Note: Descriptions are shown in the official language in which they were submitted.
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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
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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~
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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.
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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
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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.
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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
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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.
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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.
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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
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- 10-
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
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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
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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~
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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
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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
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-- 15 --
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
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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
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- 1 7 -
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.
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- 18-
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
CA 02218529 1997-11-05
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- 1 9 -
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.
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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
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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:
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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
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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
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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.
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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.
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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.
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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
CA 02218529 1997-11-05
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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
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- 29 -
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
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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
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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,
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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
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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.
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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
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- 35 -
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.
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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
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- 37 -
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.
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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
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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
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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
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-41 -
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
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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.
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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.).
