CA2224646A1

CA2224646A1 - Chimeric leptin fused to immunoglobulin domain and use

Info

Publication number: CA2224646A1
Application number: CA002224646A
Authority: CA
Inventors: Jeffrey Hugh Robinson; Conrad Gerald Chapman; Michael Joseph Browne; Helen Elizabeth Clinkenbeard
Original assignee: SmithKline Beecham Ltd
Current assignee: Individual
Priority date: 1995-06-13
Filing date: 1996-06-11
Publication date: 1997-01-03
Also published as: AU6011096A; WO1997000319A3; GB9511935D0; WO1997000319A2; EP0832219A2; JPH11507547A

Abstract

Chimeric leptin which are proteins comprising leptin or a mutant or a variant thereof fused to a human immunogobulin domain. One favoured immunoglobulin domain is the human immunoglobulin Fc domain. The chimeric derivatives of leptin have, despite their large molecular size, good pharmacological activity combined with prolonged clearance rates. These derivatives of leptin are therefore indicated to be particularly useful for the treatment or prophylaxis of obesity or diseases and conditions associated with obesity such as atherosclerosis, hypertension and type II diabetes.

Description

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.

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

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.

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

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.

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 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 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.

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.

,~ .

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

AGGATCAATGACATTTCACACACGCAGTCAGTCTCCTCCAAACAGAAAGTCACCGGTTTG

GACTTCATTCCTGGGCTCCACCCCATCCTGACCCTGTCCAAGATGGACCAGACACTGGCA

GTCTACCAACAGATCCTCACATCGATGCCTTCCAGAAACGTGATCCAAATATCCAACGAC

~0 CTGGAGAACCTCCGGGATCTTCTTCACGTGCTGGCCTTCTCTAAGAGCTGCCACTTGCCC

TGGGCCAGTGGCCTGGAGACCTTGGACAGCCTGGGGGGTGTCCTCGAGGCTTCAGGCTAC

TCCACAGAGGTGGTGGCCCTGAGCAGGCTGCAGGGGTCTCTGCAGGACATGCTGTGGCAG

CTGGACCTCAGCCCCGGGTGCGAGTCCAAATATGGTCCCCCATGCCCATCATGCCCAGCA

CA 02224646 l997-l2-l2 CCTGAATTTCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTC

~ ATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCC

GAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCG

CGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAG

GACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCATCG

ATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTG

CCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGC

TTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC

AAGACCACGCCTCCCGTGCTGGACTCCGACGGATCCTTCTTCCTCTACAGCAGGCTAACC

GTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCT

CTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA

Table 2. Amino acid sequence of leptin/IgG4 chimera, 396aa RINDISHTQS

STEVVALSRL

LFPPKPKDTL

REEQFNSTYR

QPREPQVYTL

KTTPPVLDSD t 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.

Table 3. DNA sequence of IgG4 PE variant, 984bp S
SEQnD No:l GCTAGTACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAG
AGCACgGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG

TGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA

GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACC

TACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCC

AAATATGGTCCCCCATGCCCAcCATGCCCAGCgCCTGAaTTtgaGGGGGGACCATCAGTC

TTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACG

TGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGAT

GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTAC

CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAG

TGCAAGGTCTCCAACAAAGGCCTCCCGTCaTCgATCGAGAAAACCATCTCCAAAGCCAAA

GGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAG

AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAG

TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC

GACGGaTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGG

AATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGC

CTCTCCCTGTCTCTGGGTAAATGA

Table 3A: DNA sequence of ob/IgG4PE chimera, 1188bp ATGCATTGGGGAACCCTGTGCGGATTCTTGTGGCTTTGGCCCTATCTTTTCTATGTCCAA
GCTGTGCCCATCCAAAAAGTCCAAGATGACACCAAAACCCTCATCAAGACAATTGTCACC

AGGATCAATGACATTTCACACACGCAGTCAGTCTCCTCCAAACAGAAAGTCACCGGTTTG

GACTTCATTCCTGGGCTCCACCCCATCCTGACCCTGTCCAAGATGGACCAGACACTGGCA

GTCTACCAACAGATCCTCACATCGATGCCTTCCAGAAACGTGATCCAAATATCCAACGAC

CTGGAGAACCTCCGGGATCTTCTTCACGTGCTGGCCTTCTCTAAGAGCTGCCACTTGCCC

TGGGCCAGTGGCCTGGAGACCTTGGACAGCCTGGGGGGTGTCCTCGAGGCTTCAGGCTAC

TCCACAGAGGTGGTGGCCCTGAGCAGGCTGCAGGGGTCTCTGCAGGACATGCTGTGGCAG

CTGGACCTCAGCCCCGGGTGCGAGTCCAAATATGGTCCCCCATGCCCAcCATGCCCAGCg '' 10 CCTGAATTTGAGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTC

ATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCC

GAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCG

CGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAG

GACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCATCG

ATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTG

CCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGC

TTCTACCCCAGCGACATCGCCGTGGAGTTGGAGAGCAATGGGCAGCCGGAGAACAACTAC

AAGACCACGCCTCCCGTGCTGGACTCCGACGGATCCTTCTTCCTCTACAGCAGGCTAACC

GTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCT

CTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA

Table 4: Amino acid sequence of ob 1-167/IgG4 hinge-CH2-CH3 PE variant chimera 396aa SEQ ID No: 2 201 MISRTPEVTC VVVDVSQEDP EVQFNWYVDG VEVHNAKTKP REEQFNSTYR.

50301 PPSQEEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY K l-l ~PVLDSD

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.

~ ~ ~ :

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

AGGATCAATGACATTTCACACACGCAGTCAGTCTCCTCCAAACAGAAAGTCACCGGTTTG
15. 180 GACTTCATTCCTGGGCTCCACCCCATCCTGACCCTGTCCAAGATGGACCAGACACTGGCA

GTCTACCAACAGATCCTCACATCGATGCCTTCCAGAAACGTGATCCAAATATCCAACGAC

CTGGAGAACCTCCGGGATCTTCTTCACGTGCTGGCCTTCTCTAAGAGCTGCCACTTGCCC

TGGGCCAGTGGCCTGGAGACCTTGGACAGCCTGGGGGGTGTCCTCGAGGCTTCAGGCTAC

TCCACAGAGGTGGTGGCCCTGAGCAGGCTGCAGGGGTCTCTGCAGGACATGCTGTGGCAG

CTGGACCTCAGCCCCGGGTGCGAGCCCAAATCGGCCGACAAAACTCACACATGCCCACCG

TGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG

GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC

GAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG

ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC

CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC

CCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG

TACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCG

GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGGAATGGGCAGCCGGAG

S AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGC
1080 r AAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG

CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAG

Table 6. Amino acid sequence of leptin/IgG1 chimera, 399aa MHWGTLCGFL WLWPYLFYVQ AVPIQKVQDD TKTLIKTIVT RINDISHTQS
~1 VSSKQKVTGL DFIPGLHPIL TLSKMDQTLA VYQQILTSMP SRNVIQISND

151 QGSLQDMLWQ LDLSPGCEPK SADKTHTCPP CPAPELLGGP SVFL~ K

251 TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PA~K l lSK AKGQPREPQV
301 YTLPPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNY~l~ L

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.

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

GCTGTGCCCATCCAAAAAGTCCAAGATGACACCAAAACCCTCATCAAGACAATTGTCACC

AGGATCAATGACATTTCACACACGCAGTCAGTCTCCTCCAAACAGAAAGTCACCGGTTTG

GACTTCATTCCTGGGCTCCACCCCATCCTGACCCTGTCCAAGATGGACCAGACACTGGCA

~0 GTCTACCAACAGATCCTCACATCGATGCCTTCCAGAAACGTGATCCAAATATCCAACGAC

CTGGAGAACCTCCGGGATCTTCTTCACGTGCTGGCCTTCTCTAAGAGCTGCCACTTGCCC

TGGGCCAGTGGCCTGGAGACCTTGGACAGCCTGGGGGGTGTCCTCGAGGCTTCAGGCTAC

TCCACAGAGGTGGTGGCCCTGAGCAGGCTGCAGGGGTCTCTGCAGGACATGCTGTGGCAG

CTGGACCTCAGCCCCGGGTGCGGTACCGAGCCCAAATCGGCCGACAAAACTCACACATGC
. 540 CCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAA

CCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTG

AGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAAT

GCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTC
~35780 ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA

GCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCA

CAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACC

TGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG

CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC

TATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCC

~5 GTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT

2~G

s Table 8. Amino acid sequence of leptin/IgG1 'GT' chimera, 401aa GPSVFLFPPK
20 201 PKDT,LMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN
AK'l'K~EEQY

SKAKGQPREP

PENN~K'l-l'~

Claims

Claims:

1. Chimeric leptin or a chimeric mutant or derivative of leptin.

2. A chimera according to claim 1, wherein the leptin is human leptin.

3. A chimera according to claim 1 or claim 2, wherein the leptin or a mutant or variant thereof is fused to a human immunoglobulin domain or a mutant or variant thereof.

4. A chimera according to any one of claims 1 to 3, wherein the chimeric proteincomprises one human immunoglobulin domain.

5. A chimera according to claim 4, wherein the human immunoglobulin domain is fused to the C-terminus of leptin.

6. A chimera according to any one of claims 1 to 4, which comprises a human immunoglobulin Fc domain.

7. A chimera a according to claim 6, wherein the human immunoglobulin Fc domain is an IgG4PE variant, an IgG4, IgG1 or an IgG1GT variant, in particular the hinge-CH2-CH3 region in each case.

8. A chimera according to claim 7, wherein the variant a hinge-CH2-CH3 variant.

9. Chimeric leptin selected from the list consisting of:
leptin 1-167/IgG4 hinge-CH2-CH3;
leptin 1-167/IgG4 hinge-CH2-CH3 PE variant;
leptin 1-167/IgG1 hinge-CH2-CH3; and leptin 1-167/IgG1 hinge-CH2-CH3 GT linker variant.

10. A process for preparing a chimera according to any one of claims 1 to 8, which process comprises expressing DNA encoding said compound in a recombinant host cell and recovering the product

11. A process according to claim 10, which process comprises the steps of:
i) preparing a replicable expression vector capable, in a host cell, of expressing a DNA polymer comprising a nucleotide sequence that encodes said chimera;
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 chimera; and iv) recovering said chimera.

12. A DNA polymer comprising a nucleotide sequence that encodes a chimera according to any one of claims 1 to 8.

13. A vector which comprises a DNA polymer according to claim 12.

14. A host cell transformed or transfected with a DNA polymer according to claim 12 or a vector according to claim 13.

15. A pharmaceutical composition comprising a chimera as claimed in claim 1 and a pharmaceutically acceptable carrier.

16. A chimera according to claim 1, for use as an active therapeutic substance.

17. A chimera according to claim 1, for use in the treatment of obesity or diseases associated with obesity.

18. A method for the treatment of obesity or diseases associated with in human or non-human mammal, which method comprises administering to the sufferer an effective, non-toxic amount of a chimera as claimed in claim 1.

19. A chimera as claimed in claim 1, for use in the cosmetic treatment of human or non-human mammals.

20. A method for the cosmetic treatment of a human or non-human mammal, which treatment comprises administering an effective, non-toxic amount of a compound of the invention to a human or non-human mammal in need thereof.