CA2218529A1 - Nucleic acids for treating obesity - Google Patents

Abstract

A nucleic acid molecule is provided that can be expressed in a host cell to produce a biologically active ob polypeptide that can effectively inhibit food intake and/or weight gain. Vectors and host cells containing the nucleic acid molecule are also provided, as well as methods for producing the ob protein and other ob polypeptides, methods of induction of the production of the ob polypeptides, such as by in vivo or ex vivo gene therapy, and methods for inhibition of food intake and/or weight gain. Further provided are antibodies to the ob polypeptides and methods of using such antibodies, such as for identification or detection of other ob polypeptides or homologs thereof, and for inhibition of ob polypeptide activity. A method for identification, detection, or isolation of an ob receptor is provided as well as methods for production of antibodies to the ob receptor. The antibodies and polypeptides herein can be incorporated in kits for immunoassays. Pharmaceutical compositions containing the ob polypeptide and antibodies to the ob polypeptide or to the ob receptor can be used for administration to animals and humans.

Description

CA 022l8529 l997-ll-05 WO 96135787 PCT/US~6/0660 Nucleic acids for treating obesity Field of the Invention This invention relates to the discovery that an ob polypeptide can be c~e-;~ivcly 5 cA~lessed in a host cell using a nudeic acid moleclllp~ that places the ob gene under re~ tc ry control of an eA~l ession control sequence that is not naturally ~cso~ i~tçd with the ob gene. Moreover, the lecollll~ill~llly ~,A~lcssed ob poly-peptide is biologically active in inhihiting food intake and/or weight gain. This invention also relates to vectors and host cells comprising the nudeic acid moleclllP, mPtho~c for pro~l~lcti~n of an ob 10 polypeptide, for induction of production of an ob polypeptide, for inhihition of weight gain and/or food intake, for ke~tmPnt of obesity, for inhibition of the activity of the ob polypeptide, for idPntific~ti~n detection and isolation of an ob lecep~or, and for itlPntific~ti~n or detection of an ob polypeptide or a homolog thereo~ This invention further relates to an ob poly-peptide produced in accold~lcc with the mpthods as15 mPntionp~ antibodies to ob polypeptides, a method for icQl~ti~n of antibodies to ob ccc~lol~, kits Colll~ illg such antibodies, and pharm~ce~ltir~1 compositions co...~ p ob polypeptides.

Bac~ oul~d of the Invention Obesity, the con~iti~n ofcAces~;v-e~ccllmll1~tion of fat, or cAces~i~e adiposity, affects 30% ofthe human poplll~tion and is a serious health hazard. It is generally believed that appetite, energy ba1ance and body weight gain are modlll~ted by diverse neuro~ h~mi~ ~l and neuroendocrine signals from di~cl cnl organs in the body and diverse regions in the brain. The hypoth~l~mllc plays an illlpolL~lL fim~tion in this process, acting through a variety of ~y~Lellls that involve a close interaction between nutrients, amines, neulopeplides and ho. ~..on~c~ as noted in Leibowitz, Trends in Neurosciences (1992) 15:
491-497.
~ Two peptides in the brain have r-ccc;vt;d cQn~;dPrable ~tpntiQn with respect to their role in modll1~ting behavioral and physiological fi ~ c çcs~ l to ,.. -l - ;~ and 30 energy b~l~nce They are neuropeptide Y (NPY), a 36 amino acid peptide mPmber ofthe pancreatic polypeptide family, and galanin (GAL), a 29 amino acid chain that is ~mi~l~ted WO 96/35787 PCT/US~)G/OG60~) at the COOH t~ s Both peptides are dense1y conrPntrated in the brain and particularly in the Lyln~ll.Al~mll~, which also co~ .c high levels of the rnRNA and rect;~lor sites for these peptides as desc-il,ed in Leibowitz, MODELS OF
NEI~RO~ E ACTION, Ann. N.Y. Acad Sci. (1984) 739: 12.
s In ~ltlitinn other e~eeLo,~ have been found to have a role in mo~ ting mltri~nt ing~etion and metabolism, inrl~lrlin~ the gl~lr,ocorticoid corticosterone (CORT), as described inJ. Nezlroer~ (1994) 6: 479-501; the d~lo~l~on~l peptide cholecysto'inin (CCK), as desc-il,ed in Smith and Gibbs (1992), MULTIPLE
CHOLECYSTOKININ RECEPTORS IN THE CNS (Oxford Univ. Press, Oxford) pp.
10 166-182; and insulin, as dp~rrihed in Scl-w~u ~ et al. (1994), EndocrinoL Rev. 2(1): 109-113.
Other researchers have approached the problem of underst~n-lin~ the re~ tinn of food intake and energy output by genetic analysis of ar~imals c~u-ying mutant genes. The first ofthe recessive obesity ~llt~tinn~, the obese mllt~tinn (ob) was i~lentified and 15 rl~rrihed in 1950 by Ingall et aL, J. Hered (1950) 41: 317-318. .~.l,se.~ ly, 5 single-gene mllt~tiQn~ in mice have been observed to produce an obese phenotype, as ~es~ ~ ;bed in Frilo~lm~n et al. (1990), Cell 69: 217-220. More recently, the mouse obese gene and its human homologue have been cloned, as descrihed in Zhang et al. (1994), Nature 372:
425.
Zhang ef al. reports the clor~ing and sequ~nring ofthe mouse ob gene, which ~nrodF!e a 4.5 kiloh~ce ~lirose tissue ~ ccPl~er RNA (rnRNA), with a highly conse. ved 167 amino acid open reading frame. Based upon the nudeotide seq~lenr-e and the de(11~ced amino acid sequenre ofthe open reading frame, Zhang ef al. post~ tes that the product ofthe ob gene is a 167 amino acid ob protein, pl~ bly having a signal peptide 25 at the N-tel-----.us of the ob protein. Zhang ef al. does not show, hc,w~ver, that the 167 amino acid polypeptide is indeed secreted, or what, if any, part of this polypeptide ~s actually sec-eled. Mo~e~el, Zhang ef al. ~so does not show that the polypeptide product of the ob gene has any particular bioactivity or fi~nrtir n Strategies for he~ for obesity in the past have inrl~ded dieting, surgery 30 (lipectomy), and drug tl-~ os incl~l-ling the insulin norrn~li7~tion drug Ro 23-7637, antilipolytic agents such as SDZ WAG 994 developed at Sandoz Research Tn~

CCK-A ~pnnict~ such as FPL 15849KF developed at Fisons Pharm~ce~ltic~l~ and those ~ developed at Glaxo Research ~n~tit~te, the ano,~;~ic d~ - rr-.n-.. ~l~le developed at Lederle Laboratories, the S_.OtOl.lll uptake inhihitQr, fllloYPtinp~ developed at Lilly Research Laboratories, the drug ;~ uL~ e developed at Boots Pharm~ceutic~l~, the5 anti~ hetiC of ~-adrenergic receptor ~oniet~ developed at Bristol-Myers Squibb and American Cy~n~m;~, and ente;l o~ and therm~ ~Pnic agents developed at Amylin p~ "";~ stl~
To date, despite these various mPtho~l~ of tre~tm~ont long term ~ cco~ely from the cQnr1itirn of obesity is rare. Further, ~lth~f~llgh the ob protein has been irl~ntifiy1 in mice 0 and its hsm~lc~-e identifiPd in hllm~n~ an exact d~lel . . .;~ ;on of the amino acid seqllence ofthe mature native ob protein has not been made, nor has the exact sequ~n~e ofthe serum form ofthe molec-lle been determined. Notably, the rel~tion~hir or interaction bcLwt;ell the ob protein and other factors that affect beh~vc lial and physiological fim-~ti-~n~ es~nti~l to mltrient and energy ba1ance is still very much a 15 mystery.

Summary of the Invention It is, lllt;lef~le~ an object ofthe present invention to provide a better underst~n~ling ofthe filn~tion ofthe ob protein and a better undt;l~ g ofthe 20 re~ tory mec.l-~t-;l~govt~ lg food intake and/or energy output.
It is also an object of the present invention to provide a nucleic acid molccllle that can be eA~Iessed in a cell to provide a po1ypeptide that can be used for conkol of obesity and the problems ~so~i~ted with obesity, such as type II di~h~;lf~ ~ The polypeptide ~nCodecl by such a nucleic acid molcclllP, is r~Ç~Ilt;d to herein as the ob polypeptide.
It is another object ofthe present invention to provide a nucleic acid mrl-_ le as above that ~drliti-f~n~lly co..~ a secretion leader coding seqllPnce to allow secretion of the ob polypeptide from a host cell upon eA~ress;on.
~ It is an~ el object of the present invention to provide an ~ ;,;,;oll vector co..~ such nucleic acid mrle -~lles and host cells c~ P such vectors.
It is also an object ofthe present invention to provide a mPthod for prodll~in~ the ob polypeptide, for ~ , by rec~ DNA techniques.

It is still another object of the present invention to induce the prod~lction of the ob polypeptide.
It is yet another object of the present invention to provide a method for inh;l-itinn of food intake andlor a method for inhihiti~n of weight gain.
It is also another object of the present invention to provide a method for blocking the activity of the ob polypeptide.
It is also an object of the present invention to provide antibodies to the ob polypeptide, ph~rm~re~ltir~1 compositions co~ such antibodies for thel~p~uL;c purposes, and kits for detectil~n of an ob polypeptide, or a homolog thereof, co. .1 ;.; .-:- .p o such antibodies.
It is still another object of the present invention to provide mPthods for ico1~tiQn j~lPntifir~tinn detecti~n~ or prod~lction of a ~ tic~Lor of the ob polypeptide, that is, an ob receptor, a method for idPntifir~tiQn and/or detectinn of an ob polypeptide or a homolog thereof, a method for producti- n of ~ntibo~lip~s to the ob leceplor.
It is yet another object of the present invention to provide a method for inh;llitirn of the activity of an ob polypeptide.
In accordance thereto, there is provided herein a nucleic acid mslec~lP that CQnt~inc a first mlrleoti~le sequPnre that Pnr~ocles an c~y,t:~ion control sequPnre and a second nucleotide sequenr,e that encod~Ps an ob polypeptide, the second nucleotide sequP-nre being under re~ll~t~ry control ofthe first nllrlPotide sequence, and the first mlrleoti~le seqlJenre is not naturally ~ccori~ted with the second rnlrleotirle se~l~r ~ e In acccs-d~lce to another object ofthe present invention, there is provided a nucleic acid mnleclllP as above, further co~ a third nllrhpotirle seqllPnrP, the third n~lrleotide sequence Pnr.o-1in~ a secretion leader sequ~nre that is sllffir;ent for secretion of the ob polypeptide upon cA~,~e~;on ofthe nucleic acid msle~llP, in a host cell.
In acco-d~lce to another object ofthe present invention, there is provided herein an ~,A~ ;on vector and a host cell co--l ~ the CA~ ;OII vector which CQ--l i.;~ ~C the nucleic acid m-~~~,lllP as above.
In acco,dallce to a further object of the present invention, there is provided ameth9d of production of an ob polypeptide by providing the nucleic acid mslccllle as CA 022l8529 l997-ll-05 above, introdllcin~ the nucleic acid mol~r,~le into a cell and allowing eAples~ion ofthe ob polypeptide in the cell.
There is further provided, in accordal-ce to another object of the present invention, a method of prod~ction of the ob polypeptide by providing the vector as above, 5 introd-~ring the vector into a host cell, and allowing ~,~ression of the ob polypeptide in the cell.
There is also provided herein a mPtho~l for prod~lctiQn of an ob polypeptide by providing a host cell ~ ro..,~ed with the vector as above and allowing cA~,e.,;,;on ofthe ob polypeptide in the cell.
o In accordallce to still another object ofthe present invention, there is provided a mPth~l for in~ ctiQn of pro~l~lcfir,n of an ob polypeptide in vivo by a~ the nudeic acid molPc~llP, as above, either directly or by viral or non-viral means, or by a~minieterin~ the vector as above.
In accoldallce to yet another object ofthe present invention, there is provided an ob polypeptide produced by the process of providing a host cell l~ ru~ ed with the nucleic acid molcclllp~ or the vector as above, and aUowing the eA~"cs:~ion of the ob polypeptide in the host cell.
In accordallce to still another object ofthe present invention, there is provided a mPthod for inhi1~ition of weight gain and/or a method of inhibitirJn of food intake by ~ ion of a lht;l~ l;r~lly effective amount ofthe ob polypeptide.
In acco. dallce to yet a further object of the present invention, there is provided a method for identifir,ation, i~ol~tion detecti~n or prod~lcti~m of an ob Iccc~lor by providing a labeled ob polypeptide, allowing the labeled ob polypeptide to react with the ob lcceplor to form a binding pair, and dclc ..,inillg the identity ofthe binding pair, in 25 particular, the identity ofthe mole ~le binding to the labeled ob polypeptide.
In accordance to still anoll-cr object of the present invention, there is provided an antibody to the ob polypeptide as above and a method of producing an antibody to an ob rcc~lor, the antibody to the ob polypeptide being capable of r~J....;..g a specific binding pair with the ob polypeptide, and the antibody to the ob lecc~ lor being capable of r~....;.~g 30 a specific binding pair with the ob rcceplor.

WO 96135787 PCTIUS~6/0~09 In acco-dal~ce to another object ofthe present invention, there is provided a method for blocking the activity of the ob polypeptide ~,vith the use of inh;l-itors thereto, such as ~ntiho~lies to the ob polypeptide.
In accordance to a further object of the present invention, there is provided a 5 method for id~ntifi~tion or detection of an ob polypeptide or a homolog thereo~, involving cont~ctin~ an antibody directed to an ob polypeptide as above ~,vith a sample suspected of CQ~ p an ob polypeptide or a homolog thereof, allo-,ving the ll~lule to react to form a specific binding pair, and del~ .. .;. .;. .~ the pl esence of a specific binding pair, the antibody being labeled with a detect~ble marker to f~rilit~te easy irl~ntifir,~tinn or o detection There is further provided, in acco. d~ce with another object of the present invention, a kit for detection of an ob polypeptide or a homolog thereof, and a kit for detection of antibodies to the ob polypeptide, the kits co.~l ~;. .il .~ either labeled antibodies or labeled ob polyp.eE!titl~e There is also provided, in acco-d~lce with a further object ofthe present invention, a ph~....~ce.,~ l composition co..l~ the ob polypeptide and a pharm~ce~ltir~lly ~cept~le carrier and a rh~rm~ce~tir~l comrosition c~..l~;-.;..Smtihorlies to the ob polypeptide and a rh~rm~ceutir~lly ~cc~l;~blc carrier.
Further objects, re~lules, and advantages ofthe present invention will becomP!
20 ~)p~ellL from the following det~iled desc-rirtion It should be understood, however, that the det~iled description, while in-lir,~tin~ pl~relled embo~im~nts ofthe present invention, is given by way of illustration only, since various r~ ,e5 and mt~r1ifir~tir~n.e within the spirit and scope ofthe invention will become a~)alelll to those skilled in the art from this det~iled description.

Brief Description of the Dl ~w~ll~,S
Figure 1 shows diagr~m~tir-~11y the DNA constructs each COIllpl;Slllg a rnlf lootide seq~l~nre that ~nrodes an ob polypeptide. The llullll)el~ on the left: #1122, #1123, #1124, #1132, #1130, #1131, #1119, #1129, #1127, #1150 and #1128 are the de~ l~d 30 constructs "u",ber. The term "CMV" in~ te~s that the plc,lllolcr in the Co~ hu~;l iS

derived from ~;yk~---Ps~loVirUS ("CM~'). The term "T7" in~lic~tPe that the promoter in those constructs is a T7 p,u"-oler.
Figure 2 shows diag.~------~l;r~ y other DNA constructs each COlll~l;Sill~; a nucleotide seq~enre that encodes an ob polypeptide. The ~-un~ on the left: #1144, 5 #1142, #1143, #1145, and #1147 are the deci~ted constructs ~-u~l~ber. The term "SR~x"
indir~tPe the source of the p~c .-.o~c (SV4ûtH~V hybrid promoter). Constructs #1145 and #1147 each cc ..,I,.ises viral sequPnces from ~nlcnP-y murine lel~kPmi~ virus.
Figure 3 reflects the effect of intravenous ~ el ~ ~lion of the ob protein C~pl essed from construct #1127 on the weight of treated CD rats as CO~pd- cd to that of o the u~lLlealed controls.
Figure 4 shows the amount of food co~ ~-..plion by the CD rats treated with ob protein as cc ~.,p~cd to that of u"l, c~led controls.
Figure S shows the weight of the fecal matter excreted by CD rats treated with ob protein as co...pa~ed to that of u~ ealed conl~ols.
Figure 6 shows the urine output of CD rats treated with ob protein as co...~ cd to that of u--t ealed controls.
Figure 7 shows the amount of water intake by CD rats treated with ob protein as co---palcd to that of u~ aled controls.
Figures 8a and 8b show the ~ ~:u...c.-l map of DNA construct #1122.
Figure 9 shows the nucleotide sequenres of DNA constructs #113û, #1131 and #1132, ~G~I.e~ rely, and ~li~.--.---.l~ ofthese seq~lPnrPs Figure 10 shows the ~:u---c ~I map of DNA construct #1119.
Figure 11 shows the ~ v u--lw~t map of DNA construct #1127.
Figure 12 shows the ~;u---c ~I map of DNA construct #1150.
Detailed Desc,i~.Lion ofthe Plerc--cd Embodi...~ s The invention dpsr-rihed herein draws on previously p~lblich~pd work and pPnrlinf~
patent apFlir~finnc By way of; . '-, such work co~ of sc;~nfific papers, patentsor p~ patent applir~tionc All pllhliched work inr~ in~ p~tPntC and patent 30 applic~tionc cited herein are hereby i~lco.~o.~led by ~crc.cnce.

CA 022l8529 l997-ll-05 WO 96/35787 PCTIUS~6/0 J~O9 The inventors herein have discovered that a nucleic acid molecule encoding an obpolypeptide can be cA~Icssed in a ,eco~ 1 cA~"cssion system to produce the obpolypeptide. The nucleic acid mnlec lle can also be used in the context of an cAl les:,;o vector that contqinC one or more ~A~Ic~sion control sequences for cA~.Ic~ ;on ofthe ob 5 polypeptide in a host cell. Mo.cvvcr, the nucleic acid mslcclllp~ can be used for gene therapy purposes for pro~h~ctirn or inrl~ctisn of pro~lctisn of the ob polypeptide, for example, in ex vivo or in vivo gene therapy, the nucleic acid msleclllç to be delivered either directly or by viral or non-viral means.
The ob polypeptide produced herein is useful for inhibition of food intake, and/or o inhibition of weight gain and, in PcCPnr,P., in the ~ ll of obesity or CQnceql lPnrPc Of obesity inr~ in~ type II ~liq-hetec The ob polypeptide can, furthermore, be used for prod~lction of mnnorlonql or polyclonal antibodies which, in turnJ can be used, for PYqmplç, in immllnsqcs<q,ys for cletectisn or idPntifi~,q,tisn of an ob polypeptide or a homolog thereo~ Inhibitors to the ob polypeptide, such as antibodies, can be used to 15 block the activity of the ob polypeptide. Such blorl~in~ activity is useful, for PYqmrle, for stimlllqtinE appcliLe in s.,l,;cvl ~ sllffrrinp from poor food intake and/or poor mltritinn res~llting from, for s , ~- ~t;~eqCçs or chronic con~l;l;ol~c such as anol.,Aia nervosa, psy.,l~iallic con~litinnc~ or during recovery from surgery. The qntiho~liP~s to the ob polypeptide, therefore, can be i Ico",o,~Led into a kit that can contain other cc"~ l;nnq-20 reagents for immlmsqccqys or into a pharm~qce~ltir,ql composition for Lllcl~cuLicq~lminictration. The ob polypeptide can also be labeled with an i-lPnfifiqhle marker, such as a ra~1ioq-ctive marker, and be used to detect the prcsence of an ob ~cccplor that sperifirq-lly binds the ob polypeptide, ru~ g a binding pair. phqrmqre~tirql compositinnc CG~ g the ob polypeptide can also be made for ~ lion and 25 Il,;;;.l,,,,~,,l The ob rece~lor idçntified in this manner can be sequenced, and used to make a probe for pl~ing a cDNA library to obtain a coding sequence. This ob l~c~lol coding sequence can then be used to make the ob ,ecel,~or ~cco...l.;.~"lly in the same manner as for the ob polypeptide. Polyclonal and mnnnrlonql antibodies to the ob lcce~lor can be 30 made which can also be used in a kit for ~etectinn or i(~Pntifirqtion of an ob l~ceplur.

_ 9 _ Notably, the inventors herein have found that a mature ob polypeptide can be ~ effectively cAl"cssed using ~ec~ DNA te---hnology and that after p-lrifir~qti~n or partial purifir-qti~r~ the c Al.,cssed ob polypeptide has biological activity. Such biolo~
activity in~ d~Ps the ability to inhibit weight gain and/or food intake. No toxic effects of the qrlminictration ofthe ob protein have been observed.
The present invention may be better understood in light of the following finitione inco,~o,~led herein.

Definitions o A "nucleic acid moleclllP," or a "coding seq~Pnce," as used herein, refers to either RNA or DNA that Pncodes a specific amino acid sequPn-- e or its cQmrlP~ strand.
The term "an cA~,c:j~ion control sequPn--,e" refers to a sequPn--,e that is co"vc~.l;on-qlly used to effect cA~"e~i,;on of a gene that en--,oclPs a polypeptide and include one or more CO~IpOl-c IIS that affect eA~.t~:~;ol~, inr~ in~ scliplion and tl;~ l;nn signals. Such a sequen-- e in.- h~d~Pe for PY~mple~ one or more of the following: a pl~ ùltl seqUPn~p~ an çnh~n-- rr sequ~Pnce, an upsl, w"- activation seq lencP, a duw~
t~ m sequPnc-P, a polyadc ,ylation sequ~PncP, an optimal 5' leader se~ e to Opl,l~c initiation of l,~ ;ol- in ".,~ n cells, and a Shine-Dalgarno se~ e. The eA~Ic~:~;on control seq~lPn--,e that is apprûp-ialc for _Aplc~ion ofthe present polypeptide differs depending upon the host system in which the polypeptide is to be eA~-c;~ ~cd. For pl- ~ in prok~yoles, such a control sequPn--,e can include one or more of a pl ullloler seqllPn~e, a ribosomal binding site, and a ll~s-,liplion te~ .. ~;-. ~l ;on seqUPnt~e~ In euk~yoles, for ~. , 'e, such a seqluPn~e can include a p~olllolel sçqu~Pnce~ and a ll~lscli~lion tell---~ n seqUPnce If any ll~cPCc-~y comronPnt of an eAples~;oll control sequPn-- e is lacking in the nucleic acid mqlccllls of the present hlvelllion~ such a CC~ POI1C11I can be supplied by the cAl,lession vector to effect CA1J1C~;~;OI~ EA~1C~ iOII
control sequences .s~liti1ble for use herein may be derived from a prokai-yotic source, an eukai-yotic source, a virus or viral vector or from a linear or circular pl~mirl Further details rcg~ding eAprcssion control seq~lences are provided below.
The term "ob plUtcill" refers to the putative murine polypeptide cQ~ g a sequPnce of 167 amino acid residues predicted from the i~ol~ted cDNA Pn.-,o-ling the ob CA 022l8529 l997-ll-05 gene, as weU as its homolog in .. ~ n and non-.~.. ~li~n spec;ee as descrihed in Zhang et al., mPntionPd above.
A "mature ob protein" refers to the ob protein as above except that it lacks thepuL~livt; signal peptide sequence.
The term "ob polypeptide" inrl~ldes the ob protein and the mature ob protein as defined above and further include tr~n~ ~tione variants, aUelles, analogs and derivatives thereo~ Unless spe~ifir~lly mPntioned otherwise, such ob polypeptides possess one or more of the bioactivities of the ob protein, such as those discuvt~red herein. This term is not l~mited to a specific length of the product of the ob gene. Thus, polypeptides that are lo icl~nti~ ~l or contain at least 60%, p-~r~l~bly 70%, more preferably 80%, and most pl~rc:l~bly 90~/0 h~mnl~y to the ob protein or the mature ob protein, ~vl-e,t;vel derived, from human or nonhllm~n sources are in~ ded within this dPfiniti-:>n ofthe ob polypeptide. Also in~l~lde~l therefore, are alleles and ValiallL~i ofthe product ofthe ob gene that contain amino acid ~.l,s~ .l;one dPhPtinne or insertions. The amino acid s~lbstit ltione can be conselv~Live amino acid s~lbstihltione or s~il,sl;l.~l;onc to ~ le non-eccenti~l amino acid residues such as to alter a gly~ûsylalion site, a phosl)hnlylation site, an act;lylalion site, or to alter the folding pattern by altering the position of the ~,y:,~eil~e residue that is not necP,~y for filnctinn etc. Conservative amino acid ;~.l,s~ ;one are those that p. est;l ve the general charge, hy-ll ul hnbi~ ;Iyt}l~dl ophilicity and/or steric bulk ofthe amino acid ~ubsl;l~le~1 for ~ ",pl~ l;onc b~L~n the mPmhPrs ofthe foUowing groups are conservative s Ibsfihltionc Gly/Ala, Valme/Leu, Asp/Glu, LystArg, Asn/Gln, and Phe/TrptTyr. Analogs include peptides having one or more peptide mimirs, also known as peptoids, that possess ob protein-like activity.
TnClnded within the d~l~..-l;n~ are, for; . '~, poly~c:p~ides co..lS~ P one or more 25 analogs of an amino acid (in~ lin~ for ~ . le, Ul~llalU1;3l amino acids, etc.), polypeptides with ,~,l,~l;l.,~ed lin~Pe, as well as other mn-lifi~ti~me known in the art, both naturally OC~iul~ g and ~ lly oc~ P The term "ob polypeptide" also does not exclude post ~ lession mo~1ific~tiQnc ofthe polypeptide, for ~ .~.nl,k, ~;lyco~ylations, acetylations, phosphorylations and the lilce.
The term "leader seq~lPnce" refers to either a ~ ed amino acid seq~lpn~
5' to the N-t~ s of a polypeptide sP~q~lPn~e to be t,A~I .;ssed, or an CA 022l8529 l997-ll-05 u~LI~llcl~ted nucleotide sequPnre This term inrlu(les at least one ofthe following and can be a co---bi--alion thereof: a secretion leader sequP-nrP~ as defined below, a fusion protein leader sequpnre~ and an unl~ ed rnlrleotitls seqllPnee The tr~n~l~ted amino acidleader seqllpnce can be used herein to oy~ ~e secretion, as in a secretion leader 5 seq~lPnrP ~ItPrn~tively~ the tr~n~l~tPd amino acid leader sequPnce can be used to c,pLi,-"~,e initi~tinn of l~;~n~l ~l ;nn, as by the use of all or part of a tk leader seq~l~nre.
Moreover, the amino acid sequence used to oyli~l~e initi~tion of l~ on can be used in colllh;ll~l;on with a secretion leader sequence. A fusion protein leader seq~lP-nce can be used to oyli~ e intr~Cp~ r pro~uction of a polypeptide such as an ubiq-~itin/ob lo polypeptide fusion protein for intracelllll~r ~,Ay,es~ion in yeast. A 5' unl~ led leader sequenre can be used to oyli--~e l-a ~sc,iylion~ if desired.
The term "secretion leader sequence" refers to a polypeptide that, when enro~1edat the N-terminus of a protein, causes the protein to be secreted from the site of ~ e:~;
typically the endoplasmic reticl-lllm, to another loc~tinn, such as the peripl~emic space in 15 proLq,yoles or extr~rPlllll~rly into the culture mPrlium in which the host is being prop~g~le~l The secretion leader sequence can be a signal peptide sequence or can include other seq lPnres that include glycosylation sites or proCP,s~inF~ sites for pro~luctinn of a mature protein. Such sequPnr,es can be derived from any source that is sllit~'c for cAylession in the desired host or can be hybrid sequ~Pnres or synthetic seq~Pnces For 20 eY~mrlP~ sl~it~ble secretion leaders for use in yeast include the Sacc~v".~ces c~ ;ae a-factor leader (U.S. Pat. No. 4,870,008), a-factor leader sequPncP" ~ e~e~l a-factor leader sequ~pncp~ yeast killer toxin leader seqUPnrP, and a-amylase or ~ co~..ylase leader sequPnre Hybrid leader sequence can inrl~l~P, for PY~mpl~ a signal peptide sequPnre linked to a p,oc~~ g site for pro~uctinn of a mature polypeptide upon deavage at the 25 procPe~in~ site, for PY~mrtC~ a yeast i-,~, ~se signal s~PquP-nce can be used in co...l~i~u~l ;on with a KEX 2 deavage site ~Lys-Arg) to produce a hybrid leader sequPnre More~ r,random peptide seq-lPnres for any host eAy~;ss;ol- system such as those g~n~led by a co~ 1 library can be scl~,ned for ones that are useful as leader seqUpnrpc for a desired host. RaCtPt ;~1 leader se4~e~-ces useful herein are ones that lead to the productinn 3 0 of a polypeptide that is se~ ~;led into the p~ ;pl ~" ~c space. For eY~mple~ e~
signal peptide sequence. M~mm~ n leader seq lPnre~s include leader sequPncPs of CA 022l8529 l997-ll-05 pluleillS that are normally seweled into the serum inr1l-riinF~ for; , '~ prùle"~s such as zllhllmin, immllnoglobulin, Factor VII, secreted ho~ >~pc blood-borne factors such as insulin, growth factors or can be sequ~nces derived from fat cells. The following genes also have leader sequences that f~rilit~te secretion from ...~ .n~ n cells and can be used 5 for the secretion of heterologous proteil.s in ...~ n cell systems: human inflllPn7~
virus A, human p~e~ù;..~ in~ and bovine growth hn....ol1P" among others. Further details ~êgalding secretion leader seqU~Pne~ps are provided below.
The term ~ gain in1--hilu,y amount" refers to that ~mollnt that is e~e~;Live forproductionofinhihitionofweightgainofanindividual~ Thepreciseil-1.;h;~c,.yamount o varies depending upon the health and physical con~itinn ofthe individual to be treated, the capacity of the individual's ability to adjust to the change in metabolism and body size, the form-~1~fiQn, and the ~ttpntlin~ phy~ ,e~ ofthe mP~lir~1 sitl~finn~ and other relevant factors. It is PYpected that the amount will fall in a ~ ~,laLiv~ ly broad range that can be ~le~ (1 through routine trials.
The term "food intake ;~.h h;lul~l ~.. ou.. ~" refers to that amount that is ene~,livc for pro~ ction of inhihitinn of food intake of an individual. The precise i.-k.h;l o. y amount varies depend.l~g upon the health and physical con~itinn of the individual to be treated, the capacity of the individual to adjust to the inhihifion of food intake, the fonn~ tinn and the ~tt~Pn-lin~ pl~y.,;~ 's ~r7~ .e-.l ofthe mPr~ it..~fiQn and other relevant factors. It is e.~eeled that the amount will fall in a lelalively broad range that can be ~lel~ e~l through routine trials.
A Ll.e.a~cuLically effective allluulll" is generically that amount that will genelale the desired thel~eulic ù~llcnl~p and inr~ p~ for PY~mp1 ~, a "weigll~ gain ;~k.~ Q~y ~WUIl~'' and a "food intake inl~ oly z~mm~nt ~
The term "ob réce~or~7 refers to a structure, generally a protein, located on or in a cell Illeu~ e that sperifir~11y recognizes a seq~uPnr,e of amino acids of the obpolypeptide so as to bind to it with a higher af~inity than to a random polypeptide, r a binding pair. Such an interaction between the ob polypeptide and the ob lècelJlor is l~ec~ecl to trigger an intr~cP~ r .c;,~onse.
The term "binding pair" refers to a pair of mo1ec1-1P~ usually ler~ g to a protein/protein pair, but does not PYc1~e a protein/DNA pair, in which the cn~

ofthe pair bind specific~lly to each other with a higher affinity than to a random m-~ler~ "
~ such that upon bin~lin~ for c _ p'e, in case of a ligand/.ece~lor interaction, the binding pair triggers a cellular or an intr~cto~ r re~,~ol se. An eY~mple of a ligand/~-,cel~lol binding pair is a pair formed between PDGF (platelet derived growth factor) and a PDGF
5 ~~ceplor. An ~ p'c of a di~;lw-l binding pair is an antigen/antibody pair in which the antibody is gtne.aled by ;.. ;,~ n of a host with the ~nti~n Specific bindingin-lir~tes a binding i.,~e ~;~ion having a low rlieeori~ti~n co~ ..l which .l;.;l;.,g,.;~ e specific binding from non-specifir, bac~u-l--d, binding.
The term "kit" refers to a p~cl~ge CQ..I~i,.i.~g the specified material and incl~de$
10 printed instructions for use ofthe material. For rY~mrl~, the kit can be an immlml~ee~y kit cc..~ ntibodies to detect an ~nti~Pn, such as an ob polypeptide or an obrecel~lor, or it can be an assay kit cc,~ ntig~ne to detect ~ntiho~ies "Printed instructions" may be written or printed on paper or other media, or co..~ d to electronic media such as ~ elir, tape, co...l~"er-readable disks or tape, CD-ROM, and 15 the like. Kits may also include plates, tubes, dishes, ~ nte solvents, wash fluid or other co..~ ;on~1 reage..L~,.
The term l~h~....~r,e~ltir~lly ~ e~:~b!c carrier" refers to a carrier for minietration of a the-~;u~ic agent, such as antibodies or an ob polypeptide, in vivo, and refers to any ph~ ~ r";,l I ;r~l carrier that does not itself induce the pro~lctiQn of 20 antibodies h~rmfil1 to the individual receiving the co---posilion, and which may be minietrred without undue toxicity. ~S~h~ble carriers may be large, slowly metabolized ...ac.o...o!ec~les such as p-oleil-s, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ord.na ~ skill in the art. Pl-~ .~ ~~ce~tic~lly ~- ~ eFt~b!~ salts can 25 be inrl~lded therein, for ~ e, mineral acid salts such as hydrorhk!ndre hydrobromides, ph-~sl.k~l ~c s~1f~t~c and the like; and the salts of organic acids such as acet~t~C, propion~tçC, m~lon~t~C, b~ oa~ec~ and the like. Athorough ~liccl1c~;On of pharm~ce~tir~11y arceFt-~le ~Yripientc is available in REM~GTON'S
PHARMACEUTICAI, SCIENCES ~Mack Pub. Co., N.J. l99l). p1.%....%~ 1;. ~ny 30 acceptable carriers in Ihc~culic co~pos;l;onC may contain liquids such as water, saline, glycerol and eth~n~ ition~lly, au,Yiliary ~ c~c such as wc~Li.. g or c ~-ulsirying CA 022l8529 l997-ll-05 WO 96/35787 PCT/US~6/OC~O~

agents, pH b~ P :~ul ~ rPQ and the lilce, may be present in such vehicles. Typically, the therapeutic comrositicnQ- are l)r~,ed as inject~hles, either as liquid so1~tionQ. or susp~lQ;onc; solid forms suitable for so1ution in, or Sl~ en~;Oll in, liquid vehicles prior to injection may also be p-~ed.
,~lthn~lgh the mPthodol~gy descrihed below is believed to contain sllffr;~nt details to enable one skilled in the art to practice the present invention, other constructs not sperific~11y PYPmr1ifiP~tl such as p1~cm;~lc, can be constructed and purified using standard leco...l.il-A~ DNA techni1uPs d~pscribed in, for; . '~, Sambrook et al. (1989), MOLECULAR CLON~G: A LABORATORY MANUAL, 2d edition (Co1d Spring 0 Harbor Press, Cold Spring Harbor, N.Y.), under the current re~-i~tionc des.;~ibed in United States Dept. of XEW, NATIONAL lNSTITUTE OF HEALTH ~
GUIDELINES FOR RECOMBINANT DNA RESEARCH. These references indude procedures for the following ~ d~'d mPtho-1c cloning procedures with P1~Cm;~Q
h~r~ ahon of host cells, plasmid DNA pllrifir~tion phenol eYtraction of DNA, ethanol p~ ;l; l;on of DNA, agan~se gel clc~;hc~phoresis~ pllrifiC~tinn of DNA frSI~PntQ
from agarose gels, and restrirti~n Pn-lomlr1~Qe and other DNA-modifying en~ne re7~csi~nc The coding sequence ofthe ob protein for ~u-~oses herein can be ob~ d based on the DNA sequenre of the ob protein tlicr1osed in Zhang et aL, cited bove, using any number of co--~e~.liQn~l terhni1uec such as poly~w~ase chain reaction ("PCR"). One ~1~ of such a lec~ e is the reverse hansci ;~lion PCR ("RT-PCR"). Under this mPshod~logy~ poly A+ RNA can be i~n~tPd from ~iirose tissue and reversed h~nc~ ed to produce a first strand cDNA, using a reverse primer and reverse l"-l-c~ t~e. The reverse primer CQ~ c nucleotides of a portion ofthe nonr~otlin strand ofthe ob gene.
For eY~mrlc, s~it~ble for use herein is reverse primer #553 c~ g nucleotides 593-616 ofthe non-coding strand PYtPn~led with nllchPoti-lPc of a linker. This PCR reaction ~lu~e can then be used for ~mr1ifir~tion using the same reverse primer and a rc,l w~l primer c~ .p. nllr1eotitlPc from a portion of the coding strand of the ob gene. For PY~mrle, rO-wa~d primer #552, co~ g nucleotides 115-134 ofthe coding strand PYtP~ Pd with nllr1eotirlPc for a linlcer can be used herein. The ~mrlified DNA can be CA 022l8529 l997-ll-05 WO 96/35787 PCT/US~)GI066C9 purified and used as a te~ e for generation of other ob DNA constructs for protein ~ e,~les:,ion in prok~yoles and euk~yoles.
Variations ofthis ob construct can be made by conv~ntion~l terhni~ e$ in PCR or site directed mllt~gPnP~i~ These variants can be made to create, a l-~ e~5 protein, such as an ob protein minus its signal seq~lenr~, to insert restriction sites or linker sP~q~pnr~es~ and to add a tag to f~ ilit~te ~etection of the construct, for ~ , nd~1itir~n of a Myc or HA (inflllen7~ virus hPm~ finin) seq~lPnre which can be detectP~ by use of anti-Myc or anti-HA ~nfiho-liP~ respectively.
As an ~ p'e, a l~ ed ob polypeptide lacking amino acids 1-21, the 10 pl t::iu--~ed signal sequence, can be made by use of the full-length cDNA construct, a rO-w~-] primer such as primer #560 co..~ nucleotides 178-197 ofthe coding strand and a linker seq l~nr~ and the reverse primer #558, mpntisnp~d above. An i~ lor codon for c ~ ession in prok~yoles can be added in the form of a linker.
DNA constructs co..~ P the tags for identifir~tion purposes can be synthP~i7~od using PCR For ~ D-NA construct #1150 Pn~otl~ps an ob polypeptide that cc..~ .c an epitope tag for purposes of antibody recognition. This construct can be made using the full length ob DNA construct, a rul w~d primer, such as primer #560, and a reverse primer, such as #559 co~ g nucleotides 602 to 616 ofthe noncoding strand, ~Ytp-nr1ed with a SmaI rPct~ictirln site, and amplifying the DNA by PCR using standard protocol.
20 The ~mplifiPd DNA fragment can then be ligated into a vector that co-~ a sequence coding for heart muscle kinase and the Myc epitope, for ~ r 1~~
The DNA construct made as above described can be ligated to an CA~ ;on pl~m;~1 co-~ g an applup-iale p-ù---u~r for c,.~ iion in a desired host ~ yi~s:~ion system. E~ ion rl~cmids with various pl O~wltl :i are ~,u- ~ y available c~ - - ....~ c;ally.
25 For eY~mrle, the pl~cmid pET23 can be purchased from Novagen (~rlicon W~. This plasmid utilizes a T7 pro.. lùlel seq~nre for CA~ s:,;oll in baule ;a. Co.. ~ c;ally available ~--,-------~li~n eAI~Ie;,~;on pl~cmitlc can also be used for the present ~u-l oses. In the present i~ cç, the plasmid pCG used herein is o~ od and is available from Qianjin Hu at the University of C~liforni~ San Fr~nricco, CA. This plasmid is a d~livalive of pEVRF, and directs cA~les~ioll in ~--~ n cells from the human CMV

CA 022l8529 l997-ll-05 promoter/~nh~n~er region. Further details regald.llg c,~lesslon sy~ lls are provided below.

Expression in Bacterial Cells Bacterial CAPI ession systems can be used with the present constructs. Control el~PmRntc for use in bacteria include pro---ù~e.:i, optionally CQ..Ii.i..;,~P ope.~lor seq~upn and ribosome binding sites. Useful pLOIllOLel:i include seqmPnces derived from sugar metaboli_ing e.~y."es, such as ~P1~Ctosp; lactose (lac) and m~ltose. ~1t1itir~n~1; " '~r include promoter sequences derived from biosy"ll-etic l,.~y--~es such as l~y~lopha~l (f~p), o the ~ çtPm~ee (bla) plc,...o~el system, bActPriophage ~PL, and T7. In ~ 1iti~nsynthetic promoters can be used, such as the tac p-ulllole~. The ~ ct~m~ee and lactose promoter ~y~enls are described in Chang etal., Nalure (1978) 275: 615, and ~oecldPl et al., Nature (1979) 281: 544; the ~1k~line phosphAt~eP; l~yl~lophall (trp) pro...oter system are described in Goeddel ef al., NucleicAcidsRes. (1980) 8: 4057 and EP 36,776 and hybrid promoters such as the fac pr~.-.o~er is ~lPc~rihed in U.S. Patent No. 4,551,433 and deBoer et al., Proc. Natl. Acad. Sci. USA (1983) 80: 21-25. However, other knownba~ .;Al plU~ ,)lel:i useful for cAyl~ssion of eukaryotic p,o~e -,s are also ~- ''s A
person skilled in the art would be able to operably ligate such ~u~uLel~ to the present ob coding sequences, for e- t~ ,le, as described in ~SiebPnli.et et aL, Cell (1980) 20: 269, using linkers or adaptors to supply any re~lui~ ~d restriction sites. P~o-.lol~ for use in bacterial sy:iLt;---s also generally will contain a Shine-n~lgPrn- (SD) seq~Pn~,e operably linked to the DNA en~Qt1inf~ the target polypeptide. For prok~yulic host cells that do not reco~i7P~ and process the native target polypeptide signal seq~encP, the signal seq~-Pn~e can be ~1JS~ ed by a prokaryotic signal se.luence sPl~cte~l for p ~ plr, from the group ofthe ~lk~linP pht-srh~t~eP~ p~ ~illin~cP, Ipp, or heat stable e.-Lelo~ II leaders. The origin of repli~tion from the plasmid pBR322 is suitable for most Gram-neg~liv~
b~
The roregc,il.g ~t~ --s are particularly c~...p~ with Escherichia coli.
However, llulllelous other ~l~; --s for use in b~i~ 1 hosts in~ 1in5~ Gram-nc~aLi~_ or 30 Gram-positive o.~ such as Rnrj771~ spp., Sfrepf~?cocc2~ spp., Slr~c~l ,".~ces spp., Pse~ nas species such as ~. a., u2 i,..7s~, Salmonella t~ r~u"" or Serratia marcescans, among others. l~etho~1~ for introd~cin~ exogenous DNA into these hosts ~ typically include the use of CaC12 or other agents, such as divalent cations and DMSO.
DNA can also be introduced into bacterial cells by eleel,upola~ion, nuclear injectiQn or ~lutoplast fusion as described generally in Sambrook et al. (1989), cited above. These f~Y~mrl-oc are illusL,dliv~; rather than limitin~ Preferab1y, the host cell should secrete minims~l amounts of proteolytic enzymes. Alternatively, in vitro methnrl~ of ~lonirl~ e.g., PCR or other nucleic acid polymerase rç~ctisn~ are s~lit~ble.
Prokaryotic cells used to produce the target polypeptide of this invention are cultured in s~lit~ble media, as described generally in Sambrook et al., cited above.

Expression in yeast cells E~l,.es~ion and ~ ru~ ion vectors, either extrachromssom~1 replicQn~ or il,~e~li"g vectors, have been developed for l,~,sru""~lion into many yeasts. For ~ - d~ ei,~;on vectors have been developed for, among others, the following yeasts: Sacc~ ",~ces cerevisiae ,as described in Hinnen et al., ProG Nafl. Acad. Sci. USA (1978) 75: 1929; Ito et al., J. Bacteriol.
(1983) 153: 163; Candida albicans as described in Kurtz et al., Mol. Cen Biol. (1986) 6: 142;
Candida ~ ~fQcn as ~ ibed in Kunze et al., J. BasicMicrobiol. (1985) 25: 141; Nansenula polymorpha, as desc,il,ed in ~-Jlepson etal., J. Gen. MicrobioL (1986) 132: 3459 and Ro~enl~mr etal.,Mol. Gen. Genet. (1986)202 :302);KluyveromycesJragilis, as describedin Dasetal., J.
Bacteriol. (1984)158: 1165;Kluyveromyceslactis, as desc~ihed ~DeLou~,e.uoul~etal., J.
Bacteriol. (1983) 154: 737 and Van den Berg et al., Bio/Technology (1990) 8: 135; Pic*ia guill~, ,~i, as des.ilil,cd in Kunze et al., J. Basic Microbiol. (1985) 25:141; Pichia pastoris, as described in Cregg et aL, Mol. CelL Biol. (1985) 5: 3376 and U.S. Patent Nos. 4,837,148 and 4,929,555; Schi n~ ch~ ~ ces pombe~ as desclil,ed in Beach and Nurse, Nature (1981) 300:
706; and Yarrowia lipolytica, as described in Davidow et al., Curr. Genet. (1985) 10: 380 and Gaillardin et al., Curr. Genet. (1985) 10: 49, Aspergillus hosts such as A. nidulans, as desc~ ;l.ed in R~ n~e ef al., Biochem. Biophys. Res. C'~ un. (1983) 112: 284-289; Tilburn et al., Gene - (1983) 26: 205-221 and Yelton et al., Proc. Nafl. Acad. Sci. USA (1984)_81_ 1470-1474, and A.
niger, as described in Kelly and Hynes, E~O J'. (1985) 4: 475479; l;ich~ reesia, as ~les~rihed in EP 244,234, and r.~ fung~ such as, e.g, Nez~,o.. ~o,a, Penici~
Tolypoc7~ m as decc~ ed ~n WO 91/00357.

CA 022l8529 l997-ll-05 WO 96/35787 PCT/US~G/OG6C9 Control seq~Pnc~oS for yeast vectors are known and include promoters regions from genes such as alcohol dehydrogenase (ADH), as descrihed in EP 284,044, en~l~ce~ )col~n~c~
glucose-6-phnsph~te iSOIllel~Se~ glyceraldehyde-3-phnsph~te-dehydrogen~ce (GAP or GAPDH), hPY~kin~c~; phnsphs~r l~ctQkin~ce~ 3-phosrhnglyl~e~e mutase, and ~yl~lv~Le kinase OE'yK), as desrrihed in EP 329,203. The yeast PH05 gene, ~nror1ing acid pho~ h~I ~ce, also provides usefi~l p-ul~lOIe- seql~nrçc as described in Myanohara ef aL, ProG NafL Acad. Sci. USA (1983) 80: 1.
Other s~lit~hl~ p,c,l--oLer sequ~onres for use with yeast hosts include the promoters for 3-phosrhoglycel~Le kinase, as described in Tr.l ~ n ef al., J. BioL Chem. (1980) 255: 2073, or other glycolytic el~y...es, such as pyruvate decarboxylase, triosephosph~te iso-,-c,~se, and phosrhnglucose isolllel ~se, as desc~ ;hed in Hess ef aL, J. A~. Enzyme Reg (1968) 7 149 and ~nll~nrl ef aL, Bfoc*emisfry (1978) 17: 4900. Tnt~ ;hle yeast promoters having the ~l~litinn~l advantage of l,~sc,iL~Lion controlled by growth con~litionc include from the list above and others the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phs)~l~h~ ce~
degradative el~yllles ~c.co~ ted with nitrogen mtot~holicm~ m~,t~lloth;c!ln~in, glyceraldehyde-3-phnsrh~te dehyd-u~on~cR and enymes re ,~ar ~'~ for m~ltQse and ~ ctose ti1i7, tion .~llit~ vectors and promoters for use in yeast eA~ ion are further d~c-,- ;hed in , EP 073,657. Yeast Pnh~mcprs also are adv~nt~ul.~ly used with yeast p~ el~
~d~1ition~ ~lltllGl,;c plOIlluLe :i which do not occur in nature also filn~tinn as yeast plo"-ule ~. For , u~le~un activating sequences (UAS) of one yeast p,u",~.~e, may be joined with the l,~ l;nn activation region of another yeast p,u"-olel, creali,lg a synthetic hybrid promoter.
FY~mp~~ of such hybrid piulllO~el:~ include the ADH regulatory sequPnce linked to the GAP
s~;,ip~ion activation region, as des~ ~ ;l,ed in U.S. Patent Nos. 4,876,197 and 4880,734. Other PY Imrle~ of hybrid plOlllO~el:~ include promoters which consist ofthe r~;,~l-lQ-y sequpncç~ of either the ADH2, GAL4, GAL10, or PN05 genes, co. ..l~ pd with the ~ ;n~l ac~ a~ion region of a glycolytic enzyme gene such as GAP or PyK, as dp~s~rihed in EP 164,556.
Fu- I I .~.. ~. G, a yeast promoter can include naturally oc~ p pl'OlllO~el :i of non-yeast origin that have the ability to bind yeast RNA POIY"~G~ ~se and initiate ~l if ..c~-. ;pl ir~n Other control ~1omPnt~ which may be in~ ded in the yeast ~A~J~G;~ ;)n vectors are t~ ,alOl~, for ~ ple, from GAPDH and from the enolase gene, as de~ ed in ~t~ nrl et al., J. Biol. Chem. (1981) 256: 1385, and leader sequen~es which encode signal seq~lPnces for secretion. DNA Pnço~1in~ suitable signal sequPnces can be derived from genes for se~le~ed y_ast plO~S, such as the yeast invertase gene as described in EP 012,873 and lP 62,096,086 and the a-factor gene, as described in U.S. Patent Nos. 4,588,684, 4,546,083 and 4,870,008; EP 324,274;
and WO 89/02463. ~ItP-rnRtively~ leaders of non-yeast origin, such as an hlLe.relon leader, also provide for secretion in yeast, as desrribed in EP 060,057.
~etho-l~ of intro~lcinf~ PYr~gPnr)ll~ DNA into yeast hosts are well known in the art, and typically include either the h ~.Sro....~lion of spheroplasts or of intact yeast cells treated with alkali cations.
T.~.~ro.~ 1;on~ into yeast can be carried out accold-ng to the method des~ e~
in Van Solingen et al., J. Bact. (1977) 130: 946 and Hsiao ef al., Proc. Natl. Acad. Sci.
0 USA (1979) 76_ 3829. However, other mçthods for introd~ring DNA into cells such as by nuclear injection, ele~,Llopol~lion, or proloplast fusion may also be used as desrrihed generally in Sambrook et al., cited above.
For yeast secretion the native target polypeptide signal seq ~nce may be ~,I,s~ ed by the yeast i,.ve.~se, a-factor, or acid rhr~SphRtRqe leaders. The origin of replirRtir)n from the 2~ plasmid origin is s~itR~I~ for yeast. A s~it~ble sPlecti~ n gene for use in yeast is the hpl gene present in the yeast pl~m;d desc. il,ed in Ri ~-Ps. . ~A 1- et al., Gene (1979) 7: 141 or Ts~ r et al., Gene (1980) 10 157. The bpl gene provides a sPlection marker for a mutant strain of yeast lacking the ability to grow in L-y~Lophan.
Similarly, Leu2-dçfir;~nt yeast strains (ATCC 20,622 or 38,626) are compl~--..~-.led by 20 known plR~m;~l~ bearing theLeu2 Gene.
For intracP~ r prod~lction ofthe present polypepti~lPs in yeast, a sequPnçe ~nror~ a yeast protein can be linked to a coding seq~Pnçe of the ob polypeptide to produce a fusion protein that can be cleaved intraçPll~-lRrly by the yeast cells upon e,~ ion. An ~ - ;....pl~, of such a yeast leader seqllPnçe is the yeast ubi-l ~itin gene.
- E~yres;~;on in Insect Cells Baculovirus cA~les~ion vectors (BEVs) are recol.;~l insect viruses in which the coding seq~Pnçe for a foreign gene to be c,~. esscd is ins_. Led behind a baculovirus pro...o~ in place of a viral gene, e.g., polyhedrin, as d~P5~ ed in Smith and .S~
U.S. Pat. No., 4,745,051.

CA 022l8529 l997-ll-05 An eA~ ision construct herein inr~ Ps aDNAvectoruseful as an ;, lf ."e~ le for the infpctinn or Ll ~ r~ l ;on of an insect cell system~ the vector genera DNA coding for a baculovirus h~lsc~ Lional plo-l-oler, optionally but p,erel~bly, followed dow--sl- c~n by an insect signal DNA seq~Pnce capable of di. ~Lil~g secretion of 5 a desired protein, and a site for insertion of the foreign gene Pnr orlin~ the foreign protein, the signal DNA sequenre and the foreign gene being placed under the ~ ef . ;pL;f)n~l control of a baculovirus plolnoLer, the foreign gene herein being the coding seq~Pnce of the ob polypeptide The promoter for use herein can be a baculovirus ~ scli~ional promoter region 10 derived from any ofthe over 500 baculoviruses generally ;..rc~,;..g insects, such as, for PY~mpl~ the Orders Lepidoptera, Diptera, Orthoptera, Coleoptera and IIylllenfj~Le~
inr~ ling for e~ but not limited to the viral DNAs of Autographo c~ ""ica MNPV, Bomby~ mori NPV, rrichoplusia ni MNPV, Rachlplusia ou MNPV or Galleria mellonella MNPV, Aedes aegypfi, Drosophila mel~,.o~ " Spodopterafrugiperda, and 15 Trichoplusia ni. Thus, the baculovirus L.~s-,.ipLional pllo~llOLcl can be, for ~ , a baculovirus immerli~te-early gene lEI or lEN prollloLc, an ;.. ..f~ e-early gene in cc,.~.l.;..~l;on with a baculovirus delayed-early gene promoter region sPl~cted from the groupcon;~;1; .,~ofa39KandaHindm~mPntCf~ i . adelayed-earlygene;ora baculovirus late gene pr~..,oLcn The immrrli~tP~-early or delayed-early promotPrs can be f nh~nced with L.~ scl;~Lional Pl~h~l~rf Plomf-nte Particularly suitable for use herein is the strong polyhedrin promoter of the baculovirus, which directs a high level of eA1JI e;,~;on of a DNA insert, as des~, ;l e~ in Friesen et al. (1986) "The ~ef~ tion of Baculovirus Gene EA~J1CS: j;On~ in: THE
MOLECULAR BIOLOGY OF BACULOVIRUSES (W Doerfler, ed ); EP 127,839 and EP 155,476; and the ploll-oLer from the gene enr,orling the plO protein, a~e ~leer~ e~ in Vlak etal., J. Gen. Virol. (1988) 69: 765-776 The pl~emi~:l for use herein usually also co. Ii~; .c the polyl.edlin polyadenylation signal, as desclil,ed in Miller et al., Ann. Rev. Microbiol. (1988) 42: 177 and a proc~y~,Lic ~mp:~illin-l.~ re (amp) gene and an origin of rel)lir"l;rm for sP1er~;QI- and prop~gPtiol- inE. coli. DNA ~nro~;ng suitable signal se.~ res can also be inr1~lde~ and is generally derived from genes for seclcLcd insect or baculovirus proLcLls~ such as the CA 022l8529 l997-ll-05 baculovirus polyhedrin gene, as describPd in Carbonell ef al., Gene (1988) 73: 409, as ~ well as .. -~ n signal sequences such as those derived from genes encoding human a-clrclùil as described in Maeda et al., Nafure (1985) 315: 592-594; human gastrin-re~ cing pepff~lP, as desclil,ed in Lebacq-Verheyden et al., Mol. Cell. Biol. (1988) 8:
3129; human IL 2, as t3PSC. il,ed in Smith et al., Proc. Natl. Acad. Sci. USA (1985) 82:
8404; mouse IL 3, as desc.rihed in Miy~ la et al., Gene (1987) 58: 273; and human ~IIlcoc~ eblûs d~cP~, as ~3es~ ~ ;I,e~ in Martin et al., DNA (1988) 7:99.
Numerous baculoviral strains and variants and co"cs~,olldil~g pc""is~ivc insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (.--os~ o), Aedes albopictus (mosq-~ito)~ D~ ~s~hila melanogaster (fruitfly), and Bombyx mori host cells have been idPntifiP~d and can be used herein. See, for t~Y~mpl~, the description in Luclcow et al., Bio/Tec*nologvy (1988) 6: 47-55, Miller et al., in GENETIC ENGINEERlNG (Setlow, J.K. et al.
eds.), Vol. 8 (Plenum P~ !iCt~ p 1986), pp. 277-279, and Maeda et al., Nature, (1985) 315:
592-594. A variety of such viral strains are publicly available, e.g., the ~1 variant of Az~lo~r.~
caliJ'~ ica NPV and the Bm-5 strain of Bombyx mori NPV. Such viruses may be used as the virus for h ~ - .crecl ;on of host cells such as Spodoptera // u,~ ,~ cells.
Other baculovirus genes in ~dtlition to the po1yl,e.1,i" p,u,,,olc, may be emp10yed to advantage in a baculovirus cAy'cs~ion system. These include ;~ e~ le-early (alpha), delayed-early (beta), late (gamma), or very 1ate (delta), accordillg to the phase of the viral inft~ction during which they are cA~,c~sed. The cAylc~ion ofthese genes occurs sequt~-nti~lly~ plobably as the result of a "ç~cc~de" ...~ ... oft,~lsc,i~,Lional re~ll~ti~ n Thus, the immt~tli~te_early genes are cA~,essed immt~tli~tt-ly after infP~ion in the absence of other viral fimt~tionc, and one or more of the r~ gene products ind~ces l"-~ " ;l-l;t n ofthe delayed-early genes. Some delayed-early gene products, in turn, induce hi~ " ;plit~" of late genes, and finally, the very late genes are 25 ~,A~,cssed under the control of previously ~,A~,,ci,sed gene products from one or more ofthe earlier classes. One relatively well defined colllpollclll ofthis l~~ c,ly c~cc~de is IEI, a prerc~cd immrtli~te early gene of Aulo~,..~ho caliJ~",.ica nuclear polyhedrosis virus (AcMNPV). IEI is pressed in the ~hsence of other viral filnrtionc and enrodes a product that stim~ t~s the h;1.-C. I~p~ion of several genes ofthe delayed-early class, inr~ i~ the p~crc~lcd 39K gene, as ~lesrribecl in Guarino and ~ , J. Virol. (1986) 57: 563-571 and J. KiroL (1987) 61:

WO 96/35787 PCT/US~)G/056C9 2091-2099 as well as late genes, as described in Guanno and .~llmm~rs, Virol. (1988) 162:
444-451.
T........ ~f~ ç-early genes as desc il,ed above can be used in cQ.. l.~ ;nn with a baculovirus gene promoter region ofthe delayed-early caLegclr. Unlilce the immçtli~te-early genes, such s delayed-early genes require the presence of other viral genes or gene products such as those ofthe immptli~te-early genes. The co.,l~inà~ion of immp~ te-early genes can be made with any of several delayed-early gene plo,-,oter regions such as 39K or one ofthe delayed-early gene promoters found on the Hindm fragment of the baculovirus g~n~ mP. In the present i~ cP; the 39 K
promoter region can be linked to the foreign gene to be ~A~Jl essed such that eA~l es~;on can be o further controlled by the presence of IEI, as described in L. A. Guarino and Sllmm~ors (1986a), cited above; Guarino & .~llmmçrs (1986b) J. Virol., (1986) 60: 215-223, and Guarino et al.
(1986c), J. ViroL (1986) 60: 224-229.
Additionally, when a cOIllbillalion of immçrli~te-early genes with a delayed-early gene promoter region is used, f~nh~ n~ e. . .~ of the cA~l es.,ioll of heterologous genes can be realized by 15 the pl~cP~-~e of an el~h~l~c~r seq~lPnce in direct cis linkage with the delayed-early gene promoter region. Such ~nh~n~r seq IPn~P~ are char~clP~ d by their ~nh~ l of delayed-early gene ~,Aplession in ~itll~tion~ where the ;~ p~-early gene or its product is limited. For ~ lp!e, the hrS ~nh~n~Pr sequence can be linked directly, in cis, to the delayed-early gene p~u...olel region, 39K, thereby Pnh~n~in~ the eA~ ;oll ofthe doned heterologous DNA as described in ~l~rino and S!~ i (1986a), (1986b), and (~l~rino etal. (1986).
The polyl-ed-i n gene is ~ ifi~d as a very late gene. The~;ru~e, I-~ls~ i~lion from the polyhedrin plo---o~er le.luires the previous eA~,le~iûn of an u~lh~uw~, but p.obably large number of other viral and cellular gene products. Reç~l-se of this delayed ~,A~ ion of the polyl.e,l,i"
~.o.nùLt;r, state-of-the-art BEVs, such as the ~ y BEV system desc il,ed by Smith and 2S Summers in, for eY~mrle, U.S. Pat. No., 4,745,051 will express foreign genes only as a result of gene CA~I ~SSiOll from the rest of the viral ~nomP, and only after the viral infe~tif~n is well underway. This ~ cprese~ a limit~fion to the use of ~ g BEVs. The ability of the host cell to process newly sy..ll.P~ d proleills decrtases as the baculovirus infectiQn progresses. Thus, gene eAI~l es:~ion from the polyhedrin prc,...oler occurs at a time when the host cell's ability to process 30 newly :iy~ p~l proleills is pot~ lly .l;~ .Pd for certain p-olei,1s such as human tissue pl~ gen acliv~lor. As a con~e(~ nee; the eA~,.es~ion of seclelo-y gly~oplolei~s in BEV

sy~lel~ls is comr1ir-Ated due to i~ o...pl~te secretion ofthe cloned gene product, lL~ t appi--g - the cloned gene product within the cell in an il~o~pletp~ly procesced form.
While it has been reco~ed that an insect signal sequence can be used to express a foreign protein that can be cleaved to produce a mature protein, the present invention is 5 prt;relably practiced with a ~A~ AliAn signal seq~lçnce for eYAmrle the ob signal seq~enr,~
An eYPmrl~ry insect signal seq~l~nre c litAh~e herein is the sequPnre Pnco~ling for a Lepidopteran adipokinptic hormone (AKH) peptide. The AKH fa-m-ily co-.c:cl s of short blocked n~.~.upepLides that legula~e energy :~ul~:iL~ale mr~bili7Ation and metabolism in 10 insects. In a plere,.~;d embo~imPnt a DNA seq~Pnre coding for a Lepidopteran Manduca sexfa AKH signal peptide can be used. Other insect AKH signal peptides, such as those from the Ol lllùpLel~ Schistocerca gregaria locus can also be employed to advantage. Another ~ pl~y insect signal sequence is the seq~lPnre coding for D-osophila cuticle p-oleins such as CPI, CP2, CP3 or CP4.
Cu--enlly, the most co.~.. ol.ly used l~ rer vector that can be used herein for introd~lr;~
foreign genes into AcNPV is pAc373. Many other vectors, known to those of skill in the art, can also be used herein. Materials and m~tho-lc for baculovirusrmsect cell cA~ression ~Le;llls are co.. ~ cially available in a kit form from coi.. p~ ~;es such as Invitrogen (San Diego CA) (~MAy~RAc~ kit). The l. ' ~ ~ uPs utilized herein are generally known to those skilled in the art and 20 are fully described in Sl~mmers and Smith, AMANUAL OF METHODS FORBACULOVIRUS
VECTORS AND lNSECT CELL CULTURE PROCEDURES, Texas .~griclllhlra~ l,r . ;.~.h..l Station Bulletin No. 1555, Texas A&M University (1987); Smith et al., Mol. Cen Biol. (1983) 3:
2156, and Luckow and .S~ (1989). These inrl~ldP~ for eY-Ample, the use of pVL985 which alters the polyhedrin start codon from ATG to ATT, and which introduces a BamHI cloning site 32 25 bACe,pA;. j duw~ - from the ATT, as d~sl~ ~ il.ed in Luckow and .~ " Yirology (1989) 17:31.
Thus, for I A - ~ ~plc, for insect cell ~ .;,;un of the present polyFP,pti~ec the desired DNA
se.lu~ ce can be ~--se-l~d into the ll~srer vector, using known te~h~ u~Ps An insect cell host can be COll A- ~.~;r5J~ d with the I ~-srt;- vector CO~I A;~ P the i.lse- ~ed desired DNA to,~pthpr with the 30 ge~o~ ~ ~ C DNA of wild type baculovirus, usua11y by cuL. A n~rt~.il ;on The vector and viral g~ ~n ~.r~
are allowed to lecQ~..k;~e r~ p in a 1eCO...1~ A..l virus that can be easily idPnfifiP~d and pl~r fied The par~ed It;co~ virus can be used to infect insect host cells to express the ob polypeptide.
Other mPtho~le that are applicable herein are the ~ dard methorls of insect cellculture, co~ r~;on and p-~a ~lion of p1~emids are set forth in S~lmmprs and S-m--ith 5 (1987), cited above. This rt;re.t;nce also pertains to the standard mPthnde of cloning genes into AcMNPV ll~.srer vectors, plasmid DNA ieo1~tirn tr~nefP-rrin~ genes into the Acm~PV P~o...ç, viral DNA pllrifir~tion r~ bPlinF~ leco...h~ proleills and p~ Lion of insect cell culture media. The procedure for the cultivation of viruses and cells are desrrihed in VoL~nan and S~mmtors, J. Virol (1975) 19: 820-832 and Vol~m~n 10 al., J. Virol. ( 1976) 19: 820-832.

Expression in l!~mm~ n Cells The ob polypeptides of the present invention can be c~ressed in .. ,- -.. ~li~n cells, such as adipocytes, or fat cells, using p.u---ote s and enh~nrPrs that are fim~ti-~n~l in the such cells. For ~ "~ the 422(aP2) gene and the stearoyl-CoA desaLul~se 1 (SCDl) gene contain s~it~hle adipocyte-specific promoters, as d~e~ribed in Christy et al., Genes Dev. (1989) 3:1323-1335. Synthetic non-natural plc.lllolel:i or hybrid promoters can also be used herein. For ~ ..p~e, a T7T7/T70B p.c molel can be constructed and used, in accordance with Chen et al., Nucleic Acids Res. 22: 2114-2120 (1994), where the T7 20 polylllt;l~ se iS under the re~l~tory control of its own plollloler and drives the ll~scli~lion ofthe ob coding seq~l~ncp; which is placed under the control of another T7 promoter. The plhll~y ~el~ for the fat-specific ~,A~res~ion is an ~~-h~l~c~l located at about > S kb u~LI-,alll ofthe ll~s.;li~Lional start site, as d~e~rihed inRoss etal., Proc.
NatL Acad. Sci.USA (1990) 87:9590-9594 and Graves etal., GenesDev. (1991) ~:428-25 437. Also suitable for use herein is the gene for the CCAAT/e~-h~r~-r-binding protein C/EBPa, which is highly e.~,lG~ed when 3T3-L1 ~ et commit to the difIGI~ ;nn paLIlway and in mature post-mitotic adipocy-tes, as descl il,ed in R;- k Pl ~- ~ ~;e' et al., Genes Dev. (1989) 3: 1146-1156. The lecGllLly i~ol~ted ~ s.,li~lion factor PPARy2" A~lGs~ed exclusively in adipocyte tissues, as described in TO~ .l QI~O, et al., Cell (1994) 79: 1147-30 1156, can also be used herein.

Typical p~umoLe~:i for ~n~ AliAn cell cA~res~ion include the SV40 early promoter, the CMV promoter, the mouse .. ~ y tumor virus LTR ploll~olt~, t,he adenovirus major 1ate promoter (Ad MLP), and the herpes eimpl~Y virus plul"oLel, among others. Other non-viral prolllùLel:i, such as a pro...ûlel derived from the murine metallothionP!in gene, will also find use in 5 ~ AliAn constructs. l~AmmAliAn cA~ iûn may be either con~ e or reg~ te~ (inducible), depending on the p-u--~ùLer. Typically, I-~s~ Lion t~- ...;- .~l ;on and polyadenylation seq~lenr~,s will also be present, located 3' to the L ;~n~ n stop codon. Prer~.~bly, a seq~ence for opl ;. . .;,P,l ion of initiAtion of L~n.~ ;on located 5' to the ob polypeptide coding sequPncP; is also present. FY~mrles of L-~1sclil,Lion t~ ol/pûlyadenylation signals include those derived from 0 SV40, as described in Sarnbrook et al. (1989), cited previously. Introns, GQ~ splice donor and acceptor sites, may also be deei~n~l into the constructs of the present invention.
FnhAnr~r ~l~mrnts can also be used herein to increase cAl lession levels ofthe ,..~..",.~ n constructs. F ~;....plf.e include the SV40 early gene ~l~h~nc~, as desc.il,ed in Dijkema et al., E~O J. (1985) 4: 761 and the ~nh~re./plo~"oLer derived from the long t~rrninAl repeat ~LTR) of the Rous Sarcoma Virus, as described in Gorman et aL, Proc. Natl. Acad. Sci. USA (1982b) 79:
6777 and human ~iy(o~ lovirus~ as des~ il,ed in Boshart et al., Cell (1985) 41: 521. A leader sequence can also be present which inrl~lflPe a sequence encoding a signal peptide, to provide for the secretion ofthe foreign protein in ~ AliAn cells. P.er~ bly, there are proc~eein~ sites encoded between the leader fragment and the gene of interest such that the leader se~ ce can be cleaved either in vivo or in vitro. The adenovirus l-ip~ile leader is an example of a leader seq~ nre that provides for secretion of a foreign protein in l.. ~.. AliAn cells.
There exist cA~Iess;ûn vectors that provide for the h ~s;,;--L CA~ ;On in ~ AliAn cells of DNA enro~ing the target polypeptide. In general, h~-~ ,..L cA~-e~ion involves the use of an ~,A~-ession vector that is able to replir~te ~ffiri~ntly in a host cell, such that the host cell 25 ~Accllm-llAt~,e many copies ofthe cA~,.es~;ûn vector and, in turn, s~ r~ s high levels of a desired polypeptide ~nr,oded by the ~ AylcS~;Oll vector. T~ nL ~,Al.lcs~;on s~:.le--.s, CO...~ , a suitable CA~ ession vector and a host cell, aUow for the convenient positive irlrntifir~tinn of polypepti~e ~ ~nCoded by cloned DNAs, as well as for the rapid s-,-cenil~ of such polypeptides for desired biological or physiological prope lies. Thus, transient .,A~- es~;on ~len-s are particularly useful 3 o for purposes of identifying analogs and variants of the target polypeptide that have target polypeptide-like activity.

WO 96/35787 PCT/US~)6/0660 Once comrhPt~ the .. h.. ~ n, A~.esxion vectors can be used to Lld~l~ru.. any of several .. i.. ~li~n cells. Methn~l~ for introd~lction of heterologous polynucleotides into ."",~ n cells are known in the art and include dextran-mP~i~t~Pd l,~nxrr,~ n c~W~
phosphate p~e~ l;Qn polybrene mp~ ted ~,~..xrr,,l;on, p-ù~oplast fusion, ele~,L.c,l)oration, 5 f~ px. -l~ti~n of the poly~llcleoti~1e(s) in liposome~, and direct miclu~ ~el ;on of the DNA into nuclei. General aspects of ...A..~ n cell host system ll~.,ru-~ l;nn.~ have been described by Axel in U.S. 4,399,216.
M~mm~ n cell lines available as hosts for ~ ,ression are a1so known and include many immortalized cell lines available from the AmP,rir.~n Type Culture CollectiQn (ATCC), in~ 1in~ but 10 not limited to, ChinPse h~ll:iLel ovary (CHO) cells, HeLa cells, baby hs~ xler kidney (BHK) cells, monkey Wdney cells (COS), human hPp~tocPlllll~r cal.,inu.lla cells (e.g., Hep G2), human elllll~yonic Wdney cells, baby h ~ ..~lel Wdney cells, mouse sertoli cells, canine kidney cells, buffalo rat liver cells, human lung cells, human liver cells, mouse ...~ tumor cells, as well as others.
The l~.. ~li~n host cells used to produce the target polypeptide ofthis invention may be cultured in a variety of media. Ccsm~ ,;ally available media such as Ham's F10 (Sigma), Minim~l Fx~Pnti~l ~e~ m (~ , Sigma), RP~-1640 (sigma), and Dulbecco's ~ ified Eagle's Me~i.lm ([DMEMI, Sigma) are suitable for cllltllrin~ the host cells. In ~tltlitinn any of the media dPc~.. ;be~d in Ham and Wallace, Meth Enz. (1979) 58_ 44, Barnes and Sato, Anal. Biochem. (1980) 102 255, U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, or 4,560,655, WO 90/103430, WO 87/00195, and U.S. RE
30,985, may be used as culture media for the host cells. Any ofthese media may be suppl~ ed as nPc~cc~ . y with hol lllol-es and/or other growth factors such as insulin, Il;~ ~-~rr- I ;n or epidPrm~l growth factor, salts (such as sodium chlnri~1P; ç~lr;.lm m~ P~ --.. and phosl~h~e)~ buffers (such as HEPES), nn~leos;~l~Ps (such as ~enncin~
25 and thymidine), ~ntihiotics (such as ~ ..ycin(tm) M drug), trace PlPmPntC (defined as il~ol a fic compounds usually present at final col~ alions in the micromolar range), and glucose or an equivalent energy source. Any other l~ec~c~ y sUp~~-npntc may a1so be in~1llrled at a~pl~liale col-c~ ions that would be known to those skilled in the art.
The culture con(litinnc such as lelllpel~lule~ p~ and the like, are those previously used 30 with the host cell sPlected for ~A~ ;on~ and will be a~p~ to the ~I-lina~ily skilled artisan.

WO 96/35787 PCT/US~)G/056C9 Gene Therapy Applications The nucleic acid constructs that contain the ob polypeptide coding seq~lpnce ("ob coding sequence"), with or without the signal sequPn~e, can be used for inhibitir~n of food 5 intake and weight gain, such as for tre~tmpnt of obesity or the problems ~c~oç;~ted with obesity, by ~ lion thereof via gene therapy. Gene therapy strategies for delivery of such constructs can utilize viral or non-viral vector approaches in in vivo or ex viw modality. ~res:iion of such coding sequPnce can be in-luce~ using PnrlQ~;enmlc ... ~ .. ~li~n or heterologous promoters. ~I,res~ion of the coding sequPnce in vivo can be o either con~titlltive or re~1~te~
For delivery using viral vectors, any of a number of viral vectors can be used, as desc- ;I,ed in JoLy, Cancer Gene Therapy 1: 51 -64 (1994). For ~ ~ ~ " .ple, the ob coding sequPnce can beh,selled into pl~cmifls dçsi n~d for ~,Al,-e~:,ion in retroviral vectors, as described in Kimura et al., Human Gene Therapy (1994) 5: 845-852, adenoviral vectors, as d~c~ ~ ;bP~d in Connelly ef aL, Numan Gene Therapy (1995) 6: 185-193, adeno-~cori~ted viral vectors, as dPC- ~ ;1 ~ed in Kaplitt et al., Nafure Genefics (1994) 6: 148-153 and sindbis vectors. Plu..lole.~ that are C~lif~hlo for use with these vectors include the ~olcnPy retroviral LTR, CMV p.u,..oler and the mouse albumin promoter. l~Pp~ ti~n Cc~ free virus can be produced and injected directly into the animal or hllm~n~ or 20 by tr~ncductiQn of an autologous cell ex vivo, followed by injection in vivo as desc.il,ed in tlQllksll etal., Proc. Nafl. Acad. Sci. USA (1994) 91: 5148-5152.
The ob coding sequPn~e can also be L.selk;d into pl~mid for c.~ ;on ofthe ob polypeptide in vivo or ex vivo. For in vivo therapy, the coding sequ~nce can be delivered by direct injecti~n into tissue or by intravenous infil~;on Promoters s~lit~ble for use in this 25 manner include endogenous and heterologous plUlllOIel:i such as CMV. Further, a synthetic T7T7/T70B pro..-oler can be constructed in acco..l~lce with Chen et aL(1994), Nucleic Acids Res. 22: 2114-2120, where the T7 polymerase is under the re~ tory control of its own p~o-llolt~ and drives the l-~-scliylion of the ob coding s~q~l~nC~; which is also placed under the control of a T7 prollloler. The coding seqU~on 30 can be i~;e~ d in a rO~ ln~;On COIll~ Llg a buffer that can stablize the coding sequence and f~rilh~te tr~n~d~lction thereof into cells and/or provide ~ ~,cLh~g, as desc- il,ed in Zhu etal., Science (1993) 261: 209-211 Expression of the ob coding seq ~enre in vivo upon delivery for gene ll,c. ~y purposes by either viral or non-viral vectors can be re~ ted for l~nx;~ 31 efficacy and 5 safety by use of re~ ted gene ~,A~.- ession promoters as d esc. ;bed in Gossen et al., Proc.
Natl. Acad. Sci. USA (1992) 89:5547-5551. For 1~ .n~ ple, the ob coding seq~Pnre can be re~ ted by tetracycline Ic:j~,ons-~e promoters These promoters can be re~ll~terl in a positive or negative fashion by l.~~..e~ ~ with the .~ lnlor m~ cl~le For non-viral delivery of the ob coding seq~lPnr,r the sequence can be ~--se- Led into co,.v~ l;on~l vectors that contain conv~ntion~l control sequences for high level eA~-ession, and then be inc~lb~ted with synthetic gene L-~l~rel molecl~les such as polymeric DNA-binding cations lilce polylysine, plùl~ " and albumin, linked to cell L--~g ligands such as asialooroso. ~ ~Coi~1, as desc~ibed in Wu and Wu, J. Biol. Chem.
(1987) 262: 4429-4432; insulin, as desc~ ed in ~ ~cl~ed ef al., Biochem. P~ ~o~ 40:
253-263 (1990); g~l~r,tosP; as desc il,ed in Plank et al., Bioc ",j~~le C*em. 3:533-539 (1992); lactose, as desc.;l,ed in Midoux et al., Nucleic Acids Res. 21: 871-878 (1993); or llnn.~;rt ~ , as described in Wagner ef al., Proc. NatL Acad. Sci. USA 87:3410-3414 (1990) Other delivery systems include the use of liposomes to cn~ .s ~l~te DNA
CO~ lisil.g the ob gene under the control of a variety of tissue-specific or ~biq~it active l~.o.-.oLc.~, as desc.il~ed inNabel etal., Proc. Natl. Acad. Sci. USA 90: 11307-11311 (1993), and Philip et al., MoL Cell BioL 14: 2411-2418 (1994) Further non-viral delivery sllit~ble for use inrl~des .~ c~l delivery systems such as the b;oli~ir, approach, as descnbed in Wnrren~ et al., Proc. NatL Acad. Sci. USA (1994) 91(24):
11581-11585 Mo.cover, the ob coding sequence and the product of ~ res:,io.l of such can be delivered through depositinn of photopoly~ ~ e, ;,~d l.r.l-ugel materials Other co..v~ ;nn~l m.othorl~ for gene deli~c-r that can be used for delivery ofthe ob coding seql~enre inrh~de, for ~ ple, use of hand held gene L-~.~rc particle gun, as d~ ~ . ;l ed in U. S 5,149,655; use of ioni7in~ r~ tit~n for a~Liv~L~ h all ~rtll cd gene, as ~ rribed in U S 5,206,152 and PCT applir~tinn WO 92/11033 Upon c,.~rcs~ion ofthe ob polypeptide in vilro in any ofthe above-des- il,ed .ression sy~Lt;llls, and after recovery and, optionally, folding, and p~lnfir~tion of the ob WO 96t35787 PCT/US~6/0~609 polypeptide, in accordallce with conv~ontion~1 m~thods, the ob polypeptide can be used in ~ a variety of ways. For ~ . ,pl~, the ob polypeptide can be ~lminictered h~LIa~elluusly, s~lbc~ eol~cly, peritoneally, i,.l.P....~sc~ 1y, or orally in a ther~reutir~1ly .,Lr~li~
~ ~mollnt for inhihitinn of weight gain and/or inhihition of food intake. The specific ~molmt 5 to be given is a weight-gain inhihitory amount or a food-intake inlf,bilo~ mnunt as previously dPfinPcl rlc:re~bly, the ob polypeptide is ~ cl~ed i-.ha~renously or sub~ .n~o~~cly in the form of a pharm~ceutir~l composition which",rt;rt;,~bly, ~ a pharm~ceutic~lly acceptable carrier.
Furthe.",ole, the ob protein, with or willluul its natural secretion leader sequ~-nr~., 10 can be used to identify an ob recel.lor, having specific af~inity for the ob protein. For this purpose, the ob protein can be labeled with a conv~ntion~l marker, such as a r~rlio~ctive label, and the labeled ob protein is allowed to react with cells, cell extracts, or cell ."b-~les belon inS~ to one or more cell types. The ~~lu,e is then ~ d for presence of specific binding to the labeled ob protein. The binding pairs formed can be 5 separated by conv~ntion~l techniques, such as by use of solvents or d~ P reagent or by p~Cc~ge through a column that selectively bind one .~ J~ of the pair, and eluting the opposite member, i.e., the ob receplor, with an app,up,iale solvent.
The ob ~Gce~,lor can be purified by convention~l techniques and the amino acid sequenr,e thereof ~le- ~ R~1 Based upon the amino acid sequP-nre icl~ntifie-l an20 oligomlrleQtide probe can be made to probe a cDNA or g~nomir~ DNA library. Clones that hybridize to the probe can be ~mplified and sequenced. A cDNA clone that encodes a full length ob leceplor can be used for ~ccc,~ procluction of large 4~ ;es ofthe .~c~lor, useful for further studies into the ...~ cm of of re~ tinn of obesity and to obtain ~onictc and ~ Pol~ ; thereto.
The ob polypeptide and the ob l~ce~lor of the present invention can further be used to gene,ale mnnnrl~n~l or polyclonal antibodies. ~ntibo~ s to the p.~,leins ofthe inventionl both polyclonal and monoclon~l may be pl~a.t;d by conv~onfion~l m~thn~$~ In general, the protein is _rst used to ;.. ~ e a sl~it~ble animal, prert;rably a mouse, rat, rabbit or goat. Rabbits and goats are plerc .ed for the prcpa ~lion of polyclonal sera due 30 to the volume of serum obl~ )1e, and the availability of labeled anti-rabbit and anti-goat antibodies. Tmmllni7~fi~n is generally pc,r~,...-ed by mixing or emulsifying the protein in WO 96J35787 PCT/US~6/0~09 saline, preferably in an adjuvant such as Freund's com~ te adjuvant, and injecting the ~Lure or ~mllleion p~e~Le~ally (generally s~b.~ u~ or ""l;.. ~.~l~rly). A dose of 50-200 llgrmjection is typically sllffir;~nt ~.. --.;,~.l ;nn is generally boosted 2-6 weeks later with one or more injectionc of the protein in saline, y~ erel ~bly using Freund's incomplete adjuvant. One may alternatively genel~e antibodies by in vitro ;.. ~ ;o using methot1e known in the art, which for the purposes of this invention is con~de~ed equivalent to in vivo ;.. - ~ ;on Po1yclonal antisera is ob~ d by blee ling the ;-------~-;~ed animal into a glass or plastic CQ~ r, inc~lb~tinf~ the blood at 25~C for one hour, followed by inri~ g at 4~C for 2-18 hours. The serum is recovered by crntrifilg~tion for eY~mple, at about 1,000 x g for 10 .~ es About 20-50 rnl per bleed may be obtained from rabbits.
Mnnnclc!n~l antibodies (~Abs) are pley~ed using the method of Kohler and l!~iietrin Nature (1975) 2~6: 495-96, or a mndifir~finn thereo~ Typically, a mouse or rat is ;.. ~ d as described above. However, rather than blee~lin~ the animal to extract 5 serum, the spleen, and optionally several large Iymph nodes, is removed and ~liccori~ted into single cells. If desired, the spleen cells may be scleelled, after removal of llollspecilically adherent cells, by applying a cell ~ on to a plate or well coated with the protein ~ntigen B-cells c ,~res~g melllbl~le-bound immllnn~l( bll1in specific for the antigen bind to the plate, and are not rinsed away with the rest of the ~ ~l .-' on 20 p~elllfing B-cells, or all diesori~ted spleen cells, are then inflllced to fuse with myeloma cells to form hybridom~c, and are ~ Lul'ed in a selective ...e~ .. such as one co~ g~
e.g., Ly~oY~ e~ an~illopLelill, and thymidine (a "HAT" ...~1;---..). The r~slllting hybridom~c are plated by limiting rlilllfinn and are assayed for the prod~ctinn of antibodies which bind specifir~lly to the ;.. ~.;,;.. ,~ ~ntig~.n, and which do not bind to 25 unrelated ~ ;ge~-c The sP1ected MAb-secleLil.g hybridom~e are then ~;ul~uled either in vitro, e.g., in tissue culture bottles or hollow fiber re&_Lol~, or in vivo, as ascites in mice If desired, the ~nfihorliec whether polyclonal or monorlon~l may be labeled using convPntinn~l te~l~..;4~les .~l-h~hle labels include fluorophores, ~,Iu-ol.lophores, r~lio~ctive atoms (particularly 32p and l25I), elee,lloll-dense ré~r-~l~; e ~yllleS, and ligands having 30 specificbindingpa,lll~ . Es~ymesare IYr-- ~Iy detectedbytheiractivity. For c ple7 horseradish peroxidase ~RP) is usually detected by its ability to convert CA 022l8529 l997-ll-05 3,3',5,5'-t~L.~~ ylb~n7~ n~ (TMB) to a blue pi m~nt, q~nfifi~h1c ~,vith a ~e~iLIopholo...~l~r. "Specific binding p&lLlwl refers to a protein capable of binding a ligand molccl-lP with high speçifirity~ as for ~Y~mple in the case of an antigen and a mon~ rlon~l antibody specific llleler.,r. Other specific binding p~hhlt;l~ include biotin and 5 avidin or streptavidin, IgG and protein A, and the lllllelous rec~lor-ligand cc, 'es known in the art. It should be understood that the above description is not meant to c; l ~g~ e the various labels into distinct classes, as the same label may serve in several dirrt~c;llL modes. For ~ 25I may serve as a r~liQ~ctive label or as an electron-dense reagent. HRP may serve as en_yme or as antigen for a MAb. Further, one may collll)ine various labels for desired effect. For ~Y~mrlr, MAbs and avidin also require labels in the practice of this invention: thus, one might label a MAb with biotin, and detect its plesence with avidin labeled with l25L or with an anti-biotin MAb labeled with HRP. Other p~ ;on~ and possibilities will be readily a~)p~elll to those of ol.l"l~y skill in the art, and are cQn~ ered as equivalents within the scope of the instant invention.
The antibodies ge.lel~Led in this manner can be used in any coll~ n~l applir~tion~ inrllldin~ for ~liagnçstir~ and thel~t;ulic purposes. For; ~'e, as a ~1i53 ngstir it can be used in an immllno~ y for i~entifir~ti(~n or detecti~n of an ob polypeptide or a homolog thereof in a sample suspe~;L~d of CQ~ p such. For this l)ul~osc;, the antibodies can be labeled with a sllit~hle marker, such as a r~lio~cfive label, 2 0 and allowed to react with the sample. After an applopl,~e length of time, the sarnple can be ~Y~min~d for the presence of specific binding pairs. Plesellce of specific binding s~e~t~ that an ob polypeptide or a homolog thereof is present in the sample.
The antibodies to the ob polypeptide, polyclonal or mnnorlon~l, and plcr~
monorl~nal, can be used for Ihc;l~euLic l,ul~,oses for b'cct~in~ the in vivo activity ofthe 25 ob polypeptide. Such antibodies will be cc~p~l; hlf, to the host to be treated. For e, for Llc .l ..-~ of hllm~n~, the antibodies can be human mnn~rl~n~l antibodies or l.. -.. ~l.;,ed ~ntihorliloc, as the term is generally known in the art. The l.. ~ d ~ntiho~ s can be made by any number of coll~ ..l;on~l mf~thntl~, For ~ , by cdr (ccmpl~ y deLclIllil~lgregion)gl~;llg, V~ f'e-~ P, phagelibraydisplay,orbyuse 3 0 of xeno-mouse. In cdr grafting, the coding regions of the cdr of murine antibodies are linked to the coding regions of the Ll~ullcwoll~ regions of human ~ntibo-lif~ In ~f -~ef-~ ;uP, WO 96/35787 PCT/US~6/06fJ0 the c~nf nic~1 regions ofthe antibodies, inf11lflin~ parts ofthe cdr and pa-rts ofthe murine framework regions that are exposed on the surface of the mq~eclllp; are ~A;~ P~l as well as the murine cdr regions. The ~ntihodips to be ~ el~ ed can be given in a the~ c~ 11y t;~e.;Live ~mo11nt and can be in the form of a pharm~cPIlti~ ~1 o omposition.
Design of the immllno~es~ys is subject to a great deal of variation, and a variety of these are known in the art. Protocols for the imm11nn~ce~y may be based, for PY~mp~e~
upon competition, or direct rP~ ion or sandwich type assays. Protocols may a1so, for ~",~ use solid ~uppo.Ls, or may be by ;~~------.op..~ 1 ;fm The assays herein involve the use of labeled antibodies to the ob polypeptide or labeled ob polypepti~lPe The labels may be, for PY~mrhP,, fluo-escenL, f~hrmi11~ esce~.l radio~ctive, or dye mo~ecu1Ps Assays which amplify the signals from the probe can alsobe used; eY~mples of which are assays which utilize biotin and avidin, and enzyme labeled and mrr1i~ted imml-nn~es~ys, such as ELISA assays.
The enzyme-linked imm1mosorbent assay (ELISA) can be used, for ~PY~mr1e~ to measure either ~nti~Pn the ob polypeptide, conr~ Lion or the antibody to the ob polypeptide cQncPntration~ This method ~lepPnt1e upon conj~ tion of an enyme to either the antigen or to the antibody, and uses the bound enyme activity as a .~ l;ve label. To measure the antibody col-r~ ion, the antigen is fiYed to a solid phase, such as a microplate or plastic cup, i~ b~ed with tli1ution~ ofthe sample to be tested. The miYture iS then washed, inr~1b~ted with anti-imm1m~glob111in labeled with an enzyme, and washed again. Enymes suitable for l~hP1;.~ are known in the art, and inch~dp~ for c "'-, horseradish peroYid~ee. Enyme activity bound to the solid phase is ~-w~uled by adding the specific subsLI~le, and ~el~ ;..P product form~ti~n or ;.~sLI~Le ~1ti1i7~ti~n colorimptrir~lly The enzyme activity bound is a direct fimrtil~n of the amount 25 of antibody bound.
To measure antigen co--r~ lion, that is, ob polypeptide conrPnt~ation, a known specific antibody is f~xed to the solid phase, the test material cc~ g antigen is added.
After incub~tion the solid phase is washed, and a second enzyme-labeled antibody is added. After washing, ~ub~ le is added, and enyme activity is ~ " .1. d co1c,.;.. ~l~ically, and related to antigen co~c~ lion. T~ n~o~esc~u~G assays can also be pelr~ ed with such ~ntibo~ies and ~nti~rnc, as described in ~chidQ et al., Biochem. Biophys. Res. Comm. (1992) 187(3): 1241-1248.
Kits sllit~hle for immlmotli~gnrJciC and cc~ ;nin~ the app.ol,.iaLe labeled reagents are constructed by pacl~ ing the approp.iale materials, inrlu~ling the polypeptides ofthe 5 present invention or antibodies ~ eeled against the ob polypepti~les in sllit~hle col ~;fL.
along with the r~ .; n~ reagents and materials rt~luired for the cQntlllct of the assay, as well as a sllit~ble set of assay instn~cti~nC The other materials or rtagel.L~ inrlud~ for li11l.ontc, wash and other re~gPntc applo~ le co~ ;nfL, :i such as tubes, plates, etc.
o The present invention will now be illustrated by reference to the following eY~mp1çs which set forth particularly adv~nt~geous embo~ However, it should be noted that these ~mho~impntc are illustrative and are not to be construed as restricting the invention in any way.

E~i~nl~1e 1 For easy re~elence, the nucleic acid constructs made herein and as shown in FIG. 1 and FIG. 2 are s~ ~ iGed below. In ~d~lition to the s-.. ~ , further details ~cg~ding the cQnchlcti~n ofthe nucleic acid mrl~cllles follow.
Construct #1122, as shown in FIG. 1, is a srhf .m~tir, I tprf~ Sf -~ l ;rJn of an ob 20 coding region in pCG cA~ ;on pl~cmirl Nullll~f l:i on top in~ Rte amino acid positir~nc in the ob protein. The letters "MASR" intlic~te four a~ itiQn~l amino acids, leplesenled by its one letter code, fused to the ob seq~l~nce by virtue of the cloning procedure used.
These amino acids are part of the up~ fk leader region and ensure o~
initi~ti~n of 11 ~n-l~l ion of the ob mRNA. The ob coding region is fianked by I ~yl ~ ;- I;on 25 enzyme recognition sequences forX~aI and R~T The bar in the box lepleselll~ the yulalivë signal seqllenre cleavage site.
Constructs #1123 and #1124, as shown in FIG. 1, are s~ - . .h ~ ;r, l e~ s~n~ ;nnc similar to that of construct #1122 except that the ob stop codon was removed andreplaced with a linker Colll~ illg a SmaI recQ~niti~n sequ~nce followed by mlrl~oti~ie 30 sequ~nrf. c for either the Myc or the HA epitope.

Constructs #1130- 1132, as shown in FIG. 1, are sçl ~r~ ;c ~ e~ .;se~ l ;onC of the ob coding region lacking the putative signal sequence. The ob coding regions r-nrorling amino acids 21 to 167, 22 to 167, or 25 to 167 are each preceded by sequr--nce enrorling five ~rlrlition~l amino acids "MASRX" each .~rese.-Led by its one-letter code, again due 5 to the construction method used, as inrlic~tecl above. In ~l~lition the stop codons in these constructs were ré...oved and replaced with a linker c~ a SmaI recognition sequence, followed by m-rleotirle sequenres for the Myc epitope. Construct #1132c~ c a conservative ~ub:iLiLuLion of leucine to valine at amino acid position 111.
Construct #1119, as shown in FIG. 1, is a s~ 1 ir, r ep~cr~ ;. I ;nn of the ob o coding region that inrl~ldes a mnt~tir~n at amino acid 59 gene-L~Led by the TGA m-rleotide sequr~nre This construct is not utilized for eA~-e~ion of a biolo~c~lly active ob polypeptide but, instead, is utilized for control and co..-p~ison purposes to mimic an obese ml lt~ti~n An NdeI recognition sequenre is located at the N-terminus of this ob polypeptide, linking the T7 promoter and the signal sequence ofthe molecllle ABam~
site is located at the C-l~ ;-. -c ofthe molecl~le.
Constructs #1127, #1128, and #1129, as shown in FIG. 1, are scl-r=~ l;ri rep~-cr~ l;r~nc of the ob coding region c~ying L, . ..~r ~l ;O..c of codons for 21, 24, and 20 amino acids, r e~l~e~ilively~ at the N-termini of the ob polypeptide. These constructs were made in view of the uilCc; IzL~l~Ly of the length of the signal seqllenr,e. The construct #1127 20 was used to produce the ob polypeptide for the cA~elilllent involving injection ofthe ob polypeptide into rats.
Construct #1150, as shown in FIG. 1, is a s- .I ~ ir repl ~c~ ;r~n of the ob coding region enriorlin~ amino acid 22 to 167 ofthe ob protein. In a~lrlitiQn~ the coding region is PYtPnded with nllrl~otide sequPnr,es Pnrorli~ the ~cog..~;on sequP-nr,e for heart 25 muscle kinase followed by the Myc epitope sequence.
Constructs #1142, #1143, and #1144, as shown in FIG. 2, are a sc-l~r-~ ir, lepl ~c~- .(; l ;on of all or part of the ob coding region linked to a hybrid plullloler~ SRa.
#1144 encodes the full-length ob protein with an X~aI site at the N terminus, and a BamHI site at the C te- ...;-.-~. Construct #1142 Pncodes the full-length ob protein with 30 an ~aI site at the N lelllulluS, a SmaI site as well as a Myc tag at the C l~lllillUs.
Construct #1143 Pnrod~Ps a l~ c-~ed form ofthe ob protein, lacking amino acids 1-21 at WO 96/35787 PCT/US~6/01i;6;09 the N-terminus and co..l~s a SmaI site as well as a Myc tag at the C-tP-rrninllc- The ~ Iength of the polypeptides Pncoded by construct #1142 and construct #1143 was tested by l~n-.cre~ g construct #1142 co~ .P the signal seq~lPn~e and #1143 lacking the signal sçquPnr,e into ~.. h.. ~ n cells (COS cells). Total ~r~,L~.s were s~a-~led by SDS-s PAGE and L-~l~r~--ed to nitrocelllllose filters. Ob polypeptides were ~letecte~l using monoclonal Myc antibodies. Two signals were obt~in~d for construct #1142, one coIle~onding to the p-e-;uI ~or form of ob protein and a faster migrating band which co-migrated with the product from construct #1143. The eAI Ies~ion product of each of constructs #1123 and #1124 was similarly tested to determine the size of the polypeptides frlrrn~d Based on the comigration of the processed forms with the product of #1143, it is re~con~ble to conclude that the signal sequence concictC of 21 amino acids.

Cloning of the Mouse ob Gene The mouse ob gene was cloned by reverse-LI~ls.;li~ion PCR ("RT-PCR") as follows. RNA was iCol~tçd from mouse adipose tissue and polyA+ mRNA was icQl~tedusing oligo-dT beads puIchased from Dynabead (Dynal A.S., Norway). To synthpci7e the first strand cDNA, 1 llg of this polyA+ mRNA was reverse h~l~e~,- ;berl using 1 ~g of reverse primer #553, having a sequence as follows:
5 '- GCGGATCCTCATGCGCATTCAGGGCTAACATCCAACT-3 ' . This reverse primer co~ P~d nll~leotides 593 to 616 ofthe noncoding strand, as inflir~tpd in the underlined portion of the sequence above, ~Ytpnded with a R~T rPct-ictiQn site, as inrlir,~ted in the non-lmrlprlinp~d portion. Added thereto were 10 Units 1~ ~ nP,y murine It;--k~ virus (M-MuLV) reverse ll~ ,Lase (Roel..;.~ge~ M~nnhPim ~ .I..~ and 25 ~ dNTPs. The reaction n~Lu~e was i~c.~ ed at 42~C for 60 ... ~ les Three ~1 of 25 the reverse l~ c~ e reaction were used for PCR ~mplifir~tion using the above ;ollPd reverse primer and rO- w~d primer # 552 having a sequenre as follows:
5'-CGCATATGTGCTGGAGACCCCTGT-3'. This ~Iw~d primer c~J~ fA
nucleotides 115 to 134 of the coding strand, as in~lic~tçd in the l~nderlined portion of the seqllenr,e above, beP;I-l~il-g with an NdeI restriction site, as in~1ir~tçd in the non- ~nderlinPd 30 portion ofthe sequence. The first codon ofthe ob mRNA, the mPthinnine codon ATG, is part of the NdeI restriction site.

The ~mrlifiçd DNA was gel purified and used as a te~ l e for generation of various ob constructs for protein cAyl~es~ion in prol~yoles and eul~yoles, as ~Y~mrlifiPd below The ob cA~Jlession constructs in FIG. 1 and FIG. 2 show either full-length ob protein or tr~lnr~ted versions thereof lacking variously 1-20, 1-21, or 1-24 of the N-terminal amino acid residues of the ob protein. In ad~litinn in some of the constructs, the ob coding region was fused at the C-tf!~ ",;~ e to adrlitinn~l nllcl~oti~e seq~l~nrçs co~ ing epitope such as Myc or HA (inflll~n7~ virus h~m~l.~l...;~) for recognition with anti-Myc or anti-HA ~ntihorli~s or for l~hf~lin~ with heart muscle kinase in the pl~esenc~ of r~-lio~rtive gamma-[32P]ATP

I~A~IIP1~ 2 Construction of the ob EA~ S~;On Plasmids for Ex~. es~;on of the ob Protein in Bacterial and ~mm~ n Cells ~lrlitiQn~l DNA constructs were made that ~nr,oded the full length ob protein aswell as 1. ~ d forms of it lacking a portion ofthe N-te.. ,.al region or signal seq~lPnre ofthe ob protein were made Construct #1138 (not shown) was made as follows: The coding sequence ofthe full length ob protein con.eieting of amino acids 1 through 167 was synth~ei7Pd by PCR, using (1) the above-~1~ocr ~ ;1 .ed ob cDNA, (2) rUI W~ primer #557 having a seq~l~nce 5'-GCTCTAGAATGTGCTGGAGACCCCTGTG-3', which co ~ çdn~ eotiles115to135ofthecodingstrand,as indic~tçd bythe~m~l~rlin~d portion of the above seql~enr,r, and b~ ...; g with an XbaI rÇ~striction site, as inr~ ted by the non-underlined portion of the sequence, and (3) reverse primer #558 having asequence: 5'-GCGGATCCTCAGCATTCAGGGCTAAC-3', which co .~ d nucleotides 602 to 616 ofthe nnncotling strand, as in~ te.d by the underlined portion, 25 and PYten~led with a BamHI re~trictinn site, as in~lir~ted by the non-lm~l~rlin~d portion The the full length coding seqll~once was ~mrlifi~d by PCR using a ~ dud PCR
protocol The ~mp1ified DNA product was ligated into eAylc~ ;oll plasmid pET23a from Novagen (M~flieon WI) This plasmid co ~ c a T7 p-u~oler seqllenre, for ~ JreSS;OII
3 0 in ba il ~ In this construct #1 13 8, the c~ ;oll of the ob protein is under the control ofthe T7 poly.,.c,~se which directs llP~e~ from the T7 pl~,lllolel For eAyles~ion in eukaryotes, construct #1122 was made, ecc~ y in the same way as for construct #1138, except that the DNA fragment encoding the full length ob protein was ligated into eukaryotic c,~ ssion plasmid pCG. Plasmid pCG, which is a pEVRF derivative, as ~Pc~ribed in ~tthi~c et al., Nucleic Acids Res. (1989) 17: 6418, 5 has a mn~lified polylinker, and directs cAyiess;on in ~h~ n cells from the human ~;yLo~legalovirus pl~nloLel/Pnh~ncpr region. In this construct, h ~ ;nn initi~tinn is controlled by the 5' ullllnll~ ed region of the herpes s , ~Y virus thymidine kinase gene.
Trlln--~t~Pd ob polypeptides lacking a portion of the N-ter ninal region of the ob protein were made. The constructs in~ de(l are those col~ nucleotide seq~lPncçs 0 enco~ling amino acids 21 to 167 (construct #1129), 22 to 167 (constructs #1127 and #1150), or 25 to 167 (construct #1128). Construct #1127 was sy.~ll.p~ d by PCR using (1) the above described full-length ob cDNA, (2) r~,l w~d primer #560 co.~ P a sequence as follows:
5'-GCTCTAGACATATGGTGCCTATCCAGAAAGTCC-3'. This primer co--~ ed mlcieotines i78 to i97 ofthe coding strand, as in~li~ted by the lm~~e~ p~d portion ofthe sequence, begi....;..;~. with~aI andNdeI restriction sites and (3) reverse primer #558, as deccrihed above. This primer co~ pd mlrleoti~l~Ps 602 to 616 ofthe nnnro~in~ strand, as inr1ic~ted by the underlined portion, and ~ytpnrl~pd with a SmaI
restriction site, as in~ic~ted by the non-underlined portion. The DNA was ~ pl'~7~1 by 2 0 PCR using a :jL~Idal ~1 PCR protocol.
The ~mplifiecl DNA fragment of construct #1127 was ligated into T7 CAY~ C~SiOll p1~cmir1 pHB40P. The ;..;I;~Ior mPSh;oninP~ for ~ Jre5;~;011 in prok~yc~Les was provided by the NdeI restriction site and was not part of the native protein. Vector p~340P is a derivative of pET pl~cmi~ desc~ibed in Studier et al., Mef*ods in Enymol. (1990) 185:
25 60, and co..l~ c a di~elellL polylinker col"paled to the pET vector. E~ylession ofthe ob polypeptides in this construct is under control ofthe T7 poly"w,~se which directs l,~s~i,iylion from the T7 plonloler.
For ~,Aylc;ssion in .--~ n cells, the DNA ~ e~L enCof~ the tr~m~te~
form ofthe ob protein was ligated into the euk~yoLc cAyles~ion pl~cmi~l pCG as 30 ~lF e.,~ ;l,ed above for the ~",yrtS~iOII of the full length form of the protein.

CA 022l8529 l997-ll-05 W096/35787 PCTrUS~6/OC~O~

Constructs to provide for e,~res~;on of the ob polypeptides with either an HA orMyc epitope tag were made as follows. Expression construct #1150i~ ~ in FIG. 1 was ~y~ d by PCR using (1) the above de,crrihed full length ob cDNA, (2) fc,l w~d primer #560, described above, and (3) reverse primer #559 CQ~ . a seq~l~nçe as follows: 5'-CGCCCGGGGCATTCAGGGCTAAC-3'. The DNA was ~mr1ifiÇd by PCR
using a standard PCR protocol.
For e,~res:iion in prok~yoles, the ~mrlifiçd DNA La~llc;lll was ligated into vector pHB40P, described above, co..~ g the seqllence for heart muscle kinase and the Myc epitope, 5'-CCCGGGGGAC GCAGAGCTTC CGTGGAGCAG AAGCTGATTT
lo CCGAGGGAGG ACCTGAACTGA. The final construct is irl~ntified in FIG. 1 as #1150.
Constructs #1123 and #1124, as in-lir,~tçd in FIG. 1, were synth~èi7~d by PCR
using (1) the above-descl;l,ed full length ob cDNA, (2) folw~d primer#557, deerrihed above, and (3) reverse primer #559, d~e~ ed above. For c,~y~ ion in ~ n cells, the DNA fr~ m~ntc were ligated into a pCG vector, des.i~ ;l,ed above, co..l~ g either 15 the epitope seqll~nce for the monoclonal antibody against HA or Myc. The final constructs # 1123 and #1124 are identified in FIG. 1.
Constructs #1142, #1143, and #1144 were made for c,.~ ion in cuk~yo~es, and cont~inçd no ad(lition~l amino acids from the vector, as shown in FIG. 2. The mlrlçoti~lç
sequence enro~ling the full-length ob protein (construct #1144) and versions lacking the 20 putative signal seq~len~e wilLoul ~d~ n~l amino acid residues were constructed in plasmid pBJ-1. Plasmid pBJ-1 is a pcDL-SRa296 deliv~live, as desclil,ed in Takebe ef al., MoL Cell. Biol. (1988) 8: 466-472, with a mo~lifiçd polylinker, and directs ~ ;o in . . .~ n cells from the SRa promoter. SRa is composed of the simian virus 40 (SV40) early prc,llloler and the R se m~nt and part ofthe U5 sequence (R-U5') ofthe 25 long terminal repeat (LTR) of human T-cell lellkf~.mi~ virus type 1. Construct #1144 and #1142 ~ selll the full-length ob protein with and without the Myc tag, ~ especli~ ~ ly.
Construct #1143 repl~sellls ob protein lacking the first 21 amino acids. This construct also co--~ .c the Myc tag. Constructs #1142 and #1143 further COIl.~ S a SmaI
restriction site 5' to the Myc epitope tag.
30 Constructs #1147 and #1145, identifi~cl in FIG. 2, co.. l~ d the coding se.~ofthe full-length ob protein, with or willluul Myc epitope tag, re~e~ ely~ illselled into WO 96/35787 PCT/US~6/OG~;09 the retrovirus vector pBabePuro. This vector was des.i-il,ed in Mo.)~ e:-- and Land, NucleicAcidsRes. (1990) 12: 3587-3596. E~ es~ion ofthe ob polypeptide with the pBabePuro vector is under the control of the M-MuLV long terminal repeat and can be ~dAition~lly reg~ ted with a T7 promoter. The "Puro" seq~len~ e enroAin~ ~,uro---y~-s ~e~:c~ re is under the re~ t~ry control of a SV40 promoter.

E~l"~ 3 Expression ofthe ob Protein in Bacteria: Isolation ofthe ob Protein and Generation of Polyclonal Antibodies A~ainst the ob Protein The ob DNA constructs CQ.. ~ g the T7 promoter, inrl~lAin~ the DNA encoding the full length and the l ~ c~led forms ofthe ob protein, were transformed, respectively into E. coli strain BL21 (pLysS), which was described in Studier et al., Mefhods in Enzymol. (1990) 185: 60, for ~Ayres~ion. O~e ~,A~ressed ob protein was i~l~ted from inrl~lc~on bodies by dissolving the cell pellet in rhr)sph~tp~-saline buffer co..~ P about 5 5 mM PMSF (phe..yl---c:ll,ylsulfonyl fllloriAe) and about 2 mM DTT. The suspension was s~n:~~teA ~dj-~cted to a final cQnrP~ alion of about 0.5 M NaCI and 1.0% Triton-X-100.
The Iysate was cleared by cçntrifi-g~tiQn at about 12 K rpm for about 10 ..~ es at 4~C.
The pellet was washed with phnsph~te saline buffer to remove traces of Triton-X-100.
Tnr~lC;~m bodies were dissolved in about 7 M ~l~niAillm-hydrorhlnriAe in about 100 mM
2O phosl.h~e buffer and 10 rnM Tris-HCI (pH 7.5). Tnco~ le particles were removed by centrifugation. Sol~kili7ed ob polypeptides were dialyzed against 7 M urea c~
about 10 mM NaCl and 20 mM Tris-HCI (pH 7.5) in several steps. The ob polypeptid~Ps were purified by ion t ..~h~l~ge Ch1U~ Q raphy using Q-seph~ose ~u--,l-ased fromPh~rm~r;~ (Piscak~w~y, N.J.) For rPfol~ling the ob polypeptides were dialyzed against 2 S phosElh~te-saline buffer ccsn~ -P about 1 M NaCl at a conr-e~ lion of about 10 to about 150 ~g/ml. After dialysis, against only phosFh~tP,_saline buffer, the protein was conc~ led to about 1 mg/ml.
S~ 11Y purified ob polypeptides were used to raise polyclonal ~ntihorlips inrabbits using standard teçhn;~ Pc Such polyclonal antibodies were raised by E.L Labs (Soquel, CA) in accol-iance with the following p,otocol: Three animals were ;.. -~ A
by i--l-~ r injections into the hind leg at two sites and by s~bc~ -e~ c injection at WO 96/35787 PCT/US~G/CC~09 the scruff of the back. This mode of i. . -~ ;on has been app~ ed by the NIH. The inocula con~i~ted ofthe Anti~Fn the ob protein, in 0.5 ml saline and an equal volume of adjuvant. Complete Freund's adjuvant was used for the first ;"""..~ n and i"ro~ cte Freund's adjuvant was used for all other boosts. Animals were ;...... ~ ed at 5 ap~ Y;~ F~-ly 3 week intervals. Test bleeds were pe~ r... I..Pd via the central ear artery, and ~Ampl~~ of about 5 to 10 ml are collP,cted Test bleeds were taken once as a p..l)!e e d, and then at weeks 4, 5 and 7 after ;~ AI;Oll Animals were ~Y~n~,~;"-lF~ once the titer in the sera was acceptable.

o Example 4 Expression of the ob Protein in Insect Cells For CAIJIe~;On ofthe ob protein in insect cells, the wild-type mouse ob seq~lFnre was excised from the pCG vector by cutting with XbaI and R~ This insert was thendoned into the PAcC13 vector using the XbaI and BamF~ sites. This construct was l~ rs;eled into SF9 cells using the following procedure. The ob coding sequenr,e was eco.~ d into theA~lo~ a californica baculovirus, AcNPV, via the pAcC13 rer vector, as described in MllnF cmit~l ef al., Mol. Cell. Biol. (1990) 10: 5977-5982, by co-L~ ;rpel;~g about 2 ~g oftransfer vector with about 0.5 ,ug of 1;1~F~ 1, wild-type viral DNA into SF9 cells as descl;lled in Kitts et al., Nucleic Acids Res. (1990) 18: 5667-5672. P~eco~ baculovirus was i~sl~ted by plaque p~ ;on as des~rihe~ in Smith etal., Mol. Cell. Biol. (1983) 3: 2156-2165. .S~pFn~ion ~;ul~ulés of about 1.5 x 106 SF9 cells per ml were halve~led for ligan binding following about 48 hours infFrtinn with the relevant baculovirus at m.o.i. (mllltirlicity of infection) of about 2-10, in serum-free me~ m, as described inMalorellaetal., Biotec*noL (1988) 6: 1406-1510.
A~er about 48 hours, secretion of ob polypeptide into the culture .. ~1;-.. .ofthe SF9 was obselved. The ob polypeptide was v~ li7P~d by Coc,~ - c stain and also by Western analysis using ob antibody. A two-step ~ l ;OI- procedure was employed.
First the protein was purified -via DEAE anion ~ CLI ~ J~ I O~J a~hy. The protein fractions were pooled and applied to a .~eph~yl 100 column and eluted with ph~ le 30 bu~elèd saline (PBS). The ob-co~ g fractions were vi~l~li7pd by ~ with Coom~;e. The ob protein was purified as one product. A slightly faster migrating CA 022l8529 l997-ll-05 WO 96/3~;787 PCT/US~GIOC6C9 species was seen on sodium dodecyl sulfate (SDS) gel electrophoresis in the ~l~sP~-r~ of Aith;oll.. cilol. This most likely cc,.L,I,led the prcsencc of one ~lie~llfide bond in the ob polypeptide.

Ex~ c S
In Vitro Kinase Reaction of the ob Protein Tagged with Heart Muscle Kinase Recogll,Lion Sequence and Detection of the ob Protein in Eukaryotic Ce11s Heart muscle kinase tagged ob polypeptide was p--rified from ba-;lcl;a, as described above. Applox;...~ly 2 llg of purified ob polypeptide was labeled in about 20 0 ~11 of 20 mM Tris-HCI (pH 7.5), 0.1 M NaCI, 12 mM MgC12 and 1 mM DTT with about 100 ~Ci t~ 32P]ATP purchased from A---c~ , which had an activity of about >6000 Ci/mMol, together with about 10 Units of heart muscle kinase from Sigma. The soh-tinn was i~e~lbz~e~ for about 30 .~ les at 37~C. T ~helod ob polypeptide was sep~led from ul-hlcoll~ol~led [~ 32P]ATP by ion ~ ,P, cl~rolllalography~ as desclil,ed above using Q-sepharose.
This labeled ob polypeptide is used to react with cells, cell C~ CIS, cell mt;...~ es or portions thereof to look for specific binding of a r tceplor to the labeled ob polypeptide. The specific binding pair obt~inPd in this manner is s~led from thereaction mi, and is ~ cori~ted by convçntinn~l techni1u~Ps The putative Icce~lor20 Illcll~l~el ofthe binding pair is then cl-~ t-iLcd, for eY~mr~e, to delP- ~ e m~ r size, and all or a portion ofthe amino acid sequence. Based upon the i..r~ ;nn obtained from amino acid sequ-onre analysis, the coding seq ~Pnre ofthe ob Icc~lor is cloned, in accoldal~ce with convPnti~ n~1 te~ P~ Thus, an olis~oî ~ eotide probe is constructed and labeled with an i-1Pntifi~bl,o, marker, such as a M~io~c~ive label. This 25 labeled oligonllrlootide probe is then used to probe a cDNA or ~onom;C library to look - for specific hybri-li7~tiQn A clone signifying the pl.,s~.lce of a full length coding region for an ob rcceplor is then i~ol~ted and its nllrleoti~le seq~Pnce dcltlll~ ed. This clone is used for l~cc.~ prod~ction ofthe ob rcccl~lor.
The ob constructs co.~ g the CMV promoter/e..1-~l-cf 1 were ll~n. .~lly 30 ll~lsrul,lled into COS-7 cells. For ;.~ -oblotting, cell extracts were boiled in sample buffer, as desclil,ed in T.~Pmm1; ef al., Nafure (1970) 227: 680, s~led by SDS-PAGE

CA 022l8529 l997-ll-05 WO 96/35787 PCT/US~6/06609 and L~lsrelled to nitroc~ se filters. Filters were b'~ Pd in TBST buffer cQneietins~ of about 10 mM Tris-HCI (pH 7.5), 130 mM NaCl, 0.05% Tween20, and 0.2% sodium azide co~ P about 3% non-fat dried milk. The filters were i~ "b~ed with polyclonal antibodies against the ob polypeptide or mnnocl~n~l ~ntiho~ s ~lilec~ed against epitope 5 tagged ob ploleins. Bound antibodies were detected with an ~lk~line phosph~t~ee conj~ te~ anti-rabbit or anti-mouse antibody from Promega.

"";le 6 Intravenous ~.l. "..~isl . ~lion of the ob Protein in CD Rats The bacterially ~,A~ ssed and purified ob polypeptide ofthe construct #1127, ntified in FIG. 1, was ~ ele~ ~d to each of 3 CD rats. Three other CD rats were not treated but serve as controls. The rats were all male rates about 7 weeks old. All of them were housed in metabolic cages for ~.~ol.;lo~ g body weight, food col.~ .lion, fecal weight, water intake, and urine output. Urinalysis was done daily to d~ -e15 protein, ~lcosP, kPtQn~e nitrite, ~lnhilinogen~ bilirubin, blood, PX and leukocyte levels.
More spe~ifi~lly the animals were housed in metabolic cages for 24 hours before alion ofthe ob polypeptide. The polypeptide was ~-1....n~ ed twice a day daily for 4 days (BID X4) at about 1 mg/kg per day by jugular c~.".~ c Food was removed for 2-3 hours before dosing and for 1-2 hours after dosing. Body weight, food co~ lion, fecal weight, water intake, and urine output were mo~ r~ t;d daily.
Urinalysis was done daily to dc:lel~ e the protein, gl~cose, k.otonf-e nitrite, urobilino~n bilirubin, blood, PX and 1t;~ku~1e levels.
The results of this study are reflecte~ in the graphs in FIGs. 3-7. FIG. 3 is a graph of the weight of treated CD rats. FIG. 3 shows that upon ~ Iion of the ob polypeptide, eA~ressed by the DNA construct #1127, the treated rats, treated on days 1, 2, 3, 4, and S, showed inhibition of weight gain during the 5 days of I ~
Thereafcer, the weight gained by the treated animals paralleled that gained by the control animals during an observation period of up to day 16. This result s~ ost~ that a periodic dosingl~ n suchasweeldyor b ~ hy,GOl~ of4to5daysof ?~ aliOII
ofthe ob polypeptide at each dosing, may be ~;~re~ in ~ a low weight-gain profile.

WO 96/35787 PCTIUS~6/0660 FIG. 4 shows the amount offood co~ ocl in grams by the treated and u--L-eaLcd ~ rats. Results show that con~ l;on of food in the ob-treated rats was inhibited as co...l,~ed to the unllealed controls.
FIG. S shows the weight of fecal matter eA~ilcLed by the ob-treated and u~ aaled5 controls. Results show that the fecal weight from ob-treated rats is reduced as co...p~ed to the untreated controls. In general, the graphs of the fecal weight versus days show the same general pattern co..-p~cd to the graphs offood conC~mption versus days.
FIG. 6 shows no st~tictir-~l ci~ifir~nce in urine output bcLween the ob-treated rats and the ullL- ~led rats.
FIG. 7 shows the water intake pattern of ob-treated and u~L~Led rats.
These results refiect that the CD rats that were ~ ed ob protein did not gain weight, and ob protein appears to reduce the animals' food intake and fecal output;
water intake ofthe ob treated rats was slightly dep~cs~ed and the urinary output appcalcd normal. The control rats gained weight, and had higher food intake and fecal weights 5 than the ob-treated rats.
The present invention has been d~Ct ~ e~ with Itir~rellcc to specific ~mhod;~
However, this applir~ti~n is int~n~led to cover those rl~-P~s and substitutiQnc which may be made by those skilled in the art without depal L-ng from the spirit and the scope of the appended claims.

Deposition of Cultures The constructs made herein that encode the full-length ob polypeptide and tr~n~ted versions thereofin pl~cmi~l$ pCG, pET23a, and pBabePuro vector have been deposited at the A.T.C.C. (Pa~ wll Drive, Rockville, MD, U.S.A.).

Claims (38)

WHAT IS CLAIMED IS:

1. A nucleic acid molecule comprising a first nucleotide sequence that encodes an expression control sequence and a second nucleotide sequence that encodes an ob polypeptide, wherein the second nucleotide sequence is under regulatory control of the first nucleotide sequence, and the first nucleotide sequence is not naturally associated with the second nucleotide sequence.

2. The nucleic acid molecule of claim 1, further comprising a third nucleotide sequence, wherein the third nucleotide sequence encodes a secretion leader sequence that is sufficient for secretion of the ob polypeptide upon expression of the nucleic acid molecule in a host cell.

3. The nucleic acid molecule of claim 2, wherein the secretion leader sequence is not naturally associated with the ob polypeptide.

4. The nucleic acid molecule of claim 1, wherein the expression control sequence is one selected from the group consisting of a prokaryotic cell promoter and an eukaryotic cell promoter or a viral promoter.

5. An expression vector comprising the nucleic acid molecule of claim 1 and a third nucleotide sequence that encodes a marker.

6. The vector of claim 5, further comprising a fourth nucleotide sequence, wherein the fourth nucleotide sequence encodes a secretion leader sequence that is sufficient for secretion of the ob polypeptide upon expression of the nucleic acid molecule in a host cell.

7. The vector of claim 6, wherein the secretion leader sequence is not naturally associated with the ob polypeptide.

8. The vector of claim 5, wherein the vector comprises a nucleic acid molecule selected from the group consisting of constructs #1122, #1123, #1124, #1130, #1131, #1132, #1127, #1128, #1129, #1150, #1142, #1143, #1144, #1145, and #1147.

9. A host cell comprising the vector of claim 5.

10. The host cell of claim 9, wherein the vector further comprises a third nucleotide sequence wherein the third nucleotide sequence encodes a secretion leader sequence that is sufficient for secretion of the ob polypeptide upon expression of the nucleic acid molecule in a host cell.

11. The host cell of claim 9, wherein the cell is selected from a group consisting of a prokaryotic cell and an eukaryotic cell.

12. The host cell of claim 9, wherein the cell is a prokaryotic cell, and prokaryotic cell is Escherichia coli.

13. The host cell of claim 11, wherein the cell is an eukaryotic cell, and the eukaryotic cell is selected from the group consisting of a mammalian cell, an insect cell, and a yeast cell.

14. The host cell of claim 13, wherein the eukaryotic cell a mammalian cell and the mammalian cell is a human cell.

15. A method for the production of an ob polypeptide comprising:
a) providing the nucleic acid molecule of claim 1;
b) introducing the nucleic acid molecule into a cell that is capable of expressing the ob polypeptide; and c) allowing the expression of the ob polypeptide in the cell.

16. A method for production of an ob polypeptide comprising:
a) providing the vector of claim 5;
b) introducing the vector into a host cell; and c) allowing expression of the ob polypeptide in the cell.

17. A method for production of an ob polypeptide comprising:
a) providing the host cell of claim 9; and b) allowing expression of the ob polypeptide in the cell.

18. A method for induction of production of an ob polypeptide in a mammal comprising administering to a mammal the nucleic acid molecule of claim 1.

19. The method of claim 18, wherein the nucleic acid molecule is delivered directly or by viral or non-viral means.

20. A method for induction of production of an ob polypeptide in a mammal comprising administering to the mammal the vector of claim 5.

21. An ob polypeptide produced by the method comprising:
a) providing a host cell comprising the nucleic acid molecule of claim 1; and b) allowing the expression of the ob polypeptide.

22. A method for inhibition of weight gain comprising administering a weight-gain-inhibitory amount of the ob polypeptide of claim 21.

23. A method for the inhibition of food intake comprising administering a food-intake-inhibitory amount of the ob polypeptide of claim 21.

24. A method for production of an ob receptor comprising:
a) providing a labeled ob polypeptide, wherein the ob polypeptide that is labeled is the polypeptide of claim 21;
b) allowing the labeled ob polypeptide to react with cells or portions or extracts thereof to form a binding pair; and c) separating from the binding pair an ob receptor that binds to the labeled ob polypeptide.

25. An antibody comprising an amino acid sequence, wherein the sequence is capable of binding to the ob polypeptide of claim 21 to form a binding pair.

26. The antibody of claim 25, wherein the antibody is a mammalian antibody or a humanized antibody.

27. The antibody of claim 26, wherein the antibody is a murine antibody or a human antibody.

28. A method for identification of an ob polypeptide or an ob polypeptide homolog comprising contacting a labeled antibody with a sample suspected of containing an ob polypeptide or an ob polypeptide homolog to allow formation of a binding pair, and determining the identity of the binding pair, wherein the antibody that is labeled is the antibody of claim 25.

29. A method for production of an antibody to an ob receptor comprising administering to an animal the ob receptor of claim 30, and collecting from the animals either serum containing antibodies or spleen cells for the production of monoclonal antibodies.

30. An ob receptor produced by the method of claim 24.

31. A method for detection of an ob receptor comprising:
a) providing a labeled antibody to an ob receptor, wherein antibody that is labeled is the antibody of claim 29;
b) allowing the labeled antibody to react with a cell or portions or extracts thereof to allow formation of a binding pair, and c) determining the presence of the binding pair.

32. A method for the detection of an ob polypeptide comprising:
a) providing a labeled antibody to an ob polypeptide, wherein the antibody that is labeled is the antibody of claim 25;
b) allowing the labeled antibody to react with an ob polypeptide to form a binding pair; and c) determining the presence of the binding pair.

33. A kit for detection of an ob polypeptide comprising the antibody of claim 25.

34. A kit for detection of an ob receptor comprising the antibody of claim 29.

35. A kit for the detection of an ob receptor comprising a labeled ob polypeptide, wherein the polypeptide that is labeled is the polypeptide of claim 21.

36. A kit for the detection of antibodies to an ob polypeptide comprising a labeled ob polypeptide, wherein the polypeptide that is labeled is the polypeptide of claim 21.

37. A pharmaceutical composition the ob polypeptide of claim 21 and a pharmaceutically acceptable carrier.

38. A method of blocking the activity of ob polypeptide in a mammal by administering the antibody of claim 25.