CA2160154A1 - Methods of using insulin-like growth factor binding proteins - Google Patents

Abstract

The invention relates to methods of using insulin-like growth factor binding proteins ("IGFBPs"), including IGFBP-1 or modified forms of IGFBP-1 as therapeutic agents. Modified forms include IGFBP-1 attached to a polymer or two IGFBP-1 molecules attached to opposite ends of a polymer. The methods involve administering IGFBPs, including IGFBP-1 or a modified form of IGFBP-1 to a patient having an IGF associated condition sufficient to cause a therapeutic effect. The invention also relates to non-phosphorylated IGFBPs useful in the methods.

Description

WO 9~/22466 21~ J ~ PCT/US94/03755 METHODS OF USING INSULIN-LIKE GROWTH FACTOR
BINDING PROTEINS

FIELD OF THE INVENTION
This invention relates to uses of insulin-like growth factor binding protein 1 (IGFBP-l or BP-l) as a th~ eul;c agent.
BACKGROUND OF T~; INVENTION
Circul~ting insulin-like growth factors I and II.(IGF-I and IGF-II) are 7 kDa proteins that are related in structure to each other and to insulin. IGF-I and IGF-II
are growth and dirr~nl;~l;o~- factors for most cells in the body and are present at high c~ncçnl~t;nn~ in serum (about 300 ng/ml for IGF-I and 1000 ng/ml for IGF-II).
Circ~ ting levels of IGF-I are de~-,-incd prim~n1y by growth horrnone (GH), which stiml~ls~s the liver to make IGF-I. Most of the growth-pr~".o~ g effects of growth hGl."one are believed to be .~ d by IGF-I.
Tissue IGF's also exist. Tissue IGF-I has a larger a~palcnt molec~ r mass (applc ~ tely 26 kDa), as d~ f~ by gel c}lu...~ .g..~[-hy, than circul~ting IGF.Rom, W.N. et al., J. Clin. Invest., 82: 1685-1693 (1988).
IGF-I and IGF-II have been shown to play a role in a large ~u-nbel of disease c4nA;I;onc These in-,lude, for example, breast cancer, colon cancer, lung cancer, ovarian cancer, GSt~s~ -. a, ~liom~, liver cancer, pr~st~te cancer, rhabdomyosar~...as, r~tc~-os;~ acr~...egaly, obesity, tumor-in~luce~ hypoglycemia, pulmon~ry fibrosis, ~i~be-ti~ n~hr~pat}ly and diabetic retinopathy.
The roles of insulin-like growth factors in human tumors are ~i~cussed in D~ugh~y, Endocrinol. 127:1~ (1990). For eY~mple, IGF-I and IGF-II are believed 5 ~

to function as auloc~ e or paracrine growth factors for a variety of human cancers, including breast cancers, colon cancers, lung cancers, ovarian cancers, osteosar~",as, neuroblastomas, gliomas, Wilm's tumors, and rhabdomyosarcomas as reported in Cullen et al., Cancer Inve~ti~tion, 9:443-454 (1991). A wide variety s of primary tumors have been shown to ove,eApl~ss IGF-I or IGF-II and to express receptors for these growth factors, as reported in Yee et al, Cancer Res. 48:6691-6696 (1988) and Osborne et al, Mol. Endocrinol. 3:1701-1709 (1989). Numerous m yitro studies have shown that human cell lines derived from some of the above cancers proliferate in re~ron~e to IGF-I and IGF-II. In some cases the cell lines have been shown to produce IGF-I or IGF-II and to possess cell surface receplol ~ for IGF-I
and IGF-II. In some in~ n ~;S, particularly in breast cancer, stromal cells ;..lllou~ ng the tumor have been shown to secrete IGF-I and IGF-II, res~-lting in a p~ e growth re1~ti- nchir.
Breast, lung and colon cancers are the three most collllllon cancers in the U.S., aK~cling nearly 500,000 people. The strongest evidence for a role of IGF-Iand IGF-II in growth of these cancers comes from e l~ ;...Pnt~ showing that ~-ltil~o 1iP~ to the IGF-I r~p~or block tumor formation in nude mice by a number of cell lines derived from human tumors of these types.
Analyses of biops;cs from human colon cancers showed o~ e~ression of IGF-II 1~ to 50-fold in 40% of the tumors analyzed, as l~ol~d in Tricoli et al.,Cancer ResP~arch. 46:6169-6173 (1986). The level of IGF-II ov~r~ssion cGllelated with the degree of invasion of the bowel wall.

WO 94/22466 21~ 1 PCT/US94/03755 IGF-I can mPAi~tP auloc,ine proliferation of human small cell lung cancer cell lines NCI-H345 and NCI-N417, as re~lled in N~k~ni~hi, et al., J. Clin. Invest., 82:354-359 (1988).
Ovarian cancer cell lines OVCAR-3, OVCAR-7 and PE04 express IGF-I
s mRNA. Primary and mPt~ct~tic ovarian cancer tissues also express IGF-I mRNA as well as Type I IGF receplor mRNA, as reported in Yee, et al, Cancer Res.. 51:5107-5112 (1991).
Both normal and neoplastic bone cells secrete and respond to IGF-I, as lepo,~d in Blatt, et al Biochem. Biophys. Res. Commun. 123:373-376 (1984) and C~n~ . J. Clin. Invest. 66:709-719 (1980).
IGF-II mRNA eApless;on is dcv~ nlally regulated in liver tissue.
Inc,eased IGF-II mRNA levels have been ~e~led in liver cancers of woodchucks, l----.-anc and rats, as n,po ~d in C~ri~ni, et al., J. Hepatolo~y, 13:220-226 (1990).
Three human p~st~lic cancer cell lines, PC-3, DU-145, and LNCa.FGC, produce ;~-bs~nl;~l ~"ounls of IGF-I and display cofic~;lu~ ely auto-phosphorylated IGF-I ~p~,~. Pi~hLh)..~ki, Z. et al., Cancer Research, 53: 1102-1106 (1993).
PiehLl~y et al. report that growth of all three of these cell lines was inhibited by an ~nti~Pn~ oligodeoAy,..,cl~tide to IGF-I ,~eplor RNA or by peptide analogs of - IFG-I that co---~te with IGF-I binding to its leceptor.

2 o Rhabdol,lyo~l-la is the most col.. on soft tissue sarcoma of childhood and ap~ to arise from developing striated Illuscle-forming cells. Elevated levels of IGF-II mRNA and have been r~pol~d in rhabdo"lyos~ll,a tumors as described in - El-Badry, et al., Cell Growth & Dirre.enlialion, 1:325-331 (1990).

21601~4 WO 9~/22466 PCT/US94/03755 AS inriir~ted above, the insulin-like growth factors have been associated with certain non-cancerous disorders such as acromegaly and restenosis, for example.
Acromegaly results from an excess production of growth hormone (GH). Growth hormone acts by stimul~tin~ production of IGF-I. Thus, acromegaly has been ~cccci~tP~ with inc~ased levels of IGF-I.
Restenosi~, reocclusion of the artery, occurs within 6 months in approximately 25-55 % of p~tientC who undergo angioplasty surgery. Thi~l~ening of the intimal layer of the artery is the primary cause of restenosi~. Intimal thi~ ning occurs as a result of smooth muscle cell ~li~dtion and secretion of extr~rç~ r matrix co,l,ponents.
It has been shown that IGF-I gene cA~ ,i.i,ion is induc~d 9-fold in the denuded artery following angioplasty surgery, as .,_~olled in Cercek et al Circulation Research, 66: 1755-1760 (1990). The timing and level of IGF-I gene e,~lession closely parallels that of smooth muscle cell prr~lifP,~tir~n. Hybri~li7~tiQn studies inrlir~te that the dividing smooth muscle cells are the cells that exhibit increased IGF-I gene l~l, ess;on, as lepolted in Khorsandi et al, J. Clin. Invest.. 90:1926-1931 (1992).
Other studies have shown that ~nim~lc with low circ~ tin~ IGF-I levels (due to removal of their p;~ ;fs) have greatly reduced intimal thirl~Pning following ~n~ pl~c~y surgery, as le~ d in Khorsandi et al, Atherosclerosis. 93:115-122 - (1992).
2 o Hypogylcemia ~C~i~d with certain tumors has long been known. Unusually high levels of IGF-II mRNA and of IGF-II i ~ no~ ~active peptide were observed in a leio",yos~l"a removed from a patient with r~;ul.c;,lt hypoglycemia. D~lgh~y, W.H., et al., New F.t~g]~nd Journal of ~PAiCine~ Vol. 319, No. 22: 1434-1440 (1988). After the leiomyo~co",a was removed, the hypoglycemia subsided. Id.

WO 94t22466 216 ~)13 a~ PCT/US94/03755 Others studying tumor-induc~d hypoglycemia report finding elevated plasma IGF-IIlevels before the tumors were treated and prompt reduction of IGF-II levels and resolution of the hypoglycemia after the tumors were treated. Axelrod, L. and Ron, D., New F.ngl~nd Journal of ~edir-ine~ Vol. 319, No. 22: 1477-1479 (1988).
In vivo ~-lmini~tration of IGF-l can also induce hypogylcemia. Lewitt, M. S., et al., Endocrinology, Vol. 129, No. 4: 2254-2256 (1991). Lewitt at al. also report that in vitro studies have shown IGFBP-l inhibition of glucose incorporation into fatty acids of rat adipose tissue.
In plll".~n~.y fibrosis, there is an increased number of activated alveolar macrophages and an eY~ge dted ~cum~ tion of fibroblasts in the alveolar walls.
The fibroblasts secrete an e~t~cPll~ r c~ll~ç-nous matrix. The fibroblasts and the matrix s~lions cause the alveolar walls to thicken and cause a loss of alveolar-capillary units. Activated alveolar maelophagcs have been shown to release an IGF-l that signals ~epljr~tion of fibroblasts. Rom, W.N., et al., J. Clinic. Invest., 82:
1685-1693 (1988). Alveolar ~-,acro~hages from ~t;~ with in~r~ilial lung disorders have been shown to s~ -c~u~ secrete this IGF-I. Id.; I3;t~....an, P.B.et al., J. Clin. Invest., 72: 1801-1813 (1983). Current ll~t.". .~t~ for the Cal~Ce,f~US and non ca~c~.~ ùs disorders include surgery, ~ tion~ chc.--olherapy and hormone therapy. For ~ r le, various cancers, such as breast, lung, ovarian, colon and o~t~s~col--as are treated with Su~ , r?'li~tinn and Cl e~ !h~ euLic agents.
Che~ ~ul;r, agents used for treating these cancers include nuo~p~ in~s and alkylating agents. Both of these groups eYhibit ~ignifir~nt t~Y-irh;es, including, for eY~mple, myelosuy~ ;on, i~-----u- osul)pr~ssion, neuL.opellia, gas~oinle~li.lal toYicity, renal toYicity and perirheral neulop~ll.;es ~ddition~lly, there is no known WO 94/22466 ~ PCT/US94/03755 2 1 6 ~
effective chemothela~cuLic agent for tf~t~ t of liver cancer. Surgery is highly invasive and unpredictable, while radiation is non-specific at the point of loc~li7~tion.
Hormone therapy has undesirable side effects such as unwanted hair growth and mood changes.
Antibodies to the type I IGF~ ceptor have been shown to block growth of IGF-I-responsive carcinoma cell lines in vitro. Studies have shown that antibodies to the IGF-I ~ ol block growth of certain breast and lung carcinomas transplanted into immunodçfi~ient nude mice as reported in Arteaga et al., J. Clin. Invest., 84:1418-1423, (1989); and Zia et al., Proc. Amer. Assoc. for Cancer Research~
33:270, Abstract 1616 (1992).
~nti~n~, se~ue:nces to the IGF-I gene have been shown to block growth of a m~lign~nt rat glioma cell line t.i1..~1~n!~ into rats as described in Trojan et al., ~uc~lin~ of the NaLonal Academy of Science, 89:4874-4878 (1992). However, using ~nti~nce ~u nc~s for gene lL~"a~ is still in the early development stage.
Thus, a need exists for an agent which would inhibit the action of IGFs in the above cancers and disease cond;~ n~ The present invention provides such an IGF
inhi~.;~.)~ agent, namely IGFBP-1 for the inhibition of the inappr~pliate action of IGFs in these disease con~ u~
- SOMMARY OF T~IE INVI~TION
2 0 This invention relates to .. ~1l.~S of using insulin-like growth factor binding proteins (IGFBPs) as a ~he ~l~u~ ;c agent to treat or p~ent IGF-~.sori~t~d conditions.
In particular embo~1i...- -t~ of the invention, the binding protein is IGFBP-1, also tenned "BP-1. n This invention also relates to m~tho~s of using a m~ifiP~ forms of IGFBPs as a Ih~ eu~;c agent. For e~mple~ m~ified forms of IGFBP-1 include ~O 94/22466 216 ~ ~ 3 ~1 PCT/US94/03755 IGFBP-l ~Qtt~rhP~ to a polymer or 2 or more IGFBP-l molecules ~st~hed to a polymer. The methods involve ~dminictPring IGFBPs, including for example, IGFBP-l or a modified form of IGFBP-l to a patient having an IGF associated con~litiQn sllffiriP-nt to cause a therapeutic effect. For IGFBP-l, it is contemplated - 5 that a thelapeu~ic effect can be achieved when circulating levels of IGFBP-l range from about 0.l ~g to about 300 ~g per ml in the bloodstream of the patient.
F~ . nplPs of collrlitir-~n~ wl-. le;n ~Q~mini~trQtion of IGFBPs, in particular IGFBP- l may be useful include tl~~l...ent or prevention of breast cancer, colon cancer, lung cancer, ovarian cancer, os~ ~,.-a, glinm~, liver cancer, prostate cancer, rhQh~u,.. yc,sdl,io~las, r~stenos;s, acl~,---egaly, obesity, tumor-induced hypoglycemia, pul...~ fibrosis, ~iiQhetic neph,opall,y and diabetic retinopathy. ThisirM~n also relates to l~h~ ul;rQl co...~ ;onC- conl~inil-g IGFBPs, and in particular, IGFBP-l or a ...~;I;~d form of IGFBP-l and ...~.~c of treating or preventing IGF
QC~ :~1~ ~nrl;L;nnc using the ph~-ll ~ Q1 cU~"~ nc DETAn ~) DESCRIPIION OF TIIE INVI~TION
Inappi~pliate eApl~3s;0n or utili7Qtion of IGF-I or IGF-II is a contributing factor in rnany disease con~litir~n~. It is c~n~e ..~ ed that Qdmini~tration of IGFBPs, and in particular IGFBP-1, may be a useful th~ .ll;c in disease cn n-liti~n~ which - are QC.C~ ~1 with inapplo~liate ~ ssion or utili7Qtinn of IGFs, particularly IGF-I
or IGF-II. Thus, the present invention is dil~t~d to mPtho-1s of treating a patient having an IGF ~ t~ cQn~litinrl or of preventing an IGF-~c~:~led condition by ~lminicterin~ an arnount of IGFBP, inclu~in~ IGFBP-l or a mo-lified form of IGFBP-1, suffiri~Pnt to cause a lh~ eul;G effect.

wo g~/22466 2 1 6 ~ 1~ 4 PCT/US94/03755 Terrns used throughout this spe~ifi~tion are defined as follows:
The terrn "acceptable pharmareutical carrier" refers to a physiologically-c~l"patible, aqueous or non-aqueous solvent.
The terrn "IGF-I" refers to a protein containing the sarne amino acid sequence as naturally oc iu~ g IGF-I, or a protein conl;1ining the same amino acid sequence as naturally oc-;u ~ ;i-g IGF-I with the ~lition of an N-terminal methionine (met-IGF-I), unless otherwise ~ifi~.
The term "IGF" refers to any polypeptide that binds to the IGF type I
R~ptor, inc~ ing, for eY~mple, IGF-I, IGF-II, (desl-3)IGF-I, met-IGF-I, insulin, lo and any active rl~.. ~nl~ which bind to the type I Receptor. This horrnone family is descrihe~ in Rh~ndell and ~umbel, Nature, 287:781-787 (1980).
The t~rm "IGF Zc$~:~ con~lition" refers to an eYictin~ or l,o~ntial adverse phy~;olc~l con~litinn which results from or is ~ d with an overproduction or wld~""~xlu~-1;0ll of IGF, IGF binding protein or IGF ~ceplor, inapprop~iale or inadequate binding of IGF to binding p~teins or l~eplo,~ and any disease in which IGFBP, particularly IGFBP-l, ~1mini~tration alleviates or ~lucec disease Syll~ptOIllS.
An IGF s;C~i~tPd Cnntlitinn also refers to a con-lition in which ~dmini~tr~tion of IGFBPs ;~-c~lud;ng IGFBP-l to a normal patient has a desired effect. FY~mples of IGF ~c~:~ co~lition~ include breast cancer, colon cancer, ~e~.co---a, glioma, 2 o lung cancers"habdG~I~yosa~ as~ ovarian cancer, liver cancer, ac,~,.. egaly, obesity, tumor-;~.-Juced hy~oglyce.~lia, pll1...o~-s. y fibrosis, restenosi~, diRh~eti~ nephrop~ y and ~i~hetic lclinopal}ly.
The term "patient" refers to any ~nim~l, including humans, in need of L.~l ..- .~l for an IGF ~csoci~d c;ondition.

O 94/22466 21 C ~1 S ~ PCT/US94/03755 The ter--m- "IGFBP" refers to any of the six known IGF binding proteins or to r.~..~ of these binding proteins which bind to IGF. IGF-I and IGF-II circulate in blood bound to specific binding proteins of which six are now known. The binding plolelns bind 95 % or more of the IGFs in blood. One theory is that when bound by binding proteins, IGF-I and IGF-II are prevented from interacting with certain cell surface lecc~ol~ meAi~ting their biological functions.
IGF binding protein- 1 is a 23 kDa IGF binding protein. IGFBP- 1 is ~ Aplcssed in vivo during periods of growth arrest (e.g., starvation and diabetes), suggesting that IGFBP-l acts as an IGF-I inhibitor. Oh et al., Endocrinol. 132:1337-1344, 1993, report IGF-I and IGF-II are e-c~nl;Ally equipotent in their ~ffinities for IGFBP-1.
Amniotic fluid is a natural source of IGFBP-l and cont~inc both pho~hol~lated and non-ph-~s~ol~laled forms of this binding protein. Jones, J.I. et al., Proc. Natl. Acad. Sci., 88: 7481-7485 (1991). Pl.o~hol~lated BP-l has a higher affinity for IGF-l than the non-phos~.hol~lat~ form. Jones, J.I. et al., J. Biol.
Chem., 268, 2: 1125-1131 (1993). Jones et al. pl~J~se that the phosphorylated form of BP-l is inhi~itory to cell growth, while non-pho~hol~lated BP-l is stimul~tory to cell growth. Rec~ll,binalllly produced BP-l e"~ d in b~tP-riA is non-p1to~.kol~1ated and has been shown to po~ e the effects of IGF-l. Ladin, D. et 2o al., J. C~lhll~r Bioch~!,.;sl,~, Supplement 17E: 127 (1993).
Dataprovided herein, however, de~ .cl.~1cs thatnon-phos~horylated IGFBP-1 can also be inhil)ilul~ to cell growth in vitro and in vivo. Spe~ifi~lly, it has been found that b~t -;Ally-derived feco,llbin~l BP-I inhibits the ~~m~ing cell growthwhich attends certain IGF-~ssoc~ d con-litions.

wO 94/22466 2 ~ PCT/US94/03755 Accordingly, the IGFBP-1 useful in the methods of the present invention can be pho~ho.ylated or non-phosphorylated. Thus, the BP-l useful in the present invention can be purified from natural sources such as amniotic fluid, or can bepluduced in accor~ce with recombinant procedures well known in the art. The amino amino acid sequence of mature IGFBP-1 is:
Ala-Pro-Tr~Gln-Cys-Ala-Pro-Cys-Ser-Ala-Glu-Lys-Leu-Ala-Leu-Cys-Pro-Pro-Val-Ser-Ala-Ser-Cys-Ser-Glu-Val-Thr-Arg-Ser-Ala-Gly-Cys-Gly-Cys-Cys-Pro-Met-Cys-Ala-Leu-Pro-Leu-Gly-Ala-Ala-Cys-Gly-Val-Ala-Thr-Ala-Arg-Cys-Ala-Arg-Gly-Leu-Ser-Cys-Arg-Ala-Leu-Pro-Gly-Glu-Gln-Gln-Pro-Leu-His-Ala-Leu-Thr-Arg-Gly-Gln-1 o Gly-Ala-Cys-Val-Gln-Glu-Ser-Asp-Ala-Ser-Ala-Pro-His-Ala-Ala-Glu-Ala-Gly-Ser-Pro-Glu-Ser-Pro-Glu-Ser-Thr-Glu-Ile-Thr-Glu-Glu-Glu-Leu-Leu-Asp-Asn-Phe-His-Leu-Met-Ala-Pro-Ser-Glu-Glu-Asp-His-Ser-Ile-Leu-Tr~Asp-Ala-Ile-Ser-Thr-Tyr-Asp-Gly-Ser-Lys-Ala-Leu-His-Val-Thr-Asn-Ile-Lys-Lys-Trp-Lys-Glu-Pro-Cys-Arg-Ile-Glu-Leu-Tyr-Arg-Val -Val-Glu-Ser-Leu-Ala-Lys-Ala-Gln-Glu-Thr-Ser-Gly-Glu-Glu-Ile-Ser-Lys-Phe-Tyr-Leu-Pro-Asn-Cys-Asn-Ly s-Asn-Gly-Phe-Tyr-His-Ser-Arg-Gln-Cys-Glu-Thr-Ser-Met-Asp-Gly-Glu-Ala-Gly-Leu-cys-Tr~Cys-Val-Tyr-Phe-Trp-Asn-Gly-Lys-Arg-Ile-Pro-Gly-Ser-Pro-Glu-Ile-Arg-Gly-Asp-Pro-Asn-Cys-Gln-Ile-Tyr-Phe-Asn-Val-Gln-Asn (SEQ ID NO.:l).
- The amino acid se~uence of the signal s~uence is: Ser-Glu-Val-Pro-Val-Ala-2 o Arg-Val-T~Leu-Val-Leu-Leu-Leu-Leu-Thr-Val-Gln-Val-Gly-Val-Thr-Ala-Gly (SEQ
ID No.:2).
Using SEQ ID NO.:1, one skilled in the art can chPmir-Ally synth~o~i7P a DNA
enr~;ng IGFBP-1. ~llJ ..A~;~ely, one slcilled in the art can design oligonucleotide probes based upon SEQ ID NO. :1 to isolate a genomic DNA or mRNA and generate ~o 9~/22466 2 ~ 6 ~ PCT/US94/03755 a cDNA. The DNA enco ling the IGBBP-1 can be used to transform a host for recombinant production.
For example, BP-l can be t;A~ ;.sed in E. coli BL21/DE3 using the 17 ~lu"lo~l system as an insoluble protein in inclusion bodies. BL21/DE3 is described by Studier, F.W., and Moffatt, B., J. Mol. Biol, 189: 113-30 (1986). Alternatively, a TAC promoter system can be employed. In E. Coli, the recombinantly e~ essed BP- l is co~ ~; ned in the isoluble f~~tioll . The insoluble protein is iu-l)l~el ly folded and inactive. BP-l can be denalured and folded into its proper conformation by dissolving the protein in 6M gu-q-ni~ine and a re~uring agent, ~ilu*ng the ~ix~ure 10-fold and allowing the BP-l to refold overnight. IGFBP-l cont-q-ins 18 cysteine reo;~iues, all of which are believed to participate in forming diclllfide bridges.
Despite the large n.-...bey of c~ ine residues in BP-1, the protein refolds to a single major s~s. The ~folded protein can be purified using s4u.,nlial Q s~harose and butyl s.,~h~u~ c~ mnc The yield of pu-ifi~d BP-1 per 10L fermenter run is about l.5g.
IGFBP-1 can also be t;..l"~ssed in ,..~-..n.q1iqn e,-~l ssion systems as set forth in Jones, J.I. et al., Proc. Natl. Acad. Sci., 88: 7481-7485 (1991) and in Jones, J.I.
etal, J. Biol. Chem., 268, 2: 1125-1131 (1993), both spe~ifirqlly incol~lulated herein by r~f~lce. For ~ ss;on in ,..~....nqliqn systems, a DNA e~ g both the 2 o mature protein and the signal se~uc~ce should be employed. One skilled in the art can choose any ~ ~"ate vector and ~_A~ssion system as desired.
The tll~lap~uliC utility of IGFBPs, inc~ ling IGFBP-1, may be enh-qnce~l by increasing their circul-qting half-lives. In.;,eas,ng the mol~cl)lqr weight of a protein, for e~mrl~ by covalently bonding an inert polymer chain such as polyethylene glycol 21601~ 4 WO 9~122466 PCT/US94/03755 (PEG) to the protein, is known to increase the circulating half-life of the protein in the body. See, for example, Davis et al, Biomedical Polymers: Polymeric Materials and Pharm~ceuticals for Biomedical Use, p. 441-451 (1980). Covalent ~st~hment of PEG to protein is termed herein "PEGylation." The term "PEGylated" means conjugated to polymer.
One useful method of PEGylation involves creation of a mutein having a cysteine residue available for ~tt~hment to a polymer activated with a thiol-specific reactive group. Mutein~ can be prepared using mutagenesis techniques well known in the art. For eY~mp'e, the IGFBP-1 mutein is created by repl~ing one or more s~-ific amino acids with c~,ne reci~ues, or by ~sc.ling a cysteine residue between amino acids or at the N or C te Illinus. It is eApected that such non-native cysteine residues will be "free," i.e. not involved in intr~mol~cul~r di~l~lfi~e bonds. Non-native cysteine l~,;dues can be s.~bs1;luled or L~ d in regions of the IGFBP-1 mQl~l-le that are e.~posed on the protein's surface, and which are not involved in r~cel)lor bin~ing or binding to IGF. One site for insertion or substitutiol- of the e may occur in the middle of the BP-1 protein. It is believed that cysteine can be s~slilul~d or ins~ d from amino acid 60 to 180, with the residue numbering based upon SEQ ID NO.: 1. Particularly useful muteins include the substitution of a cysteine residue at positions 98 and 101 for the naturally oc.;u. . ;ng serine found at 2 o those loc~tirn~.
All~r~ t of the inert pol~m~r chain mol~ule to one or more IGFBP-1 mol~llles creates a fu~her mo lified form of IGFBP-1, an IGFBP-1-polymer conjugate also called "PEGylated IGFBP-ln. Coupling of thiol-specific reactive groups to polymers is ~ cu~d PCT Applir~tinn Publiratinn No. WO92/16221, WO 94/22466 21 6 ~ PCT/US94/03755 incolpoldted herein by lefc~ ce. If a cysteine mutein is coupled to the polymer via a thiol-specific reactive group, the conjugate formed is expected to be attached to the protein at the non-native cysteine residue. During refolding of the mutein, however, the non-native cysteine might become involved in a diculfi~e bond and thereby free - 5 a native cysteine for PEGylation. In such cases, the polymer is ~tt~r-hed at that native cysteine residue. Using peptide mapping, one can determine the specific PEGylation site.
Also col-t~- "pl~t~P~ are "dumbbell" molecules which contain 2 IGFBP-l mol~cules, one at each ends of the polymer molecule.
One skilled in the art using convention~l mPtho lc and following the te~chingc of PCT App1ir~tion Publir~tion No. WO92/16221, incol~ldted herein by reference, and the tP~~-hing~ provided herein, can readily de~.llline the a~rop~iate pH, con~-~ t;r~n of protein, and ratio of protein to polymer useful for making thesemr~ 1ifiPd forms of IGFBPs, inclu~ g IGFBP-1.
The present invention further provides a pharm~c~utir~l coll,position con'~;ning the IGFBPs, inr,lu~ g of IGFBP-1, in an acceptable pharm~ceutir~l carrier. One carrier is ~h~siological saline solution, but it is con~".~ Pcl that other ~~rept~ble ~...~~~u~;r~l C~l;~.~ may also be used. In one emho~imPnt it is - envisioned that the carrier and the IGFBP-1 conclilu(~ a phyciologjr~lly-colll~ ible, slow-release form~ tinn. The plilU~ solvent in such a carrier may be either ayueo~s or non-aqueous in nature. In ~ litioll, the carrier may contain other ph~lllacologically-~ept~ble eYç;piP-ntC for modifying or ~ inl~;nillg the pH, osmol~rity~ viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the form~ tiom Simil~rly, the carrier may contain still other pharmacologically-2i~o~

acceptable P~çiI-ients for modifying or maintaining the stability, rate of dissolution, release, or absorption of the IGFBP-l. Such excipients are those substances usually and customarily employed to formulate dosages for ~t~minictration in either unit dose or multi-dose form.
Once the theldpeuLic colll?osiLion has been formulated, it may be stored in sterile vials as a solution, sUcpencion~ gel, emulsion, solid, or dehydrated or lyophili7~d powder. Such formul~tionc may be stored either in a ready to use form or l~uiling reconctitutiol- immedi~tely prior to ~dminict ation. The lJrefe~ed storage of such form~ tion~ is at le...~ u~s at least as low as 4~C and preferably at -70~C.
It is also plef~,led that such formul~tion~ n~ ning IGFBP-l are stored and ~minictPred at or near phy~;olQgj~l pH. It is ~ ltly believed that storage and ~mini~tr~tion in a formul~tion at a high pH (i.e. greater than 9) or at a low pH (i.e.
less than 4) is und~ e~
The ph~... ~~~utir~l c~...po,;l;~nc of the present invention can be ~dminict~Pred by intravenous, p~u~nt~,~l, ;nl A.. ~ r, ~,.,bcul~n~us, inLl~Licular injection or inru~;on, inh~l~nt mists, orally active forml~l~tirJnc~ or SuppG~;Iu. ;PS. To achieve and ,..~;n~ the desired dose of IGFBP~ p~ doses may be ~minict~pred. This method is h~nde~ to create a pl~l~t~l conr~-ll~alion range of IGFBP-l in the patient's blood strearn. It is believed that the ~sint~pn~nce of circul?~ting concP~ l;nn~ of IGFBP-l of 0.l ~g to 300 ~g per ml in the bloodsL~ealll may be effective in llcalil g an IGF :~C~ ~tJ~ con~ on. The frequency of dosing will depend on pharm~rûkinP-tic p~llelel~ of the IGFBP-l in the formulation used.
The methods of the present invention are based, in part, on experiments desr,-ihe~ in the following F~mpl~s Briefly, the present inventors discovered that ~0 94/22466 ~ PCTIUS94/03755 IGFBP-l blocks the ",itogenic effects of IGF-I and IGF-II on breast cancer, colon and o,s~ col"a cancer cells in vitro. Estrogen stimulates growth of breast cancers at least in part by causing the cells to secrete IGF-I or IGF-II.
Conc~ntrations of BP-l of 1-10 ~g/ml were required to inhibit growth of the colon cancers. One of the colon carcinoma cell lines grew in serum free media, pre~,u"~ably because it produced its own growth factors. Growth of this cell line in serum free media was inhihitP~ at least 50% by high col-c~ dtions of BP-l.
The present inventors have dçmon~trated that IGFBP-l inhibits the mitogenic effects of IGF-I on o.,~ "a cells. An a~~ te 12-fold molar excess of IGFBP-l inhibited the l~ -ic effect of 50 ng/ml IGF-I by 50% on rat ost*~.co",a cells.
IGFBP-l was also shown to inhibit the proliîel~ti.~e l~ s~)ol-~e of smooth muscle cells to IGF-I. IGFBP~ mini~tered after an~ioplasly in rats sig~ifir~ntlyinhibited the intimal thir~ in~ that results from smooth muscle proliferation and ~ r matrix ~ l-. These results in~ir~t~ that IGFBP-l is useful in t-h-e t ...'i~ or prevention of ~t~ nQs;s~
In hy~phy~l~llli~d rats, IGFBP-l inhibited the growth pro",oting effects of IGF-I and of growth hol,l,one. In - l~itiol-, IGFBP-l, its muteinC, and PEGylated - IGFBP-l inhibited IGF-I stim~ tio~ of growth of mouse 3T3 fibroblast cells.
The following ~,.,plf5 are il~t~.~d~ to illustrate but not limit the present invention.

WO 91/2~; 013 ~c PCT/US94/03755 Example 1 A. Purification & Refolding of IGFBP-1 E. coli cells eAp~ssing the IGFBP-1 were suspende~ in Buffer A (50 mM
Tris, pH 7.5, 20 mM NaCl and 1 mM DTT) at a concentration of 40 ml/10 g cell paste, and were disrupted at 1800 psi using a French p~s~ure cell. The suspen~ion was cenl,iruged 20,000 x g for 30 lllinu~s, and aliquots of the pellet & supernatant were analyzed by SDS-PAGE. A major band coll~s~n~in~ to the IGFBP-1 was present in the pellet, but not the ~. ~",at~ ~. The pellet was suspen~ed in Buffer A
(40 ml/10 g cell paste), and re-c. nL,ifuged at 20,000 x g for 30 ~"inu~s. This wash proc~lul~ was l'epeAIfed 2 times. The final pellet cont~ -it-g the IGFBP-1 was su~nded in 6M gllAni~ine, 50 mM Tris, pH 7.5, 6 mM DTT (25 ml/10 g cells) using a ground glass ho...ogf n;~f ~. The ~uspenc:on was incub~t~ at room lr~ W Al~ for 15 ~ v~-5. The Imdi~solved protein was removed by cenllifugation at 20,000 x g for 30 ",inu~s. Final conc~ lion of the IGFBP-1 was 1.0 mg/ml.
SDS-PAGE analysis of the pellet and s~ s-~nt showed that IGFBP-1 was present in the S.l~- ,.5~Ant only.
The duulul~d and laluced IGFBP-1 was ~ubje led to a three-step refolding - procedure.
a) Oxidized gl~ ne, the mixed-di~ fide proclucing reagent (GSSG), was added to the su~ tAnt to a final concentration of 25 mM, and incub~t~d at room te",~ldlure for 15 "~il ules.

wo 94/22466 w 16 ~ 9 PCT/US94/03755 b) The solution was then diluted 10 fold gradually with 50 mM tris. pH
9.7 and phenylmethylsulfonylfluoride was added to final concentration of lmM.
Final concentration of protein was lOO~g/ml.
c) The refolding ~ ure was incubated overnight at 4~C, and then cenlliruged at 20,000 x g for 15 minutes. SDS-PAGE analysis of the pellet and supelllat~nt showed that the supelllat~nt was coll.posed of relatively homogeneous IGFBP-l.
Aliquots (50~1) of the ~.~pe ..~t;~t were diluted to 200~1 with Buffer C (0.05 %
TFA), injected onto a reverse phase column (RP-4, 1 x 250mm, Synchrom), and eluted with 80% a~to~;t~;le, 0.042% TFA (Buffer D) using a linear gradient (increace of 1% Buffer D/minute) at a flow rate of 0.1 mVminute.
A single major peak l~ples~ h~g refolded IGFBP-l eluted at 68 minutes. The retentinn time of the refolded IGFBP-l shifted to 71.0 ...i~ s after being completely l~luced and denalulGd in 5 M guqni~inç~ 50 mM Tris pH 7.5, 100 mM DTT. These results in~ir~te that IGFBP-l refolds to a single pr~do.. ,inant species under the contl;l;Qnc ~e~;hed N t~ll,i.nal s~uencG analysis of IGFBP-l eluting at 68.0 .,~;nl~tes gave the sequence Met Ala Pro Trp Gln Cys Ala Pro... (SEQ ID NO 3), which ..,~teh~ the N t .,.,inal amino acid S~U~I1CG of human IGFBP-l (SEQ ID NO.:
1) except for an extra methionine residue at the N te~ us of the recombinant 2 o protein.
B. Isolation of Refolded IGFBP-1 The refold IllLl~lUlG (15000 ml) p,ep~Gd from 590g of E. coli paste con~il~ing the c~lleclly refolded IGFBP-l was con~nt~led to 1800 ml, dialyzed against 20 mM
sodium phosrh~t~, pH 6.0, cer~l~ir~lged at 10,000 x g for 30 .llil,ul~s to remove WO 94/22466 21~ 4 PCT/US9410375~

p~c.;ipitated E. coli proteins and loaded onto an Q-Sepharose (Pharmacia/LKB, Piscataway, NJ) column (5.0 x 60 cm) previously equilibrated with the same buffer.
The bound protein was eluted with a 5000 ml linear gradient to 0.5M NaCl at a flow rate of 20 ml/minute. 25 ml fractions were collected. A single major peak elutedat 0.3-0.4 M NaCl; 100 ~1 aliquots of each fraction were analyzed separately by a reverse phase chro---atography column (RP~ 1 x 250 mm Synchrom). Fractions cont~inil-g predo...in~nlly correctly refolded IGFBP-l (dele --.ined from RP-4 analysis), were pooled (900 ml), the pH was adjusted to 7.5, the conductivity was adjusted to 1 mM NaCl (95 mOhm), and loaded onto a Toyopearl butyl-650 S
hydl~phobic int~rtiol- column (5 x 5 cm) (Supelco, Bellefonte, PA), previously equilibrated with 20 mm ~S, pH 7.5, 1.0 M NaCl at a flow rate of 30 ml/minllt~
The protein was eluted with a 1500 ml linear gradient to 20 mM HEPES, pH
7.5 at a flow rate of 40 ml/minut~ A single broad peak eluted at 5 - 15% eth~nol.
Aliquots (10~1) of each peak rl~elion were analyzed by RP-4 reverse phase clu~ c.~ hy and SDS-PAGE. Fr~rti--n~ co~ i n;ng pure (95 %) correctly refolded IGFBP-1 were pooled, crnce~ ted to ~8 mg/ml and assayed for bioactivity.
In all of the following e-lx~ lL~ using IGFBP-l, the recombinant E. Coli.-e~yl~ssed IGPBP-1 was used.
Example 2 IGFBP-1 inhibits the growth promoting effects of IGF-I and growth ho~none in rats Hypophy~clollly (removal of the ~ ui~y) removes the source of growth hol,l,one from the body and results in growth ces~tion. Hypophy~lo"fized ~nim~l~

WO 94122466 2 i ~ PCT/US94/03755 can be stimulated to grow by injection of exogenous growth hormone or IGF-I, as described in Schoenle et al., Nature, 296: 252-253 (1982). The effects of subcutaneously ~dminictPred IGFBP-1 were tested on IGF-I and growth hormone-stimulated growth in this model. Growth was ~csP-ssp~ by measuring body weight gain and tibial epiphyseal width.
A. IGF-I experiments Male Sprague Dawley rats that were surgically hypophy~lo.,.ized at 120-130 grams of body weight were obtained from a commercial source (Charles River, Wilmingt(n, MA). The body weights of the rats were monitored for two to three weeks before the be~;n~ -g of the t~ --e~lC in order to verify compl~-tP-nP-sc of the hy~phy~c~ollly. Rats gaining more than 2 grams of body weight per week were e~clude~d from the study. Rats were injected s. b~.lt~nP~usly twice a day at the nape of the neck for eight c~n~u~;~e days with 0.2 ml of vehicle solution (40 mM
~S, 100 mM NaCl), IGF-I (80 ~Lg) alone, IGFBP-l alone, or IGF-I (80 ~g) co---b~led with various molar ratios of BP-l. The molar ratios of IGFBP-l:IGF-I
tested ranged from 0.04:1 to 5:1. Body weights were determined daily. The rats were ~rificed 12 hours after receiving their final injection. Their right and left tibias were removed, fixed with formalin, split at their proximal ends in a sagittal - plane and stained with silver nitrate, as descnbed in Gr~-~n, Endocrinolo~y.
2 o 45:455-463 (1949). The c~ fi~ tissue was stained dark brown and the prolirelaling zone of car~lage al,~Gd as a clearly defined white band. The car~laginous epiphyseal plate was measul~ with a st~lGo...ic~scope equil,ped with a calibrated mic~l..e~r e~.r ~c~ Appro~;...~t~y ten individual re~-lingc were made across each WO 91/2z?4~6~ (i 01 j l~ PCTIUS94/03755 epiphysis. The mean of the combined re~-ling~ from the right and left tibias wascalculated for each rat.
The results of several expenmçnt~ are compiled in Table 1. Vehicle treated rats did not gain weight over the eight day test period whereas IGF-I treated rats gained an average of about six grams per rat over that time period. Rats in the S: l IGFBP-l:IGF-I group showed no significant weight gain, in~ ting that excess IGFBP-l blocked the growth~ ,l,oling effects of IGF-I in this model. The ~lmini~tr~tion of IGFBP-l at molar ratios of 1:1 and 0.2:1 relative to IGF-I caused S0 to 75 % inhibition of IGF-I-stimulated growth. No çnh~nc~mçnt of growth abovethat stim~ by IGF-I alone was measured in any group receiving IGFBP-1. In litiQn, the ~ lminictration of IGFBP-l alone had no signific~nt growth promotingeffects. St~ti~ti~lly ~ignific~nt inhibition of IGF-I-stimulated enlargement of the tibial epiphy~al width oc~ cd with the ~lmini~tr~tiQIl of IGFBP-l at molar ratios of S:l and 1:1 relative to IGF-I ~Table 1). These data show IGFBP-l inhibits bone and car~lage growth stimul~t~ by IGF-I.

wo 94/22466 21 li 01~ ~ PCT/US94/03755 EFFECTS OF SUBCUTANEOUSLY ADMI~ KED IGFBP-1 ON IGF-I-STIMULATED INCREASES IN BODY WEIGHT
AND TIBIAL ~Y~;AL Wll~l~l MEASUREMENTS

IGF~-IGFW-l- ICFW-l:INCREASE IN TUUAL
~o/ ~/ UOF~BODY ~fl3CHT-- _ .. .~EAL ~nDTH--h~c~on~h~c~orl~Md-r R-~o l~r m-~ Imm~
O 0 0:00.9 ~ 0.8 0.124 + 0.003 0 0:15.6 1 0.8 0.175 ~ 0.006 9.~0.04:14.7 ~ 0.5 0.165 ~ 0.010 48 0.2:11.8 ~ 0.8 0.181 + 0.007 240 1:12.~ ~ 0.9 0.144 1 0.008 1200 5:11.4 1 0.3 0.133 1 0.006 O 9.~0.04 0 -2.ô 1 O.ô 0.126 1 0.007 0 48 0.2:0-0.5 ~ 0.5 0.136 ~ 0.005 2 0 0 240 1:0-0.25 1 0.~ 0.132 ~ 0.005 0 1200 5:01.3 1 0.4 0.124 ~ 0.003 * Rats received two ;~je~ ns per day.
** Values are means t st~nd~d crrors of the mean for 8 to 15 rats per group.

B. Growth ho....~ eAVC.;~ C
The design of this ~ was the same as the IGF-I ~ ,.;n~M-~s exccpt that rats ~;vcd inje~ionc of growth hul,.,one rather than IGF-I. Growth hol.none and IGFBP-l were inject~d s~Jk;~ 4uc1y at ~'-i);Z';~f injection sites. IGFBP-1 was ...;n;~h.~ twice a day at 10 mg/kg per injection, a dose that was equivalent to that ~minict~red in the 5:1 molar eAcess ratio in the ~A~ ; described above.

Human lJiluil~erived growth h~.. on~ (Sigma ~h~mir~l Co~ any, St. Louis, SUBSTITUTE SHEET (RULE 26) WO 94/22466 21~; 0 1~ Ll PCT/US94/03755 MO.) was ~minictered twice daily at 15 mU per injection. This dose of growth hormone stimulated a sllol~gel growth response in the rats than did the dose of IGF-I
used in the previous eA~ t. The growth hormone-treated rats gained 12 grams per rat on average during the SiA day ~~lminictration period (Table 2). Weight gain was inhibited by about 75 % in the IGFBP-1-treated rats. Growth hormone stimulated an apploAi.~ R two-fold inclease in tibial epiphyseal width relative to vehicle-treated ~nim~lc (Table 2). The growth h.,. ".f-n~c~i.. l~~~~ inel~sR in tibial epiphyse~ width was inhibit~ by about 75 % by co-~minict~ti~n of IGFBP-1 (Table 2).

E~ECTS OF SUBCUTANEOUSLY ADM~l~l~;KED IGFBP-l ON GROVVm HORMON~STIMULATED INCI~F~CF-~ IN
BODY VVEIGHT AND TIBIAL ~ l~YS~;AL Wll~lll MEASUREMEN~

INCREA~E U~l TIBIAL
GROWTH HORMONE-IGFW 1-BODY WEI~HT-- ~rIY~EAL WIDTH--lp-r h~ r m ) Imm~
0 0 -1 2 ~ 09 0 154 ~ 0009 15mU 0 12 l 1 3 0314 ~ 0014 15mU 1200 34~ 1 1 0195 l 0010 * Rats received two injec-tit~n.C per day.
** Value. are means ~ da~ errors of the means for 5 rats per group.
The above ~ .. e:nl~ indi~te that IGFBP-1 is capable of inhibiting the growth lJrolllolii g effects of both IGF-I and growth hormone in rats.

SUBSTITUTE SHEET (RU~E 26) wo 94/22466 2 i ~ ~1 J ~ PCT/US94/03755 Example 3 IGFBP-1 inhibits grov~th of a hlm~n breast carcinoma cell line in vitro The biological effects of IGF-I, IGF-II and IGFBP-1 were determined on a human breast cancer cell line. The human breast carcinoma cell line, MCF7, was obtained from the ~mçric~n Type Culture Collection located in Rockville,MD
(catalogue number HTB 22). The cells were "~ A;nPA in Eagle's minim~l essential " ,PA;, .... (available from MeAi~ h, Herndon,VA) co~ ining 10% fetal bovine serum, 10 ~g/ml insulin, 2 mM glu~ ;n~ mM sodium pyruvate, 100 units/ml penieillin, 100 ~g/ml ~ p~---ycin and non~s~nl;~l amino acids (Irvine Scientific). For cell proliferation assays, the MCF7 cells were det~rhP~d from plates by brief l-eatl-lellt with trypsin and EDTA. Cells were plated in 96 well tissue culture plates (Costar Co~ ;nn, Cambridge, MA) at 2 x 104 per well in a serum free IllediUIII (Eagle's millimAl eC~ n~;~A1 ~-I~;VIII CQ~ItAin;i~g 1 mg/ml bovine serum albumin, 2 mM
gl"l;1.. ;nF, 1 mM sodium pyruvate, 100 Units/ml Fenirillin, 100 ~g/ml sLl~lolllycin and non e,C~nt;~l amino acids). Varying ~ tionc of IGF-I, IGF-II or IGFBP-1 were added to the wells in a final volume of 200 ~Ll. After 4 days at 37~C, 20 ~1 of a 5 mg/ml solution of MTT (3-t4,5~i~ yl~ 1-2-yl]-2,5-diphenyl~ll;.7Olil~m bromide; available from Sigma ~hemicr~l CO-Ilp uly, catalogue # M5655) was added 2 o to each well. The cells were incub ~l~d for an ~tliti-~n~l 6 hours at 37~C. The cells and the hydroly_ed MTI were solubili7~d by the addition of S0 ~1 of a solution of 50% di",~ yl ~~ ...;de, 20% sodium dodecyl sulfate, pH 4.7. After overnight ;nc~lbAI;on at 37~C, the hydrolyzed MTT was ~luA~.~;LA~P~ by mP~lring the optical density of the liquid at 570 nm and subtracting the 650 nm optical density background WO 94/22466 ~ 1 ~ 0 ~ PCT/US9S/03755 with a VMAX kinetic microplate reader (Molecular Devices Corporation, Palo Alto,CA ).
Both IGF-I and IGF-II caused proliferation of MCF7 cells, as evidenced by an increase in the optic~ densities of the cell cultures (Table 3). IGF-I was about 5 times more potent than IGF-II at stim~ ting proliferation of MCF7 cells. MCF7 cell proliferation oc.;ulred at IGF-I concentrations ranging from about 1 to 120 ng/ml and at IGF-II conc~ .dtions r~nging from about 10 to 1200 ng/ml. IGFBP-l inhibited IGF-I and IGF-II stimulated proliferation of MCF7 cells in a dose dependent manner ~ables 4 and 5). This was de~,.uined by incub~ting MCF7 cells with 60 ng/ml IGF-I, or 300 ng/ml IGP-II, in the pl~ sellce of increasing amounts of IGFBP-l. The tissue culture .,.~Ai~l... used was the same as that used to ...~;~"~in the cells except that it did not contain serum or insulin.
For IGF-I, the IGFBP-l conc~ l ;onc tested ranged from 6 to 13,600 ng/ml.
App~J~;---~t~ly 50% growth inhibition OC~;U11~I at a IGFBP-l con~ntration of about 180 ng/ml, which COllCSpOll~S to about a 1:1 molar ratio of IGF-I:IGFBP-l (Table 4). F~Pnti~lly complete growth inhibition Occull~ at IGFBP-l conc~-ntrations rYC~ing 4000 ng/ml, which COll~fSI on~s to an appl~, .i...~tP 20-fold molar excess of IGFBP-l.
The IGFBP-l con~ .t.~ion~ tested for IGF-II ranged from about 30 ng/ml to about 23,000 ng/ml. Appro,.i.. ~lely 50% inhibition of the IGF-II growth response OC~iwl~t:d at a IGFBP-l co~ nl~lion of about 840 ng/ml, which is slightly greater than a 1:1 molar ratio of IGF-I:IGFBP-l ~able 5). F~Pnti~lly col,lplcte growth inhibition oc~;wr~d at IGFBP-l conc~nl~;on~ e c~Aing 22,000 ng/ml, which coll~s~x)nlls to slightly greater than a 2~fold molar excess of IGFBP-l.

WO 94/22466 ~ 1 6 0 L !~ 4 PCT/US94/03755 BY IGF-I AND IGF-II

IGF-I* O.D.** IGF-II* O.D.**
S (ng/ml) 57~650 mn (ng/ml) 57~650 nm 0 0.121 0 0.121 0.140 10 0.139 2 0.140 19 0.149 4 0.150 38 0.181 8 0.169 75 0.195 0.189 150 0.216 0.217 300 0.256 0.273 600 0.321 120 0.330 1200 0.411 * IGF-I and IGF-II co~ ;m~ rounded to the nearest whole ~ulll~r.
** Optical density at 570 nm minus optical density at 650 nm. Means of trir~ te wells. Standard deviations were less than 11 % of the means.

SUBSTITUTE SHEET (RULE 26) WO 94/22466 21~ 313 4 ~ PCT/US94/03755 ~IIBITION OF IGF-I-STIMULATED MCF-7 R~FA.~T CANCER CELI, GROWT~ BY IGFBP-l IGFBP-1 ~ IGF-I O.D.*~
(ng/ml) (ng/ml) 570-650 nm % .. ~ ION
0 0 0.260 0 60 0.610 0%
6 60 0.543 19%
19 60 0.472 39%
56 60 0.489 35%
168 60 0.443 48%
504 60 0.372 68%
1,511 60 0.330 80%
4,533 60 0.286 93%
13,600 60 0.242 105%

* IGFBP-l con~f~ nC rounded to the nearest whole nwll~l.
** Optical density at 570 nm minus optical density at 650 nm. Means of tripli~t~ wells. Standard deviations were less than 8% of the means.

SUBSTITUTE SHEET tRULE 26) WO 94/22466 21~ 015 ~ PCT/IJS94/03755 INHIBmON OF IGF-II STIMULATED MCF-7 RI~F.A~T CANCER CELL GROWTH BY IGFBP-l IGFBP-1 IGF-II O.D. ~
(ng/ml) ~ng/ml)570-650 nm % INHIBITION

~ 0 0.126 0 300 0.357 0%
31 300 0.345 5%
93 300 0.299 25%
280 300 0.277 35%
839 300 0.243 49%
2,519 300 0.181 76%
7,556 300 0.161 85%
22,667 300 0.144 92%

* Optical density at 570 nm minus optical density at 650 nm.Means of ;p~ e wells. Standard deviations were less than 11% of the means.

l;~n...ple 4 IGFBP-l inhibits gro~rth of human colon carcinoma cell lines jn ~itro The biologjr~l effects of IGF-I and IGFBP-l were tested on a l~u~-~b~r of human colon cancer cell lines. SL1~ human colon cal.,inGnla cell lines were oblai~ed from the ~meric~n Type Culture CQll~ti~n located in Rockville, MD. These cell lines were SK-CO-l (HTB 39), LS 174T (CL 188), DLD-l (CCl 221), HT-29 (HTB 38), COL~205 (CCL 222) and Caco 2 (HTB 37). The de~g~tiQn~ within pare,ntheses refer to the ATCC catalog llu~nber. These cell lines were s~l~ted SUBSTITUTE SHEET (RU~E 26) WO 94122466 21~ 01 S 4 PCT/US94/03755 because they all form tumors in nude mice according to the descriptions provided in the American Type Culture Collection catalogue. The cells were ma ntained in _agle's Minimal F.~Pnti~l Medium (Me~i~tP~, Herndon, VA) containing 10%
fetal bovine serum, 2 mM glul~....ne, 100 Units/ml peni~illin and 100 ug/ml sllc~t~",ycin The effect of IGF-I on these six colon cancer cell lines was determined as follows. When the cells reached 90-100% confluency, the cells were det~ch~P~
from the plates by hcaling them briefly with a trypsin/EDTA solution. The cells were washed several times, counled and resuspPn~P~I in serum-free media (Eagle's ~inim~l F~Pnti~ PAi~lm c~ ing 2 mM glu~ ;ne, 100 units/ml penicillin and 100 ~g/ml sll~pt~",ycin) at a col-~nl~alion of lxlO5/ml. 100 ~1 of the cell s ~ .C;on was added per well of a 96 well tissue culture plate (Corning Glass Works, Roc-h~t~-~, NY). 100 ~1 of serum-free media CC~nt;~ h~g varying amounts of IGF-I were added to the wells and the cultures mixed gently by piretting. The plates were incul~l~d at 37~C for 3 days. At this time, cell nu",bel was nl;L~I~d using a crystal violet dye assay. Media was suctionPd off the cells and 150 ~1 of crystal violet stain [2g of crystal violet (Aldrich ChPmi~l Company,Inc., Milwaukee, WI) dissolved in a sol~tion co~ ining 270 ml 37%
formaldehyde and 20 ml of pol; C~ ho~h~ pH 7.0] was added per well.
l~renty .. ~;n~ 5 later the liquid was sucti~ nP~ off and the wells washed 3 times with phosph~tP burL~ed saline. 200 ~1 of eYt~~tion buffer (50% ethanol, 0.1 M
sodium citrate pH 4.2) was added per well and the plates left overnight at room l~.l-pe.~lu~. The optical density of the wells at 570 nm was determined the next day using a miclopla~e reader (~Iolcc~ r Devices, Palo Alto, CA).

''~O 94/22466 21~ 01 ~J 4~ PCT/US94/03755 All six cell lines prolifel~led in response to IGF-I (Tables 6 and 7), as evidenced by an increase in the optical dçncitiP-s of the cell cultures. The Caco-2 cell line grows well in serum-free media, suggesting that it produces one or more endogenous growth factors. Growth of the Caco-2 cell line in serum-free media was çnh~nc-pd by IGF-I (Table 6). The other colon cancer cell lines e ~mine~ didnot show ci~nifir~nt cell proliferation in serum-free media under the con-litionc tested. However, they all pr~lifc.a~d in l~on~, to IGF-I as evidPn~d by an increase in the optical ~enCitips of the cultures (Tables 6 and 7).

STIMULATION OF GROVVTH OF IIUMAN COLON CANCER CELL
LINES BY IGF-I

O.D.~~
570 nm IGF-I~
~ng/ml) SK-C0-1 CaC0-2 DLD-1 COL0-205LS 174T
0 0.156 1.253 0.474 0.138 0.144 1 0.174 1.402 0.493 0.154 0.165 4 0.218 1.472 0.497 0.163 0.195 16 0.337 1.796 0.532 0.178 0.242 63 0.368 1.923 0.558 0.189 0.236 250 0.391 2.022 0.634 0.219 ' 0.225 1000 0.376 1.943 0.721 0.218 0.212 * IGF-I c4~ ;nn~ luunded to the nearest whole ~ e .
** Optical density at 570 nm. Means of trirli~t~ wells.

SUBSTITUTE SHEET (RllL~ 26) WO 94/22466 216 ~13 1l PCT/US94/03755 CELLS
BY IGF-I

IGF-I $ O.D.**
(ng/ml) 570 nm 0 0.434 0.557 0.627 13 0.694 53 0.665 213 0.631 850 0.627 * I(i~-l CQI~ nl ~Lions rounded to t~ e nearest whole number.
** Optical density at 570 nm. Means of triplir~t~ wells.

The effect of IGFBP-1 on growth of these cell lines was then dete".,ined.
Rather than de~-..ine the effect of IGFBP-1 on IGF-I-stimulated growth of the cells, it was det~.---,ned whetha IGPBP-1 could inhibit growth of the cells in the pl~. ce of serum, which may be more l~pr~~ re of the in vivo ~ tinn.
Cells were d~ d from plates by brief trypsin/EDTA L-eA~ nt, washed, pen~ed in Eagle's ~inimAl F~nti~l ]UeAillm C~ A;I~;ng 4% fetal bovine serum, 2 mM gl~ ...;ne, 100 units/ml pPnirillin and 100 ~g/ml ~ ...ycin at a con~ntration of 1 x 105/ml and 100 ~Ll of the cell suspension added per well of a 96 well tissue culture plate. IGFBP-1 was diluted to varying conc~ntrations in serum-free Eagle's MinimAl F~PntiAl media cor.lA;I~il-g 2 mM glutAmine, 100 WO 94/22466 ~ PCT/US94/03755 , units/ml peni~-illin and 100 ~g/ml sl,~l~l,lycin and 100 ~1 of the ~ ule added per well of the 96 well plate. The cell cultures were mixed by gentle pipetting and incubated for 3 days at 37~C. The final serum conr~-ntration was 2%. Cell n~,lllber was ~lu~nt;l~lPd at this time using the crystal violet dye assay described above. IGFBP-l caused si~nifi~nt inhibition of the growth response of four of the cell lines (Caco-2, COLO-205, HT-29 and SK-CO-l) in the presence of 2% serum (Tables 8 and 9). Little growth inhih~itil-n was seen until IGFBP-l levels reached several hundred ng/ml. The ...~;...--n~ growth inhibitinn observed was between 30% and 100% (at IGFBP-l levels of 10-20 ~g/ml). IGFBP-l had little effect on serum-~im~ t~ growth of the LS 174T and DLD-l cell lines (m~iml)m inl~ibilion of 9 and 22%, r~c~ ely, at 10-20 ug/ml IGFBP-l). The conc~ n~ of free IGF-I and IGF-II in the serum were not deo~

ON OF SERllM-STlMULATED GROWT~ OF IIUMAN COLON
CANCER CELL L'INES BY IGFBP-1 ~ERUM IGFEP l- O.D.-- % IN O.D.--% IN- O.D.-- % IN-~%~/n~/ml~ 670 nm HlBllnON 670 nmHUUTUDN 670 nm HlBllnON
0 0 1.253 0.138 0.145 2 0 1.9990% 0.574 0% 0.556 0%
2 0 2 3 2.076-10% 0.591 - 4% 0.510 11%
2 16 2.023- 3% 0.542 7% 0.483 18%
2 80 1.92610% 0.581 - 2% 0.497 14%
2 400 1.74135% 0.~6 25% 0.407 36%
2 2,000 1.526~3% 0.386 43% 0.344 52%
2 5 2 10,000 1.26698% 0.304 ~2~ 0.300 ~2%
* IGFBP-l cQnC~fi~ ns roui~de~ to the nearest whole ** Optical density at 570 nm. Means of trirli~te wells.

SUBSTITUTE SHEET (RULE 26) WO 9~/22466 PCT/US94/03755 21~015 ~

INHIBlTION OF SERUM-STIMULATED GROWTH OF IIUMAN

SERUM IGFBP-1 ~ O.D.~ %
~%) (ng/ml) 570nm INHIBITION
o 0 0.156 2 0 0.507 0%
2 1 0.519 -3%
2 6 0.514 -2%
2 32 0.496 3%
2 160 0.432 21%
2 800 0.406 29%
2 4,000 0.395 32%
2 20,000 0.386 34%
* IGFBP-l con~-nt-~lions r~undc;d to the nearest whole nu,l,ber.

** Optical density at 570 nm. Means of t ipli~tP- wells.

F.;...U?lf S
IG~BP-l inhibits gro~th of a human osteosarcoma cell line in ntro The ability of BP-1 to inhibit IGF-I stim~ tPA growth of an os~.cc",a was det~l,l,ned using a rat ost~ collla cell line, UMR-106 (CRL 1661), o~ined from the ~mPric~n Type Culture CollP~tinn (Rock~ille, MD). The cells were ~I~AinlA;n~A in Ham's F12 . .~;.J ~ (available from MPA;a~ , ~P- ndQn, VA) conl;-;n;ng 7% fetal bovine serum, 100 Units/ml pPnit~illin, 100 ~g/ml ~ ~lo",ycin and 2 mM g1~ .. ;nP~. The cells proliferate in response to IGF-I.

SUBSTITUTE SHEET (RULE 26) WO 94/22466 21~ 0 ~ ~ d~. PCTIUS94/03755 well of a 48 well tissue culture plate (Costar Co~ dlion, Cambridge, MA).
When the cells became confluent (after ap~)foximately 3 days at 37~C), the cells were washed twice with phosphate buffered saline (PBS) and pre-incubated for 24 hours in the above medium lacking fetal bovine serum. After the pre-incubation, the medium was removed and replaced by 0.5 ml of serum-free Ham's F12 .,.ediu-.. cont;~inil-g serial dilutions of IGF-I (1 to l,000 ng/ml). The plates were incu~ted for an ~ltijtion~l 20-24 hours at 37~C. Each well was then pulsed with 0.5 ~Ci of 3H-thymidine (NEN ~ h products, Dupont Co., Boston,NA) for 4 hours at 37~C, then washed three times with cold PBS. DNA was ~ i~t~d by adding cold 7% trichloroacetic acid a.T.Baker Inc., Philli~Js~ulg,NJ) to the cells.
After rinsing with 95% eth~nol, the cells were solubilized by the addition of 0.3 M NaOH. Aliquots were removed and c~unted in a sc-intill~tion counter to u~nt;t~l~ the ~mount of 3H-thymidine incol~lated into DNA. All assays were ~.rul,.,ed in ~
As shown in Table l0, IGF-I caused a dose-de~ndent increase in 3H-thymidine incol~latGd into DNA. The ..,~ l le,~ol~ce was about six times over the level seen in the ~ of IGF-I. The ED50 for IGF-I ranged from 4-20 ng/ ml in dirr.,.G,~

~ ~ ~ PCT/US94/03755 WO 94/22466 2 1 ~

CELL GROWTH BY IGF-I

IGF-I*
(ng/ml) ~ S~*

0 34,624 40,636 2 54,814 4 99,715 8 145,525 16 141,274 31 187,174 63 189,257 125 194,254 250 184,857 500 212,929 1000 197,268 * IGF-I eQnC~ntrations roun~ed to the nearest whole number.
** Counts per minute. Means of triplicate wells.

wo 9~/22466 2 1 ~ PCT/US94/03755 The effect of IGFBP-1 on IGF-I stimulated growth of UMR-106 cells was dele~ ed using the above assay except for the following changes. After the pre-incubation step the cells were incub~ted with serum-free Ham's F12 medium containing 50 ng/ml IGF-I and varying concentrations of BP-1 (200 to 16,000 s ng/ml). The medium also cont~inP~ the 2 mM glut~mine, 100 Units/ml penicillin and 100 ~g/ml ~l,eplon~ycin. After 20-24 hours at 37~C, the cells were pulsed with 0.5 ~Ci of 3H-thymidine for 4 hours, rinsed three times with cold PBS, and DNA plcc;~ tPd with cold 7% trichloroacetic acid. The cells were rinsed with 95% eth~nol, solubilized in 0.3 M NaOH and aliquots counled in a scintillation counter.
The results of one of these experiments are shown in Table 11. The data indi~t~ that IGFBP-1 inhibits the ~ g~ ~ic effect of IGF-I on os~collla cells.
An ap~ te 12-fold molar excess of IGFBP-1 (2,000 ng/ml) inhibited the ...;l~g~ ..ic effect of 50 ng/ml IGF-I by 50%. F~SP.nt;~11Y complete inhibition of the Il,ilogenic effect of 50 ng/ml IGF-I was seen with 50-100 fold molar excess of IGFBP-1 (8,000 -16,000 ng/ml). The ~mollnt of IGFBP-1 required to inhibit effects of IGF-I in these ~ is greater than that observed in other c-~pc-;.. f~nl~ and with other cell lines. This probably is due to the fact that 50 ng/ml IGF-I gave a m~sim~1 Mitogenic response in this experimPnt wo 91/22466 21~ ~1 S 4 PCTIUS94/03755 INHIBlIION OF IGF-I-STIMULATED GROWTH OF
RAT OSTEOSARCOMA UMR-106 CELLS BY IGFBP-l (ng/ml) (ng/ml) cpms~ % INHIBITION
0 0 32,145 0 188,933 ~%
200 172,513 10%
400 173,234 10%
800 157,475 20%
2,000 112,265 49%
4,000 78,041 71 %
8,000 51,470 88~~
16,000 34,448 99%
* Counts per minllte~ Means of ~ licate wells.

P~ le 6 IG~BP-l inhibits growth of smooth muscle cells IGFBP-l was tested to det~,u~e if it could inhibit the ~ re~ re respo~ce of smooth muscle cells to IGF-I. A rat smooth muscle cell-like cell line, Al0, was ob~ined from the ~m~.ri~n Type Culture CQ~ ti~n located in Rockville, MD (catalogue # CRL 1476). The Al0 cell line has been cl~ ~ by B.W. Kimes and B.L. Brandt, F~..;~r.rnt;~l Cell Research, 98:349-366 (1976). The cells were ~ r~l in DMEM ,..P.l;,~ .. (Dulbecco's Mo~1ifir~tir n of Eagle's MPAi-lm, available from M~;~t~.l-~ Inc. ~e ndc-n, VA) con~ il-g 10% fetal bovine serum, 2 mM gl-~ ~. ;ne, 100 Units/ml peni~ in and 100 ~g/ml sL,~pto,-"~cin. For prolir~alion assays the cells were det~-hPd from plates by brief ll~t~ -nl with a trypsin/EDTA solution, washed once with serum conl~ini~-g ,"PAi,,.", twice with serum-free .~ .. and counted using a hemo~;~lo",eter. The cells were resusl~,nded at a cone~-nl~lion of 2x105/ml in SUBSTITUTE SHEET (RULE 26) wo 94/22466 211~ 3 4 PCT/US94/03755 serum-free DMEM medium co,-l~ining 2 mM glut~mine, 100 Units/ml penicillin . and 100 ~g/ml ~l,~to",ycin. 100 ~1 of the cell suspe.n~ion was aliquoted per well of a 96 well tissue culture plate (Corning Glass Works, Roc.hester, NY). 100 ~l of serum-free medium containing increasing amounts of IGF-I (0, 2, 20, 200, or 2000 ng/ml) were added to appr~iate wells and the plates incubated for 3 days at37~C. At this time cell number was quantitated using the crystal violet dye assay described in Example 3.
IGF-I caused a dose-dependent increase in cell number, as measured by an increase in the optical density of the wells (Table 12). The maximum proliferative response occurred at an IGF-I concentration of 100 ng/ml.

CELL GROWTH BY IGF-I

IGF-I O.D.*
lS (ng/ml) 570 nm 0 0.318 0.317 0.451 2 o 1000 0.536 * Optical density at 5 /0 nm. Means of 6 wells.

The effect of recombinant IGFBP-l on IGF-I-stim~ ted growth of A-10 cells was de;l~.",ined using the above cell proliferation assay. The assay was p~,rul,ned in an identi~l manner except that the test wells cont~ined 100 ng/ml IGF-I. Some wells also cor,l~ined IGFBP-l at concel,t,dlions ranging from 1-10,000 ng/ml.
IGFBP-l caused a dose-dependent decrease in cell number as evidenced by a decrease in ~e optical densities of ~e cell cultures (Table 13). At a wo 9~/22466 21~ l PCT/US94/03755 cQnc~nt~tion of 1000 ng/ml, IGFBP-l reduced the cell number to that seen without any exogenous IGF-I (lc~e.-ed to as b~line proliferation). At a conc~ tion of 10 ~g/ml, IGFBP-l reduced the cell number to below that seen in serum-free media, sugges~ing that rat A-10 cells produce endogeno~.s IGF-I or IGF-II. These data also in-lic~t~ that IGFBP-l inhibits the proliferative response of rat smooth muscle cells to IGF-I.

ON OF IGF-I-STIMULATED GROVVTH OF
RAT A-10 SMOOTH MUSCLE CF~ 1-C BY IGFBP-1 10IGFBP-1 IGF-I O.D.~ % IN- O.D.~ % IN-(ng/ml) Ing/ml) 570nm HlBlTlON 570nm HIBITION
0 0 0.192 0.230 0 100 0.290 0 0.353 0 100 0.253 38% 0.334 19%
100 100 0.249 42% 0.282 59%
1,000 100 0.197 95% 0.236 95%
10,000 100 0.157 136% 0.183 137%
* Optical density of 570 nm. Means of trirli~te wells.

?le 7 2 o Preparation of IGFBP-1 muteins A. Construction of IG~BP-1 muteins Two IGFBP-l mutein~, C98 and C101, were constructed by mutagenesis of the IGFBP-l DNA sequence cQI~Ain~ in pl~mi~l pJU1021, ATCC Acce~ n No.
67730. In the C98 mutPin, the serine at po~iti~n 98 of the mature protein sequence has been chang~d to a .;~ in e residue. In the C101 mlJtein, the serineat position 101 of the mature protein sequence has been changed to a cysteine SUBSTITUTE SHEET (RULE 26) 216-~15~

residue. Residue numbering is based upon SEQ ID No.: 1. Mutagenesis was done utilizing the polymerase chain reaction (PCR) technique.
The C98 mutein was made using plasmid T88IQ:IGFBP-l DNA as the star~ing template DNA. Plasmid pT88:IGFBP-l contains the wild type IGFBP-l coding sequence in plasmid pT88IQ.
1 0 The e~p~ession vector pT88IQ is a derivative of the expression vector pT3XI-2. The vector pT3XI-2 was constructed in the following manner. The starting plasmid for this construction was plasmid pKK223-3 purchased from Pharmacia.
Plasmid pKK223-3 carries a partial gene for tetracycline resistance. This nonfunctional gene was replaced by a complete tetracycline resict~nce gene carried on plasmid pBR322. Plasmid pKK223-3 was digested completely with SphI and partially with BamHl. A 4.4 kilobase pair fragment was gel purified and combinedwith a synthetic adapter:
(SEQ ~ NO:4) 5' GATCTAGAATTGTCAl~l-llGACAGCTTATCAT 3' (SEQ ~ NO:ll) 3' ATCTTAACAGTACAAACTGTCGAATAGTAGC 5' 2 o BglII ClaI
and a 539 b~cep~ir fragment of DNA from a ClaI, SphI digest of the tetracycline resistance gene of pBR322 (pT Bioc}l~omi~lc, 27-4891-01). The res--lting plasmidwas design~ted pCJl.
Next, a XhoI linker purchased from New England Biolabs (Beverly, Massachusetts) was inserted into pl~cmid pCJl's PvuII site to form plasmid pCJX-l.
This insertion disrupts the rop gene which controls plasmid copy number. Next, an EcoRI fragment cont~ining the lacI gene was purified from plasmid pMC9 (Calos et al., 1983), then inserted into the XhoI site with XhoI to EcoRI adapters. Thepolylinker region in pl~cmitl pKK223-3 was next replaced with a polylinker containing 3 o additional sites by cutting with EcoRI and PstI:
(SEQ ~ NO.5) 5' AATTCCCGGG TACCAGATCT GAGCTCACTA GTCTGCA 3' (SEQ ~ NO.12) 3' GGGCCC ATGGTCTAGA CTCGAGTGAT CAG 5' The plasmid vector so obtained is deci~n~ted pCJXI-l.
SUBSTITUTE SHEET

VO 9~/22466 21 G ~15 4 PCT/US94/03755 Finally, the tetracycline rçsict~nce gene was replaced with a similar gene which had the lecognilion sites for restriction enzymes HindIII, BamHl, and SalIdestroyed by bisulfite mutagenesis. The following procedure was used to mutate the tetracycline resistance gene of pBR322. Plasmid pBR322 was cut with Hin~m~ then mutagenized with sodium bisulfite (Shortle and Botstein, 1983). The mutagenized DNA was ligated to form circular DNA, then cut with HindIII to linearize any plA~cmid that esc~pPd mutagenesis. This digestion mixture was usedto transform E. coli JM109 (Yanisch-Perron et al., 1985). Tetracycline-resistantcolonies were icol~tPd and c-hPcl~Pd for loss of the Hin-lTTT site in the tetracycline 0 recict~nce gene of the plasmid. A succescfully mutated plasmid was ~ecign~t~d pTl. A similar pr~lulc was followed to mutagenize the BamH1 site in pTl, yielding Flqcmid pT2. Plasmid pT2 in turn was mutagenized to remove the SalI
site, forming p1qcmid pT3. A ClaI-StyI fr~mPnt of pT3 carrying the mut~tP~
tetracycline rP~cict~nc~ gene was j~lqtP~ and used to replace the homologous fr~gmf-nt of pCJXI-1 to form pT3XI-2. The n~u~ d tetracycline reSict~nc~ gene still enc~d s for a rlJncl;on~l protein. Downstream of the tac l~lu~otel region, a polylinker was inh~duced which co~t~inC~ among other sites, BamH1 and KpnI
rest ictiQ~ sites useful for cloning genes for CA~ ess;on in E. coli as described below.
As in pT3XI-2, the cAyl~s~ion of the cloned gene col~l;.inil-g the pT88IQ
vector is driven by the tac promoter. Tr~n~!lqtion starts at the ATG of the unique NdeI lccognilion sequence CATATG (a do-wllsL~ NdeI site was el;",;n~ted so that this start site NdeI s4ucnce would be unique). There is a polylinker duwnsLl~ll of the NdeI site to f~rilit; tJ~ insertion of the desired gene. In addition, the XhoI Ldgl~lcnl c~n~ in~ the lacI region is re.pl~r~d by a trun~ted fragment which elilllin~tes the lacZ pr~"lo~l and the Opclator region which is a binding site for the lac repl~ssor. The lacI region in the replac~mf--nt also carries the lacIq mut~tion -- a single base ~ubsLiLuLion which results in an increase in lac ~ ssor pro~uctio~l (Muller-Hill et al., Proc. Nat'l Acad. Sci. ~IJ.S.A.) 59:1259-1264 3 o (1968)).

VO 94/22466 2 ~ 6 ~ ~ S !~ PCT/US9410375~

The spe~ific differences between pT3XI-2 and pT88IQ are as follows:
1. The cloning site region.
Between the EcoRI site upstream of the polylinker and the HindIII site at the downstream end of the polylinker, the following 135-mer sequence was ~ub~lituled (SEQ ID NO:6):
5' > CACAACG~i'l'l'l'~CCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGA
TATACATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCCC
GGGTACCGTCGACGAGCTCTTCGAACTAGTCCGCGGT > 3' lo This sequence cont~in.~ an NdeI site (underlined) at the start codon for ~;Aplesi~ion and a polylinker conl~ining ~ ;on sites for BamHI, XmaI, KpnI, SalI, SacI, BstBI, SpeI and SacII.
2. The dowllsLl~am NdeI site.
There is an NdeI site in pT3XI-2 about 2.4 Kb downstream of the cloning region. This site w~ eliminqted so that the NdeI site at the start codon ~ desc~ibed above w~ unique in pT88IQ. The site was changed from 5' > CATATG > 3' to 5' > CATATATG > 3', el;...h-~;ng the NdeI recognition sequence.
3. The lacIq region.
2 o The region in pT3XI-2 b~ ~n the two XhoI sites conl~il-ing the lacI region was rep~ by the 1230 base sequence shown below:
lacIq sequence of pT88IQ (1230 BP) (SEQ ID NO:7) CCATGGCTGG TGCCTAATGA GTGAGCTAAC TCACATTAAT
TGCGTTGCGCTCACTGCCCG ~ CAGTC GGGAAACCTG
TCGTGCCAGC TGCATTAATG AATCGGCCAA CGCGCGGGGA
GAGGCGGTTT GCGTATTGGG CGCCAGGGTG ~llll-lCTTT
TCACCAGTGA GACGGGCAAC AGCTGATTGC CCTTCACCGC
CTGGCCCTGA GAGAGTTGCA GCAAGCGGTC CACGCTGGTT
TGCCCCAGCA GGCGAAAATC CT~1-ll(3ATG GTGGTTGACG
GCGGGATATA ACATGAGCTG TCTTCGGTAT CGTCGTATCC

WO 9~/22466 21~ 01~ ~1 PCT/US94/03755 CACTACCGAG ATATCCGCAC CAACGCGCAG CCCGGACTCG
GTAATGGCGC GCATTGCGCC CAGCGCCATCTGATCGTTGG
CAACCAGCATCGCAGTGGGA ACGATGCCCTCATTCAGCAT
TTGCATGGTT TGTTGAAAAC CGGACATGGC ACTCCAGTCG
CCTTCCCGTT CCGCTATCGG CTGAATTTGA TTGCGAGTGA
GATATTTATG CCAGCCAGCC AGACGCAGAC GCGCCGAGAC
AGAACTTAAT GGGCCCGCTA ACAGCGCGATTTGCTGGTGA
CCCAATGCGA CCAGATGCTC CACGCCCAGTCGCGTACCGT
CTTCATGGGA GAAAATAATA ~ lGATGG GTGTCTGGTC
AGAGACATCA AGAAATAACG CCGGAACATT AGTGCAGGCA
GCTTCCACAG CAATGGCATC CTGGTCATCC AGCGGATAGT
TAATGATCAG CCCACTGACG CGTTGCGCGA GAAGATTGTG
CACCGCCGCTTTACAGGCTT CGACGCCGCTTCGTTCTACC
ATCGACACCA CCACGCTGGC ACCCAGTTGA TCGGCGCGAG
A'l-l-lAATCGC CGCGACAATTTGCGACGGCG CGTGCAGGGC
CAGACTGGAG GTGGCAACGC CAATCAGCAA CGACT~'l-l-l~
CCCGCCAGTT ~l-l~'l'~CCAC GCGGTTGGGA ATGTAATTCA
GCTCCGCCAT CGCCGCTTCC A~ll-l-l-lCCC GC~'l-l'l-l'CGC
AGAAACGTGG CTGGCCTGGTTCACCACGCG GGAAACGGTC
TGATAAGAGA CACCGGCATA CTCTGCGACA TCGTATAACG
TTACTGGTTT CACATTCACC ACCCTGAATT GACTCTCTTC
CGGGCGCTAT CATGCCATAC CGCGAAAGGTTTTGCACCAT
TCGATGGTGT CGGAATTAATTCAGCCATGG
This s~sliluled region el;-.-in~t~s the lacZ p~llloter and the opelator region which is a binding site for the lac l~l.,ssor. It also cont~in~ the lacIqmu~tion which causes an increase in lac l~lessor synthesis (Muller-Hill et al., ~YJ?EI-)-The IGFBP-1DNA sequence was i~ol~d from plasmid pJU1021 by stioll with the restriction e,LG~",es ~ I and T-Tin-lTTT and purified by agarosegel elec~r~phol~,s;s using NA-45 paper (Sr.hlPichPr and Schuell, Keene, NH), following the manufacturer's instructions. The i.~ol~ted IGFBP-1DNA fragment was cloned into pl~mi~ pT88IQ that had been digested with XbaI and HindIII and ~o g~/22466 216 01 ._,J ~ PCTIUS9~103755 gel-purified as above. A correctly r~or.s~ cted plasmid was named pT88IQ:IGPBP-l. The 5' oligonucleotide primer (IGFBP-1-5') used in the PCR
mutagenesis reaction has the sequence 5' CCCTCTAGAAATAATITTGTTTAACTTTAAGAAGGA 3' (SEQ ID NO.: 8).
The 3' oligonucleotide primer (IGFBP-l-C98) has the sequence 5' CAGGAGCTCCTCCTCAGTTATCTCCGTGCTCTCTGGGCATTCAGGGCTCC
CTGCCTCTGCAGCATGGGG 3' (SEQ ID NO.: 9). The PCR was pGlro--,-ed in a 50 ~1 reaction IlliAlU~ COnlA;ninf~ 10 mM Tris-HCl pH 8.3, 50 mM KCL, 2.5 mM MgCl2, 0.001 % gelatin, 500 ~M each of dATP, dCTP, dGTP and TTP, 30 lo picomoles each of the IGFBP-1-5' and IGFBP-l-C98 primers, 1-10 ng pT88IQ:IGFBP-1 plasmid DNA and 5 Units "AmpliTaq" Taq DNA polymerase (Perkin-Elmer Cetus, ~ c~d by Roche Molec~ r Systems, Inc., Branchburg, NJ). The PCR contliti~nC were an initial 3 min incub~tion at 96~C, 35 cycles of (96~C for 1 min, 66~C for 1 min, 72~C for 1.5 min) and a final 10 min incubationat 72~C.
The C101 mutein was made using an agarose gel-purified DNA fragment co~ ining the wild type IGFBP-l coding s~qucncG as the starting ~çmpl~te DNA.
The IGFBP-l coding sequence was obt~ined by ~1ig~ostin~ pl~cmid pJU1020 with ~sLeI and ~in~m and purifying the appfo~ e 0.8 kb IGFBP-l-coding DNA
fragment by agarose gel elecl,uphoresis. The 5' oligonucleotide primer used in the PCR mutagenesis reaction was the same as was used to construct the S98C
mutein (IGFBP-1-5'). The 3' oligonucleoti~e primer (IGFBP-l-C101) hac the se~ucl oe 5' CCCGAGCTCCTCCTCAGTTATCTCCGTGCACTCTGGGCTTTCAGGGCTCC
CTGC 3' (SEQ ID NO.: 10) The PCR was pe~ro~mGd in a 100 ul reaction IllLXlUlG cQnl~ini~ 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgC12, 0.001 % gelatin, 200 uM each of dATP, dCT~, dGTP and TTP, 20 picomoles of the IGFBP-1-5' and IGFBP-l-C101 primers and 2.5 Unit. of "AmpliTaq" Taq DNA poly",tl~sc. The PCR cQn~itionC were 30 cycles of (95~C for 1 min, 50~C
for 1 min, 72~C for 1 min) followed by a 10 min incub~l;on at 72~C.
After the PCR, the reaction n~ ulGs were passed through ChromaSpin 100 columns (ClonTech,Palo Alto,CA, catalogue number K1332-2) to remove wo 94/224~6 216 01~ 4 PCT/US94/03755 nucleotides and uninco,~ldled DNA primers. The DNA fragments were digested with XbaI and SacI and bands of the correct size (approximately 0.43 kb) purified by agarose gel electrophoresis as described above. The purified DNA fragments were ligated to XbaI + ~I digested pT88IQ:IGFBP-1 plasmid DNA. The ligation ~ lu~cs were used to transform E. coli strain DH5 alpha (available formClonTech Labo,dto,ies, Inc., Palo Alto, CA) and plated on LB agar plates cont~ g 50 ug/ml ampicillin. Plasmid DNAs were pr~ared from several colonies reslllting from each transformation and sequenced on both strands across the inserted region. A plq~mid with the correct sequence was select~d for each mutein. They are named clones C101-3 (C101 mutein) and C98-12 (C98 mutein).
The mutated IGFBP-l genes were then transferred back into plasmid pT5T
(Fi~nbP.g, S.P. et al., Nature, 343: 341-346 (1990). This was done by digesting pl~mi~ DNA from clones C101-3 and C98-12 with NdeI and HindIII, gel-pu,irying the appro~ le 800 bp band cQn~inin~ the mutant IGFBP-1 gene, and ligqtin~ them to pT5T plq~mid DNA that had been digested with the same restrictiQn enzymes. The ligqtio~ u-.,s were used to transform E. coli strain BL21/DE3 and plated on LB agar plates c~n~ining 50 ug/ml ~mp-~illin. Plasmid DNAs were ~ d from several colonies res~lting from each transformation.
Clones with the correct s~u~.~ces were named pT5T:IGFBP-1-C98 (C98 mutein) and pT5T:IGFBP-l-C101 (C101 mutein).
B. Preparation of Washed Inclusion Bodies E. Coli cells eAl,-~s;ng either the C98 or C101 mutein were grown in a 10 liter Çe.lll n~r. Cells oblained from the fer,llcnl~. were resusp~nded in breaking buffer (50 mM Tris, 25 mM NaCl, 1 mM Dithiot}~;lol (nDTT"), pH 7.5) at a ratio of 6ml buffer / g cells. The cells were disrupted at 10,000 PSI using a French pr~ -e cell. The sll~pencion was cenl-irùged at 17,700 x g for 30 I".nules. The pellet, which cont~in~ the inClusion bodies, was washed by resnspen~ion in the breaking buffer and ,~c~nt~;ruged at 17,700 x g. The washed and ,~ .iruged pellet can be stored frozen until pr~c~s~;ng. Appro~illlately 80% of the protein con~in?~d in the pellet was m~ltPin.

VO 94122466 PCT/US94/0375~
- 2~6~1';4 C. Mutein Refolding Each mutein was first denatured by solubilizing the pellet in 6M Guanidine HCl, 50 mM Tris, 6mM DTT, pH 7.5 (lg pellet for every 10 ml buffer) using a cell homogeni~r.
Refolding was iniL;AnP~ by adding oxidized glutathione ("GSSG") to the solubili~d pellet to a final concentration of 23 mM. The solution was incubated at room te",peldlule for 15 minutes and then diluted gradually with 50 mM Tris, pH 9.7 to a final protein concentration of 100 ~g/ml and a final guanidine con~ntration of 0.6M. The protein concer,tldLion was deLe.l"ined by the o Coomassie Blue protein assay. (Pierce, Rockford, IL). Cysteine and phenyl-melhAne~ lfonyl fluoride were then added to final concentrations of 5.6mM and lmM, l~p~clively. The refold solution was ;nc~lb~ed at 4~C overnight.
Refolding was monitored by analyzing a 100 ~l aliquot of the refold solutinn on a C4 reverse phase column (RP-4 lX250mm, Syllchl~"l, Lafayette, In). The C4 column was equilibrated with 2% ar~to~ ;le (CH3CN), 0.05%
I.;nuoli-'Pt;c acid ("TFAn). The 1O0~L1 aliquot of the refold solution was injected onto the equilibrated column and eluted using a flow rate of 0.25 ml/min with a linear grA~liPnt to 60% CH3CN, 0.05% TFA, ch~nging buffer B at 2%/min. For each mutein, the refolded protein eluted as a sharp peak approY-im~tely 2 l~inules earlier than the reduced denaluled but non-refolded proteins.
D. Purifîcation of Refolded Muteins The refold solution was c4n-~nl.~t~ a~ ely 10 fold with an Amicon SlOY3 IllCmbl~llle which has a 3kDa cutoff (Amicon division of WR
Grace and Co., Beverly M~ çku~ll,) and dialyzed into 20 mM sodium pho~hAte, pH 6Ø The dialyzed solution was ceht,ifuged at 17,700 x g for 30 es and the ;~ 5t;~ was filtered through a 0.2 micron filter. The filtered protein was loaded at 20 ml/minute onto a Q-Sepha~se anion ~Yç~nge column (Sx30cm, Pharmacia Riote~ll,Piscataway, NJ) previously equilibrated with 20 mM
Sodium Pl.osyh~ pH 6Ø The bound protein was eluted with a linear gradient (5 column volumes) to 20 mM Sodium l,ho~.h~e, O.5M NaCl, pH 6.0 at a flow rate of 20 ml/min. 25ml fractions were C~ ted. Each mutein eluted at app~ tp~ly 0.2-0.25M NaCl. Fractions were analyzed on a C4 reverse phase 21601~-3 ll WO 9~122466 PCT/US94/03755 ~ column (RP-4 lX250mm, Synchrom, Lafayette, IN) using the same conditions desclibed above for ~I~oniloling refolding or by SDS-PAGE.
Fractions co~ inil~g the refolded mutein (fractions eluting at 0.2 - 0.25M
NaCl) were pooled and dialyzed into 20 mM Tris HCI, lM NaCl, pH 7.5. The s dialy~d m~tPn~l was loaded at 25 mliminute onto a Butyl Sepharose (Supelco, Bellefonte, Pa) hydrophobic interaction column (5 x 8 cm Pharmacia Biotech,Pis~t~way, NJ) previously equilibrated with 20 mM Tris HCl, lM NaCl, pH 7.5. The bound protein was eluted with a linear gradient (10-15 column volumes) to 20 mM Tris, 0 NaCl, 25% eth~nol, pH 7.5 at a flow rate of 20 ml/min. Each mutein eluted as an asymmetrical peak at ap~io~imately 15-20%
ethanol.
1O0lL1 aliquots of the protein-conl~;l-ing fr~~tion~ (eluting at 15-20%
ethanol) were analyzed on a C4 reverse phase column (RP-4 lX250mm, S~..chro--" I~f~clle, In) using the same con~litiQn~ described above for ",~nil-.;ng refolding or by SDS-PAGE. Fr~tion~ co~ .n;l-g purified muteins were pooled and c~ tPd to apylu~ y 0.8 mg/ml and dialyzed into 20 mM Tris, 250 mM NaCl, pH 7.4. The purified refolded mutPin~ were assayed for bioactivity as dPsc~ibed in F-~m~'- 9.
FY~m~le 8 PEGylation of IG~BP-l Muteins The C98 and C101 muteins were PEGylated using monomethoxy-PEG, with an average mol~~ r weight of 20kDa, having a thiol-specific m~lPimidP
reactive group ~tt~rh~d (CH30-(CH2CH20)n-NHCOCH2CH2-N ), where n is the number of monomeric units. ~dlion and use of other suitable PEG-m~lPimi~e ,~gent~ is t~ llcoe~ in PCT Applic~tion P~lbli~tion No. WO92/16221, incol~lated herein by l~fe~nce.
During refolding, the ~sliluled cysteine residue (CYS 98 or CYS 101 in C98 and C101, le~ ely) can form mi~ed ~ lfi~les with glut~thjone, to form Cys-S-S-GSH, or with cysteine, to form Cys-S-S-Cys. Accordingly, there may be 3 o no free thiol available for reaction with the PEG reagent. Therefore, the punfied muteins were partially reduced prior to reaction with the PEG reagent.

~0 94/22466 2 ~. ~ a ~ PCT/US94103755 The partial reduction was accomplished by reacting the purified mutein (0.45mg/ml) with DTT at a molar ratio of DTT to protein of 5.625 to 1 in 20 mM
Tris, pH 7.4, 250 mM NaCl for 2 hours at room ~elllpCldtUrC. Reduction was stopped by acidification to pH 5.5. The Dl~ was removed by dialysis into 10 s mM sodium acet-qtP, pH 5.5.
The partially reduced muteins were each reacted with the PEG reagent, at a 4 to 1 molar ratio of PEG to protein (final protein concentration of 0.33 mg/ml) in 15mM sodium q-r~t-q-te, 26mM sodium phosphate, pH 7.0, 120mM NaCl for 4 hours at room tel"pcldlur~. SDS-PAGE analysis of the reaction mixture showed lo approximately 50% of the partially reduced muteins were converted to a mono-PEGylated species (C98-PEG, C101-PEG) having an a~lu~dmate ~l.arc.-t molcculqr weight of about 67 kDa. The large al"~a,enl mo~ lqr weight was due in part to PEG h~t~.ac~ions with the gel.
The reaction ll~lul~ was adjusted to 20 mM sodium phosph-qte pH 6.5.
~A Q Sepha~se anion e~c-hqnge column (5 x 10 cm, Pl,~,l,acia Biotech, Pisc~l~way, NJ) was equilibr~tP~ with 20 mM sodium phQSphqte pH 6.5 and the reaction ll~lul~ was loaded at at 20 ml/min~lte~ The bound protein was eluted with a linear gr:~iPnt (10 column volumes) to 20 mM sodium ph~h~te, lM
NaCl, pH 6.5 at a flow rate of 20 ml/min. Each of the C98 and C101 PEGylated ... ~-leil~s eluted at appro~i.. ~tPly 0.2 M NaCl. 25 ml fractions were collected and aliquots were analyzed by SDS-PAGE. Fractions C~ g PEGylated C98 or C101 which gave a single p,~to",inant band on non-re~luçing SDS PAGE
coll~n~;ng to the PEGylated mutein were pooled and assayed for Bioactivity as described in F~-- ple 9-Exa~ )le 9 IGFBP-1 and its muteins inhibit IGF-l stimulated growth of 3T3 cells A crystal violet dye assay was used to measure cell proliferation. Assays were pc.rol",ed in 96 well gelatin-coated plates. Balb/c 3T3 fibroblasts were plated at 25,000 cells/well in 200 ~11 of serum-free DMEM (Dulbecco's m~lific~tion of Eagle's media, ~eAi~rh, Herndon, VA) and 0 - 850 ng/ml IGF-1. Cells were incub~ted for 72 hours at 37~C . At this time, the media was wo g~/22466 21 f~ 01 S 4 PCT/US9~/0375~

replaced with 150 ~l of 0.2% cr,vstal violet, 10% formaldehyde, 10 mM potassium phosph~tP pH 7Ø After a 20 minute incub~tion at room le-~-pe.dlllre, the wellswere washed 3 times with phosphate buffered saline ("PBS"), and the cell-bound dye released by incubation with 200 ~l/wéll of 50% ethanol/0. lM sodium citrate,pH 4.2. Abso,bance at 570 nm was read the next day. The results, set forth in Table 13, show that recombinant IGF-l stimulates proliferation of 3T3 fibroblastcells in a dose depPndent manner. ~xim~l proliferation occurred at a IGF-l concentration of about 20-60 ng/ml. The ED50 was approximately 5-20 ng/ml.
The effect of IGFBP-l, the C98 and C101 mutPin~ and the PEGylated muteins on IGF-l-stimulated proliferation of 3T3 fibroblasts was determined by co-incuh~ting the cells with a set amount of IGF-l and increasing amounts of thebinding proteins. Balb/c 3T3 fibroblasts were plated at 25,000 cells/well in 200 ~l of serum-free DMEM c41 ~-ning 21ng/ml IGF-l, and varying amounts of IGFBP-1 and the various ~Uleinc (0 ng/ml - 11,520 ng/ml). The cells were incub~ted for an ~A~litiQn~l 72 hours and l,ç~ d as desçribe~ above.
The results show that the bioactivities of both the unPEGylated and PEGylated C98 and C101 mlltçinC are co---p~ le to the activity of wild type bilant IGFBP-l (Tables 14-18). The conc~ A~ion of IGFBP-l required to inhibit 50% of the activity of 21ng/ml IGF-I (IC50) under the conditions described is a~rv~ill-a~ly 200 - 300 ng/ml for wild type IGFBP-l, the IGFBP-l muteins, and for PEGylated IGFBP-l.

'1VO 94122466 21~ a 1 s S PCT/US94/03755 -IGF-I Absorbance* S.E.**
(ng/ml) (570nm) 850.00 1.587 0.076 212.50 1.627 0.053 53.13 1.662 0.039 13.28 1.495 0.029 3.32 1.112 0.037 0.83 0.920 0.019 0.21 0.915 0.020 0.05 0.897 0.014 0.01 0.862 0.011 0.00 0.781 0.016 * A~so,l,al~ce values are ~e mean of 12 samples ** S.E. ~ ndanl error of the mean.

SUBSTITUTE SHEET (RULE 26) WO 94122466 21~ 0 ~ 5 ~ PCT/US94103755 TABLE 15.
Dose-dependent inhibition of 3T3 fibroblast gro~th by the C98 mutein C98 mut~in ~A~ L ~ce 570nm ~S.E.
~ng/ml) ~.
11520.00 0.941 0.085 2880.00 1.001 0.033 720.00 1.161 0.028 180.00 1.580 0.013 45.00 1.775 0.023 11.25 1.787 0.006 2.81 1.797 0.016 0.70 1.747 0.011 0.18 1.818 0.004 0 1.800 0.016 * Abso~ ce values are ~e mean of triplil~t~ samples ** SE l~,~lls i.t~nda~d error of ~e mean.

SUBSTITUTE SHEET (RULE 26) wo 9~/22466 216 ~ ~ ~ '1 PCTIUS94/03755 Dose-dependent inhibition of 3T3 fibroblast growth by the C101 mutein C101 mutein Absorbance 570nm S.E.
(ng/ml) 11520.00 0.930 0.058 2880.00 1.032 0.033 720.00 1.108 0.052 180.00 1.413 0.052 45.00 1.656 0.057 11.25 1.620 0.039 2.81 1.641 0.064 0.70 1.593 0.015 0.18 1.584 0.050 0 1.516 0.052 Absc,~ ce values are the mean of ! - ;p1;~ e samples. SE n,p~nts standard error of the mean.

SUBSTITUTL SHEET (RlJLE 26) 2161)1S '~

Dose dependent inhibition of 3T3 fibroblast growth by the C98 PEGylated mutein C98-PEG mutein Absorbance 570nm S.E.
(nglml) 11520.00 0.915 .015 2880.00 0.957 .019 720.00 1.049 0.044 180.00 1.302 0.034 45.00 1.473 0.028 11.25 1.595 0.031 2.81 1.620 0.028 0.70 1.581 0.019 0.18 1.585 0.015 0 1.699 0.024 Dose-dependent inhibition of 3T3 fibroblast growth by the C101 PEGylated mutein C101-PEG mutein Absorbance 570nm S.E.
(ng/ml) 11520.00 0.956 0.041 2880.00 1.~08 0.016 720.00 1.082 0.027 180.00 1.299 0.030 45.00 1.505 0.061 11.25 1.596 0.020 2.81 1.589 0.011 0.70 1.564 0.003 0.18 1.577 0.020 0 1.614 0.016 SUBSTITUTE SHEET (RULE 26) '--''VO 94/22466 21~ ~ ~ S 4 PCT/US94/03755 Absoll,ance values are the mean of trip~ te ~mples~ SE l~nts standard error of the mean.

Dose-dependent inhibition of 3T3 fibroblast growth by wild type IGFBP-1 -WT IG~BP-lAbsorbance 570nn S.E.
(ng/ml) 11520.00 0.74 0.015 2880.00 1.799 0.036 lo 720.00 1.015 0.010 180.00 1.213 0.026 45.00 1.351 0.045 11.25 1.432 0.048 2.81 1.475 0.074 0.70 1.538 0.020 0.18 1.530 0.039 0 1.583 0.022 r.;...Wl~ 10 Pharmacokinetics of IG~BP-1 and PEGylated IGFBP-1 Four male Sprague Dawley rats were used to det~,.litle phs.~ ~;n.~;c . Two rats were injected with an I.V. bolus of lmg/kg recombinant human IGFBP-l and two rats w_re injf~t~d with an I.V. bolus of lmg/kg of the PEGylated IGPBP-l C101 mutPin The C101 mutein had been plG~ared and PEGylated as des-cnbed in F~amplçs 7 and 8. Tail vein blood samples were taken 2s at 0.016, 0.083, 0.033, 0.075, 1.5, 2, 3, 5, 6, 8, 10, 12, 24, and 48 hours after the injection. The blood was cQll~t~d in EDTA-coated tubes and c~ iîuged to collect the plasma r.,.~ n. The conc~ ation of IGFBP-l in the plasma samples was dG~I.~ined by ELISA using the Medix Pi~çhPmi~ (K~..ni~in~l~, Finland) IGFb~l test kit, Catalog No. 10831ETMB.
3 o The plasma cQn~nt~dtions for the two rats in each test group were averaged and fit to a two or three c;~ Onf--~t;~l curve using Rstrip II (Miclullldt~l Soflwal'e, Salt SUBSTITUTE SHEET (RUEE 26) WO 94/22466 21~ 01 S 4 PCT/US94/03755 Lake City, Utah). Data from the fitted curves app~ in Table 20. ELISA-dete~t~hle plasma IGFBP-l disappea ed triexponentially and biexponentially afterinjection of the wild type IGFBP-l and PEGylated C101 mutein, ~s~ /ely.
Using the fitted curve, standard pharmacokinetic parameters were calculated as set forth in Pharmacokinetics~ C~ li, M., and Perrier, D.; Swarbrick ed., 1975.
These ~IAIll tG~ appear in Table 21. The results show that PEGylation improves the pharnl~r~kinetic pelfoll.,al~ce of IGFBP-l by increasing the circulation time because of decreased plasma clearance.

10Time/hr 1 ~IGFBP-l (ng/ml)l~a PEG IGFBP-1 (ng/ml) 0.017 9057 + 410 18120 ~t 430 0.108 4937 i 150 17060 i 280 0.363 2032 :~ 59 15340 i 2000 0.751 1060 i 32 14550 :~ 1100 15 1.50 385.0 ~ 35 12920 + 12 2.03 261.0 ~ 20 11590 i 510 2.99 142.7 + 0.85 10110 ~ 150 4.97 39.84 ~ 3.3 8503 i 400 5.97 20.19 + 1.8 7224 ~ 590 20 7.97 ND* 4916 _ 250 9.97 ND 3196 + 180 12.0 ND 2258 i 130 26.0 ND 450.0 i 30 50.0 ND 83.24 + 1.2 25*Not d~te~ ble SUBSTITUTE SHEET (RULE 26) W O 94t22466 ~ L ~ d PCTrUS94/03755 T~iBL E 21 wild type PEGylated parameter$ IGFBP-1IG~BP-1 initial distribution volume, L/kg 0.095 0.063 steady-state distribution volume, Llkg 0.19 0.074 s plasma c~ ~nc~, mL/min/kg 6.2 0.15 fast (initial) half-life, min 2.9 in~.. f~iq-lf, half-life, hr 0.24 3.9 slow (t~lll.. nal) half-life, hr 1.1 13 plasma mean reci~1Pnr~ time, hr 0.52 8.2 *Mean values for two rats per group.

IGFBP-1 inhibits restenosis in the rat IGFBP-l was tested for cap~cily to modify the ploli~ ,~nc~ in carotid arteries of rats following balloon ~ ;op'~rty. ~ t~n Sprague Dawley rats weighing ~ ly 375g und~... _nt S~E,_,y to ;.. pl~nt jugular cq~ in the right jugular vein. One week hter the c~h- b~ ~ were tested for patency and c~nl;nuo~c saline i~ru~on was in;l;~ via a tell~c~d ir.ru~on system. The tethered infusion system is set forth in Francis, P.C., et al., "CO~ VQU,S Intravenous I~ru~ion in Fisher 344 rats for Si~ ~onth~ A Fe~Q;~ility Study,~ To~icolo~y Methods, Vol.2, pp.l-13 (1992), ~ifi-qlly incolpul~ted herein by lef~nce. Three days later, all qnimqlc had balloon al~giopla~ly surgery via n arte~iotomy int.i~;~)n in the left PYtornql carotid as set for~h in F~1mqn, E.R. and Karnovsky, M.J., Circulation, Vol 89, No.2, pp. 77~776 (1994) s~ifir-qlly inc~l~laled herein by l~f~ ce. A 2F
I~og~LIly balloon C~tl.--tY-~ Edwards ~ 5, Santa Ana, Calif.) was 2 5 adv, nced to the aortic . rch and pulled back with the balloon ~ ten~ with suffic~ent air to gen- ~, tP re~ t-q-nc~ and denude the e--do1l.r];.,... This l,r~cedulc was l.~ ted 6 times to insure suffi~.ent ~--~e to the artery n~ r to induce a proliferative rc~ ~ in the vessel wall. The eYt~rnql carotid was then ligated. The qnimql~ were divided into 2 groups and Llr~ were ;~ ~ immPAi~tely after angioplasty and SUBSTITUTE SHEET (RULE 26) WO 94/22466 211~ 4 PCT/US94/03755 continued for 14 days. Group 1 (N=9) consisted of control ~nim~l~ which were infused IV with isotonic saline (0.25 ml/hr). Group 2 (N=10) animals were treated with IGFBP-l by continuous IV infusion at a dose of 179~g/kg/hr. Blood samples were collected from the tail vein at 3, 9 and 14 days post angioplasty for s dete""ination of plasma levels of IGFBP-l. The concentration of IGFBP-l in the plasma sa-llples was deLel",ined by ELISA using the Medix Biochemica (~llni~in~n, Finland) IGFbp-l test kit, Catalog No. 10831ETMB. Infusions were discontinued atthe time of blood collection (approximately 15 to 30 minutes).
At the tC~ n~;nn of the study on day 14, c~ e~ patency was confirmed by Brevital testing as set forth in Francis et al. The ~nim~ls were ~nP.stheti7P~ with a combination of Ace ~",azh~e, Rompun and Kes~mine and perfused with 10%
neutral burf~ formalin via cardiac punclule. Both carotid arteries were removed and placed in formalin for 2 a~ition~l days prior to proc~Pscing for paraffin emhed~ing. Three s~cti~nc of carotid, cnCG~p~ccing almost the entire length of the lesion, were embPdded from each animal to insure that all areas of the tissue were evaluated. Adtlition~l tissues (lung, heart, liver, kidney, spleen and adrenals) from all ~nim~lc were collPct~d for hictolQgic ev~ tion to deL~""ine systemic effects (if any) of c~ntinuouC infusion of IGFBP-l. The carotid artery sectionc were stained with h~-n~t-J"ylin and eosin, ~ccon~s trichrome and tol~ ine blue. Other tissues were2 o stained with hc."atoAylin and eosin only.
Mean plasma levels of IGFBP-l at the ~,..-;n-l;on of the study were 3.69 +
0.64 ~g/ml.
The effects of treating the ~nim~lc with IGFBP-l were dete""ined by gross scoring, and by meas~l"~,n~ of n~intim~ (~Lm and pixels), ne~il.L;...~ plus media - 2s (pixels), and media (pixels). The ratio of n~inl;.. ~ (pixels) to media (pixels) was also c~lcul~tP~d. Those scores and ~ ."cr,t~ appear in Table 22. Wilcoxon Rank-Sum (Mann-Whitney U) tests were ~,r~,l"~ed for each of those six response measures and p-values de~""hled using the table set forth in Natrella, M.G., Experiment~l St~tistiss. N~tion~l Bureau of Standards Handbook 91, p. T-80 (1967).
The p-values from the Wilcoxon Rank-Sum (Mann-Whitney U) tests are reported below. The data show that IGFBP-l si~nific~ntly reduces restenosis in the rat.

wo 94/22466 2 ~ ~ a ~ ~ ~ PCTIUS94l03755 A gross qccPccmPnt of treatment-related effects was done by qcci~ning scores of 0, 1, 2, 3 or 4 to the lesioned carotids with 0=no observable thickening and 4=severe thir~P-ning. The mean (+ standard error) scores from treated and control ~nimAlc were 1.5 + 0.27 and 2.67 + 0.24, l~s~;li~ely. These values were found to be st-qtictir-q-lly ~cigllifir-qnt (p < 0.05). In this gross qCc~sc~ nt~ the inhibition of thic~Pning in the IGFBP-l treated qnimqlc was 44%.
The nP~intimq. was also measured. The ~ict-qnce from the medial side of the neointima to the luminal side of the neointima was measured at 4 points opposite and perpen~icul-qr to each other and the average of 3 sPctionC de~,lllined. The mean +
SE of neointimq. in ~m for treated and control qnimqlc was 78.50 i 10.68 and 139.33 + 8.91, ~spec~vely (p ~ 0.01). This measurement also showed a 44% inhibition of thic1~Pnin~ in the IGFBP-1 treated ~nimAlc Image analysis using the Image 1 System (available from S & M Microscopy, Colorado Springs, CO) was used to dele.lll~e the area of nP~intim~q, plus media,n~o.i.l;.. A alone, and media alone for treated and control qnimAlc (3 se~tinnc/animal).
The area (mean i SE) of n~oil~t;...7 plus media in pixels for treated and control qnimqlc was 25,160.30 i 1817.42 and 35,271.11 i 1,403.16"~ /ely (p <
0.01). Using this analysis, treAtmPnt with IGFBP-l reduced the n~intimql thirl~nPcc by 29%. The mean area in pixels of ~ t;...q alone was 14,015.40 i 1,834.24 forthe treated qnimqlc and 23,119.11 + 1,200.39 for the control qnimqlc or 39%
inhibition of thirl~pnin~ in the IGFBP-1 treated qnimqlc (p < 0.01).
Finally, the ratio of n~il.t;...7q to media was c-q-lrul~ted. This parameter also ..,nfi~ tPd the ben~firi~l effect of tre~tm~nt with IGFBP-1. The ratio in treated ~nimAlc was 1.25 i 0.17 and 1.91 i 0.13 in the control AnimAlc Using this ratio,inhibition of lestt~nocic was 35% (p < 0.05). The ratio of n~ointim7 to media isgenerally the most accepled method of ev~ Ating l~ -.f nt-related effects in this rat model. F~ mAn and Karnovsky, Circulation~ Vol. 89, No. 2 (1994).

WO 94/22466 21~ ~1 S ~ PCT~US94/03755 i TABLE 22 ANn~IAL GROSSINTIMA- MED+ ~ INT~IA MEDIA INT:MED
NO AREA llm T PDCELS PD~ELS IA
GROUP PD~ELS
2C 3 168 38693 26473 12220 2.2 3C 2 87 28927 16199 12729 1.27 4C 4 147 36516 23357 13159 1.78 6C 2 161 33042 23822 9220 2.6 7C 3 141 43016 28659 15357 1.83 8C 2 127 33144 21367 11777 1.81 9C 3 168 35767 24672 11095 2.23 lOC 2 114 31331 20312 11019 1.84 llC 3 141 37004 23211 13794 1.66 MEA~ 2.C7 139.33 35271.11 23119.11 12263.33 1.91 SE 0.24 8.91 1403.16 1200.39 593.32 0.13 21BP 2 121 29893 19613 10280 1.9 22BP 0 27 17316 4755 12561 0.37 23BP 2 74 23501 13717 9785 1.39 24BP 2 87 25148 13986 11161 1.25 25BP 2 87 29361 17851 11510 1.52 26BP 0 47 18330 8432 9898 0.83 27BP 2 34 17132 5676 11456 0.5 28BP 2 127 31113 20359 10754 1.88 29BP 2 94 30737 18021 12896 1.4 30BP 1 87 29072 17744 11328 1.5 MEAN 1.50 78.50 25160.30 14015.40 11162.90 1.25 SE 0.27 10.68 1817.42 1834.24 327.23 0.17 wo 94/22466 21~ a 1 ~ ~ PCT/US94/03755 Although this invention has been described with respect to specific embodiments, it is not intended to be limited thereto and modifications made by those skilled in the art are considered to fall within the spirit and scope of the instant invention.

Claims (36)

What is claimed is:

1. A method of treating a patient having an IGF associated condition comprising administering a therapeutically-effective amount of IGFBP-1 or a modified form thereof.

2. The method of claim 1 comprising administering IGFBP-1 to said patient.

3. The method of claim 2, wherein the patient is administered an amount of IGFBP-1 sufficient to reach a concentration of IGFBP-1 in the range from about 0.1 µg to about 300 µg per ml in the bloodstream of the patient.

4. The method of claim 1 comprising administering a modified form of IGFBP-1 to said patient.

5. The method of claim 4, wherein the modified form of IGFBP-1 is IGFBP-1 attached to a polymer.

6. The method of claim 5, wherein said polymer is polyethylene glycol.

7. The method of claim 4, wherein the modified form of IGFBP-1 comprises 2 IGFBP-1 molecules, wherein said first IGFBP-1 is attached to one end of the polymer and said second IGFBP-1 is attached to the opposite end of the polymer.

8. The method of claim 1, wherein the IGF associated condition is selected from the group consisting of breast cancer, colon cancer, lung cancer, ovarian cancer, osteosarcoma, glioma, liver cancer, rhabdomyosarcomas, restenosis, acromegaly, obesity, diabetic nephropathy and diabetic retinopathy.

9. The method of claim 8, wherein the IGF associated condition is breast cancer.

10. The method of claim 8, wherein the IGF associated condition is colon cancer.

11. The method of claim 8, wherein the IGF associated condition is osteosarcoma.

12. The method of claim 8, wherein the IGF associated condition is acromegaly.

13. The method of claim 8, wherein the IGF associated condition is restenosis.

14. A pharmaceutical composition comprising IGFBP-1 or a modified form of IGFBP-1 in an acceptable pharmaceutical carrier.

15. The pharmaceutical composition of claim 14,comprising IGFBP-1 in an acceptable pharmaceutical carrier.

16. The pharmaceutical composition of claim 14, comprising a modified form of IGFBP-1 in an acceptable pharmaceutical carrier.

17. The pharmaceutical composition of claim 16, wherein the modified form of IGFBP-1 is IGFBP-1 attached to an inert polymer chain.

18. The pharmaceutical composition of claim 17, wherein the inert polymer chain is polyethylene glycol.

19. The pharmaceutical composition of claim 16, wherein the modified form of IGFBP-1 is IGFBP-1 attached to opposite ends of an inert polymer chain.

20. A method of treating or preventing restenosis comprising administering, to a patient in need thereof, a therepeutically-effective amount of a protein capable of binding IGF.

21. The method of claim 20, wherein said protein is IGFBP-1.

22. The method of claim 21, wherein said protein is a modified form of IGFBP-1.

23. The method of claim 22, wherein said protein is IGFBP-1 attached to a polymer.

24. The method of claim 23, wherein the polymer is polyethylene glycol.

25. The method of claim 23, wherein the modified form of IGFBP-1 comprises 2 IGFBP-1 molecules, wherein said first IGFBP-1 is attached to one endof the polymer and said second IGFBP-1 is attached to the opposite end of the polymer.

26. The method of claim 1, wherein the IGFBP-1 is not phosphorylated.

27. The method of claim 21, wherein the IGFBP-1 is not phosphorylated.

28. The method of claim 26, wherein the IGFBP-1 is recombinantly produced in E. Coli.

29. The method of claim 27, wherein the IGFBP-1 is recombinantly produced in E. Coli.

30. An insulin-like growth factor binding protein (IGFBP) which is not phosphorylated and which is capable of inhibiting the activity of IGF.

31. The IGFBP of claim 30, wherein the IGFBP is recombinantly produced in E. Coli.

32. The IGFBP of claim 30, wherein said IGFBP is IGFBP-1 or a modified form thereof.

33. A use of a therapeutically-effective amount of IGFBP-1 or a modified form thereof, for treating a patient having an IGF associated condition.

34. A use of a therapeutically-effective amount of IGFBP-1 or a modified form thereof, for the production of a medicament for treating a patient having an IGF associated condition.

35. A use of a therapeutically-effective amount of protein capable of binding IGF for treating or preventing restenosis in a patient in need thereof.

36. A use of a therapeutically-effective amount of protein capable of binding IGF for the production of a medicament for treating or preventing restenosis in a patient in need thereof.