CHIMERIC LEPTIN FUSED TO IMMUNOGLOBULIN DOMAIN AND USE

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: CHIMERIC LEPTTN FUSED TO IMMUNOGLOBULIN DOMAIN 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 pharmaceutical composition comprising such a compound and the use of such a compound in medicine, especially for the treatment of obesity and associated diseases. European Patent Application, Publication number 0 464 533 discloses fusion proteins comprising various portions of the constant region of immunoglobulin molecules together with another human protein or part thereof. European Patent Application, Publication number 0 297 882 discloses fusion proteins comprising various portions of the plasminogen molecule with part of another human protein. Zhang et al. (Nature: 372, 425 - 432; 1994) describe the positional cloning of a mouse obese gene and its human homologue. The sequence of the Open Reading Frame (ORF) of the mouse gene predicts a polypeptide of 167 amino acids and Zhang et al. predicted the presence of a signal sequence which would lead to the production of a mature protein of 146 residues. 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 product on addition of microsomal membranes, consistent with the production of a pre-protein and the removal of an N-terminal signal sequence. Zhang et al also disclose the potential use of the human obese gene product (hereinafter 'leptin') in the treatment of obesity. For effective, practical treatment of obesity a paπicularly desirable property of an obesity agent is a clearance rate in humans commensurate with patient acceptable treatment regimens, especially regimens for injectable therapies. Zhang et al. do not disclose information relating to the clearance rate of the active molecule in either mouse or humans. The precise mechanism of action of leptin is currently unknown, however it is considered that in order to provide the observed pharmacological effects, leptin must interact with one or more receptors in the brain. We have now discovered certain chimeric derivatives of leptin which surprisingly, despite their large molecular size, have good pharmacological activity combined with prolonged clearance rates. These chimeric derivatives of leptin are therefore indicated to be particularly useful for the treatment or prophylaxis of obesity and for the treatment or prophylaxis of diseases and conditions associated with obesity, such as atherosclerosis, hypertension and, especially, Type II diabetes. In paπicular these compounds are considered to be useful for administration by injection. These compounds are also considered to be useful in cosmetic treatments for the improvement of body appearance. Accordingly, the invention provides a chimeric leptin or a chimeric mutant or derivative of leptin. One particular chimeric leptin is a protein comprising leptin or a mutant or variant thereof fused to a human immunoglobulin domain or a mutant or variant thereof. Suitably, the chimeric protein comprises one human immunoglobulin domain. Favourably , the human immunoglobulin domain is fused to the C-terminus of leptin. One favoured human immunoglobulin is an human immunoglobulin Fc domain. An example of a human immunoglobulin Fc domain is an IgG4PE variant in paπicular IgG4 hinge-CH 2 -CH 3 .PE. Other examples are IgG4, IgGl and IgGlGT, in particular the hinge-CH2-CH3 region in each case. The term "mutant or variant" used with respect to a paπicular protein encompasses any molecule such as a truncated 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 IgGl, IgG3 or IgG4. The said constant domain(s) or fragment thereof may be derived from the heavy or light chain or both. The invention encompasses mutations in the immunoglobulin component which eliminate undesirable propeπies of the native immunoglobulin, such as Fc receptor binding and/or introduce desirable propeπies such as stability. For example, Angal S., King D.J., Bodmer M.W., Turner A., Lawson A.D.G., Robeπs G., Pedley B. and Adair R., Molecular Immunology vol30ppl05-108, 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) from leucine to glutamate in IgG3 and from glutamate to leucine in mouse IgG2b. Substitution of leucine for glutamate in the former decreases the affinity of the immunoglobulin molecule concerned for the FcγRI receptor, and substitution of glutamate 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 pan of the constant region of the heavy chain of human IgG. The IgG component 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 component is derived from IgG4 it includes cysteine residues 8 and 11 of the IgG4 hinge region (Pinck J.R. and Milstein C, Nature vo!216pp941-942, 1967). Preferably the IgG4 component consists of amino acids coπesponding 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 glutamate (E). DNA polymers which encode mutants or variants of the human immunoglobulin 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 er 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 fuπher aspect, the invention provides a process for preparing a compound according to the invention which process comprises expressing 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 pan of the invention. The process of the invention may be performed by conventional recombinant 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 paπicular, the process may comprise 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 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 compound; and iv) recovering said compound. The invention also provides a process for preparing the DNA polymer by the condensation of appropriate mono-, di- or oligomeric nucleotide units. The preparation may be carried out chemically, enzymatically, or by a combination of the two methods, in vitro or in vivo as appropriate. Thus, the DNA polymer may be prepared by the enzymatic ligation of appropriate DNA fragments, by conventional methods such as those described by D. M. Robeπs et al in Biochemistry 1985, 24, 5090-5098. The DNA fragments may be obtained by digestion of DNA containing the required sequences of nucleotides with appropriate 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 appropriate buffer at a temperature of 20°-70°C, generally in a volume of 50μl or less with 0.1- lOμg 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 dTTP as required at a temperature of 10°-37°C, generally in a volume of 50μl or less. Enzymatic ligation of DNA fragments 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 50μl or less. The chemical synthesis of the DNA polymer or fragments 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 Fragments - 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. Bannwaπh, Tetrahedron Letters, 1983, 24, 5771; M.D. Matteucci and M.H Caruthers, Tetrahedron Letters, 1980, 21, 719; M.D. Matteucci 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 synthesizer is employed. The DNA polymer is preferably prepared 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 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 an. The expression of the DNA polymer encoding the compound in a recombinant host cell may be carried out by means of a replicable expression vector capable, in the host cell, of expressing the DNA polymer. The expression vector is novel and also forms pan 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 ligating conditions. 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 pan 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. filamentous 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 plasmids, bacteriophages, cosmids and recombinant viruses derived from, for example, baculoviruses, vaccinia or Semliki Forest virus. The preparation of the replicable expression vector may be carried out conventionally with appropriate enzymes for restriction, polymerisation and ligation of the DNA, by procedures described in, for example, Maniatis ej ai., 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 appropriate buffer at a temperature of 20°-70°C, generally in a volume of 50μl or less with 0.1-10μg 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. 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 CaCl2 (Cohen et al, Proc. Nat. Acad. Sci., 1973, 69, 2110) or with a solution comprising a mixture of RbCl, MnCl2, potassium acetate and glycerol, and then with 3-[N-morpholino]- propane-sulphonic acid, RbCl and glycerol. Mammalian cells in culture may be transformed by calcium co-precipitation of the vector DNA onto the cells. The invention also extends to a host cell transformed or transfected with a replicable expression vector of the invention. Culturing the transformed host cell under conditions permitting expression of the DNA polymer is carried out conventionally, 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 temperature 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 physically, chemically or enzymatically and the protein product isolated from the resulting 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 mammalian, the product may generally be isolated from the nutrient medium. The DNA polymer may be assembled into vectors designed for isolation of stable transformed mammalian cell lines expressing the product; e.g. bovine papillomavirus vectors or amplified vectors in Chinese hamster ovary cells (DNA cloning Vol.II D.M. Glover ed. IRL Press 1985; Kaufman, R.J. ≤i al., Molecular and Cellular Biology 5, 1750-1759, 1985; Pavlakis G.N. and Hamer, D.H., Proceedings of the National Academy of Sciences (USA) 80, 397-401, 1983; Goeddel, DN. et al., European Patent Application No. 0093619, 1983). The activity of the chimeric leptin is determined by injecting it intraperitoneally, intravenously or subcutaneously into test animals such as rodents, for example mice or rats, or primates, 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 generically obese (ob/ob) mouse. The effect of the active compound is seen as a reduction in food intake or increase in metabolic rate or oxygen consumption. Multiple injections of the active compound - at most twice daily - over a period of a week for rodents or a month for primates, 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, for example ELISA methodology. As indicated above the compounds of the present invention have useful pharmaceutical propeπies, in paπicular anti obesity activity and also for the treatment of diseases associated with obesity, such as atherosclerosis, hypeπension and, especially, Type LI diabetes. In use the compound will normally be employed in the form of a pharmaceutical 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 administration. The active compound may be formulated for administration by any suitable route and is preferably in unit dosage form. Advantageously, the composition is suitable for oral, rectal, topical, parenteral, intravenous or intramuscular administration or through the respiratory 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, reconstitutable powders, or liquid preparations such as oral or sterile parenteral solutions or suspensions. Topical formulations are also envisaged where appropriate. The invention therefore further provides a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable carrier. The dosage ranges for administration 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 contraindications, if any. For example in the treatment 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 treatment of diseases associated with obesity such as atherosclerosis, hypeπension 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, suspensions or emulsions, or dry powders which are dissolved or suspended in a suitable vehicle prior to use. Fluid unit dosage forms are prepared utilising the compound and a pyrogen-free sterile vehicle. The compound, depending on the vehicle and concentration used, can be either dissolved or suspended in the vehicle. Solutions may be used for all forms of parenteral administration, and are panicularly 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 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 using 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 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 compound may be isolated in a sterile state or alternatively it may be sterilised after isolation, e.g. by gamma iπadiation. Advantageously, a suspending agent for example polyvinylpyrrolidone is included in the composition to facilitate uniform distribution of the compound. Compositions suitable for administration via the respiratory tract include aerosols, nebulisable solutions or microfine powders for insufflation. In the latter case, panicle size of less than 50 microns, especially less than 10 microns, is preferred. Such compositions may be made up in a conventional manner and employed in conjunction with conventional administration devices. In a fuπher aspect there is provided a method of treating obesity or diseases associated with obesity, such as atherosclerosis, hypeπension and, especially, Type LT diabetes, in human or non-human mammals which comprises administering to the sufferer an effective, non-toxic amount of a compound of the invention. Suitable non-human mammals are domestic mammals such as dogs and cats. The invention funher provides a compound of the invention for use as an active therapeutic substance, in paπicular for use in treating obesity or diseases associated with obesity, such as atherosclerosis, hypertension and, especially, Type LT 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, hypeπension and, especially, Type LT diabetes. As indicated above the invention also encompasses cosmetic treatments. Accordingly, there is also provided a compound of the invention for use in the cosmetic treatment of human or non-human mammals. There is also provided 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. 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 compound of the invention and a carrier therefor. Compositions of the invention including cosmetic compositions are formulated using known methods, for example those described in standard text books of pharmaceutics and cosmetics, such as Harry's Cosmeticology published by Leonard Hill Books, Remington's Pharmaceutical Sciences, the British and US Pharmacopoeias. No unexpected toxicological effects are expected when compounds of the invention are administered 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 fusion protein leptin l-167/IgG4 hinge-CH2-CH3 The gene coding for a fusion protein comprising human leptin and the hinge-CH2-CH3 region of human IgG4 is created by recombinant DNA technology, preferably by a two-step recombinant PCR method. The human Ob' gene has been prepared 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 at 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 502-1188 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. Friedman. Nature 372:425-432), and IgG4 hinge-CH2-CH3 168-396 (sequence as Kabat). The fusion protein was expressed transiently in Cosl cells using the pCDN vector system, as described in Intemationai Patent Application Publication number WO 96/04388. The mature protein was exponed 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 1. DNA sequence of ob/IgG4 chimera, 1188bp ATGCATTGGGGAACCCTGTGCGGATTCTTGTGGCTTTGGCCCTATCTTTTCTATGTCCAA 60 GCTGTGCCCATCCAAAAAGTCCAAGATGACACCAAAACCCTCATCAAGACAATTGTCACC 120 AGGATCAATGACATTTCACACACGCAGTCAGTCTCCTCCAAACAGAAAGTCACCGGTTTG 180 GACTTCATTCCTGGGCTCCACCCCATCCTGACCCTGTCCAAGATGGACCAGACACTGGCA 240 GTCTACCAACAGATCCTCACATCGATGCCTTCCAGAAACGTGATCCAAATATCCAACGAC 300 CTGGAGAACCTCCGGGATCTTCTTCACGTGCTGGCCTTCTCTAAGAGCTGCCACTTGCCC 360 TGGGCCAGTGGCCTGGAGACCTTGGACAGCCTGGGGGGTGTCCTCGAGGCTTCAGGCTAC 420 TCCACAGAGGTGGTGGCCCTGAGCAGGCTGCAGGGGTCTCTGCAGGACATGCTGTGGCAG 480 CTGGACCTCAGCCCCGGGTGCGAGTCCAAATATGGTCCCCCATGCCCATCATGCCCAGCA 540 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 MH GTLCGFL WLWPYLFYVQ AVPIQKVQDD TKTLIKTIVT RINDISHTQS 51 VSSKQKVTGL DFIPGLHPIL TLSKMDQTLA VYQQLLTSMP SRNVIQISND 101 LENLRDLLHV LAFSKSCHLP WASGLETLDS LGGVLEASGY STEVVALSRL 151 QGSLQDMLWQ LDLSPGCESK YGPPCPSCPA PEFLGGPSVF LFPPKPKDTL 201 MISRTPEVTC VVVDVSQEDP EVQFNWYVDG VEVHNAKTKP REEQFNSTYR 251 VVSVLTVLHQ DWLNGKEYK CKVSNKGLPSS LEKTISKAKG QPREPQVYTL 301 PPSQEEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD 351 GSFFLYSRLT VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGK Example 2. Construction of DNA coding for fusion protein ob l-167/IgG4 hinge-CH2-CH3 PE variant 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 mutated 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 l-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 5' 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 prepared 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 5 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., Turner A., Lawson A.D.G., Robeπs G., Pedley B. and Adair R., Molecular Immunology vol30ppl05-108, 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 Cosl cells using the pCDN vector system, as described in International Patent Application Publication number WO 96/04388. The mature protein was exponed 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 SEQ ID No: 1 GCTAGTACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAG 60 AGCACgGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG 120 TGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA 180 GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACC 240 TACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCC 300 AAATATGGTCCCCCATGCCCAcCATGCCCAGCgCCTGAaTTtgaGGGGGGACCATCAGTC 360 TTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACG 420 TGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGAT 480 GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTAC 540 CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAG 600 TGCAAGGTCTCCAACAAAGGCCTCCCGTCaTCgATCGAGAAAACCATCTCCAAAGCCAAA 660 GGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAG 720 AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAG 780 TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC 840 GACGGaTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGG 900 AATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGC 960 CTCTCCCTGTCTCTGGGTAAATGA 984 Table3A: DNAsequenceofob/IgG4PEchimera, 1188bp ATGCATTGGGGAACCCTGTGCGGATTCTTGTGGCTTTGGCCCTATCTTTTCTATGTCCAA 60 GCTGTGCCCATCCAAAAAGTCCAAGATGACACCAAAACCCTCATCAAGACAATTGTCACC 120 AGGATCAATGACATTTCACACACGCAGTCAGTCTCCTCCAAACAGAAAGTCACCGGTTTG 180 GACTTCATTCCTGGGCTCCACCCCATCCTGACCCTGTCCAAGATGGACCAGACACTGGCA 240 GTCTACCAACAGATCCTCACATCGATGCCTTCCAGAAACGTGATCCAAATATCCAACGAC 300 CTGGAGAACCTCCGGGATCTTCTTCACGTGCTGGCCTTCTCTAAGAGCTGCCACTTGCCC 360 TGGGCCAGTGGCCTGGAGACCTTGGACAGCCTGGGGGGTGTCCTCGAGGCTTCAGGCTAC 420 TCCACAGAGGTGGTGGCCCTGAGCAGGCTGCAGGGGTCTCTGCAGGACATGCTGTGGCAG 480 CTGGACCTCAGCCCCGGGTGCGAGTCCAAATATGGTCCCCCATGCCCAcCATGCCCAGCg 540 CCTGAATTTGAGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTC 600 ATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCC 660 GAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCG 720 CGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAG 780 GACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCATCG 840 ATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTG 900 CCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGC 960 TTCTACCCCAGCGACATCGCCGTGGAGTTGGAGAGCAATGGGCAGCCGGAGAACAACTAC 1020 AAGACCACGCCTCCCGTGCTGGACTCCGACGGATCCTTCTTCCTCTACAGCAGGCTAACC 1080 GTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCT 1140 CTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA 1188 Table 4: Amino acid sequence of ob l-167/IgG4 hinge-CH2-CH3 PE variant chimera 396aa SEQ LD No: 2 1 MHWGTLCGFL WLWPYLFYVQ AVPIQKVQDD TKTLIKTIVT RINDISHTQS 51 VSSKQKVTGL DFIPGLHPIL TLSKMDQTLA VYQQLLTSMP SRNVIQISND 101 LENLRDLLHV LAFSKSCHLP WASGLETLDS LGGVLEASGY STEVVALSRL 151 QGSLQDMLWQ LDLSPGCESK YGPPCPPCPA PEFEGGPSVF LFPPKPKDTL 201 MISRTPEVTC VVVDVSQEDP EVQFNWYVDG VEVHNAKTKP REEQFNSTYR 251 VVSVLTVLHQ DWLNGKEYKC KVSNKGLPSS LEKTISKAKG QPREPQVYTL 301 PPSQEEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD 351 GSFFLYSRLT VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGK Example 3. Construction of DNA coding for fusion protein leptin 1-167/IgGl hinge-CH2-CH3 The gene coding for a fusion protein comprising human leptin and the hinge-CH2-CH3 region of human IgGl is created by recombinant DNA technology, preferably by a two-step recombinant PCR method. The human 'ob' gene has been prepared 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 at the 3' end to the 5' end of the hinge-CH2-CH3 region of the cDNA coding for the human IgGl protein, shown as nucleotides 502-1197 in the DNA sequence below. (Table 1.) The encoded protein sequence of the leptin/IgGl chimera is given in Table 2. Leptin 1-167 (numbering asY. Zhang, R. Proenca, M. Maffei, M. Barone, L. Leopold & J. Friedman. Nature 372: 425-432) and IgGl hinge-CH2-CH3 shown as amino acids 168-399. The gene coding for the human IgGl contains a number of nucleotide substitutions compared to the IgGl molecule described by Ellison J.W., Berson B.J. and Hood L.E., Nucleic Acids Research vol 10 No. 13 pp4071-4079, 1982. The IgGl 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 compared 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 (resulting 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 Cosl cells using the pCDN vector system, as described in International Patent Application Publication number WO 96/04388. The mature protein was exponed 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 5. DNA sequence of ob/IgGl chimera 1197bp ATGCATTGGGGAACCCTGTGCGGATTCTTGTGGCTTTGGCCCTATCTTTTCTATGTCCAA 60 GCTGTGCCCATCCAAAAAGTCCAAGATGACACCAAAACCCTCATCAAGACAATTGTCACC 120 AGGATCAATGACATTTCACACACGCAGTCAGTCTCCTCCAAACAGAAAGTCACCGGTTTG 180 GACTTCATTCCTGGGCTCCACCCCATCCTGACCCTGTCCAAGATGGACCAGACACTGGCA 240 GTCTACCAACAGATCCTCACATCGATGCCTTCCAGAAACGTGATCCAAATATCCAACGAC 300 CTGGAGAACCTCCGGGATCTTCTTCACGTGCTGGCCTTCTCTAAGAGCTGCCACTTGCCC 360 TGGGCCAGTGGCCTGGAGACCTTGGACAGCCTGGGGGGTGTCCTCGAGGCTTCAGGCTAC 420 TCCACAGAGGTGGTGGCCCTGAGCAGGCTGCAGGGGTCTCTGCAGGACATGCTGTGGCAG 480 CTGGACCTCAGCCCCGGGTGCGAGCCCAAATCGGCCGACAAAACTCACACATGCCCACCG 540 TGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG 600 GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC 660 GAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG 720 ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC 780 CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC 840 CCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG 900 TACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCG 960 GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG 1020 AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGC 1080 AAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG 1140 CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAG 1197 Table 6. Amino acid sequence of leptin/IgGl chimera, 399aa 1 MHWGTLCGFL WLWPYLFYVQ AVPIQKVQDD TKTLIKTIVT RINDISHTQS 51 VSSKQKVTGL DFIPGLHPIL TLSKMDQTLA VYQQLLTSMP SRNVIQISND 101 LENLRDLLHV LAFSKSCHLP WASGLETLDS LGGVLEASGY STEVVALSRL 151 QGSLQDMLWQ LDLSPGCEPK SADKTHTCPP CPAPELLGGP SVFLFPPKPK 201 DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS 251 TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKΗSK AKGQPREPQV 301 YTLPPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 351 DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK Example 4. Construction of DNA coding for fusion protein leptin 1-167/IgGl hinge-CH2-CH3 GT linker variant The gene coding for a fusion protein comprising human leptin and the hinge-CH2-CH3 region of human IgGl with a 'GT' two amino acid linker between the 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 prepared 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 5' end of the hinge-CH2-CH3 region of the IgGl cDNA (nucleotides 508-1203). The two amino acid linker between the two pans of the fusion is encoded by the nucleotide sequence GGTACC (502-507). See Table 1. The encoded protein sequence of the leptin/LgG 1 (GT) chimera is given in Table 2. Lept (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 IgGl H-CH2-CH3 170-401. The gene coding for the human IgGl contains a number of nucleotide substitutions compared to the IgGl molecule described by Ellison J.W., Berson B.J. and Hood L.E., Nucleic Acids Research vol 10 No. 13 pp4071-4079, 1982. The IgGl 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 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 compared to "TGT" in the Ellison et 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 mutation) 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 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 Cosl cells using the pCDN vector system, as described in International Patent Application Publication number WO 96/04388. The mature protein was exponed 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 Westem blotting analysis under both reducing and nonreducing conditions. Table 7. DNA sequence of ob/IgGl'GT' chimera , 1203bp ATGCATTGGGGAACCCTGTGCGGATTCTTGTGGCTTTGGCCCTATCTTTTCTATGTCCAA 60 GCTGTGCCCATCCAAAAAGTCCAAGATGACACCAAAACCCTCATCAAGACAATTGTCACC 120 AGGATCAATGACATTTCACACACGCAGTCAGTCTCCTCCAAACAGAAAGTCACCGGTTTG 180 GACTTCATTCCTGGGCTCCACCCCATCCTGACCCTGTCCAAGATGGACCAGACACTGGCA 240 GTCTACCAACAGATCCTCACATCGATGCCTTCCAGAAACGTGATCCAAATATCCAACGAC 300 CTGGAGAACCTCCGGGATCTTCTTCACGTGCTGGCCTTCTCTAAGAGCTGCCACTTGCCC 360 TGGGCCAGTGGCCTGGAGACCTTGGACAGCCTGGGGGGTGTCCTCGAGGCTTCAGGCTAC 420 TCCACAGAGGTGGTGGCCCTGAGCAGGCTGCAGGGGTCTCTGCAGGACATGCTGTGGCAG 480 CTGGACCTCAGCCCCGGGTGCGGTACCGAGCCCAAATCGGCCGACAAAACTCACACATGC 540 CCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAA 600 CCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTG 660 AGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAAT 720 GCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTC 780 ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA 840 GCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCA 900 CAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACC 960 TGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG 1020 CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC 1080 TATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCC 1140 GTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT 1200 AAG 1203 Table 8. Amino acid sequence of leptin/IgGl 'GT' chimera, 401aa 1 MHWGTLCGFL WLWPYLFYVQ AVPIQKVQDD TKTLIKTIVT RINDISHTQS 51 VSSKQKVTGL DFIPGLHPIL TLSKMDQTLA VYQQILTSMP SRNVIQISND 101 LENLRDLLHV LAFSKSCHLP WASGLETLDS LGGVLEASGY STEVVALSRL 151 QGSLQDMLWQ LDLSPGCGTE PKSADKTHTC PPCPAPELLG GPSVFLFPPK 201 PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY 251 NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPLEKTI SKAKGQPREP 301 QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP 351 VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG 401 K

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 protein comprises 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 according to claim 6, wherein the human immunoglobulin Fc domain is an IgG4PE variant, an IgG4, IgGl or an IgGlGT variant, in particular the hinge-CH2- CH3 region in each case. 8. A chimera according to claim 7, wherein the variant a hinge-CH 2 -CH 3 varianL 9. Chimeric leptin selected from the list consisting of: leptin l-167/IgG4 hinge-CH2-CH3; leptin l-167/IgG4 hinge-CH2-CH3 PE variant; leptin 1-167/IgGl hinge-CH2-CH3; and leptin 1-167/IgGl 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 claiml3. 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.

Inventors: BROWNE MICHAEL JOSEPH, CHAPMAN CONRAD GERALD, CLINKENBEARD HELEN ELIZABETH, ROBINSON JEFFREY HUGH, BROWNE, MICHAEL, JOSEPH, CHAPMAN, CONRAD, GERALD, CLINKENBEARD, HELEN, ELIZABETH, ROBINSON, JEFFREY, HUGH
Applicants: BROWNE MICHAEL JOSEPH, CHAPMAN CONRAD GERALD, CLINKENBEARD HELEN ELIZABETH, ROBINSON JEFFREY HUGH, SMITHKLINE BEECHAM PLC, BROWNE, MICHAEL, JOSEPH, CHAPMAN, CONRAD, GERALD, CLINKENBEARD, HELEN, ELIZABETH, ROBINSON, JEFFREY, HUGH
Application year: 1996
Main classification: C12N15
IPC codes: A61K38, A61P3, C07K14, C07K16, C07K19, C12N1, C12N5, C12N15, C12P21