CA2245635A1 - Cardiotrophin and uses therefor - Google Patents

Abstract

Isolated CT-1, isolated DNA encoding CT-1, and recombinant or synthetic methods of preparing CT-1 are disclosed. CT-1 is shown to bind to and activate the receptor, LIFR.beta.. These CT-1 molecules are shown to influence hypertrophic activity, neurological activity, and other activities associated with receptor LIFR.beta.. Accordingly, these compounds or their antagonists may be used for treatment of heart failure, arrhythmic disorders, inotropic disorders, neurological disorders, and other disorders associated with the LIFR.beta..

Description

CA 0224~63~ 1998-08-0 CARDIOTROPHIN AND USES THEREFOR
TECH~IICAL FIELD
This 5,l.pli-~;;..., relates to a cardiae hy~.uupl,y factor (also known as CT-I) for modulating eardiac - - funetion in the L~,aLI~L.It of heart failure, for modulating neural funetion in the Ll~,d~ ,.lt of neurologieal S disorders. and for llc;dLul-,.lL of a variety of other disorders related to a CT-I receptor, particularly the LIFR~.
BACKGROUND
~' Heart failure affects alJlJlu~-hlldLt~ly three million Americans, developing in about 400,000 each year.
It is currently one of the leading ~lmiccinn .~i~ nnc~c in the U.S. Recent advances in the ~ agr~,ent of acute cardiac diseases, including acute myocardial infarction, are resulting in an ~ andulg patient pop~ tior, that will eventually develop chronic heart failure.
Current therapy for heart failure is primarily directed to using angiotensin-converting enzyme (ACE) inhibitors and diuretics. While ~ ngin~ survival in the setting of heart failure, ACE inhibitors appear to slow the IJlu~l~,i.aiùn towards end-stage heart failure, and s~lhct~nti~l numbers of patients on ACE inhibitors have fimntjon~l class III heart failure. Moreover. ACE inhibitors cullaiah,l.lly appear unable to relieve symptoms in more than 60% of heart failure patients and reduce mortality of heart failure only by dlJ~JI u;~h-,ately 15-20%.
Heart u.~ -. is limited by the availability of donor hearts. Further, with the exception of digoxin, the chronic administration of positive inotropie agents has not resulted in a useful drug without ac~u...l.a..ying adverse side effects, such as increased arrhythmogen~cis sudden death, or other deleterious side effects related to survival. These dLri~.~ ... ;r~ in current therapy suggest the need for additional therapeutic a~ JIuaullcs.
A wide body of data suggests that pathological hy~,~. Llul~hV of cardiac musele in the setting of heart failure can be dcl~,t.,l iuui" l,Lala~.t~,. ;~d by dilation of the v.,.lLI i-,ulal chamber, an increase in wall tension/stress, an increase in the length vs. width of cardiac muscle cells, and an ~ecn~ ,;ing deerease in cardiac p~,~ rul l--a-~-,e and function. Studies have shown that the activation of physiological or c~ u.y h~ .LIu~ can be bPn~fi~ in the setting of heart failure. In faet, the effects of ACEhlllibiLula have been purported not only to unload the heart, but also to inhibit the p~tho' ~,=, I h~ lut)llic response that has been presumed to be linked to the localized renin-angiotensin system within the myu~ald;u-l Cardiac muscle hypertrophy is an illll~UI LalIL adaptive response of the hearL to injury or to an increased demandforcardiacoutput. Thishy~c-LIuphicresponseis~ la~it~ dbythereactivationofgenesnormally ,d during fetal heart d~i~,lop..,c.,L and by the ~ inn of adl~ulll~fic proteins in the absence of DNA
replication or cell division (Rockman et al., Circulation, 87:V1114-VII21 (1993~; Chien, FASEB J., 5:3037-3046 (1991); Shubeita et aL, .~. Biol. Chem., 265:20555-20562 (1990)).
On a molecular biology level. the heart functions as a syncytium of myocytes and su. . uwl-lillg support cells, called non-myocytes. While non-myocytes are primarily fibrobl~a(/ulc~ l.ymal cells, they also inelude ~n-lnth~ l and smooth muscle cells. Indeed, although myocytes make up most of the adult myocardial mass, they represent only about 30% of the total cell numbers present in heart. Beeause of their elose rel~tiQnch ip with eardiae myocytes in vivo, non-myocytes are eapable of i"n"~ g myocyte growth and/or development. This hlki.aLliull may be mediated directly through cell-cell eontact or indirectly via production of a paraerine factor.
Sueh ~oci~tinn in vivo is i~ ul L~.~L since both non-myocyte numbers and the extracellular matrix with which WO 97/30146 PCT/US97/0267~;

they interact are increased in myocardial hy~,~,. Llupl.y and in response ~o injury and infarction. These changes are ~Ccoci~tpd with abnormal myocardial function.
Cardiac myocytes are unabie to divide shortly after birth. Further growth occurs through h~ ,. IIUIJIIY
- - = of the individual cells. Cell culture models of myocyte h~e. Il u~.hy have been developed to understand better the . . ~ for cardiac myocyte hypertrophy. Simpson et aL, Circ. Res., 51 :787-801 (1982): Chien et al., FASEB J., 5:3037-3046 (1991). Most studies of heart myocytes in culture are designed to minimi7e cont~min~tir~nbynon-myocytes. See,forexample,Simpsonetal.,Cir.Cres.,50:101-116(1982);Libby,J.Mol.
CelL Cardiol., 16:803-811 (1984); Iwaki etal., J. Biol. Chem., 265:13809-13817 (1990).
Shubaita et aL, J. BioL Chem..265:20555-20562 (1990) do~llm~nt~od the utility of a culture model to 10 identify peptide-derived growth factors such as ....1.,ll.r!;..-l that can activate a hypertrophic response. Long et aL, Cell Regulation, 2: 1081- 1095 (1991) inv_;.Lig. -,d the effect of the cardiac non-myocytcs on cardiac myocyte growth in culture. Myocyte hy~ UuhiC growth was .l i....,i~l ~ in high-density cultures with increased numbers of non-myocytes and in co-cultures with increased numbers of non-myocytes. This effect of non-myocytes on myocyte size could be reproduced by serum-free medium corJrlitinnPd by non-myocyte cultures. The major 15 myocyte growth-promoting activity in the cultures was heparin binding. The ~. U~.,l L;eS of this growth factor were LullJ~alc;d to various growth factors known to be present in m~,u~,~udiu..., including fibroblast growth factor (FGF), platelet derived growth factor (PDGF~, tumor necrosis factor-alpha (TNF-a), and transforrning growth factor-betal (TGF-~ 1). The growth factor of Long et aL was found to be larger than these other known growth factors and to have a different heparin-Set,halua~i elution profile from that of all these growth factors except 20 PDGF. Further, it was not neutralized by a PDGF-specific antibody. The authors proposed that it defines a paracrine relationship ;Il~ ul LallL for cardiac muscle cell growth and d., ~ elo}J.~._..L.
Not only is there a need for an illll~lU ~ ,lIL in the therapy of heart failure such as congestive heart failure, but there is also a need to offer effective treatment for neurological disorders. N~,,u utl uphic factors such as insulin-like growth factors, nerve growth factor, brain-derived n_...uL~uphic factor, n_.l,vlluyllin-3, -4, and 25 -5, and ciliary n_~uLluuLc factor have been proposed as potential means for enh~n~ing neuronal survival, for example.asat..,dl,.,_.,Lforneurodc,5~ .aLivediseasessuchasamyorrophiclateralsclerosis,Alzheirner'sdisease, stroke, epilepsy, IT....I;..~IU~ disease, Pal~ UII'5 disease, and peripheral fie.llù,uaLII~ It would be desirable to provide an ~hinn~l therapy for this purpose.
In addition, there is a need for i~ f)n of and improvement in the therapy of diseases for which 30 cytokines, their ~ ; or agonists play a role. The IL-6 family of .,yI~illcS (IL-6/LIF/CNTF/OSM/IL- I I ) has a wide range of growth and ~lilr~lGIl~ia~iul~ activities on many cell types including those from the blood, liver, and nervous system (Akira et al., Adv. ~ .~1 54:1-78 (1993); Kieh;mntn et aL, Science. 258:593-597 (1992)). The biological effects induced by IL-6 and related proteins are mediated by a family of structurally similar cell surface receptors, the cytokme receptor family, that includes the receptors for growth hormone and 35 prolactin as well as for many cytokines (Cosman et al. ~ Trends Biochem. Sci, 15:265-270 (1990): Miyajima e~
aL,Ann Rev. ImmunoL, 10:295-331 (1992); Tagaet al., FASEB J, 6:3387-3396 (1992); Bazan, Immunol. Today, 11:350-354(1990)). ThelL-6receptorsubfamilyis~ ..p~ dofmulti-subunitc..,.,~ thatshareacommon signaling subunit, gp130 (Davis et aL, Curr. Opin. Cell BioL, 5:281-285 (1993); Stahl et al., Cell. 74:587-590 (l993);KichinlntnetoL~ Cell,7~:253-262(1994)). SomemembersofthelL-6cytokirlefarnily(1L-6andlL-ll) CA 0224~63~ 1998-08-0~

induce the homodimerization of gp 130 (Murakami e~ al., Science, 260: 1808- 1810 (1993); Hilton et al., EMBO
J.,13:4765-4775 (1994)), while others (LIF, OSM and CNTF) induce gpl30 h~hludil~.,. ro....aLiu., with the 190 kDa LIF receptor (Davis et aL, Science, 260:1805-1808 (1993)). Following dh~c~i~aLion of the signaling - - components, these receptors induce a number of intracellular signaling events inclllriing activation of the ~Id-~s~ lion factor~ NF-IL6, probably via the ras-MAP kinase cascade (ICichimotc et aL. Cell. 76:253-262 (1994~, and a~ aLiun ofthe Jak/STAT signaling pathway (Darnell et al., Science, 264: 1415-1421 (1994)). The latter pathway includes the tyrosine phosphorylation and activation of the intracellular tyrosine kinases, .~akl, Jak2,andTyk2(Li~ttickenetaL,Science,263:89-92(1994);Stahletal., Science,263:92-95(1994);YinetaL, ~cp. ~ematol., 22:467-472 (1994); Narazaki et aL, Proc. NatL Acad. US~, 91 :2285-2289 (1994)) and of the I.a.ls~ ion factors, STATI and STAT3 (T i~ttirkPn et aL, Science, 263:89-92 (1994); Zhong et aL, Science, 264:~5-98 (1994); Akira et aL, Cell, 77:63-71 (1994)).
Accu. diu-~ly, it is an object of the present invention to provide an improved therapy for the prevention and/or llcdlllll,lll of heart failure such as congestive heart failure, particularly the promotion of phycio'ogi~
forms of hypertrophy or inhibition of pathological forms of hypertrophy, for the prevention and/or l, catlll.,nl of 15 neurological disorders such as peripheral r.~,,u~aLlly~ and for the 1~ iuand treatment of disorders in which cytokines, pal ~ UIdl Iy the IL-6/LIF/CNTF/OSM/IL- I I cytokine family, their ~ their agonists, or their receptors play a role.
These and other objects of the invention will be apparent to the ordinarily skilled artisan upon consideration of the a~ irl-,dliUII as a whole.
SUMMARY OF THF. INVENTION
An in vitro neonatal rat heart hypertrophy assay has been developed that allows for expression cloning and protein ~,u. ;~n~alion of the cardiac hypertrophy factor (referred to as CHF, more preferably cardiotrophin- I
or CT-I) disclosed herein. The assay capacity of 1000 single samples a week coupled with the small sample size l ~ Uil~ ll of 100,uL or less has enabled ~ aiull cloning and protein ~u~ ir~,aliun that would have been impossible using the currently ~Jubl; .Led methods. Hence. in one ~ . .1.o.l;. . .~ ~ .I the invention provides a method for assaying a test sample for hypertrophic activity cc,...~.. i ,i..g.
(a) plating 96-well plates with a sl~cpencion of myocytes at a cell density of about 7.5 x 104 cells per mL in Dulbecco's modified Eagle's medium (D-MEM)/F-12 medium culll~liaillg insulin, llauaf~,llill, and aprotinin;
(b) culturing the cells;
(c) adding the test sample (such as one ~ t~d of cont~inine a CT-I) to the cultured cells;
(d) culturing the cells with the test sample; and (e) ~IPtl?rrnining if the test sample has hypertrophic activity.
Besides the assay, the invention provides isolated CT-I polypeptide. This CT-I polypeptide is preferably ~ lly homogenl~o~c, may be glycosylated or unglycosylated, and may be selected from the group cOllsialillg of the native sequence polypeptide, a fragment polypeptide, a variant polypeptide, and a chimeric polypeptide. A~i~liti-~n~lly, the CT-I polypeptide may be selected from the group concicting of the polypeptide that is isolated from a mammal, the polypeptide that is made by lecu...l:ii..a..L means, and the CA 0224~63~ 1998-08-0~
WO 97/30146 PCT/US97/~2675 -polypeptide that is made by synthetic means, Further. this CT-I polypeptide may be selected from ihe group consisting of the polypeptide that is human and the polypeptide that is non-immunogenic in a human.
In another aspect. the isolated CT- i polyt,~,~Jtidc shares at least 75% amino acid sequence identity with -- the ~ ala~:d CT-l sequence shown in Fig. 1. In a further aspect. the polypeptide is the mature numan CT-I
5 having the n A~ I CT- I sequence shown in Fig. 5.
In a still further aspect, the invention provides an isolated polypeptide encoded by a nucleic acid having a sequence that hybridizes under moderately stringent conditions to the nucleic acid sequence provided in Fig.
1. Preferably, this polypeptide is biologically active.
In another aspect, the invention provides a chimera COIIIpl;aillg CT-I fused to a heterologous I 0 polypeptide.
In a still further aspect, the invention provides a cu...posiliu.. ~UI~ JI iahlg biologically active CT- I and a l~h~ Y ArreptAhlc carrier or cu.. ~.i,i.. g biologically active CT-I fused to an immnnogPnic polypeptide.
In yet another aspect, the invention provides an isolated antibody that is capable of binding CT-I and 15 a method for detecting CT-I in vitro or in vivo CullllJIia;llg rontA~A~ine the antibody with a sample or cell 5llcpect~d of c~)ntAir~in~ CT-I and detecting if binding has occurred, as with an ELISA.
Instillanotheraspect~theinventionprovidesamethodforpurifyingCT-I .,u...~.;ail-gpassingamixture of CT-I over a column to which is bound the AntihoAi~c and recovering the fraction cr ntAinine CT-I .
In other aspects, the invention cu.n~ ,s an isolated nucleic acid molecule encoding CT-I, a vector 20 COIII~Iiaillg the nucleic acid molecule, preferably an cA.~.r~,~ai.,.. vector cu...~,.; .i..g the nucleic acid molecule operably linked to control sc~ .g. . i, d by a host cell l~ l u.l..ed with the vector, a host cell cu...!,. iai..g the nucleic acid mol 'o, inrl~lAing IIIAIIIIIIAI;-~I and bacterial host cells, and a method of using a nucleic acid moleculeencodingCT-I toeffectthe~--u-lu-.~iunofCT-I,cu---~ i--gculturingahostcellcu---~-iaingthenucleic acid molecule. Preferably the host cell is I~ f~ A to express CT-I nucleic acid and the CT-I is recovered 25 from the host cell culture, and if secreted~ recovered from the culture medium.
In ArlAitiAnAl aspects, the invention provides an isolated nucleic acid molecule COIIIIJI ishlg the open reading frame nucleic acid sequence shown in Fig. I or Fig. 5. The invention also provides an isolated nucleic acid molecule selected from the group co..c;~ g of:
(a) a cDNA clone co---~. iahlg the nnc!~ul;~l~r sequence of the coding region of the CT-I gene shown 30 in Figure I or Figure 5;
(b) a DNA sequence capable of hybridizing under stringent Collrlitir nc to a clone of (a); and (c) a genetic variant of any of the DNA se~ln~ of (a) and (b) which encodes a polypeptide p, ccoccing a biological property of a native CT-I polypeptide.
The invention also provides an isolated DNA molecule having a sequence capable of hybridizing to the 35 DNA sequence provided in Fig. I or Fig. S under moderately stringent conrlitionc, wherein the DNA molecule encodes a biologically active CT-I polypeptide, ~Ychl~line rat CT-I .
In yet another aspect, a method is provided of ~ . . . i, . ;. .g the presence of a CT- I nucleic acid molecule in a test sample c.~ e c, -~n l; ~e the CT-I nucleic acid molecule with the test sample and determining WO 97/30146 PCT/US97/û2675 -whether h~!bl i ii, .~ic,u has occurred, or Culll~l i 7illg hybridizing the CT- I nucleic acid molecule to a test sample nucleic acid and determining the presence of CT-I nucleic acid.
In still another aspect, the invention provides a method of amplifying a nucleic acid tesl sampie Culll~ illg priming a nucleic acid polymerase chain reaction in the tesl sample with the CT-I nucleic acid 5 molecule.
In a still further aspect~ the invention provides a CT~ g.~ and a method of identifying such ~- ~nta, onicr CUlll~l i 7illg using celH.~ as the test sample in the hypertrophy assay as described above and screening for molecules that antagonize the hypemophic activity of a CT-I demonstrated in such assay.
In a still further aspect, the invention provides a method for treating a mammal having or at risk for heatt 10 failure, an inotropic disorder, or an arrhythmic disorder Colll,~Jli.illg ~.l",i.~;~t~.. hlg to a mammal in need of such treatment a th~ ly effective amount of a l-lu. . " ~ I cul.lpo .ilion ~,olll~l i .i.lg the CT- l or a CT- I
~nt~grmict in a plla-.~ ;r~lly arccpt~hle carrier.
The invention also provides a method for treating a mammal having or at risk for a neurological disorder cc,myli7illg ~iminict~ring to a mammal in need of such ll~,aLIlll,.lL a 111 la~ li.,ally effective amount of a 15 pharm~eutic~i eu",l,n~;l;..n COlll~lliaillg the CT-I in a l~hall~ lly acceptable carrier.
The invention also provides a method for treating a mammal having or at risk for a disorder in which cytokines~ palliculally the IL-6/LIF/CNTF/OSM/IL-I I cytokine family, more preferably LIF and OSM, more preferably LIF, their ~ 'r,0'.;'15 or their agonists, and most preferably a LlF-Receptor ,~ subunit that interacts with gpl30, play a role. The methods comprise ~ irninict.-rjng to a mammal in need of such Ll~,aLIl~ a 20 - ~ y effective amount of a plia. " ,~ l cull.,vo .iliull culll~. i .illg CT- I, its ~ u~g~ l or its agonist, in a pLal.~ lly acc~.Lal,le carrier. In a most preferred embodilllcllL the disorders involve a pathway regulated or induced by the activation of LIFR~ by CT-I binding and 5..l.ae~lu~..l interaction with gpl30.
In a still further aspect, the invention provides a CT-I antagonist and a method of identifying such "..l t~....;~l culll~Jliaillg using cell ,..~ or purified or synthetic compounds as the test sample in an assay 25 in which CT-I has a demonstrated biological activity, receptor binding activity, or signaling pathwav induction activity, preferably in a microassay, and screening for molecules that antagonize the activitv of a CT-I
demonstrated in such an assay.
In ~ tion~l embodiments, the invention supplies a method of identifying a receptor for CT-I
~.UIIIIJl iahlg using labeled CT- 1, preferably r~liol~hel-od CT- I, in a cellular receptor assay, allowing the CT- I
30 to bind to cells, or using the labeled CT-I to pan for cells that contain the receptor.
Bl~TF.F DESCRTPTION OF THE DRAWrNGS
Figures I A and IB depict the ml~l~otifl~ sequence (sense and anti-sense strands) (SEQ ID NOS: I and 2) and deduced amino acid sequence ~SEQ ID NO: 3) of a mouse CT-I DNA clone. The underlined complc.l~l.~y nn~l~oti~ at position 27 show the start of another mouse CT-I clone used to obtain the full-35 length clone.
Figure 2 aligns the llall ,lak;d amino acid sequence of the mouse CT-l clone (chf.781) (SEQ ID NO:

3) with the amino acid sequence of human ciliary n~ ullupllic factor (humcntf) (SEQ ID NO: 4) to show the extent of sequence identity.

WO 97/30146 PCT/tJS97/02675 Figure 3 shows a graph of atrial nall iul~,Lic peptide (ANP) reiease for phenylephrine (s~andard curve) and transfections into 293 cells in a neonatal cardiac h~ ,. k u~,hy assay.
Figure 4 shows a graph of survival of live ciliary ganglion neurons (I~..,a~ulc;d by cell count) as a - ~ = function of either the ciliaTy ne.lLI uL- u,uLi-~ factor (CNTF) standard (in nglmL) or the h all .rt~ d 293 c~ n~lhi-~nPd S medium (in fraction of assay volume), using a CNTF standard (circles), medium from a CT- I DNA L~ rr~ I ion of 293 cells (triangles), and medium from a control DNA l~ r~ . of 293 cells (squares).
Figures 5A and 5B depict the nucleotide sequence (sense and anti-sense strands) (SEQ ID NOS: 6 and 7) and deduced amino acid sequence (SEQ ID NO: 8) of a human CT- I DNA clone.
Figure 6 aligns the 1. allslal~;i amino acid sequence of the human CT- I cione (humct I ) (SEQ ID NO:
10 8) with the llall .I.. t~,d amino acid sequence of the mouse CT- I clone (chf.781) ~SEQ ID NO: 3) to show the extent of ceqllPnre identity.
Figures 7A and 7B depict activity of CT-I in h- ~ o~oietic cell assays. The induction by the human ~h) or mouse (m) cytokines was performed as described in the Example VI, Materials and Methods. Figure 7A
shows ctim~ ti~ of 3H-thymidine incull-ulaliull in the mouse hybridoma cell line, B9, with an EC50 [IL-6] =
15 0.13 (10.03) nM. Figure 7B shows inhibition of 3H-thymidine illcul~ulaLion in the mouse myeloid leukemia cell line, M I, with an EC50 [CT- 11 = 0.0076 (+ 0.0006) nM~ EC50 [LIF~ = 0.048 (+ 0.004) nM .
Figures 8A, 8B, and 8C depict activity of CT-I in neuronal cell assays. The in-lllrtjon by mouse (m) or rat (r) cytokines was p~, rullll~,d as described in Example Vl, Materials and Methods. Figure 8A shows the switch in transmitter phenotype of rat symr~th~tic neurons. Tyrosine hydroxylase (TH3 and choline 20 acet5~1Ll~ Ç~lase (Ch4 13 activities were ~ d in rlllrlic~fP Figure 8B shows survival of rat d~ ic neurons. Plotted are the average and standard deviation of triplicate determin~ti--n ~ Figure 8C shows survival of chick ciliary neurons with an EC50 [CT-I] = 10 (+ 8.2) nM and EC50 ~CNTF] = O.Oû74 (+ 0.0049) nM.
Figure 9 depicts activity of CT-I in embryonic stem cells d~, icl. ~ Mouse embryonic stem cells were cultured in the presence of the mouse (m) cytokines as described in Example Vl, Materials and Methods.
Figures IOA, I OB, IOC and I OD depict binding and cross-competitic-n of CT- I and LIF to mouse M I
cells. Assays çr...~ .rd 0.047 nM 1251-mouse CT-I (1251-mCT-I) and llnl~hPIPfi mouse (m) CT-I. Figure IOA, orl~nl~hPll?dLlFtFigureloB;ûro.o42nMl2sl-mouseLlF(l2sl-mLIF3andunhhelçd CT-l,FigurelOC,or LIF, Figure I OD. Shown are crmr~otiti~ and Scatchard (i~sert) plots of the data. For the labeled CT- I binding, Kd [CT-I] = 0.61 (+ 0.11) nM, 1500 (+ 220) siteslcell; Kd [LIF] = 0.19 (+ 0.05) nM, 1800 (+ 150) sites/cell.
30 For labeled LIF binding, Kd [CT-I ] = 0.83 (+ O.13) nM, 1300 (_ 80) sites/cell; Kd [LIFl = 0.26 (+ O.10) nM, 1200 (+ 300) sites/cell.
Figure 11 depicts cross-linking of CT- I and LIF to Ml Cells. 1251-mouse CT- I ( l 251-mCT-I ) or 125I-mouse Lli' (1251-mLlF3 were bound and cross-linked to Ml cells in the absence (None) or presence of a 100 fold excess of the indicated mouse (m3 cytokine, and the reaction products anaiyzed by SDS gel clc~-o~hù~ ,. The 35 mobility of molecular weight - ~1~, is inrlir~P-I
Figures 12A depicts inhibition of CT-I binding to Ml cells by an anti-gpl30 mr~nocl~n~l antibody.
Assays c~nt~ined 0.12 nM 1251-mouse CT- I and antibodies as inrii~ tP~1 For the anti-gp 130 antibody, EC50 = 44 ( 8) nM. Figure 12B depicts cle.,Llu~,Lul~lic mobility shift of the DNA element SIE induced by CT-I
binding to M I cells. M I cells were ;. .~ d without (-) or with (+) S nM mouse lm) CT- I or LIF, Iysed, and CA 0224~63~ 1998-08-0~

the cell extract assayed for binding to the DNA element SIE as described in the Materials and Methods. Binding specificity was ~l~t~rrnin~d by the addition of .lnlAh~olpd SIE DNA (Cold Oligo). The specific DNA complex is indicated (arrow).
Figure 13A and 13B depict binding and cross-comretitinn of CT-I and LIF to rat primarv cardiac ~' 5 myocytes. Duplicate assays c~.. a~ti.,~d either 0.047 nM 125i-mouse CT-I (1251-mCT-I) or 0.042 nM 1251-mouse LIF (1251-mLI~';) and ImlAhel~d mouse Im) CT-I or LIF as in~lirat~
Figures 14A, 14B, 14C and 14D depict binding of CT-I to purified, soluble LIF recepIor and gpl30.
Figures 14A-C show per cent binding of 1251-mouse CT-I (0.089 nM) to soluble mouse LIF receptor (smLlFR) and soluble mouse gpl30 (smgp130) in the absence (-) or presence (+) of 164 nM unlabeled mouse CT-I (mCT-10 1). Figure 14A depicts binding to h.cleasi.lg cullcc.lllalic,lls soluble LIF receptor alone: FIgure 14B depicts binding to increasing Cull~,cllkaLiOll~ of soluble gpl30 alone; Figure 14C depicts binding at one soluble LIF
receptor ~o.lc-,lltlaLion with hl~ ,a ~hlg collccllllaLions of soluble gp I 30. Plotted is the average and half the ii~l~,~ce of duplicate ~L ~ . . .,i. ~A1 innc The results for 0.84 nM soluble LIF receptor are shown twice for clarity.
Figure 14D depicts cnmretiti-n binding of 1251-mouse CT-I (0.089 nM) to the soluble LIF receptor (2.8 nM) 15 with increasing concellllaLions of llnlAheled CT-I. Kd ~CT-I] = 1.9 (+ 0.2) nM.
Figures 15A and 15B depict sirn~larity of IL-6 family ligands and subunit structure of their receptors.
Figure 15A shows per cent amino acid identity of the mature form of the IL-6 family ligands: (m) mouse, (h) human, (c) chicken. The bottom row gives the per cent identity of the cytokine to its human homologue. Shown in bold are the pcl ~llL~_s greater than 40 %. Figure 15B is a diagram of the IL-6 family receptors. The subunit 20 t- ' ~ ry of the various c ~mi ' is not known in most cases, although recent work has led to a ronr!~ nn that the IL-6 receptor complex is a hexamer Cul~ i. .g two IL-6 molecules, two IL-6 receptors. and two gp 130 signaling subunits. Ward et ~L, J. BioL Chem., 269:23286-23289 (1994).
Figure 16 depicts Alignm~nt of the protein sequence of human CT- I, LIF and CNTF. Encoded amino acid sequence of human CT-I (hCT-I) aligned with that of human LIF (hLlF) and human CNTF (hCNTF).
25 Overliningindicatesthelocationoffour, ,.l.l.il.,ll.;-,helicesbasedontheirproposedlocationsinCNTF(Bazan, Nez~ron. 7: 197-208 (199 I )).
Figuresl7Aandl7Bdepictthec~....l.~lil;..l.forthebindingofhumanLlFtomouseMlorhumanHela cell. For Figure 17A 1251-human LIF was bound in duplicate to M 1 (5 million cells per reaction3 in the presence ofthe indicated c~....l.~ lil.~,~ For Figure 17B 1251-human LIF was bound in duplicate to i~ela cells (2.5 million 30 per reaction) in the presence of the indicated c~ ~-- .l ,~ l il~ ~ CM is cnnfiitinn.-d medium from 293 cells llall:~f~,.,t~,;i with human CT-I.
Figure 18 depicts the binding of mouse CT- I to human Hela cells. Duplicate assays containing 0.23 nM 1251-mouse-CT-I and 9 miliion cells were p~.rollll~,d as de;,cl;L,ed in the Examples. The insert is a Scatchard plot of the data. Kd=0.75 (+/-0.15) nM, 860~+/-130 sites per cell).
Figure 19 depicts the comreritil~n for the binding of human OSM to human Wl-26 cells. 1251-human OSM was bound in duplicate to Wl-26 VA4 cells (2.4 million cells per reaction) in the presence of the indicated '' cu~ ,LiLu~ as d~ lil ed in the Examples.
Figure 20 depicts CA~ liUII of CT-I in human tissues, Northern blots conr~ining poivA+RNA from the indicated tissues were hybridized with a human CT-I cDNA probe as described in the ~.Y~mplPc CA 0224~63~ 199X-08-0~
WO 97/30146 PCr/US97/0267S

Figure 21 is a 5rhPm~tir depicting several biological activities of CT- I .
DETA~LFn DT~SCl~TPTION OF THE PR~FERRED F.l\~IBODIMFNTS
1. r)efinitions -- In generaL the following words or phrases have the indicaled ~l~finition when used in the des.,. ilJtiOII~
5 examples, andclaims:
"CHF" (or "cardiac hypertrophy factor" or "~JiuLIu~hin" or "caldiul,u~.hin-l " or "CT-I ") is defined herein to be any polypeptide sequence that possesses at leasl one biological property (as defined below) of a naturally occurring polypeptide Cu~ illg the polypeptide sequence of Fig. I or the human equivalent thereof shown in Fig. S. It does not include the rat homolog of CT-l, i~. CT-I from the rat species. This ~cfinitinn 10 cncolllpa:,~cs not only the polypeptide isolated from a native CT-I source such as murine embryoid bodies described herein or from another source, such as another animal species except rat, inrhlflin~ humans, but also the polypeptide prepared by r~ c~lllbill~..lL or synthetic methods. It also includes variant forms inr~ ing filnrtinn:~l derivatives, alleles, isoforms and ~n~logll~c thereof.
A "CT-I rlcl~ll_.lL is a portion of a naturally occurring mature full-length CT-I sequence having one IS or more amino acid residues or carbohydrate units deleted. The deleted amino acid residue(s) may occur anywhere in the polypeptide, including at either the N-terminal or C-terminal end or intemally. The fragment will share at least one biological property in common with CT-l. CT-I Ga~lll_,lta typically will have a cull~__uli~/e sequence of at least lû, 15, 20, 25, 30, or 40 atnino acid residues that are identical to the se~l~,f ~~re S
of the CT- I isolated from a mammal inrt- ~rling the CT- I isolated from murine embryoid bodies or the human CT-20 1.
"CT-I variants" or "CT-I sequence variants" as defined herein mean biologically active CT-ls as defned below having less than 100% sequence identity with the CT-I isolated from ,~."...1,'.."..1 cell culture or from murine embryoid bodies having the deduced sequence described in Fig. I, or with the human equivalent described in Fig. 5. Ordinarily, a biologically active CT-I variant wlll have an amino acid sequence having at 25 least about 70% amino acid sequence identity with the CT- I isolated from murine embryoid bodies or the mature human CT-I (see Figs. I and 5), preferably at least about 75%, more ~ ;r~.~ly at least about 80%. still more preferably at least about 85%, even more preferably at least about 90%, and most preferably at least about 95%.
A"chimericCT-l"isapolypeptidec.,...~ ,gfull-lengthCT-l oroneormore r.~..~ ;thereoffused or bonded to a second protein or one or more Ga~ll~l~ts thereof. The chimera will share at least one biological 30 properLy in common with CT- 1. The second protein will typically be a cytokine, growth factor, or hormone such as growth hormone, IGF-I, or a n.,.lluLlu~l.ic factor such as CNTF, nerve growth factor (NGF), brain-derived u~ JLIuilllic factor (BDNF), n_~lluL-ulJllill-3 (NT-3), neurotrophin-4 (NT~), nc.llc"l u~hin-S (NT-5), NT-6, or the like.
"Isolated CT-I", "highly purified CT-I" and "~b~ lly homogeneous CT-I" are used 35 i~lt~ h~ geably and mean a CT-I that has been purified &om a CT-I source or has been prepared by or synthetic methods and is ~ y &ee of other peptides or proteins ( l ) to obtain at least l 5 and preferably 20 amino acid residues of the N-terminal or of an internal amino acid sequence by using a spinning cup s~ ( " or the best Cullull_,~ ;ally available amino acid S~ .. marketed or as modified by published methods as of the filing date of this application, or (2) to homogeneity by SDS-PAGE under non-CA 0224~63~ 1998-08-0~
WO g7/30146 PCT/US97/0267S

reducinQ or reducing c.,. .l;~ -c using Coomassie blue or. preferably, silver stain. HnmogenPi~y here means less than about 5% contAminAtinll with other source proteins.
"Biological properLy" when used in conj--nrtion with either "CT-I" or "isolated CT-I" means having - - myocardiotrophic~ inotropic, anti-arrhythrnic. or n~ L~ ic activity or having an in vivo effector or antigenic S function or activity that is directly or indirectly caused or performed by a CT- I (whether in its native or d~l~dtul~d conformation) or a fragment thereof. Effector functions include receptor binding and any carrier binding activity, agonism or ~ g~ --- of CT-I, especially L"...~.l..~ I jon of a proliferative signal inrlnrling replication, DNA
regulatorv function, motllllAfion of the biological activity of other growth factors, receptor activation~
deactivation, up- or down-.~ A1 ;n.~ cell growth or di~l.,llLiaLion, and the like. However, effector functions 10 do not include pO~ ,.,.,;ull of an epitope or antigenic site that is capable of cross-reacting with antibodies raised against native CT-I.
An "allLi~,~llic function" means pO~a~.~iiOn of an epitope or antigenic site that is capable of cross-reacting with ,-- ~~ O~ raised against the native CT- I whose sequence is shown in Fig. I or another mAmm siliAn native CT-I, including the human homolog whose sequence is shown in Fig. 5. The principal antigenic function of a 15 CT- I polypeptide is that it binds with an affinity of at least about I o6 L/mole to an antibody raised against CT- I
isolated from mouse embryoid bodies or a human homolog thereof. Ordinarily, the polypeptide binds with an affinityofatleastabout 107L/mole. Most~ ,f~,lal Iy,thea~.l;g~ llyactiveCT-I polypeptideisapolypeptide that binds to an antibody raised against CT-I having one of the above-described effector functions. The ;1 .o.l;~s used to define 1, ' ~v "y activity" are rabbit polyclonal Antihor~if?c raised by forrnulating the CT- I
20 isolated from recombinant cell culture or embryoid bodies in Freund's complete adjuvant, s~h.~ .ù~.l.y injecting the fnrrnnlAtinn, and boosting the immune response by i~L~ a~ u~eal injection of the fonnl- l-Ation until the titer of the anti-CT- I antibody plateaus.
"Biologically active" when used in rnnillnctinn with either "CT-I" or "isolated CT-I" mean a CT-I
polypeptide that exhibits hypertrophic, illuLI.~ c, anti-arrhythmic, or n.,l.. ulluyhiC activity or shares an effector 25 functionofCT-I isolatedfrommurineembryoidbodiesorproducedin..~l..-b~ cellculturedescribedherein, and that may (but need not) in addition possess an antigenic function. One principal effector function of CT-I
or CT-I polypeptide herein is h~flu.,ll~ g cardiac growth or hy~..,lLIu~Jl.y activity, as measured, e.g, by atrial nat iul~LiC peptide ~ANP) release or by the myocyte hypertrophy assay described herein using a specific plating medium and plating density, and ~ ,f~,l d,ly using crystal violet stain for readout. The desired function of a CT- I
30 (or CT- I a~ g~ is to increase physiological (b~nPfiriAl) forms of hy~,~.. L-~ lly and decrease pAthologirAl hypertrophy. In addition, the CT-I herein is expected to display anti-arrhythrnic function by promoting a more normal el~,L~ Lysiological phenotype. Another principal effector function of CT-I or CT-I polypeptide herein is ,1; .. l~ l ;.. g the proliferation of chick ciliary gangiion neurons in an assay for CNTF activity.
~"i~,. .,i. ~lly active CT-I is defined as a polypeptide that possesses an antigenic function of CT-I and 35 that may (but need not) in addition possess an effector fimction.
In preferred ~mbo.l;.. 1~. ~nfigl-nirAlly active CT-I is a polypeptide that binds with an affinity of at least about 106 L/mole to an antibody capable of binding CT-I. Ordinarily, the polypeptide binds with an affinity of at least about 107 L/mole. Isolated antibody capable of binding CT-I is an antibody that is id~ntifif~d and separated from a ~,J...pol~ of the natural en~dl~ -.,l-l in which it may be present. Most preferably, the CA 0224563 j 1998 - 08 - 05 antigenically active CT-1 is a polypeptide that binds to an antibody capable of binding CT-I in its native cùllrcllll.aLiol1. CT-1 in its native co..rulllla~ion is CT-I as found in nature that has not been denatured by chaolropic agents, heat, or other treatment that c~lbCt~nti~11y modifies the three-~limPnci~ nz~l structure of CT-I
- - as tl~ ri, for example, by migration on non-reducing, non-denaturing sizing gels. Antibody used in this S d~ n is rabbit polyclonal antibody raised by form~ tin~ native CT-I from a non-rabbit species in Freund's complete adjuvant, ,-~ eo~ y injecting the form~ tinn and boosting the immune response by a~ ullcal injection of the formul~tion until the titer of anti-CT-I antibody plateaus.
"Percent amino acid sequence identity" with respect to the CT-1 sequence is defined herein as the pc~ c of amino acid residues in the c~nriirl~t~ sequence that are identical with the residues in the CT-I
10 sequence isolated from murine embryoid bodies having the deduced amino acid sequence described in Fig. I or the deduced human CT-I amino acid sequence t~Ps~rihcd in Fig. 5, after aligning the $~ cec and introducing gaps, if necessary, to achieve the ll~a~ ulll percent sequence identity, and not coll~id~_. hlg any conservative s~ c as part of the sequence identity~ None of N-terminal, C-terminal, or internal extensions, ~iPkPtion c or h.sc,~i..ll . into the CT-I sequence shall be cu..~ilued as affecting sequence identity or homology. Thus, 15 c,~clll~Jlaly L '~g ily active CT-I polypeptides co~ cl~,d to have identical ~ - ..ct ~ include prepro-CT-I, pro-CT-I, and mature CT-I.
"CT-I mi.,... ~ " may be accomplished by any al~lJIu~JIia~ standard ~.lc,.,clulc provided the u.,cdul~ is sensitive enough. In one such method, highly purified polypeptide obtained from SDS gels or from a final HPLC step is 5~ .t~t~d directly by ~ Edman (phenyl isothiocyanate) dc~sladaLion using a model 470A Applied Biosystems gas-phase se I ~-, equipped with a 120A phenylthiohydantoin (PTH) amino acid analyzer. ~cit1ition~11y~ CT-I La~ prepared by chemical (e.g, CNBr, hydroxylamine, or 2-nitro-5-thioc~ oh .~ ) or enzymatic ~e.g., trypsin, clv~ Ja,.l, or staphylococcal protease? digestion followed by r.a~;lllellL purification (e.g., HPLC) may be similarly sequPnt~et1 PTH amino acids are analyzed using the ChromPerfectTM data system (Justice Innovations, Palo Alto, CA). S~ quPnce illt~ aLiOI~ is performed on a VAX 11/785 Digital ~tl--irmPnt Co. COIIIyut~,~ as .le;,~.lil,e,l by ~enzel et al., J: C~romarography, 404:41-52 (1987). Optionally, aliquots of HPLC fractions may be clc~,k~JIJllvl~sed on 5-20% SDS-PAGE, ele.,l~ul.all .I~ ,d to a PVDF ll.c~ lallc (ProBlott, AIB, Foster City, CA) and stained with Coomassie Brilliant Blue. M~.t~uld;ala, J. BioL Cfiem., 262:10035-10038 (1987). A specific protein identified by the stain is excised from the blot and N-terminal se~ ~ g is carried out with the gas-phase sc~l..e~ ,.- described above. For internal protein s~ i HPLC fractions are dried under vacuum (SpeedVac), resll~p~n~ied in à~l~luplial~
buffers~ and digested with cyanogen bromide, the Lys-specific enzyrne Lys-C (Wako (~hPm ir~ Ic, Richmond, VA), or Asp-N (Boehringer hl~nnht~im, Ind;~l~oLs, IN). After t~i~e~tion the resultant peptides are setlllent~ed as a mixrure or after ~PLC resolution on a C4 column .I~ ,lol,cd with a propanol gradient in 0.1% trifluoroacetic acid (TFA) prior to gas-phase :~C~ -g "Isolated CT-I nucleic acid" is RNA or DNA c~ nf~ining greater than 16 and preferably 20 or more 5~ nti~ u~ bases that encodes biologically active CT-I or a fragrnent thereo~, is c~ mplPnnPnt~ry to the RNA or DNA, or hybridizes to the RNA or DNA and remains stably bound under moderate to stringent conditions. This RNA or DNA is free from at least one contRminRting source nucleic acid with which it is normally ~u. i -t' d in the natural source and preferably b~ ti~lly free of any other mRmmRliRn RNA or .

CA 0224~63~ 1998-08-0~

DNA. The phra~se "free from at least one c~ e ~ g source nucleic acid with which it is norrnally ~u~
includes the case where the nucleic acid is present in the source or natural cell but is in a different cL .. os~
location or is otherwise flanked by nucleic acid se~lu Al~ es not norrnally found in the source cell. An example - - of isolated CT-I nucleic acid is RNA or DNA that encodes a biologically active CT-I sharing at least 75%! more r 5 preferablv at lea~st 80%, still more preferably at least 85%. even more preferably 90%, and most preferably 95%
sequence identity with the murine CT-I or with the human CT-I.
"Control se~ . .r~ 7" when referring to ~A,ulGaaiull means DNA se~ necessary for the expression of an operably linked coding sequence in a particular host organism. The control seq~Rn~PC that are suitable for prokaryotes. for example, include a ~Jlvlllulel. optionally an operator se(luRn~P a ribosome binding site, and possibly, other as yet poorly understood sPqllpn~pc Eukaryotic cells are known to utilize promoters, polyadenylation signals, and c.lllallc.~,~ a.
"Operably linked" when referring to nucleic acids means that the nucleic acids are placed in a filn~tion~l re!~tinnchir with another nucleic acid seqmPncP For example, DNA for a pr~oseq~ience or secretory leader is operablv linked to DNA for a polypeptide if it is e,~l,-c;.a~,d as a ~ JlUIt ill that pa~Li-,i~Jal-_~S in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the llalla~ .Lion of the sP(IuPnre~ or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate ll~laldliull~ Generally, "operably linked" means that the DNA ~ c~ s being linked are Cvllti~;uuua and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be conti~ouC Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the s~r.fhefie oligu~ eo~Li~ a~apt~c o~ ~inkers aie u~ed irl~deeGrd wiffi~ eon-~,.-tiu-,al praetic~.
"F ~ogPnmlc" when referring to an element means a nucleic acid sequence that is foreign to the cell, or homologous to tbe cell but in a position within the host cell nucleic acid in which the element is ordinarily not found.
"Cell," "cell line," and "cell culture" are used u~t-,.L,Lall~scably herein and such d~psign~tionc include all progeny of a cell or cell line. Thus, for example, terms like l~ rul~al~" and "1, aLIarul Illed cells" include the primary subject cell and cultures derived ~h~.~,rlulll without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent c Mutant progeny that have the same function or biological activity as screened for in the originally t~.... .... ..Drulll.cd cell are included. Where distinct rlPcign~tionc are intended, it will be clear from the context.
"Plasmids" are antnnnmnusly replicating circular DNA molecules possessing in(1PpPn~lP~t origins of replication and are flpcipnzltpd herein by a lower case "p" preceded and/or followed by capital letters and/or numbers. The starting plasmids herein either are cu~ ,ially available, are publicly available on an U~ ,1 basis, or can be cullatl ul,t~d from such available plasmids in accul dall.,~ with ~.ul.lial.e i yl uccdul ~a.
In addition, other equivalent plasmids are known in the art and will be apparent to the ordinary artisan.
3s "Restriction enzyme digestion" when referring to DNA means catalytic cleavage of internal pht~crho~liPctRr bonds of DNA with an enzyme that acts only at certain locations or sites in the DNA sP~lnPnce Such enzymes are called "l~a~ ,Lull ~ Ir~CeS " Each restriction Pnr~ n~ a specific DNA
sequence called a ", ~a~ ion site" that exbibits two-fold symmetry. The various l ~tl i~tiun enzymes used herein are cvl~ .~,ially available and tbeir reaction con~litinnC cofactors, and other r~ ~Uil~ as established by the == ~ = ~=

enzyme suppliers are used. Restriction enzymes commonly are ~l~ciEnAtpd by abbreviations composed of a capital letter followed by other letters ~ illg the microorganism from which each restriction enzyme originally was obtained and then a numbe m i~ .g the pa~ ,ul~ enzyme. In general, about I ,ug of plasmid or I~NA fragment is used with about 1-~ units of enzyme in about 20 IlL of buffer solution. Al.~Jl Ul,. ;al~: buffers 5 and substrate arnounts for particular ~ .Lli.,liull enzymes are specified by the r~ l. InruhAtion for about I hour at 37~C is ordinarily used. but may vary in acculdal.cc with the supplier's i~l:.llu-,Lions. After incubation, protein or polypeptide is removed by extraction with phenol and chloroform, and the digested nucleic acid is recovered from the aqueous fraction by ~ n with ethanol. Digestion with a restriction enzyme may be followed with bacterial alkaline phoc~ c.~ hydrolysis of the terminal 5' pho~llales to prevent the two 10 Ic,~hi~Liull-cleaved ends of a DNA fragment from "ch~,ulali,illg" or forming a closed loop that would impede insertion of another DNA fragment at the l-,~ ,LiOIl site. Unless otherwise stated, digestion of plasmids is not followed by S' terminal (lerhncrhnrylation. rlv~dul~ and reagents for ~l l~h~l .h. ~ ylation are conventional as described in sections 1.56-1.61 of Sambrook et aL, Mol~~ 7r Cloning: A Laborato~y Manual (New York: Cold Spring Harbor Laboratory Press, 1989).
''Recovery''or''isolation''ofagivenfragmentofDNAfromal~h;~liulldigestmeansseparationofthe digest on polyacrylamide or agarose gel by electrophoresis, i,l. ..liri. _li..ll of the fragment of interest by cull.~,ali~unofitsmobilityversusthatofmarkerDNAr~ ,, ofknown -' ' weight,removalofthegel section c-,~ the desired fi7t~n~nt and separation ofthe gel from DNA. This ~lu-,edul~ is known generally.
Forexarnple,seeLawnetaL,Nucleic,4cidsRes.,9:6103-6114(1981)andGoeddeletaL,Nucleic,4cidsRes., 20 8:4057 ( I 980).
"Southem analysis" or "Southern blotting" is a method by which the presence of DNA s~ s in a IC.~.li~,liUII Pnd~ u' k~c digest of DNA or a DNA-conts~ininE Cul~ O .ilion is cnnfirrn~d by hybridization to a known~ labeled oliEnnnrl~otide or DNA fragrnent. Southern analysis typically involves cl.~ uphoretic s~rz~rAtion of DNA digests on agarose gels, d .-l ~ ;u. . of the DNA after ele~,l- UpllOl ~,liC ~ alaLiOIl, and transfer 25 of the DNA to nitrocelhllnc~ nylon, or another suitable ~ ,llblallc support for analysis with a r?rlinl~hele~
biotinylated, or enzyme-labeled probe as dc,s.,lii,ed in sections 9.37-9.52 of Sambrook et al., supra.
"Northern analysis" or "Northern blotting" is a method used to identify RNA c~ - .c~ c that hybridize to a known probe such as an o1 i~ - k-,l irlP" DNA fi~f~nPnt, cDNA or fragment thereof, or RNA fragment. The probe is labeled with a radioisotope such as 32p, or by biotinylation, or with an enzyme. The RNA to be 30 analyzed is usually electrophoretically separated on an agarose or polyacryiamide gel, llall~f~ d to nitrocell--lnsP, nylon, or other suitable ... ~..1.., ~.~ and hybridized with the probe, using standard fPrhniril-r c well known in the art such as those described in sections 7.39-7.52 of Sambrook et al., supra.
"Ligation" is the process of forming rhncrho~lipstpr bonds between two nucleic acid fragments. For ligationofthetwo rla~l~ , theendsofthe r.,.~ mustbe f~ k witheachother. Insomecases,the 35 ends will be directly .~~ , ' ' after ~n~ cP ~ligPcfi tn However, it may be necessary frst to convert the :~La~ ,;i ends commonly l~oducc;i after Pnri~ e digestion to blunt ends to make them cc-lll!Jatil,lc for ligation. For blunting tbe ends, the DNA is treated in a suitable buffer for at least 15 minutes at 15~ C with about 10 units of the Klenow fragment of DNA polymerase I or T4 DNA polymerase in the presence of the four deoxyribonucleotide trirhnsphs~t~c The DNA is then purified by phenol-chloroform extraction and ethanol CA 0224~63~ 1998-08-0~

-~Jl C i~ n l ;. ~1. The DNA G a~~ that are to be iiga~ed together are put in solution in about equimoiar amounts.
The solution will also contain ATP, ligase buffer. and a ligase such as T4 DNA ligase at about 10 units per 0.5 llg of DNA. If the DNA is to be ligated into a vector, the vector is first linearized by digestion with the d~ lu~liaL~ restriction Pndon~-rie~ce(s). The linearized fragment is then Ireated with bacterial alkaline '' 5 pl~ e or calf intestinal ph~ to prevent self-ligation during the ligation step.
"Pll lJa~aliùll" of DNA from cells means isolating the plasmid DNA from a culture of the host cells.
Commonly used methods for DNA ~Jlci~alalion are the large- and small-scale plasmid ~ Jalaiio..s desc}ibed in sections 1.25-1.33 of Sambrook et al., supra. After L~ Jalali~:~ll of the DNA, it ean be purified by methods well known in the art such as that described in section 1.40 of Sambrook et aL, supra.
"Oligv-~--rle~JI ;-les" are short-length, single- or double-stranded polydeoxynucleotides that are ...ically s~llLIl~ ;,.~d by known methods such as phccrhl)triester, phosphite, or pho:.~,hola.llidite chemistry, using solid-phase ~. ~ h..;.~ c such as d~ .il, d in EP 266.032 published 4 May 1988, or via deoxyn-lcl~os~
H-phf~ t~ t~ ~ as described by Froehler et al., NueL Acids Res., 14:5399-5407 (1986). Further methods include the polyrnerase chain reaction defined below and other auLu~3l hllel methods and oli~nnnl~lPot;(le 15 syntheses on solid supports. All of these methods are described in Engels et al., Agnew. Chem. ~nt. E;d Engl., 28:716-734 (1989). These methods are used if the entire nucleic acid sequence of the gene is known, or the sequence of the nucleic acid c~m-F' y to the coding strand is available. Alternatively, if the target amino acid sequence is known, one may infer potential nucleic acid seq~ n~ ~c using known and preferred coding residues for each amino acid residue. The olig.. ~Irul;~ies are then purified on polyacrylamide gels.
"Polymerase chain reaction" or "PCR" refers to a ~luccJulr or ho~hnirl~ in whieh minute amounts of a specific piece of nucleic acid, RNA and/or DNA, are amplified as described in U.S. Patent No. 4,683,195 issued 28 July l 987. Generally, sequence i.. ro....~ ., . from the ends of the region of interest or beyond needs to be available, such that olig.,...~rl~uli~ primers can be ~l~cignP~I these primers will be identical or similar in sequence to opposite strands of the template to be ~mpl ifi-o~l The 5' terminal r-. ~ Ir ul ;~ of the two primers may 25 coincide with the ends of the amplified material. PCR can be used to amplify specific iWA sc~lu. ,cec specific DNA sc~u~.lces from total genomic DNA. and cDNA ~ lil)ed from total cellular E~NA. ba.~- .iulJha~,c; or plasmid se.~ , etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biof., 51 :263 (1987);
Erlich, ed., PCR Technology, (Stockton i'ress, NY,1989). As used herein, PCR is Cull~id~,..,;l to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sampie 30 Cull~ lg the use of a icnown nucleic acid as a primer and a nucleic acid polymerase to amplify or generate a specific piece of nucleic acid.
"Stringent cl~n~litionc" are those that (Chien et al., ~nnu. Rev. P~ysioL, 55:77-95 (1993)) employ low ionic strength and high n ...l...,.l...~ for washing, for example, O.û15 M NaCV0.0015 M sodium citrate/0.1%
NaDodSO4 (SDS) at 50~C, or (2) employ during hybridization a d~lalulillg agent such as formamide, for example,50% (voi/vol) forrn~ with 0.1 % bovine serum albumin/0.1% Ficoli/0.1% polyvinylpyrrolidone/50 mM sodium pllu "JhaL~ buffer at pH 6.5 with 750 mM NaCI, 75 mM sodium citrate at 42~ C. Another example is use of 50% r.. ~ 5 x SSC (0.75 M NaCI,0.075 M sodium citrate),50 mM sodium ph.. ~"l.~lr (pH 6 8), 0.1% sodium p~l . .1 .h~ , 5 x Denhardt's solution, sonicated saimon sperm DNA (50 1lg/mL), 0.1% SDS, and 10% dextran sulfate at 42~ C, with washes at 42~ C in 0.2 x SSC and 0.1 % SDS.

WO 97/30146 PCT/lUS97/02675 "Moderately stringent conditions" are described in Sambrook et al.. supra, and include the use of a washing solution and hylJ.;di~aLi~)ll cnnAi~ionC (e.g., t~ .,.alulc~ ionic strength, and %SDS) less stringent than described above. An example of moderately stringent con~liti~mC is a condition such as overnight inr-lh~ti-~r, at 37~C in a solution cullll,li7;llg: 20% finrrn~nnitlf~, 5 X SSC (I50 mM NaCI, 15 mM trisodium citrate), 50 mM
sodium phocph~t~ (pH 7.6), 5 x Dcllha~dl 7 solution, 10% dextran sulfate. and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in I x SSC at about 37-50~C. The skilled artisan will o2~li~ how to adjust the ~1ll~ aLul c, ionic strength, etG. as necessary to accommodate factors such as probe ~7 length and the like.
",~ntihorlii~c" (Abs) and "irnnnllnogl-~hllltn~" (Igs) are gl~1v~lut~llls having the same structural 10 ~,La~a~ i71ic7. While~ntiho~ ?cexhibitbinding,~.c~,,fi~,ilytoaspecificantigen,i~.. l.. f~l--bulinsincludeboth.
. ,1 ;l)o~ li.; and other antibody-like mol~ ' which lack antigen s,~iri~;ly. Polypeptides of the latter kind are, for example, ~,vducc.~ at low levels by the Iymph system and at increased levels by myelomas. "Native ,...I;l,o(l;rsand;~ r~lrlblllincllareusuallyh~t~lut~Lla~ licgly~vlJlu~4lll7ofaboutl5o~ooodaltons~co~ s~
of two identical light (E) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain 15 by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of differem ,. ""...,o~lnbulin isotypes. Each heavy and light chain also has regularly spaced intr~rh:~in disulfide bridges. Each heavy chain has at one end a variable domain ~VH) followed by a number of constant domains.
Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the frst constant domain of the heavy chain, and the light chain variable 20 domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains (Clothia et aL, J MoL BioL,186:651 -663 (1985); Novotny etaL, Proc. Natl. Acad ScL USA, 82:4592-4596 (1985)).
The term ''variable" refers to the fact that certain portions of the variable domains differ extensively in se4uence among antibodies and are used in the binding and ",~,ifl~,ily of each particular antibody for its 25 particular antigen. However, the variability is not evenly di~llibl-lcd throughout the variable domains of ~ntj~horliec It is collcc.lLl..t~:l in three segm~nt~ called complementarity-~l~trrTnininE regions (CDRs) or hy~J~,- Vdl idble regions both in the light-chain and the heavy-chain variable domains. The more highly consc~ ,d portions of variable domains are called the r a~".,~ ik (FR). The variable domains of native heavy and light chains each comprise four FR regions~ largely adopting a ~-sheet conrl~7ulalion~ c~ d by three cDRs~ which 30 form loops ,~-~-.e~l;"g and in some cases forming part of, the ,e-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, Cullll ibule to the r~,l,..alicnoftheantigen-bindingsiteof~ntihoAie~(seeKabat etaL,Se.~ .e~sofProteinsofImmunological Interesr, Fifth Edition, National Institute of Elealth, Reth~c-l~ MD (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector fimrtir,nc such as !Jal ~ JaLio 35 of the antibody in antibody-fipppnrl~nt cellular toxicity.
Papain digestion of ~ ;ho~ produces two identicai antigen- binding fragments, called "Fab"
rla~.l.,... ,~ each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin 1- caLIll~,.lL yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

CA 0224~63~ 1998-08-0~

-"Fv" is the miniml-m antibody fragment which contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent slecociqtifln It is in this Cu--ri~,u. a~iOIl that the three CDRs of each variable domain interact to define an antigen-- - binding si~e on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding Sl.c.,irl-,ily r 5 to the antibody. However, even a single variable domain (or half of an Fv Cu~ l iahlg only three CDRs specific for an antigen) has the ability to ~c~,oE;--i~ and bind antigen, although at a lower affinity than the entire binding site.
llle Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' rlaElm_~lb differ from Fab r~....,l.~5 by the addition of a few residues at the 10 carboxy terminus of the heauy chain CHI domain inrlnf~inp one or more cysteines from the antibody hinge region. Fab'-SH is the d~ .., herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody La~llcllb originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
The "light chains" of a.lliboL~ (;.. " "- --o~ b~ ) from any vu. t~,l,laLe species can be assigned to one 15 of two clearly distinct types, called kappa (lC) and lambda (~), based on the amino acid seq~e~cpc of their constant domains.
D~,".,..li..g on the amino acid sequence of the constant domain of their heavy chains, imm~nr globulins can be assigned to different classes. There are five major classes of imm~mf glf bulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into sl~h~ c~s (isotypes), e.g, IgG-I, IgG-2, IgG-3, IgG-4, 20 IgA- 1, and IgA-2. The heavy-chain constant domains that c~ ,ud to the different classes of immnnoglobulins are called a, ô, ~, y, and ~1, respectively. The subunit aLIu~Lul~,~ and three-flimPncif~n~l configurations of different classes of immunoglobulins are well known.
The term "antibody" is used in the broadest sense and cpecifir~lly covers single mnnoCl~ns~ ;l.orii~
(inr~ ing agonist and ~7~a~onict antibodies) and antibody cc,--,~ ;onc with polyepitopic aye~iri~ y.
The term "monor~ l antibody" as used herein refers to an antibody obtained from a F ~t -' ' ~ of Ul~ t;;~lly homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring ",~ that may be present in minor amounts. Monoclonal ~ .i ;l .o.~
are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different 30 ~1. t~,ll;ll,.lll~ (epitopes), each mnnoclon~l antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advc...k.geoua in that they are s~ Leai~,d by the hybridoma culture, ~u~ inz~tf~d by other imml-nogloblllinc The m- ~- -o- 1~ ~--~1, - ~~ ;1 ~o-l;-, herein include hybrid and recombinant antibodies produced by splicing a variable (including hypervariable) domain of an anti-CT- I antibody with a constant domain (e.g. "1,.. ~.. ;, . 1"
35 ~ .o.l;r~), or a light chain with a heavy chain, or a chain from one species with a chain from another species, or fusions with heterologous proteins, ~cigaldle~a of species of origin or ;...~n....~glolJulin class or subclass decign~ti~m as well as antibody rla~lll.,ub (e.g, Fab, F(ab')2, and Fv), so long as they exhibit the desired biological activity. (See, e.g. Cabilly, et aL, U.S. Pat. No. 4,816,567; Mage et al., Monoclonal Antibody Production Techniques and App~ications, pp.79-97 (Marcel Dekker, Inc., New York, 1987).) CA 0224~63~ 1998-08-0~
WO 97/30146 - PCT/~JS97/0267~;

Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a lly homogeneous population of antibodies. and is no~ to be construed as requiring production of the antibody by any particular method. For example, the mnnoclon~l antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et aL, Nature, 256:495 5 (197S). or may be made by ~ ulllbilla ll DNA methods (Cabilly el ai. . supra). The monoclnn~l ~ntihofli~c herein specifir~lly include "chimeric" ~.Lil,odies (;..""~...n~lnb.lline) in which a portion of the heavy and/or light chain is ideMical with or homologous to cu~ .p: se~ , in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to cull~ onding seflllPnfpc in ~ntihof~;Pc derived from another species or belonging to another 10 antibody class or subclass, as well as Lid~..e,.i, of such ~ntihoAiPc so iong as they exhibit the desired biological activity(CabillyetaL,supra; MorrisonetaL,Proc. NatL Acad Sci. USA, 81:6851-6855 (1984)).
.; - I" forms of non-human (e.g., murine) ~ntihofliPs are specific chimeric i..""u"oglobulins, immlmoglnbulin chains or fia~,llle.ll~ thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding a ~ r ~~ - lCf s of antihof~iPC) which contain minimal sequence derived from non-human immlmoglnbulin. For I S the most part, hllnn~ni7pd ,..,l il .o-l;f ~ are human immunoglobulins (recipient antibody) in which residues from a compl~...c..la. y-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affunity, and capacity.
In some inct~nrPC, Fv rla~ Jlh residues of the human immunflglobulin are replaced by cull ~I,onfling non-human residues. Furthermore, hllm~ni7Pd ~ntibof~ c may comprise residues which are found neither in the 20 recipient antibody nor in the imported CDR or Galll~ )lh se 1~ f i These mc~flifir~tionc are made to further refine and optimize antibody p~.rul..la l.,c. In general, the l,~ d antibody will comprise cllhct~nti~lly all of at least one, and typically two, variable domains, in which all or c-~hsrorlt~ y all of the CDR regions correspond to those of a non-human ;. .~"~ n~lnbll lin and all or ,~ lly all of the FR regions are those of a human imml-noglnb--lin c~ c seqllpnr~p~ The hllm~ni7r d antibody optimally also will comprise at least 25 a portion of an i~lnu~v~lobulin constant region (Fc), typically that of a human immlmogloblllin For further detailssee: Jonesetal.,Nature,321:522-525(1986)~ -h...~...etaL,Nature,332:323-329(1988):andPresta, Curr. Op. S~rucL BioL. 2:593-596 (1992).
"Non-imm-mogPnic in a human" means that upon CullLa~ llg the polypeptide in a l~h~ lly a~ c~Jt~l le carrier and in a th ,. ~'I ~,- .1 ;- ~lly effective amount with the ~pp,v~, ;ak tissue of a humam no state of 3û sensitivity or l~ia~l~e to the polypeptide is ~1- .,","~ upon the second admilliaLlaLioll of the polypeptide after an al,},.u~,iale latent period (e g, 8 to 14 days).
'rNeurological disorder" refers to a disorder of neurons, including both peripheral neurons and neurons from the central nervous system. Examples of such disorders include all n~,~l,udc~ aLi~e diseases, such as peripheral n~ u~alllies (motor and sensory), amyotrophic lateral sclerosis (ALS), Alzheimer's disease, 35 P~uhill~ull'a disease, stroke, Hl ~ u ~'a disease, epilepsy, and ophth~lmnlogic diseases such as those involving the retina, e.g, diabetic le~illu~JaLIl~, retinal dystrophy, and retinal deg.,.,c.~Lio.l caused by infantile m~lign~nt 03~0~ 1uaia~ ceroid-lilJuL 1~ Oa;a, or r~hr- l or caused by photo-~eg. . .~ ;nn, trauma. axotomy, ne.l~ uLu~;C-excitatory dcg~ aLion, or ischemic neuronal degeneration.

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WO 97/30146 PCT/US97/0267~;

"Peripheral n.. luyalhy" refers to a disorder affecting the peripherai nervous system. most often ; rr, ~I r(l as one or a collli;ulaLiull of motor. sensory, 5~ 01 illlùLùl, or autonomic neural dysfi ~n~tj~m The wide variety of morph~lo~,i.s exhibited by peripheral n~u u~lalhies can each be attributed uniqueiy to an equally wide number of causes. For example, peripheral neu.u~aLhies can be geneticaiiy acquired. can result from a systemic t' 5 disease. or can be induced by a toxic agent. Examples include but are not limited to distal sensorimotor n~.luluaLlly~ or autonomic n~.lupaLllies such as reduced moti}ity of the ~a~LIuill~ tract or atony of the urinary bladder. Ei;xamples of neuropathies associated with systemic disease include post-polio syndrome;
examples of h~dikuy m~..u~alllies include Charcot-Marie-Tooth disease~ Refsum's disease, Ah~tnl;l.u~ t~ 1- 1l;,. Tangierdisease, Krabbe's disease7 MrLd~,lllu~la~ic leukodystrophy, Fabry's disease, and iO Dejerine-Sottas syndrome; and examples of ncllluyall~ caused by a toxic agent include those caused by Ll-,aLIll.llt with a chemofh~ l;c agent such as vill~ e.
"Heart failure" refers to an ~bnf nn~lity of cardiac function where the heart does not pump blood at the rate needed for the ~~,~ui. ~,.ll~,.li~ of metabolizing tissues. Heart failure includes a wide range of disease states such as congestive heart faiiure, myocardial infarction, and tachyarrhythmia.
"Tl~iallllcllL" refers to both thclalJ.alic treatment and prophylactic or ~ ive measures. Those in need of treatment include those aiready with the disorder as well as those prone to have the disorder or those in which the disorder is to be ~ t~,;i.
"Mammal" for purposes of Ll~d~ ,llL refers to any animal ~l~c~ifi~od as a mammal, including humans.
domestic and farm animals, and zoo, sports, or pet animals. such as dogs, horses, cats, cows. etc. Preferably, the 20 - mammal herein is human.
As used herein, "ACE inhibitor" refers to ~ ;ut- .~ .-converting enzyme inhibiting drugs which prevent the conversion of ~ngiflt-on~in I to ~ ;ol~ II. The ACE inhibitors may be beneficial in congestive heart failure by reducing systemic vascular l c~ ,e and relieving circulatory ~ u . ~gr~ The ACE inhibitors include but are not limited to those ~ t~ d by the ~ Accupril~ Iquinapril), Altace~ (ramipril), Capoten~
25 (captopril), Lotensin~E9 (brn~l-.,"lil), Monopril~ (fosinopril), Prinivil~ ~lisinopril3, Vasotec~ (enalapril), and Zestril~9 (lisinopril). One example of an ACE inhibitor is that sold under the l-ad~,.lla~h Capo~en~}). Generically referred to as captopril, this ACE inhibitor is .k ~ rd rhf mir~lly as I -[(2S)-3-mercapto-2-methylpropionyl]-L-proline.
Tl. Modes for Practicin~ the Invention 30 1. CT-I PolypeDtides Preferred polypeptides of this invention are s~ ti~lly h-....og. ~,-rol~c CT-I polypeptide~s), having the biological ~lu~.Lies of being myocyte h~,u~,lllu~hic and of 5tim~ ting the dcvelu~,l,..lll of chick ciliary neurons in a CNTF assay. More preferred CT-ls are isolated m~nnmz~ n protein(s) having hypertrophic, anti-arrhy~mic, illUllU~I ., and neurological activity. Most preferred polypeptides of this invention are mouse and 35 human CT-ls inclllrling La~ b thereof having hypertrophic, anti-arrhythmic, inotropic, and neurological activity. Optionally these murine and human CT-ls lack glycosylation. WO 9529237. which published November, 02, 1995, and which is inco.~.u..t.,d herein by reference, discloses CT-I nucleic acid and protein sc~ c and certain uses of CT-I.

CA 0224~63~ 1998-08-0~

Optional preferred polypeptides of this invention are biologicallv active CT- I variant(s) with an amino acid sequence having at least 70% amino acid sequence identity with the murine CT-I of Fig. i, preferably at least 75~/O. more ~Icf~.ably at least 80%~ still more preferably at least 85%. even more preferably at least 90%, - - and most preferably at least 95% (ie., 70-100%, 75-100%, 80-100%, 85-100%, 90-100%, and 95-100%
S sequence identity, respectively). Altematively, the preferred ' -'~gjr~lly active CT-I variant(s) have an amino acid sequence having at least 70%, preferably at least 75%, more preferably at least 80%, still more preferably at least 85%, even more preferably at least 90%, and most pl~,f~ .ably at least 95% amino acid sequence identity with the human CT-I sequence of Fig. 5 (i.e., 70-100%, 75-100%, 80-100%, 85-100%, 90-100%. and 95-100%
sequence identity, .~ ..,ly).
The CT- I cloned from murine embryoid bodies has the following CIIOI a~l~. i .li- s.
( I ) It has a ...ole~ ula. weight of about 21-23 IcD as measured by reducing SDS-PAGE;
(2) It shows positive activity in the CNTF chick ciliary neuron assay and in the myocyte hy~,LIu~ and ANP-release hy~ L~ul~hy assays.
More pre~erred CT-I polypeptides are those encoded by genomic DNA or cDNA and having the amino 15 acid sequence of murine CT-I described in Fig. I or the amino acid sequence of human CT-I described in Fig.
5.
Other preferred naturally occurrin~ biologically active CT-I polypeptides of this invention include prepro-CT-I, pro-CT-1, pre-CT-I, mature CT-I, and glycosylation variants thereof.
Stiil other preferred polypeptides of this invention include CT- I sequence variants and chimeric CT- I s .
20 Ordinarily, preferred CT-I sequence variants are L) -1~gi~11y active CT-I variants that have an amino acid sequence having at least 70% amino acid sequence identity with the human or murine CT- I, preferably at least 75%, more preferably at least 80~/O, still more ~ r~ ~ably at least 85%, even more preferably at least 90%, and most preferably at least 95%. An ~ laly preferred CT-I variant is a C-terminal domain CT-I variant in which one or more of the basic or dibasic amino acid residue(s) (e.g., R or K) is s.~ ;I with a non-basic 25 amino acid residue(s) (e.g, hydlu~ll l c, neutral, acidic, aromatic, gly, pro and the like).
Another ~ lllyLuy preferred CT-I sequence variant is a "domain chimera" that consists of the N-terminal residues s~ ;i with one or more~ but not all~ ofthe human cNTF residues alJlJlu~illlal~ly alignedas shown in Fig. 2. In this ~mborlimpnt the CT- I chimera would have individ ual or blocks of residues from the human CNTF sequence added to or s~ t~ d into the CT-I sequence at positions CUII ~ g to the 30 ~lignm~nt shown in Fig. 2. For example, one or more of those segments of CNTF that are not hnmnlngn~ could be ~ d into the CUII- .~ linp segments of CT-I. It is cQnfomr~ that this "CT-I-CNTF domain chimera" will have mixed h~ ,L u,~.Lic/anti-arrhyth~nic/illuL O~J;C/ ~~ IuL.~"~hic biological activity.
Otherprefierred polypeptides ofthis invention include CT-I Ga~ .-~ having a cu-l.,e~.uLbte sequence .of at least 10, 15, 20, 25, 30, or 40 amino acid residues, ~ f~ ,ably about 10-150 residues, that is identical to the 35 sequenceoftheCT-I isolatedfrommurineembryoidbodiesortothatofthecull~ .ghuman CT-I. Other preferred CT-I r a~.,. .~t~ include those produced as a result of chemical or enzymatic hydrolysis or digestion of the purified CT- I .
AnotheraspectoftheinventionisamethodforpurifyingCT-lmal l~cu...~ ,i..grnnt~rtinp,aCT-I
source ront~ining the CT-I molPrnlPs to be purified with an immobilized receptor or antibody polypeptide. under .
CA 0224~63~ 1998-08-0~
WO 97~30146 PCT/US97/0267S
-c~ .nc whereby the CT-I molecules to be purified are selectively adsorbed onto the immobilized receptor or antibody polypeptide, washing the immobilized support to remove non-adsorbed material, and eluting the mr 1PC~ C to be purified from the immobilized receptor or antibody polypeptide to which they are adsorbed with - - an elution buffer. The source contAAining the CT-I may be a cell SU~ IIS;OII of embryoid bodies.
Alternatively,thesourcerc,l.lA;.. i.. gtheCT-I is.~cu.,.L.l.al.Lcellculturewheretheconc~ laliollofcT-I in either the culture medium or in cell Iysates is generally higher than in plasma or other natural sources. In ~,~ this case the above-described i.""".. ,oArl;llity method, while still useful, is usually not necessary and more traditional protein purification methods known in the art may be applied. Briefly, the preferred purification method to provide ~ Al(l;AllY h..("n~,. ,e~Ju~ CT-I culu~ c~ removing particulate debris by, for example, 10 centrifugation or ultrafiltration; optionally cull~,.,(lLlalil.g the protein pool with a commercially available protein cull-,~lluatiull filter; and thereafter purifying the CT-I from cnntAminAnt soluble proteins and polypeptides, with the following ~. u.,clu.~,~ being ~,A~,...~,lal ,y of suitable ~ ,a~iùn ~- u-,~lul .,~. by G ~ n on ~ uA ~ y or ion-exchange columns; ethanol ~ l l; reverse phase HPLC; clll ullla~O~,Iat~lly on silica or on a cation-exchanAe resin such as DEAE; chromatofocusing; SDS-PAGE; allllllulliulll sulfate ~ J;ia~ioll; Toyopearl and 15 MONO-Q or MONO-S ~,hlul-~atu~ a~Ly; gel filtration using, for example, S~phA~1PY G-75; chromatography on columns that bind the CT-I, and protein A Scpllaluse columns to remove cQntArninAntc such as IgG. One preferred purification scheme for both native and l~,culllbillallL CT-I uses a Butyl Toyopearl column followed by a MONO-Q column and a reverse-phase C4 column as described further below.
In anotherpreferred c-"l-o~l;l,, ,l, this invention provides an isolated antibody capable of binding to the 20 CT-I. A preferred isolated anti-CT-I antibody is monoclonal (Kohler et al., Na~ure, 256:495-497 (1975);
~'Arnpbell, Laboratory T~"h,..41~ in Bio~ y and Molecular Biology, Burdon et aL, Eds, Volume 13, Elsevier Science Publishers, Allls~,rdalll (1985); and Huse et~l., Science, 246:1275-1281 (1989)). Preferred isolated anti-CT-I antibody is one that binds to CT-I with an affinity of at least about lo6 L/mole. More preferably, the antibody binds with an affinity of at least about 107 ~/mole. ~ost preferably, the antibody is 25 raised against a CT-I having one of the above-dc~c. iLcd effector functions. The isolated antibody capable of binding to the CT-I may optionally be fused to a second polypeptide and the antibody or fusion thereof may be used to isolate and purify CT-I from a source as described above for immobilized CT-I polypeptide. In a further preferred aspect of this ~ "l .o~ the invention provides a method for detecting the CT- I in vitro or in vivo cul~ g CV~ p the antibody with a sample, especially a serum sample, 1~ d of AontAining 30 the CT- I and detecting if binding has occurred.
The invention also provides an isolated nucleic acid molecule encoding the CT-I or ~a~ll~,.ll~ thereof~
which nucleic acid molecule may be labeled or lmlAhPl~d with a d~,L~,~,iaL'- moiety, and a nucleic acid molecule having a sequence that is compl y to, or hybridizes under stringent or moderately stringent con-litinn~
with, a nucleic acid molecule having a sequence encoding a CT-1. A preferred CT-I nucleic acid is RNA or 35 DNA that encodes a biologically active CT-I sharing at least 75%, more preferably at least 80%, still more preferably at least 85%, even more ~l~r~.ably 90%, and most preferably 95%, sequence identity with the murine or human CT- I . More preferred isolated nucleic acid molecules are DNA se~ ~ encoding biologically active CT-I, selected from: (a) DNA based on the coding region of a mAnnmAliAn CT-I gene (e.g, DNA COU iug the n~ eOti~ sequence provided in Fig. I or Fig.5, or r ag-ll~ thereof); (b) DNA capable of hybridizing to a DNA of (a) under at least moderately stringent con~iitirlnc and ~c) DNA that is dc~ _. a~c; to a DNA defined in(a)or(b)whichresultsfromdcge.,_.a~yofthegeneticcode. Itisco-l~.."~ 'thatthenovelCT-lsdescribed herein may be members of a family of ligands having suitable sequence identity that their DNA may hybridize with the DNA of Fig. I or Fig. 5 (or r~ aglllC~ 7 thereof) under low to moderate stringency conditions. Thus, a S further aspect of this invention includes DNA that hybridi~es under low to moderate ~,l, hl~,_.ll y conditions with DNA encoding the CT-I polypeptides.
Preferably, the nucleic acid molecule is cDNA encoding the CT- I and further CVIIIIJI ia~,S a replicable vector in which the cDNA is operably linked to control sPqaPnces recogni7Pd by a host l~a.lsrul.ned with the vector. This aspect further includes host cells l.allsrulllled with the vector and a method of using the cDNA to 10 effect~lu-lucliul, of CT-I, Cul~ i7illg ~,,u,,l~,.u-gthe cDNA encoding the CT-I in a culture ofthe L-a--,rw---ed host cells and recovering the CT-I from the host cell culture. The CT-I prepared in this manner is preferably lly homogeneous murine or human CT-1.
The ill~ iUII further includes a preferred method for treating â mammal having heart failure, or an arrhythmic, inotropic, orr._ulul~ic~l disorder, culll~ illg S~clminictpring a LL_.~ irzllly effective amount of 15 a CT-I tù the mRmmRI Optionally, the CT-I is Rtiminictpred in combination with an ACE inhibitor. such as captopril, in the case of congestive heart failure, or with another myù~ al d;ULIU,JII;C~ anti-a rhythmic. or inotropic factor in the case of other types of heart failure or cardiac disorder, or with a n~,u.ul.u"hic molecule such as, e.g~
IGF-I, CNTF, NGF, NT-3, BDNF, NT4, NT-5, etc. in the case of a neurological disorder.
2. I~ Jal aLi(lll of Natural-Seguence CT-I and VariRntc 20 Mostofthelli~.u~ .. belowpertainsto~,ludu.,~iullofCT-IbyculturingcellsLIdl~ro~-~-edwithaYector c,",l 1;.~ g CT-I nucleic acid and recovering the polypeptide from the cell culture. It is further envisioned that the CT-I ofthis invention may be ~ luduce~ by homologous reccmbin~tirn as provided for in WO 91/06667 published 16 May 1991. Briefly, this method involves L.al-:.r(,...ling primary m~nnm~ n cells containing Pn~r~nmlc CT-I gene (e.g, human cells if the desired CT-I is hurnan) with a construct (i.e., vector) Culll~lisil-g 25 an ~ nplifi~hlP gene (such as dihydrofolate . ~ dh_La~c [DHFR] or others discussed below) and at least one flanking region of a length of at least about 150 bp that is homologous with a DNA sequence at the locus of the coding region ofthe CT-I gene to provide ~rnplifir~tion ofthe CT-I gene. The amplifiable gene must be at a site that does not interfere with ~ iVII of the CT-I gene. The L al.~ru....aLion is crnfi--rtPd such that the consrruct becomes homologously iult~lal. l into the genome of the primary cells to define an amplifiabie region.
i'rimary cells UUIII~ g the construct are then selected for by means of the amplifiable gene or other marker present in the construct. The presence of the marker gene ~ the presence and hlltE5lalioll of the construct into the host genome. No further selection of the primary cells need be made, since seiection will be made in the second host. If desired, the o- uull~,nce of the hol..ologol.s l~i.ulllbillaLiùll event can be determined by ~ ' yillg PCR and either sequencing the resulting amplified DNA s~ p ~ or fl t~ the a~ lv~l iaL~
35 length ofthe PCR fragment when DNA from correct Lvlllologvu~ slal~La is present and PYp~nriing only those cells c~ ;-.;..g such rla~lll_lll i. Also if desired, the selected cells may be amplified at this point bv stressing the cells with the al~lJIulJI iaL~ amplifying agent (such as methotrexate if the amplifiabie gene is DHFR), so that multiple copies of the target gene are obtained. Preferably, however, the amplification step is not cr nrll Irt~d until after the second transforrnation des. l il.ed below.

CA 0224;i63;i 1998-08-O;i After the selection step, DNA portions of the genome, 5~ffirirnrly large to include the entire a~ Jlirlablc region. are isolated from the selected primary cells. Secondary m~nnm~ n c~l.lc~"iu" host cells are then rO....rd with these genomic DNA portions and cloned, and clones are selected that contain the amplifiable - - region. The amplifiable region is then amplified by means of an amplifying agent, if not already amplified in 5 the prirnary cells. Finally, the secondary ~A~ 7~7iUll host cells now Culuyl i~,i.-g multiple copies of the ~ul~lir.al)'r region crnt~ining CT-I are grown so as to express the gene and produce the protein.
A. Isolation of DNA Encodin~ CT-I
The DNA encoding CT-I may be obtained from any cDNA library prepared from tissue believed to possess the CT-l mRNA and to express it at a detectable level. The mRNA is suitably prepared, for example, 10 from seven-day dirf~ ;aL~l embryoid bodies. The CT-I gene may also be obtained from a genomic library or by in vitro olignmloleotide synthesis as defined above ~ccllming the complete nucleotide or amino acid sequence is known.
Libraries are screened with probes designed to identify t-h-e gene of interest or the protein encoded by it. For cDNA expression libraries, suitable probes include, e.g.: monoclonal or polyclonal antibodies that 1 S l ~coy,llM, and specifically bind to the CT- l; oli~ k ~ f ~ of about 20-80 bases in length that encode known or s~ - kd portions of the CT-I cDNA from the same or different species; and/or compl~,.ll~.lLaly or homologous cDNAs or La~,lll~,.ll, thereof that encode the same or a similar gene. A~ Iù~Jl;ale probes for screening genomic DNA libraries include, but are not limited to, oli~.. - If ~ C, cDNAs, or r,a~lll~,.l~, thereof that encode the same or a similar gene, and/or hnmr 'og genomic DNAs or fragments thereof. Screening the 20 cDNA or genomic library with the selected probe may be confl~rted using standard procedures as described in chapters 10-12 of Sambrook et al.. supra.
An alternative means to isolate the gene encoding CT-I is to use PCR methodology as described in section 14 Of Sambrook et aL, supra. This method requires the use of olig~ lf o~ probes that will hybridize to the CT-I. Strategies for selection of oligo~ lnv~;Aec are described below.
A preferred method of ~ g this invention is to use carefully selected olignm-rleoti~i~ seqnrnf ~os to screen cDNA libraries from various tissues, preferably m~mm~ n dilrtl~.l,id-t~d embryoid bodies and placental, cardiac~ and brain cell lines. More preferably, human embryoid, placental. cardiac, and brain cDNA
libraries are screened with the olig. . ~. .. If vl if If probes.
The oligonucleotide Sf.~ selected as probes should be of sulrl~;~,llL length and sufficiently 30 ~-. b~ uu~thatfalsepositivesare~ fl Theactual..~lrv~ f,~ ufe(s)isusuallybasedonconserved or highly homologous ....- 1~ .,1 if if c~ f ~ The oligom~rl~otifiçs may be flf g. . . -~ - at one or more pocifinne The use of dcg~ la~t: oli~ vlidf,s may be of particular importance where a library is screened from a species in which ~ ,f~ idl codon usage is not known.
The oligl nnf leotifif- must be labeled such that it can be detected upon hybridization to DNA in the 35 library being screened. The preferred method of labeling is to use 32P-labeled ATP with pol~ r Irvl ;(1f kinase, as is well known in the art, to radiolabel the ol;g~ r~JI ;-1f However, other methods may be used to label the oli~unuclc~lLide, including, but not limited to, biotinylation or enzyme labeling.
Of particular interest is the CT- I nucleic acid that encodes a full-length polypeptide. In some preferred Pmhorlimf ntc the nucleic acid sequence includes the native CT- I signal s~ ql~nre Nucleic acid having all the CA 0224;i63;i 1998-08-O;i WO 97/30146 PC~T/US97/02675 protein coding sequence is obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessar,v, using conventional primer Pxr~n~i~7n procedures as des~,.il,cd in section 7.79 of Sambrook et al., supra, to detect ~ ,Ul:~ul:l and l lu~,_Saillg - intermediales of mRNA that may not hsve been reverse-LIan~,l il,ed into cDNA.
B. Amino Acid Sequence Variants of Native CT-l Amino acid sequence variants of native CT-I are prepared by introducing a~Jlu~JliaLe n~ Lot;~ic changes into the native CT-I DNA, or by in vitro synthesis of the desired CT-I polypeptide. Such variants include. for example, deletions from, or i..~ ions or sllhcfihltinnc of, residues within the amino acid sequence shown for murine CT-I in Figure I and for human CT-I in Figure 5. Any combination of deletion, insertion, 10 and cllhctitlltir7n is made to arrive at the final construct, provided that the final construct possesses the desired ~L~al,t~ ics. Excluded from the scope of this invention are CT- l variants or polypeptide sf~oll lPnf~Pc that are the rat homolog of CT-I . The amino acid changes also may alter post-~ ;v~i processes of the native CT-I, such as changing the number or position of glycosylation sites.
For the design of amino acid sequence variants of native CT- 1, the location of the mutation site and the 15 nature of the mutation will depend on the CT- I ellal a~.t~ LiC~s) to be modified. For example. cS~n~iirk7-t~ CT- I
,...~gr~n;~l~ or super agonists will be initially selected by locating sites that are identical or highly conserved among CT-I and other ligands binding to members of the growth hormone (GH)/cytokine receptor family, especially CNTF and leukemia inhibitory factor ~LIF). The sites for mutation can be modified individually or inseries~e~g~by(l)sl~ ll;llgflrstwithcoll~t;lvaLi~ aminoacidchoicesandthenwithmoreradicalsr~ ;oll~
20 f~ I;"g upon the results achieved, (2) deleting the target residue, or (3) inserting residues of the same or a different class adjacent to the located site, or cullll)illaLions of options 1-3.
A useful method for i. 1~ .1; r~ ull of certain residues or regions of the native CT- I polypeptide that are preferred locations for ..~ ;. n -:~ is called "alanine scan ~ing m~ ,c." -;~ " as described by fllnninghf7nn et aL, Science, 244:1081-1085 (1989). Here, a residue or group of target residues are jr7f~ntifi~d ~e.&, charged 25 residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the illL~:la~,Liull of the amino acids with the surrounding aqueous cll~ ulullc.l~ in or outside the cell. Those domains ~l~ "~"~ i"g r~ 7-l sensitivity to the ~ )nc then are refined by illLIudu~,iulg fur~her or other variants at or for the sites of 5~7hctit7ltir7n Thus. while the site for hlllullllculg an amino acid sequence variation is ~ ,d~~~,.lllined, the nature of the mutation per se need not be 30 pl~d~,~..lllined. For example, to optimize the l;clrulll,ance of a mutation at a given site, a}anine scanning or random ",~e~ is r,....~ r~ d at the t-_rget codon or region and the CT-I variants produced are screened for the optimal combination of desired activity.
There are two principal variables in the co ll~lu~,Lull of amino acid sequence variants: the location of the mutation site and the nature of the mutation. These are variants from the Fig. I or Fig. 5 Sf~f,7llf~nre and may 35 ru~ .lt naturally o~l,ullhlg alleles (which will not require ms7nipul~7tirn of the native CT-I DNA) or IJI. .7~ r, ",i,.r~d mutant forms made by mutating the DNA, either to arrive at an allele or a variant not found in nature. In general, the location and nature of the mutation chosen will depend upon the CT-I ~hala.,t~ Lic to be modified.

CA 0224~63~ l998-08-0~

Amino acid sequence deletions generally range from about I to 30 residues. more preferabiy about l to 10 residues, and typically are conrigllouc Contig~ c deletions ordinarily are made in even numbers of residues, but single or odd numbers of deletions are within the scope hereof. Deletions may be i,u- u iu~ed into - - regions of low homology among CT- I and other ligands binding to the Gi l/cytokine receptor family which share the most sequence identity to the human CT- I amino acid sequence to modify the activity of CT- 1. Deletions from CT-I in areas of ~..1 .,.~.,n~l homology with one of the receptor binding sites of other ligands that bind to f the Gi~/cytokine receptor family will be more likely to modify the biological activity of CT-I more cignifir~:lntly The number of co..cec~ /e deletions will be selected so as to preserve the tertiary structure of CT-I in the affected domain, e.g., beta-pleated sheet or alpha helix.
Amino acid sequence il~ n5 include a~,nino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides c~ , ;"p. a hundred or more residues, as well as ;" ", .~ . ce insertions of single or multiple amino acid residues. IULI. ~ .,- e insertions (i.e., insertions within the mature CT-I sequence) may range generally from about I to 10 residues, more preferably I to 5, most preferably I to 3. Insertions are preferably made in even numbers of residues, but this is not required. Examples of terminal insertions include mature CT-I with an N-terminal methionyl residue, an artifact of the direct production of mature CT-I in recombinant cell culture, and fusion of a heterologous N-terminal signal sequence to the N-terminus of the mature CT-I molecule to facilitate the secretion of mature CT-I from IL~''I'l' '~'ll hosts. Such signal 5~ ej generally will be obtained from, and thus hL~ olo~ou. to, the intended host cell species. Suitable se~
include STII or Ipp for E. coli, alpha factor for yeast, and viral signals such as herpes gD for m:~mms-li ~n cells.
Other insertional variants of the native CT- I molecule include the fusion to the N- or C-terminus of native CT-I
of ;",~ -,a". ~,;c polypeptides, ~g, bacterial polypeptides such as beta-l ~ In~ f- or an enzyme encoded by the ~;. coli trp locus, or yeast protein, and C-terminal fusions with proteins having a long half-life such as ;",....,.,n~l~.l,..iin constant regions (or other imnnl-nnglnblllin regions), albumin, or ferritin. as described in WO
89/02922 published 6 April 1989.
A third group of variants are amino acid ~ variants. These variants have at least one amino acid residue in the native CT-I molecule removed and a different residue inserted in its place. The sites of greatest interest for ~ i. ." ~ i ", . ~ include sites identified as the active site(s) of native CT- I and sites where the amino acids found in the known ~n~iogllf s are subst7mti~lly different in terms of side-chain bulk, charge, or hy~ ol)iclLy, but where there is also a high degree of sequence identity at the selected site within various animal CT-I species, or where the amino acids found in known ligands that bind to members of the GH/cytokine receptor family and novel CT-I are ~b~ lly different in terms of side-chain bulk, charge, or hydlu~Lolf._;ly, but where there also is a high degree of sequence identity OE the selected site within various anirnal ~n~logl~s of such ligands (~.g, among all the animal CNTF molecules). This analysis wiil highlight residues that may be involved in the dirr~ a~io" of activity of the cardiac hypertrophic, anti-arrhythmic, inotropic, and n~.llu~ Jllic factors, and therefore. variations at these sites may affect such activities.
Other sites of interest are those in which particular residues of the CT-I obtained from various species are identical among all animal species of CT-I and other ligands binding to GH/cytokine receptor family molecules, this degree of cullr~llllldtion ~. .g~ l ;..g ull~ul Lallcc in achieving biological activity common to these enzymes. These sites~ especially those falling within a sequence of at least three other identically conserved sites, are '~ l t d in a relatively conservative manner. Such conservative ~ ;n.)~ are shown in Table I under the heading of preferred ~..h~ If such 5nhstihlti~nc result in a change in biological activitv, then more ,,l;...l;,.i changes, d~ ..i.. ~1 exemplary ,,.l.~lil..l;~.,.c in Table 1, or as further described below in reference to amino acid classes, a}e inhroduced and the products screened.
Table I
Original FY~mpi~ry Preferred Residue Substihutions ~l.hctih-rions Ala (A) val; leu; ile val Arg (R) Iys; gln; asn Iys 10 Asn (N) gln; his; Iys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp t5 Gly (G) pro pro His (H) asn; gln; Iys; arg arg lle (I) leu; val; met; ala; phe;
norleucine leu Leu (L) nollc.l.,i.. e, ile; val;
met; ala; phe ile Lys (K) arg; gln; asn arg Met(M) leu; phe; ile leu Phe(F) leu; val; ile; ala leu Pro (P) gly gly 25 Ser (S) thr thr Thr (T) ser ser Trp (W) tyr tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe;
ala; nu~ lc leu Su~ ;r~ ;.. C in function or imrnunological identity ofthe native CT-I are a.,-,ul.l~ L~
byselecting~..l.~l;l..li-.~.cthatdiffer,:~..;ri.~ yintheireffectonm~int~ining(a)thestructureofthepolypeptide backbone in the area of the 5~h5tihltion~ for example, as a sheet or helical conr(Jlllldtio.l, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naburally occurring residues 35 are divided into groups based on common side-chain I~lVp~..
(I) hy~hu~.hobic. norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;

(4) basic: asn, gln, his, Iys, arg;
40 (5) residues that influence chain u~ dLiun gly, pro; and CA 0224~63~ 1998-08-0~
WO 97/30146 PCT/US97/û2675 -(6) aromatic: trp, tyr, phe.
Non-conservative snhstitl~tione will entail e~ch~neine a member of one of these classes for another.
such ~ d residues also may be i~ ùduced into the co~ v~Li~/e ~ sites or~ more preferably~ into - the remaining (non-conserved) sites.
In one ~ ~ - .ho. i ;.. 1 of the invention, it is desirable to inactivate one or more protease cleavage sites that are present in the molecule. These sites are identified by ;..~I.e~ .. ofthe encoded amino acid seqn~nc~ in the case of trypsin, e.g, for an arginyl or Iysinyl residue. When protease cleavage sites are i~l~ontifi~ they are rendered inactive to proteolytic cleavage by ~,..I.~I;n.~;.,g the targeted residue with another residue, preferably a basic residue such as p l l ~-- " ;"e or a hyd- u~Jhoi, - residue such as serine; by deleting the residue: or by inserting 10 a prolyl residue immr~ t~ly after the residue.
In another ~,I..bo i"".,l.L, any methionyl residues other than the starting methionyl residue of the signal seqll~nr~- or any residue located within about three residues N- or C-terminal to each such methionyl residue, is s- ~~ ,d by another residue (~. ~,f~,l~ly in accord with Table I ) or deleted. Alternatively, about 1-3 residues are inserted adjacent to such sites.
Any cysteine residues not involved in -S-; ~ the proper Cullr(J~ aliun of native CT- I also may be i, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant k ;~e Nucleic acid molecules encoding amino acid se~uence variants of native CT-I are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in 20 - the case of naturally occurring amino acid sequence variants) or ~ u aLion by oiiennllcleoti~l~mediated (or site-directed) ...~ PCR .~ and cassette ....-' ~g.?~ of an earlier prepared variant or a non-variant version of native CT- I .
Olig--l~ mediated ~ is a preferred method for preparing cllhstihltion deletion, and insertion variants of native CT-l DNA. This 1~ ~ I...i-l- ~ is well icnown in the art as described by Adelman et aL, 25 DNA, 2:183 (1983). Briefly, native CT-I DNA is altered by hybridizing an oli~.. koli~ encoding the desired mutation to a DNA template, where the template is the single-stranded form of a plasmid or bacteriophage e~ the unaltered or native DNA sequence of CT-1. After hybridization, a DNA polymerase is used to synthesi~anentiresecondc , ' ~ystrandofthetemplatethatwilithus~,.cu~,u.aL~theolienn--r~oti~i~
primer, and will code for the selected alteration in the native CT-I DNA.
Generally, oii~ rul ;~ of at least 25 nucleotides in length are used. An optimal olig-,"~cleol i.~
will have 12 to 15 nllrl~oti-~e that are co---l,lct~,ly cu...~! y to the template on either side of the mlcleoti~le(s) coding for the mnt~tion This ensures that the olignn-lr!Potirl~ will hybridize properly to the single-stranded DNA template molecule. The oli~.. If ol ;~lr C are readily S~.ILh~d using terhni~ln~e icnown in the art such as that described by Crea et al., Proc. NatL Acad Sci. USA, 75:5765 (1978).
The DNA template can be grn~or7lted by those vectors that are either derived from ba~ ,l iophage M 13 vectors(thecu.. ~l-,;dllyavailableMl3mpl8andMl3mpl9vectorsaresuitable),orthosevectorsthatcontain a single-stranded phage origin of replication as described by Viera et al., Meth. FnzymoL, 153 :3 ( 1987). Thus, the DNA that is to be mutated may be inserted into one of these vectors to generate single-stranded template.
Production of the single-stranded template is described in Sections 4.21-4.41 of Sambrook et aL, supra.

CA 0224~63~ 1998-08-0~

Alternatively, single-stranded DNA tempiate may be gPnP~tPd by dellaLul ulg double-stranded plasmid (or other) DNA using standard terhni~ Pc For alteration of the native DNA sequence (to generate amino acid sequence variants, for example), the oiig~ ide is hybridized to the single-stranded template under suitable hybridization conr~itir~nC A DNA
polymerizing en_yme, usually the Klenow fragment of DNA polymerase 1, is then added to synthesize the ?
compl~,ln.,lLal y strand of the template using the oli~onl~rlçotirlp as a primer for synthesis. A heteroduplex molecuie is thus formed such that one strand of DNA encodes the mutated form of native CT- I, and the other t strand (the original template) encodes the native. unaltered sequence of CT- I . This heteroduplex molecule is then ~ .. f A into a suitable host cell, usually a proka~yote such as E. coli JM 101. After the cells are grown, 10 they are plated onto agarose plates and screened using the oliGom~clçotirlP primer radiolabeled with 32p to identify the bacterial colonies that contain the mutated DNA. The mutated region is then removed and placed in an a~J~ lu~ t~, vector for protein 7JIudu-,lioll, generally an e~ 'e.~aiOn vector of the type typically employed for Ll,. .~r ~ ;nl- of an a~lu~n;aLe host.
The method described i.. ~r~ t Iy above may be modified such that a homoduplex molecule is created 15 wherein both strands of the plasmid contain the ~ 1(5). The mor7ifirAtionc are as foliows: The single-stranded olig~.-...r!fol;rlP is annealed to the single-stranded template as described above. A mixture of three deox~ r Ul ;r7-Pc deox~,. ;hoA~ (dATP), deox~/. ;l .o~ .r (dGTP), and deoxyribothymidine (dT~P), is cu.,.i,....,l with a modified thio-deoxyribocytosine called dCTi'-(aS) (which can be obtained from the Amersham Corporation). This mixture is added to the template-olig.. rlrolidr- complex. Upon addition of 20 DNA polymerase to this mixture, a strand of DNA identical to the template except for the mutated bases is generated. In addition, this new strand of DNA will contain dCTi'-(aS) instead of dCTP, which serves to protect it from I~Lli~Li(,ll Pnrlnmlr7~P~cP rligectir n After the template strand of the double-stranded h~lel I , ' is nicked with an a~vlul~l ;ale l~,~LI i~
en~yme, the template strand can be digested Witil EiroIII nuclease or another a~lJI U~JI iaLe nuclease past the region 25 that contains the site(s) to be .... Il: G~ The reaction is then stopped to leave a moiecule that is only paltially single-stranded. A complete doubie-slranded DNA homoduplex is then formed using DNA polymerase in the presence of all four deoxyril ~ If ol;~l~ Ll ;llllo~llll t~ ', ATi', and DNA ligase. This h~mollllrlex molecule can thenbellall~rullllcdintoasuitablehostcellsuchas~.coliJMlol~asdescribedabove.
DNA Pncorling mutants of native CT-I with more than one amino acid to be ~llh~ t~;; may be 30 generated in one of several ways. If the amino acids are located close together in the polypeptide chain, they may be mutated ~ .f )~1y u5ing one Gl;~ o~ that codes for all of the desired amino acid ~ c If, however, the amino acids are located some distance from each other (separated by more than about ten amino acids), it is more difficult to generate a single oiig... k )Li(ie that encodes all of the desired changes. Instead, one of two alternative methods may be employed.
In the first method, a separate olig.. -- ~ is ~ d for each amino acid to be subctihltr~rl The o~ ol;r~pc are then annealed to the single-stranded template DNA cimnll ...r ~ ly, and the second shand of DNA that is synthPci7Pd from the template will encode all of the desired amino acid s..l .~ ..l ;.,..c Thealternativemethodinvolvestwoormoreroundsof....~l ~,.., .i~toproducethedesiredmutant. The first round is as described for the single mutants: wild-type DNA is used for the template, an oli~ clcol ;rlP

CA 0224~63~ I998-08-0~
WO 97/30146 PCT/US97/0267~;

encoding the first desired amino acid 5~-hctit--fir~n(5) is annealed to this template, and the heteroduplex DNA
molecule is then generated. The second round of ~...":1~,,....;~ utilizes the mutated DNA produced in the first round of m~ f.~ as the template. Thus. this template already contains one or more mut~tionc The oli~ rv~ encoding the ~ ti~ desired amino acid 5nhctihltit~n(5) is then annealed to this template, and 5 the resulting strand of DNA now encodes .- -. ~l,.l ;....~ from both the first and second rounds of, . - ~ "P ~;c This resultant DNA can be used as a template in a third round of Illula~ e~;s, and so on.
~r PCR .. ~ is also suisable for making amino acid variants of native CT-I . While the following ol- refers to DNA, it is understood that the tPçhninlnf also finds application with RNA. The PCR
technique generally refers to the following ~.ucc.lu.~ (see Erlich, supra, the chapter by R. Higuchi, p. 61-70):
10 When small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the ~u~ l;..g region in a template DNA can be used to generate relatively large 4.. ~
of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template. For introduction of a mutation into a plasmid DNA, one of the primers is designed to overlap the position of the mutation and to contain the mntPf;on; the sequence of the other primer must be identical to 15 a stretch of sequence of the opposite strand of the plasmid. but this sequence can be located anywhere along the plasmid DNA. It is preferred, however, that the sequence of the second primer is located within 2ûO n-lnl.~otiflPc from that of the first, such that in the end the entire amplified region of DNA bounded by the primers can be easily s~ .- e~ PCR amplification using a primer pair like the one just described results in a pop~ ti--n of DNA G avl--~,-lb that differ at the position of the mutation specified by the primer, and possibly at other positions, 2û as template copying is sullle~.LaL error-prone.
If the ratio of template to product material is extremely low, the vast ma~ority of product DNA
fragments incorporate the desired mutation(s). This product material is used to replace the cu... ~l .u.~.l;..g region in the plasmid that served as PCR template using standard DNA technology. Mutations at separate positions can be illlludu~,e;l ~imlllt~n~oucly by either using a mutant second primer, or p~,.r~,l.llillg a second PCR with different 25 mutant primers and ligating the two resulting PCR rl a~lll~,.ll~ cimll It~n90ucly to the vector fragment in a three (or morej-part ligation.
In a specific example of PCR, . . . ~l .g, . ., ~;~, template pl~mid DNA ( I llg) is lineari~d by digestion with a l~ ion en~ .,r!~ that has a unique recogniti~n site in the plasmid DNA outside of the region to be ~mrlifiP~I Of this material, 100 ng is added to a PCR mixture cont~ining PCR buffer, which contains the four 30 deoxynucleotide tl;l.l~ s and is included in the GeneAmp~) kits (obtained from Perkin-Elmer Cetus, Norwalk. CT and Emeryville, CA), and 25 pmole of each olig~-nn~leoti~ primer, to a final volume of 50 ,uL.
The reaction mixture is overlayed with 35 ~lL mineral oil. The reaction mixture is denatured for five minutes at I OûD C, placed briefly on ice, and then I IlL T*ermus c~yuu~ (Taq) DNA polymer~e (5 units/~uL, purch~ed from Perkin-Elmer Cetus) is added below the mineral oil layer. The reaction mixture is then inserted into a DNA
35 Thermal Cycler (I ul-,hased from Perkin-Elmer Cetus) l~u~,laullulcd as follows:
2 min. 55~ C
30 sec. 72~C, then 19 cycles ofthe following:
30 sec. 94~C
30 sec. 55~ C, and WO 97/3Q146 PC'r/US97/0267S
-30 sec. 72~C.
At the end of the program, the reaction vial is removed from the thermal cycler and the aqueous phase L a~:,f~ ,d to a new vial. extrac~ed with phenoVchloroform (50:50 vol), and ethanol ~ Lcd. and the DNA
is recovered by standard ~.v, cdu.~ This material is :."lu,c~lu~ ly subjected to the d~ Iu~liale treatments for S insertion into a vector.
Another method for }~IclJal i..g variants. cassette .. .,,I;.g~ ;c is based on the technique described by Wells et aL, Gene, 34:315 ( 1985). The starting material is the plasmid (or other vector) co---~ i..g the native CT-I DNA to be mutated. The codon(s) in the native CT-I DNA to be mutated are i~lentif~ There must be a unique ~ LliCliu~ "-rk~ce site on each side of the identified mutation site(s). If no such l~an i~,Liull sites 10 exist, they may be ~,_n.,.ah,d using the above-described olig-nl~clrol;.l~-mediated mnt~gPn~Sic method to introducethematal,l,lu~.iak;locationsinthenativeCT-l DNA. Afterthe-~,~L i~Liunsiteshavebeeni--Lud~,ed into the plasmid, the plasmid is cut at these sites to linearize it. A double-stranded olig.~.. Ie~ k encoding the sequence of the DNA between the restriction sites but cnnt~ining the desired mnt~ti~m(S) is synthesi_ed using standard ~oloceJu~,;,. The t~vo strands are synth~ci7Pd scpal..t~,ly and then hybridized together using standard 15 terhnillu~c This double-stranded olig....~ kolirl~ is referred to as the cassette. This cassette is designed to have 3' and 5' ends that are cOllllJalilJl with the ends of the L,~al i~,l plasmid, such that it can be directly ligated to the plasmid. This plasmid now contsins the CT-~ DNA sequence mutated from native CT-I.
C. In~lortion of Nucleic Acid into Replicable Vector The nucleic acid (eg, cDNA or genomic DNA) encoding CT-I is inserted into a replicable vector for 20 further cloning ( ~ r~ of the DNA) or for ~A~l c ~:~iull~ Many vectors are available, and selection of the a~J~uul~l ialc vector will depend on I ) whether it is to be used for DNA annplific ~tinn or for DNA expression, 2) the size of the nucleic acid to be inserted into the vector, and 3) the host cell to be ~ r.... cd with the vector.
Each vector contains various c~ o~ ; dep~n~1ing on its function (amplification of DNA or ~A~ iUn of DNA) and the host cell with which it is ,i r ' ~ ~ The vector CUIII~JUII~,~IL i generally include, but are not limited 25 to, one or more of the following: a signal ~Pqn~nr~, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a llan~ JLiùll termination sequence.
(i) Signal Seauence C~
The CT- ls of this invention may be produced not only directly, but also as a fusion with a heterologous polypeptide"~ ,rt;.al,ly a signal sequence or other polypeptide having a specific cleavage site at the N-terminus 30 ofthematureproteinorpolypeptide. Ingeneral,thesignalsequencemaybea~-....~.u,. ~.1 ofthevector,oritmay be a part of the CT-I DNA that is inserted into the vector. The heterologous signal sequence selected should beonethatis.c~ ..;,.dandl..o~c~c,l(i.e.,cleavedbyasignalpeptidase)bythehostcell. Forprokaryotichost cells that do not r~;~,u~l~., and process the native CT-I signal seq-lPnr~, the signal sequence is ~ d by a prokaryotic signal sequence selected, for example, from the group conci~ting of the alkaline pl.o~l.h -l ~
35 IJ .;~ , lpp, or heat-stable ~ t~,.UIUAill 11 leaders. E~or yeast secretion the native signal sequence may be ~- h,~ d by, e.g, the yeast invertase leader, yeast alpha factor leader (including Sae~l,u,~,...v~~s and Kl~v~ -factor leaders, the latter described in U.S. Patent No. 5,010,182 issued 23 April 1991), yeast acid plln~ leader, mouse saliva~y arnylase leader, carboxypeptidase leader, yeast BARI leader. Humicola l,.. ~,;.. u~,- lipase leader, the C al~icans glucoamylase leader (EP 362,179 ~,ul,li~ d 4 April 1990), or the signal CA 0224~63~ 1998-08-0~

-described in WO 90/13646 published 15 November 1990. In mAmmAIi~n cell ~ iUI~ the native human signal se~uence (ie., the CT~ . .re that normally directs secretion of native CT-I from human cells in vivo) is ,~ rh~ y~ although other mAmn~liAn signal ar~l~lr~ may be suitable. such as signal se~ r C from other animai CT-ls, signal s~u- .~ from a ligand binding to another GH/cytokine receptor family member, and 5 signal ~ r ~ from secreted polypeptides of the same or related species, as well as viral secretory leaders, for example, the herpes simplex gD signal.
The DNA for such pl~:~,ulau~ region is ligated in reading frame to DNA encoding the mature CT-I.
(ii) Ori~in of Rer~lication CUIIIPOIICII~
Both ~ -y. ~aiull and cloning vectors contain a nucleic acid sequence that enables the vector to replicate I û in one or more selected host cells. Generally, in clonin r vectors this sequence is one that enables the vector to replicate inflep. "d~ .lly of the host chromosomal DNA, and includes origins of replication or autonomously ..,~,!i- ~1;~g sF(I~ ; Such $~ c are well known for a variety of bacteria, yeast, and viruses. The origin of ~ iCaliull from the plasmid pBR322 is suitable for most Gram-negative bacteriat the 2~1 plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV, or BPV) are useful for cloning 15 vectors in mAmmAliAn cells. Generally, the origin of replication culll~,ull.,.ll is not needed for mAmm~1iAn ri~yl~ai~JIl vectors (the SV40 origin may typically be used only because it contains the early promoter).
Most ~A~ aiu.l vectors are "shuttle" vectors, i.e., they are capable of replication in at least one class of Ul~all;.~lll;~ but can be ~ re~l~d into another organism for e,~ aion. For example, a vector is cloned in E.
coli and then the same vector is l~a-.~r~ ~ ~rd into yeast or ~mmA1iAn cells for ~ aion even though it is not 20 -capable of replicating inrl~pen~l~ntly of the host cell chromos(-m~
DNA may also be amplified by insertion into the host genome. This is readily accomplished using Bacillus species as hosts, for example, by including in the vector a DNA sequence that is compl.,.ll.,.l~y to a sequ~ nce found in Bacillus genomic DNA. Transfection of Bacillus with this vector results in homologous I ~ul.ll,...d~ion with the genome and insertion of CT-I DNA. However, the recovery of genomic DNA encoding 25 CT-I is more complex than that of an exogenously replicated vector because restriction enzyme digestion is required to excise the CT-I DNA.
(iii) Selection Gene C~)..svull~
~ aiu~l and cloning vectors should contain a selection gene, also termed a selectable marker. This gene encodes a protein necessary for the survival or growth of tt ,-- -~ rl ~ d host cells grown in a selective culture 30 medium. Host cells not ~ r -- .-~~d with the vector c- ~ ;..g the selection gene will not survive in the culture medium. Typical selection genes encode proteins that (a) confer r~ial~llce to antibiotics or other toxins, e.g., ampicillin, neomycin, Ill~tllo~ e~ or tetracycline, (b) complement auxotrophic d~,rci~ ,iei, or (c) supply critical nutrients not available from complex media. e.g, the gene encoding D-alanine racemase for Bacilli.
One example of a selection scheme utilizes a drug to arrest g}owth of a host cell. Those cells that are 35 s~rcrccfi-lly~ r..l.,~dwithaheterologousgeneproduceaprotein~ullL.lillgdrugl~ia~lceandthussurvive the selection regimen. ~xamples of such ~ minAnt selection use the drugs neomycin (Southern et al.. J. Molec.
A Appl. Genet., 1:327 (1982)), mycophenolic acid (Mulligan et al., Science, 209:1422 (1980)), or hygromycin (Sugden et al., Mol. CelL BioL, 5:410-413 (1985)). The three examples given above employ bacterial genes WO 97/30146 PCTlUS97/02675 -under eukaryotic control to convey l~,aia~ancc to the ap~Jlu~ e drug G418 or neomycin (gene~icin), xgpt ~mycophenolic acid), or hygromycin, respectively.
Another example of suitable selectable markers for m:~nnm~ n cells are those that enable the ntifir~tion of cells colll"c~.ll to take up the CT-1 nucleic acid. such as DHFR or thymidine kinase. The m~mm~liAn cell Llanarull~ a are placed under selection pressure that only the lla--sru.. ndl.la are uniquely r adapted to survive by virtue of having taken up the marker. Selection pressure is imposed by culturing the Ll~lsrulllldllla under cr~n-1iti-nc in which the concc,.lllaLion of selection agent in the medium is successively '~
changed, thereby leading to amplification of both the selection gene and the DNA that encodes CT-I.
r~ ;0,. is the process by which genes in greater demand for the production of a protein critical for growth 10 are reiterated in tandem within the ._l~luulusùl--es of successive generations of l~.,ulllbhlalll cells. Increased ntiti~c of CT-I are 5~ from the amplified DNA. Other examples of amplifiable genes include m~-t~llothinnPin-l and -Il, preferably primate mPt~llothi- nPin genes, ~ n~sine ~ nlin~se~ ornithine decarboxylase, etc.
For example, cells L~a~arull~lcd with the DHFR selection gene are f¢st identified by culturing all of the 15 llallafullll~ults in a culture medium that contains methotrexate (Mtx), a cul.l~LiLi~e antagonist of DHPR. An alJ~Jlu~fi~.lG host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHER activity, prepared and ~ ,agak;d as des~,l il,~d by Urlaub et aL, Proc. NatL Acad. Sci. US~, 77:4216 (1980). The Llallaful~..c~ cells are then exposed to increased levels of l~,LLuL~cAalc. This leads to the synthesis of multiple copies of the DHFR gene, and, c~nromit~ntly, multiple copies of other DNA CUlll~liaillg the 20 expression vectors, such as the DNA encoding CT-I. This amplification technique can be used with any otherwisesuitablehost,e.g,ATCCNo.CCL61CHO-Kl,noL~i~ ingthepresenceofelldcgenollcDllFR
if, for example, a mutant DHFR gene that is highly resistant to Mtx is employed (EP 117,060).
All~,.-ld~ ly, host cells (particularly wild-type hosts that contain ~n~k~genouc DHFR) l~allafulllled or co-lla~arulll~,1 with DNA s~ ~. F 5 encoding CT-l ~ wild-type DHFR protein, and another selectable marker 25 such as aminoglycoside 3 ph~ ,1.ull a~lar~a ~, (APH) can be selected by cell growth in medium cont~inine a selection agent for the CP~ marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199.
A suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 (Stinchcomb etaL, Nature, 282:39 (1979), Kingsman etal., Gene, 7:141 (1979); orTa~ etaL, Gene, 30 10: 157 (1980)). The trp I gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No.44076 or PEP4-1 (Jones, Genetics, 85: 12 (1977)). The presence of the gl21 lesion in the yeast host cell genome then provides an effective environment for detecting Llall;.ru.~--alion by growth in the absence of ~ Jpl.a... Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38.626) are ..c d by known plasmids bearing the Leu2 gene.
In addition, vectors derived from the 1.6,um circuiarplasmid pKDI can be used for Llall~ru-.--aLion of iYIUyv~/u~ycG:~ yeasts. Bianchiet al., Curr.Genet..12:185(1987). Morerecently,anexpressionsystemfor large-scale production of .~, u--~Li~ l calf chymosin was reporLed for K lactis. Van den Berg, Bio/Techn- loE~.
~: 135 (1990). Stable multi-copy G~r~,aa;un vectors for secretion of mature .. culllbillallL human serum albumin WO 97/3~\146 PCT/US97/0267S

by industrial strains of Kluyveromyces have also been fiicclrlseA Fleer et al., Bio/Technolo v. 9: 968-975 (1991).
(iv) Promoter Compcnent Expression and cloning vectors usually contain a promoter that is ~~co~,, .;~rd by the host organism and 5 is operably linked to the CT- I nucleic acid. Promoters are untranslated s~ . c s located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the Lla~ Lion and translation of particular nucleic acid sequPnrP~ such as the CT-I nucleic acid scql~f-ncç, to which they are operably linked. Such ~JlullluLcl~ typically fall into two classes, inducible and con~liluLivc. Inducible l,lulllo~
are promoters that initiate increased levels of llall~ vLion from DNA under their control in response to some 10 change in culture c~ ,..c, e.g, the presence or absence of a nutrient or a change in ~ c. At this time a large number of promoters I~cO~..i,rd by a variety of potential host cells are well known. These IJlunlut~,la are operably linked to CT-I-encoding DNA by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector. Both the native CT-I IJIUIIIUICI
sequence and many heterologous promoters may be used to direct amplification andlor expression of the CT-I
15 DNA. However, L-,t.,lulogùus promoters are preferred. as they generally permit greater l.a~ "ion and higher yields of recombinantly produced CT- I as compared to the native CT- 1 promoter.Promoters suitable for use with prokaryotic hosts include the ,B-1,.~ cs and lactose promoter systems (Chang et al., Naturet 275: 615 (1978); and Goeddel et aL, Nature. ~: 544 (1979)), alkaline r~h~ cf-, a tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res.~ 8: 4057 (1980) and EP 36,776), and hybrid 20 promoters such as the tac l,rulllot~,l (deBoer et aL, Proc. Natl. Acad. Sci. USA~ 80: 21-25 (1983)). However, other known bacterial promoters are suitable. Their mlrlpotifip sc ~ have been published. thereby enabling a skilled worker operably to ligate them to DNA encoding CT-I (Siebenlist et aL, Cell~ 20: 269 (1980)) using linkers or adaptors to supply any required restriction sites. Promoters for use in barterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding CT-I.
Promoter ~ are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located a,ulJlu~illlalcly 25 to 30 bases upstream from the site where ilal~ ,lion is initiated. Another sequence found 70 to 80 bases upstream from the start of llall~ iull of many genes is a CXCAAT region where X may be any .- ~cl~ lr At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of ~e poly A tail to the 3' end of the coding seql~f nr~o All of these ,~ r.c are suitably inserted into 30 cu~;~yuLi-, C~ iOn vectors.
Examples of suitable promoting se~ c for use with yeast hosts include the ~Jrulllut~ for 3-~h~ .l.n~,lycerate kinase (Hitzeman et aL, J~iol. ~hPnn ~: 2073 (1980)) or other glycolytic enzymes (Hess et al., J. Adv. En7,vme Re~.. 7: 149 (1968); and Holland, Biorhf,mi,ctry~ 17: 4900 (1978)), such as enolase, glyceraldehyde-3-rhncrh~tf- dehy~Lug~lla~e~ h~ ~ nL i~ pyruvate dc~allJu~ylase~ phosphur~ nL ~ f~ g 35 6-l.l.~l.h-~isulll.,la~,3-~ o~l.l.nglyceratemutase,pyruvatekinase,~ hc.~ iaullle.a,c,phocrh--plnrose i~UIII~ a ~C, and pl~lrol~ cP
Other yeast IJIUlllVt~ , which are inducible promoters having the ~flfiitjon:ll advantage of ll all5~ lion controlled by growth rnnfiitionc are the promoter regions for alcohol dehydlu~,cl,asf; 2, isoc,vtochrome C, acid G phflcrhzlt~cp~ ;la~laliveenzymes5lccori~tp~lwithnitrogenlll~al~oli~ mf~tsllloth~ p~ glyceraldehyde-3-phos-WO 97/30146 PCTlUS97/02675 phate dehy-llug.,..ase. and enzymes ,c~.ol,~ible for maltose and galactose utili7ation Suitabie vectors and ,UIUI~ for use in yeast C~ ;UII are further described in Hitzeman et aL, EP 73.657. Yeast enh~nr~rg also are ad~alllageuusly used with yeast promoters.
CT-I llans~ ioll from vectors in m~mm~ n hosl cells is controlled, for example. by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous m~mm~ n plUlllVt~ , e g., the actin promoter or an immunoglobulin promoter, from heat-shock promoters. and from the promoter normally ~o~ ~t~l with the CT-I sequence, provided such promoters are ~,ulll~dLiblc with the host cell systems.
The early and late ~JIUIIIU ~ of the SV40 virus are conveniently obtained as an SV40 l~LI;.,Liull fragment that also contains the SV40 viral origin of replication. Fiers et aL, ~ ~, 273 :1 13 ( 1978), Mulligan andBerg, Science.209: 1422-1427 (1980); PavlakisetaL, Proc. Natl. Acad. Sci. USA. 78: 7398-7402 (1981).
The . ~ 1; 5~ early promoter of the human cy~,lllc~lvv ~ us is conveniently obtained as a HindlII 1~ l ~;,LI i~liull 15 fragment. Glc~ awayeta/.,5~, 18:355-360(1982). Asystemforc.. l,lc~,il.gDNAin~ll,.. l~l.lAii~.. lhostsusing the bovine pal.illullla virus as a vector is disclosed in U.S. Patent No. 4,419,446. A mo. l;r.~ of this system is described in U.S. Patent No. 4,601,978. See also Gray et aL, Nature. 295: 503-508 (1982) on expressing cDNA encoding immune illt~,~ Ç,l~ll in monkey cells: Reyes et aL, Nature~ ~ Z: 598-601 ( 1982) on C~ iUII
of human ~-h,t~,r~.ull cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus: Canaani and Berg, Proc. h~z~tl Acad. Sci. USA. Z2: 5166-5170 (1982) on e,~ ;~;UII ofthe human interferon ~ I gene in cultured mouse and rabbit cells; and Gorman et aL, Proc. Natl. Acad. Sci. USA. 7~: 6777-6781 (1982) on c,.~ ,ion of bacterial CAT se l- -r~ C in CV-I monkey kidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa cells, and mouse NIH-3T3 cells using the Rous sarcoma virus long terminal repeat as a promoter.
~v~ Enhancer E~lementCo~
Tlans~,l il,Lion of a DNA encoding the CT-I of this invention by higher tUka~,~U~ is often increased by inserting an enhancer sequence into the vector F.nh~n~P-~c are cis-acting elements of DNA, usuallv about from 10 to 300 bp, that act on a promoter to increase its llall~ ioll. Enhancers are relatively orientation and position i..~ having been found 5' (Lai-m-ins et aL, Proc. Natl. Ar~i Sçi. USA~ :Z8: 993 ( 1981 )) and 3' 30 (Lusky et aL, Mol. Cell Bio., 3: 1108 (1983)) to the ~ unit, within an intron (Banerji et aL, ~lL ;~:
729 (1983)), as well as within the coding sequence itself (Osborne et al., Mol. Cell Bio.~ 4: 1293 (1984)). Many enhanccr 5~ln~ cc are now known from m~nnm~ n genes (globin~ elastase, albumim ~-r.,Lol~lu~ , and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. 1~ F' include the SV40 enhancer on the late side of the replirzlt~ origin (bp 100-~70), the cytomegalovirus early promoter enhancer.
35 the polvoma enhancer on the late side of the replication origin, and adenovims enhancers. See also Yaniv, Nature~ 297: 17-18 ( 1982) on ~~nh~nring elements for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5' or 3' to the CT- I -encoding se~enc~, but is 1,l ~lably located at a site 5' from the promoter.

-(vi) Tlalla~livlion Ter~nina~ion Cul...-onc.-L
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human. or nucleated cells from other mnltit~el~ r ul~,al~iallls) will also contain se-t~ .fP~ necessary for the termination of hduo~ )Liull and for 5t~hiii7ing the mRNA. Such se~ Ps are commonly available from the 5' and, 5 ocrpcirmplly 3~"~ .""~ r~ regions of eukaryotic or viral DNAs or cDNAs. These regions contain nn--lPoti-lP
segments llalls~,l ii,ed as polyadenylated fragments in the untranslated portion of the mRNA encoding CT-I .
(vii) Construction and Analvsis of Vectors Construction of suitable vectors cu..l~;.,i..g one or more of the above listed culll~Julu~ a employs standard ligation ~ ~ 1,. . i. l~ If ' Isolated plasmids or DNA G a~lut;llLa are cleaved, tailored. and religated in the form 10 desired to generate the plasmids required.
For analysis to confirm correct se~u~,~.ccs in plasmids cullaLlu~.t~,d. the ligation mixtures are used to Llailarullll E. coli K12 strain 294 (ATCC 31,446) and ~"cc~rl~l ilallar~""-a-,La seiected by ampicillin or t~ a~ , n~Oia~l-,e where alJIJIU~ ,. Plasmids from the trPncfnrmPntc are prepared, analyzed by lcoLli~Liull ecti~n, and/or 5~ ed by the method of Messing et aL, Nucleic Acids Res.~ 2: 309 (1981) 15 or by Ihe method of Maxam et al., Methods in Enzvmology. 65: 499 (1980).
(viii) Transieltt F.~cv~ iol~ Vectors Pal Li-,ulal ly useful in the practice of this invention are eA~ ,Oaiun vectors that provide for the transient ~A~ oaiOl~ in n~rnm~liPn cells of DNA encoding CT-I . In general, transient ~ aion involves the use of an expression vector that is able to replicate err..,;.,..-ly in a host cell, such that the host cell acc~rn~ tPs many 20 - copies of the expression vector and, in turn, synthesizes high levels of a desired polypeptide encoded by the expression vector. Sambrook et al., supra, pp. 16.17 - 16.22. Transient ~A~ aaiol2 systems, Culll~ lg a suitable expression vector and a host cell, allow for the con~ ,llL positive i(l~-ntifie~tion of polypeptides encoded by cloned DNAs, as well as for the rapid s~ ,.,..i--g of such polypeptides for desired biOlogicâl or physiological 1~ u~ .3. Thus, transient ~A~ aaiull systems are ~.~ Lil,UI~ Iy useful in the invention for purposes 25 of identifying analogs and variants of native CT-I that are biologically active CT-I .
(ix) Suitable Exem~iary Vertebrate Cell Vectors Other methods, vectors, and host cells suitable for adaptation to the synthesis of CT-I in recombinant V_l L~,bl aL~ cell culture are described in Gething et aL, Nature~ 93: 620-625 (1981); Mantei e~ al., Nat~tre, 281:
40-46 (1979); EP 117,060; and EP 117,058. A particularly useful plasmid for m~nnm~ n cell culture 30 production of CT-I is pRK5 (EP 307,247) or pSVI6B (WO 91/08291 published 13 June 19gl). The pRK5 derivative pRKSB (Holmes et al., Sctence. 253: 1278-1280 (1991)) is particularly suitable herein for such expression.
D. Selection ;m-l Transfortnation of Host Cells Suitable host cells for cloning or expressing the vectors herein are the prokaryote! yeast. or higher 35 eukaryote cells described above. Suitable prokaryotes for this purpose include cuba~.t~,~ ia, such as Gram-negative or Gram-positive Ol~,allialllS~ for example, E-lt~,.ui,a~.L~liaccae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, KIPks;PIl~7, Proteus, Salmonella, e.g., Salmonella typh,r,.u,.u.,., Serratia, e.g, Serrat~a m~,~ ~,~, and Shigella, as well as Bacilli such âS B. subtilis and B. Iicheniformis (e.g., B. Iicheniformis 4 I P disclosed in ~ DD 266.710 ~Jul~liOLed 12 April 1989), Pseudomonas such as P. a~, u~i~.. Jsa, and Streptomyces. One preferred -~ coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as El. coli B, ~ coli X 1776 (ATCC
31,537), ~ coli DH5a. and ~. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting. Strain W3110 is one particularly preferred host or parent host because it is a common host strain for leculllbulalll DNA product ~ Preferably, the host cell secretes minimal amounts of proteolytic 5 enzymes. ~or example. strain W3110 may be modified to effect a genetic mutation in the genes encoding proteins PnAngPnoll~ to the host, with exarnples of such hosts including ~; coli W3110 strain I AZ, which has the complete genotype tonAa; ~. coli W311û strain 9E4, which has the complete genotype tonA~J pfr3; E. coli W311û strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA~IE15 ~IfargF-lac)169 adegP ~lompTkanr; E. coli W311û strain 37D6, which has the complete genotype tonA ptr3 phoAaE15 10 ~(argF-lacJ169 ~degP ~ompT ~rbs7 ilvG kanr; E; coli W311û strain 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion rmlt:-tinn and an ~ coli strain having mutant periplasmic protease disclosed in U.S. Patent No.4,946,783 issued 7 August 199û. Alternatively, in vitro methods of cloning, e.g, PCR or other nucleic acid polymerase reactions, are suitable.
In addition to prokaryotes, eukaryotic microbes such as fi1~ fungi or yeast are suitable cloning 15 or expression hosts for CT- I -c..codillg vectors. saLe~ .~c~ c~ C'Vi~ " or common baker's yeasL is the most commonlyusedamonglowereukaryotichostllli~luul~5all~ s~ However,anumberofothergenera,species,and strains are commonly available and useful herein, such as Schizosa. cll..,v".yces pom6e (Beach and Nurse, _a~, ~Q: 140 (1981); EP 139,383 IJubli;ihed 2 May 1985); Klu~ ~e~ hosts (U.S. Patent No.4,943,529;
Fleer et al., supra) such as, e.g, K lactis (MW98-8C, CBS683, CBS4574; Louvencourt et aL, J. Bacteriol.~ 737 20 (1983)), Kfragilis (ATCC 12,424), K bulgaricus (ATCC f 6.045), K. wic~eramii(ATCC 24~178), K waltii (ATCC 56,500), K drosophilarum (ATCC 36,906; Van den Berg et al., supra), K . thermofolerans, and K
marxianus;yarrowia(EP402,226);Pichiapastoris(EP 183,û70;Sl~.,hi~hna.etal.,J.E~cicMicrobiol.~.
265-278 (1988)); C~ndida; ~ d~, - reesia (EP 244.234); N~ v~f~u~a crassa (Case et aL, Proc. l'rs~tl Açad.
Sci. USA. Z~: 5259-5263 (1979)); Sc~ ., . y~ such as S~ ces occidentalis (EP 394,538 25 pl~hlichrd 31 October l 990); and I il~ . - .1(,. .~ fungi such as, e.g, I J~u, Vi~ , Penicillium, Tolypocladium (WO
91/00357 published 10 January 1991), and Aspergillus hosts such as A. nidulans ~Ballance et aL, Biorh~ m Bio~hvs. Res. C~mm~-n . 112: 284-289 (1983); Tilburn et aL, Gene, 2~: 205-221 (1983); Yelton et aL, Proc.
N:ltl Acad. Sci. USA. 81: 1470-1474 (1984)) and A. niger (Kelly and Hynes, FMBO J.. _: 475-479 (1985)).
Suitablehostcellsforthe~,ludu-;liollofCT-larederivedfrommllltirelllll~r...~;,1..;~..,~ Suchhostcells 30 are capable of complex ~lu~,e~ and glycosylation activities. ~n principle, any higher eukaryotic cell culture is workable, whether from V~,.t~,bl~k; or in~ L~Llale culture. Examples of i~ t~,lJlaL~ cells include plant and insect cells. Numerous baculoviral strains and variants and cullc;~yollding permissive insect host cells ~om hosts such as .~ro~rt~raf, u~ , ~ (caterpillar). Aedes aegypti (mosquito), Aedes aMopic~us (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been i~ ntifiP~1 See, e.g, Luckow et al., Bio/Technolo~y. 6:
35 47-55 (1988); Miller et aL, in Genetic F.l~;.. ~ ~ ;"~ Setlow,3.K. et al., eds., Vol. 8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et aL, Nature. 315: 592-594 (1985). A variety of viral strains for transfection are publiclyavailable,e.g,theL-I variantofAutographacalifornicaNPVandtheBm-5strainofBomb~cmori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for tlall~re~lion of Spodopterafrugiperda cells.

Plant cell cultures of cotton, corn, potato. soybean~ petunia~ tomato. and tobacco can be utilized as hosts Typically, plant cells are hall~r~,~,L~,~ by inr-lhAfiorl with certain strains of the ba.,L~.;ulll Agrobacferium which has been previously .,.,...i~ d tO contain the CT-I DNA. During inrllhAtion of the plant cell cullure with A. ,~ .," the DNA encoding the CT-l is ~lall~rcll~,d to the plant cell host such that it is 5 trAncfPr~p~i~ and will, under a~ iale con~iitionC, express the CT-I DNA. In addition, regulatorv and signal se~ cu...l,dtil,le with plant celis are available, such as the nopaline synthase promoter and polyadenylation signal c,.~ Pc Depickere~aL, J. Mol. Aprl. Gen ~ 1: 561 (1982). In addition, DNA segments isolated from the upstream region of the T-DNA 780 gene are capable of activating or ill~,lca~illg Ll~-~ ,lio ~ Ievels of plant-expressible genes in recombinant DNA-contAinin~ plant tissue. EP 321,196 published 21 June 1989 However, interest has been greatest in v~ ,blaLc cells, and ~lu~a~alion of ~,.t~b.ale cells in culture (tissue culture) has become a routine l~rucclu~c in recent years (Ticcllp Culture. Academic Press, Kruse and Patterson, editors (1973)). FYAnnrlPc of useful mAmmAli~n host cell lines are a monkey kidney CVI cell line Llal.~ru.l,ledbySV40(COS-7,ATCCCRL 1651);ahumanembryonickidneyline(293 or293 cellscl~hrlonPd for growth in ~ culture, Graham et al., J. Gen Virol..36: 59 (1977)); baby hamster kidney cells (BHK, 15 ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl Acad Sci. USA.
77: 4216 (1980)3; mouse sertoli cells (TM4, Mather, Biol. Reprod.. 23: 243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A. ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065);
20 mouse mammary tumor cells (MMT 060562, ATCC CCLS I); TRI cells (Mather et al., Annals N Y Acad. Sci ~: 44-68 (1982)); MRC S cells; FS4 cells; and a human hepAtr.mA line (Hep G2).
Host cells are ll,~ f~lrd and preferably transformed with the above-described expression or cloning vectors of this invention and cultured in conventional nutrient media modified _s alJ~Jlu~JIialc for inducing ~IUIIIUt~.~, selecting L.a.l~r~ll"a,l4 or amplifying the genes encoding the desired s~lu~
Transfection refers to the taking up of an ~ iOII vector by a host cell whether or not any coding s~ 1.... ....~ PC _re in fact expressed. Numerous methods of Llall~r~,~.liull are known to the ordinarily skilled artisan.
for example, CaP04 and ele~llul.u,alioll. Successful transfection is generally l~.~o~.li,. d when any inclirAtion of the operation of this vector occurs within the host cell.
T.~ .cr~ .. means introducing DNA into an organism so that the DNA is replicable~ either as an 30 c,~Llaclllu."osomal element or by chromosomal integrant. Depending on the host cell used, transformation is done using standard tPrhni~lllPc a~ JIialc to such cells. The calcium Ll~,a~ll.c.l- employing calcium chloride, as described in section 1.82 of Sambrook et al., supra, or cl~ lu~ulaLion is generally used for prokaryotes or other cells that contain, .~ I;AI cell-wall barriers. Infection with Agroba~,iu". t~~tefn~ s is used for llall~r~lllllalion of certain plant cells, as dci,-,-;l,ed by Shaw et al., Gene, ;~: 315 (1983) and WO 89/05859 35 ~ul~liahed 29 ~une 1989. In addition, plants may be llall~r~,~,t~.d using ultrasound Ll~a~ .. L as described in WO
91/00358 published 10 January 1991. For mPrnm~ n cells without such cell walls, the calcium rhf~crh:lte ;r~n method of Graham and van der Eb, Virolo~v. ~: 456-457 (1978) is preferred. General aspects of ms-nnm~ n cell host system Ll~u. .r~...laLiul.s have been described by Axel in U.S. Patent No. 4.399.216 issued 16 August 1983 Tlall~rull,laLions into yeast are typically carried out according to the method of Van Solingen etal., J. Bact..130: 946 (1977) and Hsiao ef aL, Proc. Natl. Acad. Sci. (USA). 76: 3829 (1979). However, other methods for introducing DNA into cells~ such as by nuclear micrninjc~Atinn, ele~hul~u~a~ion~ bacterial pl'utopla~
fusion with intact cells, or polycations, e.g., polybrene. polynrnithin~, e~c.. may also be used. For various for~l~laru.,.,i"gmAmmAli~n cells,seeKeownetaL, Methods in En7ymoloAv. 185: 527-537 (1990) and Mansour et aL, Nature.336: 348-352 (1988).
E. ~ulturin~ the ~ost Cellc Prokaryotic cells used to produce the CT-I polypeptide of this invention are cultu}ed in suitable media as described generally in Sambrook et aL, supro.
The mAmmAliAn host cells used to produce the CT-I of this invention may be cultured in a variety of 10 media. Cu,."~ ,lly available media such as Ham's F-10 (Sigma), F-12 (Sigma), Minimal Essential Medium ([MEM], Sigma), RPMI-1640 (Sigma), Dulbecco's Modifled Eagle's Medium ([D-MEM], Sigma), and D-hIEM/F- 12 (Gibco BRL) are suitable for culturing the host cells. In addition, any of the media de~.,- ii,ed, for example. in Harn and Wallace, Methods in F.n7yrnolo~y, 58: 44 (1979); Barnes and Sato, AnRI Biochem.. 1~:
255 (1980); U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 5,122,469; or 4,560,655; U.S. Patent Re. No.
15 30.985; WO 90/03430; or WO 87/00195 may be used as culture media for the host cells. Any of these media may be ~ d as necessary with h<..,..on~ ~ and/or other growth factors (such as insuiin, ~,~,art"i.., aprotinin~ and/or epidermal growth factor [EGF]3, salts (such as sodium chloride, calcium""AL~ .., and pho~ ), buffers (such as HEPES), nuclro~.d~ c (such as A;flPnrcinr and thymidine), antibiotics (such as GentamycinTM drug), trace elements ~deftned as inorganic c~ .vu. ..1~ usually present at final concentrations in 20 the micromolar range), and glucose or an ~ui~/al~ energy source. Any other necessary s~ b ~ may also be included at ~ V~ conc.,llL dlions that would be known to those skilled in the art. The culture rontliti~mc such as ~ ;, pH, and the like, are those previously used with the host cell selected for (;~ oiVII~ and will be apparent to the ordinarily skilled artisan.
In general, principles, protocols, and practical f~ ~ho;~ c, for m~imi~ing the productivitv of in vifro 25 m~rnmRiiRn cell cultures can be found in M~ . Cell Biotechnolo~y: a Practical Approach. M. Butler, ed.
(IRL Press, 1991).
The host cells referred to in this disclo~u. ~ c ~ cells in in vitro culture as well as cells that are within a host animal.
F. l:~etectin~ ~ene A~ lifi~a~ion/F~ OOivll Gene amplification and/or C~ oSiUII may be measured in a sample directly, for exarnple, by cu..~ iu~lal Southem blotting, northem blotting to ~ ;l f~ the halls.,l i~.Lion of mRNA (Thomas, Proc. NRtl Acad. Sci. USA. 77: 5201-5205 (1980)), dot blotting (DNA analysis), or in sifu hybridization. using an alJ~lv~fidl~ly labeled probe, based on the sequences provided herein. Various labels may be employed, most commonly rddioiau~v~ , particularly 32p However, other 1erhniquPc may also be employed, such as uslng 35 biotin-modified nllrleoti~lpc for introduction into a pnlyn~rl~Qti~ The biotin then serves as the site for binding to avidin or Rntiho~1i.oc which may be labeled with a wide variety of labels, such as radionllr~ c~ fluorescers, enzymes, or the like. Altematively, allLilJolli.,~ may be employed that can .~co~.i~ specific duplexes. including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The ~nrikofli~s in turn may be labeled and the assay may be carried out where the duplex is bound to a surface. so that upon the f ~r~nzltinn .~f flllnleY f~n th~ cllrf~ .e thP nrecenee of ansihndv h~und t~ the dunlex can be detected.

CA 0224~63~ 1998-08-0~
WO 971301'16 PCT/US97/0267S

TFA up to 80%. The activity frActionAtPc at about 15-30 kDa on gel filtration columns. It is expected that a chaotrope such as guanidine-HCI is required for resolution and recovery.
CT-1 variants in which residues have been deleted, inserted, or s~hctit--f.od are recovered in the same fashionasnativeCT-I,takingaccountofany~..l,~u..~li-lchangesinpropertiesoc~ dbythevariation. For S example. ~ ala~iUII of â CT-I fusion with another pro~ein or polypeptide. e.g, a bacterial or viral antigen, facilitates !,ufiri~,aLiv,l; an immnnoAfflnity column c~..,l,.;.,h.g antibody to the antigen can be used to adsorb the fusion poiypeptide. 1.. ~._ rr;.. ;I y columns such ~ a rabbit polyclonal anti-CT- I column can be employed to absorb the CT-I variant by binding it to at least one remaining immune epitope. A protease inhibitor such as those defined above also may be useful to inhibit proteolytic degradation during ~u, ifi-,aLiull, and anlib Jti~,~ may 10 be included to prevent the growth of adv~,.lLiLious cnntAnninAntc One skilled in the art will a~ ,ciale that i,... ;1~. _1 ;n.. methods suitable for native CT-I may require mociifi~Afinn to account for changes in the character of CT-I or its variants upon i~lvducliull in IL.~,Vllli~;llal~t cell culture.
H. Covalent MOdifi~,aliulls of CT-I Polypeptides Covaient mo~l;r~ of CT-I polypeptides are included within the scope of this invention. Both 15 native CT- 1 and amino acid sequence variants of native CT- I may be CV ~ Y modified. One type of covalent mo~lifirAtilln included within the scope of this invention is the preparation of a variant CT- I frAgmPnt Variant CT-I L~ c,lb having up to about 40 arnino acid residues may be conveniently prepared by chemical synthesis or by enzymatic or chemical cleavage of the full-length or variant CT- I polypeptide. Other types of covalent mo-lif jrAtionc of the CT- I or r. ~r~ thereof are illL. vll ~.,cd into the molecule by reacting targeted amino acid 20 residues of the CT- I or G~ c.lt~ thereof with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues.
Cysteinyl residues most commonly are reacted with a-hAloA~etAf~s (and cu,l~ ollding amines), such as chlc,luac.,ti., acid or chlol.J~ ...;.L~, to give carboxymethyl or carboxyAnni~lomethyl derivatives. Cysteinyl residues also are de~i~rali~d by reaction with blvlllvLIinuulod~,~,tulle, a-bromo-,B-(5-imidozoyl)propionic acid, 2S chloroacetyl phO~llale. N-alkylm~ mi-lPc 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chlol ull -~,. uul ;l . . ~ l r, 2-chlu, ulllc;l ~ul i-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa- 1 ,3-diazole.
Histidyl residues are derivatized by reaction with diethylp~u~,~l,u,,ale at pH 5.5-7 0 because this agent is relatively specific for the histidyl side chain. Para-brr.mophPnAryl bromide also is useful; the reaction is preferably pc.rull,led in 0.1 M sodium cacodylate at pH 6Ø
Lysinyl and amino-terminal residues are reacted with succinic or other carboxylic acid anhydrides.
Derivatization with these agents has the effect of reversing the charge of the Iysinyl residues. Other suitable reagents for derivatizing a-amino-cl~ u;ll;llg residues include imi~loestPr.c such as methyl picolinimi~i~t~, pyridoxal phocphAtP, pyridoxal, chloroborohydride, t~ it~Vt~ ..lfi.l~ic acid, O-methylisourea, 2,4-p~ ..P.li~nP, and trAncAnninRc~catalyzed reaction with glyoxylate.
Arginyl residues are modified by reaction with one or several conventional reagents. among them phenylglyoxal, 2,3-b-~AnP~iinnP 1,2-cyrloh~ .e l;ù~ and ninhydrin. Derivatization of arginine residues requiresthatthereactionbep~,rul.l.cdinalka}inec~...~l;l;...,~becauseofthehighpKaoftheg.-AnirlinPfi-n~tionAl group. Furthermore, these reagents may react with the groups of Iysine as well as the arginine epsilon-amino group.

CA 0224~63~ 1998-08-0~

The specific mnllific~tinn of tyrosyl residues may be made. with particular interest in introducing spectrallabelsintotyrosylresiduesbyreactionwitharomatic~ 7nni--mcv.,,l,u,~ ortcLl~~ Most commonly, N-acetylimidizole and tetranitrometh~nP are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated using 125I or 131 I to prepare labeled proteins for use 5 in radioimmllnn~cc~y~ the chloramine T method described above being suitable.
Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R-N=C=N-R'), where 1~ and R' are different alkyl groups, such as l-cyclohexyl-3-(2-morpholinyl-4-ethyl) ~,a.l,o.lii..lide or l-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl ~nd glutamyl residues are Cull~ ,d to aa~Jalagillyl and glutaminyl residues by reaction with ammonium ions.
Derivatization with biru~ iunal agents is useful for crocclinking CT-I to a water-insoluble support matrix or surface for use in the method for purifying anti-CT-I antibodies, and vice-versa. Commonly used crncclinking agents include, ~g, I,l-bis(.li~ cetyl)-2-phenylethane, glutaraldehyde~ N-hydroxys-~ccirlimi~lç
esters, for example, esters with 4-~7iAocalifylic acid, homobifunctional imi-lnPctPrs, inrln~1ing ~licllrcinim esters such as 3,3'-dithiobis(s~ JIV~ ), and bi~u~ iunal .,~ "iflf c such as bis-N-m~lPirni~in-l78-15 octane. Derivatizing agents such as methyl-3-[(p-~7i~lophPnyl)dithio]propioimidate yield photoactivatable hlL~,,Illcdiàles that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive ~ub:.LIaLcs dcs.,. ibcd in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,33û,440 are employed for protein immobilization.
Glutaminyl and a~lJala~;h.yl residues are r~c~lu~,llLly ~IP~mi~tPd to the coll~ ,onding glutamyl and aspartyl residues, respectively. These residues are fiP~mi~i~t~d under neutral or basic cnn~itinnc The ~mi-l~tPd form of these residues falls within the scope of this invention.
Other mn(lir~ include hydroxylation of proline and Iysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the a-amino groups of Iysine, arginine, and histidine side chains 25 (T.E. Creighton, Proteins: Structure and MoleculOE ~lv~ Li~s~ W.~. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and ~mi~l~finn of any C-terminal carboxyl group.
Another type of covalent modification of the CT-I polypeptide included within the scope of this invention Cvlll~ cs altering the native glycosylation pattern of the polypeptide. By altering is meant deleting one or more carbohydrate moieties found in native CT-I, and/or adding one or more glycosylation sites that are 30 not present in the native CT-I.
Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the ~
of the carbohydrate moiety to the side chain of an a~alagillc residue. The 11 ipc~,Li~;h, sequPncPc a ",ala~ hlc-X-serine and a~ala~,ulc-X-threonine, where X is any amino acid except proline, are the reco~niticln seq~encrc for en~ymatic ~ltf~rhm.ont of the carbohydrate moiety to the ~I!al ~.~,hle side chain. Thus, the presence of either of 35 these ~ ,Lide 5~ in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the ~ of one of the sugars N-aceylg;~ 1 u~ ,r, g~l~rtosP or xylose to a hydroxyarnino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
Addition of glycosylation sites to the CT-I polypeptide is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide s~ c (for N-linked glycosylation sites~. The alteration may also be made by the addition of, or aubalilùliOII by, one or more serine or threonine residues to the native CT- I sequenee (for O-linked glycosylation sites). For ease. the native CT- I
amino acid sequence is preferably altered through changes at the DNA level, p~li~ulally by mutating the DNA
encoding the native CT-I polypeptide at ~ sel~ Icd bases such that codons are generated that will translate into S the desired amino acids. The DNA mllr~ti-~n(s) may be made using methods described above under Section 2B.
Another means of increasing the number of carbohydrate moieties on the CT-I polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. These yl uceJu. ~s are adv~ ro~ in that they do not require production of the polypeptide in a host cell that has glycosylation capabilities for N- or O-linked glyeosylation. D. ~ ,l iil.g on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine, 10 (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine. tyrosine, or tryptophan, or (f) the amide group of y,lulalllillc. These methods are described in WO 87/05330 published 11 S~ ....h.. 1987, and in Aplin and Wriston, CRC Crit. Rev. Bioch~m pp. 259-306 ~1981).
Removal of any carbohydrate moieties present on the CT-I polypeptide may be accomplished 15 ch~oTnirAlly or enzyrnatically. Chemical deglycosylation requires exposure of the polypeptide to the compound llinuul~ lfonic acid, or an eu,uivàl~ cflmrolm~l This treatment results in the cleavage of most or all sugars except the linking sugar (N-aeet~l~h ICO~ .r or N-acetyl~lAr~osAnnin~?), while leaving the polypeptide intact. Chemical degly~o~la~io.. is described by I T~L~imll-l~iin, et aL, Arch. Biochem. i3iophvs..259: 52 (1987) and by Edge e~ al., An~l Biochem.. 118: 131 (1981). Enzymatic cleavage of carbohydrate moieties on 20 polypeptides can be achieved by the use of a variety of endo- and exo-gly~ oa;.laa. s as described by Thotakura etal., Meth. Pn71ymol.. 138: 350 (1987).
Glycosylationatpotentialglycosylationsitesmaybepreventedbytileuseoftheromro~n~itunicarnycin as described by Duskin etal., J. Bio3. Chem.. ~: 3105 (1982). Tunieamycin blocks the fo~n~tinn of ,UIU~ ;.. N
glycoside linkages.
Another type of covalent mod; 1;~ of CT-I cu.. ~.. ;a~,~ linking the CT- I polypeptide to one of a variety of null~vlu~ uu~ polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyaikylenes, in themannersetforthinU.S.PatentNos.4,640,835;4,496,689;4,301,144;4,670,417;4,791,192Or4,179,337.
CT-I also may be c-lLIà~ ~cî in ~--i- .u~ prepared, for example, by CuaCc~ val;ull 1~ ~ h~ or by interfacial polymerization (for example, hydroxymethylcelhllc-se or gelatin-llli- Iuca~Jaules and poly-30 [methylrnethacylate] Illi-,lU~ C, respectively), in colloidal drug delivery systems (for example, li~osu---~,s, albumin mi.-~ L~,~3, micro~Tnnle;~nc nano-particles and n~nnc~p5~ s), or in ...a~ru .,.~ici~.n~ Such t~rhni~ s are disclosed in 1~ --'$ Pllal~ 1 Sciences. 16th edition, Oslo, A., Ed., (1980).
CT-I iulc~alaliùlls are also useful in gcll~.aLil~g ~ ibod;~ ~, as standards in assays for CT-I (~g., by labelingCT-lforuseasastandardinaradio;.. ~ .y,enzyme-linked;~ y,or-aLl;u.. ~Jwlassay), 35 in affinity purification terllni~ c and in cu---~,.,lili~e-type receptor binding assays when labeled with radioiodine, enzymes, fluorophores, spin labels, and the like.
Sinceitisoftendifflculttopredictinadvancethecll~a~ ia~icsofavariantcT-l~itwillbea~ ;al~d that some screening of the recovered variant will be needed to select the optimal variant. One can screen for ~nh~nrPd cardiac hypertrophic, anti-arrhythmic, inotropic, or n~ulollu,~,lli~; activity, posaejaiùll of CT-I

CA 0224~63~ 1998-08-0~

-..~ .g~ activity, increased c,~i~Jl~a:,iol3 levels. oxidative stability, ability to be secreted in elevated yields, and the like. For example, a change in the immunological character of the CT- I molecule. such as aff3nity for a given antibody. is measured by a cu~ /e-tvpe ;,.. ~ ,o~ y. The variant is assayed for changes in the ~UiJ~ ;Oll or ~ .1 of its hypertrophic, anti-arrhythmic. inotropic, and nc-u~ull o,L)lIic activities by cul.l~,al ;~ull to the 5 respective activities observed for native CT-I in the same assay (using, for example, the hy~ LluiJlly and n~u~.JLI~lL~hic assays described in the examples below.) Other potential mcl~iifif ~tions of protein or polypeptide properties such as redox or therrnal stability, hy llui hol ici~y, susceptibility to proteolytic degradation, or the tendency to ag~ , with carriers or into multimers are assayed by methods well known in the art.
I. A~ of CT-l Ant~Eonicfc to CT-I can be prepared by using the predicted famiiy of receptors for CT-I (the GH/cytokine receptor family, including the CNTF, LIF, and ~ u~ . M receptor subfamily, most pl~r~ably the LIFRi'3 or a LIFRi'3/gpl30 complex). Thus, the receptor can be expression cloned; then a soluble fomm of the receptor is made by identifying the eYfr~ r domain and excising the 1- .- ~ ~ Ih~ c domain ~ .rlu~
The soluble form of the receptor can then be used as an ~~ . or the receptor can be used to screen for small 15 molecules that would S~ CT-1 activity. T.a..sr~,.,L.,d cells expressing r~,.,olllbillallt receptor find use in screening molecules both for receptor binding and receptor activation agonism or ~ .gl~
Altematively, using the murine sequence shown in Figure I or the human sequence shown in Figure 5, variants of native CT- I are made that act as ~ Since the GH/cytokine receptor family is known to have two binding sites on the ligand, the receptor binding sites of CT- I can be determined by binding studies and one 20 - of them climinAt~d by standard ~ in~ (deletion or radical sllhstitlltion3 so that the molecule acts as an ~nt~g~nict For example, as ~iiccllcced herein, Figure 16 indicate regions that can act as ~nt~Eonictc ~nf~E- nict activity can be detemmined by several means, including the hy~ uiJll~/ assay, the nc,.......... ..uLlulJhic assay, and the other CT-I assays ~ L~d herein.
J~ H~V~ 1Ui~h~ Assay A miniatured assay is preferably used to assay for hy~ u~L;c activity. In this assay the medium used allows tl1e cells to survive at a low plating density without serum. By plating directly into this medium. washing steps are l?lim in~t~d 50 that fewer cells are removed. The plating dens;ty is i~ JUl ~allL. many fewer cells and the survival is reduced; many more cells and the myocytes begin to self-induce hyi!~lL uiJhy.
The steps involved are:
(a) plating 96-well plates with a, ~ - of myocytes at a cell density of about 7.5 x 104 cells per mL in D-MEM/F-12 medium suiJ~l ~ with at least insulin, L.all~r~,l.i.., and aprotinin;
(b) culturing the cells;
(c) adding a 5~lhct~nre to be assayed (such as one 5~lcperted of containing a CT-I);
(d) culturing the cells with the sl-hst~nrP and (e) I~l~aaulillg for h~ lulJl-~.
The medium can be ~ul,L,' 1 with at~Aiti~-n~l elements such as EGF that ensure a longer viability of the cells, but such supplements are not essential. D-MEM/F-12 medium is available from Gibco BRL, Gaithersburg, MD, and consists of one of the following media (Table 2):
~ TABLE 2 Com- 11320 11321 11330 11331 12400 12500 ponent I x I x I x I x Powder ~Q~çr Liquid Liquid Liquid k~i~ (mg/L) (mg/L) (mg/L) (mglL) (mg/L) (mg/L) .. .
AMINO "
ACIDS:
L-Ala-nine 4.45 4.45 4.45 4.45 4.45 4.45 L-Arg- 147.50 147.50 147.50 147.50 147.50 147.50 inine ~HCi L-Asp-ara- 7.50 7.50 7.50 7.50 7.50 7 50 gine H-~O
L-Asp- 6.65 6.65 6.65 6.65 6.65 6.65 artic acid L-Cys- 17.56 17.56 17.56 17.56 17.56 17.56 teine HCI H?O
L-Cys-tine 31.29 31.29 31.29 31.29 31.29 31.29 L-Glu- 7.35 7.35 7.35 7.35 7.35 7.35 tamic scid L-Glu- 365.00 365.00 365.00 365.00 365.00 365.00 tamine 2S Gly-cine 18.75 18.75 18.75 18.75 18.75 18.75 L-His- 31.48 31.48 31.48 31.48 31.48 31.48 tidine HCI
H~O
L-lso-leu- 54 47 54 47 54 47 54 47 54 47 54 47 cine L-Leu-cine 59.05 59.05 59.05 59.05 59 05 59 05 L-Lys-ine 91.25 91.25 91.25 91.25 91.25 91.25 HCI
L-Meth- 17.24 17.24 17.24 17.24 17.24 17.24 ionine L-Phen- 35.48 35.48 35.48 35.48 35.48 35.48 ylala-nine L-i'ro-line 17.25 17.25 17.25 17.25 17.25 17.25 L-Ser-ine 26.25 26.25 26.25 26.25 26.25 26.25 L-Thre- 53.45 53.45 53.45 53.45 53.45 53.45 onine -CA 0224~63~ 1998-08-0~

L-Trvp- 9.02 9.02 9.02 9.02 9.02 9.02 tophan L-Tyro- 55.79 55.79 55.79 55.79 55.79 55.79 sine ~, 5 ~2Na 2H ~O
L-Val-ine 52.85 52.85 52.85 52.85 52.85 52.85 INOR-GANIC
10 SALTS:
CaCI~ 116.60 116.60 116.60 116.60 116.60 116.60 anhyd.
CuSO4 0.0013 0.0013 0.0013 0.0013 0.0013 0.0013 .sH?o Fe 0.05 0.05 0.05 0.05 0.05 0.05 (NO3)3 9H~O
FeSO4 0.417 0.417 0.417 0.417 0.417 0.417 ~7H,O
KCI 311.80 311.80 311.80 311.80 311.80 311.80 MgCI-, 28.64 28.64 28.64 28.64 28.64 28.64 MgSO1 48.84 48.84 48.84 48.84 48.84 48.84 NaCI 6999.50 6999.50 6999.50 6999.50 6999.50 6999.50 NaHCO~ 2438.00 2438.00 2438.00 2438.00 -- --NaH PO4 62.50 62.50 62.50 -- 62.50 62.50 ~H,,O
Na~HPO~ 71.02 71.02 71.02 -- 71.02 71.02 ZnSO4 0.432 0.432 0.432 0.432 0.432 0.432 7H~O
OTHER
COMPO-NENTS:
D-Glu- 3151.00 3151.00 3151.00 3151.00 3151.00 3151.00 cose HEPES -- -- 3574.50 3574.50 3574.50 --Na 2.39 2.39 2.39 2.39 2.39 2.39 hypo-xan-thine Lino-leic 0.042 0.042 0.042 0.042 0.042 0.042 acid Lipoic 0.105 0.105 0.105 0.105 0.105 0.105 acid Phenolred 8.10 8.10 8.10 8.10 8.10 8.10 Pu- 0.081 0.081 0.081 0.081 0.081 0.081 tres-cine 2H-,O
Sodium 55.00 55.00 55.00 55.00 55.00 55.00 pyru-vate VITA-MINS:
Biotin 0.0035 0.0035 0.0035 0.0035 0.0035 0.0035 D-Ca 2.24 2.24 2.24 2.24 2.24 2.24 panto-then-ate Cho-line 8.98 8.98 8.98 8.98 8.98 8.98 chlor-ide Folic acid 2.65 2.65 2.65 2.65 2.65 2.65 i-lno-sitol 12.60 12.60 12.60 12.60 12.60 12 60 Nia-cin- 2.02 2.02 2.02 2.02 2.02 2.02 amide Pyrid-oxal 2.00 2.00 -- 2.00 2.00 HCI
Pyrid- 0.031 2.031 0.031 2.031 0.031 0.031 oxine HCI
Ribo- 0.219 0.219 0.219 0.219 0.219 0.219 flavin Thi- 2.17 2.17 2.17 2.17 2.17 2.17 amine HCI
Thy- 0.365 0.365 0.365 0.365 0.365 0.365 midine Vi- 0.68 0.68 0.68 0.68 0.68 0.68 tamin B l ,~
The preferred h~ ".l.y assay c~ p. ;~ ~
35 (a)p.l~oaLu.gthewellsof96-welltissuecultureplateswithamediumr~ .;.. gcalfserum,~-~,f~,.~ly D-MEM/F-12 medium cu..l;,;..;..g 4% fetal calf serum, wherein ~ .r~.al,ly the wells are j""..l".t~ ;I with the medium for about eight hours at about 37~ C;
(b) removing the medium;
(c) plating a ~ ;o~ of myocytes in the inner 60 wells at 7.5 x 104 cells per mL in D-MEM/F- 12 40 medium ,u~ cl with insulin, llal- ,f~,. i.., and aprotinin;

(d) culturing the myocytes for at least 24 hours;
(e) adding the test ,.,1,~";,... e, -(f) culturing the cells with the test 5..1.,~ c (preferably for about 24-72 hours, more ~ .ably for about 48 hours3; and (g) ~ ,aaul hlg for hy,u.,. Ll uivL~y, preferably with crystal violet stain.
Preferably the medium used in step (c) is a serum-free medium also contoining penicillin/au~ylolllycin S (pen/strep) and ~ n-";~-r Most plu~lalJly, the medium contains l OO mL D-MEM/F- 12, lOO uL Lld laf; .l i,l (10 mg/mL),20 IlL insulin (5 mg/mL), 50 IlL aprotinin (2 mg/mL), I mL pen/strep (JRH Bios~i ,c~s No.59602-77P), and I mL L-gl~~t~minP (200 mM).
The assay capacity of 1000 single samples a week coupled with the small sample si~ re.~uil~,.ll.,..l of 100 LIL or less has enabled an expression cloning and protein purification that would have been impossible to 10 accullll,liaL using the current methods available.
Another method for assaying h~i4.,.Liu~-hy involves Illcdaulillg for atrial natriuretic peptide (ANP) release by means of an assay that fl~ t. ." " ;". ~ the c-, "~ " for binding of 1251-rat ANP for a rat ANP receptor A-IgGfusionprotein. Themethodsuitableforuseissirnilartothatusedfomkt- ",;";"ggpl20usingaCD4-lgG
fusion protein dcs~,lil,~,d by Chamow et aL, Bio~ y. ~: 9885-9891 (1990).
The basis for the isolation and ~,l-dla~t~ dlion of the novel hypemophy factor. CT- l, is the milliaLu.i~d high through-put h~ ,lLIu~ y assay system, which was developed in a 96 well format, in which h~ luully is scored on individual myocardial cells following crystal-violet staining of neonatal rat cardiac myocytes. This assay was used in cc ~bi~ io with an in vitro model of embryonic stem cell cardiogenesis (Miller-Hance et aL, Journal of Biological Chemistry, 268:25244-25252 (1993)). These tuLilJu~l~ stem cells 20 - can li~ .lLialc into multi-cellular cystic embryoid bodies (EBs) when cultured in the absence of a fibroblast feeder layer, or without LIF. Since these embryoid bodies sl~v~ ously beat and display cardiac specific markers, it has been .,~ d that they may seNe as a vital source of novel factors that can induce a hylJ~,lLioiJllic response in Yitro )Miller-Hance et aL, .lournal of Biological Chemisny, 268:25244-25252 (1993);
Chien, Science, 260:916-917 (1993)). By dual immunonuol~s~cllce staining of cultured myocardial cells 25 incubated with EB rl~nflifi~n~od medium, it was observed that embryoid bodies elaborate a factor that can induce ari in vitro h~ ,l Llu~Jhic response in the cultured assay system. Tlhis response includes an increase in myucyte size, inrh-ctjon of the c~ ion of ANF, and the assembly of sai.,ol"cli., proteins (MLC-2v) into organized contractile units. The h~ ,llluivhy assay system was then used to c,clul~,i,aiull clone this factor, which proved to be the novel cytokirie, CT-1. These studies .lo.~ the utility of using expression cloning a~"u~uaches to 30 identify novel growth factors and cytokines from this in vitro model of embryonic stem cell ~ ,LiaLion. This assay system will be of interest in the isolation of other novel cytokines derived from ~ ,u,.,u,, of other d~ Ld cell types found in EBs~ i.e., neurogenic, skeletal myogenic, and hematopoietic ~ ui ,o,~,.
K. N~ vl~ oi41-iC Assav The assay used for ciliary ganglion ncu~uLiu~ ic activity described in Leung, Neuron, ~: 1045-1053 35 (1992) is suitable herein. Briefly, ciliary ganglia are dissected from E7-E8 chick embryos and dissociated in try!psin-EDTA (Gibco 15400-013) diluted ten fold in phocph~te-buffered saline for 15 minutes at 31~C. The ganglia are washed free of trypsin with three washes of growth medium (high glucose D-MEM supplemented with 10% fetal bovine serum, 1.5 mM gllllnl~ S, lOO ,ug/mL penicillin, and 100 llg/mL strepomvcin). and then gently triturated in I mL of growth medium into a single-cell sllcpPncii~n Neurons are enriched by plating this CA 0224~63~ 1998-08-0~

cell mixture in 5 mL of growth media onto a ! 00-mm tissue culture dish for 4 hours a~ 37~ C in a tissue culture incubator. During this time the non-neuronal cells ~..er~ Lially stick to the dish and neurons can be gently washed free at the end of the inruhAtiAIn The enriched neurons are then plated into a 96-well platé previously - coated with collagen. In each well, 1000 to 2000 cells are plated, in a final volume of 100 to 250 IlL, with 5 dilutions of the CT-l to be tested. Following a 2-4-day i~ at 37~ C, the number of live cells is assessed by staining live cells using the vital dye metallothionine (MTT). One-fifth of the volume of 5 mg/mL MTT
(Sigma M2 128) is added to the wells. After a Z-4-hour il~l;ubaLiull at 37~ C, live cells (filled with a dense purple .i,uilalt:) are counted by phase ll~ lUsco~Jy at 100X IllA~ . A~ n 3. Uses Anrl ThC~ Compositions Anri Administration of CT-I
As disclosed herein, CT-I activates dùwll~LIGalll cellularl~ ,unses via the h~t~,.u-lil~c.i~dLion of gpl30 and LIFR~. The expression pattern of CT-I and p' oLIu~vic activities suggest that it may have i...pulL~.
ru.l~,Liul-s, not only in the cardiac context, but in extra-cardiac tissues as well. CT-I acts to maintain normal embryonic growth and mc,.~.l.o~,~,.l~,;,i:" as well as physiological hu~ in the adult.
CT-I isbeiievedtofinduseasadrugfortreatmentofmArnmAIc(e.g,animalsorhumans)invivohaving 15 heart failure, arrhythmic or inotropic disorders. and/or pCl i,uh~. al ll.al ~ a.LI~ and other neuroiogical disorders involving motor neurons or other neurons in which CNTF is active. CT- I has additional uses as shown herein.
For example, CT-I may be useful in treating congestive heart failure in cases where ACE inhibitors cannot be employed or are not as effective. CT- I optionally is combined with or arimin ict~ red in concert with other agents for treating congestive heart failure, incl~ inE ACE inhibitors.
The effective a nount of ACE inhihitor to be A-lminict.~-ed, if employed, will be at the physician's or v.,t~,. illal ;~..'s discretion. Dosage administration and adju~L...~,~L is done to achieve optimal ..~ g~ 1 of congestive heart failure and ideally takes into account use of diuretics or digitalis, and con~itionc such as hy~ut~ and renal i,.~ The dose will AriAitif~nAIIy depend on such factors as the type of inhibitor used and the specific patient being treated. Typically the amount employed will be the same dose as that used 25 if the ACE inhibitor were to be Arlrninict~red without CT-I.
Thus, for example, a test dose of enalapril is 5 mg, which is then ramped up to 10-20 mg per day, once a day, as the patient tolerates it. As another example, captopril is initially A- i' " ;. . ;'~' ,. ~,d orally to human patients in a test dose of 6 5 mg and the dose is then escalated, as the patient tolerates it, to 2'i mg twice per day (BID) or three times per day ~TID) and may be titrated to 50 mg BID or TID. Tolerance level is ~ d by 30 ~l ~-"-i ;~,p.whetherdecreaseinbloodpressureisA~c~n~ bysignsofh~i~Jut.l~;on If inriirAt~rl thedose may be increased up to 100 mg BID or TID. Captopril is produced for administration as the active i.l~ ,..L, in c ~ " "l. - ,-l ;. ..~ with hydrochlorothiazide, and as a pH stabilized core having an enteric or delayed release coating which protects captopril until it reaches the colon. Captopril is available for administration in tablet or capsule form. A ~ io., of the dosage, â~LIilli ~Ll aLiOn~ in~ tionC and cA nn~in-lirAti~nc associated with captopril and 35 other ACE inhibitors can be found in the Pll,vsicians Desk Reference, Medical Economics Data Production Co., Montvale, NJ. 2314-2320 (1994).
CT-I is also potentially useful in the ~;-.I-.aLiOll, maturation, and survival of oligodendrocytes in vitro for ~. u'~,~,liu~l of oligodendrocytes against natural and tumor necrosis factor-induced death, in the survival and dir~.e..LiaLion of astrocytes and the inductiûn of type-2 astrocyte development, and in the 5tim~l1Ation of the CA 0224~63~ 1998-08-0 WO 97/30146 PCT/US97/0267~

recombinant production of low-affinity nerve growth factor receptor and CD-4 by rat central nervous system (CNS) microglia.
CT-I is also potentially useful in having a trophic effect on denervated skeletal muscle. In addition, - - it is expected to have the proliferative IC~OllSC.? and binding properties of hematopoietic cells Lla-lsr~lrd with low-affinity receptors for leukemia inhibitory factor, ~,. ,c-~"~ . M, and ciliary r.~ u- uLlul.liic factor~ to regulate r.b.i..o~ gene expression in hepatocytes by binding to the interleukin-6 receptor. to have trophic actions on ., murine embryonic càl. illull-a cells, to be an Pn~logçn--ue pyrogen, and to have a mitogpnir~ effect on human IMR
32 nc,llubl, Olu.lla cells.
In addition, CT- 1 is expected to enhance the response to nerve growth factor of cultured rat sym r~thptic neurons. to maintain Illolullc;ululls and their target muscles in developing rats, to induce motor neuron sprouting in vivo, to promote the survival of neonatal rat cc l Lic~ l neurons in vitro, to prevent degc..~ .aLiull of adult rat ?~ nigra dop~nlinçrgic neurons in vivo, to alter the threshold of hi,.po- a.ll~al pyramidal neuron sensitivity to excitotoxin damage, to prevent neuronal deg. Il~ .alion and promote low-affinity NGF receptor production in the adult rat CNS, and to enhance neuronal survival in embryonic rat hi~,~oca~ ,al cultures.
CT- I induces a phenotypic switch in symr ithPtic neurons and it promotes the survival of rif~r~rn inprgic neurons from the central nervous system and ciliary neurons from the periphery These activities translate into the L~dL~ of all r.~,~.vdeg~.lclaiive diseases by CT-I, including p~,l i~,l.~. al ne.u ul,aLl-ies (motor and sensory), ALS, Al~l.~,....~,. ', disease, Pal hil..,oll', disease, stroke, k'~ s, disease. and ophthalmologic diseases, for example, those involving the retina.
As shown herein CT-I shares at least some of the growth inhibitory activities of the IL-6 family cytohines. CT-I has the potential for use as a th~,.al,~.uLic non-proliferative agent for su~ Oaillg some forms ofmyeloidl~ ~--;-aswellasareagentfor,.lo.liryi,lg...a-..upl.agefunctionandother.~O~,ullacstoi..f.~
CT- I was 6 fold more potent than LIF in inhibiting the uptake of 3H-thymidine by M I cells and thus the growth of the myeloid leukemia cell line. CT- I inhibits the growth of the mouse myeloid leukemia cell line, M I, and 25 induces its dirr~ ion into a .,.a~..u,uhage-like phenotype. CT-I does not mimic the activity of IL-6 in ulllulillg B cell PYp~nci~n Unlike IL-6, CT- I has the advantage of not stim~ ting the growth of several B cell l~,...~,i.-... ~~ myelomas, and plasmacytomas. Thus, CT-I will find use in treating Iymrh~-m~c and lcll~pmi~c~
preferably B-cell and myeloid ~ L' ..;~' and patients with certain il.re~lions. Since CT-I is useful the treatment of patients with some forms of myeloid l~ and patients with certain ;., r~ the present invention also 30 extends to p l --....~- ~ l ;. -I cU~ uOiliollo c Ul~ l iOi,lg CT-I ~ particularly human CT- 1~ either Cullly' 'y or in part, produced for example using cloned CT-I-encoding DNA se-~ es or by chemical synthesis, and to r~ c~ ;u.~ of ~ of CT-l, for example produced by chemical synthesis or derived by ... r L~ . c;, of aforesaid cloned CT-I-encoding DNA sc-~ c~ The ,~.hal...~ c~l CUII~JU~;I;UII~ may also contain at least one other biological regulator of blood cells, such as G-CSF or GM-CSF. Furthermore, the 35 invention also extends to .I;~-~ u.~ ;c reagents for use in detecting genetic I callal~ lents, alterations or lesions ~c~OI ~Ird with the human CT-1 gene in diseases of blood cell formation, inrhlrlinv IPllk7.Pmis- and cnngPrlit~l diseases ~o~ d with :,uscc~Jlil ility to infection. CT-I can be used in the Ll~a~ clll of a wide variety of nc~".l~Lic cu..~ ;....c~ such as carcinomas. sarcomas, mcLu-u.llas, Iymphom~c, IPnk~mi~c which may affect a wide variety of organs, including the blood, lungs, Illallllllclly organ, prostate. intestine, liver, heart, skin, WO 97/301"6 PCT/US97/02675 pancreas. and brain. CT-I can be used in vttro to eliminate m~lign~nt cells from marrow for autologous marrow Llall 7IJlall~7 or to inhibit ~,lulif~,.alion or eliminate m~lign~nr cells in other tissue, e.g. blood, prior to reinfusion.
CT- I can also be used as â ll Ga~ L in disorders of the h~.llaLo~o:_lic system, especially as a means - ~Of ctim~ ting hPm~t~ ~ ~ in patients with ,u,u~ ,.,s d bone marrow function, for example~ patients suffering 5 from aplastic anemia, inherited or acquired immune deficiency, or patients undergoing radiotherapy or f hPmothPrapy.
E7~ toCT-I canalsobeusedfortreatingawidevarietyofwounds incl-lfling".,h~ y all r~"m...- ~,..c wounds, corneal wounds, and injuries to the epithelial-lined hollow organs of the body and those involving myocytes and neurons. Wounds suitable for treatment include those resulting from trauma such as 10 burns, abrasions, cuts, and the like as well as from surgical ,JlV~,GdUI-,.7 such as surgical incisions and skin grafting. Other con~itinnq suitable for treatmem w,th the CT-I ~ I_g~ include chronic conditions, such as chronic ulcers, diabetic ulcers, and other non-healing (trophic) conf1itir,nq Preferably, a CT-I ~nt~onict is illeVI ~JUI ~.t~.d in physiologically-acu~ ' carriers for local or site-specific ~ ,aLiull to the affected area. The nature of the carriers may vary widely and will depend on the intended location of application. If desired, it will 15 be possible to illCul~)ulale CT-I ~.n~,.J..;~1 c~ ;fmc in bandages and other wound dressings to provide for co -l ;w~uuc exposure of the wound to the, peptide. Aerosol f~ Jlh,a~iol~s also find use. The anizlgonict will be present in an amount effective to suppress CT-l inhih;~ion of epithelial cell ~lvliF~.la~ion. The compositions will be applied topically to the afFected area, typically as eye drops to the eye or as creams, ointments or lotions to the skin. In the case of eyes, frequent ~l~dtlll~ is desirable, usually being applied at intervals of 4 hours or less.
20 On the skin, it is desirable to c~ ntiml~lly maintain the IIG~ COIl]~v~ iull on the affected area during healing, with applications of the Ll~,aLI~ c~....l.o~ n from two to four times a day or more G~u~ ly.
CT-I .. ~ a s.;..~the Ulldia-GI ~ t~dphenotypeofembryonicstemcells. CT-I canpromote c ellsurvival and acts as an anti-apoptotic factor during mouse embryogenesis. Thus CT-l will find use in l. . l...;.l..P~ in which Ulldi~ ES cells are useful as well as t~prhniflupc in which control of their dilr.,....l~iaLion is useful For 25 example, CT-l will find use to maintain the undirr~.Gll~ia~ed state of embryonic stem cells during recombinant DNA transformation and their syn~;Llulli~d dirr~ ia~ion in methods such as gene cloning and creating ~ncgPnifanimals. CT-Ialsofinduseinartificial;..~ ..,;,.,..;~..,l~.l"~ Pc Thus,inonepreferredc.l,bo-lilll~,.l~, CT-I is used in the f-nh~nf PmPnt of .1~ ~.1 and ~ .. m ~e of animal or m~mm~ n embryos and to enhance illl~ .Lion.
A major difficulty aqqoci~t~d with present in vitro fertilization (IVF) and embryo transfer (ET) prograrns, particularly in humans, is the success rate "a~L;~ ,;i" on i~ of fertilized embryos. Currently, in human IVF pluglallls, the il~ lLaliul~ rate may be as low as 10%, leading to the present practice of using up to four fertilized embryos in each treatment which, in turn, leads oc~ lly to multiple births. Accordingly, there is a need to improve the implantation rate in human IVF programs. Similarly, in IVF and ET Ll~a~
35 in domestic animals such as sheep, cattle, pigs and goats, it is highly desirable for econ~mir reasons to have as high an ;",pl~ rate as possible so as to reduce the numbers of fertilized embryos lost and ".,~.,rcf -~.'ul ll~ allll.,.lL ,ulucc1ul~ .. perfûrrned. Furthermore, as with human IVF plu,,elulc:s, the practice of t~ rt,lillg more than one embryo to the recipient animal to ensure ~Jl~,~lall~,y can result in wl~allt~.d multiple births. One major c..l~cl~ with embryo transfer is the need to hold embryos in culture media for either reiatively short periods CA 0224~63~ 1998-08-0~

of time, perhaps only a few hours prior to transfer or for longer periods of some days, after micl .," ,,.~ ls.tinn In the dcv~,h-.iJ...c.ll of a ",~ , embryo, the fertilized egg passes through a number of stages inr~ iing the morula and the blastocyst stages. In the blastocyst stage, the cells form an outer cell layer known as the trophP~ tc.~i~rm (which is the ~ ,ulaOl of the placenta~ as well as an inner cell mass (from which the whole of 5 the embryo proper is derived). The blastocyst is au-luulldc-d by the zona pellucida~ which is ~..i.,. .l- ,lI,y lost when the blastocyst "hatches". The cells of the Llu~ 1 are then able to come into close contact with the -~ wall of the uterus in the implantation stage. Prior to formation of the embryo proper by the inner cell mass by ~ulaLion, the whole cell mass may be referred to as "pre-embryo." Embryo mortality has been attributed to incomrlet~ hatching of the blastocyst from the zona pellucida and/or Im~lcc~c~rlll implantation of the embryo 10 to the uterine wall, possibly due to srnntAn~ollC di~.._l-LiaLion of the embryonic stem cells (ES) during their period in culture prior to ~ fi1 ;~n CT- I can be included in an in vi~o embryo culture medium to enhance the hatching process leading to an increased number of embryos c.=llli I g the d~;v~:lu.~ c.~L stage by uu~d~ ,c g d~l.Ji""- ~,li-l changes s.ccocif~t~d with i,..l,~ . Tt.us, CT-I is an embryo protective agent. As aresult,thei...~.lal.LaLit..ratesforlVFandETprogramscanbe~;g";ri~llyimprovedbytheuseofCT-I inthe 15 in vitro embryo culture medium. FU.L~ ...U.~ media c~ .;"Sg CT-l is suitable for use in early manipulative ~u-,edw~.. on the oocyte/embryo such as in vitro ferti~li7sitiori~ embryo splitting and nuclear transfer where survival rates of embryos are low. CT- I also has i-"i .~.- LallL a~ .aliOlls in the growth of luLi,uuL~,.IL stem cell lines for cloning for inclusion into the media used for the transport of cooled or frozen embryos/semen. Thus a method for ~ -.g the i --~ ,~laLiun rate in an animal with one or more embryos is provided which cu---~Jfi~s 20 the steps of ,-- ~ ,;"g and/or developing the embryos in a medium con~slining an effective amount of CT-I for 5~r~ time and under ay~lulJIiate cfinrlitionc and then hll,JlallLil,g the embryos into the animal. By "illl~l)l~,~laLieill~ means the rate of sllrc~ccfill i~ ;0~ lC and ~ ;e~ I development of â fertilized embryo.
Also provided is a method for ~ i.";-;., ;"~ embryos or pre-embryos in culture while retaining viability for use in embryo transfer and/or genetic msmiplllsltinri which method includes culturing the embryos in a medium 25 c~ .,g an effective ~nount of CT-I for sllfficient time and under a~ JIial~ conAitinnc This method of ms imslinin~ the viability of embryos in culture has potential for allowing genetic msmirlllsltion of the whole embryo. Such ~ r~l genetic ms nirl~lsltic n is restricted at the present time due to the limited amount of time available to perform ~;~u~..i,--c~-L~ on viable embryos. The method also may be advsmtslg-oonc in ~ g viability of embryos under transport cull.liLiulls and may also be beneficial in the storage of embryos when 3û compared to te~hnin~ c currently employed. Another aspect of the present invention relates to a method for f-nhsinring the in vitro development of a ",~....,.5.1;,... embryo to the ;".~ -.0~l;0n stage, which method ~,u...~
thestepofculturingtheembryoinvitroinaculturemediumr~ ;";..ganeffectiveamountofmsinnmsilisinCT-I.
As is demonstrated below the inclusion of CT-I in the culture medium prior to the ~ullllaliull of tlhe blastocyst, or both prior to and following blastocyst fnrm~tion, also increases the number of pre-embryos co---~' g the 35 d~ l.J~ l stage by undergoing ;I~,~clopl..c.lL changes ~o~i t- d with implsintsition The addition of CT-I
also reduces the number of pre-embryos .1~ Ic~al g while in culture. As a result, the illlulallLaLion rate for IVF
and ET programs can be cignifi~Antiy improved by use of CT-I in the in vitro culture medium. The present invention, also extends to a method for in vitro fertilization _nd ~ ,lallLaliull of a mA nmAliAn embryo which is ~,Lala~,t~,fi~d in that the embryo is cultured in vitro in a culture medium c~mtAininv an effective amount CA 0224~63~ 1998-08-0~
WO 97/30146 PCT/US97/~675 of m~mm~ n CT-I prior to transfer into animal or ms-mmAiiz-n host, where "host" is defined as a suitably receptive female animal or mammal. A further aspect of the present invention relates to a non-human animal and in particular a chimeric non-human animal or llal~ ic progeny of said animal generated by icnown te~hniquFc - - ~ using ES cells which have been ....;.,~ 1 in vitro in CT-I-cont~ining culture medium. In accu.ddl,~e with this 5 aspect of the present invention, ES cells are derived from animal embryos passaged in a culture medium rv--l~;--i--s CT-I wherein said ES cells have ~i.l;l;....~l genetic material inserted therein. The transgenic animals c- ntpmrlDtpd include n.. l... n~Dmn.~lc such as livestock and ruminant animals and domestic animals. The present invention is also directed to c- ....I.n~:l inn Cu~ J.; 7hlg an effective amount of CT-I in combination with an animal (e.g. m~mm~ n) embryo m~int~ining medium. The present invention also provides a compocitirm 10 having embryotrophic and/or embryo protective properties Cullll~li,;llg CT-I. The amount of CT-I used in accordance with the present invention is that required to maintain andlor develop embryos and/or enhance ~iOll. GenerallyitisintherangeofO.I ng/mlto 10,000nglml,preferably I nglmlto 1000nglml.
CT- I also finds use to produce a m~mn~D~ plul ilJolclllial embryonic stem cell ~.. l .. ~ :l ;~l~ which can be ... ~ ;.-Fd on feeder layers and give rise to embryoid bodies and multiple ~ lLàl~ d cell phenotypes in 15 monolaver culture. Provided is a method of making a pluli~u~.lLial embryonic stem cell by ~riminictr~ring a growth ~ ~ amount of basic fibroblast growth factor, CT-I, membrane Z~ccociDtp~i steel factor, and soluble steel factor to l~l illlul d;al germ cells under cell growth cr~n~ c~ thereby making a plul ;lJot~ ;al embryonic stem cell. A ''plul;yu~lllial embryonic stem cell" as used herein means a cell which can give rise to many lLi ~d cell types in an embryo or adult, including the gerrn cells (sperm and eggs). This cell type is also 20 referred to as an "ES cell." Only those m~m~lc which can be induced to form ES cells by the described methodsarewithinthescopeoftheinvention. Althoughnotal~ uilc~ forapplicationofthispmboflimpnt of the invention, the ES cells may be capable of indefinite n IA ~ P~ typically at least 15 days. Once the ES
cells are ~ ,I .I.I;~I.~A they can be gPnPtic~lly m~nir~ tpd to produce a desired ~ Lala~t~,liaLic. For example, the ES cells can be mutated to render a gene non-r---- li--- ~1 e.g. the gene a~u~ d with cystic fibrosis or an 25 nnCogpnp Alt~ ~lldti~ ly, ri ' genes can be inserted to allow for the production of that gene product in an animal. e.g. growth hollllull~s or valuable proteins. The invention also provides a ~ ion Colll!JIi:.illg pluli~JuL~,lLal ES cells and/or primordial germ cells and/or embryonic ectoderm cells and CT-I, an FGF.
n.~"lll,lalle ~ oG U,;l SF, and soluble SF wherein the factors are present in amounts to enhance the growth of and allow the c~ntinllPd proliferation of the cell. Growth and proliferation . .l~ g amounts can vary.
3û Generally, 0.5 to 500 ng factorlml of culture solution is ~dPq~ tP Preferably, the amount is between 10 to 20 ng/ml. Alternatively, CT-I can be used to maintain ES cells. In this case, the amounts of CT-I, FGF, and SF
necessaly to maintain ES cells can be much less than that required to enhance growth or ~Jluli~ ,aliun to become ES cells. In addition, CT-I, FGF, or SF may not be required for .. ;.: .t.. e of ES cells. The invention also provides a method of making a p' , . ' ES cell cu.l~ i.lg z--lminictPring a growth ~ ..l....~ :~lg amount of 35 CT-I. basic FGF, Ill. Illb~ e ~o~ :.u~ d SF, and soluble SF to ~lilllul.lial germ cells and/or embryonic ectoderm cells under cell growth con~litionc thereby making a plul;l.ut~ l ES cell. This method can be practiced utilizing any anima] cell, especially mammal cells including mice, rats, rabbits, guinea pigs, goats. cows, pigs, humans~ etc. The ES cell produced by this method is also eontemplated. "Cell growth conrlitic)ncl~ are set forth in the Examples. However, many alterations to these c~n-liti-nc ean be made and are routine in-the art. Sinee .
CA 0224~63~ 1998-08-0~

the invention provides ES eells generated for virtually any animal, the invention provides a method of using the ES eells to CUIILI;1JULG to ehimeras in vivo Culll~,l;>illg injeeting the eell into a blastoeyst and growing the blastoeyst in a foster mother. Alternatively, ag~lG~3aLillg the eell with a morula stage embryo and growing the - - embryo in a foster mother ean be used to produee a chimera. The ES eells can be mAnirnlAfPd to produce a 5 desired effeet in the ehimerie animal. Alternatively, the ES eells can be used to derive cells for therapy to treat an abnormal condition. For example, derivatives of human ES cells could be placed in the brain to treat a ., neurodc~ .lc.alive disease.
CT-I will stimulate the proliferation of satellite eells and the snhsp~flll~nt development of myoblasts.
Accc l.lill~ly, provided are methods of c~ E the proliferation and/or dilT~,.,..Lidion of mAmmAliAn satellite 10 eells into myoblasts whieh ineludes the steps of cu- .~ said eells with a ctimlllAtinn-effective atnount of CT- I
for a tirne and under enn~iitinnC sufficient for said satellite eells to ululif~,.alG andlor dil~,~,,lLiale into myoblasts.
The ctiTn~llAfinn-effective amount of CT- I ean be A- 1. .. ;. .;~l r~ Gd - - I II IIIA- -~ UU~IY or in cf qll~nti~l CUI ~ ~ ~;"AI ;UI~ with oneormoreothercytokines~foratimeandundercnntiitinncsufflcientforsaidsatellitecellstoiJlulir~aleandlor ~li~.~ ,lliale into myoblasts. Also provided are methods of myoblast transfer therapy which include the steps 15 of eont~tingm:~TnmAIi~nsatellitecellswithaproliferation-and/ordirrGI~,,lLidiull-effectiveamountofCT-l for a time and under enn ihinnc sufficient for said satellite eells to proliferate and/or dil~.-,.lLalG into myoblasts and then A.l."illi~ g said Ill~U~6~1~ at multiple sites into muscles. In an alternative to this ennbofiimpnt~ CT-I is used in ~ - ....1~ ~PU~ ~ or cp~lupntiAl com I ~ with one or more other cytokines. Al,-,ul d~.lgly, a eell activating cu..~p~ .cu.ll~u.i~illgCT-l incombinationwithoneormoreothercytokines,andoneormorephy~iolog~ lly 20 aeceptable earriers and/or diluents is provided. And there is provided a pl~ e.llifAl culllpG.ilion for sl ;l l ~- IlAI ;- ~, the proliferation and/or diirr~ ,.ILaLiull of satellite cells which ineludes CT- I and one or more other cytokines and one or more i hA .. IA~ . .1;. Ally aceeptable carriers and/or diluents. In one preferred PmhoriimPnt the cytokines in optional comhinAtinn with CT-I include IL-6 and/or TGF alpha and/or FGF. The methods and e~ o~-~;nnC find use espeeially in relation to primary, gPnPtie~lly dGl~lllillcd, musele myopathies, the most 25 severe and the most eommon of which is Duchenne museular dystrophy (DMD). Beeause of the size and eomplexity of the DMD gene, it is unlikely that genetic mAniplllAtion will be possible in the near future.
However. an effective approaeh involves the growing of myoblasts in eulture derived from normal ., ..~ ~,.. .Aic and inieeting them, at multiple sites, into museles of the patient to result in the museles eontAining dystrophin whereas iuusl~ there was little or none. Thus human myoblasts, grown in eulture, are injeeted at multiple sites into 3û museles of DMD. This approaeh is al .~ Ir to all primary myopathies, not only DMD. At present, t~r hniriuf-s of culturing myoblasts utilize medium to long term eulture witi1 varying c ~ innc of the eA ~ reagent fetal ealf serum. Thus, a~,.,el.,.dti~-g myoblast ~li~e. ~,..lia~ion and growth should be .~;p . . i .~i- A- .l advance toward redueing the eost of myoblast produetion and faeilitate therapy. CT-I alone, or in eombination with other eytokines sueh as IL-6 and/or TGF alpha and/or FGF, will provide this accel~,,alion. Aeeordingly, provided 35 herein is a method (and cU...l.u~;l;ùn:~ for same) of 5fim~ tinP the proliferation and/or dii~ ,.lLialion of mAmm~liAn satellite eells into myoblasts whieh ineludes the steps of CI~IIIA- I;~.g said satellite cells with a stimulation-effective amount of CT-I, alone or in combination with other eytokines sueh as IL-6 and/or TGF
alpha and/or FGF, for a time and under conditions sl-ffl~ nt to stimulate the satellite cells. In these methods the satellite cells are most preferably from the same mammal to be treated, less preferably from the same species CA 0224;i63;i 1998-08-O;i of mammal. and least preferably from different m~mm~lc The mammal can be human, mouse. a livestock or a pet animal. Most preferably CT-l and the satellite cells are from the same species of m~mm7i CT-I can be provided at a cu...,c..-. l.Lion of from about 0.1 to about 1000 ng/ml, and more preferably from a cn~ n ,-l ;on of from about I to 100 ng/ml.
CT-l can be involved in the repair of injured muscle and the .~ t~ of cellular hn~ For example. the ,UlUlllil~C.ll G~ iVII of CT-I in skeletal muscle indicates that CT-I will serve to promote the survival of skeletal muscle cells during periods of muscle injury. This is GQn';'~' ~.1 with the finding that in skeletal muscle, LIF and CNTF were found to be involved in the repair of injured muscle (Barnard er aL, J
Neurol Sci., 123:108-113 (1994); Helgren et aL, Cell, 76:493-504 ~19g4)). This function of CT-I is cn~
10 withtheenlargingroleofthegpl30cl ~ cytokinesin~.vvlil.gcellsurvival. Inaddition.thelevelofCT-I eA~ aiv.~ in the mature heart and other tissues is c~ .I with its supportive role to maintain tissue survival in these tissues. In Ihis regard, previous studies have cie.llv.~ ,d that LIF and CNTF can promote neuronal cell survival in vitro (0~ - . . et al., Science, 251: 1616- 1618 ( I 991); Martinou et al., Neuron, 8:737-744 (1992)). In addition, analysis of LIF deficient mice suggests that LIF may be required for the microenvironmen~
15 to maintain the survival of hematopoietic cells (Escary et aL, Nature, 363:361-364 (1993)). Although the members of the IL-6 family share a great degree of ~ .n~ l ;. ."~1 reA--nA~nry, individual family members may have their own specific target tissues and divergent filn~tjonc~ based upon the localized ii:,L~ ivuliull and density of the cytokines and their receptors. CT-l can block viral induced apoptosis of neonatal cardiac muscle cells following infection with cardiomyopathic viruses.
As shown herein, CT-I is a multi-fi-nction~l cytokine which shares several biological activities with other members of the IL-6 cytokine family. CT- I and LIF have similar activities in the in vitro assay systems t~ min~d thus far. Accu. iil~gly, CT-l is expected to find use in the medical L.~,~Lm~l.L uses known for LIF.
Figure 21 is a s. h . - -,-~ ;' that ~Illlllll~ i.~.~ the diverse bioactivities of CT-I in a wide variety of cell types. These activities include the ability of CT-I to inhibit embryonic stem cell dirr~l~,llLi~.Lion and aortic endothelial cell 25 proliferation, thus CT-l will function in reg~ ting development. Like other IL-6 family members, CT-I
induces acute phase proteins in hepatocytes and thus will modulate local ;"n, ----- ,.~Uly ~lu~,~Sal,5. and play a roleasanacutephasemediatorinvivo(seealsoPetersetaL, ~EBSLetters,372:177-180(1995)). CT-I orits will be useful in the treatrnent of arthritis and infl7mm7tQry pathologies. During the ;.. n,.~ . .ly reaction., I.~ modifications occur in the synthesis of a group of plasma proteins called acute-phase 30 proteins. Some of these proteins-including rLI illO~ , reactive protein C, h~p~- globin are increased during the acute-phase reaction, whereas others such as albumin and Lld,l~l~llill are reduced. The alteration of these proteins, in p~u li.,ul~ fibrinogen, is l ~uonsiL~- for the mntiifi~ ~ti~n~ in the plasma viscosity and for the increase in thespeedofs~ li.. li.ti.. ~ which areobservedin the ;.111,.. ~ .linn Becauseoftheircorrelation with clinical pdl~n~t~l:,duringthedcv~k,l,lll~llLandthel~ .G~ ;u..~observedinl~ ..i.iarthritis,someofthese 35 acute-ph~se proteins have been used as a criterion for evaluating the disease (Mallya et aL. J. RheumatoL 9:224-8 (1982); Thompson et aL, ,4rthritis Rheum. 30:618-23 (1987)). Accordingly, a method is provided for treating a mamunal affiicted with arthritis or an in ll~ "~ ,, y disease, including those reiated to autoimmune diseases.
The method includes the step of ~rlminict-?rjng to the mammal an amount of compound which is effective for alleviation of the con~lition Infl,.. ,.l-.. y states in m~mm~lc include, but are not limited to, allergic and --s2--CA 0224~63~ 1998-08-0~

asthmatic n.~lir~ linnc dermatological diseases. ;.. n~.. ,e.. ~ y diseases. collagen diseases~ reperfusion in3ury and stroke, infeetions, and lupus eryt~ l."~-~ Treatment of both acute and chronic diseases are possible.
Preferred diseases for LlcaLIll~ are arthritis, asthma, allergic rhinitis, i..n~ y bowel disease (ILD), psoriasis. . ~ . ru~;ull injury and stroke. Other disorders involving acute phase proteins are acute l~ oblzO~ic 5 leukemia (ALL), acute graft versus host disease ~aGvHD), chronic Iymphoeytic leukemia (CLL), ~u~
T-cell Iymphoma (CTCL), type I diabetes, aplastic anemia (AA), Crohn's Disease, and sclc.ud~ -a. Additional ;.,n ~ ,..." c~nrlifinnc include patients with severe burns, kidney transplants~ acute infections of the central nervous system and septic shock.
Furthermore, CT- I like LIF, inhibits the proliferation and induces the ~ lLiaLiull of a mouse myeloid 10 leukemiacellline. SimilartotheactivityseenforLIFandCNTF,CT-I hasneuronalfunction,inthatitpromotes the survival of cultured dopaminergic neurons and ciliary ganglion neurons and induces a switeh in the Lla.lOllliLt. l phenotype of sY..~ ll.- lic neurons. Thus, while CT-I was initially isolated on the basis of its actions on eardiac muscle eells, it may also have pleiuLIopic functions in other organ systems that overlap to a ~:~ .; ri~
extent with the activities other IL-6 family cytokines, preferably those of LIF and OSM, and more preferably 15 those of LIF.
As shown herein, CT-I signals through and induces tyrosine phosphorylation of the gp 1 30/LIFR~-h~,~lu~lilll~l in cardiac myocytes and other cell types. This does not exclude the possibility that CT-I may use an alternative signaling pathway via an ~rlrlition~l private receptor in some cell types. Members of the IL-6 cytokine family inrlrl-iin~, IL-I 1, LIF, CNTF, and OSM trigger du...l~.lcaul signaling pathways in multiple eell 20 - types through the homodill.c.iLc.Lion of gpl30 or through the heterod.l--c-i~Lion of gpl30 and a related tr~ncmPmhrane signal L~ ducel, the LIF receptor subunit LIFR~ (Figure 15B; Gearing et al., Science, 255:1434-1437~1992);1petal.,Cell, 69:1121-1132(1992);MurakamietaL,Science,26û:1808-1810(1993);
Davis e~ aL, Science, 260:1805-1808 (1993). An anti-gp l 30 monoclonal antibody was used to determine its effeetonCT-I bindingtoMI cells. Thisneutrali~ingantibodyinhibitedCT-I bindingtoMI cellsin~ ting 25 that gpl30 is a cu~ of the CT-1 receptor complex. CT- l and LlF also cross-compete for binding to rat cardiac myocytes and mouse Ml cells inrlics-ting that these two ligands act on these cells via the LIF receptor.
In addition, c-fos induction by CT- I and LIF in cardiac myocytes was ~ g~-~ .;, ~ d by the anti-gp 130 m nnc~clnn~l antibody as well as by a mutated human LIF protein, acting as a L}FR ,1}-.... 1 ~g, ... i~l A direct demonstration that CT-I interacts with LIFR~ and gpl3û has been shown by the binding of CT-1 to purified soluble gp 130 and 30 LIFR,~. Accu-~li..gl~y, CT-I will find use in disorders, diseases or Cun-liLiull relating to cells ~ i--g the LIFR,(~ and to its signaling pathways.
As demonstrated by ;-----.--~ lion with a polyclonal anti-gpl30 cytoplasmic peptide antibody and 5~-hCpquent anti-phosphotyrosine immu..obluLli..g, ctim~ tinn of cardiomyocytes with CT-I. LIF, and a cul.lbil.clLion of IL-6 and soluble IL-6 receptor (slL-6R) resulted in the rapid tyrosine ~JhO:~JIlul ~lation of gp 130.
35 These data indicate that tyrosine phosphorylation of the receptor ~,ulllpul.cl-L gpl3û is an early step in CT-I
Si~n~ing as has previously been shown for the other members of the IL-6 cytokine farnily (Ip et al., CeU, 69:1121-1132 (1992); Yin et al., Journal of Immunology, 151:2555-2561 (1993); Taga e~ al., Proc. Na~l ,4cad. Sci. USA, 89:10998-1 1001 (1992)). As determined by immnnQ-hlotting with an anti-phosphotyrosine antibody, LIF induced the tyrosine phosphorylation of an ~ itinr~ 200 kDa protein. which was not WO 97/30146 PCT/~JS97tO267S

phosphorylated upon 5tim~ fion with the IL-6/slL-6R complex. Based on previous results~ this protein most likely co.~ .ollds to the LIF receptor subunit LIFR,B (Ip et al., Cell, 69:1121-1132 ~1992); Davis et aL, Seienee.260:1805-1808(1993);Boultonetal., JournalofBiologicalChemistry.269:11648-11655~1994)).
As shown herein, ctim~ tinn of cardiac cells with CT- l also resulted in the tyrosine pho~ (,.ylation of a protein, 5 in~i~tin~ r in size from the LIFR,B. And an LIFR13 ~ntzlgnnict blocked the action of CT-I in cardiomyocytes. Accu,dil~gly, CT-I, like LIF, induces the tyrosine phosphorylation of LIFR~.
Since CT- I and LIF appear to have filn~fi~nsll ~ .,lunddll-,y in these assay systems, the possibility exists that CT- I c~ r~ rd for the complete loss of LIF during embryonic dcv~lo~ ..L and adulthood in these LIF
defieient embryos. Alternatively, since LIF is not ~ sL1 at very high levels in the embryo, CT-I may be the 10 r- ~firlg~ ligand which normally performs this function during mouse embryonie dc~r~,lop."~,l,L. If the latter isthecase,onemightexpeetsevereembryoniedefectsinCT-I defieientembryos,~n~log~-uctoeithertheLIFR,B
defieientorgpl30defieientphenotypes. CT-I willbeinvolvedinthell ,-; ~ eofnormaleardiacgrowth, l,,...llhn~ and hypertrophy, which can be analyzed in the basal state and in response to the hll~u~iLiol~ of a ~ ~ l ~ h~ ~ ;f ;~1 stimulus for hypertrophy via Illi..i~Lul i~d physiological technology (Rockman et aL, Proc. Natl 15 Acad. Sci USA, 88:8277-8281 (1991)). This system will allow screening and i~ ntifj~ti~n of CT-I agonists and _.ai g,...;~ Il.t.~ Lill~ly, a large disparity between the phenotypes seen in mice lacking the CNTF receptor and mice lacking CNTF have been reported (DeChiara et al., Cell, 83:313-322 (1995)). While animals which . ~ Iy lack the CNTF receptor display pl ulllhl~ motor neuron deficits at birth, mice that lack CNTF appear to be relatively ~ n d (DeChiara et al., Cell, 83:313-322 (1995)), and do not display any notable 2û abnorrnalities in the developrng nervous system. In addition, LlFR,B deficient neonates also display similar profound motor neuron deficits (Li et al, Nature, 378:724-727 (1995)). These studies strongly suggest the possibility that there may be an alternative ligand to CNTF that binds to the CNTF receptor and LIFR~ that is requrred to maintain normal nervous system d.~.lo~ I While the CNTF receptor is not required for CT-I
binding to the gpl30/ LIFR,B complex and int~ tinn of CT-I with the CNTF receptor has not been 25 demonstrated, CT-I may be this alternative ligand.
CT-1 may also be useful as an adjunct Ll~aLlll~,llL of neurological disorders together with such r.c.lluLIuLJhic factors as. e.g, CNTF, NGF, BDNF, NT-3, NT~, and NT-5.
The nucleic acid encoding the CT- I may be used as a ~ 1;~,", .~ for tissue-specific typing. For example, such procedures as in situ hyl,l idi~Livn, northern and Southern blotting, and PCR analysis may be used to 30 ~let~rrnine whether DNA and/or RNA encoding CT-1 is present in the cell type(s) being c~aluaL~d~
Isolated CT-1 polypeptide may also be used in ~ - a ili~ gnnstic assays as a standard or control againstwhichsamplesc-..a;.;..i..gunknown~ -.lilir~ofCT-I maybeprepared.
Th,.la~ ILic forrnnl~tinnc of CT- 1 for treatrng heart failure, r..,~ ulo~;,icdl disorders, and other disorders are prepared for storage by mixing CT-I having the desired degree of purity with optional physiologically 35 aceeptable carriers, excipients, or stabilizers (Remin~ n's P~ n~ac~ l Sciences. supra~, in the form of Iyophilized cake or aqueous solutions. A~ ' ' carriers, ."~ .-ls, or stabilizers are non-toxic to l~ s at the dosages and conc~"~LI~.Lions employed, and include buffers such as ph. .~ , citrate, and other organic acids; ~mioyi~ ntc i~.~l.,.1;"~ ascorbic acid; low molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as CA 0224~63~ 1998-08-0~

polyvinylpyrrolidone; amino acids such as glycine, gll.lnll.;..c, aalJald~ c, arginine or Iysine; monoaac.,l,alides, di .ac~,Lal ides, and other carbohydrates including glucose~ mannose, or dextrins; chelating agents such as EDTA;
sugar alcohols such as mannitol or sorbitol; salt-forming Cuullt~. ;on5 such as sodium; and/or nonionic aw ~a~, - - such as Tween, Pluronics, or polyethylene glycol (PEG).
CT-I to be used for in vivo al~lminictration must be sterile. This is readily accomplished by filtration through sterile filtration ~ ~ ~ ~ . Ihl .~ c, prior to or following Iynrhil i~Rti,~n and ~ ~c~ .. . CT- I ordinarily will be stored in Iyophilized form or in solution.
Thc.a~ Lic CT-I c.. ~ ;l ionc generally are placed into a cu,.L~ having a sterile access port, for example. an intravenous solution bag or vial having a stopper pierceable by a hypoderrnic injection needle.
The route of CT-I or CT-I antibody a~L,,i,,;aLIaLion is in accord with known methods. e.g, injection or infusion by illlla~ vua~ i.. lla~c~ neal, i.lL-ac~,l l.,al, ill.li.. ~ ulRr~ intraocular, illllaalt~,l;âL or intralesional routes, or by cllct~insd-relea5e systems as noted below. CT-I is RfiminictPred cU.,l; .~o..~ly by infusion or by bolus injection. CT-I antibody is ~fiminictPred in the same fashion, or by ~1minictr~ti~m into the blood stream or Iymph. Most ,ol~f~,.ably, CT-I or its Rnt~gQnict is R-lminictPred locally or site-specifically to better obtain a 15 local or site-specific effect. Such suitable delivery methods are known in the art including implants~ pumps, patches~ direct injection, and trRncm~ Qc~l delivery. Site-specific delivery can be obtained by gene delivery vectors and viruses and by llalla~lalllalion of cells e~ ,aaillg CT-I or an _~ g~
Suitable ~.~al.~,lcs of sustained-release preparations include semiper~neable matrices of solid hy~Lul)hol)ic polymers c~ e the protein, which matrices are in the form of shaped articles~ e.g, films, or 20 Illil,lu~ c Examples of sustained-release matrices include polyesters, hydrogels (e.g, poly(2-hydroxyethyl-methacrylate) as described by Langer et al., J. Biomed. Mater. Res.~ 15: 167-277 ~1981) and Langer, Chem.
Tech.~ 12: 98-105 (1982) or poly(vinylalcohol)), polylactides (U.S. Patent No. 3,773,919, EP 58,481), copolymersofL-glutamicacidandgammaethyl-L-~ t--- (Sidmanetal.,Biopolvmers.22:547-556(1983)~, non-de~;,alal,le ethylene-vinyl acetate (Langer et aL, supra), degradable lactic acid-glycolic acid copolymers 25 such as the Lupron DepotTM (hljc~lable mi-,lva~ h~"~s CullllJo..c;i of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid (EP 133,988).
While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When PncRrs~ tpd proteins remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37" C, 30 resulting in a loss of biological activity and possible changes in i.. n,,.. icity. Rational strategies can be devised for protein stabilization ~ -i;..g on the mP- h ~iqm involved. For example, if the ag~ aliol, mPchRnicm is discovered to be intermolecular S-S bond fu~ a~iull through thio-disulfide interchange, f,l;~ i..ll may be achieved by modifying sulfhydryl residues, Iyophilizing from acidic solutions controlling moisture content, using alJ~JIv~.ia~l; additives, and d~,elo~illg specific polymer matrix cu~ JoaiLiulls.
Sustained-release CT-I culll~Josi~ions also include lipl-c~-mRlly c.lLIa~v~Jcd CT-I. T irosomPc c~ nt~inine CT-1 arepreparedbymethodsknownperse: DE3,218,121;Epsteinetal.,proc.Natl.Acad.Sci.USA~82:
3688-3692 (1985); Hwang etaL, Proc. ~Zlfl Acad. Sci. USA. 77: 4030-4034 (Ig80): EP 52,322: EP 36.676; EP
88,046; EP 143,949; EP 142,641; Japanese patent application 83-118008; U.S. Patent Nos. 4.485,045 and 4,544,545; and EP 102,324. Ordinarily the liposomes are of the small (about 200-800 Angstroms! unilamellar type in which the lipid content is greater than about 30 mol. % cholesterol, the selected pl u~ul LiOII being adjusted for the optimal CT-I therapy.
An effective amount of CT-I to be employed ther~pelltir~lly will depend. for example. upon the ~= IL~ay~ ic objectives, the route of administration, and the condition of the patient. Accordingly~ it will be 5 necessary for the therapist to titer the dosage and modify the route of aflminictr~tif)n as required to obtain the optimal ll~ LiC effect. A typical daily dosage might range from about I llg/kg to up to 100 mg/kg of patient body weight or more per day, f~epPnrling on the factors ...~ cd above, preferably about 10 ~lg/kg/day to 10 g/kg/.lay. Typically,theclinicianwill~f~mini~t ~CT-I untiladosageisreachedthatachievesthedesiredeffect for treatment of the heart, neural, or other dycfimrtif~n For example, the amount would be one which increases 10 ~ sLI i-,ulal Cùl~lla~lili~.y and decreases peripheral vasculamc,~ ~lce or ameliorates or treats conflitif)nc of similar importance in collge~Li~, heart failure patients. The progress of these therapies is easily monitored by conv~,l.,iullal assays.
4. CT- I Antibody Preparation (i) ~tslrtinp Materials and Methods I,-",l~--,nglobulin5 (Ig) and certain variants thereof are known and many have been prepared in ,U..-l.~ L cell culture.3~or example, see U.S. Patent No. 4.745,055; EP 256,654; EP 120.694; EP 125,023;
EP 255,694; EP 266,663; WO 88/03559; Faulkner ef al., Nature, 298: 286 (1982); Morrison, J. Immun.. ~:
793 ~1979); Koehler et aL, Proc. Natl. Acad. Sci. USA. 77: 2197 (1980); Raso et aL, ('~nrPr Res.. 41: 2073 (1981); Morrison e~aL, ~nn Rev. Immunol..2: 239 (1984); Morrison, Science. ~: 1202 (1985); and Morrison 20 etaL, Proc. N~tl Acad. Sci. USA. 81: 6851 (1984). Reassorted immnnoglcb~l;n chains are also known. See.
for example, U.S. Patent No. 4,444,878; WO 88/03565; and EP 68,763 and l~r.,.~,.lces cited therein. The i....,,.)..f~l..l,..l;., moiety in the chimeras of the present invention may be obtained from IgG-I, IgG-2, IgG-3, or IgG-4 subtypes, IgA, IgE, IgD, or IgM, but ~l~,f~,lably from IgG-1 or IgG-3.
(ii) Polvc~f~n~l alllil Odi~s 2SPolyclonal ,..... ~ O~ to CT-I polypeptides or CT-I Ga~ll~;llL~ are generally raised in animals by multiple ~. .l .~ ~ . ~ . . .r ~ (SC) or illLI a~ UII.,dl (ip) injections of CT- I or CT- I fragment and an adjuvant. It mav be useful to conjugate CT-I or a fragment c~ the target amino acid sequence to a protein that is imml~mfigenic in the species to be imml-ni7~, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifi-nrtion~l or d~l ivaLi~hlg agent, for example~
30 m~ imitlobenzoyl s~llr.~.fc; .;...;-1~ ester (conjugation through cysteine residues), N-hydroxycnrcirlimiA~
(through Iysine residues), glutaraldehyde, succinic anhydride, SOC12, or RIN=C=NR, where R and Rl are different alkyl groups.
Animals are i............ i,. d againstthe CT-l polypeptide or CT-I fragment, i.,.. og.. ic cfmjllE~t~c or derivatives by combining I mg or I ~lg of the peptide or c~-nj--g~t.~ (for rabbits or mice, respectively) with 3 35 volutnes of Freund's complete adjuvant and injecting the solution hlLI ad~ .ally at multiple sites. One month later the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by ~ - ~r u ~~ injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for CT-I or CT-I fragment antibody titer. Animals are boosted until the titer plateaus. Preferably, theanimalisboostedwiththec~ ofthesameCT-I orCT-I fragment,butcf.. j.. ~ dtoadifferentprotein CA 0224~63~ 1998-08-0~

and/or through a differem cross-linking reagent. Cvllju~,at~.~ also can be made in recombinant cell culture as protein fusions. Also, a~ alillg agents such as alum are suitably used to enhance the immune response.
(iii) Monocion~ ;ho~lies - - ~hlonoclnn~l ~ntihorlil-c are obtained from a population of cllhst~nti~lly homogeneous antibodies, i.e., S the individual allLl,odies cullll~liaillg the population are identical except for possible naturally occurring " ...l~l ;....~ that may be present in minor amounts. Thus, the modifier "monnc~nn~l" indicates the character of the antibody as not being a mixture of discrete antibodies.
For example, the CT-I monoclnn~l antibodies of the invention may be made using the hybridoma method furst described by Kohler and Milstein, ~a~, 256: 495 ( 1975), or may be made by recombinant DNA
I û methods (Cabilly et al., supra).
In the hybridoma method, a mouse or other a~,prulJ~ ialG host animal, such as a hamsIer, is il ". . .n.~
as hereinabove described to elicit Iymphocytes that produce or are capable of producing ~ntibo~ s that will crerifir~lly bind to the CSF or CSF fragment used for ;.. i,~l;nn Alternatively, Iymphocytes may be immnni7.~d in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as 15 polyethyleneglycol,toformahybridomacell(Goding,M~,I.o~lo,.,.lAntibodies PrinciplesandPractice.pp.59-103 [Academic Press, 1986)).
The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more ~ that inhibit the growth or survival of the unfused, parental myeloma cells. For example. if the parental myeloma cells lack the en_yme hy~.o~ guanine phosphoribosyMIall~r~la~e 20 - (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypvY~ minopt~-rin~
and thymidine (HAT medium), which 51.h~ c prevent the growth of HGPRT-deficient cells.
Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-I I
25 mouse numors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 cells available from the American Type Culture Collection, Rockville, Maryland USA.
Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against CT-I. Preferably, the binding ~I,e~,irl, iLy of ml noc!nn~l antibodies produced by hybridoma cells is ~ by il ~ ;Lali~Jll or by an in vitro binding assay~ such a (RIA) or enzyme-linked ;.. - ~-Ah~ assay (ELISA).
The binding affinity of the nnonoçlnn~l antibody can, for example, be ~1Pt~nin~od by the Scatchard analysis of Munson and Pollard, Anal. Biochem.. 107: 220 (1980).
After hybridoma cells are i~l~ntifiPd that produce allLilJodi~s of the desired ~ ir.~i~", affunity, and/or activity, the clones may be ~h~ I ...~ I by limiting dilution ~u-,elu~ and grown by standard methods (Goding, 35 supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition. the hybridoma cells may be grown in vivo as ascites tumors in an animal.
The mulloclùllal s~ntiho~ s secreted by the ,..1.~1-... c are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Se~Jhaluse, hydroxyapatite chromatography, gel electrophoresis. dialysis. or affinity clllullla~o~;la~h~/.

-DNA encoding the monoclonal antibodies of the invention is readily isolated and :,c~ cl using conventional p~uCGIu~c~ (e.g, by using olig.,...~ probes that are capable of binding specifirSllly to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a - - preferred source of such DNA. Once isolated. the DNA may be placed into e,~ aiul- vectors. which are then S llall:~c u d into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal a.lLilJo.li~s in the l~cunllJillallL host cells. Review articles on recombinant expression in bacteria of DNA
encoding the antibody include Skerra et aL, Cllrr~ Opinion in Immllnnl~ 5: 256-262 (1993) and Pliickthun, Jmmlmol. Revs.. 130: 151-188 (1992).
10 The DNA also may be mn~ifi.--l, for example, by, ~h~ ;.. g the coding sequence for human heavy-and light-chain constant domains in place of the hnm~l~g murine s~ , (Morrison, et al., Proc. Nat.
Ar~i Sci..81:6851 (1984)),orbycovalentlyjoiningtotheimml~nnglnb~lincodingsequenceallorpartofthe codingsequenceforanon-i-"""-.,o~lnbl-linpolypeptide. Intbatmanner~ l,in,~ic"or"hybrid" ~~ lo~l~care prepared that have the binding specificity of an anti-CT~ c I~J nAI antibody herein.
15 Typically such non-immunoglobulin polypeptides are ,.... ~ d for the constant domains of an antibody of the invention, or they are ~ lrd for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody cu,,,,... iaiulg one antigen-combining site having a~JG~.irlcily for a CT-I and another antigen-cu...b;..iu.g site having :"Jecirl. iLy for a different antigen.
Chimeric or hybrid ~ o-l;~s also may be prepared in vitro using known methods in synthetic protein 20 chemistry, including those involving ~ i"g agents. For example, i" .... ~ " .~ may be cu"~LI u~,L~d using a disulfide-exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include imin~lthiol~ and methyl-4-,..~ .- a~lul uLyrimidate.
For fii~nctie a~ iC~ILiunS, the ~ltiho~ s of the invention typically will be labeled with a detectable moiety. The detectable moiety can be any one which is capable of p-u-lu. i,~g, either directly or indirectly, a d~ L,le signal. For example, the d~t~ le moiety may be a ~adioi~uLulJc, such as 3H, 14C,32p,35S, or 1251;
a fluorescent or rh~m jl~ cu...,vuu.,d, such as nuUl~ cill isothiocyanate, rhodamine~ or luciferin;
radioactive isotopic labels, such as, e.&~ 1251, 32p, 14C, or 3E~; or an enzyme, such as alkaline phûcrh~t~c~
beta-~l Irtoci~l~ce or hw ~c.~di ~ll peroxidase.
Any method known in the art for separately c....j..~1 ;..g the antibody to the detectable moiety may be employed, including those methods described by Hunter e~ aL, Nature, 144: 945 (1962); David et al., Bi-~. h. n~ / 13: 1014 (1974); Pain etal., J. Immnnol. M~th 4Q: 219 (1981); and Nygren, J. Hic-torh5ln. :~n~
~ytochPm ~: 407 (1982).
The antibodies of the present invention may be employed in any known assay method, such as ~u--~ /e binding assays, direct and indirect sandwich assays, and immunu~ ,it~tion assays. Zola, Monsclnn~l Antih(~ c A M~n~l of Techniques. pp. 147-158 (CRC Press, Inc., 1987).Competitive binding assays rely on the ability of a labeled standard (which may be a CT-I or an immunologically reactive portion thereof) to compete with the test sample analyte (CT-I) for binding with a limited amount of antibody. The amount of CT-I in the test sample is inversely proportional to the amount of standard that becomes bound to the zlnfihorli~c To facilitate determining the amount of standard that becomes CA 0224~63~ 1998-08-0~
W O 97/30146 PCTrUS971~2675 bound~ the ~ ;ho~ generally are insolubilized before or after the cnmretitinn) so that the standard and analyte that are bound to the ~ il,odi~s may conveniently be separated from the standard and analyte which remain unbound.
- - Sandwich assays involve the use of two ~ntiho~ c~ each capable of binding to a different i~ Ul~O~ .ic 5 portion. or epitope, of the protein (CT-I) to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody which is immobili_ed on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex. David and Greene~ U.S. Patent No. 4,376,110. The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be ~,.e~u,~d using an anti-i..,...~.o~loL, ~lin antibody that is labeled with a d._~,LhiJle moiety (indirect sandwich assay). For example, 10 one type of sandwich assay is an ELISA assay, in which case the dct~,~,lhble moiety is an enzyme (e.g, horseradish peroxidase).
(iv) Hllmz~ni7~l alllil)o~ s Methods for hnnn~ni7inp non-human antibodies are well known in the art. Generally, a l.,.."_..;,~d antibodv has one or more amino acid residues i~,l. vdu-,ed into it from a source which is non-human. These non-15 human amino acid residues are often referred to as "import" residues. which are typically taken from an "import"variable domain. I i,...,~.,i, lin.l can be essentially performed fol lowing the method of Winter and co-workers (Jones et al., Nature 321, 522-525 (1986); l~i~- h ..~.u~ et aL, Nature .332. 323-327 (1988); Verhoeyen et aL, Science ;~2. 1534-1536 (1988)), by ,..h~ .g rodent CDRs or CDR je~ ei for the cu", ;,i~v~lding of a human antibody. Accc -v~.~ ,Iy, such Ill.. " ;,~d" ...l il .o(l;l~ are chimeric antibodies (Cabilly et 20 al., ~, wherein ~llb~ lly less than an intact human variable domain has been ~h.l;l~ d by the cu..~l,v,,di,.g sequence from a non-human species. In practice, l..l"-~ d antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are ~ sl;l~ d by residues from golle sites in rodent ~-lil,od;~ s.
The choice of human variable domains, both light and heavy, to be used in making the h""-~ .;,- d 25 .,.l ;h oriif c is very i...po, Lall~ to reduce ~ ,l ;g....i. i~y. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sc~ ; The human sequence which is closest to that of the rodent is then accepted as the human G~ "k (FR) for the l"...~ d antibody (Sims e~ aL, J. Imm-~nnl.. 151: 2296 (1993); Chothia and Lesk, J
Mol.Biol..196:901 (1987)). Anothermethodusesapa,Li~ula~ Ga...~ ..u.hderivedfromthe~ sequence 30 of all human i~. ,1 ;1.o~l ;. c of a particular subgroup of light or heavy chains. The same G~..c .. .~ may be used for several different 1.~ -;,- d s~ntihOrli~C (Carter et al., Proc. Natl. Acad. Sci. USA~ 89: 4285 (1992); Presta et aL, J. Immnol.. 151: 2623 ( I 993)).
Itisfurtheri~,l,v,L~lLthat~ll;lloll: ~beh~--~ni7~dwithretentionofhighaffmityfortheantigenand other favorable biological ~lUiJ~lL;~. To achieve this goal, according to a preferred method, 1-1lll,~-,;~1 35 antibodies are prepared by a process of analysis of the parental se~l"- , ~c and various conceph~l hllm~ni7 -d products using three-~ l models of the parental and hnm~ni7l-d se~ Pnc~qc Three-~li,..--,.~.;~,.~i immllnoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-di,,,~ iu,,al conformational ~LIU~.Lul~;S of selected t s~nriiri~t~ immllnogl( blllin se~lu~.llCe:s. incpection of these displays permits analysis of the likely role of the residues in the r,~ e ofthe candidate i...,..l...n~;lobuljn sequence, ie.. the analvsis of residues that influence the abiiitv of the r m ii~l~f~ immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the C~ and impo}t ~ so that the desired antibody ~,hala~,L~ ;, such as increased affinitv forthe target antigen(s), is achieved. In general, the CDR residues are directly and most ,..I,s~ lly 5 involved in influencing antigen binding.
(v) H--m~n antibodies Human m- nnrlon~l ~ntiho~ c can be made by the hybridoma method. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example. by Kozbor, J. Imm-lnol. ~, 300i (1984); Brodeur, et al., Monoclonal Antihodv Production 10 T~rhni~ c ~n~ aLiu~ pp 51-63 (Marcel Dekker~ lnc~ New York~ 1987); and Boerner er aL, J. Immlmn 147: 86-95 (1991).
It is now possible to produce transgenic animals (e.g, mice) that are capable, upon i~ ;o~, of producing a full r~ ; of human all~il,odies in the absence of ~ .. in~" ...~. .c immlmnglnblllin production. For example. it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) 15 gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody pro~ctinn Transfer of the human ger~n-line immnl~oglnb~iin gene array in such germ-line mutant mice will result in the iJIUd\1~,LiUII of human ~ il ,u~ c upon antigen rh~llPnge See, e.g., Jakobovits et al., Proc. Natl . Acad. Sci. USA~
90: 2551 (1993); Jakobovits et aL, ~ature, ~Ç~: 255-258 ( 1993); Bl ~g&_~ llla lll et al., yP~r in Tmml-l1n.~ Z: 33 (1993).
Alternatively, the phage display t~ hnoloE;~ (McCafferty et aL, ~ature. 348: 552-553 (1990)) can be used to produce human ,...1 ;I.û 1;- ~ and antibody Ga~ll~,~.~ in vitro, from immnnoglnb-llin variable (V) domain gene ICI~ ,-tUi.~d from l---;------.---i,..;i donors. According to this n . ~ e~ antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filal~.c.ltvua ba~,t~,~ iol,hage, such as M 13 or fd, and displayed as fimrtinn~l antibody r,a~.._.lL~ on the surface ofthe phage particle. Because the ~i,.... .l...~ particle contains a single-stranded DNA copy ofthe phage genome, s~l~. I;r~ based on the filnrt;nn~ lu~ ofthe antibodv also result in selection of the gene encoding the antibody exhibiting those i~ ,. Li~,s. Thus. the phage mirnicks some of the 1~l u~ ,5 of the B-cell. Phage display can be p~,. rOl ".cd in a variety of forma~s; for their review see, e.g, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structur;~l Biolo~v. 3: 564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et aL, ~, ~,: 624-628 ( 1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of Vgenesderivedfromthespleensof;.. ,.. i,.dmice. Al~_.Lui~ofVgenesfrom~ .. dhumandonors can be col"~,u-,t~,d and antibodies to a diverse array of antigens (i-,clu.li"g self-antigens) can be isolated essentially following the teçhni~ln~c described by Marks et al., J. Mol. Biol.. 2~ ~: 581-597 (1991). or Griffith et al., EMR~ J.. ~: 725-734 ( 1993).
In a natural immune response, antibody genes ~rcnm--l~te mutations at a high rate (somatic h~ e.i ;. ..~). Some of the changes introduced will confer higher affinity, and B celis displaying high-affinity surface immnnoglobulin are p.~r~ ,..Lidlly replicated and differentiated during ~ antigen rh~ ng~
This naturai process can be mimicked by employing the ~-orhn~ e known as "chain shuffling" (Marks et al..
Bio/T~hnol.. 10: 779-783 (1992)). In this method, the affinity of "primary" human antibodies obtained by -CA 0224~63~ 1998-08-0~

..
phage display can be improved by sequ~?nti~lly replacing the heavy and light chain V region genes with uu~,O of naturally occurring variants (~ uu~,~) of V domain genes obtained from --nimmnni7~od donors.
This t~. lUliuuc allows the ~JIudu- Liull of antibodies and antibody fragments with affinities in the nM range. A
- - strategy for making very large phage antibody l~cllu . ~ has been described by W~tPrhonce et aL, ~ucl. Acids ~ç~, 21: 2265-2266 (1993).
Gene shuffling can also be used to derive human _-,I;l.o~ c from rodent antibodies. where the human 7 antibody has similar aff~nities and ~ - c to the starting rodent antibody. According to this method, which is also referred to as "epitope itnprinting", the heavy or light chain V domain gene of rodent antibodies obtained by phage display t-~hniquc is replaced with a I~ LU;AC of human V domain genes, creating rodent-human Ahim~r-c Selection on antigen results in isolation of human variable capable of restoring a functional antigen-binding site, i.e. the epitope governs (imprints) the choice of partner. When the prûcess is repeated in order to replace the I clllaulillg rodent V domain, a human antibody is obtained (see PCT WO 93/06213, published 1 April 1993). Unliketraditionall.-~ ;.,..ofrodent Ar~ o~ sbyCDRgrafting,this ~rhnir~ providesculu~l 'y human alllil)odi~s~ which have no Galll. ~.ulk or CDR residues of rodent origin. (vi) Bic~rific antibodies Bispecific Antiho~i~c are monoclonal, preferably human or hllmAni7~ antibodies that have binding .; r. ;; i~-c for at least two different antigens. In the present case, one of the binding ~l~e~ir~ s is for a CT- I, the other one is for any other antigen, and plcr~ ~ably for another ligand that binds to a GH/cytokine receptor family member. For example, b;~,.e~irc A. ~l ;l ~o~ l ;. c ~ ly binding a CT- I and nc..l vLI OphiC factor, or two 20 - different types of CT- I polypeptides are within the scope of the present invention.
Methods for making b;;",~ir~; allLibo.l;~;s are known in the art.
TrArlitit~nAlly~ the recombinant production of bi~l ecirlc antibodies is based on the co-expression of two immnnnglobl-lin heavy chain-light chain pairs, where the two heavy chains have different sl,e~ c (Milstein and Cuello, ~, 305: 537-539 (1983)). Because of the random a~ul ~ .IL of immnn-lgloblllin heavy and 25 lightchains,thesehyl,lidc,lllas(yua.Lulll~)produceapotentialmixtureof10differentantibodymolecules,of which only one has the correct bispecific structure. The purification of the correct molecule~ which is usually donebyaffinity.,luulllaLu~auLysteps,israther~ .,h ~ ,andtheproductyieldsarelow. Similar~,luceJ~.,~
are disclosed in WO 93/08829 ~ubli,hed 13 May 1993, and in T~au"eck~l et uL, EMRO J.. 10: 3655-3659 (1991).
According to a different and more preferred approach, antibody-variable domains with the desired binding ~c~,iL~,iLies (antibody-antigen culllbulillg sites~ are fused to illullu~oglobulin constant-domain se- 1~ c The fusion ~I- r~-ably is with an; -,-~~--o~ in heavy-chain constant domain, COIII~ il.g at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI), contAininC
the site necessary for light-chain binding, present in at least one of the fusions. DNAs encoding the immlmn_lobulin heavy chain fusions and, if desired, the ;.. ,.. ~ ~;lob~lin light chain, are inserted into separate tA.~ iull vectors~ and are co-llall~r~ k:d into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide La~ in embodilll~ when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding se~ for two or all three polypeptide chains in one ~ iU~l vector when the production of at Ieast two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular ciEnifi~nre In a preferred embodiment of this approach. the bispecific ~ntiborties are composed of a hybrid ; ,.. ~ ~nylftblllin heavy chain with a first binding sl,c~ir~-,iLy in one arrn. and a hybrid immllnopk~hulin heavy - - chain-liPht chain pair (providing a second binding a~c.iri~,ily) in the other arm. It was found that this asymmetric 5 structure facilitates the separation of the desired bispecific c~mpolm-l from ull~al~t~,d imn -lnoPloblllin chain combinations, âS the presence of an jmmllnngklbuljn light chain in only one half of the bispecific molecule provides for a facile way of ,~ . For further details of gvnclclLu.g bispecific ~ ilol l;~s~ see~ for example~
Suresh e~ al., Mf~thn~lc in Fn7ymolo~v~ .L~.: 210 (1986).
(vii) Heteroconjn.~ate ~ntibodies Heterocnnj--vatP al.lil,odies are also within the scope of the present invention. Heteroconjugate ~l;bo~ arec~ rdoftwocovalentlyjoined~ntiho~ c Such---l;lo~ ,have,forexample,beenproposed to target immune system cells to u~ L~d cells (U.S. Patent No.4,676,980~, and for llcat.ll~,..t of HIV infection (WO 91/00360; WO 92/00373; and EP 03089). Il~,t~,~u~ a;l.O~ c may be made using any cu..~e.li.,l.
cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Patent 15 No.4.676~980, along with a number of cross-linking t~rhn j~ Pc 5 Uses of CT-I Antibodies CT- I --- .l ;l .o. I; c are useful in ~ ;c assays for CT- I, e.g, its production in specific cells, tissues, or serum. The ~ntihorli~s are labeled in the same fashion as CT-I described above and/or are immobilized on an insoluble matrix. In one A- .~ho.l;. ~11 ~.1 of a receptor-binding assay, an antibody ~,c,...posiLion that binds to all 20 or a selected plurality of CT-ls is immobilized on an insoluble matrix, the test sample is co~ d with the immobilized antibody cu...l.o~;Liu.. to adsorb all CT-ls, and then the immobili_ed CT-ls are c.,. ~ d with a pluralityof~ il.o.~ specificforeachCT-l,eachofthe,..~l;l.o.l:Pcbeingindividuallyidentifiableasspecific for a ~ d.,t~,ll..ined CT-I, as by unique labels such as discrete nuuluphu~ or the like. By determining the presence and/or amount of each unique label, the relative proportion and amount of each CT-I can be 25 ~r~.",;"p,, The al.LilJodi~,s of this invention are also useful in passively immlmi7inP patients.
CT-In--l;ho.l;~calsoareusefulfortheaffinitypurificationofCT-I fromrecombinantcellcultureor natural sources. CT- I ~ l .o~ that do not detectably cross-react with the rat CT- I can purify CT- I free from such protein.
Suitable ~ ,,noctir assays for CT-I and its ~-Libo.lic~ are well known per se. ln addition to the bioassays ~IPel rihed in the examples below wherein the candidate CT-I is tested to see if it has hy~J~ILlu~llic~ anti-alllly~lllllic, inotropic, or ncl.-u~ l-ic activity, cu,~ e, sandwich and steric inhibition immnnolcc~y are useful. The ~,u---~,~,iti~e and sandwich methods employ a phasc S~.pala~iull step as an integral part ofthe method, while steric inhihiti~-n assays are ~ ~ ....1... t d in a single reaction mixture. F~ lly, the same 35 I,-u.,clu-~s are used forthe assay of CT-I and for c..l.~ 5 that bind CT-I, although certain methods will be favored ri~op~nflin~ upon the molecular weight of the sllhst~nre being assayed. Therefore, the sllhct~nre to be tested is referred to herein as an analyte, i..~!,c~;Li~e of its status ~,LI.clwis-; as an antigen or antibody, and proteins ehat bind to the analyte are dPnomin~t~d binding partners, whether they be antibodies, cell-surface receptors. or antigens.

CA 0224~63~ 1998-08-0~

Analyticai methods for CT- I or its antibodies all use one or more of the following reagents: labeled analyte anaiogue, immobilized analyte r ~ ' ~gn-', labeled binding partner, immobili~d binding par~ner. and steric cu.j~g, ~- ~ The labeled reagents also are known as "tracers."
The label used (and this is also useful to label CT-I nucleic acid for use as a probe) is any detectable 5 filnçtjon llity that does not interfere with the binding of analyte and its binding partner. Numerous labels are knownforusein;..,..~ .o-~-y,examplesincludingmoietiesthatmaybedetecteddirectly,suchasfluc,,u..luu.l.c, r.hPmilnmincc~ont and radioactive labels, as well as moieties, such as enzymes, that must be reacted or derivati~d to be detected. FY~mrlPc of such labels include the radioisotopes 32p, 14C, 1251, 3H, and 1311; fluorophores such as rare earth chelates or lluul~ cchl and its derivatives; rhnt1~mine and its derivatives; dansyl;
10 umh~lliferone; I~ rt-,-~ s. e.g, firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737.456); luciferin;
2,3-dihydrnphth~ malate deh~L us~,.a .~i~ urease; p~,l u,~i.lase such as hul~7~,l adi ~1l p~l u~idase (H~P);
alkaline ~ n~l~l"~ e; ,B-p~ ùc .I_~c, glucoamylase; Iysozyme; saccharide oxidases, e.g, glucose oxidase, galactose oxidase, and glucose-6-pl-r.~ deh~.l.u~,~l,a.~, heterocyclic oxidases such as uricase and xanthine ûxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye ~ uu.~u. such as E~RP, 15 lactoperoxidase, or l~iu~u~lu~idase; biotin/avidin; spin labels; ba,,Lt;. iu~)hagc labels; stable free radicals; and the like.
Those of ordinary skill in the art will know of other suitable labels that may be employed in a~,culda~l~,c with the present invention. The binding of these labels to CT-I, antibodies, or La~ .llL. thereof can be accomplished using standard terhni~lu~c cnmmonly known to those of ordinary skill in the art. For instance, 20 coupling agents such as dialdehydes, .,~Lo~l;;...;~k ~ ~lim~ ; niArc bis-imidates, bis-t1iz~7nti7~ d b ..,;.li..l~ and thelilcemaybeusedtotagthepolypeptidewiththeabove-describedfluorescent,rht~mil.. ;.. ~sc~.. l andenzyme labels. See, for example, U.S. Patent Nos. 3,940,475 (fluorimetry) and 3,645,090 (enzymes); Hunter et al., Nature. i 44: 945 (1962); David et aL, Biochemistrv. 13: 1014- 1021 (1974), Pain et aL, J. Immunol. Methods.
40: 219-230 (1981); Nygren, J. ~i~t~chc-m and Cvtochem.. 3Q: 407-412 (1982); O'Sullivan et aL, "Methods for 25 the 1'~ t~aLùn of Enzyme-antibody Conjng~t~s for Use in Enzyme I- .. -.. n_~ in Methods in F.n7 yrnoloQv.
ed. J.J. Langone and H. Van Vunakis, Vol. 73 (Academic Press, New York, New York. 1981), pp. 147- 166:
Kennedy et aL, Clin. Chim. Acta~ 70: 1-31 (1976); and Schurs et aL, Clin. Chim. Acta. 81: 1-40 (1977).
Coupling ~ s mPntinn~d in the l_llrl ....~1 reference are the glutaraldehyde method, the periodate method, the riimzt~ method, and the m-m~ mi-lnbPn7vl-N-hydroxysuccinimide ester method.
3û In the practice of the present invention, enzyme labels are a preferred .. I-o~ .. I No single enzyme is ideal for use as a label in every cu~ rdtv'o assay. Instead, one must fi~t~rminP which enzyme is suitable for a pau L~,ulal assay system. Criteria i..,~,c" L~ll for the choice of enzymes are turnover number of the pure enzyme (the number of substrate molecules converted to product per enzyme site per unit of time), purity of the enzyme preparation, sensitivity of detection of its product~ ease and speed of detection of the enzyme reaction. absence 35 of h.l~,lr~.u.g factors or of enzyme-like activity in the test fluid, stability of the enzyme and its conju~ L~, availability and cost of the enzyme and its conjugate, and the like. Included among the enzymes used as preferred labels in the assays ofthe present invention are alkaline ph.~ , HRP, beta-g~l~rtocifl~cl~ urease, glucose oxidase, glucoamylase, malate dehyd,u~ ,dse, and glucose-6-phosphate dehyll-u~ ds~. Urease is -CA 0224~63~ 1998-08-0~

among the more preferred enzyme labels. particulariy because of chromogenic pH indicators that make its activity readily visible to the naked eye.
Immobilization of reagents is required for certain assay methods. ~mm--hili7 ~tinn entails .~IJdlaLl.g the - binding partner from any analyte that remains free in solution. This conventionally is accomplished by either ' ' ;li~lg the binding paltner or analyte analogue before the assay 1~ uc~,lu- ~;, as by adso. ~,Lion to a water-insoluble matrix or surface (Bennich et aL . U.S. Patent No. 3,720,760), by covalent coupling (for exarnple, using glutaraldehyde cross-linking), or by insolubilizing the partner or analogue dntl-.dld, e.g, by Other assay methods, known as cu..l~ re or sandwich assays, are well established and widely used 10 in the collllll~,.c;dl ~ o~ , industry.
Competitive assays rely on the ability of a tracer analogue to compete with the test sample analyte for a limited number of binding sites on a common binding partner. The binding partner generally is insolubilized before or after the cl ~ . .1 ~ l il i. ~n and then the tracer and analyte bound to the binding partner are S~r~t~d from the unbound tracer and analyte. This s~,fJalaLiull is ~rcnmrlich~-d by rl~ c~ ~ing (where the binding partner was 15 preinsolubilized)orbyc~l.L iru~,u.g(wherethebindingpartnerwas~ ,dafterthecu...l,~l;l;vereaction).
The amount of test sample analyte is inversely proportional to the amount of bound tracer as ..lcasu- ~d by the amount of marker ,. ~ . .. e Dose-response curves with known amounts of analyte are prepared and cu...~ d with the test results to 4ua~LiL;~ ely ~lPtP~rn ine the amount of analyte present in the test sample. These assays are called ELISA systems when enzymes are used as the ~PtPc~l le markers.
Another species of c~.. ;.e~ e assay, called a "hnn~n~.. -.. ~,.. ~' assay, does not require a phase ., l i.... Here, a CC~ l r of an enzyme with the analyte is prepared and used such that when anti-analyte binds to the analyte, the presence of the anti-analyte modifies the enzyme activity. In this case, CT-I or its immunologically active r.a2~ ,l.b are conjugated with a bifim~tinn~l organic bridge to an enzyme such as p~,.u~idase. Conjugates are selected for use with anti-CT-I so that binding of the anti-CT-I inhibits or 25 p5,t~ ~ .l i..'' ' the enzyme activity of the label. This method per se is widely practiced under the name of EMIT.
Steric c....j..~,.,t~ ~ are used in steric hindrance methods for homngeneol-c assay. These c-, ,j,.~ c are synthesized by covalently linking a low-molecular-weight hapten to a small analyte so that antibody to hapten ly is unable to bind the c~ u~ at the same time as anti-analyte. Under this assay ~.. u-,cdu-c; the analyte present ;n the test sample will bind anti-analyte, thereby allowing anti-hapten to bind the c~...j..g,.l~, 30 resulting in a change in the character of the cnnil~gptp hapten, e.g, a change in nuOI~ ,c;llCC when the hapten is a nu.~ u- ~.
Sandwich assays particularly are useful for the d~ -dLion of CT- I or CT- I antibodies. In 5Pq~lPnti~l sandwich assays an immobili7Pd binding partner is used to adsorb test sample analyte, the test sample is removed as by washing, the bound analyte is used to adsorb labeled binding partner, and bound material is then separated 35 from residual tracer. The amount of bound tracer is directly proportional to test sample analyte. In ~ ;....llu~ ull~ sandwich assays the test sample is not separated before adding the labeled binding partner. A
sPquPnti:ll sandwich assay using an anti-CT-I mnnn~ lnn~l antibody as one antibody and a polyclonal anti-CT-I
antibody as the other is useful in testing samples for CT- I activity.

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The foregoing are merely exemplary ~ vnostic assays for CT- I and antibodies. Other methods now or hereafter developed for the d~ laLion of these analytes are included within the scope hereof. including the bioassays described above.
The following examples are offered by way of ilhlctr~tinrl and not by way of limi1~tinn The disclosures 5 of all citations in the ~,e- il;- -I;on are expressly illcul~Jula~,d herein by reference.
EXAMPT F I
- Id~ irl~,alion and In Yitro Activity of a CT- I
A. Assay for E~ si(J"-Clûned Material The assay used for hypertrophy is an in vitro neonatal rat heart hypertrophy assay described in general lû as follows:
I . Pre~aration of the Myocyte Cell Su~v~ iùn The ~Jll,lJ~aliOll of the myocyte cell sllcp~nci~n is based on methods outlined in Chien et aL, ~ilL
Invest.. 75: 1 770-178û (1985) and Iwaki et al., supra. Ventricles from the hearts of 1-2 day Sprague-Dawley rat pups were removed and trisected. The minced ventricles were digested with a series of se~l ~Pnti~l coll~ge~qc~P
15 ~ LIII~.~I6. Purification of the resulting single-cell ~ 011 on a discu.lLilluous Percoll gradient resulted in a ellcpPncinn of 95% pure myocytes.
2. Pl~tin~ ~nd Culture of the Mvocvtes Two l,ul,li~hcd methods for plating and culturrng the myocytes are as follows: ( I ) Long et aL, supra, preplated the cell s--epPnci~-n for 30 min. in MEM/5% calf serum. The u~ n~ d myocytes were then plated 20 - in serum-free MEM ~u~ d with insulin, L-~l ,r~,lli l, BrdU, and bovine serum albumin in 35-mm tissue-culture dishes at a density of 7.5 x 104 cells per mL. (2) Iwaki et aL, supra, plated the cell s~lcpl~nciorl in D-MEM/I 99/5% horse seruml5% fetal calf serum in 1 0-cm tissue-culture dishes at 3 x 105 cells per mL. After 24 hr in culture the cells were washed and i"~ d in serum-free D-MEM/199.
A different protocol has been d.,~ ~,Iul, d in accul d~l~,c with this invention for plating and culturing these 25 cells to increase testing capacity with a miniaturized assay. The wells of 96-well tissue-culture plates are precoated with D-MEM/F12/4% fetal calf serum for 8 hr a~ 37~C. This medium is removed and the cell s"~ oll is plated in the inner 6û wells at 7.5 x 104 cells per mL in D-MEM/F-12 ~u~ ,d with insulin, ,r~ ll, and aprotinin. The medium ~ypically also contains an antibiotic such as p~ni~illin/~ ulllycin and t~min~- This medium allows these cells to survive at this low plating density without serum. Test ,~
30 are added directly into the wells after the cells have been in culture for 24 hours.
3. Readout of H~v~ lu~Jh~
After ~ , with alpha a-L~,Ic.~,ic agonists or elldull.~,lill, neonatal rat myocardial cells in culture display several features of the in vivo cardiac muscle cell hypertrophy seen in congestive heart failure. including an increase in cell size and an increase in the assembly of an individual contractile protein into organized 35 contractile units. Chien et aL, FASEB J.. s2~pra. These changes can be viewed with an inverted phase microscope and the degree of hy~ ,u~.L~f scored with an arbitrary scale of 7 to û, with 7 being fully - hypertrophied cells and 3 being non-ctimlll~t~d cells. The 3 and 7 states may be seen in Simpson ef al., Circ~ ti~n Research. ~: 787-801 ~1982), Figure 2~ A and B, respectively. To facilitate the mi~lu~-,ol~i., readout of the 96-well cultures and to generate a permanent record, the myocytes are fixed and stained after the JlU~Jlidl~ testing period with crystal violet stain in methanol. Crystal violet is a commonly used protein stain for cultured cells.
AriAirionAliy, an aliquot can be taken îrom the 96-well plates and monitored for the eA~ S~iOn of protein markers of the response such as release of ANF or ANP.
S B. F~x~ ùn Clonir~
Poly(A)+ RNA was isolated (Aviv and Leder, Proc. Natl. Acad. Sci. USA~ 69: 1408-1412 (i972);
Cathala et al., DNA. ~: 329-335 (1983)) from day 7 mouse embryoid bodies. Embryoid bodies were generated by the .lirf~ liun of I~IUI i~.vt.,..L embryonic stem (ES) cells (Do~ l . ~A et ol., ~. Emhrvol. Ex~. Morphol..
87: 27-45 (1985)). The embryonic stem cell line ES-D3 (ATCC No. CRL 1934) was ...Ai..lA;-.rd in an 10 ulldirf~ .lLidt~dstateinamedium~ i.. ELIF(WilliamsetaL,~I~h~,336:684-687(1988)). Thismedium cnnt~inPdD-MEM(highglucose), 1%gl~ --r70.1 mM2-l~ A ol,penicillin-streptomycin, 15%heat-inactivated fetal bovine serum, and 15 ng/mL mouse LIF. When these cells were put into ~ n culture in the same medium without LIF and c~ ;--;--g 20% heat-inactivated fetal bovine serum (day 0), they agE;~ ak;d and .lirr~ idL~d into mllltirPII~ aLIul,lw~,;. called embryoid bodies. By day 8 of culture, beating primordial 15 heart-like all UI~IUI~ formed on a fraction of the bodies. The embryoid bodies were evaluated for the production of CT-I activity by changing the dirft.~ Li~iulg lFS cells to serum-free medium (D-MEM/F-12, 1% gllltAminP, penicillin-streptomycin, cnnt~ininr. 0.03% bovine serum albumin) for a 24-hour AAcllmnlAtion Prior to assay, the cnn~irinnpd medium was collc~.lLI_ ' 10 fold with a 3-K ultrafiltration membrane (Amicon), and dialyzed against assay medium. Mediurn ..~... i ;l ;....rd for 24 hours starting at day 3 gave a hyl,~,. LI~ LY score of 4.5-5 .5, 2Q and starting at day 6 a score of 5.5-7.5.
A cDNA library in the plasmid eA~ ,ion vector, pRKSB (Holmes et al., ~ç~, 253: 1278-1280 (1991)),waspreparedfollowingavectorprimingstrategy(Strathdeeetal.,Nature~ 356: 763-767(1992)). The vector, pRE~5B, was linearized at the Notl site, treated with alkaline ~I-r.~l,l".l,.c~ and ligated to the single-stranded oligonllclPoti-hP, ocdl.l.3, having the S~ c 25 5'-GCGGCCGCGAGCTCGAAl 11''111'11-111111-111-1111111111-1-11111 (SEQ ID NO: 5). The ligated product was then cut with BstXI, and the 4700-bp vector fragment was isolated by agarose gel electrophoresis. The vector was further purified by oligo dA cLlulllaLuy~lal~h!/.
The eA~ iUII library was CUII:~LI u.,~,d using I ~g of the poly (A)+ RNA, S llg of vector primer, and reagents from AmPrsh~m Following first- and second-strand synthesis and T4 DNA polyrnerase fill-in reactions, 30 the material was sized for inserts of greater than 500 bp by gel cle~LlulJllul~ ia and uiluulali~d by blunt-end ligation without the addition of linkers. The ligations were used to L~allarul~ . coli strain DH5~ by e~ LlupulaLion. From I llg of poly(A)~ RNA, 499 ng of double-stranded cDNA were ~ l Seventeen nanograms of cDNA were ligated, and 3.3 ng were h ~ arulll.cd to yield 780,000 clones. 83% of which had inserts with an average size of 1470 bp.
DNA was isolated from pools of 75-15,000 clones and L,al. ,r.,~L~d into human embryonic kidney 293 cells by T ;pof~ ....i..P transfection (Gibco BRL). Two mi.,lu~.allls of DNA were used to transfect ~200,000 cells in 6-well dishes. The cells were inrllh~t~d in 2 mL of serum-free assay medium for four days. This medium consisted of lOû mL D-MEM/F-12, 100 IlL Llculaf~ (10 mg/mL), 20 ~lL insulin (5 mg/mL), 50 IlL aprotinin ~2 mg/mL), I mL pen/strep (~RH Rios~ No. 59602-77P), and I mL L-~ (200 mM). Transfection CA 0224~63~ 1998-08-0~

and expression efficiency was ll~ulli~u-~,d by the inclusion of 0.2 ~lg of DNA for a plasmid e~ ail-g a secreted form of alkaline pho~ e (Tate et al., FASEB J.. _: 227-231 (1990)).
One hundred microliters of conriitinnPd culture medium from each lldll~r~.,Led pool was assayed for - hyperLrophy in a final volume of 200 IlL. For some pools the cor ~iitioned medium was co.lcGl-l-~t~d 4-5 fold before assay with Centricon 3~M mi.,.~,c-,-l.,. ~. (Amicon). Ninety pools of 10.000-15,000 clones, 359 pools of 1000-5800 clones, and 723 pools of 75-700 clones were lldllsre~Led and assayed for hyperLrophy activity. Of - these 1172 pools, t~vo were found to be positive. Pool 437 (a pool of 187 clones) and pool 781 (a pool of 700 clones) gave scores of 4. A pure clone (dP~;~..,.lrd pchf.437.48) from pool 437 was isolated by retransfection of positive pools co. ~ E fewer and fewer numbers of clones until a single clone was obtained. A pure clone from pool 781 (~k ~ t pchf.78 1) was isolated by colony h~b~idi~dliull to the insert from clone pchf.437.48.
The sequence for the insert of clone pchf.78 1 is provided in Figure I (SEQ ID NOS: I, 2, and 3 for the two n--rlPotidP strands and amino acid se~lupnce~ respectively). The sequence of the insert of clone pchf.437.48 matches clone 781 statting at base 27 (underlined).
The first open reading frarne of clone pchf.78 1 (see Lldll~ldLion, Fig. I ) encodes a protein of 203 amino acids (translated MW = 21.5 kDa). This protein contains one cysteine residue, one potential N-linked glycosylation site, and no h~ l ~ N ~,....L..al secretion signal seq--PnrP The 3' u..~ d..sl.. ~d region of clone pchf.78 I contains a common mouse repeat known as b I (bp ~895-1015). Hybridization of 7-day embryoid body poly(A)+ '~NA with a probe from clone pchf.78 I shows a single band of -1.4 kb, which is about the same size as the insert from the cDNA clones.
The encoded sequence is not highly similar (> 35% amino acid identity) to any known protein se.~
in the Dayhoff database. It does, however, show a low degree of similarity to a family of distantly related proteins inrt~lriinp CNTF, hit~ .Jkill-6 (IL-6), illt~ ,..ki-l-l I (IL-I 1), LIF, and nnCost~tin M (OSM) (Bazan, Neuron, 7: 197-208 (1991)). Mouse CT-I has 24% amino acid identity with mouse LIF (Rose and Todaro, WO
93/05169) and 21% amino acid identity with human CNTF (McDonald et aL, Biochim. Biophvs. Acta. 1090:
70-80(1991)). SeeFigure2foran~1i~mPntofmouseCT-landhumanCNTFse~ CNTF,IL-6,1L-II,LIF~ and OSM use related receptor signaling proteins inrh1riing gp 130 that are members of the GH/cytokine receptorfamily(Kishimotoetal.,Cell,76:253-262(1994)). CNTF,likeCT-l,lacksanN-terminalsecretion signal sequ~nre.
C. Identitv and Activitv of Clone To demonstrate that clone pchf.781 encodes a CT-I, expression studies were p~,.ru.. ed both by iu~ of 293 cells and by utilizing a coupled in vitro SP6 l~ vLion/tr,anslation system. 3SS-I1I~Lh;UII;IIC
and cysteine labeling of the proteins ~J- u-luced by pchf.78 1 -lldll~re~ d 293 cells (in CollllJdl ;~u.. with vector-Ll~ular~t~tcells~showedthatthecr~ J~rdmediumcnntAintdalabeledproteinofabout2l 8kDa~andthat the cell extract showed a protein of 22.5 kDa. C....~ .,.rd media from these trSIncf~ctic-nc gave a morphology 35 score of 6 when assayed for cardiac h~ y at a dilution of 1:4 using the assay described above.
Con~ihirmrd media from llni:lhel~d Llall~re~lions gave a morphology score of 5.5-6.5 at a dilution of 1:1.
These assays were also positive for a second measure of cardiac hypertrophy--ANP release. See Figure 3. Thisassaywasp~,,rul...edbyrJet~nnin~tirnofthec~ inn forthebindingof 1251-ratANPforaratANP
- receptor A-lgG fusion protein. This method is similar to that used for the d~L~ .. illa~ion of gp 120 using a CD4-CA 0224;i63;i 1998-08-0;i -IgG fusion protein (Chamow et al., Bioc l,~ 4: 9885-9891 (1990)). Briefly, microtiter wells were coated with 10011L of rat anti-human IgG antibody (2 ~lg/mL) overnight at 4~ C. After washing with plIn~ t~ buffered saline cont~ining 0.5% bovine serum albumin, the wells were ;..c~ rl with 100 ,uL of 3 ng/mL rat ANP
-- receptor A-lgG (produced and purified in a manner analogous to the human ANP receptor A-lgG (Bennett et S aL, J. Biol. ('h~-m 266: 23060-23067 (1991)) for one hour at Z4~ C. The wells were washed and incubated with 50 IlL of rat ANP standard or sample for one hour at 24~ C. Then 50 IlL of 1251-rat ANP (Amerch~m) was added for an ~t1r1iti~n~1 one-hour ;- ~. --1.,n i~" - The wells were washed and counted to determine the extent of binding c n. . ,l ~ l il ;-,., ANP cu.,~ Lions in the samples were ~ n .,, . ;. .,~ by cu..~ ,u.. to a rat ANP standard curve.
35S-I~<llliv~ r labeling of the proteins made by SP6-coupled in vitro ~lau~ iulJtranslation (materials from Promega~ of clone pchf.781 showed a labeled protein of 22.4 kDa. The labeled translation product was active when assayed for cardiac h~ ul~Ly at a dilution of 1 :200 ~morphology score 5-6~. To verifythatthe22.4-kDa-labeledbandwas.~ ,v.l~;blefortheh~ .u,ul.yactivity~thelabeled~ ullproduct wasappliedtoareverse-phaseC4column(S~ ;L~ul~akRO-4-4000)f~ t~ 1in 10%ac~u,~iL il~0~1%TFA~
15 and eluted with an acetonitrile gradient. Coincident peaks of labeled protein and hypertrophy activity eluted from this colurnn at -55% acetonitrile.
A cardiac myocyte h~,.L~uplly activity has been reported and partially purified from rat cardiac ril" ubl~L~. Long et aL, supra. To investigate further the identity of the CT- I herein, rat cardiac r,bl ubl~b were cultured. Cnn-~ n~d medium from these primary cultures does have cardiac hy~.~,lLIupl~y in the in vitro 20 neonatal rat heart h~ u~Jhy assay herein. Blot hylLi~ iOII of rat r.l"ul,la:,l mRNA isolated from these cuitures shows a clear band of 1.4 kb when probed with a coding region fragment of clone pchf.781.
(H~ li~Lic,.. was p~ in S x SSC, 20% forrn~mi~lp at 42~C with a final wash in 0.2 x SSC at 50~ C.
D. Pul;~aLivll of Factor The culture medium cn~ n~d by cells ~ r- . t~ d with clone pchf.781 Or a human clone is adjusted 25 to 1.5 M NaCI and applied to a ToyopearlTM Butyl-650M column. The column is washed with 10 mM TRIS-HCl,pH7.5~ I MNaCLandtheactivityelutedwith 10mMTRIS-HCl,pH7.5,10mMZwiLt~,l~c;-llTM3-10. The peak of activity is adjusted to 150 mM NaCI, pH 8.0, and applied to a MONO-Q Fast Flow column. The column iswashedwith 10mMTRlS-HCl,pH8.0, 150mMNaCI,0.1%octylglucosideandactivityisfoundintheflow-through fraction. The active material is then applied to a reverse phase C4 column in 0.1% TFA, 10%
30 ~ , and eluted with a gradient of 0. l% TFA up to 80%. The activity fr?ntinn~t~s at about 15-30 kDa on gel-filtration columns. It is expected that a chaullu~,~, such as gn~ni~lin~-Hcl is required for resolution and recovery.
EXAMPLE Il T~cting for in vivo H~ ,.llu!,l,y Activity 35 A. Nonn~ tc The purified CT-I from Example I is tested in normal rats to observe its effect on cardiovascular p~r~nn~t~rs such as blood pressure, heart rate, systemic vascular recict~nnr, contr~ftility, force of heart beat.
cùncl,...l ;-, or dilated hypertrophy, lef~ ventricular systolic pressure, left ventricular mean pressure. Ieft ~ lLI i~,ulal end-diastolic pressure, cardiac output, stroke index, histological pal~ll~t~ , ventricularsize, wall thi~kn~, etc.

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B. Pressure-Overload Mouse Model The purified CT-I is also tested in the pressure-overload mouse model wherein the pulmonary artery is culla~ ed, resulting in right ventricular failure.
- - C. RV Murine Dysfi-n~ti~-nal Model A retroviral murine model of ventricular dysfunction can be used to test the purified CT-I, and the dP/dt, ejection fraction, and volumes can be assayed with the hypertrophy assay described above. ln this model, ~! the pulmonary artery of the mouse is C.JI.sll iul~d so as to generate pulmonary hypertrophy and failure.
D. Trans~enic Mouse Model Tlaulsgcllic mice that harbor a muscle actin promoter-lGF-I fusion gene display cardiac and skeletal muscle hypertrophy, without evidence of myopathy or heart failure. Further, IGF-I- gene-targeted mice dispiay defects in cardiac myogenesis (as well as skeletal) including markedly decreased expression of ~,~,.n. ;~,Uldl muscle contractile protein genes. The purified CT-I is tested in these two models.
~lrliti~nsll genetic-based models of dilated cardiomyopathy and cardiac dycfim~ fi--n, without necrosis, can be d~ ,d in ~ .l-c and gene-targeted mice (MLC-ros mice; aortic banding of hetero_ygous IGF-I-deficient mice).
Post-Myocardial Inral~,liùl~ Rat Modrel The purified CT-I is also tested in a post-myocardial il.ral.,lion rat model, which is predictive of human congestive heart fai}ure in ~1 u-lu~illg liaL~ iUl<,li~, peptide. Specifically, male Sprague-Dawley rats (Charles River Breeding Labo.~lu.ies, Inc., eight weeks of age) are ~rclim~t.~d to the facility for at least one week before 20 surgery. Rats are fed a pelleted rat chow and water ad libitum and housed in a light- and l~,p~,. alul ~-controlled room.
1. Coronary Arterial l i~ti~m Myocardial infarction is produced by left coronary arterial ligation as described by Greenen et al., L
~pl. Physiol.. 2~: 92-96 (1987) and Buttrick et al., Am. J. Physiol.. 260: 11473-11479 (1991). The rats are 25 ~nPcfhPti7Pd with sodiurn ~ ~,.llul~ àl (60 mg/kg, ill~ unc;àlly), intubated via ~,a.,LeoLuy, and ventilated by a l.~J al~)l (Harvard Apparatus Model 683). After a left-sided thOIacolull~y, the left coronary artery is ligated JIu~ lal~ly 2 mm from its origin with a 7-0 silk suture. Sham animals undergo the same procedure except that the suture is passed under the coronary artery and then removed. All rats are handled according to the "Position of the American Heart ~ccoci~~iorl on Research Animal Use" adopted 11 November 1984 by the American Heart ~ccori-~tinn Four to six weeks after ligation, myocardial infarction could develop into heart failure in rats.
In clinical patients, myocardial il~r~-,liuil or coronary artery disease is the most common cause of heart failure. Congestive heart failure in this model reasonably mimics congestive heart failure in most human patients.
2. Ele~ u~al dio~rams One week after surgery, ele.,l. u~,al -lio~- alllS are obtained under light metofane ~nPcth~ci~ to ~locnm ~nt the d~ ,lu~,.ll.,l,l of infarcts. The ligated rats of this study are aub~ulJ d according to the depth and p~laia~lce of p~tholoE~ Qwaves acrossthe IJIr_cOI-liàl leads. Buttricke~aL,supra; Kloneretal., Am. HeartJ.. 51: 1009-1013 (1983). This provides a gross estimate of infarct size and assures that large and small infarcts are not -differently d;auil/uLed in the ligated rats treated with CT-I or CT-I ~ g.~ l and vehicle. Confi~mAtiA~n is made by precise infarct size ~ au.~,..-ent.
3. CT-l or CT-I Antap--nict Arlminic1ration - Four weeks after surgery, CT-I or CT~ Ag". .;J ( 10 11g/kg to 10 mg/kg twice a day for 15 days) or 5 saline vehicle is injccted a.~h~ ly in both ligated rats and sharn controls. Body weight is measured twice a week during the ll~,a~ ell~. CT-I or CT-l A~ i iS Ariminictpred in saline or water as a vehicle.
4 (~Ath ~t." i, A I; nll After l3-day Ll~all~lc~lL with cT-l ~ cT-~ g~ or vehicle~ rats are Anpsfhpti7pd with p~lnuba~
sodium (50 mg/kg, illLIalJ~,.iL~ eally). A catheter (PE 10 fused with PE 50) filled with heparin-saline solution 10 (50/U/mL) is i~ lallled into the Ah~lom inAl aorta through the right femoral artery for Ul~.aaUI ~,Ill.,l~L of arterial pressure and heart rate. A second catheter (PE 50) is implanted into the right atrium through the right jugular vein for .l.caaul~,lll.,llL of right atrial pressure and for saline ini~p~Ation For mca,ull,..-~,..l of left ~.,.lLIi.,ulal pressures and c~ f Al 1 ;I ;I,y (dP/dt), a third catheter (PE 50) is implanted into the left ventricle through the right carotid artery. For the ~ u~ ,.lL of cardiac output by a thP7 mnr~ tinn method, a thP rnirtor catheter (Lyons 15 Medical Illall ullle~L CO., Sylmar, CA) is inserted into the aortic arch. The catheters are e~t~. ;o. i cd at the back of the neck with the aid of a stainless-steel wire tunneled ~7U~ aA~IPOIICIY and then fixed. Following catheter j",l,lA...Al;~,.., all rats are housed individually.
5. HPrnOdYnamiÇ MPA~
One day after catherization, the lh~ l' catheter is ylucesa~id in a mi~,.ucu~.y ~ svstem (Lyons 20 Medical IllaLI Ull~.li Co.) for cardiac output ~ k t~ .... ;. IAI ;~ ~' ~, and the other three catheters are cnnnPct~-d to a Model CP-IO pressure tlallsdu~.el (Century Technology Company, Inglewood, CA) coupled to a Grass Model 7 polygraph (Grass IllaLlulll~,llLa~ Quincy, MA). Mean arterial pressure (MAP), systolic arterial pressure (SAP), heart rate (HR), right atrial pressure (RAP), left v~ li-,ulal systolic pressure (LVSP), left ventricular mean pressure (LVMP), left ventricular end-diastolic pressure (LVEDP~, and left ventricular IllA~d..~lllll (dP/dt) are 25 Illc~.ll~,d in conscious, un-~,~Lla;..ed rats.
For Ill-,aau~ ent of cardiac output, 0.1 mL of isotonic saline at room ~ . . .l .. . A1 ~1 l ~ is injected as a bolus via the jugular vein catheter. The thPTTno~ tirJrl curve is monitored by VR-I 6 ~ullulLla~,e recorders (Honeywell Co., NY) and cardiac output (CO) is digitally obtained by the ~ uC.~ --l-ul- ~ . Stroke volume (SV)=CO/HR;
Cardiac index (CI)=CO/BW; Systemic vascular . ~.~iaLallCc (SVR)=MAP/CI.
After lllcaa.~ ,.lL of these hemodynamic pa alllct~,. a, I mL of blood is collected throu-A h the arterial catheter. Serum is separated and stored at -70~C for Illea~ lL of CT-I levels or various biorhPnnirAl parameters if desired.
At the cnnr!~ tl of the ~ ,~,.,. illl~,.lL:i, the rats are ~nPsthPti7~d with pentobarbital sodium (60 mg/kg) and the heat~ is arrested in diastole with intra-atrial injection of KCl (I M). The heart is removed. and the atria and great vessels are trimmed from the ventricle. The ventricle iâ weighed and fxed in 10% buffered formalin.
All e~Ly~ llLal ylu~,cdulcis are approved by the ln~titl~tionAl Animal Care and Use ColnTnittPe of GenPntrrh j Inc. before initiation of the study.

-6. Infarct si7e M~aau~
The right ventricular free wall ;s dissected from the left ventricle. The left ventricle is cut in four Llal,a~.ac slices from apex to base. Five micrometer sections are cut and stained with Massons' trichrome stain - - and mounted. The endocardial and epicardial ~ ,ulllÇ~ cs of the infarcted and non-infarcted left ventricle 5 are d~t~r ninrd with a pLllilllct~,l Digital Image Analy_er. The infarcted circumference and the left ventricular circumference of all four slices are summed separately for each of the epicardial and endocardial surfaces and the sums are ~ a~,..l as a ratio of infarcted circumference to left ventricular circumference for each surface.
These two ratios are then averaged and ~,.~,..,~sed as a p.,l-,~"Lge for infarct size.
7. ~t~tictit~ l Analvsis Results are expressed as mean + SEM. Two-way and one-way analysis of variance (ANOVA) is p~ u,---cd to assess .lirr.l~,l..,es in pala.ll~ among groups. !~ignificzmt dirr~-c;-,.,e~ are then subjected to post hoc analysis using the Newman-Keuls method.
p < 0.05 is considered cignjfirs~nt 8. Results The mean body weight before and after Ll~aLlll~,llL with CT-1 or CT-I ~nt~gonict or vehicle is not expected to be different among the t;~l,~i. illlc lllal groups. Infarct size in ligated rats is not expected to differ between the vehicle-treated group and the CT-1- or CT-I- Ant~nnict-treated group.
It is expected that administration of CT-I or CT-I ~nt~gonict to the ligated rats in the doses set forth above would result in improved cardiac h~ ,lLIuyLy by increasing ventricular contractility and de~ ,âa;llg 20 p~ LI lal vascular resistance over that observed with the vehicle-treated sham and ligated rat controls. This expected result would demonstrate that admilliahaLiu.. of CT-1 or CT-l ~ntagnnict improves cardiac function in cw,~5e~Li~, heart failure. In sham rats, however, CT-I or CT-I ,~n.~ lminicfr~til7rl at this dose is not expected to alter ,;~;"il~ ly cardiac function except possibly slightly lowering arterial pressure and p~ /lll,.al vascular .~
It would be reasonably expected that the rat data herein may be c.~LIayc~laL~d to horses, cows, humans, andotherm~mm~lc;correctingforthebodyweightofthemammalinaccL..da.l..ewith,~o~,";,.dveterinaryand clinical l--u~,~du~i,. Using standard protocols and ~-ù~,~,lu.~,~, the V~.tl_fi~ lll or clinician will be able to adjust the doses, srh~ linE~ and mode of a~L~h~ aLioll of CT- I or a CT-I :~nt:lgl~nict to achieve maximal effects in the desired mammal being treated. Humans are believed to respond in this manner as well.
3û FXAMPLL III
Proposed Clinir~l Treatment of Dilated Cardiom,yop ~tl~y A. T.,t~ lion Patient self-admi..iaL.aLiu.. of CT-I or CT-I ,...l~g~ at an initial dose of 10-150 ,ug/kg/day is proposed. The dose would be adiusted downward for adverse effects. If no beneficial effects and no limiting 35 adverse effects are flett rmined at the time of re-evaluation, the dose would be adjusted upward. Con.,u..e..t mf~ til~n doses (e.g, captopril as an ACE inhibitor and diuretics) would be adjusted at the diaul~liull of the study physician. After the .,."~i"""" dose is z~lminicf~red for 8 weeks, the CT-I or CT-I antagonist a~ i..iail aLion is stopped, and re-evaluation is performed after a similar time period off treatment (or a placebo).

B. Inclncion Criteria Patients would be conaldcl ~,d for the study if they meet the following criteria:
-Dilated cardiomyopathy ~DCM). Idiopathic DCM, or ischemic DCM without discrete areas of akinesis dyskinesis of the left ventricle (LV) on contrast ventriculography or 2D echocardiography. Evidence 5 for impaired systolic function to include either LV end-diastolic ~ n (EDD) > 3.2 cm/m2 BSA or EDV
> 82 mLim2 on 2D e~ oc~ Jiu~alJl.y, LV fractional shortening < 28% on echocal .lio~ ~"~l. y, or ejection fraction (by contrast ventriculography or ..~ . li iP ~lgio~,la~hy~ < 0.49.
-Symptoms. New Yor}c Heart ~c~O~ class III or peak exercise VO2 ~ 16 mL/kg/min. (adjusted for agej. stable for at least one month on digoxin, diuretics, and ~ co~ u~ a ~ACE inhibitors).
10 -Concu.. cl,t ACE inhibitor therapy.
-Adequate c~l.ocaldiographic "windows" to permit a~ - .i of left ventricular volume and mass.
-Ability to self-a~iminictPr CT-1 or CT-I rnt~gorlict accol.lillg to the dosage s~hP i--ie, and to return reliably for follow-up i~
-Consent of patient and patient's primary physician to ~)al ~ alc.
-Absence of ~yrillcinn criteria.
C. ~xcl~ n Criteri~ l Patients would be excluded from cona;.l.,.~ltion for any of the following ressons:
-Dilated cardiomyopathy resulting from valvular heart disease (operable or not), spccific treatable etiolo~ies (including alcohol, if ~ has not been r~t~nnptPd), or operable coronary artery disease.
-Exercise limited by chest pain or obstructive p~ cl~.l vascular disease.
-Chronic obstructive lung disease.
-Diabetes mellitus or impaired glucose tol -History of carpal tunnel syndrome, or evidence for positive Tinel~s sign on PY:~ nin~tj~n -History of kidney stones.
-S~ ;r vat.,Oa~ .;Lia.
-Inability to consent for or pal~ ...c in serial bicycle el~u...clly with invasive hemodynamic monitoring (as described below).
-Active mS~lign~nr D. p~tiPnt Accpccment 1 ) Major ~CcPccnnpnt Points: baseline; after peak stable CT- I or CT- I ,.. , I.. g.. i~l dose .. ,. ;.. l ~ ;.. rd for 8 weeks: after equal period after drug . l;~ Ul' I ;~ 1 ;on.
-It is - .1;. ;~ d that patients would remain in the hospital for two to three days at the onset of active treatment, with daily weights and labula~wy data including electrolytes, phosphorus, BUN, creatinine, and glucose. Following thiâ, they would be m.,..iLc .~,d on the Clinical Research Center floor daily for an additional~5 two to three days.
i. Physical eY~min~tion ii. Symptom Point Score (Kelly et aL, AmPr. HP~rt J.. 1 19: 11 1 1 ( 1990)).

CA 0224~63~ 1998-08-0~

iii. Laboratory data: CBC; electrolytes (including Mg+2 and Cat3; BUN; ~ ati~ lc;
phosphorus; fasting glucose and lipid profile (total cholesterol, HDL-C, LDL-C, triglycerides); liver function tests (AST, ALT, alkaline ~ho~l .h,ll~fL total bilirubin); total protein; albumin; uric acid: and CT- I .
- - iv. 2D,M-mode,anddopplere-,hoc~diog~ l-y,inr~ in~ diastolicandsystoiicdimensions 5 at the papillary muscle level; ejection fraction estimate by area pl~.i~ y from apical 2-chamber and 4-chamber views~ estimated systolic and diastolic volumes by Simpson's rule method, and ectim~tf~d left ventricular mass;
doppler ~cc. ~ of mitral valve inflow profile (IVRT, peak E, peak A, decclc, ~iion time, A wave duration), and pulmonary vein flow profile (systolic flow area, diastolic flow area, A reversal duration, and velocity).
v. Rest and exercise hemodynamics and measured oxygen cullau~ "ion. using bicyele 10 ergometry with p~ Gv~ly inserted puimonary artery and arterial catheters. Perceived exertion level would be scored on the Borg scale, and ~ ,a~ llc~lL~ of puimonary artery systolic, diastolic, and mean pressures~ as well as arterial pressures and IJullll<Jilaly capillary wedge pressure would be .l.ca~u~.,d at each increment of workload, along with arterial and mixed venous oxygen content for rs~lr,ll~ ing cardiae output.
vi. A''f ~...- .1 of body fat and lean body mass, as well as skeietal muscle strength and 15 e,ldul a ....,e.
2) Interim AC'~'' -.~ ~.1 Points: weekly i. Physieal ~Y~nnin~fi~n ii. Symptom Point Score.
iii.T~hor.~rydata: electrolytes,BUN,-,l~,aLilille,pl~u~ ,.u:,,fastingglucose,c.. ~l~.. f li.. -20 - C, and CT- I .
F Potential Benefi~c 1) Improved sense of well-being.
2) Increased exereise toleranee.
3) Inereased muscle strength and lean body mass.
4) Decreased systemic vascular ~~
S) Fnh~nred cardiac p~,lr~3....al.ce.
6) F~nh~nred C-...~l.f ~ y myocardial h)~ uu~ y.
P.XAMPLE IV
Tf ctinp for in vitro Nc.l-~ " hic ActivitY
An assay used for ciliary ganglion h~ l ul-u~llic activity was p~,. r.,lllled as described in Leung, Neuron.
~: 1045-1053 (1992). Briefly, ciliary ganglia were dissected from E7-E8 chick embryos and ~ o.: -~rd in trypsin-EDTA (Gibco 15400-013) diluted ten fold in pl~o~ l. buffered saline for 15 minutes at 37~C. The ganglia were washed free of trypsin with three washes of growth medium (high glucose D-MEM supplemented with }0% fetal bovine serum, 1.5 mM gll~t~min~.100 llg/mL penicillin, and 100 ~g/mL strepomycin), and then gently triturated in I mL of growth medium into a single-cell ~ " . Neurons were enriched by plating this cell mixture in 5 mL of growth media onto a 100-mm tissue culture dish for 4 hours at 37~ C in a tissue culture i~ incubator. During this time the non-neuronal cells ~lef~ lLially stuck to the dish and neurons were gently washed free at the end of the incubation.

-The enriched neurons were then plated into a 96-well plate previously coated with collagen. In each well, 1000 to 2000 cells were plated. in a final volume of 100 to 250 IlL, with dilutions of the conrlifion~d medium from the pchf.781 ~ r~ ~ Ird 293 cells of Example 1. The cells were also plated with the 11 ~,n~r. ..Led - - 293 conditioned medium as a comrol, and with a CNTF standard as a COl~ oll. Following a 2-4-day 5 in~llhAti~n at 37~ C, the number of l jve cells was assessed by staining live cells using the vital dye metA ~ th ion inp (M~l ). One-fifth of the volume of S mg/mL MTT (Sigma M2128) was added to the wells. After a 2-4-hour ;.-- .,l-Al;-~nat37~C,livecells(filledwithadensepurple~ , )werecountedbyphasemicroscopyat 100X
m ~Agnifil~Atic n The results of the assay are shown in Figure 4. It can be seen that the pchf.781 Llall:,r~ .liull (triangles~
10 increased survival of the live neurons (measured by cell count3 as the fraction of assay volume of l- a....r~t~ 293 d medium increased. This is similar to the pattem for the CNTF standard (circles), and is in contrast to the control 1, . r ~ I ;ul- (squares), which showed no increase in survival as a function of increased fraction of assayvolume of c.~...l;l;u,.cdmedium. This indicatesthatCT-I isusefulasan~ .llvL~u~l.ic agent, havingasimilar effect to that observed with CNTF.
F.XAMPLE V
AsourceofmRNAencodinghumanCT-I (alsoknownashuman~-liukupl-in-l (CT-l])wasidentified by screening poly(A)+~NA from several adult tissues with a probe from the mouse CT-I cDNA clones. Heart, skeletal muscle, colon, ovary, and prostate showed a 1.8 kb band upon blot hybridization with a 180-bp mouse CT-l probe (h~ ..l...g from 19 bp sl of the initiatirlg ATG through amino acid so) in 2o% f~rm~mirl~ s x ssc at 42 D C with a final wash at 0.25 X SSC at 52~ C. Clones encoding human CT- I were isolated by s~ . c.,.. ,,.g a human heart cDNA library (Clontech) with the same probe and conditions (final wash at 55~ C).
Eleven clones were isolated from l million screened. The EcoR~ inserts of several of the clones were suhcl~ nr~l into plasmid vectors and their DNA ~ d t~ .ed.
The DNA sequence from clone h5 (SEQ ID NOS: 6 and 7 for the sense and anti-sense strands, respectively) is shown in Figure S and includes the whole coding region. Clone hS (pBSSK+.hu.CTl.h5) was deposited on July 26,1994 in the American Type Culture Coll~-cfinn as ATCC No. 75,841. The DNA sequence of another clone, ~ t' d h6, matches that of clone hS in the region of overlap. Clone h6 begins at base 47 of clone hS and extends 3' of clone hS for an additinal 521 bases. The encoded protein sequence of human CT- I
(SEQ ID NO: 8) is 79% identical with the mouse CT-1 sequence (SEQ ID NO: 3), as evident from Figure 6, 3Q wherein the former is .1.~ d "humct l " and the latter is ~ d "chf.781. "To show that human CT-I encoded by clone hS is b ~Iogi~ lly active, the EcoRI fragment was cloned into the mzn~n~ n eA~ ;77iOII vector pRKS (EP 307,247) at the unique EcoRI site to give the plasmid pRKS.hu.CTI . This plasmid was transfected into human 293 cells, and the cells were .,.~;,,u.;,.~A in serum-free medium for 3-4 days. This medium was then assayed for cardiac myocyte hy~. Ll u~hy as described above for mouse CT- I . The L~ rl ~ L~;d 293 c~ d mediu}n was clearly active in this assay (hypertrophy score of 5~5 at a dilution of l :20; Table 3). Other cytokines were also tested for h~"~. LIU~JhY activity (Table 3) .

--Table 3 HY~ UYIIY assay of CT-I-related cytokines Cytokine Conc., nM Hypertrophy Score*
- - None ~ 3 CT-I fusion 0.05 6 0.1 5 0.25 6 0.5 6.5 1.0 7 Mouse LIF 0.05 4 0.25 5.5 2.5 6 Human IL-II 0.1 3.5 û.2 4.5 0.5 4.5 1.0 4.5 2.0 5.5 Eluman OSM 6.25 4.5 12.5 4.5 Mouse IL-6 50 3-5 100 3.5 Rat CNTF 25 4 *A score of 3 is no h~ Ll u~,hy; 7 is maximal h~"~,. L. uphy (see Materials and Methods).
The mouse and human CT-I encoded by these clones have 80% amino acid identity and are about 200 aminoacidsinlengthcu.. ~ gtoar~lr~ t~-dmolcc~ rmassof21.5kDA. BothhumanandmouseCT-I
lack a ~ull~/c.-tiu~al hydrophobic amino terminal secretion Se<~ nr~?, however, they are found in the medium of 15 Lla~l 71;~LL;I m7lmm~ n cells. The coding regions of human and mouse CT-I are cont~ined on three separate exons that span 6-7 kbp of genomic DNA. The human CT-I gene was localized by fluorescent in sit~
hS,L. i.li~ion and by somatic cell h~L. idi~iu,. to chrrlm~-c- m~ 1 6p l I . I - p l 1.2.
The t~ .7~iUII pattern of mouse CT-I was determined by Northern blot analysis. CT-I
mRNA is widely (but not universally) expressed in adult mouse tissues including heart, kidney, skeletal muscle, 20 and liver. A single 1.4 kb CT-I mRNA species was detected in the adult mouse heart, skeletal muscle, liver, lung, and kidney. Lower amounts of mRNA were seen in testis and brain, while no ~ ,;. ,iu,- was observed in the spleen. The CT-I transcript was also detected in seven-day embryoid body mRNA, which was the RNA used to prepare the cDNA eA~ liUn library. In a survev of human adult tissues (Figure 20), high levels of CT- I
mRNA ( 1.7 kb mRNA) were seen in heart, skeletal muscle, prostate and ovary. Lower levels were observed in lung, kidney, pancreas, thymus, testis and small intestine. Little or no c~ aiUII was seen in the brain, placenta, liver spleen, colon or p." i~L~,~al blood leukocytes. High levels of ~.A~ iUII were also seen in human fetal heart.
5 lung, and kidney, ~..g~ T, that CT-I might be mvolved in embryonic dcv~,lv~,l".,." of these organs. In situ analysis of CT- 1 expression during mouse embryogenesis reveals widespread e,~ iun in a variety of non-cardiac systems. The high level of expression in these other adult tissues suggests the possibility of fi~nctif)nAAl roles for CT-I in a wide variety of adult organ systems, outside of the cardiovascular system. The pattern in humans and mouse are similar with the ~ Li~" . of CA~ iVII in the liver, which is wcakly positive in human 1 0 samples.
Like CNTF, CT-I lacks a cull~ iu,.al amino-terminal secretion signal s~ ~, it is. however, found in the medium of lla~ .d ~ cells.
The predicted tertiary structure of CT-I is cr~ with its containing four amphirAAthic helices that are features of a large number of cytokines and other proteins incl.lrling growth hormone. (For reference see IS AbdclM_~,uidetal., Proc.Nat/AcadSciU~4,84:6434-6437(19873andBazan,l~euron,7:1g7-208(1991)).
Although these cytokines share binlf gjrAAl activities and receptor subunits, AAli~nnf~Tlt of the amino acid sequence of human CT-I and other members of the IL-6 cytokine family, reveals that they are only distantly related in primary sequence (15~/~25% identity) Figure 16. There is little conservation of the cysteine residues and only a partial I..O;l.t~ c of the exon-intron boundaries. Based on the sequence identity culll~al ;sun d~t~.""i"f d 20 herein, studies analyzing the crystal structure and biologirAl function of mouse LIF and their l.,l.,~ancc to receptor binding (Robinson et aL, Cell, 77:1 lûl-l 1 16 (1994~) suggest useful subunit regions of CT-I. As .1~ ~ ~1l;ll~dbyX-rdycrystdllographyata2.ûAresolution.themainchainfoldofmouseLlFconformstothefour a-helixbundle~ )Q~IO~ ythathasbeennotedforothermembersofthelL-6cytokinefamily~ .AliEnm~-ntofthe se-l~ f ~ for filnrtif n~Ally-related mol lf. ~ such as .~ f~ ;ll M and CNTF, and conceq~f nf mapping to the 25 LIF strucmre, indicated regions of conserved surface character. A series of human and mouse LIF chimeras have if df nfifif d the fourth helix and the l.l.,cc1hlg loop as potentially illlpUl Lalll sites for ill~t~d~,Liull with the LIF
receptor (Robinson et aL, Cell, 77:1101-1116 (1994)). Although LIF and CT-I display a high degree of ...,c in primary sequence within these regions, the similar domains within CT-I are likely hll~vllf~ in m~nfAining the illl~,. a~;~iUI15 of CT-I with the LIF receptor. Peptides derived from these regions will find use 30 as CT-I agonists (see Figure 16 for exarnple). Similar a~.l"uacl.~,~ to generate mouse LIF/CT-I chimeras will be of vâlue.
HnmAn ~T-I bin~i~ to the msuse 1,1F receptor. As .I;~-,u~ d herein, human CT-I was ~A~ ,s~ed by ~nhC ~rlnin~ the coding region from plasmid pBSSK+.hu.CTI .h5, which coll~ lcd all of the cDNA protein codin_ region. tO give plasmid pRK5.hu.CTI. Clarified c-~n-iitit~np~i medium was obtained from human 293 cells rt;l t~d with this plasmid and ..~ ~;..u.~ d in serum-free medium for four days. Binding to M I cells (ATCC
TIB 19Z), Hela cells and WI-26 VA4 (ATCC CCL-95.1) cells was p~"r~ .ed for 2 hours a 4 degrees C and analyzed as described herein. For the Hela cell binding, CM was cOllc;~ laL~d 10 fold and added at a 3-fold 5 dilution tO the binding assays. For the Wl-26 binding the contlitit)-lPd medium was used without conc~,;lll aLion.
This c- n(liti-~ncd medium competed for labeled human LIF (iodinated with IODO-BEAD from Pierce or ia~,~vlJ~,. u~iuaac meinods to a specific activiy of i 000- i 500 Ci~'mmoi as described nerein) as did purified mouse and human LIF and mouse CT-l. CM from vector l~ r .. ~d cells failed to compete (Figure 17A). While booth mouse and human LIF bind and activate the mouse LIF receptor, mouse LIF fails to bind the human LIF
10 receptor. As shown herein, human LIF competes for the binding of labeled human LIF to Hela cells while mouse LIF does not (Figure 17B). Mouse CT-I and conditioned medium form 293 cells llalla~ d with the human CT-I ~ ioll vector compete for this binding as well. (Figure 17B). However, the binding of labeled mouse CT-I is ~ ly c~ d by ~-ni~hPIPd human LIF. Thus, both human and mouse CT- I bind to human LIF
receptor~ and CT- I lacks the species a~c~,ifi~,iL~ of binding found for LIF. The affunity of mouse CT- I for human 15 LIF receptor was determined (Figure 18). A single binding cup~ ~I was observed with an affinity (Kd approxØ75 nM), about equal to that for the mouse LIF receptor as shown herein.
Human CT-I does not bind the specific OSM Receptor. Although n l~f~U~ 1 M binds and functions via the LIF receptor (Gearing et al. (1992) New Biologist 4:61 -65), but as shown herein CT- I is not a ligand for the OSM specific receptor, the .,- - t~ M receptor, which has been ~ n~ifi-~d in and cloned from the human 20 lung cell line Wl-26 VA4. Both purified mouse CT- I and the CM from 293 cells Ll~lar~,L~d with human CT-I
cDNA failed to compete for labeled OSM binding (Figure 19).
CT-I induces a distinct form of mvocardial cell hypertrophy cllalac~.i~d bv ~al~ullu,~ic assemblv in The CT-I induced hypertrophic phenotype is distinct from the h~ ,Llu~hic phenotype observed following G-protein d~ ,..lt;llL ~timnl:-ti~n with a~ ,lE;ic agonists (Knowlton et al. (Journal of Biological 25 Chemistry, 266:7759-7768 (1991); Knowlton et aL, Journal of Biological Chemistry, 268: 15374- 15380 (1993), endothelin-l, Shubeita et aL. Journal of Biological Chemistry, 265:20555-20562 (1990), and angiotensin 11 (S~lochim~ et aL, Circ. Res., 73:413-423 (1993)). On a single cell level, h~l~.luLlil~l.,,ic G-protein d~ ~- .,.1. .u pathways induce a form of h~ ;l Ll u~Ly with a relatively uniform increase in myocyte size and the addition of new myofibrils in parallel (Knowlton et aL, Journal of Biological Chemistry, 268:15374-15380 (1993);
30 Shubeita et aL, Jownal of Biological Chemistry, 265:20555-20562 (1990); Iwaki et aL, Journal of Biological Chemistry, 265, 13809-13817 (1990)). In contrast, CT-I induces an increase in myocyte size ~hala~,t~ ;d by a marked increase in cell length, but little or no change in cell width. Consistent with the resuits presented herein for CT- I . LIF iâ also capable of activating a similar paKern of hypertrophy in the cultured myocardial cell assay WO 97/30146 PCT/US97/0267~;

system, while IL-6 and CNTF had little effeet, ~ u-l-al,ly because of the lack of cA~Jrl,Dsioll of the private receptor in cultured myoeardial eells. LIF signals through the gpl30/LIFR~B eomplex, through whieh CT-I also functions as shown herein.
To~ ala~ i~theeffectsofgpl3olLlFRi3-~ ctiml~lAti-nllonthemyofibrillarcytoarchitecture~
5 eardiomyocytes were dual-stained for thick (~3MHC) and thin (F-actin) myofilAm. Pntc and viewed by confocal laser~ luD~u~y(Messerli etal., HistocJ.G",i,.,.v, 100:193-202(1993)). Cardiomyocytes ~;III..i,~1..d with CT-I
and LIF displayed a high degree of myofibrillar ul~ ;n. . myofibrils were organized in a strictly Dal~,olll., ic pattem, oriented along the l....~;lu~ l cell axis, and extended to the cell periphery. Illl~JulLalllly, the inerease in eell size and length was not ~c~ .ied by a ehange in the average Da.-,ull...~ iength, strongly s~ ,e~ p.
10 thatthecelle~ ,g,~l;-,-,inresponsetogpl30/LlFR13-sti~ lAtlnnresultsfromanadditionofnew~al~ull~ units in series. The morphologie ehanges indueed by gpl30/LIFR~ Ctim~lAtinn in vi~ro are r~ minicrPnt of the ehanges observed in eardiae myocytes isolated from hearts subjeeted to ehronie volume overload (Anversa et aL, Circ Res., 52:57-64 (1983); Gerdes et aL, Lab Invest., 59:857-861 (1988)). By eontrast, the pattern of eardiomyoeytehypertrophyindueedbya-a.L."..,~ic,~ ;n-.moreeloselyresemblesapressureoverload-liice 15 phenotype (Morkin, Science, 167: 1499- 1501 (1970); Anversa et al., J. Am. ColL Cardiol., 7: 1140- 1149 (1986)).
On a molecular level, gpl30 ~ J .~fi~ l C1imlll~finn and a-adl.,,~ ,ic stim~llAtion result in distinet patterns of embryonic gene, MLC-2v, and ;...".~ early gene expression. The reactivation of an embryonic pattem of gene l~A~ DD;UII is a central feature of cardiomyocyte hypertrophy (Chien et aL, Faseb J, 5:5037-3046 (1991~). Members ofthe embryonie gene program, such ~ ANF and skeletal a-actin are Al.~l...l,...l1y ~A~ ;.Ded 20 in the v~,lLli.,ul~ m~ut,aldiulll during embryonic develorm~?nt but their eA~ DDiUII is down-regulated shortly after birth. Stimnl~tinn of eardiomyocytes with CT-I or LIF induced prepro-ANF mRNA expression, and perinuclear ~cc~mlllAticn and secretion of immunoreactive ANF. However, in contrast to ~-adrenergic 5timnl~ n CT-I and LIF did not induce skeletal a-actin ~Ayl~DiOll. Growth faetors, signaling through G-protein eoupled reeeptors, inc Ill-ling a-aL."~,.gic agonists, ~~nrlclthplin-l~ and An~iot~ncin Il, induee ANF and 25 skeletal a-aetin in a eoordinate fashion (Knowlton et al., Journal of Biological Chemistry, 266:7759-7768 (1991); Bishoprie ef aL, Jozwnal of Clinical l,.~ ,u~ion, 80:1194-1199 (1987); .CA/1Oeh inn A et aL, Circ Res, 73:413423(1993)). Areeentstudyculll~,al ;itheeA~ ,DDiullpatternofdistinctmembersoftheembryoniegene prograrn in pressure overload versus volume overload hypertrophy in vivo in the rat myu.,aldiulll (Calderone et al., Circulation, 92:2385-2390 (1995)). As shown previously (Izumo et aL, Proc. l'latl Acad Sci. US~, 85:339-30 343 (1988)) pressure overload resulted in the couldLIlat~ induction of ANF and skeletal a-actin. However, volume overload hypertrophy was ~O~: ~t- d with a seleetive inerease in ANF t:A~ ,DDiUn, and no induetion of skeletal a-aetin, sn ggl~cting that the r~uldtiull of distinct embryonic genes in vivo is related to the hypertrophic stimulus (Calderone et aL, Circulation, 92:2385-2390 (1995)). The pattern of embryonic gene l~ DDiUn CA 0224~63~ 1998-08-0~

induced by CT-I and LIF in cardiomyocyte culture therefore resembles the pattern observed in volume overload h~ ul~hy in vivo.
******
, Deposit of Material The following plasmid has been ~lepl~Cit~i with the American Type Culture Collection,12301 Parklawn Drive, Rockville, MD, USA ~ATCC):
Plasmid ATCC Dep. No. DeDosit Date pBSSK+.hu.CTl .h5 75,841 July 26. 1994 This deposit was made under the provisions of the Budapest Treaty on the International RPcognitif n 10 oftheDepositofMi.,.uu.~ sforthePurposeofPatentr~u.,~ andtheRPgl-iltinncthereunder(Budapest Treaty). Thisassures..,~;-.t.-~ eofaviablecultureofthedepositfor30yearsfromthedateofdeposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty, and subject to an ag.~;~...c.-l between GPnPntp~ h~ Inc. and ATCC, which assures F . ~ and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public 15 of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one ~letprmimpd by the U.S. C~-mmiccinnpr of Patents and Tr~ n~:lrke to be entitled thereto accu.di.lg to 35 USC
122 and the Cnmmicr;~mPrls rules pursuant thereto (inr~ ing 37 CFR I .14 with particular reference to 886 OG 638).
The assignee of the present ,.~ ;nn has agreed that if a culture of the plasmid on deposit should die 20 or be lost or destroyed when cultivated under suitable cnn~itione, the plasmid will be promptly replaced on n--tifir~rir,n with another of the same plasmid. Availability of the depc-eited plasmid is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.
In respect of those tiPcign~tirne in which a European patent is sought, a sample of the dProeited 25 microorganism will be made available until the publication of the mention of the grant of the European patent or until the date on which the a~ ,aliull has been refused or withdrawn or is deemed to be withdrawn, only by the issue of such a sample to an expert nnminz~tcd by the person rf (lu~eting the sample. (Rule 28(4) EPC) EXAMPLE Vl MATERr~LS AND MFTHODS.
Human IL-6 was from Gen~yme, mouse LIF was from R & D Systems and Genentech ,.,~.. r~ ,.. hlg, andratCNTFandGDNF, PoulsenetaL,Neuron, 13:1245-1252(1994)wereproducedbyG~n~nt~rh Mouse CT-I was ~ n~e~ed and purifed as a fusion protein as described. This protein results in a 34 amino acid N-terminal extension that encodes a portion of the herpes simplex virus gl~u~ t~ D and a factor Xa cleavage CA 0224~63~ 1998-08-0~

site. In some cases an alternative fusion protein was used that ~b~lil,.l. ~ a different site for the Factor Xa cleavage site giving the amino acid sequence . . . DQLLEGGAAHY followed by the CT-I sequence MSQREGSL . . . CT -I and LIF were iodinated by the iodo-bead (Pierce) and la-,Lu~elu,.iLiase (Gladek et al., Arch. Immunol. Ther. ~xp., 31 :541 -553 (1983)) methods to specific activities of 900- 1100 Ciimmol.
Hem~t~poietic. neuronal. and d~ vlu~-lllcllld.l ~c~z~,ys. Proliferation of the mouse hybridoma cell line, B9 (Aarden e- aL, ~ur. ~ Immunol., 17: 1411 - 1416 (1987)) w~ assayed by 3H-thymidine illCul ~ul aLion 84 h after the addition of cytokine as described (Nordan et al., Science, 233:566-569 (1986)). Inhibition of the proliferation ofthe mouse myeloblast cell line, Ml (T-22), was assayed by 3H-thymidine ill~ul~Jc.laLioll 72 h after the addition of cytokine as described (Lowe et al., DNA, 8:351-359 (1989)). The data were fit to the four pA~ alll~ , e~uation, y=d-((d-a)/(l +(x/c)b)), where the palalll~t~" c is the EC50.
Fortheassayofthetransmitterphenotype,newbornrats~,...l~All.- ~i~ neuronswerepreparedasdescribed (Hawrot et al., Meth. ~nzymoL, 58:574-583 (1979)). Superior cervical ganglia were fl;~o~ ;i with trypsin (0.08%) and plated in serum free F- 12 medium c ~ l lg nerve growth factor and additives as described (Davies et aL, Neuron, 11:555-574 ~1993)). Neurons were plated at 30,000 per well in 24 well plates precoated with 15 poly-omithine and ECL cell AllA~ 1....~ a matrix (Promega) and allowed to grow for ten days in the presence of indicated factors. Tyrosine hydroxylase and choline acetyll.all:.r."ase activities were assayed as described (Reinhard et aL, Life Sci., 39:2185-2189 (1986); Fonnum, Biochem. J., 115:465-472 (1969)).
The survival of rat 11. ~IJAl " i~ .. . ~i~, neurons was assayed as described (Poulsen et aL, Neuron, 13: 1245-1252 (1994)). Ciliary neuron survival assays were p~,.rulll-cd with neurons isolated from E8 chick embryos as described (Mallll.ull,e ef al., (Rush, R., eds.) Vol. pp.31 -56, John Wiley & Sons (1989)). Survival was assessed by counting live neurons after staining with the vital dye MTT (Mosmann, .r. ImmunoL Meth. 65 :55-63 (1983)).
The data were fit to the four ~alalll_tel equation described above.
For the assay of embryonic stem cell iirf~ Liation~ passage 15 embryonic stem cells. ES.D3 (Gossler et al., Proc. NatL Acad Sci. US,4, 83:9065-9069 (1986)) were mAAinrAinf~d in DMEM (GIBCO, high glucose, no sodium pyruvate), C ~ 23.83 g/l HEPES,500 mg/1 p~ni~ illin~ 500 mg/l streptomycin,4 g/1 L-~ l~ . e ~
I g/l gentAAn~ sulfate, I mM 2~ Apl~vll -- ol, 15% fetal bovine serum, and 1.2 Munits/l mouse LIF
(GIBCO). Cells were trypsinized, plated in duplicate at 1000 cells per well in 24-well tissue culture plates in the above culture medium Witil or without LIF or CT-I, and scored 9 days later. No change in colony numbers was observed except in the no addition group where the cells had flattened and di~l~,llLiaLcd.
Cell bin-lin.~ Antl cross-linkin Binding was pv- rul u~ed in RPMI- 1640 containing 0.1 % bovine serum albumin with 7.5-10 miilion Ml cells (TIB 192, ATCC) in a volume of 250 Il] fo} 2 h on ice with shaking.
Reactions were layered on 250111 of RPMI CLn~ .;. .g O. I % albumin and 20 % sucrose. .,c,ll~ u~,_d at 4000 rpm .
CA 0224~63~ l998-08-0~

for I min at 4~ C, and the cell pellet counted. The data were fit to a one-site binding model as described (Munson etaL, AnaL Biochem., 107:220-239 (1980)). Lines shown in the figures are from the curve fits.
Anti-gpl30 antibody inhibition rYI., . ;..., -t~ were p~lru l-lcd with a rat anti-mouse gpl30 monoclonal antibody (RX435)2 or a rat anti-gpl20 control antibody (C~Pnl?nt~h 6D8.1E9) in a volume of 150 IlL Reactions S were inrllh:lt~od on ice 2 h, c- .. 1. ir.. ~. d at 12,500 rpm, and washed with I ml of cold pho:"Jha~e buffered saline cnnt:~ining 0.1 % albumin. The data were fit to the four parameter equation described above.
Binding to neonatal rat cardiac myocytes was IJ~ ~ rul ...ed as for M I cells, but cells isolated as described herein and plated for 16 h. Assays were p~" ru...led with I million cells in a volume of 100 ,ul.
Cross-linking was p~,Arullllcd with 10 million Ml cells in phocph~t~ buffered saline col-t~ining 0.1%
10albumin. 7.2 nM 1251-mouse CT-I or 2.2 nM ~Z51-mouse LIF, with or without a 100 fold molar excess of the unlabeled ligands in a volume of 250 111. After I h at room It;llll~.,.a~ulc, 10 mM l-ethyl-3-(3-dimethylaminopropyl)~,a l,o.l;;..,i~l~ hydrochloride (EDC) and 5 mM N-hydroxysulfos~lccinimirlP (sulfo-NHS) (Pierce) were added and the incub~ti~n cu-.l;....Pd for 30 min at room Lellly.,.dlu~. The samples were then ~.lucess~dasdescribed(Greenlundetal.,J. Biol. Chem.,268:18103-18110(1983)).
15DNA binding activity. Two hundred thousand Ml cells were inrllh~f.od in I ml of RPMI-1640 in 12-well dishes with ligand for 30 min at 37 C. After ~timnl~ti~n, the cells were collected by c~ ;ru~;a~ion, cll~pPn-ied in 200 ul of h.. ~ l ;.. butter ( 10 mM HEPES (pH 7.2), 10 mM KCI, 0.1 mM EDTA, 0.1 mM
EGTA, ImM DTT, I mM phenylmethylsulfonylfluoride, 10 ug/ml lP--peptin, 10 ug/ml aprotinin), and incubated at O C for l 5 min. Cells were lysed by the addition of NP-40 to 0.1%, and cell extracts prepared by incnh~tion 20 at 0 C for 15 min, c.,.. l- iru~alion at 100 x g for 5 min, and retention of the ~ IIal~ll. DNA binding activity in the cell extracts was assayed by elc~ ù~l.u.~lic mobility shift assay as described (Greenlund et aL. l~MBO J, 13:1591-1600(1994)). Briefly,bindingreactionscontained 10 mM Tris-HCI buffer(pH 7.5), 100 mM KCI, 5 mM MgCI2, I mM DTT, 6 7 % glycerol, 0.067 g/l poly(dldC)(dldC), 0.5 ng (25,000 cpm) 3~P-SIE DNA (5'-CTAGAGTCGACATTTCCCGTAAATCT and 5'-CTAGAGATTTACGGGAAATGTCGACT, high affinity 25 m67(SadowskietaL,Science,261:1739-1744(1993);Wagneretal.,~Ml~OJ.,9:4477-4484(1990)),and3ul of cell extract in a final volume of 15 ul. Some reactions included 100 ng of lln l~hDl~d SIE DNA. The reactions were ;..~ rd 30 min at 22 C and analyzed by polyacrylamide gel electrophoresis and autoradiography.
Biadin~ to soluble LIF recevtor and soluble ~p130. DNA encoding the extracellular domain of the mouseLlFreceptor(aminoacids 1-826)andmousegpl30(1-617)wasgeneratedbyPCRofMI cell(above) 30 mRNA and of a mouse lung cDNA library (Clontech). These seg~enrPs were cloned with a C-terminal tag encoding 6 histidine residues in the m~rnm~ n expression vector, pRK5 (Suva el aL, Science, 237:893-896 (1987)) to give the plasmids, pRK5µslifr and pRK5µsgpl30. DNA se~u~ g of the coding regions confirmed that these plasmids encode proteins that match the published amino acid sequence (Tomida et al., J

-CA 0224563' 1998-08-0' WO 97/30146 ~'CT/US97/02675 Bioc~em.. 115:557-562 (1994); Saito etal., J. ImmunoL~ 148:4066-4071 (1992)), with the exception ofthe . of lysine for arginine at amino acid 326 of gp l 3o~ a change that was found for three rl ai~~ from both sources. The plasmids were L.,..~.r ~ l~d into human 293 cells, and the proteins isolated from 4-day c.. ---l;~;n.,-clmediumbyNi~-NTA-agarose(Qiagen~afffunitypurification. Briefly,thec.,.,.l;l;.. dmediumwas S c.- ~ ~.ed ~ 18 fold (Centriprep 10, Amicon), and the tagged protein purified by binding to the Ni--resin for 2 h at room t~ ,. d~UI ~. Following two washes with rh-- 5phf t~ buffer saline cctntf~ining S mM imidazole, the proteins were eluted with l.l..,~,l.~la buffer saline c~ ;..;..g 200 mM ' ' l; '- and ~ d by colu.il...,L. ic assay (BioRad). Analysis of the proteins by SDS-polyacrylamide gel clec.~ u- .,~.s showed single bands of 120 kDa for the soluble LIF receptor and 85 kDa for soluble gp 130. Amino acid s.~.~u~n~,illg gave the expected 10 amino terminal sequence for the soluble LIF receptor bcginning at amino acid 44 (Tomida et aL, J. Biochem., 115:557-562 (1994); von Heijne, NucL Acids Res., 14:4683-4690 (1986)); the amino terminus of gpl30 is expected to be blocked (Saito et aL, J. ImmunoL, 148:4066-4071 (1992); von Heijne, Nz~cL Acids Ales., 14:4683-4690 (1986)) and amino terminal protein se~ g gave no sequence for soluble gpl30.
Binding to the soluble LIF receptor and soluble gpl30 was performed in a manner similar to that 15 previously described (Layton et al., ~ BioL Chem., 269: 17048- 17055 (1994)). Briefly, assays were performed in 96-well Mulli ,~ .,.. -HV filtration plates with 0.45 llm PVDF .. ,.. ~.. ~,s (Millipore) in ,oho~ buffered saline c~ ;. .g O. I % bovine serum albumin and including 25,ul of ~ o~ buffer saline-washed Ni~-NTA-Agarose (Qiagen) in a final volume of 175 ~1. Plates were i.-- ~ Ird at room t~,,ll,J~"~Lulc: overnight with agitation. Foilowing vacuum filtration and one wash with 200 ,~II of cold pl,..~l~h.-~ buffer saline? the individual 20 assay welis were cut from the plate and counted. The data were analyzed as described above for M I binding.
~F.!~uLTS
As shown herein some members of the IL-6 cytokine family (LIF, OSM, and IL-I I ) induce cardiac myocyte hypertrophy in vitro like CT-I . The previously known members of this farnily have a wide range of LUPO ;-:, neuronal, and dcv~ i activities (Kishimoto et aL, Sc~ence, 258:593-597 (1992)). CT- I
25 was assayed for its activity in these biolQgir~l systems.
Hematopoietic assays. IL-6 ~lv111~3L~;~ the proliferation and di~r.,l.,lllialion of B cells into antibody producing cells following antigen stim~ tinn (Akira et al., A~v. ImmunoL, 54: 1 -78 (1993)). In the order to termin.o whether CT-I could also mediate these effects, CT-I was tested on the mouse hybridoma cell line, B9 (Aarden et aL, Eur. .~. ~mmunol., 17:1411-1416 (1987)). While IL-6 ctimnl~tl~s the pro!iferation of B9 cells as 30 indicated by an increase in 3H-thymidine i..col~.olaLion, CT-I and LlF were inactive (Figure 7A). even at co---,c;..Ll~lions as high as 2 uM (data not shown). Thus, CT-I does not mimic the activity of IL-6 in promoting B cell r ~

-While }L-6 -- ' the growth of several B cell Iymphr m~c myelomas, and plasmacytomas, it also has growth inhibitory effects on certain B Iymphoma and myeloid leukemia cells (Akira et al.. A~v. Immunol., 54:1-78 (1993)). IL-6 (as well as LIF and OSM) inhibits the growth of the mouse myeloid leukemia cell line, r Ml, and induces its dirr~ Lia~ion into a l-,a~,.ul,hagt-like phenotype (Akira et al., Adv. Immunol., 54:1-78 (1993): Rose et aL, Proc. NatL Acad Sci. USA, 88:8641-8645 (1991)). CT-I was 6 fold more potent than LIF
in inhibiting the uptahe of 3H-thymidine by Ml cells (Fig. 7B). Thus, CT-I does share at least some of the growth inhibitory activities of the IL-6 family cytokines .
Neuronal ~cc~ys~ Members of the IL-6 cytokine family modulate the phenotype and promote the survival of neuronal cells (Patterson, Proc. Natl. Acad Sci. USA, 91:7833-7835 (1994)). LIF and CNTF can induce a switch in the ~ phenotype of s~ neurons from nula~ to cholinergic7 a change that is a c-~ ---ird by the in~ cti~n of several n~u~u~ s inc~ ng ~ub~ CC- P, sU~ and vasoactive intestinal polypeptide (Rao, J. NeurobioL, 24:215-232 (1992)). The ability of CT- I to induce this switch in the transmitter phenotype was d~lt,..-i..ed with cultured rat symp~thetir neurons. CT-I inhibited the tyrosine hydroxylase activity (a nu.a~l.~,...,.~;ic marker) and stirn~ t~d su-~ .l-al the choline acet~,ll-a.~rt.~e 15 activity (a cholinergic marker) of these cells, effects that palalleled the actions of LIF (Fig. 8A). Thus, CT- I is active in mr)r~ ting the phenotype of 5~ ;c neurons.
P~uhi..so..'s disease is caused by the dcg_.~c.aliu~l of dopaminergic neurons of the midbrain (Hirsch et aL, Nature, 334:345-348 (1988)). While proteins ofthe n.,.--ul-u~,lL,- family (brain-derived n~.l-uLIu~hic factor and r.cu.uL-ul-hin-4/5) as well as ofthe TGF-~ family (GDNF, TGF-~2 and TGF-~3) promote the survival of cultured dop~min-~rgic neurons (Poulsen et aL, Neuron, 13:1245-1252 (1994)) many other growth factors and cytokines. including CNTF, do not. Unlike CNTF, CT- I was found to promote the survival of rat rl~p~rr~ in.~rgic neurons. although it was not as potent as GDNF (Fig. 8B).
While inactive on ~flp~Tnin~rgic neurons, CNTF does promotes the survival of ciliary neurons (Ip et aL, Prog. Growth FactorRes., 4:139-155 (1992)). CT-I was tested for its activity in ~,,u...uLi..g the survival of 25 chick ciliary neurons (Fig. 8C). While at maxirnal c~ c CT-I was as active as CNTF, the potency of CT-I in y-u~ g ciliary neuron survival was about 1000 fold less than that of CNTF (Fig. 8C~. Thus, CT-I
shares some neuronal activities with the IL-6 family cytokines such as CNTF.

Embrvonic develoDment assav. The presence or absence of soluble factors plays a key role during embryonic and fetal dcv~ 1. ,1,.. .- .I For example, embryonic stem cells require the c~ntin--rluC presence of soluble 3û factors secreted by fibroblasts to maintain their undil~ l.LiaLed, plu.il,ut~,..t phenotype. LIF (Williams e~ aL.
Nature, ,36:688-69û ( 1988); Smith et aL, Nature, 336:688-69û ( 1988)), CNTF (Conover et aL, Developmen~, 119:559-565 (1993)), and OSM (Rose et aL, Cytokine~ 6:48-54 (1994))--but not IL-6 without the soluble IL-6 receptor (Yoshida et al.. Mech. Dev., 45:163-171 (1994))--can replace these fibroblast-derived factors in WO 97/3~146 PCT/US97/02675 -.,.~;..l, ;..i..g the plul;l~u~ phenotype of embryonic stem cells in culture. CT-I was also found to inhibit the diLr~ Lion of mouse embryonic stem cells (Fig. 9); it was as effective as LIF at the coneentrations tested.
Thus, the data from seven in vitro biological assays indicate that CT-1 is active in assays where LIF is aetive and vice versa. Acccldillgly, these assays sys~ems (and others in whieh CT-I has a d.,~l~Ollallalt:d aetivity S as shown herein) ean be used to screen for and identify CT-I agonists and antagonists useful for treating disorders depPnrl!~nt upon or resultinf from the biological aetivity (or loss. reduction or overproducction of the aetivity) r~ t~ ;i in these assays. These data also show that CT- I is aetive in assays where CNTF is active, but that the converse is not always the case, and that CT- I is inactive in IL-6 specific assays, assays in which LlF
is also inactive. Since the activity profiles of members of this cytokine family are determined by the receptors 10 expressed on target cell P~~ these data are c~ with the hypothesis that CT-I binds and u ansdu.,.,~, its biological effects via the LIF receptor.
CT- I hinrlin p to M I ~ lc In order to show directly that CT- I functions via the LIF receptor, binding was perforrned on Ml eells, where LIF binding has been previously ~h~ L~ ;I (Hilton et aL, Proc. Natl.
Acad. Sci. US~, 85:5971-5975 (1988)). Both CT-1 and LIF inhibit the growth of this cell line (see above).
15 Labeled CT-I was cp~eifi~ 11y bound to M1 cells (Fig. IOA~, and this binding was eomp' Iy comret~d by unlabeled LIF (Fig. lOB). Similarly, labeled LIF binding was comret~od by both llnl~hekfl LIF and CT-I (Fig.
IOC and 10D). These data suggest that CT-I and LIF bind to the same receptor on Ml cells. Scatchard analysis yields a single class of binding sites in all cases; the binding pal.,~ ,t~,l . are similar regardless of the labeled ligand--Kd (CT-I] ~ 0.7 nM, Kd [LIF] ~ 0.2 nM, and - 1500 sites per cell.
Cross-linkinp of CT- I on M I cellc To analyze the protein(s) that bind CT- I on the cell surface, labeled CT-I and LIF were bound to Ml cells, ~~hcmir~lly cross-linked, and the solubilized proteins analyzed by SDS
gel cle~L-uphul~ (Fig. I l). Both ligands gave one specific band with a mobility of~ 200 kDa. and in both cases this cross-linked band was cc.l-,p~t~d by either nnl~heled ligand. Thus, CT-I and LIF interact with a protein of the same size on the surface of M I cells; this protein has a mobility expected for the LIF receptor 25 (Davis etal., Science, 260:1805-1808 (1993); Gearing etaL, ~MBO J., 10:2839-2848 (1991)).
Inhibiti~n of CT-I binflin~ to M I eellc bv an anti-~r 130 monoelon~l ~ntihody. In order to show that gpl30, the eommon signaling subunit shared by all receptors for ligands of the IL-6 cytokine family, is a part ofthereceptorbindingeomplexforCT-I,theeîfectofananti-gpl30monoclonalantibodyonCT-I bindingwas .l~l....,;...~d(Fig. 12A). Thisn~ i7ingantibodyinhibitedover8o%ofthespecificcT-l bindingtoMI eells;
30 no inhibition was found with CUIII~aI~IC c~n~ c of a eontrol antibody. These data indicate that gp l 30 is a cc,~ .u..~ of the CT-I receptor complex.
CT-l induçtion of D~A hin(lin~ activitv in Ml cells. To show that CT-I induces intracellular signaling events like those found for other cytokines that signal via gp 130 (Yin et aL, ~;p. Hematol.. 22:467-472 (1994):

Narazaki et al., Proc. NatL Acad USA, 91 :22B5-2289 ( 1994); Zhong et aL, Science. 264:95-98 ( 1994); Akira etaL. Cell, 77:63-71 (1994)) DNA mobility shift assays wee performed with cell extracts from Ml cells (Fig.
12B). CT-I, like LIF, induced a shift in the mobility of the DNA element, SIE. Addition of the lml~hel.od element showed that the shifted band was specific. Thus, CT-I induces the activation of a DNA binding activity 5 like that expected for signaling via gp 130 and activation of the ~ak/STAT pathway.
CT-1 bin~1in~ to cardiac mvocvtes. The binding of labeled CT-I and LIF was also deterrnined for rat cardiac myocytes, the cells used for the original assay and isolation of CT-I. Both ligands specifically bound and cross-cu~ ,.,~d for binding to these cells (Fig. 13A and 13B), as was the case for Ml cells. These data suggest that CT- I and LIF bind and induce cardiac myocyte hy~ u~ y via the LIF receptor.
10CT-I bin~li~ to the soluble LTF receDtor. In order to clarify whether CT-I can bind directly to the LIF
receptor or gpl30 without the need for an ~ltliti~n~l membrane-bound coll.pol..,.ll (as is the case for CNTF), binding eA~ were p~;:l rul llled with purified, soluble forms of the mouse LlF receptor and gp l 3o cA~ d as their eAtracellular domains c~ .;..g a C-terminal histidine tag. Such eA~ c.ll~ have recently shown that OSM binds directly to soluble gp 130 (Kd ~ 44 nM for the human proteins) (Saadat et aL, J. Cell Bio~., 108:1807-151816 (198g)). On the other hand, LIF binds directly to the LIF binding protein, a naturally occurring soluble form of the LIF receptor (Kd ~ 2 nM for the mouse proteins) (Layton et al., J. BioL Chem., 269:17048-17055 (1994); Layton et al., Proc. NatL Acad. Sci USA, 89:8616-8620 (1992)). The soluble mouse LIF receptor and gpl30 were eA~ aed in ...~ l; - - cells, purified by Ni~ chelate l~hlUlll~llU~ ly7 and judged to be at least 90 % pure by SDS gel cle.,ll u~horesis (data not shown). Binding CA~J.~. illl~,.lb with labeled CT- ] show that it 20 specifi~ llly binds to the soluble LIF receptor (Fig. 14A), as does labeled LIF (data not shown). CT-I failed to bind to soluble gp 130 at gp 130 ~,on~,.lLI ~Liulla as high as 350 nM (Fig. 14B). The binding of CT- I to the soluble LIF receptor was enhanced by the addition of soluble gpl30 (Fig. 14C), suggesting that CT-1. soluble LIF
receptor, and soluble gp 130 form a tripartite complex as would be expected for the CT- I activation of the LIF
receptor complex. Cc,lll~.,.iliun binding ~,AI.t;l ;llu,.lla show that CT-I binds to the soluble LIF receptor with a 25 reasonable affinity, Kd = 1.9 nM (Fig. 14D). This affinity is about the same as that found for the binding of LIF
(Kd = 1.5 nM, data not shown) and is the same as that found previously for LIF binding to the naturally occurring forrn of the soluble LIF receptor (Kd = 1-4 nM (48)). These data d~.llu,l~Ll alt~ that CT- I interacts directly with the soluble LIF receptor without the need for an ~l,litil.n~l binding Cclll~Jull~lL The results suggest that CT-I
(like LIF) binds first with a relatively low affinity to the LIF receptor on the cell ~ e and then forms a 30 h~,t~,. uL~ ;--lcric complex with a higher apparent affinity upon interaction with gp 1 3û.
DISCUSSION
In vitro hematopoietic, neuronal, and developmental assays have been used herein ~o show that CT- I
has a range of activities in addition to the in~ tion of cardiac myocyte h~ Lrul~h~ for which it was initially -isolated. As disclosed herein, CT-I is more potent than Ll~ in inhibiting the growth of tha myeloid leukemia cell line, Ml; it induces a phenotypic switch in symp~fhPtic neutrons; it promotes the survivai of dopaminergic neurons from the central nervous system and ciliary neurons from the periphery; and it ,,.~,..l;.;.,c the undirf~ Lir~L~d phenotype of embryonic stem cells. CT-I and LIF share a common activity profile--both inhibit 5 the growth of Ml cells, induce the switch in symrslthPtic neuron phenotype, inhibit the dilI;.~.,Lialion of embryonic stem cells, and induce cardiac myocyte hypertrophy. CT-I is active in assays where CNTF is active--bothinducetheswitchinsy...l. lh-~icneuronphenotype(SaadatetaL,J.Ce/lBioL,108:1807-1816(1989)) promote the survival of ciliary neurons, and inhibit the dilrt;l~ Lia~ioll of embryonic stem cells (Conover et al., Development, 1 19:559-565 (1993)). On the other hand, CT-l is active in several assays where CNTF is inactive-10 -inhibition of Ml cell growth (CNTF activity requires the inclusion of soluble CNTF receptor (Davis et aL, Science,259:1736-1739(1993)),~lulllotiollof(1~-l,,--";..~ ,neuronsurvival,andinductionofcardiacmyocyte hy~. Llu~hy. CT- I is inactive, as are LIF and CNTF (Davis et aL, Science, 259: 1736-1739 (1993); Kitamura et aL, Irend~ Endo. MetaboL,5:87744-14 (1994)) in the stimlllsltinn B9 cell growth, an assay that is relatively specific for IL-6.
~ of the amino acid c~ . .. e ~ of CT-l and other members of the lL-6 cytokine family show that while these cytokines share biological activities and receptor subunits, they are only distantly related in primary sequence (14-24 % identity for the m~nnmzlli~n proteins, Fig. 15A). There is little conservation of the cysteine residues and only a partial ". ~;..l.~~.~ .re of the exon-intron bowld~u ies (Bruce et aL ~ Prog. Growt*
Factor ~es., 4: 157- 170 (1992); Bazan. Neuron, 7: 197-208 (1991)). More soFhictic~tPd analyses (;. ,~ the 20 crystalstructureofLIF(RobinsonetaL,Cell,77:1101-1116(1994))showthattheseproteinsshareacommon structural~.,hiL~L~ offouralphahelices(forreferenceseeBazan,Neuron,7:197-208(1991)). Theindividual family members are more related across species. The human and mouse seq~PncPc for CT-I, LIF. CNTF, or IL-11 are 79-88 % identical (Fig.15A); the IL-6 homologues are 41 ~/O identical. Some unc~i. Li~ y remains as to whether the chick protein, irhqntifipd as GPA, is the avian homologue of CNTF or another family member for 25 which no m~mm~ n hnmo!rlgn~- has yet been identified (Leung et aL, Neuron,8: 1045- 1053 (1992); Richardson, P*armacol. ~her., 63:187-198 (1994)). CT-I does not appear to be the m~rnm~ n homologue of GPA, as chicken GPA is more similar in amino sequence to mouse CNTF than to mouse CT- I (46 verses 26 % identity, Fig. lSA). On the other hand, there are ~ among CT-l, CNTF, and GPA--all lack a conventional amino terminal, secretion signal ceqllPn~p ll~t~ ly, CT-I and GPA appear to be secreted from cells while CNTF
30 is not (Leung et al., Neuron, 8: 1045- 1053 (1992); Stockli et aL, Nature,342:920-923 ~1989); Lin et aL. Science, 246:1023-1025 (1989)).
As is shown dia~ .. ,.~ lly in Fig. l 5B, the receptors for cytokines of the IL-6 family are composed of related subunits some of which are cytokine specific and some of which are shared (Davis et aL . Curr. Opin.

~ Cell Biol.. 5:281-285 (1993); Stahl et aL. Cell, 74:587-590 (1993); Kishimoto et al.. Cell, 76:253-~62 (1994);
Hilton et al., EMBO.~., 13:4765-4775 (1994)). All the }eceptors in this family have in common the all" signaling subunit, gpl30. The binding of IL-6 to the 80 kDa IL-6 receptor ~ subunit leads to the d~ alion of gpl30 as the first step in signal tr~n~ lrtic-n Similarly, the binding of IL-I I to the IL-I I
S receptor also leads to gpl 30 ~ aLiom LIF. OSM. and CNTF induce the heterodimeriztion of gp 130 and with another signaling subunit, the LIF receptor. LIF and OSM bind directly to the LIF receptor or gp 130 and induce di..lt,.;,,-lion without a ligand-specific ~Y subunit, while CNTF binds first to the GPI-linked CNTF
receptor. While the formation of receptor complexes c~ ;. .;..g homo- or heterodimers of gp 130 is believed to be an essential signaling event, the exact ~ LI.y of the subunits in the complex is not known in most cases.
10 For the IL-6 receptor, a recent report c~".~ that the signaling complex is a hexamer c~mtslininE~ two 20 kDa ligands, two 80 kDa IL-6 receptors, and two 130 kDa gpl30 molecules (Ward et al., J. BioL Chem.,269:23286-23289 (1994)). The ligand-induced dilln,.i~ation of gpl30 or gpl30 and LIF receptor leads to the tyrosine phosphorvlation and activation of ~c~oci~ed tyrosine kinases of the Jak family (Jakl. Jak2, and Tyk2) followed by the activation of llall~ lion factors of the STAT family (STATI and STAT3) (T .iHtirk~n et al., Science, 15 263:89-92 (1994); Stahl et aL, Science, 263:92-95 (1994); Yin et aL, Exp. ~ematoL, 22:467-472 (1994);
Narazaki et aL, Proc. NatL Acad USA, 91:~85-2289 (1994); Zhong et aL, Science, 264:95-98 (1994); Akira etaL,Cell.77:63-71 (1994)). Althoughnotmeanttobelimiting,itisproposedthattheactivationoftheJak-STAT pathway is probably one of the key steps in the signal Lldu:,duuLion ml~ch~nicm for most if not ali the actions of the IL-6 family cytokines, inrll-~lin~ CT-I.
The presence or absence of the different subunits of the IL-6 family receptors dictates the u~ of variouscellstothedifferentcytokines(Tagaetal.~FASEBJ~6:3387-3396(1992);Kishimoto etaL, Cell.76:253-262(1994)). Thus,allresponsivecellsarebelievedtoexpressgpl30, B9cellsfailtorespond to LIF and CNTF because they lack LIF receptor, lL-6 is inactive on embryonic stem cells because these cells lack the IL-6 receptor e subunit, LIF is active on Ml cells because both gpl30 and LIF receptor âre present, 25 while CNTF is inactive due to a lack of CNTF receptor o~, etc. Based on the profile of CT-I activities reported here, CT-I functions via the LIF receptor. This is established directly herein as follows. First, as shown herein, CT-I and LIF cu---~,k.t._ly cross-compete for binding to Ml cells, a cell line where LIF binding has been previously well ~,Lua~t~ ;d, Kd [LIF3 = 0.1 -0.2 nM (Hilton et aL, Proc. NatL Acad Sci. USA, 85 :5971 -5975 (1988); Gearing et aL, New Biologist, 4:61-65 ~1992)). Regardless of which ligand is used as the label or 30 competitor, an af~mity for CT-l, Kd ~ 0.7 nM which is 3-4 fold less than that found for LIF, Kd - û.2 nM is found. Secondly, cross-linking data show that CT-I and LIF specifically interact with a protein of ~ 200 kDa, a protein about the size expected for the LIF receptor (Davis et al., Science, 260: 1805- 1808 (1993); Gearing etaL,EMBOJ..10:2839-2848(1991)). Third,asshownherein.ananti-gpl30m~-n(1cl~-n~1antibodyspecifically CA 0224~63~ 1998-08-0~

inhibits the binding of labeled CT-I to Ml cells, showing that gpl30 is a CU~ III of the CT-I receptor complex. Fourth, CT- I induces the activation of a DNA binding activity, an intracellular signaling event induced by LIF and other members of the IL-6 cytokine family in the course of activation of the Jak/STAT pathway (T.ii11irk.-n et al.. Science, 263:89-92 (i994); Yin et aL, ~:xp. HematoL. 22:467 ~72 (1994); Zhong et aL, Science~264:95-98 (1994); AkiraetaL, Cell, 77:63-71 (1994)). These data demonstrate thatCT-I can bindto and activate the LIF receptor complex. This finding does not exclude the possibility that some cells have an A~ tinnAlcT-lspecificreceptororreceptorsubunitthatformsaheterodimerwithgpl3o~ashasbeenreported for OSM (Mosley et aL, Cytokine, 6:554 (1994)).
As shown herein, CT-1 and LIF also cross-compete for binding to rat cardiac myocytes. This finding 10 is cor~cict~nt with the hypothesis that these two ligands act on these cells via the LIF receptor, as ~ ,o~ .Pd herein for Ml cells.
While LIF and OSM induce the heterodimerization of the sarne receptor subunits. LIF receptor and gp 130. the affinity of these two ligands for the individual receptor components differs. LIF binds to the LIF
receptor (Kd ~ 2 nM (Gearing et aL, EMBO J., 10:2839-2848 (1991)) but does not interact with gpl30 in the 15 absence of the LIF receptor. Conversely, OSM binds to gpl30 (Kd ~ I nM (Liu et aL, J. Biol. Chem., 267:16763-16766(1992))butdoesnotbindtotheLlFreceptoralone(GearingetaL,EMBOJ., 10:2839-2848 (1991)). Soluble forrns of these two receptor subunits, c~ of their extracellular domains. are found in the circulation (Layton et aL, Proc. NatL Acad Sci. US~, 89:8616-8620 (1992); Narazaki et aL, Blood, 82: 1120-1126 (1993)). The soluble LIF binding protein binds LIF with a Kd ~ 2 nM (for the mouse proteins) (Layton et 20 aL,J.BioLChem.,269:17048-17055(1994)),whilea.~c. ~- formofsolublegpl30bindsOSMwithaK~
~ 44 nM (for the human proteins) (Sporeno et aL, J. BioL Chem., 269: 10991 - 10995 (1994)). As shown herein.
CT-I binds to the soluble LIF receptor with about the same affinity as LIF (Kd ~ 2 nM, for the mouse proteins) and in the absence of other proteins. CT-I does not bind to soluble mouse gp 130 even at high CullC.,ullaLiOIlS.
The addition of soluble gpl30 does increase the binding of CT-I to the soluble LIF receptor. however.
25 ~ ,. Ullldbly by the l;,- lll~lLu-- of a t~ ul- illlcl ic complex. The C~ lLlaliull of soluble gp 130 required for this effect ~~ 100 nM), ~vhile high by solution binding ~L~.dc..-ls, is readily aLL~ ble on the surface of a cell. For example.500moleculesofgpl30~i,.".~ lonthesurfaceofacellofl0,umdiameterwouldhaveaneffective cun.,~.lL aLiu.. of~ 300 nM in a 100 A shell surrounding the cell, see (Ward et al., J. BioL Chem., 269:23286-23289 (1994)). Thus, these results indicate that CT-I binds to the LIF receptor in the same manner as LIF, by 30 first binding with low affinity to the LIF receptor subunit, an interaction that does not require Arl~litionAl ,olll~.oll~llL" and second by lt;~,luiLi~g gpl30 to form a high affinity signaling complex. Although CT-I was isolatcd based on its ability to induce cardiac myocyte hy~L~ul~l-y, it clearly has a much wider range of activities.asisfoundfortheothercytokinesofthelL-6family(Kishimotoetal..Science,258:593-597(1992):

CA 0224~63~ 1998-08-0~

WO 97t30146 rCT/US97102675 -Kichimorn et aL. Cell. 76:253-262 (1994)). The receptor data pl'ei,e.lled here predict that CT-I will mimic the many effects of LIF in vitro and in vivo. Some of the functions of LIF, and thus targets for CT-I and its or agonists, are obtained from the targeted deletion of the LIF gene in mice, which leads to animals that are outwardly normal although they do exhibit a reduced growth rate, a decrease in h~ -aloyoietic cells, and S a failure of proper embryo implantation (Escary et aL, Nature, 263:361-364 (1993)). These studies are ~, c~ with the in vitro data iJles~ d herein and the uses of CT-I and its AntAgnni~tc and agonists.
The foregoing written specification is collaid~ ,d to be s~ffi~ ont to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the construct deposited. since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any con ,l- ....~
10 that are r.,, ~ .. "~lly c~U;v~ are within the scope of this invention. The deposit of material herein does not cnn~tit--t-o an A inniCcinn that the written des~ Lioll herein c~ntAin~d is ;l.Afi~ ,llr to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it .~ ,.lL~. Indeed, various m~lrlificAtinn~ of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the description herein 15 and fall within the scope of the ~ppPnfi~d claims.

CA 02245635 l998-08-05 WO 97/30146 PCT~US97/02675 -~U~N~ LISTING
(1~ GENERAL INFORMATION:
(i) APPLICANT: Genentech, Inc.
The Regents of the University o~ California (ii) TITLE OF lNV~NllON: Cardiotrophin and Uses There~or (iii) NU~3ER OF ~U~:N~S: 8 (iv~ CORRESPuN~N-~ ADDRESS:
(A) ADDRESSEE: G~n~ntech~ Inc.
(B) STREET: 460 Point San Bruno Blvd (C) CITY: South San Francisco (D) STATE: California (E) CUUN 'LK Y: USA
(F) ZIP: 94080 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5 inch, 1.44 Mb ~loppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: WinPatin (G~n~nte~h) (vi) ~uKR~N-l APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) AllOKN~:Y/AGENT INFORMATION:
(A) NAME: Torchia, PhD., Timothy E.
(B) REGISTRATION NUMBER: 36,700 (C) REFERENCE/DOCKET NUMBER: P0994PCT
(ix) TEL~CC.~ IJN 1 CATION INFORMATION:
(A) TELEPHONE: 415/225-8674 (B) TELEFAX: 415/952-9881 (C) TELEX: 910/371-7168 (2) INFORMATION FOR SEQ ID NO:1:
(i) ~Uu~N-~ C~ARACTERISTICS:
(A) LENGTH: 1352 ba~e pairs (B) TYPE: Nucleic Acid (C) STRA~n~nN~s: Single (D) TOPOLOGY: Linear (xi) ~Qu~N~ DESCRIPTION: SEQ ID NO:1:

. CA 02245635 l998-08-05 WO 97130146 PCT~US97/02675 CGCCAGACAC ACAACCTTGC CCGCCTCCTG ACCA~ATATG CAGAACAACT 150 TCTGGAGGAA TACGTGCAGC AACAGGGAGA GCC~111~GG CTGCCGGGCT 200 CA~11~GGG CCCTGGGCGC CGCGGTGGAG ACAGTGCTGG CCGCGCTGGG 450 GGGCCAGCTG GTGCCAGGGG GC~1~GC~1~ AGAGTGAATA ~'L 1 1 L'l'~'L'l'G 650 TAAGCTCGCT ~1~1~1CGCC TCTTTGGCTT CA~ATTTTCT ~ L~ 1~1 CCAT 7 0 0 ~l~l~lC~lG 'l'~'L~'ll~'l'lG GG~1~1~CC~1~ A'1~'1"1''L~'1'GC A'L'1"L~'L~'1'GG 750 '1'C'L~'L~'L~1"1' CTG~'L~'1'C~'L CTCTGCAGGG AG~'L'L~'L'L'1''1' TTCCA~CAGT 800 TT~ L C~ LL1L ~'1'~'L~''L~''1'CC A~ ~AAC A~'L"1''1"L~'L~'L CCGAGAGGTC 850 'L~''L'L'L'L'1~'1''1' TC~11~'L~'L~ 'L1G~L1~LL1 CTTTGCTTGC TTG~'1''1'G~'L'1' 900 G~''1'1G~'1"L~'1' TGTTGAGACA GGGTCTCACC ATATAGCTCT GGATGGCCTG 9 50 CCTCCGACTC CCAATTTCCC CA~ LC C~ ~ATCC ATATGGGTAT 1050 GTGTAACCCT TA~L1L~1~1~ CATGGAGGTG ACAATTTTTC TCCCTTCAGT 1100 TT~''LL'L~'1''LC TTTACTGACC AGA~AAGTGC CTACTTGTCC CCTGGTGGCA 1150 AGGCCATTCA CCTTAGGACC TTCCCACCAG '1LC~L11~1A GGCAAATCCC 1200 25 TCCCCCTTTG AG~1C~ C~ CTTTCATACC GCCCTAGGCT GGTCAATGGA 1250 CA 0224~63~ 1998-08-0~
W O 97/30146 PCTrJS97/02675 AA~llLl~ A AATAAAATGT TTAACAATAA AACTAAACTT TTATGAAAAA 1350 (2) INFORMATION FOR SEQ ID NO 2 ( i ) ~ U~N~'~ CHARACTERISTICS
(A) LENGTH 1352 base pairs (B) TYPE Nucleic Acid (C) STRANv~vN~SS Single (D) TOPOLOGY Linear (Xi) ~UU~:N~' DESCRIPTION SEQ ID NO 2 CCTATTCGGA CCCCG~LC~l ACTCGGTCTC C~1CC~11~A GAC~lL--LGG 50 lG~-l~lGACT GAGGAGTTAG AGTAAGGATG GGGTA~ACCT CCG~llulAG 100 GCGGTCTGTG TGTTGGAACG GGCGGAGGAC lG~ L"l l'ATAC ~'1~'1'1~ll~A 150 AGAC~l~ll ATGCACGTCG ll~lCC~~ rÇr,r~A~rCC GACGGCCCGA 200 AGAGTGGTGG CGrrC-~rr-r-C GACCGGCCGG ACTCACCGGG CCGAGGCTCG 250 GTA~LCCC~ ATGGCCACAG GCTCGCCGAC GCC~lC~l~C GTCGGCGGGA 300 AGTGCCGGTT ~lC~l'~ACGT CCGTAGAAGA ~lCG~llCCA CGACCCCAAG 550 GTGCACACGC CGGAGATACC GCTCACCCAC TCGGCGTGTC lCCCG~lGGA 600 CCCGGTCGAC CACGGTCCCC CGCAGCGGAC TCTCACTTAT GA~AAAGAAC 650 2S GACACAGGAC ACACAAGAAC CCGACAGGGA TAGAAAGACG T~A~r~rArr 750 AGAGAGAGAA GACGAGAGGA GAGACGTCCC TCGAAGAAAA AAG~ll~l~A 800 AAGAGCAAAA CAGAGAGAGG TCAGAACTTG TGALAACAGA GG~l~lC~AG 850 , CA 0224~63~ l998-08-0~
WO 97/30146 PCTrUS97/02675 AGAAA~ACAA AGGAACAGAG AACCAAGAAA GAAACGAACG AACGAACGAA 90O

CTTGAACGAT ACALCCG~LC CGACCGGAGG TCGAGTATCT CTAGGTGAAC 1000 5 CACATTGGGA ATGAAACAGA GTACCTCCAC TGTTA~AAAG AGGGAAGTCA 1100 AAGAAACAAG A~ATGACTGG l~lllL~ACG GATGAACAGG GGACCACCGT 1150 TCCGGTAAGT GGAATCCTGG AAGG~lG~lC AAGGA~ACAT CCGTTTAGGG 1200 AGGGGGA~AC TCCAGGAAGG GA~AGTATGG CGGGATCCGA CCAGTTACCT 1250 ~'l~'LUl''l''l'CC ~l'~'L'L'L'L'L~'L AGA~ATTTCT CA~AATA~AC TCTTATTTAA 1300 (2) INFORMATION FOR SEQ ID NO:3:
(i~ ~u~N~ CHARACTERISTICS:
(A) LENGTH: 203 amino acids 15(B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) S~uu~ DESCRIPTION: SEQ ID NO:3:
Met Ser Gln Arg Glu Gly Ser Leu Glu Asp His Gln Thr Asp Ser Ser Ile Ser Phe Leu Pro His Leu Glu Ala Lys Ile Arg Gln Thr His Asn Leu Ala Arg Leu Leu Thr Lys Tyr Ala Glu Gln Leu Leu Glu Glu Tyr Val Gln Gln Gln Gly Glu Pro Phe Gly Leu Pro Gly Phe Ser Pro Pro Arg Leu Pro Leu Ala Gly Leu Ser Gly Pro Ala Pro Ser His Ala Gly Leu Pro Val Ser Glu Arg Leu Arg Gln Asp _ 80 85 go -Ala Ala Ala Leu Ser Val Leu Pro Ala Leu Leu Asp Ala Val Arg Arg Arg Gln Ala Glu Leu Asn Pro Arg Ala Pro Arg Leu Leu Arg Ser Leu Glu Asp Ala Ala Arg Gln Val Arg Ala Leu Gly Ala Ala Val Glu Thr Val Leu Ala Ala Leu Gly Ala Ala Ala Arg Gly Pro Gly Pro Glu Pro Val Thr Val Ala Thr Leu Phe Thr Ala Asn Ser Thr Ala Gly Ile Phe Ser Ala Lys Val Leu Gly Phe is Val Cy~

Gly Leu Tyr Gly Glu Trp Val Ser Arg Thr Glu Gly Asp Leu Gly Gln Leu Val Pro Gly Gly Val Ala (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 200 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) ~u~ DESCRIPTION: SEQ ID NO:4:
Met Ala Phe Thr Glu His Ser Pro Leu Thr Pro His Arg Arg Asp Leu Cys Ser Arg Ser Ile Trp Leu Ala Arg Lys Ile Arg Ser Asp Leu Thr Ala Leu Thr Glu Ser Tyr Val Lys His Gln Gly Leu Asn Lys Asn Ile Asn Leu Asp Ser Ala Asp Gly Met Pro Val Ala Ser Thr Asp Gln Trp Ser Glu Leu Thr Glu Ala Glu Arg Leu Gln Glu Asn Leu Gln Ala Tyr Arg Thr Phe His Val Leu Leu Ala Arg Leu -CA 02245635 l998-08-05 go Leu Glu Asp Gln Gln Val His Phe Thr Pro Thr Glu Gly Asp Phe Y His Gln Ala Ile Xi8 Thr Leu Leu Leu Gln Val Ala Ala Phe Ala Tyr Gln Ile Glu Glu Leu Met Ile Leu Leu Glu Tyr Lys Ile Pro Arg Asn Glu Ala Asp Gly Met Pro Ile Asn Val Gly Asp Gly Gly Leu Phe Glu Lys Lys Leu Trp Gly Leu Lys Val Leu Gln Glu Leu Ser Gln Trp Thr Val Arg Ser Ile His Asp Leu Arg Phe Ile Ser Ser His Gln Thr Gly Ile Pro Ala Arg Gly Ser His Tyr Ile Ala Asn Asn Lys Lys Met (2) INFORMATION FOR SEQ ID NO:5:
( i ) ~Uu~N~ C~ARACTERISTICS:
(A) LENGTH: 50 base pairs (B) TYPE: Nucleic Acid (C) ST~PN~N~S: Single (D) TOPOLOGY: Linear (xi) ~QU~N~ DESCRIPTION: SEQ ID NO:5:
25 GCGGCCGCGA GCTCGAATTC 'L'l-l-L-l"L-Lll'-l L1-1-1-1'-1-1-1'-1''1' 'Ll'-l"l'-l-l'-l-l'-l-l' 50 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1018 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
.~

CA 0224~63~ 1998-08-0~

GTGAAGGGAG CCGGGATCAG CCAGGGGCCA GCATGAGCCG &AGGGAGGGA 50 GGAGGCCAAG A1CC~L~AGA CACACAGCCT TGCGCACCTC CTCACCAAAT 150 5 GGGCTGCCCA G~11~1CGCC GCCGCGGCTG CCGGTGGCCG GCCTGAGCGC 250 CCCGGCTCCG AGCCACGCGG GG~LGC~AGT GCACGAGCGG CTGCGGCTGG 3 0 0 ACGCGGCGGC GCTGGCCGCG CTGCCCCCGC TGCTGGACGC A~'L'~'1'~'1'L~C 3 5 0 GGACGCGGCG CGCCAGGCCC GGGCCCTG&G CGCCGCCGTG GAGGCCTTGC 4 5 0 GCCACCGCCT CAGCCGCCTC CGCr~rCGGG ~ CCCCG CCAAGGTGCT 550 GGGGCTCCGC ~L"l 1GCGGCC TCTACCGCGA GTGGCTGAGC CGCACCGAGG 600 GCGACCTG&G CCAGCTGCTG CCCGGGGGCT CGGCCTGAGC GCCGCGGGGC 6 5 0 AGCTCGCCCC GC~'1'C~'LCCC GCTGG&TTCC ~L~L~LC~LL CCG~Ll'~L'l l 700 ~'1'~'L'1''1 ~L~'l' GCCGCTGTCG ~l'~L~L~L~l' GTCTGCTCTT AG~1~1~LCC 750 ATTGCCTC&G C~11~11LGC ~111L1~LGGG GGAGAGGGGA GGGGACGGGC 8 0 0 AGG~.~-1~1~ TCGCCCAGGC TGGGGTGCAG TGGCGCGATC CCAGCACTGC 8 5 0 ~AGCTGGGACT ACAGGCACGC GCCACCACAG CCGG~'1'AATT TTTTATTTAA 950 'I LLLlL~'LAG AGACGAGGTT TCGCCATGTT GCCCAGGCTG ~1~LL~AACT 1000 ( 2 ~ INFORMATION FOR SEQ ID NO: 7:
(i) S~:QU~'N~'~ CXARACTERISTICS
(A) LENGT~ 1018 base pairs (B) TYPE: Nucleic ACid (C) STR~NI ~1-:1 )NIC~ S Single (D) TOPOLOGY: Linear CA 0224~63~ 1998-08-0~
W O 97/30146 PCTrUS97/02675 (Xi) S~QU~N~ DESCRIPTION SEQ ID NO 7 CACTTCCCTC GGCCCTAGTC G~L~CCCCG~1 CGTACTCGGC ~LCC~1FCCT 50 TCAGACCTTC TGGGG~1~1G ACTAAGGAGT CAGAGTGAAG AAGGGGTGAA 100 CCTCCGGTTC TAGGCAGTCT ~L~1~1CGGA ACGCGTGGAG GA~lG~lllA 150 CCC~-~rr-GGT CGAAGAGCGG CGGCGCCGAC GGCCACCGGC CGGACTCGCG 2 5 0 GGGCCGAGGC TCGGTGCGCC rCr-~rGGTCA C~1G~1C~CC GACGCCGACC 300 GCG~1CCGGC TCGACTTGGG CGCGCGCGGC GCGGACGACG CGGCGGACCT 4 0 0 CG~LG~ACCC GGTCGACGAC GGGCCCCCGA GCCGGACTCG CGGCGCCCCG 650 TCGAGCGGGG CGGAGGAGGG CGACCCAAGG CAGAGAGGAA GGCGAAGA~A 700 TAACGGAGCC GGAAGA~ACG AAAAACACCC C~ 1~1 CCC~ 1 CCCCTGCCCG 8 0 0 A~AAAACATC 1~1G~1C~AA AGCGGTACAA CGGGTCCGAC CAGAACTTGA 1000 ( 2 ) INFORMATION FOR SEQ ID NO: 8:
(i~ SEQUENCE CHARACTERISTICS
(A) LENGTH 201 aminO aCidS
(B) TYPE AminO ACid CA 0224~63~ l998-0X-0~
W O 97/30146 PCTrUS97/0267S

(D) TOPOLOGY: Linear (xi) ~u~ DESCRIPTION: SEQ ID NO:8:
Met Ser Arg Arg Glu Gly Ser Leu Glu Asp Pro Gln Thr Asp Ser Ser Val Ser Leu Leu Pro His Leu Glu Ala Lys Ile Arg Gln Thr His Ser Leu Ala His Leu Leu Thr Lys Tyr Ala Glu Gln Leu Leu Gln Glu Tyr Val Gln Leu Gln Gly Asp Pro Phe Gly Leu Pro Ser Phe Ser Pro Pro Arg Leu Pro Val Ala Gly Leu Ser Ala Pro Ala Pro Ser His Ala Gly Leu Pro Val His Glu Arg Leu Arg Leu Asp Ala Ala Ala Leu Ala Ala Leu Pro Pro Leu Leu Asp Ala Val Cy~

Arg Arg Gln Ala Glu Leu Asn Pro Arg Ala Pro Arg Leu Leu Arg Arg Leu Glu Asp Ala Ala Arg Gln Ala Arg Ala Leu Gly Ala Ala Val Glu Ala Leu Leu Ala Ala Leu Gly Ala Ala Asn Arg Gly Pro Arg Ala Glu Pro Pro Ala Ala Thr Ala Ser Ala Ala Ser Ala Thr Gly Val Phe Pro Ala Lys Val Leu Gly Leu Arg Val Cys Gly Leu Tyr Arg Glu Trp Leu Ser Arg Thr Glu Gly Asp Leu Gly Gln Leu Leu Pro Gly Gly Ser Ala

Claims (28)

WHAT IS CLAIMED IS:

1. A method of enhancing the maintenance of pregnancy in a mammal into which an embryo has been introduced, the method comprising prior to said introducing, culturing at least one embryo in a medium containing an amount of CT-1 for sufficient time and under appropriate conditions so as to effect an enhancement of the maintenance of pregnancy in said mammal.

2. The method according to claim 1, wherein said mammal is selected from the group consisting of human, sheep, pig, cow, goat, donkey, horse, dog and cat.

3. The method according to claim 1, wherein CT-1 is of human or murine origin.

4. The method according to claim 1, wherein the medium for maintenance of the embryo is SOF
or M2 medium.

5. A composition, comprising pluripotential embryonic stem cells and CT-1, a fibroblast growth factor, membrane associated steel factor, and soluble steel factor, the factors present in amounts to enhance the growth of and allow the continued proliferation of the cells.

6. A composition, comprising primordial germ cells and CT-1, a fibroblast growth factor, membrane associated steel factor and soluble steel factor, the factors present in amounts to enhance the growth of and allow the continued proliferation of the cells.

7. A composition, comprising embryonic ectoderm cells and CT-1, fibroblast growth factor, membrane associated steel factor and soluble steel factor, the factors present in amounts to enhance the growth of and allow the continued proliferation of the cells.

8. A composition, comprising CT-1, fibroblast growth factor, membrane associated steel factor, and soluble steel factor in amounts to enhance the growth of and allow the continued proliferation of primordial germ cells.

9. A composition, comprising CT-1, a fibroblast growth factor, membrane associated steel factor, and soluble steel factor in amounts to promote the formation of pluripotent embryonic stem cells from primordial germ cells.

10. A method of making a mammalian pluripotential embryonic stem cell, comprising administering a growth enhancing amount of CT-1, a basic fibroblast growth factor, membrane associated steel factor, and soluble steel factor to primordial germ cells under cell growth conditions, thereby making a pluripotential embryonic stem cell.

11. A method of making a pluripotential embryonic stem cell comprising administering a growth enhancing amount of CT-1, a basic fibroblast growth factor, membrane associated steel factor, and soluble steel factor to embryonic ectoderm cells under cell growth conditions, thereby making a pluripotential embryonic stem cell.

12. A method of stimulating the proliferation and differentiation of mammalian satellite cells into myoblasts, which method comprises contacting said cells with a stimulation-effective amount of CT-1 for a time and under conditions sufficient for said satellite cells to proliferate and differentiate into myoblasts.

13. The method according to claim 1 which further comprises the addition of one or more other cytokines in simultaneous or sequential combination with CT-1.

14. A method of myoblast transfer. comprising contacting mammalian satellite cells with a proliferation- and differentiation-effective amount of CT-1 for a time and under conditions sufficient for said satellite cells to proliferate and differentiate into myoblasts and then administering said myoblasts at multiple sites into muscles.

15. A method of treating a neoplastic disorder, comprising administering to a population of cells that comprise neoplastic cells of a patient in need of such treatment a therapeutically effective amount of CT-1.

16. The method of claim 16, wherein the neoplastic disorder is selected from the group consisting of a carcinoma, sarcoma, melanoma, lymphoma. and leukemia.

17. The method of claim 16, wherein the neoplastic cells are in vitro.

18. The method of claim 17, wherein the CT-1 is administered bone marrow to eliminate malignant cells from marrow for autologous marrow transplants.

19. A method of treating a mammal afflicted with arthritis or an inflammatory disease, comprising administering to the mammal in need of such treatment an amount of CT-1 antagonist which is effective for alleviation of the condition.

20. A method of treating a neuron other than a ciliary ganglion neuron, comprising providing the neuron with an amount of CT-1 affective to promote neuronal survival, growth, regeneration, sprouting or a change in neuronal type.

21. The method of claim 20, wherein the neuron is a central nervous system neuron.

22. The method of claims 20 or 21, wherein the change in neuronal phenotype is in the transmitter phenotype of the neuron.

23. The method of any one of claim 20 to 23, wherein the neuron is in vivo.

24. Use of an effective amount of CT-1 in the preparation of a medicament for the treatment of a neuron other than a ciliary ganglion neuron, to promote neuronal survival, growth, regeneration, sprouting or a change in neuronal type.

25. Use of an effective amount of CT-1 according to claim 24 wherein the neuron is a central nervous system neuron.

26. Use of an effective amount of CT-1 according to claim 24 of claim 25 wherein the change in neuronal phenotype is in the transmitter phenotype of the neuron.

27. Use of an effective amount of CT-1 according to any one of claims 24 to 26 wherein the neuron is in vivo.

28. The method of treating a neuron other than a ciliary ganglion neuron, comprising providing the neuron with an amount of CT-1 effective to promote neuronal survival, growth, regeneration, or sprouting.