(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 13 August 2009 (13.08.2009) WO 2009/100255 A2 (51) International Patent Classification: (74) Agent: WALLEN, John, W., Ill; 10975 North Torrey C07K 14/705 (2006.01) Pines Road, Suite 100, La JoUa, CA 92037 (US). (21) International Application Number: (81) Designated States (unless otherwise indicated, for every PCT/US2009/033277 kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (22) International Filing Date: CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, 5 February 2009 (05.02.2009) EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, (25) Filing Language: English HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, (26) Publication Language: English MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, (30) Priority Data: NZ, OM, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, 61/027,4 14 8 February 2008 (08.02.2008) US SK, SL, SM, ST, SV, SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (71) Applicant (for all designated States except US): AM- BRX, INC. [US/US]; 10975 North Torrey Pines Road, (84) Designated States (unless otherwise indicated, for every Suite 100, La JoUa, CA 92037 (US). kind of regional protection available): ARIPO (BW, GH, GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, (72) Inventors; and ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, (75) Inventors/Applicants (for US only): KRAYNOV, TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, Vadim [US/US]; 5457 White Oak Lane, San Diego, CA ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, 92130 (US). PUTNAM, Anna-Maria, A., Hays MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, TR), [US/US]; 11522 Cesped Drive, San Diego California OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, 92124 (US). KNUDSEN, Nick [US/US]; 10709 Matinal MR, NE, SN, TD, TG). Circle, San Diego, CA 92127 (US). PINKSTAFF, Jason [US/US]; 2095 Wandering Road, Encinitas, CA 92024 Published: (US). MYLER, Heather [US/US]; 5312 Forecastle — without international search report and to be republished Court, Carlsbad, CA 92008 (US). SULLIVAN, Lorraine upon receipt of that report (Rule 48.2(g)) [US/US]; 4612 Monongahela Street, San Diego, CA — with sequence listing part of description (Rule 5.2(a)) 921 17 (US). (54) Title: MODIFIED LEPTIN POLYPEPTIDES AND THEIR USES (57) Abstract: Modified human teptin polypeptides and uses thereof arc provided. Modified Leptin Polypeptides and Their Uses CROSS-REFERENCB TO RELATED APPLICATIONS This application claims priority to and benefit of U.S. Provisional Patent Application Serial No. 61/027,414, filed February 8, 2008 and U.S. Provisional Patent Application Serial No. 61/1 12,671, filed November 7, 2008, the specifications and disclosures of which arc incorporated herein in its entirety. FIELD OF THE INVENTION This invention relates to leptin polypeptides modified with at least one non-naturally-encoded amino acid. BACKGROUND OF THE INVENTION [01] The obese (ob) gene product, leptin, is an important circulating signal for the regulation of body weight (Zhang Y el al (1994) Nature 372: 425-432). Mice homozygous for a nonfunctional ob gene become morbidly obese and diabetic, due to overeating and increased metabolic efficiency. In 1995, Tartaglia L A et al (Cell 83: 1263-1271) described a high affinity receptor for murine leptin (OB-R). Evidence suggests that the weight-reducing effects of leptin may be mediated by signal transduction through OB-R in the hypothalamus (Lee G II et al (1996) Nature 379: 632-635). [02] Regulation in the expression of splice variants can have an important role in the activity of signal transduction molecules and has been implicated in the pathogenesis of several diseases (Khachigian L M ct al (1992) Pathology 24: 280-290). For example, mutations that create new splice variants of the sulfonylurea receptor gene segregate with familial persistent hyperinsulinemic hypoglycemia (Thomas P M et al (1995) Science 268: 426-429). [03j At least 9 alternatively spliced forms of mouse OB-R have been described (Lee et al, supra). A splice variant, B219, is expressed in the mouse yolk sac, early fetal liver, enriched hematopoietic stem cells, a variety of lympho-hematopoietic cells lines, and in adult reproductive organs and may be directly involved in hematopoiesis and reproduction (Cioffi J Λ ct al (1996) Nature Medicine 2 ; 585-589). Additional support for leptin and a leptin receptor role in reproduction comes from Chchab F F et al, who report that treatment with leptin corrects a sterility defect in ob/ob female mice (1996, Nature Genet 12: 318-320). The researchers showed that leptin brings on fertility by restoring necessary hypothalmic and pituitary hormone levels rather than by fat reduction. [04J Λn OB-R mutation that creates an alternatively spliced transcript is responsible for the severely obese phenotype of db/db mice (Chen Ϊ I et al (1996) Cell 84: 491-495). Based on synteni between human and mouse chromosomes, the human version of OB-R is likely to map to human chromosome Ip3 1 (Lee et al, supra). [05] Genome sequencing efforts in Caenorhabditis elegans and Saccharomyces cerevisiae have revealed putative open reading frames (ORFs) C30B5.2 and YJR044c, respectively (Wilson R et al, (1994) Nature 368: 32-38; Huang M R et al (1995) Yeast 11: 775-781). YJR044c and C30B5.2 are 27% identical and 71% similar in amino acid sequence and share a similar pattern of hypdrophobicity. YJR044c has been characterized as a putative membrane associated protein (Wilson et al, supra). The C30B5.2 amino acid sequence has a consensus pattern (CCxxHxxC) for phospholipase Λ2, a family of enzymes that release fatty acids from the second carbon group of glycerol. [06] The activity of many signal transduction molecules, such as the leptin receptor, is thought to be regulated by the expression of splice variants of the molecule. A new leptin receptor-related protein could provide the basis for diagnosis and treatment of disease states related to signal transduction events associated with metabolic disorders, such as obesity and diabetes, and reproductive disorders, including infertility. [07] Covalent attachment of a hydrophilic polymer poly(ethylene glycol), abbreviated PEG, is a method of increasing water solubility, bioavailability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending the circulation time of many biologically active molecules, including proteins, peptides, and particularly hydrophobic molecules. PEG has been used extensively in pharmaceuticals, on artificial implants, and in other applications where biocompatibility, lack of toxicity, and lack of immunogenicity are of importance In order to maximize the desired properties of PEG, the total molecular weight and hydration state of the PIiG polymer or polymers attached to the biologically active molecule must be sufficiently high to impart the advantageous characteristics typically associated with PEG polymer attachment, such as increased water solubility and circulating half life, while not adversely impacting the bioactivity of the parent molecule. [08] PEG derivatives are frequently linked to biologically active molecules through reactive chemical functionalities, such as lysine, cysteine and histidine residues, the N- terminus and carbohydrate moieties. Proteins and other molecules often have a limited number of reactive sites available for polymer attachment. Often, the sites most suitable for modification via polymer attachment play a significant role in receptor binding, and are necessary for retention of the biological activity of the molecule. Λs a result, indiscriminate attachment of polymer chains to such reactive sites on a biologically active molecule often leads to a significant reduction or even total loss of biological activity of the polymer- modified molecule. R. Clark et al, (1996), J . Biol. Chem., 271 :21969-21977. To form conjugates having sufficient polymer molecular weight for imparting the desired advantages to a target molecule, prior art approaches have typically involved random attachment of numerous polymer arms to the molecule, thereby increasing the risk of a reduction or even total loss in bioactivity of the parent molecule. [09] Reactive sites that form the loci for attachment of PEG derivatives to proteins are dictated by the protein's structure. Proteins, including enzymes, are composed of various sequences of alpha-amino acids, which have the general structure H2N—CHR--COOII. The alpha amino moiety (H N-) of one amino acid joins to the carboxyl moiety (--COOII) of an adjacent amino acid to form amide linkages, which can be represented as -(NII-CHR-CO) n —, where the subscript "n" can equal hundreds or thousands. The fragment represented by R can contain reactive sites for protein biological activity and for attachment of PEG derivatives. [10] For example, in the case of the amino acid lysine, there exists an -NH moiety in the epsilon position as well as in the alpha position. The epsilon —NH is free for reaction under conditions of basic pH. Much of the art in the field of protein derivatization with PEG has been directed to developing PEG derivatives for attachment to the epsilon -NH 2 moiety of lysine residues present in proteins. "Polyethylene Glycol and Derivatives for Advanced PEGylation", Ncktar Molecular Engineering Catalog, 2003, pp. 1-17. These PEG derivatives all have the common limitation, however, that they cannot be installed selectively among the often numerous lysine residues present on the surfaces of proteins.