cte~i~l e~lGssion, using the phagemid constructs in a supO strain and in~ çing 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 e~lGssion vector which enables us tomove fr~gm~rlt.c encoding residues 4-48 of uPAI 48 in a single step from the ph~gemitl vectors. This was accomplished as follows: Plasmid pAGo~G,

4) 94/28145 ~ ~ ~2 8 ~ 5 PCT/US94/05669 i(ientic~l to pCBR except for a small deletion of an Xba fr~gmçnt in the ADH2-GAPDH promoter, was digested with Sacl, which cleaves once within the promoter, and then treated with Mung Bean nllc~ e which destroys the site.
SubsequP.nt religation yielded plasmid pAGc~G-Sac. Digestion with BglII and

5 treatment with Alk~lin~ phosphatase yielded a vector into which was ligated the BgllI fragment col.e~yonding to the synthetic gene for uPAI 48. Transformation of E. coli strain HB101 to ampicillin resi~tAnce and restriction analysis yielded the correct clone. The 2.4 kB BamHI fr~mlont from this plasmid (pAG~G-Sacl-48synth), cc ..~ g the e,~y-Gssion cA~ette, was isolated and 10 ligated into pAB24, which had been treated with BamHI and AlkAlin~.
phosphatase. The res~ltin~ plasmid has unique Sacl and Xhol sites which can be used for l.dn~r~r of the phagemid 1-48 genes. This is accomplished by digesting the phagemid with RAm~, ll~ating with Mung Bean Nuclease, digesting with Sacl and isolating the 145 bp frAgmPnt The vector is ~iigested with Xhol, treated 15 with Mung Bean Nuclease, digested with Sacl,and treated with Alk~line phosphatase. Ligation then yields the correct recombinants in a single step in the yeast e~le~ion vector. T.ar.~l.,lalion of yeast strain ABl 10 then yields high levels of secreted 1-48 variants for analysis.
Using this construct, one can express a library of uPA variations for 20 sc~nil~g. Variations may be constructed by a variety of meth-)~.c, inc]u~ling low-fidelity PCR (which introduces a large number of random point mut~tioll~), site-specific mutation, primer-based mutagenesis, and ligation of the uPAI 48 se~uellce (or portions thereof) to a random oligonucleotide se~u~nce (e.g., by ~tt~ hing (NNS)X to the uPAI 48 coding sequçnce, or sub~ ;..g NNS for one or more 25 uPAI 48 codons). Gen~r~tinn of random oligonucleotide sequences is detailed in Devlin, WO91/18980, inco"~oldled herein by r~fe,~;nce. Phage displaying uPAl 48 variants (having one or more amino acid substitutions) are screened accordingto the protocol ~escrihed above (using, e.g., r~M~ as a positive control) and selected for improved binding.

Example 5 (l~ormulation of huPAl~8) huPAl 48 formulations suitable for use in chemotherapy are prepared as follows:
5 A) Injectable Formulation:
huPAI ~18 7.0 mg Na2HPO4 (0.5 M) 0.5 mL
m~nnitol (25%) 2.5 mL
sodium laureate (1 %) 2.5 mL
pH 7.5 PBS qs 20 mL

This formulation is ~c~d~d following the procedure set forth in US
4,816,440, inco.yulalGd herein by l~,f~ nce. The formulation is ~dmini~ered by 5 ~arGI~t~,~al injection at the site to be trGatGd. The formulation is also generally suitable for ~ dlion as ~11U~S directly to the conjunctiva, or by intranasal~dmini~tration as an aerosol. ~Ite.rn~tively, a conce~ d formulation (e.g., reducing the phosphate buffered saline to 2 mL) may be used to fill an Alzet~
Illih~ullll), and the ~ u~ ) i npld..led at the site to be treated.
2 0 B) Ophth7~lmi~ G~)aldlion huPAl ~8 1 mg fil~une~;LiJ~ 69 mg albumin 37.5 mg water qs 3.0 mL
HCl (0.01 M) ~s pH 4.0 This dosage form is plep~cd following the ~J~ucelur~ set forth in US
5,124,155, incorporated herein by ~c~cnce. The fibrûnectin and albumin are dissolved in water to form a 3.0 mL solution, and HCl added to a pH of 4.0, 3 0 causing the fibronectin to flocculate. The flocculent is filtered, and combined with the huPAl 48. The ~ lule is then placed in a contact lens mold, and the mold closed for 30 min to form a corneal "shield" in the shape of a contact lens.

1~ 94/28145 2 PCT/US94/05669 The shield releases huPAI 48 over a period of time, and is useful for preventingangiogenesis of corneal tissue following ophth~lmic surgery.

The present invention has been described with reference to specific 5 embodiments. However, this application is intended to cover those changes and substitutions which may be made by those skilled in the art without departing from the spirit and the scope of the appended claims.

WO 94/28145 . PCTAJS94/05669 ~yu~N~ LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Ro~enberg, Steven Stratton-Thomas, Jennifer R.
(ii) TITLE OF lNv~N-llON: Expression of Urokinase PlA~mlnngen Activator Inhibitors (iii) NUMBER OF S~U~N~S: 19 (iv) CORR~SPONDENCE ADDRESS:
(A) ADDRESSEE: Chiron Corporation (B) STREET: 4560 Horton Street (C) CITY: Emeryville (D) STATE: CA
(E) COUN-1KY: USA
2û (F) ZIP: 94608 (v) C~_~U1~K READABLE FORM:
(A MEDIUM TYPE: Floppy disk (B C~ ~ul~: IBM PC compatible (C OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version ~1.25 (vi) ~UKK~Nl APPLICATION DATA:
(A) APPLICATION N~MBER: US
3û (B) FILING DATE:
(C) CLASSIFICATION:
(viii) ArlvKN~Y/AGENT INFORMATION:
(A) NAME: Green, Grant D.
(B) REGISTRATION ~TMRR~: 31,259 tC) R~K~N~/DOCKET ~TMRRR: 0939.001 (ix) TRT~RCnMMTT~ICATION INFORMATION:
(A) TELEPHONE: 510-601-2706 4û (B) TELEFAX: 510-655-3542 (2) INFORNATION FOR SEQ ID NO:1:

(i) ~yU~N~ CHARACTERISTICS:
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRA-dDEDNESS: single (D) TOPOLOGY: linear 5û
(ii) MOLECULE TYPE: cDNA
(iii) ~Y~uln~lCAL: NO

(Xi ) ~yU~N~ DESCRIPTION: SEQ ID NO:1:
ATGCTAGATC TAATGAACTT CATCAGGTAC CATCG
6û 35 ~ 94/28145 2 ~ 6 2 ~ ~ 5 PCT~USg4/05669 (2) INFORMATION FOR SEQ ID NO:2:
u~N~ CHARACTERISTICS:
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO

(Xi) S~U~N~ DESCRIPTION: SEQ ID NO:2:
CGATAGATCT TTATTTTGAC TTATCTATTT CACAG

(2) INFORMATION FOR SEQ ID NO:3:
( i ) S~U~N~ CHARACTERISTICS:
(A) LENGTH: 953 base pairs (B) TYPE: nucleic acid (C) sTRpNnRnNRss: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
3û (iii) ~Y~Ol~b:llCAL: NO

(vii) IMMEDIATE SOURCE:
(B) CLONE: ~lFlag-BGF-pIII fusion (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 25..903 (Xi ) S~YU~N~ DESCRIPTION: SEQ ID NO:3:
CCATGGCTAC AGAGGAATAT TA~A ATG AAT AAG GCA APA ACT TTA CTC TTC

~et Asn Lys Ala Lys Thr Leu Leu Phe l 5 ACT GCG CTA GCT TTT GGT TTA TCT CAT CAA GCT TTA GCG GAC TAC APA

Thr Ala Leu Ala Phe Gly Leu Ser His Gln Ala Leu Ala Asp Tyr Lys GAC GAT GAC GAT AAG AAT TCT GAC AGT GAA TGC CCG CTG AGC CAC GAC

Asp Asp Asp Asp Lys Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp GGC TAC TGC CTG CAC GAC GGT GTT TGC ATG TAC ATC GAA GCT CTA GAC

Gly Tyr Cys Leu His Asp Gly Val CYB Met Tyr Ile Glu Ala Leu Asp WO 94/28145 PCTnJS94105669 AAG TAC GCA TGC AAC TGC GTT GTT GGG TAC ATC GGT GAG CGC TGC CAG

Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cy6 Gln TAC CGA GAT CTT AAG TGG TGG GAA CTC CGT GGG CCC TTC GTT TGT GAA

Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg Gly Pro Phe Val Cys Glu TAT CAA GGC CAA TCG TCT GAC CTG CCT CAA CCT CCT GTC AAT GCT GGC

Tyr Gln Gly Gln Ser Ser Asp Leu Pro Gln Pro Pro Val Asn Ala Gly GGC GGC TCT GGT GGT GGT TCT GGT GGC GGC TCT GAG GGT GGT GGC TCT

Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly Ser GAG GGT GGC GGT TCT GAG GGT GGC GGC TCT GAG GGA GGC GGT TCC GGT

Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Gly GGT GGC TCT GGT TCC GGT GAT TTT GAT TAT GAA AAG ATG GCA AAC GCT

Gly Gly Ser Gly Ser Gly Asp Phe Asp Tyr Glu Lys Met Ala Asn Ala AAT AAG GGG GCT ATG ACC GAA AAT GCC GAT GA~ AAC GCG CTA CAG TCT

Asn Lys Gly Ala Net Thr Glu Asn Ala Asp Glu Asn Ala Leu Gln Ser GAC GCT A~A GGC AAA CTT GAT TCT GTC GCT ACT GAT TAC GGT GCT GCT

Asp Ala Lys Gly Lys Leu Asp Ser Val Ala Thr Asp Tyr Gly Ala Ala ATC GAT GGT TTC ATT GGT GAC GTT TCC GGC CTT GCT AAT GGT AAT GGT

Ile Asp Gly Phe Ile Gly Asp Val Ser Gly Leu Ala Asn Gly Asn Gly GCT ACT GGT GAT TTT GCT GGC TCT AAT TCC CAA ATG GCT CAA GTC GGT

Ala Thr Gly Asp Phe Ala Gly Ser Asn Ser Gln Met Ala Gln Val Gly GAC GGT GAT AAT TCA CCT TTA ATG AAT AAT TTC CGT CAA TAT TTA CCT

Asp Gly Asp Asn Ser Pro Leu Met Asn Asn Phe Arg Gln Tyr Leu Pro TCC CTC CCT CAA TCG GTT GAA TGT CGC CCT TTT GTC TTT AGC GCT GGT

Ser Leu Pro Gln Ser Val Glu Cys Arg Pro Phe Val Phe Ser Ala Gly 94/28145 ~ 21~ PCT~US94/05669 A~A CCA TAT GAA TTT TCT ATT GAT TGT GAC A~A ATA AAC TTA TTC CGT

Lys Pro Tyr Glu Phe Ser Ile Asp Cy5 Asp Lys Ile Asn Leu Phe Arg GGT GTC TTT GCG TTT CTT TTA TAT GTT GCC ACC TTT ATG TAT GTA TTT

Gly Val Phe Ala Phe Leu Leu Tyr Val Ala Thr Phe Met Tyr Val Phe TCT ACG TTT GCT AAC ATA CTG CGT AAT AAG GAG TCT TAATCATGCG

Ser Thr Phe Ala Asn Ile Leu Arg Asn Lys Glu Ser CGCTCACTGG CC~lC~l-l-ll ACAACGTCGT GACTGGGAAA

(2) INFORMATION FOR SEQ ID NO:4:
(i) ~yU~N~ CHARACTERISTICS:
(A) LENGTH: 293 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (Xi ) ~yU~N~ DESCRIPTION: SEQ ID NO:4:
Net Asn Lys Ala Lys Thr Leu Leu Phe Thr Ala Leu Ala Phe Gly Leu Ser His Gln Ala Leu Ala Asp Tyr Lys Asp Asp Asp Asp Lys Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly 4û Val Cy8 Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg Gly Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp Leu Pro Gln Pro Pro Val Asn Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly 5 Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Asp Phe Asp Tyr Glu Lys Met Ala Asn Ala Asn Lys Gly Ala Met Thr Glu 6û
Asn Ala Asp Glu Asn Ala Leu Gln Ser Asp Ala Lys Gly Lys Leu Asp WO 94/28145 PCTrUS94/05669 ~

Ser Val Ala Thr Asp Tyr Gly Ala Ala Ile Asp Gly Phe Ile Gly Asp Val Ser Gly L~u Ala Asn Gly Asn Gly Ala Thr Gly Asp Phe Ala Gly Ser Asn Ser Gln Met Ala Gln Val Gly Asp Gly Asp Asn Ser Pro Leu Met Asn Asn Phe Arg Gln Tyr Leu Pro Ser Leu Pro Gln Ser Val Glu 5 Cy8 Arg Pro Phe Val Phe Ser Ala Gly Lys Pro Tyr Glu Phe Ser Ile Asp Cys Asp Lys Ile Asn Leu Phe Arg Gly Val Phe Ala Phe Leu Leu Tyr Val Ala Thr Phe Met Tyr Val Phe Ser Thr Phe Ala Asn Ile Leu Arg Asn Lys Glu Ser (2) INFORMATION FOR SEQ ID NO:5:
(i) S~U~N~ CHARACTERISTICS:
(A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRPNn~nN~-~S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) nY~ln~llCAL: NO

(~ii) IMMEDIATE SOURCE:
(B) CLONE: ~U~CKl (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CTCATCAAGC TTTAGCGGAC TACA~AGACG ATGACGATAA GAGCAATGAA CTTCATCAAG

(2) INFORMATION FOR SEQ ID NO:6:
(i) S~U~N~ CHARACTERISTICS:
(A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

6û (iii) nY~uln~llCAL: N-O

94/28145 21~ 5 PCTIUS94105669 (vii~ IMMEDIATE SOURCE:
(B) CLONE: EUKGPCR1 (xi) S~YU~N~ DESCRIPTION: SEQ ID NO:6:
CTCATCAAGC TTTAGCCGAA TACATGCCAA TGGA~AGCAA TGAACTTCAT CAAG

lû (2) INFORMATION FOR SEQ ID NO:7:
( i ) S~YU~N~ CHARACTERISTICS:
(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRPNn~n~S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
2 0 ( i i i ) hY~O~ CAL: NO

(vii) IMMEDIATE SOURCE:
(B) CLONE: EUKPCR2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CACCGGAACC GGATCCACCC TATTTTGACT TATC

(2) INFORMATION FOR SEQ ID NO:8:
( i ) S~YU~N~ CHARACTERISTICS:
(A) LENGTH: 52 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO

(vii) IMMEDIATE SOURCE:
(B) CLONE: SROl (Xi) S~YU~:N~ DESCRIPTION: SEQ ID NO:8:
GAPATAGATA AGTCA~AATA GG~l~TCC G~l-lCCG~lG ATTTTGATTA TG

(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear W O 94/28145 PCTrUS94/05669 ~

C~ ~ ~285~

(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO

(vii) IMMEDIATE SOURCE:
(B) CLONE: EUKCPCR1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:g:
GA~ACCATCG ATAGCAGCAC CG

(2) INFORMATION FOR SEQ ID NO:10:
(i) S~QU~N~ CHARACTERISTICS:
(A) LENGTH: 779 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO

(~ii) IMMEDIATE SOURCE:
(B) CLONE: MlFlag uPA1-48 - pIII fusion (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 25..729 (xi) S~YU~N~ DESCRIPTION: SEQ ID NO:10:
CCATGGCTAC AGAGGAATAT TAAA ATG AAT AAG GCA AAA ACT TTA CTC TTC
4û Met Asn Lys Ala Lys Thr Leu Leu Phe ACT GCG CTA GCT TTT GGT TTA TCT CAT CAA GCT TTA GCC GAC TAC AAA

Thr Ala Leu Ala Phe Gly Leu Ser His Gln Ala Leu Ala Asp Tyr Lys GAC GAT GAC GAT AAG AGC A~T GAA CTT CAT CAA GTT CCA TCG AAC TGT

Asp Asp Asp Asp Lys Ser Asn Glu Leu His Gln Val Pro Ser Asn Cys GAC TGT CTA AAT GGA GGA ACA TGT GTG TCC AAC AAG TAC TTC TCC AAC

Asp Cys Leu Asn Gly Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser Asn ATT CAC TGG TGC AAC TGC CCA A~G A~A TTC GGA GGG CAG CAC TGT GAA

Ile His Trp Cys Asn CyS Pro Lys Lys Phe Gly Gly Gln His Cys Glu 94/28145 ~ ~ ~ 2 ~ ~ PCTrUS94/05669 ATA GAT AAG TCA AAA TAG GGT GGA TCC GGT TCC GGT GAT TTT GAT TAT

Ile A6p Lys Ser Lys * Gly Gly Ser Gly Ser Gly Asp Phe A6p Tyr GAA A~G ATG GCA AAC GCT AAT AAG GGG GCT ATG ACC GAA AAT GCC GAT

Glu Lys Met Ala Asn Ala Asn Lys Gly Ala Met Thr Glu Asn Ala Asp GAA AAC GCG CTA CAG TCT GAC GCT A~A GGC AAA CTT GAT TCT GTC GCT

Glu Asn Ala Leu Gln Ser Asp Ala Lys Gly Lys Leu Asp Ser Val Ala ACT GAT TAC GGT GCT GCT ATC GAT GGT TTC ATT GGT GAC GTT TCC GGC

Thr Asp Tyr Gly Ala Ala Ile Asp Gly Phe Ile Gly Asp Val Ser Gly CTT GCT AAT GGT AAT GGT GCT ACT GGT GAT TTT GCT GGC TCT AAT TCC

Leu Ala Asn Gly Asn Gly Ala Thr Gly Asp Phe Ala Gly Ser Asn Ser CAA ATG GCT CAA GTC GGT GAC GGT GAT AAT TCA CCT TTA ATG AAT AAT

Gln Met Ala Gln Val Gly Asp Gly Asp Asn Ser Pro Leu Met Asn Asn TTC CGT CAA TAT TTA CCT TCC CTC CCT CAA TCG GTT GAA TGT CGC CCT

Phe Arg Gln Tyr Leu Pro Ser Leu Pro Gln Ser Val Glu Cys Arg Pro TTT GTC TTT AGC GCT GGT AAA CCA TAT GAA TTT TCT ATT GAT TGT GAC

Phe Val Phe Ser Ala Gly Lys Pro Tyr Glu Phe Ser Ile Asp Cys Asp AAA ATA AAC TTA TTC CGT GGT GTC TTT GCG TTT CTT TTA TAT GTT GCC

Ly6 Ile Asn Leu Phe Arg Gly Val Phe Ala Phe Leu Leu Tyr Val Ala ACC TTT ATG TAT GTA TTT TCT ACG TTT GCT AAC ATA CTG CGT AAT AAG

Thr Phe Met Tyr Val Phe Ser Thr Phe Ala Asn Ile Leu Arg Asn Lys GAG TCT TAATCATGCG CGCTCACTGG CC~1C~1-11L ACAACGTCGT GACTGGGA~A

Glu Ser (2) lN~O~L.TION- FOR SEQ ID NO:ll:
( i ) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 235 amino acids (B) TYPE: amino acid W O 94/28145 PCTrUS94/05669 2~ &~

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) ~yu~N~ DESCRIPTION: SEQ ID NO:11:
Met Asn LYB Ala Lys Thr Leu Leu Phe Thr Ala Leu Ala Phe Gly Leu Ser His Gln Ala Leu Ala Asp Tyr Lys Asp Asp Asp Asp Lys Ser Asn Glu Leu His Gln Val Pro Ser Asn Cys Asp Cys Leu Asn Gly Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser Asn Ile His Trp Cys Asn Cys Pro Lys Lys Phe Gly Gly Gln His Cys Glu Ile Asp Lys Ser Lys * Gly Gly Ser Gly Ser Gly Asp Phe Asp Tyr Glu Lys Met Ala Asn Ala Asn Lys Gly Ala Met Thr Glu Asn Ala Asp Glu Asn Ala Leu Gln Ser Asp . 100 105 110 Ala Lys Gly Lys Leu Asp Ser Val Ala Thr Asp Tyr Gly Ala Ala Ile 3û
Asp Gly Phe Ile Gly Asp Val Ser Gly Leu Ala Asn Gly Asn Gly Ala Thr Gly Asp Phe Ala Gly Ser Asn Ser Gln Met Ala Gln Val Gly Asp Gly Asp Asn Ser Pro Leu Met Asn Asn Phe Arg Gln Tyr Leu Pro Ser û Leu Pro Gln Ser Val Glu Cys Arg Pro Phe Val Phe Ser Ala Gly Lys Pro Tyr Glu Phe Ser Ile Asp Cys Asp Lys Ile Asn Leu Phe Arg Gly Val Phe Ala Phe Leu Leu Tyr Val Ala Thr Phe Met Tyr Val Phe Ser Thr Phe Ala Asn Ile Leu Arg Asn Lys Glu Ser (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 773 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

~ 94/28145 216 ~ 8 ~ ~ PCTrUS94/05669 (iii) HYPOTHETICAL: NO

(vii) IMMEDIATE SOURCE:
(B) CLONE: Glu-tag uPAl-48 - pIII fusion (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 25..723 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
CCATGGCTAC AGAGGAATAT TA~A ATG AAT AAG GCA AAA ACT TTA CTC TTC

Met Asn Lys Ala Lys Thr Leu Leu Phe ACT GCG CTA GCT TTT GGT TTA TCT CAT CAA GCT TTA GCC GAA TAC ATG

Thr Ala Leu Ala Phe Gly Leu Ser His Gln Ala Leu Ala Glu Tyr Met CCA ATG GAA AGC AAT GAA CTT CAT CAA GTT CCA TCG AAC TGT GAC TGT

Pro Met Glu Ser Asn Glu Leu His Gln Val Pro Ser Asn Cys Asp Cys CTA AAT GGA GGA ACA TGT GTG TCC AAC AAG TAC TTC TCC AAC ATT CAC

Leu Asn Gly Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser Asn Ile His TGG TGC AAC TGC CCA AAG A~A TTC GGA GGG CAG CAC TGT GAA ATA GAT

Trp Cys A~n Cys Pro Lys Lys Phe Gly Gly Gln His Cys Glu Ile Asp AAG TCA AAA TAG GGT GGA TCC GGT TCC GGT GAT TTT GAT TAT GAA AAG

Lys Ser Lys * Gly Gly Ser Gly Ser Gly A~p Phe Asp Tyr Glu Lys ATG GCA AAC GCT AAT AAG GGG GCT ATG ACC GAA AAT GCC GAT GAA AAC

Met Ala Asn Ala Asn Lys Gly Ala Met Thr Glu Asn Ala Asp Glu Asn GCG CTA CAG TCT GAC GCT A~A GGC A~A CTT GAT TCT GTC GCT ACT GAT

Ala Leu Gln Ser Asp Ala Lys Gly Lys Leu Asp Ser Val Ala Thr Asp TAC GGT GCT GCT ATC GAT GGT TTC ATT GGT GAC GTT TCC GGC CTT GCT

Tyr Gly Ala Ala Ile Asp Gly Phe Ile Gly Asp Val Ser Gly Leu Ala AAT GGT AAT GGT GCT ACT GGT GAT TTT GCT GGC TCT AAT TCC CAA ATG

Asn Gly Asn Gly Ala Thr Gly Asp Phe Ala Gly Ser Asn Ser Gln Met W 0 94/28145 PCTrUS94/05669 GCT CAA GTC GGT GAC GGT GAT AAT TCA CCT TTA ATG AAT AAT TTC CGT

Ala Gln Val Gly Asp Gly Asp Asn Ser Pro Leu Met Asn Asn Phe Arg CAA TAT TTA CCT TCC CTC CCT CAA TCG GTT GAA TGT CGC CCT TTT GTC

Gln Tyr Leu Pro Ser Leu Pro Gln Ser Val Glu Cys Arg Pro Phe Val TTT AGC GCT GGT A~A CCA TAT GAA TTT TCT ATT GAT TGT GAC A~A ATA

Phe Ser Ala Gly Lys Pro Tyr Glu Phe Ser Ile Agp CYB A6p Lys Ile AAC TTA TTC CGT GGT GTC TTT GCG TTT CTT TTA TAT GTT GCC ACC TTT

Asn Leu Phe Arg Gly Val Phe Ala Phe Leu Leu Tyr Val Ala Thr Phe ATG TAT GTA TTT TCT ACG TTT GCT AAC ATA CTG CGT AAT AAG GAG TCT

Met Tyr Val Phe Ser Thr Phe Ala Asn Ile Leu Arg Asn Lys Glu Ser TAATCATGCG CGCTCACTGG CC~lC~ l ACAACGTCGT GACTGGGAAA

(2) LN~O~L!TION FOR SEQ ID NO:13:
(i) ~U~N~ CHARACTERISTICS:
A) LENGTH: 233 amino acids B) TYPE: amino acid D) TOPOLOGY: linear (ii) MOhECULE TYPE: protein (xi) SEQ~ENCE DESCRIPTION: SEQ ID NO:13:
Met Asn Lys Ala LYB Thr Leu Leu Phe Thr Ala Leu Ala Phe Gly Leu Ser His Gln Ala Leu Ala Glu Tyr Net Pro Net Glu Ser Asn Glu Leu His Gln Val Pro Ser Asn Cys Asp Cys Leu Asn Gly Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser Asn Ile His Trp Cys Asn Cys Pro Lys Lys ~ 50 55 60 Phe Gly Gly Gln Hi6 Cy8 Glu Ile Asp Lys Ser Lys * Gly Gly Ser Gly Ser Gly Asp Phe Asp Tyr Glu Lys Met Ala Asn Ala Asn Lys Gly Ala Met Thr Glu Asn Ala Asp Glu Asn Ala Leu Gln Ser Asp Ala Lys 2 ~
~ 94/28145 PCTrUS94/05669 -Gly LYB Leu Asp Ser Val Ala Thr Asp Tyr Gly Ala Ala Ile Asp Gly Phe Ile Gly Asp Val Ser Gly Leu Ala Asn Gly Asn Gly Ala Thr Gly Asp Phe Ala Gly Ser Asn Ser Gln Net Ala Gln Val Gly Asp Gly Asp Asn Ser Pro Leu Met Asn Agn Phe Arg Gln Tyr Leu Pro Ser Leu Pro Gln Ser Val Glu Cy8 Arg Pro Phe Val Phe Ser Ala Gly Lys Pro Tyr Glu Phe Ser Ile Asp CYB Asp Lys Ile Asn Leu Phe Arg Gly Val Phe Ala Phe Leu Leu Tyr Val Ala Thr Phe Met Tyr Val Phe Ser Thr Phe Ala Asn Ile Leu Arg Asn Lys Glu Ser (2) lN~O~TION FOR SEQ ID NO:14:
(i) S~U~N~ CHARACTERISTICS:
(A) LENGTH: 773 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) ~Y~Ol~b:llCAL: NO

(vii) IMMEDIATE SOURCE:
(B) CLONE: Glu-tag uPAl-48 synth. - pIII map (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 25... 723 (xi) ~U~N~ DESCRIPTION: SEQ ID NO:14:
CCATGGCTAC AGAGGAATAT TA~A ATG AAT AAG GCA A~A ACT TTA CTC TTC

Met Asn LYB Ala Lys Thr Leu Leu Phe ACT GCG CTA GCT TTT GGT TTA TCT CAT CA~ GCT TTA GCG GAA TAC ATG

Thr Ala Leu Ala Phe Gly Leu Ser His Gln Ala Leu Ala Glu Tyr Met ~û CCA ATG GAA AGC AAT GAG CTC CAT CAA GTA CCA TCG AAC TGT GAC TGT

W O 94/28145 PCTrUS94/05669 Pro Met Glu Ser Asn Glu Leu His Gln Val Pro Ser Asn Cy6 Asp Cy5 CTA AAT GGA GGT ACC TGT GTG TCC AAC AAG TAC TTT TCG AAC ATT CAC

Leu Asn Gly Gly Thr Cy8 Val Ser Asn Lys Tyr Phe Ser Asn Ile His TGG TGC AAT TGC CCA AAG AAA TTC GGA GGG CAG CAC TGT GAA ATC GAT

Trp Cys Asn Cys Pro Lys Lys Phe Gly Gly Gln His Cys Glu Ile Asp AAG TCA ~AA TAG GGT GGA TCC GGT TCC GGT GAT TTT GAT TAT GAA AAG

Lys Ser Lys * Gly Gly Ser Gly Ser Gly ABP Phe Asp Tyr Glu Lys ATG GCA AAC GCT AAT AAG GGG GCT ATG ACC GAA AAT GCC GAT GAA AAC

Met Ala Asn Ala Asn LYB Gly Ala Net Thr Glu A6n Ala Asp Glu Asn GCG CTA CAG TCT GAC GCT AAA GGC AAA CTT GAT TCT GTC GCT ACT GAT

Ala Leu Gln Ser Asp Ala Lys Gly Lys Leu A6p Ser Val Ala Thr Asp TAC GGT GCT GCT ATC GAT GGT TTC ATT GGT GAC GTT TCC GGC CTT GCT

Tyr Gly Ala Ala Ile Asp Gly Phe Ile Gly Asp Val Ser Gly Leu Ala 125 130 . 135 AAT GGT AAT GGT GCT ACT GGT GAT TTT GCT GGC TCT AAT TCC CAA ATG

Asn Gly Asn Gly Ala Thr Gly A~p Phe Ala Gly Ser Asn Ser Gln Met GCT CAA GTC GGT GAC GGT GAT AAT TCA CCT TTA ATG AAT AAT TTC CGT

Ala Gln Val Gly Asp Gly Asp Asn Ser Pro Leu Met Asn Asn Phe Arg CAA TAT TTA CCT TCC CTC CCT CAA TCG GTT GAA TGT CGC CCT TTT GTC

Gln Tyr Leu Pro Ser Leu Pro Gln Ser Val Glu Cys Arg Pro Phe Val TTT AGC GCT GGT A~A CCA TAT GAA TTT TCT ATT GAT TGT GAC AAA ATA

Phe Ser Ala Gly Lys Pro Tyr Glu Phe Ser Ile Asp Cys Asp Lys Ile AAC TTA TTC CGT GGT GTC TTT GCG TTT CTT TTA TAT GTT GCC ACC TTT

A6n Leu Phe Arg Gly Val Phe Ala Phe Leu Leu Tyr Val Ala Thr Phe ATG TAT GTA TTT TCT ACG TTT GCT AAC ATA CTG CGT AAT AAG GAG TCT

Met Tyr Val Phe Ser Thr Phe Ala Asn Ile Leu Arg Asn Lys Glu Ser ~ 94/28145 . 2 ~ ~ 2 ~ ~ ~ PCT~US94/05669 TAATCATGCG CGCTCACTGG CC~lC~l~ l ACAACGTCGT GACTGGGAAA

(2) lN~O~L!TION FOR SEQ ID NO:15:
(i) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 233 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Net Asn Lys Ala Lys Thr Leu Leu Phe Thr Ala Leu Ala Phe Gly Leu 2û
Ser His Gln Ala Leu Ala Glu Tyr Met Pro Met Glu Ser Asn Glu Leu His Gln Val Pro Ser Asn Cys Asp Cys Leu Asn Gly Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser Asn Ile His Trp Cys Asn Cys Pro Lys Lys Phe Gly Gly Gln His Cys Glu Ile Asp Lys Ser Lys * Gly Gly Ser Gly Ser Gly Asp Phe Asp Tyr Glu Lys Met Ala Asn Ala Asn Lys Gly Ala Met Thr Glu Asn Ala Asp Glu Asn Ala Leu Gln Ser Asp Ala Lys Gly Lys Leu Asp Ser Val Ala Thr Asp Tyr Gly Ala Ala Ile Asp Gly Phe Ile Gly Asp Val Ser Gly Leu Ala Asn Gly Asn Gly Ala Thr Gly Asp Phe Ala Gly Ser Asn Ser Gln Met Ala Gln Val Gly Asp Gly Asp Asn Ser Pro Leu Met Asn Asn Phe Arg Gln Tyr Leu Pro Ser Leu Pro Gln Ser Val Glu Cys Arg Pro Phe Val Phe Ser Ala Gly Lys Pro Tyr Glu Phe Ser Ile Asp Cys Asp Lys Ile Asn Leu Phe Arg Gly Val Phe Ala Phe Leu Leu Tyr Val Ala Thr Phe Met Tyr Val Phe Ser Thr Phe 6û Ala Asn Ile Leu Arg Asn Lys Glu Ser W O 94/28145 PCTrUS94/05669 ~ 5 - 38 -(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
A) LENGTH: 39 base pairs B) TYPE: nucleic acid C) STRANDEDNESS: single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) ~Y~O~ CAL: NO

(vii) IMMEDIATE SOURCE:
(B) CLONE: SRO35 (xi) ~yu~N~ DESCRIPTION: SEQ ID NO:16:
AGCTTTAGCG GAATACATGC CAATGGAAAG CAATGAGCT

(2) lN~O~L!TION FOR SEQ ID NO:17:
(i) ~yu~N~ CHARACTERISTICS:
'A) LENGTH: 31 base pairs B) TYPE: nucleic acid C) STRANDEDNESS: single ,D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) ~Y~ul~llCAL: NO

(vii) IMMEDIATE SOURCE:
(B) CLONE: SRO36 (xi) ~yu~N~ DESCRIPTION: SEQ ID NO:17:
CATTGCTTTC CATTGGCATG TAl-lCCG~lA A

(2) lN~L.TION FOR SEQ ID NO:18:
(i) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: ~ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO

(vii) IMM~DIATE SOURCE:
(B) CLONE: SRO37 ~p 94/28145 216 2 8 ~ 5 PCT/US94/05669 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
CGATAAGTCA A~ATAGGGTG

(2) INFORMATION FOR SEQ ID NO:19:
(i) S~yUhN~ CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) sTR~NnRnN~s ~ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
( i i i ) ~lY ~Ul'~l~; l lcAL: NO

(vii) IMMEDIATE SOURCE:
2û (B) CLONE: SRO38 (Xi ) S~yu~NC~: DESCRIPTION: SEQ ID NO:19:
GATCCACCCT ATTTTGACTT AT

Claims (20)

WHAT IS CLAIMED:

1. A method for producing a non-fucosylated polypeptide con-sisting essentially of the EGF-like domain of human urokinase-type plasminogen activator, said method comprising:
providing a yeast host transformed with an expression vector, said vector comprising a transcriptional promoter operably linked to an oligonucleotide encoding a huPAR antagonist polypeptide consisting essentially of the EGF-like domain of human urokinase-type plasminogen activator or an active analog thereof;
culturing said yeast host under conditions which promote expression of said polypeptide; and isolating said polypeptide.

2. The method of claim 1, wherein said oligonucleotide further encodes a signal leader polypeptide operatively connected to said huPAR
antagonist polypeptide or analog, operative in said host cell to direct secretion of the expressed polypeptide.

3. The method of claim 2, wherein said signal leader comprises yeast .alpha.-factor leader.

4. The method of claim 3 wherein said yeast .alpha.-factor leader is S. cerevisiae .alpha.-factor leader.

5. The method of claim 1, wherein said host cell is Saccharomyces cerevisiae.

6. The method of claim 1, wherein said huPAR antagonist polypeptide consists essentially of huPA1-48.

7. A huPAR antagonist polypeptide composition comprising a non-fucosylated polypeptide consisting essentially of the EGF-like domain of human urokinase-type plasminogen activator or an active analog thereof.

8. The composition of claim 7, wherein said non-fucosylated polypeptide consists essentially of huPA1-48.

9. The composition of claim 7, further comprising a pharma-ceutically acceptable excipient.

10. A method for treating a uPA-mediated disorder, said method comprising:
providing a composition comprising a non-fucosylated polypeptide con-sisting essentially of the EGF-like domain of human urokinase-type plasminogen activator or an active analog thereof; and administering an effective amount of said composition to a patient having a uPA-mediated disorder.

11. The method of claim 10, wherein said polypeptide consists essentially of huPA1-18.

12. The method of claim 10, wherein said uPA-mediated disorder is selected from the group consisting of metastasis, inappropriate angiogenesis, and chronic inflammation.

13. The method of claim 12, wherein said uPA-mediated disorder is selected from the group consisting of Kaposi's sarcoma, diabetic retinopathy, and rheumatoid arthritis.

14. The method of claim 10, wherein said composition is administered by instillation in the eye.

15. A method for pre-enriching a monovalent phage display mixture prior to screening for binding to a target, comprising:
(a) providing a mixture of monovalent display phage and non-displaying phage, wherein said monovalent display phage display both a candidatepeptide and a common peptide, wherein said common peptide is identical for each monovalent display phage, and wherein said candidate peptide is different for different monovalent display phage; and (b) separating all phage displaying a common peptide from phage not displaying a common peptide.

16. The method of claim 15, wherein said candidate peptide is huPA1-48 or an active analog or active portion thereof.

17. The method of claim 15, wherein said common peptide com-prises an antibody epitope.

18. The method of claim 17, wherein said epitope comprises Glu-Tyr-Met-Pro-Met-Glu.

19. The method of claim 15, further comprising:
(c) contacting said separated phage displaying said common peptide with said target; and (d) separating phage which bind said target from phage which do not bind said target.

20. The method of claim 19, wherein said target comprises huPAR.