Leptin receptor agonist antibody for use in treatment of metabolic dysfunction or hypoleptinemia

Abstract

To provide therapeutic methods of treating metabolic dysfunction and therapeutic methods of restoring insulin sensitivity in leptin deficiency and lipodystrophy.SOLUTION: The invention relates to a method for treating or preventing a metabolic dysfunction or hypoleptinemia, or a disease or condition associated with metabolic dysfunction or hypoleptinemia, or one or more symptoms of the disease or condition, the method comprising administering a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds human leptin receptor (LEPR) and activates LEPR signaling, and a pharmaceutically acceptable carrier or diluent, to a subject in need thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to therapeutic methods for treating metabolic dysfunction and restoring insulin sensitivity in leptin deficiency and lipodystrophy using agonistic antibodies and antigen-binding fragments of agonistic antibodies that bind to the human leptin receptor (LEPR).

[0002] Sequence Listing An official copy of the Sequence Listing has been submitted electronically via EFS-Web as an ASCII formatted Sequence Listing contemporaneously with the present specification, with the filename "10436WO_SEQ_LIST_ST25," a creation date of April 5, 2019, and a size of approximately 105 kilobytes. The Sequence Listing contained in this ASCII formatted document is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0003] Leptin is a polypeptide hormone primarily expressed by adipose tissue and involved in regulating metabolism, neuroendocrine function, immunity, energy balance, and food intake. Leptin activity is mediated by interaction with and signaling through the leptin receptor. The leptin receptor (also known as LEPR, WSX, OB receptor, OB-R, and CD295) is a single-transmembrane receptor of the class I cytokine receptor family with a large (818 amino acid) extracellular domain. Leptin deficiency, leptin resistance, and specific LEPR signaling-deficient / impaired mutations are associated with obesity, type 2 diabetes, dyslipidemia, lipodystrophy, hepatic steatosis, nonalcoholic and alcoholic fatty liver disease, severe insulin resistance, leprechaunism / Donahue syndrome, Rabson-Mendenhall syndrome, and related complications. Therapeutic approaches to address leptin resistance, leptin deficiency, and hypoleptinemia (e.g., lipodystrophy) have primarily focused on delivering supplemental leptin or leptin analogs to affected individuals. However, such approaches have generally demonstrated limited efficacy, particularly in leptin-resistant individuals, and are frequently associated with adverse side effects. Thus, there is a need in the art for alternative approaches to treating leptin resistance and other conditions associated with leptin deficiency or hypoleptinemia. Summary of the Invention

[0004] The present invention provides antibodies and antigen-binding fragments thereof that bind to the human leptin receptor (LEPR). The antibodies of the present invention are agonistic antibodies. That is, binding of the anti-LEPR antibodies of the present invention to the LEPR results in activation of leptin receptor signaling, particularly in cells. In certain embodiments, the antibodies of the present invention do not compete with leptin for binding to the LEPR. The antibodies of the present invention are useful, for example, to mimic, substitute for, or complement the biological activity of normal leptin in a subject. Thus, the antibodies and antigen-binding fragments of the present invention are useful for therapeutic treatment of diseases and disorders associated with leptin resistance and leptin deficiency.

[0005] Antibodies of the invention may be full-length (e.g., IgG1 or IgG4 antibodies) or may comprise only the antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments), and may be modified to affect function, e.g., to eliminate residual effector function (Reddy et al., 2000, J. Immunol. 164:1925-1933).

[0006] Exemplary anti-LEPR antibodies of the invention are listed in Tables 1 and 2 herein. Table 1 lists the amino acid sequence identifiers for the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) of exemplary anti-LEPR antibodies. Table 2 lists the nucleic acid sequence identifiers for the HCVR, LCVR, HCDR1, HCDR2 HCDR3, LCDR1, LCDR2, and LCDR3 of exemplary anti-LEPR antibodies.

[0007] The present invention provides antibodies or antigen-binding fragments thereof that specifically bind to LEPR, wherein the HCVR comprises an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0008] The present invention also provides an antibody or antigen-binding fragment thereof that specifically binds to a LEPR comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0009] The present invention also provides antibodies, or antigen-binding fragments thereof, that specifically bind to LEPR, comprising an HCVR and LCVR amino acid sequence pair (HCVR / LCVR) comprising any of the HCVR amino acid sequences listed in Table 1, paired with any of the LCVR amino acid sequences listed in Table 1. According to certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, comprising an HCVR / LCVR amino acid sequence pair contained within any of the exemplary anti-LEPR antibodies listed in Table 1. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 10, 26 / 10, 34 / 10, 42 / 10, 50 / 10, 58 / 66, 74 / 66, and 82 / 66.

[0010] The present invention also provides an antibody or antigen-binding fragment thereof that specifically binds to LEPR, wherein the antibody or antigen-binding fragment comprises a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0011] The present invention also provides an antibody or antigen-binding fragment thereof that specifically binds to LEPR, wherein the antibody or antigen-binding fragment comprises a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0012] The present invention also provides an antibody or antigen-binding fragment thereof that specifically binds to LEPR, wherein the antibody or antigen-binding fragment comprises a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0013] The present invention also provides an antibody or antigen-binding fragment thereof that specifically binds to LEPR, wherein the antibody or antigen-binding fragment comprises a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0014] The present invention also provides an antibody or antigen-binding fragment thereof that specifically binds to LEPR, wherein the antibody or antigen-binding fragment comprises a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0015] The present invention also provides an antibody or antigen-binding fragment thereof that specifically binds to LEPR, wherein the antibody or antigen-binding fragment comprises a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0016] The present invention also provides antibodies, or antigen-binding fragments thereof, that specifically bind to LEPR, comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3), comprising any of the HCDR3 amino acid sequences listed in Table 1 paired with any of the LCDR3 amino acid sequences listed in Table 1. According to certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3 / LCDR3 amino acid sequence pair contained in any of the exemplary anti-LEPR antibodies listed in Table 1. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 8 / 16, 24 / 16, 32 / 16, 40 / 16, 48 / 16, 56 / 16, 64 / 72, 80 / 72, and 88 / 72.

[0017] The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind to LEPR, comprising a set of six CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) contained in any of the exemplary anti-LEPR antibodies listed in Table 1. In certain embodiments, the set of amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 is selected from the group consisting of SEQ ID NOs: 4, 6, 8, 12, 14, 16; 20, 22, 24, 12, 14, 16; 28, 30, 32, 12, 14, 16; 36, 38, 40, 12, 14, 16; 44, 46, 48, 12, 14, 16; 52, 54, 56, 12, 14, 16; 60, 62, 64, 68, 70, 72; 76, 78, 80, 68, 70, 72; and 84, 86, 88, 68, 70, 72.

[0018] In related embodiments, the present invention provides antibodies or antigen-binding fragments thereof that specifically bind to LEPR, comprising a set of six CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) contained within an HCVR / LCVR amino acid sequence pair defined by any of the exemplary anti-LEPR antibodies listed in Table 1. For example, the present invention includes antibodies or antigen-binding fragments thereof that specifically bind to LEPR, comprising a set of amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 contained within an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 10, 26 / 10, 34 / 10, 42 / 10, 50 / 10, 58 / 66, 74 / 66, and 82 / 66. Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specific HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the position of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within antibodies.

[0019] The present invention also provides nucleic acid molecules encoding anti-LEPR antibodies or portions thereof. For example, the present invention provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0020] The present invention also provides nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0021] The present invention also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0022] The present invention also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0023] The present invention also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0024] The present invention also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0025] The present invention also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0026] The present invention also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0027] The present invention also provides nucleic acid molecules encoding HCVRs, wherein the HCVRs comprise a set of three CDRs (i.e., HCDR1, HCDR2, and HCDR3), wherein the set of amino acid sequences of HCDR1, HCDR2, and HCDR3 are as defined by any of the exemplary anti-LEPR antibodies listed in Table 1.

[0028] The present invention also provides a nucleic acid molecule encoding an LCVR, wherein the LCVR comprises a set of three CDRs (i.e., LCDR1, LCDR2, LCDR3), wherein the set of amino acid sequences of LCDR1, LCDR2, and LCDR3 are as defined by any of the exemplary anti-LEPR antibodies listed in Table 1.

[0029] The present invention also provides nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises the amino acid sequence of any of the HCVR amino acid sequences listed in Table 1, and wherein the LCVR comprises the amino acid sequence of any of the LCVR amino acid sequences listed in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments according to this aspect of the invention, the nucleic acid molecule encodes an HCVR and an LCVR, wherein both the HCVR and LCVR are derived from the same anti-LEPR antibody, as listed in Table 1.

[0030] The present invention also provides recombinant expression vectors capable of expressing polypeptides comprising the heavy chain variable region or light chain variable region of an anti-LEPR antibody. For example, the present invention includes recombinant expression vectors comprising any of the above-described nucleic acid molecules, i.e., nucleic acid molecules encoding any of the HCVR, LCVR, and / or CDR sequences as set forth in Table 1. Also included within the scope of the present invention are host cells into which such vectors have been introduced, as well as methods for producing antibodies or portions thereof by culturing the host cells under conditions that permit the production of the antibody or antibody fragment, and methods for recovering the antibodies and antibody fragments so produced.

[0031] In another aspect, the invention provides a pharmaceutical composition comprising a recombinant human antibody or fragment thereof that specifically binds to LEPR and a pharmaceutically acceptable carrier. In a related aspect, the invention features a composition that is a combination of an anti-LEPR antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-LEPR antibody.

[0032] As used throughout this disclosure, the term "subject" is interchangeable with the term "patient." A subject or patient may be an adult. Pediatric patients are also expected to benefit from the methods and compositions provided herein.

[0033] In yet another aspect, the present invention provides therapeutic methods for enhancing or stimulating LEPR signaling using an anti-LEPR antibody of the present invention, or an antigen-binding portion of an antibody of the present invention. The therapeutic method according to this aspect of the present invention comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an antibody of the present invention or an antigen-binding fragment of an antibody of the present invention. The disorder to be treated is any disease or condition that is ameliorated, improved, inhibited, or prevented by stimulating or activating LEPR signaling or by otherwise mimicking the natural activity of leptin in vitro or in vivo.

[0034] In some aspects, provided herein are therapeutic methods for treating or preventing metabolic dysfunction or hypoleptinemia, the methods comprising administering to a subject in need thereof a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to the human leptin receptor (LEPR) and activates LEPR signaling, and a pharmaceutically acceptable carrier or diluent.

[0035] In some aspects, provided herein are therapeutic methods for treating or preventing metabolic dysfunction or hypoleptinemia, or a disease or condition associated with metabolic dysfunction or hypoleptinemia, or one or more symptoms of said disease or condition, comprising administering to a subject in need thereof a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to human LEPR and activates LEPR signaling and a pharmaceutically acceptable carrier or diluent.

[0036] In some embodiments, the condition is selected from the group consisting of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis (NASH), female infertility, amenorrhea, hormone cycle abnormalities, immune dysfunction, hypothyroidism, obesity, monogenic obesity, type I diabetes, type II diabetes, lipodystrophy, congenital lipodystrophy, generalized lipodystrophy, acquired lipodystrophy, partial lipodystrophy, congenital partial lipodystrophy, congenital generalized lipodystrophy, acquired partial lipodystrophy, and acquired generalized lipodystrophy.

[0037] In some embodiments, the one or more symptoms of a disease or condition associated with metabolic dysfunction or hypoleptinemia are selected from the group consisting of adiposity, obesity, hyperphagia, hyperglycemia, hypertriglyceridemia, hypercholesterolemia, insulin resistance, dyslipidemia, growth retardation, delayed pubertal growth spurt, impaired growth hormone secretion, elevated HbA1c, low bone mineral density (or low bone mass), low bone mineral content, and low lean body mass. Symptoms of a disease or condition associated with metabolic dysfunction or hypoleptinemia can be prevented, ameliorated, or reduced in severity and / or duration or reduced following administration of an antibody or antigen-binding fragment thereof that binds to human LEPR.

[0038] In yet another aspect, provided herein are methods for treating metabolic complications of lipodystrophy. The method includes administering to a subject in need thereof a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to the human leptin receptor (LEPR) and activates LEPR signaling and a pharmaceutically acceptable carrier or diluent. In some embodiments, treatment alleviates hyperglycemia, reduces insulin resistance, reduces hypertriglyceridemia, lowers circulating cholesterol levels, and / or lowers HbA1c levels in the subject. Lipodystrophy can include acquired partial lipodystrophy, acquired generalized lipodystrophy, congenital partial lipodystrophy, and congenital generalized lipodystrophy.

[0039] Congenital leptin deficiency is a rare disease characterized by pathogenic variants in LEPR or leptin. Some subjects have circulating leptin, but the protein is nonfunctional due to gene mutations, such as p.N103K, which encodes a biologically inactive form of leptin. Some subjects have little or no circulating leptin. Other genes, including LMNA, PPARG, PLIN1, AKT2, CIDEC, LIPE, and ADRA2A, may also be involved in impaired leptin signaling, and the anti-LEPR antibodies and antigen-binding fragments thereof provided herein are useful for reducing the effects of these mutations on leptin signaling.

[0040] In some aspects, provided herein are methods for treating congenital leptin deficiency. The method includes administering to a subject in need thereof a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to the human leptin receptor (LEPR) and activates LEPR signaling and a pharmaceutically acceptable carrier or diluent. In some embodiments, the subject has lipodystrophy and has failed metreleptin treatment. In some embodiments, symptoms associated with congenital lipodystrophy are prevented, ameliorated, or reduced in severity and / or duration after administration of the antibody or antigen-binding fragment thereof that binds to human LEPR. In some embodiments, the treatment reverses or reduces one or more of hyperphagia, obesity, hyperinsulinemia, dyslipidemia, and hepatic steatosis in the subject. In some embodiments, the subject's blood glucose is reduced, the subject's weight is reduced, the subject exhibits reduced food intake, the subject's fat mass is reduced, the subject's lean mass is increased, and / or the subject's bone mass is increased.

[0041] In some aspects, provided herein are therapeutic methods for the treatment of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis (NASH). In some aspects, the subject has hypoleptinemia, lipodystrophy, or leptin deficiency. According to this aspect, the method comprises administering to a subject in need thereof a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to human LEPR and activates LEPR signaling and a pharmaceutically acceptable carrier or diluent. In some embodiments, the subject's liver weight is reduced after treatment. In some embodiments, the symptoms of non-alcoholic fatty liver disease, including non-alcoholic hepatic steatosis, are alleviated in the subject after treatment. In some embodiments, the plasma level of alanine transaminase (ALT) and / or the plasma level of aspartate transaminase (AST) is reduced in the subject.

[0042] In yet another aspect, provided herein are methods for treating female infertility, amenorrhea, or restoring normal hormone cycles associated with metabolic dysfunction or hypoleptinemia. The methods include administering to a subject in need thereof a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to human LEPR and activates LEPR signaling and a pharmaceutically acceptable carrier or diluent. In some embodiments, administration of an antibody or antigen-binding fragment thereof that binds to human LEPR can increase fertility and / or increase the chance of pregnancy. In some embodiments, the subject becomes pregnant. In some embodiments, treatment can restore or initiate normal menstrual cycles.

[0043] In some aspects, provided herein are methods for treating immune dysfunction associated with metabolic dysfunction or hypoleptinemia. The method comprises administering to a subject in need thereof a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to human LEPR and activates LEPR signaling and a pharmaceutically acceptable carrier or diluent. In some embodiments, administration of the antibody or antigen-binding fragment thereof that binds to human LEPR results in an increase in CD4+ T cell count.

[0044] In another aspect, provided herein is a method for increasing bone mass in a subject with metabolic dysfunction or hypoleptinemia, comprising administering to a subject in need thereof a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to human LEPR and activates LEPR signaling and a pharmaceutically acceptable carrier or diluent.

[0045] In another aspect, provided herein is a method for treating adiposity or obesity, or a method for reducing body weight. According to this aspect, the method comprises administering to a subject in need thereof a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to human LEPR and activates LEPR signaling, and a pharmaceutically acceptable carrier or diluent. In some embodiments, the treatment reduces fat mass but does not reduce lean mass.

[0046] In some embodiments, the subject in need thereof has hypoleptinemia, lipodystrophy, or leptin deficiency. In some embodiments, the subject in need thereof is not hypoleptinemia or leptin deficiency. In some embodiments, the metabolic dysfunction, adiposity, or obesity is not associated with or caused by a signaling-deficient or signaling-impaired LEPR mutation.

[0047] In some embodiments, administration of an antibody or antigen-binding fragment that binds to human LEPR and activates LEPR signaling in accordance with the methods provided herein stimulates hypothalamic STAT3 signaling or enhances leptin-induced or leptin-independent STAT3 signaling.

[0048] In some embodiments, administration of an antibody or antigen-binding fragment thereof that binds to human LEPR and activates LEPR signaling lowers circulating plasma triglycerides and / or lowers circulating plasma total cholesterol.

[0049] In another aspect, provided herein are methods for treating hyperphagia, hyperglycemia, insulin resistance, dyslipidemia, nonalcoholic steatohepatitis (NASH), or nonalcoholic fatty liver disease by stimulating hypothalamic STAT3 signaling. The methods include administering to a subject in need thereof a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to the human leptin receptor (LEPR) and activates LEPR signaling, and a pharmaceutically acceptable carrier or diluent. In some embodiments, the treatment reduces circulating plasma triglycerides. In some embodiments, the treatment reduces circulating plasma total cholesterol.

[0050] In yet another aspect, provided herein is a method for treating growth retardation, lack of pubertal growth spurt, and / or growth hormone deficiency associated with congenital leptin deficiency, comprising administering to a subject in need thereof a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to human LEPR and activates LEPR signaling and a pharmaceutically acceptable carrier or diluent.

[0051] In yet another aspect, provided herein is a method for treating hypothyroidism associated with congenital leptin deficiency, comprising administering to a subject in need thereof a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to human LEPR and activates LEPR signaling and a pharmaceutically acceptable carrier or diluent.

[0052] In yet another aspect, provided herein are methods for treating low bone mineral density and / or low bone mineral content associated with hypoleptinemia and / or leptin deficiency, comprising administering to a subject in need thereof a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to human LEPR and activates LEPR signaling and a pharmaceutically acceptable carrier or diluent.

[0053] One or more additional therapeutic agents may be administered to a subject described herein along with an antibody or antigen-binding fragment thereof that binds to human LEPR. The second therapeutic agent may be recombinant human leptin, a PCSK9 inhibitor, a statin, ezetimibe, insulin, an insulin variant, an insulin secretagogue, metformin, a sulfonylurea, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, a GLP-1 agonist / analog, a glucagon (GCG) inhibitor, a glucagon receptor (GCGR) inhibitor, an angiopoietin-like protein (ANGPTL) inhibitor, phentermine, orlistat, topiramate, bupropion, topiramate / phentermine, bupropion / naltrexone ( Naltrexone, Bupropion / Zonisamide, Pramlintide / Metreleptin, Lorcaserin, Cetilistat, Tesofensine, Velneperit, anticonvulsants, digoxin, Coumadin, Vitamin D, Thyroxine, thyroid supplements, vitamin supplements, calcium supplements, carnitine, Coenzyme Q10, anti-constipation medications, anti-allergy medications, Gabapentin, anesthetics, ketamine, lidocaine, or venlafaxine hydrochloride.

[0054] The present invention also provides methods of treating, preventing, or ameliorating (i) lipodystrophy (any type) and / or monogenic obesity; (ii) a condition associated with lipodystrophy and / or monogenic obesity; or (iii) a symptom of (i) or (ii) in a patient, the methods comprising administering to a patient in need thereof an agonistic antibody (e.g., H4H17319P2) or an antigen-binding fragment thereof that specifically binds to LEPR. For example, in embodiments of the present invention, the condition associated with lipodystrophy and / or monogenic obesity is very early-onset obesity; hyperphagia and impaired satiety; impaired immune function (CD4 +number); insulin resistance; nonalcoholic fatty liver disease; NASH, dyslipidemia; diabetes; reproductive dysfunction; hypogonadism; missed pubertal growth spurt; hypothyroidism; thyroid dysfunction; low bone mineral density and / or low bone mass. In an embodiment of the invention, the symptoms are enlarged liver, elevated liver enzymes, elevated blood levels of alanine aminotransferase (ALT), elevated blood levels of aspartate aminotransferase (AST), advanced steatosis; body mass index greater than the 85th percentile for age and sex; abnormal food-seeking behavior; abnormal food-aggression behavior; recurrent and potentially fatal infections; hyperinsulinemia; hepatic steatosis; progression to NASH (lipodystrophy); hypertriglyceridemia; elevated HbA1c; high blood glucose levels; impaired glucose tolerance; delayed pubertal development; decreased onset of secondary sexual characteristics; amenorrhea or menstrual irregularities; infertility; dwarfism; abnormal growth hormone secretion; altered T3; altered TSH; and / or altered free thyroxine levels.

[0055] In embodiments of the invention, an agonistic anti-LEPR antibody or antigen-binding fragment thereof (e.g., H4H17319P2; see WO2017 / 66204) is administered as follows: (i) one or more intravenous doses of about 5 mg / kg body weight, followed by (ii) one or more subcutaneous doses of about 250-300 mg, e.g., 250 mg or about 300 mg, once per week, followed by (iii) optionally, one or more subcutaneous doses of about 250 mg or about 300 mg once per month or about once every 28 days. For example, (i) one or more intravenous doses of about 5 mg / kg body weight, followed by (ii) one or more subcutaneous doses of about 250 mg or about 300 mg once per week. In embodiments of the invention, the antibody is administered as follows: (i) a single intravenous dose of 5 mg / kg body weight (on day 1); then (ii) four subcutaneous doses of about 250 mg or about 300 mg weekly (e.g., on days 4, 11, 18, and 25); then (iii) one or more subcutaneous doses of about 250 mg or about 300 mg monthly or every 28 days (e.g., on days 53, 81, 109, 137, 165, and 193). For example, in embodiments of the invention, the first subcutaneous dose is administered on about day 4, which is about three days after the intravenous dose administered on day 1.

[0056] Other embodiments will become apparent from consideration of the following detailed description. [The present invention 1001] A method for treating or preventing metabolic dysfunction or hypoleptinemia, or a disease or condition associated with metabolic dysfunction or hypoleptinemia, or one or more symptoms of said disease or condition, comprising administering to a subject in need thereof a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to the human leptin receptor (LEPR) and activates LEPR signaling, and a pharmaceutically acceptable carrier or diluent. [The present invention 1002] 1001. The method of claim 1001, wherein said condition is selected from the group consisting of non-alcoholic fatty liver disease, NASH, female infertility, amenorrhea, hormone cycle abnormalities, immune dysfunction, hypothyroidism, obesity, monogenic obesity, type I diabetes, type II diabetes, lipodystrophy, congenital lipodystrophy, generalized lipodystrophy, acquired lipodystrophy, partial lipodystrophy, congenital partial lipodystrophy, congenital generalized lipodystrophy, acquired partial lipodystrophy, and acquired generalized lipodystrophy. [The present invention 1003] The method of the present invention 1002 for treating or preventing non-alcoholic fatty liver disease. [The present invention 1004] The method of the present invention 1002 for treating or preventing congenital lipodystrophy. [The present invention 1005] The method of the present invention 1002 for treating or preventing generalized lipodystrophy. [The present invention 1006] The method of the present invention 1002 for treating or preventing acquired lipodystrophy. [The present invention 1007] The method of the present invention 1002 for treating or preventing partial lipodystrophy. [The present invention 1008] The method of the present invention 1002 for treating or preventing monogenic obesity. [The present invention 1009] the condition associated with metabolic dysfunction or hypoleptinemia is congenital lipodystrophy; The method of the present invention 1002, wherein the symptoms associated with congenital lipodystrophy are prevented, ameliorated, or reduced in severity and / or duration or reduced after administration of the antibody or antigen-binding fragment thereof that binds to human LEPR. [The present invention 1010] The method of claim 1009, wherein after administration of the antibody or antigen-binding fragment thereof that binds to human LEPR, the subject's blood glucose is reduced, the subject's body weight is reduced, the subject exhibits reduced food intake, the subject's fat mass is reduced, the subject's lean mass is increased, and / or the subject's bone mass is increased. [The present invention 1011] The method of claim 1002, wherein the condition is non-alcoholic fatty liver disease and, after treatment, the subject's liver weight is reduced, the subject's plasma level of alanine transaminase (ALT) is reduced, and / or the subject's plasma level of aspartate transaminase (AST) is reduced. [The present invention 1012] 1003. The method of claim 1002, wherein said condition is female infertility. [The present invention 1013] The method of claim 1012, wherein said condition is female infertility and said subject's hormone cycle is restored and / or said subject becomes pregnant. [The present invention 1014] The method of claim 1002, wherein said condition is amenorrhea. [The present invention 1015] The method of claim 1014, wherein said condition is amenorrhea and said subject begins to have normal hormone cycles. [The present invention 1016] 1002. The method of claim 1002, wherein said condition is an immune dysfunction. [The present invention 1017] 1016. The method of claim 1016, wherein said condition is immune dysfunction and said subject has an increased CD4+ T cell count. [The present invention 1018] one or more symptoms of a disease or condition of or associated with metabolic dysfunction or hypoleptinemia, which are one or more selected from the group consisting of adiposity, obesity, hyperphagia, hyperglycemia, hypertriglyceridemia, hypercholesterolemia, insulin resistance, dyslipidemia, growth retardation, delayed pubertal growth spurt, abnormal growth hormone secretion, elevated HbA1c, low bone mineral density (or low bone mass), low bone mineral content, and low lean body mass 1001. A method of treating or preventing a disease according to claim 1001. [The present invention 1019] A method for increasing bone mass in a subject having low bone mass that is a symptom of a metabolic dysfunction or hypoleptinemia from which the subject is suffering, comprising administering to the subject in need thereof an antibody or antigen-binding fragment thereof that binds to the human leptin receptor (LEPR) and activates LEPR signaling, and a pharmaceutically acceptable carrier or diluent. [The present invention 1020] The method of any of claims 1001 to 1019, wherein said subject in need thereof has leptin deficiency. [The present invention 1021] The method of any of claims 1001 to 1019, wherein said subject in need thereof does not have a leptin deficiency. [The present invention 1022] The method of any of claims 1001 to 1021, wherein said condition is obesity, and said obesity is not associated with or caused by a signaling-deficient or signaling-impaired LEPR mutation. [The present invention 1023] The method of any one of claims 1001 to 1022, wherein said treatment reduces fat mass but does not reduce lean mass. [The present invention 1024] Any of the methods of claims 1001 to 1023, wherein the treatment with the antibody or antigen-binding fragment thereof that binds to human LEPR and activates LEPR signaling stimulates hypothalamic STAT3 signaling or enhances leptin-induced or leptin-independent STAT3 signaling. [The present invention 1025] The method of any one of claims 1001 to 1024, wherein said treatment reduces circulating plasma triglycerides. [The present invention 1026] The method of any one of claims 1001 to 1025, wherein said treatment reduces circulating plasma total cholesterol. [The present invention 1027] In patients, (i) lipodystrophy and / or monogenic obesity, (ii) a condition associated with lipodystrophy and / or monogenic obesity, or (iii) Symptoms of (i) or (ii) 1. A method for treating, preventing, or ameliorating a disorder comprising administering to said patient in need thereof an agonistic antibody that specifically binds to LEPR. [The present invention 1028] The conditions associated with lipodystrophy and / or monogenic obesity include very early onset obesity; hyperphagia and impaired satiety; impaired immune function (CD4 + number); insulin resistance; non-alcoholic fatty liver disease; NASH, dyslipidemia; diabetes; reproductive dysfunction; hypogonadism; missed pubertal growth spurt; hypothyroidism; thyroid dysfunction; low bone mineral density or low bone mass. [The present invention 1029] The method of the present invention 1027, wherein the symptoms are enlarged liver, elevated liver enzymes, elevated blood levels of alanine aminotransferase (ALT), elevated blood levels of aspartate aminotransferase (AST), advanced steatosis; body mass index above the 85th percentile for age and sex; abnormal food-seeking behavior; abnormal food-aggression behavior; recurrent and potentially fatal infections; hyperinsulinemia; hepatic steatosis; progression to NASH (lipodystrophy); hypertriglyceridemia; elevated HbA1c; high blood glucose levels; impaired glucose tolerance; delayed pubertal development; decreased expression of secondary sexual characteristics; amenorrhea or menstrual irregularities; infertility; dwarfism; abnormal growth hormone secretion; altered T3; altered TSH; or altered free thyroxine levels. [The present invention 1030] The method of any of claims 1001 to 1029, wherein said subject has failed metreleptin treatment. [The present invention 1031] 10. The method of any one of claims 1001 to 1030, wherein the antibody is administered as follows: (i) one or more intravenous doses of about 5 mg / kg body weight; then (ii) one or more subcutaneous doses of about 250 mg or about 300 mg once weekly; then (iii) optionally, one or more subcutaneous doses of about 250 mg or about 300 mg once a month or about once every 28 days. [The present invention 1032] 10. The method of any one of claims 1001 to 1030, wherein the antibody is administered as follows: (i) one or more intravenous doses of about 5 mg / kg body weight; then (ii) One or more subcutaneous doses of about 250 mg or about 300 mg once weekly. [The present invention 1033] 10. The method of any one of claims 1001 to 1031, wherein the antibody is administered as follows: (i) a single intravenous dose of 5 mg / kg body weight; then (ii) four subcutaneous doses of about 250 mg or about 300 mg once weekly; then (iii) One or more subcutaneous doses of about 250 mg or about 300 mg once a month or once every 28 days. [The present invention 1034] The method of any of claims 1031 to 1033, wherein the first subcutaneous administration is performed 3 days after said intravenous administration. [This invention 1035] the antibody or antigen-binding fragment thereof (a) a complementarity determining region (CDR) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 74, or SEQ ID NO: 82; and (b) CDR of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 66 Any of the methods of 1001 to 1034 of the present invention, comprising: [The present invention 1036] Any of the methods of claims 1001 to 1034, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain CDR and a light chain CDR of an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 26 / 10, 34 / 10, 42 / 10, 50 / 10, 58 / 66, 74 / 66 and 82 / 66. [This invention 1037] Any of the methods of claims 1001 to 1034, wherein the antibody or antigen-binding fragment thereof comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 26 / 10, 34 / 10, 42 / 10, 50 / 10, 58 / 66, 74 / 66 and 82 / 66. [The present invention 1038] The method of any of claims 1001 to 1034, wherein the antibody or antigen-binding fragment thereof comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 26 / 10. [This invention 1039] The method of any of claims 1001 to 1034, wherein the antibody or antigen-binding fragment thereof comprises a combination of HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 amino acid sequences of SEQ ID NOs: 28 / 30 / 32 / 12 / 14 / 16. [The present invention 1040] The method further comprises administering to the subject a second therapeutic agent, wherein the second therapeutic agent is selected from the group consisting of recombinant human leptin, PCSK9 inhibitors, statins, ezetimibe, insulin, insulin variants, insulin secretagogues, metformin, sulfonylureas, sodium-glucose cotransporter 2 (SGLT2) inhibitors, GLP-1 agonists / analogs, glucagon (GCG) inhibitors, glucagon receptor (GCGR) inhibitors, angiopoietin-like protein (ANGPTL) inhibitors, phentermine, orlistat, topiramate, bupropion, and topiramate. / phentermine, bupropion / naltrexone, bupropion / zonisamide, pramlintide / metreleptin, lorcaserin, cetilistat, tesofensine, velneperit, anticonvulsants, digoxin, coumadin, vitamin D, thyroxine, thyroid supplements, vitamin supplements, calcium supplements, carnitine, coenzyme Q10, anti-constipation medications, anti-allergy medications, gabapentin, anesthetics, ketamine, lidocaine, and venlafaxine hydrochloride. [Brief explanation of the drawings]

[0057] [Figure 1] FIG. 1 shows the binding of dimeric human LEPR to human leptin in the presence of increasing concentrations of test anti-LEPR antibodies or control molecules, as measured by ELISA (absorbance at 450 nm).

[0058] [Figure 2] Figures 2A-2C show the extent of LEPR signaling in HEK293 cells expressing either wild-type LEPR (circles), a signaling-deficient LEPR mutant (A409E, squares), or a signaling-impaired LEPR mutant (P316T, triangles). LEPR signaling is expressed as the ratio of pSTAT3-Y705 / STAT3 and was measured by densitometry of Western blots prepared from cells treated with increasing concentrations of leptin (Figure 2A), H4H16650 (Figure 2B), or H4H16679 (Figure 2C).

[0059] [Figure 3] FIG. 3 shows the average daily food intake of leptin-deficient mice administered either 3 mg / kg of an isotype control antibody or 3 mg / kg of a LEPR antibody selected from H4H16650P2, H4H16679P2, H4H17319P2, or H4H17321P2.

[0060] [Figure 4] FIG. 4 shows the mean percent change in body weight for mice administered either 3 mg / kg of an isotype control antibody or 3 mg / kg of a LEPR antibody selected from H4H16650P2, H4H16679P2, H4H17319P2, or H4H17321P2.

[0061] [Figure 5] FIG. 5 shows the mean fat mass of animals in each antibody treatment group as quantified by μCT 1 day before (unshaded bars) and 6 days after (shaded bars) antibody treatment, expressed as the mean±SEM.

[0062] [Figure 6] FIG. 6 shows the percent change in body weight of mice given 30 mg / kg of an antibody selected from H4H18482P2, H4H18487P2, H4H18492P2 or an isotype control.

[0063] [Figure 7] Figures 7A-7B. Figure 7A shows the fat mass of mice before administration of anti-LEPR antibodies H4H18482P2, H4H18487P2, or H4H18492P2. Figure 7B shows the fat mass of mice treated with 30 mg / kg of H4H18482P2, H4H18487P2, or H4H18492P2.

[0064] [Figure 8] Figure 8. Figure 8 shows that the validated anti-LEPR antibodies activated monkey (Mf) LEPR in the IMR-32 / STAT3-luc / Mf LEPR cell line.

[0065] [Figure 9A] Figures 9A-9C. Leptin deficiency was induced in 18-week-old male Leprhu / hu mice by HDD of mLepr.ECD on day 0. Seven days after HDD, mice were stratified into two groups based on the relative percent change in body weight and received a single 10 mg / kg SC dose of control monoclonal antibody (gray circles or bars) or H4H17319P2 (black circles or bars). Data are mean ± SEM. *, P<0.05 for the comparison of H4H17319P2 and control monoclonal antibody at the indicated time points. $, P<0.05 between pre-HDD (day -1) and any day after HDD (day 6 or 13) within each treatment group. &, P<0.05 between pre-monoclonal antibody (day 6) and post-monoclonal antibody (day 13) within each treatment group. For the groups indicated by each font color, there were no significant differences (ns) from the day 0 baseline on the indicated days. Figure 9A shows the body weight (left) and daily food intake (right) of Leprhu / hu mice over the course of the experiment, demonstrating that induction of leptin deficiency results in rapid weight gain and hyperphagia. N = 14 per group. Figure 9B shows body composition analysis by micro-CT imaging performed 1 day before HDD (day -1), 1 day before monoclonal antibody administration (day 6 after HDD), and 6 days after administration (day 13 after HDD). Quantification of fat mass (left) and lean mass (right) is shown for the control monoclonal antibody-treated group (N = 14) and the H4H17319P2 (N = 14)-treated group (gray and black bars, respectively). Figure 9C. Analysis of plasma obtained 6 days after treatment (day 13) from inducible leptin-deficient Leprhu / hu mice treated with a single dose of control monoclonal antibody (gray bars, N=14) or H4H17319P2 (black bars, N=14). [Figure 9B] See the legend to Figure 9A. [Figure 9C] See the legend to Figure 9A.

[0066] [Figure 10A]1 shows gene targeting to generate humanized Leprhu / hu mice of the leptin receptor extracellular domain. [Figure 10B] Micro-CT quantification of body weight (9 weeks of age) and body composition (9–12 weeks of age) in male wild-type (Lepr+ / +, gray bars) and Leprhu / hu (black bars) mice. Data are mean ± SEM. N = 6–10 mice per group. [Figure 10C] Insulin tolerance test (0.75 U / kg, IP) in 8-11 week old male Lepr+ / + (gray circles) and Leprhu / hu (black circles) mice. Data are mean ± SEM. N = 8-9 mice per group. [Figure 10D] Serum leptin levels in male Lepr+ / + (gray bars) and Leprhu / hu (black bars) mice aged 9–13 weeks. Data are mean ± SEM. N = 8–9 mice per group.

[0067] [Figure 11] Figures 11A-11B. Figure 11A. Body weight (left), percent change in body weight from baseline on day 0 (center), and cumulative food intake (right) in 8-week-old male C57BL / 6N mice after hydrodynamic DNA delivery (HDD) on day 0 of a plasmid encoding the mouse leptin receptor extracellular domain (mLeprECD.hFc, black circles) or a control plasmid (Control-hFc, gray circles). Data are means ± SEM. N = 6 per group. Figure 11B. Micro-CT quantification of body composition 7 days after HDD of a plasmid encoding the mouse leptin receptor extracellular domain (mLeprECD.hFc, black bars) or a control plasmid (Control-hFc, gray bars). Data are means ± SEM for fat mass, lean mass, bone mass, bone mineral content, and bone mineral density (left to right). N = 6 per group.

[0068] [Figure 12A]Figures 12A-12C. Leptin deficiency was induced on day 0 by HDD of mLepr.ECD in 17- to 20-week-old female or 18-week-old male Leprhu / hu mice. Seven days after HDD, mice were stratified into two groups based on the relative percent change in body weight and then received a single 10 mg / kg SC dose of control monoclonal antibody (gray circles or bars) or H4H17319P2 (black circles or bars). Data are mean ± SEM. *, P<0.05 for the comparison of H4H17319P2 and control monoclonal antibody at the indicated time points. $, P<0.05 between pre-HDD (day -1) and any day after HDD (day 6 or 13) within each treatment group. &, P<0.05 between pre-monoclonal antibody (day 6) and post-monoclonal antibody (day 13) within each treatment group. There were no significant differences (ns) from the day 0 baseline for the groups indicated by each font color on the indicated days. Figure 12A. Body weight (left) and food intake (right) of female mice over the course of the experiment demonstrate that induction of leptin deficiency results in rapid weight gain and hyperphagia. Weight gain continues after administration of a control monoclonal antibody (N = 14). N = 10–11 mice per group. Figure 12B. Body composition analysis by micro-CT imaging of female mice was performed 1 day before HDD (day -1), 1 day before monoclonal antibody administration (day 6 after HDD), and 6 days after administration (day 13 after HDD). Data are mean ± SEM for fat mass, lean mass, bone mass, bone mineral content, and bone mineral density (left to right). N = 10–11 mice per group. Figure 12C. Body composition analysis by micro-CT imaging of male mice was performed 1 day before HDD (day -1), 1 day before monoclonal antibody administration (day 6 after HDD), and 6 days after administration (day 13 after HDD). Data are mean ± SEM for bone mass, bone mineral content, and bone mineral density (left to right). N = 14 per group. [Figure 12B] See the legend to Figure 12A. [Figure 12C] See the legend to Figure 12A.

[0069] [Figure 13A] Figures 13A-13D. Leptin deficiency was induced on day 0 in 17- to 20-week-old female Leprhu / hu mice by HDD with mLepr.ECD. For comparison, a group of mice underwent HDD with a control vector. Seven days after HDD, mice receiving mLepr.ECD were stratified into three groups based on body weight and administered 3 mg / kg SC twice (days 7 and 13) of either a control monoclonal antibody (gray circles or bars) or H4H17319P2 (dark gray circles or bars). Alternatively, mice were pair-fed (light gray circles or bars) using the amount of food consumed by inducible leptin-deficient mice treated with H4H17319P2. Data are means ± SEM. *, P<0.05 for each group indicated by the symbol color or bar, compared with HDD mLeprECD administered with a control monoclonal antibody at the indicated time point. # indicates statistical significance for pair-fed animals in HDD mLepr ECD treated with H4H17319P2 compared with HDD mLepr ECD. $, P<0.05 for each group indicated by the symbol color or bar compared with HDD control mice administered a control monoclonal antibody at the indicated time points. Figure 13A shows the change in body weight (left) and cumulative food intake (right) of mice from pre-HDD. N=6-11 mice per group. Figure 13B shows micro-CT quantification of body composition for Leprhu / hu mice before HDD on day 13 (7 days after treatment). Fat mass, lean mass, bone mass, bone mineral content, and bone mineral density (left to right). N=5-10 mice per group. Figure 13C shows insulin tolerance testing on day 10 after HDD (3 days after treatment). Mice were fasted for 4 hours and then treated with insulin (1.0 U / kg, IP) at 0 min. Blood glucose levels and glucose area under the curve (AUC) from the insulin tolerance test are shown (left and right, respectively). N=6-11 animals per group. Figure 13D shows plasma lipid chemistry analysis obtained on day 16 or 17. N=6-11 animals per group. [Figure 13B] See the description of Figure 13A. [Figure 13C] See the description of Figure 13A. [Figure 13D]See the description of Figure 13A.

[0070] [Figure 14A]Figures 14A-G. Male lipodystrophic aP2-nSrebp1cTg / +; Leprhu / hu mice (Tg) were administered 10 mg / kg (SC) of control monoclonal antibody (gray circles or bars) or H4H17319P2 (black circles or bars) once a week from 27 to 30 weeks of age. As a reference, male non-transgenic Leprhu / hu mice (non-Tg) were also administered 10 mg / kg (SC) of control monoclonal antibody (white diamonds and white bars) once a week from 27 to 30 weeks of age. All data are mean ± SEM. *, P<0.05 for each group indicated by the symbol color or bar compared to non-Tg mice administered control monoclonal antibody at the indicated time points. #, P<0.05 for each group indicated by the symbol color or bar compared to Tg mice administered control monoclonal antibody at the indicated time points. $, P<0.05, day 27 compared with day -5 for each group. Figure 14A, left and right, show body weight and cumulative food intake, respectively. N = 8–9 mice per group. Figure 14B, left and right, show lean mass and fat mass quantified by micro-CT imaging before dosing on day -5 and after treatment on day 27, respectively. N = 9 mice per group. Figure 14C shows blood glucose levels during the study (left) and percent hemoglobin A1c levels on day 28 (right). N = 9 mice per group. Figure 14D shows blood glucose levels and the area under the glucose curve (AUC) obtained from an insulin tolerance test (0.5 U / kg IP) on day 23. N = 9 mice per group. Figures 14E and 14F show that circulating lipids (E) and liver enzyme levels (F) were reduced in H4H17319P2-treated lipodystrophic Leprhu / hu (Tg) mice. Chemical analysis of plasma obtained on day 28 for lipids (triglycerides, cholesterol, LDL-C, and HDL-C) and liver enzyme levels (alanine transaminase, ALT, and aspartate transaminase, AST). N=9 per group. Figure 14G shows liver weight, liver triglyceride content, and representative hematoxylin-eosin stained liver sections (left, center, and right, respectively) collected on day 30. N=5 per group. [Figure 14B] See the legend to Figure 14A. [Figure 14C] See the legend to Figure 14A. [Figure 14D] See the legend to Figure 14A. [Figure 14E] See the legend to Figure 14A. [Figure 14F] See the legend to Figure 14A. [Figure 14G] See the legend to Figure 14A.

[0071] [Figure 15A] Figures 15A-C. Phenotypic analysis of male non-Tg Leprhu / hu (black circles or black bars) and aP2-nSrebp1cTg / + Leprhu / hu mice (black circles and black bars) at the indicated ages. Data are means ± SEM. *p<0.05 compared with Leprhu / hu mice at the indicated time points. Figure 15A. Left: Body weight at 12-24 weeks of age. Center: Fat mass quantified by micro-CT imaging at approximately 15-20 weeks of age. Right: Plasma leptin levels measured at 19-21 weeks of age. N = 17-28 mice per group. Figure 15B. Blood glucose levels during an insulin tolerance test (0.75 U / kg insulin, IP) at 18-20 weeks of age. N = 12-13 mice per group. Figure 15C. From left to right, plasma levels of triglycerides, cholesterol, LDL-C, and HDL-C at 15–17 weeks of age. N = 20–28 animals per group. [Figure 15B] See the legend to Figure 15A. [Figure 15C] See the legend to Figure 15A.

[0072] [Figure 16A]Figure 16A. Male lipodystrophic aP2-nSrebp1cTg / +; Leprhu / hu mice (Tg) were administered 10 mg / kg (SC) of a control monoclonal antibody (gray bars) or H4H17319P2 (black bars) once a week from 27 to 30 weeks of age. As a reference, male non-transgenic Leprhu / hu mice (non-Tg) at 27 to 30 weeks of age were also administered 10 mg / kg (SC) of a control monoclonal antibody (white bars) once a week. All data are mean ± SEM. *, P<0.05 for each group compared with non-Tg mice administered the control monoclonal antibody at the indicated time points. #, P<0.05 for each group compared with Tg mice administered the control monoclonal antibody at the indicated time points. $, P<0.05. Day 27 compared with day -5 for each group. Figure 16A. Body composition by micro-CT imaging showing bone mass, bone mineral content, and bone density (left to right) quantified pre-dose on day -5 and post-treatment on day 27. N=9 per group.

[0073] [Figure 17-1]Figures 17A-17E. Data in Figure 17A are from male lipodystrophic aP2-nSrebp1cTg / +;Leprhu / hu mice (Tg), 32-38 week-old lipodystrophic (Tg) mice that received a single dose (10 mg / kg SC) of control (gray bars) or H4H17319P2 (dark gray bars), or leptin infusion (30 μg / day SC; black bars). For data in Figures 17B-17E, 27-30 week-old male Tg mice were administered 10 mg / kg (SC) of control monoclonal antibody (gray circles or bars) or H4H17319P2 (dark gray circles or bars), or leptin infusion (30 μg / day SC; black bars) once a week. Where indicated, 27-30 week-old male nontransgenic Leprhu / hu mice (non-Tg) were also administered 10 mg / kg (SC) of a control monoclonal antibody (white diamond and white bar) once a week. All data are mean ± SEM. *, P<0.05 for each group indicated by the symbol color or bar compared with Tg mice administered the control monoclonal antibody. #, P<0.05 for each group indicated by the symbol color or bar compared with Tg mice administered H4H17319P2. Figure 17A shows immunohistochemical staining of pSTAT3 Y705 in the arcuate nucleus (Arc) and ventromedial hypothalamus (Vmh) at approximately -1.52 mm from the anterior section, demonstrating an increased number of pSTAT3 Y705+ cells in male 32-38 week-old lipodystrophic (Tg) mice. Brain sections were taken 3 days after treatment with a single dose of control or H4H17319P2 (10 mg / kg SC) or leptin infusion (30 μg / day SC). Representative photomicrographs and quantification of pSTAT3 Y705+ cells in the Arc and Vmh (left, center, and right, respectively) are shown. N = 4–5 mice per group. The left panel of Figure 17B shows blood glucose levels during the experiment. The center and right panels show blood glucose levels and the area under the curve for glucose levels during the insulin tolerance test on day 9. N = 8–9 mice per group. Figure 17C shows body weight (left), change in body weight (center), and cumulative food intake (right). N = 8–9 Tg mice per group.For non-Tg mice, N=5. Figure 17D shows chemistry analysis of plasma obtained on day 13 for triglycerides, cholesterol, LDL-C, and HDL-C, demonstrating that H4H17319P2 treatment reduced plasma triglycerides and cholesterol. N=8-9 Tg mice per group. For non-Tg mice, N=5. Figure 17E shows liver weight (left) and hepatic triglyceride content (right) of liver obtained on day 14. N=6-9 Tg mice per group. For non-Tg mice, N=5. [Figure 17-2] See the description of Figure 17-1. [Figure 17-3] See the description of Figure 17-1.

[0074] [Figure 18A]Figures 18A-18D. Data shown in Figures 18A and 18B are from 32-week-old female Leprhu / hu mice given a single dose of control monoclonal antibody (10 mg / kg, SC; gray circles), H4H17319P2 (3 mg / kg, SC; white circles), or H4H17319P2 (10 mg / kg; black circles). Data shown in Figure 18C are from lean male and female cynomolgus monkeys given a single dose of control (SC; gray circles), H4H17319P2 (3 mg / kg, SC; white circles), or H4H17319P2 (10 mg / kg; black circles). Data shown in Figure 18D are from high-weight (approximately 6.0 kg) male and female cynomolgus monkeys that received two doses of control (IV; gray circles) or H4H17319P2 (30 mg / kg IV; black circles). Data are mean ± SEM. *, P<0.05 for H4H17319P2 (10 mg / kg) compared to the respective control at the indicated time point. #, P<0.05 for H4H17319P2 (3 mg / kg) compared to the respective control at the indicated time point. $, P<0.05 for H4H17319P2 (3 mg / kg) compared to H4H17319P2 (10 mg / kg) at the indicated time point. &, P<0.05 for H4H17319P2 (30 mg / kg IV) compared to the respective control. Figure 18A shows the percent change in body weight (left) and food intake (right) from pre-dose on day 0 during the study in lean female mice. N = 6-7 mice per group. Figure 18B shows quantitative NMR analysis of the percent change in fat mass (left) and lean mass (right) from pre-dose on day 0 during the study in lean female mice. N = 6-7 mice per group. Figure 18C shows the percent change in body weight from pre-dose on day -1 during the study in lean mice. N = 12 mice per group. Figure 18D shows the percent change in body weight (left), fat mass (center), and lean mass (right) from pre-dose averages on days -14, -7, and -1, as quantified by DEXA (Dual Energy X-Ray Absorptiometry for measuring body composition) during the study in overweight obese monkeys.N=4 and 8 for the control and H4H17319P2-treated groups, respectively. [Figure 18B] See the legend to Figure 18A. [Figure 18C] See the legend to Figure 18A. [Figure 18D] See the legend to Figure 18A.

[0075] [Figure 19] FIG. 19 shows the single-patient protocol utilized in the compassionate use clinical trial.

[0076] [Figure 20A-1] FIG. 20A is a table showing the evaluation schedule for patients treated with H4H17319P2 during Treatment Period 1. [Figure 20A-2] See the description of Figure 20A-1. [Figure 20B-1] FIG. 20B is a table showing the evaluation schedule for patients treated with H4H17319P2 during Treatment Period 2 and the Extension Treatment Period. [Figure 20B-2] See the description of Figure 20B-1.

[0077] [Figure 21]Figures 21A-21C show the effects of H4H17319P2 on blood glucose, body weight, and food intake in a mouse model of congenital leptin deficiency. Figure 21A shows blood glucose in mg / dL, Figure 21B shows body weight in grams, and Figure 21C shows cumulative food intake in grams. Grey circles represent Leprhu / hu mice receiving the IgG4P control (N=5). Black squares represent Leprhu / huLep- / - mice receiving the IgG4P control (N=7). White squares represent Leprhu / huLep- / - mice receiving H4H17319P2 (N=8). Data are presented as mean + SEM. *, P<0.05 for the Leprhu / huLep- / - + H4H17319P2 group compared with the Leprhu / hu,Lep- / - + IgG4P control by two-way ANOVA with Tukey's post-hoc test. For food intake assessment, two mice were excluded from the Leprhu / huLep- / - + IgG4P control group and one mouse was excluded from the Leprhu / huLep- / - + H4H17319P2 group due to excessively shredded food in their cages.

[0078] [Figure 22]Figures 22A-22C show μCT body composition analysis of fat mass, bone mass, and lean mass in a mouse model of congenital leptin deficiency. Figure 22A shows fat mass, Figure 22B shows bone mass, and Figure 22C shows lean mass in grams. Mice underwent baseline scans on day -5 before the start of the study. Post-mAb administration scans were performed on day 35 of the study. Gray bars represent Leprhu / hu mice administered the IgG4P control (N=5). Black bars represent Leprhu / huLep- / - mice administered the IgG4P control (N=7). White bars represent Leprhu / huLep- / - mice administered H4H17319P2 (N=8). Mice received weekly subcutaneous injections of either 10 mg / kg H4H17319P2 or an isotype control antibody. Data are presented as mean + SEM. *, P<0.05 compared to baseline. #, P<0.05 compared with the Leprhu / hu + IgG4P control at each time point. +, P<0.05 compared with the H4H17319P2 group at each time point. Statistical analysis was performed by two-way ANOVA with Tukey's post-hoc test.

[0079] [Figure 23]Figures 23A-23C show the effects of H4H17319P2 on blood glucose, body weight, and food intake in a mouse model of congenital leptin receptor deficiency. Figure 23A shows blood glucose in mg / dL, Figure 23B shows body weight in grams, and Figure 23C shows cumulative food intake in grams. Grey circles represent Leprhu / hu mice (N=7-8) administered the IgG4P control. Black squares represent LeprA409E / A409E mice (N=9-10) administered the IgG4P control. White squares represent LeprA409E / A409E mice (N=10) administered H4H17319P2. Data are expressed as mean + SEM. *, P < .05 for the LeprA409E / A409E + H4H17319P2 group compared with the LeprA409E / A409E + IgG4P control by a mixed-effects model with Sidak's post-hoc test. For food intake assessment, one mouse was excluded from the Leprhu / hu + IgG4P control group and one mouse was excluded from the LeprA409E / A409E + IgG4P control group due to death during the study. For food intake assessment, one mouse was excluded from the Leprhu / hu + IgG4P control group and three mice were excluded from the LeprA409E / A409E + IgG4P control group due to excessively shredded food during the study.

[0080] [Figure 24]Figures 24A-24C show μCT body composition analysis of fat mass, bone mass, and lean mass in a mouse model of congenital leptin receptor deficiency. Figure 24A shows fat mass, Figure 24B shows bone mass, and Figure 24C shows lean mass in grams. Mice underwent a baseline scan on day -1 before the start of the study. Post-mAb administration scans were performed on day 41 of the study. Gray bars represent Leprhu / hu mice administered the IgG4P control (N = 7-8). Black bars represent LeprA409E / A409E mice administered the IgG4P control (N = 9-10). White bars represent LeprA409E / A409E mice administered H4H17319P2 (N = 10). Mice received weekly subcutaneous injections of either 10 mg / kg H4H17319P2 or an isotype control (IgG4P) antibody. Data are expressed as mean + SEM. *, P<0.05 compared to baseline. #, P<0.05 compared to Leprhu / hu + IgG4P control at each time point. +, P<0.05 compared to LeprA409E / A409E + H4H17319P2 at each time point. All statistical analyses were performed using a mixed-effects model with Sidak post-hoc test.

[0081] [Figure 25-1] Figure 25 shows the schedule of events for the Part A cohort of the first-in-human clinical trial, including the procedures performed at each visit for screening, treatment, and follow-up visits. [Figure 25-2] See the description of Figure 25-1.

[0082] [Figure 26-1] Figure 26 shows the schedule of events for the Part B cohort of the first-in-human clinical trial, including procedures performed at each visit for pre-screening, screening, and baseline determination. [Figure 26-2] See the description of Figure 26-1. [Figure 26-3] See the description of Figure 26-1.

[0083] [Figure 27-1]Figure 27 shows the second event schedule for the Part B cohort of the first-in-human clinical trial, including procedures performed at each treatment and follow-up visit. [Figure 27-2] See the description of Figure 27-1. DETAILED DESCRIPTION OF THE INVENTION

[0084] Leptin is an adipose tissue hormone that regulates energy balance and metabolic and neuroendocrine functions (Flak and Myers, Mol Endocrinol. 2016;30:3-12; Zhang et al. Nature. 1994;372:425-432). Under conditions of energy deficiency, low circulating leptin levels induce adaptive responses, including increased hunger and energy conservation through modulation of neuroendocrine pathways. Leptin regulates energy and metabolic balance by engaging the leptin receptor (LEPR), a member of the class I cytokine receptor family (Tartaglia et al., 1995). LEPR is encoded by a single gene, and alternative splicing generates multiple splice isoforms of LEPR, which differ in their C-terminal sequences (Baumann et al., 1996). Of these splice isoforms, LEPR-b is the basal isoform that mediates the effects of leptin and is the only isoform that stimulates JAK-STAT signaling (Baumann et al., 1996; Tartaglia et al., 1995; White and Tartaglia, 1996). LEPR-b-expressing neurons in the brain are the primary target and mediator of leptin's actions on energy, metabolic, and neuroendocrine homeostasis. This suggests that Lepr db / db This finding is supported by experimental evidence showing that selective neuronal expression of Lepr-b in mice rescues obesity, diabetes, and reproductive phenotypes (de Luca et al., 2005). Furthermore, neuronal deletion of the Lepr gene results in the reversal of Leprdb / db The obesity and hyperglycemia phenotype in animals has been replicated (Cohen et al., 2001). Leptin deficiency is caused by a loss-of-function genetic mutation in the Lep gene, which results in hyperphagia, obesity, insulin resistance, dyslipidemia, and neuroendocrine dysfunction in mice, which are reversed by leptin treatment (Barash et al., 1996; Campfield et al., 1995; Chehab et al., 1996; Halaas et al., 1995; Pelleymounter et al., 1995). Clinically, the leptin analog metreleptin reverses obesity and metabolic and reproductive dysfunction in patients with monogenic obesity due to leptin deficiency (Farooqi et al., 1999; Farooqi et al., 2002). Similar to primary leptin deficiency, secondary hypoleptinemic disease states are associated with glucose and lipid metabolic dysfunction that can be reversed with leptin treatment. Congenital and acquired generalized lipodystrophies are rare and severe disorders characterized by the near-complete loss of adipose tissue stores (Brown et al., 2016; Patni and Garg, 2015). In these patients, very low circulating leptin levels result in conditions of hyperphagia, hypertriglyceridemia, hypercholesterolemia, hepatic steatosis, insulin resistance, and diabetes (Brown et al., 2016; Patni and Garg, 2015). A severe complication of hypertriglyceridemia, especially when TG levels exceed 500 mg / dL–1000 mg / dL, is acute, recurrent pancreatitis (Yadav and Pitchumoni 2003), which, when combined with complications such as infection or organ failure, can be life-threatening, with a mortality rate of over 40% (UK Guidelines 2005). Ectopic lipid accumulation in the liver (hepatic steatosis) can result in steatohepatitis. Steatohepatitis is characterized by fat accumulation, cellular damage, and liver inflammation and is one of the most common causes of cirrhosis (El-Zayadi 2008, Federico 2006, and Festi 2004).

[0085] In Tg-aP2-nSrebp1c mice, which exhibit characteristics of generalized lipodystrophy with near-complete loss of fat accumulation, leptin treatment reduced hyperphagia and improved dyslipidemia, hepatic steatosis, and glycemic control (Shimomura et al., 1999; Shimomura et al., 1998). Metreleptin attenuates metabolic dysfunction in patients with generalized lipodystrophy (Oral et al., 2002). However, it is not approved for the treatment of patients with partial lipodystrophy (Ajluni et al., 2016).

[0086] Patients with lipodystrophy often experience other serious comorbidities, such as chronic kidney disease, cardiovascular complications, autoimmune diseases, and peripheral T-cell lymphoma, acute lymphoblastic leukemia, and Hodgkin's lymphoma.

[0087] Patients with congenital leptin deficiency exhibit rapid weight gain and immune abnormalities during the first few months of life and are at significantly increased risk of death within the first 10 to 20 years of life (Funcke, et al., Monogenic forms of childhood obesity due to mutations in the leptin gene. Mol Cell Pediatr. 2014;1(1):3); (Dubern, et al., Leptin and leptin receptor-related monogenic obesity. Biochimie. 2012;94(10):2111-5); (Paz-Filho, et al., Ten years of leptin replacement therapy. Obesity reviews. 2011;12:e315-e323).Although there are no approved treatments for congenital leptin deficiency, in several small, open-label studies of patients with monogenic obesity caused by loss-of-function mutations in leptin, leptin treatment significantly reduced appetite, body weight, adiposity, metabolic abnormalities, bone age-chronological age gap, and hormonal and immune abnormalities (Wabitsch, et al., Severe Early-Onset Obesity Due to Bioinactive Leptin Caused by a p.N103K Mutation in the Leptin Gene. J Clin Endocrinol Metab. 2015;100(9):3227-3230); (Farooqi, et al., Effects of recombinant leptin therapy in a child with congenital leptin deficiency. N Engl J Med. 1999;341(12):879-84); (Licinio, et al., Phenotypic effects of leptin replacement on morbidity) obesity,diabetes mellitus,hypogonadism,and behavior in leptin-deficient adults.Proc Natl Acad Sci USA.2004;101(13):4531-6);(Gibson et al.,Congenital Leptin Deficiency Due to Homozygosity for the Delta133G mutation:report of another case and evaluation of response to four years of leptin therapy.J Clin Endocrin.& Metab.2004;89(10):4821-4826).Other reported leptin treatments have resulted in rapid and sustained increases in plasma thyroid hormone levels, promoted timely pubertal development, and improved circulating CD4(+) T cell counts, T cell proliferation, and cytokine release (Farooqi et al., Beneficial effects of leptin on obesity, T cell hyporesponsiveness, and neuroendocrine / metabolic dysfunction of human congenital leptin deficiency. J Clin Invest. 2002;110(8):1093-1103). However, in some cases, neutralizing anti-metreleptin antibodies develop, leaving patients without targeted treatment options (Ozsu, et al., Early-onset severe obesity due to complete deletion of the leptin gene in a boy. J Pediatr Endocrinol Metab. 2017;30(11):1227-1230). Provided herein are methods of treating patients with congenital leptin deficiency using leptin receptor agonists such as H4H17319P2 (see WO2017 / 66204).

[0088] Provided herein are agonistic monoclonal antibodies that activate human LEPR with efficacy similar to that of leptin. Monoclonal antibody-mediated activation of LEPR is effective in reversing severe weight gain and metabolic dysfunction in mouse models of primary and secondary leptin-deficiency disorders. Furthermore, LEPR agonistic monoclonal antibodies reduce adiposity and body weight in normal-weight mice and in non-human primates with normal and high body fat, and stimulate LEPR in the presence of circulating leptin.

[0089] It is to be understood that this disclosure is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure is limited only by the appended claims.

[0090] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs.As used herein, the term "about" when used in relation to a specific referenced numerical value means that the value can vary from the referenced value by 1% or less.For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (for example, 99.1, 99.2, 99.3, 99.4, etc.).

[0091] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

[0092] definition As used herein, the terms "leptin receptor," "LEPR," and similar terms refer to a human leptin receptor comprising the amino acid sequence set forth in SEQ ID NO: 113 (see also UniProtKB / Swiss-Prot Accession No. P48357). Alternative names for LEPR used in the scientific literature include "OB receptor," "OB-R," and "CD295." LEPR is also referred to as "WSX" (see, e.g., U.S. Patent No. 7,524,937). The term "LEPR" includes both monomeric and multimeric (e.g., dimeric) LEPR molecules. As used herein, the term "monomeric human LEPR" refers to a LEPR protein or portion thereof that does not contain or include any multimerization domain and exists under normal conditions as a single LEPR molecule without direct physical connection to another LEPR molecule. An exemplary monomeric LEPR molecule is a molecule designated herein as "hLEPR.mmh," which comprises the amino acid sequence of SEQ ID NO: 114 (see, e.g., Example 3 herein). As used herein, the term "dimeric human LEPR" refers to a construct comprising two LEPR molecules connected to each other via a linker, a covalent bond, a non-covalent bond, or a multimerization domain, such as an antibody Fc domain. An exemplary dimeric LEPR molecule is a molecule designated herein as "hLEPR.mFc," which comprises the amino acid sequence of SEQ ID NO: 115 (see, e.g., Example 3 herein), or "hLEPR.hFc," which comprises the amino acid sequence of SEQ ID NO: 116. As used herein, unless specifically indicated otherwise, the terms "anti-LEPR antibody," "antibody that specifically binds to LEPR," "LEPR-specific binding protein," and the like refer to a molecule that binds to full-length human LEPR, monomeric human LEPR, dimeric human LEPR, or other constructs comprising or consisting of the LEPR extracellular domain.

[0093] All references herein to proteins, polypeptides, and protein fragments are intended to refer to the human version of the respective protein, polypeptide, or protein fragment, unless explicitly specified as being from a non-human species. Thus, the term "LEPR" refers to human LEPR, unless specified as being from a non-human species, e.g., "mouse LEPR," "monkey LEPR," etc.

[0094] As used herein, the phrase "cell surface-expressed LEPR" refers to one or more LEPR proteins, or extracellular domains thereof, that are expressed on the surface of a cell in vitro or in vivo, such that at least a portion of the LEPR protein is exposed to the extracellular side of the cell membrane and accessible to the antigen-binding portion of an antibody. A "cell surface-expressed LEPR" can include or consist of a LEPR protein that is expressed on the surface of a cell that normally (e.g., in a natural or wild-type state) expresses the LEPR protein. Alternatively, a "cell surface-expressed LEPR" can include or consist of a LEPR protein that is expressed on the surface of a cell that does not normally express human LEPR on its surface but has been artificially engineered to express LEPR on its surface.

[0095] As used herein, phrases such as "anti-LEPR antibody" or "antibody that binds to the human leptin receptor" include both monovalent antibodies having a single specificity, as well as bispecific antibodies comprising a first arm that binds to LEPR and a second arm that binds to a second (target) antigen, wherein the anti-LEPR arm comprises any of the HCVR / LCVR or CDR sequences set forth in Table 1 herein.

[0096] The term "antibody," as used herein, refers to any antigen-binding molecule or molecular complex that contains at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., LEPR). The term "antibody" encompasses immunoglobulin molecules that contain four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (herein referred to as HCVR or V). H The heavy chain constant region comprises a C H 1. C H 2, and C H The light chain comprises three domains: a light chain variable region (LCVR or VVR) and a light chain variable region (VVV). L The light chain constant region comprises one domain (C L 1) V H Area and V L The regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present invention, the FRs of an anti-LEPR antibody (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0097] As used herein, the term "antibody" also includes antigen-binding fragments of a complete antibody molecule. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies may be derived from complete antibody molecules using any suitable standard method, such as, for example, proteolytic or recombinant genetic engineering techniques, which involve the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0098] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" as used herein.

[0099] Antigen-binding fragments of antibodies typically contain at least one variable domain, which may be of any size or amino acid composition and generally contain at least one CDR adjacent to or in frame with one or more framework sequences. L V bound to the domain H In an antigen-binding fragment having a domain, V H Domains and V L The domains can be arranged relative to each other in any suitable configuration. For example, the variable region is a dimer, with the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may contain a dimer of monomeric V H or V L It may contain domains.

[0100] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the invention include: (i) a V H -C H 1, (ii) V H -C H 2, (iii) V H -C H 3, (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3, (vii)V H -C L、 (viii)V L -C H 1, (ix)V L -C H 2. (x)V L -C H 3. (xi) V L -CH 1-C H 2, (xii)V L -C H 1-C H 2-C H 3, (xiii)V L -C H 2-C H 3, and (xiv) V L -C L In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to one another or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present invention may be linked to one another and / or to one or more monomeric V H Or V L It may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above in non-covalent association with the domains (e.g., via disulfide bonds).

[0101] As with intact antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two different variable domains, each capable of specifically binding to a separate antigen or a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use with antigen-binding fragments of antibodies of the present invention using routine techniques available in the art.

[0102] In certain embodiments of the present invention, the anti-LEPR antibodies of the present invention are human antibodies. The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences, for example, in the CDRs, particularly CDR3 (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0103] In some embodiments, the antibodies of the present invention may be recombinant human antibodies. The term "recombinant human antibody," as used herein, is intended to include all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transduced into a host cell (described in more detail below), antibodies isolated from a recombinant, combinatorial human antibody library (described in more detail below), antibodies isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means, including splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, when animals transgenic for human Ig sequences are used), thereby increasing the V H Area and V L The amino acid sequence of the region is human germline V HArray and V L While derived from and related to sequences, they may not naturally occur in the human antibody germline repertoire in vivo.

[0104] The present invention provides a hinge, C H 2nd area or C H The present invention encompasses antibodies with one or more mutations in the three regions, which may be desirable, for example, to improve the yield of a desired antibody type in manufacturing.

[0105] The antibody of the present invention may be an isolated antibody. As used herein, an "isolated antibody" refers to an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally occurs or is naturally produced, is an "isolated antibody" for purposes of the present invention. An isolated antibody includes an antibody in situ within a recombinant cell. An isolated antibody is an antibody that has undergone at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0106] The present invention includes variants of the anti-LEPR antibodies disclosed herein that contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibody is derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies derived from any of the amino acid sequences disclosed herein, and antigen-binding fragments thereof, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V LAll of the framework and / or CDR residues within a domain are mutated back to the residue found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated to the original germline sequence, e.g., only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while other remaining residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed by the present invention.

[0107] The present invention includes anti-LEPR antibodies and antigen-binding fragments that comprise amino acid sequences that are substantially similar or substantially identical to the amino acid sequences of one or more variable domains or CDRs present in any of the exemplary anti-LEPR antibodies disclosed herein.

[0108] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences, when optimally aligned using predefined gap weights, such as by the programs GAP or BESTFIT, share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or degree of homology may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331. Examples of groups of amino acids with side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, (2) aliphatic-hydroxyl side chains: serine and threonine, (3) amide-containing side chains: asparagine and glutamine, (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, (5) basic side chains: lysine, arginine, and histidine, (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0109] Sequence similarity for polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present invention to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, with default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410, and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

[0110] As used herein, the term "subject" refers to an animal, preferably a mammal, more preferably a human, in need of amelioration, prevention, and / or treatment of a disease or disorder associated with leptin deficiency. The subject may be an adult or an infant. The subject may have a metabolic dysfunction, such as generalized lipodystrophy or partial lipodystrophy. The subject may have congenital leptin deficiency or acquired leptin deficiency. Subjects with congenital leptin deficiency include subjects with genetic mutations that result in little or no circulating levels of leptin, or in circulating but biologically inactive leptin. The subject may have one or more symptoms associated with leptin deficiency. As used herein, the term "subject" is interchangeable with the term "patient." In some embodiments, the subject has neutralizing antibodies against metreleptin.

[0111] As used herein, the terms "treat," "treating," or "treatment" refer to the reduction in severity or amelioration of at least one symptom of leptin deficiency by administering a therapeutic agent, such as an antibody of the invention, to a subject in need thereof. The term includes the inhibition of progression of the associated disease or the inhibition of the worsening of a condition or symptom associated with the disease. The term also includes a positive prognosis of the disease; that is, administration of a therapeutic agent, such as an antibody of the invention, may render the subject symptom-free. A positive prognosis may include the alleviation of any of the following conditions: hyperphagia, hyperglycemia, insulin resistance, dyslipidemia, or hepatic steatosis.

[0112] The terms "prevent," "preventing," or "prevention" refer to the inhibition of the onset of any symptom, condition, or symptom associated with leptin deficiency.

[0113] Therapeutic agents may be administered to a subject in therapeutic doses. By the phrase "therapeutically effective amount" is meant an amount that is administered to produce a desired effect. The precise amount will depend on the purpose of the treatment and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding), and are discussed in detail herein.

[0114] Anti-LEPR antibodies containing Fc variants According to certain embodiments of the invention, anti-LEPR antibodies are provided that include an Fc domain that includes one or more mutations that enhance or decrease antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the invention provides an anti-LEPR antibody that includes an Fc domain that includes one or more mutations that enhance or decrease antibody binding to the FcRn receptor at acidic pH compared to neutral pH. H 2 or C HAnti-LEPR antibodies include those containing mutations in the 3 region that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., endosomes at pHs ranging from about 5.5 to about 6.0). Such mutations can result in increased serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T), or at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y), or modifications at positions 250 and / or 428, or at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include a 428L (e.g., M428L) and a 434S (e.g., N434S) modification, a 428L, a 259I (e.g., V259I), and a 308F (e.g., V308F) modification, a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification, a 252, a 254, and a 256 (e.g., 252Y, 254T, and 256E) modification, a 250Q and a 428L modification (e.g., T250Q and M428L), and a 307 and / or a 308 modification (e.g., 308F and / or 308P).

[0115] For example, the invention includes anti-LEPR antibodies comprising an Fc domain containing one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the foregoing Fc domain mutations, and other mutations in antibody variable domains disclosed herein, are contemplated as being within the scope of the invention.

[0116] The anti-LEPR antibodies of the invention may comprise an altered Fc domain with reduced effector function. As used herein, a "altered Fc domain with reduced effector function" refers to any Fc portion of an immunoglobulin that has been altered, mutated, or truncated relative to a wild-type, native Fc domain such that the molecule comprising the altered Fc exhibits a reduction in the severity or extent of at least one effect selected from the group consisting of cell killing (e.g., ADCC and / or CDC), complement activation, phagocytosis, and opsonization, relative to a comparator molecule comprising a native, wild-type Fc portion. In certain embodiments, a "altered Fc domain with reduced effector function" is an Fc domain with reduced or attenuated binding to an Fc receptor (e.g., FcγR).

[0117] In certain embodiments of the present invention, the modified Fc domain is a variant IgG1 Fc or variant IgG4 Fc comprising a substitution in the hinge region. For example, a modified Fc for use in the context of the present invention may comprise a variant IgG1 Fc in which at least one amino acid in the IgG1 Fc hinge region has been substituted with the corresponding amino acid in an IgG2 Fc hinge region. Alternatively, a modified Fc for use in the context of the present invention may comprise a variant IgG4 Fc in which at least one amino acid in the IgG4 Fc hinge region has been substituted with the corresponding amino acid in an IgG2 Fc hinge region. Exemplary modified Fc regions that may be used in the context of the present invention are described in U.S. Patent Application Publication No. 2014 / 0243504.

[0118] Other modified Fc domains and Fc modifications that may be used in the context of the present invention include any of the modifications described in US 2014 / 0171623, US 8,697,396, US 2014 / 0134162, WO 2014 / 043361. Methods for constructing antibodies or other antigen-binding fusion proteins comprising the modified Fc domains described herein are known in the art.

[0119] Biological characteristics of antibodies The present invention includes antibodies and antigen-binding fragments thereof that bind to human LEPR and activate LEPR signaling. Such antibodies may be referred to herein as "agonistic antibodies." In the context of the present invention, "activation of LEPR signaling" refers to stimulation of intracellular effects that normally result from the interaction of LEPR with leptin in cells that express LEPR. In certain embodiments, "activation of LEPR signaling" refers to the transcriptional activation of STAT3, which can be detected using any method that can directly or indirectly measure or identify the activity of STAT3, for example, using a labeled form of STAT3 expressed in a reporter cell line. For example, the present invention includes antibodies and antigen-binding fragments that activate LEPR signaling in a cell-based reporter assay or a substantially similar assay, for example, using the cell-based assay format set forth in Example 7 herein. Cell-based reporter assays that detect LEPR activation, such as the assay described in Example 7 herein, can be used to detect EC 50 In certain exemplary embodiments of the invention, antibodies can produce a detectable signal that can be expressed in units of an EC value (i.e., the antibody concentration required to produce 50% of maximal signaling) and / or as a percentage of maximal signaling observed in the presence of leptin. In certain exemplary embodiments of the invention, antibodies can produce an EC value of less than about 12.0 nM in a cell-based reporter assay using, for example, the assay format set forth in Example 7 herein or a substantially similar assay. 50 In certain exemplary embodiments of the present invention, anti-LEPR antibodies are provided that activate LEPR signaling at a maximal percentage activation for leptin signaling of greater than about 65% in a cell-based reporter assay using, for example, the assay format set forth in Example 7 herein, or a substantially similar assay.

[0120] The present invention includes antibodies and antigen-binding fragments thereof that bind to monomeric human LEPR with high affinity. For example, the present invention provides antibodies and antigen-binding fragments thereof that have a K of less than about 150 nM as measured by surface plasmon resonance or a substantially similar assay at 25° C. or 37° C. using, for example, the assay format set forth in Example 3 herein. D and binds to monomeric human LEPR (e.g., hLEPR.mmh, SEQ ID NO: 114) at a concentration of less than about 150 nM, less than about 140 nM, less than about 130 nM, less than about 120 nM, less than about 110 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 5 ... K less than 0 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, or less than about 300 pM D and binds to monomeric human LEPR at 25°C.

[0121] The present invention further includes antibodies and antigen-binding fragments thereof that bind to monomeric human LEPR (e.g., hLEPR.mmh, SEQ ID NO: 114) with a dissociation half-life (t) of greater than about 50 minutes when measured by surface plasmon resonance or a substantially similar assay at 25°C or 37°C, e.g., using the assay format set forth in Example 3 herein. According to certain embodiments, anti-LEPR antibodies are provided that bind to monomeric human LEPR at 25°C with a t of greater than about 50 minutes, greater than about 55 minutes, greater than about 60 minutes, greater than about 65 minutes, or longer when measured by surface plasmon resonance or a substantially similar assay, e.g., using the assay format set forth in Example 3 herein.

[0122] The present invention further includes antibodies and antigen-binding fragments thereof that bind with high affinity to dimeric human LEPR (e.g., hLEPR.mFc, SEQ ID NO: 115). For example, the present invention provides antibodies and antigen-binding fragments thereof that have a K of less than about 1.5 nM as measured by surface plasmon resonance or a substantially similar assay at 25°C or 37°C, e.g., using the assay format set forth in Example 3 herein. D According to certain embodiments, the anti-LEPR antibodies have a K of less than about 150 nM, less than about 130 nM, less than about 110 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, or less than about 10 nM as measured by surface plasmon resonance or a substantially similar assay, e.g., using the assay format set forth in Example 3 herein. D and binds to dimeric human LEPR at 25°C.

[0123] The present invention further includes antibodies and antigen-binding fragments thereof that bind to dimeric human LEPR (e.g., hLEPR.mFc, SEQ ID NO: 115) with a dissociation half-life (t) of greater than about 10 minutes when measured by surface plasmon resonance or a substantially similar assay at 25°C or 37°C, e.g., using the assay format set forth in Example 3 herein. According to certain embodiments, anti-LEPR antibodies are provided that bind to dimeric human LEPR at 25°C with a t of greater than about 10 minutes, greater than about 15 minutes, greater than about 20 minutes, greater than about 25 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, or more, when measured by surface plasmon resonance or a substantially similar assay, e.g., using the assay format set forth in Example 3 herein.

[0124] The present invention further includes antibodies and antigen-binding fragments thereof that bind to LEPR complexed with human leptin ("LEPR complexed with human leptin" is sometimes expressed as "leptin:LEPR"). For example, the present invention includes antibodies and antigen-binding fragments thereof that have the ability to bind to a preformed complex comprising hLEPR and human leptin. That is, according to certain embodiments, the interaction between an anti-LEPR antibody and LEPR is not inhibited by the presence of leptin complexed with LEPR. Similarly, according to this aspect of the invention, the interaction between leptin and LEPR is not inhibited by the presence of an anti-LEPR antibody. An exemplary assay format for determining whether an antibody or antigen-binding fragment thereof binds to LEPR complexed with human leptin is described in Example 4 herein.

[0125] Similarly, the present invention also includes antibodies and antigen-binding fragments thereof that bind to LEPR and do not block the LEPR:leptin interaction. For example, the present invention includes antibodies and antigen-binding fragments thereof that have the ability to bind to LEPR, thereby producing an antibody:LEPR complex, wherein the resulting antibody:LEPR complex has the ability to interact with leptin to produce a three-membered complex comprising the antibody, LEPR, and leptin. An exemplary assay format for determining whether an antibody or antigen-binding fragment thereof has the ability to bind to LEPR in a manner that does not block or interfere with the interaction between LEPR and leptin is described in Example 5 herein.

[0126] The present invention also includes antibodies and antigen-binding fragments thereof that bind to cell-surface-expressed LEPR in the presence and / or absence of human leptin. Cell-surface-expressed LEPR refers to LEPR or a portion thereof (e.g., the extracellular portion of LEPR) that is expressed on the surface of cells, either naturally occurring or engineered, such that an antibody or its antigen-binding fragment can bind to the LEPR molecule. In certain embodiments, cell-surface-expressed LEPR comprises a recombinant complex comprising the extracellular domain of LEPR linked to the cell via a tag or anchor (e.g., a GPI anchor as exemplified in Example 6 herein). According to this aspect of the invention, antibodies are provided that can bind to cell-surface-expressed LEPR in the absence of leptin and can also bind to cell-surface-expressed LEPR in the presence of leptin (i.e., under conditions in which leptin can bind to cell-surface-expressed leptin). That is, according to certain embodiments, the interaction between an anti-LEPR antibody and cell-surface-expressed LEPR is not inhibited by the presence of leptin complexed with cell-surface-expressed LEPR. Antibodies according to this aspect of the invention have the ability to form a three-membered complex on the cell surface comprising the antibody, cell surface-expressed LEPR, and leptin. An exemplary assay format for determining whether an antibody or antigen-binding fragment thereof has the ability to bind to cell surface-expressed LEPR in the presence and absence of human leptin is described in Example 6 herein.

[0127] Antibodies of the invention may possess one or more of the foregoing biological characteristics, or any combination thereof. The foregoing list of biological characteristics of the antibodies of the invention is not intended to be comprehensive. Other biological characteristics of the antibodies of the invention will be apparent to those of skill in the art from a review of this disclosure, including the working examples herein.

[0128] Epitope mapping and related techniques The present invention also includes anti-LEPR antibodies containing variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein, having one or more conservative substitutions. For example, the present invention includes anti-LEPR antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences set forth in Table 1 herein. In certain embodiments, the present invention provides anti-LEPR antibodies comprising variant HCVR, LCVR, and / or CDR amino acid sequences (e.g., containing conservative amino acid substitutions) relative to the sequences set forth in Table 1 herein, wherein the variant antibody still exhibits one or more functions and / or properties of the exemplary anti-LEPR antibodies disclosed herein.

[0129] The extracellular domain of human LEPR contains an N-terminal cytokine receptor homology domain (CRH-1), an immunoglobulin-like (Ig) domain, and a second CRH domain (CRH-2), termed the leptin-binding domain (LBD) (Carpenter et al. (2012) Structure 20:487-97). Furthermore, LEPR shares the highest homology with granulocyte colony-stimulating factor (GCSF) and glycoprotein 130 (gp13), as well as similar extracellular domain size and organization (Haniu et al. (1998) J Biol Chem 273(44):28691-699).

[0130] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on the antigen and have different biological effects. Epitopes may be either conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes generated by adjacent amino acid residues within a polypeptide chain. In certain circumstances, epitopes may include sugar, phosphoryl, or sulfonyl moieties on the antigen.

[0131] The present invention includes anti-LEPR antibodies that interact with one or more epitopes present within amino acids M1-D839 of human LEPR (SEQ ID NO: 113). As described in Example 11, peptide 201 from human LEPR showed significantly reduced deuterium incorporation when bound to the H4H16650P2 antibody. When bound to H4H16650P2, peptides corresponding to amino acids 162-169 (human LEPR, amino acids LYVLPEVL of SEQ ID NO: 113) and amino acids 170-191 (human LEPR, amino acids EDSPLVPQKGSF of SEQ ID NO: 113) showed slower rates of deuteration, suggesting that this antibody binds to at least two human LEPR epitopes having the sequences LYVLPEVL or EDSPLVPQKGSF (amino acids 162-169 or amino acids 170-191 of SEQ ID NO: 113, respectively).

[0132] The epitope to which an antibody of the invention binds may consist of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of the LEPR protein. Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) of the LEPR. In some embodiments, the epitope is located on or near the leptin-binding domain of the LEPR. In other embodiments, the epitope is located in a region distinct from the leptin-binding domain of the LEPR, e.g., at a location on the surface of the LEPR where the antibody, when bound to the epitope, does not interfere with leptin binding to the LEPR.

[0133] Various techniques known to those skilled in the art can be used to identify amino acids within an epitope recognized by a particular antibody. Exemplary techniques include alanine scanning mutation analysis, peptide blot analysis, and peptide cleavage analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, the hydrogen / deuterium exchange method involves labeling the target protein with deuterium and then binding the antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, allowing hydrogen-deuterium exchange to occur at all residues except those protected by the antibody (which remain deuterium-labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis to reveal the deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-ray crystallography of an antibody complexed with an antigen can also be used to identify the amino acids within the polypeptide with which the antibody interacts.

[0134] The present invention further includes anti-LEPR antibodies that bind to the same epitope as certain exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences described in Table 1 herein). Similarly, the present invention also includes anti-LEPR antibodies that compete for binding to LEPR with any of the specific exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences described in Table 1 herein).

[0135] Using routine methods known in the art and exemplified herein, it is possible to determine whether an antibody binds to the same epitope as a reference anti-LEPR antibody or competes for binding with the reference anti-LEPR antibody. For example, to determine whether a test antibody binds to the same epitope as a reference anti-LEPR antibody of the present invention, the reference antibody is bound to a LEPR protein. The ability of the test antibody to bind to a LEPR molecule is then evaluated. If the test antibody can bind to LEPR after saturation binding with the reference anti-LEPR antibody, it can be concluded that the test antibody binds to a different epitope from the reference anti-LEPR antibody. On the other hand, if the test antibody cannot bind to a LEPR molecule after saturation binding with the reference anti-LEPR antibody, the test antibody may bind to the same epitope as the epitope bound by the reference anti-LEPR antibody of the present invention. Additional routine experiments (e.g., peptide mutation and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether the observed lack of binding is due to steric blocking (or another phenomenon). This type of experiment can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of the invention, two antibodies bind to the same (or overlapping) epitope if, for example, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits binding of the other antibody by at least 50%, but preferably by 75%, 90%, or even 99%, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antibodies are considered to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other. Two antibodies are considered to have "overlapping epitopes" if only a subset of the amino acid mutations that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other.

[0136] To determine whether an antibody competes for binding (or cross-competes for binding) with a reference anti-LEPR antibody, the above-described binding technique is performed in two ways: in the first way, the reference antibody is bound to the LEPR protein under saturating conditions, and then the binding of the test antibody to the LEPR molecule is assessed. In the second way, the test antibody is bound to the LEPR molecule under saturating conditions, and then the binding of the reference antibody to the LEPR molecule is assessed. In both ways, if only the first (saturating) antibody is able to bind to the LEPR molecule, it is concluded that the test antibody and the reference antibody compete for binding to LEPR. As will be understood by those skilled in the art, an antibody that competes for binding to a reference antibody does not necessarily have to bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding an overlapping or adjacent epitope.

[0137] Preparation of human antibodies The anti-LEPR antibody of the present invention can be a fully human antibody.Methods for generating monoclonal antibodies, including fully human monoclonal antibodies, are known in the art.Any such known method can be used in the context of the present invention to generate human antibodies that specifically bind to human LEPR.

[0138] For example, using the VELOCIMMUNE™ method or any other similar known method for generating fully human monoclonal antibodies, high-affinity chimeric antibodies against LEPR having human variable regions and mouse constant regions are first isolated. As in the experimental section below, the antibodies are characterized and selected for desired characteristics, including affinity, ligand-blocking activity, selectivity, epitope, etc. If necessary, the mouse constant region is replaced with a desired human constant region, such as wild-type or modified IgG1 or IgG4, to generate a fully human anti-LEPR antibody. While the constant region selected can vary depending on the specific application, the high-affinity antigen-binding and target specificity characteristics reside in the variable region. In a specific example, fully human anti-LEPR antibodies are isolated directly from antigen-positive B cells.

[0139] biological equivalent The anti-LEPR antibodies and antibody fragments of the present invention include proteins having amino acid sequences that differ from those of the described antibodies but retain the ability to bind to human LEPR. Such variant antibodies and antibody fragments contain one or more additions, deletions, or substitutions of amino acids compared to the parent sequence, but exhibit biological activity that is essentially equivalent to that of the described antibody. Similarly, DNA sequences encoding the anti-LEPR antibodies of the present invention include sequences that contain one or more additions, deletions, or substitutions of nucleotides compared to the disclosed sequences, but encode anti-LEPR antibodies or antibody fragments that are essentially biologically equivalent to the anti-LEPR antibodies or antibody fragments of the present invention. Examples of such variant amino acid sequences and DNA sequences are discussed above.

[0140] Two antigen-binding proteins, or antibodies, are considered to be bioequivalents if they are pharmaceutical equivalents or pharmaceutical substitutes that do not exhibit significant differences in the rate and extent of absorption when administered, for example, at the same molar dose under similar experimental conditions, either in single or multiple doses. Antibodies that are comparable in their extent of absorption but not in their rate of absorption are considered to be equivalents or pharmaceutical substitutes, and furthermore, are considered to be bioequivalents because any such differences in absorption rate are intentional and reflected in the labeling, are not essential to achieving effective body drug concentrations, for example, in chronic use, and are not considered medically significant for the particular drug product being studied.

[0141] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity, or efficacy.

[0142] In one embodiment, two antigen binding proteins are bioequivalent if a patient can switch one or more times between the reference product and the biological product without an expected increase in risk of adverse effects, including clinically significant changes in immunogenicity, or a decrease in efficacy, compared to continuous therapy without such switching.

[0143] In one embodiment, two antigen binding proteins are bioequivalent if they both act by one or more common mechanisms of action for one or more conditions of use, to the extent that such mechanisms are known.

[0144] Bioequivalence may be demonstrated by in vivo and in vitro methods. Bioequivalence measurements include, for example, (a) in vivo studies in humans or other mammals in which the concentration of an antibody or its metabolites is measured as a function of time in blood, plasma, serum, or other biological fluids, (b) in vitro studies that correlate with and reasonably predict in vivo bioavailability data in humans, (c) in vivo studies in humans or other mammals in which the relevant acute pharmacological effect of the antibody (or its target) is measured as a function of time, and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0145] Biologically equivalent variants of the anti-LEPR antibodies of the present invention can be constructed, for example, by making various substitutions of residues or sequences or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or substituted with other amino acids to prevent unnecessary or incorrect intramolecular disulfide bridge formation during renaturation. In other situations, biologically equivalent antibodies may include anti-LEPR antibody variants that contain amino acid changes that alter the glycosylation characteristics of the antibody, for example, mutations that eliminate or remove glycosylation.

[0146] Species selectivity and cross-species reactivity According to certain embodiments, the present invention provides anti-LEPR antibodies that bind to human LEPR but not to LEPR from other species. The present invention also provides anti-LEPR antibodies that bind to human LEPR and to LEPR from one or more non-human species. For example, an anti-LEPR antibody of the present invention may bind to human LEPR and, in some cases, may or may not also bind to one or more of the LEPRs of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee. According to certain exemplary embodiments of the present invention, an anti-LEPR antibody that specifically binds to human LEPR and cynomolgus monkey (e.g., Macaca fascicularis) LEPR is provided. Other anti-LEPR antibodies of the present invention bind to human LEPR but do not bind, or only weakly bind, to cynomolgus monkey LEPR.

[0147] multispecific antibodies The antibodies of the present invention may be monospecific or multispecific (e.g., bispecific). Multispecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen-binding domains specific for multiple target polypeptides. See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The anti-LEPR antibodies of the present invention can be linked to or coexpressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be operatively linked (e.g., by chemical coupling, genetic fusion, or noncovalent association, or vice versa) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with a second binding specificity.

[0148] The present invention includes bispecific antibodies in which one immunoglobulin arm binds to human LEPR and the other immunoglobulin arm is specific for a second antigen. The LEPR-binding arm may comprise any of the HCVR / LCVR or CDR amino acid sequences set forth in Table 1 herein.

[0149] An exemplary bispecific antibody format that can be used in the context of the present invention is H 3 domain and second Ig C H 3 domains, in which the first and second Ig C H The three domains differ from each other by at least one amino acid, and in this case, the at least one amino acid difference reduces binding of the bispecific antibody to Protein A compared to a bispecific antibody lacking the amino acid difference. H The 3 domain binds to protein A and the second IgC H The C3 domain contains a mutation that reduces or eliminates Protein A binding, for example, the H95R (according to IMGT exon numbering; H435R in EU numbering) modification. H 3 may further contain a Y96F modification (Y436F in EU according to IMGT). H Additional modifications that may be found in 3 include: D16E, L18M, N44S, K52N, V57M, and V82I for IgG1 antibodies (D356E, L358M, N384S, K392N, V397M, and V422I in EU by IMGT), N44S, K52N, and V82I for IgG2 antibodies (N384S, K392N, and V422I in IMGT, EU), and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I for IgG4 antibodies (Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I in EU by IMGT). Variations on the bispecific antibody formats described above are contemplated within the scope of the present invention.

[0150] Other exemplary bispecific formats that may be used in the context of the present invention include, but are not limited to, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knobs-into-holes, common light chains (such as common light chains with knobs-into-holes), CrossMab, CrossFab, (SEED) bodies, leucine zippers, duobodies, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab 2 Bispecific formats include (e.g., Klein et al. 2012, mAbs 4:6, 1-11 and references cited therein for a summary of such formats). Bispecific antibodies may also be constructed using peptide / nucleic acid conjugation. For example, unnatural amino acids with orthogonal chemical reactivity are used to create site-specific antibody-oligonucleotide conjugates that then self-assemble into multimeric complexes with defined composition, valency, and structure. (See, e.g., Kazane et al., J. Am. Chem. Soc. [Published online December 4, 2012]).

[0151] Therapeutic Formulation and Administration The present invention provides pharmaceutical compositions comprising the anti-LEPR antibodies or antigen-binding fragments thereof of the present invention. The pharmaceutical compositions of the present invention are formulated with suitable carriers, excipients, and other agents that improve transportation, delivery, tolerability, etc. Numerous suitable formulations can be found in a formulary known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsified carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0152] The dosage of an antibody administered to a patient may vary depending on the patient's age and size, the target disease, condition, route of administration, etc. Preferred dosages are typically calculated according to body weight or body surface area. For adult patients, it may be beneficial to administer the antibody of the present invention intravenously at a dose of about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight, usually as a single dose. The frequency and duration of treatment can be adjusted depending on the severity of the condition. Effective doses and schedules for administering anti-LEPR antibodies can be determined empirically; for example, patient progress can be monitored by periodic evaluation, and the dosage adjusted accordingly. Furthermore, interspecies dosage scaling can be performed using methods known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0153] For example, for patients, such as pediatric patients, it may be beneficial to administer the antibody intravenously at a therapeutically effective dose of about 5 mg / kg body weight, or about 1 mg / kg to about 20 mg / kg body weight, or about 1 mg / kg to about 15 mg / kg body weight, or about 5 mg / kg to about 10 mg / kg body weight. For example, an intravenous (IV) loading dose of about 5 mg / kg may be selected to achieve an antibody serum concentration of 100 mg / L or greater. It may also be beneficial to administer the antibody subcutaneously at a dose of about 250 mg, or about 300 mg, or about 100 mg to about 500 mg, or about 200 mg to about 300 mg. For example, a weekly subcutaneous (SC) maintenance dose of 250 mg or 300 mg of H4H17319P2 maintains a serum trough concentration of 100 mg / L or greater. In some embodiments, the SC dosing regimen is initiated several days after the IV loading dose to best maintain target serum trough concentrations. In some embodiments, the first SC dose is administered 2 to 7 days after the loading dose, e.g., 2, 3, 4, 5, 6, or 7 days after the loading dose. In some embodiments, the SC dose is administered every 3 to 14 days, e.g., every 3, 4, 5, 6, or 7 days after the loading dose. In some embodiments, the SC dose is administered once every 3 to 14 days, e.g., once every 3, 4, 5, or 6 days, once weekly, once every 10 days, or once every two weeks, e.g., after the first SC dose, three weekly SC doses are administered, followed by monthly (approximately every 28 days). In embodiments of the invention, the therapeutically effective dose of antibody (H4H17319P2) is depicted in Figure 19 herein, and is optionally continued after the final monthly dose shown in Figure 19.

[0154] In some embodiments, it is desirable to maintain a serum trough concentration of about 50 mg / L to about 200 mg / L, or about 100 mg / L, or about 150 mg / L, or greater than 50 mg / L, or greater than 100 mg / L, or greater than 150 mg / L.

[0155] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention. For example, liposomes, microparticles, encapsulation in microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis, etc. (See, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal mucosa, and intestinal mucosa), and can be administered together with other biologically active agents. Administration can be systemic or local.

[0156] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, for subcutaneous delivery, a pen delivery device is easily used to deliver the pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, disposable pen delivery devices are sold in a pre-filled state, with the pharmaceutical composition held in a reservoir inside the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0157] A number of reusable pen and autoinjector delivery devices find use in the subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany). Examples of disposable pen delivery devices having use in the subcutaneous delivery of the pharmaceutical compositions of the invention include, but are not limited to, the SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA™ Pen (Abbott Labs, Abbott Park, IL).

[0158] In certain circumstances, pharmaceutical compositions can be delivered in a controlled-release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used. See Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Press, Boca Raton, Florida. In yet another embodiment, a controlled-release system can be placed in proximity to the target of the composition, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled-release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0159] Injectable preparations may include administration forms such as intravenous, subcutaneous, intradermal, and intramuscular injections, infusions, and the like. These injectable preparations may be prepared by known methods. For example, injectable preparations may be prepared by dissolving, suspending, or emulsifying the above-described antibody or its salt in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injection include, for example, physiological saline, isotonic solutions containing glucose, and other auxiliary agents, which may be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Oily media include, for example, sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injection solution prepared in this manner is preferably filled into an appropriate ampule.

[0160] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared in dosage forms with unit doses suitable for adjusting the dose of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained in each dosage form is generally about 5 to about 500 mg per unit dose, and particularly in the form of injection, the antibody is preferably contained in an amount of about 5 to about 100 mg, and for other dosage forms, the antibody is preferably contained in an amount of about 10 to about 250 mg.

[0161] Therapeutic uses of antibodies The present invention includes administering to a subject in need thereof a therapeutic composition comprising an anti-LEPR antibody (e.g., an anti-LEPR antibody comprising any of the HCVR / LCVR or CDR sequences set forth in Table 1 herein). The therapeutic composition may comprise an anti-LEPR antibody or antigen-binding fragment thereof disclosed herein and a pharmaceutically acceptable carrier or diluent.

[0162] The antibodies and antigen-binding fragments provided herein are particularly useful for treating, preventing, and / or ameliorating any disease or disorder associated with or mediated by metabolic dysfunction or hypoleptinemia, such as nonalcoholic fatty liver disease, NASH, female infertility, amenorrhea, hormone cycle abnormalities, immune dysfunction, hypothyroidism, obesity, monogenic obesity, type I diabetes, type II diabetes, lipodystrophy, congenital lipodystrophy, generalized lipodystrophy, acquired lipodystrophy, partial lipodystrophy, congenital partial lipodystrophy, congenital generalized lipodystrophy, acquired partial lipodystrophy, and acquired generalized lipodystrophy, or those treatable by stimulating or activating LEPR signaling or by mimicking the natural activity of leptin in vitro or in vivo. For example, the antibodies and antigen-binding fragments thereof are useful for treating lipodystrophic conditions. Exemplary lipodystrophic conditions treatable by the antibodies and antigen-binding fragments of the invention include, for example, congenital generalized lipodystrophy, congenital partial lipodystrophy, acquired generalized lipodystrophy, familial partial lipodystrophy, acquired partial lipodystrophy, abdominal lipodystrophy centrifugally, lipoatrophia annularis, focal lipodystrophy, and HIV-associated lipodystrophy, and symptoms associated with such conditions.

[0163] The anti-LEPR antibodies and antigen-binding fragments thereof provided herein are useful for treating, preventing, and / or ameliorating monogenic obesity and / or lipodystrophy. Monogenic obesity and lipodystrophy can be associated with a number of pathological conditions. For example, it may be associated with: very early-onset obesity, in which the subject has a BMI above the 85th percentile for age and sex; binge eating and impaired satiety, in which the subject exhibits food-seeking and food-aggressive behaviors; impaired immune function, with reduced CD4+ T-cell counts and recurrent (and potentially fatal) infections; insulin resistance and hyperinsulinemia; progression to non-alcoholic fatty liver disease, hepatic steatosis, and lipodystrophy; dyslipidemia leading to hypertriglyceridemia; diabetes mellitus with elevated HbA1c and / or hyperglycemia and impaired glucose tolerance; hypogonadism, reproductive dysfunction leading to delayed pubertal development; reduced expression of secondary sexual characteristics; amenorrhea or menstrual irregularities, and infertility; dwarfism, absence of the pubertal growth spurt resulting in abnormal growth hormone secretion; hypothyroidism or thyroid dysfunction; altered T3, TSH, or free thyroxine levels; and variable bone changes, including bone density and bone mineral content. The spectrum of conditions and symptoms associated with monogenic obesity and / or lipodystrophy can vary depending on the underlying causative gene, e.g., AGPAT2, LMNA, BSCL2, or others. A given mutation can result in loss of function of leptin or LEPR, which can vary in endocrine severity, e.g., menstrual irregularities versus complete amenorrhea.

[0164] The antibodies and antigen-binding fragments provided herein are also useful for treating, alleviating, or preventing one or more symptoms of diseases or conditions associated with metabolic dysfunction or hypoleptinemia, including adiposity, obesity, hyperphagia, hyperglycemia, hypertriglyceridemia, hypercholesterolemia, insulin resistance, dyslipidemia, growth retardation, delayed pubertal growth spurt, impaired growth hormone secretion, elevated HbA1c, low bone mineral density (or bone mass), low bone mineral content, and low lean body mass.

[0165] The present invention also includes anti-LEPR antibodies and antigen-binding fragments thereof useful for restoring leptin signaling to cells, tissues, and organs expressing one or more LEPR mutations. Such mutations may be associated with metabolic dysfunction or hypoleptinemia, and diseases or conditions associated with metabolic dysfunction or hypoleptinemia, such as obesity, congenital lipodystrophy, infertility, and non-alcoholic fatty liver disease. For example, certain LEPR variants have been identified that exhibit no or reduced signaling in the presence of leptin and are associated with obesity and related disorders. As used herein, LEPR variants that do not exhibit signaling in the presence of leptin are referred to as "signaling-deficient LEPR variants." An exemplary signaling-deficient LEPR variant is LEPR-A409E (Farooqi et al., 2007, N Engl J Med 356(3):237-247). As used herein, LEPR mutants that exhibit reduced signaling (compared to wild-type LEPR) in the presence of leptin are referred to as "signaling-impaired LEPR mutants." An exemplary signaling-impaired LEPR mutant is LEPR-P316T (Mazen et al., 2011, Mol Genet Metab 102:461-464). Accordingly, the present invention includes anti-LEPR antibodies and antigen-binding fragments thereof that are useful for treating, preventing, and / or ameliorating diseases and disorders caused by or associated with one or more signaling-defective (e.g., A409E) LEPR mutants and / or one or more signaling-impaired (e.g., P316T) LEPR mutants.

[0166] The present invention also includes anti-LEPR antibodies and antigen-binding fragments thereof that are useful for restoring leptin signaling by alleviating mutations in the leptin gene. Some subjects have circulating leptin, but the protein is nonfunctional due to a genetic mutation that encodes a biologically inactive form of leptin, such as the p.N103K mutation in the leptin gene. Some subjects have little or no circulating leptin. Other genes, including LMNA, PPARG, AGPAT2, BSCL2, PLIN1, AKT2, CIDEC, LIPE, and ADRA2A, may also be involved in impaired leptin signaling, and the anti-LEPR antibodies and antigen-binding fragments thereof provided herein are useful for alleviating the effects of such mutations on leptin signaling.

[0167] The anti-LEPR antibodies and antigen-binding fragments thereof of the present invention are also useful for treating or preventing one or more conditions, diseases, or disorders selected from the group consisting of obesity, monogenic obesity, metabolic syndrome, diet-induced bulimia, functional hypothalamic amenorrhea, type 1 diabetes, type 2 diabetes, female infertility, amenorrhea, immune dysfunction, hypothyroidism, insulin resistance, severe insulin resistance including severe insulin resistance due to insulin receptor mutations, severe insulin resistance not due to insulin receptor mutations, severe insulin resistance caused by mutations in downstream signaling pathways or induced by other causes, non-alcoholic and alcoholic fatty liver disease, non-alcoholic steatohepatitis (NASH), Alzheimer's disease, leptin deficiency, leptin resistance, lipodystrophy, leprechaunism / Donahue syndrome, and Rabson-Mendenhall syndrome.

[0168] The LEPR agonistic antibodies provided herein are useful for treating metabolic dysfunction. The method includes administering to a subject in need thereof a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to the human leptin receptor (LEPR) and activates LEPR signaling, and a pharmaceutically acceptable carrier or diluent.

[0169] The LEPR agonistic antibody provided herein is useful for reducing adiposity or obesity, or body weight.In some embodiments, treatment reduces fat mass in treated subjects, but does not reduce lean mass.In some aspects, treatment causes subjects to consume fewer calories or reduce food intake.

[0170] The LEPR agonistic antibodies provided herein are useful for treating female infertility associated with leptin deficiency or restoring normal hormone cycles. In some embodiments, the treatment can increase fertility and / or increase the chances of pregnancy. In some embodiments, the treatment can restore normal menstrual cycles. Methods for restoring normal menstrual cycles disrupted at least in part by leptin deficiency are also part of the present invention.

[0171] As provided herein, the methods are useful when a subject in need thereof has or does not have hypoleptinemia or leptin deficiency. The methods are useful when the metabolic dysfunction, adiposity, or obesity is or is not associated with, or caused by, a signaling-deficient or a signaling-impaired LEPR mutation.

[0172] The LEPR agonistic antibody provided herein is useful for treating non-alcoholic fatty liver disease or non-alcoholic steatohepatitis (NASH) in patients with hypoleptinemia, lipodystrophy, or leptin deficiency.Treatment can reduce the symptoms of non-alcoholic fatty liver disease in subjects, such as hepatic steatosis.In some cases, the plasma level of alanine transaminase (ALT) and / or the plasma level of aspartate transaminase (AST) are reduced in subjects after receiving treatment.

[0173] The LEPR agonistic antibodies provided herein are useful for treating hyperphagia, hyperglycemia, insulin resistance, dyslipidemia, nonalcoholic steatohepatitis (NASH), or nonalcoholic fatty liver disease by stimulating hypothalamic STAT3 signaling. Treatment can reduce circulating plasma triglycerides and / or circulating plasma total cholesterol.

[0174] The LEPR agonistic antibodies provided herein are useful for treating lipodystrophy, which treatment reduces hyperglycemia, decreases insulin resistance, and / or lowers HbA1c levels in treated subjects.

[0175] The LEPR agonistic antibodies provided herein are useful for treating infertility and / or amenorrhea associated with metabolic disease or hypoleptinemia.The treatment can regulate hormone cycles and improve pregnancy rates in treated female subjects.The treatment can restore normal menstrual cycles.

[0176] The LEPR agonistic antibodies provided herein are useful for treating immune dysfunction, such as reduced CD4+ T cell counts, associated with hypoleptinemia and / or leptin deficiency. Treatment can improve immune function, for example, by increasing CD4+ T cell counts.

[0177] The LEPR agonistic antibodies provided herein are useful for treating growth retardation, lack of pubertal growth spurt, and / or defective growth hormone secretion associated with congenital leptin deficiency. Treatment can improve growth, promote the pubertal growth spurt, and / or improve growth hormone secretion.

[0178] The LEPR agonistic antibodies provided herein are useful for treating hypothyroidism associated with congenital leptin deficiency. Treatment can improve symptoms associated with hypothyroidism.

[0179] The LEPR agonistic antibodies provided herein are useful for treating low bone mineral density and / or low bone mineral content associated with hypoleptinemia and / or leptin deficiency. Treatment can improve bone mineral density and / or improve bone mineral content.

[0180] In the context of the therapeutic methods described herein, anti-LEPR antibodies may be administered as monotherapy (i.e., as the only therapeutic agent) or in combination with one or more additional therapeutic agents, examples of which are described elsewhere herein.

[0181] Combination Therapies and Formulations The present invention includes compositions and therapeutic formulations comprising any of the anti-LEPR antibodies described herein in combination with one or more additional therapeutically active ingredients, and includes methods of treatment comprising administering the compositions to a subject in need thereof.

[0182] The anti-LEPR antibodies of the invention may be co-formulated with and / or administered in combination with one or more additional therapeutically active ingredients, such as, for example, pharmaceutical agents prescribed for the treatment of obesity, hypercholesterolemia, hyperlipidemia, type 2 diabetes, type 1 diabetes, appetite control, amenorrhea, infertility, etc. Examples of such additional therapeutically active ingredients include, for example, recombinant human leptin (e.g., metreleptin [MYALEPT]), PCSK9 inhibitors (e.g., anti-PCSK9 antibodies [e.g., alirocumab, evolocumab, bococizumab, roderucizumab, ralpancizumab, etc.]), statins (e.g., atorvastatin, rosuvastatin, cerivastatin, pitavastatin, fluvastatin, simvastatin, lovastatin, pravastatin, etc.), ezetimibe, insulin, insulin variants, insulin secretagogues, metformin, sulfonylureas, sodium-glucose cotransporter 2 (SGLT2) inhibitors (e.g., dapaglifozin, canaglifozin, empagliflozin, etc.), GLP-1 agonists / analogs (e.g., extendi n)-4, exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, etc.), glucagon (GCG) inhibitors (e.g., anti-GCG antibodies), glucagon receptor (GCGR) inhibitors (e.g., anti-GCGR antibodies, small molecule GCGR antagonists, GCGR-specific antisense oligonucleotides, anti-GCGR aptamers [e.g., Spiegelmers], etc.), angiopoietin-like protein (ANGPTL) inhibitors (e.g., anti-ANGPTL3 antibodies, anti-ANGPTL4 antibodies, anti-ANGPTL8 antibodies, etc.), phentermine, orlistat, topiramate, bupropion, topiramate / phentermine, bupropion / naltrexone, bupropion / zonisamide, pramlintide / metreleptin, lorcaserin, cetilistat, tesofensine, verneperit, etc.Further examples include, for example, fish oil, pioglitazone, setmelanotide, fibrates (e.g., fenofibrate), prednisone, niacin, anticonvulsants, digoxin, Coumadin, vitamin D, thyroxine, thyroid supplements, vitamin supplements, calcium supplements, carnitine, coenzyme Q10, anti-constipation medications, anti-allergy medications, gabapentin, anesthetics, ketamine, lidocaine, and venlafaxine hydrochloride. In an embodiment of the invention, the anti-LEPR antibodies of the invention are not formulated or administered with an appetite suppressant.

[0183] The additional therapeutically active ingredient, such as any of the agents listed above or derivatives thereof, may be administered immediately prior to, simultaneously with, or immediately following administration of the anti-LEPR antibody of the invention (for purposes of this disclosure, such administration regimens will be considered administration of the anti-LEPR antibody "in combination with" the additional therapeutically active ingredient). The present invention includes pharmaceutical compositions in which the anti-LEPR antibody of the invention is co-formulated with one or more of the additional therapeutically active ingredients described elsewhere herein.

[0184] The present invention also includes methods of using compositions and therapeutic formulations comprising any of the anti-LEPR antibodies described herein in combination with a therapeutic procedure, such as, for example, plasma exchange.

[0185] Dosing regimen According to certain embodiments of the invention, multiple doses of an anti-LEPR antibody (or a pharmaceutical composition comprising a combination of an anti-LEPR antibody and any of the additional therapeutically active agents mentioned herein) may be administered to a subject over a defined time course. The method according to this aspect of the invention comprises sequentially administering multiple doses of an anti-LEPR antibody of the invention to a subject. As used herein, "sequentially administering" means that each dose of an anti-LEPR antibody is administered to a subject at different times, such as on different days separated by predetermined intervals (e.g., hours, days, weeks, or months). The invention includes methods comprising sequentially administering to a patient a single initial dose of an anti-LEPR antibody, followed by one or more second doses of the anti-LEPR antibody, and optionally followed by one or more third doses of the anti-LEPR antibody.

[0186] The terms "initial dose," "second dose," and "third dose" refer to the temporal order of administration of an anti-LEPR antibody of the invention. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose," "loading dose," "starting dose," etc.), a "second dose" is a dose administered after the initial dose, and a "third dose" is a dose administered after the second dose. The initial, second, and third doses all contain the same amount of anti-LEPR antibody, but typically may differ from one another in terms of administration frequency. However, in certain embodiments, the amount of anti-LEPR antibody contained in the initial, second, and / or third doses differs from one another (e.g., adjusted upward or downward) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered as "loading doses" at the beginning of a treatment regimen, with subsequent doses administered on a less frequent basis (e.g., "maintenance doses").

[0187] Diagnostic and analytical uses of antibodies The anti-LEPR antibodies of the present invention can also be used to detect and / or measure LEPR or LEPR-expressing cells in a sample, e.g., for diagnostic purposes. For example, an anti-LEPR antibody or fragment thereof may be used to diagnose a condition or disease characterized by aberrant expression (e.g., overexpression, underexpression, lack of expression, etc.) of LEPR. An exemplary diagnostic assay for LEPR includes, e.g., contacting a sample obtained from a patient with an anti-LEPR antibody of the present invention, wherein the anti-LEPR antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-LEPR antibody can be used in diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be, for example, 3 H, 14 C. 32 P, 35 S, or 125 The LEPR may be a radioisotope such as I, a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine, or an enzyme such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure LEPR in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorting (FACS), and positron emission tomography (PET) imaging.

[0188] Samples that can be used in the LEPR diagnostic assay of the present invention include any tissue or fluid sample obtainable from a patient that contains a detectable amount of LEPR protein or a fragment thereof under normal or pathological conditions. Generally, to first establish a baseline or standard LEPR level, the LEPR level is measured in a particular sample obtained from a healthy patient (e.g., a patient not suffering from a disease or condition associated with abnormal LEPR levels or activity). This baseline level of LEPR can then be compared with the LEPR level measured in a sample obtained from an individual suspected of having a LEPR-related disease or condition. [Example]

[0189] The following examples are provided so as to fully disclose to those skilled in the art how to make and use the methods and compositions of this invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, and temperature is in °C (Celsius) at or near atmospheric pressure.

[0190] Example 1: Production of an antigen-binding protein that specifically binds to the leptin receptor (LEPR) Anti-LEPR antibodies were obtained by immunizing VELOCIMMUNE® mice (i.e., engineered mice containing DNA encoding the variable regions of human immunoglobulin heavy and kappa light chains) with an immunogen containing the extracellular domain of LEPR. Antibody immune responses were monitored by LEPR-specific immunoassays. Fully human anti-LEPR antibodies were isolated and purified using previously reported techniques.

[0191] The specific biological properties of exemplary anti-LEPR antibodies generated according to the methods of this example are described in detail in the Examples set forth below.

[0192] Example 2: Amino acid and nucleic acid sequences of the heavy and light chain variable regions Table 1 shows the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-LEPR antibodies of the invention. The corresponding nucleic acid sequence identifiers are listed in Table 2.

[0193] Table 1: Amino acid sequence identifiers TIFF2025166129000001.tif95156

[0194] Table 2: Nucleic acid sequence identifiers TIFF2025166129000002.tif95156

[0195] Antibodies are generally referred to herein according to the following nomenclature: an Fc prefix (e.g., "H4H," "H1M," "H2M," etc.), followed by a numerical designator (e.g., "16650," "16679," etc.), followed by a "P" or "N" suffix. Thus, according to this nomenclature, antibodies may be referred to herein as, for example, "H4H16650P2," "H4H16679P2," etc. The Fc prefixes (H4H, H1M, and H2M) to antibody names used herein indicate the isotype of the particular Fc region of the antibody. For example, an "H4H" antibody has a human IgG4 Fc, while an "H1M" antibody has a mouse IgG1 Fc (all variable regions are fully human when an initial "H" appears in the antibody name). As will be appreciated by those skilled in the art, an antibody with a particular Fc isotype can be converted to an antibody with a different Fc isotype (e.g., an antibody with a murine IgG1 Fc can be converted to an antibody with a human IgG4 Fc, etc.), but in either case, the variable domains (including the CDRs) - which are indicated by the numerical identifiers shown in Tables 1 and 2 - will remain constant, and the binding characteristics are expected to be the same or substantially similar regardless of the nature of the Fc domain.

[0196] As used in the examples herein, "comparator mAb" refers to Fab9F8, described in Fazeli et al. (2006) J Immunol Methods 312:190-200, and Carpenter et al. (2012) Structure 20(3):487-97.

[0197] See International Patent Application Publication WO2017 / 66204.

[0198] Example 3: Surface Plasmon Resonance-Derived Binding Affinity and Rate Constants of Human Monoclonal Anti-LEPR Antibodies The equilibrium dissociation constant (K) for the binding of LEPR to purified anti-LEPR antibodies D The binding activity (RI) of the LEPR-1 antibody was determined using a real-time surface plasmon resonance biosensor with a Biacore4000 instrument. All binding studies were performed at 25°C and 37°C in a running buffer of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant Tween-20, pH 7.4 (HBS-ET). The Biacore sensor surface was first derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (GE, #BR-1008-39) to capture the anti-LEPR monoclonal antibody. Binding studies were performed on the following LEPR reagents: human LEPR extracellular domain expressed with a C-terminal myc-myc hexahistidine tag (hLEPR.mmh; SEQ ID NO: 114), cynomolgus monkey LEPR extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (mfLEPR.mmH; SEQ ID NO: 117), human LEPR extracellular domain expressed with a C-terminal mouse IgG2a Fc tag (hLEPR.mFc; SEQ ID NO: 115), mouse LEPR extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (mLEPR.mmh; SEQ ID NO: 118), and rat LEPR extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (rLEPR.mmh; SEQ ID NO: 119). Different concentrations of LEPR reagent were first prepared in HBS-ET running buffer (100 nM to 3.7 nM; 3-fold serial dilutions) and injected over the anti-human Fc-captured anti-LEPR monoclonal antibody surface at a flow rate of 30 μL / min for 4 min. Dissociation of the monoclonal antibody-bound LEPR reagent was monitored for 10 min in HBS-ET running buffer. Kinetic rates (k) were calculated by fitting the real-time binding sensorgrams to a 1:1 binding model with mass transport limitation using Scrubber 2.0c curve-fitting software. a ) and dissociation rate (k d The binding-dissociation equilibrium constant (K D) and dissociation half-life (t1 / 2) were calculated from the kinetic rate constants as follows:

[0199]

number

[0200] The binding kinetic parameters of hLEPR.mmh, mfLEPR.MMH, or hLEPR.mFc binding to various anti-LEPR monoclonal antibodies of the present invention at 25°C and 37°C are shown in Tables 3 to 8.

[0201] Table 3: Binding kinetic parameters of hLEPR-MMH binding to LEPR monoclonal antibodies at 25°C. TIFF2025166129000004.tif148149 * NB indicates that no binding was observed under the current experimental conditions.

[0202] Table 4: Binding kinetic parameters of hLEPR-MMH binding to LEPR monoclonal antibodies at 37°C. TIFF2025166129000005.tif149149 * NB indicates that no binding was observed under the current experimental conditions.

[0203] Table 5: Binding kinetic parameters of mfLEPR.MMH binding to LEPR monoclonal antibodies at 25°C. TIFF2025166129000006.tif148149 * NB indicates that no binding was observed under the current experimental conditions. * The IC indicates that the observed binding is global and could not be fitted to real-time binding data.

[0204] Table 6: Binding kinetic parameters of mfLEPR.MMH binding to LEPR monoclonal antibodies at 37°C. TIFF2025166129000007.tif149149 *NB indicates that no binding was observed under the current experimental conditions. * The IC indicates that under the current experimental conditions, the observed binding is global and it was not possible to fit the real-time binding data.

[0205] Table 7: Binding kinetic parameters of hLEPR.mFc binding to LEPR monoclonal antibodies at 25°C. TIFF2025166129000008.tif149149 * NB indicates that no binding was observed under the current experimental conditions.

[0206] Table 8: Binding kinetic parameters of hLEPR.mFc binding to LEPR monoclonal antibodies at 37°C. TIFF2025166129000009.tif149149 * NB indicates that no binding was observed under the current experimental conditions.

[0207] At 25°C, anti-LEPR monoclonal antibodies exhibited K values ​​ranging from 7.93 nM to 148 nM, as shown in Table 5. D At 37°C, the anti-LEPR monoclonal antibodies bound to hLEPR-MMH with K values ​​ranging from 14.8 nM to 326 nM, as shown in Table 4. D values, bound to hLEPR-MMH.

[0208] Ten of the twelve anti-LEPR monoclonal antibodies of the present invention bound to mfLEPR.MMH. At 25°C, the anti-LEPR monoclonal antibodies exhibited K values ​​ranging from 2.27 nM to 139 nM, as shown in Table 7. D At 37°C, the anti-LEPR monoclonal antibodies bound to mfLEPR.MMH with K values ​​ranging from 5.18 nM to 264 nM, as shown in Table 8. D values, bound to mfLEPR.MMH.

[0209] At 25°C, anti-LEPR monoclonal antibodies have K values ​​ranging from 613 pM to 5.7 nM, as shown in Table 7. D At 37°C, the anti-LEPR monoclonal antibodies bound to hLEPR-mFc with K values ​​ranging from 1.16 nM to 12.8 nM, as shown in Table 8. D values ​​and bound to hLEPR-mFc.

[0210] None of the anti-LEPR monoclonal antibodies of the present invention bound to mLEPR.MMH or rLEPR.MMH at 25° C. or 37° C. (data not shown).

[0211] Example 4: Anti-LEPR antibodies of the invention bind to LEPR in the presence of leptin:LEPR binding. Blockade of anti-LEPR antibody binding to LEPR by human leptin was assessed using a real-time surface plasmon resonance biosensor on a Biacore T200 instrument. The entire experiment was performed at 25°C in 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant Tween-20 (HBS-ET running buffer). The Biacore CM5 sensor surface was first derivatized by amine coupling of human leptin (R&D Systems, #398-LP) using standard EDC / NHS surface chemistry. Complexes of human LEPR and human leptin were formed by injecting 20 nM of human LEPR extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (hLEPR-MMH; SEQ ID NO: xx) over a human leptin-immobilized Biacore sensor surface at a flow rate of 10 μL / min or 25 μL / min for 4 minutes, achieving a binding response of approximately 200 RU. To assess whether antibody binding to hLEPR-MMH could be blocked by human leptin, 200 nM of anti-LEPR monoclonal antibodies were injected over the preformed hLEPR-MMH:human leptin complex at a flow rate of 50 μL / min or 25 μL / min for 4-5 minutes. All of the anti-LEPR antibodies of the present invention bound to the hLEPR-MMH:human leptin complex (leptin:LEPR) with approximately the same signal intensity. The observed binding, expressed in RU, is reported in Table 9. This result indicates that human leptin does not block the binding of hLEPR-MMH to the anti-LEPR antibodies tested.

[0212] Table 9. Binding of anti-LEPR monoclonal antibodies to preformed complexes of hLEPR-MMH and human leptin. TIFF2025166129000010.tif39128

[0213] Example 5: Human leptin receptor blockade ELISA For ELISA, human leptin (hLeptin; R&D Systems, #398-LP-01M) was coated onto 96-well microtiter plates at a concentration of 5 μg / mL in PBS overnight at 4°C. Nonspecific binding sites were then blocked using 0.5% (w / v) BSA in PBS. A 10 nM aliquot of the extracellular domain of the LEPR protein expressed with a C-terminal human Fc tag (hLEPR.hFc, SEQ ID NO: 116) was titrated with serial dilutions ranging from 8.5 pM to 500 nM of anti-LEPR antibody, human leptin protein, or isotype control antibody. These antibody-protein or protein-protein complexes were then incubated at room temperature (RT) for 1.5 hours. The complexes were then transferred to the human leptin-coated microtiter plates and incubated for 2 hours at room temperature. Wells were washed, and plate-bound hLEPR.hFc was detected using a horseradish peroxidase-conjugated anti-human IgG polyclonal antibody (Jackson ImmunoResearch Inc., #109-035-098). Samples were developed with TMB solution (BD Biosciences, #555214; substrate A and B mixed in a 1:1 ratio according to the manufacturer's instructions) to produce a colorimetric reaction, then neutralized with 1 M sulfuric acid before measuring absorbance at 450 nm on a Victor X5 plate reader.

[0214] Data analysis was performed using a sigmoidal dose-response model in Prism™ software (GraphPad). The percentage of blocking at the highest concentration of antibody tested was calculated as an indication of the antibody's ability to block the binding of 10 nM hLEPR.hFc to human leptin on the plate. In the calculation, the binding signal of 10 nM hLEPR.hFc in the absence of antibody was designated 100% binding or 0% blocking. And the baseline signal of buffer alone in the absence of hLEPR.hFc was designated 0% binding or 100% blocking. Blocking data at an antibody concentration of 500 nM are summarized in Table 10.

[0215] As shown in Table 10, none of the anti-LEPR antibodies of the present invention blocked hLEPR.hFc binding to the human leptin-coated surface by more than 28%. However, the comparator antibody and human leptin as positive controls were able to block 99% of hLEPR.hFc binding to the human leptin-coated surface. The isotype control antibody showed no measurable blocking at concentrations up to 500 nM.

[0216] Table 10. Blocking ELISA of hLEPR.hFc binding to human leptin by anti-LEPR antibodies TIFF2025166129000011.tif173128

[0217] Example 6: Cell binding by FACS analysis using HEK293 / Mycx2-hLepR(ecto)-GPI-fixed cells The leptin receptor (LEPR) is a single-transmembrane receptor of the class I cytokine receptor family (Tartaglia et al. (1997) J Biol Chem 7:272(10):6093-6). LEPR can bind to leptin, which is primarily expressed in adipose tissue, where it is involved in regulating food intake and metabolism (Friedman et al. (2014) J Endocrinol 223(1):T1-8).

[0218] To assess cell binding by anti-LEPR antibodies, HEK293 stable cell lines were generated. One cell line, hereafter referred to as HEK293 / hLEPR-GPI, stably expressed the extracellular domain of human LEPR (amino acids 22-839 of Accession No. P48357 (SEQ ID NO: 113), isoform B) with an N-terminal myc-myc tag and a C-terminal peptide sequence derived from human carboxypeptidase M, which directs the addition of GPI (glycosylphosphatidylinositol) so that the protein is membrane-tethered (Deddish et al. (1990) J. Biological Chemistry 265:25:15083-89). Another HEK293 cell line was engineered to stably express full-length human LEPR (amino acids 1-1165 of Accession No. P48357 (SEQ ID NO: 113), isoform B) along with a luciferase reporter (Stat3-luciferase, Stat3-luc, SA Bioscience, #CLS-6028L), hereafter referred to as HEK293 / Stat3-luc / hLEPR-FL. HEK293 cells containing only the Stat3-luciferase reporter were also engineered as a control cell line (HEK293 / Stat3-luc).

[0219] For FACS analysis, HEK293 parental cells and HEK293 / hLEPR-GPI cells were dissociated and plated at 5x10 in 96-well v-bottom plates in PBS containing 2% FBS (FACS buffer). 5Cells were seeded at 1000 x g / well. To test whether the ability of anti-hLEPR antibodies to bind to cells was affected by the presence of leptin, FACS buffer containing or not containing 1 μM human leptin (R&D Systems, #398-LP) was incubated with the cells for 30 minutes at 4°C, and then anti-LEPR or control antibodies were added at 10 nM in FACS buffer. The cells were then incubated for 30 minutes at 4°C, washed, and incubated with 16 μg / mL Alexa Fluor®-647-conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc., #109-547-003) for 30 minutes at 4°C. The cells were then fixed using BD CytoFix™ (Becton Dickinson, #554655), filtered, and analyzed on a HyperCyt Flow Cytometer (Beckman Coulter). Unstained and secondary antibody-only controls were also tested for all cell lines. Results were analyzed using ForeCyt (IntelliCyt) and FlowJo version 10 software to determine the geometric means of fluorescence for live cells. The geometric means of fluorescence for each sample were then normalized to the geometric means of unstained cells to obtain relative binding per condition, termed the "binding ratio." These binding ratios were recorded for each antibody tested.

[0220] As shown in Table 11, nine anti-LEPR antibodies of the present invention tested at 10 nM exhibited binding to HEK293 / hLEPR-GPI cells in the absence of leptin with binding ratios ranging from 824- to 3374-fold. Anti-LEPR antibodies also exhibited binding ratios of 398 and 4184-fold in the presence of 1 μM leptin. As shown in Table 11, a comparator antibody tested at 10 nM exhibited binding to HEK293 / hLEPR-GPI cells in the absence of leptin with a binding ratio of 2349-fold, but in the presence of 1 μM leptin, the binding ratio was 112, indicating a significant decrease in binding to the cells. Anti-LEPR antibodies exhibited little binding to HEK293 parental cells, with binding ratios ranging from 1- to 9-fold in the presence or absence of 1 μM leptin. Isotype control antibody and secondary antibody alone samples also showed little binding to either cell line in the presence or absence of leptin, with binding ratios ranging from 1 to 6-fold.

[0221] As shown in Table 12, in the absence of leptin, the four antibodies of the present invention tested at 70 nM exhibited binding to HEK293 / hLEPR-GPI cells with binding ratios ranging from 707-1131-fold and to HEK293 / Stat3-luc / hLEPR-FL cells with binding ratios ranging from 42-51-fold. The anti-LEPR antibodies exhibited little binding to HEK293 / Stat3-luc cells, with binding ratios ranging from 1-8-fold. The isotype control antibody and secondary antibody alone samples also exhibited little binding to any of the cell lines tested, with binding ratios ranging from 1-2-fold.

[0222] Table 11. Binding of 10 nM anti-LEPR antibodies to HEK293 / hLEPR-GPI cells and HEK293 parental cells + / - 1 μM human leptin TIFF2025166129000012.tif149144 * The classification of antibodies as "agonists" or "potentiators" is based in part on the results observed in Examples 7 and 8 herein.

[0223] Table 12. Binding of 70 nM anti-LEPR antibodies to HEK293 / hLEPR-GPI, HEK293 / Stat3-hLEPR-FL, and HEK293 / Stat3-luc parental cells. TIFF2025166129000013.tif97147

[0224] Example 7: Anti-LEPR antibodies of the present invention activate LEPR signaling in the presence or absence of leptin We developed a bioassay to detect STAT3 transcriptional activation via LEPR activation using a reporter cell line stably expressing full-length human LEPR (hLEPR; accession number NP_002294.2, amino acids 1–1165) in the human neuroblastoma cell line IMR-32, along with a luciferase reporter (STAT3-Luc, Qiagen, #CLS-6028L). The resulting stable cell line, designated IMR-32 / STAT3-Luc / hLEPR, was isolated and maintained in MEM-Earl's medium supplemented with 10% FBS, NEAA, 1 μg / mL puromycin, 100 μg / mL hygromycin B, and penicillin / streptomycin / L-glutamine (complete medium).

[0225] The resulting bioassay was used to measure the effect of anti-LEPR antibodies of the present invention on LEPR signaling in the presence or absence of leptin. For the bioassay, IMR-32 / STAT3-Luc / hLEPR cells were seeded in complete medium at a density of 20,000 cells / 100 μl / well in a 96-well format. The following day, the cells were replaced with an appropriate volume of Opti-MEM medium supplemented with 1% BSA and 0.1% FBS (assay buffer) for 30 minutes. To measure the effect of antibodies of the present invention in the absence of leptin, anti-LEPR antibodies or isotype control antibodies and human leptin (hLeptin; R&D Systems, #398-LP) were serially diluted in half-logarithmic amounts in assay buffer to final concentrations ranging from 100 nM to 300 fM. These were then added to the cells and incubated overnight at 37°C in 5% CO2.

[0226] To measure the effect of the antibodies of the present invention in the presence of leptin, a fixed concentration of 200 pM human leptin in assay buffer was added to the cells, followed immediately by the addition of anti-LEPR antibodies or isotype control antibodies in half-log serial dilutions to final concentrations ranging from 100 nM to 300 fM. Samples were then incubated overnight at 37°C in 5% CO2. OneGlo reagent (Promega, #E6051) was then added to the samples, and luciferase activity was measured on an Envision Multilabel Plate Reader (Perkin Elmer) in luminescence mode. Relative light unit (RLU) values ​​were obtained, and the results were analyzed using nonlinear regression in GraphPad Prism software (GraphPad). The maximum RLU value obtained from the human leptin dose-response was defined as 100% activation in the IMR-32 / STAT3-Luc / hLEPR assay.

[0227] As shown in Table 13, in Study 1, in the absence of human leptin, all anti-LEPR antibodies tested showed weak stimulation of IMR-32 / STAT3-Luc / hLEPR cells. 50 The values ​​ranged from 134 pM to 11.9 nM, with maximal activation ranging from 5% to 13% of the maximal activation obtained from the human leptin dose-response. In Study 2, in the absence of human leptin, the four anti-LEPR antibodies tested demonstrated stimulation of IMR-32 / STAT3-Luc / hLEPR cells. EC 50 The values ​​ranged from 61.9 pM to 206.9 pM, with maximal activation ranging from 65% to 68% of the maximal activation obtained from the human leptin dose-response. In Study 1, in the presence of 200 pM human leptin, all tested anti-LEPR antibodies demonstrated stimulation of IMR-32 / STAT3-Luc / hLEPR cells. EC 50The values ​​ranged from 20.2 pM to 523 pM, with maximal activation ranging from 66% to 107% of the maximal activation obtained from human leptin dose-response, respectively. Because these antibodies potentiated leptin-induced LEPR signaling, these antibodies were classified as "potentiators," as defined herein. In Study 2, in the presence of 200 pM human leptin, the four anti-LEPR antibodies tested demonstrated stimulation of IMR-32 / STAT3-Luc / hLEPR cells. EC 50 Values ​​ranged from 51.9 pM to 257.3 pM, with maximal activation ranging from 76% to 88% of that obtained from human leptin dose-response. LEPR signaling was not appreciably enhanced by these antibodies in the presence of leptin. Isotype control antibodies showed no measurable stimulation of IMR-32 / STAT3-Luc / hLEPR cells in any of the assays.

[0228] Table 13. Activation of hLEPR by anti-LEPR antibodies TIFF2025166129000014.tif146128

[0229] Example 8: Anti-LEPR antibodies of the invention activate signaling in cells expressing signaling-deficient or signaling-impaired LEPR mutants LEPR variants have been identified that exhibit defects or impairments in leptin-mediated signaling and are associated with early-onset obesity. For example, LEPR-A409E is a signaling-defective mutant LEPR protein that does not transmit leptin signals to STAT3. The A409E variant was originally identified as a monogenic cause of early-onset obesity (Farooqi et al., 2007, N Engl J Med 356(3):237-247). LEPR-P316T is a signaling-impaired mutant LEPR protein that has also been shown to be associated with early-onset obesity (Mazen et al., 2011, Mol Genet Metab 102:461-464).

[0230] In this example, the ability of anti-LEPR antibodies of the present invention to stimulate LEPR signaling was evaluated in cell lines expressing signaling-deficient or signaling-impaired LEPR mutants. Specifically, reporter cell lines (HEK293) expressing either wild-type LEPR, LEPR-A409E (signaling-deficient), or LEPR-P316T (signaling-impaired) were constructed. Cells were treated with either vehicle alone, recombinant human leptin, control IgG, or an agonistic anti-LEPR antibody of the present invention (H4H16650 or H4H16679), and the extent of LEPR signaling (measured by Western blotting detection of pSTAT3-Y705 expression relative to STAT3 expression) was determined.

[0231] In these experiments, the agonistic anti-LEPR antibodies of the present invention (H4H16650 and H4H16679) were shown to stimulate LEPR signaling (measured by STAT3 expression) in a dose-dependent manner in cells expressing the LEPR-A409E or LEPR-P316T mutant (Figure 2, panels B and C). In contrast, leptin treatment induced only moderate signaling in cells expressing the LEPR-P316T mutant but not in cells expressing the LEPR-A409E mutant (Figure 2, panel A). Furthermore, LEPR signaling was not detected in any cell line treated with vehicle or an IgG control antibody (data not shown). Other signaling-deficient or signaling-impaired LEPR mutants tested in this assay were not activated by the anti-LEPR mutants (data not shown). This suggests that this restorative effect is mutant-dependent.

[0232] The results of this example indicate that the agonistic anti-LEPR antibodies of the invention may be useful for treating diseases and disorders (e.g., early-onset obesity) caused by or associated with certain signaling-deficient or signaling-impaired LEPR mutants (e.g., LEPR-P316T or LEPR-A409E).

[0233] Example 9: Octet cross-competition between various anti-LEPR monoclonal antibodies. Binding competition between a panel of various anti-LEPR monoclonal antibodies was determined using a real-time label-free biolayer interferometry assay on an Octet HTX biosensor platform (Pall ForteBio Corp.). The entire experiment was performed at 25°C with shaking plates at 1000 rpm in a buffer containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant Tween-20, 1 mg / mL BSA, pH 7.4 (HBS-EBT). To assess whether two antibodies could compete with each other for binding to their respective epitopes on recombinant human LEPR (hLEPR.mmh; SEQ ID NO: 114) expressed with a C-terminal myc-myc-hexahistidine tag, approximately 0.25 nm or 0.34 nm of hLEPR-MMH was first captured on an anti-penta-His antibody-coated Octet biosensor chip (Fortebio Inc., #18-5122) by immersing the biosensor chip in a well containing 20 μg / mL hLEPR-MMH for 5 minutes. The biosensor chip was then saturated with a first anti-LEPR monoclonal antibody (hereafter referred to as mAb-1) by immersing the captured chip in a well containing 50 μg / mL mAb-1 solution for 210 seconds. The biosensor chip was then immersed in a well containing 50 μg / mL of a second anti-LEPR monoclonal antibody (hereafter referred to as mAb-2) for 150 seconds. The biosensor chip was washed with HBS-EBT buffer between steps of the experiment. Real-time binding responses were monitored throughout the entire course of the experiment, and the binding responses were recorded at the end of each step. The binding responses of mAb-2 to pre-complexed hLEPR-MMH with mAb-1 were compared to determine the competitive / non-competitive behavior of various anti-LEPR monoclonal antibodies, as shown in Tables 14 and 15.

[0234] Table 14. Cross-competition between anti-LEPR monoclonal antibodies TIFF2025166129000015.tif190128

[0235] Table 15. Cross-competition between anti-LEPR monoclonal antibodies TIFF2025166129000016.tif119128

[0236] Example 10: In vivo efficacy of LEPR agonistic antibodies H4H16650P2, H4H16679P2, H4H17319P2, and H4H17321P2 in an inducible mouse model of leptin deficiency. The effects of four specific agonistic anti-LEPR antibodies of the present invention, H4H16650P2, H4H16679P2, H4H17319P2, and H4H17321P2, on food intake, body weight, and adiposity were assessed in a genetically engineered LEPR mouse model. Hu / Hu This was determined in an inducible model of leptin deficiency in mice. The mice express a leptin receptor composed of the human LEPR extracellular domain sequence instead of the mouse LEPR extracellular domain sequence. The leptin deficiency model was induced by hydrodynamic DNA delivery (HDD) of a plasmid encoding an hFc-tagged mouse LEPR extracellular domain (referred to herein as mLEPR.hFc or "leptin trap"; SEQ ID NO: 120). When expressed, leptin trap is secreted and binds to circulating leptin. After HDD of 50 μg of a DNA construct encoding leptin trap, mice exhibited increased food consumption, as well as increased adiposity and body weight.

[0237] Baseline daily food intake was measured 7–4 days before administration of leptin trap (days −7–−4). On day 0, 35 male LEPR mice aged 13–17 weeks were used. Hu / HuMice successfully underwent HDD using a leptin trap. Blood was collected retroorbitally on days 6 and 13 after HDD, and body composition, including adiposity, was quantified by μCT. On day 7 after HDD, mice were randomized into five groups (7 mice per group) based on the percent change in body weight from day 0. Each group received a single dose of 3 mg / kg of isotype control antibody, 3 mg / kg of H4H16650P2, 3 mg / kg of H4H16679P2, 3 mg / kg of H4H17319P2, or 3 mg / kg of H4H17321 via subcutaneous injection. The isotype control antibody did not bind to any known mouse protein. Food intake and body weight were measured for each animal throughout the study. Figure 3 summarizes the average daily food intake for each treatment group. In Figure 3, the dotted line represents the average baseline food intake before HDD injection. The percent change in body weight from day 0 was calculated for each animal at each time point. Figure 4 summarizes the mean percent change in body weight for animals in each antibody treatment group. Figure 5 summarizes the mean fat mass of animals in each antibody treatment group, as quantified by μCT 1 day before and 6 days after antibody treatment. All results are expressed as mean ± SEM.

[0238] As shown in Figures 3 and 4, similar increases in food intake and rates of weight change were observed between mouse groups before antibody treatment after a leptin trap HDD. As shown in Figure 3, mice treated with 3 mg / kg of H4H16650P2 or H4H16679P2 antibody exhibited significantly reduced food intake from day 1 after antibody treatment (day 8 after HDD) and at all subsequent measurement points compared to mice injected with an isotype control antibody. Mice treated with 3 mg / kg of H4H17319P2 or H4H17321P2 antibody exhibited significantly reduced food intake from day 2 after antibody treatment (day 9 after HDD) and at all subsequent measurement points compared to mice injected with an isotype control antibody. As shown in Figure 4, mice treated with 3 mg / kg of H4H16650P2 antibody exhibited a significantly reduced rate of weight change on day 1 after antibody treatment (day 8 after HDD) and at other subsequent time points compared to mice injected with an isotype control antibody. On day 8, one day after antibody treatment, mice treated with an isotype control antibody showed a weight gain of 21.16 ± 1.27% from day 0. Meanwhile, mice treated with H4H16650P2 showed a weight gain of 15.57 ± 0.9% from day 0. Mice treated with 3 mg / kg of H4H16679P2, H4H17319P2, or H4H17321P2 antibodies exhibited a significantly reduced rate of weight change on day 2 after antibody treatment (day 9 after HDD) and at other subsequent time points compared to mice injected with an isotype control antibody. On day 9, the percent weight change from day 0 was 23.18 ± 1.22%, 13.17 ± 1.05%, 12.95 ± 1.26%, 15.98 ± 1.78%, and 15.83 ± 2.01% for mice treated with isotype control, H4H16650P2, H4H16679P2, H4H17319P2, or H4H17321P2, respectively. As shown in Figure 5, mice treated with 3 mg / kg of isotype control antibody showed a significant increase in fat mass 6 days after antibody treatment (post-HDD, day 13) compared to 1 day before antibody treatment (post-HDD, day 6).Mice treated with 3 mg / kg of H4H16650P2, H4H16679P2, H4H17319P2, or H4H17321P2 antibodies did not show an increase in fat mass after antibody treatment compared to before antibody treatment. Six days after treatment (day 13 after HDD), mice treated with 3 mg / kg of H4H16650P2, H4H16679P2, or H4H17319P2 antibodies showed a significant decrease in fat mass compared to mice treated with 3 mg / kg of an isotype control antibody.

[0239] Example 11: Epitope mapping for H4H16650P2 binding to the human leptin receptor (hLEPR.mmh) by hydrogen-deuterium exchange. Experiments were conducted to determine the amino acid residues of hLEPR.mmh (amino acids M1-D839 of SEQ ID NO: 114) with which H4H16650P2 interacts. To this end, H / D exchange epitope mapping including mass spectrometry was performed. A general description of the H / D exchange method is described, for example, in Ehring (1999) Analytical Biochemistry 267(2):252-259; and Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0240] Experimental procedure HDX-MS experiments were performed on an integrated Waters HDX / MS platform consisting of a Leaptec HDX PAL system for deuterium labeling, a Waters Acquity M-Class (Auxiliary solvent manager) for sample digestion and loading, a Waters Acquity M-Class (μBinary solvent manager) for analytical column gradients, and a Synapt G2-Si mass analyzer for digested peptide mass measurement.

[0241] Labeling solutions were prepared in 10 mM PBS buffer in DO at pH 7.0 (equivalent to pH 6.6). For deuterium labeling, 3.8 μL of hLEPR.mmh (8 pmol / μL) or hLEPR.mmh premixed with antibody at a 2:1 molar ratio was incubated with 56.2 μL of DO labeling solution for various times (e.g., undeuterated control = 0 s, labeling = 1 and 20 min). Samples were quenched by transferring 50 μL of pre-chilled quench buffer (0.2 M TCEP, 6 M guanidine chloride in 100 mM phosphate buffer, pH 2.5), and the combined sample solution was incubated for 2 min at 1.0 °C. The quenched samples were then injected into the Waters HDX Manager for online pepsin / protease XIII digestion. The digested peptides were trapped on an ACQUITY UPLC BEH C18 1.7-μm, 2.1 x 5 mm VanGuard precolumn at 0 °C and eluted onto an ACQUITY UPLC BEH C18 1.7-μm, 1.0 x 50 mm analytical column using a 9-minute gradient of 5% to 40% B (mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile). The mass spectrometer was configured with a cone voltage of 37 V, a scan time of 0.5 s, and a mass / charge range of 50 to 1700 Th.

[0242] For the identification of peptides derived from human LEPR, LC-MSE data from undeuterated samples were processed and searched against a database containing human LEPR, pepsin, and their randomized sequences via Waters ProteinLynx Global Server (PLGS) software. Identified peptides were imported into DynamX software and filtered by two criteria: 1) minimum product per amino acid of 0.2, and 2) duplicate file threshold of 3. DynamX software then automatically determined the deuterium incorporation of each peptide based on retention time and high mass accuracy (<10 ppm) from multiple time points, including three replicates per time point.

[0243] result .MSE Using an online pepsin / protease XIII column coupled with data acquisition, a total of 201 peptides from human LEPR were reproducibly identified in the presence or absence of antibodies, representing 70% sequence coverage. As shown in Table 16, five peptides showed significantly reduced deuterium incorporation upon binding to H4H16650P2 (centroid delta values ​​>0.4 Daltons, p-values ​​<0.05). Reported peptide masses correspond to the average centroid MH+ mass from three replicates. These peptides, corresponding to amino acids 162-169 (human LEPR; amino acids LYVLPEVL of SEQ ID NO: 113) and 170-181 (human LEPR; amino acids EDSPLVPQKGSF of SEQ ID NO: 113), showed a reduced deuteration rate upon binding to H4H16650P2. These identified residues also correspond to amino acid residues 162-169 and 170-181 of human LEPR as defined by Uniprot entry P48357 (SEQ ID NO: 113; human leptin receptor).

[0244] Table 16. Human leptin receptor peptides that are significantly protected by binding to the H4H16650P2 antibody TIFF2025166129000017.tif59141

[0245] Example 12: Validation of in vivo efficacy of LEPR-enhancing antibodies in humanized LEPR mice The effects of the three specific enhancing anti-LEPR antibodies of the present invention, H4H18482P2, H4H18487P2, and H4H18492P2, on body weight and adiposity were evaluated in single-housed genetically engineered LEPR mice. Hu / Hu This was determined in mice expressing a leptin receptor composed of the human LEPR extracellular domain sequence in place of the mouse LEPR extracellular domain sequence (mLEPR.hFc, SEQ ID NO: 120).

[0246] On day -19, body composition, including adiposity, was quantified by μCT. On day 0, 48 14- to 16-week-old female LEPR rats were used. Hu / HuMice were randomized based on body weight into four groups of 12 mice per group. On days 0 and 11, mice in each group received a single dose of 30 mg / kg isotype control antibody, 30 mg / kg H4H18482P2, 30 mg / kg H4H18487P2, or 30 mg / kg H4H18492P2 via subcutaneous injection. The isotype control antibody does not bind to any known mouse protein. Each animal was weighed throughout the study. The percent change in body weight from day 0 was calculated for each animal at each time point. Figure 6 summarizes the average percent change in body weight for animals in each treatment group. Figure 6 summarizes the average fat mass of animals in each antibody treatment group, as determined by μCT 19 days before and 11 days after antibody treatment. All results are expressed as mean ± SEM.

[0247] As shown in Figure 6, a decrease in the rate of weight change was observed after administration of the LEPR-enhancing antibody, but not the isotype control antibody. As shown in Figure 6, mice treated with 30 mg / kg of H4H18482P2 antibody exhibited a significant decrease in the rate of weight change from day 2 after treatment (day 2) onward at other time points compared to mice injected with the isotype control antibody. Mice treated with 30 mg / kg of H4H18487P2 antibody exhibited a significant decrease in the rate of weight change from day 2 onward at other time points compared to mice injected with the isotype control antibody. Mice treated with 30 mg / kg of H4H18492P2 antibody exhibited a significant decrease in the rate of weight change on days 4, 5, and 17, but not at other time points, compared to mice injected with the isotype control antibody. Mice treated with 30 mg / kg H4H18482P2 antibody exhibited a significant decrease in weight change rate compared to mice injected with H4H18492P2 antibody on day 6, but not on days 7, 14, and 17. Mice treated with 30 mg / kg H4H18487P2 antibody exhibited a significant decrease in weight change rate at other time points from day 3, but not on days 4 and 5, compared to mice injected with H4H18492P2 antibody.

[0248] As shown in Figure 7A, there was no difference in fat mass between the groups before treatment (day -19). As shown in Figure 7B, mice treated with H4H18482 and H4H18487 antibodies at 30 mg / kg, but not H4H18492 antibody, showed a statistically significant decrease in fat mass 17 days after treatment (day 12) compared to the isotype control antibody.

[0249] Example 13: Effect of anti-LEPR antibodies of the present invention on monkey LEPR signaling To evaluate the transcriptional activation of the monkey leptin receptor, a stable cell line was developed. IMR-32 cells (human neuroblastoma, ATCC) were generated stably expressing the extracellular domain of the cynomolgus monkey LEPR (MfLEPR; accession number XP_005543194.1, amino acids 22–837, with a threonine-to-alanine change at position 827) fused to the transmembrane and cytoplasmic domains of the human LEPR (hLEPR; accession number NP_002294.2, amino acids 840–1165) along with a luciferase reporter (STAT3-Luc; SABiosciences, #CLS-6028L). The resulting cell line, hereafter referred to as IMR-32 / STAT3-Luc / MfLEPR, was isolated and maintained in MEM-Earl medium supplemented with 10% FBS, NEAA, 1 ug / mL puromycin, 100 ug / mL hygromycin B, and penicillin / streptomycin / L-glutamine.

[0250] Bioassays were performed to measure the effect of anti-LEPR antibodies of the present invention on monkey LEPR signaling in the absence of leptin. For the bioassays, IMR-32 / STAT3-Luc / MfLEPR cells were seeded at 10,000 cells / well in 96-well plates in Optimem with penicillin / streptomycin and 0.1% FBS (assay buffer) and incubated overnight at 37°C with 5% CO2. The next day, human leptin (hLeptin), anti-LEPR antibodies, or isotype controls were serially diluted in assay buffer from 50 nM to 0.8 pM (plus a sample containing buffer alone without the test molecule) and added to the cells. After 5.5 hours at 37°C with 5% CO2, luciferase activity was measured using OneGlo™ reagent (Promega, # E6031) and a Victor™ X multilabel plate reader (Perkin Elmer). The results were analyzed using nonlinear regression (four-parameter logistic) with Prism™ 6 software (GraphPad Inc.) to determine EC 50 The antibody activation ratio was calculated as the maximum range of RLU achieved by the antibody compared to the maximum range of RLU achieved by human leptin.

[0251] As shown in Table 17, in the absence of human leptin, all anti-LEPR antibodies tested demonstrated activation of monkey LEPR signaling in IMR-32 / STAT3-Luc / mfLEPR cells. 50 The values ​​ranged from 266 pM to 368 pM, with a maximum activation range of 76% to 82%. 100% activation was obtained with human leptin, which has an EC of 333 pM. 50 The isotype control antibody showed no measurable stimulation of IMR-32 / STAT3-Luc / mfLEPR cells.

[0252] Table 17. Activation of cynomolgus monkey LEPR by anti-LEPR antibodies TIFF2025166129000018.tif67128

[0253] Example 14: Epitope binding to the full length extracellular domain of human LEPR using Luminex MFI signal To determine the epitopes on human LEPR that are bound by the anti-LEPR antibodies of the present invention, Luminex FLEXMAP (FM3DD, LuminexCorp) flow cytometry-based analysis was used to characterize the interaction of recombinant human LEPR protein domains with anti-LEPR antibodies. For the assay, approximately 3 million carboxylated Microplex R Microspheres (Luminex, Cat# LC1000A) were washed, vortexed, and sonicated in 0.1 M NaPO4, pH 6.2 (activation buffer), followed by centrifugation and removal of the supernatant. The microspheres were resuspended in 120 μL of activation buffer, and the carboxylate groups (-COOH) were activated by adding 15 μL of 50 mg / mL N-hydroxysuccinimide (NHS, Thermo Scientific, Cat# 24500). Then, at 25°C, 15 μL of 50 mg / mL 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC, Thermo Scientific, Cat# 22980) was added. After 10 min, the pH of the reaction mixture was lowered to 5.0 by adding 600 μL of 50 mM MES, pH 5 (coupling buffer). The microspheres were vortexed and centrifuged to remove the supernatant. The activated beads were immediately mixed with 500 μL of 20 μg / mL anti-myc monoclonal antibody containing either mouse IgG or human IgG in coupling buffer and incubated for two hours at 25°C. The coupling reaction was quenched by adding 50 μL of 1 M TRIS-HCl, pH 8.0, and the microspheres were rapidly vortexed, centrifuged, and washed four times with 1 mL of DPBS to remove uncoupled proteins and other reaction components.

[0254] Human LEPR extracellular domain expressed with a C-terminal myc-myc hexahistidine tag (human LEPR-MMH, SEQ ID NO: 113), human LEPR CRH1(D1) expressed with a C-terminal myc-myc hexahistidine tag (human LEPR CRH1(D1)-MMH, amino acids 1-208 of SEQ ID NO: 113 with a myc-myc hexahistidine tag at amino acids 209-236), human LEPR CRH1(D1,D2) domain expressed with a C-terminal myc-myc hexahistidine tag (human LEPR-CRH1(D1,D2)-MMH, amino acids 1-318 of SEQ ID NO: 113 with a myc-myc hexahistidine tag at amino acids 319-346), human LEPR expressed with a C-terminal myc-myc hexahistidine tag CRH1-Ig(D1, D2, D3) domain (human LEPR CRH1(D1, D2, D3)-MMH, amino acids 1-278 of SEQ ID NO: 113, with a myc-myc hexahistidine tag at amino acids 279-306), human LEPR CRH1-Ig(D2, D3) domain expressed with a C-terminal myc-myc hexahistidine tag (human LEPR CRH1-Ig(D2, D3)-MMH, amino acids 1-198 of SEQ ID NO: 113, with a myc-myc hexahistidine tag at amino acids 199-226), human LEPR Ig(D3) domain expressed with a C-terminal myc-myc hexahistidine tag (human LEPR Ig(D3)-MMH, amino acids 1-88 of SEQ ID NO: 113, with a myc-myc hexahistidine tag at amino acids 89-116; human LEPR CRH2 domain expressed with a C-terminal myc-myc hexahistidine tag (human LEPR CRH2-MMH, amino acids 1-207 of SEQ ID NO: 113, with a myc-myc hexahistidine tag at amino acids 208-235); human LEPR FNIII domain expressed with a C-terminal myc-myc hexahistidine tag (human LEPR FNIII-MMH, amino acids 1-204 of SEQ ID NO: 113, with a myc-myc hexahistidine tag at amino acids 205-232); human LEPR Ig-CRH2-FNIII domain expressed with a C-terminal myc-myc hexahistidine tag (human LEPRTransiently expressed LEPR proteins, including Ig-CRH2-FNIII-MMH (amino acids 1-510 of SEQ ID NO: 113, with a myc-myc hexahistidine tag at amino acids 511-538), were suspended in serum-free CHO-S-SFM II medium (Thermo Fisher Scientific, Cat. # 31033020) and then clarified by centrifugation. Aliquots of microspheres with immobilized anti-myc monoclonal antibodies prepared as described above were added individually to 1 mL of each of these protein supernatants. The microspheres were gently mixed, incubated at 25°C for 2 hours, washed twice with 1 mL of DBPS, centrifuged to remove the supernatant, and finally resuspended in 1 mL of DPBS buffer. 48 μL of anti-myc IgG-coupled microspheres from individual reactions with full-length human LEPR and each human LEPR domain protein were withdrawn and mixed together in 3.6 mL of PBS + 20 mg / mL BSA + 0.05% sodium azide (blocking buffer).

[0255] From this mixed pool, microspheres were seeded at 75 μL per well of a 96-well filter plate (Millipore, Cat. No. MSBVN1250), mixed with 25 μL of individual anti-human LEPR monoclonal antibodies (0.5 or 5 μg / mL), incubated at 25°C for two hours, and then washed twice with 200 μL of DPBS containing 0.05% Tween 20 (wash buffer). To detect and quantify the amount of anti-LEPR antibody bound to individual microspheres, either 2.5 μg / mL R-Phycoerythrin-conjugated goat F(ab')2 anti-human kappa (Southern Biotech, Cat. No. 2063-09) in 100 μL of blocking buffer or 1.25 μg / mL R-Phycoerythrin AffiniPure F(ab')2 fragment goat anti-mouse IgG, F(ab')2 fragment specific (Jackson Immunoresearch, Cat. No. 115-116-072) in 100 μL of blocking buffer was added and incubated for 30 minutes at 25°C. After 30 minutes, samples were washed twice with 200 μL of wash buffer and resuspended in 150 μL of wash buffer. The median fluorescence intensity (MFI) of the microspheres was measured using a Luminex analyzer.

[0256] Table 18. Luminex MFI signals of anti-LEPR antibodies binding to the myc-tag-captured full-length human LEPR extracellular domain and isolated human LEPR domain. TIFF2025166129000019.tif207163

[0257] The results of the Luminex-based analysis are listed in Table 18. The Luminex MFI signal intensities indicate that the 12 anti-LEPR antibodies of the present invention bound to the complete human LEPR extracellular domain. Anti-LEPR antibodies H4H18417P2, H4H18438P2, and H4H18492P2 bound to epitopes within the CRH1 D2 domain of human LEPR. Anti-LEPR antibodies H4H18449P2, H4H16650P, and H4H16679P bound to epitopes within the CRH1 (D1-D2) domain of human LEPR. Comparator monoclonal antibodies for the anti-LEPR antibodies bound to epitopes within the CRH2 domain of human LEPR. Anti-LEPR antibody H4H18445P2 bound to an epitope within the FNIII domain of human LEPR. The anti-LEPR antibodies H4H18446P2, H4H18482P2, and H4H18487P2 bound to epitopes within the Ig-CRH2-FNIII domain of human LEPR.

[0258] Examples 15 to 19: Animal Experiment Protocol All animal studies were conducted in accordance with and approved by the Institutional Animal Care and Use Committee (IACUC) of Regeneron Pharmaceuticals, Inc. Monkey studies were also conducted in accordance with and approved by the IACUC of Covance Laboratories, Inc.

[0259] Mouse experiments Hydrodynamic DNA delivery Hydrodynamic DNA delivery (HDD)-based in vivo transfection is a protocol involving the rapid injection of a large volume of solution containing naked plasmid DNA to express foreign proteins in live animals (Suda, 2007). Expression vectors were freshly prepared by weighing mice and suspending them in a final volume of saline equal to one-tenth of the body weight (v / w). The vectors were then delivered by injection through the lateral tail vein. For the leptin attenuation model validation experiment (Figure 11), 8-week-old C57BL / 6N mice (Taconic) were administered 50 μg DNA per mouse of the mLeprECD expression vector (pRG977.mROR.mLepR.ecto.hFc) or control vector (pRG977.hFc). For the H4H17319P2 evaluation experiment (Figure 9), 17- to 20-week-old male and female Lepr hu / hu Mice were administered 50 μg DNA per mouse of the mLeprECD expression vector (pRG977.mROR.mLepR.ecto.hFc).

[0260] Measurement of body weight and food intake Body weight was measured by placing the mouse in a container on a tared digital laboratory scale. The average weight during a 3-second dynamic weighing was used. Food intake was determined by measuring the amount of food in the food hopper using a digital laboratory scale. Food intake was calculated as the difference between the weight of food placed in the hopper and the weight of food remaining in the hopper.

[0261] Body composition Micro-computed tomography (micro-CT) imaging was performed on live mice anesthetized with isoflurane gas using a Quantum Micro-CT imaging system (Perkin Elmer) according to the manufacturer's instructions. Body composition (fat mass, lean mass, bone mass, bone mineral content, and density) was measured using the Quantum Micro-CT imaging system (Perkin Elmer) according to the manufacturer's instructions. Scans were then analyzed using AnalyzeDirect imaging software. Fat mass, lean mass, and bone mass were calculated by multiplying the recorded tissue volume by the following respective mass densities: 0.92, 1.05, and 1.7 g / cm. 3 Quantitative nuclear magnetic resonance (qNMR) using an EchoMRI 100 instrument (EchoMRI) was performed on conscious, live mice to measure fat, lean mass, free water, and total water.

[0262] Blood glucose measurement Postprandial blood glucose levels were measured from a tail vein nick in conscious mice using a glucometer (AlphaTrak2, Zoetis) and glucose test strips (Zoetis).

[0263] Insulin Tolerance Test After a 4-hour fast, animals were injected intraperitoneally (IP) with 0.5, 0.75, or 1.0 U / kg insulin (Humulin®, Eli Lilly and Company) doses, as indicated. Glucose measurements were performed using an AlphaTRAK2 blood glucose meter and test strips (Zoetis) at 0, 15, 30, 60, and 120 minutes after insulin injection. hu / hu In the characterization experiments, Accu-Chek® Compact Plus blood glucose meters and test strips (Roche Diabetes Care, Inc.) were used.

[0264] Chemical analysis and hormone assays Unless otherwise specified, mice were fasted for 4 hours, after which blood was collected via retro-orbital bleed. For serum isolation, blood was transferred to serum separator tubes (Sarstedt AG & Co.), allowed to clot on wet ice for at least 30 minutes, and then centrifuged at 10,000 x g for 5 minutes. Serum was removed and stored at -80°C until processed for leptin quantification. For plasma measurements, blood was transferred to K3EDTA-coated tubes (Sarstedt AG & Co.). A cocktail of dipeptidyl peptidase 4 (DPP4) inhibitors and protease inhibitors was added to the blood samples, which were then stored on ice until processed. Plasma was obtained by centrifugation at 2000 x g for 10 minutes. The plasma was aliquoted and stored at -80°C until used for quantification of plasma lipids, liver enzymes, and leptin levels. HbA1c was measured in fresh whole blood collected in K3EDTA-coated tubes. HbA1c, plasma lipids, and liver enzymes were quantified using a chemistry analyzer (Advia Chemistry XPT, Siemens). Serum or plasma leptin levels were measured using an immunoassay kit (Milliplex MAP, Millipore) according to the manufacturer's recommended protocol.

[0265] Liver triglyceride determination Liver triglyceride content was quantified from 100–200 mg of liver. The tissue was ground to a powder using a liquid nitrogen-cooled mortar and pestle. The powdered liver tissue was weighed and homogenized in PBS using a bead homogenizer (FastPrep 24-5G, MP Biomedical). The homogenate was transferred to a glass tube containing 5 mL of Folch's solution (2:1 chloroform:methanol), vortexed, and centrifuged at 1500 x g for 20 minutes. The lower layer was collected, drawn down to 5 mL, and vortexed. Specific volumes (25–50 μL) of sample and triolein standard (Verichem) were transferred to a 96-well polypropylene plate, mixed with 10 μL of a 1:1 chloroform:Triton X-100 mixture, and air-dried. To the dried samples and standards, 300 μL of triglyceride reagent (Thermo Scientific) was added, and the plate was shaken for 5 minutes and then incubated for 20 minutes at 37° C. 200 μL of each reaction was transferred to a new clear polypropylene 96-well plate, and the absorbance at 500 nm was measured using a plate reader (Molecular Devices).

[0266] Fixation perfusion and immunohistochemistry Mice were anesthetized with sodium pentobarbital (110 mg / kg, IP) and transcardially perfused with 2 mL of saline, followed by 150 mL of 4% paraformaldehyde in 0.1 M borate buffer, pH 9.5. Livers were post-fixed for 2 hours, transferred to 70% ethanol, paraffin-embedded, sectioned, and stained with hematoxylin and eosin in Histserv. Brains were post-fixed for 2 hours at 4°C and then immersed in 15% sucrose in potassium phosphate-buffered saline overnight at 4°C. Whole brains were prepared on a freezing sliding microtome (Leica), sectioned at 30 μm thickness, collected in evenly spaced series, and preserved in cryoprotectant (20% glycerol and 30% ethylene glycol in 0.1 M phosphate buffer) and stored at −20°C in cryoprotectant (20% glycerol and 30% ethylene glycol in 0.1 M phosphate buffer).

[0267] Serial brain sections spaced 150 μm apart from each experimental group were processed simultaneously to ensure comparable staining across animal and treatment groups. For all immunostaining, brain sections were blocked, and primary and secondary antibodies were diluted in a solution containing 1% donkey serum (Equitech), 0.03% Triton X-100, and 0.05 M phosphate-buffered saline. Avidin and biotin blocking was performed according to the manufacturer's protocol (Vector Labs). Immunohistochemical staining of pStat3 (Y705) (#9145, Cell Signaling Technologies, 1:1000, 16 hours at 4°C) was performed on equally spaced serial brain sections using the avidin-biotin complex method with the chromogen diamine-benzidine (Vector Labs). For p-STAT3 (Y705) immunohistochemistry (IHC), sections were pretreated with 1% H2O2 in 1% NaOH for 10 min, 0.3% glycine in K-PBS for 10 min, and 0.03% SDS in K-PBS for 10 min.

[0268] Brightfield images were acquired by whole-slide scanning using a 40x objective on a Scanscope XT (Aperio). Sections were matched for histological comparison using the mouse brain atlas of Franklin and Paxinos. Neuroanatomical regions and distances from the anterior section were estimated based on this atlas. The number of pSTAT3 (Y705)-immunoreactive cells was quantified bilaterally for a given region at the indicated rostral-caudal level using Halo software (Indica Labs).

[0269] Monkey experiments All experiments with cynomolgus monkeys were performed at Covance Laboratories, Inc. Monkeys were fed Certified Primate Diet #5048 (PMI, Inc.) twice daily and given free access to fresh water.

[0270] body weight Monkeys were weighed prior to dosing on the day of dosing and at least once a week for the remainder of the study, if applicable.

[0271] Dual-energy X-ray absorptiometry (DEXA) At the veterinarian's discretion, whole-body scans were performed on fasted monkeys anesthetized with ketamine and dexmedetomidine using a Discovery A densitometer (Hologic).

[0272] Example 15: Anti-LEPR antibodies reverse obesity induced by leptin deficiency Experiments were conducted to determine whether H4H17319P2 is effective in vivo. Because H4H17319P2 does not bind to mouse LEPR, genetically modified mice were generated using the VelociGene® method (Valenzuela et al., 2003). These mice contained a portion of the mouse Lepr gene encoding the LEPR extracellular domain replaced with the corresponding human LEPR genomic sequence (Lepr hu / hu ; Figure 10A). Lepr hu / hu Mice showed significant differences in body weight, body composition, insulin sensitivity, and serum leptin levels compared with Lepr + / + showed no difference from mice (Fig. 10B, C, and D).

[0273] A mouse model of leptin deficiency was developed by hydrodynamic DNA delivery (HDD) of a plasmid encoding the hFc-tagged mouse Lepr ectodomain (mLeprECD) to prevent leptin signaling by suppressing endogenous circulating mouse leptin, mimicking leptin signaling. C57BL / 6N mice fed a diet containing mLeprECD rapidly gained weight, significantly increasing from day 3 after HDD compared with mice receiving a control hFc HDD (Figure 11A). At the end of the study (day 10 after HDD), body weight increased by 24% after HDD with mLeprECD, whereas only a modest increase of 3% was observed in the control group (Figure 11A). Aligned with body weight data, cumulative food intake after HDD with mLeprECD was significantly increased from day 3 after HDD compared with the control group (Figure 11A). To confirm whether the weight gain reflected increased adiposity, micro-CT imaging was performed 7 days after HDD and showed a significant 2-fold increase in fat mass after HDD in the mLeprECD compared to the control group (Figure ​(Figure11B). 11B). All other body composition parameters were similar between groups (Figure 11B).

[0274] To evaluate the efficacy of H4H17319P2 in vivo, we administered the drug to food-fed male and female Leprosy mice. hu / hu HDD of mLeprECD induced leptin deficiency in mice. As expected, expression of mLeprECD inhibited the production of Lepr hu / hu Rapid weight gain was promoted in both male and female mice (Figs. 9A and 12A). After 7 days of HDD, based on the relative change in body weight, Lepr hu / huMice were stratified and administered a single 10 mg / kg SC dose of control or H4H17319P2. Both male and female mice administered the control monoclonal antibody (hereafter referred to as control mAb) continued to gain weight, increasing by 40.5 ± 2.7% and 44.1 ± 4.7%, respectively, 14 days after the HDD (Figures 9A and 11A). In contrast, mice treated with a single dose of the LEPR agonistic monoclonal antibody lost weight and returned to their initial pre-HDD weight (Figures 9A and 11A). Because H4H17319P2 binds to human LEPR but not mouse LEPR, we can exclude the possibility that the improvement in body weight was secondary to interference by the LEPR mAb with the ability of mLeprECD to sequester leptin. The weight loss was associated with a decrease in food intake (Figures 9A and 11A). Before treatment, daily food intake was similar between the two groups. However, mice treated with H4H17319P2 showed a significant decrease in food intake compared with the control monoclonal antibody group (Figures 9A and 11A). The body weight and food intake-reducing effects observed with a single dose of H4H17319P2 eventually weakened. In male and female mice treated with H4H17319P2, food intake remained significantly lower than in mice treated with the control monoclonal antibody until 12 and 9 days (days 19 and 16), respectively (Figures 9A and 11A). The body weights of male and female mice treated with H4H17319P2 remained similar to baseline weight until 16 and 13 days (days 23 and 20), respectively, after which weight gain occurred (Figures 9A and 11A).

[0275] The weight loss induced by H4H17319P2 treatment reflected a decrease in adiposity and lean mass. MicroCT analysis revealed that both male and female treated groups of mice exhibited similar body compositions before HDD on day -1 and before treatment on day 6 (Figures 9B, 12B, and 12C). Consistent with the induction of leptin deficiency, on day 6 both groups showed significant increases in fat mass and lean mass, but not bone mass, compared to before HDD on day -1 (Figures 9B, 12B, and 12C). After 6 days of treatment (day 13), control monoclonal antibody-treated mice showed a further increase in fat mass compared to day 6. However, no further increase in adiposity was detected after H4H17319P2 treatment. Consistent with the changes in body weight, after 7 days of treatment, both fat mass and lean mass were decreased in H4H17319P2-treated mice compared to control monoclonal antibody-treated mice. No treatment-related effects on bone mass, bone mineral content, or bone density were observed (FIGS. 12B and 123C).

[0276] Next, we evaluated H4H17319P2 for its ability to alter circulating lipids in mice with induced leptin deficiency. Plasma chemistry analysis revealed that after 6 days of treatment, H4H17319P2 reduced circulating plasma triglycerides and total cholesterol, including HDL-cholesterol (HDL-C) and LDL-cholesterol (LDL-C), compared with male mice administered a control monoclonal antibody (Figure 9C).

[0277] Given that leptin promotes energy expenditure in leptin-deficient ob / ob mice (Halaas et al., 1995; Pelleymounter et al., 1995), we assessed the effect of pair-feeding to clarify whether the weight-reducing effect of H4H17319P2 in induced leptin deficiency was entirely explained by reduced food intake. hu / hu Mice were treated with Lepr hu / huCompared to mice treated with mLeprECD, mice treated with H4H17319P2 rapidly gained weight and were hyperphagic (Figure 13A). After dosing on days 7 and 13, mice treated with the control monoclonal antibody consumed more food and continued to gain weight, whereas mice treated with H4H17319P2 decreased their food intake and lost weight (Figure 13A). Even in pair-housed mice, mice expressing mLeprECD were significantly more phagic than mice treated with the control monoclonal antibody. hu / hu Weight loss was enhanced in leptin-deficient mice. Notably, pair-fed mice consumed the same amount of food as H4H17319P2-treated mice, but did not lose weight to the same extent (Figure 13A). Consistent with these data, pair-fed mice had reduced adiposity compared with mice treated with a control monoclonal antibody, but the adiposity was significantly higher than that of mice treated with H4H17319P2 (Figure 13B). In contrast, a similar effect was observed between pair-fed and H4H17319P2 treatment on the loss of lean mass compared with leptin-deficient mice treated with a control monoclonal antibody (Figure 13B). No effect was observed on bone mass, bone mineral density, or mineral content (Figure 13B).

[0278] Leptin also improves insulin sensitivity in leptin-deficient ob / ob mice (Muzzin et al., 1996; Pelleymounter et al., 1995). Therefore, the relative effects of H4H17319P2 treatment and pair-feeding on insulin sensitivity were determined after induction of leptin deficiency. Insulin tolerance tests were performed 3 days after monoclonal antibody administration or pair-feeding. As shown in Figure 13C, mLeprECD-expressing Lepr mice treated with a control monoclonal antibody showed significantly improved insulin sensitivity. hu / hu Mice were treated with Lepr hu / hu Compared with mice with inducible leptin deficiency, Lepr mice showed decreased insulin sensitivity. hu / hu In mice, treatment with H4H17319P2 restored insulin sensitivity, whereas pair-feeding had no effect (FIG. 13C).

[0279] Finally, we confirmed that the effect of H4H17319P2 in lowering circulating lipids was primarily due to reduced food intake: H4H17319P2, but not pair-fed, significantly reduced plasma triglycerides in leptin-deficient mice, whereas total plasma cholesterol and HDL-C were reduced by both H4H17319P2 and pair-fed mice (Figure 13D).

[0280] Taken together, our data demonstrate that H4H17319P2 not only reversed hyperphagia and obesity but also improved insulin resistance in an inducible leptin deficiency model. Notably, while the weight loss and reduced adiposity observed with H4H17319P2 were attributable to reduced food intake, reduced food intake was insufficient to improve insulin sensitivity or lower plasma triglyceride levels.

[0281] Example 16: H4H17319P2 ameliorates hyperglycemia, insulin resistance, dyslipidemia, and hepatic steatosis in lipodystrophic mice Next, we tested H4H17319P2 to determine whether it attenuates hyperphagia, metabolic dysfunction, and hepatic steatosis in mice with secondary hypoleptinemia caused by generalized lipodystrophy. To test this hypothesis, we transformed aP2-nSrebp1c mice, which express a phenotype characterized by generalized lipodystrophy. Tg / + Mice (Shimomura et al., 1998) were used as Lepr hu / hu aP2-nSrebp1c mice were crossed with Tg / + Mice express nuclear Srebp1c in adipose tissue via the aP2 promoter and were used in the classic experiment that provided the first evidence that leptin reverses the metabolic complications caused by low leptin levels in generalized lipodystrophy (Shimomura et al., 1999). Tg / + ;Lepr hu / hu Transgenic (Tg) mice were transgenic for aP2-nSrebp1c + / + ;Lepr hu / huThe aP2-nSrebp1c Tg mice were more severely affected than non-Tg animals (Figure 15A). However, consistent with previous reports, Tg mice exhibited reduced adiposity and lower leptin levels compared to non-Tg mice (Figure 15A). Tg / + ;Lepr hu / hu The mice also exhibited significant insulin resistance and mild dyslipidemia, with elevated plasma levels of triglycerides, total cholesterol, and LDL-C compared to non-Tg mice (FIGS. 15B and 15C).

[0282] Male lipodystrophic Tg mice were treated with either a control monoclonal antibody or H4H17319P2 at 10 mg / kg (SC) once a week. Additionally, male non-Tg mice were treated with a control monoclonal antibody at 10 mg / kg (SC) once a week to serve as a reference for wild-type metabolic parameters. On day 0, before treatment, Tg mice exhibited significantly higher body weights than non-Tg mice (Figure 14A). Three days after the start of treatment, the body weights of lipodystrophic mice treated with H4H17319P2 were significantly lower than those of lipodystrophic mice treated with a control monoclonal antibody (Figure 14A). Tg mice treated with a control monoclonal antibody were hyperphagic and consumed more food than non-lipodystrophic non-Tg mice treated with a control monoclonal antibody (Figure 14A). However, Tg mice treated with H4H17319P2 consumed less food than Tg mice administered a control monoclonal antibody (FIG. 14A).

[0283] To determine the underlying causes of the observed weight changes, body composition was quantified by micro-CT. These analyses revealed that the changes in body weight between genotypes and treatments were primarily explained by differences in lean mass. Before treatment (day -5), lean mass was significantly elevated in lipodystrophic Tg mice compared with non-Tg mice (Figure 14B). Conversely, before treatment (day -5), Tg mice had decreased fat mass compared with non-Tg mice (Figure 14B). After 4 weeks of weekly treatment, H4H17319P2 significantly reduced fat mass and lean mass in Tg mice compared with those in Tg mice treated with a control monoclonal antibody (Figure 14B). Bone mass and bone mineral content were elevated in Tg mice compared with wild-type mice, but not significantly within the treatment group at pretreatment (Figure 16A). Furthermore, no differences in bone mass and bone mineral content were observed between lipodystrophic mice administered control monoclonal antibody or H4H17319P2 (Figure 16A). Similarly, no genotype- or treatment-related effects were detected on bone mineral density (Figure 16A). These data indicate that H4H17319P2 reduces lean and fat mass in lipodystrophic mice but does not affect bone mass, bone mineral content, or bone mineral density.

[0284] Consistent with previous reports, lipodystrophic Tg mice exhibited marked hyperglycemia compared with non-Tg mice before treatment on day 0 (Figure 14C). After 3 days of treatment with H4H17319P2, ad libitum blood glucose levels were normalized in Tg mice and were comparable to those in non-Tg mice. Notably, with weekly H4H17319P2 treatment, Tg mice maintained normoglycemia until the end of the experiment. Meanwhile, Tg mice treated with a control monoclonal antibody remained hyperglycemic throughout the experiment (Figure 14C). Consistently, hemoglobin A1c (HbA1c) levels were reduced in lipodystrophic mice on day 28 compared with those before treatment or treatment with a control monoclonal antibody (Figure 14C). Furthermore, the reduction in blood glucose levels by H4H17319P2 treatment was associated with improved insulin sensitivity. Insulin tolerance tests on day 23 showed that lipodystrophic mice treated with the control monoclonal antibody were insulin resistant, whereas lipodystrophic mice treated with H4H17319P2 were as insulin sensitive as non-Tg mice treated with the control monoclonal antibody (Figure 14D). Overall, these results indicate that H4H17319P2 attenuates hyperglycemia, insulin resistance, and lowers HbA1c levels in a mouse model of generalized lipodystrophy.

[0285] Plasma chemistry analysis performed at the end of the study (day 28) further revealed that H4H17319P2 reduced hypertriglyceridemia and hypercholesterolemia in mice with generalized lipodystrophy. At the end of the study, plasma levels of triglycerides, total cholesterol, and LDL-C were significantly elevated in Tg mice treated with the control monoclonal antibody compared with non-Tg mice treated with the control monoclonal antibody (Figure 1E). Notably, plasma triglyceride and cholesterol levels were significantly reduced in Tg mice treated with H4H17319P2. Thus, H4H17319P2 improves dyslipidemia in mice with generalized lipodystrophy.

[0286] In addition to diabetes, patients with generalized and partial lipodystrophy may develop insulin resistance, dyslipidemia, and nonalcoholic fatty liver disease (including hepatic steatosis). Therefore, the effects of H4H17319P2 on liver enzyme levels, liver weight, and hepatic steatosis were examined. On day 28, Tg mice administered a control monoclonal antibody showed significantly increased circulating levels of ALT and AST compared with Tg mice treated with H4H17319P2 or non-Tg mice administered a control monoclonal antibody (Figure 14F). Importantly, the improved liver enzyme profile was associated with beneficial effects on liver weight and hepatic steatosis (Figure 14G). Specifically, the livers of lipodystrophic mice administered a control monoclonal antibody were significantly heavier than those of non-Tg mice administered a control monoclonal antibody, weighing 3.6 ± 0.7 g (Figure 14G). The livers of Tg mice treated with the control monoclonal antibody also had higher triglyceride content compared with both non-Tg mice treated with the control monoclonal antibody and lipodystrophic mice treated with H4H17319P2 (Figure 14G). Of note, the livers of Tg mice treated with H4H17319P2 were only 0.6 ± 0.1 g heavier than those of non-Tg mice treated with the control monoclonal antibody, and did not exhibit increased hepatic triglyceride content (Figure 14G). The improvement of hepatic steatosis with H4H17319P2 was also evident in hematoxylin and eosin-stained liver sections (Figure 14G).

[0287] Example 17: H4H17319P2 activates hypothalamic STAT3 signaling LEPR activation in the hypothalamic Arc plays a central role in the management of energy and metabolic balance (Coppari et al., 2005; Cowley et al., 2001). H4H17319P2 is involved in the activation of LEPR in lipodystrophic Leprosy. hu / huGiven that H4H17319P2 reduced hyperphagia and metabolic complications in mice, we further explored whether it induces STAT3 activation in the Arc, similar to leptin. We performed immunostaining for pSTAT3 Y705 in matched brain sections from male lipodystrophic Tg mice that received a single dose of 10 mg / kg (SC) control or H4H17319P2, or a continuous infusion of 30 μg / day (SC) human leptin for 3 days. The leptin dose was chosen based on previous studies showing that 5 μg / day (SC) was effective in lipodystrophic mice (Shimomura et al., 1999), and that maximal effects were observed in mice at doses of 10–42 μg / day (SC) (Denroche et al., 2013; Halaas et al., 1997; Harris et al., 1998). These analyses revealed that in lipodystrophic mice treated with a control monoclonal antibody, Arc cells showed little pSTAT3 Y705 staining. In contrast, both leptin and H4H17319P2 induced pSTAT3 Y705 in Arc, and the detected pSTAT3 Y705 + The cell numbers were similar (Fig. 17A). Although we initially focused on the Arc because of its established role in mediating leptin action and its location near the median eminence, a periventricular organ lacking the blood-brain barrier, it was notable that both leptin and H4H17319P2 induced pSTAT3 Y705 in the Vmh. However, H4H17319P2 induced pSTAT3 staining in a greater number of Vmh cells than was detected with leptin treatment (Fig. 17A). Collectively, these data indicate that leptin and H4H17319P2 induced pSTAT3 Y705 in similar numbers of cells in the Arc, but that H4H17319P2 had a more pronounced effect in the Vmh.

[0288] A drawback of monoclonal antibodies as CNS therapeutics is their limited ability to cross the blood-brain barrier, with approximately 0.1% of their circulating concentration detected in the CSF (Zuchero et al., 2016). Despite this limitation, we found that H4H17319P2 not only induces STAT3 phosphorylation in the hypothalamic Arc, but also stimulates STAT3 signaling in the Vmh. The Arc has been reported to receive inflow via fenestrated blood vessels, but to a lesser extent than the adjacent median eminence (Ciofi et al., 2009; Norsted et al., 2008). Currently, there is no literature evidence that the Vmh is privileged for direct exposure to blood-borne molecules. While not wishing to be bound by theory, a possible explanation is that the monoclonal antibody reaches the Vmh via diffusion from the Arc. Alternatively, the H4H17319P2 antibody may exhibit signaling properties distinct from those of leptin. Although functional LEPR-b is expressed in Vmh, another possibility is that the H4H17319P2 monoclonal antibody indirectly stimulates STAT3 signaling in Vmh. Nevertheless, it was unexpected that H4H17319P2 induced more neuronal STAT3 signaling in Vmh than leptin.

[0289] Example 18: H4H17319P2 is at least as effective as leptin in reducing metabolic and hepatic dysfunction in lipodystrophic mice. Because H4H17319P2 induces STAT3 signaling in Arc and Vmh cells to a similar extent or better than leptin, we compared the efficacy of H4H17319P2 and leptin treatment in lipodystrophic mice. Male transgenic mice were treated with 10 mg / kg (SC) of a control monoclonal antibody or LEPR agonist once a week, or with a continuous infusion of 30 μg / day (SC) of leptin for 14 days. As a reference, male non-transgenic mice were also treated with 10 mg / kg (SC) of a control monoclonal antibody once a week. Before treatment on day 0, transgenic mice were significantly hyperglycemic (Figure 17B). Compared with treatment with the control monoclonal antibody, H4H17319P2 or leptin infusion reduced blood glucose levels to a similar extent (Figure 17B). Blood glucose levels decreased two days after the initiation of H4H17319P2 or leptin treatment and remained low until the end of the study (Figure 17B). To verify whether improved insulin sensitivity contributed to the glucose-lowering effect, an insulin tolerance test was performed on day 9. Indeed, the insulin tolerance test revealed that Tg mice administered a control monoclonal antibody were resistant, whereas H4H17319P2- and leptin-treated Tg mice were insulin-sensitive (Figure 17B). No differences were observed between H4H17319P2 and leptin treatment in terms of blood glucose reduction or insulin sensitivity.

[0290] Consistent with previous data, H4H17319P2 significantly promoted weight loss in lipodystrophic mice compared with control monoclonal antibody treatment, starting 4 days after treatment (Figure 17C). In Tg mice, H4H17319P2 significantly reduced cumulative food intake compared with control monoclonal antibody treatment (Figure 17C). Interestingly, leptin reduced body weight and food intake in Tg mice, although the weight loss was smaller than that observed with H4H17319P2 treatment (Figure 17C).

[0291] Compared with leptin, H4H17319P2 provided better benefits in lowering plasma lipids and reversing hepatomegaly in lipodystrophic mice. Plasma chemistry analysis on day 13 showed that H4H17319P2, but not leptin, reduced plasma triglyceride and cholesterol levels in Tg mice compared with the control monoclonal antibody (Figure 17D). No significant changes in other lipids were evident with H4H17319P2 treatment compared with the control monoclonal antibody in Tg mice. H4H17319P2 also reduced liver mass and reversed hepatic steatosis compared with the control antibody treatment in Tg mice (Figure 17E). These effects were similar in leptin-treated mice, although a greater reduction in liver mass was observed with H4H17319P2 (Figure 17E). Liver mass was normalized by H4H17319P2, but not by leptin treatment, and was similar to that in non-Tg mice receiving a control monoclonal antibody (FIG. 17E).

[0292] Example 19: H4H17319P2 reduces body weight and adiposity in lean mice and normal and high body fat monkeys In vitro binding and functional assay experiments demonstrated that H4H17319P2 did not compete for leptin binding and induced greater LEPR activation in the presence of leptin than leptin alone. We therefore determined whether H4H17319P2 could induce weight loss under normal weight homeostasis. hu / huMice were administered a single dose of either control monoclonal antibody (10 mg / kg, SC) or H4H17319P2 at 3 or 10 mg / kg on day 0. Significant weight loss was observed 1-2 days after H4H17319P2 treatment at both dose levels compared to control monoclonal antibody (Figure 18A). Concomitantly, both dose levels of H4H17319P2 also reduced food intake compared to control monoclonal antibody treatment (Figure 18A). Body weight changes were associated with a decrease in fat mass, but not lean mass (Figure 18B). At both dose levels, H4H17319P2 treatment induced a -32% decrease in fat mass. Control monoclonal antibody treatment, on the other hand, resulted in a minimal change of -2% in fat mass (Figure 18B). Of note, no significant differences in lean mass were observed between control monoclonal antibody and H4H17319P2 treatment (Figure 18B). Both dose levels of H4H17319P2 resulted in similar changes in body weight and fat mass, although the duration of effect differed, possibly reflecting differences in pharmacokinetics (Figures 18A and 18B).

[0293] Next, we evaluated the effect of H4H17319P2 treatment on promoting weight loss in non-human primates. First, we determined the effect of H4H17319P2 on the body weight of lean cynomolgus monkeys. Monkeys were administered either a control solution or 3 mg / kg or 10 mg / kg H4H17319P2 once weekly for 13 weeks. H4H17319P2 treatment resulted in a dose-dependent weight loss compared to monkeys administered the control solution (Figure 18C). After the 13-week study, monkeys administered the control solution had an increase of 9.7 ± 0.9% of their initial body weight. Meanwhile, treatment with 3 and 10 mg / kg H4H17319P2 resulted in a minimal weight change of 0.3 ± 1.6% and a weight loss of -6.0 ± 1.6%, respectively (Figure 18C).

[0294] Because H4H17319P2 dose-dependently reduced body weight in lean monkeys, the effects of H4H17319P2 treatment on body weight and body composition were examined in cynomolgus monkeys with high body fat. Animals were administered either a control solution or 30 mg / kg of an LEPR agonistic antibody once a week for 2 weeks. H4H17319P2 reduced body weight compared to control solution injection. At the end of the study (day 56), monkeys receiving both doses of H4H17319P2 showed a 3.6 ± 1.9% weight loss from baseline, while monkeys receiving the control solution gained 6.4 ± 1.5% weight. Quantitative parallel analysis by dual-energy X-ray absorptiometry (DEXA) demonstrated that H4H17319P2 treatment reduced fat mass, but not lean mass, compared to control solution (Figure 18D). Monkeys receiving the control solution exhibited a 28.0±1.5% increase in adiposity by the end of the study. In contrast, monkeys treated with the LEPR agonist H4H17319P2 showed a significant decrease in adiposity of 13.7±6.4% (FIG. 18D). Collectively, these data suggest that the H4H17319P2 antibody significantly reduces adiposity in normal-weight Lepr mice. hu / hu It has been shown to promote weight loss through selective reduction of adiposity in both mice and lean to high body fat non-human primates.

[0295] Example 20: Double-blind study using anti-LEPR antibodies in humans Obesity affects 13% of people worldwide and is a risk factor for developing type 2 diabetes, cardiovascular disease, various types of cancer, and orthopedic disorders. In Western countries, over 20% of people are obese (Ng et al., Lancet. 2014; 384: 766-781), and in the United States, the prevalence of obesity is currently over 35% (Flegal et al., JAMA. American Medical Association. 2016; 315(21): 2284-2291). While diet and exercise are effective in some people, they often only result in modest weight loss, and the majority of people remain obese (≥ 30 kg / m 2) or overweight (defined as a body mass index [BMI] of 25 to <30 kg / m 2 (defined as BMI of 18.5 or 20.5).

[0296] Current weight-loss medications have only a modest effect on body weight and / or are poorly safe and / or well-tolerated, limiting the adoption of these interventions by payers, physicians, and patients (Zhang et al., Obes Sci Pract. 2016;2(2):104-114). Current standard medications, such as liraglutide, orlistat, naltrexone HCl / bupropion HCl, phentermine / topiramate, and lorcaserin HCl, achieve an average weight loss of approximately 3-10% from baseline. Typically, weight loss plateaus, and treatment is discontinued within one year (Sjostrom et al., Lancet. 1998;352(9123):167-72) (Smith et al., N. Engl. J. Med. 2010;363(3):245-56). Bariatric surgery is recommended for the most severely obese patients (BMI ≥ 40 kg / m). 2 or BMI ≥ 35 kg / m with comorbidities 2 ), with over 200,000 cases per year in the United States (American Society for Metabolic and Bariatric Surgery) and fewer than 150,000 cases per year across Europe (Angrisani et al., Obes Surg. 2017, 27:2279-2289). There is a large unmet medical need for safe and effective therapies for the treatment and prevention of obesity and obesity-related metabolic complications.

[0297] Leptin is a circulating adipose-derived hormone that binds to the hypothalamic leptin receptor (LEPR) to regulate food intake, energy expenditure, and glucose / lipid metabolism (Allison et al., J Endocrinol 2014, 223(1):T25-35). Individuals with primary leptin deficiency, caused by very rare homozygous loss of function mutations in the leptin (LEP) gene, or with impaired leptin signaling due to homozygous mutations in LEPR, develop severe obesity, diabetes, susceptibility to infection, and infertility (Montague et al., Nature 1997, 387(6636):903-908) (Clement et al., Nature 1998, 392(6674):398-401). Administration of recombinant human leptin to children and adults with monogenic obesity caused by leptin loss due to functional mutations results in stable weight loss and improvement of metabolic complications (Farooqi et al., N Engl Jo Med 1999,341(12):879-884) (Licinio et al., Proc Nat Acad Sci 2004,101(13):4531-4536).

[0298] Secondary leptin deficiency also occurs in the rare disorder lipodystrophy, which results from the genetic or acquired loss of leptin-producing adipose tissue from various regions of the body.

[0299] Patients with generalized and partial lipodystrophy develop varying degrees of diabetes, severe insulin resistance, hypertriglyceridemia, and fatty liver (Brown et al., J Clin Endocrinol Metab. 2016;101(12):4500-4511). Subcutaneous delivery of recombinant human leptin once daily has been shown to reduce hemoglobin A1c (HbA1c), glucose, triglycerides, and hepatic steatosis in patients with generalized lipodystrophy (Oral et al., N Engl J Med. 2002;346(8):570-578) (Ebihara et al., J Clin Endocrinol Metab 2007,92(2):532-541). The majority of patients with generalized lipodystrophy have serum leptin levels below 4 ng / mL, and subgroup analyses suggest that the reduction in HbA1c and triglycerides (TG) with leptin treatment is greater in patients with generalized lipodystrophy and partial lipodystrophy whose leptin levels are below 4 ng / mL (Brown et al., J Clin Endocrinol Metab. 2016;101(12):4500-4511) (FDA Advisory Committee Meeting 2013).

[0300] Metreleptin (recombinant methionyl leptin; Novelion®) is currently approved in the United States under the Risk Evaluation and Mitigation Strategies (REMS) program for the treatment of complications of leptin deficiency in patients with generalized congenital and acquired lipodystrophy, and in Japan for all subtypes of lipodystrophy. Metreleptin requires daily administration, and adverse effects include the risk of immunogenicity, resulting in the development of binding antibodies in approximately 85% of patients, and the risk of developing neutralizing antibodies that cross-react with endogenous leptin in approximately 9% (Chan, Clin Endocrinol. 2016;85(1):137-149).

[0301] In contrast to hypoleptinemia or leptin deficiency disorders, a BMI of 30–35 kg / m 2 Overweight or obese individuals typically have high levels of leptin, with median values ​​of 10 ng / mL in men and 24 ng / mL in women, in subjects in the range of 20-25 kg / m (Ruhl and Everhart, Am J Clin Nutr. 2001;74(3):295-301). 2Circulating leptin levels in male and female adults with a normal BMI are median values ​​of 3 and 9 ng / mL, respectively (Ruhl et al., Am J Clin Nutr 2001;74(3):295-301), with a normal range of 1.2-9.5 ng / mL for men and 4.1-25 ng / mL for women (Quest Lab Reference Range). Many studies have shown correlations between circulating leptin levels, BMI, and body fat percentage (Ruhl et al., Am J Clin Nutr. 2001, 74(3):295-301) (Considine et al., N Engl J Med. 1996, 334(5):292-295), but leptin levels among individuals with similar BMI are highly variable (Buettner et al., J Endocrinol. 2002;175(3):745-756). Leptin levels exhibit diurnal variations (Gavrila et al., J Clin Endocrinol Metab. 2003, 88(6):2838-43) (Schoeller et al., J Clin Invest. 1997, 100(7):1882-87) and vary depending on nutritional status. In normal weight and obese individuals, leptin levels rapidly decline by 35-60% after a 24-hour fast and continue to decline even after extended fasting (Chan et al., J Clin Invest. 2003;111(9):1409-1421) (Schurgin et al., 2004, J Clin Endocrinol Metab, 89(11):5402-5409) (Boden et al., J Clin Endocrinol Metab. 1996;81(9):3419-3423).Leptin levels also decrease during weight loss (Considine et al., N. Engl J Med, 1996;334(5):292-295) (Herrick et al., J Obes. Hindawi. 2016;2016(2):8375828-5) (van Dielen et al., J Clin Endocrinol Metab. 2002;87(4):1708-1716) and increase during weight gain (Ravussin et al., Cell Metab. 2014;20(4):565-572), consistent with changes in fat mass. Administration of recombinant human leptin to obese subjects results in minimal weight loss (weight loss minus 3% PBO) (Heymsfield et al., JAMA. 1999;282(16):1568-1575) (Hukshorn et al., J Clin Endocrinol Metab. 2000;11(12):1163-1172) (Ravussin et al., Obesity. 2009;17(9):1736-1743), likely due to saturation of leptin receptor signaling by high levels of endogenous leptin. Several population studies suggest the existence of a subset of obese patients who have relatively low leptin levels (Ruhl and Everhart, Am J Clin Nutr. 2001;74(3):295-301) (Buettner et al., J Endocrinol. 2002;175(3):745-756) and whose leptin receptors may not be saturated. An important question to address is whether restoring leptin signaling reduces appetite, food intake, and body weight in obese subjects with relatively low baseline leptin levels.

[0302] H4H17319P2 is a human anti-LEPR antibody that acts as a LEPR agonist and binds to human LEPR with nanomolar affinity. In preclinical studies, H4H17319P2 activates LEPR signaling in the presence or absence of leptin. Weekly administration of H4H17319P2 (10 mg / kg subcutaneously [SC]) improved glycemic control, insulin sensitivity, dyslipidemia, food intake, body weight, liver mass, and hepatic steatosis in lipodystrophic humanized LEPR mice. H4H17319P2 also reduced body weight and steatosis in humanized LEPR mice with inducible leptin deficiency, but not in diet-induced obese humanized LEPR mice. In a Good Laboratory Practice (GLP) toxicity study in which lean cynomolgus monkeys were treated with H4H17319P2 by subcutaneous injection (3, 10, or 30 mg / kg) or intravenous injection (100 mg / kg) once weekly for 13 weeks, H4H17319P2 exposure resulted in suppression of body weight gain or induction of weight loss. H4H17319P2-treated monkeys lost a maximum of 8.7% body weight (group mean compared to pre-treatment weight), whereas parallel control animals receiving placebo gained a mean of 8.3% body weight over the same period. The magnitude of observed weight loss did not appear to have a clear dose- or exposure-response in H4H17319P2-treated animals. Weight changes were observed to reverse upon cessation of exposure during a treatment-free recovery period.

[0303] As described above, H4H17319P2 administration was well tolerated at all dose levels and both administration routes, with no adverse clinical effects. Transient decreases in insulin and circulating absolute lymphocyte counts (decreased T cell counts) were observed approximately 30 days into the treatment period, but these findings were not observed at the end of the treatment period, indicating that the effects were not dose-responsive. Decreased thymus weight and decreased thymic cortical cellularity were observed in the H4H17319P2-treated group during the treatment period and were fully or partially reversible during the recovery period. The thymic changes and transient decreases in T cell counts are likely related to reduced nutritional intake and weight loss in growing monkeys and are unlikely to be observed in adult humans with normal nutritional status.

[0304] This is a first-in-human (FIH) randomized, double-blind, placebo-controlled, two-part study designed to evaluate the safety, tolerability, pharmacokinetics (PK), and pharmacology (PD) of intravenously (IV) and SC administered doses of H4H17319P2 in healthy participants. The objective of Part A is to evaluate the safety, tolerability, PK, and PD of single ascending IV and SC doses in healthy male and female subjects. Interim analyses of PK / PD, safety, and tolerability in Part A will be used to select dosing regimens for evaluation in Part B. Subjects enrolled in Part A will not be eligible for Part B. For Part B, new overweight or obese subjects will be enrolled to evaluate the safety, tolerability, PK, and PD of 12-week repeat doses of H4H17319P2 (single dose level) or placebo. The effect of H4H17319P2 on biomarkers such as food intake, appetite, body composition, and body weight will be evaluated in four different cohorts defined by baseline leptin levels.

[0305] the purpose The primary objective of this study is to evaluate the safety and tolerability of H4H17319P2 in healthy subjects. Secondary objectives of the study are: Characterization of the single- and multiple-dose pharmacokinetic (PK) profile of H4H17319P2; · Estimation of the effect of repeated administration of H4H17319P2 on body weight; · Evaluation of the effect of repeated administration of H4H17319P2 on ad libitum energy intake in overweight and obese subjects; Evaluating the effects of single and repeated administration of H4H17319P2 on soluble lipid regulatory protein (sLEPR and ANGPTL3) levels over time; - Evaluation of the immunogenicity of single and multiple doses of H4H17319P2.

[0306] Other exploratory objectives of this study are: 1. Estimation of the effect of a single dose of H4H17319P2 on body weight and serum / plasma glycemic and lipid parameters. 2. Estimated effects of repeated administration of H4H17319P2 over 12 weeks on: · Serum / plasma glycemic and lipid parameters. Patients reported appetite assessments (e.g., hunger, bloating, and satiety) that may influence eating behavior. · Total and distribution of fat mass and lean mass by dual-energy X-ray absorptiometry (DXA) imaging. · Quantification of SC fat and visceral fat (including liver fat) by magnetic resonance imaging (MRI). Other exploratory biomarkers include leptin, thyroid hormones (T3, T4, thyroid-stimulating hormone [TSH]), luteinizing hormone (LH), testosterone, estradiol, cortisol, and adiponectin.

[0307] reason: Part A is a single ascending-dose FIH design in which up to 88 healthy subjects will be randomized 3:1 to receive H4H17319P2 or placebo in up to seven ascending-dose cohorts (up to five IV and two SC) with a 112-day follow-up period to evaluate the safety, tolerability, and pharmacokinetics of single ascending doses of H4H17319P2. Eight subjects will be randomized to receive H4H17319P2 or placebo (six active: two placebo) at each dose level in each of the seven ascending-dose cohorts. The study design also includes two additional optional cohorts (16 subjects will be randomized to receive H4H17319P2 or placebo (12 active: four placebo in each cohort)). If interim analyses from the escalating dose cohorts suggest possible covariate effects (e.g., age, weight, or sex) on the PK profile, optional cohorts will be enrolled to collect additional data at one or more doses (up to the maximum dose of 30 mg / kg IV in the study) to obtain further estimates of covariate effects on PK.

[0308] Part A of the study consists of a screening period (days -21 to -2), a pre-baseline visit (day -1), and a follow-up period (days 3 to 113) that includes the end of the study visit (day 113). At the pre-baseline visit, subjects are approved for a 2-day clinic stay (for subjects receiving IV dosing and subjects receiving SC dosing in the safety monitoring block) or a 1-day clinic stay (for subjects receiving SC dosing).

[0309] Evaluation of safety and tolerability is the primary objective of Part A, and safety will be carefully evaluated throughout the study. The starting and maximum doses administered in this study are expected to be approximately 10,000-fold and 20-fold lower, respectively, than the exposures observed in toxicity studies. Throughout the clinical trial, safety assessments will include vital signs, physical examination, electrocardiogram (ECG), laboratory tests including blood / differential, and monitoring for adverse events (AEs). Body weight will also be assessed. Pharmacological measures will also be collected in this study; however, meaningful changes in PD markers are not expected to be observed in the lean / overweight subject group based on the minimal effect of body weight (less than 1 kg) observed with metreleptin in lean individuals (Heymsfield, 1999).

[0310] Pharmacological measurements include body weight, metabolic parameters (glucose, lipids), and ANGPTL3. ANGPTL3 is a potential marker that may be regulated by leptin and / or insulin (Muniyappa, 2017) (Nidhina Haridas, 2015). Body weight will be carefully assessed at each visit. If a single dose of H4H17319P2 treatment has a clinically meaningful effect on body weight in lean / overweight subjects, the timing of safety / dose-escalation decisions may be modified, and additional safety data may be collected before making dose-escalation decisions (e.g., waiting for the day 15 safety assessment).

[0311] Part B is a single-dose, repeat-dose study with a 12-week treatment period to evaluate the safety, PK, and effect of H4H17319P2 on body weight in overweight / obese subjects. Several population studies suggest that there is a subset of obese patients with relatively low leptin levels (Ruhl, 2001) (Buettner, 2002), whose leptin receptors may not be saturated. The key question addressed in Part B is whether restoring leptin signaling reduces appetite, food intake, and body weight in overweight or obese subjects with relatively low baseline leptin levels.

[0312] Therefore, Part B will evaluate the effects of H4H17319P2 on body weight, food intake, metabolic parameters, and body composition in subjects with varying body weight and relatively low baseline leptin levels. Healthy subjects with overweight or obesity (BMI range 25-40 kg / m) 2 ) will be enrolled into four separate cohorts defined by prescreening leptin levels. Enrollment into the four separate cohorts will be conducted to ensure an adequate number of subjects are studied across a relatively low baseline leptin and BMI range. Cohort stratification will occur at the time of randomization to H4H17319P2 or placebo. A maximum of approximately 20 subjects will be enrolled in each of the four cohorts, with a total sample size of up to 81 subjects in Part B, as defined below: Leptin levels and BMI will be measured at the prescreening visit and initially assessed for study eligibility and eligibility for one of the four cohorts. Enrollment into the study will occur at the time of screening for eligible subjects. If enrollment into a particular cohort reaches the maximum number permitted, subjects will no longer be eligible for enrollment. Because some cohorts may be difficult to enroll given the low leptin levels and low obesity rates, sponsors may choose to stop enrollment within certain cohorts.

[0313] The four cohorts in Part B are defined as follows: Cohort 1: Patients with a fasting leptin level of <5 ng / mL at pre-screening and a mean of 28.0 to 40.0 kg / m at pre-screening 2 Male and female subjects with a BMI of Cohort 2: Patients with a fasting leptin level of <5 ng / mL at pre-screening and a mean of 25.0 to <28 kg / m 2 Male and female subjects with a BMI of Cohort 3: Patients with fasting leptin levels of 5.0 ng / mL or higher and 8.0 ng / mL or lower at pre-screening and a mean of 28.0 to 40.0 kg / m 2 Male subjects with a BMI of Cohort 4: Patients with fasting leptin levels ≥ 5.0 ng / mL and ≤ 24.0 ng / mL at pre-screening and ≥ 28.0 to 40.0 kg / m 2 Female subjects with a BMI of

[0314] Note: Eligibility for one of the above cohorts is based on BMI and leptin values ​​at the pre-screening visit. If BMI and / or leptin values ​​are not within the range specified for the cohort, the subject is not eligible for enrollment. If BMI or leptin values ​​at pre-screening are borderline for enrollment in one or more cohorts, BMI and leptin measurements may be repeated once during the pre-screening window. For repeated measurements, the lowest leptin value will be used for enrollment into one of the cohorts. If enrollment into a particular cohort reaches the maximum number allowed, the subject will no longer be eligible for enrollment.

[0315] Initiation of Part B will be guided by an interim analysis of available safety, PK, and PD data from Part A, which will determine the appropriate dose, dosing frequency, and administration mode (IV or SC) for Part B. Subjects enrolled in Part A are not eligible for Part B. The Part B study design consists of a prescreening period (days -60 to -14) to assess BMI and fasting leptin levels and assess eligibility for one of four cohorts; a screening period (days -32 to -14); a baseline period (days -29 to -1); a treatment period (days 1 to 85); and a treatment-free follow-up period (days 107 to 191). Subjects will be allowed a 2-day clinic visit for accurate appetite assessments during the baseline period (scheduled on days -14 to -1) and at two time points during the treatment period (days 29 to 30 and 84 to 85), as well as an ad libitum food intake assessment. The clinic stay is necessary to accurately assess calorie intake during ad libitum food intake assessment under controlled conditions, with standard meals provided and minimization of cues other than appetite that may influence food intake (e.g., time of day, other people's eating behavior, portion size, etc.). Subjects will also be granted a 2-day clinic stay on Day -1. On Day 1 of the treatment period, subjects will receive their first dose of investigational drug or placebo, have blood samples taken for a series of PK and other clinical laboratory measurements, and remain overnight to complete a 24-hour PK assessment on Day 2.

[0316] Treatment visits for blood sampling will occur weekly. The dose and frequency of study drug will be determined by an interim analysis of PK data from Part A and will be determined to be every 4 weeks or every 2 weeks (maximum frequency of no more than once a week).

[0317] Body composition by DXA and quantification of liver, abdominal, and thigh fat by MRI will be performed at baseline, near the end of the treatment period, and during the follow-up period. After the treatment period, subjects will be followed during a 16-week washout period to assess safety and PD efficacy. Evaluation of the safety and tolerability of repeat dosing at the single dose level (based on safety and tolerability from Part A) is the primary objective of Part B of the study. Throughout the study, safety assessments will include vital signs, physical examinations, ECGs, laboratory tests, and monitoring for AEs. Anti-drug antibodies will also be assessed. Additionally, secondary objectives evaluating body weight, ad libitum food intake, and metabolic parameters such as glucose and lipids will be carefully evaluated throughout the study.

[0318] Pharmacological measures in Part B will include baseline and treatment assessments of parameters potentially affected by increased leptin receptor signaling. These PD assessments include patient-reported appetite measurements, quantitative food intake measurements in a controlled inpatient setting, accurate body composition and fat mass measurements by DXA, and accurate weight measurements using a calibrated scale. Additionally, metabolic parameters such as leptin, glucose, insulin, homeostasis model assessment-estimated insulin resistance (HOMA-IR), HbA1c, and lipids will be measured at baseline, during treatment, and at the end of the treatment period to assess the effects of H4H17319P2 on insulin sensitivity and lipid metabolism. Liver MRI will also be performed at baseline and after treatment to determine whether H4H17319P2 affects hepatic steatosis.

[0319] Evidence for pharmacological and biomarker variables Assessment of ad libitum food intake (Part B only) Treatment with H4H17319P2 increases hypothalamic leptin receptor signaling and is hypothesized to have downstream effects on neurons that influence feeding behavior. H4H17319P2 treatment resulted in significant reductions in food intake and body weight in inducible leptin-deficient mice. Although quantitative food intake assessments were not performed in the cynomolgus monkey study, H4H17319P2 treatment had significant effects on body weight and fat mass. In this study, the effects of H4H17319P2 on food intake in overweight / obese subjects with relatively low leptin levels will be assessed at baseline and at two time points during the treatment period using a rigorous quantitative inpatient ad libitum food intake assessment, as previously reported (Krishna, 2009) (Addy, 2008). Briefly, subjects will be fasted and admitted to the clinical trial unit and provided with a standardized breakfast, lunch, and dinner to establish a standardized baseline energy intake. After an overnight fast, subjects are offered an ad libitum breakfast, lunch, and dinner, and food / energy intake is quantified. Subjects consume all test meals in a specialized room where perception of time / time of day and social cues is masked.

[0320] Meals of known calorie density are served in 4-5x excess amounts, with the total number of calories consumed concealed. Subjects are asked to eat as much as they like. To quantify food intake, meals are weighed before and after food intake assessment, and calorie intake is calculated.

[0321] Appetite assessment (Part B only) Leptin's effect on appetite is central to its mechanism of action, and therefore this is a direct assessment of the predicted mechanism of action of H4H17319P2. Individuals with hypoleptinemia, either due to mutations in LEP or generalized lipodystrophy, exhibit insatiable appetite, leading to food intake and obesity. Treatment of these individuals with metreleptin increases satiety and reduces food intake (Farooqi, 1999) (Farooqi, 2007) (McDuffie, 2004). The effect of H4H17319P2 on appetite will be assessed at baseline and during follow-up using questionnaires measuring various components of appetite (e.g., hunger, satiety) (Flint, 2000) (Dalton, 2015).

[0322] Appetite will be assessed before and after a standard calorie load during clinic visits, and additional appetite questionnaires will be completed daily during the baseline, treatment, and follow-up periods.

[0323] ANGPTL3 Angiopoietin-like protein 3 (ANGPTL3) regulates lipoprotein levels, such as triglycerides (TG) and low-density lipoprotein C (LDL-C), through the inhibition of lipoprotein lipase (reviewed by Tikka, 2016). ANGPTL3 levels can be regulated by feeding, leptin, and / or insulin (Minicocci, 2012) (Nidhina, 2015). ANGPTL3 levels are elevated in patients with lipodystrophy and decrease after treatment with metreleptin (Muniyappa, 2017). ANGPTL3 reduction may be mediated by enhanced lipase clearance of TG-rich lipoproteins. ANGPTL3 and TG levels are also increased in mouse models of lipodystrophy and normalized after treatment with H4H17319P2. Therefore, ANGPTL3 may be a pharmacological marker that can be decreased under conditions of enhanced leptin receptor signaling. ANGPTL3 will be measured at baseline and at several time points after treatment in Parts A and B.

[0324] Soluble LEPR Leptin circulates in the bloodstream as a free form and can also bind to sLEPR, which is generated through shedding of the LEPR extracellular domain (Sinha, 1996) (Lammert, 2001). Soluble LEPR can regulate leptin bioavailability and / or clearance (Lou, 2010). Target saturation may depend on sLEPR saturation, and therefore, variations in linear and nonlinear target-mediated kinetics may be affected by sLEPR values. Therefore, in Parts A and B, sLEPR will be measured at baseline and at various time points during treatment (corresponding to sampling for antibody pharmacokinetics).

[0325] Imaging (Part B only) DXA and MRI imaging were performed at baseline, near the end of treatment, and at the end of the study to estimate changes from baseline in total and regional fat distribution, SC and visceral fat in the thigh / abdominal region, and liver fat content. In a pilot study in cynomolgus monkeys, preclinical data showed that H4H17319P2 reduced body weight by reducing total fat mass (as assessed by DXA) without affecting lean mass. DXA has been shown to be a useful quantitative biomarker of total fat mass and regional fat distribution in clinical trials. For example, significant differences in regional fat distribution using DXA (trunk fat % compared to leg fat % (fat mass ratio [FMR]) 1.78 ± 0.53) were observed in patients with partial lipodystrophy compared to normal subjects (Aijluni, 2017). DXA has also been used to quantify changes in total and regional fat mass in trials of weight loss agents, such as GLP-1 agonists ( Jendle, 2009 ) and cognitive therapy ( Ponti, 2018 ).

[0326] Magnetic resonance imaging (MRI) has been shown to be an effective tool for measuring fat content in various tissues, including liver (Aijluni, 2017) and muscle (Burakiewicz, 2017), either through whole-organ imaging using dedicated pulse sequences or through spectroscopy in limited tissue volumes. MRI has also demonstrated high-contrast imaging for measuring abdominal fat mass and distribution (Klopfenstein, 2012), where patients with familial lipodystrophy type 2 had a 2.5-fold higher visceral fat percentage than control patients (Al-Attar, 2007). Muscle fat fraction can also be assessed using quantitative MRI, and as shown in multiple muscular dystrophy studies, spectroscopy and pulse sequences allow for anatomical imaging and fat fraction estimation, demonstrating efficacy in assessing disease progression (Burakiewicz, 2017).

[0327] In this study, abdominal and thigh MRI will be performed to quantify regional changes in SC fat, visceral fat, and liver fat at baseline and after treatment with H4H17319P2.

[0328] Rationale for dose selection The intravenous and SC doses for Part A were selected based on efficacy, safety, and pharmacokinetic data from preclinical studies in mouse models of lipodystrophy and monogenic obesity, as well as from toxicity studies in monkeys. The highest dose in this FIH study was ≤30 mg / kg, with a starting dose of approximately 0.3 mg / kg. In a GLP toxicity study in cynomolgus monkeys, H4H17319P2 was well tolerated at 100 mg / kg IV once weekly for up to 12 weeks, with a no-observed-adverse-effect level (NOAEL) of 100 mg / kg. Based on the predicted serum exposure of H4H17319P2 in humans, the planned maximum dose of 30 mg / kg in this FIH study has an exposure multiple less than 20-fold above the NOAEL. The initial starting dose of 0.3 mg / kg has a safety margin of over 10,000, lower than the highest dose tested in GLP toxicology studies, and is predicted to provide H4H17319P2 concentrations in humans above the limit of quantitation, and therefore is predicted to provide useful PK information.

[0329] The objectives of Part B dosing selection include ensuring a broad exposure range to assess tolerability and facilitating characterization of exposure-response relationships, as well as elucidating a pharmacokinetic profile that allows for characterization of both linear and nonlinear pharmacokinetics. Furthermore, dose selection must ensure exposure above and below putative PD marker thresholds of potential interest (e.g., sLEPR saturation). Part B dosing will be based on interim safety, PK, and PK / PD data from Part A. Selection of dose level, dosing interval, and route of administration (IV or SC) will be based on safety, pharmacokinetic, and, if available, PK / PD data from Part A and animal preclinical studies. Part B dosing will not exceed that assessed in Part A, and dosing will likely be every 4 weeks or every 2 weeks, but no more frequently than weekly.

[0330] Dosing regimens do not exceed exposures observed in toxicity studies.

[0331] standard A maximum of 169 subjects (maximum 88 for Part A and maximum 81 for Part B) will be enrolled in the target population. The target populations are healthy lean or overweight men and women for Part A and healthy overweight or obese men and women with various baseline leptin levels for Part B.

[0332] Important Registration Criteria Subjects must meet the following criteria at screening to be eligible for enrollment in Part A of the study: 1. Men and women aged 18 to 50 years old. 2.18.5~<30.0kg / m 2 Body mass index (BMI). 3. Subject is in good health and free of major comorbidities as determined by the investigator based on medical history, physical examination, and clinical laboratory safety tests performed at screening and / or prior to the first dose of study drug. 4. Willing and able to comply with clinic visits, study-related procedures, and dietary instructions. 5. Willingness to maintain normal diet and exercise regimen throughout the study. 6. Able and willing to provide signed informed consent.

[0333] Subjects must meet all of the following criteria at screening to be eligible for enrollment in Part B of the study (except for BMI and leptin eligibility, which will be determined at pre-screening): 1. Men and women aged 18 to 65. 2. Have a pre-screening body mass index (BMI) and fasting leptin level as defined below by one of the cohorts: Once enrollment into a particular cohort reaches the maximum number allowed, the subject will no longer be eligible for enrollment. 3. Subjects are determined by the investigator to be free of major comorbidities based on medical history, physical examination, and clinical laboratory safety tests performed at screening and / or prior to the first dose of study drug. Subjects may have a history of mild hyperlipidemia and / or mild hypertension, but must have been receiving stable treatment with lipid-lowering or blood pressure-lowering medications for at least 2 months prior to screening. 4. Willing and able to comply with clinic visits, study-related procedures, and dietary instructions. 5. Willingness to maintain normal diet and exercise regimen throughout the study. 6. Able and willing to provide signed informed consent.

[0334] Exclusion criteria Subjects who meet any of the following criteria at screening will be excluded from Part A of the study: 1. History of clinically significant cardiovascular disease (e.g., history of hypertension, myocardial infarction, stroke, peripheral vascular disease, heart failure, refractory arrhythmia), respiratory disease, liver disease, kidney disease, gastrointestinal disease, endocrine disease (e.g., hyperlipidemia), blood disease, or neurological disease. 2. History of type 1 diabetes, type 2 diabetes, or prediabetes stage, or fasting blood glucose (FBG) >100 mg / dL at screening, or HbA1c >5.7% at screening. 3. Fasting LDL-C ≥ 130 mg / dL and TG > 250 mg / dL. 4. Clinically significant complete blood count abnormalities, clinical chemistry, urinalysis, or urine drug screen at screening. Minor deviations in laboratory test results are acceptable. Note: Any laboratory test abnormality (e.g., creatinine phosphokinase (CPK) within 3x of the upper limit of normal (ULN) and presumably due to strenuous physical activity) may be repeated once during the screening period.

[0335] Subjects who meet any of the following criteria at screening will be excluded from Part B of the study: 1. History of clinically significant cardiovascular disease (e.g., moderate to severe hypertension, myocardial infarction, stroke, peripheral vascular disease, heart failure, refractory arrhythmia), respiratory disease, liver disease, kidney disease, gastrointestinal disease, endocrine disease, blood disease, or neurological disease. 2. History of type 1 or type 2 diabetes, or FBG >126 mg / dL at screening, or HbA1c >6.5% at screening. A diagnosis of "prediabetes stage" is acceptable. 3. Fasting LDL-C >160 mg / dL or TG >500 mg / dL. 4. Clinically significant complete blood count abnormalities, clinical chemistry, urinalysis, or urine drug screen at screening. Mild lipid and glycemic abnormalities are excluded as described above. Minor deviations in laboratory test results are acceptable. Note: Any laboratory test abnormality (e.g., CPK within 3x of ULN presumably due to strenuous physical activity) may be repeated once during the screening period. 5. Restrictive eating habits (e.g., vegetarian or vegan), aversions to specific food categories used in food intake assessments, or eating behaviors that interfere with or confound the interpretation of food intake, appetite, or food control assessments.

[0336] Subjects who meet any of the following criteria at screening will be excluded from Parts A and B of the study: 1. Hospitalization for any reason (i.e., >24 hours) within 60 days of the screening visit. 2. The subject has any physical examination findings and / or any medical history that, in the opinion of the investigator, would confound the results of the study or place the subject at additional risk by participating in the study. 3. History of hypothalamic amenorrhea or lipodystrophy. 4. Weight change of more than 5% over the past 3 months prior to screening. 5. History of bariatric surgery for obesity (e.g., sleeve gastrectomy, gastric bypass, banding, etc.). 6. Weight loss surgery (e.g., liposuction) or body contouring surgery within the past 6 months. 7. Treatment with medications (over-the-counter [OTC] or prescription) for weight loss in the past 3 months (e.g., lorcaserin, phentermine / topiramate, naltrexone HCl / bupropion HCl, liraglutide). 8. History of major psychiatric disorder or eating disorder (e.g., bulimia, anorexia). 9. Current smokers or former smokers (cigarettes or e-cigarettes) who quit within 3 months prior to screening. 10. History of recreational drug (including marijuana) or alcohol dependence (>2 drinks per day) within 1 year prior to the screening visit. 11. History of hepatitis B infection or hepatitis B surface antigen positivity (HbsAg+) at the time of screening. 12. History of HIV infection or HIV seropositivity at the time of the screening visit. 13. History of hepatitis C infection or positive hepatitis C antibody test result at the time of screening. 14. Any malignancy within the past 10 years, except for basal or squamous cell carcinoma of the skin, or carcinoma in situ of the cervix or anus, which has been excised and has had no evidence of metastatic disease for 3 years. 15. History of active or latent tuberculosis (TB). Note: History of latent TB is defined as either a positive tuberculin skin test (TST; defined as a skin induration >5 mm, regardless of Bacillus Calmette-Guerin (BCG) or other vaccine history) or a positive (not equivocal) QuantiFERON® TB Gold test. 16. For Part A: At least two measurements of sitting or supine blood pressure (>140 / 90 or <90 / 60) and resting pulse (<45 or >125) or orthostatic variation (decrease in systolic blood pressure >20 mm Hg and / or decrease in diastolic blood pressure >10 mm Hg) at the screening visit and at the baseline visit. For Part B: At least two measurements of sitting or supine blood pressure (>150 / 90 or <90 / 50) and resting pulse (<45 or >125) or orthostatic variation (decrease in systolic blood pressure >20 mm Hg and / or decrease in diastolic blood pressure >10 mm Hg) at the screening visit. If blood pressure values ​​are high, blood pressure measurements may be repeated or the subject may be re-screened once. 17. Subject has a blood pressure of <60 mL / min / 1.73 m at screening. 2 have an estimated glomerular filtration rate (using the MDRD formula) of 18. Clinically significant ECG abnormalities or abnormal intervals confirmed on at least two measurements (QTcF >450 msec for men and >470 msec for women; PR <120 msec or >220 msec, QRS >100 msec). 19. Hypersensitivity to doxycycline (or any drug of the tetracycline class) or any other component of the formulation. 20. History of acute hypersensitivity and / or anaphylaxis to protein therapeutics. 21. History of severe allergies (including latex or anaphylactic reactions or allergies) that, in the opinion of the investigator, may pose a substantial risk to the subject. 22. Participation in any clinical trial evaluating another investigational drug (including biologics) or investigational therapy within 90 days or at least 5 half-lives of an investigational biologic (whichever is longer), or within at least 4 weeks for other investigational products, or within at least 6 months for immunotherapy, at the time of the screening visit. 23. Pregnant or lactating women. 24. Women of childbearing potential who are not willing to use highly effective contraception before the first dose / initiation of first treatment, during the study, and for at least 4 months after the last dose. * Highly effective contraceptive methods include: a. Stable use of combined (estrogen and progesterone-containing) hormonal contraceptives (oral, intravaginal, transdermal) or progesterone-only hormonal contraceptives (oral, injectable, implant) associated with ovulation inhibition initiated for two or more menstrual cycles prior to screening. b. Intrauterine device (IUD); intrauterine hormone-releasing system (IUS). c. Tubal ligation. d. A partner who has had a vasectomy. e. and / or sexual abstinence†,‡ * Postmenopausal women must be amenorrheic for at least 12 months to be considered fertile. Pregnancy testing and contraception are not required for women with documented proof of hysterectomy or tubal ligation. †Sexual abstinence is considered highly effective only when defined as abstinence from heterosexual intercourse for the entire period of risk associated with the study drug. ‡Periodic abstinence (calendar, symptom-temperature, postovulation), withdrawal (withdrawal), spermicide only, and lactational amenorrhea (LAM) are not acceptable methods of contraception. Female and male condoms should not be used together. 25. Sexually active men who are unwilling to use the following medically acceptable forms of contraception during study drug treatment and for 4 months after the last dose of study drug: vasectomy with evaluation for medical surgical success or consistent use of condoms. Sperm donation is prohibited during the study and for 4 months after the last dose of study drug. 26. Use of concomitant medications except those listed in the acceptable medications or dietary supplements.

[0337] Description of Study Cohorts and Dose Escalation Seven sequential escalating dose cohorts are planned, ranging from 0.3 mg / kg to a maximum dose of 30 mg / kg. Each dose cohort will consist of eight subjects: six randomized to receive H4H17319P2 and two randomized to receive placebo. To optimize safety, eight subjects (six active, two placebo) will be divided into two blocks in each of Cohort 1 (0.3 mg / kg IV), Cohort 2 (1 mg / kg IV), Cohort 3 (3 mg / kg IV), Cohort 4 (300 mg SC), and Cohort 5 (10 mg / kg IV). Two subjects (one active, one placebo) will be enrolled in Block 1 as a safety monitoring group, and the remaining six subjects will be enrolled in Block 2 (five active, one placebo). Subjects in Block 1 will be enrolled first and will be dosed on the same day. Enrollment of subjects in Block 2 will begin only after both Block 1 subjects have safely completed at least 24 hours of safety assessments, the safety data have been reviewed by the Investigator and Sponsor's Medical Monitor, and it is agreed by the Investigator and Sponsor's Medical Monitor that enrollment of subjects in Block 2 can begin. All Block 2 subjects may be dosed on the same day.

[0338] Cohort 6 (600 mg SC) and Cohort 7 (30 mg / kg IV) each comprise 8 subjects (6 active: 2 placebo) and will be divided into two blocks of 4 subjects (3 active: 1 placebo). Dosing of each block will occur on a different day. Escalating dose cohorts will be enrolled as follows: Cohort 1: H4H17319P2 0.3 mg / kg IV, single dose Cohort 2: H4H17319P2 1 mg / kg IV, single dose Cohort 3: H4H17319P2 3 mg / kg IV, single dose Cohort 4: H4H17319P2 300 mg SC, single dose Cohort 5: H4H17319P2 10 mg / kg IV, single dose Cohort 6: H4H17319P2 600 mg SC, single dose Cohort 7: H4H17319P2 nominal dose of 30 mg / kg IV, single dose

[0339] If PK variability is greater than expected, additional subjects will be enrolled and additional cohorts will be required to examine the role of specific covariates, such as age, weight, and sex. Cohort 8: H4H17319P2 nominal dose of 30 mg / kg IV, single dose Cohort 9: H4H17319P2 nominal dose of 30 mg / kg IV, single dose. A maximum dose of 30 mg / kg IV has been assigned to Cohorts 8 and 9, but lower doses may be administered depending on emerging PK data.

[0340] Dose Escalation: The safety / dose escalation team includes the principal investigator, medical / study director, biostatistician, and risk management lead. The principal investigator and the sponsor's investigational team, including other site personnel and medical monitors, will also be blinded to the treatment being administered. The sponsor may have unblinded individuals, but these unblinded individuals will not be part of the sponsor's investigational team.

[0341] Dose escalation to Cohort 2 (1 mg / kg) may proceed once all subjects in the previous cohort have completed the Day 8 safety assessment and the blinded safety data have been reviewed at the Safety / Dose Escalation Team meeting.

[0342] Dose escalation to Cohort 3 (3 mg / kg IV) and Cohort 4 (300 mg SC) may proceed once all subjects in the previous cohort have completed the Day 8 safety assessment and the blinded safety data have been reviewed at the Safety / Dose Escalation Team meeting. Dosing of Cohort 4 may occur concurrently with dosing of Cohort 3.

[0343] Dose escalation to Cohort 5 (10 mg / kg IV) may proceed once all subjects in Cohort 3 have completed the Day 8 safety assessment and the blinded safety data have been reviewed at the Safety / Dose Escalation Team meeting.

[0344] Dose escalation to Cohort 6 (600 mg / kg SC) may proceed in parallel with Cohort 5 only once all subjects in Cohort 4 have completed the Day 8 safety assessment and the blinded safety data have been reviewed at the Safety / Dose Escalation Team meeting.

[0345] A nominal dose of 30 mg / kg IV has been assigned to Cohort 7, but a lower dose may be administered depending on newly available PK data. Prior to dose escalation to 30 mg / kg IV, all subjects in Cohort 5 completed a Day 8 safety assessment, and blinded safety data was reviewed at a safety / dose escalation team meeting. If the pharmacological effect on body weight is greater than expected, the timing of the safety / dose escalation decision may be modified and additional safety data may be collected. For example, if at least three of six subjects lose more than 3% of their body weight by Day 8, the observation period will be extended to Day 15, after which any dose escalation decision will be made. If at least three of six subjects lose more than 5% of their body weight over two weeks, the observation period will be extended to four weeks, after which a decision on dose escalation will be made.

[0346] Table 19. Dosing cohorts in Parts A and B of the study TIFF2025166129000020.tif162156

[0347] Test Design This is a Phase 1, randomized, double-blind, placebo-controlled, two-part study evaluating the safety, tolerability, PK, and pharmacology (PD) of single and repeat doses of H4H17319P2 in healthy participants. In Part A, healthy, lean, or overweight subjects will be enrolled to evaluate the safety, tolerability, PK, and PD of single ascending intravenous (IV) and subcutaneous (SC) doses. Interim PK and safety information from Part A will be used to select the dose level, dosing frequency, and mode of administration (IV or SC) for Part B. In Part B, subjects with a body mass index (BMI) of 25-40 kg / m will be enrolled. 2 of overweight / obese subjects will be enrolled to evaluate the safety, tolerability, PK, and PD of repeat doses of H4H17319P2 in four separate cohorts defined by baseline leptin levels.

[0348] In Part A, up to 88 subjects will be randomized into up to seven sequential ascending single-dose (up to five IV and two SC) cohorts (Cohorts 1, 2, 3, 4, 5, 6, and 7) and two optional single-dose cohorts (Cohorts 8 and 9). The seven sequential single-dose cohorts will randomize eight subjects to receive H4H17319P2 or placebo (6 active: 2 placebo) at each dose level. The two additional optional single-dose cohorts will randomize up to 16 subjects to receive H4H17319P2 or placebo (12 active: 4 placebo). Up to five single IV dose levels (0.3, 1.0, 3, 10, and 30 mg / kg) and two SC dose levels (300 and 600 mg) will be evaluated in a single ascending fashion. The decision to escalate the dose will be based on an analysis of safety parameters and AEs. Interim analyses of antibody concentrations over time, exploratory PD measurements, and, if applicable, PK / PD relationships between dose cohorts may also inform dose escalation decisions. The decision to enroll two optional cohorts at dose levels up to 30 mg / kg will be based on an interim analysis of the variability in the PK profile.

[0349] Optional Cohorts: Cohorts 8 and 9 will be enrolled when higher-than-expected PK variability is observed among subjects and when additional subjects are needed to understand the impact of specific covariates on the PK profile. Subjects within specific subpopulations within the population defined by inclusion / exclusion criteria, such as prespecified numbers of men, women, age ranges, weights, or specific BMI cutpoints, will be enrolled. A nominal dose of 30 mg / kg IV has been assigned to Cohorts 8 and 9, but lower doses may be administered depending on newly available PK data. Cohorts 8 and 9 will each enroll 16 subjects (4 assigned to placebo and 12 to H4H17319P2). Prior to dose escalation to 30 mg / kg IV, all subjects in Cohort 5 completed the Day 8 safety assessment, and blinded safety data were reviewed at a safety / dose escalation team meeting. The study design for Part A consists of a screening period (days -21 to -2), a pre-baseline visit (day -1), a follow-up period (days 3 to 113), and an end of study visit (day 113). At the pre-baseline visit, subjects are admitted to a clinic stay with a 2-night stay (for subjects receiving IV dosing and subjects receiving SC dosing in the safety monitoring block) or a 1-day clinic stay (for other subjects receiving SC dosing). Throughout the study, safety assessments include vital signs, weight, physical examination, ECG, clinical laboratory tests, and monitoring of AEs, PK measurements, and various PD assessments.

[0350] In Part B, BMI between 25 and 40 kg / m 2 Up to 81 subjects will be enrolled in four cohorts (maximum approximately 20 subjects per cohort) defined by baseline leptin levels and randomized (3:1 or 6:1 H4H17319P2:placebo, depending on cohort assignment) to a placebo-controlled, double-blind, 12-week, multiple-dose study. The choice of dose, dosing interval, and mode of administration (IV vs. SC) will be based on safety, PK, and, where applicable, PK / PD data from Part A. The PK profile of H4H17319P2 from Part A will be used to predict serum concentrations after multiple doses.

[0351] The study consists of a pre-screening period (days -60 to -14), a screening period (days -32 to -14), a baseline period (days -29 to -1) to obtain baseline measurements of body weight, body composition by DXA and MRI, and a clinic visit for collection of baseline measurements of fasting leptin, body weight, appetite assessment, and ad libitum food intake assessment. Subjects are admitted to the clinic for a 2-day visit on day -1. On day 1, subjects receive their first dose of study drug or placebo, undergo serial blood sampling for pharmacokinetic measurements, stay overnight for 24-hour pharmacokinetic (PK) sampling, and are discharged on day 2. Study drug may be administered every 4 weeks or every 2 weeks, but not more frequently than weekly during the treatment period (dosing frequency will be determined by the interim analysis of PK data in Part A).

[0352] Visits during the treatment period will occur up to weekly, and accurate and repeated measurements of body weight and serum metabolic parameters (glucose, insulin, HOMA-IR, and lipids) will be collected. Follow-up assessments of appetite and ad libitum food intake will be conducted during the clinic stay at week 4 and at the end of the treatment period (12 weeks). Subjects will also complete daily appetite questionnaires during the baseline, treatment, and follow-up periods. Follow-up assessments with DXA and MRI imaging will also be conducted. After the treatment period, subjects will be followed for a 16-week washout period. Safety assessments throughout the study will include vital signs, physical examinations, ECGs, clinical laboratories, and monitoring for AEs. Drug concentrations, target engagement markers (sLEPR), and exploratory biomarkers will also be measured throughout the study.

[0353] Exam period Part A of the study will last approximately 19 weeks for subjects and will include a screening period. Part B of the study will last approximately 35 weeks for subjects and will include a prescreening / screening / baseline period. End of study will be defined as the last visit of the last subject in Part B.

[0354] Therapeutic drug dose / route / schedule H4H17319P2 will be supplied as a lyophilized powder in sterile, single-use 20 mL glass vials for either IV or SC administration. The matching placebo for H4H17319P2 will be prepared with the same formulation without added protein. For Part A, single doses will be administered IV and SC. For Part B, the choice of dose, dosing interval, and mode of administration (IV vs. SC) will be based on safety, PK, and, if applicable, PK / PD data from Part A.

[0355] Procedures and Evaluation Safety will be assessed by monitoring / assessing TEAEs, vital signs, physical examination, electrocardiogram (ECG), and clinical laboratory tests. High-density, low-volume samples will be collected for measurement of serum H4H17319P2 concentrations at pre-specified time points to assess pharmacokinetics. Pharmacology will be assessed by measuring body weight and waist circumference, food intake and appetite assessment, and body composition using DXA and MRI.

[0356] Primary outcome measures: 1. Number of treatment-emergent adverse events (TEAEs) [Time frame: Week 12 (end of treatment period)]

[0357] Secondary outcome measures: 1. Serum H4H17319P2 concentrations over time [Time frame: up to week 27 (end of study)] 2. Percent change in weight in overweight or obese subjects [Time frame: Baseline to Week 12] 3. Absolute change in body weight in overweight or obese subjects [Time Frame: Baseline to Week 12] 4. Change from baseline in caloric intake in response to a standard diet in overweight or obese subjects [Time Frame: Baseline to Week 12] 5. Changes in lipid-regulatory protein levels over time after a single dose of H4H17319P2 [Time frame: up to 16 weeks] 6. Changes in lipid-regulatory protein levels over time after repeated administration of H4H17319P2 [Time frame: up to 27 weeks] 7. Incidence of anti-drug antibodies to H4H17319P2 over time after a single dose of H4H17319P2 [Time frame: up to week 16] 8. Incidence of anti-drug antibodies to H4H17319P2 over time after repeated administration of H4H17319P2 [Time frame: up to week 27]

[0358] Pharmacokinetic variables In addition to time, the concentration of total H4H17319P2 is measured. Pharmacokinetic parameters include, but are not limited to: AUC last - The computed area under the curve (AUC) from time zero to the time of the last positive concentration AUC 0-τ - the computed AUC during a dosing interval of length τ C max - Peak Concentration · t max - C max Time until CL - Clearance C trough - Trough concentration

[0359] Note that for Part B, the selection of these (and other) parameters is based on the final sampling schedule selected and the resulting data obtained.

[0360] Antidrug antibody variables Anti-drug antibody (ADA) variables include ADA response and titer as follows: A treatment-emergent response is defined as a positive ADA assay response after any dose when the baseline result was negative. A treatment-enhanced ADA response is defined as any post-dose positive ADA assay response that is 9-fold or greater than the baseline titer when the baseline ADA assay was positive. Potency value Potency category - Low (titer <1,000) - Medium (1,000≦titer≦10,000) - High (titer > 10,000)

[0361] Pharmacological and other biomarker variables Pharmacological and biomarker variables include body weight, ad libitum food intake assessment, appetite assessment, serum / plasma glycemic (e.g., fasting glucose, insulin, HbA1c) and lipid parameters (e.g., total cholesterol, TG, LDL-C, HDL-C), DXA measurements of total and regional body fat and lean mass, MRI quantification of regional SC and visceral fat, ANGPTL3, leptin, and sLEPR. Additional exploratory biomarkers include the effect of H4H17319P2 on thyroid hormones (T3, T4, TSH), luteinizing hormone (LH), testosterone, estradiol, cortisol, and adiponectin.

[0362] Validity Procedures Body weight will be assessed at designated study visits during screening and throughout the study. Weight will be assessed in triplicate using a precision calibrated digital scale before any other study assessments are performed. Subjects must urinate (bladder empty) before weight assessment is performed. Subjects should wear only underwear and no shoes during weight assessment. Weight will be recorded to the nearest 0.1 kg.

[0363] All anthropometric measurements should be performed in triplicate, and the final reported value is the average. Triceps, subscapularis, suprailiac, and thigh skinfold thickness should be taken from the right side of the body in an area of ​​dry, intact skin, unless deformity or missing limbs require alternative methods. To measure waist circumference, subjects are instructed to stand upright and relaxed, with arms at their sides and feet together and facing forward. The iliac crest and lowest costal margin are identified by palpation, and the skin overlying these areas is marked with a pen. The midpoint between these skin marks is then identified and marked with a tape measure. Waist circumference at the midpoint is then measured using the tape measure upon a gentle, full exhalation. Height is measured while standing fully upright upon a full inhalation using a calibrated stadiometer and recorded to the nearest 0.1 cm. For Part B and any cohorts in Part A, height will be measured as a single measurement at the pre-screening or screening visit, respectively. Body mass index will be calculated by dividing the mean weight (kilograms) by the square of the height (meters). For Part A (except for any cohorts), mean height will be used in the calculation. For Part B and any cohorts in Part A, the values ​​for height (single measurement at pre-screening or screening, respectively) and weight (average of three measurements) at each visit will be used to calculate BMI.

[0364] Ad libitum food intake will be assessed. Subjects will be excluded at screening if they have eating behavior disorders or aversions to foods used in the food intake assessment. Energy intake (breakfast + lunch + dinner) using the ad libitum food intake assessment will be quantified at baseline, after 4 weeks of treatment, and at the end of the treatment period (week 12) in Part B. One day before the clinic visit where the food assessment ...

Claims

1. A method for treating or preventing metabolic dysfunction or hypoleptinemia, or a disease or condition associated with metabolic dysfunction or hypoleptinemia, or one or more symptoms of said disease or condition, comprising administering to a subject in need thereof a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to the human leptin receptor (LEPR) and activates LEPR signaling, and a pharmaceutically acceptable carrier or diluent.

2. 2. The method of claim 1, wherein the condition is selected from the group consisting of non-alcoholic fatty liver disease, NASH, female infertility, amenorrhea, hormone cycle abnormalities, immune dysfunction, hypothyroidism, obesity, monogenic obesity, type I diabetes, type II diabetes, lipodystrophy, congenital lipodystrophy, generalized lipodystrophy, acquired lipodystrophy, partial lipodystrophy, congenital partial lipodystrophy, congenital generalized lipodystrophy, acquired partial lipodystrophy, and acquired generalized lipodystrophy.

3. 3. The method of claim 2 for treating or preventing non-alcoholic fatty liver disease.

4. 3. The method of claim 2 for treating or preventing congenital lipodystrophy.

5. 3. The method of claim 2 for treating or preventing generalized lipodystrophy.

6. 3. The method of claim 2 for treating or preventing acquired lipodystrophy.

7. 3. The method of claim 2 for treating or preventing partial lipodystrophy.

8. 3. The method of claim 2 for treating or preventing monogenic obesity.

9. the condition associated with metabolic dysfunction or hypoleptinemia is congenital lipodystrophy; The method of claim 2, wherein the symptoms associated with congenital lipodystrophy are prevented, ameliorated, or reduced in severity and / or duration or reduced after administration of the antibody or antigen-binding fragment thereof that binds to human LEPR.

10. The method of claim 9, wherein after administration of the antibody or antigen-binding fragment thereof that binds to human LEPR, the subject's blood glucose is reduced, the subject's weight is reduced, the subject exhibits reduced food intake, the subject's fat mass is reduced, the subject's lean mass is increased, and / or the subject's bone mass is increased.

11. 3. The method of claim 2, wherein the condition is non-alcoholic fatty liver disease, and after treatment, the subject's liver weight is reduced, the subject's plasma level of alanine transaminase (ALT) is reduced, and / or the subject's plasma level of aspartate transaminase (AST) is reduced.

12. 3. The method of claim 2, wherein the condition is female infertility.

13. 13. The method of claim 12, wherein the condition is female infertility and the subject's hormone cycle is restored and / or the subject becomes pregnant.

14. 3. The method of claim 2, wherein the condition is amenorrhea.

15. 15. The method of claim 14, wherein the condition is amenorrhea and the subject begins to have normal hormone cycles.

16. 3. The method of claim 2, wherein the condition is an immune dysfunction.

17. 17. The method of claim 16, wherein the condition is immune dysfunction and the subject has an increased CD4+ T cell count.

18. one or more symptoms of a disease or condition of or associated with metabolic dysfunction or hypoleptinemia, which are one or more selected from the group consisting of adiposity, obesity, hyperphagia, hyperglycemia, hypertriglyceridemia, hypercholesterolemia, insulin resistance, dyslipidemia, growth retardation, delayed pubertal growth spurt, abnormal growth hormone secretion, elevated HbA1c, low bone mineral density (or low bone mass), low bone mineral content, and low lean body mass The method of claim 1 for treating or preventing

19. A method for increasing bone mass in a subject having low bone mass that is a symptom of a metabolic dysfunction or hypoleptinemia from which the subject suffers, comprising administering to the subject in need thereof an antibody or antigen-binding fragment thereof that binds to the human leptin receptor (LEPR) and activates LEPR signaling, and a pharmaceutically acceptable carrier or diluent.

20. The method of any one of claims 1 to 19, wherein the subject in need thereof is leptin deficient.

21. The method of any one of claims 1 to 19, wherein the subject in need thereof is not leptin deficient.

22. 22. The method of any one of claims 1 to 21, wherein the condition is obesity, and wherein the obesity is not associated with or caused by a signaling-deficient or signaling-impaired LEPR mutation.

23. 23. The method of any one of claims 1 to 22, wherein the treatment reduces fat mass but does not reduce lean mass.

24. The method of any one of claims 1 to 23, wherein the treatment with the antibody or antigen-binding fragment thereof that binds to human LEPR and activates LEPR signaling stimulates hypothalamic STAT3 signaling or enhances leptin-induced or leptin-independent STAT3 signaling.

25. 25. The method of any one of claims 1 to 24, wherein the treatment lowers circulating plasma triglycerides.

26. 26. The method of any one of claims 1 to 25, wherein the treatment lowers circulating plasma total cholesterol.

27. In patients, (i) lipodystrophy and / or monogenic obesity, (ii) a condition associated with lipodystrophy and / or monogenic obesity, or (iii) Symptoms of (i) or (ii) 10. A method for treating, preventing, or ameliorating a disease comprising administering to said patient in need thereof an agonistic antibody that specifically binds to LEPR.

28. The conditions associated with lipodystrophy and / or monogenic obesity include very early onset obesity; hyperphagia and impaired satiety; impaired immune function (CD4 ＋ 28. The method of claim 27, wherein the underlying condition is a condition characterized by: insulin resistance; non-alcoholic fatty liver disease; NASH, dyslipidemia; diabetes; reproductive dysfunction; hypogonadism; missed pubertal growth spurt; hypothyroidism; thyroid dysfunction; low bone mineral density or low bone mass.

29. 28. The method of claim 27, wherein the symptoms are enlarged liver, elevated liver enzymes, elevated blood levels of alanine aminotransferase (ALT), elevated blood levels of aspartate aminotransferase (AST), advanced steatosis; body mass index greater than 85th percentile for age and sex; abnormal food-seeking behavior; abnormal food-aggression behavior; recurrent and potentially fatal infections; hyperinsulinemia; hepatic steatosis; progression to NASH (lipodystrophy); hypertriglyceridemia; elevated HbA1c; high blood glucose levels; impaired glucose tolerance; delayed pubertal development; decreased onset of secondary sexual characteristics; amenorrhea or menstrual irregularities; infertility; dwarfism; abnormal growth hormone secretion; altered T3; altered TSH; or altered free thyroxine levels.

30. 30. The method of any one of claims 1 to 29, wherein the subject has failed metreleptin treatment.

31. The method of any one of claims 1 to 30, wherein the antibody is administered as follows: (i) one or more intravenous doses of about 5 mg / kg body weight; then (ii) one or more subcutaneous doses of about 250 mg or about 300 mg once weekly; and (iii) optionally, one or more subcutaneous doses of about 250 mg or about 300 mg once a month or about once every 28 days.

32. The method of any one of claims 1 to 30, wherein the antibody is administered as follows: (i) one or more intravenous doses of about 5 mg / kg body weight; then (ii) One or more subcutaneous doses of about 250 mg or about 300 mg once weekly.

33. 32. The method of any one of claims 1 to 31, wherein the antibody is administered as follows: (i) a single intravenous dose of 5 mg / kg body weight; then (ii) four subcutaneous doses of about 250 mg or about 300 mg once weekly; then (iii) One or more subcutaneous doses of about 250 mg or about 300 mg once a month or once every 28 days.

34. 34. The method of any one of claims 31 to 33, wherein the first subcutaneous administration is administered three days after the intravenous administration.

35. the antibody or antigen-binding fragment thereof (a) a complementarity-determining region (CDR) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:42, SEQ ID NO:50, SEQ ID NO:58, SEQ ID NO:74, or SEQ ID NO:82; and (b) a CDR of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 66 The method of any one of claims 1 to 34, comprising:

36. 35. The method of any one of claims 1 to 34, wherein the antibody or antigen-binding fragment thereof comprises the heavy chain CDRs and light chain CDRs of an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 26 / 10, 34 / 10, 42 / 10, 50 / 10, 58 / 66, 74 / 66 and 82 / 66.

37. 35. The method of any one of claims 1 to 34, wherein the antibody or antigen-binding fragment thereof comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 26 / 10, 34 / 10, 42 / 10, 50 / 10, 58 / 66, 74 / 66 and 82 / 66.

38. The method of any one of claims 1 to 34, wherein the antibody or antigen-binding fragment thereof comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 26 / 10.

39. The method of any one of claims 1 to 34, wherein the antibody or antigen-binding fragment thereof comprises a combination of the amino acid sequences HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 of SEQ ID NOs: 28 / 30 / 32 / 12 / 14 / 16.

40. The method further comprises administering to the subject a second therapeutic agent, wherein the second therapeutic agent is selected from the group consisting of recombinant human leptin, PCSK9 inhibitors, statins, ezetimibe, insulin, insulin variants, insulin secretagogues, metformin, sulfonylureas, sodium glucose cotransporter 2 (SGLT2) inhibitors, GLP-1 agonists / analogs, glucagon (GCG) inhibitors, glucagon receptor (GCGR) inhibitors, angiopoietin-like protein (ANGPTL) inhibitors, phentermine, orlistat, topiramate, bupropion, and topiramate.

40. The method of any one of claims 1 to 39, wherein the active ingredient is selected from the group consisting of phentermine, bupropion / naltrexone, bupropion / zonisamide, pramlintide / metreleptin, lorcaserin, cetilistat, tesofensine, velneperit, anticonvulsants, digoxin, coumadin, vitamin D, thyroxine, thyroid supplements, vitamin supplements, calcium supplements, carnitine, coenzyme Q10, anti-constipation medications, anti-allergy medications, gabapentin, anesthetics, ketamine, lidocaine, and venlafaxine hydrochloride.

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