Note: Descriptions are shown in the official language in which they were submitted.
CA 02224646 1997-12-12
WO 97/00319 PCT/GB96/01388
C~nuPDTr LEPTTN hUSED TO nM~nUNCK;LOBULnN ~ MAnN AND USE
The present invention relates to a novel compound being a novel chimeric protein,
to a process for the preparation of such a compound, a pharmaceutieal composition
5 comprising such a compound and the use of such a compound in medicine, especially for
the tre~tm~nt of obesity and associated diseases.
European Patent Application, Publieation number 0 464 533 discloses fusion
proteins comprising various portions of the constant region of immunoglobulin moleeules
together with another human protein or part thereof. European Patent Applieation,
Publieation number 0 297 882 diseloses fusion proteins eomprising various portions of
the plasminogen moleeule with part of another human protein.
Zhang et àl. (Nature: 372, 425 - 432; 1994) deseribe the positional eloning of amouse obese gene and its human homologue. The sequenee of the Open Reading Frame(ORF) of the mouse gene prediets a polypeptide of 167 amino aeids and Zhang et al.
15 predicted the presence of a signal sequence which would lead to the production of a
mature protein of 146 reci~ es The human homologue was disclosed as having a similar
size of 146 amino acids for the mature protein. Zhang et al. showed the presence of a
primary translation product of approximate size of 18 kilodaltons (kD) with truncation to
a 16kD produet on addition of mierosomal membranes, eoncist.-nt with the produetion of a
20 pre-protein and the removal of an N-terminal signal sequenee. Zhang et al also diselose
the potential use of the human obese gene produet (hereinafter 'leptin') in the treatment of
obesity.
For effeetive, praetieal tre~rment of obesity a particularly desirable ~-upel~y of an
obesity agent is a clearance rate in hum~nc commensurate with patient acceptable25 treatment regim.-nc, especially regimens for injectable therapies. Zhang et al. do not
diselose information relating to the elearanee rate of the aetive moleeule in either mouse or
hnm~3n.c
The preeise meehanism of aetion of leptin is eurrently unknown, however it is
eonsidered that in order to provide the observed pharmaeologieal effeets, leptin must
30 in~eraet with one or more reeeptors in the brain.
We have now diseovered eertain ehimerie derivatives of leptin whieh surprisingly,
despite their large moleeular size, have good pharmaeological activity combined with
prolonged clearance rates. These ehimerie derivatives of leptin are therefore in(li~t~ to
be partieularly useful for the treatment or prophylaxis of obesity and for the treatment or
35 prophylaxis of diseases and eonditions assoeiated with obesity, such as atherosclerosis,
hypertension and, especially, Type II diabetes. In partieular these eompounds are
eonsidered to be useful for ~lminictration by injeetion.
CA 02224646 1997-12-12
W O 97/00319 PCT/GB96/01388
These compounds are also considered to be useful in cosmetic tre~tm~ontC for theimprovement of body appearance.
Accordingly, the invention provides a chimeric leptin or a chimeric mutant or
derivative of leptin. r
One particular chimeric leptin is a protein comprising leptin or a mutant or variant
thereof fused to a human immnnQglobulin domain or a mutant or variant thereof.
Suitably, the chimeric protein cc,~ .ises one human immlln(~globulin domain.
Favourably, the human immnnoglobulin domain is fused to the C-terminllc of
leptin.
One favoured human immllnf-globulin is an human immnnoglobulin Fc ~ m~in
An example of a human immunoglobulin Fc domain is an IgG4PE variant in
particular IgG4 hinge-CH2-CH3 PE. Other examples are IgG4, IgG1 and IgGlGT, in
particular the hinge-CH2-CH3 region in each case.
The term "mutant or variant" used with respect to a particular protein
encomp~ cses any molecule such as a trllnc~te~l or other derivative of the relevant protein
which retains substantially the same activity in humans as the relevant protein. Such
other derivatives can be prepared by the addition, deletion, substitution, or rearrangement
of amino acids or by chemical modifications thereof.
The immunoglobulin may be of any subclass (IgG, IgM, IgA, IgE), but is
preferably IgG, such as IgG 1, IgG3 or IgG4. The said constant domain(s) or fragment
thereof may be derived from the heavy or light chain or both. The invention
encomp~cses mutations in the immnnoglobulin component which elimin~te undesirable
~,~,pe~Lies of the native immunoglobulin, such as Fc receptor binding and/or introduce
desirable ~-~,pe- Lies such as stability~ For example, Angal S., King D.J., Bodmer M.W.,
Turner A., Lawson A.D.G., Roberts G., Pedley B. and Adair R., Molecular Immunology
vol30pplO5-108, 1993, describe an IgG4 molecule where residue 241 (Kabat numbering)
is altered from serine to proline. This change increases the serum half-life of the IgG4
molecule. Canfield S.M. and Morrison S.L., Journal of Experimental Medicine
voll73ppl483-1491, describe the alteration of residue 248 (Kabat numbering) fromleucine to glllt~m~te in IgG3 and frorn ghlt~m~te to leucine in mouse IgG2b.
Suhstit~ltion of leucine for glut~m:~te in the former decreases the affinity of the
immunoglobulin molecule concerned for the Fc yRI receptor, and substitution of
gl~lt~m~te for leucine in the latter increases the affinity. EP0307434 discloses various
mutations including an L to E mutation at residue 248 (Kabat numbering) in IgG.
The constant domain(s) or fragment thereof is preferably the whole or a
substantial part of the constant region of the heavy chain of human IgG. The IgG
CA 02224646 1997-12-12
W O 97/00319 PCT/GB96101388
cc,lllponent suitably comprises the CH2 and CH3 domains and the hinge region including
cysteine residues contributing to inter-heavy chain disulphide bonding.
For example when the IgG co,l,ponent is derived from IgG4 it incltl~s cysteine
residues 8 and 11 of the IgG4 hinge region (Pinck J.R. and Milstein C., Nature
f 5 vol216pp941-942, 1967). Preferably the IgG4 component consists of amino acids
corresponding to residues 1- 12 of the hinge, 1-110 of CH2 and 1-107 of CH3 of IgG4
described by Ellison J., Buxbaum J. and Hood L., DNA vollppl 1-18, 1981. In one
example of a suitable mutation in IgG4, residue 10 of the hinge (residue 241, Kabat
numbering) is altered from serine (S) in the wild type to proline (P) and residue 5 of CH2
(residue 248, Kabat numbering) is altered from leucine (L) in the wild type to gll~r~m~te
(E).
DNA polymers which encode m-lt~ntC or variants of the human immllnoglobulin
may be prepared by site-directed mutagenesis of the cDNA which codes for the required
protein by conventional methods such as those described by G. Winter et al in Nature
1982, 299, 756-758 or by Zoller and Smith 1982; Nucl. Acids Res., 10, 6487-6500, or
deletion mutagenesis such as described by Chan and Smith in Nucl. Acids Res., 1984, 12,
2407-2419 or by G. Winter et al in Biochem. Soc. Trans., 1984; 12, 224-225 or
polymerase chain reaction such as described by Mikaelian and Sergeant in Nucleic Acids
Research, 1992, 20, 376.
When used herein 'compound of the invention' or 'compounds of the invention'
relates to the above mentioned chimera.
In a further aspect, the invention provides a process for preparing a compound
according to the invention which process comprises ek~-~s~ g DNA encoding said
compound in a recombinant host cell and recovering the product.
The DNA polymer comprising a nucleotide sequence that encodes the compound
also forms part of the invention.
The process of the invention may be performed by conventional recomhin~nt
techniques such as described in Maniatis et. al., Molecular Cloning - A Laboratory
Manual; Cold Spring Harbor, 1982 and DNA Cloning vols I, II and III (D.M. Glover ed.,
IRL Press Ltd).
In particular, the process may comprise the steps of:
ing a replicable expression vector capable, in a host cell, of expressing a
DNA polymer comprising a nucleotide sequence that encodes said compound;
ii) transforming a host cell with said vector;
iii) culturing said transformed host cell under conditions permitting expression of
said DNA polymer to produce said c~ ound; and
iv) recovering said compound.
CA 02224646 1997-12-12
WO 97/00319 PCT/GB96/01388
The invention also provides a process for preparing the DNA polymer by the
con-len~atiQn of a~lopliate mono-, di- or oligomeric nucleotide units.
The preparation may be carried out chemically, enzym~tir~lly, or by a
combination of the two mt~tho~ls, in vitro or in vivo as a~rol,liate. Thus, the DNA
S polymer may be l,r~ ed by the enzymatic ligation of ay~lol,liate DNA fr~gment~, by
convensionzll methods such as those described by D. M. Roberts et al in Biochemistry
1985, 24, 5090-5098.
The DNA fr~gm~ tc may be obtained by digestion of DNA containing the
required sequences of nucleotides with a~,ulo~liate restriction enzymes, by chemical
synthesis, by enzymatic polymerisation on DNA or RNA templates, or by a combination
of these methods.
Digestion with restriction enzymes may be performed in an al,~lo~liate buffer at a
temperature of 20~-70~C, generally in a volume of 50~1 or less with 0.1- lO,ug DNA.
Enzymatic polymerisation of DNA may be carried out in vitro using a DNA
polymerase such as DNA polymerase I (Klenow fragment) in an appropriate buffer
containing the nucleoside triphosphates dATP, dCTP, dGTP and d~P as le~ui-~,d at a
L~ pelature of 10~-37~C, generally in a volume of 50~1 or less.
Enzymatic ligation of DNA fr~gment~ may be carried out using a DNA ligase
such as T4 DNA ligase in an appropriate buffer at a temperature of 4~C to ambient,
generally in a volume of 50111 or less.
The chemical synthesis of the DNA polymer or fr~gm~ntc may be carried out by
conventional phosphotriester, phosphite or phosphoramidite chemistry, using solid phase
techniques such as those described in 'Chemical and Enzymatic Synthesis of Gene
Fr~gm.-nec - A Laboratory Manual' (ed. H.G. Gassen and A. Lang), Verlag Chemie,
Weinheim (1982),or in other scientific publications, for example M.J. Gait, H.W.D.
Matthes, M. Singh, B.S. Sproat, and R.C. Titmas, Nucleic Acids Research, 1982, 10,
6243; B.S. Sproat and W. Bannwarth, Tetrahedron Letters, 1983, 24, 5771; M.D.
~tteucci and M.H Caruthers, Tetrahedron Letters, 1980, 21, 719; M.D. M~tteucci and
M.H. Caruthers, Journal of the American Chemical Society, 1981, 103, 3185; S.P.
Adams et al., Journal of the American Chemical Society,1983, 105, 661; N.D. Sinha, J.
Biernat, J. McMannus, and H. Koester, Nucleic Acids Research, 1984, 12, 4539; and
H.W.D. Matthes et al., EMBO Journal, 1984, 3, 801. Preferably an automated DNA
synthesi7.or is employed.
The DNA polymer is preferably ~ p~cd by ligating two or more DNA
molecules which together comprise a DNA sequence encoding the compound. A
particular process in accordance with the invention comprises ligating a first DNA
CA 02224646 1997-12-12
WO 97/00319 PCT/GB96/01388
molecule encoding a said leptin or variant and a second DNA molecule encoding a said
immunoglobulin domain or fragment thereof.
The DNA molecules may be obtained by the digestion with suitable restriction
enzymes of vectors carrying the required coding sequences or by use of polymerase chain
5 reaction technology.
The precise structure of the DNA molecules and the way in which they are
obtained depends upon the structure of the desired product. The design of a suitable
strategy for the construction of the DNA molecule coding for the compound is a routine
matter for the skilled worker in the art.
The expression of the DNA polymer encoding the compound in a recombinant
host cell may be carried out by means of a replicable e~lession vector capable, in the
host cell, of e~ ;ssing the DNA polymer. The expression vector is novel and also forms
part of the invention.
The replicable expression vector may be prepared in accordance with the
invention, by cleaving a vector compatible with the host cell to provide a linear DNA
segment having an intact replicon, and combining said linear segment with one or more
DNA molecules which, together with said linear segment, encode the compound, under
llganng condlnons.
The ligation of the linear segment and more than one DNA molecule may be
carried out simultaneously or sequentially as desired.
Thus, the DNA polymer may be preformed or formed during the construction of
the vector, as desired.
The choice of vector will be determined in part by the host cell, which may be
prokaryotic, such as E. coli, or eukaryotic, such as mouse C127, mouse myeloma,
chinese hamster ovary, Cosl or Hela cells, fungi e.g. fil~7mentous fungi or unicellular
yeast or an insect cell such as Drosophila. The host cell may also be a transgenic animal.
A preferred host cell is Cosl.
Suitable vectors include pl~.cmill$~ bacteriophages, cosmids and recombinant
viruses derived from, for example, baculoviruses, vaccinia or Semliki ~orest virus.
The preparation of the replicable expression vector may be carried out
conventionally with ap~ iate enzymes for restriction, polymerisation and ligation of
the DNA, by procedures described in, for example, Maniatis et_l., cited above.
Polymerisation and ligation may be performed as described above for the preparation of
the DNA polymer. Digestion with restriction enzymes may be performed in an
a~ lvpliate buffer at a temperature of 20~-70~C, generally in a volume of 50~1 or less
with 0.1-lO~Lg DNA.
CA 02224646 1997-12-12
W O 97/00319 PCT/GB96/01388
The recombinant host cell is prepared, in accordance with the invention, by
transforming a host cell with a replicable expression vector of the invention under
transforming conditions. Suitable transforming conditions are conventional and are
described in, for example, Maniatis et al., cited above, or "DNA Cloning" Vol. II, D.M.
5 Glover ed., IRL Press Ltd, 1985.
The choice of transforming conditions is determined by the host cell. Thus, a
bacterial host such as E. coli may be treated with a solution of CaC12 (Cohen et al, Proc.
Nat. Acad. Sci., 1973, 69, 2110) or with a solution comprising a mixture of RbCl,
MnC12, potassium acetate and glycerol, and then with 3-[N-morpholino]-
10 propane-sulphonic acid, RbCl and glycerol. ~mm~ n cells in culture may be
transforrned by calcium co-precipitation of the vector DNA onto the cells.
The invention also extends to a host cell transforrned or transfected with a
replicable e~l,rts:,ion vector of the invention.
Culturing the transformed host cell under conditions permitting expression of the
15 DNA polymer is carried out convenlionally, as described in, for example, Maniatis et al
and "DNA Cloning" cited above. Thus, preferably the cell is supplied with nutrient and
cultured at a Le~ cl~ture below 45~C.
The expression product is recovered by conventional methods according to the
host cell. Thus, where the host cell is bacterial, such as E. coli it may be lysed
20 physically, chemic~lly or enzym~tiç~lly and the protein product isolated from the
res~ in~ lysate. If the product is to be secreted from the bacterial cell it may be
recovered from the periplasmic space or the nutrient medium. Where the host cell is
..-~-.... ~liz~n, the product may generally be isolated from the nutrient m~ m
The DNA polymer may be assembled into vectors designed for isolation of stable
transformed m~mm~ n cell lines c~ cssing the product; e.g. bovine papillomavirusvectors or amplified vectors in chinese hamster ovary cells (DNA cloning Vol.II D.M.
Glover ed. IRL Press 1985; K:~nfm~l-, R.J. et al., Molecular and Cçlllll~r Biology 5,
1750-1759, 1985; Pavlakis G.N. and Hamer, D.H., P~occedings of the National Academy
of Sciences (USA) 80, 397-401, 1983; Goeddel, D.V. et al., European Patent Application
No. 0093619, 1983).
The activity of the chimeric leptin is determined by injecting it in~ c-itoneally,
intravenously or subcutaneously into test ~nim~l~ such as rodents, for example mice or
rats, or prim~rçs, for example rhesus monkeys. In order to maximise activity, the test
animals are preferably overweight or obese animals that have been made overweight by
feeding them on a high fat or other palatable diet, or have acquired fat through the ageing
process. In the case of mice, however, the ideal strain is the genetically obese (ob/ob)
mouse. The effect of the active compound is seen as a reduction in food intake or increase
CA 02224646 1997-12-12
W O 97/00319 PCT/GB96/01388
in metabolic ra~e or oxygen consumption. Multiple injections of the active collipou"d - at
most twice daily - over a period of a week for rodents or a month for prim~tçs, also cause
a reduction in body weight and in the size of discrete adipose tissue depots.
Clearance rates are determined by conventional plasma assay using ob-antibodies,~ 5 forexarnple ELISA methodology.
As in~liç~te~l above the compounds of the present invention have useful
J pharmaceutical properties, in particular anti obesity activity and also for the tre~tm~nt of
~lice~es associated with obesity, such as atherosclerosis, hypertension and, especially,
Type II diabetes.
In use the compound will normally be employed in the form of a pharm~ceuric~l
composition in association with a human pharmaceutical carrier, diluent and/or excipient,
although the exact form of the composition will depend on the mode of a~ministration.
The active compound may be formulated for ~imini~tration by any suitable route
and is preferably in unit dosage form. Advantageously, the co-lJl~o~ition is suitable for
oral, rectal, topical, parenteral, intravenous or intramuscular a-lmini~tration or through the
lcs~ilat~ly tract. Preparations may be designed to give slow release of the active
ingredient.
The compositions of the invention may be in the form of tablets, capsules, sachets,
vials, powders, granules, lozenges, suppositories, recon~ritut~ble powders, or liquid
preparations such as oral or sterile parenteral solutions or suspensions. Topical
formulations are also envisaged where a~lu~liate.
The invention therefore further provides a pharm~eutical composition
comprising a compound of the invention and a pharm~ceutically acceptable carrier.
The dosage ranges for ~-1mini~rration of the compounds of the present invention
are those to produce the desired therapeutic effect. Dosage will generally vary with age,
extent or severity of the medical condition and contr~in~ tions, if any. For example in
the tre~tment of obsity the unit dosage can vary from less than lmg to 300mg, but
typically will be in the region of 1 to 20mg per dose, in one or more doses, such as one to
six doses per day, such that the daily dosage is in the range 0.02-40mg/kg.
Dosages and compositions for the tre~tmçnt of diseases associated with obesity
such as atherosclerosis, hypertension and, especially, Type II diabetes are selected from an
equivalent range to that used in the treatment of obesity.
Compositions suitable for injection may be in the form of solutions, suspensionsor emulsions, or dry powders which are dissolved or suspended in a suitable vehicle prior
to use.
Fluid unit dosage forrns are prepared utilising the compound and a pyrogen-free
sterile vehicle. The compound, depending on the vehicle and concentration used, can be
CA 02224646 l997-l2-l2
W 097/00319 PCT/GB96/01388
either dissolved or suspended in the vehicle. Solutions may be used for all forrns of
parenteral ~rlminictration, and are particularly used for intravenous infection. In
preparing solutions the compound can be dissolved in the vehicle, the solution being
made isotonic if necessary by addition of sodium chloride and sterilised by filtration
5 through a sterile filter using aseptic techniques before filling into suitable sterile vials or
ampoules and sealing. Alternatively, if solution stability is adequate, the solution in its
sealed containers may be sterilised by autoclaving. Advantageously additives such as
buffering, solubilising, stabilising, preservative or bactericidal, suspending or
emulsifying agents and/or local anaesthetic agents may be dissolved in the vehicle.
Dry powders which are dissolved or suspended in a suitable vehicle prior to use
may be prepared by filling pre-sterilised drug substance and other ingredients into a
sterile container ùsing aseptic technique in a sterile area. Alternatively the drug and other
ingredients may be dissolved in an aqueous vehicle, the solution is sterilised by filtration
and distributed into suitable containers using aseptic technique in a sterile area. The
15 product is then freeze dried and the containers are sealed aseptically.
Parenteral suspensions, suitable for intramuscular, subcutaneous or intradermal
injection, are prepared in substantially the same manner, except that the sterile compound
is suspended in the sterile vehicle, instead of being dissolved and sterilisation cannot be
accomplished by filtration. The cornpound may be isolated in a sterile state or
20 alternatively it may be sterilised after isolation, e.g. by gamma irradiation.
Advantageously, a suspending agent for example polyvinylpy;rolidone is included in the
composition to facilitate uniform distribution of the compound.
Compositions suitable for ~minictration via the respiratory tract include aerosols,
nebulisable solutions or microfine powders for incllffl~tion. In the latter case, particle
25 size of less than 50 microns, especially less than 10 microns, is ~lcfc-lcd. Such
compositions may be made up in a conventional manner and employed in conjunctionwith conventional ~rlminictration devices.
In a further aspect there is provided a method of treating obesity or ~lice~cçc
~Csoci~te~l with obesity, such as atherosclerosis, hypertension and, especially, Type II
30 diabetes, in human or non-human m~mm~lc which comprises ~minictering to the
.urrcl~. an effective, non-toxic amount of a cc,~ ound of the invention.
Suitable non-human m~mm~lc are domestic m~mm~lc such as dogs and cats.
The invention further provides a compound of the invention for use as an active
therapeutic substance, in particular for use in treating obesity or tlice~ces associated with
35 obesity, such as atherosclerosis, hypertension and, especially, Type II diabetes.
CA 02224646 1997-12-12
W O 97/00319 PCT/GB96/01388
The invention also provides the use of a compound of the invention in the
manufacture of a medicament for treating obesity or diseases associated with obesity,
such as atherosclerosis, hypertension and, especially, Type ~ diabetes.
As in~lic~tecl above the invention also encompasses cosmetic tre~tm~ontc
Accordingly, there is also provided a compound of the invention for use in the
cosmetic tre~tment of human or non-human m~mm~lc
There is also provided a method for the cosmetic treatment of a human or non-
human ~ 1, which treatment comprises ~ministering an effective, non-toxic
arnount of a compound of the invention to a human or non-human " "" "" ,~l in need
thereof.
Cosmetic treatment suitably includes treatment for the improvement of body
appearence, such as weight reduction treatment.
The invention also extends to a cosmetic composition, comprising a co~ ound of
the invention and a carrier therefor.
Compositions of the invention including cosmetic compositions are fo~nl~te~l using
known methods, for example those described in standard text books of pharmaceutics and
co~metics, such as Harry's Co~mt~ticology published by Leonard Hill Books, Remington's
Pharm~ce~ltical Sciences, the British and US Pharmacopoeias.
No unexpected toxicological effects are expected when co~ ou~lds of the
invention are ~lmini~tered in accordance with the present invention.
The following Examples illustrate the invention but do not limit it in any way.
,~ .
CA 02224646 1997-12-12
W O 97/00319 PCT/GB96/01388
Example 1.
Construction of DNA coding for f~sion protein leptin 1-167/IgG4 hinge-CH2-CH3
The gene coding for a fusion protein comprising human leptin and the
S hinge-CH2-CH3 region of human IgG4 is created by recombinant DNA technology, r
preferably by a two-step recombinant PCR method.
The human 'ob' gene has been ~ d synthetically based on the amino acid sequence
of Zhang et al, and assembled in the pcDNA3 vector.
The cDNA encoding full length human leptin, nucleotides 1-501 is joined ae the
3' end to the 5' end of the hinge-CH2-CH3 region of the cDNA coding for the human
IgG4 protein, shown as nucleotides ~02-1188 in the DNA sequence below. (Table 1.)
The encoded protein sequence of the leptin/IgG4 chimera is given in Table 2.
Leptin 1-167 (numbering as Y. Zhang, R. Proenca, M. Maffei, M. Barone, L. Leopold
& J. Frierlm~n Nature 372:425-432)7 and IgG4 hinge-CH2-CH3 168-396 (sequence as
Kabat).
The fusion protein was expressed transiently in Cos 1 cells using the pCDN vector
system, as described in International Patent Application Publication number WO 96/04388.
The mature protein was exported from the cells into the culture m~ m and was ~letect~fi by
anti-leptin antibody. It was shown to to have a size consistent with the predicted structure by
Western blotting analysis under both reducing and nonreducing conditions.
Table 1. DNA sequence of ob/IgG4 chimera, 1188bp
ATGCATTGGGGAACCCTGTGCGGATTCTTGTGGCTTTGGCCCTATCTTTTCTATGTCCAA
GCTGTGCCCATCCAAAAAGTCCAAGATGACACCAAAACCCTCATCAAGACAATTGTCACC
120
AGGATCAATGACATTTCACACACGCAGTCAGTCTCCTCCAAACAGAAAGTCACCGGTTTG
180
GACTTCATTCCTGGGCTCCACCCCATCCTGACCCTGTCCAAGATGGACCAGACACTGGCA
240
GTCTACCAACAGATCCTCACATCGATGCCTTCCAGAAACGTGATCCAAATATCCAACGAC
300
~0 CTGGAGAACCTCCGGGATCTTCTTCACGTGCTGGCCTTCTCTAAGAGCTGCCACTTGCCC
360
TGGGCCAGTGGCCTGGAGACCTTGGACAGCCTGGGGGGTGTCCTCGAGGCTTCAGGCTAC
420
TCCACAGAGGTGGTGGCCCTGAGCAGGCTGCAGGGGTCTCTGCAGGACATGCTGTGGCAG
480
CTGGACCTCAGCCCCGGGTGCGAGTCCAAATATGGTCCCCCATGCCCATCATGCCCAGCA
540
CA 02224646 l997-l2-l2
WO 97/00319 PCT/GB96/01388
CCTGAATTTCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTC
600
~ ATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCC
660
GAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCG
720
CGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAG
780
GACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCATCG
840
ATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTG
900
CCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGC
960
TTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC
1020
AAGACCACGCCTCCCGTGCTGGACTCCGACGGATCCTTCTTCCTCTACAGCAGGCTAACC
1080
GTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCT
1140
CTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA
1188
Table 2. Amino acid sequence of leptin/IgG4 chimera, 396aa
1 MHWGTLCGFL WLWPYLFYVQ AVPIQKVQDD TKTLIKTIVT
RINDISHTQS
51 VSSKQKVTGL DFIPGLHPIL TLSKMDQTLA VYQQILTSMP SRNVIQISND
101 LENLRDLLHV LAFSKSCHLP WASGLETLDS LGGVLEASGY
STEVVALSRL
151 QGSLQDMLWQ LDLSPGCESK YGPPCPSCPA PEFLGGPSVF
LFPPKPKDTL
201 MISRTPEVTC VVVDVSQEDP EVQFNWYVDG VEVHNAKTKP
REEQFNSTYR
CA 02224646 1997-12-12
WO 97/00319 PCT/GB96/01388
251 VVSVLTVLHQ DWLNGKEYK CKVSNKGLPSS IEKTISKAKG
QPREPQVYTL
301 PPSQEEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY
KTTPPVLDSD t
351 GSFFLYSRLT VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGK
CA 02224646 1997-12-12
W O 97/00319 PCT/GB96/01388
ExamDle2.
Construction of DNA coding for fusion protein ob 1-167/IgG4 hinge-CH2-CH3
PE variant
3 5 The gene coding for a fusion protein comprising the human 'ob' protein and the
Hinge-CH2-CH3 region of human IgG4 PE (a form of IgG4 m~lt~te~ as below) is
created by recombinant DNA technology, preferably by a two-step recombinant PCR
method.
The cDNA coding for the complete human leptin, amino acids 1-167(numbering as
Y. Zhang, R. Proenca, M. Maffei, M. Barone, L. Leopold & J. Friedman. Nature 372:
425-432) is joined at the 3' end to the S' end of the hinge-CH2-CH3 region of the cDNA
coding for the human IgG4 (PE variant) protein, shown as amino acids 168-396 in the
protein sequence below.
The human 'ob' gene has been ylc;p~ed synthetically based on the amino acid sequence
of Zhang et al, and assembled in the pcDNA3 vector. The encoded protein sequence is
given in Table 2.
Human IgG4 heavy chain PE variant. In IgG4 PE, residue 10 of the hinge
(residue 241, Kabat numbering) is altered from serine (S) in the wild type to proline (P)
and residue S of CH2 (residue 248, Kabat numbering) is altered from leucine (L) in the
wild type to glutamate (E). Angal S., King D.J., Bodmer M.W., Tumer A., Lawson
A.D.G., Roberts G., Pedley B. and Adair R., Molecular Immunology vol30pplO5-108,1993, describe an IgG4 molecule where residue 241 (Kabat numbering) is altered from
serine to proline. This change increases the serum half-life of the IgG4 molecule.
The IgG4 PE variant was created using PCR mutagenesis on the synthetic human
IgG4 heavy chain cDNA. The sequence of the IgG4 PE variant is described in Table 1.
The residues of the IgG4 nucleotide sequence which were altered to make the PE variant
are as follows:
referring to Table 1:
residue 322 has been altered to "C" in the PE variant from "T" in the wild
type;
residue 333 has been altered to "G" in the PE variant from "A" in the wild
type; and
residues 343-344 have been altered to "GA" in the PE variant from "CT" in
the wild type.
The fusion protein was expressed transiently in Cos 1 cells using the pCDN vector
system, as described in International Patent Application Publication number WO 96/04388.
The mature protein was exported from the cells into the culture medium and was detected by
anti-leptin antibody. It was shown to to have a size consistent with the predicted structure by
Western blotting analysis under both reducing and nonreducing conditions.
CA 02224646 1997-12-12
W O 97/00319 PCT/GB96/01388
Table 3. DNA sequence of IgG4 PE variant, 984bp
S
SEQnD No:l
GCTAGTACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAG
AGCACgGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG
120
TGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA
180
GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACC
240
TACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCC
300
AAATATGGTCCCCCATGCCCAcCATGCCCAGCgCCTGAaTTtgaGGGGGGACCATCAGTC
360
TTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACG
20420
TGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGAT
480
GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTAC
540
CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAG
600
TGCAAGGTCTCCAACAAAGGCCTCCCGTCaTCgATCGAGAAAACCATCTCCAAAGCCAAA
660
GGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAG
30720
AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAG
780
TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC
840
GACGGaTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGG
900
AATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGC
960
CTCTCCCTGTCTCTGGGTAAATGA
40984
Table 3A: DNA sequence of ob/IgG4PE chimera, 1188bp
ATGCATTGGGGAACCCTGTGCGGATTCTTGTGGCTTTGGCCCTATCTTTTCTATGTCCAA
GCTGTGCCCATCCAAAAAGTCCAAGATGACACCAAAACCCTCATCAAGACAATTGTCACC
120
AGGATCAATGACATTTCACACACGCAGTCAGTCTCCTCCAAACAGAAAGTCACCGGTTTG
180
GACTTCATTCCTGGGCTCCACCCCATCCTGACCCTGTCCAAGATGGACCAGACACTGGCA
240
GTCTACCAACAGATCCTCACATCGATGCCTTCCAGAAACGTGATCCAAATATCCAACGAC
300
14
CA 02224646 1997-12-12
W O 97/00319 PCT/GB96101388
CTGGAGAACCTCCGGGATCTTCTTCACGTGCTGGCCTTCTCTAAGAGCTGCCACTTGCCC
360
TGGGCCAGTGGCCTGGAGACCTTGGACAGCCTGGGGGGTGTCCTCGAGGCTTCAGGCTAC
420
TCCACAGAGGTGGTGGCCCTGAGCAGGCTGCAGGGGTCTCTGCAGGACATGCTGTGGCAG
480
CTGGACCTCAGCCCCGGGTGCGAGTCCAAATATGGTCCCCCATGCCCAcCATGCCCAGCg
540
'' 10 CCTGAATTTGAGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTC
600
ATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCC
660
GAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCG
15720
CGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAG
780
GACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCATCG
840
ATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTG
900
CCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGC
960
TTCTACCCCAGCGACATCGCCGTGGAGTTGGAGAGCAATGGGCAGCCGGAGAACAACTAC
251020
AAGACCACGCCTCCCGTGCTGGACTCCGACGGATCCTTCTTCCTCTACAGCAGGCTAACC
1080
GTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCT
1140
CTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA
1188
Table 4: Amino acid sequence of ob 1-167/IgG4 hinge-CH2-CH3 PE variant chimera
396aa
SEQ ID No: 2
1 MHWGTLCGFL WLWPYLFYVQ AVPIQKVQDD TKTLIKTIVT RINDISHTQS
4051 VSSKQKVTGL DFIPGLHPIL TLSKMDQTLA VYQQILTSMP SRNVIQISND
101 LENLRDLLHV LAFSKSCHLP WASGLETLDS LGGVLEASGY STEVVALSRL
151 QGSLQDMLWQ LDLSPGCESK YGPPCPPCPA PEFEGGPSVF LFPPKPKDTL
201 MISRTPEVTC VVVDVSQEDP EVQFNWYVDG VEVHNAKTKP REEQFNSTYR.
251 VVSVLTVLHQ DWLNGKEYKC KVSNKGLPSS IEKTISKAKG QPREPQVYTL
50301 PPSQEEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY K l-l ~PVLDSD
CA 02224646 1997-12-12
WO 97/00319 PCT/GB96/01388
351 GSFFLYSRLT VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGK
Examl)le 3.
Construction of DNA coding for fusion protein leptin 1-167/IgG1 hinge-CH2-CH3
The gene coding for a fusion protein comprising human leptin and the
hinge-CH2-CH3 region of human IgG1 is created by recombinant DNA technology,
preferably by a two-step recombinant PCR method.
The human 'ob' gene has been prepared synthetic~lly based on the amino acid sequence
of Zhang et al, and assembled in the pcDNA3 vector.
The cDNA encoding full length human leptin, nucleotides 1-501 is joined at the 3' end
to the 5' end of the hinge-CH2-CH3 region of the cDNA coding for the human IgG 1protein, shown as nucleotides 502-1197 in the DNA sequence below. (Table 1.)
The encoded protein sequence of the leptin/IgG1 chimera is given in Table 2. Leptin
1-167 (numbering asY. Zhang, R. Proenca, M. Maffei, M. Barone, L. Leopold & J. Friçflm~n
Nature 372: 425-432) and IgG1 hinge-CH2-CH3 shown as amino acids 168-399.
The gene coding for the human IgG1 contains a number of nucleotide substit--tions
co~ alcd to the IgG1 molecule described by Ellison J.W., Berson B.J. and Hood L.E.,
Nucleic Acids Research vol 10 No. 13 pp4071-4079, 1982. The IgG1 nucleotides which
differ from the Ellison J.W. et al published sequence and the resulting amino acid
substitutions are as follows ( nucleotide numbering as in table 1)
nucleotide 513 is "G" in this variant co~ al~d to "T" in the Ellison et al sequence
(silent mutation)
nucleotides 514-516 are "GCC" in this variant compared to "TGT" in the Ellison
et al sequence (resulting in substitution of Ala for Cys in this variant, amino acid 172
in table 2)
nucleotide 759 is "T" in this variant compared to "G" in the Ellison et al sequence
(silent mutation)
nucleotide 924 is "G" in this variant compared to "T" in the Ellison et al sequence
(res--lting in substitution of Glu for Asp in this variant, amino acid 308 in table2)
nucleotide 928 is "A" in this variant compared to "C" in the Ellison et al sequence
(resulting in substitution of Met for Val in this variant, amino acid 310 in table 2)
nucleotide 1077 is "T" in this variant compared to "C" in the Ellison et al sequence
(silent mutation)
nucleotide 1197 is "G" in this variant compared to "A" in the Ellison et al sequence
(silent mutation)
The fusion protein was expressed transiently in Cos 1 cells using the pCDN vector
system, as described in International Patent Application Publication number WO 96/04388.
- 16-
~ ~ ~ :
CA 02224646 1997-12-12
W O 97/00319 PCT/GB96/01388
The mature protein was exported from the cells into the culture medium and was detected by
anti-leptin antibody. It was shown to to have a size con.cictent with the predicted structure by
Western blotting analysis under both reducing and nonreducing conditions.
Table 5. DNA sequence of ob/IgGl chimera 1197bp
ATGCATTGGGGAACCCTGTGCGGATTCTTGTGGCTTTGGCCCTATCTTTTCTATGTCCAA
GCTGTGCCCATCCAAAAAGTCCAAGATGACACCAAAACCCTCATCAAGACAATTGTCACC
120
AGGATCAATGACATTTCACACACGCAGTCAGTCTCCTCCAAACAGAAAGTCACCGGTTTG
15. 180
GACTTCATTCCTGGGCTCCACCCCATCCTGACCCTGTCCAAGATGGACCAGACACTGGCA
240
GTCTACCAACAGATCCTCACATCGATGCCTTCCAGAAACGTGATCCAAATATCCAACGAC
300
CTGGAGAACCTCCGGGATCTTCTTCACGTGCTGGCCTTCTCTAAGAGCTGCCACTTGCCC
360
TGGGCCAGTGGCCTGGAGACCTTGGACAGCCTGGGGGGTGTCCTCGAGGCTTCAGGCTAC
420
TCCACAGAGGTGGTGGCCCTGAGCAGGCTGCAGGGGTCTCTGCAGGACATGCTGTGGCAG
30480
CTGGACCTCAGCCCCGGGTGCGAGCCCAAATCGGCCGACAAAACTCACACATGCCCACCG
540
TGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG
600
GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC
660
GAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG
720
ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC
45780
CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC
840
CCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG
900
TACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCG
960
-17-
CA 02224646 1997-12-12
WO 97/00319 PCT/GB96/01388
GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGGAATGGGCAGCCGGAG
1020
S AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGC
1080 r
AAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG
1140
CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAG
1197
Table 6. Amino acid sequence of leptin/IgG1 chimera, 399aa
MHWGTLCGFL WLWPYLFYVQ AVPIQKVQDD TKTLIKTIVT RINDISHTQS
~1 VSSKQKVTGL DFIPGLHPIL TLSKMDQTLA VYQQILTSMP SRNVIQISND
101 LENLRDLLHV LAFSKSCHLP WASGLETLDS LGGVLEASGY STEVVALSRL
151 QGSLQDMLWQ LDLSPGCEPK SADKTHTCPP CPAPELLGGP SVFL~ K
25 201 DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS
251 TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PA~K l lSK AKGQPREPQV
301 YTLPPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNY~l~ L
351 DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK
Examl~le 4.
Construction of DNA coding for fusion protein leptin 1-16i/IgG1 hinge-CH2-CH3
GT linker variant
The gene coding for a fusion protein comprising human leptin and the
45 hinge-CH2-CH3 region of human IgG 1 with a 'GT' two amino acid linker betweenthe two parts of the fusion molecule, is created by recombinant DNA technology,
preferably by a two-step recombinant PCR method.
The human 'ob' gene has been ~-c~aled synthetically based on the amino acid sequence
of Zhang et al, and assembled in the pcDNA3 vector.
- 18 -
CA 02224646 1997-12-12
W O 97/00319 PCT/GB96/01388
The cDNA encoding the full length human leptin (nucleotides 1-501) is joined at the 3'
end to the S' end of the hinge-CH2-CH3 region of the IgGl cDNA (nucleotides 508-1203).
The two arnino acid linker between the two parts of the fusion is encoded by the nucleotide
sequence GGTACC (502-507). See Table 1.
S The encoded protein sequence of the leptin/IgG l (GT) chimera is given in Table 2. Leptin 1- 1
(numbering asY. Zhang, R. Proenca, M. Maffei, M. Barone, L. Leopold & J. Friedman.
Nature 372:425-432), followed by the GT linker (168-169) and IgG1 H-CH2-CH3 170-401.
The gene coding for the human IgG1 contains a number of nucleotide subsht-ltionsco~ ,d to the IgG1 molecule described by Ellison J.W., Berson B.J. and Hood L.E.,
Nucleic Acids Research vol 10 No. 13 pp4071-4079, 1982. The IgG1 nucleotides which
differ from the Ellison J.W. et al published sequence and the resulting amino acid
substitll~iQns are as follows ( nucleotide numbering as in table 1)
nucleotide 519 is "G" in this variant compared to "T" in the Ellison et al sequence
(silent mutation)
nucleotides 520-522 are "GCC" in this variant co-ll~u~ed to "TGT" in the Ellisonet al sequence (resulting in substitution of Ala for Cys in this variant, amino acid 174
in table 2)
nucleotide 759 is "T" in this variant compared to "G" in the Ellison et al sequence
(silent mllt~tion)
nucleotide 924 is "G" in this variant compared to "T" in the Ellison et al sequence
(resulting in substitution of Glu for Asp in this variant, amino acid 308 in table2)
nucleotide 928 is "A" in this variant colllpaled to "C" in the Ellison et al sequence
(resulting in substitution of Met for Val in this variant, amino acid 310 in table 2)
nucleotide 1077 is "T" in this variant compared to "C" in the Ellison et al sequence
(silent mut~tion)
nucleotide 1197 is "G" in this variant co~ d to "A" in the Ellison et al sequence
(silent mutation)
The fusion protein was expressed transiently in Cos 1 cells using the pCDN vector
system, as described in International Patent Application Publication number WO 96/04388.
The mature protein was exported from the cells into the culture medium and was detecte~l by
anti-leptin antibody. It was shown to to have a size consistent with the predicted structure by
Western blotting analysis under both reducing and nonreducing conditions.
Table 7. DNA sequence of ob/IgGl'GT' chimera, 1203bp
ATGCATTGGGGAACCCTGTGCGGATTCTTGTGGCTTTGGCCCTATCTTTTCTATGTCCAA
- 19-
CA 02224646 1997-12-12
W O 97/00319 PCT/GB96/01388
GCTGTGCCCATCCAAAAAGTCCAAGATGACACCAAAACCCTCATCAAGACAATTGTCACC
120
AGGATCAATGACATTTCACACACGCAGTCAGTCTCCTCCAAACAGAAAGTCACCGGTTTG
180
GACTTCATTCCTGGGCTCCACCCCATCCTGACCCTGTCCAAGATGGACCAGACACTGGCA
240
~0 GTCTACCAACAGATCCTCACATCGATGCCTTCCAGAAACGTGATCCAAATATCCAACGAC
300
CTGGAGAACCTCCGGGATCTTCTTCACGTGCTGGCCTTCTCTAAGAGCTGCCACTTGCCC
360
TGGGCCAGTGGCCTGGAGACCTTGGACAGCCTGGGGGGTGTCCTCGAGGCTTCAGGCTAC
420
TCCACAGAGGTGGTGGCCCTGAGCAGGCTGCAGGGGTCTCTGCAGGACATGCTGTGGCAG
20480
CTGGACCTCAGCCCCGGGTGCGGTACCGAGCCCAAATCGGCCGACAAAACTCACACATGC
. 540
CCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAA
600
CCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTG
660
AGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAAT
720
GCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTC
~35780
ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA
840
GCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCA
900
CAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACC
960
TGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG
1020
CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC
501080
TATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCC
1140
~5 GTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT
1200
-20-
CA 02224646 1997-12-12
W O 97/00319 PCT/GB96/01388
2~G
1203
s
Table 8. Amino acid sequence of leptin/IgG1 'GT' chimera, 401aa
1 MHWGTLCGFL WLWPYLFYVQ AVPIQKVQDD TKTLIKTIVT
10 RINDISHTQS
51 VSSKQKVTGL DFIPGLHPIL TLSKMDQTLA VYQQILTSMP SRNVIQISND
101 LENLRDLLHV LAFSKSCHLP WASGLETLDS LGGVLEASGY
15 STEVVALSRL
151 QGSLQDMLWQ LDLSPGCGTE PKSADKTHTC PPCPAPELLG
GPSVFLFPPK
20 201 PKDT,LMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN
AK'l'K~EEQY
251 NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI
SKAKGQPREP
301 QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ
PENN~K'l-l'~
351 VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY
30 TQKSLSLSPG
401 K
