INTRODUCTION

[001] Excess body weight is a primary underlying risk factor for a variety of human diseases including type 2 diabetes, cardiovascular disease, and most types of cancer. Over 30% of adults and 20% of adolescents in the United States are now categorized as obese, with prevalence increasing steadily. Although a variety of dietary and pharmacological interventions have emerged in the past several decades to combat obesity, therapeutics capable of safely promoting significant and sustained weight loss are still needed.

[002] Leptin is an adipocyte-derived protein hormone discovered in the 1990s as a critical regulator of body weight in mammals. Genetic loss of leptin results in increased food intake and severe early onset obesity in both mice and humans, and administration of recombinant leptin is sufficient to restore normal body weight in this context. Although these observations led to substantial interest in the clinical use of leptin for obesity, exogenous leptin treatment is not effective in most obese patients, nearly all of whom already exhibit significantly elevated plasma leptin levels but diminished leptin responsiveness.

[003] Leptin exerts its satiety-promoting effects by activating the leptin receptor (LepR) on the surface of a subset of hypothalamic neurons. Activation of LepR in turn drives the phosphorylation and activation of the Signal Transducer and Activator of Transcription 3 (STAT3), which drives production of anorexigenic peptides that suppress food intake and increase energy expenditure. Physiologically, leptin is produced by adipocytes such that levels of circulating leptin are proportional to the amount of adipose tissue, thereby serving as a homeostatic feedback mechanism to correlate food intake with organismal energy stores. In the context of obesity, however, this pathway is dysregulated, resulting in a failure to reduce food intake despite excess adiposity and high levels of circulating leptin. Although the mechanism of this observed leptin resistance is not fully understood, substantial evidence suggests that it is driven by chronic hyperleptinemia and the resulting desensitization of LepR to leptin, due in large part to the expression and recruitment of cytosolic LepR antagonists such as Suppressor of Cytokine Signaling 3 (SOCS3). Given the high incidence of apparent leptin resistance in human obesity, therapeutic modalities capable of bypassing leptin resistance to promote satiety are of high clinical interest.

SUMMARY OF THE INVENTION

[004] Compositions and methods are provided for preventing or treating obesity and/or overweight, and managing body weight. The compositions comprise partial agonists of the leptin receptor (lepr), which partial agonists elicit sub-maximal signaling at saturating ligand concentrations, and bias the response to STAT3 signaling. Human leptin protein variants, for example leptin modified to increase affinity at site 2 binding site(s) and decrease binding at site 3 binding site(s), preferentially suppress phosphorylation of SHP2, ERK, and STAT1 relative to STAT3, resulting in a biased signal that selectively activates pathways associated with satiety, resulting in decreased activation of pathways associated with leptin resistance. The variant proteins find use in the suppression of appetite; and can provide for therapeutic weight loss.

[005] In some embodiments a partial agonist of lepr is a variant of leptin protein, comprising one or more amino acid modifications, e.g. amino acid substitutions, that alter the binding to the lepr receptor. Partial agonists may elicit a maximal STAT3 response of from about 25% to about 75% relative to the wild-type ligand, e.g. wild-type leptin. A partial agonist of interest induces STAT3-biased signaling, where maximal phosphorylation of phosphatase SHP2, ERK, or STAT1 is less than about 25% of the phosphorylation obtained with the wild-type ligand, less than about 10%, less than about 5%, less than about 1%.

[006] In some embodiments, amino acid substitutions are made at one or more “site 3” residues of human leptin, which residues are shown in FIG. 10, and are, relative to the mature human wildtype protein, SEQ ID NO:1 , K33, Q34, K35, T37, S1 17, Y1 19, S120. The combined effect of amino acid modifications at site 3 residues reduce binding to the site 3 residues, e.g. by reducing from about 2-fold to about 500-fold relative to wild-type, from about 5-fold to about 100-fold, from about 5-fold to about 50-fold, from about 5-fold to about 10-fold. These amino acid modifications thus generate a partial agonist of lepr. In some embodiments, amino acid modifications, e.g. a substitution to an amino acid other than the wild-type; or an amino acid deletion, is provided at one, two, three, four, five or more residues selected from K33, Q34, K35, T37, S117, Y1 19, and S120.

[007] In some embodiments amino acid modifications are made at K33, including without limitation K33A, K33E, K33D, K33I, K33L, K33T, K33S, K33V. In some embodiments amino acid modifications are made at Q34, including without limitation Q34A, Q34E, Q34D, Q34I, Q34L, Q34T, Q34S, Q34V, Q34K, Q34R. In some embodiments amino acid modifications are made at K35; including without limitation K34A, K34E, K34D, K34I, K34L, K34T, K34S, K34V. In some embodiments amino acid modifications are made at T37, including without limitation T37A, T37R, T37N, T37D, T37Q, T37E, T37K, T37I, T37L, T37V. In some embodiments amino acid modifications are made at S1 17, including without limitation S117A, S1 17E, S117D, S1 17I, S1 17L, S117V, S117K, S117R, S1 17N, S117Q. In some embodiments amino acid modifications are made at Y119, including without limitation Y1 19A, Y119E, Y1 19D, Y119I, Y1 19L, Y119V, Y1 19K, Y119R, Y1 19N, Y1 19Q, Y1 19F, Y1 19W. In some embodiments amino acid modifications are made at S120, including without limitation S120A, S120R, S120N, S120D, S120Q, S120E, S120K, S120I, S120L, S120V. In some embodiments amino acid modifications are made at E122, including without limitation E122A, E122R, E122K, E122I, E122L, E122T, E122S, E122V. In some embodiments, modifications are made at each of K33, Q34, K35. In some embodiments modifications are made at S1 17; and may be made at each of K33, Q34, K35 and S1 17.

[008] In some embodiments, in addition to site 3 modifications, one or more amino acid modifications are made at one or more “site 2” residues, which residues are shown in FIG. 10, and are, relative to the human wild-type protein, SEQ ID NO:1 , K5, D9, L13, K15, T16, R20, N72, Q75, N82, D85, and L86. A consideration for the therapeutic LepR agonists is that the serum levels of endogenous leptin are highly elevated in the context of human obesity. It is therefore desirable to include in protein modifications one or more modifications that enhance affinity of binding to site 2 residues, e.g. by increasing from about 2-fold to about 100-fold relative to wildtype, from about 2-fold to about 10-fold, from about 2-fold to about 5-fold. In some embodiments, amino acid modifications, e.g. a substitution to an amino acid other than the wild-type; or an amino acid deletion, is provided at one, two, three, four, five or more residues selected from K5, D9, L13, K15, T16, R20, N72, Q75, N82, D85, and L86. In some embodiments an amino acid modification is made at D23, including without limitation D23L, which may be combined with modifications at each of K33, Q34, K35, at S1 17; or at each of K33, Q34, K35 and S117.

[009] The present disclosure provides for variant leptin proteins as disclosed above, pharmaceutical formulations comprising such proteins, and for methods of using such proteins. In particular, the present invention provides a pharmaceutical formulation comprising a therapeutically effective amount of a variant partial agonist leptin protein, alone or in combination with a pharmaceutically acceptable carrier. The formulation may be provided in a unit dose, comprising an effective dose of the protein.

[0010] In some embodiments, a formulation is provided, comprising an effective dose of a variant leptin partial agonist and a pharmaceutically acceptable excipient, where the therapeutically effective amount of the variant leptin partial agonist is in the range of from about 0.1 mg/kg to about 100 mg/kg. In some embodiments the effective dose is at least about 0.1 mg/kg, at least about 0.5 mg/kg, at least about 1 mg/kg, at least about 5 mg/kg, at least about 10 mg/kg, at least about 20 mg/kg, at least about 50 mg/kg, up to about 100 mg/kg, in some embodiments the effective dose is from about 1 to 25 mg/kg. Dosing may be daily, every 2 days, every 3 or more days, e.g. weekly, semi-weekly, bi-weekly, monthly, etc. Dosing may be parenteral, including sustained release formulations.

[0011] Further provided is a method for weight management, which may be associated with treating or delaying the progression or onset of diabetes and metabolic syndrome, especially type II diabetes, which include reducing complications of diabetes such as retinopathy, neuropathy, nephropathy and delayed wound healing, and related diseases such as insulin resistance (impaired glucose homeostasis), hyperglycemia, hyperinsulinemia, elevated blood levels of fatty acids or glycerol, hyperlipidemia including hypertriglyceridemia, Syndrome X, atherosclerosis and hypertension, wherein a therapeutically effective amount of a variant leptin partial agonist is administered to a mammalian, e.g., human, patient in need of treatment.

[0012] In some embodiments, a method is provided for treating obesity and related diseases as defined herein, wherein a therapeutically effective amount of a variant leptin partial agonist is administered to a mammalian, e.g., human, patient in need of treatment. In some such embodiments, the reduced food intake observed with administration the variant leptin partial agonist is associated with weight loss, e.g. loss of 1 % body weight, 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30% or more, depending on the initial weight of the subject.

[0013] These methods of treatment may be combined with one or more other types of therapeutic agent, such as an antidiabetic agent, a hypolipidemic agent or anti-obesity agent, e.g. metformin, sulfonylureas, e.g. glyburide, glipizide, glimepiride; glinides, e.g. repaglinide and nateglinide; thiazolidinediones, e.g. rosiglitazone, pioglitazone; DPP-4 inhibitors, e.g. sitagliptin, saxagliptin, linagliptin; GLP-1 receptor agonists, e.g. exenatide, liraglutide, semaglutide; SGLT2 inhibitors, e.g. canagliflozin, dapagliflozin, empagliflozin, etc. is administered to a human patient in need of treatment. The other therapeutic agents, when employed in combination with the compounds of the present invention may be used, for example, in those amounts indicated in the Physician's Desk Reference, as in the patents set out above or as otherwise determined by one of ordinary skill in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0015] FIGS. 1 A-1 D: Cryo-EM structure of the leptin receptor signaling complex. (A) Schematic depicting LepR ECD domain architectures, and the regions analyzed for structure determination using cryo-EM. (B) Cryo-EM 2-dimensional class averages of assembled leptin-bound LepR ^{D1 D7} and LepR ^{D3 D7} complexes. (C) Overlaid segmented density maps of the Leptin-LepR ^{D1 D7} and Leptin-LepR ^{D3 D7} complexes resolved to 5.9 A and 4.5 A resolution, respectively. (D) Three views of the leptin-LepR ^{D1 D7} structural model, with leptin in salmon and LepR in purple.

[0016] FIGS. 2A-2E: Structural homology between leptin and IL-6 family cytokine receptor complexes. (A) Side and top views of leptin-bound LepR structural model (PDB ID: 8DH9), showing the leptin-binding domains (D3-D5) of LepR, with leptin in salmon and LepR in purple. (B) Side and top views of the hexameric IL-6 receptor complex (PDB ID: 1 P9M) showing IL-6 in pink, gp130 in green, and IL6Rct in gray. (C) Side and top views of the IL-27 receptor complex (PDB ID: 7U7N), showing IL-27 subunits p28 and Ebi3 in yellow and gray, respectively, gp130 in green, and IL-27Ra in blue. (D) Schematic showing how LepR mutants L503S/L504S (LepR- 2KO) and L370S (LepR-3KO) assemble to exclusively form an asymmetric 1 :2 leptin-LepR complex, in the same conformation as the partially open 2:2 complex observed in our structure. (E) Immunoblot of lysates prepared from HEK-293T cells transiently expressing the indicated LepR constructs and stimulated with 10 nM recombinant leptin for 20 min.

[0017] FIGS. 3A-3K: Structural basis for leptin dependent LepR dimerization. (A) Front view of the segmented density map of the leptin-LepR ^{D3 D7} complex resolved to 4.5 A resolution (transparent) with the focus refined map encompassing leptin and the leptin-binding domains of LepR, resolved to 3.8 A resolution (solid). (B, C) Close-up views of the leptin-LepR site 2 binding interface. Hydrogen bonds and salt-bridges are shown as black dashed-lines. (D) Immunoblot of lysates prepared from HEK-293T cells transiently expressing the indicated LepR constructs and stimulated with the indicated concentration of recombinant leptin for 20 min. (E) Top view of the segmented density maps shown in (A). (F) Close-up view of the leptin-LepR site 3a binding interface. (G) Immunoblot of lysates prepared and analyzed as in (C). (H) Comparison of the apoleptin structure (PDB ID: 1 AX8) and LepR-bound leptin structure (this paper, PDB ID: 8DHA), showing leptin in salmon, LepR D3 in purple. (I) Close-up view of the leptin-LepR site 3b binding interface. (J) LepR-dependent ordering of the leptin AB loop residues 24-39, shown in green. (K) Immunoblot of lysates prepared from HEK-293T cells stably expressing wild-type LepR and stimulated with the indicated leptin variants for 20 min.

[0018] FIGS. 4A-4I : Biased leptin analogs decouple activation of STAT3 from LepR negative regulators. (A) Cartoon model of LepR signaling in which STAT3 recruited by phosphorylation of LepR-Y1141 , whereas LepR negative regulators are recruited by phosphorylation of LepR-Y986. (B) Phospho-STAT3 dose-response curves for WT or mutant leptin in LepR-expressing HEK 293T cells, analyzed by flow cytometry and shown as a percent of maximal WT leptin mean fluorescent intensity (MFI; mean ± SD, n>2). (C) Immunoblot of lysates prepared from HEK-293T cells stably expressing wild-type LepR that were serum starved for 18 hours and then stimulated with the indicated leptin variants for 20 min. (D) Quantification of immunoblots prepared as in (C) (100 nM leptin, mean ± SEM, n=4, two-sided t-test). (E) Schematic showing engineering strategy to create high affinity partial LepR agonists, through increasing affinity of leptin for LepR at site 2 and decreasing affinity at site 3. (F) Phospho-STAT3 signaling in LepR-expressing HEK 293T cells stimulated with 10 nM WT Leptin and the indicated concentration of leptin variants, analyzed as in (B) (mean ± SEM, n=6). (G) Relative expression of SOCS3 from LepR-expressing HEK 293T cells stimulated with 10 nM WT Leptin and 100 nM of the indicated leptin variants for 6 hours, analyzed by RT-qPCR. (mean ± SEM, n=six independent replicates analyzed in triplicate, two-sided t-test). (H) Immunoblot of lysates prepared from HEK-293T cells stably expressing wild-type LepR that were serum starved for 18 hours and then stimulated with 10 nM WT leptin and the indicated concentration of the indicated leptin variants for 20 min. (I) Quantification of immunoblots prepared as in (H) (100 nM leptin variants, mean ±SEM, n=4, students two-sided t- test).

[0019] FIGS. 5A-5D: Assembly and purification of the mouse leptin receptor complex, (a-d) Sizeexclusion chromatography profiles and corresponding SDS-PAGE gels of the assembled Leptin- LepRD1-D7 complex (a), crosslinked Leptin-LepRD1-D7 complex (b), Leptin-LepRD3-D7 complex (c), and crosslinked Leptin-LepRD3-D7 complex (d). Green bars indicate the Leptin- LepR complex, orange bars indicate free Leptin, and red box indicates fractions that were collected for further processing and structural characterization.

[0020] FIGS. 6A-6F: Leptin-LepRD1 -D7 complex cryo-EM data processing, (a) Representative cryo- EM micrograph of the leptin-LepRD1-D7 complex, (b) Reference free 2D class averages of the Leptin-LepRD1 -D7 complex, (c) Workflow for processing Leptin-LepRD1 -D7 complex cryo-EM dataset, (d) Local resolution of the Leptin-LepRD1 -D7 complex cryo-EM density map, ranging from 4.0 A to 7.5 A resolution, (e) Euler angle orientation distribution for Leptin-LepRDI - D7 complex cryo-EM dataset, (f) Half-set gold-standard FSC and map vs model FSC curves for Leptin-LepR ^{D1 D7} complex.

[0021] FIGS. 7A-7I: Leptin-LepRD3-D7 complex cryo-EM data processing, (a) Representative cryo- EM micrograph of the Leptin-LepRD3-D7 complex, (b) 2D class averages of the Leptin- LepRD3-D7 complex, (c) Workflow for processing Leptin-LepRD3-D7 complex cryo-EM dataset, (d) Local resolution of the Leptin- LepRD3-D7 complex cryo-EM density map, ranging from 2.0 A to 6.0 A resolution, (e) Euler angle orientation distribution for Leptin-LepRD3-D7 complex cryo- EM dataset, (f) Half-set gold-standard FSC and map vs model FSC curves for Leptin-LepRD3- D7 complex, (g) Local resolution cryo-EM density map of the focus refined region of the Leptin- LepRD3-D7 complex, ranging from 2.0 A to 4.5 A resolution, (h) Euler angle orientation distribution for the focus refined region of Leptin-LepRD3-D7 complex cryo-EM dataset. (I) Halfset gold-standard FSC and map vs model FSC curves for the focus refined region of Leptin- LepRD3-D7 complex.

[0022] FIGS. 8A-8D: Analysis of cryo-EM map quality, (a) Molecular model of the leptin-LepRDI - D7 complex fit into its 5.9 A resolution cryo-EM density map. (b) Sample cryo-EM density map and fitted molecular model for key regions of the 4.5 A resolution cryo-EM density map of the leptin-LepRD3-D7 complex. (c,d) Sample cryo-EM density map and fitted molecular model for key regions at the focus refine d 3.8 A resolution map of the leptin-LepR interface, showing that secondary structure and bulky side chain s can be resolved at the current resolution.

[0023] FIGS. 9A-9C: Analysis of “open” LepR complex, (a) Quantification of immunoblot in Figure 2e. (b) Top and side views for the Leptin-LepRD3-D7 complex reported here, highlighting the asymmetric, “open” conformation of the one LepR D3 domain, and absence of steric hindrance at the membrane proximal D7 domains, (c) Top and side view of Leptin-LepR model in which the second LepR D3 domain is closed to form a site 3 contact with the second bound Leptin, analogous the symmetric receptor complex observed in the IL-6 receptor complex structure. This rotation results in a significant steric clash between the membrane proximal D7 domains of the two LepR chains.

[0024] FIGS. 10A-10B: Conservation of the Leptin-LepR binding interface, (a) Sequence alignment of mouse (SEQ ID NO:2) and human (SEQ ID NO:1 ) Leptin with positions according to increasing sequence identity. Residues involved in LepR binding at site 2 and site 3 are indicated, (b) Sequence alignment of the Leptin binding domains of mouse and human LepR (D3 (SEQ ID NO:3 and SEQ ID NO:6), D4 (SEQ ID NO:4 and SEQ ID NO:7), and D5 (SEQ ID NO:5 and SEQ ID NO:8) with positions colored according to increasing sequence identity. Residues involved in Leptin binding at site 2 and site 3 are indicated.

[0025] FIGS. 1 1 A-1 1 E: Functional Characterization of Leptin and LepR mutants, (a, b) Quantification of immunoblots in Figure 3d (a) and 3g (b). (C) Coomassie-stained SDS-PAGE gel of recombinant leptin variants, (c) Coomassie-stained SDS-PAGE gel of recombinant leptin variants, (d) Quantification of immunoblots from Figure 3k. (d) Quantification of immunoblots from Figure 3K. (e) Table of calculated EC50 and Emax values for all leptin mutants tested on mLepR expressing 293T cells.

[0026] FIGS. 12A-12E: Characterization of a biased mouse LepR analog, (a) Coomassie-stained SDS- PAGE gel of recombinant mouse serum albumin (MSA)-conjugated leptin variants, (b) Phospho-STAT3 dose- response curves for WT or mutant leptin in mouse LepR-expressing HEK 293T cells, analyzed as in (mean ± SEM, n=3 independent replicates). (C) Quantification of immunoblots of lysates prepared from HEK-293T cells stably expressing wild-type mouse LepR that were serum starved for 18 hours and then stimulated with 10 nM leptin (mean ± SEM, n=3, two-sided t-test, ***P<0.001 ). (d, E) Eight-week-old B6.Cg-Lepob/J mice were treated with either vehicle (PBS) or 80 pg of MSA-conjugated leptin variant D23L/S117A twice daily via I.P. injection for seven days (mean ± SEM, n=5 mice/group, two-way ANOVA, ***P<0.001 ).

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0027] Compositions and methods are provided for preventing or treating obesity and/or overweight; and managing body weight. The compositions comprise partial agonists of the leptin receptor (lepr), which partial agonists elicit sub-maximal signaling at saturating ligand concentrations; and bias the response to STAT3 signaling. Human leptin protein variants provided herein preferentially suppress phosphorylation of SHP2 and ERK relative to STAT3, resulting in a biased signal that selectively activates pathways associated with satiety, with decreased activation of pathways associated with leptin resistance. The variant proteins find use in the suppression of appetite, and can provide for therapeutic weight loss. [0028] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0029] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supercedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0031] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.

[0032] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0033] Leptin is a hormone that regulates food intake, body mass, and reproductive function. It is encoded by the obese (ob) gene, and expressed by fat cells in white adipose tissue. The leptin receptor is the homodimeric leptin receptor (LEP-R), which is structurally similar to the class I family of cytokine receptors. Leptin regulates appetite and metabolism by inhibiting the synthesis and release of neuropeptide Y (NPY) in the arcuate nucleus (ARC). Leptin can inhibit neural pathways activated by appetite stimulants (orexigenic) to reduce energy intake and activate pathways targeted by anorexigenic to suppress appetite. Among other activities, leptin affects the transcription of proopiomelanocortin POMC, whose a-MSH product is released into the synapse to activate neurons via binding to the melanocortin receptor (MGR) and leads to appetite-suppression. Reference sequences for human leptin can be accessed at Genbank, e.g. NM_000230.

[0034] The mature human leptin protein has the reference sequence (SEQ ID NO:1 ) VPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAV YQQILTS MPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALS RLQG SLQDMLWQLDLSPGC.

[0035] The mature mouse leptin protein has the reference sequence (SEQ ID NO:2) VPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAV YQQILTS MPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALS RLQG SLQDMLWQLDLSPGC.

[0036] The leptin-mediated dimerization of LepR results in the JAK2-dependent phosphorylation of multiple tyrosine residues on the intracellular domain (ICD) of LepR, each of which have distinct biological roles. Phosphorylation of Tyr1141 drives activation of STAT3 and the subsequent satiety promoting effects of leptin, whereas phosphorylation of Tyr986 activates the SHP2/ERK pathway and is required for recruitment of cytosolic LepR antagonists that drive leptin resistance.

[0037] Leptin is overexpressed at the gene level in the adipose tissue of individuals with obesity, and strong positive associations exist between plasma leptin levels and body fat percentage. Leptin resistance refers to states in which leptin fails to promote its anticipated effects, frequently coexisting with hyperleptinaemia. Leptin resistance is closely associated with obesity and also observed in physiological situations such as pregnancy and in seasonal animals. Leptin resensitisation refers to the reversion of leptin-resistant states and is associated with improvement in endocrine and metabolic disturbances commonly observed in obesity and a sustained decrease of plasma leptin levels, possibly below a critical threshold level. In obesity, leptin resensitisation can be achieved with treatments that reduce body adiposity and leptinaemia, or with some pharmacological compounds, while physiological leptin resistance reverts spontaneously. Although the mechanism of leptin resistance is not fully understood, substantial evidence suggests that it is driven by chronic hyperleptinemia and the resulting desensitization of LepR to leptin, due in large part to the expression and recruitment of cytosolic LepR antagonists such as Suppressor of Cytokine Signaling 3 (SOCS3). [0038] Sites at which leptin binds to its receptor are shown in Figure 10, including those marked as binding sites II and binding sites III. The lepr sites are consistent with the art, e.g. for example see Peelman et al. (2004) Protein Structure and Folding 279(39): 41038-41046, herein specifically incorporated by reference. Reference may be made herein to amino acid modifications in leptin at binding site III, or “site 3” residues, which comprise relative to the human wild-type protein, SEQ ID NO:1 , K33, Q34, K35, T37, S117, Y119, S120. In general these modifications act to reduce the affinity of leptin to lepr. Reference may be made to amino acid modifications at binding site II, or “site 2” residues, which comprise, relative to the human wildtype protein, SEQ ID NO:1 , K5, D9, L13, K15, T16, R20, N72, Q75, N82, D85, and L86. In general these modification increase the affinity of leptin to lepr.

Polypeptides

[0039] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

[0040] The term "sequence identity," as used herein in reference to polypeptide or DNA sequences, refers to the subunit sequence identity between two molecules. When a subunit position in both of the molecules is occupied by the same monomeric subunit (e.g., the same amino acid residue or nucleotide), then the molecules are identical at that position. The similarity between two amino acid or two nucleotide sequences is a direct function of the number of identical positions. In general, the sequences are aligned so that the highest order match is obtained. If necessary, identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990).

[0041] By "protein variant" or "variant protein" or "variant polypeptide" herein is meant a protein that differs from a wild-type protein by virtue of at least one amino acid modification. Variant polypeptide may refer to the polypeptide itself, a composition comprising the polypeptide, or the amino sequence that encodes it. Preferably, the variant polypeptide has at least one amino acid modification compared to the parent polypeptide, e.g. from about one to about ten amino acid modifications, and preferably from about one to about five amino acid modifications compared to the parent.

[0042] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma- carboxyglutamate, and O-phosphoserine. “Amino acid analogs” refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. “Amino acid mimetics” refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0043] As used herein, the terms “peptide residue” and “peptidic structure” are intended to include peptides comprised of naturally-occurring L-amino acids and the corresponding D-amino acids, as well as peptide derivatives, peptide analogues and peptidomimetics of the naturally- occurring L-amino acid, structures. Approaches to designing peptide analogues, derivatives and mimetics are known in the art. For example, see Veber and Freidinger 1985 T/NS p. 392; Evans, et al. 1987 J. Med. Chem. 30:229. Peptidomimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent or enhanced therapeutic or prophylactic effect, by methods known in the art and further described in the following references: Spatola, A. F. 1983 in: Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267; Holladay, et al. 1983 Tetrahedron Lett. 24:4401 -4404.

[0044] Systematic substitution of one or more amino acids of a consensus sequence with a D- amino acid of the same type (for example, D-lysine in place of L-lysine) may be used to generate more stable peptides. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo, et al. 1992 Ann. Rev. Biochem. 61 :387, incorporated herein by reference in their entireties); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide, adding cyclic lactam bridge, or the use of flexible 6- aminohexanoic acid (Ahx), rigid aminoisobutyric acid (Aib) or D-amino acid residues to alter the stability of the helix.

[0045] As used herein, a “derivative” of a compound, for example, a peptide or amino acid, refers to a form of that compound in which one or more reactive groups in the compound have been derivatized with a substituent group. Examples of peptide derivatives include peptides in which an amino acid side chain, the peptide backbone, or the amino- or carboxy-terminus has been derivatized (for example, peptidic compounds with methylated amide linkages or hydroxylated amino acids or amino acid residues).

[0046] As used herein, the term “amino acid structure” is intended to include the amino acid, as well as analogs, derivatives and mimetics of the amino acid that maintain the functional activity of the compound. For example, the term “phenylalanine structure” is intended to include phenylalanine as well as pyridylalanine and homophenylalanine. The term “leucine structure” is intended to include leucine, as well as substitution with valine, isoleucine or other natural or nonnatural amino acid having an aliphatic side chain, such as norleucine.

[0047] The amino- and/or carboxy-terminus of the peptide compounds disclosed herein can be standard amino and carboxy termini as seen in most proteins. Alternatively, the amino- and/or carboxy-terminus of the peptide compound can be chemically altered by the addition or replacement of a derivative group. Amino-derivative groups which can be present at the N- terminus of a peptide compound include acetyl, aryl, aralkyl, acyl, epoxysuccinyl and cholesteryl groups. Carboxy-derivative groups which can be present at the C-terminus of a peptide compound include alcohol, aldehyde, epoxysuccinate, acid halide, carbonyl, halomethane, diazomethane groups and carboxamide.

[0048] As used herein, "modified" refers to a polypeptide which retains the overall structure of a related polypeptide but which differs by at least one residue from that related polypeptide. As used herein a "modified C-terminus" is a C-terminus of a polypeptide that has a chemical structure other than a standard peptide carboxy group, an example of such a modified C-terminus being a C-terminal carboxamide.

[0049] As used herein, "pharmaceutically acceptable carrier" refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredients and which is not toxic to the host or patient.

[0050] As used herein, the terms "peptide residue" and "peptidic structure" are intended to include peptides comprised of naturally-occurring L-amino acids and the corresponding D-amino acids, as well as peptide derivatives, peptide analogues and peptidomimetics of the naturally- occurring L-amino acid structures. Approaches to designing peptide analogues, derivatives and mimetics are known in the art (see Farmer, P.S. in: Drug Design E.J. Ariens, ed. Academic Press, New York, 1980, vol. 10, pp. 1 19-143; Ball J.B. & Alewood, P.F. 1990 /. Mol. Recognition 3:55; Luthman, et al. 1996 A Textbook of Drug Design and Development, 14:386-406, 2nd Ed., Harwood Academic Publishers; Joachim Grante, Angew. 1994 Chem. Int. Ed. Engl. 33: 1699- 1720). Peptidomimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent or enhanced therapeutic or prophylactic effect, by methods known in the art (see Spatola, A.F. 1983 in: Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267; Spatola, A.F. 1983 Vega Data, Vol. 1 , Issue 3, Peptide Backbone Modifications (general review); Jennings-White, et al. 1982 Tetrahedron Lett. 23:2533; Holladay, et al. 1983 Tetrahedron Lett. 24:4401 -4404; and Hruby, 1982 Life Sci. 31 : 189-199).

[0051] In addition, constrained peptides may be generated by methods known in the art (Rizo, et al. 1992 Ann. Rev. Biochem. 61 :387); for example, by adding internal cysteine residues or organic linkers capable of forming intramolecular bridges which cyclize the peptide, adding cyclic lactam bridge, or the use of flexible 6-aminohexanoic acid (Ahx), rigid aminoisobutyric acid (Aib) or D-amino acid residues to alter the stability of the helix.

[0052] Synthetic or non-naturally occurring amino acids refer to amino acids which do not naturally occur in vivo but which, nevertheless, can be incorporated into the peptide structures described herein.

[0053] Optionally, polypeptides of the disclosure are modified by covalent linkage to a heterologous moiety, which moiety may comprise a polymer, an Fc, an FcRn binding ligand, immunoglobulin, albumin, a collagen-binding motif, etc. A covalently linked polymer may be selected from the group consisting of a polyethyleneglycol (PEG) moiety; a polypropylenglycol (PPG) moiety; a PAS moiety; which is an amino acid sequence comprising mainly alanine and serine residues or comprising mainly alanine, serine, and proline residues, the amino acid sequence forming random coil conformation under physiological conditions [US No. 2010/0292130 and WO 2008/155134]; and a hydroxyethylstarch (HES) moiety [WO 02/080979]; an Fc immunonoglobulin sequence; a FcRn binding ligand; albumin and an albumin-binding ligand as well as an XTEN moiety (see Schellenberger, et al., 2009, Nature Biotechnology 27(12): 1186-1192).

[0054] An "Fc region" can be a naturally occurring or synthetic polypeptide that is homologous to an IgG C-terminal domain produced by digestion of IgG with papain. IgG Fc has a molecular weight of approximately 50 kDa. The leptin polypeptide may be fused to the entire Fc region, or a smaller portion that retains the ability to extend the circulating half- life of a chimeric polypeptide of which it is a part. In addition, full-length or fragmented Fc regions can be variants of the wildtype molecule. That is, they can contain mutations that may or may not affect the function of the polypeptides; as described further below, native activity is not necessary or desired in all cases.

[0055] In other embodiments, the leptin polypeptide can comprise a polypeptide that functions as an antigenic tag, such as a FLAG sequence. FLAG sequences are recognized by biotinylated, highly specific, anti-FLAG antibodies, as described herein (see also Blanar et al., Science 256: 1014, 1992; LeClair et al., Proc. Natl. Acad. Sci. USA 89:8145, 1992). In some embodiments, the chimeric polypeptide further comprises a C-terminal c-myc epitope tag.

[0056] Where the covalent linkage is to PEG, the PEG molecular weight may be between about 1 kDa and about 100 kDa for ease in handling and manufacturing. For example, the PEG may have an average molecular weight of about 200, 500, 1000, 2000, 4000, 8000, 16,000, 32,000, 64,000, or 100,000 kDa. In some embodiments, the PEG may have a branched structure (U.S. Pat. No. 5,643,575; Morpurgo et al. AppL Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al, Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al, Bioconjug. Chem. 10:638-646 (1999)). [0057] Optionally, modified polypeptide derivatives comprise one or more substitutions of disulfide bonds with lactam bridges to increase the metabolic stability of the peptides. Cystathiones are resistant towards thiol reduction. Therefore, substitutions of disulfides with thioethers, or selenosulfide, diselenide and ditelluride bridges can provide protection against reduction [Knerr et al., ACS Chem Biol , 6(7), 753-760, 2011 ; Muttenthaler et al. J Med Chem., 53(24), 8585-8596, 2010]. Peptide disulfide bond mimics based on diaminodiacids can also be used to improve the stability of analogs (Cui et al., Angew Chem, 125, 9737-9741 , 2013). The disulfide bridge can also be modified either by the insertion of linkers or bridges of a different nature.

[0058] Optionally, polypeptides are modified by the addition of one or more alkane, cholesterol, or PEG-cholesterol moieties to increase the metabolic stability of the peptides. Stapled peptides, via the introduction of a synthetic brace (staple), can be synthesized using ring-closing metathesis to lock a peptide in a specific conformation and reduce conformational entropy.

[0059] 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, i.e. will differ by at least one amino acid, and may differ by at least two but not more than about ten amino acids. The sequence changes may be substitutions, insertions or deletions, including truncation at the corboxy 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: (glycine, alanine); (valine, isoleucine, leucine); (aspartic acid, glutamic acid); (asparagine, glutamine); (serine, threonine); (lysine, arginine); or (phenylalanine, tyrosine).

[0060] Modifications of interest that do not alter primary sequence include chemical derivatization of polypeptides, e.g., acetylation, amidation, acylation, or carboxylation. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences that have phosphorylated amino acid residues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine.

[0061] Also included in the subject invention are polypeptides that have been modified using ordinary molecular biological techniques and synthetic chemistry so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. For examples, the backbone of the peptide may be cyclized to enhance stability (see Friedler et al. (2000) J. Biol. Chem. 275:23783-23789). Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids. [0062] Those skilled in the art of peptide chemistry are aware that amino acid residues occur as both D and L isomers, and that the instant invention contemplates the use of either or a mixture of isomers for amino acid residues incorporated in the synthesis of the peptides described herein.

[0063] The present invention includes within its scope pharmaceutical compositions comprising, as an active ingredient, a therapeutically effective amount of at least one lepr partial agonist, alone or in combination with a pharmaceutical carrier or diluent. Optionally, polypeptides of the present invention can be used alone, in combination with other compounds of the invention, or in combination with one or more other therapeutic agent(s), e.g., an antidiabetic agent or other pharmaceutically active material.

[0064] If desired, various groups may be introduced during synthesis or during expression, which allow for linking to other molecules or to a surface. Thus cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.

[0065] 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). These procedures can also be used to synthesize peptides in which amino acids other than the 20 naturally occurring, genetically encoded amino acids are substituted at one, two, or more positions of any of the modified peptides as disclosed herein. For instance, naphthylalanine can be substituted for tryptophan, facilitating synthesis. Other synthetic amino acids that can be substituted into the peptides of the present embodiments include L-hydroxypropyl, L-3, 4-dihydroxy-phenylalanyl, d amino acids such as L-d-hydroxylysyl and D-d-methylalanyl, L-a-methylalanyl, |3-amino acids, and isoquinolyl. D amino acids and non- naturally occurring synthetic amino acids can also be incorporated into the peptides of the present embodiments (see Roberts, et al. 1983 Unusual Amino/ Acids in Peptide Synthesis 5:341 -449). In some embodiments, the naturally occurring side chains of the 20 genetically encoded amino acids, or any other side chain as disclosed herein can be transposed to the nitrogen of the amino acid, instead of the a-carbon as typically found in peptides.

[0066] The peptides can be synthesized in a stepwise manner on an insoluble polymer support (also referred to as "resin") starting from the C-terminus of the peptide. A synthesis is begun by appending the C-terminal amino acid of the peptide to the resin through formation of an amide or ester linkage. This allows the eventual release of the resulting peptide as a C-terminal amide or carboxylic acid, respectively. Alternatively, in cases where a C-terminal amino alcohol is present, the C-terminal residue may be attached to 2-Methoxy-4-alkoxybenzyl alcohol resin (SASRIN.TM., Bachem Bioscience, Inc., King of Prussia, Pa.) as described herein and, after completion of the peptide sequence assembly, the resulting peptide alcohol is released with LiBH.sub.4 in THF (see J. M. Stewart and J. D. Young, supra, p. 92). [0067] The syntheses of the peptides described herein can be carried out by using a peptide synthesizer, such as an Advanced Chemtech Multiple Peptide Synthesizer (MPS396) or an Applied Biosystems Inc. peptide synthesizer (ABI 433A). If the MPS396 was used, up to 96 peptides were simultaneously synthesized. If the ABI 433A synthesizer was used, individual peptides were synthesized sequentially. In both cases the stepwise solid phase peptide synthesis was carried out utilizing an Fmoc/t-butyl protection strategy.

[0068] Peptides with the desired purity can be obtained by purification using preparative HPLC, for example, on a Waters Model 4000 or a Shimadzu Model LC-8A liquid chromatograph. The solution of crude peptide is injected into a YMC S5 ODS column and eluted with a linear gradient of MeCN in water, both buffered with 0.1 % TFA, using a flow rate of 14-20 mL/min with effluent monitoring by UV absorbance at 220 nm. The structures of the purified peptides can be confirmed by electro-spray MS analysis.

[0069] The polypeptides may also be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. For the most part, the compositions which are used will be substantially pure, e.g. the peptide of interest will comprise at least 20% by weight of the desired product, more usually at least about 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes, usually at least about 99.5% by weight, or more, in relation to contaminants related to the method of preparation of the product and its purification. The percentages may be based upon total protein.

[0001] 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).

[0002] It will be understood that there are medically accepted definitions of obesity and overweight. A patient may be identified by, for example, measuring body mass index (BMI), which is calculated by dividing weight in kilograms by height in meters squared, and comparing the result with the definitions. The recommended classifications for BMI in humans, adopted by the Expert Panel on the Identification, Evaluation and T reatment of Overweight and Obesity in Adults, and endorsed by leading organizations of health professionals, are as follows: underweight <18.5 kg/m ² , normal weight 18.5-24.9 kg/m ² , overweight 25-29.9 kg/m ² , obesity (class 1 ) 30-34.9 kg/m ² , obesity (class 2) 35-39.9 kg/m ² , extreme obesity (class 3) >40 kg/m ² (Practical Guide to the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults, The North American Association for the Study of Obesity (NAASO) and the National Heart, Lung and Blood Institute (NHLBI) 2000). Modifications of this classification may be used for specific ethnic groups. [0003] Another alternative for assessing overweight and obesity is by measuring waist circumference. There are several proposed classifications and differences in the cutoffs based on ethnic group. For instance, according to the classification from the International Diabetes Federation, men having waist circumferences above 94 cm and women having waist circumferences above 80 cm are at higher risk of diabetes, dyslipidemia, hypertension and cardiovascular diseases because of excess abdominal fat. Another classification is based on the recommendation from the Adult Treatment Panel III where the recommended cut-offs are 102 cm for men and 88 cm for women. However, the methods, combinations and compositions of the invention may also be used for reduction of self-diagnosed overweight and for decreasing the risk of becoming obese due to life style, genetic considerations, heredity and/or other factors.

[0070] The term "obesity-related condition" refers to any disease or condition that is caused by or associated with (e.g., by biochemical or molecular association) obesity or that is caused by or associated with weight gain and/or related biological processes that precede clinical obesity. Examples of obesity-related conditions include, but are not limited to, type 2 diabetes, metabolic syndrome, fatty liver disease such as NASH, hyperglycemia, hyperinsulinemia, impaired glucose tolerance, impaired fasting glucose, hyperlipidemia, hypertriglyceridemia, insulin resistance, hypercholesterolemia, atherosclerosis, coronary artery disease, peripheral vascular disease, and hypertension.

[0071] Syndromes with associated obesity include, without limitation, 5p13 microduplication syndrome; 16p1 1 .2 deletion; Albright hereditary osteodystrophy/PHP Type 1 a; Alstrom syndrome; Bardet Biedel syndrome (BBS); Borjeson-Forssman-Lehmann Syndrome; Carpenter syndrome; CHOPS syndrome; Chudley-Lowry syndrome; Cohen syndrome; Kabuki syndrome/Niikawa-Kuroki syndrome; Kleefstra syndrome; MORM syndrome; Prader-Willi Syndrome; Rubinstein-Taybi syndrome; Shashi-X-linked mental retardation; Smith Magenis Syndrome; WAGRO syndrome; OBHD; Ulnary Mammary syndrome; Bannayan-Riley-Ruvalcaba syndrome; Beckwith-Weidemann syndrome; Klippel-Trenaunay-Weber syndrome; Parkes Weber syndrome; Proteus syndrome; Silver-Russell syndrome; Simpson-Golabi-Behmel syndrome; Sotos syndrome; Weaver syndrome; Camera-Marugo-Cohen Syndrome; Clark- Baraitser Syndrome; MEHMO syndrome; MOMES syndrome; MOMO syndrome; Morgagni- Stewart-Morel Syndrome; 1 p36 deletion syndrome; and 2p25.3 deletion syndrome. See, for example, Thaker (2017) Adolesc Med State Art Rev. 28(2): 379-405, herein specifically incorporated by reference.

[0072] Diabetes is a metabolic disease that occurs when the pancreas does not produce enough of the hormone insulin to regulate blood sugar (“type 1 diabetes mellitus”) or, alternatively, when the body cannot effectively use the insulin it produces (“type 2 diabetes mellitus”).

[0073] According to recent estimates by the World Health Organization, more than 200 million people worldwide have diabetes, whereby 90% suffer from type 2 diabetes mellitus. Typical long term complications include development of neuropathy, retinopathy, nephropathy, generalized degenerative changes in large and small blood vessels and increased susceptibility to infection. Since individuals with type 2 diabetes still have a residual amount of insulin available in contrast to type 1 diabetic individuals, who completely lack the production of insulin, type 2 diabetes only surfaces gradually and is often diagnosed several years after onset, once complications have already arisen.

[0074] Insulin resistance occurs in 25% of non-diabetic, non-obese, apparently healthy individuals, and predisposes them to both diabetes and coronary artery disease. Hyperglycemia in type II diabetes is the result of both resistance to insulin in muscle and other key insulin target tissues, and decreased beta cell insulin secretion. Longitudinal studies of individuals with a strong family history of diabetes indicate that the insulin resistance precedes the secretory abnormalities. Prior to developing diabetes these individuals compensate for their insulin resistance by secreting extra insulin. Diabetes results when the compensatory hyperinsulinemia fails. The secretory deficiency of pancreatic beta cells then plays a major role in the severity of the diabetes.

[0075] Type II diabetes mellitus as diagnosed according to criteria published in the Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus whereby fasting plasma glucose level is greater than or equal to 126 milligrams per deciliter, and latent autoimmune diabetes mellitus of adults. It is characterized by insulin resistance and hyperglycemia, which in turn can cause retinopathy, nephropathy, neuropathy, or other morbidities. Additionally, diabetes is a well-known risk factor for atherosclerotic cardiovascular disease. Metabolic syndrome refers to a group of factors, including hypertension, obesity, hyperlipidemia, and insulin resistance (manifesting as frank diabetes or high fasting blood glucose or impaired glucose tolerance), that raises the risk of developing heart disease, diabetes, or other health problems; (Grundy et al, Circulation. 2004; 109:433-438).

[0076] There is a well-characterized progression from normal metabolic status to a state of impaired fasting glucose (IFG: fasting glucose levels greater than 100 mg/dL) or to a state of impaired glucose tolerance (IGT: two-hour glucose levels of 140 to 199 mg/dL after a 75 gram oral glucose challenge). Both IFG and IGT are considered pre-diabetic states, with over 50% of subjects with IFG progressing to frank type II diabetes within, on average, three years (Nichols, Diabetes Care 2007. (2): 228-233).

[0077] The term "metabolic syndrome" refers to metabolic disorders, particularly glucose and lipid regulatory disorders, including insulin resistance and defective secretion of insulin by pancreatic beta cells, and may further include conditions and states such as abdominal obesity, dyslipidemia, hypertension, glucose intolerance or a prothrombotic state, and which may further result in disorders such as hyperlipidemia, obesity, diabetes, insulin resistance, glucose intolerance, hyperglycemia, and hypertension.

[0004] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal being assessed for treatment and/or being treated. In some embodiments, the mammal is a human. The terms “subject,” “individual,” and “patient” encompass, without limitation, individuals having a disease or a condition such as obesity. Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g., mice, rats, etc.

[0005] The term “sample” with reference to a patient encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The term also encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations, such as diseased cells. The definition also includes samples that have been enriched for particular types of molecules, e.g., nucleic acids, polypeptides, etc. The term “biological sample” encompasses a clinical sample, and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and the like. A “biological sample” includes a sample obtained from a patient’s diseased cell, e.g., a sample comprising polynucleotides and/or polypeptides that is obtained from a patient’s diseased cell (e.g., a cell lysate or other cell extract comprising polynucleotides and/or polypeptides); and a sample comprising diseased cells from a patient. A biological sample comprising a diseased cell from a patient can also include non-diseased cells.

[0006] The term “diagnosis” is used herein to refer to the identification of a molecular or pathological state, disease or condition in a subject, individual, or patient.

[0007] As used herein, the terms “treatment,” “treating,” and the like, refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect on or in a subject, individual, or patient. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of effecting a partial or complete cure for a disease and/or symptoms of the disease. “Treatment,” as used herein, may include treatment of obesity in a mammal, particularly in a human, and includes achievement of weight loss, or prevention of weight gain.

[0008] As used herein, a "therapeutically effective amount" refers to that amount of the therapeutic agent sufficient to treat or manage a disease or disorder. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease. Further, a therapeutically effective amount with respect to a therapeutic agent of the invention means the amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease.

Leptin Variants

[0078] In some embodiments a partial agonist of lepr is a variant of leptin protein, comprising one or more amino acid modifications, e.g. amino acid substitutions, that alter the binding to the receptor. Partial agonists may elicit a maximal STAT3 response of from about 25% to about 75% relative to the wild-type ligand, e.g. wild-type leptin. A partial agonist of interest induces STAT3- biased signaling, where maximal phosphorylation of phosphatase SHP2, ERK, or STAT1 is less than about 25% of the phosphorylation obtained with the wild-type ligand, less than about 10%, less than about 5%, less than about 1%.

[0079] The modifications may comprise amino acid substitutions in site 3 residues that decrease affinity. Site 3 binding residues comprise, relative to the human wild-type protein, SEQ ID NO:1 , K33, Q34, K35, T37, S117, Y119, S120. The combined effect of amino acid modifications at site 3 residues reduce binding to the site 3 residues, e.g. by reducing from about 2-fold to about 500- fold relative to wild-type, from about 5-fold to about 100-fold, from about 5-fold to about 50-fold, from about 5-fold to about 10-fold. These amino acid modifications thus generate a partial agonist of lepr. In some embodiments, amino acid modifications, e.g. a substitution to an amino acid other than the wild-type; or an amino acid deletion, is provided at one, two, three, four, five or more residues selected from K33, Q34, K35, T37, S117, Y1 19, S120.

[0080] In some embodiments amino acid modifications are made at K33, including without limitation K33A, K33E, K33D, K33I, K33L, K33T, K33S, K33V. In some embodiments amino acid modifications are made at Q34, including without limitation Q34A, Q34E, Q34D, Q34I, Q34L, Q34T, Q34S, Q34V, Q34K, Q34R. In some embodiments amino acid modifications are made at K35; including without limitation K34A, K34E, K34D, K34I, K34L, K34T, K34S, K34V. In some embodiments amino acid modifications are made at T37, including without limitation T37A, T37R, T37N, T37D, T37Q, T37E, T37K, T37I, T37L, T37V. In some embodiments amino acid modifications are made at S1 17, including without limitation S117A, S1 17E, S117D, S1 17I, S1 17L, S117V, S117K, S117R, S1 17N, S117Q. In some embodiments amino acid modifications are made at Y119, including without limitation Y1 19A, Y119E, Y1 19D, Y119I, Y1 19L, Y119V, Y1 19K, Y119R, Y1 19N, Y1 19Q, Y1 19F, Y1 19W. In some embodiments amino acid modifications are made at S120, including without limitation S120A, S120R, S120N, S120D, S120Q, S120E, S120K, S120I, S120L, S120V. In some embodiments amino acid modifications are made at E122, including without limitation E122A, E122R, E122K, E122I, E122L, E122T, E122S, E122V.

[0081] In some embodiments, in addition to site 3 modifications, one or more amino acid modifications are made at one or more “site 2” residues, which residues are shown in FIG. 9, and are, relative to the human wild-type protein, SEQ ID NO:1 , K5, D9, L13, K15, T16, R20, N72, Q75, N82, D85, and L86. A consideration for the therapeutic LepR agonists is that the serum levels of endogenous leptin are highly elevated in the context of human obesity. It is therefore desirable to include in protein modifications one or more modifications that enhance affinity of binding to site 2 residues, e.g. by increasing from about 2-fold to about 100-fold relative to wildtype, from about 2-fold to about 10-fold, from about 2-fold to about 5-fold. In some embodiments, amino acid modifications, e.g. a substitution to an amino acid other than the wild-type; or an amino acid deletion, is provided at one, two, three, four, five or more residues selected from K5, D9, L13, K15, T16, R20, N72, Q75, N82, D85, and L86. In some embodiments an amino acid modification is made at D23, including without limitation D23L.

[0082] Selection of amino acid modifications at binding site 2 and binding site 3 may utilize methods of mutagenesis, surface display and selection. For example and without limitation, yeast surface display can be used to select for desired changes at the specified residues, by generating a library of polypeptides comprising amino acid substitutions at targeted residues, displaying the library of polypeptides on the surface of yeast cells, and selecting for desired binding characteristics. Labeling with soluble ligands enables rapid and quantitative analysis of yeast- displayed libraries by flow cytometry, while cell-surface selections allow screening of libraries with insoluble or even as-yet-uncharacterized binding targets. Affinity and specificity are key parameters governing a protein's function as a diagnostic or therapeutic agent, and yeast surface display can be applied for improving or altering these binding properties. For example see Gee et al., 2018, Cell 172, 549-563; Bozovicar et al. Int. J. Mol. Sci. 2020, 21 , 215; doi:10.3390/ijms21010215; Sibener et al. Cell 2018 Jul 26;174(3):672-687.e27. doi: 10.1016/j. cell.2018.06.017; Mendoza et al. (2017) Immunity. 46(3):379-392; each herein specifically incorporated by reference.

Methods of Treatment

[0083] Methods are provided for treating obesity and related diseases as defined herein, wherein a therapeutically effective amount of a variant leptin polypeptide as disclosed herein, e.g. a lepr partial agonist, is administered to a mammalian, e.g., human, patient in need of treatment. In some such embodiments, the reduced food intake observed with administration of a lepr partial agonist is associated with weight loss, e.g. loss of 1 % body weight, 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30% or more, depending on the initial weight of the subject.

[0084] Methods are also provided for treating or delaying the progression or onset of diabetes and metabolic syndrome, especially type II diabetes, including complications of diabetes, including retinopathy, neuropathy, nephropathy and delayed wound healing, and related diseases such as insulin resistance (impaired glucose homeostasis), hyperglycemia, hyperinsulinemia, elevated blood levels of fatty acids or glycerol, obesity, hyperlipidemia including hypertriglyceridemia, Syndrome X, atherosclerosis and hypertension, and for increasing high density lipoprotein levels, wherein a therapeutically effective amount of a variant leptin polypeptide as disclosed herein, e.g. a lepr partial agonist is administered to a mammalian, e.g., human, patient in need of treatment, for a period of time sufficient to effect treatment.

[0085] These methods of treatment are optionally combined with one or more other types of therapeutic agent, such as an antidiabetic agent, a hypolipidemic agent or anti-obesity agent, e.g. metformin, sulfonylureas, e.g. glyburide, glipizide, glimepiride; glinides, e.g. repaglinide and nateglinide; thiazolidinediones, e.g. rosiglitazone, pioglitazone; DPP-4 inhibitors, e.g. sitagliptin, saxagliptin, linagliptin; GLP-1 receptor agonists, e.g. exenatide, liraglutide, semaglutide; SGLT2 inhibitors, e.g. canagliflozin, dapagliflozin, empagliflozin, etc. is administered to a human patient in need of treatment. The other therapeutic agents, when employed in combination with the compounds of the present invention may be used, for example, in those amounts indicated in the Physician's Desk Reference, as in the patents set out above or as otherwise determined by one of ordinary skill in the art.

[0009] A lepr partial agonist, e.g. a variant leptin polypeptide as disclosed herein, is effective to induce at least "minimal weight loss" when the compound induces, over a period of time from 12 to 52 weeks, a statistically significant and placebo-adjusted decrease in mean body weight of at least about 2.5%, but less than about 5.0%, in a cohort of subjects with a baseline mean BMI > 27 kg/m ² .

[0010] A lepr partial agonist, e.g. a variant leptin polypeptide as disclosed herein is effective in "treatment of obesity" or "to induce weight loss" when the composition induces, over a period of time from 12 to 52 weeks, a statistically significant and placebo-adjusted decrease in body weight of at least about 5.0% in a cohort of subjects with a baseline mean BMI>27 kg/m ² .

[0011] An effective dose of a variant leptin may vary according to gender and body weight, and may be comparable to the dosing for metreleptin. For example, an initial dose may be from about 0.01 mg/kg/day, about 0.025 mg/kg/day, about 0.05 mg/kg/day, about 0.1 mg/kg/day, and the dose may be up to about 500 mg/kg/day, up to about 250 mg/kg/day, up to about 100 mg/kg/day, up to about 75 mg/kg/day, up to about 50 mg/kg/day, up to about 25 mg/kg/day, up to about 10 mg/kg/day. Administration may be, for example daily, or extended release formulations can be utilized to provide the same effective daily dose, by released over a period of about 1 week, 2 weeks, 3 weeks, one month, etc.

[0012] Efficacy of treatment for obesity can be readily determined by weight loss, for example the reduced food intake observed with administration of a lepr partial agonist is associated with weight loss, e.g. loss of 1 % body weight, 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30% or more, depending on the initial weight of the subject. [0013] Along with weight loss, there can be an improvement in insulin sensitivity. Insulin sensitivity can be monitored with various methods known to the art, to determine an improvement in insulin sensitivity (or decrease in insulin resistance), where an improvements may be, for example, 5%, 20%, 15%, 20%, 25%, 30%, 40%, 50% or more improvement. Hyperinsulinemic euglycemic clamp (HEC) is known to be the “gold standard” for the measurement of insulin sensitivity. However simplified assays can be used in quantification of insulin sensitivity. There are two major groups of insulin sensitivity indices: (1 ) Indices calculated by using fasting plasma concentrations of insulin, glucose and triglycerides, (2) indices calculated by using plasma concentrations of insulin and glucose obtained during 120 min of a standard (75 g glucose) OGTT. The former group include homeostasis model assessment-insulin resistance (HOMA-IR), QUIKI INDEX, and McAuley index while latter include, Matsuda, Belfiore, Cederholm, Avignon and Stumvoll index. For clinical uses HOMA-IR, QUIKI, and Matsuda are suitable while HES, McAuley, Belfiore, Cederholm, Avignon and Stumvoll index are suitable for epidemiological/research purposes.

[0014] The HEC-derived index of insulin sensitivity (ISIHEC, ml/kg/min/plU ml) is obtained during a steady state period of HEC. ISIHEC = MCR/I _me an where, l _me an - average steady state plasma insulin response (plU/ml), MCR: Metabolic clearance rate of glucose (ml/kg/min). MCR = Mmean/(Gmean x 0.18), where Mmear,: Metabolized glucose expressed as average steady state glucose infusion rate per kg of body weight (mg/kg/min) Gmean:Average steady state blood glucose concentration (mmol/l) 0.18 -conversion factor to transform blood glucose concentration from mmol/l into mg/ml.

[0015] HOMA is a model of the relationship of glucose and insulin dynamics that predicts fasting steady-state glucose and insulin concentrations for a wide range of possible combinations of insulin resistance and p-cell function. The HOMA model has proved to be a robust clinical and epidemiological tool for the assessment of insulin resistance., where I RHOMA = (lo x Go)/ 22.5(mathematically:e ^lrix =1 / x).

[0016] Quantitative insulin sensitivity check index (QUICKI) is an empirically-derived mathematical transformation of fasting blood glucose and plasma insulin concentrations that provide a consistent and precise ISI with a better positive predictive power. It is a variation of HOMA equations, as it transforms the data by taking both the logarithm and the reciprocal of the glucose-insulin product, thus slightly skewing the distribution of fasting insulin values. It employs the use of fasting values of insulin and glucose as in HOMA calculations. QUICKI is virtually identical to the simple equation form of the HOMA model in all aspects, except that a log transform of the insulin glucose product is employed to calculate QUICKI. The QUICKI can be determined from fasting plasma glucose (mg/dl) and insulin (plU/ml) concentrations. [0017] McAuley index is used for predicting insulin resistance in normoglycemic individuals. Regression analysis was used to estimate the cut-off points and the importance of various data for insulin resistance (fasting concentrations of insulin, triglycerides, aspartate aminotransferase, basal metabolic rate (BMI), waist circumference). A bootstrap procedure was used to find an index most strongly correlating with insulin sensitivity index, corrected for fat-free mass obtained by HEC (Mffm/I).

[0018] Matsuda index derives an ISI from the OGTT. In these methods, the ratio of plasma glucose to insulin concentration during the OGTT is used. The OGTT ISI (composite) is calculated using both the data of the entire 3 h OGTT and the first 2 h of the test. The composite wholebody insulin sensitivity index (WBISI) is based on insulin values given in microunits per milliliter (pU/mL) and those of glucose, in milligrams per deciliter (mg/L) obtained from the OGTT and the corresponding fasting values.

[0019] Dosage and frequency of dosing may vary depending on the half-life of the agent in the patient. It will be understood by one of skill in the art that such guidelines will be adjusted for the molecular weight of the active agent, the clearance from the blood, the mode of administration, and other pharmacokinetic parameters. The dosage may also be varied for localized administration, e.g. intranasal, inhalation, etc., or for systemic administration, e.g. i.m., i.p., i.v., oral, and the like.

[0020] An active agent can be administered by any suitable means, including topical, oral, parenteral, intrapulmonary, and intranasal. In some embodiments administration is subcutaneous. Parenteral infusions include intramuscular, intravenous (bolus or slow drip), intraarterial, intraperitoneal, intrathecal or subcutaneous administration. An agent can be administered in any manner which is medically acceptable. This may include injections, by parenteral routes such as intravenous, intravascular, intraarterial, subcutaneous, intramuscular, intratumor, intraperitoneal, intraventricular, intraepidural, or others as well as oral, nasal, ophthalmic, rectal, or topical. Sustained release administration is also specifically included in the disclosure, by such means as depot injections or erodible implants.

[0021 ] As noted above, an agent can be formulated with an a pharmaceutically acceptable carrier (one or more organic or inorganic ingredients, natural or synthetic, with which a subject agent is combined to facilitate its application). A suitable carrier includes sterile saline although other aqueous and non-aqueous isotonic sterile solutions and sterile suspensions known to be pharmaceutically acceptable are known to those of ordinary skill in the art. An "effective amount" refers to that amount which is capable of ameliorating or delaying progression of the diseased, degenerative or damaged condition. An effective amount can be determined on an individual basis and will be based, in part, on consideration of the symptoms to be treated and results sought. An effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.

[0022] An agent can be administered as a pharmaceutical composition comprising a pharmaceutically acceptable excipient. The preferred form depends on the intended mode of administration and therapeutic application. The compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

[0023] As used herein, compounds which are "commercially available" may be obtained from commercial sources including but not limited to Acros Organics (Pittsburgh PA), Aldrich Chemical (Milwaukee Wl, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), Avocado Research (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester PA), Crescent Chemical Co. (Hauppauge NY), Eastman Organic Chemicals, Eastman Kodak Company (Rochester NY), Fisher Scientific Co. (Pittsburgh PA), Fisons Chemicals (Leicestershire UK), Frontier Scientific (Logan UT), ICN Biomedicals, Inc. (Costa Mesa CA), Key Organics (Cornwall U.K.), Lancaster Synthesis (Windham NH), Maybridge Chemical Co. Ltd. (Cornwall U.K.), Parish Chemical Co. (Orem UT), Pfaltz & Bauer, Inc. (Waterbury CN), Polyorganix (Houston TX), Pierce Chemical Co. (Rockford IL), Riedel de Haen AG (Hannover, Germany), Spectrum Quality Product, Inc. (New Brunswick, NJ), TCI America (Portland OR), Trans World Chemicals, Inc. (Rockville MD), Wako Chemicals USA, Inc. (Richmond VA), Novabiochem and Argonaut Technology.

[0024] Compounds useful for co-administration with the active agents of the invention can also be made by methods known to one of ordinary skill in the art. As used herein, "methods known to one of ordinary skill in the art" may be identified through various reference books and databases. Suitable reference books and treatises that detail the synthesis of reactants useful in the preparation of compounds of the present invention, or provide references to articles that describe the preparation, include for example, "Synthetic Organic Chemistry", John Wiley & Sons, Inc., New York; S. R. Sandler et al., "Organic Functional Group Preparations," 2nd Ed., Academic Press, New York, 1983; H. O. House, "Modern Synthetic Reactions", 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L. Gilchrist, “Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, New York, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanisms and Structure”, 4th Ed., Wiley-lnterscience, New York, 1992. Specific and analogous reactants may also be identified through the indices of known chemicals prepared by the Chemical Abstract Service of the American Chemical Society, which are available in most public and university libraries, as well as through on-line databases (the American Chemical Society, Washington, D.C., may be contacted for more details). Chemicals that are known but not commercially available in catalogs may be prepared by custom chemical synthesis houses, where many of the standard chemical supply houses (e.g., those listed above) provide custom synthesis services.

[0025] The active agents of the invention and/or the compounds administered therewith are incorporated into a variety of formulations for therapeutic administration. In one aspect, the agents are formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and are formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. As such, administration of the active agents and/or other compounds can be achieved in various ways, usually by oral administration. The active agents and/or other compounds may be systemic after administration or may be localized by virtue of the formulation, or by the use of an implant that acts to retain the active dose at the site of implantation.

[0026] In pharmaceutical dosage forms, the active agents and/or other compounds may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds. The agents may be combined, as previously described, to provide a cocktail of activities. The following methods and excipients are exemplary and are not to be construed as limiting the invention.

[0027] Formulations are typically provided in a unit dosage form, where the term "unit dosage form," refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active agent in an amount calculated sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage forms of the present invention depend on the particular complex employed and the effect to be achieved, and the pharmacodynamics associated with each complex in the host.

[0028] The term "sustained-release", as in a sustained-release form, sustained-release composition or sustained-release formulation, is intended to include a form of an active ingredient, or formulation for an active ingredient, which has an extended in vivo half-life or duration of action. A sustained-release form may result from modification of the active ingredient, such as modifications that extend circulation residence time, decrease rates of degradation, decrease rates of clearance or the like, or may result from formulations or compositions which provide for extended release of the active ingredient, such as use of various liposomes, emulsions, micelles, matrices and the like. A controlled-release form or formulation is a type of sustained-release form or formulation. [0029] In some embodiments a unit dose is at least about 0.1 mg/kg, at least about 0.5 mg/kg, at least about 1 mg/kg, at least about 5 mg/kg, at least about 10 mg/kg, at least about 20 mg/kg, at least about 50 mg/kg, at least about 100 mg/kg, in some embodiments the effective dose is from about 1 to 50 mg/kg. Dosing may be daily, every 2 days, every 3 or more days, e.g. weekly, semi-weekly, bi-weekly, monthly, etc. Dosing may be parenteral, including sustained release formulations. Dosing may be maintained for long periods of time, e.g. months, or years, to maintain desirable glucose and fatty acid levels.

[0030] The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are commercially available. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are commercially available. Any compound useful in the methods and compositions of the invention can be provided as a pharmaceutically acceptable base addition salt. "Pharmaceutically acceptable base addition salt" refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[0031 ] For oral preparations, the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

[0032] In some embodiments, pharmaceutical compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized SepharoseTM, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).

[0033] A carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group, and non-covalent associations. Suitable covalent-bond carriers include proteins such as albumins, peptides, and polysaccharides such as aminodextran, each of which have multiple sites for the attachment of moieties. The nature of the carrier can be either soluble or insoluble for purposes of the invention.

[0034] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG). Formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0035] The active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0036] Compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97- 119, 1997. The agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient. The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0037] Toxicity of the active agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in further optimizing and/or defining a therapeutic dosage range and/or a sub-therapeutic dosage range (e.g., for use in humans). The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.

[0038] As used herein, the term “dosing regimen” refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0039] "In combination with", "combination therapy" and "combination products" refer, in certain embodiments, to the concurrent administration to a patient of the engineered proteins and cells described herein in combination with additional therapies, e.g. surgery, radiation, chemotherapy, and the like. When administered in combination, each component can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.

[0040] "Concomitant administration" means administration of one or more components, such as engineered proteins and cells, known therapeutic agents, etc. at such time that the combination will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of components. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration.

[0041 ] The use of the term "in combination" does not restrict the order in which prophylactic and/or therapeutic agents are administered to a subject. A first prophylactic or therapeutic agent can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second prophylactic or therapeutic agent to a subject with a disorder.

[0086] Examples of suitable anti-diabetic agents for use in combination with the compounds of the present invention include biguanides (e.g., metformin or phenformin), glucosidase inhibitors (e.g,. acarbose or miglitol), insulins (including insulin secretagogues or insulin sensitizers), meglitinides (e.g., repaglinide), sulfonylureas (e.g., glimepiride, glyburide, gliclazide, chlorpropamide and glipizide), biguanide/glyburide combinations (e.g., Glucovance.RTM.), thiazolidinediones (e.g., troglitazone, rosiglitazone and pioglitazone), PPAR-alpha agonists, PPAR-gamma agonists, PPAR alpha/gamma dual agonists, glycogen phosphorylase inhibitors, inhibitors of fatty acid binding protein (aP2), DPP-IV inhibitors, and SGLT2 inhibitors.

[0087] Thiazolidinediones include Mitsubishi's MCC-555 (disclosed in U.S. Pat. No. 5,594,016), Glaxo-Welcome's GL-262570, englitazone (CP-68722, Pfizer) or darglitazone (CP-86325, Pfizer, isaglitazone (MIT/J&J), JTT-501 (JPNT/P&U), L-895645 (Merck), R-119702 (Sankyo/WL), NN- 2344 (Dr. Reddy/NN), or YM-440 (Yamanouchi).

[0088] Suitable PPAR alpha/gamma dual agonists include AR-HO39242 (Astra/Zeneca), GW- 409544 (Glaxo-Wellcome), KRP297 (Kyorin Merck) as well as those disclosed by Murakami et al, "A Novel Insulin Sensitizer Acts As a Coligand for Peroxisome Proliferation-Activated Receptor Alpha (PPAR alpha) and PPAR gamma. Effect on PPAR alpha Activation on Abnormal Lipid Metabolism in Liver of Zucker Fatty Rats", Diabetes 47, 1841 -1847 (1998), and in U.S. application Ser. No. 09/644,598, filed Sep. 18, 2000, employing dosages as set out therein, which compounds designated as preferred are preferred for use herein.

[0089] Suitable aP2 inhibitors include those disclosed in U.S. application Ser. No. 09/391 ,053, filed Sep. 7, 1999, and in U.S. application Ser. No. 09/519,079, filed Mar. 6, 2000, employing dosages as set out therein.

[0090] Suitable DPP4 inhibitors that may be used in combination with the compounds of the invention include those disclosed in WO99/38501 , WO99/46272, WO99/67279 (PROBIODRUG), WO99/67278 (PROBIODRUG), WO99/61431 (PROBIODRUG), NVP-DPP728A (1 -[[[2-[(5- cyanopyridin-2-yl)amino]ethyl]amino]acetyl]-2-cyano-(S)-pyrr o- Udine) (Novartis) as disclosed by Hughes et al, Biochemistry, 38 (36), 1 1597-1 1603, 1999, TSL-225 (tryptophyl-1 , 2,3,4- tetrahydroisoquinoline-3-carboxylic acid (disclosed by Yamada et al, Bioorg. & Med. Chem. Lett. 8 (1998) 1537-1540, 2-cyanopyrrolidides and 4-cyanopyrrolidides, as disclosed by Ashworth et al, Bioorg. & Med. Chem. Lett., Vol. 6, No. 22, pp 1163-1166 and 2745-2748 (1996) employing dosages as set out in the above references.

[0091] Suitable meglitinides include nateglinide (Novartis) or KAD1229 (PF/Kissei).

[0092] Examples of other suitable glucagon-like peptide-1 (GLP-1 ,) compounds that may be used in combination with the GLP-1 mimics of the present invention include GLP-1 (1 -36) amide, GLP-1 (7-36) amide, GLP-1 (7-37) (as disclosed in U.S. Pat. No. 5,614,492 to Habener), as well as AC2993 (Amylin), LY-315902 (Lilly) and NN-2211 (NovoNordisk).

[0093] Examples of suitable hypolipidemic/lipid lowering agents for use in combination with the compounds of the present invention include one or more MTP inhibitors, HMG CoA reductase inhibitors, squalene synthetase inhibitors, fibric acid derivatives, ACAT inhibitors, lipoxygenase inhibitors, cholesterol absorption inhibitors, ileal Na.sup.+/bile acid cotransporter inhibitors, upregulators of LDL receptor activity, bile acid sequestrants, cholesterol ester transfer protein inhibitors (e.g., CP-529414 (Pfizer)) and/or nicotinic acid and derivatives thereof.

[0094] MTP inhibitors which may be employed as described above include those disclosed in U.S. Pat. Nos. 5,595,872, 5,739,135, 5,712,279, 5,760,246, 5,827,875, 5,885,983 and 5,962,440.

[0095] The HMG CoA reductase inhibitors which may be employed in combination with one or more compounds of SEQ ID NO:X include mevastatin and related compounds, as disclosed in U.S. Pat. No. 3,983,140, lovastatin (mevinolin) and related compounds, as disclosed in U.S. Pat. No. 4,231 ,938, pravastatin and related compounds, such as disclosed in U.S. Pat. No. 4,346,227, simvastatin and related compounds, as disclosed in U.S. Pat. Nos. 4,448,784 and 4,450,171. Other HMG CoA reductase inhibitors which may be employed herein include, but are not limited to, fluvastatin, disclosed in U.S. Pat. No. 5,354,772, cerivastatin, as disclosed in U.S. Pat. Nos. 5,006,530 and 5,177,080, atorvastatin, as disclosed in U.S. Pat. Nos. 4,681 ,893, 5,273,995, 5,385,929 and 5,686,104, atavastatin (Nissan/Sankyo's nisvastatin (NK-104)), as disclosed in U.S. Pat. No. 5,01 1 ,930, visastatin (Shionogi-Astra/Zeneca (ZD-4522)), as disclosed in U.S. Pat. No. 5,260,440, and related statin compounds disclosed in U.S. Pat. No. 5,753,675, pyrazole analogs of mevalonolactone derivatives, as disclosed in U.S. Pat. No. 4,613,610, indene analogs of mevalonolactone derivatives, as disclosed in PCT application WO 86/03488, 6-[2-(substituted- pyrrol-1 -yl)-alkyl)pyran-2-ones and derivatives thereof, as disclosed in U.S. Pat. No. 4,647,576, Searle's SC-45355 (a 3-substituted pentanedioic acid derivative) dichloroacetate, imidazole analogs of mevalonolactone, as disclosed in PCT application WO 86/07054, 3-carboxy-2- hydroxy-propane-phosphonic acid derivatives, as disclosed in French Patent No. 2,596,393, 2,3- disubstituted pyrrole, furan and thiophene derivatives, as disclosed in European Patent Application No. 0221025, naphthyl analogs of mevalonolactone, as disclosed in U.S. Pat. No. 4,686,237, octahydronaphthalenes, such as disclosed in U.S. Pat. No. 4,499,289, keto analogs of mevinolin (lovastatin), as disclosed in European Patent Application No.0142146 A2, and quinoline and pyridine derivatives, as disclosed in U.S. Pat. Nos. 5,506,219 and 5,691 ,322.

[0096] Hypolipidemic agents include pravastatin, lovastatin, simvastatin, atorvastatin, fluvastatin, cerivastatin, atavastatin and ZD-4522. In addition, phosphinic acid compounds useful in inhibiting HMG CoA reductase, such as those disclosed in GB 2205837, are suitable for use in combination with the compounds of the present invention.

[0097] Squalene synthetase inhibitors suitable for use herein include, but are not limited to, a- phosphono-sulfonates disclosed in U.S. Pat. No. 5,712,396, those disclosed by Biller et al, J. Med. Chem., 1988, Vol. 31 , No. 10, pp 1869-1871 , including isoprenoid (phosphinyl- methyl)phosphonates, as well as other known squalene synthetase inhibitors, for example, as disclosed in U.S. Pat. Nos. 4,871 ,721 and 4,924,024 and in Biller, S. A., Neuenschwander, K., Ponpipom, M. M., and Poulter, C. D., Current Pharmaceutical Design, 2, 1-40 (1996). Other squalene synthetase inhibitors suitable for use herein include the terpenoid pyrophosphates disclosed by P. Ortiz de Montellano et al, J. Med. Chem., 1977, 20, 243-249, the farnesyl diphosphate analog A and presqualene pyrophosphate (PSQ-PP) analogs as disclosed by Corey and Volante, J. Am. Chem. Soc., 1976, 98, 1291 -1293, phosphinylphosphonates reported by McClard, R. W. et al, J.A.C.S., 1987, 109, 5544 and cyclopropanes.

[0098] The fibric acid derivatives which may be employed in combination with one or more compounds of SEQ ID NO:X include fenofibrate, gemfibrozil, clofibrate, bezafibrate, ciprofibrate, clinofibrate and the like, probucol, and related compounds, as disclosed in U.S. Pat. No. 3,674,836, probucol and gemfibrozil being preferred, bile acid sequestrants, such as cholestyramine, colestipol and DEAE-Sephadex (Secholex.RTM., Policexide.RTM.), as well as lipostabil (Rhone-Poulenc), Eisai E-5050 (an N-substituted ethanolamine derivative), imanixil (HOE-402), tetrahydrolipstatin (THL), istigmastanylphos-phorylcholine (SPG, Roche), aminocyclodextrin (Tanabe Seiyoku), Ajinomoto AJ-814 (azulene derivative), melinamide (Sumitomo), Sandoz 58-035, American Cyanamid CL-277,082 and CL-283,546 (disubstituted urea derivatives), nicotinic acid, acipimox, acifran, neomycin, p-aminosalicylic acid, aspirin, poly(diallylmethylamine) derivatives, such as disclosed in U.S. Pat. No. 4,759,923, quaternary amine poly(diallyldimethylammonium chloride) and ionenes, such as disclosed in U.S. Pat. No. 4,027,009, and other known serum cholesterol lowering agents.

[0099] The ACAT inhibitor which may be employed in combination with one or more compounds of SEQ ID NO:X include those disclosed in Drugs of the Future 24, 9-15 (1999), (Avasimibe); "The ACAT inhibitor, CI-1011 is effective in the prevention and regression of aortic fatty streak area in hamsters", Nicolosi et al, Atherosclerosis (Shannon, Irel). (1998), 137 (1 ), 77-85; "The pharmacological profile of FCE 27677: a novel ACAT inhibitor with potent hypolipidemic activity mediated by selective suppression of the hepatic secretion of ApoBI OO-containing lipoprotein", Ghiselli, Giancarlo, Cardiovasc. Drug Rev. (1998), 16 (1 ), 16-30; "RP 73163: a bioavailable alkylsulf inyl-diphenylim idazole ACAT inhibitor", Smith, C., et al, Bioorg. Med. Chem. Lett. (1996), 6 (1 ), 47-50; "ACAT inhibitors: physiologic mechanisms for hypolipidemic and anti-atherosclerotic activities in experimental animals", Krause et al, Editor(s): Ruffolo, Robert R., Jr.; Hollinger, Mannfred A., Inflammation: Mediators Pathways (1995), 173-98, Publisher: CRC, Boca Raton, Fla.; "ACAT inhibitors: potential anti-atherosclerotic agents", Sliskovic et al, Curr. Med. Chem. (1994), 1 (3), 204-25; "Inhibitors of acyl-CoA:cholesterol O-acyl transferase (ACAT) as hypocholesterolemic agents. 6. The first water-soluble ACAT inhibitor with lipid-regulating activity. Inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). 7. Development of a series of substituted N-phenyl-N'-[(1 -phenylcyclopentyl)methyl]ureas with enhanced hypocholesterolemic activity", Stout et al, Chemtracts: Org. Chem. (1995), 8 (6), 359-62, or TS-962 (Taisho Pharmaceutical Co. Ltd).

[00100] The hypolipidemic agent may be an upregulator of LD2 receptor activity, such as MD-700 (Taisho Pharmaceutical Co. Ltd) and LY295427 (Eli Lilly). Examples of suitable cholesterol absorption inhibitor for use in combination with the compounds of the invention include SCH48461 (Schering-Plough), as well as those disclosed in Atherosclerosis 1 15, 45-63 (1995) and J. Med. Chem. 41 , 973 (1998).

[00101 ] Examples of suitable ileal NaVbile acid cotransporter inhibitors for use in combination with the compounds of the invention include compounds as disclosed in Drugs of the Future, 24, 425- 430 (1999).

[00102] The lipoxygenase inhibitors which may be employed in combination with one or more compounds of SEQ ID NO:X include 15-lipoxygenase (15-LO) inhibitors, such as benzimidazole derivatives, as disclosed in WO 97/12615, 15-LO inhibitors, as disclosed in WO 97/12613, isothiazolones, as disclosed in WO 96/38144, and 15-LO inhibitors, as disclosed by Sendobry et al "Attenuation of diet-induced atherosclerosis in rabbits with a highly selective 15-lipoxygenase inhibitor lacking significant antioxidant properties”, Brit. J. Pharmacology (1997) 120, 1199-1206, and Cornicelli et al, ”15-Lipoxygenase and its Inhibition: A Novel Therapeutic Target for Vascular Disease”, Current Pharmaceutical Design, 1999, 5, 1 1 -20.

[00103] Examples of suitable anti-hypertensive agents for use in combination with the compounds of the present invention include beta adrenergic blockers, calcium channel blockers (L-type and T-type; e.g. diltiazem, verapamil, nifedipine, amlodipine and mybefradil), diuretics (e.g., chlorothiazide, hydrochlorothiazide, flumethiazide, hydroflumethiazide, bendroflumethiazide, methylchlorothiazide, trichloromethiazide, polythiazide, benzthiazide, ethacrynic acid tricrynafen, chlorthalidone, furosemide, musolimine, bumetanide, triamtrenene, amiloride, spironolactone), renin inhibitors, ACE inhibitors (e.g., captopril, zofenopril, fosinopril, enalapril, ceranopril, cilazopril, delapril, pentopril, quinapril, ramipril, lisinopril), AT-1 receptor antagonists (e.g., losartan, irbesartan, valsartan), ET receptor antagonists (e.g., sitaxsentan, atrsentan and compounds disclosed in U.S. Pat. Nos. 5,612,359 and 6,043,265), Dual ET/AII antagonist (e.g., compounds disclosed in WO 00/01389), neutral endopeptidase (NEP) inhibitors, vasopepsidase inhibitors (dual NEP-ACE inhibitors) (e.g., omapatrilat and gemopatrilat), and nitrates.

[00104] Examples of suitable anti-obesity agents for use in combination with the compounds of the present invention include a NPY receptor antagonist, a MCH antagonist, a GHSR antagonist, a CRH antagonist, a beta 3 adrenergic agonist, a lipase inhibitor, a serotonin (and dopamine) reuptake inhibitor, a thyroid receptor beta drug and/or an anorectic agent.

[00105] The beta 3 adrenergic agonists which may be optionally employed in combination with compounds of the present invention include AJ9677 (Takeda/Dainippon), L750355 (Merck), or CP331648 (Pfizer,) or other known beta 3 agonists, as disclosed in U.S. Pat. Nos. 5,541 ,204, 5,770,615, 5,491 ,134, 5,776,983 and 5,488,064, with AJ9677, L750,355 and CP331648 being preferred.

[00106] Examples of lipase inhibitors which may be optionally employed in combination with compounds of the present invention include orlistat or ATL-962 (Alizyme), with orlistat being preferred.

[00107] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Experimental

Example 1

Leptin analogs to treat obesity

[00108] Leptin is an adipocyte-derived protein hormone that promotes satiety and energy homeostasis by activating the leptin receptor (LepR)-STAT3 signaling axis in a subset of hypothalamic neurons. Leptin signaling is dysregulated in obesity, however, where appetite remains elevated despite high levels of circulating leptin, due to leptin resistance. As a result, therapeutic use of leptin as an anti-obesity medication has been limited to rare cases of leptin deficiency. To gain insight into the mechanisms of leptin signaling and resistance, we determined the structure of a stabilized leptin-bound LepR signaling complex using single particle cryo-EM. The structure reveals an asymmetric complex architecture in which a single leptin induces LepR dimerization via two distinct receptor-binding sites. Analysis of the leptin-LepR binding interfaces reveals the molecular basis for human obesity-associated mutations in both leptin and LepR. Structure-based design of leptin mutants that destabilize the LepR dimer yielded both partial and biased agonists that decouple stimulation of STAT3 from activation of LepR negative regulators, even in the presence of wild-type leptin. Together, these results reveal the structural basis for leptin signaling and provide therapeutic LepR agonists that overcome leptin resistance in the context of obesity.

[00109] Cryo-EM structure of the leptin receptor signaling complex. To gain molecular insight into the mechanisms of leptin signaling and resistance, we sought to determine the structure of the active LepR signaling complex. Previous attempts to resolve the structure of leptin-bound LepR however have been hindered by the instability of the 2:2 leptin-LepR complex in vitro. To overcome this, we engineered a construct comprising the complete extracellular domain (D1 -D7) of LepR with the transmembrane (TM) domains replaced by dimerizing leucine zippers (LepR ^{D1- D7} -zip) , thereby enhancing the avidity of LepR for leptin to stabilize the leptin-LepR complex (Fig. 1 a).

[00110] Recombinantly expressed mouse LepR ^{D1 D7} -zip formed a stable complex with mouse leptin in vitro as assessed by co-elution over size-exclusion chromatography (FIG. 5a). Analysis of this complex using single particle cryo-electron microscopy (cryo-EM) yielded a 3D reconstruction of the leptin-LepR ^{D1 D7} complex to 5.9 A resolution (Fig. 1a, b; Fig. 6a-e). Given that flexibility of the membrane distal D1 and D2 domains of LepR appeared to limit the resolution of these reconstructions, we next purified and performed cryo-EM analysis on a truncated leptin— LepR ^{D3 D7} -zip complex, which yielded an improved 4.5 A resolution 3D reconstruction (Fig. 1 a,b; Fig. 7a, b and Fig. 8a-e). Using AlphaFold models of mouse leptin and monomeric LepR, these maps enabled high confidence docking and refinement of the complete leptin-LepR signaling complex (Fig. 1c).

[00111] Analysis of the resulting molecular model of the 2:2 leptin-LepR complex unexpectedly revealed an asymmetric, partially open conformation in which one leptin simultaneously binds both LepR chains, whereas the second leptin engages only a single LepR subunit (Fig. 1c). Consistent with previous mutagenesis studies, the high affinity leptin-LepR interaction, referred to as “site 2,” is formed by the hinge of the second cytokine homology region (CHR2) of LepR (D4-D5), which engages helices A and C of leptin (Fig. 1 c). The low affinity site 3 interface by contrast is formed by the immunoglobulin (Ig) domain of LepR (D3), which interacts with the top of leptin helix D and loop AB. Notably, the canonical “site 1 ” interface, which participates in high affinity receptor binding in most cytokine receptor complexes, is unoccupied for both leptin molecules in the leptin-LepR complex (Fig. 1d). [00112] Above the leptin-binding regions, the membrane distal CHR1 domains (D1 and D2) bend upwards, resulting in a highly elongated structure with a total estimated length of approximately 180 A projecting from the cell surface (Fig. 1 c,d). Below the leptin binding interface, the two- membrane proximal fibronectin domains (FNIII, D6 and D7) of LepR bend inwards towards one another, forming an approximately 90° angle between D6 and D7 which is predicted to bring to TM domains of each LepR monomer in close association. Notably, there is no direct interaction between the two LepR ECDs, suggesting that receptor dimerization is ligand-dependent, as is the case for other class 1 cytokine receptors.

[00113] Structural homology between leptin and IL-6 family cytokine receptor complexes. Based on both sequence and structural homology, leptin is most closely related to the IL-6 family cytokines, which exert diverse biological effects through the shared receptor gp130 (Fig. 2a-c). Comparison of the cryo-EM structure of leptin-LepR reported here with the hexameric complex of IL-6 bound to IL-6Ra and gp130 reveals that the docking modes of the site 2 and site 3 interactions between leptin and LepR are similar to those formed between IL-6 and gp130 (Fig. 2a, b). However, in the IL-6 structure, the Ig domains of both gp130 subunits bend back in to engage the adjacent IL-6 molecules, forming two site 3 interactions in a symmetric, closed receptor conformation (Fig. 2b). By contrast, the Ig domain of only a single LepR engages leptin at site 3, whereas the Ig domain of the second LepR projects outward, away from the second bound leptin which is unoccupied at site 3 (Fig. 2a). Thus, despite being a 2:2 homodimeric receptor complex, the architecture of the leptin-bound LepR complex more closely resembles heterodimeric IL-6 family receptor complexes, such as IL-27, in which a single ligand dimerizes two different receptors, gp130 and IL27Ra, to form a similarly open and asymmetric receptor complex (Fig. 2c).

[00114] The asymmetric conformation observed in our structure predicts that a single leptin molecule is sufficient to dimerize two LepR chains and activate downstream STAT3 signaling. To test this hypothesis, we transfected HEK-293T cells with cDNA encoding either WT LepR, LepR lacking site 2 binding (LepR-2KO, L503S/L504S), and/or LepR lacking site 3 binding (LepR-3KO, L370S, Fig. 2d). Whereas cells expressing LepR-2KO or LepR-3KO alone failed to respond to recombinant leptin, cells co-expressing both mutant receptors exhibited full leptin responsiveness, as assessed by phosphorylation of STAT3 (Fig. 2e). Given that cells coexpressing these two receptor mutants can only engage leptin in an “open” 1 :2 orientation (Fig. 2d), these data demonstrate that the asymmetric conformation observed in our structures represents an active signaling complex. Moreover, modelling a “closed” LepR complex by rotating the Ig domain of the second LepR results in a substantial clash between the membrane proximal D7 domains of the two LepR chains (Fig. 8a, b). Thus, although we cannot rule out the formation of a closed LepR complex on the cell surface, perhaps transiently or in a dynamic equilibrium with the asymmetric complex, this would require significant rearrangement of LepR domains compared to what is observed in our structures.

[00115] Structural basis for leptin-dependent LepR dimerization. Focused refinement of the LepR ^{D3 D7} -zip complex centered on the leptin-LepR interface yielded an additional 3.8 A resolution map comprising leptin together with the three interacting domains of LepR (D3 and D4/D5), enabling molecular analysis of the site 2 and site 3 binding interfaces (Fig. 3a-f and Fig. 8d-e). At the high affinity site 2, the A and C helices of leptin engage several loops in the CHR2 (D4 and D5) of LepR, burying 750 A ² of surface area (Fig. 3a). The interaction appears to be mediated in large part by hydrophobic contacts, such as between Leu13 and Leu86 of leptin and Leu503 and Leu504 of LepR (Fig. 3b). This is consistent with previous reports that mutation of Leu503 and Leu504 in LepR abolishes leptin responsiveness (Fig. 2e). In addition, several apparent polar and electrostatic contacts are also formed between leptin and LepR at this interface, including between leptin residues Asp9, Thr16, and Asp85, which appear to contact LepR residues Tyr470, Glu563, and Ser468, respectively. Moreover, leptin residues Arg20 and Gln75, which were previously reported to be required for LepR binding, form apparent hydrogen bonds with Thr441 in LepR (Fig. 3c).

[00116] At the low affinity site 3 interface, the Ig domain of LepR (D3) engages the top of helix D and the CD loop of leptin (Fig. 3, D and E), burying 775 A ² . The primary contact appears to be mediated by Tyr1 19 within the CD loop of leptin, which inserts into an aromatic cluster consisting of mouse LepR residues Phe403, Tyr405, Tyr409, and Tyr420 (“site 3a,” Fig. 3e). Consistent with a key role for these aromatic contacts in LepR activation, mutation of the corresponding aromatic residues in human LepR including F405A/Y407A (mouse F403/Y405), Y411 A (mouse 409), or Y422A (mouse Y420), showed greatly diminished leptin responsiveness as assessed by phosphorylation of STAT3 in HEK-293T cells (Fig. 3f). Immediately adjacent to Tyr1 19, leptin residue Ser120 forms apparent hydrogen bonds with both the hydroxyl of Tyr409 and the backbone carbonyl of His418, which is also contacted by the neighboring leptin residue Ser117 (Fig. 3e).

[00117] A second site 3 contact with LepR is formed by the leptin AB loop, which engages the LepR Ig domain via a primarily backbone-mediated p-sheet interaction formed between LepR residues 415-417 and leptin residues 35-37 (“site 3b”, Fig. 3g). The side chains of neighboring leptin residues Gln34 and Lys33 appear also form additional contacts with Cys416 and Gln414 of LepR, respectively (Fig. 3g). Notably, a large portion of the leptin AB loop (residues 24-39), including the region implicated in the site 3B interaction, is disordered in the crystal structure apoleptin, but ordered upon LepR binding (Fig. 3h). The ordering of the AB loop in the LepR-bound structure is accompanied by a substantial rearrangement of leptin helix E and the CD loop, which swing outward to accommodate the repositioning of the AB loop along the LepR Ig domain. Thus, LepR binding appears to induce a large conformational change in leptin to enable the site 3 interaction and resulting LepR dimerization.

[00118] To assess the functional importance of the leptin residues implicated in site 3 binding in our structure, we mutated corresponding residues in human leptin and analyzed their effect on LepR signaling in HEK-293T cells expressing WT LepR. Notably, mutation of site 3a residues Tyr1 19 or Ser120 to alanine (Y1 19A and S120A) abolished leptin signaling activity as assessed by phosphorylation of STAT3 (Fig. 3i). Mutation of the neighboring Ser1 17 to Ala (S117A) however had negligible impact on LepR signaling, although mutation to Asn (S117N) resulted in partially reduced STAT3 activation, likely by creating a steric clash with His418 of LepR (Fig. 3e and h). Simultaneous mutation of three site 3b contact residues in the AB loop of leptin (K33A/Q34A/K35A, corresponding to mouse residues K33/Q34/R35) similarly resulted in partially reduced STAT3 activation (Fig. 3i). Together, these results suggest that leptin-mediated LepR dimerization is primarily driven by the aromatic and polar contacts formed by leptin residues Tyr1 19 and S120 at site 3a, with the binding-induced conformational change of the leptin AB loop playing a secondary role to further stabilize the LepR dimer and enable maximal STAT3 activation.

[00119] Analysis of the site 2 and site 3 leptin-LepR interfaces also provides insight into the molecular basis of several human obesity-associated mutations in leptin, including D79Y, N82K, R84W, and S120C (Fig. 3b and e). Notably, Asn82 of leptin lies in near the center of the site 2 interface and makes key hydrogen bonds contacts with LepR residues Ser505 and Leu503, suggesting a loss of site 2 binding associated with the M82K'mutation (Fig. 3b). The neighboring residues Asp79 and Arg84 are also near the site 2 interface, but do not directly contact LepR, instead making apparent intramolecular contacts with leptin residues Arg20 and Gln62 (Fig. 3b and c), likely indirectly stabilizing the site 2 interaction. Although most obesity-associated mutations in LepR are distal to the leptin binding sites and likely act to simply destabilize LepR, two previously identified human LepR mutations (A409E and Y422H) both occur at the site 3 binding interface and are predicted by our structure to disrupt the key aromatic contacts formed by leptin Tyr1 19 (Fig. 3h).

[00120] Engineered biased agonists decouple activation ofSTAT3 from LepR negative regulators. The leptin-mediated dimerization of LepR results in the JAK2-dependent phosphorylation of multiple tyrosine residues on the intracellular domain (ICD) of LepR, each of which have distinct biological roles. Specifically, phosphorylation of Tyr1 141 (Y1138 in mice) drives activation of STAT3 and the subsequent satiety promoting effects of leptin, whereas phosphorylation of Tyr986 (Y985 in mice) activates the SHP2/ERK pathway and is required for recruitment of cytosolic LepR antagonists that drive leptin resistance (Fig. 4a). Consistent with this, mice expressing a Y1138S LepR mutant are extremely obese and hyperphagic due to loss of leptin- mediated STAT3 activation. By contrast, mice expressing a Y985F LepR mutant are lean and do not become leptin resistant, phenocopying loss of hypothalamic SOCS3 expression.

[00121] Previously, we showed that engineered partial agonists of the cytokine receptor IL-22R elicit biased downstream signaling by inducing differential tyrosine phosphorylation on the receptor ICD. To test whether a similar approach could be used for LepR, we first assessed whether various leptin site 3 mutants exhibited partial agonism (i.e., sub-maximal signaling at saturating ligand concentrations), by performing comprehensive STAT3 dose-response analysis using phospho-flow cytometry. The human leptin mutants K33A/Q34A/K35A (KQK-AAA) and S117N in particular displayed pronounced partial agonism, with maximal STAT3 responses of approximately 75% and 40% of WT leptin, respectively (Fig. 4B). Critically, these partial agonists also induced STAT3-biased signaling, with minimal phosphorylation of the phosphatase SHP2 or its downstream effector ERK when compared to WT leptin, suggesting reduced phosphorylation of LepR ^Y98S relative to LepR ^Y1141 (Fig. 4c).

[00122] An important consideration for the development of therapeutic LepR agonists is that the serum levels of endogenous leptin are highly elevated in the context of human obesity, ranging from 5 ng/ml (0.3 nM) in lean individuals to as high as 100 ng/ml (6 nM) in obesity. Therefore, we next assessed how the leptin partial agonists KQK-AAA and S1 17N impacted LepR signaling in the presence of high levels of WT leptin. To enhance the ability of these variants to compete with WT leptin for LepR binding, we incorporated an additional mutation at site 2, D23L, which reduces the off rate of leptin for LepR (Fig. 4d). These engineered variants were sufficient to enforce sub- maximal STAT3 activation and reduced induction of the LepR antagonist SOCS3, even in the presence of 10 nM WT leptin, thereby functioning as “receptor signaling clamps” (Fig. 4e and f). Moreover, these variants preferentially suppressed phosphorylation of SHP2 and ERK to a much greater extent than STAT3, with the S1 17N variant driving a complete inhibition of SHP2/ERK signaling despite maintaining partial STAT3 activation (Fig. 4G). Thus, engineered high affinity partial agonists preferentially suppress signaling upstream of LepR antagonists while retaining the capacity to induce STAT3 activation, even in the presence of WT leptin.

[00123] Together with previous biochemical studies, the cryo-EM structures reported here support a two-step mechanism of LepR activation in which leptin first binds LepR via the high affinity site 2 interface to form a 1 :1 complex on the cell membrane. Subsequent trans-interaction between leptin and the LepR Ig domain forms the site 3 interface, which dimerizes LepR to form signaling competent 2:2 complexes. Surprisingly, our cryo-EM data reveals that a partially open, asymmetric 2:2 complex in which only one leptin forms a site 3 contact with LepR is the most stable dimer conformation in solution. Moreover, LepR complexes that can form only one site 3 interaction exhibit full signaling activity in cells, suggesting that engagement of a single leptin at site 3 is sufficient for receptor activation. The asymmetric receptor homodimerization observed here is reminiscent of the asymmetry seen in some homodimeric receptor tyrosine kinases, including Insulin Receptor and Epithelial growth factor receptor (EGFR), but is unique among known cytokine receptor complexes. Given the extremely low affinity of the leptin-LepR site 3 interaction, it is likely that the complex dynamically interchanges between 1 :1 and partially open 2:2 complexes, with minimal sampling of the fully closed 2:2 conformation.

[00124] Consistent with the site 3 leptin-LepR interaction being the rate-limiting step for leptindependent signal transduction, our mutagenesis data reveals that the modulation of the leptin site 3 binding affinity can “tune” LepR signal strength across a wide dynamic range (Fig. 4b). Moreover, some leptin site 3 mutants also exhibited biased agonism, selectively promoting signaling through the LepR ^Tyr1141 -STAT3 axis with diminished signaling through the LepR ^Tyr986 - SHP2-ERK axis. Although we are unable to directly assess site-specific LepR ICD phosphorylation with available reagents, these results indicate that phosphorylation of Tyr1 141 may occur more efficiently than phosphorylation of Tyr986, such that reducing stability of the 2:2 complex more substantially impacts phosphorylation at Tyr986. Alternatively, the phosphotyrosine binding domains of STAT3 and SHP2 may have different affinities for each LepR phospho-tyrosine, such that even uniform reduction of LepR ICD phosphorylation may differentially impact these two pathways. Distinguishing between these and other possible mechanisms will require further biochemical analysis of the LepR ICD interactions and may be informative for future development of biased LepR agonists.

[00125] The observation that the structure-based modulation of the leptin-LepR interaction can yield partial and biased agonism even in the presence of WT leptin has important implications for the targeting of LepR therapeutically. Recently, Zhao et al. demonstrated that the partial suppression of leptin signaling with an anti-leptin antibody is sufficient to restore leptin sensitivity and promote weight loss in a mouse model of leptin resistant obesity. The use of engineered LepR partial agonists described here provide an analogous path to restoring leptin sensitivity, by directly re-calibrating LepR signaling to a submaximal level while also promoting STAT3-biased signaling activity. Overall, these results provide new mechanistic insights into LepR activation and new therapeutic modalities that harness leptin signaling in the treatment of obesity.

Materials and Methods:

[00126] Protein production and purification. For structural studies, mouse LepR ^{D1 D7} (M. musculus, residues 23-839) and LepR ^{D3 D7} (M musculus, residues 330-839) were cloned into a pD649 mammalian expression vector containing an N-terminal HA signal peptide, C-terminal GCN4 leucine zipper (EELLSKNYHLENEVARLKK), and C-terminal 6xHis-tag. DNA was transiently transfected into Expi-293F cells (Thermo) using Expifectamine transfection reagent (Thermo). Expi293F cells were grown in serum free Expi293 expression media (Thermo) and maintained at 37°C with 5% CO2 with gentle agitation. 72 hours after transfection, cell supernatant was harvested and proteins were purified with Ni-NTA resin (Qiagen) followed by size-exclusion chromatography (SEC) on a Superdex 200 column (GE) in Phosphate-buffered saline (PBS, 135 mM NaCI, 2.5 mM KOI, 8.0 mM Na ₂ HPO ₄ , 30 mM KH ₂ PO ₄ , pH 7.2).

[00127] Purified receptor complexes were incubated overnight at 4° C with E. coli produced M. musculus leptin with a leptin:LepR ratio of 4:1. The leptin-LepR complex was then re-purified by SEC on a Superdex 200 column in PBS. Purified sample was crosslinked with 1 mM bis(sulfosuccinimidyl)suberate (BS3) (Thermo) for 45 min at room temperature and quenched with 20 mM Tris-HCI pH 8. Crosslinked complex was re-purified by SEC on a Superdex 200 column in PBS and concentrated to 1 -2 mg/ml for cryo-EM analysis.

[00128] For signaling experiments, wild-type or mutant leptin (/-/. sapiens, residues 23-167) was cloned into a pET28a E. coli expression vector containing a C-terminal 6xHis-tag. DNA was transformed into BL21 (DE3) competent cells and grown at 37°C in LB media supplemented with Kanamycin (40 pg/mL) until the culture reached log-phase growth. Protein expression was induced by adding IPTG at a final concentration of 1 mM. The culture was shaken at 37°C for 4 hours. Cells were harvested by centrifugation at 6000xg for 6 minutes, and frozen at -20°C.

[00129] Cell pellets were resuspended in Lysis Buffer (50 mM Tris-HCI pH 8.0, 1% (v/v) TritonX- 100, 100 mM NaCI, 5 mM MgCI ₂ , 1 mg Benzonase) and lysed by sonication. Inclusion bodies were isolated by centrifugation at 10,000xg for 15 min. Inclusion bodies were washed 3x in Wash Buffer 1 (50 mM T ris-HCI pH 8.0, 0.5% T ritonX-100, 100 mM NaCI, 1 mM Na EDTA, 1 mM DTT) and 1 x in Wash Buffer 2 (50 mM Tris-HCI pH 8.0, 100 mM NaCI, 1 mM Na EDTA, 1 mM DTT), with centrifugation at 10,000xg for 15 min between each wash. Inclusion bodies were then solubilized by rotating in Denaturing Buffer (20 mM Tris-HCI pH 8.0, 8 M Urea, 1 mM DTT) at room temperature for 24 hours. Solubilized samples were then centrifuged at 30,000xg for 30 minutes, and supernatant was frozen at -80 for further processing.

[00130] Leptin proteins were refolded by dropwise addition into cold Refolding Buffer (100 mM Tris-HCI pH 8.0, 400 mM L-Arginine, 1 mM Oxidized Glutathione, and 10 mM reduced Glutathione) to a final concentration of approximately 0.05 mg/ml and stirred gently at 4° C for 48 hours. Samples were then filtered through a 0.22 uM Millipore Express© PLUS filter, concentrated 30-fold before being purified by SEC on a Superdex 75 column in Tris-buffered saline (TBS, 20 mM Tris-HCL pH 8.0, 150 mM NaCI). Proteins were maintained in TBS, concentrated to approximately 1 mg/mL before being flash-frozen in liquid nitrogen and stored at -80° C for future use.

[00131 ] Cryo-EM specimen preparation and data collection. Aliquots of 3 pL of the LepR ^{D1 D7} and LepR ^{D3 D7} receptor complex were supplemented with 0.01 % fluorinated octyl maltoside (Anatrace) and immediately applied to glow-discharged Quantifoil® (1 .2/1 .3) grids. The grids were blotted for 3 seconds at 100% humidity with an offset of +3 and plunge frozen into liquid ethane using a Vitrobot Mark IV (Thermo Fisher). Grids were imaged on a 300 keV Titan Krios cryo-electron microscope (Thermo Fisher) equipped with a K3 camera (Gatan). Additionally, an energy filter (Gatan) was used during imaging of the LepR ^{D1 D7} complex. Movies were collected at a calibrated magnification corresponding to 0.653 A and 0.8521 A per physical pixel, for LepR ^{D1 D7} and LepR ^{D3 D7} complexes, respectively. The dose was set to a total of 53 electrons per A ² over an exposure of 1 .518 and 2.545 seconds, for LepR ^{D1 D7} and LepR ^{D3 D7} complexes, respectively. Automated data collection was carried out using SerialEM with a nominal defocus range set from 0.8 to 2.0 pM. 1 1 ,292 movies were collected for the complex with LepR ^{D1 D7} and 21 ,1 12 movies were collected for the complex with LepR ^{D3 D7} .

[00132] Cryo-EM Data processing and 3D reconstruction. All movies were processed using cryoSPARC v3.1 .0 unless otherwise specified. Movies were motion corrected, had contrast transfer functions (CTFs) determined, and particles picked using the cryoSPARC live processing functions. During this processing, micrographs were binned to the physical pixel size.

[00133] For the LepR ^{D1 D7} receptor complex, a prior test collection using no BS3 crosslinker in sample preparation was used in successive rounds of reference-free 2D classification, leaving 607,681 particles in well-defined classes. These particles were used to generate one good and two bad ab initio models. These ab initio models were then used in six rounds of iterative heterogenous refinement with 1 ,735,072 particles of the crosslinked data. This resulted in a class with 270,721 particles which had a resolution of 6.4 A when refined with non-uniform refinement. Local Refinement, with a generous mask around defined regions of density, was then used to generate a final map at 5.9 A resolution, which was sharpened using LAFTER.

[00134] For the LepR ^{D3 D7} receptor complex, successive rounds of reference-free 2D classification of 17,952,333 raw particles were performed, leaving 168,307 particles in well-defined classes. These particles were used to generate ab initio models. These ab initio models were then used models were used in seven rounds of heterogenous refinement. This resulted in a class with 137,338 particles which had a resolution of 5.2 A when refined with non-uniform refinement. Local Refinement, with a generous mask around defined regions of density, was then used to generate a final map at 4.5 A resolution, which was sharpened with DeepEMhancer.

[00135] To obtain higher resolution detail for the leptin-LepR binding interface, a mask was generated around leptin and each domain of the LepR complex which was contacting LepR. This was then used to perform focused, non-uniform, refinement on the 444,608 particles from the third iteration of the heterogenous refinements. This resulted in a map which had a resolution of 3.8 A, which was sharpened with cryoSPARC.

[00136] Model building and refinement. AlphaFold models of mouse leptin and LepR were docked into the various maps using UCSF Chimera. The resultant model was then refined using Phenix real space refine and manual building in Coot. The model for the full-length receptor had its sidechains truncated to C|3. Figures of cryo-EM maps and structural models were generated using UCSF ChimeraX. [00137] Lentivirus production and lentiviral transduction. Lentiviruses were produced by transfection of HEK-293T cells with pLV-EF1 a-IRES-Puro vector containing an N-terminal Myc- tag in combination with the pMD2G envelope and psPax2 packaging plasmids. Forty-eight hours after transfection, the virus-containing supernatant was collected and centrifuged for 5 minutes at 300xg to remove cell debris. Virus was concentrated by incubation with 1 x PEG-IT (SBI) at 4° C for 12-24 hours. The solution was then centrifuged a 1 ,500xg for 30 min and virus pellet was resuspended with 10% of the initial virus volume in serum free DMEM. Target cells were plated in 6-well plates containing DMEM supplemented with 10% v/v fetal bovine serum, penicillinstreptomycin, 1 mM sodium pyruvate, 10 nM HEPES and 2 mM GlutaMAX™ (Gibco). Concentrated virus was added to the media together with 8 pg/mL polybrene. Forty-eight hours later, the media was changed to fresh media containing puromycin for selection.

[00138] Cell signaling assays. For analysis of human LepR mutants, HEK-293T cells were plated in six-well culture dishes coated with fibronectin (Millipore) at 0.7x10 ⁶ cells per well in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10% v/v fetal bovine serum, penicillinstreptomycin, 1 mM sodium pyruvate, 10 nM HEPES and 2 mM GlutaMAX™ (Gibco). Forty-eight hours later, cells were transfected using FuGene 6 (Promega) with pD649 vector containing HA- or FLAG-tagged full-length human LepR, WT or mutant. Twenty-four hours after transfection, cells were treated with recombinant human Leptin for 20 minutes at 37°C. Cells were then rinsed once with ice-cold PBS and immediately lysed with Triton lysis buffer (1% v/v Triton, 20 mM HEPES pH 7.4, 150 mM NaCI, one tablet of PhosSTOP phosphatase inhibitor cocktail (Roche), and one tablet of EDTA-free protease inhibitor (Roche) (per 10 ml buffer). The cell lysates were cleared by centrifugation at 15,000 g at 4°C for 10 min. Cell lysates were denatured by the addition of SDS sample buffer and boiling for 5 min., resolved by SDS-PAGE, and analyzed by immunoblotting.

[00139] For immunoblot-based analysis of human Leptin variants, HEK-293T cells stably expressing Myc-tagged full-length human LepR were plated in six-well culture dishes coated with fibronectin (Millipore) at 1 x10 ⁶ cells per well in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10% v/v fetal bovine serum, penicillin-streptomycin, 1 mM sodium pyruvate, 10 nM HEPES and 2 mM GlutaMAX™ (Gibco). Twenty-Four hours later, cells were treated with recombinant human Leptin for 20 minutes at 37°C. Cells were then rinsed one time with ice-cold PBS and immediately lysed and analyzed as described above. For experiments analyzing phosphorylation of ERK and SHP2, twenty-four hours after transfection cell media was replaced with serum free DMEM for an additional twenty-four hours before addition of recombinant Leptin. Antibodies used for immunoblots were obtained from Cell Signaling Technologies and include: Phospho-STAT3 (Y705, Antibody #9131 ), Phospho-SHP2 (Y542, Antibody #3751 ), Phospho- ERK1/2 (T202/Y204, Antibody #9101 ), STAT3 (clone 79D7), SHP2 (Antibody #3752), ERK1/2 (clone 137F5), HA (clone C29F4). [00140] For flow-cytometry-based signaling experiments, HEK-293T cells stably expressing Myc- tagged full-length human LepR were plated in 96-well plates and stimulated with WT or mutant Leptin for 20 min at 37°C, followed by fixation with paraformaldehyde (Electron Microscopy Sciences) for 10 min at room temperature. The cells were permeabilized for intracellular staining by treatment with ice cold methanol (Fisher) for 30 min at -20°C. The cells were then incubated with Alexa Fluor 647 conjugated Anti-Stat3 (pY705) antibody (1 :100, BD, clone 4/P-STAT3) and anti-c-Myc-Alexa Fluor 488 (1 :100, CST, clone 9B1 1 ) for 1 hour at room temperature in autoMACS buffer (Miltenyi). Data was acquired using CytoFlex, flow cytometer instrument (Beckman Coulter). The MFI values were background subtracted and represented as a percent of the maximal WT Leptin value within each experiment and plotted in Prism 8 (GraphPad). The dose-response curves were generated using the “sigmoidal dose-response” analysis.

[00141] Gene expression analysis. For gene expression analysis by qPCR, HEK-293T cells stably expressing Myc-tagged full-length human LepR were treated with PBS or Leptin variants for 5 hours. Cells were isolated and lysed using Qiashredder columns (Qiagen) per the manufacturer’s instructions. RNA was isolated using RNAeasy plus mini kit (Qiagen) per the manufacturer’s instructions. 1 pg RNA for each sample was used for cDNA generation with iScript Reverse Transcription Supermix (BioRad). Relative gene expression was measured by SYBR-green based qPCR using the comparative AC _t method and normalized to GAPDH expression. All samples were run in triplicate. The following mouse qPCR primers were obtained from IDT: GAPDH: (5’GGA AAC TTG CTG TGG GTG A3’, 5’CAA GGA CGG AGA CTT CGA TTC3’), SOCS3: (5TGT AGT TGA GGT CAA TGA AGG G3’, 5’ACA TCG CTC AGA CAC CAT G3’).

[00142] Data Availability. Cryo-EM maps and atomic coordinates for LepR ^{D1 D7} receptor complex, LepR ^{D3 D7} receptor complex, and the focused interaction have been deposited in the EMDB (EMD-27432, EMD-27433, EMD-27434) and PDB (8DH8, 8DH9, 8DHA) respectively.

Data Table 1. Cryo-EM data collection refinement, and validation statistics.

Leptin-LepR ^{01 07} complex Leptin-LepR ^{D3 D7} complex Leptin-LepR ^{01 07} complex - (PDB 8DH8/EMD-27432) (PDB 8DH9/EMD-27433) focused refinement

(PDB 8DHA /EMD-27434) Data collection and processing

Voltage (keV) 300 300

Electron exposure (e /A ² ) 53 53

Defocus range (pm) -0.8 to -2.0 -0.8 to -2.0

Pixel size (A) 0 653 0.8521

Symmetry imposed C1 C1

Initial particle images 3,150,756 17,952,333

Final particle images 270,721 137,338 460,818

Map resolution FSC threshold (A) 0.143 0.143 0.143

Map resolution (A) 5.9 4.5 3.8

Refinement Initial model used (PDB) AlphaFold AlphaFold

Model resolution FSC threshold (A) 0.5 0.5 0.5

Model resolution (A) 8 1 4 3 4 3

Map sharpening B-factor (A ² ) 288.6 118.5 150 0

Model Composition

Non-hydrogen atoms 7,644 10,229 3,510

Protein residues 1 ,541 1 ,281 439

Ligands 2

B-factors (A ² )

Protein 477 59 186.15 204.60

Ligand 180.15

R.m.s. deviations

Bond lengths (A) 0.005 0.003 0.004

Bond angles (°) 1 .157 0.649 0.789

Validation

MolProbity score 1 .96 1 .84 2.00

Clashscore 13.09 13.96 14.65

Retainer outliers (%) 0.08 0.49

Ramachandran plot

Favoured (%) 95.17 93.86 95.15

Allowed (%) 4 63 2 91 4 85

Outliers (%) 0.20 0.24 0.00

Leptin receptor sequences (from FIG. 10)

Mouse

REGION D3 (SEQ ID NO:3)

QDVVYFPPKILTSVGSNASFHCIYKNENQIISSKQIVWWRNLAEKIPEIQYSIVSDR VSKVTFSNL

KATRPRGKFTYDAVYCCNEQACHHRYAELYVID

REGION D4 (SEQ ID NO:4)

DVNINISCETDGYLTKMTCRWSPSTIQSLVGSTVQLRYHRRSLYCPDSPSIHPTSEP KNCVLQR

DGFYECVFQPIFLLSGYTMWIRINHSLGSLDSPPTCVLPDSVV

REGION D5 (SEQ ID NO:5)

PPSNVKAEITVNTGLLKVSWEKPVFPENNLQFQIRYGLSGKEIQWKTHEVFDAKSKS ASLLVSN

LCAVYVVQVRCRRLDGLGYWSNWSSPAYTLVM

Human receptor

REGION D3 (SEQ ID NO:6)

QDVIYFPPKILTSVGSNVSFHCIYKKENKIVPSKEIVWWMNLAEKIPQSQYDVVSDH VSKVTFFN

LNETKPRGKFTYDAVYCCNEHECHHRYAELYVID

REGION D4 (SEQ ID NO:7) DVNINISCETDGYLTKMTCRWSTSTIQSLAESTLQLRYHRSSLYCSDIPSIHPISEPKDC YLQSD

GFYECIFQPIFLLSGYTMWIRINHSLGSLDSPPTCVLPDSVV

REGION D5 (SEQ ID NO:8)

PPSSVKAEITINIGLLKISWEKPVFPENNLQFQIRYGLSGKEVQWKMYEVYDAKSKS VSLPVPDL

CAVYAVQVRCKRLDGLGYWSNWSNPAYTVVM

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[00143] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[00144] The present invention has been described in terms of particular embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. Moreover, due to biological functional equivalency considerations, changes can be made in protein structure without affecting the biological action in kind or amount. All such modifications are intended to be included within the scope of the appended claims.