Leptin variants and methods of use thereof

Abstract

Provided herein are leptin protein variants comprising cysteine substitutions. Also provided herein are methods of producing and using the leptin protein variants.

Description

LEPTIN VARIANTS AND METHODS OF USE THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims benefit of and priority to U.S. Application No. 63 / 653,650, filed May 30, 2024, U.S. Application No. 63 / 653,655, filed May 30, 2024, U.S. Application No. 63 / 653,657, filed May 30, 2024, U.S. Application No. 63 / 653,662, filed May 30, 2024, U.S. Application No. 63 / 653,666, filed May 30, 2024, and U.S. Application No. 63 / 653,672, filed May 30, 2024, the entire contents of which are hereby incorporated by reference for all purposes.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which is hereby incorporated by reference in its entirety. Said XML copy, created on May 27, 2025, is named GHR- 001WO_SL.xml, and is 9,094 bytes in size.BACKGROUND

[0003] Leptin is an adipocyte-derived protein hormone that activates the leptin receptor (LepR)-STAT3 signaling axis in hypothalamic neurons to promote satiety and energy homeostasis. Leptin signaling is a negative feedback signal from adipose tissue to the brain that decreases appetite and increases energy expenditure. Leptin signaling is often dysregulated in obesity.SUMMARY

[0004] In some aspect, provided herein are one or more isolated proteins comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises an addition of an N-terminal cysteine (C) and wherein the variant is capable of binding a leptin receptor.

[0005] In some aspect, provided herein are one or more isolated proteins comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) for isoleucine (I) amino acid substitution at position 3 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor.

[0006] In some aspect, provided herein are one or more isolated proteins comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) forserine (S) amino acid substitution at position 31 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor.

[0007] In some aspect, provided herein are one or more isolated proteins comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) for histidine (H) amino acid substitution at position 46 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor.

[0008] In some aspect, provided herein are one or more isolated proteins comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) for alanine (A) amino acid substitution at position 101 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor.

[0009] In some aspect, provided herein are one or more isolated proteins comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) for tryptophan (W) amino acid substitution at position 100 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor.

[0010] In some embodiments, the polypeptide comprises an amino acid sequence having at least 90-99%, 92-98%, 93-97%, 94-96%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence as set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises a glutamic acid (E) for tryptophan (W) amino acid substitution at position 100 of the sequence set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or more additional amino acid mutations as compared to the sequence as set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises 3 additional amino acid mutations as compared to the sequence as set forth in SEQ ID NO: 7.

[0011] In some embodiments, the polypeptide comprises the sequence as set forth in SEQ ID NOs 1-6. In some embodiments, the polypeptide comprises a moiety conjugated to at least the additional cysteine residue(s) located at the N-terminus of SEQ ID NO: 1 or the amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located at position 3 of SEQ ID NO: 2 or the amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located atposition 31 of SEQ ID NO: 3 or the amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located at position 46 of SEQ ID NO: 4 or the amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located at position 101 of SEQ ID NO: 5 or the amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located at position 100 of SEQ ID NO: 6 or the amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7.

[0012] In some embodiments, the moiety is a peptide, a lipid, a PEG molecule, or a polymer. In some embodiments, the moiety is a PEG molecule, a lipid, an Fc domain, or albumin.

[0013] In some aspect, provided herein are fusion proteins comprising the protein disclosed herein, fused to an Fc domain or albumin. In some embodiments, the Fc domain or albumin is fused to the N terminus or the C terminus of the variant leptin polypeptide

[0014] In some aspect, provided herein are isolated polynucleotides or set of polynucleotides encoding the protein disclosed herein.

[0015] In some aspect, provided herein are vectors or set of vectors comprising the polynucleotide or set of polynucleotides disclosed herein.

[0016] In some aspect, provided herein are host cells comprising the polynucleotide or set of polynucleotides or the vector disclosed herein.

[0017] In some aspect, provided herein are methods of producing a protein comprising expressing the protein with the host cell disclosed herein and isolating the expressed protein.

[0018] In some aspect, provided herein are methods of producing a protein comprising synthesizing or having synthesized the protein disclosed herein.

[0019] In some aspect, provided herein are pharmaceutical compositions comprising the protein disclosed herein and a pharmaceutically acceptable excipient.

[0020] In some aspect, provided herein are methods of treating obesity in a subject comprising administering the protein disclosed herein to a subject.

[0021] In some aspect, provided herein are methods of increasing half-life, solubility, and / or decreasing aggregation of the protein disclosed herein, comprising conjugating a moiety to the cysteine residue(s). In some embodiments, the moiety is a PEG molecule, a lipid, an Fc domain, or albumin.

[0022] In some aspect, provided herein are methods of increasing half-life, solubility, and / or decreasing aggregation of the protein disclosed herein, comprising fusing an Fc domain or albumin to the polypeptide. In some embodiments, the Fc domain or albumin is fused to the N terminus or the C terminus of the variant leptin polypeptide.BRIEF DESCRIPTION OF DRAWINGS

[0023] FIGs. 1A-1F show 6 lipidated cysteine variants of leptin, assessed for their in vitro potency in a stable HEK293 cell line co-expressing the human leptin receptor (hLepR) and a STAT3-luciferase (STAT3-luc) reporter. FIG. 1A shows the potency of the (-l)C / W100E leptin analog to induce LepR signaling after incubation with the reporter cells in either HSA or OVA. FIG. IB shows the potency of the I3C / W100E leptin analog to induce LepR signaling after incubation with the reporter cells in either HSA or OVA. FIG. 1C shows the potency of the S31C / W100E leptin analog to induce LepR signaling after incubation with the reporter cells in either HSA or OVA. FIG. ID shows the potency of the H46C / W100E leptin analog to induce LepR signaling after incubation with the reporter cells in either HSA or OVA. FIG. IE shows the potency of the A101C / W100E leptin analog to induce LepR signaling after incubation with the reporter cells in either HSA or OVA. FIG. IF shows the potency of the W100C leptin analog to induce LepR signaling after incubation with the reporter cells in OVA. The figures illustrate the potency of the variants in the presence of 1% human serum albumin (HSA; left panels) or 1% ovalbumin (OVA; right panels), relative to human recombinant leptin (hLep).

[0024] FIGs. 2A-2F show the in vivo potency of the leptin analogs in the B6.Cg-Ze / ?ofe / J (ob / ob) rodent model. The figures illustrate each variant’s ability to suppress food intake (FI; left panels) and reduce body weight (BW; right panels), relative to vehicle-treated controls. FIG. 2A shows the food intake (FI, left panel) and % body weight change (BW, right panel) for mice treated with the (-l)C / W100E leptin analog. FIG. 2B shows the food intake (FI, left panel) and % body weight change (BW, right panel) for mice treated with the I3C / W100E leptin analog. FIG. 2C shows the food intake (FI, left panel) and % body weight change (BW, right panel) for mice treated with the S31C / W100E leptin analog. FIG. 2D shows the food intake (FI, left panel) and % body weight change (BW, right panel) for mice treated with the H46C / W100E leptin analog. FIG. 2E shows the food intake (FI, left panel) and % body weight change (BW, right panel) for mice treated with the A101C / W100E leptin analog. FIG. 2F shows the food intake (FI, left panel) and % body weight change (BW, right panel) for mice treated with wild type human leptin.DETAILED DESCRIPTIONDefinitions

[0025] Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0026] The term “ameliorating” refers to any therapeutically beneficial result in the treatment of a disease state, e.g., obesity, including prophylaxis, lessening in the severity or progression, remission, or cure thereof.

[0027] The term “in situ” refers to processes that occur in a living cell growing separate from a living organism, e.g., growing in tissue culture.

[0028] The term “in vivo” refers to processes that occur in a living organism.

[0029] The terms “ex vivo” and “in vitro” refer to processes that occur outside of a living organism.

[0030] The term "obesity" means the condition of excess body fat (adipose tissue), including by way of example in accordance with the National Institutes of Health Federal Obesity Clinical Guidelines for adults, whereby body mass index ("BMI") calculated by dividing body mass in kilograms by height in meters squared is equal to or greater than twenty-five (25).

[0031] The term “mammal” as used herein includes both humans and non-humans and include but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0032] The term “percent identity,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the “percent identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

[0033] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0034] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0035] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0036] The term “sufficient amount” means an amount sufficient to produce a desired effect, e.g., an amount sufficient to modulate protein aggregation in a cell.

[0037] The term “therapeutically effective amount” is an amount that is effective to ameliorate a symptom of a disease. A therapeutically effective amount can be a “prophylactically effective amount” as prophylaxis can be considered therapy.

[0038] The term "substantially purified" refers to a construct described herein, or variant or analogue thereof that may be substantially or essentially free of components that normally accompany or interact with the protein as found in its naturally occurring environment, i.e. a native cell, or host cell in the case of recombinantly produced proteins comprising a variant leptin polypeptide (e.g., a leptin analogue) that in certain embodiments, is substantially free of cellular material includes preparations of protein having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating protein. When the variant leptin polypeptide (e.g., a leptin analogue) is recombinantly produced by the host cells, the protein in certain embodiments is present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cells. When the variant leptin polypeptide (e.g., a leptin analogue) is recombinantly produced by the host cells, the protein, in certain embodiments, is present in the culture medium at about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, about 750 mg / L, about 500 mg / L, about 250 mg / L,about 100 mg / L, about 50 mg / L, about 10 mg / L, or about 1 mg / L or less of the dry weight of the cells. In certain embodiments, "substantially purified" variant leptin polypeptide (e.g., a leptin analogue) produced by the methods described herein, has a purity level of at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, specifically, a purity level of at least about 75%, 80%, 85%, and more specifically, a purity level of at least about 90%, a purity level of at least about 95%, a purity level of at least about 99% or greater as determined by appropriate methods such as SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0039] As used herein, “Leptin,” “leptins,” “leptin protein,” and “leptin polypeptide” encompass wild type leptin and leptin polypeptides comprising one or more amino acid substitutions, additions or deletions. These terms include, but are not limited to, leptin proteins including those comprising one or more amino acid substitutions, additions, or deletions as well as analogues, variants, fusions, mutants, fragments, agonists, antagonists, dimers, multimers, polypeptides covalently bound to polymers, polypeptides that share 90% or greater amino acid sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity) to human leptin, and polypeptides that possess the four helical bundle structure. Leptin polypeptides disclosed herein may comprise modifications (e.g., substitutions) with one or more natural amino acids. Exemplary substitutions of amino acid positions in naturally-occurring leptin polypeptides include, but are not limited to, substitutions that modulate one or more of the biological activities of the leptin polypeptide, such as but not limited to, increase agonist activity, increase solubility of the polypeptide, convert the polypeptide into an antagonist, etc. and are encompassed by the term “leptin polypeptide.” The term “leptin polypeptide” also includes the pharmaceutically acceptable salts and prodrugs, and prodrugs of the salts, polymorphs, hydrates, solvates, biologically-active fragments, biologically active variants and stereoisomers of the naturally- occurring leptin as well as agonist, mimetic, and antagonist variants of the naturally- occurring leptin and polypeptide fusions thereof. Fusions comprising additional amino acids at the amino terminus, carboxyl terminus, or both, are encompassed by the term “leptin polypeptide.” The terms include plural reference unless the context clearly indicates otherwise.

[0040] The terms “analogue” and “variant” are interchangeable when used in the context of a leptin protein. E.g., “leptin variant” and “leptin analogue” both refer to a human leptin protein comprising one or more mutation disclosed herein, as compared to wild type human leptin.

[0041] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.Proteins and Polypeptides

[0042] Human leptin (HGNC: 6553, NCBI Gene: 3952, UniProtKB / Swiss-Prot: P41159) is encoded by the Lep gene, and is a protein hormone predominantly made by adipocytes. Leptin also has several endocrine functions, and is involved in the regulation of immune and inflammatory responses, hematopoiesis, angiogenesis, reproduction, bone formation and wound healing.

[0043] Those of skill in the art will appreciate that amino acid positions corresponding to positions in SEQ ID NOs: 1, 2, 3, 4, 5, 6 or 7 can be readily identified in any other leptin molecule such as leptin fusions, analogues, variants, fragments, etc. For example, sequence alignment programs such as BLAST can be used to align and identify a particular position in a protein that corresponds with a position in SEQ ID NOs: 1, 2, 3, 4, 5, 6 or 7. Substitutions, deletions or additions of amino acids described herein in reference to SEQ ID NOs: 1, 2, 3, 4, 5, 6 or 7 are intended to also refer to substitutions, deletions or additions in corresponding positions in leptin fusions, analogues, variants, fragments, etc. described herein or known in the art and are expressly encompassed by the present invention.

[0044] Leptin polypeptides may comprise secretion signal sequences. Examples of secretion signal sequences include, but are not limited to, a prokaryotic secretion signal sequence, an eukaryotic secretion signal sequence, an eukaryotic secretion signal sequence 5 '-optimized for bacterial expression, a novel secretion signal sequence, pectate lyase secretion signal sequence, Omp A secretion signal sequence, and a phage secretion signal sequence.Examples of secretion signal sequences, include, but are not limited to, STII (prokaryotic), Fd Gill and Ml 3 (phage), Bgl2 (yeast), and the signal sequence bla derived from a transposon.

[0045] The sequence of leptin may be altered in various ways known in the art to generate targeted changes in sequence. The polypeptide will usually be substantially similar to the sequences provided herein, e.g., will differ by at least one amino acid, and may differ by at least three amino acids. The sequence changes may be substitutions, insertions or deletions, including truncation at the carboxyl or the amino terminus. Scanning mutations that systematically introduce alanine, or other residues, may be used to determine key amino acids. Conservative amino acid substitutions typically include substitutions within the following groups: (aliphatic: glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L));(acidic and amides: aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q)); (hydroxyl or sulfur containing: serine (S), threonine (T), cysteine (C), methionine (M)); (basic: lysine (K), arginine (R), histidine (H)); and (aromatic: phenylalanine (F), tyrosine (Y), tryptophan (W)).

[0046] Polypeptides described herein can be prepared by, for example, by using standard solid phase techniques, or by expression in a cell through recombinant methods. (See Merrifield, 1963. Am. Chem. Soc. 85:2149; J.M. Stewart and J.D. Young, 1984 Solid Phase Peptide Syntheses 2nd Ed., Pierce Chemical Company).

[0047] Provided herein are one or more isolated proteins comprising a variant leptin polypeptide (e.g., a leptin analogue) comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises an addition of an N-terminal cysteine (C) and wherein the variant is capable of binding a leptin receptor. In some embodiments, one or more isolated proteins comprising a variant leptin polypeptide (e.g., a leptin analogue) comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) for isoleucine (I) amino acid substitution at position 3 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor. In some embodiments, one or more isolated proteins comprising a variant leptin polypeptide (e.g., a leptin analogue) comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) for serine (S) amino acid substitution at position 31 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor. In some embodiments, one or more isolated proteins comprising a variant leptin polypeptide (e.g., a leptin analogue) comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) for histidine (H) amino acid substitution at position 46 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor. In some embodiments, one or more isolated proteins comprising a variant leptin polypeptide (e.g., a leptin analogue) comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) for alanine (A) amino acid substitution at position 101 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor. In some embodiments, one or more isolated proteins comprising a variant leptin polypeptide (e.g., a leptin analogue) comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptidecomprises a cysteine (C) for tryptophan (W) amino acid substitution at position 100 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor.

[0048] In some embodiments, the polypeptide comprises an amino acid sequence having at least 90-99%, 92-98%, 93-97%, 94-96%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence as set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises amino acid sequences as set forth in SEQ ID NOs: 1-6.

[0049] Additional mutations can be made to the variant leptin polypeptide. For example, the polypeptide can comprise 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or more additional amino acid mutations as compared to the sequence as set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or more additional amino acid mutations as compared to the sequence as set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises 3 additional amino acid mutations as compared to the sequence as set forth in SEQ ID NO: 7.

[0050] The polypeptide can also comprise a glutamic acid (E) for tryptophan (W) amino acid substitution at position 100 of the sequence set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises a glutamic acid (E) for tryptophan (W) amino acid substitution at position 100 of the sequence set forth in SEQ ID NO: 7.Fusion Proteins

[0051] In some embodiments, the polypeptide comprises a fusion protein comprising the leptin variant polypeptide (e.g., a leptin analogue) fused to a peptide, an Fc domain, albumin (HGNC: 399, NCBI Gene: 213, UniProtKB / Swiss-Prot: P02768), an XTEN peptide, an albumin-binding peptide, or an elastin-like polypeptide (ELP). In some embodiments, the peptide moiety can be an albumin-binding protein, peptide, or small molecule (e.g., an albumin binding domain polypeptide (ABD)). Exemplary albumin-binding proteins, peptides, or small molecules are disclosed in International Patent Publications W02008068280A1, W02009016043A2, WO2011095545A1, WO2018104444A1, WO2019016237A1 and in Zorzi A, et al, Non-covalent albumin binding ligands for extending the circulating half life of small biotherapeutics, Med. Chem. Commun., 2019, 10, 1068, each of which are hereby incorporated by reference in its entirety. In some embodiments, the peptide moiety can be an XTEN polypeptide. Exemplary XTEN polypeptides are disclosed in Schellenberger et al, Nat Biotechnol. 2009 Dec;27(12): l 186-90, and US Patent Publication 9,371,369, both of which are hereby incorporated by reference in its entirety. In some embodiments, the peptide moietycan be an elastin-like polypeptide (ELP). Exemplary ELPs are disclosed in Despanie J., et al. J Control Release. 2016 Oct 28;240:93-108, and Guo, Y., et al. J Nanobiotechnol 21, 418 (2023), both of which are hereby incorporated by reference in their entirety.

[0052] In some embodiments, the leptin variant polypeptide can be fused to an albuminbinding protein or peptide (e.g., an albumin binding domain polypeptide (ABD)). In some embodiments, the leptin variant polypeptide can be fused to an XTEN polypeptide. In some embodiments, the leptin variant polypeptide can be fused to an elastin-like polypeptide (ELP). The peptide, Fc domain, albumin, an XTEN peptide, albumin-binding protein or peptide, or elastin-like polypeptide (ELP) can be fused to the N terminus or C terminus of the leptin variant polypeptide.Conjugation Moieties

[0053] In certain embodiments, the leptin polypeptide includes at least one post-translational modification, e.g., a moiety conjugated to an amino acid residue. In some embodiments, the polypeptide comprises a moiety conjugated to the additional N-terminal cysteine of SEQ ID NO: 1. In some embodiments, the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located at position 3 of SEQ ID NO: 2. In some embodiments, the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located at position 31 of SEQ ID NO: 3. In some embodiments, the polypeptide comprises moiety conjugated to at least the cysteine residue(s) located at position 46 of SEQ ID NO: 4. In some embodiments, the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located at position 101 of SEQ ID NO: 5. In some embodiments, the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located at position 100 of SEQ ID NO: 6. Such moieties include, but are not limited to, a peptide, a lipid, a fatty acid, a PEG molecule, a polymer, an XTEN peptide, an albumin-binding peptide, or an elastin-like polypeptide (ELP). For example, the moiety can be a PEG molecule, a lipid, a fatty acid, an Fc domain, or albumin. In some embodiments, the moiety is a PEG molecule, a lipid, a fatty acid, an Fc domain, albumin, an XTEN peptide, an albumin-binding peptide, or an elastinlike polypeptide (ELP). In some embodiments, the moiety an mTOR inhibitor. In some embodiments, the mTOR inhibitor is rapamycin (sirolimus) or a rapamycin analog, or rapalog, such as everolimus, temsirolimus, or ridaforolimus.

[0054] In some embodiments, the fatty acid or lipid is a C20 fatty diacid (eicosanedioic acid). In some embodiments, the C20 fatty diacid comprises a linker and / or a cysteine reactive moiety. In some embodiments, the C20 fatty diacid section is linked to a glutamic acid. Insome embodiments, the C20 fatty diacid section is linked to one, two, or more (2-(2- aminoethoxy)ethoxy)acetic acid unit(s). In some embodiments, the C20 fatty diacid section is linked to a glutamic acid and two (2-(2-aminoethoxy)ethoxy)acetic acid units. In some embodiments, the lipid or fatty acid comprises the structure as shown in Formula VII.Formula VII:

[0055] In some embodiments, the leptin polypeptide comprises the conjugated polypeptide ofSEQ ID NO: 1 as shown in Formula I:

[0056] In some embodiments, the leptin polypeptide comprises the conjugated polypeptide ofSEQ ID NO: 2 as shown in Formula II:

[0057] In some embodiments, the leptin polypeptide comprises the conjugated polypeptide ofSEQ ID NO: 3 as shown in Formula III:

[0058] In some embodiments, the leptin polypeptide comprises the conjugated polypeptide ofSEQ ID NO: 4 as shown in Formula IV:

[0059] In some embodiments, the leptin polypeptide comprises the conjugated polypeptide ofSEQ ID NO: 5 as shown in Formula V:

[0060] In some embodiments, the leptin polypeptide comprises the conjugated polypeptide ofSEQ ID NO: 6 as shown in Formula VI:Nucleic Acids, Vectors, and Cells

[0061] In some embodiments, the present disclosure contemplates nucleic acid(s), modules, cassettes, or DNA template inserts encoding a protein or variant leptin polypeptide. In some embodiments, the nucleic acid(s), modules, cassettes, or DNA template inserts are codon optimized. A variety of codon optimization tools are known in the art, see e.g., the Integrated DNA Technologies (IDT) codon optimization tool at idtdna.com / pages / tools / codon- optimizati on-tool or Genescript’s codon optimization tool at genscript.com / gensmart-free- gene-codon-optimization.html and disclosed in W02020024917A1, hereby incorporated by reference.

[0062] In one embodiment, an isolated nucleic acid encoding a variant leptin polypeptide described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the variant leptin polypeptide. In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. Exemplary vectors are discussed in Nora LC, et al, The art of vector engineering: towards the construction of nextgeneration genetic tools. Microb Biotechnol. 2019 Jan; 12(1): 125-147, hereby incorporated by reference in its entirety. In a further embodiment, a host cell comprising such a vector or nucleic acid is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the variant leptin polypeptide.

[0063] For recombinant production of the variant leptin polypeptide, a nucleic acid encoding a variant leptin polypeptide, e.g., as described above, can be synthesized and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid may be synthesized using conventional procedures, e.g., via in vitro DNA synthesis methods as described in Hughes RA, et al, Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology. Cold Spring Harb Perspect Biol. 2017 Jan 3;9(l):a023812, hereby incorporated by reference. Alternatively, the nucleic acid encoding a variant leptin polypeptide can be genetically engineered via conventional methods of recombinant DNA generation to isolate a wild type leptin nucleic acid (e.g., by using oligonucleotide probes that are capable of binding specifically to a gene encoding the leptin nucleic acid) and introduce the variant nucleic acid sequence via recombinant DNA technology (see, e.g., Celie PH, et al, Recombinant cloning strategies for protein expression. Curr Opin Struct Biol. 2016 Jun;38: 145-54, hereby incorporated by reference). In some embodiments, the host cell is prokaryotic, e.g., an E. coli cell. In some embodiments, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell, human embryonic kidney (HEK) cell, or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of making a protein comprising a variant leptin polypeptide is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the protein comprising a variant leptin polypeptide, as provided above, under conditions suitable for expression of the protein comprising a variant leptin polypeptide, and optionally recovering the protein from the host cell or a host cell culture medium.

[0064] Suitable host cells for cloning or expression of leptin polypeptide-encoding vectors include prokaryotic or eukaryotic cells described herein.

[0065] A "recombinant host cell" or "host cell" refers to a cell that includes an exogenous nucleic acid, regardless of the method used for insertion, for example, direct uptake, genetic editing, transduction, f-mating, or other methods known in the art to create recombinant host cells. The nucleic acid encoding the protein comprising a variant leptin polypeptide may be maintained as a nonintegrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome. Host cells can include bacterial host cells, yeast host cells, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, E. coli, CHO, derivatives of CHO, NSO, Sp2O, CV-1, VERO-76, HeLa, HepG2, Per.C6, or BHK.

[0066] As used herein, the term "eukaryote" refers to organisms belonging to the phylogenetic domain Eucarya such as animals (including but not limited to, mammals, insects, reptiles, birds, etc.), ciliates, plants (including but not limited to, monocots, dicots, algae, etc.), fungi, yeasts, flagellates, microsporidia, protists, etc.

[0067] As used herein, the term "prokaryote" refers to prokaryotic organisms. For example, a non-eukaryotic organism can belong to the Eubacteria (including but not limited to, Escherichia coli (E. coli), Thermus thermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, etc.) phylogenetic domain, or the Archaea (including but not limited to, Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium such as Haloferax volcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pemix, etc) phylogenetic domain.

[0068] For example, the protein comprising a variant leptin polypeptide may be produced in bacteria, in particular when glycosylation is not needed. For expression of polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,789,199, and 5,840,523, each of which are hereby incorporated by reference. After expression, the protein comprising a variant leptin polypeptide may be isolated from the bacterial cell paste in a soluble fraction and can be further purified. For a review of E. coli cell lines suitable for protein production, see, e.g., Baeshen MN et al, Production of Biopharmaceuticals in E. coli: Current Scenario and Future Perspectives. J Microbiol Biotechnol. 2015 Jul;25(7):953-62, hereby incorporated by reference.

[0069] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of proteins with a partially or fully human glycosylation pattern. See Gemgross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215(2006), each of which are hereby incorporated by reference. For a review of yeast cell lines suitable for protein production, see, e.g., Vieira Gomes AMet al, Comparison of Yeasts as Hosts for Recombinant Protein Production. Microorganisms. 2018 Apr 29;6(2):38, hereby incorporated by reference.

[0070] Suitable host cells for the expression of glycosylated protein comprising a variant leptin polypeptide are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0071] Plant cell cultures can also be utilized as hosts. See, e.g., Feng Z, Li X, Fan B, Zhu C, Chen Z. Maximizing the Production of Recombinant Proteins in Plants: From Transcription to Protein Stability. Int J Mol Sci. 2022 Nov 4;23(21): 13516, hereby incorporated by reference.

[0072] Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23 :243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3 A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells, each of which are hereby incorporated by reference. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA '1T.Y2A6 (1980) hereby incorporated by reference); and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N. J.), pp. 255-268 (2003) and Hacker DL et al, Recombinant protein production from stable mammalian cell lines and pools. Curr Opin Struct Biol. 2016 Jun;38: 129-36, each of which are hereby incorporated by reference.

[0073] In one embodiment, the protein comprising a variant leptin polypeptide described herein are produced in prokaryotic cells, by a method comprising: transforming at least one prokaryotic cell with: nucleic acid encoding the protein comprising a variant leptinpolypeptide, in a predetermined ratio; and expressing the nucleic acid in the at least one prokaryotic cell. In some embodiments, the predetermined ratio of nucleic acid is determined in transient transfection experiments to determine the relative ratio of input nucleic acids that results in the highest percentage of the protein comprising a variant leptin polypeptide in the expressed product.

[0074] In one embodiment, the protein comprising a variant leptin polypeptide described herein are produced in E. coli cells, by a method comprising: transforming at least one E. coli cell with: nucleic acid encoding the protein comprising a variant leptin polypeptide, in a predetermined ratio; and expressing the nucleic acid in the at least one E. coli cell. In some embodiments, the predetermined ratio of nucleic acid is determined in transient transfection experiments to determine the relative ratio of input nucleic acids that results in the highest percentage of the protein comprising a variant leptin polypeptide in the expressed product.

[0075] In some embodiments is the method of producing a protein comprising a variant leptin polypeptide in cells described herein, said method comprising identifying and purifying the desired the protein comprising a variant leptin polypeptide. In some embodiments, the said identification is by one or both of liquid chromatography and mass spectrometry.

[0076] If required, the protein comprising a variant leptin polypeptide can be purified or isolated after expression. Proteins may be isolated or purified in a variety of ways known to those skilled in the art. Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reversed-phase, carried out at atmospheric pressure or at high pressure using systems such as FPLC and HPLC. Purification methods also include electrophoretic, immunological, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. Purification can often be enabled by a particular fusion partner. For example, the variant leptin polypeptides may be purified using glutathione resin if a GST fusion is employed, Ni+2affinity chromatography if a His-tag is employed or immobilized anti-flag antibody if a flag-tag is used. For general guidance in suitable purification techniques, see, e.g. incorporated entirely by reference Protein Purification: Principles and Practice, 3rdEd., Scopes, Springer-Verlag, NY, 1994, incorporated entirely by reference. The degree of purification necessary will vary depending on the use of the variant leptin polypeptides. In some instances no purification is necessary.

[0077] In certain embodiments the proteins comprising a variant leptin polypeptide are purified using Anion Exchange Chromatography including, but not limited to, chromatography on Q-sepharose, DEAE sepharose, poros HQ, poros DEAF, Toy opearl Q,Toyopearl QAE, Toyopearl DEAE, Resource / Source Q and DEAE, Fractogel Q and DEAE columns.

[0078] In specific embodiments the proteins comprising a variant leptin polypeptide are purified using Cation Exchange Chromatography including, but not limited to, SP-sepharose, CM sepharose, poros HS, poros CM, Toyopearl SP, Toyopearl CM, Resource / Source S and CM, Fractogel S and CM columns and their equivalents and comparables.

[0079] In addition, the protein comprising a variant leptin polypeptide described herein can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W. H. Freeman & Co., N.Y and Hunkapiller et al., Nature, 310: 105-111 (1984) hereby incorporated by reference). For example, a polypeptide can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4diaminobutyric acid, alphaamino isobutyric acid, 4aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, alanine, fluoro-amino acids, designer amino acids such as methyl amino acids, C-methyl amino acids, N-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).Pharmaceutical compositions

[0080] Methods for treatment of obesity are also encompassed by the present disclosure. The methods of the disclosure include administering a therapeutically effective amount of a protein comprising a leptin variant polypeptide to a subject in need thereof. The protein comprising a leptin variant polypeptide can be formulated in pharmaceutical compositions. These compositions can comprise, in addition to one or more of the leptin variant polypeptides, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other materials well known to those skilled in the art. Such materials should generally be non-toxic or interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can depend on the route of administration.

[0081] Administration can be in a “therapeutically effective amount” or “prophylactically effective amount’ ’(as the case can be, although prophylaxis can be considered therapy), thisbeing sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of disease being treated. Prescription of treatment, e.g. decisions on dosage, etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0082] A composition can be administered alone or in combination with other treatments.Methods of Treating Obesity

[0083] In some aspects, provided herein are methods of treating obesity in a subject comprising administering a protein comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises an addition of an N-terminal cysteine (C) and wherein the variant is capable of binding a leptin receptor. In some embodiments, provided herein are methods of treating obesity in a subject comprising administering a protein comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) for isoleucine (I) amino acid substitution at position 3 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor. In some embodiments, provided herein are methods of treating obesity in a subject comprising administering a protein comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) for serine (S) amino acid substitution at position 31 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor. In some embodiments, provided herein are methods of treating obesity in a subject comprising administering a protein comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) for histidine (H) amino acid substitution at position 46 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor. In some embodiments, provided herein are methods of treating obesity in a subject comprising administering a protein comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forthin SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) for alanine (A) amino acid substitution at position 101 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor. In some embodiments, the polypeptide comprises an amino acid sequence having at least 90-99%, 92-98%, 93-97%, 94-96%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence as set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises a glutamic acid (E) for tryptophan (W) amino acid substitution at position 100 of the sequence set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or more additional amino acid mutations as compared to the sequence as set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises 3 additional amino acid mutations as compared to the sequence as set forth in SEQ ID NO: 7.

[0084] In some embodiments, the polypeptide comprises the sequence as set forth in SEQ ID NO: 1.. In some embodiments, the polypeptide comprises the sequence as set forth in SEQ ID NO: 2. In some embodiments, the polypeptide comprises the sequence as set forth in SEQ IDNO: 3. In some embodiments, the polypeptide comprises the sequence as set forth in SEQ IDNO: 4. In some embodiments, the polypeptide comprises the sequence as set forth in SEQ IDNO: 5. In some embodiments, the polypeptide comprises a moiety conjugated to at least the additional cysteine residue(s) located at the N-terminus of SEQ ID NOs: 1. In some embodiments, the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located at position 3 of SEQ ID NO: 2. In some embodiments, the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located at position 31 of SEQ ID NO: 3. In some embodiments, the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located at position 46 of SEQ ID NO: 4. In some embodiments, the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located at position 101 of SEQ ID NO: 5. In some embodiments, the moiety is a peptide, a polymer, a PEG molecule, a lipid, a fatty acid, an Fc domain, albumin, an XTEN peptide, an albuminbinding peptide, or an elastin-like polypeptide (ELP).

[0085] In some aspects, provided herein are methods of treating obesity in a subject comprising administering a protein comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) for tryptophan (W) amino acid substitution at position 100 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor. In some embodiments, the polypeptide comprises an amino acid sequence having at least 90-99%, 92-98%, 93-97%, 94-96%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence as set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or more additional amino acid mutations as compared to the sequence as set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises 3 additional amino acid mutations as compared to the sequence as set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises the sequence as set forth in SEQ ID NO 6. In some embodiments, the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located at position 100 of SEQ ID NO: 6. In some embodiments, the moiety is a PEG molecule, a peptide, a polymer, a lipid, a fatty acid, an Fc domain, albumin, an XTEN peptide, an albumin-binding peptide, or an elastin-like polypeptide (ELP)..

[0086] Body mass index (“BMI”), also called the Quetelet number or Quetelet index, is currently the most widely accepted calculation of excess body fat for humans. Developed by Adolphe Quetelet, BMI is calculated by dividing the subject's weight by the square of his / her height (BMI=W / h.sup.2). In SI units, BMI is typically given as kg / m2; in English units, BMI is typically given as lb / in2. For example, a person who weighs 75 kilograms and stands 1.8 meters tall would have a BMI of 75 / (1.82)-23.148 and thus would not be in need of weight loss. However, a person who weighs 100 kilograms and stands 1.8 meters tall would have a BMI of 100 / (1.8)2=30.864 and therefore would both be in the “obese” range, and thus in need of weight loss.

[0087] The methods disclosed herein may be used to treat humans having a BMI above the recommended body mass index, i.e., at least in the “overweight” range, or at least in the “obese” range. In one embodiment, a human subject is considered in need of weight loss when his or her BMI is 25 or above. In other embodiments, the methods of the invention may be used for the purpose of treating humans having a body mass index of at least about 25, above 25, at least about 30, or above 30.

[0088] As used herein, the term “obese” is when a mammal is at least 20 percent above its ideal weight. In another embodiment, a human subject is obese when his or her body mass index (BMI) is about 30 or above. In another embodiment of any of the disclosed methods, the obese subject has a BMI of between about 30 and about 35. Alternately, the obese subject has a BMI of about 35 or higher. Men with a waist measurement exceeding 40 inches are considered at risk. Women are at risk with a waist measurement of 35 inches or greater.

[0089] As used herein, the term “obesity” is meant to encompass all of the above definitions of obesity.

[0090] Obesity -induced or obesity-related co-morbidities include, but are not limited to, diabetes, non-insulin dependent diabetes mellitus-type II (2), impaired glucose tolerance, impaired fasting glucose, insulin resistance syndrome, dyslipidemia, hypertension, hyperuricacidemia, gout, coronary artery disease, myocardial infarction, angina pectoris, sleep apnea syndrome, Pickwickian syndrome, metabolic syndrome, fatty liver; cerebral infarction, cerebral thrombosis, transient ischemic attack, orthopedic disorders, arthritis deformans, lumbodynia, menstrual disorders, and infertility. In particular, co-morbidities include: hypertension, hyperlipidemia, dyslipidemia, glucose intolerance, cardiovascular disease, sleep apnea, diabetes mellitus, and other obesity-related conditions.

[0091] The leptin polypeptides disclosed herein may be used for prevention of weight regain, prevention of weight gain and use for weight maintenance. Prevention of weight regain, prevention of weight gain and use for weight maintenance refer to the administration of the compounds or combinations of the present invention to reduce or maintain the body weight of a subject at risk of obesity. One outcome of prevention may be preventing body weight regain of body weight previously lost as a result of diet, exercise, or pharmacotherapy. Another outcome of prevention may be preventing obesity from occurring if the treatment is administered prior to the onset of obesity in a subject at risk of obesity. Another outcome of prevention may be decreasing the occurrence and / or severity of obesity-related disorders if the treatment is administered prior to the onset of obesity in a subject at risk of obesity. Moreover, if treatment is commenced in already obese subjects, such treatment may prevent the occurrence, progression or severity of obesity-related disorders.

[0092] “ Treatment” of obesity and obesity-related disorders refers to the administration of the leptin polypeptides or combinations thereof as disclosed herein to reduce food intake, to reduce body weight, or to maintain the body weight of an obese subject. One outcome of treatment may be reducing the body weight of an obese subject relative to that subject's body weight immediately before the administration of the leptin polypeptide or combinations thereof disclosed herein. Another outcome of treatment may be preventing body weight regain of body weight previously lost as a result of diet, exercise, or pharmacotherapy. Another outcome of treatment may be decreasing the occurrence of and / or the severity of obesity-related diseases. Another outcome of treatment may be to maintain weight loss. The treatment may suitably result in a reduction in food or calorie intake by the subject, including a reduction in total food intake, or a reduction of intake of specific components of the diet such as carbohydrates or fats; and / or the inhibition of nutrient absorption; and / or the inhibition of the reduction of metabolic rate; and in weight reduction in patients in needthereof. The treatment may also result in an alteration of metabolic rate, such as an increase in metabolic rate, rather than or in addition to an inhibition of the reduction of metabolic rate; and / or in minimization of the metabolic resistance that normally results from weight loss.

[0093] “Prevention” of obesity and obesity-related disorders refers to the administration of the leptin polypeptides or combinations thereof as disclosed herein to reduce food intake, to reduce body weight, or to maintain the body weight of a subject at risk of obesity. One outcome of prevention may be reducing the body weight of a subject at risk of obesity relative to that subject's body weight immediately before the administration of the leptin polypeptides or combinations thereof as disclosed herein. Another outcome of prevention may be preventing body weight regain of body weight previously lost as a result of diet, exercise, or pharmacotherapy. Another outcome of prevention may be preventing obesity from occurring if the treatment is administered prior to the onset of obesity in a subject at risk of obesity. Another outcome of prevention may be decreasing the occurrence and / or severity of obesity-related disorders if the treatment is administered prior to the onset of obesity in a subject at risk of obesity. Another outcome of prevention may be to prolong resistance to weight gain. Another outcome of prevention may be to prevent weight regain. Moreover, if treatment is commenced in already obese subjects, such treatment may prevent the occurrence, progression or severity of obesity-related disorders, such as, but not limited to, arteriosclerosis, Type II diabetes, polycystic ovarian disease, cardiovascular diseases, osteoarthritis, dermatological disorders, hypertension, insulin resistance, metabolic syndrome, hypercholesterolemia, hypertriglyceridemia, and cholelithiasis.

[0094] The obesity-related disorders herein are associated with, caused by, or result from obesity. Examples of obesity-related disorders include overeating and bulimia, hypertension, diabetes, elevated plasma insulin concentrations and insulin resistance, dyslipidemias, hyperlipidemia, endometrial, breast, prostate and colon cancer, osteoarthritis, obstructive sleep apnea, cholelithiasis, gallstones, heart disease, abnormal heart rhythms and arrythmias, myocardial infarction, congestive heart failure, coronary heart disease, sudden death, stroke, polycystic ovary disease, craniopharyngioma, the Prader-Willi Syndrome, Frohlich's syndrome, GH-deficient subjects, normal variant short stature, Turner's syndrome, and other pathological conditions showing reduced metabolic activity or a decrease in resting energy expenditure as a percentage of total fat-free mass, e.g, children with acute lymphoblastic leukemia. Further examples of obesity-related disorders are metabolic syndrome, also known as syndrome X, insulin resistance syndrome, reproductive hormone abnormalities, sexual and reproductive dysfunction, such as impaired fertility, infertility, hypogonadism in males andhirsutism in females, fetal defects associated with maternal obesity, gastrointestinal motility disorders, such as obesity-related gastro-esophageal reflux, respiratory disorders, such as obesity-hyperventilation syndrome (Pickwickian syndrome), breathlessness, cardiovascular disorders, inflammation, such as systemic inflammation of the vasculature, arteriosclerosis, hypercholesterolemia, hyperuricaemia, lower back pain, gallbladder disease, gout, kidney cancer, and increased anesthetic risk. The leptin polypeptides or combinations thereof as disclosed herein are also useful for reducing the risk of secondary outcomes of obesity, such as reducing the risk of left ventricular hypertrophy.

[0095] The term “treating” is used to refer to either prophylactic and / or therapeutic treatments.Methods of Increasing Half Life, Solubility., and / or Decreasing Aggregation of Leptin Protein

[0096] In some aspects, provided herein are methods of increasing the half-life or solubility and / or decreasing aggregation of a protein comprising a variant leptin polypeptide as disclosed herein, comprising conjugating a moiety to at least the additional cysteine residue(s) located at the N-terminus of SEQ ID NOs: 1. In some embodiments, provided herein are methods of increasing half-life or solubility and / or decreasing aggregation of a protein comprising a variant leptin polypeptide as disclosed herein, comprising conjugating a moiety to at least the cysteine residue(s) located at position 3 of SEQ ID NO: 2. In some embodiments, provided herein are methods of increasing half-life or solubility and / or decreasing aggregation of a protein comprising a variant leptin polypeptide as disclosed herein, comprising conjugating a moiety to at least the cysteine residue(s) located at position 31 of SEQ ID NO: 3. In some embodiments, provided herein are methods of increasing halflife or solubility and / or decreasing aggregation of a protein comprising a variant leptin polypeptide as disclosed herein, comprising conjugating a moiety to at least the cysteine residue(s) located at position 46 of SEQ ID NO: 4. In some embodiments, provided herein are methods of increasing half-life or solubility and / or decreasing aggregation of a protein comprising a variant leptin polypeptide as disclosed herein, comprising conjugating a moiety to at least the cysteine residue(s) located at position 101 of SEQ ID NO: 5. In some embodiments, provided herein are methods of increasing half-life or solubility and / or decreasing aggregation of a protein comprising a variant leptin polypeptide as disclosed herein, comprising conjugating a moiety to at least the cysteine residue(s) located at position 100 of SEQ ID NO: 6. In some embodiments, the moiety is a peptide, a lipid, a fatty acid, a PEG molecule, a polymer, an XTEN peptide, an albumin-binding peptide, or an elastin-likepolypeptide (ELP). In some embodiments, the moiety is a PEG molecule, a lipid, a fatty acid, an Fc domain, albumin, an XTEN peptide, an albumin-binding peptide, or an elastin-like polypeptide (ELP).

[0097] In some embodiments, the protein comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises an addition of an N-terminal cysteine (C) and wherein the variant is capable of binding a leptin receptor. In some embodiments, the protein comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) for isoleucine (I) amino acid substitution at position 3 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor. In some embodiments, the protein comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) for serine (S) amino acid substitution at position 31 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor. In some embodiments, the protein comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) for histidine (H) amino acid substitution at position 46 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor. In some embodiments, the protein comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) for alanine (A) amino acid substitution at position 101 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor. In some embodiments, the polypeptide comprises an amino acid sequence having at least 90-99%, 92-98%, 93-97%, 94-96%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence as set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises a glutamic acid (E) for tryptophan (W) amino acid substitution at position 100 of the sequence set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or more additional amino acid mutations as compared to the sequence as set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises 3 additional amino acid mutations as compared to the sequence as set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises the sequence as set forth in SEQ ID NO: 1. In some embodiments, the polypeptide comprisesthe sequence as set forth in SEQ ID NO 2. In some embodiments, the polypeptide comprises the sequence as set forth in SEQ ID NO 3. In some embodiments, the polypeptide comprises the sequence as set forth in SEQ ID NO 4. In some embodiments, the polypeptide comprises the sequence as set forth in SEQ ID NO 5. In some embodiments, the polypeptide comprises a moiety conjugated to at least the additional cysteine residue(s) located at the N-terminus of SEQ ID NO: 1. In some embodiments, the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located at position 3 of SEQ ID NO: 2. In some embodiments, the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located at position 31 of SEQ ID NO: 3. In some embodiments, the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located at position 46 of SEQ ID NO: 4. In some embodiments, the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located at position 101 of SEQ ID NO: 5.

[0098] In some embodiments, the protein comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) for tryptophan (W) amino acid substitution at position 100 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor. In some embodiments, the polypeptide comprises an amino acid sequence having at least 90-99%, 92-98%, 93-97%, 94-96%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence as set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or more additional amino acid mutations as compared to the sequence as set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises 3 additional amino acid mutations as compared to the sequence as set forth in SEQ ID NO: 7. In some embodiments, the polypeptide comprises the sequence as set forth in SEQ ID NO 6. In some embodiments, the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located at position 100 of SEQ ID NO: 6.

[0099] In some embodiments, the conjugation moiety comprises a peptide, a lipid, a PEG molecule, or a polymer. For example, the moiety can be a PEG molecule, a lipid, a fatty acid, an Fc domain, or albumin. In some embodiments, the moiety is a PEG molecule, a lipid, a fatty acid, an Fc domain, or albumin. In some embodiments, the moiety is rapamycin (sirolimus) or a rapamycin analog, or rapalog, such as everolimus, temsirolimus, or ridaforolimus.

[0100] In some aspects, provided herein are methods of increasing solubility and / or decreasing aggregation of a protein comprising a variant leptin polypeptide as disclosedherein, comprising fusing a peptide, an Fc domain, albumin, an XTEN peptide, an albuminbinding peptide, an elastin-like polypeptide (ELP), rapamycin (sirolimus) or a rapamycin analog, or rapalog, such as everolimus, temsirolimus, or ridaforolimus to the variant leptin polypeptides as disclosed herein (e.g., any one of the variant leptin polypeptides provided in SEQ ID NOs: 1-6). The peptide, Fc domain, albumin, rapamycin (sirolimus) or rapamycin analog, or rapalog can be fused to the N terminus or C terminus of the leptin variant polypeptide. In some embodiments, the moiety is a C20 fatty diacid (e.g., as shown in Formula VII).

[0101] Aggregation and other biophysical properties can assessed using a variety of methods. Differential Scanning Fluorimetry (DSF) can be used to assay thermal stability. Aggregation assays can include fluorometric assays such as PROTEOSTAT® protein aggregation assay (Enzo Life Sciences), dynamic light scattering (DLS), analytical sizeexclusion chromatography (SEC), and flow cytometry.

[0102] Proteolytic stability can be assessed by incubating the leptin analogs in blood plasma and monitoring degradation via protein assays (e.g., immunoblot, fluorogenic probes, chromogenic probes) or via metabolite identification (MetID) assays using LC-MS on in vitro or in vivo samples. MetID assays can be performed by multiple vendors, including LabCorp, Evotec, BioIVT, and WuXi.

[0103] In some aspects, the protein comprising a variant leptin polypeptide as disclosed herein has an increased half-life as compared to wild type leptin. The half life (or pharmacokinetics) of the variant leptin polypeptide can be assessed via any assay known to one of skill in the art, including, but not limited to mass spectroscopy (MS, LC-MS / MS), multiple reaction monitoring, MesoScale, and / or ELISA.Kits and Articles of Manufacture

[0104] The present application provides kits comprising the variant leptin polypeptide compositions described herein along with instructions for use. The instructions for use can be present in the kits as a package insert, in the labeling of the container of the kit or components thereof, or can be in digital form (e.g. on a CD-ROM, via a link on the internet). A kit can include one or more of the variant leptin polypeptide. Additional components within the kits are also contemplated, for example, buffer (such as reconstituting buffer, stabilizing buffer, diluting buffer), and / or one or more control vectors.

[0105] In some embodiments, the kits further contain a component selected from any of secondary antibodies, reagents for immunohistochemistry analysis, pharmaceuticallyacceptable excipient and instruction manual and any combination thereof. In one specific embodiment, the kit comprises a pharmaceutical composition comprising any one or more of the variant leptin polypeptide compositions described herein, with one or more pharmaceutically acceptable excipients.

[0106] The present application also provides articles of manufacture comprising any one of the variant leptin polypeptide compositions or kits described herein. Examples of an article of manufacture include vials (including sealed vials).EXAMPLES

[0107] Below are examples of specific embodiments for carrying out the present disclosure. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present disclosure in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0108] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.);Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rdEd. (Plenum Press) Vols A and B(1992).Example 1: Generation and Characterization of Variant Leptin Polypeptides

[0109] Protein Expression

[0110] An N-terminal cysteine, a cysteine mutation of the isoleucine at amino acid position 3, a cysteine mutation of the serine at amino acid position 31, a cysteine mutation of the histidine at amino acid position 46, a cysteine mutation of the alanine at amino acid position 101 and a cysteine mutation of the tryptophan at amino acid position 100 were introduced to the leptin protein sequence of SEQ ID NO: 7, to produce a leptin variants (SEQ ID NOs: 1-6, respectively), for use as a conjugation residue to attach a moiety to test for improved pharmacokinetic properties, altered solubility and / or aggregation of the leptin variant. The coding sequence for the variant protein was synthesized and cloned into thepETl la bacterial expression plasmid. Additional mutations (e.g., one to six additional amino acid substitutions) can also be incorporated into the leptin variants as provided in SEQ ID NOs: 1-6.

[0111] Rosetta2(DE3)pLysS E. coli was transformed with the leptin expression plasmid. LB medium with ampicillin and chloramphenicol was inoculated with the transformed E. coli and incubated overnight. The next day, the overnight starter E. coli was transferred to Terrific Broth (TB) medium with ampicillin and chloramphenicol, and incubated at 37°C for approximately 7-8 hours. IPTG was added to a final concentration of 0.5 mM and the inoculation was incubated overnight. The bacteria were harvest the next day via centrifugation at 5000 rpm for 15 min.

[0112] Protein Refolding

[0113] The harvested cell pellet was resuspended in lOOmM Tris, pH 9.0, 8M urea, 10 mM DTT and sonicated to lyse the cells. After lysis, the cell lysate was stirred for 2 hours and then diluted 2 fold with MilliQ water, stirred for an hour, and diluted and stirred twice more for a total of 8 fold dilution. Cysteamine was added to a final concentration of 5 mM. The mixture was stirred overnight at 4°C. The solution was clarified by centrifugation at 8000 imp for 30 min. The supernatant pH was adjusted to pH 8 and loaded on to a Poros 50HQ column (100ml). The column was washed with wash buffer (20 mM Tri, pH 8.0, 50 mM NaCl). The leptin protein was eluted with a elution buffer (20 mM Tri, pH 8.0, 500 mM NaCl). The eluted sample from the 50HQ column was loaded onto a Ni-NTA column. The column as washed with wash buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1% triton-XlOO, 0.3% tributyl phosphate and 20 mM Tris, pH 8.0, 300 mM NaCl, 10 mM imidazole). The leptin protein was eluted with 20mM Tris, pH 8.0, 300 mM NaCl, 500 mM imidazole.

[0114] 1 mg of Ulpl enzyme was added to the Ni column eluted leptin and incubated for2 hours at room temperature or overnight at 4°C. The buffer was changed to 20 mM sodium phosphate, pH 7.4, 50 mM NaCl by a desalting column. The leptin protein was further purified with a Source 15Q column.

[0115] PEGylation of the leptin variant protein

[0116] 2.5 mL of the leptin protein in phosphate buffer at 3mg / mL (7.5mg) is diluted with 2.5mL NaOAc buffer (lOOmM, pH5.0). 60uL of 50% AcOH is added to adjust pH to 4.9. 14mg of mPEG-ALD, MW20kDa (Creative PEGWorks, PJK-241) is added and mixture is briefly vortexed to dissolve PEG. An additional 0.5mL of the acetate buffer is used for wash and added to the reaction. NaCNBH3 (1 lOuL of IM solution in water) is added for thefinal concentration of 20mM. Reactions proceed at 4°C, resulting in the conjugation of PEG to the leptin variant protein.

[0117] Biophysical characterization of leptin protein variants

[0118] Aggregation and other biophysical properties can assessed using a variety of methods. Differential Scanning Fluorimetry (DSF) can be used to assay thermal stability. Aggregation assays can include fluorometric assays such as Proteostat, dynamic light scattering (DLS), analytical size-exclusion chromatography (SEC), and flow cytometry.Example 2: Generation and Characterization of Variant Leptin Polypeptides

[0119] Native leptin has a short half-life of approximately 0.5 hours. In order to increase leptin half-life, multiple positions on the leptin molecule were explored as attachment points for an exemplary pharmacokinetic lipid protractor (a C20 fatty diacid with a linker and a cysteine reactive group), by incorporating cysteine mutations in those positions. The cysteine leptin variants (e.g., analogues) were produced and covalently conjugated to a C20 fatty diacid lipid (i.e. lipidated) using thioether chemistry. The resulting lipidated analogs were evaluated by an in-vitro LepR-STAT3 activity assay with or without the presence of human albumin, which can affect the affinity and potency of lipidated molecules. The leptin analogs were further tested in the B6.Cg-Ze / ?ofe / J (ob / ob) rodent model for their efficacy in food intake (FI) suppression and body weight (BW) loss.Protein Expression

[0120] An N-terminal cysteine (-1(C)), a cysteine mutation of the isoleucine at amino acid position 3 (13 C), a cysteine mutation of the serine at amino acid position 31 (S31C), a cysteine mutation of the histidine at amino acid position 46 (H46C), a cysteine mutation of the alanine at amino acid position 101 (A101C), or a cysteine mutation of the tryptophan at amino acid position 100 (W100C) were introduced to the wild type human leptin protein sequence of SEQ ID NO: 7, to produce leptin analogs (SEQ ID NOs: 1-6, respectively) for use as a conjugation residue to attach a moiety to test for altered solubility and aggregation of the leptin analog. All cysteine mutants, which the exception of the W100C analog, also comprised a W100E mutation. The coding sequences for the leptin analogs was synthesized and cloned into the pETl la bacterial expression plasmid. Additional mutations (e.g., one to eight or more additional amino acid substitutions) can also be incorporated into the leptin analog, e.g., as provided in SEQ ID NOs: 1-6.

[0121] Competent E. coli was heat shock transformed with the leptin expression plasmid. LB medium was inoculated with the transformed E. coli for 30 minutes. The transformed E. coli was spread on LB agar plates with ampicillin and incubated overnight. The next day, a single E. coli colony was selected and resuspended in LB medium with ampicillin, and incubated at 37 °C overnight. The starter culture was inoculated into LM medium with ampicillin and incubated at 37 °C until OD600 of 0.6 was reached. IPTG was added to a final concentration of 0.4 mM and the inoculation was incubated overnight at 20°C. The bacteria were harvest the next day via centrifugation at 12,000 rpm for 10 min. The cell pellet was resuspended in PBS buffer containing a protease inhibitor cocktail and lysed with a high pressure homogenizer. The lysed samples were centrifuged at 12,000 g for 40 minutes and the supernatant collected for protein purification.Protein Purification

[0122] An Ni -agarose column was washed and equilibrated by running 200 ml of His Elution Buffer (20 mM ISMLPCU, 500 mM NaCl, 250 mM imidazole, pH 8.0) followed by 500 ml of His Binding Buffer (20 mM ISMLPCU, 500 mM NaCl, 10 mM imidazole, pH 8.0) through the column. The supernatant (clarified lysate) was flowed through the prepared Ni- agarose column. The column was washed with His Binding Buffer, and the protein eluted with His Elution buffer. The protein was exchanged into 20 mM Citrate, 50 mM NaCl, 3% mannitol, pH 7.4 and the protein concentration was adjusted to 1 mg / mL. Reducing and nonreducing SDS-PAGE gels were run to analyze the purity of the protein.

[0123] The 6xHis-SUMO Tag was removed with Ulpl enzyme. His-tagged Ulpl enzyme was added to the protein in citrate buffer at a 1 :33 Ulpl :protein ratio. The protein was digested for 4 hours at 20°C. The digested sample was run through the Ni column and the flowthrough saved. The purity of the tag-free protein was determined via reducing and nonreducing SDS-PAGE.

[0124] The target protein was further purified via a Q column. The Q resin was transferred to a column and 10 CV 0.2 M NaOH was flowed through the column followed by 10 CV of ultrapure water. The column was equilibrated with 20 mM PB pH 6.9 buffer. The pH of the protein sample was adjusted to 7-9 pH by adding 20 mM PB pH 6.9 buffer. The protein sample was added to the Q column, and eluted with 10%, 30%, and 100% buffer 20 mM PB, 1 M NaCl pH 6.9. Each elution fraction was saved. Reducing and non-reducing SDS-PAGE gels were run to analyze the purity of the protein. The purified protein wasconcentrated into a pre-determined buffer with an Ultrafiltration tube and sterilized via 0.22 pm filtration.Fatty Acid Acylation (lipidation) of the leptin analog

[0125] (-l) C

[0126] The leptin analog typically exists as a mixture of monomer and covalent dimer with the extra cysteine residue that was incorporated for lipidation. To selectively break apart dimers without affecting intramolecular disulfide bond(s), leptin protein (in 20 mM Tris buffer, pH 8.5, 1.3 mg / mL) was treated with bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium salt (2.9 mg, 2.0 equiv.) dissolved in 20 pL of water and added to leptin protein (25 mL, 32 mg) in Tris buffer (25 mL, 20 mM, pH 8.5). The reaction was incubated at 4 °C for 18 hours and conversion to monomer was confirmed by LCMS (Method A). Lipidation was done by adding a lipid reagent (e.g., a C20 fatty diacid, 2.9 mg, 1.5 equiv.) dissolved in methanol. The lipidated leptin was purified by HPLC using XBridge Protein Column (XBridge Protein BEH C4 OBD Prep Column, 300A, 5 pm, 10 mm X 250 mm) and a 30-50% acetonitrile gradient (buffer A is 20 mM ammonium bicarbonate pH 8). The clean fractions were determined by LCMS analysis (Method A) and were pooled. Product was dialyzed with PBS using 10 kDa MWCO SLIDE -A-LYZER™ G3 cassettes. Final product was characterized by LCMS (Method B): Rt= 5.05 min, m / z [M+14H]14+1211.1, m / z [M+13H]13+1304.2. Expected MW average: 16942 Da; Observed: 16941.

[0127] The lipidated (-l)C leptin analog is shown as Formula I:

[0128] 13 C

[0129] The 13 C leptin analog was lipidated as described above. Product was dialyzed with PBS using lOkDa MWCO SLIDE- A-LYZER™ G3 cassettes. Final product wascharacterized by LCMS (Method B): Rt= 5.14min, m / z [M+14H]14+1203.0, m / z [M+13H]13+1295.5. Expected MW average: 16829 Da; Observed: 16829.

[0130] The lipidated I3C leptin analog is shown as Formula II:

[0131] S31C

[0132] The S31C leptin analog was lipidated as described above. Product was dialyzed with PBS using lOkDa MWCO SLIDE- A-LYZER™ G3 cassettes. Final product was characterized by LCMS (Method B): Rt= 5.26min, m / z [M+14H]14+1204.9, m / z [M+13H]13+1297.5. Expected MW average: 16855 Da; Observed: 16855.

[0133] The lipidated S31C leptin analog is shown as Formula III:

[0134] H46C

[0135] The H46C leptin analog was lipidated as described above. Product was dialyzed with PBS using 10 kDa MWCO SLIDE- A-LYZER™ G3 cassettes. Final product was characterized by LCMS (Method B): Rt= 5.33 min, m / z [M+14H]14+1201.3, m / z [M+13H]13+1293.6. Expected MW average: 16805 Da; Observed: 16804.

[0136] The lipidated H46C leptin analog is shown as Formula IV:

[0137] W100C

[0138] The W100C leptin analog was lipidated as described above. Final product was characterized by LCMS (Method B): Rt= 5.06 min, m / z [M+14H]14+1201.8, m / z [M+13H]13+1294.2. Expected MW average: 16813 Da; Observed: 16811.

[0139] The lipidated W100C leptin analog is shown as Formula V:

[0140] A101C

[0141] The A101C leptin analog was lipidated as described above. Final product was characterized by LCMS (Method B): Rt= 5.27 min, m / z [M+14H]14+1206.0, m / z [M+13H]13+1298.7. Expected MW average: 16871 Da; Observed: 16870.

[0142] The lipidated A101C leptin analog is shown as Formula VI:LCMS analysis

[0143] Analysis was performed on Agilent 1260 HPLC, 6120 Quad MSD using Kinetex column (2.6 pm C8, 100A, 100 x 4.6mm). Eluent A is 100% water, 0.1% formic acid and Eluent B is 100% MeCN, 0.1% formic acid. Method A: 30-80% B over 4 minutes at the flow rate of 2 mL / min. Method B: 20-100% B over 10 minutes at the flow rate of 1 mL / min. Eluted proteins were detected by absorption at 214 nm and 280 nm, and MS scan in 200-2000 amu range.In vitro potency testing of leptin variants

[0144] A leptin potency assay was established using a stable HEK293 cell line coexpressing the human leptin receptor (hLepR) and a STAT3 -luciferase (STAT3-luc) reporter. Briefly, hLepR and STAT3-luc were stably integrated into HEK293 cells via lentiviral transduction. Single-cell clones expressing hLepR and STAT3-luc were selected, evaluated, and expanded to a larger cell pool. On Day 1, 4 x 104hLepR-STAT3-luc cells were seeded per well in a 96-well plate with 100 pL of MEM supplemented with 10% FBS, L-GlutaMAX, penicillin / streptomycin, G418, and puromycin, and incubated overnight. On Day 2, leptin variants and a reference standard (RS, human recombinant leptin (hLep), PeproTech Catalog#: 300-27-5MG) were serially diluted in base media (100% MEM) to generate eight full and half-log concentrations. Then, 10 pL of each 10x drug dilution was added to the wells in duplicate, yielding a final concentration range of 30 nM to 1 fM. For modified leptin variants (e.g., lipidated), dilutions were prepared in either 1% ovalbumin (OVA) or 1% human serum albumin (HSA), with both conditions tested separately. The W100C leptin variant was only tested with 1% OVA. On Day 3, after ~20 hours of stimulation, 100 pL ofONE-Step Component A (Luciferase Reagent Buffer) was mixed with 10 pL of Component B (Luciferase Reagent Substrate), and 110 pL of this mixture was added to 110 pL of each well. Plates were covered with foil to protect from light and incubated at room temperature on a shaker for 30 minutes. Luminescence was then measured at 450 nm using an Agilent Gen5 BioTek Synergy Neo 2 plate reader. Data were exported to Microsoft Excel, normalized to background, and plotted as logarithmic dose-response curves using GraphPad Prism. ECso values were calculated and are provided in Table 1 and 2.Table 1. Potency (ECso) of leptin variants in the presence of 1% OVA or 1% HSATable 2. Potency (ECso) of leptin variant W100C in the presence of 1% OVA

[0145] FIGs. 1A-1F show the potency of the variants in the presence of 1% human serum albumin (HSA; left panels) or 1% ovalbumin (OVA; right panels), relative to human recombinant leptin (hLep). FIG. 1A shows the potency of the (-l)C / W100E leptin analog to induce LepR signaling after incubation with the reporter cells in either HSA or OVA. FIG. IB shows the potency of the I3C / W100E leptin analog to induce LepR signaling after incubation with the reporter cells in either HSA or OVA. FIG. 1C shows the potency of the S31C / W100E leptin analog to induce LepR signaling after incubation with the reporter cells in either HSA or OVA. FIG. ID shows the potency of the H46C / W100E leptin analog to induce LepR signaling after incubation with the reporter cells in either HSA or OVA. FIG. IE shows the potency of the A101C / W100E leptin analog to induce LepR signaling after incubation with the reporter cells in either HSA or OVA. FIG. IF shows the potency of the W100C leptin analog to induce LepR signaling after incubation with the reporter cells in OVA.

[0146] As shown in FIGs. 1A - IF and Tables 1 and 2, the leptin analog S31C and A101C had the best relative in vitro potency EC50 in both HSA and OVA of the leptin analogs tested. Without wishing to be bound by theory, the overall potency of the analogs may be best assessed in vivo, since reduced potency is possible for lipidated peptides relative to the non-lipidated peptide counterparts. Importantly, reduced in vitro activity does not necessarily result in diminished in vivo efficacy. Furthermore, without wishing to be bound by theory, in certain cases reduced potency may be desired as it results in slowed receptor- mediated clearance and extended overall duration of action.In vivo potency testing of leptin variants

[0147] In vivo potency testing of the leptin analogs (-l)C, I3C, S31C, H46C, and A101C was conducted as a single subcutaneous (SC) injection in the B6.Cg-Lepob / J mouse model (leptin deficient). Body weight and food intake were recorded daily for a total of 8 days. Study groups consisted of 4-6 mice (3-12 months old, sourced from Jackson Laboratories), which were weight-matched prior to dosing. Animals had ad libitum access to water and standard chow throughout the study. The required dose of each leptin analog was calculated per group and diluted with vehicle. Animals were administered a single dose of 100 nmol / kg in a volume of 2 ml / kg of body weight. The mice were injected subcutaneously in the interstitial space of the back with a 30 G insulin syringe. Body weight and food consumption were recorded daily, and data were analyzed using GraphPad Prism, presented as percent change in body weight (BW) and daily food intake (FI) (g). Control mice were injected with vehicle alone or human recombinant leptin (hLep) alone.

[0148] In vivo potency testing of the leptin analog W100C is conducted as a single subcutaneous (SC) injection in the B6.Cg-Lepob / J mouse model (leptin deficient) as described above. Reduced food intake and decrease in body weight are observed in mice treated with the leptin analog W100C as compared to the mice treated with vehicle alone.

[0149] FIG. 2A shows the food intake (FI, left panel) and % body weight change (BW, right panel) for mice treated with the (-l)C / W100E leptin analog. FIG. 2B shows the food intake (FI, left panel) and % body weight change (BW, right panel) for mice treated with the I3C / W100E leptin analog. FIG. 2C shows the food intake (FI, left panel) and % body weight change (BW, right panel) for mice treated with the S31C / W100E leptin analog. FIG. 2D shows the food intake (FI, left panel) and % body weight change (BW, right panel) for mice treated with the H46C / W100E leptin analog. FIG. 2E shows the food intake (FI, left panel) and % body weight change (BW, right panel) for mice treated with the A101C / W100E leptinanalog. FIG. 2F shows the food intake (FI, left panel) and % body weight change (BW, right panel) for mice treated with the human recombinant leptin control.

[0150] All mice treated with the leptin analogs (-l)C, I3C, S31C, H46C, and A101C had reduced food intake and a decrease in body weight as compared to the mice treated with vehicle alone. Mice treated with the control human recombinant leptin did not have reduced FI or BW after day 1 (e.g., on days 2-5) as compared to the mice treated with vehicle alone.

[0151] Comparison of the five lipidated cysteine mutants of leptin in ob / ob rodents indicated a substantial difference in their performance, with mice treated with the (-l)C and 13 C analogs showing the greatest reduction in FI and BW, and the S31C analog resulting in a moderate reduction in FI and BW as compared to the (-l)C and 13 C analogs and A101C and H46C analogs. However, the A101C and H46C analogs did still result in reduced FI and BW in the treated mice as compared to vehicle alone.

[0152] Without wishing to be bound by theory, lipidation near the N-terminus of the leptin molecule - e.g., the variants with (-l)C and I3C - provided an improvement in efficacy (as measured by daily FI and BW loss) as compared to leptin analogs with lipidation at S31C, A101C, or H46C, in order of improvement. Without wishing to be bound by theory, the absence of significant variation in potencies between the analogs with the same exemplary lipid protractor (C20 fatty diacid), indicate that the difference may be driven by pharmacokinetic properties of each molecule arising from the specific location of the lipid on the molecule. Without wishing to be bound by theory, the (-l)C and I3C lipidated molecules may have improved proteolytic stability as compared to the wild type leptin.

[0153] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

[0154] All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.INFORMAL SEQUENCE LISTING

Claims

CLAIMS1. An isolated protein comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises an addition of an N-terminal cysteine (C) and wherein the variant is capable of binding a leptin receptor.

2. An isolated protein comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) for isoleucine (I) amino acid substitution at position 3 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor.

3. An isolated protein comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) for serine (S) amino acid substitution at position 31 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor.

4. An isolated protein comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) for histidine (H) amino acid substitution at position 46 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor.

5. An isolated protein comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) for alanine (A) amino acid substitution at position 101 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor.

6. An isolated protein comprising a variant leptin polypeptide comprising an amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a cysteine (C) for tryptophan (W) amino acid substitution at position 100 of the sequence set forth in SEQ ID NO: 7, and wherein the variant is capable of binding a leptin receptor.

7. The isolated protein of any one of claims 1-6, wherein the polypeptide comprises an amino acid sequence having at least 90-99%, 92-98%, 93-97%, 94-96%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence as set forth in SEQ ID NO: 7.

8. The isolated protein of any one of claims 1-5, wherein the polypeptide comprises a glutamic acid (E) for tryptophan (W) amino acid substitution at position 100 of the sequence set forth in SEQ ID NO: 7.

9. The protein of claims 1-8, wherein the polypeptide comprises 0, 1, 2, 3, 4, 5, 6, 7, 8,9. 10, 11, 12, 13, 14, or 15, or more additional amino acid mutations as compared to the sequence as set forth in SEQ ID NO: 7.

10. The protein of claim 1-8, wherein the polypeptide comprises 3 additional amino acid mutations as compared to the sequence as set forth in SEQ ID NO: 7.

11. The protein of any one of claims 1-6, wherein the polypeptide comprises the sequence as set forth in SEQ ID NOs 1-6.

12. The protein of any one of claims 1 or 7-11, wherein the polypeptide comprises a moiety conjugated to at least the additional cysteine residue(s) located at the N-terminus of SEQ ID NO: 1 or the amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7.

13. The protein of any one of claims 2 or 7-11, wherein the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located at position 3 of SEQ ID NO: 2 or the amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7.

14. The protein of any one of claims 3 or 7-11, wherein the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located at position 31 of SEQ ID NO: 3 or the amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7.

15. The protein of any one of claims 4 or 7-11, wherein the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located at position 46 of SEQ ID NO: 4 or the amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7.

16. The protein of any one of claims 5 or 7-11, wherein the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located at position 101 of SEQ ID NO: 5or the amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7.

17. The protein of any one of claims 6-7 or 9-11, wherein the polypeptide comprises a moiety conjugated to at least the cysteine residue(s) located at position 100 of SEQ ID NO: 6 or the amino acid sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 7.

18. The protein of any one of claims 12-17, wherein the moiety is a peptide, a lipid, a PEG molecule, or a polymer.

19. The protein of any one of claims 12-18, wherein the moiety is a PEG molecule, a lipid, an Fc domain, or albumin.

20. A fusion protein comprising the protein of any one of claims 1-19, fused to an Fc domain or albumin.

21. The fusion protein of claim 20, wherein the Fc domain or albumin is fused to the N terminus or the C terminus of the variant leptin polypeptide.

22. An isolated polynucleotide or set of polynucleotides encoding the protein of any one of claims 1-21.

23. A vector or set of vectors comprising the polynucleotide or set of polynucleotides of claim 22.

24. A host cell comprising the polynucleotide or set of polynucleotides of claim 22 or the vector of claim 23.

25. A method of producing a protein comprising expressing the protein with the host cell of claim 24 and isolating the expressed protein.

26. A method of producing a protein comprising synthesizing or having synthesized the protein of any one of claims 1-21.

27. A pharmaceutical composition comprising the protein of any one of claims 1-21, and a pharmaceutically acceptable excipient.

28. A method of treating obesity in a subject comprising administering the protein of any one of claims 1-21 to a subject.

29. A method of increasing half-life, solubility, and / or decreasing aggregation of the protein of any one of claims 1-21, comprising conjugating a moiety to the cysteine residue(s).

30. The method of claim 29, wherein the moiety is a PEG molecule, a lipid, an Fc domain, or albumin.

31. A method of increasing half-life, solubility, and / or decreasing aggregation of the protein of any one of claims 1-21, comprising fusing an Fc domain or albumin to the polypeptide.

32. The method of claim 31, wherein the Fc domain or albumin is fused to the N terminus or the C terminus of the variant leptin polypeptide.

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