Leptin receptor agonist antibodies for the treatment of metabolic dysfunction or hypoleptinemia

Abstract

Provided herein are therapeutic methods of treatment using an agonist leptin receptor (LEPR) antibody, antigen-binding fragment thereof, or a composition comprising the LEPR antibody or antigen-binding fragment thereof, to perform the treatment. Such therapeutic methods include treating conditions associated with metabolic dysfunction, including, for example, lipodystrophy, obesity predisposition or obesity, weight loss, nonalcoholic fatty liver disease, bulimia, hyperglycemia, insulin resistance, dyslipidemia, hepatic steatosis, and infertility.

Description

TECHNICAL FIELD

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

[0002] SEQUENCE LISTING

[0003] The official copy of the sequence listing is submitted electronically via EFS-Web in an ASCII format file named 10436WO_SEQ_LIST_ST25, created on April 5, 2019, having a size of about 105 kilobytes. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety. The contents of the entire sequence listing are incorporated herein by reference in its entirety. BACKGROUND

[0004] Leptin is a polypeptide hormone expressed primarily by adipose tissue that is involved in the regulation of metabolism, neuroendocrine function, immunity, energy balance, and food intake. Leptin activity is mediated through interaction with the leptin receptor and signaling through the leptin receptor. The leptin receptor (also known as "LEPR," "WSX," "OB receptor," "OB-R," and "CD295") is a single-pass transmembrane receptor of the class I cytokine receptor family with a large (818 amino acid) extracellular domain. Leptin deficiency, leptin resistance, and certain LEPR signaling deficiency / impairment mutations are associated with obesity, type 2 diabetes, dyslipidemia, lipodystrophy, hepatic steatosis, nonalcoholic and alcoholic fatty liver disease, severe insulin resistance, leptin deficiency syndrome / DONOHUE syndrome, Rabson-Mendenhall syndrome, and related complications. Therapeutic approaches to address leptin resistance, leptin deficiency, and hypoleptinemia (e.g., lipodystrophy) have primarily focused on delivery of leptin or leptin analogs to affected individuals. However, these approaches generally show limited efficacy, particularly in leptin-resistant individuals, and are often associated with adverse side effects. Thus, there is a need in the art for alternative approaches to treat leptin resistance and other disorders associated with leptin deficiency or hypoleptinemia. SUMMARY

[0005] The present invention provides antibodies and antigen-binding fragments thereof that bind to human leptin receptor (LEPR). The antibodies of the invention are agonist antibodies; that is, binding of an anti-LEPR antibody of the invention to LEPR results in, among other things, activation of leptin receptor signaling in cells. In certain embodiments, the antibodies of the invention do not compete with leptin for binding to LEPR. The antibodies of the invention can be used, for example, to mimic, replace, or supplement normal biological activity of leptin in a subject. Accordingly, the antibodies and antigen-binding fragments of the invention can be used in the therapeutic treatment of diseases and disorders associated with leptin resistance and leptin deficiency.

[0006] The antibodies of the invention can be full-length (e.g., IgGl or IgG4 antibodies) or can comprise only an antigen-binding portion (e.g., a Fab, F(ab')2, or scFv fragment), and can be modified to affect function, e.g., to eliminate residual effector function (Reddy et al., 2000, J. Immunol. 164: 1925-1933).

[0007] 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.

[0008] The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to LEPR, the antibody or antigen-binding fragment thereof comprising a HCVR comprising an amino acid sequence selected from any of the HCVR 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 thereto.

[0009] The present invention also provides an antibody or antigen-binding fragment thereof that specifically binds to LEPR, the antibody or antigen-binding fragment thereof comprising a LCVR comprising an amino acid sequence selected from any of the LCVR 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 thereto.

[0010] The present application also provides antibodies or antigen-binding fragments thereof that specifically bind to LEPR, comprising a 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 application provides antibodies or antigen-binding fragments thereof comprising a 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.

[0011] The present application also provides antibodies or antigen-binding fragments thereof that specifically bind to LEPR, comprising 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 thereto.

[0012] The present application also provides antibodies or antigen-binding fragments thereof that specifically bind to LEPR, comprising 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 thereto.

[0013] The present application also provides antibodies or antigen-binding fragments thereof that specifically bind to LEPR, comprising 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 thereto.

[0014] The present application also provides antibodies or antigen-binding fragments thereof that specifically bind to LEPR, comprising 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 thereto.

[0015] The present application also provides an antibody or antigen-binding fragment thereof that specifically binds to LEPR, comprising 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 thereto.

[0016] The present application also provides an antibody or antigen-binding fragment thereof that specifically binds to LEPR, comprising 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 thereto.

[0017] The present application also provides an antibody or antigen-binding fragment thereof that specifically binds to LEPR, comprising a pair of HCDR3 and LCDR3 amino acid sequences (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 application provides an antibody or antigen-binding fragment thereof comprising a pair of HCDR3 / LCDR3 amino acid sequences contained within any of the exemplary anti-LEPR antibodies listed in Table 1. In certain embodiments, the pair of HCDR3 / LCDR3 amino acid sequences is selected from the group consisting of SEQ ID NO: 8 / 16, 24 / 16, 32 / 16, 40 / 16, 48 / 16, 56 / 16, 64 / 72, 80 / 72, and 88 / 72.

[0018] The present disclosure 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 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences 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.

[0019] In a related embodiment, the present application provides an antibody or antigen-binding fragment thereof that specifically binds LEPR, comprising a set of six CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) contained within a HCVR / LCVR amino acid sequence pair defined by any one of the exemplary anti-LEPR antibodies listed in Table 1. For example, the present application includes an antibody or antigen-binding fragment thereof that specifically binds LEPR, comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences contained within a HCVR / LCVR amino acid sequence pair selected from the group consisting of: the HCVR / LCVR amino acid sequence pairs 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 specified HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, and the AbM definition. Generally speaking, the Kabat definition is based on sequence differences, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., 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.

[0020] The present application also provides nucleic acid molecules encoding anti-LEPR antibodies or portions thereof. For example, the present application provides nucleic acid molecules encoding any one of the HCVR amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecules comprise a polynucleotide sequence selected from any one of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0021] The present application also provides nucleic acid molecules encoding 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 LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0022] The present application also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1; 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0023] The present application also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1; 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0024] The present application also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1; 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0025] The present application also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1; 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0026] The present application also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1; 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0027] The present application also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1; 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 thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0028] The present application also provides nucleic acid molecules encoding a HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1, HCDR2, HCDR3), wherein the HCDR1, HCDR2, HCDR3 amino acid sequence set is as defined by any of the exemplary anti-LEPR antibodies listed in Table 1.

[0029] The present application also provides nucleic acid molecules encoding a LCVR, wherein the LCVR comprises a set of three CDRs (i.e., LCDR1, LCDR2, LCDR3), wherein the LCDR1, LCDR2, LCDR3 amino acid sequence set is as defined by any of the exemplary anti-LEPR antibodies listed in Table 1.

[0030] The present application also provides nucleic acid molecules encoding both a HCVR and a 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 thereof 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 thereof 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 application, the nucleic acid molecule encodes a HCVR and a LCVR, wherein both the HCVR and the LCVR are derived from the same anti-LEPR antibody listed in Table 1.

[0031] The present application also provides recombinant expression vectors capable of expressing a polypeptide comprising a heavy chain variable region or a light chain variable region of an anti-LEPR antibody. For example, the present application includes recombinant expression vectors comprising any of the nucleic acid molecules described above, i.e., nucleic acid molecules encoding any of the HCVR, LCVR, and / or CDR sequences set forth in Table 1. Also included within the scope of the present application are host cells into which such vectors have been introduced, as well as methods of producing antibodies or portions thereof by culturing the host cells under conditions permitting production of the antibodies or antibody fragments, and recovering the antibodies and antibody fragments so produced.

[0032] In another aspect, the present application provides a pharmaceutical composition comprising a recombinant human antibody or fragment thereof that specifically binds LEPR and a pharmaceutically acceptable carrier. In a related aspect, the present application 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.

[0033] As used throughout this disclosure, the term "subject" is interchangeable with the term "patient." A subject or patient can be an adult. Pediatric patients are also contemplated as benefiting from the methods and compositions provided herein.

[0034] In yet another aspect, the present application provides therapeutic methods for enhancing or stimulating LEPR signaling using the anti-LEPR antibodies or antigen-binding portions of antibodies of the present application. The therapeutic methods according to this aspect of the present application include administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an antibody or antigen-binding fragment of an antibody of the present application. The disorder treated is any disease or condition that is ameliorated, improved, inhibited, or prevented by stimulating or activating LEPR signaling or by mimicking the natural activity of leptin in vitro or in vivo.

[0035] In some aspects, provided herein are therapeutic methods for treating or preventing a 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 leptin receptor (LEPR) and activates LEPR signaling, and a pharmaceutically acceptable carrier or diluent.

[0036] In some aspects, provided herein are therapeutic methods for treating or preventing a metabolic dysfunction or hypoleptinemia, or a disease or condition associated with a metabolic dysfunction or hypoleptinemia, or one or more symptoms of the disease or condition. 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.

[0037] In some embodiments, the disorder is selected from the group consisting of nonalcoholic fatty liver disease, nonalcoholic steatohepatitis (NASH), female infertility, amenorrhea, abnormal hormone cycles, impaired immune function, 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.

[0038] In some embodiments, one or more symptoms of a disease or disorder associated with metabolic dysfunction or leptinopenia are selected from the group consisting of obesity predisposition, obesity, hyperphagia, hyperglycemia, hypertriglyceridemia, hypercholesterolemia, insulin resistance, dyslipidemia, growth delay, delayed precocious puberty, abnormal growth hormone secretion, elevated HbAlc, low bone mineral density (or low bone mass), low bone mineral content, and low lean body mass. Symptoms of a disease or disorder associated with metabolic dysfunction or leptinopenia can be prevented, ameliorated, or lessened in severity and / or duration, or reduced, following administration of an antibody or antigen-binding fragment thereof that binds human LEPR.

[0039] In still other aspects, provided herein are methods for treating metabolic complications of lipodystrophy. Such methods include administering to a subject in need thereof 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. In some embodiments, the treatment lessens hyperglycemia, reduces insulin resistance, reduces hypertriglyceridemia, lowers circulating cholesterol levels, and / or lowers HbAlc levels in the subject. The lipodystrophy can include acquired partial lipodystrophy, acquired generalized lipodystrophy, congenital partial lipodystrophy, and congenital generalized lipodystrophy.

[0040] 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 genetic mutations (e.g., p.N103K, which encodes a non-biologically active form of leptin). Some subjects have little or no circulating leptin. Other genes involved in impaired leptin signaling can include LMNA, PPARG, PLIN1, AKT2, CIDEC, LIPE, and ADRA2A, and the anti-LEPR antibodies and antigen-binding fragments thereof provided herein can be used to lessen the effects of such mutations on leptin signaling.

[0041] In some aspects, provided herein are methods for treating congenital leptin deficiency. Such methods include administering to a subject in need thereof a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to human leptin receptor (LEPR) and activates LEPR signaling, and a pharmaceutically acceptable carrier or diluent. In some embodiments, the subject has lipodystrophy and fails metreleptin therapy. In some embodiments, symptoms associated with congenital lipodystrophy can be prevented, ameliorated, or lessened in severity and / or duration, or reduced, following administration of the antibody or antigen-binding fragment thereof that binds to human LEPR. In some embodiments, the treatment reverses or lessens 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 body weight is reduced, the subject exhibits reduced food intake, the subject's fat mass is reduced, the subject's lean body mass is increased, and / or the subject's bone mass is increased.

[0042] In some aspects, provided herein are methods of treatment for treating nonalcoholic fatty liver disease or nonalcoholic steatohepatitis (NASH). In some aspects, the subject is hypoleptinemic, lipodystrophic, or leptin deficient. According to this aspect, 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, the subject's liver weight is reduced following treatment. In some embodiments, symptoms of nonalcoholic fatty liver disease, including nonalcoholic hepatic steatosis, in the subject are reduced following treatment. In some embodiments, the subject's plasma levels of alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST) are reduced.

[0043] In yet other aspects, provided herein are methods for treating female infertility, amenorrhea, or restoring normal hormonal cycles associated with metabolic dysfunction or hypoleptinemia. Such 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 aspects, administration of the antibody or antigen-binding fragment thereof that binds to human LEPR can increase fertility and / or increase the chances of conception. In some aspects, the subject conceives. In some aspects, the treatment can restore or initiate a normal menstrual cycle.

[0044] In some aspects, provided herein are methods for treating impaired immune function associated with metabolic dysfunction or leptinopenia. Such 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, CD4+ T cell counts are increased following administration of the antibody or antigen-binding fragment thereof that binds to human LEPR.

[0045] In other aspects, provided herein are therapeutic methods for increasing bone mass in a subject having metabolic dysfunction or leptinopenia. 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.

[0046] In other aspects, provided herein are therapeutic methods for treating a predisposition to or obesity or for reducing body weight. According to this aspect, 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, the treatment reduces fat mass but not lean body mass.

[0047] In some embodiments, the subject in need is leptinopenic, lipodystrophic, or leptin deficient. In some embodiments, the subject in need is not leptinopenic or leptin deficient. In some embodiments, the metabolic dysfunction, predisposition to or obesity is not associated with or is not caused by a signaling-deficient or signaling-impaired LEPR mutation.

[0048] In some embodiments, administration of the antibody or antigen-binding fragment thereof that binds to human LEPR and activates LEPR signaling according to the methods provided herein stimulates hypothalamic STAT3 signaling or enhances leptin-induced or leptin-independent STAT3 signaling.

[0049] In some embodiments, administration of the antibody or antigen-binding fragment thereof that binds to human LEPR and activates LEPR signaling reduces circulating plasma triglycerides and / or reduces circulating plasma total cholesterol.

[0050] In other aspects, provided herein are methods of treatment for hyperphagia, hyperglycemia, insulin resistance, dyslipidemia, nonalcoholic steatohepatitis (NASH), or nonalcoholic fatty liver disease by stimulating hypothalamic STAT3 signaling. Such methods include administering to a subject in need thereof a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to 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.

[0051] In yet other aspects, provided herein are methods for treating growth retardation, precocious puberty deficiency, and / or abnormal growth hormone secretion associated with congenital leptin deficiency. Such 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.

[0052] In yet other aspects, provided herein are methods for treating hypothyroidism associated with congenital leptin deficiency. Such 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.

[0053] In yet other aspects, provided herein are methods for treating low bone mineral density and / or bone mineral content associated with hypoleptinemia and / or leptin deficiency. Such 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.

[0054] One or more additional therapeutic agents can be administered to a subject described herein with an antibody or antigen-binding fragment thereof that binds to human LEPR. The second therapeutic agent can be selected from the group consisting of recombinant human leptin, a PCSK9 inhibitor, a statin, ezetimibe, insulin, an insulin variant, an insulin secretagogue, metformin, a sulfonylurea, a sodium glucose co-transporter 2 (SGLT2) inhibitor, a GLP-1 agonist / analogue, a glucagon (GCG) inhibitor, a glucagon receptor (GCGR) inhibitor, an angiopoietin-like protein (ANGPTL) inhibitor, phentermine, Orlistat, Topiramate, bupropion, Topiramate / phentermine, bupropion / naltrexone, bupropion / zonisamide, Pramlintide / Metreleptin, Lorcaserin, Cetilistat, Tesofensine, Velneperit, an anticonvulsant, digoxin, coumarin, vitamin D, thyroxine, a thyroid supplement, a vitamin supplement, a calcium supplement, carnitine, coenzyme Q10, an anti-constipation medication, an anti-allergy medication, gabapentin, a narcotic, ketamine, lidocaine, or venlafaxine hydrochloride.

[0055] The present application also provides a method for treating, preventing or ameliorating (i) lipodystrophy (of 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 method comprising administering to a patient in need thereof an agonist antibody or antigen-binding fragment thereof that specifically binds to LEPR (e.g., H4H17319P2). For example, in an embodiment of the present application, the condition associated with lipodystrophy and / or monogenic obesity is extreme early onset obesity; excessive appetite and impaired satiety; impaired immune function (CD4 +counting); insulin resistance; non-alcoholic fatty liver disease; NASH, dyslipidemia; diabetes; reproductive dysfunction; hypogonadism; precocious puberty deficiency; hypothyroidism; impaired thyroid function; low bone mineral density and / or low bone mass. In an embodiment of the application, the symptom is hepatomegaly, elevated liver enzymes, elevated blood levels of alanine aminotransferase (ALT), elevated blood levels of aspartate aminotransferase (AST), high propensity for obesity; body mass index > 85th percentile for age and sex; abnormal feeding behavior; abnormal food guarding behavior; recurrent and potentially fatal infections; hyperinsulinemia; liver steatosis; progression to NASH (adiposopathy); hypertriglyceridemia; elevated HbAlc; elevated glucose levels; reduced glucose tolerance; delayed puberty; reduced expression of secondary sexual characteristics; amenorrhea or irregular menses; infertility; short stature; abnormal growth hormone secretion; altered T3; altered TSH; and / or altered free thyroxine levels.

[0056] In an embodiment of the application, the agonist anti-LEPR antibody or antigen-binding fragment thereof (e.g., H4H17319P2; see WO2017 / 66204) is administered as follows: (i) one or more doses of about 5 mg / kg body weight intravenously; then (ii) one or more doses of about 250-300 mg, e.g., 250 mg or about 300 mg subcutaneously once weekly; then (iii) optionally, one or more doses of about 250 mg or about 300 mg subcutaneously once monthly or about 28 days. For example, (i) one or more doses of about 5 mg / kg body weight intravenously; then (ii) one or more doses of about 250 mg or about 300 mg subcutaneously once weekly. In an embodiment of the application, the antibody is administered as follows: (i) 5 mg / kg body weight intravenously once (on day 1); then (ii) four doses of about 250 mg or about 300 mg subcutaneously once weekly (e.g., on days 4, 11, 18, and 25); then (iii) one or more doses of about 250 mg or about 300 mg subcutaneously once monthly or about 28 days (e.g., on days 53, 81, 109, 137, 165, and 193, etc.). For example, in an embodiment of the application, the first subcutaneous dose occurs on about day 4, i.e., about three days after the intravenous dose is administered on day 1.

[0057] Other embodiments will become apparent by reference to the following detailed description. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1Binding 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) is depicted.

[0059] Figures 2A-2C The extent of LEPR signaling in HEK293 cells expressing wild-type LEPR (circles), a signaling-deficient LEPR mutant (A409E, squares), or a signaling-impaired LEPR mutant (P316T, triangles) is shown. LEPR signaling is expressed as the ratio of pSTAT3-Y705 / STAT3, which was measured by densitometry from Western blots prepared from cells treated with increasing concentrations of leptin (A), H4H16650 (B), or H4H16679 (C). Figure 2A Figure 2B Figure 2C

[0060] Figure 3 The average daily food intake of leptin-deficient mice administered 3 mg / kg of an isotype control antibody or 3 mg / kg of a LEPR antibody selected from H4H16650P2, H4H16679P2, H4H17319P2, or H4H17321P2 is shown.

[0061] Figure 4 The average percent change in body weight of mice administered 3 mg / kg of an isotype control antibody or 3 mg / kg of a LEPR antibody selected from H4H16650P2, H4H16679P2, H4H17319P2, or H4H17321P2 is shown.

[0062] Figure 5 The average fat mass of animals in each antibody treatment group, as quantified by μCT 1 day prior to antibody treatment (unshaded bar) and 6 days after antibody treatment (shaded bar), is shown, expressed as mean ± SEM.

[0063] Figure 6 The percent change in body weight of mice fed 30 mg / kg of an antibody selected from H4H18482P2, H4H18487P2, H4H18492P2, or an isotype control is shown.

[0064] Figure 7 A-7B. Figure 7 A shows the fat mass of mice prior to administration of anti-LEPR antibodies H4H18482P2, H4H18487P2, or H4H18492P2. Figure 7 ​​​B shows fat mass in mice treated with 30 mg / kg H4H18482P2, H4H18487P2, or H4H18492P2.

[0065] Figure 8 . Figure 8 shows that the anti-LEPR antibodies tested activated monkey (Mf) LEPR in the IMR-32 / STAT3-luc / Mf LEPR cell line.

[0066] Figures 9A-9C On day 0, leptin deficiency was induced in 18-week-old male Lepr hu / hu mice by HDD of mLepr.ECD. Seven days after HDD, mice were stratified into 2 groups according to relative percent change in body weight and administered a single 10 mg / kg subcutaneous dose of control monoclonal antibody (gray solid circles or bars) or H4H17319P2 (solid circles or bars). Data are mean ± SEM. *, P < 0.05 for H4H17319P2 vs. control monoclonal antibody at the indicated time points. $, P < 0.05 between pre-HDD (day -1) and any day post-HDD (day 6 or 13) in the same respective administered group. &, P < 0.05 between pre-monoclonal antibody administration (day 6) and post-monoclonal antibody administration (day 13) in the same respective administered group. No significant difference (ns) from day 0 baseline for groups represented in the respective text color on the indicated day. Figure 9A shows body weight (left) and daily food intake (right) in Lepr hu / hu mice throughout the study, showing that induction of leptin deficiency resulted in rapid weight gain and hyperphagia. N = 14 per group. Figure 9B shows body composition analysis by micro-CT imaging 1 day pre-HDD (day -1), 1 day pre- monoclonal antibody administration (day 6 post-HDD), and 6 days post-administration (day 13 post-HDD). Quantification of fat mass (left) and lean body mass (right) is shown for control monoclonal antibody (N = 14) and H4H17319P2 (N = 14) administered groups (gray and black bars, respectively). Figure 9C . shows body composition analysis 6 days post-treatment (day 13) in induced leptin deficient Lepr hu / hu mice treated with a single dose of control monoclonal antibody (gray bars, N = 14) or H4H17319P2 (black bars, N = 14).

[0067] Figures 10A-10D . Figure 10A . shows a schematic of the gene targeted for production of a leptin receptor extracellular domain humanized Lepr hu / hu mice.Figure 10B Body weight (9 weeks of age) and micro-CT quantification of body composition (9 to 12 weeks of age) of male wild-type (Lepr + / + , gray bars) and Lepr hu / hu (black bars) mice. Data are mean ± SEM. N = 6-10 per group. Figure 10C Insulin tolerance test (0.75 U / kg, i.p.) of male Lepr + / + (gray circles) and Lepr hu / hu (black circles) mice, 8 to 11 weeks of age. Data are mean ± SEM. N = 8-9 per group. Figure 10D Serum leptin levels in male Lepr + / + (gray bars) and Lepr hu / hu (black bars) mice, 9 to 13 weeks of age. Data are mean ± SEM. N = 8-9 per group.

[0068] Figures 11A-11B . Figure 11A Body weight (left), percent change in body weight from baseline at day 0 (middle), and cumulative food intake (right) in 8-week-old male C57BL / 6N mice following hydrodynamic DNA delivery (HDD) of a plasmid encoding the extracellular domain of the mouse leptin receptor (mLeprECD.hFc, black circles) or a control plasmid (control hFc, gray circles) at day 0. Data are mean ± SEM. N = 6 per group. Figure 11B Micro-CT quantification of body composition 7 days after HDD of a plasmid encoding the extracellular domain of the mouse leptin receptor (mLeprECD.hFc, black bars) or a control plasmid (control hFc, gray bars). Data are mean ± SEM for fat mass, lean body mass, bone mass, bone mineral content, and bone density (left to right). N = 6 per group.

[0069] Figures 12A-12C At day 0, 17- to 20-week-old female or 18-week-old male Lepr hu / huHDD in mice to induce leptin deficiency. Seven days after HDD, mice were stratified into 2 groups of animals according to relative percent change in body weight, respectively, and then administered a single 10 mg / kg subcutaneous dose of control monoclonal antibody (gray open circles or bars) or H4H17319P2 (black open circles or bars). Data are mean ± SEM. *, P < 0.05 for H4H17319P2 vs. control monoclonal antibody at the indicated time point. $, P < 0.05 between pre-HDD (Day -1) and any day post-HDD (Day 6 or 13) in the same respective administered group. &, P < 0.05 between pre-monoclonal antibody administration (Day 6) and post-monoclonal antibody administration (Day 13) in the same respective administered group. No significant difference (ns) from Day 0 baseline for groups represented in the respective text color at the indicated date. Figure 12A . Body weight (left) and food intake (right) in female mice throughout the study, which shows that induction of leptin deficiency results in rapid weight gain and hyperphagia. Body weight continued to increase after control monoclonal antibody administration (N = 14). N = 10-11 per group. Figure 12B . Body composition analysis by micro-CT imaging in female mice 1 day pre-HDD (Day -1), 1 day pre-monoclonal antibody administration (Day 6 post-HDD), and 6 days post-administration (Day 13 post-HDD). Data are mean ± SEM for fat mass, lean body mass, bone mass, bone mineral content, and bone density (left to right). N = 10-11 per group. Figure 12C . Body composition analysis by micro-CT imaging in male mice 1 day pre-HDD (Day -1), 1 day pre-monoclonal antibody administration (Day 6 post-HDD), and 6 days post-administration (Day 13 post-HDD). Data are mean ± SEM for bone mass, bone mineral content, and bone density (left to right). N = 14 per group.

[0070] Figures 13A-13D . On Day 0, 17- to 20-week-old female Lepr hu / huHDD of mLepr.ECD in mice to induce leptin deficiency. For comparison, a group of mice was subjected to HDD of control vector. Seven days after HDD, mice receiving mLepr.ECD were stratified into 3 groups by body weight and administered two (days 7 and 13) 3 mg / kg subcutaneous doses of control monoclonal antibody (gray solid circles or bars) or H4H17319P2 (dark gray solid circles or bars), or pair-fed (light gray solid circles or bars) with the amount of food consumed by the leptin deficiency-induced mice treated with H4H17319P2. Data are mean ± SEM. *, P < 0.05 relative to HDD mLeprECD administration of control monoclonal antibody at the indicated time point for the corresponding group represented in the symbol color or bar. #, statistical significance of HDD mLepr ECD treatment with H4H17319P2 versus pair-feeding of HDD mLeprECD animals. $, P < 0.05 relative to HDD control mice administered control monoclonal antibody at the indicated time point for the corresponding group represented in the symbol color or bar. Figure 13A Body weight change (left) and cumulative food intake (right) in mice relative to pre-HDD are shown. N = 6-11 per group. Figure 13B Lepr hu / hu Micro-CT quantification of body composition in mice. Fat mass, lean mass, bone mass, bone mineral content, and bone density (left to right). N = 5-10 per group. Figure 13C Insulin tolerance test at day 10 (3 days post-treatment) after HDD in mice fasted 4h prior to 0 min insulin treatment (1.0 U / kg, i.p.) is shown. Blood glucose levels and glucose area under the curve (AUC) of the insulin tolerance test are shown (left and right panels, respectively). N = 6-11 per group. Figure 13D Chemical analysis of plasma lipids obtained at day 16 or 17 is provided. N = 6-11 per group.

[0071] Figures 14A-14G Male lipodystrophic aP2-nSrebp1c Tg / + ; Lepr hu / hu Mice (Tg) were given once at 10 mg / kg (s.c.) control monoclonal antibody (gray circles or bars) or H4H17319P2 (black circles or bars). As a reference, male non-transgenic Lepr hu / huMice (non-Tg) were given a control monoclonal antibody once at 10 mg / kg (s.c.). All data are mean ± SEM. *, P < 0.05 for the corresponding group represented by symbol color or bar relative to non-Tg mice given control monoclonal antibody at the indicated time point. #, P < 0.05 for the corresponding group represented by symbol color or bar relative to Tg mice given control monoclonal antibody at the indicated time point. $, P < 0.05 for the corresponding group at day 27 versus day -5. Figure 14A The left and right panels show body weight and cumulative food intake, respectively. N = 8-9 per group. Figure 14B The left and right panels show lean mass and fat mass, respectively, which were quantified by micro-CT imaging at day -5 before administration and at day 27 after treatment. N = 9 per group. Figure 14C Blood glucose levels throughout the study (left) and percentage of hemoglobin Alc levels at day 28 (right) are provided. N = 9 per group. Figure 14D Blood glucose levels and area under the glucose curve (AUC) from the insulin tolerance test (0.5 U / kg i.p.) at day 23 are provided. N = 9 per group. Figure 14E and 14F showed a decrease in circulating lipids (E) and liver enzyme levels (F) in H4H17319P2-treated lipodystrophic Lepr hu / hu (Tg) mice. Chemical analysis of plasma for lipid (triglycerides, cholesterol, LDL-C and HDL-C) and liver enzyme levels (alanine aminotransferase ALT and aspartate aminotransferase AST) obtained at day 28. N = 9 per group. Figure 14G Liver weight, triglyceride content and representative hematoxylin and eosin-stained liver sections (left, middle and right panels, respectively) from livers harvested at day 30 are provided. N = 5 per group.

[0072] Figures 15A-15C . Phenotypic characterization of male non-Tg Lepr hu / hu (black open circles or black bars) and aP2-nSrebp1c Tg / + Lepr hu / hu mice (black open circles or black bars). Data are mean ± SEM. *, P < 0.05 at the corresponding time point relative to Lepr hu / hu mice. Figure 15A . Left panel, body weight at 12 to 24 weeks of age. Middle panel, fat mass quantified by micro-CT imaging at approximately 15 to 20 weeks of age. Right panel, plasma leptin levels measured at 19 to 21 weeks of age. N = 17-28 per group. Figure 15BBlood glucose levels (0.75 U / kg insulin, intraperitoneal) during insulin tolerance testing at 18 to 20 weeks of age. N = 12-13 per group. Figure 15C From left to right, these represent plasma levels of triglycerides, cholesterol, LDL-C, and HDL-C at 15 to 17 weeks of age. N = 20 to 28 per group.

[0073] Figure 16A Administer aP2-nSrebp1c weekly to males aged 27 to 30 weeks with lipodystrophy. Tg / + Lepr hu / hu Mice (Tg) were administered either a control monoclonal antibody (gray bar) or H4H17319P2 (black bar) once daily at 10 mg / kg (subcutaneous). As a reference, 27- to 30-week-old male non-transgenic Lepr mice were additionally administered the antibody weekly. hu / hu Mice (non-Tg) were administered a single dose of control monoclonal antibody (white bar) at 10 mg / kg (subcutaneously). All data are mean ± SEM. * P < 0.05 for the corresponding group compared to non-Tg mice that received control monoclonal antibody at the specified time point. # P < 0.05 for the corresponding group compared to Tg mice that received control monoclonal antibody at the specified time point. $ P < 0.05 for the corresponding group on day 27 versus day -5. Figure 16A Body composition quantified by mini-CT imaging before administration on day -5 and after treatment on day 27. The figure shows bone mass, bone mineral content, and bone density (from left to right). N=9 per group.

[0074] Figures 17A-17E : Figure 17A The data in the article refers to male lipodystrophy aP2-nSrebp1c Tg / + Lepr hu / hu Mice (Tg) were 32- to 38-week-old lipodystrophy (Tg) mice that received a single dose (10 mg / kg subcutaneously) of either the control (gray bar) or H4H17319P2 (dark gray bar) or a leptin infusion (30 g / day subcutaneously; black bar). Figures 17B-17E The data showed that 27- to 30-week-old male Tg mice were given 10 mg / kg (subcutaneously) of control monoclonal antibody (gray circle or bar) or H4H17319P2 (dark gray circle or bar) once a week, or leptin (30 g / day subcutaneously; black bar). As shown in the figure, 27- to 30-week-old male non-transgenic Lepr mice were also given weekly. hu / huMice (non-Tg) were given a single dose of control mAb (10 mg / kg, s.c.) at 10 mg / kg (s.c.) once. All data are mean ± SEM. *, P < 0.05 for the corresponding group represented by symbol color or bar, relative to Tg mice given control mAb. #, P < 0.05 for the corresponding group represented by symbol color or bar, relative to Tg mice given H4H17319P2. Figure 17A Immunohistochemical staining of pSTAT3 Y705 in Arc at approximately -1.52 mm from the lambdoid fontanel and Vmh is provided, and the graph shows pSTAT3 Y705 in Arc and Vmh in male 32- to 38-week-old lipodystrophic (Tg) mice + Cell number was increased. Brain sections from brains harvested 3 days after a single dose of control or H4H17319P2 (10 mg / kg s.c.) or leptin infusion (30 g / day s.c.) treatment. Representative micrographs and quantification of pSTAT3 Y705 in Arc and Vmh are shown + Quantification of cell number (left, middle, and right panels, respectively). N = 4-5 per group. Figure 17B The left panel provides blood glucose levels during the study. The middle and right panels, blood glucose levels and area under the curve for glucose levels during the insulin tolerance test on day 9. N = 8-9 per group. Figure 17C Body weight (left panel), body weight change (middle panel), and cumulative food intake (right panel) are provided. N = 8-9 Tg mice per group. For non-Tg mice, N = 5. Figure 17D Chemical analysis of triglycerides, cholesterol, LDL-C, and HDL-C of plasma obtained on day 13 is provided, and the graph shows reduction in plasma triglycerides and cholesterol after treatment with H4H17319P2. N = 8-9 Tg mice per group. For non-Tg mice, N = 5. Figure 17E Liver mass (left panel) and liver triglyceride content (right panel) of livers obtained on day 14 are provided. N = 6-9 Tg mice per group. For non-Tg mice, N = 5.

[0075] Figures 18A-18D : Figure 18A and 18B Data shown in FIGS. 1-6 are for 32-week-old female Lepr hu / hu mice administered a single dose of control mAb (10 mg / kg, s.c.; gray filled circles), H4H17319P2 (3 mg / kg, s.c.; open circles), or H4H17319P2 (10 mg / kg; black filled circles). Figure 18CData shown in the graph are for lean male and female cynomolgus monkeys administered a single dose of control (subcutaneous; gray solid circles), H4H17319P2 (3 mg / kg, subcutaneous; open circles), or H4H17319P2 (10 mg / kg; black solid circles). Figure 18D Data shown in the graph are for high body fat (approximately 6.0 kg) male and female cynomolgus monkeys administered two doses of control (intravenous; gray solid circles) or H4H17319P2 (30 mg / kg, intravenous; black solid circles). Data are mean ± SEM. *, P < 0.05 at the indicated time point for H4H17319P2 (10 mg / kg) vs. the corresponding control. #, P < 0.05 at the indicated time point for H4H17319P2 (3 mg / kg) vs. the corresponding control. *, P < 0.05 at the indicated time point for H4H17319P2 (3 mg / kg) vs. H4H17319P2 (10 mg / kg). &, P < 0.05 for H4H17319P2 (30 mg / kg, intravenous) vs. the corresponding control. Figure 18A Percent body weight change (left panel) and food intake (right panel) prior to Day 0 of the study in lean female mice are provided. N = 6-7 per group. Figure 18B Quantitative NMR analysis is demonstrated, and the graph shows percent change in fat mass (left panel) and lean body mass (right panel) prior to Day 0 of the study in lean female mice. N = 6-7 per group. Figure 18C Percent body weight change prior to Day -1 of the study in lean cynomolgus monkeys is demonstrated. N = 12 per group. Figure 18D Percent body weight change (left panel) and percent fat mass change (middle panel) and percent lean body mass change (right panel) from the mean values on Day -14, Day -7, and Day -1 prior to dosing as quantified by DEXA (dual-energy X-ray absorptiometry for measuring body composition) during the study in high body fat cynomolgus monkeys are demonstrated. N = 4 and 8 for the control and H4H17319P2 dosing groups, respectively.

[0076] Figure 19 A single patient protocol employed in a compassionate use clinical trial is described.

[0077] Figures 20A-20B A table providing the schedule of assessments for patients receiving H4H17319P2 treatment in Treatment Period 1 (A) and Treatment Period 2 and Extension Treatment Period (B) is provided.

[0078] Figure 21 A-21C demonstrates the effect of H4H17319P2 on blood glucose, body weight, and food intake in a murine model of congenital leptin deficiency. Figure 21A provides blood glucose in mg / dL, Figure 21 B provides body weight in grams, and Figure 21 C provides cumulative food intake in grams. Gray circles, Lepr P Control Lepr hu / hu (N=5). Black squares, Lepr P Control Lepr hu / hu Lep - / - (N=7). White squares, Lepr hu / hu Lep - / - (N=8). Data are presented as mean ± SEM. *, for Lepr hu / hu Lep - / - + H4H17319P2 group compared to Lepr hu / hu , Lep - / - + IgG4 P Control, P<.05. Two mice were excluded from food intake assessment in the Lepr hu / hu Lep - / - + IgG4 P Control group and one mouse was excluded from the Lepr hu / hu Lep - / - + H4H17319P2 group.

[0079] Figure 22 A-22C demonstrates fat mass, bone mass, and lean body mass quantified by uCT body composition analysis in a mouse model of congenital leptin deficiency. Figure 22 A provides fat mass, Figure 22 B provides bone mass, and Figure 22 C provides lean mass in grams. Mice were scanned at baseline prior to the start of the study on D-5. Post-mAb scans were performed on day 35 of the study. Gray bars, Lepr P Control Lepr hu / hu (N=5). Black bars, Lepr P Control Lepr hu / hu Lep - / - (N=7). White bars, Lepr hu / hu Lep - / -(N=8). Mice were subcutaneously injected weekly with 10 mg / kg H4H17319P2 or an isotype control antibody. Data are expressed as mean ± SEM. *, P < .05 relative to baseline; #, P < .05 relative to Lepr at the corresponding time point. hu / hu +IgG4 P For control groups, P < 0.05; for positive control groups, P < 0.05 at the corresponding time points relative to group H4H17319P2. Statistical analysis was performed using a two-way ANOVA with Tuki post-hoc test.

[0080] Figure 23 A-23C demonstrates the effects of H4H17319P2 on blood glucose, body weight, and food intake in a murine model with congenital leptin receptor deficiency. Figure 23 A provides blood glucose levels in mg / dL. Figure 23 B provides weight in grams, and Figure 23 C provides cumulative food intake in grams. Gray circle, administration of IgG4. P Lepr (comparison) hu / hu (N=7-8). Black square, administered with IgG4. P Lepr (comparison) A409E / A409E (N = 9-10). White square, applied with Lepr H4H17319P2. A409E / A409E (N=10). Data are expressed as mean ± SEM. * For Lepr data using a mixed-effects model with post-hoc Sidak test, ... A409E / A409E +H4H17319P2 group and Lepr A409E / A409E +IgG4 P Control, P < .05. One mouse was excluded from Lepr for food intake assessment due to death during the study period. hu / hu +IgG4 P In addition to the control group, one mouse was excluded from Lepr. A409E / A409E +IgG4 P Outside the control group. One mouse was excluded from Lepr because it chewed too much food during the study, affecting its food intake assessment. hu / hu +IgG4 P In addition to the control group, three mice were excluded from Lepr. A409E / A409E +IgG4 P Outside of the control group.

[0081] Figure 24 A-24C demonstrates the quantification of fat mass, bone mass, and lean body mass via uCT body composition analysis in a mouse model with congenital leptin receptor deficiency. Figure 24 A provides fat quality, Figure 24B provides the bone mass, and Figure 24 C provides the lean mass in grams. Mice were scanned at baseline on D-1 prior to study initiation. Post-mAb scans were performed on day 41 of the study. Gray bars, Lepr P Control Lepr hu / hu (N = 7-8). Black bars, Lepr P Control Lepr A409E / A409E (N = 9-10). White bars, Lepr A409E / A409E (N = 10). Mice were injected subcutaneously with 10 mg / kg H4H17319P2 or isotype control (IgG4 P ) antibody weekly. Data are presented as mean ± SEM. *, P <.05 vs. baseline; #, P <.05 vs. Lepr hu / hu + IgG4 P control at corresponding time point; +, P <.05 vs. Lepr A409E / A409E + H4H17319P2 at corresponding time point. All statistical analyses were performed using mixed effect models with Sidak’s post-hoc test.

[0082] Figure 25 An event schedule for Part A cohort of the first-in-human clinical trial is depicted and includes screening, treatment, and follow-up visit procedures to be performed at each visit.

[0083] Figure 26 An event schedule for Part B cohort of the first-in-human clinical trial is depicted and includes prescreening, screening, and baseline determination procedures to be performed at each visit.

[0084] Figure 27 A second event schedule for Part B cohort of the first-in-human clinical trial is depicted and includes treatment and follow-up procedures to be performed at each visit. DETAILED DESCRIPTION

[0085] Leptin is an adipose tissue hormone that governs energy balance as well as metabolic and neuroendocrine function (Flak and Myers, Mol Endocrinol. 2016; 30:3-12; Zhang et al., Nature. 1994; 372:425-432). In states of energy deficit, low circulating leptin levels drive 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 that differ in C-terminal sequence (Baumann et al., 1996). Among these splice isoforms, LEPR-b is the predominant isoform that mediates leptin action and is the only isoform that stimulates JAK-STAT signaling (Baumann et al., 1996; Tartaglia et al., 1995; White and Tartaglia, 1996). Neurons in the brain that express LEPR-b are the primary targets and mediators of the effects of leptin on energy, metabolic, and neuroendocrine homeostasis. This is supported by the observation that Lepr-b in Lepr db / db Selective neuronal expression in mice rescues obesity, diabetes, and reproductive phenotypes (de Luca et al., 2005). In addition, genetic deletion of Lepr from neurons phenocopies Lepr db / dbObesity and hyperglycemic phenotypes in animals (Cohen et al., 2001). Leptin deficiency in mice due to loss-of-function mutations in the Lep gene leads to hyperphagia, obesity, insulin resistance, dyslipidemia, and impaired neuroendocrine function, which can be 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, reversed obesity and metabolic and reproductive dysfunction in monogenic obese patients due to leptin deficiency (Farooqi et al., 1999; Farooqi et al., 2002). Similar to primary leptin deficiency, the disease states of secondary hypoleptinemia are associated with glucose and lipid metabolic dysfunction, which can be reversed by leptin treatment. Congenital and acquired total lipodystrophy are rare and severe diseases characterized by almost complete loss of adipose tissue depots (Brown et al., 2016; Patni and Garg, 2015). Very low circulating leptin levels in these patients lead to states 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 to 1000 mg / dL, is acute and recurrent pancreatitis (Yadav and Pitchumoni 2003), which can be life-threatening and has a mortality rate of over 40% with complications such as infection or organ failure (UK Guidelines 2005). Ectopic lipid deposition in the liver (hepatic steatosis) can lead to steatohepatitis, which is characterized by fat accumulation, cell damage, and liver inflammation, and is one of the most common causes of cirrhosis (El-Zayadi 2008, Federico 2006, and Festi 2004).

[0086] In Tg-aP2-nSrebp1c mice that develop almost complete loss of adipose depots characterized by total lipodystrophy, leptin treatment reduced hyperphagia and improved dyslipidemia, hepatic steatosis, and glycemic control (Shimomura et al., 1999; Shimomura et al., 1998). Metreleptin reduced metabolic dysfunction in patients with total lipodystrophy (Oral et al., 2002), but is not approved for the treatment of patients with partial lipodystrophy (Ajluni et al., 2016).

[0087] Fatty dystrophy patients often experience other serious comorbidities, such as chronic kidney disease, cardiovascular complications, autoimmune diseases, and peripheral T-cell lymphoma, acute lymphoblastic leukemia, and Hodgkin lymphoma.

[0088] Congenital leptin deficiency patients appear in the first months of life, with rapid weight gain and immune abnormalities, with a significantly increased risk of death in the first and second decades 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.).While no therapy for congenital leptin deficiency is approved, in several small open-label studies in patients with monogenic obesity due to loss-of-function mutations in leptin, leptin therapy significantly reduced appetite, body weight, adiposity, metabolic abnormalities, the gap between bone age and chronological age, hormonal abnormalities, 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 morbid 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 reports have shown that leptin therapy results in rapid and sustained increases in plasma thyroid hormone levels, promoting timely pubertal development, and improving the number of circulating CD4(+) T cells and 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, the development of anti- metreleptin antibodies with neutralizing activity precludes the patient from any targeted therapeutic options. 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 using a leptin receptor agonist, such as H4H17319P2 (see WO2017 / 66204), to treat patients with congenital leptin deficiency.

[0089] Provided herein are agonist monoclonal antibodies that activate human LEPR with similar potency as leptin. Monoclonal antibody-mediated activation of LEPR is effective in reversing severe weight gain and metabolic dysfunction in mouse models of both primary and secondary leptin deficiency disorders. In addition, LEPR agonist monoclonal antibodies reduce the propensity for obesity and body weight in normal weight mice, as well as in normal and high body fat non-human primates, and stimulate LEPR in the presence of circulating leptin.

[0090] It should be understood that this disclosure is not limited to the particular methodology or experimental conditions described, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the disclosure will be limited only by the appended claims.

[0091] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value can vary from the recited value by not more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0092] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are now described.

[0093] Definitions

[0094] As used herein, the expressions "leptin receptor," "LEPR," and the like refer to the human leptin receptor, which comprises an amino acid sequence as 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 expression "LEPR" includes both monomeric LEPR molecules and multimeric (e.g., dimeric) LEPR molecules. As used herein, the expression "monomeric human LEPR" means a LEPR protein or portion thereof that does not contain or have any multimerization domain and exists as a single LEPR molecule (not directly physically linked to another LEPR molecule) under normal conditions. An exemplary monomeric LEPR molecule is the molecule referred to herein as "hLEPR.mmh" comprising an amino acid sequence of SEQ ID NO: 114 (see, e.g., Example 3 herein). As used herein, the expression "dimeric human LEPR" means a construct comprising two LEPR molecules that are linked to each other by a linker, a covalent bond, a non-covalent bond, or by a multimerization domain such as an antibody Fc domain. An exemplary dimeric LEPR molecule is the molecule referred to herein as "hLEPR.mFc" comprising an amino acid sequence of SEQ ID NO: 115 (see, e.g., Example 3 herein), or the molecule referred to herein as "hLEPR.hFc" comprising an amino acid sequence of SEQ ID NO: 116. As used herein, unless specifically indicated otherwise, expressions such as "anti-LEPR antibody," "antibody that specifically binds to LEPR," "LEPR-specific binding protein," and the like refer to molecules that bind to full-length human LEPR, monomeric human LEPR, dimeric human LEPR, or other constructs comprising or consisting of a LEPR extracellular domain.

[0095] Unless explicitly stated to be derived from a non-human species, all references to proteins, peptides, and protein fragments herein are intended to refer to the human form of the corresponding protein, peptide, or protein fragment. Therefore, unless a non-human species is specified, such as “mouse LEPR,” “monkey LEPR,” etc., the expression “LEPR” refers to human LEPR.

[0096] As used herein, the term "cell surface expressed LEPR" means one or more LEPR proteins or their extracellular domains expressed on the surface of cells in vitro or in vivo such that at least a portion of the LEPR protein is exposed to the extracellular space of the cell membrane and is accessible to the antigen-binding portion of an antibody. "Cell surface expressed LEPR" can include or consist of LEPR proteins expressed on the surface of cells that normally (e.g., in their natural or wild-type state) express LEPR proteins. Alternatively, "cell surface expressed LEPR" can include or consist of LEPR proteins expressed on the surface of cells that normally do not express human LEPR but have been engineered to express LEPR on their surface.

[0097] As used herein, expressions such as “anti-LEPR antibody” or “antibody that binds to human leptin receptor” include both monovalent antibodies with single specificity and 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 listed in Table 1 herein.

[0098] As used herein, the term "antibody" means any antigen-binding molecule or molecular complex containing at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., LEPR). The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains (i.e., two heavy (H) chains and two light (L) chains interconnected by disulfide bonds), and their multimers (e.g., IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or V). H ) and the heavy-chain constant region. The heavy-chain constant region contains three structural domains: C H 1. C H 2 and C H 3. Each light chain contains a light chain variable region (abbreviated as LCVR or V in this document). L The light chain constant region contains a structural domain (C) and a light chain constant region. L 1). V H District and V L The region can be further subdivided into highly variable regions (called complementarity-determining regions (CDRs)), interspersed with more conservative regions (called framing regions (FRs)). Each VH and V L Comprises three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the application, the FRs of the anti-LEPR antibody (or antigen binding portion thereof) can be identical to the human germline sequence, or can be naturally or artificially modified. An amino acid consensus sequence can be defined according to a parallel analysis of two or more CDRs.

[0099] As used herein, the term "antibody" also includes antigen-binding fragments of full antibody molecules. As used herein, the term "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody can be derived from full antibody molecules for example, using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains, and optionally, antibody constant domains. Such DNA is known and / or is readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The 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 into a suitable configuration, or to introduce codons, create disulfide bond residues, modify, add or delete amino acids, etc.

[0100] 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 the amino acid residues that mimic the 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. Also encompassed within the expression "antigen-binding fragment," as used herein, are other engineered molecules, such as 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 shark variable IgNAR domains.

[0101] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain can be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In LDomain-associated V H In antigen-binding fragments of domains, V H Domain and V L The domains can be positioned relative to one another in any suitable arrangement. For example, the variable region can be dimeric and contain V H -V H , V H -V L or V L -V L dimers. Alternatively, an antigen-binding fragment of an antibody can contain monomeric V H or V L domains.

[0102] In certain embodiments, an antigen-binding fragment of an antibody can contain at least one variable domain covalently attached to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that can be present within an antigen-binding fragment of an antibody of the application include: (i) 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 -C H 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 LIn any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can be either directly connected to one another or can be connected by a complete or partial hinge or linker region. A hinge region can consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that create a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of the antibodies of the application can comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above that are non-covalently associated (e.g., by one or more disulfide bonds). H domains or V L domains non-covalently associated (e.g., by one or more disulfide bonds).

[0103] As with full antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitide on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of antigen-binding fragments of the antibodies of the application using routine techniques available in the art.

[0104] In certain embodiments of the application, the anti-LEPR antibodies of the application are human antibodies. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived in

[0105] In some embodiments, the antibodies of the present application can be recombinant human antibodies. As used herein, the term "recombinant human antibody" is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, as described further herein below, antibodies isolated from a recombinant, combinatorial human antibody library (also described further herein below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to 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, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V H and V L regions of the recombinant antibodies are sequences that, while derived from and related to human germline V H and V L sequences, can not naturally exist within the human antibody germline repertoire in vivo.

[0106] The present application encompasses antibodies having one or more mutations in the hinge, C H 2 or C H 3 region, which can be desirable, for example, in production to improve the yield of the desired antibody format.

[0107] The antibodies of the present application can be isolated antibodies. As used herein, "isolated antibody" means 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 exists or is naturally produced, is an "isolated antibody" for the purposes of the present application. Isolated antibody also includes an antibody in situ within a recombinant cell. An isolated antibody is one that has been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody can be substantially free of other cellular material and / or chemicals.

[0108] The present application includes variants of the anti-LEPR antibodies disclosed herein that comprise 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 antibodies were derived. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The present application includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein in which one or more amino acid mutations within one or more framework and / or CDR regions are to the corresponding residue of the germline sequence from which the antibody was derived, or to the corresponding residue of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are referred to herein collectively as "germline mutations"). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments that comprise one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V L all of the framework and / or CDR residues within the variable domain are mutated back to the residues present in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues present within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues present within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to one or more corresponding residues of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present application can contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue(s) of a different germline sequence. Once antibodies and antigen-binding fragments containing one or more germline mutations are obtained, the antibodies and antigen-binding fragments can be readily tested for one or more desired property, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties as the case can be, reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed by the present application.

[0109] The present application includes anti-LEPR antibodies and antigen-binding fragments thereof comprising an amino acid sequence that is substantially similar or substantially identical to one or more variable domain or CDR amino acid sequences present in any of the exemplary anti-LEPR antibodies disclosed herein.

[0110] The terms "substantial similarity" or "substantially similar" when applied to polypeptides means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, the differences at non-identical residue positions are conservative amino acid substitutions. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a side chain (R group) of similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not significantly alter the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity can be adjusted upwards to correct for the conservative nature of the variations. Means for making this adjustment are well known in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331. Examples of groups of amino acids that have side chains of 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, i.e., cysteine and methionine. Preferred conservative amino acids substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic- aspartic acid, and asparagine-glutamine. Alternatively, conservative substitutions are any changes in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445 that have positive values. "Moderately conservative" substitutions are any changes in the PAM250 log-likelihood matrix that have non-negative values.

[0111] Sequence similarity for polypeptides, also called sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity that take into account various substitutions, deletions and other modifications among the matched sequences. For instance, GCG software contains programs such as Gap and Bestfit that 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 a mutant version of that protein. See, e.g., GCG Version 6.1. Polypeptide sequences can also be compared using FASTA (a program in GCG Version 6.1) using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the application to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using 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.

[0112] As used herein, the term "subject" refers to an animal, preferably a mammal, more preferably a human, in need of improvement, prevention, and / or treatment of a disease or disorder associated with leptin deficiency. The subject can be an adult or a child. The subject can have a metabolic dysfunction, such as a systemic or localized lipodystrophy. The subject can have congenital leptin deficiency or acquired leptin deficiency. Subjects with congenital leptin deficiency include subjects with genetic mutations that result in low or even absent levels of circulating leptin or circulating but biologically inactive leptin. The subject can have one or more symptoms associated with leptin deficiency. As used herein, the term "subject" is interchangeable with the term "patient." In some aspects, the subject has neutralizing antibodies against metreleptin.

[0113] As used herein, the term "treatment" refers to a reduction or amelioration in the severity of at least one symptom of leptin deficiency resulting from the administration of a therapeutic agent, such as an antibody of the application, to a subject in need thereof. The term includes inhibition of the progression of the associated disease or the worsening of the conditions or symptoms associated with the disease. The term also includes active prognosis of the disease, i.e., the subject can be free of symptoms after administration of the therapeutic agent, such as an antibody of the application. Active prognosis can include remission of any of the following conditions: hyperphagia, hyperglycemia, insulin resistance, dyslipidemia, or hepatic steatosis.

[0114] The term "prevent," "preventing," or "prevention" refers to the inhibition of the manifestation of any symptom, disorder, or indication associated with leptin deficiency.

[0115] A therapeutic agent can be administered to a subject at a therapeutic dose. The phrase "therapeutically effective amount" means an amount that produces the effects for which it is administered. The exact 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 is discussed in greater detail herein.

[0116] Anti-LEPR antibodies comprising Fc variants

[0117] According to certain embodiments of the application, there are provided anti-LEPR antibodies comprising an Fc domain comprising one or more mutations that, for example, enhance or reduce binding of the antibody to the FcRn receptor at acidic pH compared to neutral pH. For example, the application includes anti-LEPR antibodies comprising an Fc domain having one or more mutations at the C H 2 or C H3. In the 3-region, one or more mutations that increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in endosomes with a pH ranging from about 5.5 to about 6.0). Such mutations can result in an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q); positions 250 and 428 (e.g., L or F); position 252 (e.g., L / Y / F / W or T), position 254 (e.g., S or T), and position 256 (e.g., S / R / Q / E / D or T); or modifications 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 modifications at positions 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P).

[0118] For example, the present application includes anti-LEPR antibodies comprising an Fc domain comprising one or more pairs or one or more sets 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 within the antibody variable domains disclosed herein are contemplated within the scope of the present application.

[0119] The anti-LEPR antibodies of the present application can comprise a modified Fc domain having reduced effector function. As used herein, "modified Fc domain having reduced effector function" means any Fc portion of an immunoglobulin that has been modified, mutated, truncated, and the like, relative to a wild-type naturally-occurring Fc domain, such that a molecule comprising the modified Fc exhibits a reduction in the severity or extent of at least one of the effects 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 the wild-type naturally-occurring version of the Fc portion. In certain embodiments, a "modified Fc domain having reduced effector function" is an Fc domain having reduced or diminished binding to an Fc receptor (e.g., FcyR).

[0120] In certain embodiments of the present application, the modified Fc domain is a variant IgGl Fc or a variant IgG4 Fc comprising substitutions in the hinge region. For example, a modified Fc for use in the context of the present application can comprise a variant IgGl Fc in which at least one amino acid of the IgGl Fc hinge region is replaced with the corresponding amino acid from the IgG2 Fc hinge region. Alternatively, a modified Fc for use in the present application can comprise a variant IgG4 Fc in which at least one amino acid of the IgG4 Fc hinge region is replaced with the corresponding amino acid from the IgG2 Fc hinge region. Non-limiting, exemplary modified Fc regions that can be used in the context of the present application are set forth in U.S. Patent Application Publication No. 2014 / 0243504.

[0121] Other modified Fc domains and Fc modifications that can be used in the context of the present application include any of the modifications set forth in US 2014 / 0171623; US 8,697,396; US 2014 / 0134162; WO 2014 / 043361. Methods of constructing antibodies or other antigen-binding fusion proteins comprising a modified Fc domain as described herein are known in the art.

[0122] Biological characteristics of antibodies

[0123] The present invention includes antibodies and antigen-binding fragments thereof that bind human LEPR and activate LEPR signaling. Such antibodies can be referred to herein as "agonist antibodies." In the context of the present invention, "activation of LEPR signaling" means stimulation of intracellular effects that would normally result from the interaction of leptin with LEPR in a cell expressing LEPR. In certain embodiments, "activation of LEPR signaling" means transcriptional activation of STAT3, which can be detected using any method that can measure or identify STAT3 activity directly or indirectly, for example, using a tagged version of STAT3 expressed in a reporter cell line. For example, the present invention includes antibodies and antigen-binding fragments thereof that activate LEPR signaling in a cell-based reporter assay, for example, using a cell-based assay format as defined in Example 7 herein, or a substantially similar assay. Cell-based reporter assays that detect LEPR activation, such as the assay shown in Example 7 herein, can produce a detectable signal that can be expressed in EC 50 values (i.e., the concentration of antibody required to produce half the maximum signaling) and / or the percentage of maximum signaling observed in the presence of leptin. In certain exemplary embodiments of the present invention, anti-LEPR antibodies are provided that activate LEPR signaling with an EC 50 values (i.e., the concentration of antibody required to produce half the maximum signaling) and / or the percentage of maximum signaling observed in the presence of leptin. In certain exemplary embodiments of the present invention, anti-LEPR antibodies are provided that activate LEPR signaling with an EC

[0124] The present invention includes antibodies and antigen-binding fragments thereof that bind monomeric human LEPR with high affinity. For example, the present invention includes anti-LEPR antibodies that bind monomeric human LEPR (e.g., hLEPR.mmh, SEQ ID NO: 114) with a KD of less than about 150 nM, the K Dat 25°C or 37°C, e.g., using the assay format defined in Example 3 herein, or a substantially similar assay. According to certain embodiments, anti-LEPR antibodies are provided that bind monomeric human LEPR (e.g., hLEPR.mmh, SEQ ID NO: 114) with a K D at 25°C or 37°C, e.g., using the assay format defined in Example 3 herein, or a substantially similar assay. According to certain embodiments, anti-LEPR antibodies are provided that bind monomeric human LEPR (e.g., hLEPR.mmh, SEQ ID NO: 114) with a K

[0125] The present application also includes antibodies and antigen-binding fragments thereof that bind monomeric human LEPR (e.g., hLEPR.mmh, SEQ ID NO: 114) with a dissociative half-life (t 1 at 25°C or 37°C, e.g., using the assay format defined in Example 3 herein, or a substantially similar assay. According to certain embodiments, anti-LEPR antibodies are provided that bind monomeric human LEPR (e.g., hLEPR.mmh, SEQ ID NO: 114) with a K 1 at 25°C or 37°C, e.g., using the assay format defined in Example 3 herein, or a substantially similar assay. According to certain embodiments, anti-LEPR antibodies are provided that bind monomeric human LEPR (e.g., hLEPR.mmh, SEQ ID NO: 114) with a K

[0126] The present application also includes antibodies and antigen-binding fragments thereof that bind dimeric human LEPR (e.g., hLEPR.mFc, SEQ ID NO: 115) with high affinity. For example, the present application includes anti-LEPR antibodies that bind dimeric human LEPR with a K DKD as measured by surface plasmon resonance, e.g., using the assay format defined in Example 3 herein, or a substantially similar assay, at 25°C or 37°C. According to certain embodiments, anti-LEPR antibodies are provided that bind dimeric human LEPR with a K D KD as measured by surface plasmon resonance, e.g., using the assay format defined in Example 3 herein, or a substantially similar assay, at 25°C or 37°C.

[0127] The present application also includes antibodies and antigen-binding fragments thereof that bind dimeric human LEPR with an off-rate (k 1 KD as measured by surface plasmon resonance, e.g., using the assay format defined in Example 3 herein, or a substantially similar assay, at 25°C or 37°C. According to certain embodiments, anti-LEPR antibodies are provided that bind dimeric human LEPR with a K 1 KD as measured by surface plasmon resonance, e.g., using the assay format defined in Example 3 herein, or a substantially similar assay, at 25°C or 37°C. According to certain embodiments, anti-LEPR antibodies are provided that bind dimeric human LEPR with a K

[0128] The present application also includes antibodies and antigen-binding fragments thereof that bind LEPR complexed with human leptin ("LEPR complexed with human leptin" can also be denoted in the expression "leptin:LEPR"). For example, the present application includes antibodies and antigen-binding fragments thereof that are capable of binding to a preformed complex comprising hLEPR and human leptin. That is, according to certain embodiments, the presence of leptin complexed with LEPR does not inhibit the interaction between an anti-LEPR antibody and LEPR; likewise, according to this aspect of the application, the presence of an anti-LEPR antibody does not inhibit the interaction between leptin and LEPR. An exemplary assay format for determining whether an antibody or antigen-binding fragment thereof binds to LEPR complexed with human leptin is set forth in Example 4 herein.

[0129] Similarly, the present application also includes antibodies and antigen-binding fragments thereof that bind LEPR and do not block LEPR:leptin interactions. For example, the present application includes antibodies and antigen-binding fragments thereof that are capable of binding LEPR to produce an antibody:LEPR complex, wherein the resulting antibody:LEPR complex is capable of interacting with leptin to produce a ternary complex comprising the antibody, LEPR, and leptin. Exemplary assay formats for determining whether an antibody or antigen-binding fragment thereof is capable of binding LEPR in a manner that does not block or interfere with interactions between LEPR and leptin are set forth in Example 5 herein.

[0130] The present application also includes antibodies and antigen-binding fragments thereof that bind cell surface-expressed LEPR in the presence and / or absence of human leptin. Cell surface-expressed LEPR means LEPR or a portion thereof (e.g., an extracellular portion of LEPR) expressed on the surface of a cell in the form of a native or engineered cell line, such that an antibody or antigen-binding fragment thereof is capable of binding to the LEPR molecule. In certain embodiments, cell surface-expressed LEPR includes a recombinant complex comprising an extracellular domain of LEPR linked to a cell via a tag or anchor (e.g., a GPI anchor as demonstrated in Example 6 herein). In accordance with this aspect of the present application, antibodies are provided that are capable of binding cell surface-expressed LEPR in the absence of leptin and are also capable of binding cell surface-expressed LEPR in the presence of leptin (i.e., in an environment where leptin is capable of binding to cell surface-expressed leptin). That is, in accordance with certain embodiments, the presence of leptin complexed with cell surface-expressed LEPR does not inhibit the interaction between an anti-LEPR antibody and cell surface-expressed LEPR. Antibodies in accordance with this aspect of the present application are capable of forming a ternary complex on the surface of a cell comprising the antibody, cell surface-expressed LEPR, and leptin. Exemplary assay formats for determining whether an antibody or antigen-binding fragment thereof is capable of binding cell surface-expressed LEPR in the presence and absence of human leptin are set forth in Example 6 herein.

[0131] Antibodies of the present application can have one or more of the foregoing biological characteristics, or any combination thereof. The foregoing list of biological characteristics of antibodies of the present application is not intended to be exhaustive. Other biological characteristics of antibodies of the present application will be apparent to those of ordinary skill in the art upon review of this disclosure, including the working examples herein.

[0132] Epitope mapping and related techniques

[0133] The present application also includes anti-LEPR antibodies comprising 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 application 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, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequence groups shown in Table 1 herein. In certain embodiments, the present application provides anti-LEPR antibodies comprising variant HCVR, LCVR, and / or CDR amino acid sequences relative to the sequences shown in Table 1 herein (e.g., comprising conservative amino acid substitutions), wherein the variant antibodies still exhibit one or more of the functions and / or properties of the exemplary anti-LEPR antibodies disclosed herein.

[0134] 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) (referred to as the leptin binding domain (LBD)). (Carpenter et al. (2012) Structure 20:487-97). In addition, LEPR shares the greatest homology with granulocyte colony-stimulating factor (GCSF) and glycoprotein 130 (gpl30) and similar extracellular domain size and organization. (Haniu et al. (1998) J Biol Chem 273(44):28691-699).

[0135] The term "epitope" refers to a determinant region of an antigen that interacts with a particular antigen-combining site (referred to as a paratope) in the variable region of an antibody molecule. A single antigen can have more than one epitope. Thus, different antibodies can bind to different regions on an antigen and can have different biological effects. Epitopes can be conformational or linear. Conformational epitopes result from the spatial arrangement of amino acids from different segments of a linear polypeptide chain. Linear epitopes result from the juxtaposition of amino acid residues on the same or adjacent stretches of a polypeptide chain. In certain instances, an epitope can include a sugar, a phosphoryl group, or a sulfonyl group moiety on the antigen.

[0136] The present disclosure includes anti-LEPR antibodies that interact with one or more epitopes present within amino acids M1-D839 (SEQ ID NO: 113) of human LEPR. As described in Example 11, 201 peptides from human LEPR had significantly reduced uptake of deuterium when bound to antibody H4H16650P2. Peptides corresponding to amino acids 162-169 (amino acids LYVLPEVL of human LEPR, SEQ ID NO: 113) and 170-191 (amino acids EDSPLVPQKGSF of human LEPR, SEQ ID NO: 113) had slower rates of deuteration when bound to H4H16650P2, indicating that this antibody binds at least two human LEPR epitopes having the sequence LYVLPEVL or EDSPLVPQKGSF (amino acids 162-169 or 170-191 of SEQ ID NO: 113, respectively).

[0137] The epitope to which an antibody of the present disclosure binds can consist of a single contiguous sequence of 3 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 can consist of multiple non-contiguous amino acids (or amino acid sequences) of LEPR. In some embodiments, the epitope is located on or near the leptin binding domain of LEPR. In other embodiments, the epitope is located in a region of LEPR that is different from the leptin binding domain, e.g., such that when an antibody binds to such an epitope on the surface of LEPR, it does not interfere with the binding of leptin to LEPR.

[0138] Amino acids within an epitope recognized by a particular antibody can be identified using various techniques known to those of ordinary skill in the art. Exemplary techniques include, e.g., alanine scanning mutational analysis, peptide blot analysis, and peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be employed (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify amino acids within a polypeptide that interact with an antibody is hydrogen / deuterium exchange detected by mass spectrometry. In general, the hydrogen / deuterium exchange method involves deuterium labeling of a protein of interest, followed by binding of the antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water to allow hydrogen-deuterium exchange to occur at all residues except those protected by the antibody (which remain deuterium-labeled). After dissociation of the antibody, protease cleavage and mass spectrometry analysis of the target protein reveals the deuterium-labeled residues, which correspond to the specific amino acids that interact with the antibody. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-ray crystallographic analysis of antibodies complexed with antigens can also be used to identify amino acids within a polypeptide that interact with an antibody.

[0139] The present application also includes anti-LEPR antibodies that bind to the same epitope as any of the particular exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences listed in Table 1 herein). Likewise, the present application also includes anti-LEPR antibodies that compete for binding to LEPR with any of the particular exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences listed in Table 1 herein).

[0140] By using routine methods known in the art and exemplified herein, it can be determined whether an antibody binds to the same epitope as, or competes for binding with, a 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 application, the reference antibody is allowed to bind to the LEPR protein. Then, the ability of the test antibody to bind to the LEPR molecule is assessed. If the test antibody is able to bind to the LEPR after saturating binding with the reference anti-LEPR antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-LEPR antibody. In another aspect, if the test antibody is not able to bind to the LEPR molecule after saturating binding with the reference anti-LEPR antibody, the test antibody can bind to the same epitope as the epitope bound by the reference anti-LEPR antibody of the application. Additional routine experimentation (e.g., peptide mutation and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is the cause of the observed lack of binding. Such experiments 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 application, 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 the binding of the other by at least 50%, but preferably 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. 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, the two antibodies are considered to have an “overlapping epitope.”

[0141] To determine whether an antibody competes for binding (or cross-competes for binding) with a reference anti-LEPR antibody, the binding method described above is performed in two directions: In the first direction, the reference antibody is bound to the LEPR protein under saturation conditions, and then the binding of the test antibody to the LEPR molecule is evaluated. In the second direction, the test antibody is bound to the LEPR molecule under saturation conditions, and then the binding of the reference antibody to the LEPR molecule is evaluated. If only the first (saturated) antibody can bind to the LEPR molecule in both directions, it can be concluded that the test antibody and the reference antibody compete for LEPR binding. As will be understood by those skilled in the art, an antibody competing for binding with the reference antibody may not necessarily bind to the same epitope as the reference antibody, but may spatially block the binding of the reference antibody through binding to overlapping or adjacent epitopes.

[0142] Preparation of human antibodies

[0143] 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 methods can be used in the context of the present invention to prepare human antibodies that specifically bind to human LEPR.

[0144] Use, for example, VELOCIMMUNE TM This technique, or any other similar known method for generating fully human monoclonal antibodies, initially isolates a high-affinity chimeric antibody against LEPR having a human variable region and a mouse constant region. As described in the Experimental Section below, desired characteristics of the antibody are characterized and selected, including affinity, ligand blocking activity, selectivity, epitopes, etc. If desired, the mouse constant region is replaced with the desired human constant region (e.g., wild-type or modified IgG1 or IgG4) to generate a fully human anti-LEPR antibody. While the selected constant region can vary depending on the specific application, the high-affinity antigen-binding and target-specific characteristics remain in the variable region. In some cases, fully human anti-LEPR antibodies are isolated directly from antigen-positive B cells.

[0145] bioequivalent

[0146] The anti-LEPR antibodies and antibody fragments of the present invention encompass proteins having an amino acid sequence that differs from that of the antibodies but which retains the ability to bind human LEPR. Such variant antibodies and antibody fragments comprise, when compared to the parent sequence, one or more additions, deletions, or substitutions of amino acids, but exhibit substantially comparable biological activity to the described antibodies. Likewise, the DNA sequences encoding the anti-LEPR antibodies of the present invention encompass sequences which comprise, when compared to the disclosed sequences, one or more additions, deletions, or substitutions of nucleotides, but which encode an anti-LEPR antibody or antibody fragment that is substantially bioequivalent to an anti-LEPR antibody or antibody fragment of the present invention. Examples of such variant amino acid and DNA sequences are discussed above.

[0147] Two antigen binding proteins or antibodies are considered bioequivalent if they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single dose or multiple doses. Certain antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption, but not in their rate of absorption, they will be considered bioequivalent, however, because of this difference in the rate of absorption is intentional and reflected in the labeling, these antibodies are not necessary for the achievement of effective in vivo drug concentration for the intended use, and are considered by the agency to have no clinical significance for the particular drug product investigated.

[0148] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0149] In one embodiment, two antigen binding proteins are bioequivalent if a patient can be switched between the reference product and the biological product once or more without an increased risk of adverse reactions expected, including a clinically significant change in immunogenicity, or reduced effectiveness, as compared to continuous therapy without such switching between the reference product and the biological product.

[0150] In one embodiment, two antigen binding proteins are bioequivalent if both act through one or more common mechanisms of action for one or more conditions of use to the extent such mechanisms are known.

[0151] Bioequivalence can be demonstrated by in vivo and in vitro methods. Bioequivalence measures include, for example, (a) in vivo testing in humans or other mammals in which the concentration of the antibody or its metabolites in blood, plasma, serum, or other biological fluid is measured over time; (b) in vitro testing that is correlated with and reasonably predicts in vivo bioavailability data in humans; (c) in vivo testing in humans or other mammals in which the appropriate acute pharmacological effects of the antibody (or its target) are measured over time; and (d) clinical trials that establish a good control for safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0152] Bioequivalent variants of the anti-LEPR antibodies of the application can be constructed, e.g., by various substitutions or deletions of terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other cases, bioequivalent antibodies can include anti-LEPR antibody variants that comprise amino acid changes that modify the glycosylation profile of the antibody, e.g., mutations that eliminate or remove glycosylation.

[0153] Species selectivity and species cross-reactivity

[0154] According to certain embodiments, the present application provides anti-LEPR antibodies that bind to human LEPR but not to LEPR of other species. The present application also includes anti-LEPR antibodies that bind to human LEPR and LEPR of one or more non-human species. For example, the anti-LEPR antibodies of the present application can bind to human LEPR and can or can not bind to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee LEPR, as the case can be. According to certain exemplary embodiments of the present application, there are provided anti-LEPR antibodies that specifically bind to human LEPR and to cynomolgus monkey (e.g., Macaca fascicularis) LEPR. Other anti-LEPR antibodies of the present application bind to human LEPR but do not bind to or only weakly bind to cynomolgus monkey LEPR.

[0155] Multispecific antibodies

[0156] Antibodies of the application can be monospecific or multispecific (e.g., bispecific). Multispecific antibodies can have specificity for different epitopes of one target polypeptide, or can contain antigen-binding domains with specificity for more than one target polypeptide. See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. Anti-LEPR antibodies of the application can be linked to or co-expressed with another functional molecule (e.g., another peptide or protein). For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) 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.

[0157] The application includes bispecific antibodies in which one arm of the immunoglobulin binds human LEPR and the other arm of the immunoglobulin has specificity for a second antigen. The LEPR-binding arm can comprise any of the HCVR / LCVR or CDR amino acid sequences listed in Table 1 herein.

[0158] Exemplary bispecific antibody formats that can be used in the context of the application involve the use of a first immunoglobulin (Ig) C H 3 domain and a second Ig C H 3 domain, wherein at least one amino acid of the first and second Ig C H 3 domains differ from each other, and wherein at least one amino acid difference reduces the binding of the bispecific antibody to protein A as compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the Ig C H 3 domain binds protein A, and the second Ig C H 3 domain contains a mutation that reduces or eliminates protein A binding, such as a H95R modification (by IMGT exon numbering; H435R by EU numbering). The second Ig C H 3 can also comprise a Y96F modification (by IMGT numbering; Y436F by EU numbering). The second Ig C HAdditional modifications in 3 include: in the case of IgGl antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU); in the case of IgG2 antibodies, N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU); and in the case of IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU). Variations of the bispecific antibody formats described above are contemplated within the scope of the application.

[0159] Other exemplary bispecific formats that can be used in the context of the application include, but are not limited to, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knob-in-hole, common light chain (e.g., common light chain with knob-in-hole, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgGl / IgG2, dual acting Fab (DAF)-IgG, and Mab 2 Bispecific formats (for a review of the foregoing formats, see, e.g., Klein et al., 2012, mAbs 4:6, 1-11, and references cited therein). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, e.g., where unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates, which then self-assemble into multimeric complexes with defined composition, valency, and geometry. (See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: December 4, 2012]).

[0160] Therapeutic formulations and administration

[0161] The present application provides pharmaceutical compositions comprising the anti-LEPR antibodies or antigen-binding fragments thereof of the present application. The pharmaceutical compositions of the present application are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in all of the reference works known to medicinal chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN TM , Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in- water and water-in-oil emulsions, emulsions carbowaxes (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0162] The dosage of antibody to be administered to a patient can vary depending on the age and size of the patient, the target disease, the condition, the route of administration, and the like. Preferred dosages are generally calculated according to body weight or body surface area. In adult patients, it can be advantageous to intravenously administer the antibodies of the present application generally in single doses 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. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. Effective doses and schedules for anti-LEPR antibody administration can be determined empirically; for example, patient progress can be monitored by periodic assessment and the dosage adjusted accordingly. Furthermore, interspecies scaling of dosages can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0163] Intravenous administration of an antibody at a therapeutically effective dose, e.g., at about 5 mg / kg body weight, or about 1 mg / kg body weight to about 20 mg / kg body weight, or about 1 mg / kg body weight to about 15 mg / kg body weight, or about 5 mg / kg body weight to about 10 mg / kg body weight, can be advantageous in patients, e.g., pediatric patients. For example, an intravenous (IV) loading dose of about 5 mg / kg can be selected to achieve an antibody serum concentration equal to or greater than 100 mg / L. Subcutaneous administration of an antibody at a dose of about 250 mg, or at a dose of about 300 mg, or at a dose of about 100 mg to about 500 mg, or about 200 mg to about 300 mg, can be additionally advantageous. For example, a subcutaneous (SC) maintenance dose of 250 mg H4H17319P2 or 300 mg H4H17319P2 per week will maintain a trough concentration in serum equal to or greater than 100 mg / L. In some aspects, the subcutaneous dosing regimen begins several days after administration of the intravenous loading dose, to best maintain the target trough concentration in serum. In some aspects, the first subcutaneous dose is administered 2 to 7 days after the loading dose, e.g., 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the loading dose. In some aspects, the subcutaneous dose is administered once every 3 to 14 days, e.g., once every 3 days, once every 4 days, once every 5 days, once every 6 days, once per week, once every 10 days, or once every 2 weeks, e.g., 3 subcutaneous doses per week after the first subcutaneous dose, followed by a monthly (about every 28 days) dose. In one embodiment of the application, a therapeutically effective dose of an antibody (e.g., H4H17319P2) is as indicated herein, but optionally continues beyond the last monthly dose indicated therein. Figure 19

[0164] In some aspects, it is desirable to maintain a trough concentration in serum of between about 50 mg / L to about 200 mg / L, or about 100 mg / L, or about 150 mg / L, or equal to or greater than 50 mg / L, or equal to or greater than 100 mg / L, or equal to or greater than 150 mg / L.

[0165] Various delivery systems are known and can be used to administer the pharmaceutical composition of the application, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant virus, receptor mediated endocytosis (see, e.g., 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 composition 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 and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local.​

[0166] The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously using standard needles and syringes. Furthermore, for subcutaneous delivery, pen-type delivery devices are readily applicable for delivering the pharmaceutical compositions of the present invention. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all 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-type delivery device can then be reused. In disposable pen-type delivery devices, there is no replaceable cartridge. In fact, disposable pen-type delivery devices have a reservoir pre-filled with the pharmaceutical composition within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0167] Various reusable pen-type delivery devices and auto-injector delivery devices are used for subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, but are not limited to, AUTOPEN. TM (Owen Mumford, Inc., Woodstock, UK), DISSETRONIC TM Pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN70 / 30 TM Pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN TM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TM (NovoNordisk,Copenhagen,Denmark)、BD TM Pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM OPTIPEN PRO TM OPTIPEN STARLET TM and OPTICLIK TM (Sanofi-Aventis, Frankfurt, Germany, etc.) Examples of disposable pen delivery devices that can be used for subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, SOLOSTAR. TM Pen (Sanofi-Aventis), FLEXPEN TMNOVOLET® (Novo Nordisk), and KWIKPEN TM EGRIFT® (Eli Lilly), SURECLICK TM AUTOPEN® (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMIRA TM PEN (Abbott Labs, Abbott Park IL), and the like.

[0168] In certain situations, the 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, polymeric materials can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed in proximity to the composition's target, 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.

[0169] Injectable preparations can include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, drip infusion, and the like. These injectable preparations can be prepared by well-known methods. For example, preparations for injection can be prepared, for example, by dissolving, suspending, or emulsifying the antibody or salt thereof described above in the sterile aqueous medium or in the oily medium conventionally used for injection. As the aqueous medium for injection, there are, for example, physiological saline, isotonic solutions containing glucose and other auxiliary agents, and the like, which can be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], and the like. As the oily medium, use is made of, for example, sesame oil, soybean oil, and the like, which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, and the like. The injection thus prepared is preferably filled in an appropriate ampoule.

[0170] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients appropriate for administration. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injection solutions (ampoules), suppositories, etc. The amount of the aforementioned antibody contained is usually about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, for other dosage forms, the amount of the aforementioned antibody contained is preferably about 5 to about 100 mg and about 10 to about 250 mg.

[0171] Therapeutic uses of antibodies

[0172] The present application includes methods comprising administering a therapeutic composition comprising an anti-LEPR antibody (e.g., an anti-LEPR antibody comprising any of the HCVR / LCVR or CDR sequences listed in Table 1 herein) to a subject in need thereof. The therapeutic composition can comprise any of the anti-LEPR antibodies or antigen-binding fragments thereof disclosed herein, and a pharmaceutically acceptable carrier or diluent.

[0173] 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 leptin deficiency, such as nonalcoholic fatty liver disease, NASH, female infertility, amenorrhea, abnormal hormone cycles, impaired immune function, 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 any disease or disorder that can be treated by in vitro or in vivo stimulation or activation of LEPR signaling or mimicking the natural activity of leptin. For example, the antibodies and antigen-binding fragments thereof can be used to treat a lipodystrophy disorder. Exemplary lipodystrophy disorders that can be treated by the antibodies and antigen-binding fragments of the present application include, for example, congenital generalized lipodystrophy, congenital partial lipodystrophy, acquired generalized lipodystrophy, familial partial lipodystrophy, acquired partial lipodystrophy, centrifugal abdominal lipodystrophy, annular lipodystrophy, partial lipodystrophy, and HIV-associated lipodystrophy, as well as symptoms associated with such disorders.

[0174] The anti-LEPR antibodies and antigen-binding fragments thereof provided herein can be used to treat, prevent, and / or ameliorate monogenic obesity and / or lipodystrophy. Monogenic obesity and lipodystrophy can be associated with a number of pathologies, including, for example: extreme early onset obesity, a subject’s BMI greater than the 85th percentile for age and gender; hyperphagia and impaired satiety, a subject exhibiting foraging and hoarding behaviors; impaired immune function with reduced CD4+ T cell counts, recurrent (potentially fatal) infections; insulin resistance and hyperinsulinemia; nonalcoholic fatty liver disease, hepatic steatosis, and progression of lipodystrophy; dyslipidemia leading to hypertriglyceridemia; diabetes with elevated HbAlc and / or glucose levels, and reduced glucose tolerance; reproductive dysfunction to hypogonadism, delayed puberty; decreased manifestation of secondary sexual characteristics, amenorrhea or oligomenorrhea, and infertility; lack of pubertal statural growth leading to short stature, abnormal growth hormone secretion; hypothyroidism or impaired thyroid function, altered T3 or TSH, or free thyroxine levels; and variable bone changes, including bone density and bone mineral content. The range of disorders and symptoms associated with monogenic obesity and / or lipodystrophy can vary depending on the underlying causative gene, e.g., AGPAT2, LMNA, BSCL2, and the like. A given mutation can result in loss of leptin or LEPR function with varying degrees of endocrine severity (e.g., oligomenorrhea versus complete amenorrhea).

[0175] The antibodies and antigen-binding fragments provided herein can also be used to treat, reduce, or prevent one or more symptoms of a disease or disorder associated with metabolic dysfunction or low leptinemia. Such symptoms include obesity predisposition, obesity, hyperphagia, hyperglycemia, hypertriglyceridemia, hypercholesterolemia, insulin resistance, dyslipidemia, growth delay, delayed pubertal statural growth, abnormal growth hormone secretion, elevated HbAlc, low bone mineral density (or low bone mass), low bone mineral content, and low lean body mass.

[0176] The present application also includes anti-LEPR antibodies and antigen-binding fragments thereof that can be used to restore leptin signaling to cells, tissues, and organs expressing one or more LEPR mutations. Such mutations can be associated with metabolic dysfunction or hypoleptinemia and diseases or conditions involving metabolic dysfunction or hypoleptinemia, such as obesity, congenital lipodystrophy, infertility, and nonalcoholic fatty liver disease. For example, certain LEPR mutants have been identified that do not exhibit or exhibit reduced signaling in the presence of leptin and are associated with obesity and related disorders. As used herein, LEPR mutants that do not exhibit signaling in the presence of leptin are referred to as “signaling-deficient LEPR mutants.” An exemplary signaling-deficient LEPR mutation is LEPR-A409E (Farooqi et al., 2007, N Engl J Med 356(3):237-247). As used herein, LEPR mutants that exhibit reduced signaling in the presence of leptin (compared to wild-type LEPR) are referred to as “signaling-impaired LEPR mutants.” An exemplary signaling-impaired LEPR mutation is LEPR-P316T (Mazen et al., 2011, Mol Genet Metab 102:461-464). Accordingly, the present application includes anti-LEPR antibodies and antigen-binding fragments thereof that can be used to treat, prevent, and / or ameliorate diseases and disorders caused by or associated with one or more signaling-deficient (e.g., A409E) and / or signaling-impaired (e.g., P316T) LEPR mutants.

[0177] The present application also includes anti-LEPR antibodies and antigen-binding fragments thereof that can be used to restore leptin signaling by mitigating mutations in the leptin gene. Some subjects have circulating leptin, but the protein is nonfunctional due to genetic mutations, e.g., p.N103K in the leptin gene, which encodes a biologically inactive form of leptin. Some subjects have little or no circulating leptin. Other genes involved in impaired leptin signaling can include LMNA, PPARG, AGPAT2, BSCL2, PLIN1, AKT2, CIDEC, LIPE, and ADRA2A, and the anti-LEPR antibodies and antigen-binding fragments thereof provided herein can be used to mitigate the effects of such mutations on leptin signaling.

[0178] The anti-LEPR antibodies and antigen-binding fragments thereof of the present application can also be used to treat or prevent one or more conditions, diseases, or disorders selected from the group consisting of obesity, monogenic obesity, metabolic syndrome, diet-induced food cravings, functional hypothalamic amenorrhea, type 1 diabetes, type 2 diabetes, female infertility, amenorrhea, impaired immune function, hypothyroidism, insulin resistance, severe insulin resistance (including severe insulin resistance due to mutations in the insulin receptor, severe insulin resistance not due to mutations in the insulin receptor, severe insulin resistance due to mutations in downstream signaling pathways, or due to other causes), nonalcoholic and alcoholic fatty liver disease, nonalcoholic steatohepatitis (NASH), Alzheimer's disease, leptin deficiency, leptin resistance, lipodystrophy, dwarfism / Donohue syndrome, Robertson-Mendenhall syndrome.

[0179] The LEPR agonist antibodies provided herein can be used to treat metabolic dysfunction. 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 leptin receptor (LEPR) and activates LEPR signaling, and a pharmaceutically acceptable carrier or diluent.

[0180] The LEPR agonist antibodies provided herein can be used to treat a predisposition to obesity or obesity, or to reduce body weight. In some embodiments, the treatment reduces fat mass in the treated subject, but does not reduce lean body mass. In some aspects, the treatment reduces the number of calories consumed or reduces food intake by the subject.

[0181] The LEPR agonist antibodies provided herein can be used to treat female infertility or to restore normal hormonal cycles associated with leptin deficiency. In some aspects, the treatment can increase fertility and / or increase the chances of conception. In some aspects, the treatment can restore normal menstrual cycles. Methods for restoring normal menstrual cycles that are interrupted at least in part due to leptin deficiency are also part of the present application.

[0182] As demonstrated herein, the methods are useful when the subject in need is a subject with hypo-leptinemia or leptin deficiency or a subject without hypo-leptinemia or leptin deficiency. The methods are useful when the metabolic dysfunction, predisposition to obesity or obesity is or is not associated with or caused by a signaling-deficient or signaling-impaired LEPR mutation.

[0183] The LEPR agonist antibodies provided herein can be used to treat nonalcoholic fatty liver disease or nonalcoholic steatohepatitis (NASH) in a leptinemic, lipodystrophic, or leptin-deficient patient. The treatment can reduce symptoms of nonalcoholic fatty liver disease, such as hepatic steatosis, in the subject. In some cases, plasma levels of alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST) are reduced in the subject after receiving the treatment.

[0184] The LEPR agonist antibodies provided herein can be used to treat hyperphagia, hyperglycemia, insulin resistance, dyslipidemia, nonalcoholic steatohepatitis (NASH), or nonalcoholic fatty liver disease by stimulating hypothalamic STAT3 signaling. The treatment can reduce circulating plasma triglycerides and / or circulating plasma total cholesterol.

[0185] The LEPR agonist antibodies provided herein can be used to treat lipodystrophy. The treatment reduces hyperglycemia, reduces insulin resistance, and / or reduces HbAlc levels in the subject receiving the treatment.

[0186] The LEPR agonist antibodies provided herein can be used to treat infertility and / or amenorrhea associated with metabolic disease or leptinemia. The treatment modulates the hormonal cycle and can improve the rate of conception in a female subject receiving the treatment. The treatment can restore a normal menstrual cycle.

[0187] The LEPR agonist antibodies provided herein can be used to treat impaired immune function, such as reduced CD4+ T cell counts associated with leptinemia and / or leptin deficiency. The treatment can improve immune function, for example, can increase CD4+ T cell counts.

[0188] The LEPR agonist antibodies provided herein can be used to treat delayed growth, lack of pubertal growth spurt, and / or abnormal growth hormone secretion associated with congenital leptin deficiency. The treatment can improve growth, can promote a pubertal growth spurt, and / or can improve growth hormone secretion.

[0189] The LEPR agonist antibodies provided herein can be used to treat hypothyroidism associated with congenital leptin deficiency. The treatment can improve symptoms associated with hypothyroidism.

[0190] The LEPR agonist antibodies provided herein can be used to treat low bone mineral density and / or bone mineral content associated with leptinemia and / or leptin deficiency. The treatment can improve bone mineral density and / or can improve bone mineral content.

[0191] In the context of the treatment methods described herein, the anti-LEPR antibodies can 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).

[0192] Combination therapy and formulations

[0193] The present application includes compositions and therapeutic formulations comprising any of the anti-LEPR antibodies described herein in combination with one or more additional therapeutically active components, as well as therapeutic methods comprising administering such combinations to a subject in need thereof.

[0194] The anti-LEPR antibodies of the present application can be co-formulated and / or administered in combination with one or more additional therapeutically active components (e.g., prescription drugs for the treatment of obesity, hypercholesterolemia, hyperlipidemia, type 2 diabetes, type 1 diabetes, appetite control, amenorrhea, infertility, etc.).Examples of such additional therapeutically active components include, for example, recombinant human leptin (e.g., metreleptin [MYALEPT]), PCSK9 inhibitors (e.g., anti-PCSK9 antibodies [alirocumab, evolocumab, bococizumab, lodelcizumab, ralpancizumab, etc.]), statins (atorvastatin, rosuvastatin, cerivastatin, pitavastatin, fluvastatin, simvastatin, lovastatin, pravastatin, etc.), ezetimibe, insulin, insulin variants, insulin secretagogues, metformin, sulfonylureas, sodium glucose co-transporter 2 (SGLT2) inhibitors (e.g., dapaglifozin, canaglifozin, empagliflozin, etc.), GLP-1 agonists / analogs (e.g., exendin-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 proteins (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 / metrelepin, lorcaserin, cetilistat, tesofensine, velneperit, etc.Additional examples include, for example, fish oil, pioglitazone, semaglutide, a fibrate (e.g., fenofibrate), prednisone, niacin, an anticonvulsant, digoxin, coumarin, vitamin D, thyroxine, thyroid supplements, vitamin supplements, calcium supplements, carnitine, coenzyme Q10, an anti-constipation medication, an anti-allergy medication, gabapentin, a narcotic, ketamine, lidocaine, and venlafaxine hydrochloride. In one embodiment of the application, the anti-LEPR antibody of the application is not formulated or administered with an anorectic agent.

[0195] One or more additional therapeutically active components, such as any of the agents listed above or a derivative thereof, can be administered prior to, concurrently with, or immediately after administration of the anti-LEPR antibody of the application; (for purposes of the present disclosure, such an administration regimen is considered to be administration of the anti-LEPR antibody “in combination” with the additional therapeutically active component(s)). The present application includes pharmaceutical compositions in which the anti-LEPR antibody of the application is co-formulated with one or more additional therapeutically active components as described elsewhere herein.

[0196] The present application also includes methods of using compositions and therapeutic formulations comprising any of the anti-LEPR antibodies described herein in conjunction with therapeutic procedures, such as plasmapheresis.

[0197] Administration Regimens

[0198] According to certain embodiments of the application, multiple doses of an anti-LEPR antibody (or a pharmaceutical composition comprising an anti-LEPR antibody and a combination of any of the additional therapeutically active agents mentioned herein) can be administered to a subject within a defined time frame. Methods according to this aspect of the application include administering multiple doses of an anti-LEPR antibody of the application to a subject sequentially. As used herein, “administered sequentially” means that each dose of the anti-LEPR antibody is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present application includes methods comprising administering a single initial dose of an anti-LEPR antibody, followed by one or more secondary doses of the anti-LEPR antibody, and optionally followed by one or more tertiary doses of the anti-LEPR antibody, to a patient sequentially.

[0199] The terms "initial dose," "second dose," and "third dose" refer to the chronological order of administration of the anti-LEPR antibodies of the present application. Thus, the "initial dose" is the dose administered at the beginning of the treatment regimen (also referred to as the "baseline dose," "loading dose," "starting dose," and the like); the "second dose" is the dose administered after the initial dose; and the "third dose" is the dose administered after the second dose. The initial, second, and third doses can all contain the same amount of anti-LEPR antibody, but generally can differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of anti-LEPR antibody contained in the initial, second, and / or third doses differs from one another (e.g., is appropriately up- or down-regulated) over the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at a "loading dose" at the beginning of the treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered at a lower frequency.

[0200] Diagnostic and analytical uses of antibodies

[0201] The anti-LEPR antibodies of the present application 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 can be used in a diagnostic to characterize a disorder or disease characterized by abnormal expression (e.g., overexpression, underexpression, lack of expression, etc.) of LEPR. An exemplary diagnostic assay for LEPR can comprise, e.g., contacting a sample obtained from a patient with an anti-LEPR antibody of the present application, wherein the anti-LEPR antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-LEPR antibody can be used in combination with a secondary antibody that is itself detectably labeled for diagnostic applications. The detectable label or reporter molecule can be a radioisotope, such as 3 H, 14 C, 32 P, 35 S or 125 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. Particular 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).

[0202] Samples that can be used in LEPR diagnostic assays according to the present application include any tissue or fluid sample obtainable from a patient under normal or pathological conditions that contains a detectable amount of LEPR protein or fragments thereof. Typically, LEPR levels in a particular sample obtained from a healthy patient (e.g., a patient not suffering from a disease or disorder associated with aberrant LEPR levels or activity) will be measured to initially establish a baseline or standard LEPR level. This baseline level of LEPR can then be compared to LEPR levels measured in samples obtained from individuals suspected of having a LEPR-related disease or disorder.

[0203] EMBODIMENTS

[0204] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the application, and are not intended to limit the scope of what the inventors regard as their application. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric pressure.

[0205] Example 1. Generation of antigen binding proteins that specifically bind leptin receptor (LEPR)

[0206] By immunizing mice (i.e., engineered mice comprising DNA encoding human immunoglobulin heavy and kappa light chain variable regions) with an immunogen comprising the extracellular domain of LEPR Antibodies against LEPR were obtained by immunizing mice (i.e., engineered mice comprising DNA encoding human immunoglobulin heavy and kappa light chain variable regions) with an immunogen comprising the extracellular domain of LEPR. Antibody immune responses were monitored by LEPR-specific immunoassays. Using techniques previously described, fully human anti-LEPR antibodies were isolated and purified.

[0207] Certain biological properties of exemplary anti-LEPR antibodies generated according to the methods of this example are described in detail in the examples shown below.

[0208] Example 2. Heavy and light chain variable region amino acid and nucleic acid sequences

[0209] 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 present application. Table 2 shows the corresponding nucleic acid sequence identifiers.

[0210] Table 1: Amino acid sequence identifiers

[0211]

[0212]

[0213] Table 2: Nucleic acid sequence identifiers

[0214]

[0215] Antibodies are referred to herein generally according to the following nomenclature: an Fc prefix (e.g., “H4H,” “H1M,” “H2M,” etc.), followed by a numerical identifier (e.g., “16650,” “16679,” etc.), followed by a “P” or “N” suffix. Thus, according to this nomenclature, antibodies can be referred to herein as, for example, “H4H16650P2,” “H4H16679P2,” etc. The Fc prefix on the antibody name used herein (H4H, H1M, and H2M) indicates the particular Fc region isotype 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, as indicated by the first ‘H’ in the antibody name). As will be appreciated by one of ordinary skill in the art, an antibody having a particular Fc isotype can be converted to an antibody having a different Fc isotype (e.g., an antibody having a mouse IgG1 Fc can be converted to an antibody having a human IgG4, etc.), but regardless, the variable domains (including the CDRs) (represented by the numerical identifiers shown in Tables 1 and 2) will remain the same, and the binding properties are expected to be the same or substantially similar, regardless of the nature of the Fc domain.

[0216] 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.

[0217] See International Patent Application Publication No. WO 2017 / 66204.

[0218] Example 3: Surface Plasmon Resonance-derived Binding Affinity and Kinetic Constants for Human Monoclonal Anti-LEPR Antibodies

[0219] The Biacore 4000 instrument was used to determine the equilibrium dissociation constant (KD) for LEPR binding to purified anti-LEPR monoclonal antibodies using real-time surface plasmon resonance biosensors. DAll binding studies were performed in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% v / v surfactant Tween-20 pH 7.4 (HBS-ET) running buffer at 25 °C and 37 °C. Biacore sensor surfaces were first derivatized by amine coupling of a monoclonal mouse anti-human Fc antibody (GE, #Br-1008-39) to capture anti-LEPR monoclonal antibodies. 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 reagents were first prepared in HBS-ET running buffer (100 nM - 3.7 nM; 3-fold serial dilutions) and injected for 4 minutes at a flow rate of 30 μL / min over the anti-human Fc captured anti-LEPR monoclonal antibody surface, while dissociation of LEPR reagents bound to the monoclonal antibody was monitored in HBS-ET running buffer for 10 minutes. Real-time binding sensorgrams were fitted to a 1 : 1 binding model with mass transfer limitations using Scrubber 2.0c curve fitting software to determine the kinetic association rate (k a ) and dissociation rate (k d ) constants. The calculation formula for the dissociation equilibrium constant (K D ) and the dissociation half-life (t 1 / 2) are constant from the kinetic rate constants:

[0220] and

[0221] Tables 3 to 8 show the binding kinetic parameters for the binding of hLEPR.mmh, mfLEPR.MMH, or hLEPR.mFc to different anti-LEPR monoclonal antibodies of the application at 25 °C and 37 °C.

[0222] Table 3: Binding kinetic parameters for the binding of hLEPR-MMH to LEPR monoclonal antibodies at 25 °C.

[0223]

[0224]

[0225] *NB indicates no binding was observed under the present experimental conditions.

[0226] Table 4: Binding kinetic parameters for hLEPR-MMH binding to LEPR monoclonal antibodies at 37 °C.

[0227]

[0228] *NB indicates no binding was observed under the present experimental conditions.

[0229] Table 5: Binding kinetic parameters for mfLEPR.MMH binding to LEPR monoclonal antibodies at 25 °C.

[0230]

[0231]

[0232] *NB indicates no binding was observed under the present experimental conditions.

[0233] *IC indicates the observed binding included end points, and

[0234] Real-time binding data could not be fitted under the present experimental conditions.

[0235] Table 6: Binding kinetic parameters for mfLEPR.MMH binding to LEPR monoclonal antibodies at 37 °C.

[0236]

[0237] *NB indicates no binding was observed under the present experimental conditions.

[0238] *IC indicates the observed binding included end points, and Real-time binding data could not be fitted under the present experimental conditions indicates the observed binding included end points, and

[0239] Table 7: Binding kinetic parameters for hLEPR.mFc binding to LEPR monoclonal antibodies at 25 °C.

[0240]

[0241] *NB indicates no binding was observed under the present experimental conditions.

[0242] Table 8: Binding kinetic parameters for hLEPR.mFc binding to LEPR monoclonal antibodies at 37 °C.

[0243]

[0244]

[0245] *NB indicates that binding was not observed under the current experimental conditions.

[0246] At 25°C, the anti-LEPR monoclonal antibody binds to hLEPR-MMH, K D The values ​​ranged from 7.93 nM to 148 nM, as shown in Table 5. At 37 °C, the anti-LEPR monoclonal antibody bound to hLEPR-MMH, K... D The values ​​range from 14.8 nM to 326 nM, as shown in Table 4.

[0247] Ten of the twelve anti-LEPR monoclonal antibodies of this invention bind to mfLEPR.MMH. At 25°C, the anti-LEPR monoclonal antibodies bind to mfLEPR.MMH, K D The values ​​range from 2.27 nM to 139 nM, as shown in Table 7. At 37 °C, the anti-LEPR monoclonal antibody binds to mfLEPR.MMH, K... D The values ​​range from 5.18 nM to 264 nM, as shown in Table 8.

[0248] At 25°C, the anti-LEPR monoclonal antibody binds to hLEPR-mFc, K D The values ​​ranged from 613 pM to 5.7 nM, as shown in Table 7. At 37 °C, the anti-LEPR monoclonal antibody bound to hLEPR-mFc, K... D The values ​​range from 1.16 nM to 12.8 nM, as shown in Table 8.

[0249] At 25°C or 37°C, the anti-LEPR monoclonal antibody of the present invention does not bind to mLEPR.MMH or rLEPR.MMH (data not shown).

[0250] Example 4. The anti-LEPR antibody of the present invention binds to LEPR in the presence of leptin:LEPR binding.

[0251] Real-time surface plasmon resonance biosensor on a Biacore T200 instrument was used to assess the inhibition of human leptin on the binding of anti-LEPR antibodies to LEPR. The entire study was performed 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) at 25 °C. 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) at a flow rate of 10 μL / min or 25 μL / min for 4 minutes on human leptin-immobilized Biacore sensor to achieve approximately 200 RU of binding response. To assess whether human leptin blocks the binding of the antibodies to hLEPR-MMH, 200 nM of anti-LEPR monoclonal antibodies were injected onto the preformed hLEPR-MMH:human leptin complex at a flow rate of 50 μL / min or 25 μL / min for 4-5 minutes. Table 9 reports that all anti-LEPR antibodies of the present application bound to the complex of hLEPR-MMH and human leptin ("leptin:LEPR") with almost similar signal strength, and the binding was observed in RU. This result indicates that human leptin does not block the binding of hLEPR-MMH to the tested anti-LEPR antibodies.

[0252] Table 9: Binding of anti-LEPR monoclonal antibodies to pre-complex of hLEPR-MMH and human leptin.

[0253]

[0254] Example 5. Human leptin receptor blocking ELISA

[0255] In ELISA, human leptin (human leptin; R&D Systems, #398-LP-01M) was coated on 96-well microtiter plates at a concentration of 5 pg / mL (dissolved in PBS) overnight at 4C. Non-specific binding sites were then blocked using a 0.5% (w / v) solution of BSA in PBS. A constant amount of 10 nM of the extracellular domain portion of LEPR protein expressed with a C-terminal human Fc tag (hLEPR.hFc; SEQ ID NO: 116) was titrated with anti-LEPR antibodies, human leptin, or isotype control antibodies (serial dilution range 8.5 pM to 500 nM). 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 at room temperature for 2 hours, the wells were washed, and the bound hLEPR.hFc was detected with an anti-human IgG polyclonal antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch Inc, #109-035-098). The samples were developed with TMB solution (BD Biosciences, #555214; substrate A and B were mixed at a 1:1 ratio according to the manufacturer’s instructions) to generate a colorimetric reaction, which was then neutralized with 1 M sulfuric acid, and the absorbance at 450 nm was then measured on a Victor X5 plate reader.

[0256] Data analysis was performed using a sigmoidal dose response model in Prism software (GraphPad). The percent block at the maximum concentration of the tested antibodies was calculated as an indicator of the ability of the antibodies 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 the antibody was referred to as 100% binding or 0% block; the baseline signal of the single buffer in the absence of hLEPR.hFc was referred to as 0% binding or 100% block. Table 10 summarizes the blocking data at 500 nM antibody concentration.

[0257] As shown in Table 10, none of the anti-LEPR antibodies of the present application exhibited >28% block of the binding of hLEPR.hFc to human leptin-coated surface. However, the comparative antibody and human leptin as positive controls were able to block 99% of the binding of hLEPR.hFc to human leptin-coated surface. The isotype control antibody did not exhibit measurable block at the highest concentration of 500 nM.

[0258] Table 10: ELISA of blocking of hLEPR.hFc binding to human leptin by anti-LEPR antibodies

[0259]

[0260]

[0261] Example 6. Cell binding measured by FACS analysis using HEK293 / Mycx2-hLepR(ecto)-GPI anchored cells

[0262] The leptin receptor LEPR is a single-pass 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, a protein expressed primarily by adipose tissue that is involved in the regulation of food intake and metabolism (Friedman et al. (2014) J Endocrinol 223(1):T1-8).

[0263] 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 expresses the extracellular domain of human LEPR with an N-terminal myc-myc tag and a C-terminal peptide sequence from human carboxypeptidase M (amino acids 22-839 of Accession No. P48357 (SEQ ID NO: 113), isoform B) that directs the addition of a GPI (glycosylphosphatidylinositol) (Deddish et al. (1990) J. Biological Chemistry 265:25: 15083-89) so that the protein can be anchored to the membrane by the GPI. Another HEK293 cell line was generated that stably expresses full-length human LEPR (amino acids 1-1165 of Accession No. P48357 (SEQ ID NO: 113), isoform B) along with a luciferase reporter gene (Stat3-luciferase, Stat3-luc, SA Bioscience, #CLS-6028L), this cell line is hereafter referred to as HEK293 / Stat3-luc / hLEPR-FL. A HEK293 cell with only the Stat3-luciferase reporter gene (HEK293 / Stat3-luc) was also generated as a control cell line.

[0264] For FACS analysis, HEK293 parental cells and HEK293 / hLEPR-GPI cells were dissociated and resuspended in PBS with 2% FBS (FACS buffer) at 5 x 105cells / mL. Cells were incubated with 10 μg / mL of anti-LEPR antibody for 1 hour at 4°C. Cells were then washed twice with FACS buffer and incubated with 1:200 dilution of goat anti-human IgG Fc specific Alexa Fluor® 488 conjugated antibody (Jackson ImmunoResearch, # 109-546-098) for 1 hour at 4°C. Cells were washed twice with FACS buffer and analyzed by flow cytometry. 5Cells were plated at 1 cell / well in 96-well v-bottom plates. To test whether the ability of the anti-hLEPR antibodies to bind to the cells was affected by the presence of leptin, FACS buffer (R&D Systems, #398-LP) with or without 1 μΜ human leptin was incubated with the cells for 30 minutes at 4°C, followed by the addition of anti-LEPR antibodies or control antibodies at 10 nM in FACS buffer. The cells were then incubated for 30 minutes at 4°C, followed by washing, then incubation with 16 g / mL Alexa conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc., #109-547-003) for 30 minutes at 4°C. The cells were then fixed using BD CytoFix / Cytoperm (Becton Dickinson, #554655), filtered, and analyzed on a HyperCyt flow cytometer (Beckman Coulter). Unstained and secondary antibody controls were also tested for all cell lines. Results were analyzed using ForeCyt (IntelliCyt) and FlowJo version 10 software to determine the geometric mean of fluorescence of live cells. The geometric mean of fluorescence for each sample was then normalized to the geometric mean of unstained cells to obtain the relative binding for each condition, referred to as the "binding ratio," and these binding ratios were recorded for each test antibody. TM (Becton Dickinson, #554655), filtered, and analyzed on a HyperCyt flow cytometer (Beckman Coulter). Unstained and secondary antibody controls were also tested for all cell lines. Results were analyzed using ForeCyt (IntelliCyt) and FlowJo version 10 software to determine the geometric mean of fluorescence of live cells. The geometric mean of fluorescence for each sample was then normalized to the geometric mean of unstained cells to obtain the relative binding for each condition, referred to as the "binding ratio," and these binding ratios were recorded for each test antibody.

[0265] As shown in Table 11, the 9 anti-LEPR antibodies of the application 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. The anti-LEPR antibodies also bound in the presence of 1 μΜ leptin with binding ratios of 398 and 4184-fold. As shown in Table 11, the comparative 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 binding to the cells was significantly reduced in the presence of 1 M leptin with a binding ratio of 112. The anti-LEPR antibodies did not exhibit any significant binding to HEK293 parental cells in the presence and absence of 1 μΜ leptin with binding ratios ranging from 1 to 9-fold. The isotype control antibody and secondary antibody samples alone also did not exhibit significant binding to any of the cell lines with or without leptin with binding ratios ranging from 1 to 6-fold.

[0266] The four antibodies of the application tested at 70 nM in the absence of leptin exhibited binding to HEK293 / hLEPR-GPI cells with a binding ratio in the range of 707 to 1131-fold, and to HEK293 / Stat3-luc / hLEPR-FL cells with a binding ratio in the range of 42 to 51-fold, as shown in Table 12. The anti-LEPR antibodies did not exhibit any significant binding to HEK293 / Stat3-luc cells, with a binding ratio in the range of 1 to 8-fold. The isotype control antibody alone and the secondary antibody sample also did not exhibit significant binding to any of the test cell lines, with a binding ratio in the range of 1-2-fold.

[0267] Table 11: Binding of 10 nM anti-LEPR antibodies to HEK293 / hLEPR-GPI and HEK293 parental cells + / - 1 mM human leptin

[0268]

[0269] *Classification of antibodies as "agonists" or "potentiators" is based in part on the results observed in Examples 7 and 8 herein.

[0270] Table 12: Binding of 70 nM anti-LEPR antibodies to HEK293 / hLEPR-GPI, HEK293 / Stat3-hLEPR-FL and HEK293 / Stat3-luc parental cells

[0271]

[0272] Example 7. Anti-LEPR antibodies of the application activate LEPR signaling in the presence or absence of leptin

[0273] A bioassay to detect STAT3 transcriptional activation via LEPR activation in the IMR-32 cell line, a human neuroblastoma cell line, was developed using a reporter cell line that stably expresses full-length human LEPR (hLEPR; amino acids 1 to 1165 of Accession No. NP_002294.2) along with a luciferase reporter gene (STAT3-Luc; Qiagen, #CLS-6028L). The resulting stable cell line, designated IMR-32 / STAT3-Luc / hLEPR, was isolated and maintained in MEM-Earl media supplemented with 10% FBS, NEAA, 1 ug / mL puromycin, 100 ug / mL hygromycin B, and penicillin / streptomycin / L-glutamine (complete media).

[0274] The resulting bioassay was used to measure the effect of the anti-LEPR antibodies of the application on LEPR signaling in the presence or absence of leptin. For the bioassay, IMR-32 / STAT3-Luc / hLEPR cells were plated in a 96-well plate at a density of 20,000 cells / 100 ul / well in complete medium and on the next day changed to the appropriate volume of Opti-MEM medium (supplemented with 1% BSA and 0.1% FBS (assay buffer)) for 30 minutes. To measure the effect of the antibodies of the application in the absence of leptin, anti-LEPR antibodies or isotype control antibodies and human leptin (human leptin; R&D Systems, #398-LP) were serially diluted half-logarithmically to a final concentration ranging from 100 nM to 300 fM in assay buffer, which was added to the cells, followed by overnight incubation at 37°C, 5% CO2.

[0275] To measure the effect of the antibodies of the application in the presence of leptin, a fixed concentration of human leptin was added to the cells at 200 pM in assay buffer, immediately followed by the addition of anti-LEPR antibodies or isotype control antibodies serially diluted half-logarithmically to a final concentration ranging from 100 nM to 300 fM. The samples were then incubated overnight at 37°C, 5% CO2. OneGlo reagent (Promega, #E6051) was then added to the samples, and luciferase activity was measured in luminescence mode on an Envision Multilabel plate reader (Perkin Elmer). Relative light unit (RLU) values were obtained, and the results were analyzed using non-linear regression using 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.

[0276] As shown in Table 13, in Study 1, in the absence of human leptin, all the test anti-LEPR antibodies exhibited weak stimulation of IMR-32 / STAT3-Luc / hLEPR cells, with EC 50 values ranging from 134 pM to 11.9 nM, and maximum activation ranging from 5% to 13%. In Study 2, in the absence of human leptin, 4 of the test anti-LEPR antibodies exhibited stimulation of IMR-32 / STAT3-Luc / hLEPR cells, with EC 50Values ranged from 61.9 pM to 206.9 pM, with maximum activation ranging from 65% to 68%. In Study 1, in the presence of 200 pM human leptin, all of the test anti-LEPR antibodies exhibited stimulation of IMR-32 / STAT3-Luc / hLEPR cells, with EC 50 Values ranged from 20.2 pM to 523 pM, with maximum activation ranging from 66% to 107%. Since these antibodies potentiated leptin-induced LEPR signaling, they were classified as "potentiators" as defined herein. In Study 2, in the presence of 200 pM human leptin, 4 of the test anti-LEPR antibodies exhibited stimulation of IMR-32 / STAT3-Luc / hLEPR cells, with EC 50 Values ranged from 51.9 pM to 257.3 pM, with maximum activation ranging from 76% to 88%. These antibodies did not significantly potentiate LEPR signaling in the presence of leptin. In no assay did the isotype control antibody exhibit any measurable stimulation of IMR-32 / STAT3-Luc / hLEPR cells.

[0277] Table 13: Activation of hLEPR by anti-LEPR antibodies

[0278]

[0279] Example 8. Anti-LEPR antibodies of the application activate signaling in cells expressing signaling-deficient or signaling-impaired LEPR mutants

[0280] LEPR mutants 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-deficient mutant LEPR protein that does not transduce leptin signals to STAT3; the A409E mutant was originally identified as a single-gene 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 also shows an association with early-onset obesity. (Mazen et al., 2011, Mol Genet Metab 102:461-464).

[0281] In this example, the ability of anti-LEPR antibodies of the application to stimulate LEPR signaling in cell lines expressing signaling-deficient or signaling-impaired LEPR mutants was assessed. Specifically, reporter cell lines (HEK293) expressing wild-type LEPR, LEPR-A409E (signaling-deficient), or LEPR-P316T (signaling-impaired) were constructed. Cells were treated with vehicle only, recombinant human leptin, control IgG, or agonist anti-LEPR antibodies of the application (H4H16650 or H4H16679), and the extent of LEPR signaling was determined (as measured by Western blot detection of pSTAT3-Y705 expression relative to STAT3 expression).

[0282] In these experiments, agonist anti-LEPR antibodies of the application (H4H16650 and H4H16679) were shown to stimulate LEPR signal transduction in cells expressing the LEPR-A409E mutant or the LEPR-P316T mutant in a dose-dependent manner (as measured by STAT3 expression) (Figure 2, panels B and C). In contrast, leptin treatment induced only modest signaling in cells expressing the LEPR-P316T mutant, and no signaling in cells expressing the LEPR-A409E mutant. (Figure 2, panel A). Moreover, no LEPR signal transduction was detected in any of the cell lines treated with vehicle or IgG control antibody (data not shown). Other signaling-deficient or signaling-impaired LEPR mutants were tested in this assay, but were not activated by the anti-LEPR mutants (data not shown), suggesting that this rescue effect can be mutant-dependent.

[0283] The results of this example demonstrate that agonist anti-LEPR antibodies of the application can be used to treat 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).

[0284] Example 9: Octet Cross-competition between different anti-LEPR monoclonal antibodies.

[0285] Binding competition between a panel of different anti-LEPR monoclonal antibodies was determined using real-time, label-free bio-layer interferometry assay on an Octet HTX biosensor platform (Pall ForteBio Corp.). The entire experiment was performed at 25 °C 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) with the plate shaking at 1000 rpm. To assess whether two antibodies were able to compete with each other for binding to their respective epitope on recombinant human LEPR (expressed with a C-terminal myc-myc-hexahistidine tag) (hLEPR.mmh, SEQ ID: 114), about 0.25 nm or 0.34 nm of hLEPR-MMH was first captured onto anti-5His antibody coated Octet biosensor tips (Fortebio Inc, #18-5122) by dipping the biosensor tips into a well containing 20 pg / mL of hLEPR-MMH for 5 minutes. Then, the antigen-captured biosensor tips were saturated with a first anti-LEPR monoclonal antibody (hereafter referred to as mAb-1) by dipping the biosensor tips into a well containing 50 pg / mL of mAb-1 solution for 210 seconds. The biosensor tips were then dipped into a well containing 50 pg / mL of a second anti-LEPR monoclonal antibody (hereafter referred to as mAb-2) solution for 150 seconds. Between each step of the experiment, the biosensor tips were washed in HBS-EBT buffer. The real-time binding reaction was monitored throughout the experiment and the binding reaction at the end of each step was recorded. The binding reaction of mAb-2 to hLEPR-MMH pre-complexed with mAb-1 was compared and the competitive / non-competitive behavior of different anti-LEPR monoclonal antibodies was determined as shown in Table 14 and Table 15.

[0286] Table 14: Cross-competition between anti-LEPR monoclonal antibodies

[0287]

[0288]

[0289] Table 15: Cross-competition between anti-LEPR monoclonal antibodies

[0290]

[0291]

[0292] Example 10: In vivo efficacy of LEPR agonist antibodies H4H16650P2, H4H16679P2, H4H17319P2, and H4H17321P2 in a leptin-deficient inducible mouse model.

[0293] In genetically engineered LEPR Hu / Hu The effects of four specific agonist anti-LEPR antibodies of the present application, H4H16650P2, H4H16679P2, H4H17319P2, and H4H17321P2, on food intake, body weight, and obesity propensity were determined in a leptin-deficient inducible model in mice expressing a leptin receptor consisting of a human LEPR extracellular domain sequence in place of the murine LEPR extracellular domain sequence. The leptin-deficient model was induced by hydrodynamic DNA delivery (HDD) of a plasmid encoding a hFc-tagged mouse LEPR extracellular domain (referred to herein as mLEPR.hFc or “leptin trap”; SEQ ID NO: 120). The leptin trap, when expressed, is secreted and binds circulating leptin. Following HDD of 50 g of the DNA construct encoding the leptin trap, the mice exhibited increased food consumption, as well as increased obesity propensity and body weight.

[0294] Baseline daily food intake was measured between 7 days and 4 days prior to administration of the leptin trap (Days -7 and -4). On Day 0, thirty-five 13- to 17-week-old male LEPR Hu / Hu The mice successfully received HDD of the leptin trap. On Days 6 and 13 post-HDD, blood was collected retro-orbitally, and body composition, including obesity propensity, was quantified by CT. On Day 7 post-HDD, the mice were randomly divided into five groups of 7 based on percent change in body weight from Day 0. Each group received a single dose of 3 mg / kg of any one of an isotype control antibody, 3 mg / kg H4H16650P2, 3 mg / kg H4H16679P2, 3 mg / kg H4H17319P2, or 3 mg / kg H4H17321 via subcutaneous injection. The isotype control antibody does not bind to any known mouse protein. Food intake and body weight were measured for each animal during the study. Figure 3 The average daily food intake was summarized for each treatment group. In Figure 3 In the graph, the dotted line represents the average baseline food intake prior to the HDD injection. The percent change in body weight from Day 0 was calculated for each animal at each time point. Figure 4 The average percent change in body weight was summarized for the animals in each antibody treatment group. Figure 5Mean fat mass of animals in each antibody treatment group was summarized, quantified by CT scans 1 day before and 6 days after antibody treatment. All results are expressed as mean ± SEM.

[0295] like Figure 3 and 4 As shown, similar increases in food intake and percentage changes in body weight were observed in mice in each group before antibody treatment following HDD treatment with leptin trap. Figure 3 As shown, compared with mice injected with isotype control antibodies, mice treated with 3 mg / kg antibodies H4H16650P2 or H4H16679P2 exhibited significantly reduced food intake from one day after antibody treatment (day 8 after HDD) and at subsequent measurement time points. Compared with mice injected with isotype control antibodies, mice treated with 3 mg / kg antibodies H4H17319P2 or H4H17321P2 exhibited significantly reduced food intake two days after antibody treatment (day 9 after HDD) and at other subsequent measurement time points. Figure 4 As shown, compared with mice injected with the isotype control antibody, mice treated with 3 mg / kg antibody H4H16650P2 showed a significantly reduced percentage change in body weight at one day after antibody treatment (day 8 after HDD) and other subsequent measurement time points. One day after antibody treatment, on day 8, mice treated with the isotype control showed a body weight increase of 21.16 ± 1.27% from day 0, while mice treated with H4H16650P2 showed a body weight increase of 15.57 ± 0.9% from day 0. Compared with mice injected with the isotype control antibody, mice treated with 3 mg / kg antibodies H4H16679P2, H4H17319P2, or H4H17321P2 showed a significantly reduced percentage change in body weight at two days after antibody treatment (day 9 after HDD) and other subsequent measurement time points. On day 9, for mice treated with isotype controls H4H16650P2, H4H16679P2, H4H17319P2, or H4H17321P2, the percentage changes in body weight from day 0 were 23.18±1.22, 13.17±1.05, 12.95±1.26, 15.98±1.78, and 15.83±2.01, respectively. Figure 5As shown, mice treated with 3 mg / kg isotype control antibody showed a significant increase in fat mass 6 days after antibody treatment (day 13 after HDD) compared to 1 day prior to antibody treatment (day 6 after HDD). Mice treated with 3 mg / kg of antibody H4H16650P2, H4H16679P2, H4H17319P2, or H4H17321P2 did not show an increase in fat mass after antibody treatment compared to prior to antibody treatment. Mice treated with 3 mg / kg of antibody H4H16650P2, H4H16679P2, or H4H17319P2 showed a significant decrease in fat mass 6 days after treatment (day 13 after HDD) compared to mice treated with 3 mg / kg isotype control antibody.

[0296] Example 11: Epitope mapping of H4H16650P2 binding to human leptin receptor (hLEPR.mmh) by hydrogen-deuterium exchange.

[0297] Experiments were performed to determine the amino acid residues of hLEPR.mmh (amino acids M1-D839 of SEQ ID NO: 114) that interact with H4H16650P2. For this purpose, H / D exchange epitope mapping using mass spectrometry was performed. General descriptions of the H / D exchange method are set forth in, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; and Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0298] Experimental procedures The 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 uploading, a Waters Acquity M-Class (μBinary solvent manager) for analytical column gradient, and a Synapt G2-Si mass spectrometer for pepsinolysis peptide mass measurement.

[0299] Labeling solution was prepared in 10 mM PBS buffer in D2O at pD 7.0 (equivalent to pH 6.6). For deuterium labeling, 3.8 μL of hLEPR.mmh (8 pmol / μL) or hLEPR.mmh pre-mixed with antibody at 2: 1 molar ratio was incubated with 56.2 μL of D2O labeling solution for different time points (e.g., un-deuterated control = 0 sec, labeled 1 min and 20 min). Deuterium quenching was performed by transferring 50 μL of sample to 50 μL of pre-chilled quenching buffer (0.2 M TCEP, 6 M guanidinium chloride in 100 mM phosphate buffer pH 2.5) and the mixed sample was incubated at 1.0 °C for two minutes. The quenched sample was then injected into Waters HDX Manager for on-line pepsin / protease XIII digestion. The digested peptides were captured on an ACQUITY UPLC BEH C18 1.7-μm, 2.1 x 5 mm VanGuard pre-column at 0 °C and eluted to an analytical column ACQUITY UPLC BEH C18 1.7-μm, 1.0 x 50 mm with a gradient of 5% - 40% B for 9 minutes (mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile). The mass spectrometer was set at cone voltage 37 V, scan time 0.5 s and mass to charge ratio range 50 - 1700 Th.

[0300] For the identification of peptides from human LEPR, LC-MSE data from un-deuterated samples were processed and searched in a database via Waters ProteinLynx Global Server (PLGS) software, which included human LEPR, pepsin and its randomized sequence. The identified peptides were imported into DynamX software and filtered with two criteria: 1) minimum product per amino acid: 0.2, and 2) duplicate file threshold: 3. Then, DynamX software automatically determined the amount of deuterium uptake for each peptide according to retention time and high quality accuracy (<10 ppm) at multiple time points, each time repeated 3 times.

[0301] Results . Using on-line pepsin / protease XIII column with MS EData collection combined, in the absence or presence of antibody, reproducibly identified a total of 201 peptides from human LEPR, representing 70% sequence coverage. As shown in Table 16, the amount of deuterium uptake for five peptides was significantly reduced (centroid delta value > 0.4 Dalton, p-value < 0.05) when bound to H4H16650P2. The recorded peptide masses correspond to the average of centroid MH+masses from three replicates. These peptides correspond to amino acids 162-169 (amino acids LYVLPEVL of human LEPR; SEQ ID NO: 113) and amino acids 170-181 (amino acids EDSPLVPQKGSF of human LEPR; SEQ ID NO: 113), for which the rate of deuteration was slowed when these peptides were bound to H4H16650P2. These identified residues also correspond to residues 162-169 and 170-181 of human LEPR as defined by Uniprot entry P48357 (SEQ ID NO. 113; human Leptin receptor).

[0302] Table 16: Human Leptin Receptor peptides with significant protection when bound to antibody H4H16650P2

[0303]

[0304] Example 12: In vivo efficacy testing of LEPR potentiating antibodies in humanized LEPR mice.

[0305] In genetically engineered LEPR Hu / Hu In mice that were single-housed and genetically engineered to express a leptin receptor consisting of a human LEPR extracellular domain sequence in place of the murine LEPR extracellular domain sequence (mLEPR.hFc, SEQ ID NO: 120), the effect of three specific potentiating anti-LEPR antibodies of the application, H4H18482P2, H4H18487P2, and H4H18492P2, on body weight and obesity propensity was determined.

[0306] On day -19, body composition, including obesity propensity, was quantified by CT. On day 0, forty-eight 14- to 16-week-old female LEPR Hu / Hu Mice were randomly divided into four groups of 12. On days 0 and 11, mice from 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. Body weight of each animal was measured during the study. The percent change in body weight from day 0 was calculated for each animal at each time point. Figure 6The average percent change in body weight of animals in each treatment group was summarized. Figure 6 The average fat mass of animals in each antibody treatment group was summarized, as quantified by CT 19 days prior to antibody treatment and 11 days after antibody treatment. All results are expressed as mean ± SEM.

[0307] As shown in Figure 6 percent change in body weight was observed following administration of LEPR-potentiating antibodies, but not isotype control antibodies. As shown in Figure 6 percent change in body weight compared to mice injected with isotype control antibodies, starting at day 2 and at other time points. Mice treated with 30 mg / kg H4H18487P2 exhibited a statistically significant percent change in body weight compared to mice injected with isotype control antibodies, starting at day 2 and at other time points. Mice treated with 30 mg / kg H4H18492P2 exhibited a statistically significant percent change in body weight compared to mice injected with isotype control antibodies at days 4, 5, and 17, but not at other time points. Mice treated with 30 mg / kg H4H18482P2 exhibited a statistically significant percent change in body weight compared to mice injected with H4H18492P2, starting at day 6 and at subsequent days (but not at days 7, 14, and 17). Mice treated with 30 mg / kg H4H18487P2 exhibited a statistically significant percent change in body weight compared to mice injected with H4H18492P2, starting at day 3 and at other time points (but not at days 4 and 5).

[0308] As shown in Figure 7 A, there were no differences in fat mass between groups prior to treatment (day -19). As shown in Figure 7 B, mice treated with 30 mg / kg of antibodies H4H18482 and H4H18487, but not H4H18492, exhibited a statistically significant reduction in fat mass at 17 days after treatment (day 12) compared to isotype control antibodies.

[0309] Example 13: Effect of Anti-LEPR Antibodies of the Invention on Monkey LEPR Signaling

[0310] To assess the transcriptional activation of monkey leptin receptor, a stable cell line was developed. IMR-32 cells (human neuroblastoma ATCC) stably expressing the extracellular domain of cynomolgus monkey LEPR (MfLEPR; amino acids 22 to 837 of Accession No. XP_005543194.1, threonine at position 827 changed to alanine) fused to the transmembrane and cytoplasmic domains of human LEPR (hLEPR; amino acids 840 to 1165 of Accession No. NP_002294.2) and a luciferase reporter (STAT3-Luc; SABiosciences, #CLS-6028L) were generated. The resulting cell line (hereinafter 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.

[0311] In the absence of leptin, a bioassay was performed to measure the effect of the anti-LEPR antibodies of the application on monkey LEPR signaling. For the bioassay, IMR-32 / STAT3-Luc / MfLEPR cells were plated at 10,000 cells / well in 0.1% FBS in 96-well plates (in Optimem with penicillin / streptococcus) and incubated overnight at 37°C in 5% CO2. The next day, human leptin (hLeptin), anti-LEPR antibodies or isotype control antibodies were serially diluted from 50 nM to 0.8 pM in assay buffer (plus samples containing only buffer but no test molecule) and added to the cells. After 5.5 hours at 37°C in 5% CO2, luciferase activity was measured using OneGlo TM reagent (Promega, #E6031) and Victor TM X multilabel plate reader (Perkin Elmer). Results were analyzed using nonlinear regression (4 parameters, log) and Prism TM 6 software (GraphPad) to obtain EC 50 values. The formula for the percent activation of the antibodies was as follows: the maximum range of RLU achieved by the antibody divided by the maximum range of RLU achieved by human leptin.

[0312] As shown in Table 17, in the absence of human leptin, all the anti-LEPR antibodies tested showed activation of monkey LEPR signaling in IMR-32 / STAT3-Luc / mfLEPR cells, with EC 50Values ranged from 266 pM to 368 pM, with maximum activation ranging from 76% to 82%, with 100% activation obtained with human leptin, EC50 50 Values ranged from 266 pM to 368 pM, with maximum activation ranging from 76% to 82%, with 100% activation obtained with human leptin, EC50

[0313] Table 17: Activation of cynomolgus monkey LEPR by anti-LEPR antibodies

[0314]

[0315] Example 14: Epitope binding to full-length extracellular domain of human LEPR using Luminex MFI signal

[0316] To determine the epitope of human LEPR to which the anti-LEPR antibodies of the present application bind, an analysis based on Luminex FLEXMAP (FM3DD, Luminex Corp) flow cytometry was employed to characterize the interaction of anti-LEPR antibodies with recombinant human LEPR protein domains. For this assay, approximately 3 million carboxylated Microplex R Microspheres (Luminex, Cat# LC1000A) were washed, vortexed and sonicated in 0.1 M NaP04, pH 6.2 (activation buffer) and then centrifuged to remove the supernatant. The microspheres were resuspended in 120 L of activation buffer and the carboxylate groups (-COOH) were activated by adding 15 L of 50 mg / mL N-hydroxysuccinimide (NHS, Thermo Scientific, Cat# 24500). 15 L of 50 mg / mL l-ethyl-3-[3- dimethylaminopropyl]carbodiimide (EDC, Thermo Scientific, Cat# 22980) was added at 25 °C. After 10 minutes, the pH of the reaction was lowered to 5.0 by adding 600 L of 50 mM MES, pH 5 (coupling buffer) and the microspheres were vortexed and centrifuged to remove the supernatant. The activated beads were immediately mixed with 500 L of 20 g / mL monoclonal anti-myc monoclonal antibody in coupling buffer, either mouse IgG or human IgG, and incubated for two hours at 25 °C. The coupling reaction was quenched by adding 50 L of 1 M Tris-HCl, pH 8.0, the microspheres were quickly vortexed, centrifuged and washed four times with 1 mL of DPBS to remove uncoupled protein and other reaction components.

[0317] Transiently expressed LEPR proteins, including human LEPR extracellular domain expressed with C-terminal myc-myc hexahistidine tag (human LEPR-MMH, SEQ ID NO: 113), human LEPR CRH1(D1) expressed with C-terminal myc-myc hexahistidine tag (human LEPR CRH1(D1)-MMH, amino acids 1-208, amino acids 209-236 of SEQ ID NO: 113 with myc-myc hexahistidine tag), human LEPR CRH1(D1, D2) domains expressed with C-terminal myc-myc hexahistidine tag (human LEPR CRH1(D1, D2)-MMH, amino acids 1-318, amino acids 319-346 of SEQ ID NO: 113 with myc-myc hexahistidine tag), human LEPR CRH1-Ig(D1, D2, D3) domains expressed with C-terminal myc-myc hexahistidine tag (human LEPR CRH1(D1, D2, D3)-MMH, amino acids 1-278, amino acids 279-306 of SEQ ID NO: 113 with myc-myc hexahistidine tag), human LEPR CRH1-Ig(D2, D3) domains expressed with C-terminal myc-myc hexahistidine tag (human LEPR CRH1-Ig(D2, D3)-MMH, amino acids 1-198, amino acids 199-226 of SEQ ID NO: 113 with myc-myc hexahistidine tag), human LEPR Ig(D3) domain expressed with C-terminal myc-myc hexahistidine tag (human LEPR Ig(D3)-MMH, amino acids 1-88, amino acids 89-116 of SEQ ID NO: 113 with myc-myc hexahistidine tag), human LEPR CRH2 domain expressed with C-terminal myc-myc hexahistidine tag (human LEPR CRH2-MMH, amino acids 1-207, amino acids 208-235 of SEQ ID NO: 113 with myc-myc hexahistidine tag), human LEPR FNIII domain expressed with C-terminal myc-myc hexahistidine tag (human LEPR FNIII-MMH, amino acids 1-204, amino acids 205-232 of SEQ ID NO: 113 with myc-myc hexahistidine tag), and human LEPR Ig-CRH2-FNIII domain expressed with C-terminal myc-myc hexahistidine tag (human LEPR Ig-CRH2-FNIII-MMH,Amino acids 1-510 of SEQ ID NO: 113 with myc-myc hexahistidine tag, amino acids 511-538). An aliquot of microspheres with immobilized anti-myc monoclonal antibody prepared as described above was added to 1 mL of each of these protein supernatants, respectively. The microspheres were gently mixed, incubated at 25 °C for two hours, washed twice with 1 mL of DPBS, centrifuged to remove the supernatant, and finally resuspended in 1 mL of DPBS buffer. The 48L anti-myc IgG coupled microspheres extracted from the individual reactions with full-length human LEPR and each human LEPR domain protein were mixed together in 3.6 mL of PBS + 20 mg / mL BSA + 0.05% sodium azide (blocking buffer).

[0318] From this mixed pool, 75L microspheres / well were plated on a 96-well filter plate (Millipore, catalog number: MSBVN1250) and mixed with 25L of individual anti-human LEPR monoclonal antibodies (0.5 or 5 g / mL) and incubated at 25 °C for two hours, then washed twice with 200L of DPBS containing 0.05% Tween 20 (wash buffer). To detect and quantify the amount of anti-LEPR antibody levels bound to individual microspheres, 100L of 2.5 g / mL R-phycoerythrin-conjugated goat F(ab')2 anti-human kappa antibody (Southern Biotech, catalog #2063-09) dissolved in blocking buffer or 100L of 1.25 g / mL R-phycoerythrin AffiniPure F(ab')2 fragment goat anti-mouse IgG, F(ab')2 fragment specific (Jackson Immunoresearch, catalog number: 115-116-072) dissolved in blocking buffer were added and incubated at 25 °C for 30 minutes. After 30 minutes, the samples were washed twice with 200L of wash buffer and then resuspended in 150L of wash buffer. The median fluorescence intensity (MFI) of the microspheres was measured in a Luminex analyzer.

[0319] Table 18: Luminex MFI signal of anti-LEPR antibodies bound to myc-tag captured full-length extracellular domain of human LEPR and isolated human LEPR domains

[0320]

[0321] The results of the Luminex-based analysis are set forth in Table 18. The Luminex MFI signal strength indicates that the twelve anti-LEPR antibodies of the application bind to the full human LEPR ectodomain. Anti-LEPR antibodies H4H18417P2, H4H18438P2, and H4H18492P2 bind to an epitope within the CRH1 D2 domain of human LEPR. Anti-LEPR antibodies H4H18449P2, H4H16650P, and H4H16679P bind to an epitope within the CRH1 (D1-2) domain of human LEPR. Anti-LEPR antibody H4H17319P2 binds to an epitope within the CRH2 domain of human LEPR. Anti-LEPR antibody H4H18445P2 binds to an epitope within the FNIII domain of human LEPR. Anti-LEPR antibodies H4H18446P2, H4H18482P2, and H4H18487P2 bind to an epitope within the Ig-CRH2-FNIII domain of human LEPR.

[0322] Examples 15-19: Animal Study Protocols

[0323] All animal studies were conducted in accordance with the guidelines and with approval from the Institutional Animal Care and Use Committee (IACUC) of Regeneron Pharmaceuticals. The monkey studies were also conducted in accordance with the guidelines and with approval from the IACUC of Covance Laboratories.

[0324] Mouse studies

[0325] Hemodynamic DNA delivery

[0326] Hydrodynamically based DNA delivery (HDD) in vivo transfection is a protocol that involves a bolus injection of a large volume of solution containing naked plasmid DNA to express a foreign protein in a live animal (Suda, 2007). Mice were weighed and expression vectors were freshly prepared by suspension in a final volume equal to 1 / 10 body weight (V / W) of saline, which were delivered by injection into the lateral tail vein. For the leptin sink model validation study (Figure 11), 8-week-old male C57BL / 6N mice (Taconic) were subjected to 50 μg DNA of mLeprECD expression vector (pRG977.mROR.mLepR.ecto.hFc) or control vector (pRG977.hFc) per mouse. For the H4H17319P2 evaluation study (Figure 9), 17- to 20-week-old male and female Lepr hu / huMice received 50 μg of DNA in the mLeprECD expression vector (pRG977.mROR.mLepR.ecto.hFc) per mouse.

[0327] Body weight and food intake measurements

[0328] Mice were weighed by placing them in containers on a calibrated digital laboratory balance. Average weight was measured using a 3-second dynamic weighing method. Food intake was determined by measuring the mass of food in the feed hopper using the digital laboratory balance. Food intake was calculated as the weight of food provided in the feed hopper minus the weight of food remaining in the feed hopper.

[0329] Body composition

[0330] According to the manufacturer's instructions, a miniature computed tomography (MCT) scan was performed on live mice anesthetized with gaseous isoflurane using the Perkin Elmer quantum miniature CT imaging system. Body composition (fat mass, lean body mass, bone mass, bone mineral content, and density) was measured using the Perkin Elmer quantum miniature CT imaging system, according to the manufacturer's instructions. The scan results were then analyzed using AnalyzeDirect imaging software. The recorded tissue volumes were multiplied by their corresponding mass densities: 0.92, 1.05, and 1.7 g / cm³. 3 Fat mass, lean body mass, and bone mass were calculated. Quantitative nuclear magnetic resonance (qNMR) was performed on conscious live mice using an EchoMRI 100 instrument (EchoMRI) to measure fat mass, lean body mass, free water mass, and total water mass.

[0331] Blood glucose measurements

[0332] Blood glucose levels after feeding were measured from a tail vein incision in conscious mice using a blood glucose meter (AlphaTrak2, Zoetis) and glucose test strips (Zoetis).

[0333] Insulin tolerance test

[0334] After a 4-hour fast, animals were administered intraperitoneal (IP) injections of insulin (Humulin R, Eli Lilly, and Company) at doses of 0.5, 0.75, or 1.0 U / kg, as directed. Glucose levels were measured at 0, 15, 30, 60, and 120 minutes post-insulin injection using an AlphaTRAK2 glucometer and test strips (Zoetis). (Lepr) hu / hu In characterization studies, using Accu-Chek® Compact Plus blood glucose meter and test strips (Roche Diabetes Care, Inc.).

[0335] Chemical analysis and hormone assays

[0336] Unless otherwise indicated, mice were fasted for 4 h and blood was collected by retro-orbital bleed. For serum isolation, blood was transferred to serum separator tubes (Sarstedt AG & Co.) and allowed to clot on wet ice for at least 30 min before centrifugation at 10,000 g for 5 min. Serum was removed and stored at -80°C until leptin quantification. For plasma measurements, blood was transferred to K3EDTA-coated tubes (Sarstedt AG & Co.). Dipeptidyl peptidase 4 (DPP4) inhibitor and protease inhibitor cocktail were added to the blood sample, which was placed on ice until plasma was obtained by centrifugation at 2000 x g for 10 min. Plasma was aliquoted and stored at -80°C until used for quantification of plasma lipid, liver enzymes, and leptin levels. HbAlc was measured in fresh whole blood collected in K3EDTA-coated tubes. HbAlc, 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) and following the manufacturer’s recommended protocol.

[0337] Liver triglyceride quantification

[0338] Liver triglyceride content was quantified from 100 to 200 mg of liver ground to powder with a mortar and pestle cooled with liquid nitrogen. The powdered liver tissue was weighed and homogenized in PBS by a bead beater (FastPrep 24-5G, MP Biomedical). The homogenate was transferred to a glass tube containing 5 mL of Folch solution (2: 1 chloroform:methanol), vortexed, and centrifuged at 1500 x g for 20 min. The lower phase was collected to a 5 mL volume and vortexed. A specific volume (25 to 50 pL) of sample and triolein standard (Verichem) was transferred to a 96-well polypropylene plate, mixed with 10 pL of a 1: 1 mixture of chloroform:Triton X-100, and air-dried. 300 pL of triglyceride reagent (Thermo Scientific) was added to the dried samples and standards, the plate was shaken for 5 min, and then incubated at 37°C for 20 min. 200 pL 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).

[0339] Fixation perfusion and immunohistochemistry

[0340] Mice were anesthetized with sodium pentobarbital (110 mg / kg, i.p.) and perfused with 2 mL of saline followed by 150 mL of 4% paraformaldehyde in 0.1 M borate buffer pH 9.5. Livers were postfixed for 2 hours, transferred to 70% ethanol and processed for paraffin embedding, sectioned at Histoserv for hematoxylin and eosin staining. Brains were postfixed at 4°C for 2 h and then immersed in 15% sucrose in potassium phosphate buffered saline at 4°C overnight. Whole brains were mounted on a cryostat sliding microtome (Leica), sectioned at 30 pm thickness, collected at equal intervals series, stored 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).

[0341] A series of brain sections at 150 pm intervals from each experimental group were processed synchronously to ensure comparable staining between animals and treatments. For all immunostaining, brain sections were blocked and primary / secondary antibodies were diluted in a solution containing 1% donkey serum (Equitech), 0.03% Triton X-100 and 0.05 M potassium phosphate buffered saline. Affinity and biotin blocking was performed according to the manufacturer’s protocol (Vector Labs). Immunohistochemical staining for pStat3 (Y705) was performed on equal interval series of brain sections using the avidin-biotin complex method and the chromogen diaminobenzidine (Vector Labs) (#9145, Cell Signaling Technologies; 1 : 1000 for 16 h at 4°C). For P-STAT3 (Y705) immunohistochemistry (IHC), sections were pretreated with 1% H2O2 in 1% NaOH for 10 min, with 0.3% glycine in K-PBS for 10 min, and with 0.03% SDS in K-PBS for 10 min.

[0342] Brightfield images were obtained by scanning the entire slide on a Scanscope XT (Aperio) with a 40x objective. Sections were matched by histological comparison with the Franklin and Paxinos mouse brain atlas. Neuroanatomical areas and distance from bregma were estimated according to this atlas. Bilateral quantification of the number of pSTAT3 (Y705) immunoreactive cells in a given area was performed at the indicated rostrocaudal levels using the Halo software (Indica Labs).

[0343] Monkey studies

[0344] All cynomolgus monkey studies were conducted at Covance Laboratories, Inc. The monkeys were fed twice daily with certified primate food #5048 (PMI, Inc.) and were allowed free access to fresh water.

[0345] Body weight

[0346] The monkeys were weighed on the day of administration and at least once a week as needed for the remainder of the study.

[0347] Dual energy X-ray absorptiometry (DEXA)

[0348] Based on the veterinarian's assessment, a whole-body scan was performed on fasting monkeys anesthetized with ketamine and dexmedetomidine using a Discovery A densitometer (Hologic).

[0349] Example 15: Leptin deficiency-induced anti-LEPR antibody reverses obesity

[0350] The study aimed to determine whether H4H17319P2 was effective in vivo. Since H4H17319P2 does not bind to mouse LEPR, [the study was conducted using...]. The technique (Valenzuela et al., 2003) was used to generate genetically modified mice in which a portion of the mouse Lepr gene encoding the extracellular domain of LePR was replaced with the corresponding human LEPR genome sequence (Lepr...). hu / hu ; Figure 10A Lepr hu / hu Mice did not show similarities to Lepr in terms of body weight, body composition, insulin sensitivity, and serum leptin levels. + / + Differences in mice ( Figure 10B (C and D).

[0351] A mouse model of leptin deficiency was developed by hydrodynamic DNA delivery (HDD) of a plasmid encoding an hFc-labeled mouse Lepr extracellular domain (mLeprECD) to isolate endogenous circulating mouse leptin, thereby acting as leptin deposits and thus blocking leptin signaling. Following HDD with mLeprECD, feed-fed C57BL / 6N mice experienced rapid weight gain, with a significant increase starting on day 3 compared to mice receiving control hFc-based HDD. Figure 11A At the end of the study (day 10 after HDD), the mLeprECD group showed a 24% increase in post-HDD weight, compared to only a small 3% increase observed in the control group. Figure 11A). Consistent with the body weight data, cumulative food intake was significantly increased in mLeprECD post-HDD compared to control post-HDD starting at day 3 post-HDD ( Figure 11A ). To confirm that the body weight gain reflected an increase in the propensity for obesity, micro-CT imaging was performed at day 7 post-HDD and showed a significant 2-fold increase in fat mass post-HDD in mLeprECD compared to control ( Figure 11B ). All other body composition parameters were similar between groups ( Figure 11B ).

[0352] To assess the in vivo efficacy of H4H17319P2, leptin deficiency was induced by HDD of mLeprECD in feed-fed male and female Lepr hu / hu mice. As expected, expression of mLeprECD promoted rapid body weight gain in both genders of Lepr hu / hu mice ( Figure 9A and 12A ). At seven days post-HDD, Lepr hu / hu mice were stratified according to percent change in body weight and administered a single 10 mg / kg subcutaneous dose of control or H4H17319P2. At 14 days post-HDD, body weight continued to increase in both male and female mice administered the control monoclonal antibody (hereinafter control mAb), with body weight gain of 40.5 ± 2.7% and 44.1 ± 4.7%, respectively ( Figure 9A and 11A ). In contrast, body weight decreased in mice treated with a single dose of LEPR agonist monoclonal antibody and returned to initial body weight prior to HDD ( Figure 9A and 11A ). Since H4H17319P2 binds human but not mouse LEPR, it is unlikely that the body weight gain was due to interference with the ability of the mLeprECD to sequester leptin. The loss of body weight was associated with a decrease in food intake ( Figure 9A and 11A ). While there was no difference in daily food intake between the first two groups, food intake was significantly decreased in mice treated with H4H17319P2 compared to the control monoclonal antibody group ( Figure 9A and 11A ). The effect of the single dose of H4H17319P2 on body weight and food intake was ultimately attenuated. Food intake remained significantly lower in male and female mice treated with H4H17319P2 compared to control monoclonal antibody administered mice until 12 and 9 days post-treatment, respectively (days 19 and 16) ( Figure 9A and 11AMale and female mice treated with H4H17319P2 maintained a similar body weight to baseline until days 16 and 13 (days 23 and 20) after treatment, after which weight gain occurred. Figure 9A and 11A ).

[0353] H4H17319P2 treatment-induced weight loss reflected a predisposition to obesity and a reduction in lean body mass. Mini-CT analysis showed similar body composition in both male and female mice before day -1 HDD and before day 6 treatment. Figure 9B , 12B (and 12C). Consistent with the induced leptin deficiency, both groups showed a significant increase in fat mass and leptin mass on day 6 compared to before day -1 HDD, but no increase in bone mass ( Figure 9B , 12B (and 12C). Six days after treatment (day 13), mice given the control monoclonal antibody showed a further increase in fat mass compared to day 6, while no further increase in obesity tendency was detected after H4H17319P2 treatment. Consistent with the changes in body weight, mice treated with H4H17319P2 showed a decrease in both fat mass and lean body mass compared to mice given the control monoclonal antibody after 7 days of treatment. No treatment-related effects on bone mass, bone mineral content, or bone density were observed. Figure 12B (and 123C).

[0354] Then, the ability of H4H17319P2 to alter circulating lipids in leptin-deficient mice was evaluated. Plasma chemistry analysis showed that, compared with male mice treated with a control monoclonal antibody after 6 days of treatment, H4H17319P2 reduced circulating plasma triglycerides and total cholesterol, including HDL-cholesterol (HDL-C) and LDL-cholesterol (LDL-C). Figure 9C ).

[0355] Given that leptin promotes energy expenditure in leptin-deficient ob / ob mice (Halaas et al., 1995; Pelleymounter et al., 1995), the effects of paired feeding were evaluated to determine whether reduced food intake could fully explain the weight loss effect of H4H17319P2 in induced leptin deficiency. This was compared with Lepr mice receiving the control vector via HDD. hu / hu Compared to mice, induced leptin-deficient Lepr hu / hu The mice gained weight rapidly and had an excessive appetite. Figure 13A Following administration on days 7 and 13, mice treated with the control monoclonal antibody continued to consume more food and gain weight, while mice treated with H4H17319P2 showed decreased food intake and weight loss.Figure 13A ) Pair-feeding also promoted a reduction in body weight in the mLeprECD-expressing Lepr hu / hu mice. Notably, pair-fed mice consumed the same amount of food as H4H17319P2-treated mice, but did not show the same degree of body weight reduction Figure 13A ) Consistent with these data, pair-fed mice had a reduced propensity for obesity compared to control monoclonal antibody-administered mice, but significantly greater than H4H17319P2-treated mice Figure 13B ) In contrast, similar lean mass-reducing effects were observed between pair-feeding and H4H17319P2 treatment relative to control monoclonal antibody-administered leptin-deficient mice Figure 13B ) No effects on bone mass, bone density, or bone mineral content were observed Figure 13B

[0356] 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 following induced leptin deficiency. Insulin tolerance tests were performed three days after monoclonal antibody administration or pair-feeding. As shown in Figure 8, mLeprECD-expressing Lepr Figure 13C mice showed reduced insulin sensitivity compared to control monoclonal antibody-administered Lepr hu / hu mice. Notably, H4H17319P2 treatment restored insulin sensitivity, while pair-feeding had no effect in the induced leptin-deficient Lepr hu / hu mice hu / hu Figure 13C

[0357] Finally, the effects of H4H17319P2 on circulating lipids were determined primarily through reduced food intake. While H4H17319P2, but not pair-feeding, significantly reduced plasma triglycerides in the absence of leptin, both H4H17319P2 and pair-feeding reduced total plasma cholesterol and HDL-C Figure 13D

[0358] In summary, the data indicate that in the induced leptin deficiency model, H4H17319P2 not only reversed hyperphagia and obesity, but also improved insulin resistance. Notably, while reduced food intake contributed to the reduced body weight and propensity for obesity observed with H4H17319P2, reduced food intake was insufficient to improve insulin sensitivity or reduce plasma triglyceride levels. ​​​​

[0359] Example 16: H4H17319P2 reverses hyperglycemia, insulin resistance, dyslipidemia, and hepatic steatosis in lipodystrophy mice.

[0360] Then, H4H17319P2 was tested to determine whether it could alleviate excessive appetite, metabolic dysfunction, and hepatic steatosis in mice with secondary leptinemia due to systemic lipodystrophy. To test this hypothesis, aP2-nSrebp1c was used. Tg / + Mice (exhibiting phenotypic features of generalized lipodystrophy) (Shimomura et al., 1998) and Lepr hu / hu Mice mating. aP2-nSrebp1c Tg / + Mice express nuclear Srebp1c in adipose tissue via the aP2 promoter and this expression was used in a classic experiment that first demonstrated that leptin could resolve metabolic complications caused by low leptin levels due to systemic lipodystrophy (Shimomura et al., 1999). Male aP2-nSrebp1c Tg / + Lepr hu / hu Mouse (Tg) mouse ratio aP2-nSrebp1c + / + Lepr hu / hu (Non-Tg) Animal weight ( Figure 15A However, consistent with previous reports, Tg mice showed reduced obesity tendency and lower leptin levels compared to non-Tg mice. Figure 15A Compared to non-Tg mice, aP2-nSrebp1c 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. Figure 15B and 15C ).

[0361] Male lipodystrophy Tg mice received either a control monoclonal antibody or H4H17319P2 once weekly at a dose of 10 mg / kg (subcutaneous). Additionally, male non-Tg mice received a control monoclonal antibody once weekly at a dose of 10 mg / kg (subcutaneous) as a reference for wild-type metabolic parameters. Prior to treatment on day 0, Tg mice showed a significant increase in body weight compared to non-Tg mice. Figure 14A Three days after the start of treatment, the body weight of lipodystrophy mice receiving H4H17319P2 was significantly lower than that of lipodystrophy mice given the control monoclonal antibody. Figure 14A Therefore, Tg mice given the control monoclonal antibody exhibited excessive appetite and consumed more food than non-lipid-malnourished non-Tg mice given the control monoclonal antibody.Figure 14A ). However, Tg mice treated with H4H17319P2 consumed less food than Tg mice administered the control monoclonal antibody ( Figure 14A ).

[0362] To determine the basis for the observed changes in body weight, body composition was quantified by micro-CT. These analyses showed that lean mass differences were primarily responsible for the body weight changes between genotype and treatment. Prior to treatment (Day -5), lean mass was significantly increased in lipodystrophic Tg mice compared to non-Tg mice ( Figure 14B ). Conversely, fat mass was reduced in Tg mice compared to non-Tg mice prior to administration (Day -5) ( Figure 14B ). After 4 weeks of once weekly administration, H4H17319P2 significantly reduced fat mass and lean mass in Tg mice compared to the fat mass and lean mass of Tg mice administered the control monoclonal antibody ( Figure 14B ). Skeletal mass and bone mineral content were elevated in Tg mice relative to wild-type mice, but were not significantly elevated in any of the treatment groups prior to administration ( Figure 16A ). Furthermore, no differences in skeletal mass or bone mineral content were observed between lipodystrophic mice administered the control monoclonal antibody or H4H17319P2 ( Figure 16A ). Similarly, no significant genotype or treatment-related effects on bone density were detected ( Figure 16A ). These data show that H4H17319P2 reduces lean mass and fat mass in lipodystrophic mice, but does not affect skeletal mass, bone mineral content, or bone density.

[0363] In line with previous findings, lipodystrophic Tg mice showed significantly higher blood glucose compared to non-Tg mice prior to treatment on Day 0 ( Figure 14C ). Three days after treatment with H4H17319P2, blood glucose levels were normalized in Tg mice and were different from the blood glucose levels of non-Tg mice. Notably, Tg mice maintained normal blood glucose through the end of the study with once weekly H4H17319P2 treatment, while Tg mice receiving the control monoclonal antibody remained hyperglycemic throughout the study ( Figure 14C ). Accordingly, the percentage level of hemoglobin Alc (HbAlc) was reduced in lipodystrophic mice on Day 28 compared to pre-treatment or control monoclonal antibody administration ( Figure 14C). In addition, the reduction in blood glucose levels by H4H17319P2 treatment was associated with improved insulin sensitivity. On day 23, insulin tolerance testing showed that control monoclonal antibody-administered lipodystrophic mice were insulin resistant, while H4H17319P2-treated lipodystrophic mice had insulin sensitivity equivalent to control monoclonal antibody-administered non-Tg mice ( Figure 14D ). Overall, these results indicate that H4H17319P2 reduced hyperglycemia, insulin resistance, and lowered HbAlc levels in a systemic lipodystrophic mouse model.

[0364] Plasma chemistry analysis performed at the end of the study (day 28) further revealed that H4H17319P2 reduced hypertriglyceridemia and hypercholesterolemia in systemic lipodystrophic mice. At the end of the study, plasma levels of triglycerides, total cholesterol, and LDL-C were significantly elevated in Tg mice that received control monoclonal antibody compared to non-Tg mice that also received control monoclonal antibody ( Figure 1 E). Notably, plasma triglyceride and cholesterol levels were significantly reduced in Tg mice treated with H4H17319P2. Thus, H4H17319P2 improved dyslipidemia in mice with systemic lipodystrophy.

[0365] In addition to diabetes, insulin resistance, and dyslipidemia, patients with systemic and localized lipodystrophy can also develop nonalcoholic fatty liver disease, including hepatic steatosis. Therefore, the effect of H4H17319P2 on liver enzyme levels, liver weight, and liver steatosis in lipodystrophic mice was investigated. On day 28, Tg mice that received control monoclonal antibody showed significantly increased circulating levels of ALT and AST compared to H4H17319P2-treated Tg mice or non-Tg mice that were administered control monoclonal antibody ( Figure 14F ). Importantly, the improved liver enzyme profile was associated with favorable effects on liver weight and liver steatosis ( Figure 14G ). Specifically, livers from lipodystrophic mice that received control monoclonal antibody were significantly heavier than livers from non-Tg mice that received control monoclonal antibody, with a weight of 3.6 ± 0.7 g ( Figure 14G ). Livers from control monoclonal antibody-administered Tg mice also had higher triglyceride content compared to both control monoclonal antibody-administered non-Tg mice and H4H17319P2-treated lipodystrophic mice ( Figure 14G ). Notably, livers from H4H17319P2-treated Tg mice increased in weight by 0.6 ± 0.1 g and did not show increased liver triglyceride content compared to livers from control monoclonal antibody-administered non-Tg mice ( Figure 14G). Hepatic steatosis was also apparent from hematoxylin and eosin-stained liver sections, where H4H17319P2 improved liver steatosis Figure 14G

[0366] Example 17: H4H17319P2 activates hypothalamic STAT3 signaling

[0367] LEPR activation in the arcuate nucleus of the hypothalamus plays a key role in the control of energy and metabolic balance (Coppari et al., 2005; Cowley et al., 2001). Since H4H17319P2 alleviated the adipisic Lepr hu / hu Hyperphagia and metabolic comorbidities in mice, it was further explored whether H4H17319P2 induced STAT3 activation in the arcuate nucleus similar to leptin. Immunostaining for pSTAT3 Y705 was performed on matched brain sections obtained from male adipisic Tg mice that received a single dose of control or 10 mg / kg (s.c.) of H4H17319P2, or a continuous infusion of 30 pg / d (s.c.) of human leptin for 3 days. The leptin dose was chosen because previous studies showed that 5 pg / d (s.c.) was effective in adipisic mice (Shimomura et al., 1999) and maximal efficacy was observed in mice at 10 to 42 pg / d (s.c.) (Denroche et al., 2013; Halaas et al., 1997; Harris et al., 1998). These analyses showed that in adipisic mice that received control monoclonal antibody, few cells in the arcuate nucleus showed pSTAT3 Y705 staining. In contrast, both leptin and H4H17319P2 induced pSTAT3 Y705 in the arcuate nucleus and similar numbers of pSTAT3 Y705 + Cells Figure 17A While the initial focus was on the arcuate nucleus, a circumventricular organ that lacks a blood-brain barrier, because of its established role in mediating the effects of leptin and its location proximal to the median eminence, it was noted that both leptin and H4H17319P2 induced pSTAT3 Y705 in the ventromedial hypothalamus. However, H4H17319P2 induced pSTAT3 staining in a greater number of ventromedial hypothalamic cells compared to that detected with leptin treatment Figure 17A In summary, these data show that leptin and H4H17319P2 induced pSTAT3 Y705 in similar numbers of cells in the arcuate nucleus, while H4H17319P2 had a more pronounced effect in the ventromedial hypothalamus.

[0368] ​A drawback of monoclonal antibodies as a CNS therapy is their limited ability to cross the blood brain barrier, with ~0.1% of the circulating concentration detected in the CSF (Zuchero et al., 2016). Despite this limitation, it was determined herein that H4H17319P2 not only induced STAT3 phosphorylation in the arcuate nucleus of the hypothalamus, but also stimulated STAT3 signaling in the ventromedial hypothalamus. The arcuate nucleus has been reported to be supplied by fenestrated 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 to suggest that the ventromedial hypothalamus has access to direct exposure to blood-borne molecules. While not wishing to be bound by theory, a possible explanation is that the monoclonal antibody reaches the ventromedial hypothalamus by diffusion from the arcuate nucleus. Alternatively, the H4H17319P2 antibody can exhibit signaling properties that are different from leptin. While functional LEPR-b is expressed in the ventromedial hypothalamus, another possibility is that the H4H17319P2 monoclonal antibody can indirectly stimulate ventromedial hypothalamic STAT3 signaling. Unexpectedly, however, H4H17319P2 induced STAT3 signaling in more neurons in the ventromedial hypothalamus compared to leptin.

[0369] Example 18: Effect of H4H17319P2 on reducing metabolic and liver dysfunction in lipodystrophic mice is at least comparable to leptin

[0370] Since H4H17319P2 is similar to or better than leptin in engaging STAT3 signaling in arcuate nucleus and ventromedial hypothalamic cells, the efficacy of H4H17319P2 and leptin treatment was compared in lipodystrophic mice. Male Tg mice received a 10 mg / kg (s.c.) dose of control monoclonal antibody once a week or a continuous infusion of 30 pg / d (s.c.) of leptin for 14 days. As a reference, male non-Tg mice received a 10 mg / kg (s.c.) dose of control monoclonal antibody once a week. Prior to treatment on day 0, Tg mice had a significantly higher blood glucose ( Figure 17B ) compared to non-Tg mice. Blood glucose levels were reduced to the same extent by H4H17319P2 or leptin infusion compared to control monoclonal antibody administration ( Figure 17B ). Two days after the start of H4H17319P2 or leptin treatment, blood glucose levels were reduced and remained low until the end of the study ( Figure 17B ). To test whether improved insulin sensitivity contributed to the blood glucose lowering effect, an insulin tolerance test was performed on day 9. Indeed, the insulin tolerance test showed that Tg mice administered with control monoclonal antibody were insulin resistant, while Tg mice treated with H4H17319P2 and leptin were insulin sensitive ( Figure 17B). No differences between H4H17319P2 and leptin treatment were observed in terms of blood glucose lowering or insulin sensitivity.

[0371] Consistent with previous data, H4H17319P2 significantly promoted body weight loss in fat malnutrition mice relative to control monoclonal antibody administration, starting at day 4 post-treatment ( Figure 17C ). H4H17319P2 reduced cumulative food intake in Tg mice compared to control monoclonal antibody ( Figure 17C ). Interestingly, while leptin reduced body weight and food intake in Tg mice, it caused less body weight loss compared to that observed with H4H17319P2 treatment ( Figure 17C ).

[0372] H4H17319P2 had a better beneficial effect than leptin in reducing plasma lipids and resolving hepatomegaly in fat malnutrition mice. Plasma chemistry analysis at day 13 showed that H4H17319P2, but not leptin, reduced plasma triglyceride and cholesterol levels in Tg mice compared to control monoclonal antibody ( Figure 17D ). There were no significant changes in other lipids in Tg mice with H4H17319P2 treatment compared to control monoclonal antibody administration. H4H17319P2 also reduced liver mass and resolved hepatic steatosis in Tg mice compared to control antibody treatment ( Figure 17E ). While these effects were similar in leptin-treated mice, the reduction in liver mass was greater with H4H17319P2 ( Figure 17E ). Liver mass was normalized by H4H17319P2, but not leptin treatment, and was similar to liver mass in non-Tg mice that received control monoclonal antibody ( Figure 17E ).

[0373] Example 19: H4H17319P2 reduces body weight and adiposity in lean mice and in normal and high body fat monkeys

[0374] In vitro binding and functional assay experiments showed that H4H17319P2 does not compete for leptin binding and induces more LEPR activation in the presence of leptin than leptin alone. Therefore, it was determined whether H4H17319P2 could cause weight loss in the context of normal body weight homeostasis. Male Lepr hu / hu Mice were administered a single dose of control monoclonal antibody (10 mg / kg, s.c.) or H4H17319P2 at 3 or 10 mg / kg on day 0. After treatment with H4H17319P2 at both dose levels, a significant decrease in body weight was observed compared to control monoclonal antibody at day 1 and day 2 post-treatment ( Figure 18A). Thus, both dose levels of H4H17319P2 reduced food intake relative to control monoclonal antibody administration Figure 18A Body weight changes were associated with reductions in fat mass but not lean mass Figure 18B At both dose levels, H4H17319P2 treatment resulted in a -32% reduction in fat mass, while control monoclonal antibody administration resulted in minimal changes in fat mass of -2% Figure 18B Notably, no significant differences in lean mass were observed between control monoclonal antibody and H4H17319P2 treatment Figure 18B While both dose levels of H4H17319P2 resulted in similar magnitude of changes in body weight and fat mass, the duration of the effects differed, which can reflect differences in pharmacokinetics Figure 18A and 18B

[0375] The effects of H4H17319P2 treatment on promoting weight loss in non-human primates were then assessed. First, the effects of H4H17319P2 on lean food fed cynomolgus monkey body weight were determined. Monkeys received 3 mg / kg or 10 mg / kg control solution H4H17319P2 once per week for 13 weeks. H4H17319P2 treatment resulted in dose-dependent reductions in body weight compared to monkeys receiving control solution Figure 18C After 13 weeks of study, monkeys administered control solution gained 9.7 ± 0.9% of their initial body weight, while 3 and 10 mg / kg H4H17319P2 treatment resulted in minimal changes in body weight of 0.3 ± 1.6% and a -6.0 ± 1.6% body weight loss, respectively Figure 18C

[0376] Since H4H17319P2 dose-dependently reduced body weight in lean monkeys, the effects of H4H17319P2 treatment on body weight and body composition were tested in high body fat food fed cynomolgus monkeys. Animals received 30 mg / kg control solution or LEPR agonist antibody once per week for 2 weeks. H4H17319P2 reduced body weight compared to control solution injections. At the end of the study (day 56), monkeys receiving two doses of H4H17319P2 had a 3.6 ± 1.9% reduction in body weight from baseline, while monkeys receiving control solution had a 6.4 ± 1.5% increase in body weight. In parallel, H4H17319P2 treatment reduced fat mass but not lean mass compared to control solution administration as quantified by dual energy X-ray absorptiometry (DEXA) Figure 18D ​​). By the end of the study, the propensity for obesity in monkeys receiving the control solution increased by 28.0 ± 1.5%. In contrast, the propensity for obesity in monkeys treated with the LEPR agonist H4H17319P2 decreased significantly by 13.7 ± 6.4% Figure 18D ). Together, these data indicate that H4H17319P2 antibodies promote weight loss in normal weight Lepr hu / hu selective loss of the propensity for obesity in mice to promote weight loss, and both tend to have high body fat non-human primates.

[0377] Example 20: Double-blind study in humans using anti-LEPR antibodies

[0378] Obesity affects 13% of the world’s population and is a risk factor for the development of type 2 diabetes, cardiovascular disease, various cancers, and orthopedic diseases. In Western European countries, more than 20% of individuals are obese (Ng et al., Lancet. 2014; 384:766-781), while in the United States, the prevalence of obesity has now exceeded 35% (Flegal et al., JAMA. American Medical Association. 2016; 315(21):2284-2291). Dietary and exercise habits are effective for some people, but often result in modest weight loss, while most people remain obese (defined as a body mass index [BMI] > 30 kg / m 2 ) or overweight (defined as a BMI equal to 25 to < 30 kg / m 2 ).

[0379] Current weight loss medications also have modest effects on body weight and / or poor safety / tolerability, which limits their use by payers, physicians, and patients (Zhang et al., Obes Sci Pract. 2016; 2(2): 104-114). Current standard of care pharmacological treatments, such as liraglutide, orlistat, naltrexone hydrochloride / bupropion hydrochloride, phentermine / topiramate, and lorcaserin hydrochloride can reduce body weight by an average of about 3% to 10% from baseline. Typically, weight loss plateaus and therapy 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 only available for the most severely obese patients (BMI > 40 kg / m 2 or BMI > 35 kg / m 2< 200,000 cases per year in the United States (American Society for Metabolic and Bariatric Surgery) and less than 150,000 cases per year in all of Europe (Angrisani et al. Obes Surg. 2017, 27:2279-2289). There is a large unmet medical need for safe and effective therapies to treat and prevent obesity and obesity-related metabolic comorbidities.

[0380] Leptin is a circulating fat-derived hormone that binds to leptin receptors (LEPR) in the hypothalamus and regulates control of food intake, energy expenditure, and glucose / lipid metabolism (Allison et al. J Endocrinol 2014, 223(1):T25-35). Individuals with primary leptin deficiency, due to a hyper-rare homozygous loss-of-function mutation in the leptin (LEP) gene or due to a homozygous mutation in LEPR, resulting in defective leptin signaling, present with 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 monogenic obese children and adult obese patients due to a leptin functional mutation resulted in significant weight loss and improvement in metabolic comorbidities (Farooqi et al. N Engl J Med 1999, 341(12):879-884) (Licinio et al. Proc Nat Acad Sci 2004, 101(13):4531-4536).

[0381] Secondary leptin deficiency also occurs in the rare disease lipodystrophy, which is caused by genetic or acquired loss of adipose tissue that produces leptin from various sites in the body.

[0382] Patients with generalized and localized 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). Daily subcutaneous delivery of recombinant human leptin has been shown to reduce hemoglobin Alc (HbAlc), glucose, triglycerides, and liver 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). Serum leptin levels in most patients with generalized lipodystrophy are <4 ng / mL, and subgroup analysis showed that leptin therapy reduced HbAlc and triglycerides (TG) more in patients with generalized and localized lipodystrophy with leptin levels <4 ng / mL (Brown et al., J Clin Endocrinol Metab. 2016; 101(12):4500-4511) (FDA Advisory Committee Meeting 2013).

[0383] Metreleptin (recombinant methionyl leptin; ) is currently approved in the United States through a Risk Evaluation and Mitigation Strategies (REMS) program for the treatment of complications of leptin deficiency in patients with generalized congenital and acquired lipodystrophy and is also approved in Japan for the treatment of all subtypes of lipodystrophy. The dose of metreleptin required for daily administration, adverse reactions include the risk of immunogenicity, about 85% of patients develop binding antibodies, about 9% of developed neutralizing antibodies cross-react with endogenous leptin (Chan, Clin Endocrinol. 2016; 85(1): 137-149).

[0384] In contrast to low leptin or leptin deficiency disorders, overweight or obese individuals generally have higher leptin levels, with a BMI of 30-35 kg / m2, and are resistant to the effects of leptin (Halaas et al., Science. 1995; 269(5224): 543-546). The leptin receptor is expressed in the hypothalamus, and the hypothalamus is the primary site of action of leptin. Leptin acts on the hypothalamus to suppress appetite and increase energy expenditure, and the hypothalamus is the primary site of action of leptin. Leptin acts on the hypothalamus to suppress appetite and increase energy expenditure. 2The median level in subjects in the range is 10 ng / mL in men and 24 ng / mL in women. (Ruhl and Everhart, Am J Clin Nutr. 2001; 74(3):295-301). The median level of circulating leptin levels in adult men and women with a normal BMI of 20-25 kg / m2is 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 to 9.5 ng / mL in men and 4.1 to 25 ng / mL in women (Quest Lab reference ranges). Numerous studies have shown a correlation between circulating leptin levels and BMI and percent body fat (Ruhl et al., Am J Clin Nutr. 2001, 74(3):295-301) (Considine et al., N Engl J Med. 1996, 334(5):292-295), although leptin levels are highly variable among individuals with similar BMIs (Buettner et al., J Endocrinol. 2002; 175(3):745-756). Leptin levels have diurnal variation (Gavrila et al., J Clin Endocrinol Metab. 2003, 88(6):2838-43) (Schoeller et al., J Clin Invest. 1997, 100(7): 1882-87) and fluctuate according to nutritional status. In normal weight and obese individuals, leptin levels decrease rapidly by 35-60% after 24 hours of fasting and continue to decrease after prolonged 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.Recombinant human leptin administration to obese subjects results in minimal weight loss (3% PBO weight loss) (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 endogenous leptin levels. Several population studies suggest that there can be a subset of obese patients with 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 the leptin receptor can not be saturated. A key question to address is whether restoration of leptin signaling would reduce appetite, food intake, and body weight in obese subjects with relatively low baseline leptin levels.

[0385] H4H17319P2 is a human anti-LEPR antibody that can act as a LEPR agonist and binds to human LEPR with nanomolar affinity. In preclinical studies, H4H17319P2 activated 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 a lipodystrophic humanized LEPR mouse. H4H17319P2 also reduced body weight and adiposity in an inducible leptin-deficient humanized LEPR mouse, but not in a diet-induced obese humanized LEPR mouse. In a good laboratory practice (GLP) toxicology study, lean cynomolgus monkeys were treated with H4H17319P2 by subcutaneous (3, 10, 30 mg / kg) or intravenous (100 mg / kg) injection once weekly for 13 weeks, and the results of H4H17319P2 exposure were inhibition of body weight gain or induction of body weight loss. Monkey body weight decreased by 8.7% (group mean compared to pre-dose body weight) in animals administered H4H17319P2, while body weight in control animals administered placebo concurrently increased by an average of 8.3% over the same period. The magnitude of body weight loss observed in H4H17319P2-treated animals did not appear to have a clear dose or exposure response. Body weight changes reversed upon cessation of exposure in the absence of a dose recovery period.

[0386] As described above, H4H17319P2 administration was well tolerated at all dose levels and by both routes of administration without any adverse clinical impact. Transient reductions in insulin and circulating absolute lymphocyte counts (T cell numbers) were observed approximately 30 days into treatment, but these findings were not observed at the end of the dosing period and the effect was not dose responsive. Lower thymus weight and reduced thymic cortical cellularity were observed in the H4H17319P2 treatment groups during treatment and were completely or partially reversed during the recovery period. The changes in thymus and transient reductions in T cell numbers can be associated with reduced nutrient intake and weight loss in monkeys that were in the growth phase and can not be observed in normal nutritional status adults.

[0387] This study is a first-in-human (FIH), randomized, double-blind, placebo-controlled, two-part study designed to evaluate the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of H4H17319P2 administered intravenously (IV) and subcutaneously in healthy participants. The objective of Part A is to evaluate the safety, tolerability, PK, and PD of single ascending intravenous and subcutaneous doses in healthy male and female subjects. Interim analysis of PK / PD, safety, and tolerability in Part A will be used to select the dose regimen evaluated in Part B. Subjects enrolled in Part A do not qualify for Part B. For Part B, new subjects who are overweight or obese will be enrolled to evaluate the safety, tolerability, PK, and PD of repeat doses of H4H17319P2 (single dose levels) or placebo for 12 weeks. The effects of H4H17319P2 on biomarkers such as food intake, appetite, body composition, and weight will be evaluated in 4 different cohorts defined by baseline leptin levels.

[0388] Objectives

[0389] The primary objective of this study is to evaluate the safety and tolerability of H4H17319P2 in healthy subjects. The secondary objectives of this study are:

[0390] • To characterize the pharmacokinetic (PK) profile of single and repeat doses of H4H17319P2;

[0391] • To estimate the effect of repeat doses of H4H17319P2 on body weight;

[0392] • To evaluate the effect of repeat doses of H4H17319P2 on free energy intake in overweight and obese subjects;

[0393] • To evaluate the effect of single and repeat doses of H4H17319P2 on soluble lipid-regulating protein (sLEPR and ANGPTL3) levels over time;

[0394] • To assess the immunogenicity of single and repeat doses of H4H17319P2.

[0395] Other exploratory objectives of this study are:

[0396] 1. To estimate the effect of a single dose of H4H17319P2 on body weight and serum / plasma glucose and lipid parameters

[0397] 2. To estimate the effect of repeat doses of H4H17319P2 over 12 weeks on the following parameters:

[0398] • Serum / plasma glucose and lipid parameters

[0399] • Patient reported assessment of appetite that will influence feeding behavior (e.g., hunger, satiety, and satiation)

[0400] • Total and distribution of fat and lean body mass by dual X-ray absorptiometry (DXA) imaging

[0401] • Quantification of subcutaneous and visceral fat (including liver fat) by magnetic resonance imaging (MRI)

[0402] • Other exploratory biomarkers include leptin, thyroid hormones (T3, T4, thyroid stimulating hormone [TSH]), luteinizing hormone (LH), testosterone, estradiol, cortisol, and adiponectin.

[0403] Rationale

[0404] Part A is a single ascending dose FIH design in which up to 88 healthy subjects are randomized 3: 1 to H4H17319P2 versus placebo to assess the safety, tolerability, and pharmacokinetics of single ascending doses of H4H17319P2 with a 112-day follow-up period in up to 7 ascending single dose cohorts (up to 5 intravenous and 2 subcutaneous). There will be 8 subjects in each of the seven ascending single dose cohorts, randomized to receive H4H17319P2 or placebo at each dose level (6 active drug: 2 placebo). The study design also includes 2 additional optional cohorts (16 subjects randomized to receive H4H17319P2 or placebo (12 active drug: 4 placebo in each cohort)). If interim analyses of the ascending dose cohorts indicate that a covariate (e.g., age, body weight, or gender) can have an impact on PK characteristics, optional cohorts can be included to collect additional data on one or more doses (up to the maximum dose in this study, 30 mg / kg intravenous) to better estimate the impact of the covariate on PK.

[0405] Part A of the study includes a screening period (Day -21 to -2), a pre-baseline visit (Day -1), in which subjects will be allowed to be hospitalized for a period of 2 days (for subjects receiving intravenous and subcutaneous doses in the safety dose range) or a clinical stay of 1 day (for subjects receiving subcutaneous administration), a follow-up period (Day 3 to Day 113), and the end of the study visit (Day 113).

[0406] Safety and tolerability assessments are the primary objective of Part A, and safety will be assessed carefully throughout the study. The starting dose and maximum dose in this study are expected to be approximately 10,000 and approximately 20 times lower, respectively, than the exposures observed in the toxicology studies. Throughout the clinical study, safety assessments include vital signs, physical examinations, electrocardiograms (ECG), laboratory tests including hematology / differential, and monitoring of adverse events (AEs). Body weight will also be assessed. Pharmacodynamic markers will also be collected in this study, but based on the minimal impact of meptinide observed on body weight (<1 kg) in lean individuals, no meaningful changes in PD markers are expected in the lean / overweight subject population (Heymsfield, 1999).

[0407] Pharmacodynamic markers include body weight, metabolic parameters (glucose, lipids), and ANGPTL3, a potential marker that can be regulated by leptin and / or insulin (Muniyappa, 2017) (Nidhina Haridas, 2015). Body weight will be assessed carefully at each visit. If single dose H4H17319P2 treatment has a clinically meaningful impact on body weight in lean / overweight subjects, the timing schedule for safety / dose escalation decisions can be modified, and additional safety data collected before making dose escalation decisions (e.g., waiting for Day 15 safety assessments).

[0408] Part B is a repeat-dose study at single dose levels for a treatment period of 12 weeks to assess the safety, PK, and impact of H4H17319P2 on body weight in overweight / obese subjects. Several population studies have shown that leptin levels are relatively low in obese patients (Ruhl, 2001) (Buettner, 2002), and that leptin receptors can be unsaturated. One key question to be addressed in Part B is whether restoration of leptin signaling reduces appetite, food intake, and body weight in overweight or obese subjects with relatively low baseline leptin levels.

[0409] Accordingly, Part B will assess the impact of H4H17319P2 on body weight change and body weight, food intake, metabolic parameters, and body composition in subjects with relatively low baseline leptin levels. Overweight or obese (BMI range 25-40 kg / m 2Healthy subjects of the H4H17319P1 study will be enrolled into 4 unique cohorts defined by leptin level prescreening. Enrollment of the 4 unique cohorts will be conducted to ensure adequate number of subjects are studied within a relatively lower baseline leptin level and BMI range. Stratification by cohort will be conducted at randomization to H4H17319P2 versus placebo. Up to approximately 20 subjects will be enrolled in each of the 4 cohorts for a total sample size of up to 81 subjects in Part B, as described below. Leptin level and BMI will be measured at the prescreening visit to preliminarily assess eligibility for the study and for 1 of the 4 cohorts. Study enrollment will be conducted if the subject is screened in. A subject will not be eligible for enrollment if enrollment in a particular cohort has reached the allowed maximum. Given the low prevalence of obesity in low leptin level, certain cohorts can be difficult to enroll; the Sponsor can elect to stop enrollment in a particular cohort.

[0410] The 4 cohorts in Part B are defined as follows:

[0411] • Cohort 1 : Male and female subjects with prescreening BMI between 28.0 and 40.0 kg / m2, inclusive, and prescreening fasting leptin level < 5 ng / mL 2

[0412] • Cohort 2: Male and female subjects with prescreening BMI of 25.0 to < 28 kg / m2and prescreening fasting leptin level < 5 ng / mL 2

[0413] • Cohort 3: Male subjects with prescreening BMI between 28.0 and 40.0 kg / m2, inclusive, and prescreening fasting leptin level between 5.0 and 8.0 ng / mL, inclusive 2

[0414] • Cohort 4: Female subjects with prescreening BMI between 28.0 and 40.0 kg / m2, inclusive, and prescreening fasting leptin level between 5.0 and 24.0 ng / mL, inclusive 2

[0415] Note: Eligibility for one of the above cohorts is based on BMI and leptin level at the prescreening visit. If the BMI and / or leptin level is not within the range defined for the cohort, the subject will not be eligible for enrollment. If the prescreening BMI or leptin level is at the threshold to enroll in one or more cohorts, a BMI and leptin measurement can be repeated once within the prescreening window. For the repeat measurement, the lowest leptin level will be used to enroll in one of the cohorts. A subject will not be eligible for enrollment if enrollment in a particular cohort has reached the allowed maximum.

[0416] ​​​​Initiation of Part B will be triggered by an interim analysis of the available safety, PK, and PD data from Part A to select the appropriate dose, frequency of dosing, and route of administration (intravenous or subcutaneous) for Part B. Subjects enrolled in Part A will not be eligible to participate in Part B. Part B study design includes a pre-screening period (Day -60 to -14 for assessment of BMI and fasting leptin levels and eligibility for 1 of 4 cohorts), a screening period (Day -32 to -14), a baseline period consisting of (Day -29 to -1), a treatment period (Day 1 to 85), and a post-treatment follow-up period (Day 107 to 191). Subjects will be allowed a 2-day clinical stay for accurate appetite and ad libitum food intake assessments at 2 time points during the baseline period (scheduled between Day -14 to -1) and the treatment period (Day 29 to 30 and Day 84 to 85). A clinical stay is necessary to assess precise caloric intake in an ad libitum food assessment in a controlled environment where there are cues that can influence food quantity other than appetite (e.g., time of day, predatory other food behaviors, portion size, etc.). Subjects will also be allowed a 2-day clinical stay on Day -1. On Day 1 of the treatment period, subjects will receive the first dose of study drug or placebo, have blood sampling for serial PK and other laboratory measurements, and an overnight on Day 2 to complete the 24-hour PK assessment.

[0417] Visits will be conducted during the treatment period so that blood is drawn once a week. The dose and frequency of study drug will be determined by an interim analysis of the PK data from Part A, possibly once every 4 weeks or every 2 weeks (with a maximum frequency of no more than once a week).

[0418] Body composition by DXA and quantification of liver, abdominal, and thigh fat by MRI will be performed at baseline, near the end of treatment, and during follow-up. After the treatment period, subjects will be evaluated for safety and PD effects 16 weeks after drug discontinuation. Safety and tolerability assessment of repeat dosing at the single dose level (based on safety and tolerability from Part A) will be the primary objective of Study B. Throughout the study, safety assessments will include vital signs, physical examinations, ECGs, laboratory tests, AE monitoring. Anti-drug antibodies will also be assessed. In addition, secondary objectives will be carefully assessed throughout the study to assess body weight, ad libitum food intake, and metabolic parameters such as blood glucose and lipids.

[0419] Pharmacodynamic endpoints in Part B include baseline and on-treatment assessments of parameters that can be influenced by leptin receptor signaling. These PD assessments include patient reported appetite measures, quantitative measures of food intake under controlled inpatient conditions, precise measures of body composition and fat mass by DXA, and precise measures of body weight using calibrated scales. In addition, metabolic parameters such as leptin, glucose, insulin, homeostatic model assessment estimated insulin resistance (HOMA-IR), HbAlc, lipids will be measured at baseline, on-treatment, and at the end of treatment to assess the effect of H4H17319P2 on insulin sensitivity and lipid metabolism. MRI of the liver will also be performed at baseline and post-treatment to determine if H4H17319P2 has an effect on hepatic steatosis.

[0420] Rationale for pharmacodynamic and biomarker variables

[0421] Free food intake assessment (Part B only)

[0422] It is hypothesized that treatment with H4H17319P2 will increase hypothalamic leptin receptor signaling and have downstream effects in neurons that influence feeding behavior. H4H17319P2 treatment resulted in a significant reduction in food intake and body weight in leptin-deficient mice that were induced. While food intake assessments were not performed in the cynomolgus monkey study, H4H17319P2 treatment had a significant impact on body weight and fat mass. In the current study, the effect of H4H17319P2 on food intake in overweight / obese subjects with relatively low leptin levels will be assessed using a rigorous quantitative inpatient free food intake assessment at baseline and 2 time points during treatment, as previously described (Krishna, 2009) (Addy, 2008). Briefly, subjects will be admitted to the clinical trial unit after an overnight fast and provided with standardized breakfast, lunch, and dinner to establish a standardized baseline energy intake. After an overnight fast, subjects will be provided with free breakfast, lunch, and dinner to conduct a quantitative / energy intake. Subjects will consume all test foods in specialized rooms that mask perception of temporal / circadian and social cues.

[0423] Foods of known caloric density will be provided in 4-5-fold excess portions and in a manner that masks the total number of calories consumed. Subjects will be asked to eat as much as desired. Meals will be weighed before and after the food intake assessment to quantify food intake and calculate calories ingested.

[0424] Appetite assessment (Part B only)

[0425] The appetite effects of leptin are central to its mechanism of action, and thus are approximated

[0426] Evaluation of the expected mechanism of action of H4H17319P2. Patients with LEP mutations or lipodystrophy syndromes that cause low leptinemia exhibit loss of appetite, leading to poor food intake and obesity. Treatment of these individuals with metreleptin can increase satiety and decrease food intake (Farooqi, 1999) (Farooqi, 2007) (McDuffie, 2004). The effect of H4H17319P2 on appetite will be assessed using a questionnaire that measures various components of appetite (e.g., hunger, satiety) (Flint, 2000) (Dalton, 2015) that will be completed daily at baseline and during follow-up.

[0427] Appetite will be assessed before and after a standard caloric load during clinic visits, and a daily appetite questionnaire will be completed at baseline, treatment, and during follow-up.

[0428] ANGPTL3

[0429] Angiopoietin-like protein 3 (ANGPTL3) regulates lipoprotein levels, such as TG, low-density lipoprotein C (LDL-C) by inhibiting lipoprotein lipase (reviewed in Tikka, 2016). Levels of ANGPTL3 can be modulated by food intake, leptin, and / or insulin (Minicocci, 2012) (Nidhina, 2015). Levels of ANGPTL3 are elevated in patients with lipodystrophy and decrease after treatment with metreleptin (Muniyappa, 2017), and decreases in ANGPTL3 can mediate increased lipase clearance of TG-rich lipoproteins. ANGPTL3 and TG levels are also increased in mouse models of lipodystrophy and return to normal after treatment with H4H17319P2. Thus, ANGPTL3 can be a pharmacodynamic marker that can decrease in increased leptin receptor signaling. ANGPTL3 will be measured at baseline and after treatment at several time points in Part A and Part B.

[0430] Soluble LEPR

[0431] Leptin circulates in the blood as a free entity and can also bind to sLEPR, which is shed from the LEPR extracellular domain (Sinha, 1996) (Lammert, 2001). Soluble LEPR can modulate the bioavailability and / or clearance of leptin (Lou, 2010). Target saturation can depend on the saturation of sLEPR, so sLEPR levels can affect linear and non-linear target-mediated changes in pharmacokinetics. Thus, sLEPR will be measured at baseline and at various time points during treatment (corresponding to sampling for antibody pharmacokinetics) in Parts A and B.

[0432] Imaging (Part B only)

[0433] DXA and MRI imaging will be performed at baseline, near the end of treatment, and at the end of the study to estimate the overall and regional fat distribution, subcutaneous and visceral fat, in the thigh / abdominal region and liver fat content, changes from baseline. In a preliminary study in cynomolgus monkeys, preclinical data suggest that H4H17319P2 reduces body weight by decreasing total fat mass without affecting lean body mass (assessed by DXA). DXA has been shown to be a useful quantitative biomarker of total fat mass and regional fat distribution in clinical studies. For example, in patients with partial lipodystrophy, significant differences in regional fat distribution were observed using DXA (% fat trunk to % fat leg (fat mass ratio [FMR]) of 1.78 ± 0.53) compared to normal subjects (Aijluni, 2017). DXA has also been used to quantify changes in total and regional fat in trials of weight loss drugs such as GLP-1 agonists (Jendle, 2009) and cognitive therapy (Ponti, 2018).

[0434] Magnetic resonance imaging (MRI) has been shown to be an effective means of imaging entire organs by specialized pulse sequences or measuring fat content in multiple tissues including liver (Aijluni, 2017) and muscle (Burakiewicz, 2017) by spectroscopy in limited volumes of tissue. MRI has also shown high-contrast images for measuring abdominal adipose volume and distribution (Klopfenstein, 2012), where patients with type 2 familial lipodystrophy had 2.5-fold higher visceral fat percentage than control patients (Al-Attar, 2007). Fat content in muscle can also be assessed by quantitative MRI as demonstrated by the Multimodal Muscle Disease Institute, where spectroscopy and pulse sequences that allow anatomical imaging and fat content estimation have shown to be promising in assessing disease progression (Burakiewicz, 2017).

[0435] In this study, MRI will be performed on the abdomen and thighs to quantify regional changes in subcutaneous, visceral, and liver fat at baseline and after treatment with H4H17319P2

[0436] Rationale for dose selection

[0437] Based on efficacy, safety, and

[0438] Pharmacokinetic data from preclinical studies in a mouse model of lipodystrophy and monogenic obesity and monkey toxicology studies were used to select intravenous and subcutaneous doses for Part A. The maximum dose in this FIH study will not exceed 30 mg / kg, and the starting dose will be approximately 0.3 mg / kg. In GLP toxicology studies in cynomolgus monkeys, H4H17319P2 was most tolerable at intravenous doses of up to 100 mg / kg for 12 weeks, with no observed adverse effect level (NOAEL) of 100 mg / kg. Based on projected human serum exposures of H4H17319P2, the maximum dose of 30 mg / kg planned in this FIH study is less than twenty-fold lower than the NOAEL. The initial starting dose of 0.3 mg / kg is more than 10,000-fold lower than the highest dose tested in the GLP toxicology studies and is projected to provide human H4H17319P2 concentrations above the limit of quantitation, thus providing useful PK information.

[0439] Objectives for Part B dose selection include ensuring broad exposure to assess tolerability and to facilitate characterization of exposure-response relationships, as well as to characterize pharmacokinetic properties that enable characterization of linear and nonlinear pharmacokinetics. In addition, dose selection should ensure exposure above and below putative PD marker thresholds that could raise concerns (e.g., sLEPR saturation). Doses for Part B will be based on interim safety, PK, and PK / PD data from Part A. Selection of dose levels, dosing intervals, and routes of administration (intravenous or subcutaneous) will be based on safety, pharmacokinetic, and, if applicable, PK / PD data from Part A, as well as preclinical studies in animals. Doses in Part B will not exceed those evaluated in Part A and can be administered once every 4 weeks or once every 2 weeks, but not more frequently than once weekly.

[0440] Dose regimens will not exceed exposures observed in toxicology studies.

[0441] Criteria

[0442] Up to 169 subjects (up to 88 in Part A and up to 81 in Part B) will be enrolled in the target population: healthy lean or overweight men and women for Part A, and healthy overweight or obese men and women (with varying baseline leptin levels) for Part B.

[0443] Key inclusion criteria

[0444] Subjects must meet the following criteria at screening to be eligible for enrollment in Part A of the study:

[0445] 1. Men and women 18 to 50 years of age, inclusive

[0446] 2. Body mass index (BMI) of 18.5 to < 30.0 kg / m22

[0447] 3. The subject is judged by the Investigator to be in good health, with no major comorbidities based on medical history, physical examination, laboratory safety tests performed at screening and / or prior to administration of the initial dose of study drug

[0448] 4. Willing and able to comply with outpatient visits, study-related procedures, and dietary instructions

[0449] 5. Willing to maintain usual dietary and exercise habits throughout the study

[0450] 6. Able and willing to provide a signed informed consent form

[0451] Subjects must meet all of the following screening criteria (except for BMI and leptin eligibility (determined at pre-screening)) to be eligible for inclusion in Part B of the study:

[0452] 1. Males and females 18 to 65 years of age, inclusive

[0453] 2. Have a body mass index (BMI) and fasting leptin level at the pre-screening visit as defined in one of the following cohorts. If enrollment in a particular cohort has reached the maximum allowed, the subject will not be eligible for inclusion.

[0454] 3. The subject is judged by the Investigator to be free of major comorbidities based on medical history, physical examination, laboratory safety tests performed at screening and / or prior to administration of the initial dose of study drug. Subjects can have a history of mild hyperlipidemia and / or mild hypertension, but should be on a stable dose of lipid-lowering or blood pressure-lowering medication for at least 2 months prior to screening

[0455] 4. Willing and able to comply with outpatient visits, study-related procedures, and dietary instructions

[0456] 5. Willing to maintain usual dietary and exercise habits throughout the study

[0457] 6. Able and willing to provide a signed informed consent form

[0458] Exclusion criteria

[0459] Subjects who meet any of the following criteria at screening will be excluded from Part A of the study:

[0460] 1. Have a history of clinically significant cardiovascular (e.g., history of hypertension, myocardial infarction, stroke, peripheral vascular disease, heart failure, arrhythmia), respiratory, hepatic, renal, gastrointestinal, endocrine (e.g., hyperlipidemia), hematologic, or neurological disease.

[0461] Type 2 diabetes mellitus or prediabetes history, or FBG > 100 mg / dL at screening, or HbAlc > 5.7% at screening.

[0462] 3. Fasting LDL-C > 130 mg / dL, TG > 250 mg / dL

[0463] 4. Clinically significant abnormal complete blood count, clinical chemistry, urinalysis, or urine drug screen test at screening. Minor deviations in laboratory results are allowed. Note: During screening, any abnormal laboratory test result can be repeated once (e.g., creatine phosphokinase (CPK) within 3 times of the upper limit of normal (ULN) and a suspected cause due to strenuous physical activity).

[0464] Subjects who meet any of the following criteria at screening will be excluded from Part B of the study:

[0465] 1. History of clinically significant cardiovascular disease (e.g., history of moderate-severe hypertension, myocardial infarction, stroke, peripheral vascular disease, heart failure, arrhythmia), respiratory, liver, renal, gastrointestinal, endocrine, hematologic, or neurological disease.

[0466] 2. Type 2 diabetes mellitus or history, or FBG > 126 mg / dL at screening, or HbAlc > 6.5% at screening. Diagnosis of “prediabetes” is allowed.

[0467] 3. Fasting LDL-C > 160 or TG > 500 mg / dL

[0468] 4. Clinically significant abnormal complete blood count, clinical chemistry, urinalysis, or urine drug screen test at screening in addition to the mild lipid or glycemic abnormalities described above. Minor deviations in laboratory results are allowed. Note: During screening, any abnormal laboratory test result can be repeated once (e.g., CPK within 3 times of the upper limit of normal (ULN) and a suspected cause due to strenuous physical activity).

[0469] 5. Dietary habits restricted (e.g., vegan or vegetarian), aversion to specific food categories used in the assessment of food intake, or dietary behaviors that would interfere with or confound the interpretation of the assessment of food intake, appetite, or food control

[0470] Subjects who meet any of the following criteria at screening will be excluded from Part A and Part B of the study:

[0471] 1. Hospitalization (i.e., > 24 hours) for any reason within 60 days after the screening visit

[0472] 2. The investigator believes that the subject has any physical examination finding and / or any history of illness that may confound the results of the study or expose the subject to additional risk because of his / her participation in the study.

[0473] 3. History of hypothalamic amenorrhea or lipodystrophy.

[0474] 4. Body weight change of more than 5% in the past 3 months prior to screening.

[0475] 5. Prior history of bariatric surgery (e.g., sleeve gastrectomy, gastric bypass surgery, lap band, etc.).

[0476] 6. Procedure for weight loss (e.g., liposuction) or body sculpting in the past 6 months.

[0477] 7. Treatment with weight loss medication (over-the-counter [OTC] or prescription) in the past 3 months (e.g., lorcaserin, phentermine / topiramate, naltrexone hydrochloride / bupropion hydrochloride, liraglutide).

[0478] 8. History of major psychiatric illness, eating disorder (e.g., bulimia, anorexia).

[0479] 9. Current smoker or former smoker (cigarettes or e-cigarettes) who stopped smoking within 3 months prior to screening.

[0480] 10. History of recreational drug (including marijuana) or alcohol abuse (>2 drinks per day) within the past year prior to screening visit.

[0481] 11. History of hepatitis B infection or positive for hepatitis B surface antigen (HbsAg+) at screening.

[0482] 12. History of HIV infection or positive HIV serology at screening.

[0483] 13. History of hepatitis C infection or positive test result for hepatitis C antibodies at screening.

[0484] 14. Any malignancy, except basal cell or squamous cell carcinoma of the skin or carcinoma in situ of the cervix or anus, has been resected within the past 10 years, but with no evidence of metastatic disease within 3 years.

[0485] 15. History of active or latent tuberculosis (TB). Note: Latent TB history is defined as a positive tuberculin skin test (TST; defined as a skin induration >5 mm, independent of Bacillus Calmette-Guerin (BCG) or other vaccination history), or positive (not indeterminate) TB Gold test.

[0486] 16. For Part A: At least 2 determinations of seated or supine blood pressure readings (> 140 / 90 or < 90 / 60) and resting pulse (< 45 or > 125) or orthostatic changes (decrease in systolic blood pressure > 20 mmHg and / or diastolic blood pressure > 10 mmHg) at screening and baseline visits. For Part B: At least 2 determinations of seated or supine blood pressure readings (> 150 / 90 or < 90 / 50) and resting pulse (< 45 or > 125) or orthostatic changes (decrease in systolic blood pressure > 20 mmHg and / or diastolic blood pressure > 10 mmHg) at screening. If blood pressure readings are elevated, blood pressure readings can be repeated or the subject can be rescreened once.

[0487] 17. Estimated glomerular filtration rate of the subject at screening (using the MDRD equation) < 60 mL / min / 1.73 m 2 .

[0488] 18. Clinically significant abnormal ECG or abnormal intervals confirmed in at least 2 determinations (QTcF > 450 msec in men, > 470 msec in women; PR < 120 msec or > 220 msec; QRS > 100 msec).

[0489] 19. Hypersensitivity to doxycycline (or tetracyclines) or other components of the formulation.

[0490] 20. History of acute hypersensitivity and / or allergy to protein therapy.

[0491] 21. History of severe allergy (including latex or anaphylaxis or anaphylaxis) that the investigator considers to represent a significant risk for the subject.

[0492] 22. Participation in any clinical study evaluating another investigational drug (including biologic agents) or therapy that should be conducted within 90 days or at least 5 half-lives of the study biologic, whichever is longer, or for other investigational products at least 4 weeks or for immunotherapies 6 months prior to the screening visit.

[0493] 23. Pregnant or lactating women.

[0494] 24. Women of childbearing potential who are unwilling to practice highly effective contraception from the first dose / start of first treatment until at least 4 months after the last dose. Highly effective contraception measures include:

[0495] a. Stable use of a combined (containing estrogens and progestogens) hormonal contraceptive (oral, intravaginal, transdermal) or a hormonal contraceptive (oral, injectable, implantable) with progestogens only associated with ovulation inhibition for 2 or more menstrual cycles prior to screening

[0496] b. Intrauterine device (IUD); Intrauterine hormone releasing system (IUS)

[0497] c. Bilateral tubal ligation

[0498] d. Vasectomy

[0499] e. Sexual abstinence

[0500] Postmenopausal women must have been amenorrhic for at least 12 months to be considered not of childbearing potential. Women with a documented hysterectomy or tubal ligation do not need to be pregnancy tested and do not need to use contraception.

[0501] Sexual abstinence is considered an effective method only if it is defined as the avoidance of heterosexual intercourse that is in the context of the overall risk related to study treatment.

[0502] Periodic abstinence (calender, ovulation, post-ovulation methods), withdrawal (coitus interruptus), spermicides only, and lactational amenorrhea methods (LAM) are not acceptable methods of contraception. Female and male condoms should not be used together.

[0503] 25. Sexually active males who are unwilling to use a medically acceptable method of birth control in the following forms during the study drug treatment period and for 4 months after the last dose of study drug: vasectomy, medical assessment of surgical success, or consistent use of a condom. Donating sperm is prohibited during the study and for 4 months after the last dose of study drug.

[0504] 26. Concomitant medication should be used in addition to the drugs listed in the allowed medications or nutritional supplements.

[0505] Description of the study cohorts and dose escalation

[0506] The plan includes seven consecutive escalating dose cohorts ranging from a dose of 0.3 mg / kg up to a maximum dose of 30 mg / kg. Each dose cohort will consist of 8 subjects: 6 subjects randomized to receive H4H17319P2 and 2 subjects randomized to receive placebo. To optimize safety, each cohort of 8 subjects (6 active drug; 2 placebo) in Cohort 1 (0.3 mg / kg intravenous), Cohort 2 (1 mg / kg intravenous), Cohort 3 (3 mg / kg intravenous), Cohort 4 (300 mg subcutaneous), and Cohort 5 (10 mg / kg intravenous) will be divided into 2 blocks. 2 subjects (1 active drug: 1 placebo) will be enrolled as a safety sentinel group in Block 1, and the remaining 6 subjects (5 active drug: 1 placebo) will be enrolled in Block 2. Subjects in Block 1 will be enrolled first and dosed on the same day. Only after both subjects in Block 1 have completed the at least 24-hour safety assessment safely, the Investigator and the Sponsor Medical Monitor review the safety data and obtain the Investigator’s and the Sponsor Medical Monitor’s agreement (it is safe to begin enrolling subjects in Block 2) will subjects in Block 2 be enrolled. All subjects in Block 2 can be dosed on the same day.

[0507] Each cohort of 8 subjects (6 active drug: 2 placebo) in Cohort 6 (600 mg subcutaneous) and Cohort 7 (30 mg / kg intravenous) will be divided into 2 blocks of 4 subjects (3 active drug: 1 placebo) each. The doses for each block will be performed on different days. The escalating dose cohorts will be enrolled as follows:

[0508] □ Cohort 1 H4H17319P2, 0.3 mg / kg intravenous, single dose

[0509] □ Cohort 2 H4H17319P2, 1 mg / kg intravenous, single dose

[0510] □ Cohort 3 H4H17319P2, 3 mg / kg intravenous, single dose

[0511] □ Cohort 4 H4H17319P2, 300 mg subcutaneous, single dose

[0512] □ Cohort 5 H4H17319P2, 10 mg / kg intravenous, single dose

[0513] □ Cohort 6 H4H17319P2, 600 mg subcutaneous, single dose

[0514] □ Cohort 7 H4H17319P2, nominal dose 30 mg / kg intravenous, single dose

[0515] Optional cohorts will be enrolled if PK variability is greater than expected and additional subjects are needed to examine the effect of specific covariates such as age, body weight, gender.

[0516] • Cohort 8 H4H17319P2, nominal dose 30 mg / kg intravenous, single dose

[0517] • Cohort 9 H4H17319P2, nominal dose 30 mg / kg intravenous, single dose, maximum dose of 30 mg / kg intravenous has been allocated to Cohort 8 and Cohort 9, but lower doses can be administered based on the most recent PK data.

[0518] Dose Escalation: The safety / dose escalation team will include the Investigator, Medical / Research Monitors, the study biostatistician, and the Risk Management Leader. The Investigator and other study site personnel, including medical monitors, the Sponsor Research Team will not know the treatment administered. There can be Sponsor unblinded individuals who will not be part of the Sponsor Research Team.

[0519] 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, the dose can be escalated to Cohort 2 (1 mg / kg).

[0520] 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, the dose can be escalated to Cohort 3 (3 mg / kg intravenous) and Cohort 4 (300 mg subcutaneous). Dosing in Cohort 4 can be performed in parallel with dosing in Cohort 3.

[0521] 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, the dose can be escalated to Cohort 5 (10 mg / kg intravenous).

[0522] 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, the dose can be escalated to Cohort 6 (600 mg subcutaneous) in parallel with Cohort 5, but only once.

[0523] A nominal dose of 30 mg / kg intravenous has been assigned to Cohort 7, but lower doses can be administered based on the latest PK data. All subjects in Cohort 5 have completed the Day 8 safety assessment and the blinded safety data have been reviewed at the safety / dose escalation team meeting prior to dose escalation to 30 mg / kg intravenous. If the pharmacodynamic effect on body weight is greater than expected, the timing of the safety / dose escalation decision can be modified, and additional safety data collected. For example, if at least 3 of 6 subjects have >3% weight loss at 8 days, the observation period will be extended to Day 15 before any dose escalation decision is made; if at least 3 of 6 subjects have further weight loss to >5% over 2 weeks, the observation period will be extended to 4 weeks before a dose escalation decision is made.

[0524] Table 19: Dosing Cohorts in Part A and Part B of the Study

[0525]

[0526]

[0527] Study Design

[0528] This is a Phase I, randomized, double-blind, placebo-controlled, 2-part study of the safety, tolerability, PK, and pharmacodynamics (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 levels, frequency, and route of administration (intravenous or subcutaneous) for Part B. In Part B, overweight / obese subjects with a body mass index (BMI) of 25-40 kg / m2will be enrolled to evaluate the safety, tolerability, PK, and PD of repeat doses of H4H17319P2 in 4 different cohorts defined by baseline leptin levels. 2

[0529] ​In Part A, up to 88 subjects will be randomized to up to 7 consecutive escalating single dose (up to 5 intravenous and 2 subcutaneous) cohorts (Cohorts 1, 2, 3, 4, 5, 6, 7) and 2 optional single dose cohorts (Cohorts 8 and 9). Seven 7 consecutive single dose cohorts will have 8 subjects, randomized to receive H4H17319P2 or placebo (6 active drug: 2 placebo) at each dose level. The 2 additional optional single dose cohorts will have up to 16 subjects, randomized to receive H4H17319P2 or placebo (12 active drug: 4 placebo). Up to 5 individual intravenous dose levels (0.3, 1.0, 3, 10, and 30 mg / kg) and 2 subcutaneous doses (300 and 600 mg) will be evaluated in a single escalating manner. The decision to escalate doses will be based on analysis of safety parameters and AEs. Interim analysis of antibody concentration over time, exploratory PD measures, and PK / PD relationships between dose cohorts (if applicable) can also contribute to the dose escalation decisions. Based on interim analysis of PK characteristic variability, a decision will be made whether to enroll the 2 optional cohorts at the maximum 30 mg / kg dose level.

[0530] Optional Cohorts: Cohort 8 and Cohort 9 will be enrolled if greater than expected PK variability is observed in subjects, additional subjects are needed to understand the impact of specific covariates on PK characteristics. Subjects can be enrolled in specific subgroups defined by inclusion / exclusion criteria such as pre-specified male, female, age range, weight, or specific BMI cut points. The nominal dose of 30 mg / kg intravenous has been assigned to Cohorts 8 and 9, but lower doses can be administered based on the most recent PK data. Each of Cohorts 8 and 9 will enroll 16 subjects (4 subjects assigned to receive placebo, 12 subjects to receive H4H17319P2), respectively. Prior to dose escalation to 30 mg / kg intravenous, all subjects in Cohort 5 have completed the Day 8 safety assessment and the blinded safety data have been reviewed at the safety / dose escalation team meeting. The Part A study design includes a screening period (Day -21 to -2), a pre-baseline visit (Day -1) in which subjects will be allowed a 2-night clinical stay (for subjects receiving intravenous and subjects receiving subcutaneous doses within the safe dose range) or a 1-day clinical stay (for other subjects receiving subcutaneous administration), a follow-up period (Day 3 to Day 113), and the end of study visit (Day 113). Throughout the study, safety assessments include vital signs, weight, physical examination, ECG, laboratory tests, and monitoring of AEs, PK parameters, and various PD assessments.

[0531] In Part B, up to 81 subjects with BMI 25-40 kg / m 2Subjects will be enrolled in 4 cohorts defined by baseline leptin levels (up to approximately 20 per cohort) and randomized (3: 1 or 6: 1 H4H17319P2 to placebo, depending on cohort assignment) to placebo control, double-blind, 12-week repeat-dose study. The choice of dose, dosing interval, and mode of administration (intravenous vs. subcutaneous) will be based on safety, PK, and PK / PD (if any) from Part A. The PK profile of H4H17319P2 from Part A will be used to predict concentrations in serum following repeat administration.

[0532] The study includes a pre-screening period (Day -60 to -14), a screening period (Day -32 to -14), a baseline period (Day -29 to -1) to obtain baseline measurements of body weight, body composition by DXA and MRI, and baseline measurements of fasting leptin, body weight, appetite assessment, and free food intake assessment at the time of clinical admission. Subjects will be allowed a 2-day clinical admission on Day -1. On Day 1, subjects will receive the first dose of study drug or placebo, undergo serial blood sampling for pharmacokinetic measurements, and an overnight 24-hour PK sampling on Day 2 post-discharge. Study drug can be administered once every 4 weeks or once every 2 weeks, but no more than once per week during the treatment period (dosing frequency will be determined by interim analysis of PK data from Part A).

[0533] Visits during the treatment period will continue once per week to collect precise and repeatable measurements of body weight and serum metabolic parameters (glucose, insulin, HOMA-IR, lipids). Follow-up assessments of appetite and free food intake will be performed at Week 4 of clinical admission and at the end of the treatment period (12 weeks). Subjects will also complete a daily appetite questionnaire at baseline, during treatment, and during follow-up. Follow-up assessments of DXA and MRI imaging will also be performed. After the treatment period, subjects will be visited 16 weeks after drug discontinuation. Throughout the study, safety assessments will include vital signs, physical examinations, ECGs, laboratory tests, and AE monitoring. Drug concentrations, target engagement markers (sLEPR), and exploratory biomarkers will also be measured throughout the study.

[0534] Study Duration

[0535] The duration of Study Part A for subjects is approximately 19 weeks, including a screening period. The duration of Study Part B for subjects is approximately 35 weeks, including a pre-screening / screening / baseline period. The end of the study is defined as the last visit for the last subject in Part B.

[0536] Treatment Dose / Route / Time Table

[0537] H4H17319P2 will be supplied as a lyophilized powder in sterile, single-use 20 mL glass vials for intravenous or subcutaneous administration. Placebos matching H4H17319P2 will be prepared in the same formulation without the addition of protein. For Part A, a single dose will be administered intravenously and subcutaneously. For Part B, the choice of dose, dosing interval, and mode of administration (intravenous vs. subcutaneous) will be based on safety, PK (if any), PK / PD, data from Part A.

[0538] Procedures and Assessments

[0539] Safety will be assessed by monitoring / assessment of TEAEs, vital signs, physical examinations, electrocardiograms (ECGs), and laboratory tests. To assess pharmacokinetics, dense and sparse samples will be collected at pre-specified time points to measure H4H17319P2 concentration in serum. Pharmacodynamics will be assessed by measurement of body weight and waist circumference, food intake and appetite assessment, and body composition using DXA and MRI.

[0540] Primary Outcome Measures:

[0541] 1. Number of treatment-emergent adverse events (TEAEs) [Time Frame: Week 12 (end of treatment period)]

[0542] Secondary Outcome Measures:

[0543] 1. Concentration of H4H17319P2 in serum over time [Time Frame: up to Week 27 (end of study)]

[0544] 2. Percent change in body weight in overweight or obese subjects [Time Frame: baseline to Week 12]

[0545] 3. Absolute change in body weight in overweight or obese subjects [Time Frame: baseline to Week 12]

[0546] 4. Change from baseline in caloric intake in response to a standardized diet in overweight or obese subjects [Time Frame: baseline to Week 12]

[0547] 5. Change in lipid-regulating protein levels over time following a single dose of H4H17319P2 [Time Frame: up to Week 16]

[0548] 6. Change in lipid-regulating protein levels over time following repeat doses of H4H17319P2 [Time Frame: up to Week 27]

[0549] 7. Incidence of H4H17319P2 anti-drug antibodies over time following a single dose of H4H17319P2 [Time Frame: up to Week 16]

[0550] 8. Incidence of H4H17319P2 anti-drug antibodies over time following repeat dosing of H4H17319P2 [time frame: up to Week 27]

[0551] Pharmacokinetic variables

[0552] In addition to time, total concentration of H4H17319P2 will be measured. Pharmacokinetic parameters can include, but are not limited to, the following:

[0553] • AUC 最后 - Area under the curve (AUC) calculated from time zero to the time of the last positive concentration

[0554] • AUC 0-τ - AUC calculated over the dosing interval of length T

[0555] • C max - Peak concentration

[0556] • t max - Time to reach C max

[0557] • CL - Clearance

[0558] • C 谷 - Trough concentration

[0559] Note that for Part B, the selection of these (and other) parameters depends on the final sampling schedule chosen and the resulting data obtained.

[0560] Anti-drug antibody variables

[0561] Anti-drug antibody (ADA) variables include ADA responses and titers as follows:

[0562] • On-treatment responses, defined as any positive ADA assay response following dosing when the baseline result is negative

[0563] • Treatment-emergent ADA responses, defined as any positive ADA assay response following dosing that exceeds the baseline titer level by 9-fold or more when the baseline in the ADA assay is positive

[0564] • Titer values

[0565] • Titer categories

[0566] - Low (titer < 1,000)

[0567] - Moderate (1,000 < titer < 10,000)

[0568] - High (titer > 10,000)

[0569] • Titer values

[0565] • Titer categories

[0566] - Low (titer < 1,000)

[0567] - Moderate (1,000 < titer < 10,000)

[0568] - High (titer > 10,000)

[0569] Pharmacodynamic and other biomarker variables

[0570] Pharmacodynamic and biomarker variables are: body weight, assessment of free food intake, assessment of appetite, serum / plasma glycemic (e.g., fasting glucose, insulin, HbAlc) and lipid parameters (e.g., total cholesterol, TG, LDL-C, HDL-C), DXA measurements of total fat mass and lean mass, and MRI quantification of body position, regional subcutaneous and visceral fat, ANGPTL3, leptin, and sLEPR. Other exploratory biomarkers will include the effect of H4H17319P2 on thyroid hormones (T3, T4, TSH), luteinizing hormone (LH), testosterone, estradiol, cortisol, and adiponectin.

[0571] Efficacy procedures

[0572] Body weight will be assessed at the designated study visits during the screening period and throughout the study. Body weight will be assessed in triplicate using a high-precision calibrated digital scale prior to other study assessments. The subject should be empty (bladder empty) prior to body weight assessment. The subject can only be in undergarments and not wearing shoes during the body weight assessment. Body weight will be recorded to the nearest 0.1 kg.

[0573] All anthropometric measurements should be performed in triplicate, with the final reported value being the mean. Triceps skinfold, subscapular skinfold, suprailiac skinfold, and thigh skinfold should be taken from the right side of the body in dry, intact skin areas, unless otherwise requested, avoiding deformities or missing limbs. To measure waist circumference, instruct the subject to stand straight and relaxed with arms at the sides and feet together pointing forward. The iliac crest and the lowest rib margin can be identified by palpation and the skin overlying these areas is marked with a pen. The midpoint between these skin marks is then determined using a tape measure and marked with a pen. The waist circumference at the midpoint mark is then measured using a tape measure at the end of a relaxed expiration. Height is measured using a calibrated distance meter at a fully upright standing position at the end of inspiration and recorded to the nearest 0.1 cm. For Part B and the optional cohort in Part A, height will be measured as a single measurement at the prescreening or screening visit, respectively. Body mass index will be calculated using the mean of body weight (kg) divided by the square of height (m). In Part A (except for the optional cohort), the mean height will be used for the calculation. In Part B and the optional cohort in Part A, BMI is calculated using height (single measurement at prescreening or screening, respectively) and body weight (mean of 3 measurements) at each visit.

[0574] Assess free food intake. Subjects will be excluded from screening if they have abnormal eating behaviors or aversions to the foods used in the food intake assessment. Energy intake (breakfast + lunch + dinner) will be assessed with free food intake at baseline in Part B, after 4 weeks of treatment, at the end of the treatment period (12 weeks). Subjects will be asked to avoid strenuous exercise and alcohol consumption the day before their clinic visit when food assessments are performed.

[0575] Subjects will enter the clinic in a fasted state and arrive in the morning. During the 4-week inpatient food intake assessment, subjects will receive their assigned treatment (study drug or placebo). They will then be provided with standardized breakfast, lunch, and dinner meals of fixed caloric and macronutrient content and then fasted overnight. The next day, they will be provided with free breakfast, free lunch, and free dinner. During the free test meal, subjects will be provided with a large excess of food (4-5 times the typical portion). The dietitian will design the meals to have a standardized macronutrient content and known caloric density. All meals and food will be secretly weighed using a dedicated calibrated scale before and after the test meal to accurately assess the amount of food consumed to within 0.1 g. Subjects will consume all meals in a private, dedicated room without a clock, radio, cell phone, and television to eliminate temporal or social cues that can influence food intake. The meals will be masked in some way to the amount of food consumed so that food intake is not influenced by visual / social cues of excess food availability. Subjects will be asked to eat as much as they want, and they will remain undisturbed during the test meal and are prohibited from engaging in leisure activities such as reading, listening to music, making phone calls, or watching videos. Subjects will be allowed to leave the test meal room when they are sufficiently sated, and if they have not left the room after 1 hour, eating will be terminated by the study staff.

[0576] Clinical Appetite Assessment and Daily Appetite Questionnaire (Part B only)

[0577] Clinical appetite will be assessed by questionnaire (Flint, 2000) and subjects will be instructed to record their answers on a visual analog scale. Subjects will complete the Clinical Appetite Assessment approximately 30 minutes before and after the standardized dinner on the first day of clinical admission, as well as the Daily Appetite Questionnaire at baseline, during treatment, and during follow-up. Care will be taken to ensure that study subjects do not share questionnaire results with other study participants. Details of the questions used in the Clinical Appetite Assessment and Daily Appetite Questionnaire in Study Part B will be described in the study manual, and these questions (finalized) will be submitted to the ethics committee for review prior to the start of Part B.

[0578] Measurement of body composition by DXA (Part B only)

[0579] DXA is widely used for clinical whole-body bone density measurements. The total examination time is short (approximately 6 to 7 minutes) and the ionizing radiation dose is minimal, approximately 0.1 mGy. Dual X-ray absorptiometry is able to provide estimates of lean body mass (LBM) and body composition and has been used for LBM measurements in clinical studies. Longitudinal studies have demonstrated the clinical validity of DXA for LBM measurements, showing a significant correlation between changes in LBM and declines in physical function (Goodpaster, 2006). X-ray absorptiometry will be performed twice, at baseline, at the end of the treatment visit, and at the end of the study. Regarding individual subject preparation for DXA scans, efforts should be made to maintain consistent hydration, consistent food intake, consistent caffeine intake, consistent activity (no strenuous activity for 24 hours prior to the scan), consistent clothing, and consistent subject positioning on the DXA table for scans throughout the study. Likewise, for any given subject, DXA scans should be obtained at the same routine time of day throughout the study.

[0580] Measurement of body composition by MRI (Part B only)

[0581] Magnetic resonance imaging is used to measure liver fat content. A multi-echo gradient-echo sequence is recommended to acquire axial images to cover the entire liver. The echo times should be such that fat and water alternate between out-of-phase and in- phase at successive echo times (TE). To minimize motion artifacts, these should be acquired during a brief breath-hold period. A complete acquisition takes 3 to 5 minutes. Scanning of the fat content on the thighs requires careful positioning of the subject and fixation using shaped foam supports and positioners. A complete thigh MRI acquisition can take up to 10 minutes. Subjects can be required to fast for 10 to 12 hours prior to the morning scan.

[0582] Study schedule of events and footnotes

[0583] Figure 25 , 26 The study assessments and procedures are provided by time period and visit in Tables 27 and 27 with the following footnotes:

[0584] 1. Clinical discharge for non-sentinel subcutaneous dosing group, but the center can choose to have the subject overnight and discharge the next day.

[0585] 2. Clinical discharge for intravenous dosing and safety sentinel subcutaneous dosing

[0586] 3. Vital signs and monitoring of AEs should also be performed prior to administration, 30 minutes prior, at the end of the study drug infusion, and 1, 2, 4, and 8 hours post-infusion on Day 1 of the IV cohort in Part A. For the subcutaneous cohort in Part A, vital signs and monitoring of AEs should also be performed prior to administration, 30 minutes prior, at the end of the study drug infusion, and 1, 2, 4, and 8 hours post-injection on Day 1. For dose administration on Day 1 in Part B, vital signs and monitoring of AEs should also be performed prior to administration, 30 minutes prior, at the end of the study drug infusion intravenous dose, and 1, 2, 4, and 8 hours post-infusion or post-injection for intravenous or subcutaneous doses, respectively. On subsequent administration days, vital signs and monitoring of AEs should also be performed prior to administration, 30 minutes prior, at the end of the study drug infusion intravenous dose, and 1, 2, and 4 hours post-infusion for intravenous and post-injection for subcutaneous doses, respectively. Vital signs also include a postural blood pressure assessment at the screening visit (Parts A and B) and on Day 1 (Part A only). For the postural assessment, blood pressure and pulse rate are measured after the subject has been supine for about 10 minutes, after standing for about 1 minute, and after standing for about 3 minutes.

[0587] 4. Blood is collected after at least 8 hours of fasting. On the days of administration, only the pre-dose sample needs to be in a fasted state.

[0588] 5. Body weight must be measured in triplicate in a fasted state using a dedicated calibrated body weight scale without shoes and only in underwear after an empty bladder (voiding bladder).

[0589] 6. Skinfold thickness should be collected in triplicate from the following three areas: deltoid, subscapular, suprailiac, and thigh to adequately characterize body fat distribution.

[0590] 7. Sample collection for drug concentration, sLEPR concentration, and ANGPTL3 concentration on Day 1 will be at pre-infusion / injection, ± 15 minutes post-infusion / injection, and 1 hour ± 15 minutes, 2 hours ± 15 minutes, 4 hours ± 15 minutes, 8 hours ± 15 minutes, 12 hours ± 15 minutes, and 24 hours ± 15 minutes post-infusion / injection. sLEPR and ANGPTL3 can only be analyzed at a subset of the time points where drug concentration is measured.

[0591] 8. DNA can be collected at each visit

[0592] 9. Overnight admission on the day after for the second day. For Part B, the subject will also be admitted and overnight on the day prior to Day 1 (Day -1). Day -1 can have procedures such as drug screening, urinalysis, and urine pregnancy test performed in addition to the assessments that must be performed after overnight fasting, ECG, PK measurements, and study drug administration on Day 1.

[0593] 10. Visit 30 must be performed exactly 1 day after visit 29.

[0594] 11. Visit 85 must be performed 1 day after visit 84.

[0595] 12. The frequency of study drug administration in Part B will depend on the results obtained in Part A.

[0596] 13. Two baseline images (DXA and MRI) will be obtained from day -29 to -14. DXA and MRI can be performed on the same day or on different days; however, the first and second DXA and the first and second MRI must be performed separately

[0597] 14. The DXA and MRI procedures for visit 18 can be performed up to 5 days prior to the visit or up to 5 days after visit 18, however, the DXA and MRI should not be performed within 24 hours of study drug administration. The DXA and MRI procedures for visit 24 can be performed up to 7 days prior to visit 24.

[0598] 15. For Part B, the actual sample collection schedule will depend on the interim review of the PK data for Part A. However, due to the repeat dosing, intensive sampling will be performed after the first dose and trough sample at other time points.

[0599] 16. Clinical appetite assessments will be performed before and after the standardized meal

[0600] 17. At each designated visit, the center will check the subject for completion of the daily appetite questionnaire.

[0601] 18. ECGs will be performed prior to dosing.

[0602] Safety

[0603] Vital signs including temperature, blood pressure, pulse, and respiratory rate will be collected at the time points according to Figures 25-27

[0604] A thorough physical examination will be performed at the time points according to Figures 25-27

[0605] At visits where blood draws are required, an ECG will be performed prior to blood draw. The ECG will be performed according to Figures 25-27 ​​A standard 12-lead ECG should be performed at the specified time points. Subjects should rest for at least 10 minutes before the 12-lead ECG is performed in the supine position. The investigator will provide a local interpretation of the ECG. Heart rate should be recorded from the ventricular rate, along with PR, QRS, RR, QTcB, and QTcF intervals. Any clinically significant abnormalities should be recorded as AE / SAE, if applicable. Each ECG follow-up will be compared and analyzed with the screening record follow-up. ECG bars or reports will be retained along with the source.

[0606] Blood samples (serum or urine) will be analyzed for hematological, chemical, urinalysis, drug screening, and pregnancy testing. Detailed instructions for blood sample collection are provided in the laboratory manual provided to the research center. Figures 25-27 Samples will be collected during the visit for laboratory testing. The tests will include: Blood chemistry Sodium, potassium, chloride, bicarbonate, calcium, glucose, albumin, total serum protein, creatinine, blood urea nitrogen (BUN), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, lactate dehydrogenase (LDH), gamma-glutamyl transferase (GGT), total bilirubin, total cholesterol (low-density lipoprotein [LDL] and high-density lipoprotein [HDL]), triglycerides, uric acid, creatine phosphokinase (CPK), LDL-C, HDL-C; Blood chemistry Hemoglobin, hematocrit, red blood cells (RBC), white blood cells (WBC), red blood cell index (mean corpuscular volume [MCV], mean corpuscular hemoglobin [MCH], mean corpuscular hemoglobin concentration [MCHC], red blood cell distribution width [RDW]), platelet count, and differentials (*if clinically significant abnormalities are observed, additional tests can be performed by flow cytometry to assess cell subsets), including neutrophils, lymphocytes, monocytes, basophils, and eosinophils; Urine analysis: Color, glucose, RBC, transparency, blood hyaluronic acid and other components, pH, bilirubin, bacteria, specific gravity, leukocyte esterase, epithelial cells, ketones, nitrite, crystals, proteins, WBC, yeast; Other laboratory tests Leptin, insulin, and endocrine hormones (such as thyroid hormones (T3, T4, TSH), luteinizing hormone (LH), testosterone, estradiol), and HbA1c will be assessed in Parts A and B. For Part B, serum leptin will be measured during pre-screening to determine eligibility for one of the four cohorts. Leptin will also be measured throughout the study, such as... Figures 25-27 As shown. Other proteins to be measured include adiponectin and cortisol.

[0607] Laboratory outliers and adverse events

[0608] All laboratory values ​​must be reviewed by the investigator or an authorized designated person. Significantly abnormal test results that appear after treatment initiation must be repeated to confirm the nature and extent of the abnormality. Appropriate ancillary investigations should be initiated if necessary. If the abnormality cannot be resolved or explained by an event or condition unrelated to the study drug or its administration, the medical / research supervisor must be consulted.

[0609] Within the scope of the disease studied, the clinical significance of abno...

Claims

1. Use of an antibody or antigen-binding fragment thereof that binds to human leptin receptor (LEPR) and activates LEPR signaling, and a pharmaceutically acceptable carrier or diluent, in the manufacture of a medicament for increasing bone mass in a subject, wherein the subject has low bone mass resulting from leptin deficiency, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3); and wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (LCVR) comprising three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) wherein, the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 28; the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 30; the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 32; the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 12; the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 14; and the amino acid sequence of the LCDR3 is set forth in SEQ ID NO:

16.

2. The use of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 26 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

10.

3. The use of claim 1, wherein the heavy chain variable region is linked to an IgG4 human constant region, and wherein the light chain variable region is linked to a kappa light chain constant region.

4. The use of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is a full-length antibody.

5. The use of claim 1, wherein the subject has amenorrhea.

6. The use of claim 1, wherein the subject has functional hypothalamic amenorrhea. ​ ​ ​ ​ ​ ​

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