RU2812670C2 - Agonistic antibody to leptin receptor for use in treatment of metabolism disorders or hypoleptinemia - Google Patents

Abstract

FIELD: pharmaceutics and medicine.

SUBSTANCE: method of increasing bone mass in a subject having low bone mass, which is a symptom of congenital leptin deficiency from which the subject suffers, including the administration of a pharmaceutical composition containing an antibody or an antigen-binding fragment that binds the receptor human leptin (LEPR) and activate LEPR-mediated signalling, containing a heavy chain variable region (HCVR), which contains HCDR1, HCDR2 and HCDR3 HCVR containing the amino acid sequence of SEQ ID NO: 26; and a light chain variable region (LCVR), which contains LCDR1, LCDR2 and LCDR3 LCVR, containing the amino acid sequence of SEQ ID NO: 10, and a pharmaceutically acceptable carrier or diluent to the subject in need thereof.

EFFECT: increase of bone mass in a subject having low bone mass, which is a symptom of congenital leptin deficiency.

4 cl, 27 dwg, 24 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

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

LIST OF SEQUENCES

[0002] An official copy of the sequence listing has been filed concurrently with the description electronically via EFS-Web as a sequence listing in ASCII format entitled 10436WO_SEQ_LIST_ST25, created April 5, 2019, and approximately 105 kilobytes in size. The sequence listing contained herein in ASCII format is part of the specification and is incorporated herein by reference in its entirety.

BACKGROUND OF THE ART

[0003] Leptin is a polypeptide hormone expressed predominantly by adipose tissue that is involved in the regulation of metabolism, neuroendocrine function, immunity, energy balance and food intake. Leptin activity is mediated by interaction with and signaling through the leptin receptor. The leptin receptor (also known as "LEPR", "WSX", "OB receptor", "OB-R" and "CD295") is a single-pass transmembrane receptor of the class I cytokine receptor family with a large (818 amino acids) extracellular domain. Leptin deficiency, leptin resistance, and certain mutations that impair or attenuate LEPR-mediated signal transduction are associated with obesity, type 2 diabetes mellitus, dyslipidemia, types of lipodystrophy, hepatic steatosis, nonalcoholic and alcoholic fatty liver disease, severe insulin resistance, and leprechaunism. / Donohue syndrome, Rabson-Mendenhall syndrome and related complications. Therapeutic approaches aimed at overcoming leptin resistance, leptin deficiency, and hypoleptinemia (eg, lipodystrophy) have primarily focused on the delivery of supplemental leptin or leptin analogues to individuals suffering from related diseases. However, such approaches have generally shown limited effectiveness, especially in individuals with leptin resistance, and are often associated with unwanted side effects. Thus, there is a need in the art for alternative approaches to the treatment of leptin resistance and other conditions associated with leptin deficiency or hypoleptinemia.

BRIEF DESCRIPTION OF THE INVENTION

[0004] The present invention provides antibodies and antigen binding fragments thereof that bind the human leptin receptor (LEPR). The antibodies of the present invention are agonistic antibodies; those. binding of the anti-LEPR antibodies of the present invention to LEPR causes, inter alia, activation of leptin receptor-mediated cell signaling. In certain embodiments, the antibodies of the present invention do not compete with leptin for binding to LEPR. The antibodies of the present invention are useful, for example, to mimic, replace or supplement the normal biological activity of leptin in a subject. Accordingly, the antibodies and antigen binding fragments of the present invention are useful for the therapeutic treatment of diseases and disorders associated with leptin resistance and leptin deficiency.

[0005] Antibodies of the present invention may be full-length (for example, an IgG1 or IgG4 antibody) or may contain only an antigen-binding portion (for example, a Fab, F(ab') ₂ or scFv fragment) and can be modified to provide an effect in regarding functionality, for example, by ensuring the elimination of residual effector functions (Reddy et al., 2000, J. Immunol. 164:1925-1933).

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

[0007] The present invention provides antibodies or antigen-binding fragments thereof that specifically bind LEPR containing HCVR that contains an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof that is characterized by at least at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[0008] The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind LEPR containing LCVR that contains an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof that is characterized by at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[0009] The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind LEPR containing a pair of amino acid sequences HCVR and LCVR (HCVR/LCVR), which contains any of the amino acid sequences of HCVR listed in Table 1, paired with any of the amino acid sequences LCVR sequences listed in Table 1. In certain embodiments, the present invention provides antibodies or antigen binding fragments thereof comprising the HCVR/LCVR amino acid sequence pair contained in any of the exemplary anti-LEPR antibodies listed in Table 1. In certain embodiments, the pair HCVR/LCVR amino acid sequences selected from the group consisting of SEQ ID NO: 2/10, 18/10, 26/10, 34/10, 42/10, 50/10, 58/66, 74/66 and 82/66 .

[0010] The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind LEPRs containing 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 thereto which has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[0011] The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind LEPRs containing 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 thereto which has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[0012] The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind LEPRs containing a heavy chain CDR3 (HCDR3) containing an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence thereto which has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[0013] The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind LEPRs containing 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 thereto which has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[0014] The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind LEPRs containing a light chain CDR2 (LCDR2) containing an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence thereto which has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[0015] The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind LEPRs containing a light chain CDR3 (LCDR3) containing an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence thereto which has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[0016] The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind LEPR containing a pair of amino acid sequences HCDR3 and LCDR3 (HCDR3/LCDR3), containing any of the amino acid sequences of HCDR3 listed in Table 1, paired with any of the amino acid sequences LCDR3 listed in Table 1. In certain embodiments, the present invention provides antibodies or antigen binding fragments thereof comprising the HCDR3/LCDR3 amino acid sequence pair contained in any of the exemplary anti-LEPR antibodies listed in Table 1. In certain embodiments, the amino acid sequence pair HCDR3/LCDR3 sequences 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.

[0017] The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind LEPR comprising a set of six CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) contained in any of the exemplary anti-LEPR antibodies, listed in Table 1. In certain embodiments, the set of amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 is selected from the group consisting of SEQ ID NO: 4, 6, 8, 12, 14, 16; 20, 22, 24, 12, 14, 16; 28, 30, 32, 12, 14, 16; 36, 38, 40, 12, 14, 16; 44, 46, 48, 12, 14, 16; 52, 54, 56, 12, 14, 16; 60, 62, 64, 68, 70, 72; 76, 78, 80, 68, 70, 72 and 84, 86, 88, 68, 70, 72.

[0018] In a related embodiment, the present invention provides antibodies or antigen-binding fragments thereof that specifically bind LEPRs comprising a set of six CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) contained within a pair of HCVR amino acid sequences /LCVR defined for any of the exemplary anti-LEPR antibodies listed in Table 1. For example, the present invention provides antibodies or antigen-binding fragments thereof that specifically bind LEPR comprising the set of amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 contained in the amino acid sequence pair HCVR/LCVR selected from the group consisting of SEQ ID NO: 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 in HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs in said HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventional methods that may be used to identify CDR boundaries include, for example, the Kabat determination, the Chothia determination, and the AbM determination. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the loop structural regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997) and Martin et al., Proc. Natl. Acad. Sci. USA 56:9268-9272 (1989). Public databases are also available to identify CDR sequences in an antibody.

[0019] The present invention also provides nucleic acid molecules encoding anti-LEPR antibodies or portions thereof. For example, the present invention provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 1; 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, that is characterized by at least 90%, at least 95%, at least 98% or at least 99% sequence identity with it.

[0020] The present invention also provides nucleic acid molecules encoding any of the LCDR amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR amino acid sequences listed in Table 2, or a substantially similar sequence thereof, that is characterized by at least 90%, at least 95%, at least 98 % or at least 99% sequence identity with it.

[0021] The present invention 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 amino acid sequences listed in Table 2, or a substantially similar sequence thereof, that is characterized by at least 90%, at least 95%, at least 98 % or at least 99% sequence identity with it.

[0022] The present invention 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 amino acid sequences listed in Table 2, or a substantially similar sequence thereof, that is characterized by at least 90%, at least 95%, at least 98 % or at least 99% sequence identity with it.

[0023] The present invention 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 amino acid sequences listed in Table 2, or a substantially similar sequence thereof, that is characterized by at least 90%, at least 95%, at least 98 % or at least 99% sequence identity with it.

[0024] The present invention 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 amino acid sequences listed in Table 2, or a substantially similar sequence thereof, that is characterized by at least 90%, at least 95%, at least 98 % or at least 99% sequence identity with it.

[0025] The present invention 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 amino acid sequences listed in Table 2, or a substantially similar sequence thereof, that is characterized by at least 90%, at least 95%, at least 98 % or at least 99% sequence identity with it.

[0026] The present invention 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 amino acid sequences listed in Table 2, or a substantially similar sequence thereof, that is characterized by at least 90%, at least 95%, at least 98 % or at least 99% sequence identity with it.

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

[0028] The present invention also provides nucleic acid molecules encoding LCVR, wherein LCVR contains a set of three CDRs (i.e., LCDR1, LCDR2, LCDR3), wherein the set of amino acid sequences LCDR1, LCDR2, LCDR3 is as defined for any from the exemplary anti-LEPR antibodies listed in Table 1.

[0029] The present invention also provides nucleic acid molecules encoding both HCVR and LCVR, wherein HCVR contains an amino acid sequence from any of the HCVR amino acid sequences listed in Table 1, and where LCVR contains an amino acid sequence from 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, that is characterized by 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 sequence substantially similar thereto having at least 90% similarity. , at least 95%, at least 98%, or at least 99% sequence identity with it. In certain embodiments, in accordance with this aspect of the present invention, the nucleic acid molecule encodes HCVR and LCVR, where both HCVR and LCVR are derived from the same anti-LEPR antibody listed in Table 1.

[0030] The present invention also provides recombinant expression vectors capable of expressing a polypeptide comprising an anti-LEPR antibody heavy or light chain variable region. For example, the present invention includes recombinant expression vectors containing any of the above-mentioned nucleic acid molecules, i.e. nucleic acid molecules encoding any of the HCVR, LCVR and/or CDR sequences shown in Table 1. Also included within the scope of the present invention are host cells into which such expression vectors have been introduced, as well as methods for producing antibodies or parts thereof by culturing the cells -hosts under conditions that ensure the production of antibodies or antibody fragments, and the isolation of the antibodies and antibody fragments thus obtained.

[0031] In another aspect, the present invention 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 invention provides 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 provides benefit when combined with an anti-LEPR antibody.

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

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

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

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

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

[0037] In some embodiments, one or more symptoms of a disease or condition associated with a metabolic disorder or hypoleptinemia is selected from the group consisting of obesity, obesity, hyperphagia, hyperglycemia, hypertriglyceridemia, hypercholesterolemia, insulin resistance, dyslipidemia, growth retardation, delayed pubertal spurt height, abnormal growth hormone secretion, elevated HbA1c, low bone mineral density (or low bone mass), low bone mineralization, and low lean body mass. Prevention, alleviation of symptoms of a disease or condition associated with metabolic disorders or hypoleptinemia, or a decrease in their severity and/or duration, or a decrease in their intensity can be achieved after administration of an antibody or antigen-binding fragment thereof that binds human LEPR.

[0038] In yet other aspects, provided herein are methods of treating metabolic complications of lipodystrophy. Such methods include administering a pharmaceutical composition containing an antibody or antigen-binding fragment thereof that binds the human leptin receptor (LEPR) and activates LEPR-mediated signaling, and a pharmaceutically acceptable carrier or diluent, to a subject in need thereof. In some embodiments, the treatment provides relief from hyperglycemia, a decrease in the severity of insulin resistance, a decrease in the severity of hypertriglyceridemia, a decrease in circulating cholesterol levels, and/or a decrease in HbA1c levels in the subject. Lipodystrophy may include acquired focal lipodystrophy, acquired generalized lipodystrophy, congenital focal lipodystrophy, and congenital generalized lipodystrophy.

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

[0040] In some aspects, provided herein are methods of treating congenital leptin deficiency. Such methods include administering a pharmaceutical composition containing an antibody or antigen-binding fragment thereof that binds the human leptin receptor (LEPR) and activates LEPR-mediated signaling, and a pharmaceutically acceptable carrier or diluent, to a subject in need thereof. In some embodiments, the subject has lipodystrophy and treatment with metra leptin is ineffective. In some embodiments, administration of an antibody or antigen-binding fragment thereof that binds human LEPR prevents, reduces, or reduces the severity and/or duration of symptoms associated with congenital lipodystrophy. In some embodiments, the treatment reverses or alleviates one or more of hyperphagia, obesity, hyperinsulinemia, dyslipidemia, and hepatosteatosis in the subject. In some embodiments, the subject's blood glucose level decreases, the subject's body weight decreases, the subject experiences a decrease in food intake, the subject's fat mass decreases, the subject's lean mass increases, and/or the subject's bone mass increases.

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

[0042] In other aspects, provided herein are methods of treating female infertility, amenorrhea, or restoring normal hormonal cycles associated with metabolic disorders or hypoleptinemia. Such methods include administering a pharmaceutical composition containing an antibody or antigen-binding fragment thereof that binds human LEPR and activates LEPR-mediated signaling, and a pharmaceutically acceptable carrier or diluent, to a subject in need thereof. In some aspects, administration of an antibody or antigen binding fragment thereof that binds human LEPR may provide increased fertility and/or increased likelihood of conception. In some aspects, the subject becomes pregnant. In some aspects, treatment can restore or initiate a normal menstrual cycle.

[0043] In some aspects, provided herein are methods of treating disorders of immune function associated with metabolic disorders or hypoleptinemia. Such methods include administering a pharmaceutical composition containing an antibody or antigen-binding fragment thereof that binds human LEPR and activates LEPR-mediated signaling, and a pharmaceutically acceptable carrier or diluent, to a subject in need thereof. In some embodiments, following administration of an antibody or antigen-binding fragment thereof that binds human LEPR, the numbers of CD4+ T cells are increased.

[0044] In other aspects, provided herein are therapeutic methods for increasing bone mass in a subject with a metabolic disorder or hypoleptinemia. The methods include administering a pharmaceutical composition containing an antibody or antigen-binding fragment thereof that binds human LEPR and activates LEPR-mediated signaling, and a pharmaceutically acceptable carrier or diluent, to a subject in need thereof.

[0045] In other aspects, provided herein are therapeutic methods for treating obesity or obesity or reducing body weight. In this aspect, the methods include administering a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds human LEPR and activates LEPR-mediated signaling, and a pharmaceutically acceptable carrier or diluent, to a subject in need thereof. In some embodiments, the treatment provides a reduction in fat mass, but not lean mass.

[0046] In some embodiments, the subject in need thereof is a subject who has hypoleptinemia, lipodystrophy, or leptin deficiency. In some embodiments, the subject in need thereof is a subject who does not have hypoleptinemia or leptin deficiency. In some embodiments, a metabolic disorder, obesity, or obesity is not associated with or caused by a LEPR mutation resulting in a signal transduction disorder or attenuated signal transduction.

[0047] In some embodiments, administration of an antibody or antigen-binding fragment thereof that binds human LEPR and activates LEPR-mediated signaling, in accordance with a method provided herein, stimulates STAT3-mediated signaling in the hypothalamus or enhances leptin-induced or leptin-independent signaling STAT3-mediated signal transduction.

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

[0049] In other aspects, provided herein are therapeutic methods for treating hyperphagia, hyperglycemia, insulin resistance, dyslipidemia, non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease by stimulating STAT3-mediated signaling in the hypothalamus. Such methods include administering a pharmaceutical composition containing an antibody or antigen-binding fragment thereof that binds the human leptin receptor (LEPR) and activates LEPR-mediated signaling, and a pharmaceutically acceptable carrier or diluent, to a subject in need thereof. In some embodiments, the treatment provides a reduction in circulating plasma triglyceride levels. In some embodiments, the treatment provides a reduction in circulating plasma total cholesterol levels.

[0050] In other aspects, provided herein are methods of treating growth retardation, failure of the pubertal growth spurt, and/or abnormal growth hormone secretion associated with congenital leptin deficiency. Such methods include administering a pharmaceutical composition containing an antibody or antigen-binding fragment thereof that binds human LEPR and activates LEPR-mediated signaling, and a pharmaceutically acceptable carrier or diluent, to a subject in need thereof.

[0051] In other aspects, provided herein are methods of treating hypothyroidism associated with congenital leptin deficiency. Such methods include administering a pharmaceutical composition containing an antibody or antigen-binding fragment thereof that binds human LEPR and activates LEPR-mediated signaling, and a pharmaceutically acceptable carrier or diluent, to a subject in need thereof.

[0052] In other aspects, provided herein are methods of treating low bone mineral density and/or bone mineralization associated with hypoleptinemia and/or leptin deficiency. Such methods include administering a pharmaceutical composition containing an antibody or antigen-binding fragment thereof that binds human LEPR and activates LEPR-mediated signaling, and a pharmaceutically acceptable carrier or diluent, to a subject in need thereof.

[0053] One or more additional therapeutic agents can be administered with an antibody or antigen-binding fragment thereof that binds human LEPR to subjects described herein. The second therapeutic agent may be selected from the group consisting of recombinant human leptin, PCSK9 inhibitor, statin, ezetimibe, insulin, insulin variant, insulin secretagogue, metformin, sulfonylurea, sodium-glucose cotransporter 2 (SGLT2) inhibitor, agonist/analog GLP-1, glucagon inhibitor (GCG), glucagon receptor inhibitor (GCGR), angiopoietin-like protein inhibitor (ANGPTL), phentermine, orlistat, topiramate, bupropion, topiramate/phentermine, bupropion/naltrexone, bupropion/zonisamide, pramlintide/metreleptin, lorcaserin, cetilistat, tesofensine, wellneperite, anticonvulsant, digoxin, coumadin, vitamin D, thyroxine, thyroid supplement, vitamin supplement, calcium supplement, carnitine, coenzyme Q10, constipation drug, antiallergy drug, gabapentin, narcotic drugs, ketamine, lidocaine or venlafaxine hydrochloride.

[0054] The present invention also provides a method of treating, preventing or reducing the manifestations of (i) lipodystrophy (any type) and/or monogenic obesity; (ii) conditions associated with lipodystrophy and/or monogenic obesity; or (iii) a symptom of (i) or (ii) in a patient, comprising administering to a patient in need thereof an agonistic antibody or antigen-binding fragment thereof that specifically binds to LEPR (eg, H4H17319P2). For example, in an embodiment of the present invention, the condition associated with lipodystrophy and/or monogenic obesity is extremely early-onset obesity; hyperphagia and impaired satiety; impaired immune function (CD4 ⁺ counts); insulin resistance; non-alcoholic fatty liver disease; NASH, dyslipidemia; diabetes; reproductive dysfunction; hypogonadism; absence of a pubertal growth spurt; hypothyroidism; dysfunction of the thyroid gland; low bone mineral density and/or low bone mass. In one embodiment of the present invention, said symptom is an enlarged liver, increased levels of liver enzymes, increased levels of alanine aminotransferase (ALT) in the blood, increased levels of aspartate aminotransferase (AST) in the blood, high levels of fat deposition; body mass index >85th percentile for age and sex; abnormal foraging behavior; abnormal feeding aggressive behavior; recurrent and potentially fatal infections; hyperinsulinemia; liver steatosis; progression to NASH (lipodystrophy); hypertriglyceridemia; increased HbA1c levels; increased glucose levels; impaired glucose tolerance; delayed sexual development; decreased expression of secondary sexual characteristics; absence or irregularity of menstruation; infertility; short stature; abnormal secretion of growth hormone; change in T3 level; change in TSH levels and/or change in free thyroxine levels.

[0055] In one embodiment of the present invention, an agonistic anti-LEPR antibody or antigen binding fragment thereof (e.g., H4H17319P2; see WO 2017/66204) is administered in dosages as follows: (i) one or more doses of approximately 5 mg/kg body weight intravenously; then (ii) one or more doses of about 250-300 mg, such as 250 mg or about 300 mg, subcutaneously once a week; then (iii) optionally one or more doses of about 250 mg or about 300 mg subcutaneously once a month or about 28 days. For example, (i) one or more doses of approximately 5 mg/kg body weight intravenously; then (ii) one or more doses of approximately 250 mg or approximately 300 mg subcutaneously once a week. In one embodiment of the present invention, the antibody is administered in dosages as follows: (i) 5 mg/kg body weight as a single intravenous dose (on day 1); then (ii) four doses of approximately 250 mg or approximately 300 mg subcutaneously once weekly (eg, on days 4, 11, 18, and 25); then (iii) one or more doses of approximately 250 mg or approximately 300 mg subcutaneously once a month or every 28 days (eg, on days 53, 81, 109, 137, 165 and 193, etc.). For example, in one embodiment of the present invention, the subcutaneous administration of the first dose occurs on approximately day 4, which is approximately three days after the intravenous administration of the dose, which occurs on day 1.

[0056] Other embodiments will be apparent from a review of the following detailed description.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

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

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

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

[0060] Figure 4 shows the average percent change in body weight of mice dosed with either an isotype control antibody at 3 mg/kg or an anti-LEPR antibody selected from H4H16650P2, H4H16679P2, H4H17319P2, or H4H17321P2 at 3 mg/kg. kg.

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

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

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

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

[0065] Figures 9A-9C. Leptin deficiency was induced on day 0 by HDD mLepr.ECD in male Lepr ^hu/hu mice at 18 weeks of age. Seven days after HDD, mice were divided into 2 groups based on the relative percentage change in body weight and SC (subcutaneously) administered a single dose of 10 mg/kg control monoclonal antibody (gray circles or bars) or H4H17319P2 (filled circles or bars). . Data represent mean ±SEM. * - P <0.05 when comparing H4H17319P2 and the control monoclonal antibody at the indicated time points. $ - P < 0.05 between pre-HDD (day -1) and any post-HDD (day 6 or 13) within the same respective dose group. & - P < 0.05 between pre-monoclonal antibody administration (day 6) and post-monoclonal antibody administration (day 13) within the same respective dosing group. For the group indicated by the corresponding symbol color, there is no significant difference (ns) on the indicated days from baseline on day 0. Figure 9A shows the body weight (left) and daily food intake (right) of Lepr ^hu/hu mice throughout the study. demonstrating that induction of leptin deficiency leads to rapid body weight gain and hyperphagia. N=14 per group. Figure 9B shows micro-CT imaging body composition analysis performed 1 day before HDD (day -1), 1 day before monoclonal antibody dosing (day 6 after HDD), and 6 days after dosing ( day 13 after HDD). Quantification of fat mass (left) and lean mass (right) is shown for control monoclonal antibody (N=14) and H4H17319P2 (N=14) dosing groups (gray and black bars, respectively). Figure 9C. Chemical analyzes of plasma obtained 6 days after treatment (day 13) from Lepr ^hu/hu mice with induced leptin deficiency that were treated with a single dose of control monoclonal antibody (gray bars, N=14) or H4H17319P2 (black bars, N=14).

[0066] Figures 10A-10D. Figure 10A. Schematic showing gene targeting to create leptin receptor ectodomain humanized Lepr ^hu/hu mice. Figure 10B. Body weight (9 weeks of age) and micro-CT quantification of body composition (9–12 weeks of age) of male wild-type (Lepr ^+/+ , gray bars) and Lepr ^hu/hu (black bars) mice. Data represent mean±SEM. N=6-10 per group. Figure 10C. Insulin tolerance test (0.75 U/kg, IP) in male Lepr ^+/+ (gray circles) and Lepr ^hu/hu (black circles) mice aged 8-11 weeks. Data represent mean ±SEM. N=8-9 per group. Figure 10D. Serum leptin levels in male Lepr ^+/+ (gray bars) and Lepr ^hu/hu (black bars) mice aged 9-13 weeks. Data represent mean ±SEM. N=8-9 per group.

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

[0068] Figures 12A-12C. Leptin deficiency was induced by HDD mLepr.ECD on day 0 in Lepr ^hu/hu mice that were 17-20 week old female or 18 week old male. Seven days after HDD, mice were divided into 2 groups of animals, each based on relative percentage change in body weight, and then SC administered a single dose of 10 mg/kg control monoclonal antibody (gray open circles or bars) or H4H17319P2 (black open circles or bars). . Data represent mean ±SEM. * - P <0.05 when comparing H4H17319P2 and the control monoclonal antibody at the indicated time points. $ - P < 0.05 between pre-HDD (day -1) and any post-HDD (day 6 or 13) within the same respective dose group. & - P < 0.05 between pre-monoclonal antibody administration (day 6) and post-monoclonal antibody administration (day 13) within the same respective dosing group. For the group indicated by the corresponding symbol color, there is no significant difference (ns) from baseline on day 0 on the indicated days. Figure 12A. Body weight (left) and food intake (right) of female mice throughout the study show that induction of leptin deficiency leads to rapid body weight gain and hyperphagia. Body weight gain continued after administration of control monoclonal antibody (N=14). N=10-11 per group. Figure 12B. Body composition analysis using micro-CT imaging in female mice was performed 1 day before HDD (day -1), 1 day before dosing of monoclonal antibodies (day 6 after HDD), and 6 days after dosing (day 13 after HDD). Data are means ±SEM for fat mass, lean mass, bone mass, bone mineralization, and bone density (from left to right). N=10-11 per group. Figure 12C. Body composition analysis using micro-CT imaging in male mice was performed 1 day before HDD (day -1), 1 day before dosing of monoclonal antibodies (day 6 after HDD), and 6 days after dosing (day 13 after HDD). Data are means ±SEM for bone mass, bone mineralization, and bone density (from left to right). N=14 per group.

[0069] Figures 13A-13D. Leptin deficiency was induced on day 0 by HDD mLepr.ECD in female Lepr ^hu/hu mice aged 17–20 weeks. For comparison, a group of mice were subjected to control vector HDD. Seven days after HDD, mLepr.ECD-treated mice were divided into 3 groups based on body weight and SC treated with two (days 7 and 13) doses of 3 mg/kg control monoclonal antibody (gray circles or bars) or H4H17319P2 ( dark gray circles or bars) or matched paired feeding (light gray circles or bars) using the amount of food consumed by leptin-deficient mice treated with H4H17319P2. Data represent mean ±SEM. * - P < 0.05 when comparing the corresponding group, indicated by symbol color or bar, and HDD mLeprECD with control monoclonal antibody administered at the indicated time point. # denotes statistical significance when comparing HDD mLeprECD animals treated with H4H17319P2 and HDD mLeprECD animals treated with equal feeding in paired groups. $ - P < 0.05 comparing the corresponding group, indicated by symbol color or bar, and HDD control mice treated with control monoclonal antibody at the indicated time points. Figure 13A shows the change in body weight of pre-HDD mice (left) and cumulative food intake (right). N=6-11 per group. Figure 13B demonstrates quantification of body composition of Lepr ^hu/hu mice by micro-CT before day 13 post-HDD (7 days post-treatment). Fat mass, lean mass, bone mass, bone mineralization, and bone density (from left to right). N=5-10 per group. Figure 13C shows insulin tolerance testing on day 10 post-HDD (3 days post-treatment) in mice that were fasted for 4 hours before insulin treatment (1.0 U/kg, IP) at 0 minutes. For the insulin tolerance test, blood glucose levels and the area under the curve (AUC) for glucose are shown (left and right, respectively). N=6-11 per group. Figure 13D shows plasma lipid chemistry data obtained on day 16 and day 17. N=6-11 per group.

[0070] Figures 14A-14G. Male mice with lipodystrophy aP2-nSrebplc ^Tg/+ ; Lepr ^hu/hu (Tg) 27-30 weeks of age received weekly (SC) doses of 10 mg/kg control monoclonal antibody (gray circles or bars) or H4H17319P2 (black circles or bars). For comparison, male Lepr ^hu/hu non-transgenic (nonTg) mice aged 27-30 weeks were also given weekly (SC) doses of 10 mg/kg control monoclonal antibody (open diamonds and white bars). All data are means ±SEM. * - P < 0.05 when comparing the corresponding group, indicated by symbol color or bar, and nonJg mice dosed with control monoclonal antibody at the indicated time point. # - P < 0.05 when comparing the corresponding group, indicated by symbol color or bar, and Jg mice dosed with control monoclonal antibody at the indicated time point. $ -P < 0.05 when comparing the value on day 27 and the value on day -5 for the corresponding group. Figure 14A shows body weight and total food intake on the left and right, respectively. N=8-9 per group. Figure 14B shows left and right lean mass and fat mass, respectively, quantified by micro-CT imaging before dosing on day -5 and post-dosing on day 27. N=9 per group. Figure 14C shows blood glucose levels throughout the study (left) and percentage hemoglobin A1c levels on day 28 (right). N=9 per group. Figure 14D shows blood glucose levels and area under the curve (AUC) for glucose as measured by insulin tolerance testing (0.5 U/kg IP) on day 23. N=9 per group. Figures 14E and 14F show that circulating lipid (E) and liver enzyme (F) levels are reduced in H4H17319P2-treated Lepr ^hu/hu lipodystrophy (Tg) mice. Chemical analyzes of blood plasma obtained on day 28 for levels of lipids (triglycerides, cholesterol, LDL-C and HDL-C) and liver enzymes (alanine transaminase, ALT, and aspartate transaminase, AST). N=9 per group. Figure 14G shows liver weight values, liver triglyceride content, and representative hematoxylin and eosin-stained liver sections from liver samples collected on day 30 (left, middle, and right, respectively). N=5 per group.

[0071] Figures 15A-15C. Phenotypic characteristics of male nonTg Lepr ^hu/hu mice (black open circles or black bars) and aP2-nSrebplc ^Tg/+ Lepr ^hu/hu mice (black open circles or black bars) at the indicated ages. Data represent mean ±SEM. * p < 0.05 when compared with Lepr ^hu/hu mice at corresponding time points. Figure 15A. Left: body weight values from 12 to 24 weeks of age. Middle: fat mass quantified using micro-CT imaging at approximately 15–20 weeks of age. Right: Plasma leptin levels measured at 19–21 weeks of age. N=17-28 per group. Figure 15B. Blood glucose levels during an insulin tolerance test (0.75 U/kg insulin, IP) at 18–20 weeks of age. N=12-13 per group. Figure 15C. From left to right: plasma levels of triglycerides, cholesterol, LDL-C and HDL-C at 15-17 weeks of age. N=20-28 per group.

[0072] Figure 16A. Male mice with lipodystrophy aP2-nSrebplc ^Tg/+ ; Lepr ^hu/hu (Tg) at 27-30 weeks of age were given weekly (SC) doses of 10 mg/kg control monoclonal antibody (gray bars) or H4H17319P2 (black bars). For comparison, male Lepr ^hu/hu non-transgenic (nonTg) mice aged 27-30 weeks were also given weekly (SC) doses of 10 mg/kg control monoclonal antibody (white bars). All data are means ±SEM. * - P <0.05 when comparing the corresponding group and nonJg mice dosed with a control monoclonal antibody at the indicated time point. # - P < 0.05 when comparing the corresponding group and Tg mice dosed with control monoclonal antibody at the indicated time point. $ - P<0.05 when comparing the value on day 27 and the value on day -5 for the corresponding group. Figure 16A. Body composition from micro-CT imaging showing bone mass, bone mineralization, and bone density (from left to right) quantified pre-dose on day -5 and post-dose on day 27. N=9 per group.

[0073] Figures 17A-17E. Data in Figure 17A are for male aP2-nSrebplc ^Tg/+ lipodystrophy mice; Lepr ^hu/hu (Tg), lipodystrophy (Tg) mice, 32–38 weeks of age, that received a single dose (10 mg/kg, SC) of control (gray bars) or H4H17319P2 (dark gray bars) or leptin infusion ( 30 μg/day, SC; black bars). To obtain the data in Figures 17B-17E, male Jg mice, 27-30 weeks of age, were dosed weekly (SC) with 10 mg/kg control monoclonal antibody (gray circles or bars) or H4H17319P2 (dark gray circles or bars) or by infusion of leptin (30 μg/day, SC; black bars). Male non-transgenic Lepr ^hu/hu (nonTg) mice aged 27-30 weeks were also shown to be dosed weekly (SC) with 10 mg/kg control monoclonal antibody (open diamonds and white bars). All data represent mean±SEM. * - P < 0.05 when comparing the corresponding group, indicated by the color of the symbol or bar, and Jg mice that were dosed with a control monoclonal antibody. # - P < 0.05 when comparing the corresponding group, indicated by the color of the symbol or bar, and Tg mice dosed with H4H17319P2. Figure 17A shows immunohistochemical staining data for pSTAT3 Y705 in the arcuate (Arc) and ventromedial (Vmh) nuclei of the hypothalamus at approximately -1.52 mm from bregma, showing an increased number of pSTAT3 Y705 ⁺ cells in aged male lipodystrophy (Tg) mice 32-38 weeks. Brain sections were prepared from brain samples collected 3 days after treatment with a single dose of control or H4H17319P2 (10 mg/kg, SC) or leptin infusion (30 μg/day, SC). Representative micrographs and quantification of the number of pSTAT3 Y705 ⁺ cells in Arc and Vmh are shown (left, middle, and right, respectively). N=4-5 per group. Figure 17B on the left shows blood glucose levels during the study. Middle and right: Blood glucose levels and area under the curve for glucose levels during insulin tolerance testing on day 9. N=8-9 per group. Figure 17C shows body weight (left), change in body weight (middle), and cumulative food intake (right). N=8-9 per group of Tg mice. N=5 for nonTg mice. Figure 17D presents chemical assays of plasma obtained on day 13 for triglycerides, cholesterol, LDL-C and HDL-C, showing a decrease in plasma triglyceride and cholesterol levels upon treatment with H4H17319P2. N=8-9 per group of Jg mice. N=5 for nonTg mice. Figure 17E shows liver weight (left) and liver triglyceride content (right) for liver samples obtained on day 14. N=6-9 per group of Jg mice. N=5 for nonTg mice.

[0074] Figures 18A-18D. Data shown in Figures 18A and 18B are for 32 week old female Lepr ^hu/hu mice treated with a single dose of control monoclonal antibody (10 mg/kg, SC; gray circles), H4H17319P2 (3 mg/kg, SC; open circles) or H4H17319P2 (10 mg/kg; black circles). Data shown in Figure 18C are for lean male and female cynomolgus monkeys that were administered a single dose of control (SC; gray circles), H4H17319P2 (3 mg/kg, SC; open circles), or H4H17319P2 (10 mg/kg; circles filled in black). Data shown in Figure 18D are for high body fat (~6.0 kg) male and female cynomolgus monkeys given two doses of control (IV; gray circles) or H4H17319P2 (30 mg/kg, IV ; circles filled in black). Data represent mean ±SEM. * - P<0.05 when comparing H4H17319P2 (10 mg/kg) and the corresponding control at the indicated time points. # - P<0.05 when comparing H4H17319R2 (3 mg/kg) and the corresponding control at the indicated time points $ - P<0.05 when comparing H4H17319R2 (3 mg/kg) and H4H17319R2 (10 mg/kg) at the indicated times time. & - P<0.05 when comparing H4H17319P2 (30 mg/kg, IV) and the corresponding control. Figure 18A shows the percentage change in body weight from pre-dose on day 0 (left) and food intake (right) during the study in lean female mice. N=6-7 per group. Figure 18B shows quantitative NMR data showing the percentage change in fat mass (left) and lean mass (right) from pre-dose on day 0 during the study in lean female mice. N=6-7 per group. Figure 18C shows the percentage change in body weight from pre-dose on day -1 during the lean monkey study. N=12 per group. Figure 18D shows the percentage change in body weight from the predose mean on days -14, -7 and -1 (left) and the percentage change in fat mass (middle) and lean mass (right) quantified by DEXA (dual energy X-ray absorptiometry to measure body composition) during a study on high body fat monkeys. N=4 and 8 for control and H4H17319R2 dose groups, respectively.

[0075] Figure 19 depicts a single patient protocol used in a clinical trial of an unapproved compassionate use drug.

[0076] Figure 20A-20B is a table that provides an evaluation plan for a patient being treated with H4H17319P2 in Treatment Period 1 (A), and Treatment Period 2, and Extended Treatment Period (B).

[0077] Figures 21A-21C demonstrate the effects of H4H17319P2 on blood glucose, body weight, and food intake in a mouse model of congenital leptin deficiency. Figure 21A represents blood glucose level in mg/dL, Figure 21B represents body weight in grams, and Figure 21C represents cumulative food intake in grams. Gray circles—Lepr ^hu/hu treated with control IgG4 ^P (N=5). Black squares - Lepr ^hu/hu Lep ^-/- treated with control IgG4 ^P (N=7). White squares - Lepr ^hu/hu Lep ^-/- , which were injected with H4H17319P2 (N=8). Data are expressed as mean ±SEM. * - P <0.05 when comparing the Lepr ^hu/hu Lep ^-/- + H4H17319P2 group and the control Lepr ^hu/hu , Lep ^-/- + IgG4 ^P using two-way ANOVA with Tukey's post hoc test. Two mice were eliminated from the Lepr ^hu/hu Lep ^−/− + control IgG4 ^P group and one mouse from the Lepr ^hu/hu Lep ^−/− + H4H17319P2 group when assessed for food intake due to excessive grinding of food in their cage.

[0078] Figures 22A-22C demonstrate fat, bone, and lean mass as measured by μCT body composition analysis in a mouse model of congenital leptin deficiency. Figure 22A represents fat mass, Figure 22B represents bone mass, and Figure 22C represents lean mass in grams. Before the start of the study, mice underwent a baseline scan in D-5. Scanning after mAb administration was performed on day 35 of the study. Gray bars - Lepr ^hu/hu treated with control IgG4 ^P (N=5). Black bars - Lepr ^hu/hu Lep ^-/- treated with control IgG4 ^P (N=7). White bars - Lepr ^hu/hu Lep ^-/- , which were injected with H4H17319P2 (N=8). Mice received weekly subcutaneous injections of 10 mg/kg of either H4H17319P2 or isotype control antibodies. Data are expressed as mean ±SEM. * - P<0.05 when compared with the baseline; # - P<0.05 when compared with the control Lepr ^hu/hu + IgG4 ^P at the corresponding time point; + - P<0.05 when compared with the H4H17319P2 group at the corresponding time point. Statistical analyzes were performed using two-way ANOVA with Tukey's post hoc test.

[0079] Figures 23A-23C demonstrate the effects of H4H17319P2 on blood glucose, body weight, and food intake in a mouse model of congenital leptin receptor deficiency. Figure 23A represents blood glucose level in mg/dL, Figure 23B represents body weight in grams, and Figure 23C represents cumulative food intake in grams. Gray circles - Lepr ^hu/hu treated with control IgG4 ^P (N=7-8). Black squares are Lepr ^A409E/A409E treated with control IgG4 ^P (N=9-10). White squares—Lepr ^A409E/A409E injected with H4H17319P2 (N=10). Data are expressed as mean±SEM. * - P <0.05 when comparing the Lepr ^A409E/A409E + H4H17319P2 group and the Lepr ^A409E/A409E + IgG4 ^P control using a mixed effects model with Sidak’s post hoc test. One mouse was excluded from the Lepr ^hu/hu + control IgG4 ^P group and one mouse from the Lepr ^A409E/A409E + control IgG4 ^P group when assessing food intake due to mortality during the study. One mouse was excluded from the Lepr ^hu/hu + control IgG4 ^P group and three mice from the Lepr ^A409E/A409E + control IgG4 ^P group when assessing food intake due to excessive grinding of food during the study.

[0080] Figures 24A-24C demonstrate fat, bone, and lean mass as measured by μCT body composition analysis in a mouse model of congenital leptin receptor deficiency. Figure 24A represents fat mass, Figure 24B represents bone mass, and Figure 24C represents lean mass in grams. Before the start of the study, mice underwent a baseline scan in D-1. Post-mAb scans were performed on day 41 of the study. Gray bars - Lepr ^hu/hu treated with control IgG4 ^P (N=7-8). Black bars - Lepr ^A409E/A409E treated with control IgG4 ^P (N=9-10). White bars - Lepr ^A409E/A409E , which were injected with H4H17319P2 (7V=10). Mice received weekly subcutaneous injections of 10 mg/kg of either H4H17319P2 or isotype control antibody ( ^IgG4P ). Data are expressed as mean ±SEM. * -P<0.05 when compared with the baseline; # - P<0.05 when compared with the control Lepr ^hu/hu + IgG4 ^P at the corresponding time point; + - P<0.05 when compared with pp}^409E/A409E+H4H17319P2 at the corresponding point in time. All statistical analyzes were performed using a mixed effects model with Sidak's post hoc test.

[0081] Figure 25 depicts a timeline of events for the Part A cohorts of a first-in-human clinical trial and includes procedures to be performed at each screening, treatment, and follow-up visit.

[0082] Figure 26 depicts a timeline of events for the Part B cohorts of a first-in-human clinical trial and includes procedures to be performed at each prescreening, screening, and baseline visit.

[0083] Figure 27 depicts a timeline of events for the Part B cohorts of a first-in-human clinical trial and includes procedures to be performed at each treatment and follow-up visit.

DETAILED DESCRIPTION OF THE INVENTION

[0084] Leptin is an adipose tissue hormone that regulates 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 deficiency, low levels of circulating leptin induce adaptive responses, including increased hunger and energy conservation through modulation of neuroendocrine pathways. Leptin modulates energy and metabolic balance by interacting with 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 results in multiple LEPR splice isoforms that differ in their C-terminal sequence (Baumann et al., 1996). Of these splicing isoforms, LEPR-b is the major isoform that mediates the effects of leptin and is the only isoform that stimulates JAK-STAT-mediated signaling (Baumann et al., 1996; Tartaglia et al., 1995; White and Tartaglia, 1996 ). Brain neurons expressing LEPR-b are the main targets and mediators of leptin's effects on energy, metabolic and neuroendocrine homeostasis. This is supported by the observation that selective expression of Lepr-b in neurons in Lepr ^db/db mice rescues obesity, diabetic and reproductive phenotypes (de Luca et al., 2005). In addition, deletion at the genetic level of Lepr in neurons ensures copying at the phenotypic level of the obesity and hyperglycemia phenotypes in Lepr ^db/db animals (Cohen et al., 2001). Leptin deficiency due to genetic loss-of-function mutations in the Lep gene results in hyperphagia, obesity, insulin resistance, dyslipidemia, and impaired neuroendocrine function in mice, which is 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 analogue metreleptin has been shown to reverse obesity as well as metabolic and reproductive dysfunction in patients with monogenic obesity due to leptin deficiency (Farooqi et al., 1999; Farooqi et al., 2002). Like primary leptin deficiency, the disease states referred to as secondary hypoleptinemia are associated with impaired glucose and lipid metabolism, which can be reversed by treatment with leptin. The types of congenital and acquired generalized 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 levels of circulating leptin in these patients lead to a state of hyperphagia, hypertriglyceridemia, hypercholesterolemia, hepatic steatosis, insulin resistance and diabetes mellitus (Brown et al., 2016; Patni and Garg, 2015). A severe complication of hypertriglyceridemia, especially when TG levels exceed 500–1000 mg/dL, is acute and recurrent pancreatitis (Yadav and Pitchumoni 2003), which can be life-threatening, with a mortality rate of over 40% if accompanied by complications such as infection or organ failure (UK Guidelines 2005). Ectopic deposition of lipids in the liver (hepatic steatosis) can lead to steatohepatitis, which is characterized by fat accumulation, cellular damage and inflammation in the liver and is one of the most common causes of cirrhosis (El-Zayadi 2008, Federico 2006 and Festi 2004).

[0085] Leptin treatment provides a reduction in hyperphagia and improvements in dyslipidemia, hepatic steatosis and glycemic control in Tg-aP2-nSrebplc mice, which develop almost complete loss of fat depots, characteristic of generalized lipodystrophy (Shimomura et al., 1999; Shimomura et al., 1998). Metreleptin provides attenuation of metabolic dysfunction in patients with generalized lipodystrophy (Oral et al., 2002), but is not approved for the treatment of patients with focal lipodystrophy (Ajluni et al., 2016).

[0086] Patients with lipodystrophy often face other serious comorbidities, such as chronic kidney disease, cardiovascular complications, autoimmune diseases and peripheral T-cell lymphoma, acute lymphoblastic leukemia and Hodgkin lymphoma.

[0087] Patients with congenital leptin deficiency demonstrate rapid weight gain and immunological abnormalities in the first few months of life, with a significantly increased risk of death during 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.). Although there are no approved therapies for congenital leptin deficiency, in several small open-label studies of patients with monogenic obesity due to loss-of-function leptin mutations, leptin treatment provided marked improvements in appetite, body weight, obesity, severity of metabolic abnormalities, bone age gap and chronological age, severity of hormonal abnormalities and immunological 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 Delta33G 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 authors have reported that leptin therapy resulted in a rapid and sustained increase in plasma thyroid hormone levels and promoted timely pubertal development, and provided improvements in the number of circulating CD4(+) T cells, 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 antibodies to metreleptin with neutralizing activity leaves patients without any options for targeted therapy (Özsu, 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 WO 2017/66204) to treat patients with congenital leptin deficiency.

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

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

[0090] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. As used herein, the expression “approximately,” when used in relation to a specific specified numerical value, means that the value may differ from the specified value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

[0091] Although any methods and materials similar or equivalent to those described herein may be used in practicing or testing the present invention, only the preferred methods and materials are described herein.

Definitions

[0092] The expression "leptin receptor", "LEPR", etc., as used herein, refers to the human leptin receptor containing the amino acid sequence presented under SEQ ID NO: 113 (see also UniProtKB accession number P48357/ Swiss-Prot). Alternative names for LEPR used in the scientific literature include "OB receptor", "OB-R" and "CD295". LEPR is also called "WSX" (see, for example, US Patent No. 7524937). The expression "LEPR" includes both monomeric and multimeric (eg, dimeric) LEPR molecules. As used herein, the expression “monomeric human LEPR” means a LEPR protein or portion thereof that does not contain or contain multimerizing domains and that exists under normal conditions as a single LEPR molecule without direct physical association with another LEPR molecule. An exemplary monomeric LEPR molecule is the molecule referred to herein as “hLEPR.mmh” containing the amino acid sequence of SEQ ID NO: 114 (see, for example, Example 3 herein). As used herein, the expression “dimeric human LEPR” means a construct comprising two LEPR molecules attached to each other by a linker, a covalent bond, a non-covalent bond, or a multimerizing domain, such as an antibody Fc domain. An exemplary LEPR dimeric molecule is the molecule referred to herein as "hLEPR.mFc" containing the amino acid sequence of SEQ ID NO: 115 (see, for example, Example 3 herein), or the molecule referred to herein as "hLEPR.hFc" "containing the amino acid sequence of SEQ ID NO: 116. As used herein, expressions such as "anti-LEPR antibody", "antibody that specifically binds LEPR", "LEPR-specific binding protein" and the like, as applicable not otherwise specified, refer to molecules that bind full-length human LEPR, monomeric human LEPR, dimeric human LEPR, or other constructs that contain or consist of an extracellular domain of LEPR.

[0093] All references to proteins, polypeptides and protein fragments herein refer to the human version of the corresponding protein, polypeptide or protein fragment unless explicitly stated to be from a non-human species. Thus, the expression "LEPR" means human LEPR unless specified to be from a non-human species, such as "mouse LEPR", "monkey LEPR", etc.

[0094] As used herein, the term “cell surface expressed LEPR” means one or more LEPR proteins or an extracellular domain thereof that are expressed on the cell surface in vitro or in vivo such that at least a portion of the LEPR protein is located on the extracellular side of the cell surface. membrane and is accessible to the antigen-binding portion of the antibody. “Cell surface expressed LEPR” may comprise or consist of a LEPR protein expressed on the surface of a cell that normally (eg, in a native or wild-type state) expresses a LEPR protein. Alternatively, “cell surface expressed LEPR” may comprise or consist of a LEPR protein expressed on the surface of a cell that does not normally express human LEPR on its surface but has been artificially engineered to express LEPR on its surface.

[0095] As used herein, expressions such as “anti-LEPR antibody” or “antibody that binds the human leptin receptor” include both monovalent antibodies with a single specificity and bispecific antibodies containing a first arm that binds LEPR and a second an arm that binds a second (target) antigen, wherein the arm to the LEPR contains any of the HCVR/LCVR or CDR sequences presented in Table 1 herein.

[0096] 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 or interacts with a particular antigen (eg, LEPR). The term "antibody" includes immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains and two light (L) chains linked by disulfide bonds, as well as their multimers (for example, IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or _VH ) and a heavy chain constant region. The heavy chain constant region contains three domains: CH ₁ , _CH 2 and _CH 3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or V _L ) and a light chain constant region. The light chain constant region contains one domain (C _L 1). The V _H and V _L regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FR). Each V _H and V _L consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments of the present invention, FR antibodies to LEPR (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. The consensus amino acid sequence can be determined based on analysis based on direct comparison of two or more CDRs.

[0097] As used herein, the term “antibody” also includes antigen-binding portions of complete antibody molecules. The terms “antigen binding portion” of an antibody, “antigen binding fragment” of an antibody, and the like as used herein include any naturally occurring, enzymatically produced, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding antibody fragments can be produced, for example, from whole antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombination-based genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and chemically manipulated or manipulated using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration or to introduce codons, insert cysteine residues, modify, add or delete amino acids, etc. .d.

[0098] Non-limiting examples of antigen binding fragments include (i) Fab fragments; (ii) F(ab') ₂ -fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv molecules (scFv); (vi) dAb fragments and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of the antibody (eg, a dedicated complementarity determining region (CDR), such as the CDR3 peptide) or a peptide with limited conformational freedom FR3- CDR3-FR4. Other engineered molecules such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, diabodies, tri-bodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc. ), small modular protein (SMIP) immunopharmaceuticals, and shark IgNAR variable domains are also included in the expression “antigen binding fragment” as used herein.

[0099] An antigen binding fragment of an antibody will typically contain at least one variable domain. The variable domain can be of any size or amino acid composition and will typically contain at least one CDR that is adjacent or in reading frame to one or more framework sequences. In antigen binding fragments containing a V _H domain associated with a V _L domain, the V _H and V _L domains may be arranged relative to each other in any suitable order. For example, the variable region may be dimeric and contain dimers V _H -V _H , V _H -V _L or V _L -V _L . Alternatively, the antigen binding fragment of the antibody may comprise a monomeric V _H or V _L domain.

[0100] In certain embodiments, an antigen binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be present in an antigen binding fragment of an antibody of the present invention include: (i) V _H -Ch1; (ii) V _H -Ch2; (iii) V _H -CH3; (iv) V _H -Ch1-Ch2; (v) V _H -Ch1-Ch2-Ch3; (vi) V _H -Ch2-Ch3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) V _L -Ch1-Ch2; (xii) V _L -Ch1-Ch2-Ch3; (xiii) VL-CH2-CH3; and (xiv) V _L -C _L . In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can either be directly connected to each other or can be connected by all or part of a hinge or linker region. The hinge region may consist of at least 2 (eg, 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible connecting element between adjacent variable and/or constant domains in a single polypeptide molecule. In addition, the antigen binding fragment of an antibody of the present invention may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other and/or with one or more monomeric _VH or V _L (eg via disulfide bond(s)).

[0101] As with full antibody molecules, antigen binding fragments can be monospecific or polyspecific (eg, bispecific). A polyspecific antigen-binding antibody fragment will typically contain at least two different variable domains, where each variable domain is capable of specifically binding to a different antigen or to a different epitope of the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of an antigen binding fragment of an antibody of the present invention using standard techniques available in the art.

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

[0103] The antibodies of the present invention may, in some embodiments, be recombinant human antibodies. As used herein, the term “recombinant human antibody” is intended to include all human antibodies produced, expressed, generated or released by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into a host cell (further described below), antibodies isolated from a recombinant human antibody combinatorial library (further described below), antibodies isolated from an animal (eg, mouse) that is transgenic for human immunoglobulin genes (see, for example, Taylor et al. (1992) Nucl. Acids Res 20:6287-6295), or antibodies produced, expressed, created or released by any other means that involve splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies contain variable and constant regions derived from human immunoglobulin germline sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, if an animal transgenic for the human Ig sequence is used, to somatic mutagenesis in vivo) and, therefore, the amino acid sequences of the V _H and V _L regions of the recombinant antibodies are sequences which, being derived from and related to human germline V _H and V _L sequences, may not naturally exist in the human germline antibody repertoire in vivo.

[0104] The present invention covers antibodies containing one or more mutations in the hinge region, C _H 2 - or C _H 3 region, which may be necessary, for example, during production, to provide improved yield of the desired form of the antibody.

[0105] Antibodies of the present invention may be isolated antibodies. As used herein, the term “isolated antibody” means an antibody that has been identified and separated from 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 from a body, or from a tissue or cell in which the antibody exists or is naturally produced is an “isolated antibody” for purposes of the present invention. The isolated antibody also includes the antibody in situ in the recombinant cell. Isolated antibodies are antibodies that have undergone at least one purification or isolation step. In certain embodiments, the isolated antibody may be substantially free of other cellular material and/or chemicals.

[0106] The present invention includes variants of the anti-LEPR antibodies disclosed herein containing one or more amino acid substitutions, insertions and/or deletions in the framework regions and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences, from which antibodies were obtained. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from publicly available antibody sequence databases. The present invention includes antibodies and antigen binding fragments thereof that are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework regions and/or CDR regions are mutated to the corresponding residue(s). s) the germline sequence from which the antibody is derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes referred to herein collectively as “germline mutations”). One of ordinary skill in the art, from the heavy and light chain variable region sequences disclosed herein, can readily produce numerous antibodies and antigen binding fragments that contain one or more individual germline mutations or combinations thereof. In certain embodiments, all residues from the framework region and/or CDRs in the V _H and/or V _L domains are backmutated to 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, such as only mutated residues present within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only mutated residues present within CDR1, CDR2, or CDR3. In other embodiments, one or more residues from the framework region and/or CDR are mutated to the corresponding residue(s) of another germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally produced). In addition, the antibodies of the present invention may contain any combination of two or more germline mutations in framework regions and/or CDR regions, for example, wherein certain individual residues are mutated to the corresponding residue of a certain germline sequence, while certain other residues that differ from the original germline sequence are retained or mutated to the corresponding residue of another germline sequence. Once produced, antibodies and antigen binding fragments that contain one or more germline mutations can be readily tested for one or more desired properties, such as improving binding specificity, increasing binding affinity, improving or enhancing biological antagonistic or agonistic properties (as appropriate) , decreased immunogenicity, etc. The present invention covers antibodies and antigen binding fragments obtained using this general method.

[0107] The present invention includes anti-LEPR antibodies and antigen binding fragments thereof comprising amino acid sequences that are 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 described herein .

[0108] In the context of polypeptides, the term “substantially similar” or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the GAP or BESTFIT programs, using default gap penalty values, are characterized by at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, the positions of the residues that are not identical differ in conservative amino acid substitutions. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by another amino acid residue containing a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). Typically, a conservative amino acid substitution will not essentially change the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage sequence identity or degree of similarity may be adjusted to higher values to correct for the conservative nature of the substitution. Means for effecting this correction are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24.307-331. Examples of groups of amino acids that contain side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic side chains with hydroxyl groups: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine and tryptophan; (5) main side chains: lysine, arginine and histidine; (6) acidic side chains: aspartate and glutamate and (7) sulfur-containing side chains are cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagine-glutamine. Alternatively, a conservative replacement is any change characterized by a positive value in the PAM250 log-likelihood function matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445. A “moderately conservative” replacement is any change characterized by a non-negative value in the PAM250 log-likelihood function matrix.

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

[0110] As used herein, the term “subject” refers to an animal, preferably a mammal, more preferably a human, in need of alleviating, preventing and/or treating a disease or disorder associated with leptin deficiency. The subject may be an adult or a child. The subject may be characterized by the presence of a metabolic disorder such as global or focal lipodystrophy. A subject may have congenital leptin deficiency or acquired leptin deficiency. Subjects who have congenital leptin deficiency include subjects with gene mutations that result in little or no circulating levels of leptin, or in circulating leptin that is biologically inactive. A subject may be characterized by the presence of 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.

[0111] As used herein, the terms “treat,” “treat,” or “treat” refer to reducing the intensity or ameliorating the severity of at least one symptom of leptin deficiency due to the administration of a therapeutic agent, such as an antibody of the present invention, to a subject in need of this. These terms include inhibiting the progression of a corresponding disease or the worsening of a condition or symptoms associated with a disease. The terms also provide for a positive prognosis of the disease, that is, the subject may be asymptomatic after administration of a therapeutic agent, such as an antibody of the present invention. A positive prognosis may include mitigation of any of the following conditions: hyperphagia, hyperglycemia, insulin resistance, dyslipidemia, or hepatic steatosis.

[0112] The terms “prevent,” “prevent,” or “prevent” refer to suppressing the occurrence of any symptoms, conditions, or indications associated with leptin deficiency.

[0113] The therapeutic agent can be administered to a subject at a therapeutic dose. The phrase "therapeutically effective amount" means an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of treatment and will be determined by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding) and is discussed in more detail herein.

Antibodies to LEPR, including Fc variants

[0114] In accordance with certain embodiments of the present invention, anti-LEPR antibodies contain an Fc domain containing one or more mutations that cause the antibody to increase or decrease binding to the FcRn receptor, for example, at an acidic pH compared to a neutral pH . For example, the present invention includes anti-LEPR antibodies containing a mutation in the C _H 2 - or C _H 3 region of the Fc domain, where the mutation(s) provide an increase in the affinity of the Fc domain for FcRn in an acidic environment (for example, in the endosome, where the pH ranges from about 5.5 to about 6.0). The result of such mutations may be an increase in the half-life of the antibody in the blood serum when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modification at position 250 (eg, E or Q); 250 and 428 (for example, L or F); 252 (for example, L/Y/F/W or T), 254 (for example, S or T) and 256 (for example, S/R/Q/E/D or T), or modification at position 428 and/or 433 (for example, H/L/R/S/P/Q or K), and/or 434 (for example, H/F or Y); or a modification at position 250 and/or 428, or a modification at position 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modification includes modification 428L (e.g., M428L) and 434S (e.g., N434S), modification 428L , 2591 (for example, V259I) and 308F (for example, V308F), modification 433K (for example, H433K) and 434 (for example, 434Y), modification 252, 254 and 256 (for example, 252Y, 254T and 256E), modification 250Q and 428L (for example, T250Q and M428L) and modification 307 and/or 308 (for example, 308F or 308P).

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

[0116] Antibodies to LEPR of the present invention may contain a modified Fc domain with reduced effector function. As used herein, the expression “modified Fc domain having reduced effector function” means any portion of the Fc domain of an immunoglobulin that has been modified, mutated, truncated, etc., relative to the naturally occurring wild-type Fc domain such that when used molecule containing a modified Fc, there is a reduction in the severity or extent of at least one effect selected from the group consisting of cell killing (eg, ADCC and/or CDC), complement activation, phagocytosis and opsonization, compared to a comparison molecule comprising a version part of the wild type Fc found in nature. In certain embodiments, a “modified Fc domain having reduced effector function” is an Fc domain with reduced or impaired binding to an Fc receptor (eg, FcγR).

[0117] In certain embodiments of the present invention, the modified Fc domain is an IgG1 Fc variant or an IgG4 Fc variant containing a substitution in the hinge region. For example, a modified Fc for use in the context of the present invention may include a variant Fc from IgG1 in which at least one Fc hinge region amino acid from IgG1 is replaced by a corresponding amino acid from the Fc hinge region from IgG2. Alternatively, a modified Fc for use in the context of the present invention may include a variant Fc from IgG4 in which at least one amino acid of the hinge region of the Fc from IgG4 is replaced by a corresponding amino acid from the hinge region of the Fc from IgG2. Non-limiting examples of modified Fc regions that can be used in the context of the present invention are presented in US Patent Application Publication No. 2014/0243504.

[0118] Other modified Fc domains and Fc modifications that can be used in the context of the present invention include any modifications presented in US 2014/0171623; US 8697396; US 2014/0134162; WO 2014/043361. Methods for constructing antibodies or other antigen-binding fusion proteins containing a modified Fc domain described herein are known in the art.

Biological characteristics of antibodies

[0119] The present invention includes antibodies and antigen-binding fragments thereof that bind human LEPR and activate LEPR-mediated signaling. Such antibodies may be referred to herein as “agonist antibodies.” In the context of the present invention, “activation of LEPR-mediated signaling” means stimulation of the intracellular effect that normally results from the interaction of leptin with LEPR in cells expressing LEPR. In certain embodiments, “activation of LEPR-mediated signaling” means activation of STAT3 transcription, which can be detected using any method in which STAT3 activity can be measured or identified, directly or indirectly, for example, using a tagged version of STAT3 expressed in the line reporter cells. For example, the present invention includes antibodies and antigen-binding fragments thereof that activate LEPR-mediated signaling in a cellular reporter gene assay, for example, using the assay format defined in Example 7 herein or a substantially similar assay. Cellular reporter gene assays that detect LEPR activation, such as the assay presented in Example 7 herein, can produce a detectable signal that can be expressed as an EC ₅₀ value (i.e., the concentration of antibody required to produce half-maximal signal transduction intensity) and/or the percentage of maximum signal transduction intensity observed in the presence of leptin. In certain illustrative embodiments of the present invention, anti-LEPR antibodies are provided that activate LEPR-mediated signaling with an EC ₅₀ value of less than about 12.0 nM in a cellular reporter gene assay, for example, using the assay format defined in Example 7 in this document, or a substantially similar analysis. In certain illustrative embodiments of the present invention, anti-LEPR antibodies are provided that activate LEPR-mediated signaling with a maximum percentage of activation relative to leptin-mediated signaling of greater than about 65% in a cellular reporter gene assay, for example, using an assay format defined in Example 7 in this document, or a substantially similar analysis.

[0120] The present invention provides 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 (eg, hLEPR.mmh, SEQ ID NO: 114) with a K _D of less than about 150 nM, measured by surface plasmon resonance at 25°C or 37 °C, for example, using the assay format defined in Example 3 herein, or a substantially similar assay. In certain embodiments, anti-LEPR antibodies are provided that bind monomeric human LEPR at 25°C with a K _D of less than about 150 nM, less than about 140 nM, less than about 130 nM, less than about 120 nM, less than less than about 110 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM , less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less less than about 400 pM or less than about 300 pM, measured using surface plasmon resonance, for example, using the assay format defined in Example 3 herein, or a substantially similar assay.

[0121] The present invention also includes antibodies and antigen-binding fragments thereof that bind monomeric human LEPR (e.g., hLEPR.mmh, SEQ ID NO: 114) with a dissociation half-life (t½) of greater than about 50 minutes, measured using surface plasmonic resonance at 25°C or 37°C, for example, using the assay format defined in Example 3 herein, or a substantially similar assay. In certain embodiments, anti-LEPR antibodies are provided that bind monomeric human LEPR at 25°C with a t½ of greater than about 50 minutes, greater than about 55 minutes, greater than about 60 minutes, greater than about 65 minutes, or greater. measured by surface plasmon resonance, for example, using the assay format defined in Example 3 herein, or a substantially similar assay.

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

[0123] The present invention also includes antibodies and antigen-binding fragments thereof that bind dimeric human LEPR (e.g., hLEPR.mFc, SEQ ID NO: 115) with a half-life (t½) of greater than about 10 minutes as measured by surface plasmonic resonance at 25°C or 37°C, for example, using the assay format defined in Example 3 herein, or a substantially similar assay. In certain embodiments, anti-LEPR antibodies are provided that bind human dimeric LEPR at 25°C with a t½ of greater than about 10, greater than about 15 minutes, greater than about 20 minutes, greater than about 25 minutes, greater than about 30 minutes, more than about 40 minutes, more than about 50 minutes, more than about 60 minutes, more than about 70 minutes or more, measured by surface plasmon resonance, for example, using the assay format defined in Example 3 herein , or essentially similar analysis.

[0124] The present invention also includes antibodies and antigen-binding fragments thereof that bind LEPR complexed with human leptin (“LEPR complexed with human leptin” may also be referred to as “leptin:LEPR”). For example, the present invention includes antibodies and antigen-binding fragments thereof that are capable of binding to a preformed complex containing hLEPR and human leptin. That is, in certain embodiments, the interaction between anti-LEPR antibodies and LEPR is not inhibited by the presence of leptin complexed with LEPR; likewise, the interaction between leptin and LEPR in accordance with this aspect of the present invention is not inhibited by the presence of an anti-LEPR antibody. An exemplary assay format for determining whether an antibody or antigen binding fragment thereof binds to LEPR in complex with human leptin is presented in Example 4 herein.

[0125] Similarly, the present invention also includes antibodies and antigen binding fragments thereof that bind LEPR and do not block the LEPR:leptin interaction. For example, the present invention includes antibodies and antigen-binding fragments thereof that are capable of binding LEPR, thereby producing an antibody:LEPR complex, where the resulting antibody:LEPR complex is capable of interacting with leptin to form a ternary complex containing the antibody, LEPR and leptin. An exemplary assay format 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 the interaction between LEPR and leptin is presented in Example 5 herein.

[0126] The present invention also includes antibodies and antigen binding fragments thereof that bind LEPR expressed on the cell surface in the presence and/or absence of human leptin. Cell surface expressed LEPR means LEPR or a portion thereof (eg, the extracellular portion of LEPR) expressed on the surface of a cell, either naturally or in an engineered cell line, such that an antibody or antigen binding fragment thereof is capable of binding to a LEPR molecule. In certain embodiments, the LEPR expressed on the cell surface includes recombinant complexes containing the extracellular domain of the LEPR attached to the cell via a tag or anchor (eg, a GPI anchor, as illustrated in Example 6 herein). In accordance with this aspect of the present invention, 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., under circumstances in which leptin is capable of binding with leptin expressed on the cell surface). That is, in certain embodiments, the interaction between anti-LEPR antibodies and cell surface expressed LEPR is not inhibited by the presence of leptin complexed with cell surface expressed LEPR. Antibodies in accordance with this aspect of the present invention are capable of forming a three-component complex on the cell surface containing the antibody, LEPR expressed on the cell surface, and leptin. An exemplary assay format for determining whether an antibody or antigen binding fragment thereof is capable of binding LEPR expressed on a cell surface in the presence and absence of human leptin is presented in Example 6 herein.

[0127] Antibodies of the present invention may have one or more of the above biological characteristics or any combination thereof. The above list of biological characteristics of the antibodies of the present invention is not exhaustive. Other biological characteristics of the antibodies of the present invention will be apparent to one of ordinary skill in the art from a review of the present invention, including the practical examples herein.

Epitope mapping and related techniques

[0128] The present invention also includes anti-LEPR antibodies containing variants of any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes anti-LEPR antibodies. containing amino acid sequences HCVR, LCVR and/or CDR with, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions with respect to any of the amino acid sequences HCVR, LCVR and/or CDR presented in Table 1 herein. In certain embodiments, the present invention provides anti-LEPR antibodies containing variant amino acid sequences HCVR, LCVR and/or CDR with respect to the sequences presented in Table 1 herein (e.g., containing conservative amino acid substitutions), wherein such variant antibodies, however less exhibit one or more of the functions and/or properties of the exemplary anti-LEPR antibodies disclosed herein.

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

[0130] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. One antigen may contain more than one epitope. Thus, different antibodies can bind to different sites on the antigen and can have different biological effects. Epitopes can be conformational or linear. A conformational epitope is formed by amino acids from different segments of a linear polypeptide chain located nearby in space. A linear epitope is an epitope formed by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope may contain saccharide moieties, phosphoryl groups, or sulfonyl groups on the antigen.

[0131] The present invention includes anti-LEPR antibodies that react with one or more epitopes present within the human LEPR amino acids M1-D839 (SEQ ID NO: 113). As presented in Example 11, 201 human LEPR peptides provided a significant reduction in deuterium uptake when bound to the H4H16650P2 antibody. Peptides corresponding to amino acids 162-169 (amino acids LYVLPEVL from human LEPR, SEQ ID NO: 113) and 170-191 (amino acids EDSPLVPQKGSF from human LEPR, SEQ ID NO: 113) had lower levels of deuteration when bound to H4H16650P2, indicating that this antibody binds at least two human LEPR epitopes having the sequences LYVLPEVL or EDSPLVPQKGSF (amino acids 162-169 or 170-191, respectively, from SEQ ID NO: 113).

[0132] The epitope to which the antibodies of the present invention bind may consist of one 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 may consist of multiple non-contiguous amino acids (or amino acid sequences) of the LEPR. In some embodiments, the epitope is located on or adjacent to the leptin-binding domain of LEPR. In other embodiments, the epitope is located in a region other than the leptin-binding domain of the LEPR, for example, at a location on the surface of the LEPR in which the antibody, when bound to such epitope, does not interfere with leptin binding to the LEPR.

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

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

[0135] Using standard methods known in the art and presented in the examples herein, it is possible to determine whether an antibody binds to the same epitope as or competes with the reference anti-LEPR antibody for binding. For example, to determine whether a test antibody binds to the same epitope as a reference anti-LEPR antibody of the present invention, allow the reference antibody to bind to the LEPR protein. The ability of the test antibody to bind to the LEPR molecule is then assessed. If the test antibody is able to bind to LEPR after saturating binding to the reference anti-LEPR antibody, then it can be concluded that the test antibody binds to a different epitope than the epitope to which the reference anti-LEPR antibody binds. On the other hand, if the test antibody is unable to bind to a LEPR molecule after saturating binding to a reference anti-LEPR antibody, then the test antibody may bind to the same epitope as the epitope that is bound by the reference anti-LEPR antibody of the present invention. Additional routine experiments (e.g., peptide mutation assays and binding assays) can then be performed to confirm that the observed lack of binding of the test antibody is due to binding to the same epitope as the reference antibody, or that the lack of observed binding is due to binding is spatial blocking (or other phenomenon). Experiments of this type can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay known in the art. In accordance with certain embodiments of the present invention, two antibodies bind to the same (or overlapping) epitope if, for example, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits the binding of the other by at least at least 50%, but preferably 75%, 90% or even 99%, when measured using a competitive binding assay (see, for example, Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antibodies are considered to bind to the same epitope if substantially all amino acid mutations in the antigen that weaken or eliminate binding of one antibody weaken or eliminate binding of the other antibody. Two antibodies are considered to have "overlapping epitopes" if only a portion of the amino acid mutations that weaken or eliminate the binding of one antibody weaken or eliminate the binding of the other antibody.

[0136] To determine whether an antibody competes for binding (or cross-competes for binding) with a reference anti-LEPR antibody, the binding assay described above is performed in two ways. In the first direction, a reference antibody is allowed to bind to the LEPR protein under saturating conditions, followed by an assessment of the binding of the test antibody to the LEPR molecule. In the second direction, the test antibody is allowed to bind to the LEPR molecule under saturating conditions, followed by an assessment of the binding of the reference antibody to the LEPR molecule. If in both directions only the first (saturating) antibody is capable of binding to the LEPR molecule, then it is concluded that the test antibody and the reference antibody are competing for binding to the LEPR. One of ordinary skill in the art will appreciate that an antibody that competes for binding to a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding to an overlapping or adjacent epitope.

Obtaining human antibodies

[0137] The anti-LEPR antibodies of the present invention may be fully human antibodies. Methods for producing 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 create human antibodies that specifically bind to human LEPR.

[0138] Using, for example, VELOCIMMUNE™ technology or any other similar known method for producing fully human monoclonal antibodies, high-affinity chimeric anti-LEPR antibodies containing a human variable region and a mouse constant region are initially isolated. As shown in the experimental section below, antibodies are subjected to characterization and selection for desired characteristics including affinity, ligand blocking activity, selectivity, epitope, etc. If necessary, the mouse constant regions are replaced with the desired human constant region, for example from wild-type IgG1 or IgG4 or from modified IgG1 or IgG4, to obtain a fully human anti-LEPR antibody. Although the selected constant region may vary depending on the particular application, the characteristics of high antigen binding affinity or target specificity are inherent in the variable region. In certain cases, fully human anti-LEPR antibodies are isolated directly from antigen-positive B cells.

Biological equivalents

[0139] Antibodies and antibody fragments to LEPR of the present invention cover proteins having amino acid sequences that differ from the sequences of the described antibodies, but which retain the ability to bind human LEPR. Such variant antibodies and antibody fragments contain one or more additions, deletions or substitutions of amino acids when compared to the original sequence, but exhibit biological activity that is substantially equivalent to that of the described antibodies. Likewise, DNA sequences encoding an anti-LEPR antibody of the present invention include sequences that contain one or more of nucleotide additions, deletions, or substitutions when compared to the disclosed sequence, but which encode an anti-LEPR antibody or antibody fragment that is substantially biologically equivalent an anti-LEPR antibody or antibody fragment of the present invention. Examples of such variant amino acid and DNA sequences are discussed above.

[0140] Two antigen-binding proteins or antibodies are considered biological equivalents if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and/or extent of absorption are not significantly different when administered at the same molar dose under similar experimental conditions, as in the case of a single dose , and in the case of several doses. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in their extent of absorption, but not in their rate of absorption, and may still be considered biologically equivalent because such differences in absorption rate are intended and reflected in the drug product information, not are necessary to achieve effective concentrations of the drug in the body, for example during long-term use, and are considered not medically significant in the case of the particular drug product being studied.

[0141] In one embodiment, two antigen binding proteins are biological equivalents if there are no clinically significant differences with respect to their safety, purity, and potency.

[0142] In one embodiment, two antigen binding proteins are biological equivalents if the patient can be switched one or more times from the reference product to the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or a decrease in efficacy compared with that of continuing therapy without such a transfer.

[0143] In one embodiment, two antigen binding proteins are biological equivalents if they both act according to a common mechanism or mechanisms of action under a condition or conditions of use, to the extent such mechanisms are known.

[0144] Biological equivalence can be demonstrated using in vivo and in vitro methods. Measurements of the degree of biological equivalence include, for example, (a) an in vivo test in humans or other mammals in which the concentration of an antibody or its metabolites is measured in blood, plasma, serum or other biological fluids as a function of time; (b) an in vitro test that correlated with and predicted the in vivo bioavailability data in humans with reasonable accuracy; (c) an in vivo test in humans and other mammals in which the relevant early pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) a well-controlled clinical trial that establishes the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0145] Biologically equivalent variants of the anti-LEPR antibodies of the present invention can be constructed by, for example, creating various substitutions of residues or sequences or deleting terminal or internal residues or sequences that are not essential for biological activity. For example, cysteine residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unwanted or inappropriate intramolecular disulfide bridges upon renaturation. In other situations, biologically equivalent antibodies may include variants of anti-LEPR antibodies containing amino acid changes that modify the glycosylation characteristics of the antibodies, for example, mutations that eliminate or remove glycosylation.

Species selectivity and interspecific cross-reactivity

[0146] In accordance with certain embodiments, the present invention provides anti-LEPR antibodies that bind to human LEPR, but not to LEPR of other species. The present invention also includes anti-LEPR antibodies that bind to human LEPR and to LEPR of one or more non-human species. For example, the anti-LEPR antibodies of the present invention may bind to human LEPR and may or may not bind, as the case may be, to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit LEPR , goats, sheep, cows, horses, camels, cynomolgus monkeys, monkeys, rhesus monkeys or chimpanzees. In accordance with certain illustrative embodiments of the present invention, anti-LEPR antibodies are provided that specifically bind human LEPR and cynomolgus LEPR (eg, Macaca fascicularis). Other anti-LEPR antibodies of the present invention bind human LEPR but do not bind or bind weakly cynomolgus LEPR.

Polyspecific antibodies

[0147] Antibodies of the present invention may be monospecific or polyspecific (eg, bispecific). Multispecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen binding domains specific for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The anti-LEPR antibodies of the present invention may be coupled to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof may be operably linked (e.g., by chemical linkage, genetic fusion, non-covalent 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 the latter binding specificity.

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

[0149] An exemplary antibody in a bispecific format that can be used in the context of the present invention involves the use of a C _H 3 domain of a first immunoglobulin (Ig) and a C _H 3 domain of a second Ig, wherein the C _H 3 domains of the first and second Ig are different. from each other by at least one amino acid, and wherein the at least one amino acid difference impairs the binding of the bispecific antibody to Protein A compared to a bispecific antibody in which there is no amino acid difference. In one embodiment, the C _H 3 domain of the first Ig binds Protein A, and the C _H 3 domain of the second Ig contains a mutation that reduces or eliminates Protein A binding, such as the H95R modification (IMGT exon numbering; H435R EU numbering). The second _CH 3 may additionally contain modification Y96F (according to IMGT; Y436F according to EU). Additional modifications that may be present within the second C _H 3 include: D16E, L18M, N44S, K52N, V57M and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M and V422I by EU) in the case of IgG1-based antibodies ; N44S, K52N and V82I (IMGT; EU N384S, K392N and V422I) in the case of IgG2-based antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I by EU) for IgG4-based antibodies. The variants of the bispecific antibody format described above are contemplated within the scope of the present invention.

[0150] Other exemplary bispecific formats that may be used in the context of the present invention include, but are not limited to, scFv- or diabody-based bispecific formats, IgG-scFv fusion products, double variable domain (DVD)-Ig, quadromy, overhang structure -to-tooth, common light chain (e.g., common light chain with peak-to-tooth structure, etc.), CrossMab, CrossFab, (SEED)-antibody, leucine zipper, DuoBody, IgG1/ IgG2, Fab (DAF)-IgG dual-action and bispecific formats based on Mab ² (for a review of the above formats, see, for example, Klein et al. 2012, mAbs 4:6, 1-11, and sources referenced herein ). Bispecific antibodies can also be constructed using peptide/nucleic acid conjugation, for example, where non-naturally occurring amino acids with orthogonal chemical reactivity are used to produce site-specific antibody-oligonucleotide conjugates, which then self-assemble into multimeric complexes with defined composition, valence and geometric shape. (See, for example, Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

Compilation and administration of therapy

[0151] The present invention provides pharmaceutical compositions containing anti-LEPR antibodies or antigen-binding fragments thereof of the present invention. The pharmaceutical compositions of the present invention can be formulated with suitable carriers, excipients and other means that provide improved transport, delivery, tolerability and the like. Many suitable formulations can be found in a reference book well known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, Calif.), DNA conjugates, anhydrous absorption pastes, “oil-in-water” and “water-in-oil” emulsions, emulsions based on carbowax (polyethylene glycols with different molecular weights), semi-liquid gels and semi-liquid mixtures containing carbowax. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238–311.

[0152] The dose of antibody administered to a patient may vary depending on the age and size of the patient, the target disease, pathological condition, route of administration, and the like. The preferred dose is usually calculated according to body weight or body surface area. For an adult patient, it may be advantageous to administer intravenously the antibody of the present invention, typically at a single dose of from about 0.01 to about 20 mg/kg body weight, more preferably from about 0.02 to about 7, from about 0.03 to about 5 or from about 0.05 to about 3 mg/kg body weight. Depending on the severity of the condition, the frequency of administration and duration of treatment can be adjusted. Effective dosages and dosages of anti-LEPR antibodies can be determined empirically; for example, the patient's progress can be monitored through periodic assessment and the dose can be adjusted accordingly. Moreover, cross-species dosage scaling can be accomplished using methods well known in the art (eg, Mordenti et al., 1991, Pharmaceut. Res. 5:1351).

[0153] For a patient, for example a pediatric patient, it may be advantageous to administer, for example intravenously, the antibody at a therapeutically effective dose of about 5 mg/kg body weight, or about 1 mg/kg body weight to about 20 mg/kg body weight , or from about 1 mg/kg body weight to about 15 mg/kg body weight, or from about 5 mg/kg body weight to about 10 mg/kg body weight. For example, an intravenous (IV) loading dose of approximately 5 mg/kg may be selected to achieve serum antibody concentrations of 100 mg/L or higher. In addition, it may be advantageous to administer the antibody subcutaneously at a dose of about 250 mg, or at a dose of about 300 mg, or at a dose of from about 100 mg to about 500 mg, or from about 200 mg to about 300 mg. For example, a weekly subcutaneous (SC) maintenance dose of 250 mg H4H17319R2 or 300 mg H4H17319R2 will maintain serum trough concentrations of 100 mg/L or greater. In some aspects, the SC dosing schedule begins several days after administration of the IV loading dose to best maintain target serum trough concentrations. In some aspects, the first SC dose is administered 2-7 days after the saturation dose, such as 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the saturation dose. In some aspects of the SC, the dose is administered once every 3-14 days, for example, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every week, once every 10 days or once every 2 weeks, eg 3 weekly SC doses after the first SC dose followed by monthly doses (approximately every 28 days). In one embodiment of the present invention, the therapeutically effective dose of the antibody (eg, H4H17319P2) is as represented in Figure 19 herein, but does not necessarily continue beyond the last monthly dose indicated therein.

[0154] In some aspects, it is necessary to maintain minimum serum concentrations of about 50 mg/L to about 200 mg/L, or about 100 mg/L, or about 150 mg/L, or at or above 50 mg/L, or at a level of 100 mg/L or higher, or at a level of 150 mg/L or higher.

[0155] Various delivery systems are known that can be used to administer the pharmaceutical composition of the present invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al., 1987, J Biol Chem 262:4429-4432). Routes of administration 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 integuments (for example, oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration may be systemic or local.

[0156] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, with regard to subcutaneous delivery, when delivering the pharmaceutical composition of the present invention, a pen-type delivery device can be freely used. This pen-type delivery device can be reusable or disposable. A reusable pen-type delivery device typically uses a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition from the cartridge has been administered and the cartridge remains empty, the empty cartridge can be immediately discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen-type delivery device can then be reused. In the case of a disposable pen delivery device, there is no replaceable cartridge. Instead, the disposable pen delivery device is pre-filled with a pharmaceutical composition contained in a reservoir within the device. After emptying the reservoir of the pharmaceutical composition, the entire device is disposed of as waste.

[0157] For subcutaneous delivery of the pharmaceutical composition of the present invention, a variety of reusable delivery devices such as pen syringes and auto-injectors are used. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burgdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, syringe -HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, Indiana), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), syringe pen BD™ (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™ and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), to name a few. Examples of disposable pen delivery devices used for subcutaneous administration of a pharmaceutical composition of the present invention include, but are not limited to, the SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk) and KWIKPEN™ (Eli Lilly), an auto-injector SURECLICK™ (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMIRA™ pen (Abbott Labs, Abbott Park, IL), to name a few of them.

[0158] In certain situations, the pharmaceutical composition can be delivered in the form of a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials may be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, FL. In yet another embodiment, the controlled release system can be placed near the target of the composition, 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 Langer, 1990, Science 249:1527-1533.

[0159] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, and the like. These injectable preparations can be prepared using generally known methods. For example, injectable preparations can be prepared, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof described above in a sterile aqueous or oily medium commonly used for injection. As the aqueous injection medium, for example, physiological saline solution, isotonic solution containing glucose and other auxiliary agents, etc. are used, which can be used in combination with a suitable solubilizing agent such as alcohol (for example, ethanol), polyhydric alcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oil medium, for example, sesame oil, soybean oil and the like are used, which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol and the like. The injectable composition obtained in this manner is preferably filled into a suitable ampoule.

[0160] Advantageously, the pharmaceutical compositions described above for oral or parenteral administration are prepared in dosage forms with a unit dose adjusted to correspond to the dose of the active ingredients. Such unit dose dosage forms include, for example, tablets, pills, capsules, injectable formulations (ampoules), suppositories, etc. The amount of the above antibody contained is generally from about 5 to about 500 mg per unit dose dosage form; in particular in the form of an injectable composition; for other dosage forms, it is preferable that the content of the above antibody be from about 5 to about 100 mg and from about 10 to about 250 mg.

Applications of antibodies in therapy

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

[0162] The antibodies and antigen-binding fragments provided herein are useful, inter alia, for the treatment, prevention and/or attenuation of the severity of any disease or disorder associated with or mediated by a metabolic disorder or hypoleptinemia, such as non-alcoholic fatty liver disease, NASH , female infertility, amenorrhea, abnormal hormonal cycle, immune function disorder, hypothyroidism, obesity, monogenic obesity, type I diabetes mellitus, type II diabetes mellitus, lipodystrophy, congenital lipodystrophy, generalized lipodystrophy, acquired lipodystrophy, focal lipodystrophy, congenital focal lipodystrophy, congenital generalized lipodystrophy, acquired focal lipodystrophy, and acquired generalized lipodystrophy, or otherwise treatable by stimulating or activating LEPR-mediated signaling or mimicking natural leptin activity in vitro or in vivo. For example, antibodies and antigen-binding fragments thereof are useful in the treatment of lipodystrophic conditions. Иллюстративные липодистрофические состояния, которые поддаются лечению с помощью антител и антигенсвязывающих фрагментов по настоящему изобретению, включают, например, врожденную генерализованную липодистрофию, врожденную очаговую липодистрофию, приобретенную генерализованную липодистрофию, семейную очаговую липодистрофию, приобретенную очаговую липодистрофию, центробежную абдоминальную липодистрофию, lipoatrophia annularis, локализованную липодистрофию and lipodystrophy associated with HIV, as well as symptoms associated with such conditions.

[0163] Antibodies to LEPR and antigen binding fragments thereof provided herein are useful for the treatment, prevention and/or amelioration of monogenic obesity and/or lipodystrophy. Monogenic obesity and lipodystrophy can be associated with many pathologies, including, for example: extremely early-onset obesity in subjects with a BMI above the ^85th percentile for age and sex; hyperphagia and impaired satiety in subjects exhibiting food-seeking and aggressive eating behavior; impaired immune function with decreased CD4+ T cell counts and recurrent (and possibly fatal) infections; insulin resistance and hyperinsulinemia; non-alcoholic fatty liver disease, hepatic steatosis and progression to lipodystrophy; dyslipidemia leading to hypertriglyceridemia; diabetes mellitus with elevated HbAlc and/or glucose levels and impaired glucose tolerance; impaired reproductive function up to hypogonadism, delayed sexual development; decreased severity of secondary sexual characteristics, absence or irregularity of menstruation and infertility; lack of a pubertal growth spurt, leading to short stature, abnormal secretion of growth hormone; hypothyroidism or thyroid dysfunction, changes in T3, or TSH, or free thyroxine levels; and various changes in bone tissue, including bone density and bone mineralization. The spectrum of conditions and symptoms associated with monogenic obesity and/or lipodystrophy may vary depending on the underlying genetic causes, such as AGPAT2, LMNA, BSCL2 or others. A particular mutation may result in loss of leptin or LEPR function, which has endocrine effects with varying degrees of severity, such as irregular menstruation versus complete amenorrhea.

[0164] The antibodies and antigen binding fragments provided herein are also useful for treating, mitigating, or preventing one or more symptoms of a disease or condition associated with a metabolic disorder or hypoleptinemia. Such symptoms include obesity, obesity, hyperphagia, hyperglycemia, hypertriglyceridemia, hypercholesterolemia, insulin resistance, dyslipidemia, growth retardation, delayed pubertal growth spurt, abnormal growth hormone secretion, elevated HbAlc, low bone mineral density (or low bone mass), low mineralization bone tissue and low lean body mass.

[0165] The present invention also includes anti-LEPR antibodies and antigen-binding fragments thereof that are useful for restoring leptin-mediated signaling in cells, tissues and organs expressing one or more LEPR mutations. Such mutations may be associated with metabolic disorders or hypoleptinemia and diseases or conditions related to metabolic disorders or hypoleptinemia, such as obesity, congenital lipodystrophy, infertility and non-alcoholic fatty liver disease. For example, certain LEPR mutant variants have been identified that exhibit decreased or absent signaling in the presence of leptin and are associated with obesity and related disorders. As used herein, a LEPR mutant that exhibits a lack of signal transduction in the presence of leptin is referred to as a “signaling defective LEPR mutant.” An example of a LEPR mutation that results in complete disruption of signal transduction is LEPR-A409E (Farooqi et al., 2007, N Engl J Med 356(3): 237-247). As used herein, a LEPR mutant that exhibits decreased signaling in the presence of leptin (compared to wild-type LEPR) is referred to as a “attenuated LEPR mutant.” An example of a LEPR mutation that results in reduced signal transduction is LEPR-P316T (Mazen et al., 2011, Mol Genet Metab 102:461-464). Thus, the present invention includes anti-LEPR antibodies and antigen-binding fragments thereof that are useful for the treatment, prevention and/or amelioration of diseases and disorders caused by one or more LEPR mutant variants characterized by impaired signal transduction (eg, A409E) and/or weakening of signal transmission (for example, P316T), or associated with them.

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

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

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

[0169] The LEPR agonistic antibodies provided herein are useful for the treatment of obesity or for reducing body weight. In some embodiments, the treatment provides a reduction in fat mass, but not lean mass, in the subject receiving the treatment. In some aspects, treatment causes a person to eat fewer calories or reduce their food intake.

[0170] The LEPR agonist antibodies provided herein are useful for treating female infertility or restoring normal hormonal cycles associated with leptin deficiency. In some aspects, treatment may provide increased fertility and/or increased likelihood of conception. In some aspects, treatment can restore normal menstrual cycles. Methods for restoring a normal menstrual cycle that has been disrupted, at least in part, due to leptin deficiency are also part of the present invention.

[0171] As shown herein, the method is applicable if the subject in need thereof is a subject who has hypoleptinemia or leptin deficiency, or who does not have hypoleptinemia or leptin deficiency. This method is applicable if metabolic disorder, obesity or obesity is associated or not associated with, or is or is not caused by, a LEPR mutation resulting in signal transduction disorder or signal transduction impairment.

[0172] The LEPR agonist antibodies provided herein are useful for the treatment of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis (NASH) in patients with hypoleptinemia, lipodystrophy or leptin deficiency. Treatment may provide a reduction in the severity of symptoms of non-alcoholic fatty liver disease, such as hepatic steatosis, in the subject. In some cases, alanine transaminase (ALT) and/or aspartate transaminase (AST) levels in a subject's blood plasma decrease after receiving treatment.

[0173] The LEPR agonist antibodies provided herein are useful for treating hyperphagia, hyperglycemia, insulin resistance, dyslipidemia, non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease by stimulating STAT3-mediated signaling in the hypothalamus. Treatment may provide a reduction in circulating plasma triglycerides and/or circulating plasma total cholesterol.

[0174] The LEPR agonist antibodies provided herein are useful for the treatment of lipodystrophy. The treatment provides relief from hyperglycemia, a decrease in the severity of insulin resistance, and/or a decrease in HbAlc levels in the subject receiving treatment.

[0175] The LEPR agonistic antibodies provided herein are useful for the treatment of infertility and/or amenorrhea associated with metabolic disease or hypoleptinemia. Treatment regulates hormonal cycles and may provide improved fertility in women receiving treatment. Treatment can restore normal menstrual cycles.

[0176] The LEPR agonist antibodies provided herein are useful for treating a disorder of immune function, such as a decrease in CD4+ T cells associated with hypoleptinemia and/or leptin deficiency. Treatment may provide improvements in immune function, such as an increase in the number of CD4+ T cells.

[0177] The LEPR agonist antibodies provided herein are useful for the treatment of growth failure, failure of the pubertal growth spurt, and/or abnormal growth hormone secretion associated with congenital leptin deficiency. Treatment may provide improvement in growth, may promote the pubertal growth spurt, and/or may provide improvement in growth hormone secretion.

[0178] The LEPR agonist antibodies provided herein are useful for the treatment of hypothyroidism associated with congenital leptin deficiency. Treatment may provide improvement in symptoms associated with hypothyroidism.

[0179] The LEPR agonist antibodies provided herein are useful for the treatment of low bone mineral density and/or bone mineralization associated with hypoleptinemia and/or leptin deficiency. Treatment may provide improvement in bone mineral density and/or may provide improvement in bone mineralization.

[0180] In the context of the treatments described herein, an anti-LEPR antibody may be administered as monotherapy (i.e., as a single therapeutic agent) or in combination with one or more additional therapeutic agents (examples of which are described elsewhere in this document).

Combination therapies and formulations

[0181] The present invention includes compositions and therapeutic formulations comprising any of the anti-LEPR antibodies described herein in combination with one or more additional therapeutically active components, and methods of treatment comprising administering such combinations to subjects in need thereof.

[0182] The anti-LEPR antibodies of the present invention may be formulated together and/or administered in combination with one or more additional therapeutically active components, such as, for example, pharmaceuticals prescribed for the treatment of obesity, hypercholesterolemia, hyperlipidemia, type 2 diabetes mellitus, type 1 diabetes, appetite control, amenorrhea, infertility, etc. Examples of such additional therapeutically active components include, for example, recombinant human leptin (eg, metreleptin [MYALEPT]), PCSK9 inhibitors (eg, 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 cotransporter 2 (SGLT2) inhibitors (eg , dapagliflozin, canagliflozin, empagliflozin, etc.), GLP-1 agonists/analogues (e.g. extendin-4, exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, etc.), glucagon inhibitors (GCG) (e.g. anti-GCG antibodies), glucagon receptor (GCGR) inhibitors (e.g., anti-GCGR antibodies, small molecule GCGR antagonists, GCGR-specific antisense oligonucleotides, anti-GCGR aptamers [e.g., Spiegelmers], etc.), angiopoietin-like protein inhibitors (ANGPTL ) (e.g. anti-ANGPTL3, anti-ANGPTL4, anti-ANGPTL8, etc.), phentermine, orlistat, topiramate, bupropion, topiramate/phentermine, bupropion/naltrexone, bupropion/zonisamide, pramlintide/metreleptin, lorcaserin, cetilistat, tesofensine, wellneperit, etc. Additional examples include, for example, fish oil, pioglitazone, setmelanotide, fibrates (eg, fenofibrate), prednisone, niacin, anticonvulsants, digoxin, coumadin, vitamin D, thyroxine, thyroid supplement, vitamin supplement, calcium supplement, carnitine , coenzyme Q10, anti-constipation drug, anti-allergy drugs, gabapentin, narcotic drug, ketamine, lidocaine and venlafaxine hydrochloride. In one embodiment of the present invention, the anti-LEPR antibodies of the present invention are not included in or administered with the anorexigenic agent.

[0183] Additional therapeutically active component(s), for example, any of the above agents or derivatives thereof, can be administered immediately before, simultaneously with, or shortly after the anti-LEPR antibody of the present invention (for the purposes of the present invention, such administration schedules are considered as administration of an anti-LEPR antibody “in combination with” an additional therapeutically active component). The present invention includes pharmaceutical compositions in which the anti-LEPR antibody of the present invention is formulated together with one or more additional therapeutically active components, as described elsewhere herein.

[0184] The present invention also includes methods of using compositions and therapeutic formulations containing any of the anti-LEPR antibodies described herein in combination with therapeutic procedures such as plasmapheresis.

Administration regimens

[0185] In accordance with certain embodiments of the present invention, multiple doses of an anti-LEPR antibody (or a pharmaceutical composition comprising a combination of an anti-LEPR antibody and any of the additional therapeutically active agents mentioned herein) can be administered to a subject over a period of time. Methods according to this aspect of the present invention involve sequentially administering multiple doses of an anti-LEPR antibody of the present invention to a subject. As used herein, “sequential administration” means that each dose of anti-LEPR antibody is administered to a subject at a separate point in time, for example, on different days separated by a predetermined interval (for example, hours, days, weeks or months). The present invention includes methods that include sequentially administering to a patient one initial dose of an anti-LEPR antibody, then one or more secondary doses of an anti-LEPR antibody, and then optionally one or more tertiary doses of an anti-LEPR antibody.

[0186] The terms "initial dose", "secondary doses" and "tertiary doses" refer to the time sequence of administration of the anti-LEPR antibody of the present invention. Thus, the "starting dose" is the dose that is administered at the beginning of the treatment regimen (also called the "starting dose", "loading dose", "starting dose", etc.); "secondary doses" are doses that are administered after the initial dose; and “tertiary doses” are doses that are administered after secondary doses. Each of the initial, secondary and tertiary doses may contain the same amount of anti-LEPR antibody, but typically may differ from each other in the frequency of administration. However, in certain embodiments, the amount of anti-LEPR antibody contained in the initial, secondary, and/or tertiary doses differs from each other (eg, adjusted upward or downward as necessary) throughout the course of treatment. In some embodiments, two or more (eg, 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as "loading doses" followed by subsequent doses that are administered at less frequent intervals (eg, "maintenance doses").

Diagnostic and analytical applications of antibodies

[0187] The anti-LEPR antibody of the present invention can also be used to detect and/or measure the content of LEPR or LEPR-expressing cells in a sample, for example, for diagnostic purposes. For example, an anti-LEPR antibody or fragment thereof can be used to diagnose a condition or disease characterized by aberrant expression (eg, overexpression, underexpression, lack of expression, etc.) of LEPR. Exemplary LEPR diagnostic assays may involve, for example, contacting a sample obtained from a patient with an anti-LEPR antibody of the present invention, wherein the anti-LEPR antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-LEPR antibody may be used in diagnostic applications in combination with a secondary antibody that is itself labeled for detection. The detectable label or reporter molecule may be a radioactive isotope 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. Specific exemplary assays that can be used to detect or measure LEPR content in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorting (FACS), and positron emission tomography (PET) scanning.

[0188] Samples that can be used in LEPR-related diagnostic assays in accordance with the present invention include a sample of any tissue or fluid obtained from a patient that contains detectable amounts of LEPR protein or fragments thereof in normal or pathological conditions. Typically, the initial determination of a baseline or standard LEPR level will measure LEPR levels in a specific sample obtained from a healthy patient (eg, a patient not affected by a disease or condition associated with abnormal LEPR levels or activity). This baseline LEPR level can then be compared with LEPR levels measured in samples obtained from individuals suspected of having a disease or condition associated with LEPR.

EXAMPLES

[0189] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to carry out and use the methods and preparation and use of the compositions of the present invention, and are not intended to limit the scope of what the inventors of the present invention considered as their invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. quantity, temperature, etc.), but some experimental errors and deviations must be taken into account. Unless otherwise noted, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

Example 1: Preparation of antigen-binding proteins that specifically bind the leptin receptor (LEPR)

[0190] Antibodies to LEPR were generated by immunizing a VELOCIMMUNE® mouse (ie, an engineered mouse containing DNA encoding the variable regions of the human immunoglobulin heavy chain and kappa light chain) with an immunogen containing the extracellular domain of LEPR. The humoral immune response was monitored using a LEPR-specific immunoassay. Fully human anti-LEPR antibodies were isolated and purified using previously described techniques.

[0191] Certain biological properties of exemplary anti-LEPR antibodies produced in accordance with the methods of this example are described in detail in the examples presented below.

Example 2 Amino Acid and Nucleic Acid Sequences of the Heavy and Light Chain Variable Regions

[0192] Table 1 provides the variable region amino acid sequence identifiers and heavy and light chain CDRs of selected anti-LEPR antibodies of the present invention. The corresponding nucleic acid sequence identifiers are presented in Table 2.

[0193] Antibodies are generally named herein according to the following nomenclature: a prefix indicating Fc (e.g., "H4H", "H1M", "H2M", etc.) is followed by a numeric identifier (e.g., "16650" , "16679", etc.), followed by the suffix "P" or "N". Thus, in accordance with this nomenclature, the antibody may be referred to herein as, for example, "H4H16650P2", "H4H16679P2", etc. The Fc prefixes in the antibody names used herein (H4H, H1M, and H2M) indicate the specific isotype of the Fc region of the antibody. For example, antibody "H4H" contains a human IgG4 Fc, while antibody "H1M" contains a mouse IgG1 Fc (all variable regions are fully human, as indicated by the first "H" in the antibody designation). One of ordinary skill in the art will appreciate that an antibody with an Fc of a particular isotype can be converted into an antibody with an Fc of a different isotype (e.g., an antibody with an Fc from a mouse IgG1 can be converted into an antibody with an Fc from a human IgG4, etc.), but in any case, the variable domains (including CDRs), which are designated by the numeric identifiers shown in Tables 1 and 2, will remain the same, and the binding properties are expected to be identical or substantially similar regardless of the nature of the Fc domain.

[0194] The term "comparative MAb" used in the examples herein 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.

[0195] See International Patent Application Publication No. WO 2017/66204.

Example 3: Surface Plasmon Resonance Binding Affinities and Kinetic Constants of Anti-Human LEPR Monoclonal Antibodies

[0196] Equilibrium dissociation constants ( _KD values) for LEPR binding to purified anti-LEPR monoclonal antibodies were determined using a real-time surface plasmon resonance biosensor using a Biacore 4000 instrument. All binding studies were performed in a running buffer consisting of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% v/v. surfactant Tween-20, pH 7.4 (HBS-ET), at 25°C and 37°C. The Biacore sensor surface was first derivatized by immobilizing the amino group of a mouse monoclonal anti-human Fc antibody (GE, no. BR-1008-39) to capture the anti-LEPR monoclonal antibody. Binding studies were performed with the following LEPR reagents: extracellular domain of human LEPR expressed with a C-terminal myc-myc-hexahistidine tag (hLEPR.mmh; SEQ ID NO: 114), extracellular domain of Macasa fascicularis LEPR expressed with a C-terminal myc- myc-hexahistidine tag (mfLEPR.mmh; SEQ ID NO: 117), extracellular domain of human LEPR expressed with the C-terminal Fc tag of mouse IgG2a (hLEPR.mFc; SEQ ID NO: 115), extracellular domain of mouse LEPR expressed with a C-terminal myc-myc-hexahistidine tag (mLEPR.mmh; SEQ ID NO: 118), and the extracellular domain of rat LEPR expressed with a C-terminal myc-myc-hexahistidine tag (rLEPR.mmh; SEQ ID NO: 119). Various concentrations of LEPR reagents were first prepared in HBS-ET running buffer (100 nM-3.7 nM; 3-fold serial dilution) and applied to the surface with anti-LEPR monoclonal antibody captured by anti-human Fc antibody for 4 minutes at flow rate 30 μl/minute, while monitoring the dissociation of the LEPR reagent bound to the monoclonal antibody was carried out for 10 minutes in HBS-ET running buffer. Kinetic rate constants for association (k _a ) and dissociation (k _d ) were determined by fitting real-time binding sensorgrams to a mass transfer-limited 1:1 binding model using Scrubber 2.0 s curve fitting software. Equilibrium binding dissociation constants (K _D ) and dissociation half-periods (t½) were calculated based on the kinetic rate constants:

[0197] Binding kinetic parameters for the binding of hLEPR.mmh, mfLEPR.MMH or hLEPR.mFc to various anti-LEPR monoclonal antibodies of the present invention at 25°C and 37°C are shown in Tables 3-8.

[0198] At 25°C, anti-LEPR monoclonal antibodies bound to hLEPR-MMH with K _D values ranging from 7.93 nM to 148 nM, as shown in Table 5. At 37°C, anti-LEPR monoclonal antibodies bound to hLEPR-MMH with K _D values ranging from 14.8 nM to 326 nM, as shown in Table 4.

[0199] Ten of the 12 anti-LEPR monoclonal antibodies of the present invention bound to mfLEPR.MMH. At 25°C, anti-LEPR monoclonal antibodies bound to mfLEPR.MMH with K _D values ranging from 2.27 nM to 139 nM, as shown in Table 7. At 37°C, anti-LEPR monoclonal antibodies bound to mfLEPR.MMH with K values _D ranges from 5.18 nM to 264 nM, as shown in Table 8.

[0200] At 25°C, anti-LEPR monoclonal antibodies bound to hLEPR-mFc with K _D values ranging from 613 pM to 5.7 nM, as shown in Table 7. At 37°C, anti-LEPR monoclonal antibodies bound to hLEPR-mFc with K _D values ranging from 1.16 nM to 12.8 nM, as shown in Table 8.

[0201] None of the anti-LEPR monoclonal antibodies of the present invention bound to mLEPR.MMH or rLEPR.MMH at 25°C or at 37°C (data not shown).

Example 4 Antibodies to LEPR of the present invention bind LEPR in the presence of leptin:LEPR binding

[0202] The blocking of anti-LEPR antibody binding to LEPR by human leptin was assessed using a real-time surface plasmon resonance biosensor on a Biacore T200 instrument. The entire 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 surface of the Biacore CM5 sensor was first derivatized by immobilization at the amino group of human leptin (R&D Systems, No. 398-LP) using standard methods of chemical surface modification using EDC /NHS. The human LEPR-human leptin complex was formed by applying 20 nM of the extracellular domain of human LEPR expressed with a C-terminal myc-myc-hexahistidine tag (hLEPR-MMH; SEQ ID NO: xx) to the surface of the Biacore sensor immobilized with human leptin at a flow rate 10 µl/min. or 25 µl/min. for 4 minutes to achieve a binding response of approximately 200 RU. To assess whether antibody binding to hLEPR-MMH is blocked by human leptin, 200 nM anti-LEPR monoclonal antibody was applied to the preformed hLEPR-MMH:human leptin complex at a flow rate of 50 μl/min. or 25 µl/min. within 4-5 minutes. All anti-LEPR antibodies of the present invention bound to the complex of hLEPR-MMH and human leptin (“leptin:LEPR”) with almost equal signal strength, and the observed binding values expressed in RU are presented in Table 9. This result indicates that that human leptin does not block the binding of hLEPR-MMH to the anti-LEPR antibodies tested.

Example 5: Human Leptin Receptor Blocking ELISA Assay

[0203] To perform the ELISA, human leptin (hLeptin; R&D Systems, #398-LP-01M) was applied at a concentration of 5 μg/ml in PBS to a 96-well microtiter plate overnight at 4°C. Nonspecific binding sites were subsequently blocked using 0.5% (w/v) BSA in PBS. A constant amount of 10 nM of the extracellular domain portion of the LEPR protein that was expressed with a C-terminal human Fc tag (hLEPR.hFc; SEQ ID NO: 116) was titrated with anti-LEPR antibody, hLeptin protein, or isotype antibody. control in serial dilution in the range from 8.5 pM to 500 nM. These antibody-protein or protein-protein complexes were then incubated for 1.5 hours at room temperature (RT). The complexes were then transferred to hLeptin-coated microtiter plates and incubated for 2 hours at RT, the wells were washed, and plate-bound hLEPR.hFc was detected using a polyclonal anti-human IgG antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch Inc, no. 109-035-098). Samples were treated with TMB solution (BD Biosciences, #555214; substrates A and B mixed 1:1 according to manufacturer's instructions) to obtain a colorimetric reaction and then neutralized with 1 M sulfuric acid before measuring absorbance at 450 nm at tablet reader Victor X5.

[0204] Data analysis was performed using a sigmoidal dose-response model in Prism™ software (GraphPad). The percentage blocking at the maximum concentration of the antibody tested was calculated as an indication of the antibody's ability to block the binding of 10 nM hLEPR.hFc to human leptin on the plate. In calculations, the binding signal of 10 nM hLEPR.hFc without the presence of antibody was designated as 100% binding or 0% blocking; and the original buffer signal alone without the presence of hLEPR.hFc was designated as 0% binding or 100% blocking. Blocking data at 500 nM antibody concentration are summarized in Table 10.

[0205] As shown in Table 10, none of the anti-LEPR antibodies of the present invention demonstrated >28% blocking of hLEPR.hFc binding to an hLeptin-coated surface. However, the reference antibody and hLeptin as a positive control were able to block 99% of the binding of hLEPR.hFc to the hLeptin-coated surface. The isotype control antibody did not demonstrate a measurable level of blocking at concentrations up to 500 nM.

Example 6 Cell Binding by FACS Analysis to HEK293/Mycx2-hLepR(ecto)-GPI-Anchored Cells

[0206] The leptin receptor, LEPR, is a single-pass transmembrane receptor of the class I cytokine receptor family (Tartaglia et al. (1997) JBiol Chem 7:272(10):6093-6). LEPR can bind to leptin, a protein expressed predominantly by adipose tissue that is involved in the regulation of food intake and metabolism (Friedman et al. (2014) J Endocrinol 223(1):T1-8).

[0207] To evaluate cell binding by anti-LEPR antibodies, stable HEK293 cell lines were generated. One cell line, hereafter known as HEK293/hLEPR-GPI, stably expressed the extracellular domain of human LEPR (amino acids 22-839 under accession no. P48357 (SEQ ID NO: 113), isoform B) with an N-terminal myc-myc tag and A C-terminal peptide sequence from human carboxypeptidase M that directs the addition of GPI (glycosylphosphatidylinositol) (Deddish et al. (1990) J. Biological Chemistry 265:25:15083-89) so that the protein can be GPI-anchored in the membrane. Another HEK293 cell line was generated to provide stable expression of full-length human LEPR (amino acids 1-1165 accession no. P48357 (SEQ ID NO: 113), isoform B) along with a luciferase reporter (StatS-luciferase, Stat3-luc, SA Bioscience, no. CLS -6028L), and is referred to hereinafter as HEK293/Stat3-luc/hLEPR-FL. HEK293 cells with only the StatS-luciferase reporter (HEK293/Stat3-luc) were also generated as a control cell line.

[0208] For FACS analysis, parental HEK293 cells and HEK293/hLEPR-GPI cells were separated and seeded into 96-well v-bottom plates at 5 x 10 ⁵ cells/well in PBS containing 2% FBS (FACS buffer) . To test whether the presence of leptin affects the ability of anti-hLEPR antibodies to bind to cells, FACS buffer with or without 1 μM human leptin (R&D Systems, #398-LP) was incubated with cells for 30 minutes at 4°C, followed by the addition of anti-LEPR antibodies or control antibodies at a concentration of 10 nM in FACS buffer. Cells were then incubated for 30 minutes at 4°C, followed by washing and then incubation with 16 μg/ml Alexa Fluor®-647-conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc., #109-547-003) for 30 minutes at 4°C. Cells were then fixed with BD CytoFix™ (Becton Dickinson, #554655), filtered, and analyzed on a HyperCyt flow pitometer (Beckman Coulter). Controls, which were separate unstained and secondary antibodies, were also tested for all cell lines. Results were analyzed using ForeCyt software (IntelliCyt) and FlowJo version 10 to determine geometric mean fluorescence values for viable cells. The geometric mean fluorescence value for each sample was then normalized to the geometric mean of unstained cells to obtain relative binding values for each condition, called “binding coefficients,” and these binding coefficients were recorded for each antibody tested.

[0209] As shown in Table 11,9, the anti-LEPR antibodies of the present invention, tested at a concentration of 10 nM, demonstrated binding to HEK293/hLEPR-GPI cells with binding coefficients ranging from 824 to 3374 without leptin. Anti-LEPR antibodies also bound in the presence of 1 μM leptin with binding coefficients ranging from 398- to 4184-fold. As shown in Table 11, the reference antibody tested at a concentration of 10 nM demonstrated binding to HEK293/hLEPR-GPI cells by a factor of 2349 in the absence of leptin, but showed significantly less binding to cells in the presence of 1 μM leptin by a factor binding 112. Anti-LEPR antibodies did not demonstrate any significant level of binding to parental HEK293 cells, with binding ratios with and without 1 µM leptin ranging from 1- to 9-fold. Isotype control and secondary antibody samples alone also did not demonstrate significant levels of binding to any of the cell lines with or without leptin, with binding ratios ranging from 1- to 6-fold.

[0210] As shown in Table 12, four antibodies of the present invention, tested at a concentration of 70 nM without leptin, demonstrated binding to HEK293/hLEPR-GPI cells with binding coefficients ranging from 707 to 1131 times, and to HEK293/ Stat3-luc/hLEPR-FL - with binding coefficients ranging from 42 to 51. Anti-LEPR antibodies did not demonstrate any significant level of binding to HEK293/Stat3-luc cells, with binding coefficients ranging from 1 to 8 once. Isotype control and secondary antibody samples alone also did not demonstrate significant levels of binding to any of the cell lines tested, with binding ratios ranging from 1- to 2-fold.

Example 7 Anti-LEPR Antibodies of the Present Invention Activate LEPR-Mediated Signaling in the Presence and Absence of Leptin

[0211] A biological assay was developed to detect transcriptional activation of STAT3 upon LEPR activation using a reporter cell line that stably expresses full-length human LEPR (hLEPR; amino acids 1 to 1165 under accession number NP 002294.2) together with a luciferase reporter (STAT3-Luc; Qiagen, No. CLS-6028L) in the IMR-32 cell line, a human neuroblastoma cell line. The resulting stable cell line, termed IMR-32/STAT3-Luc/hLEPR, was isolated and maintained in MEM with Earle's salts supplemented with 10% FBS, NEAA, 1 μg/ml puromycin, 100 μg/ml hygromycin B, and penicillin/streptomycin/ L-glutamine (complete medium).

[0212] The resulting biological assay was used to measure the effect of the anti-LEPR antibodies of the present invention on LEPR-mediated signaling in the presence or absence of leptin. For biological assays, IMR-32/STAT3-Luc/hLEPR cells were seeded at a density of 20,000 cells/100 μl/well for a 96-well format in complete medium and the next day replaced with an appropriate volume of Opti-MEM medium supplemented with 1% BSA and 0. 1% FBS (assay buffer) for 30 minutes. To measure the effect of antibodies of the present invention in the absence of leptin, semi-log serial dilutions of anti-LEPR antibodies or isotype control antibodies and human leptin (hLeptin; R&D Systems, #398-LP) were made to final concentrations ranging from 100 nM to 300 fM in leptin buffer. analysis, the resulting dilutions were added to the cells and then incubated overnight at 37°C in 5% CO2.

[0213] To measure the effect of the antibodies of the present invention in the presence of leptin, a fixed concentration of 200 pM human leptin in assay buffer was added to the cells, followed immediately by the addition of anti-LEPR antibodies or isotype control antibodies, which were subjected to semi-log serial dilution to final concentrations ranging from 100 nM to 300 fM. The samples were then incubated overnight at 37°C in 5% CO2. OneGlo reagent (Promega, no. E6051) was then added to the samples, and luciferase activity was measured on an Envision multichannel plate reader (Perkin Elmer) in luminescent mode. Relative light unit (RLU) values were obtained and the results were analyzed using nonlinear regression using GraphPad Prism software (GraphPad). The maximum RLU value obtained in response to a dose of hLeptin was taken as 100% activation in the IMR-32/STAT3-Luc/hLEPR assay.

[0214] As shown in Table 13, in Study 1, in the absence of hLeptin, all anti-LEPR antibodies tested showed 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%, respectively, of the maximum activation obtained as a result of the hLeptin dose response. In Study 2, in the absence of hLeptin, the 4 anti-LEPR antibodies tested demonstrated stimulation of IMR-32/STAT3-Luc/hLEPR cells with EC ₅₀ values ranging from 61.9 pM to 206.9 pM and maximum activation ranging from 65% to 68% accordingly from the maximum activation obtained as a result of the response to the hLeptin dose. In Study 1, in the presence of 200 pM hLeptin, all anti-LEPR antibodies tested demonstrated stimulation of IMR-32/STAT3-Luc/hLEPR cells with EC ₅₀ values ranging from 20.2 pM to 523 pM and maximum activation ranging from 66% to 107% accordingly from the maximum activation obtained as a result of the response to the hLeptin dose. Because these antibodies enhanced leptin-induced LEPR-mediated signaling, these antibodies were classified as “enhancers” as defined herein. In Study 2, in the presence of 200 pM hLeptin, the 4 anti-LEPR antibodies tested demonstrated stimulation of IMR-32/STAT3-Luc/hLEPR cells with EC ₅₀ values ranging from 51.9 pM to 257.3 pM with maximum activation ranging from 76% to 88% of the maximum activation obtained as a result of the hLeptin dose response. LEPR-mediated signaling was not markedly enhanced by these antibodies in the presence of leptin. The isotype control antibody did not demonstrate any measurable level of stimulation of IMR-32/STAT3-Luc/hLEPR cells in any of the assays.

Example 8 Antibodies to LEPR of the present invention activate signaling in cells expressing mutant variants of LEPR characterized by impaired signal transduction or reduced signal transduction

[0215] Mutant variants of LEPR have been identified that exhibit impaired or attenuated leptin-mediated signaling and are associated with early-onset obesity. For example, LEPR-A409E is a signal transduction defective mutant of the LEPR protein that does not transmit leptin-mediated signals to STAT3; the A409E mutant was initially identified as a monogenic cause of early-onset obesity. (Farooqi et al., 2007, N Engl J Med 356(3): 237-247). LEPR-P316T is a mutant variant of the LEPR protein characterized by reduced signaling that has also been shown to be associated with early-onset obesity. (Mazen et al., 2011, Mol Genet Metab 102:461-464).

[0216] In this example, the ability of the anti-LEPR antibodies of the present invention to stimulate LEPR-mediated signaling in cell lines expressing mutant LEPR variants characterized by impaired or reduced signal transduction was assessed. Specifically, reporter cell lines (HEK293) expressing wild-type LEPR, LEPR-A409E (signal transduction impaired), or LEPR-P316T (signal transduction attenuated) were constructed. Cells were treated with either vehicle alone, recombinant human leptin, control IgG, or LEPR agonist antibodies of the present invention (H4H16650 or H4H16679), and the extent of LEPR-mediated signaling was determined (measured by Western blot detection of pSTAT3-Y705 expression relative to STAT3 expression).

[0217] In these experiments, the LEPR agonist antibodies of the present invention (H4H16650 and H4H16679) were shown to stimulate LEPR-mediated signaling in cells expressing the LEPR-A409E mutant or the LEPR-P316T mutant (as measured by STAT3 expression) in a dose-dependent manner way (Figure 2, panels B and C). In contrast, leptin treatment induced only a moderate level of signaling in cells expressing the LEPR-P316T mutant, and no signaling was observed in cells expressing the LEPR-A409E mutant (Figure 2, panel A). Moreover, no LEPR-mediated signaling was detected in any of the cell lines treated with vehicle or control antibody-IgG (data not shown). In this assay, other LEPR mutant variants characterized by impaired signal transduction or attenuated signal transduction were tested, but they were not activated by anti-LEPR mutant variants (data not shown), suggesting that this rescue effect may be mutant-dependent.

[0218] The results in this example indicate that the LEPR agonistic antibodies of the present invention may be useful in the treatment of diseases and disorders (eg, early-onset obesity) that are caused by certain LEPR mutant variants characterized by impaired signal transduction or reduced transmission signal (for example, LEPR-РЗ16Т or LEPR-A409E), or associated with them.

Example 9: Octet Assay Cross-Competition Between Different Anti-LEPR Monoclonal Antibodies

[0219] Binding competition for a panel of different anti-LEPR monoclonal antibodies was determined using a real-time label-free biolayer interferometry assay on the Octet HTX biosensor platform (Pall ForteBio Corp.). The entire experiment was carried out 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) while shaking the plate at 1000 rpm. To assess whether two antibodies are able to compete with each other for binding to their respective epitopes on recombinant human LEPR expressed with a C-terminal myc-myc-hexahistidine tag (hLEPR.mmh; SEQ ID NO: 114), about 0.25 nM or 0.34 nM hLEPR-MMH was first captured on Octet biosensor tips coated with anti-penta-His antibody (Fortebio Inc, #18-5122) by immersing the biosensor tips for 5 minutes in wells containing 20 μg/ml hLEPR-MMH. The antigen-captured biosensor tips were then saturated with the first anti-LEPR monoclonal antibody (subsequently called mAb-1) by immersion into wells containing 50 μg/mL mAb-1 solution for 210 seconds. The biosensor tips were then dipped into wells containing a 50 μg/mL solution of a second monoclonal anti-LEPR antibody (subsequently called mAb-2) for 150 seconds. Biosensor tips were washed in HBS-EBT buffer between all experimental steps. Real-time monitoring of the binding response was carried out throughout the experiment and the binding response was recorded at the end of each stage. The binding response of mAb-2 to the preformed hLEPR-MMH complex and mAb-1 was compared, and the competitive/noncompetitive binding patterns of various anti-LEPR monoclonal antibodies were determined, as shown in Table 14 and Table 15.

Example 10. In vivo efficacy of H4H16650P2, H4H16679P2, H4H17319P2 and H4H17321P2, which are agonistic anti-LEPR antibodies, in an inducible mouse model of leptin deficiency

[0220] The effects of four specific LEPR agonist antibodies of the present invention, H4H16650P2, H4H16679P2, H4H17319P2 and H4H17321P2, on food intake, body weight and obesity were determined in an inducible leptin deficiency model in genetically engineered LEPR ^Hu/Hu mice that expresses the receptor leptin, which consists of a human LEPR ectodomain sequence instead of a mouse LEPR ectodomain sequence. The leptin deficiency model was induced using hydrodynamic DNA delivery (HDD) of a plasmid encoding an hFc-tagged mouse LEPR ectodomain (referred to herein as mLEPR.hFc or “leptin decoy”; SEQ ID NO: 120). When expressed, the leptin trap is secreted and binds circulating leptin. Following HDD of 50 μg of a DNA construct encoding a leptin decoy, mice exhibited increased food intake and increased adiposity and body weight.

[0221] Baseline daily food intake was measured 7-4 days before leptin trap administration (days -7 and -4). On day 0, thirty-five male LEPR ^Hu/Hu mice, 13 to 17 weeks old, were successfully subjected to HDD leptin decoys. At days 6 and 13 post-HDD, blood samples were collected from the retro-orbital sinus and body composition, including adiposity, was quantified using μCT. On day 7 after HDD, mice were randomized into five groups of 7 mice based on the percentage change in body weight from day 0. Each group received a single dose of one of the following by subcutaneous injection: isotype control antibody at 3 mg/kg, H4H16650P2 at dose of 3 mg/kg, H4H16679P2 at a dose of 3 mg/kg, H4H17319P2 at a dose of 3 mg/kg, or H4H17321 at a dose of 3 mg/kg. The isotype control antibody did not bind to any known mouse proteins. Food intake and body weight were measured for each animal throughout the study. Figure 3 provides a summary of the average daily food intake for each treatment group. In Figure 3, the dotted line represents the average baseline food intake before HDD administration. The percentage change from day 0 was calculated for each animal at each time point. Figure 4 provides a summary of the average percentage change in body weight of animals in each antibody treatment group. Figure 5 provides a summary of the average fat mass of animals in each antibody treatment group, quantified by μCT 1 day before and 6 days after antibody treatment. All results are expressed as mean ±SEM.

[0222] As shown in Figures 3 and 4, following HDD leptin decoy, similar levels of increase in food intake and percentage change in body weight were observed in the groups of mice before antibody treatment. As shown in Figure 3, mice treated with H4H16650P2 or H4H16679P2 antibodies at 3 mg/kg exhibited significant levels of reduction in food intake starting one day after antibody treatment (day 8 post-HDD) and at subsequent time points when measured against with mice injected with an isotype control antibody. Mice treated with H4H17319P2 or H4H17321P2 at 3 mg/kg had a significant reduction in food intake two days after antibody treatment (day 9 post-HDD) and at other subsequent time points when measured compared to mice injected with the antibody. isotype control. As shown in Figure 4, mice treated with H4H16650P2 antibody at 3 mg/kg showed a significant reduction in the percentage change in body weight one day after antibody treatment (day 8 post-HDD) and at other subsequent time points when measured compared to mice who were injected with an isotype control antibody. One day after antibody treatment, on day 8, mice that were treated with the isotype control had an increase in body weight of 21.16 ± 1.27% relative to day 0, while mice that were treated with H4H16650P2 had an increase in body weight of 15.57±0.9% relative to day 0. Mice treated with H4H16679P2, H4H17319P2 or H4H17321P2 antibodies at a dose of 3 mg/kg showed a significant decrease in the percentage change in body weight two days after antibody treatment (day 9 after HDD) and at other subsequent time points when measured compared to mice injected with isotype control antibody. On day 9% change in body weight relative to day 0 was 23.18±1.22, 13.17±1.05, 12.95±1.26, 15.98±1.78 and 15.83±2.01 for mice treated with isotype control, H4H16650P2, H4H16679P2, H4H17319P2, or H4H17321P2, respectively. As shown in Figure 5, mice treated with isotype control antibody at 3 mg/kg showed a significant increase in fat mass 6 days after antibody treatment (day 13 post-HDD) compared to 1 day before antibody treatment (day 6 post-HDD ). Mice treated with H4H16650P2, H4H16679P2, H4H17319P2, or H4H17321P2 antibodies at 3 mg/kg did not gain fat mass after antibody treatment compared to before antibody treatment. At 6 days post-treatment (day 13 post-HDD), mice treated with H4H16650P2, H4H16679P2, or H4H17319P2 antibodies at 3 mg/kg showed a significant decrease in fat mass compared to mice treated with isotype control antibody at 3 mg/kg.

Example 11. Epitope mapping of H4H16650P2 binding to the human leptin receptor (hLEPR.mmh) using hydrogen-deuterium exchange

[0223] Experiments were conducted to determine the amino acid residues of hLEPR.mmh (amino acids M1-D839 of SEQ ID NO: 114) with which H4H16650P2 interacts. For this purpose, epitope mapping was performed using H/D exchange using mass spectrometry. A general description of the H/D exchange method is given, for example, in Ehring (1999) Analytical Biochemistry 267(2):252-259; and Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0224] Experimental procedure. HDX-MS experiments were performed on an integrated Waters HDX/MS platform consisting of a Leaptec HDX PAL system for deuterium labeling, a Waters Acquity M-Class (auxiliary solvent management system) for sample digestion and loading, a Waters Acquity M-Class (μBinary solvent management system) ) for a gradient analytical column and Synapt G2-Si mass spectrometer to measure the mass of peptides obtained using pepsin.

[0225] The labeling solution was prepared in 10 mM PBS buffer in D20 at pD 7.0 (equivalent to pH 6.6). For deuterium labeling, 3.8 μL of hLEPR.mmh (8 pmol/μL) or hLEPR.mmh, premixed with antibody in a 2:1 molar ratio, was incubated with 56.2 μL of _D2O labeling solution at various time points (e.g. , nondeuterated control = 0 s, labeled for 1 min and 20 min). The deuteration reaction was quenched by transferring a 50 μL sample to 50 μL of pre-cooled quenching buffer (0.2 M TCEP, 6 M guanidine chloride in 100 mM phosphate buffer, pH 2.5) and the mixed sample was incubated at 1.0°C within two minutes. The quenched sample was then injected into the Waters HDX control system for real-time pepsin/protease XIII digestion. Digested peptides were captured on an ACQUITY UPLC VEN C18 1.7 μm, 2.1 × 5 mM VanGuard precolumn at 0°C and eluted onto an ACQUIT Y UPLC VEN C18 1.7 μm, 1.0 × 50 mM analytical column for 9 min. gradient separation 5%-40% B (mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile). The mass spectrometer was set to a taper voltage of 37 V, a scan time of 0.5 s, and a mass/charge value in the range of 50–1700 Ts.

[0226] To identify peptides from human LEPR, LC-MSE data obtained from the non-deuterated sample was processed and searched against a database including human LEPR, pepsin and their randomized sequences using ProteinLynx Global Server (PLGS) software from Waters. Identified peptides were imported into DynamX software and filtered using two criteria: 1) minimum number of products per amino acid: 0.2 and 2) replicate file threshold: 3. DynamX software then automatically determined the level of deuterium uptake of each peptide based on retention time and high accuracy of mass determination (<10 ppm) at several points in time with 3 repetitions at each time point.

[0227] Results. Using real-time acquisition from a pepsin/protease XIII column coupled with MSE, a total of 201 peptides from human LEPR were reproducibly identified in the absence or presence of antibody, corresponding to 70% sequence coverage. Five peptides had significantly reduced deuterium uptake (delta centroid values > 0.4 Da with p-values < 0.05) when bound to H4H16650P2, as shown in Table 16. The reported peptide mass corresponds to the average MH+ centroid mass of the three repeat. These peptides, corresponding to amino acids 162-169 (amino acids LYVLPEVL from human LEPR; SEQ ID NO: 113) and amino acids 170-181 (amino acids EDSPLVPQKGSF from human LEPR; SEQ ID NO: 113), were characterized by lower levels of deuteration upon binding to H4H16650P2. These identified residues also correspond to human LEPR acid residues 162-169 and 170-181 as defined in Uniprot entry P48357 (SEQ ID NO: 113; human leptin receptor).

Example 12 In Vivo Efficacy Testing of LEPR Enhancing Antibodies in LEPR Humanized Mice

[0228] The effects of three specific LEPR enhancer antibodies of the present invention, H4H18482P2, H4H18487P2, and H4H18492P2, on body weight and obesity were determined in single housed genetically engineered LEPR ^Hu/Hu mice that express the leptin receptor, which consists of from the human LEPR ectodomain sequence instead of the mouse LEPR ectodomain sequence (mLEPR.hFc, SEQ ID NO: 120).

[0229] On day -19, body composition, including obesity, was quantified using μCT. On day 0, forty-eight female LEPR ^Hu/Hu mice, 14 to 16 weeks old, were randomized into four groups of 12 mice based on body weight. On days 0 and 11, mice from each group received a single dose of isotype control antibody at 30 mg/kg, H4H18482P2 at 30 mg/kg, H4H18487P2 at 30 mg/kg, or H4H18492P2 at 30 mg/kg by subcutaneous injection. The isotype control antibody did not bind to any known mouse proteins. Body weight was measured throughout the study for each animal. The percentage change from day 0 was calculated for each animal at each time point. Figure 6 provides a summary of the average percentage change in body weight of animals in each treatment group. Figure 6 provides a summary of the average fat mass of animals in each antibody treatment group, quantified by μCT 19 days before and 11 days after antibody treatment. All results are expressed as mean ±SEM.

[0230] As shown in Figure 6, a decrease in percent change in body weight was observed following dosing of the LEPR enhancer antibodies, but not the isotype control antibody. As shown in Figure 6, mice treated with H4H18482P2 at 30 mg/kg exhibited a significant decrease in the percentage change in body weight starting two days after treatment (day 2) and at other time points compared to mice who were injected with an isotype control antibody. Mice treated with H4H18487P2 at 30 mg/kg showed a significant decrease in the percentage change in body weight from day 2 and at other time points compared to mice injected with the isotype control antibody. Mice treated with H4H18492P2 at 30 mg/kg showed a significant reduction in percent change in body weight on days 4, 5, and 17, but not at other time points, compared with mice injected with isotype control antibody. Mice treated with H4H18482P2 at 30 mg/kg had a significant decrease in the percentage change in body weight from day 6 onwards, but not on days 7, 14 and 17, compared to mice treated by injections of H4H18492R2. Mice treated with H4H18487P2 at 30 mg/kg had a significant decrease in percent change in body weight from day 3 and other time points, but not days 4 and 5, compared to mice treated by injection N4N18492R2.

[0231] As shown in Figure 7A, there was no difference in fat mass between the groups before treatment (day -19). As shown in Figure 7B, mice treated with H4H18482 and H4H18487, but not H4H18492, at 30 mg/kg showed a statistically significant decrease in fat mass 17 days after treatment (day 12) compared to the isotype control antibody.

Example 13 Effect of Anti-LEPR Antibodies of the Present Invention on LEPR-Mediated Signal Transduction in the Monkey

[0232] A stable cell line was developed to evaluate transcriptional activation of the monkey leptin receptor. IMR-32 cells (human neuroblastoma ATCC) were obtained by stably expressing the extracellular domain of Macaca fascicularis LEPR (MfLEPR; amino acids 22 to 837 under accession number XP 005543194.1 with the replacement of threonine at position 827 by alanine), fused with the transmembrane and cytosolic domains of human LEPR (hLEPR; amino acids 840 to 1165 under accession number NP 002294.2) together with a luciferase reporter (STAT3-Luc; SABiosciences, no. CLS-6028L). The resulting cell line, referred to herein as IMR-32/STAT3-Luc/MfLEPR, was isolated and maintained in MEMS Earle's salts supplemented with 10% FBS, NEAA, 1 μg/ml puromycin, 100 μg/ml hygromycin B, and penicillin. streptomycin/E-glutamine.

[0233] A biological assay was performed to measure the effect of the anti-LEPR antibodies of the present invention on LEPR-mediated signaling in the monkey in the absence of leptin. For biological analysis, IMR-32/STAT3-Luc/MfLEPR cells were seeded at a rate of 10,000 cells/well in a 96-well plate in 0.1% FBS in Optimem with penicillin/streptomycin (assay buffer) and incubated overnight at 37°C C in 5% CO ₂ . The next day, serial dilutions of human leptin (hLeptin), anti-LEPR antibody, or isotype control antibody ranging from 50 nM to 0.8 pM in assay buffer (plus a sample containing only buffer without the test molecule) were prepared and added to the cells. After 5.5 hours at 37°C in 5% CO _{2 ,} luciferase activity was measured using OneGlo™ reagent (Promega, No. E6031) and a Victor™ X multichannel plate reader (Perkin Elmer). Results were analyzed using non-linear (4-parameter logistic) regression using Prism™ 6 software (GraphPad) to obtain _EC50 values. The percentage of antibody activation was calculated as the maximum RLU range achieved by the antibody compared to the maximum RLU range achieved by hLeptin.

[0234] As shown in Table 17, in the absence of hLeptin, all anti-LEPR antibodies tested showed activation of simian LEPR-mediated signaling in IMR-32/STAT3-Luc/mfLEPR cells with EC ₅₀ values ranging from 266 pM to 368 pM and maximum activation ranging from 76% to 82%, where 100% activation was obtained with hLeptin. hLeptin activated with an EC ₅₀ value of 333 pM. The isotype control antibody did not demonstrate any measurable level of stimulation of IMR-32/STAT3-Luc/mfLEPR cells.

Example 14 Determination of epitope binding to the full-length extracellular domain of human LEPR from the MFI signal obtained using Luminex

[0235] To determine the human LEPR epitope to which the anti-LEPR antibodies of the present invention bind, a flow cytometry-based assay, Luminex FLEXMAP (FM3DD, LuminexCorp), was used to characterize the interaction of anti-LEPR antibodies with recombinant human LEPR protein domains. For this assay, approximately 3 million carboxylated MicroplexR microspheres (Luminex, cat. no. LC1000A) were washed, vortexed, and sonicated in 0.1 M NaPO ₄ , pH 6.2 (activation buffer), and then centrifuged to remove the supernatant. The microspheres were resuspended in 120 μl of activation buffer and the carboxyl groups (-COOH) were activated by adding 15 μl of 50 mg/ml N-hydroxysuccinimide (NHS, Thermo Scientific, cat. no. 24500) followed by the addition of 15 μl of 50 mg/ml 1-ethyl- 3-[3-dimethylaminopropyl]carbodiimide (EDC, ThermoScientific, cat. no. 22980) at 25°C. After 10 minutes, the pH of the reaction was reduced to 5.0 by adding 600 μl of 50 mM MES, pH 5 (binding buffer), and the microspheres were shaken and centrifuged to remove the supernatant. The activated beads were immediately mixed with 500 μl of 20 μg/ml anti-thuja monoclonal antibody with either mouse IgG or human IgG in binding buffer and incubated for two hours at 25°C. The binding reaction was quenched by adding 50 μl of 1 M Tris-HCl, pH 8.0, and the microspheres were quickly shaken, centrifuged, and washed four times with 1 ml of DPBS to remove unbound proteins and other components of the reaction mixture.

[0236] Transiently expressed LEPR proteins, including the extracellular domain of human LEPR expressed with a C-terminal myc-myc-hexahistidine tag (human LEPR-MMH, SEQ ID NO: 113), human LEPR CRH1 (D1) expressed with a C-terminal myc-myc-hexahistidine tag (LEPR CRH1 (D1)-MMH human, amino acids 1-208 of SEQ ID NO: 113 with myc-myc-hexahistidine tag, amino acids 209-236), human LEPR CRH1 (D1, D2) domain, expressed with a C-terminal myc-myc-hexahistidine tag (LEPR CRH1 (D1, D2)-MMH human, amino acids 1-318 of SEQ ID NO: 113 with a myc-myc-hexahistidine tag, amino acids 319-346), LEPR CRH1 domain Human -Ig (D1, D2, D3) expressed with a C-terminal myc-myc-hexahistidine tag (LEPR CRH1 (D1, D2, D3)-MMH human, amino acids 1-278 of SEQ ID NO: 113 with myc-myc -hexahistidine tag, amino acids 279-306), human LEPR CRH1-Ig (D2, D3) domain expressed with a C-terminal myc-myc-hexahistidine tag (LEPR CRH1-Ig (D2, D3)-MMH human, amino acids 1- 198 of SEQ ID NO: 113 with a myc-myc-hexahistidine tag, amino acids 199-226), human LEPR Ig (D3) domain expressed with a C-terminal myc-myc-hexahistidine tag (human LEPR Ig (D3)-MMH, amino acids 1-88 of SEQ ID NO: 113 with a myc-myc-hexahistidine tag, amino acids 89-116), human LEPR domain CRH2 expressed with a C-terminal myc-myc-hexahistidine tag (human LEPR CRH2-MMH, amino acids 1- 207 of SEQ ID NO: 113 with a myc-myc-hexahistidine tag, amino acids 208-235), human FNIII LEPR domain expressed with a C-terminal myc-myc-hexahistidine tag (human LEPR FNIII-MMH, amino acids 1-204 of SEQ ID NO: 113 with a myc-myc-hexahistidine tag, amino acids 205-232) and the human LEPR Ig-CRH2-FNIII domain expressed with a C-terminal myc-myc-hexahistidine tag (human LEPR Ig-CRH2-FNIII-MMH, amino acids 1-510 from SEQ ID NO: 113 with a myc-myc-hexahistidine tag, amino acids 511-538), suspended in serum-free CHO-S-SFMII medium (Thermo Fisher, cat. no. 31033020) and then clarified by centrifugation.

Aliquots of thuja monoclonal antibody-immobilized microspheres prepared as described above were added separately to 1 ml of each of these protein supernatants. The microspheres were mixed gently, incubated for two hours at 25°C, washed twice with 1 ml DBPS, centrifuged to remove the supernatant and finally resuspended in 1 ml DPBS buffer. Forty-eight μl of thuja IgG-bound microspheres obtained from separate reactions with full-length human LEPR and each of the human LEPR domain proteins were recovered and mixed together in 3.6 ml PBS + 20 mg/ml BSA + 0 .05% sodium azide (blocking buffer).

[0237] From this mixed pool, 75 μl of microspheres per well were seeded into a 96-well filter plate (Millipore, Cat. No.: MSBVN1250) and mixed with 25 μl of individual anti-human LEPR monoclonal antibodies (0.5 or 5 μg/ ml), incubated for two hours at 25°C and then washed twice with 200 µl DPBS with 0.05% Tween 20 (wash buffer). To detect and quantify the levels of anti-LEPR antibodies bound to individual microspheres, either 100 μl of 2.5 μg/ml R-phycoerythrin-conjugated goat anti-human kappa chain F(ab')2 (Southern Biotech, cat. no. 2063-09) in blocking buffer, or 100 μl of 1.25 μg/ml AffiniPure R-phycoerythrin-conjugated goat F(ab')2-fragment to mouse IgG, F(ab')2-fragment-specific (Jackson Immunoresearch, Cat. No.: 115-116-072) in blocking buffer, added and incubated for 30 minutes at 25°C. After 30 minutes, samples were washed twice with 200 μL wash buffer and resuspended in 150 μL wash buffer. The mean fluorescence intensity (MFI) of the microspheres was measured in a Luminex analyzer.

[0238] The results of the Luminex-based assay are presented in Table 18. The intensity of the Luminex MFI signals indicates that the twelve anti-LEPR antibodies of the present invention bind to the entire extracellular domain of human LEPR. The anti-LEPR antibodies H4H18417P2, H4H18438P2 and H4H18492P2 bound to epitopes in the CRH1 D2 domain of human LEPR. The anti-LEPR antibodies H4H18449P2, H4H16650P and H4H16679P bound to epitopes in the CRHl(Dl-2) domain of human LEPR. An anti-LEPR monoclonal antibody, a reference antibody, bound to epitopes in the CRH2 domain of human LEPR. The anti-LEPR antibody H4H18445P2 bound to epitopes in the FNIII domain of human LEPR. The anti-LEPR antibodies H4H18446P2, H4H18482P2 and H4H18487P2 bound to epitopes in the Ig-CRH2-FNIII domain of human LEPR.

Examples 15-19. Animal Research Protocols

[0239] All animal studies were conducted in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines and approval by Regeneron Pharmaceuticals. Monkey studies were also conducted in accordance with IACUC guidelines and with its approval from Covance Laboratories.

Mouse studies

Hydrodynamic DNA delivery

[0240] In vivo transfection based on hydrodynamic DNA delivery (HDD) provides a protocol involving the rapid injection of a large volume of a solution of deproteinized plasmid DNA to express foreign proteins in living animals (Suda, 2007). Mice were weighed and freshly prepared expression vector was suspended in a final volume of saline equal to 1/10 body weight (v/w), which was administered by injection through the lateral tail vein. For a validation study of the leptin acceptor model (Figure 11), male C57BL/6N mice (Taconic) at 8 weeks of age received the mLeprECD expression vector (pRG977.mROR.mLepR.ecto.hFc) or control vector (pRG977.hFc) at a rate of 50 μg of DNA on the mouse. For the H4H17319P2 evaluation study (Figure 9), male and female LEPR ^hu/hu mice, 17 to 20 weeks of age, received the mLeprECD expression vector (pRG977.mROR.mLepR.ecto.hFc) at 50 μg of DNA per mouse.

Measuring body weight and food intake

[0241] Body weight was measured by placing the mouse in a container on a tared digital laboratory scale. The average body weight obtained as a result of dynamic weighing for 3 s was used. Food intake was determined by measuring the weight of feed in the feed bin using a digital laboratory scale. The level of food consumption was calculated as the difference in the mass of feed remaining in the bin and the mass of feed placed in the bin.

Body composition

[0242] Micro-computed tomography (micro-CT) imaging was performed using a Quantum micro-CT imaging system (Perkin Elmer) according to the manufacturer's instructions on live mice anesthetized with isoflurane gas. Body composition (fat mass, lean mass, bone mass, mineralization, and bone density) was measured using a Quantum micro-CT imaging system (Perkin Elmer) according to the manufacturer's instructions. The scanned images were then analyzed using AnalyzeDirect image processing software. Fat mass, lean mass, and bone mass were calculated by multiplying the recorded tissue volumes by the corresponding mass densities: 0.92, 1.05, and 1.7 g/cm ³ . Quantitative nuclear magnetic resonance (qNMR) on the EchoMRI 100 (EchoMRI) was performed on live, awake mice to measure fat mass, lean mass, free water mass, and total water mass.

Measuring blood glucose levels

[0243] Postprandial blood glucose levels were measured in samples obtained by tail vein incision in awake mice using a glucometer (AlphaTrak2, Zoetis) and glucose test strips (Zoetis).

Insulin tolerance testing

[0244] After fasting for 4 hours, the animals were intraperitoneally (IP) injected with a dose of 0.5, 0.75, or 1.0 U/kg insulin (Humulin R, Eli Lilly and Company) as indicated. Glucose measurements were performed using an AlphaTRAK2 meter and test strips (Zoetis) at 0, 15, 30, 60, and 120 min. after insulin injection. The study used an Accu-Chek® Compact Plus blood glucose meter and test strips (Roche Diabetes Care, Inc.) to characterize LEPR ^hu/hu .

Chemical tests and quantitative tests for hormones

[0245] Unless otherwise stated, mice were fasted for 4 hours, then blood samples were collected by collecting blood from the retroorbital sinus. To separate the blood serum, the blood was transferred into serum separation tubes (Sarstedt AG & Co.) and allowed to clot for at least 30 minutes. on wet ice before centrifugation at 10,000 g for 5 min. Serum was collected and stored at -80°C until processed for leptin quantification. For plasma measurements, blood was transferred into K3EDTA-coated tubes (Sarstedt AG & Co.). A mixture of dipeptidyl peptidase 4 (DPP4) inhibitor and protease inhibitor was added to the blood sample, which was kept on ice until processed to obtain plasma by centrifugation at 2000 × g for 10 min. Blood plasma was aliquoted and stored at −80°C until used for quantitation of plasma lipids, liver enzymes, and leptin levels. HbAlc levels were measured in fresh whole blood collected in K3EDTA-coated tubes. Quantitative determination of HbAlc, plasma lipids and liver enzymes was carried out using a chemical analyzer (Advia Chemistry XPT, Siemens). Serum or plasma leptin levels were measured using an immunoassay kit (Milliplex MAP, Millipore) and following the protocol recommended by the manufacturer.

Quantitative determination of triglycerides in the liver

[0246] Quantitative determination of liver triglyceride content was performed on 100-200 mg liver samples, which were ground into powder while cooled with liquid nitrogen using a mortar and pestle. Powdered liver tissue was weighed and homogenized in PBS by bead homogenization (FastPrep 24-5G, MP Biomedical). The homogenate was transferred into a glass tube containing 5 ml of Folch solution (chloroform:methanol in a ratio of 2:1), shaken and centrifuged for 20 minutes. at 1500 × g. The lower phase was collected, brought to a volume of 5 ml and shaken. Specific volumes (25 to 50 μL) of triolein samples and standards (Verichem) were transferred to a 96-well polypropylene plate, mixed with 10 μL of 1:1 chloroform:Triton X-100, and allowed to air dry. 300 μL of triglyceride reagent (Thermo Scientific) was added to the dried samples and standards, the plate was shaken for 5 min, and then incubated for 20 min. at 37°C. 200 μl of each reaction mixture was transferred to a new 96-well clear polypropylene plate and absorbance was measured at 500 nm using a plate reader (Molecular Devices).

Fixative perfusion and immunohistochemistry

[0247] Mice were anesthetized with sodium pentobarbital (110 mg/kg, IP) and transcardially perfused with 2 ml saline followed by 150 ml 4% paraformaldehyde in 0.1 M borate buffer, pH 9.5. Liver samples were secondary fixed for 2 h, transferred to 70% ethanol and processed for paraffin embedding, and sectioned for hematoxylin and eosin staining in Histoserv. Brains were secondary fixed for 2 h at 4°C and then immersed in 15% sucrose in potassium phosphate buffered saline overnight at 4°C. Whole brains were placed on a freezing sliding microtome (Leica), sectioned at 30 μm thickness, serially collected at regular intervals, and stored in cryoprotectant (20% glycerol and 30% ethylene glycol in 0.1 M phosphate buffer) and stored in cryoprotectant ( 20% glycerol and 30% ethylene glycol in 0.1 M phosphate buffer) at -20°C.

[0248] Serial brain sections spaced 150 μm apart from each experimental group were processed simultaneously to ensure comparable staining across animals and treatments. In general, for 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. Avidin and biotin blocking were performed according to the manufacturer's protocol (Vector Labs). Immunohistochemical staining of HapStat3 (Y705) (#9145, Cell Signaling Technologies; 1:1000, 16 h at 4°C) was performed on serial brain sections at equal intervals using the avidin-biotin complex method with the chromogen diaminobenzidine (Vector Labs ). For immunohistochemistry (IHC) examination of P-STAT3 (Y705), sections were pretreated with 1% _H2O2 in 1% _NaOH for 10 min, 0.3% glycine in K-PBS for 10 min. and 0.03% SDS in K-PBS for 10 min.

[0249] Brightfield images were obtained by scanning the entire slide using a 40x objective on a Scanscope XT (Aperio). The sections were matched by comparing the tissues to the Franklin and Paxinos mouse brain atlas. Neuroanatomical regions and distance from bregma were approximated based on this atlas. The number of immunoreactive cells with pSTAT3 (Y705) was quantified bilaterally for a given region at the rostro-caudal level indicated using Halo software (Indica Labs).

Monkey studies

[0250] All studies on cynomolgus monkeys were conducted at Covance Laboratories, Inc. Monkeys were fed twice daily with certified primate diet 5048 (PMI, Inc.) and provided with free access to fresh water.

Body weight values

[0251] Monkeys were weighed pre-dose on the day of dosing and, if applicable, at least once per week for the remainder of the study.

Dual-energy X-ray absorptiometry (DEXA)

[0252] Whole body scans were performed using a Discovery A densitometer (Hologic) on fasted monkeys anesthetized with ketamine and dexmedetomidine, and at the discretion of the veterinary staff.

Example 15: Anti-LEPR Antibodies Reverse Obesity Caused by Leptin Deficiency

[0253] Studies were conducted to determine the in vivo effectiveness of H4H17319P2. Since H4H17319P2 does not bind mouse LEPR, genetically modified mice were generated using VelociGene® technology (Valenzuela et al., 2003), in which the part of the mouse Lepr gene encoding the extracellular domain of LEPR was replaced with the corresponding genomic sequence of human LEPR (Lepr ^hu/hu ; figure 10A). Lepr ^hu/hu mice do not show differences from Lepr ^+/+ mice with respect to body weight, body composition, insulin sensitivity, and serum leptin levels (Figures 10B, C and D).

[0254] A mouse model of leptin deficiency was developed by hydrodynamic DNA delivery (HDD) of a plasmid that encodes hFc-tagged mouse Lepr ectodomain (mLeprECD) to bind endogenous circulating mouse leptin, thereby acting as a leptin scavenger and thereby preventing leptin-mediated signal transduction. Following HDD mLeprECD, chow-fed C57BL/6N mice rapidly gained body weight with a significant increase starting at day 3 post-HDD compared to mice that received hFc control HDD (Figure 11A). At the end of the study (day 10 after HDD), body weight values increased by 24% after HDD mLeprECD, while only a slight increase of 3% was observed in the control group (Figure 11A). Consistent with body weight data, cumulative food intake after HDD mLeprECD was significantly increased compared to the control group starting at day 3 after HDD (Figure 11A). To confirm that the increase in body weight reflected an increase in adiposity, micro-CT imaging was performed on day 7 after HDD, which showed a significant 2-fold increase in fat mass after HDD mLeprECD compared to the control group (Figure 11B). All other body composition parameters were the same between the groups (Figure 11B).

[0255] To evaluate the effectiveness of H4H17319P2 in vivo, leptin deficiency was induced by HDD mLeprECD in chow-fed male and female Lepr ^hu/hu mice. As expected, mLeprECD expression promoted rapid body weight gain in Lepr ^hu/hu mice of both sexes (Figures 9A and 12A). Seven days after HDD, Lepr ^hu/hu mice were separated based on the relative percentage change in body weight and administered a single SC dose of control or H4H17319P2 at 10 mg/kg. Both male and female mice treated with the control monoclonal antibody (hereinafter referred to as the control mAb) continued to gain body weight with an increase in body weight of 40.5 ± 2.7% and 44.1 ± 4.7%, respectively, after 14 days after HDD (Figure 9A and 11A). In contrast, mice treated with a single dose of an agonistic anti-LEPR monoclonal antibody lost body weight and returned to baseline body weight values observed before HDD (Figures 9A and 11A). Because H4H17319P2 binds human but not mouse LEPR, we can exclude the possibility that improvements in body weight are secondary to the effect of the LEPR mAb on the ability of mLeprECD to bind leptin. A decrease in body weight was associated with a decrease in food intake (Figures 9A and 11A). Although daily food intake did not differ between the two groups before dosing, mice treated with H4H17319P2 had a significant reduction in food intake compared to the control monoclonal antibody group (Figures 9A and 11A). The body weight and food intake reduction effects observed with a single dose of H4H17319R2 were ultimately attenuated. Food intake remained significantly lower in male and female mice treated with H4H17319P2 than in mice treated with control monoclonal antibody until days 12 and 9 post-treatment (days 19 and 16), respectively (Figures 9A and 11A). The body weights of male and female mice treated with H4H17319P2 remained similar to their baseline body weights until days 16 and 13 post-treatment (days 23 and 20), after which weight gain occurred (Figures 9A and 11A).

[0256] The decrease in body weight induced by H4H17319P2 treatment reflected a decrease in adiposity and lean mass. Micro-CT analysis showed that before HDD on day -1 and before treatment on day 6, both treatment groups of male or female mice showed similar body composition (Figure 9B, 12B and 12C). Consistent with induced leptin deficiency on day 6, both groups showed a significant increase in fat and lean mass, but not bone mass, compared with pre-HDD values on day -1 (Figures 9B, 12B and 12C). Six days after treatment (day 13), mice dosed with the control monoclonal antibody showed a further increase in fat mass compared to day 6, whereas no additional increase in adiposity was detected after treatment with H4H17319P2. Consistent with changes in body weight, both fat and lean mass were reduced in H4H17319P2-treated mice compared with control monoclonal antibody-treated mice after 7 days of treatment. No treatment-related effects on bone mass, mineral composition or bone density were observed (Figures 12B and 123C).

[0257] H4H17319P2 was then assessed for its ability to alter circulating lipid levels in mice with induced leptin deficiency. Chemical analyzes of blood plasma showed that H4H17319P2 provides a decrease in the levels of circulating triglycerides and total cholesterol in the blood plasma, including HDL cholesterol (HDL-C) and LDL cholesterol (LDL-C), compared with levels in male mice treated with a control monoclonal antibody, after 6 days of treatment (Figure 9C).

[0258] Given that leptin promotes energy expenditure in leptin-deficient ob/ob mice (Halaas et al., 1995; Pelleymounter et al., 1995), the effects of equal feeding were assessed in paired groups to determine whether the reduction was complete. food intake explains the effect of H4H17319P2 on body weight reduction in induced leptin deficiency. Lepr ^hu/hu mice with induced leptin deficiency rapidly gained weight and exhibited hyperphagia compared to Lepr ^hu/hu mice that received the control vector via HDD (Figure 13A). After dosing 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 decreased food intake and lost body weight (Figure 13A). Equal feeding in paired groups also promoted weight loss in Lepr ^hu/hu mice expressing mLeprECD. It should be noted that mice treated with equal feeding in paired groups consumed the same amount of food as mice treated with H4H17319P2, but did not show the same degree of body weight loss (Figure 13A). Consistent with these data, obesity was reduced in matched-pair feeding mice compared with control monoclonal antibody-treated mice, but was significantly greater than obesity in mice treated with H4H17319P2 (Figure 13B ). In contrast, matched-pair feeding and H4H17319P2 treatment showed similar effects in reducing lean mass compared to leptin-deficient mice treated with control monoclonal antibody (Figure 13B). No effects were observed on bone mass, bone density, or bone mineralization (Figure 13B).

[0259] Leptin also provides improvements in insulin sensitivity in leptin-deficient ob/ob mice (Muzzin et al., 1996; Pelleymounter et al., 1995). Thus, the relative effects of H4H17319P2 treatment and matched feeding on insulin sensitivity were determined after induction of leptin deficiency. Insulin tolerance tests were performed three days after monoclonal antibody dosing or paired group feeding. As shown in Figure 13C, Lepr ^hu/hu mice expressing mLeprECD treated with control monoclonal antibodies showed reduced insulin sensitivity compared to Lepr ^hu/hu mice subjected to control vector HDD. Notably, treatment with H4H17319P2 restored insulin sensitivity, whereas equal feeding in paired groups had no effect in leptin deficiency-induced Lepr ^hu/hu mice (Figure 13C).

[0260] Finally, the effect of H4H17319P2 in reducing circulating lipids was confirmed, mainly through a decrease in food intake. While receiving H4H17319P2 without equal feeding in paired groups provided a significant decrease in plasma triglyceride levels in patients with leptin deficiency, simultaneous receipt of H4H17319R2 and implementing the principle of equal feeding in paired groups ensured a decrease in plasma total cholesterol and HDL-C levels ( figure 13D).

[0261] Thus, the data demonstrate that in a model of induced leptin deficiency, H4H17319P2 not only reverses hyperphagia and obesity, but also attenuates insulin resistance. It should be noted that although reduced food intake contributes to the reduction in body weight and obesity observed with H4H17319P2 treatment, the reduction in food intake is not sufficient to provide improvements in insulin sensitivity or reductions in plasma triglyceride levels. Example 16 H4H17319P2 reverses hyperglycemia, insulin resistance, dyslipidemia, and hepatic steatosis in lipodystrophic mice.

[0262] H4H17319P2 was then tested to determine whether it would alleviate hyperphagia, metabolic dysfunction, and hepatic steatosis in mice with hypoleptinemia secondary to generalized lipodystrophy. To test this hypothesis, aP2-nSrebplc ^Tg/+ mice, which develop phenotypic features characteristic of generalized lipodystrophy (Shimomura et al., 1998), were crossed with Lepr ^hu/hu mice. aP2-nSrebplc ^Tg/+ mice express nuclear Srebplc in adipose tissue via the aP2 promoter and were used in the classic experiment that provided the first evidence that leptin reverses the metabolic complications associated with low leptin levels in generalized lipodystrophy (Shimomura et al ., 1999). Male aP2-nSrebplc ^Tg/+ mice; Lepr ^hu/hu (Tg) were heavier than aP2-nSrebplc ^+/+ animals; Lepr ^hu/hu (nonTg) (Figure 15A). However, consistent with previous reports, Tg mice exhibited reduced adiposity and lower leptin levels compared to nonTg mice (Figure 15A). In P2-nSrebplc ^Tg/+ mice; Lepr ^hu/hu also observed severe insulin resistance and mild dyslipidemia with elevated plasma levels of triglycerides, total cholesterol and LDL-C compared to nonTg mice (Figure 15B and 15C).

[0263] Male Tg mice with lipodystrophy received once weekly 10 mg/kg (SC) of control monoclonal antibody or H4H17319P2. In addition, male nonTg mice were also treated once weekly with 10 mg/kg (SC) control monoclonal antibody to serve as a reference for wild-type metabolic parameters. Before treatment on day 0, Jg mice showed significantly increased body weight values compared to nonTg mice (Figure 14A). Three days after the start of treatment, the body weights of lipodystrophy mice treated with H4H17319P2 were significantly lower than those of lipodystrophy mice dosed with the control monoclonal antibody (Figure 14A). Accordingly, Jg mice treated with the control monoclonal antibody were hyperphagic and consumed more food than non-Tg mice without lipodystrophy treated with the control monoclonal antibody (Figure 14A). However, Jg mice that were treated with H4H17319P2 consumed less food than Jg mice that were given the control monoclonal antibody (Figure 14A).

[0264] To determine the underlying cause of the observed body weight changes, body composition was quantified using micro-CT. These analyzes showed that changes between body weight due to genotypes and body weight due to treatment were mainly explained by differences in lean mass. Before treatment (day -5), lean mass was significantly increased in Jg lipodystrophy mice compared to nonTg mice (Figure 14B). In contrast, Jg mice had reduced fat mass compared to nonTg mice pre-dose (day -5) (Figure 14B). After 4 weeks of once-weekly administration of H4H17319P2, there was a significant reduction in fat mass and lean mass in Jg mice compared to fat mass and lean mass in Jg mice dosed with the control monoclonal antibody (Figure 14B). Bone mass and bone mineralization were increased in Jg mice compared to wild-type mice, but were not significantly increased in pre-dose treatment groups (Figure 16A). Moreover, no differences in bone mass or bone mineralization were observed between lipodystrophic mice treated with control monoclonal antibody or H4H17319P2 (Figure 16A). Similarly, no significant genotype- or treatment-related effects were found on bone density (Figure 16A). These data show that H4H17319P2 reduces lean mass and fat mass in lipodystrophic mice but does not affect bone mass, bone mineralization, or bone density.

[0265] Consistent with previous results, Jg mice with lipodystrophy exhibited significant hyperglycemia compared to pop Jg mice before treatment on day 0 (Figure 14C). Three days after treatment with H4H17319P2, ad-libitum-fed blood glucose levels in Jg mice normalized and did not differ from blood glucose levels in nonTg mice. It should be noted that when treated with H4H17319P2 once a week, Tg mice remained normoglycemic until the end of the study, whereas Tg mice treated with the control monoclonal antibody remained hyperglycemic throughout the study (Figure 14C). Accordingly, at day 28, percentage hemoglobin Ale (HbAlc) levels were reduced in lipodystrophy mice compared with pre-treatment or control monoclonal antibody (Figure 14C). Additionally, reductions in blood glucose levels upon treatment with H4H17319P2 were associated with improved insulin sensitivity. On day 23, insulin tolerance testing showed that lipodystrophy mice treated with a control monoclonal antibody were insulin resistant, whereas lipodystrophy mice treated with H4H17319P2 were as insulin sensitive as nonTg mice treated with control monoclonal antibody (Figure 14D). Overall, these results demonstrate that H4H17319P2 provides relief from hyperglycemia, insulin resistance, and reduction in HbAlc levels in a mouse model of generalized lipodystrophy.

[0266] Plasma chemistries performed at the end of the study (day 28) further demonstrated that H4H17319P2 provided a reduction in hypertriglyceridemia and hypercholesterolemia in mice with generalized lipodystrophy. At the end of the study, plasma triglyceride levels, total cholesterol and LDL-C levels were significantly elevated in Jg mice that received the control monoclonal antibody compared with nonTg mice that also received the control monoclonal antibody (Figure 1E). Notably, plasma triglyceride and cholesterol levels were significantly lower in Tg mice treated with H4H17319P2. Therefore, H4H17319P2 provides improvement in dyslipidemia in mice with generalized lipodystrophy.

[0267] In addition to diabetes mellitus, insulin resistance and dyslipidemia, patients with generalized and focal lipodystrophies may develop non-alcoholic fatty liver disease (including hepatic steatosis). Therefore, the effects of H4H17319P2 on liver enzyme levels, liver weight, and hepatic steatosis in lipodystrophy mice were examined. At day 28, Jg mice treated with control monoclonal antibody showed a significant increase in circulating ALT and AST levels compared to Jg mice treated with H4H17319P2 or nonTg mice treated with control monoclonal antibody (Figure 14F). Importantly, an improved liver enzyme profile was associated with beneficial effects on liver weight and hepatic steatosis (Figure 14G). Specifically, liver samples from lipodystrophy mice treated with the control monoclonal antibody were significantly heavier than liver samples from nonTg mice treated with the control monoclonal antibody, weighing 3.6 ± 0.7 g (Figure 14G) . Liver samples from Tg mice treated with the control monoclonal antibody also had higher triglyceride content compared to nonJg mice treated with the control monoclonal antibody and lipodystrophic mice treated with H4H17319P2 (Figure 14G). Notably, liver samples from Tg mice treated with H4H17319P2 were only 0.6 ± 0.1 g heavier and did not exhibit an increase in triglyceride content in liver samples compared to liver samples from nonTg mice. treated with a control monoclonal antibody (Figure 14G). Improvements in hepatic steatosis provided by H4H17319P2 were also evident in hematoxylin and eosin stained liver sections (Figure 14G).

Example 17: H4H17319P2 Activates STAT3-Mediated Signaling in the Hypothalamus

[0268] Activation of LEPR in the Arc of the hypothalamus plays a key role in controlling energy and metabolic balance (Coppari et al., 2005; Cowley et al., 2001). Because H4H17319P2 provides relief from hyperphagia and metabolic complications in Lepr ^hu/hu mice with lipodystrophy, we further examined whether H4H17319P2 induces STAT3 activation in Arc similar to leptin. Immunostaining for pSTAT3 Y705 was performed on matched brain sections obtained from male Jg mice with lipodystrophy that received a single dose of control or H4H17319P2 at 10 mg/kg (SC) or a continuous infusion of human leptin at a dose of 30 μg/day (SC ) within 3 days. This dose of leptin was chosen because previous work showed that a dose of 5 μg/day (SC) was effective in mice with lipodystrophy (Shimomura et al., 1999), and maximum effectiveness in mice was observed at a dose in the range of 10-42 μg/ day (SC) (Denroche et al., 2013; Halaas et al., 1997; Harris et al., 1998). These analyzes showed that several cells in Arc exhibited pSTAT3 Y705 staining in lipodystrophic mice that received a control monoclonal antibody. In contrast, both leptin and H4H17319P2 induced pSTAT3 Y705 in Arc, with similar numbers of pSTAT3 Y705 ⁺ cells found (Figure 17A). Although initial focus was on Arc due to its established role in mediating the actions of leptin and its proximal location to the median eminence, a circumventricular organ that lacks the blood-brain barrier, both leptin and H4H17319P2 were noted to induce pSTAT3 Y705 in Vmh. However, H4H17319P2 induced pSTAT3 staining in more Vmh cells than detected by leptin treatment (Figure 17A). Thus, these data indicate that leptin and H4H17319P2 induce pSTAT3 Y705 in similar numbers of cells in Arc, while H4H17319P2 has a more pronounced effect in Vmh.

[0269] A disadvantage of monoclonal antibodies as CNS therapeutics is their limited ability to cross the blood-brain barrier, with ~0.1% of circulating antibody concentrations found in the cerebrospinal fluid (CSF) (Zuchero et al., 2016). Despite this limitation, we herein determine that H4H17319P2 not only induces STAT3 phosphorylation in the hypothalamic Arc but also stimulates STAT3-mediated signaling in the Vmh. The Arc is reported to be surrounded by porous blood vessels, but to a lesser extent than the adjacent median eminence (Ciofi et al., 2009; Norsted et al., 2008). There is currently no evidence in the literature that Vmh has the benefit of direct exposure to blood-borne molecules. Without being limited to any particular theory, a possible explanation is that the monoclonal antibodies reach Vmh by diffusion from Arc. Alternatively, the H4H17319P2 antibody may exhibit signal transduction properties that differ from those of leptin. Although functional LEPR-b is expressed in the Vmh, another possibility is that the monoclonal antibody H4H17319P2 may indirectly stimulate STAT3-mediated signaling in the Vmh. However, it is surprising that H4H17319P2 induces STAT3-mediated signaling in more neurons in the Vmh than leptin.

Example 18 H4H17319P2 is at least as effective as leptin in ameliorating metabolic disorder and liver dysfunction in lipodystrophic mice

[0270] Because H4H17319P2 engages STAT3-mediated signaling in Arc and Vmh cells as well as or better than leptin, the efficacy of H4H17319P2 and leptin treatment was compared in mice with lipodystrophy. Male Jg mice received a once-weekly dose of 10 mg/kg control monoclonal antibody or LEPR agonist (SC) or a continuous infusion of leptin at 30 μg/day (SC) for 14 days. To provide a reference, male nonTg mice received a once-weekly dose of 10 mg/kg control monoclonal antibody (SC). Before treatment on day 0, Tg mice exhibited severe hyperglycemia (Figure 17B). Blood glucose levels were reduced to a similar extent by administration of H4H17319P2 or leptin compared with administration of the control monoclonal antibody (Figure 17B). Blood glucose levels decreased 2 days after initiation of treatment with H4H17319P2 or leptin and remained decreased until the end of the study (Figure 17B). To test whether improved insulin sensitivity contributed to the glucose-lowering effect, insulin tolerance tests were performed on day 9. Indeed, insulin tolerance testing showed that Tg mice treated with a control monoclonal antibody were insulin resistant, whereas Tg mice treated with H4H17319P2 and leptin were insulin sensitive (Figure 17B). No differences were observed between H4H17319P2 and leptin treatments in reducing blood glucose or insulin sensitivity.

[0271] Consistent with previous data, H4H17319P2 promoted significant body weight loss compared to control monoclonal antibody administration in lipodystrophy mice starting at day 4 post-treatment (Figure 17C). In Tg mice, H4H17319P2 provided a reduction in total food intake compared to that of the control monoclonal antibody (Figure 17C). Interestingly, while leptin reduced body weight and food intake in Tg mice, it resulted in less body weight loss than observed with H4H17319P2 treatment (Figure 17C).

[0272] H4H17319P2 provides superior benefits over leptin in lowering plasma lipids and eliminating hepatomegaly in lipodystrophic mice. Plasma chemistries at day 13 showed that in Tg mice, H4H17319P2, but not leptin, reduced plasma triglyceride and cholesterol levels compared with those of the control monoclonal antibody (Figure 17D). No significant changes in the levels of other lipids were detected when treated with H4H17319P2 compared with those when the control monoclonal antibody was administered to Tg mice. H4H17319P2 also reduced liver weight and eliminated hepatic steatosis compared to control antibody treatment in Tg mice (Figure 17E). Although these effects were similar to those in mice treated with leptin, a greater reduction in liver weight was observed with H4H17319P2 (Figure 17E). Liver weight was normalized by treatment with H4H17319P2, but not leptin, and was similar to liver weight in nonTg mice that received the control monoclonal antibody (Figure 17E).

Example 19: H4H17319P2 Reduces Body Weight and Obesity in Lean Mice and Normal and High Body Fat Monkeys

[0273] In vitro binding and functional assay experiments demonstrate that H4H17319P2 does not compete for binding to leptin and, in the presence of leptin, causes greater LEPR activation than leptin alone. Thus, it was determined whether H4H17319P2 could cause a decrease in body weight under conditions of homeostasis with normal body weight. Chow-fed male Lepr ^hu/hu mice were administered a single dose of control monoclonal antibody (10 mg/kg, SC) or H4H17319P2 at 3 or 10 mg/kg on day 0. A significant decrease in body weight compared with that of the control monoclonal antibody was observed 1 and 2 days after treatment with H4H17319P2 at both dose levels (Figure 18A). Accordingly, both dose levels of H4H17319P2 provided a reduction in food intake compared to control monoclonal antibody administration (Figure 18A). Changes in body weight were associated with a decrease in fat mass, but not lean mass (Figure 18B). At both dose levels, treatment with H4H17319P2 caused a 32% reduction in fat mass, while administration of the control monoclonal antibody resulted in a minimal change in fat mass of -2% (Figure 18B). It should be noted that there were no significant differences in lean mass between control monoclonal antibody and H4H17319P2 treatments (Figure 18B). Although both dose levels of H4H17319P2 resulted in similar magnitudes of change in body weight and fat mass, the duration of effects differed, likely reflecting differences in pharmacokinetics (Figures 18A and 18B).

[0274] The effect of treatment with H4H17319P2 in promoting body weight loss in non-human primates was then assessed. First, the effects of H4H17319P2 on body weight in lean cynomolgus monkeys were determined. Monkeys received either control or H4H17319P2 at 3 mg/kg or 10 mg/kg once weekly for 13 weeks. Treatment with H4H17319P2 resulted in a dose-dependent reduction in body weight compared to control-treated monkeys (Figure 18C). After 13 weeks of the study, monkeys treated with the control solution had gained 9.7 ± 0.9% of their initial body weight, while treatment with H4H17319P2 at doses of 3 and 10 mg/kg resulted in a minimal change in body weight of 0. 3±1.6%, and body weight loss of -6.0±1.6%, respectively (Figure 18C).

[0275] Because H4H17319P2 provided a dose-dependent reduction in body weight in lean monkeys, the effects of H4H17319P2 treatment on body weight and body composition were tested in high body fat cynomolgus monkeys. Animals received control solution or anti-LEPR agonist antibody at a dose of 30 mg/kg once weekly for 2 weeks. H4H17319P2 provided a decrease in body weight compared to injection of the control solution. At the end of the study (day 56), monkeys that received two doses of H4H17319P2 showed a decrease in body weight of 3.6 ± 1.9% from baseline, while monkeys that received the control solution gained 6.4 ± 1.5 % body weight. In parallel, treatment with H4H17319P2 provided a reduction in fat mass, but not lean mass, compared to control solution, as quantified by dual-energy x-ray absorptiometry (DEXA) (Figure 18D). Monkeys receiving the control solution experienced a 28.0 ± 1.5% increase in obesity by the end of the study. In contrast, monkeys treated with the LEPR agonist H4H17319P2 showed a significant reduction in adiposity of 13.7±6.4% (Figure 18D). Taken together, these data indicate that the H4H17319P2 antibody promotes body weight reduction through selective loss of obesity in normal-weight Lepr ^hu/hu mice as well as in non-human primates with low or high body fat.

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

[0276] Obesity affects 13% of people worldwide and is a risk factor for type 2 diabetes mellitus, cardiovascular disease, various types of cancer and orthopedic disorders. In Western Europe, more than 20% of people are obese (Ng et al., Lancet. 2014; 384: 766-781), and in the United States, obesity prevalence now exceeds 35% (Flegal et al., JAMA. American Medical Association. 2016 ; 315(21): 2284-2291). Modifications through diet and exercise are effective for some individuals but often result in modest reductions in body weight, with most individuals remaining obese (defined as a body mass index [BMI] ≥30 kg/m ² ) or overweight (defined as BMI from 25 to <30 kg/m ² ).

[0277] Current body weight loss medications also have modest effects on body weight and/or have poor safety/tolerability, which has led to limited use of these agents by clients, physicians, and patients (Zhang et al., Obes Sci Pract. 2016 ; 2(2): 104-114). Current pharmacotherapies in standard clinical practice, such as liraglutide, orlistat, naltrexone HCl/bupropion HCl, phentermine/topiramate, and lorcaserin HCl, reduce body weight by an average of approximately 3-10% from baseline. Typically, body weight loss plateaus and therapy is discontinued within a 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 limited to the most severely obese patients (BMI≥40 kg/ ^m2 or BMI≥35 kg/ ^m2 with comorbidities) with <200,000 cases per year in the US (American Society of Metabolic and Bariatric Surgery) and <150,000 cases per year throughout Europe (Angrisani et al., Obes Surg. 2017, 27:2279-2289). There is a significant unmet medical need for safe and effective therapies to treat and prevent obesity and obesity-related metabolic complications.

[0278] Leptin is a circulating adipose tissue hormone that binds to leptin receptors (LEPR) in the hypothalamus and modulates the 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 extremely rare homozygous loss-of-function mutations in the leptin gene (LEP) or defective leptin-mediated signaling due to homozygous mutations in LEPR develop severe obesity, diabetes mellitus, susceptibility to infections, 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 pediatric and adult patients with monogenic obesity due to leptin loss-of-function mutations resulted in significant reduction in body weight and improvement in metabolic complications (Farooqi et al., N Engl Jo Med 1999, 341(12): 879-884) (Licinio et al., Proc Nat Acad Sci 2004, 101(13):4531-4536).

[0279] Secondary leptin deficiency also occurs in rare lipodystrophy disorders, which are caused by genetic or acquired loss of leptin-producing adipose tissue in various areas of the body.

[0280] Patients with generalized and focal lipodystrophy develop varying degrees of diabetes mellitus, severe insulin resistance, hypertriglyceridemia, and fatty liver (Brown et al., J Clin Endocrinol Metab. 2016; 101(12):4500-4511). Recombinant human leptin, administered subcutaneously once daily, has been shown to reduce hemoglobin A1c (HbAlc), glucose, triglycerides, and hepatic steatosis in patients with generalized lipodystrophy (Oral et al., N Engl J Med. 2002; 346(8). ):570-578) (Ebihara et al., J Clin Endocrinol Metab 2007, 92(2):532-541). Most patients with generalized lipodystrophy had serum leptin levels <4 ng/mL, and subgroup analyzes showed that the reduction in HbAlc and triglyceride (TG) levels with leptin treatment was greater in patients with generalized and focal lipodystrophy when leptin levels were <4 ng/mL (Brown et al., J Clin Endocrinol Metab. 2016; 101(12):4500-4511) (FDA Expert Panel Meeting, 2013).

[0281] Metre leptin (recombinant methionyleptin; Novelion®) is currently approved in the United States under the Risk Evaluation and Mitigation Strategies (REMS) program for the treatment of complications of leptin deficiency in patients with generalized congenital and acquired lipodystrophy, and is also approved in Japan for all subtypes of lipodystrophy. Daily dosing of metreleptin is required, and side effects include the risk of immunogenicity: -85% of patients develop binding antibodies, and ~9% develop neutralizing antibodies that cross-react with endogenous leptin (Chan, Clin Endocrinol. 2016;85(1): 137-149).

[0282] In contrast to low leptin or leptin deficiency disorders, overweight or obese individuals typically have high leptin levels, with a median level of 10 ng/mL in men and 24 ng/mL in women for subjects with a BMI in the 30 range -35 kg/ ^m2 (Ruhl and Everhart, Am J Clin Nutr. 2001; 74(3): 295-301). Circulating leptin levels in adult men and women with a normal BMI of 20–25 kg/m ² have median levels of 3 and 9 ng/mL, respectively (Ruhl et al., Am J Clin Nutr 2001; 74(3): 295–301) with reported normal ranges are 1.2 to 9.5 ng/mL in men and 4.1 to 25 ng/mL in women (Quest Lab reference range). Numerous studies have demonstrated a correlation between circulating leptin levels and BMI and body fat percentage (Ruhl et al., Am J Clin Nutr. 2001, 74(3):295-301) (Considine et al., N Engl J. Med. 1996, 334(5):292-295), although there is a high degree of variability in leptin levels among individuals with similar BMIs (Buettner et al., J Endocrinol. 2002; 175(3): 745-756). Leptin levels vary throughout the day (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 depending on nutritional status. Leptin levels decline rapidly by 35-60% after a 24-hour fast in normal-weight and obese individuals and continue to fall 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 with 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 with weight gain (Ravussin et al., Cell Metab. 2014; 20(4): 565-572 ), coinciding with changes in fat mass. Administration of recombinant human leptin to obese subjects resulted in minimal weight loss (3% body weight reduction minus EVR) (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-mediated signaling by high levels of endogenous leptin. Several population-based studies suggest that there may be subgroups of obese patients whose leptin levels are relatively low (Ruhl and Everhart, Am J Clin Nutr. 2001; 74(3): 295-301) (Buettner et al., J Endocrinol. 2002 ; 175(3): 745-756) and leptin receptors may not be saturated. A key question to address is whether restoring leptin-mediated signaling will reduce appetite, food intake, and body weight in obese subjects with relatively low baseline leptin levels.

[0283] H4H17319P2 is an anti-human LEPR antibody that acts as a LEPR agonist and binds to human LEPR with nanomolar affinity. In preclinical studies, H4H17319P2 activates LEPR-mediated signaling in the presence or absence of leptin. Weekly administration of H4H17319P2 (10 mg/kg subcutaneously [SC]) provided improvements in glycemic control, insulin sensitivity, dyslipidemia, food intake, body weight, liver weight, and hepatic steatosis in LEPR humanized lipodystrophy mice. H4H17319P2 also reduced body weight and adiposity in humanized LEPR mice with leptin deficiency, but did not reduce body weight in humanized LEPR mice with diet-induced obesity. In a Good Laboratory Practice (GLP) toxicology study in which lean cynomolgus monkeys were treated with H4H17319P2 by subcutaneous injection (3, 10, 30 mg/kg) or intravenous (100 mg/kg) once weekly over 13 weeks, exposure to H4H17319P2 resulted in suppression of weight gain or induction of body weight loss. Monkeys dosed with H4H17319P2 lost up to 8.7% of body weight (group average compared to pre-dose weight values), while control animals co-treated with placebo gained an average of 8.3% of body weight over the same period of time. The magnitude of the observed reduction in body weight did not appear to have a clear dose or effect relationship in animals treated with H4H17319P2. A reversal of body weight was observed after cessation of exposure during a non-dosing recovery period.

[0284] As described above, administration of H4H17319P2 was well tolerated at all dose levels and both routes of administration without any adverse clinical effects. A transient decrease in the amount of insulin and the absolute number of circulating lymphocytes (decrease in T cell number) was observed during the approximately 30 day treatment period, but these results were not observed at the end of the dosing period, and the effect was not dose dependent. A decrease in thymus weight values and a decrease in the saturation of the thymus cortex with cells were observed in groups receiving H4H17319P2 during the treatment period and were fully or partially reversible during the recovery period. Changes in the thymus and transient reduction in T cell numbers are likely associated with decreased food intake and levels of body weight loss in growth-phase monkeys and would likely not be observed in adults with normal nutritional status.

[0285] 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 intravenous (IV) and subcutaneously (SC) doses of H4H17319R2 in healthy participants. The purpose of Part A is to evaluate the safety, tolerability, PK, and PD of single escalating IV and SC doses in healthy men and women. The interim analysis of PK/PD, safety and tolerability in Part A will be used to select the dosing regimen for evaluation in Part B. Subjects included in Part A are not eligible for Part B. For Part B, new subjects who are overweight body or obesity, will be included in a study to evaluate the safety, tolerability, PK and PD of repeated doses of H4H17319R2 (at the single dose level) or placebo for 12 weeks. The effects of H4H17319P2 on biomarkers such as food intake, appetite, body composition and body weight will be assessed in 4 different cohorts defined by baseline leptin levels.

Goals

[0286] The primary objective of the study is to evaluate the safety and tolerability of H4H17319P2 in healthy subjects. The secondary objectives of the study were as follows.

• Characterize the pharmacokinetic (PK) profile of single and repeated doses of H4H17319P2.

• Assess the effects of repeated doses of H4H17319R2 on body weight.

• To evaluate the effects of repeated doses of H4H17319P2 on ad lib energy intake in overweight and obese individuals.

• Assess the effects of single and repeated doses of H4H17319P2 on levels of soluble forms of lipid regulatory proteins (sLEPR and ANGPTL3) over time.

• Assess the immunogenicity of single and repeated doses of H4H17319R2.

[0287] Other research objectives of this study were as follows.

1. Assess the effects of single doses of H4H17319P2 on body weight and parameters related to serum/plasma glucose and lipid levels.

2. Assess the effects of repeated doses of H4H17319R2 for 12 weeks on

• parameters related to serum/plasma glucose and lipid levels;

• patient-reported appetite ratings, which may influence eating behavior (eg hunger, satiety, and satiety);

• total body weight and distribution of fat and lean body mass according to dual-energy X-ray absorptiometry (DXA);

• quantification of SC and visceral fat (including liver fat) using magnetic resonance imaging (MRI);

• other research biomarkers, including leptin, thyroid hormones (T3, T4, thyroid-stimulating hormone [TSH]), luteinizing hormone (LH), testosterone, estradiol, cortisol, and adiponectin.

Rationale

[0288] Part A is a single escalating dose FIH design in which no more than 88 healthy subjects will be randomized 3:1 to receive H4H17319P2 or placebo into 7 escalating single dose cohorts (no more than 5 IV and 2 SC) for to evaluate the safety, tolerability and pharmacokinetics of a single escalating dose of H4H17319R2 with a follow-up period of 112 days. The seven escalating single-dose cohorts will have 8 subjects randomized to receive H4H17319P2 or placebo (6 active: 2 placebo) at each dose level. The study design also includes 2 additional optional cohorts (16 subjects randomized to receive H4H17319P2 or placebo (12 active: 4 placebo in each cohort). If interim analyzes in escalating dose cohorts indicate that there may be effects of covariates (eg, age, body weight or sex) on PK profiles, optional cohorts can be included to collect additional data at one or more doses (up to the maximum dose in this study of 30 mg/kg IV) to obtain a more precise estimate of the effects of covariates on PK.

[0289] Part A of this study consists of a screening period (days -21 to -2), a preliminary baseline assessment visit (day -1), where patients will be admitted to the clinic for a 2-day stay (for subjects receiving IV and SC doses, which are in the safety alert block) or a 1-day clinic stay (for subjects receiving the SC dose), a follow-up period (Day 3 to Day 113) with an end-of-study visit (Day 113).

[0290] Assessing safety and tolerability is the primary goal of Part A, and safety will be carefully assessed throughout the study. The initial and maximum doses administered in this study are expected to be −10,000- and ∼20-fold lower, respectively, than concentrations observed in toxicology studies. Throughout the clinical study, safety assessments include vital signs, physical examination, electrocardiograms (ECGs), laboratory tests including hematology tests/leukograms, and adverse event (AE) monitoring. Body weight will also be assessed. Pharmacodynamic parameters will also be collected in this study, but it is not expected that there will be significant changes in PD markers in this lean/overweight patient population based on the minimal effects on body weight (<1 kg) observed with metreleptin. in lean individuals (Heymsfield, 1999).

[0291] Pharmacodynamic parameters include body weight, metabolic parameters (glucose, lipids) and ANGPTL3, a potential marker that may be regulated by leptin and/or insulin (Muniyappa, 2017) (Nidhina Haridas, 2015). Body weight will be carefully assessed at each visit. In the event that single doses of H4H17319P2 treatment produce a clinically significant effect on body weight in lean/overweight subjects, the timing of safety/dose escalation decisions may be modified and additional safety data collected (eg, pending evaluation safety on day 15) before deciding to increase the dose.

[0292] Part B is a single-dose repeat dose study with a 12-week treatment period to evaluate the safety, PK and body weight effects of H4H17319P2 in overweight/obese subjects. Several population-based studies suggest that there are subgroups of obese patients in whom leptin levels are relatively low (Ruhl, 2001) (Buettner, 2002) and leptin receptors may not be saturated. The key question addressed in Part B is whether restoring leptin-mediated signaling will reduce appetite, food intake, and body weight in overweight or obese subjects with relatively low baseline leptin levels.

[0293] Therefore, Part B will evaluate the effects of H4H17319P2 on body weight, food intake, metabolic parameters, and body composition in subjects with varying body weights and relatively low baseline leptin levels. Healthy overweight or obese subjects (BMI range 25-40 kg/ ^m2 ) will be included in 4 separate cohorts defined by leptin levels at pre-screening. Enrollment in 4 separate cohorts will be conducted to ensure an adequate number of subjects are studied across a range of relatively low baseline leptin levels and BMI ranges. Cohort allocation will occur at randomization to receive H4H17319R2 or placebo. Each of the 4 cohorts will include up to approximately 20 subjects, with a total sample size of no more than 81 subjects for Part B, as defined below. Leptin levels and BMI will be measured at the prescreening visit for initial assessment of study eligibility and for 1 of the 4 cohorts. Study enrollment will occur upon screening of eligible subjects. If enrollment in a particular cohort has reached the maximum allowed number, the subject will not be eligible for enrollment. Some cohorts may be difficult to enroll due to the low prevalence of obesity with low leptin levels; the sponsor may stop enrolling in a particular cohort.

[0294] The 4 cohorts in Part B are defined as follows.

• Cohort 1: male and female subjects with a BMI at pre-screening between 28.0 and 40.0 kg/m ² inclusive, with a fasting leptin level at pre-screening <5 ng/mL.

• Cohort 2: male and female subjects with pre-screening BMI between 25.0 and <28 kg/ ^m2 and pre-screening fasting leptin <5 ng/mL.

• Cohort 3: male subjects with a BMI at pre-screening between 28.0 and 40.0 kg/ ^m2 inclusive and a fasting leptin level at pre-screening between 5.0 and 8.0 ng/mL inclusive.

• Cohort 4: female subjects with a BMI at pre-screening between 28.0 and 40.0 kg/ ^m2 inclusive and a fasting leptin level at pre-screening between 5.0 and 24.0 ng/mL inclusive.

[0295] Note: Eligibility for participation in one of the above cohorts is based on BMI and leptin levels at the prescreening visit. If BMI and/or leptin levels do not fall within the ranges defined by the cohorts, the subject is not eligible for enrollment. If BMI or leptin levels at the time of prescreening are borderline for inclusion in 1 or more than 1 cohort, BMI and leptin measurements can be repeated once during the prescreening window. For repeated measurements, the lowest leptin level will be used for inclusion in one of the cohorts. If enrollment in a particular cohort has reached the maximum allowed number, the subject will not be eligible for enrollment.

[0296] The start of Part B will be initiated by an interim review of available safety, PK, and PD data from Part A to select the appropriate dose, dosing frequency, and route of administration (IV or SC) for Part B. Subjects included in Part A are not will be eligible to participate in Part B. The Part B study design consists of a pre-screening period (days -60 to -14) to assess BMI and fasting leptin levels, and eligibility for inclusion in 1 of 4 cohorts, a screening period (days from -32 to -14), a baseline assessment period consisting of (days -29 to -1), a treatment period (days 1-85) and a non-treatment follow-up period (days 107-191). Subjects will be hospitalized for 2 days in the clinic for accurate assessment of appetite and ad lib food intake during the baseline assessment period (scheduled from days -14 to -1) and at 2 time points during the treatment period (days 29-30 and days 84 -85). A clinical stay is necessary to accurately determine caloric intake during an ad lib food assessment in a controlled setting where there are standardized meals and minimization of cues that may influence food intake other than appetite (eg, time of day, eating behavior of others , serving size, etc.). Subjects will also be admitted to the hospital for a 2-day clinic stay on day -1. On Day 1 of the treatment period, subjects will receive the first dose of study drug or placebo, have blood samples drawn for serial PK and other laboratory measurements, and will remain overnight to complete the 24-hour PK assessment on Day 2.

[0297] Treatment visits for blood draws will occur once a week. The dose and frequency of the study drug will be determined at the interim analysis of the PK data from Part A and possibly every 4 weeks or every 2 weeks (with a maximum frequency of no more than once per week).

[0298] Body composition assessment using DXA and quantification of liver, abdominal and thigh fat using MRI will be performed at baseline, towards the end of treatment and during the follow-up period. Following the treatment period, subjects will be followed for a 16-week drug-free period to assess effects regarding safety and PD. Assessing the safety and tolerability of repeated doses at the single-dose level (based on safety and tolerability from Part A) will be the primary objective of Part B of the study. During the study, safety assessments will include vital signs, physical examination, ECG, laboratory tests, and AE monitoring. Antibodies to drugs will also be assessed. In addition, secondary objectives of body weight, ad lib food intake, and metabolic parameters such as glucose and lipids will be scrutinized throughout the study.

[0299] Pharmacodynamic measures in Part B include analysis of baseline and during treatment parameters that may be affected by increased leptin receptor-mediated signaling. These PD assessments include patient-reported measures of appetite, quantitative measurements of food intake in a controlled hospital setting, accurate measurements of body composition and fat mass using DXA, and accurate measurement of body weight using calibrated scales. In addition, metabolic parameters such as leptin, glucose, insulin, homeostasis model-assessed insulin resistance (HOMA-IR), HbAlc, lipids will be measured at baseline, during and at the end of the treatment period to evaluate the effects of H4H17319R2 on sensitivity to insulin and lipid metabolism. Liver MRI will also be performed at baseline and after treatment to determine whether H4H17319P2 affects hepatic steatosis.

Rationale for pharmacodynamic variables and biomarkers

Ad libitum food intake assessment (Part B only)

[0300] Treatment with H4H17319P2 is predicted to enhance leptin receptor signaling in the hypothalamus and affect neurons that have effects on eating behavior. Treatment with H4H17319P2 resulted in a significant reduction in food intake and body weight in mice with induced leptin deficiency. Although food intake was not quantified in the cynomolgus monkey studies, treatment with H4H17319P2 resulted in a significant effect on body weight and fat mass. In the current study, the effects of H4H17319P2 on food intake in overweight/obese subjects with relatively low leptin levels will be assessed at baseline and 2 time points during treatment using a rigorous inpatient ad lib food intake quantification as previously described. (Krishna, 2009) (Addy, 2008). Briefly, after fasting, subjects will be admitted to the clinical trial unit and will be provided with a standardized breakfast, lunch, and dinner to establish a standard energy intake at baseline. After an overnight fast, subjects will be offered breakfast, lunch, and dinner ad lib to quantify food/energy intake. Subjects will consume all test meals in specialized environments that mask the perception of time/time of day and social cues.

[0301] Meals with known caloric content will be provided in 4- to 5-fold excess portions and will be presented in such a way as to mask the total number of calories consumed. Subjects will be asked to eat the desired amount. Meals will be weighed before and after food intake assessments to quantify food intake, and calories consumed will be counted.

Appetite assessment (Part B only)

[0302] The effects of leptin on appetite are central to its mechanism of action and therefore represent a precise

[0303] assessment of the predicted mechanism of action of H4H17319P2. Individuals with hypoleptinemia caused by LEP mutations or generalized lipodystrophy exhibit a voracious appetite that contributes to food consumption and obesity. Treatment of these individuals with metra leptin increases satiety and reduces food intake (Farooqi, 1999) (Farooqi, 2007) (McDuffie, 2004). The effects of H4H17319P2 on appetite will be assessed using questionnaires (Flint, 2000) (Dalton, 2015) that measure various components of appetite (e.g., hunger, satiety), at baseline and during follow-up.

[0304] Appetite will be assessed before and after a standard caloric challenge during clinic visits, and a supplemental appetite questionnaire will be completed daily during the baseline, treatment, and follow-up periods.

ANGPTL3

[0305] Angiopoietin-like protein 3 (ANGPTL3) regulates lipoprotein levels, as well as TG, low-density lipoprotein C (LDL-C), by inhibiting lipoprotein lipase (reviewed in Tikka, 2016). ANGPTL3 levels can be regulated by nutrition, leptin and/or insulin (Minicocci, 2012) (Nidhina, 2015). ANGPTL3 levels are increased in patients with lipodystrophy and decreased after metreleptin treatment (Muniyappa, 2017), and the reduction of ANGPTL3 may mediate increased lipase clearance of TG-rich lipoproteins. ANGPTL3 and TG levels are also increased in a mouse model of lipodystrophy and are normalized after treatment with H4H17319P2. Therefore, ANGPTL3 may be a pharmacodynamic marker that may be decreased under conditions of increased leptin receptor-mediated signaling. ANGPTL3 will be measured at baseline and post-treatment at multiple time points in Parts A and B.

Soluble LEPR

[0306] Leptin circulates in the bloodstream both free and bound to sLEPR, which is formed when the LEPR ectodomain separates (Sinha, 1996) (Lammert, 2001). Soluble LEPR may regulate the bioavailability and/or clearance of leptin (Lou, 2010). It is possible that target saturation may depend on sLEPR saturation, and therefore variability in linear and nonlinear target-mediated kinetics may be influenced by sLEPR levels. Therefore, in Parts A and B, sLEPR will be measured at baseline and at various time points (corresponding to sampling for antibody pharmacokinetics) during the treatment period.

Visualization (Part B only)

[0307] DXA and MRI imaging will be performed at baseline, near the end of treatment and at the end of the study to assess change from baseline in total and regional fat distribution, SC and visceral fat in the thigh/abdomen and fat content in the liver. In a pilot study in cynomolgus monkeys, preclinical data demonstrated that H4H17319P2 reduced body weight by reducing total fat mass without effects on lean mass (as 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, patients with focal lipodystrophy showed significant differences in regional fat distribution as measured by DXA (% trunk fat to % leg fat (fat mass ratio [FMR]) 1.78 ± 0.53) compared with normal subjects (Aijluni , 2017). DXA has also been used to quantify changes in total and regional fat mass in trials of weight loss agents such as GLP-1 agonists (Jendle, 2009) and cognitive therapy (Ponti, 2018).

[0308] Magnetic resonance imaging (MRI) has proven to be an effective means of measuring fat content in various tissues, including the liver (Aijluni, 2017) and muscle (Burakiewicz, 2017), by imaging entire organs using special pulsed modes or using limited spectroscopy volume of fabric. MRI also showed high-contrast images to measure the volume and distribution of abdominal fat (Klopfenstein, 2012), where patients with familial lipodystrophy type 2 had a visceral fat percentage 2.5 times greater than control patients (Al-Attar, 2007). Fat fraction in muscle can also be assessed using quantitative MRI, as has been shown in numerous studies of muscular dystrophy, where spectroscopy and pulsed imaging, allowing anatomical imaging and assessment of fat fraction, have shown potential in assessing disease progression (Burakiewicz, 2017).

[0309] The current study will conduct MRI of the abdomen and thigh to quantify regional changes in SC, visceral and liver fat at baseline and after treatment with H4H17319P2.

Rationale for dose selection

[0310] IV and SC dosages were selected for Part A based on efficacy, safety, and

[0311] pharmacokinetic data from preclinical studies in mouse models of lipodystrophy and monogenic obesity and toxicological studies in monkeys. The highest dose in this FIH study will be no more than 30 mg/kg, and the starting dose will be approximately 0.3 mg/kg. In a GLP toxicology study in cynomolgus monkeys, H4H17319P2 was well tolerated at doses up to 100 mg/kg IV weekly for 12 weeks, with a no observed adverse event level (NOAEL) of 100 mg/kg. Based on predicted exposure to H4H17319P2 in human serum, the planned maximum dose of 30 mg/kg in this FIH study has a concentration twenty times lower than the NOAEL. The original starting dose of 0.3 mg/kg has a safety margin of more than 10,000 below the maximum dose tested in the GLP toxicology study and is expected to provide human H4H17319P2 concentrations above the limit of quantitation and therefore provide useful information by PK.

[0312] The goals of dose selection for Part B include providing a broad concentration range to assess tolerability and facilitate characterization of exposure-response relationships, as well as elucidating pharmacokinetic profiles that allow characterization of both linear and nonlinear pharmacokinetics. In addition, dose selection should ensure concentrations above and below putative thresholds for PD markers that may be of interest (eg, sLEPR saturation). The dose for Part B will be based on the interim safety data, PK and PK/PD from Part A. The choice of dose levels, dosing interval and route of administration (IV or SC) will be based on the safety data, pharmacokinetics and, if available, PK /PD, from Part A, as well as from preclinical animal studies. The dose in Part B will not exceed the doses assessed in Part A, and administration will likely occur every 4 weeks or every 2 weeks, but not more than once a week.

[0313] The dosing regimen should not exceed concentrations observed in toxicology studies.

Criteria

[0314] Up to 169 subjects (up to 88 for Part A and up to 81 for Part B) will represent the target population of subjects included in the study. The target population will be healthy lean or overweight men and women for part A and healthy overweight or obese men and women with different baseline leptin levels for part B.

Key inclusion criteria

[0315] To be eligible for inclusion in Part A of the study, the subject must meet the following criteria at screening:

1. Men and women aged 18 to 50 years inclusive

2. Body mass index (BMI) from 18.5 to <30.0 kg/ ^m2

3. The investigator determines that the subject is in good health and has no significant underlying medical conditions based on medical history, physical examination, and laboratory safety tests performed at screening and/or prior to administration of the initial dose of study drug.

4. Willingness and ability to comply with clinic visits, study procedures, and dietary instructions

5. Willingness to maintain normal diet and exercise regimen throughout the study

6. Ability and willingness to provide signed informed consent

[0316] A subject must meet all of the following screening criteria (except meeting BMI and leptin criteria, which are determined prescreening) to be eligible for inclusion in Part B of the study:

1. Men and women aged 18 to 65 years inclusive

2. Presence of body mass index (BMI) and fasting leptin level at the prescreening visit, as defined below for one of the cohorts. If enrollment in a particular cohort has reached the maximum allowed number, the subject will not be eligible for enrollment.

3. The investigator determines that the subject does not have significant comorbidities based on the medical history, physical examination, and laboratory safety tests performed at screening and/or prior to administration of the initial dose of study drug. Subjects may have a history of mild hyperlipidemia and/or mild hypertension but should be on stable doses of lipid-lowering or blood pressure-lowering medications for at least 2 months prior to screening

4. Willingness and ability to comply with clinic visits, study procedures, and dietary instructions

5. Willingness to maintain normal diet and exercise regimen throughout the study

6. Ability and willingness to provide signed informed consent

Exclusion criteria

[0317] A subject who meets any of the following screening criteria will be excluded from Part A of the study.

1. A history of clinically significant cardiovascular (for example, hypertension, myocardial infarction, stroke, peripheral vascular disease, heart failure, history of arrhythmias), respiratory, hepatic, renal, gastrointestinal, endocrine (for example, hyperlipidemia), hematological or neurological diseases.

2. History of diabetes mellitus type 1 or 2 or prediabetes, or fasting blood glucose (FBG) at screening >100 mg/dL, or HbAlc at screening >5.7%.

3. LDL-C≥130 mg/dL, TG>250 mg/dL on an empty stomach.

4. Clinically significant abnormalities on the CBC, clinical chemistry panel, urinalysis, or urine drug screening test at screening. Minor deviations in laboratory test results are allowed. NOTE: Laboratory tests with any abnormal results (eg, creatine phosphokinase (CPK) level within 3 times the upper limit of normal (ULN) with a suspected cause of strenuous exercise) can be repeated once during the screening period.

[0318] A subject who meets any of the following screening criteria will be excluded from Part B of the study.

1. History of clinically significant cardiovascular (eg, moderate to severe hypertension, myocardial infarction, stroke, peripheral vascular disease, heart failure, history of arrhythmias), respiratory, hepatic, renal, gastrointestinal, endocrine (eg , hyperlipidemia), hematological or neurological diseases.

2. History of diabetes mellitus type 1 or 2, or FBG at screening > 126 mg/dL, or HbAlc at screening > 6.5%. A diagnosis of prediabetes is acceptable.

3. LDL-C>160 or TG>500 mg/dL on an empty stomach

4. Clinically significant abnormalities on a complete blood count, clinical chemistry panel, urinalysis, or urine drug screening test at screening, excluding minor abnormalities in lipid levels or glycemia as described above. Minor deviations in laboratory test results are allowed. NOTE: Laboratory tests with any abnormal results (eg, CPK within 3x ULN with suspected strenuous exercise) can be repeated once during the screening period.

5. Restrictive eating habits (eg, vegetarians or vegans), aversions to certain food categories used in food intake assessments, or eating behaviors that may interfere with or make it difficult to interpret food intake, appetite, or dietary control assessments.

[0319] A subject who meets any of the following criteria at screening will be excluded from Part A and Part B of the study.

1. Hospitalization (i.e. > 24 hours) for any reason within 60 days of the screening visit

2. The subject has any physical examination findings and/or medical history that, in the opinion of the investigator, may confound the study results or pose an additional risk to the subject's participation in the study.

3. History of hypothalamic amenorrhea or lipodystrophy.

4. Change in body weight by more than 5% in the last 3 months before screening.

5. Previous bariatric procedures for obesity (eg, sleeve gastrectomy, gastric bypass, banding, etc.).

6. Weight loss procedures (eg liposuction) or body contouring procedures in the last 6 months.

7. Treatment with medications (over-the-counter [OTC] or prescription) for weight loss (eg, lorcaserin, phentermine/topiramate, naltrexone-HCl/bupropion-HCl, liraglutide) within the past 3 months.

8. A history of serious mental disorders, eating disorders (for example, bulimia, anorexia).

9. Current cigarette smoker or former smoker (cigarettes or e-cigarettes) who quit smoking within 3 months before screening.

10. History of recreational drug use (including marijuana) or alcohol abuse (>2 drinks per day) in the year prior to the screening visit.

11. A history of hepatitis B infection or a positive test result for hepatitis B surface antigen (HbsAg+) at screening.

12. History of HIV infection or HIV seropositive status at the screening visit.

13. A history of hepatitis C infection or a positive test result for antibodies to the hepatitis C virus during screening.

14. Any malignancy within the last 10 years, except basal cell or squamous cell epithelial carcinomas of the skin or carcinoma in situ of the cervix or anus that have been removed without evidence of metastatic disease within 3 years.

15. History of active or latent tuberculosis (TB). NOTE: A history of latent TV is defined as either a positive tuberculin skin test (TST; defined as skin thickening > 5 mm, regardless of bacillus Calmette-Guerin (BCG) vaccination or other vaccination history) or a positive (not indeterminate) result test (QuantiFERON® TV Gold.

16. For Part A: sitting or supine blood pressure readings with at least 2 readings (>140/90 or <90/60) and resting heart rate (<45 or >125) or with orthostatic changes (>20 mm Hg drop in systolic pressure and/or >10 mm Hg drop in diastolic pressure) at the screening and baseline assessment visits. For Part B: sitting or supine blood pressure readings with at least 2 readings (>150/90 or <90/50) and resting heart rate (<45 or >125) or with orthostatic changes (falling) systolic pressure >20 mmHg and/or diastolic pressure >10 mmHg) at the screening visit. If blood pressure readings are elevated, collection of blood pressure readings can be repeated or subjects can be rescreened once.

17. A subject has an estimated glomerular filtration rate (using the MDRD equation) at screening of <60 mL/min/1.73 m ² .

18. Clinically significant abnormal ECG findings or abnormal intervals confirmed by at least 2 determinations (QTcF>450 ms for men and >470 ms for women; PR<120 ms or >220 ms; QRS>100 ms).

19. Hypersensitivity to doxycycline (or drugs of the tetracycline class) or other components of the composition.

20. History of acute hypersensitivity and/or anaphylaxis to protein therapeutic agents.

21. A history of severe allergic reactions (including reactions to latex or anaphylactic reactions or allergic reactions) which, in the opinion of the investigator, may pose a significant risk to the subject.

22. Participation in any clinical trial evaluating another investigational medicinal product (including biologicals) or therapy for 90 days or at least 5 half-lives (whichever is longer) of the investigational biological medicinal product, or at least 4 weeks for other investigational drugs, or 6 months for immunotherapy before the screening visit.

23. Pregnant or breastfeeding women.

24. Women of childbearing potential* who are unwilling to use highly effective contraception before the initial dose/start of first treatment, during the study, and for at least 4 months after the last dose. Highly effective contraceptives include:

a. stable use of combined (containing estrogen and progestogen) hormonal contraceptives (oral, intravaginal, transdermal) or hormonal contraceptives containing only progestogens (oral, injectable, implantable), associated with suppression of ovulation, started 2 or more menstrual cycles before screening;

b. intrauterine device (IUD); intrauterine system (IUS);

c. bilateral tubal ligation;

d. partner's vasectomy;

e. and/or sexual abstinence

[0320] *Postmenopausal women must be amenorrheic for at least 12 months before they are considered to be of childbearing potential. Women with a documented hysterectomy or tubal ligation do not require pregnancy testing or contraception.

[0321] Sexual abstinence is considered highly effective only if it is defined as abstinence from heterosexual intercourse for the entire period of risk associated with the study treatment.

[0322] Intermittent abstinence (calendar, symptothermal, postovulatory methods), coitus interruptus, spermicide only, and lactational amenorrhea method (LAM) are not acceptable methods of contraception. The female condom and the male condom cannot be used together.

25. Sexually active men who are unwilling to use the following forms of medically acceptable birth control during the period of study drug treatment and for 4 months after the last dose of study drug: vasectomy with medical assessment of surgical success OR continuous use condom. Sperm donation is prohibited during the study and for 4 months after the last dose of study drug.

26. Use of concomitant medications other than those listed among approved medications or nutritional supplements.

Description of study cohorts and dose escalations

[0323] Seven escalating dose cohorts are planned to include doses ranging from 0.3 mg/kg to a maximum dose of 30 mg/kg. Each dose cohort will consist of 8 subjects: 6 randomized to receive H4H17319P2 and 2 randomized to receive placebo. To optimize safety, 8 subjects (6 active: 2 placebo) in each of Cohort 1 (0.3 mg/kg IV), Cohort 2 (1 mg/kg IV), Cohort 3 (3 mg/kg IV) , Cohort 4 (300 mg SC) and Cohort 5 (10 mg/kg IV) will be divided into 2 blocks. Two subjects (1 active drug: 1 placebo) will be included in block 1 as the safety sentinel group, and the remaining 6 subjects (5 active drug: 1 placebo) will be included in block 2. Subjects from block 1 will be included in study first and will be dosed on the same day. Enrollment of subjects in Block 2 will begin only after both subjects in Block 1 have safely completed at least a 24-hour safety assessment, safety data have been reviewed by the investigator and the sponsor's medical observer, and agreement has been reached between the investigator and the sponsor's medical observer that that subjects in block 2 can begin to be enrolled. All subjects in block 2 can be dosed on the same day.

[0324] The 8 subjects (6 active drug: 2 placebo) in each of Cohort 6 (600 mg SC) and Cohort 7 (30 mg/kg IV) will be divided into 2 blocks of 4 subjects (3 active drug: 1 taking a placebo). Dosing will be administered on different days in each block. Escalating dose cohorts will be included in the study as follows.

Cohort 1: H4H17319R2 at 0.3 mg/kg IV, single dose

Cohort 2: H4H17319R2 at 1 mg/kg IV, single dose

Cohort 3: H4H17319R2 at 3 mg/kg IV, single dose

Cohort 4: H4H17319R2 at 300 mg SC, single dose

Cohort 5: H4H17319R2 at 10 mg/kg IV, single dose

Cohort 6: H4H17319R2 at 600 mg SC, single dose

Cohort 7: H4H17319R2 at nominal dose 30 mg/kg IV, single dose

[0325] Optional cohorts will be included in a study if PK variability is greater than expected and additional subjects are needed to examine the role of specific covariates such as age, weight, sex:

Cohort 8: H4H17319P2 at nominal dose 30 mg/kg IV, single dose

Cohort 9: H4H17319R2 at a nominal dose of 30 mg/kg IV, single dose. A maximum dose of up to 30 mg/kg IV was given to cohorts 8 and 9, but a lower dose may be administered depending on emerging PK data.

[0326] Dose increase. The safety/dose escalation team will include the investigator, medical director/study director, study biomedical statistician, and risk management manager. The investigator(s) and other study site personnel, as well as the sponsor's study team, including the medical observer, will also be blinded to treatment assignment. The sponsor may have randomized coded individuals, but these randomized coded individuals will not be part of the sponsor's research team.

[0327] Dose escalation to cohort 2 (1 mg/kg) may occur after all subjects in the previous cohort have completed day 8 safety assessments and de-identified safety data have been reviewed at a safety/dose escalation team meeting.

[0328] Dose escalation to Cohort 3 (3 mg/kg IV) and Cohort 4 (300 mg SC) may occur after all subjects in the previous cohort have completed safety assessments on Day 8 and de-identified safety data have been reviewed at the meeting safety/dose escalation teams. Dosing in Cohort 4 may occur in parallel with dosing in Cohort 3.

[0329] Dose escalation to Cohort 5 (10 mg/kg IV) may occur after all subjects in Cohort 3 have completed Day 8 safety assessments and de-identified safety data have been reviewed at a safety/dose escalation team meeting.

[0330] Dose escalation to Cohort 6 (600 mg SC) may occur in parallel with escalation to Cohort 5, but only after all subjects in Cohort 4 have completed safety assessments on Day 8 and de-identified safety data have been reviewed at a team meeting for safety monitoring/dose escalation.

[0331] A nominal dose of 30 mg/kg IV was administered to Cohort 7, but a lower dose may be administered depending on emerging PK data. Before dose escalation to 30 mg/kg IV, all subjects in cohort 5 must complete safety assessments on day 8, and anonymized safety data must be reviewed at a safety/dose escalation team meeting. The timing of safety/dose escalation decisions may be modified and additional safety data may be collected if pharmacodynamic effects on body weight are greater than expected. For example, if body weight decreases by >3% in at least 3 of 6 subjects over 8 days, the observation period will be extended to day 15 before any dose escalation decisions are made; if body weight further decreases to >5% within 2 weeks in at least 3 of 6 subjects, the observation period will be extended to 4 weeks before a dose escalation decision is made.

Study plan

[0332] This is a randomized, double-blind, placebo-controlled, 2-part Phase I study of the safety, tolerability, PK, and pharmacodynamics (PD) of single and repeated doses of H4H17319R2 in healthy participants. Part A will include healthy lean or overweight subjects to evaluate the safety, tolerability, PK, and PD of single escalating intravenous (IV) and subcutaneous (SC) doses. Interim PK and safety information from Part A will be used to select the dose level, frequency, and route of administration (IV or SC) for Part B. In Part B, overweight/obese subjects with a body mass index (BMI) of 25-40 kg / ^m2 will be included to evaluate the safety, tolerability, PK and PD of repeated doses of H4H17319R2 in 4 different cohorts defined by baseline leptin levels.

[0333] In Part A, up to 88 subjects will be randomized into 7 escalating single-dose cohorts (up to 5 IV and 2 SC) (cohorts 1, 2, 3, 4, 5, 6, 7) and 2 optional single-dose cohorts (cohorts 8 and 9). The 7 consecutive single-dose cohorts will have 8 subjects randomized to receive H4H17319P2 or placebo (6 active drug: 2 placebo) at each dose level. In 2 additional optional single-dose cohorts, there will be up to 16 subjects randomized to receive H4H17319P2 or placebo (12 active drug: 4 placebo). Up to 5 single IV dose levels (0.3, 1.0, 3, 10 and 30 mg/kg) and 2 SC doses (300 and 600 mg) will be evaluated in a single escalating dose regimen. Decisions on dose escalation will be based on analysis of safety parameters and AEs. Interim analyzes of antibody concentrations over time, study PD rates, and, if applicable, PK/PD ratios between dose cohorts may also influence dose escalation decisions. The decision to enroll 2 optional cohorts at dose levels up to a maximum of 30 mg/kg will be based on an interim analysis of PK profile variability.

[0334] Optional Cohorts: Cohorts 8 and 9 will be included in the study if there is greater than expected PK variability among subjects and additional subjects are needed to understand the effects of specific covariates on PK profiles. Subjects in specific subgroups within a population defined by inclusion/exclusion criteria such as predetermined numbers of men, women, age ranges, body weight, or specific BMI cutoff points may be included. A nominal dose of 30 mg/kg IV was given to cohorts 8 and 9, but a lower dose may be administered depending on emerging PK data. Cohorts 8 and 9 will each include 16 subjects (4 will be assigned to receive placebo and 12 will be assigned to receive H4H17319P2). Before dose escalation to 30 mg/kg IV, all subjects in cohort 5 must complete safety assessments on day 8, and anonymized safety data must be reviewed at a safety/dose escalation team meeting. The Part A study design consists of a screening period (days -21 to -2), a preliminary baseline assessment visit (day -1), when patients will enter the clinic for a 2-night stay (for subjects receiving IV and for subjects receiving the SC dose, which is in the safety alert block) or a 1-day clinic stay (for the remaining subjects receiving the SC dose), 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, body weight, physical examination, ECG, laboratory tests, and monitoring of AE, PK parameters, and various PD assessments.

[0335] In Part B, up to 81 subjects with a BMI of 25-40 kg/m ² will be enrolled in 4 cohorts (up to approximately 20 per cohort) defined by baseline leptin levels and randomized (3:1 or 6:1 for H4H17319P2 and placebo depending on cohort assignment) in a placebo-controlled, double-blind, 12-week repeat-dose study. Selection of dose, dosing interval, and route of administration (IV versus SC) will be based on safety data, PK, and, if available, PK/PD from Part A. H4H17319P2 PK profiles from Part A will be used to predict serum concentrations blood after repeated administration.

[0336] The study consists of a pre-screening period (days -60 to -14), a screening period (days -32 to -14), a baseline assessment period (days -29 to -1) to obtain baseline body weight measurements, body composition by DXA and MRI, and a clinic stay to collect baseline measurements of fasting leptin, body weight, appetite scores, and ad lib food intake scores. Subjects will present to the clinic for a 2-day clinic stay on day -1. On Day 1, subjects will receive the first dose of study drug or placebo, have serial blood drawn for pharmacokinetic measurements, and will remain overnight for 24-hour PK sampling on Day 2 when they are discharged. The study drug may be administered every 4 weeks or every 2 weeks, but will be administered no more than once per week during the treatment period (dose frequency will be determined at the interim analysis of the PK data from Part A).

[0337] Visits during the treatment period will occur at least weekly to collect accurate and repeated measurements of body weight and serum metabolic parameters (glucose, insulin, HOMA-IR, lipid levels). Assessments of appetite and ad lib food intake during the follow-up period will be conducted during the clinic stay in week 4 and at the end of the treatment period (12 weeks). Subjects will also complete a daily appetite questionnaire during the baseline, treatment, and follow-up assessment periods. DXA and MRI imaging assessments will also be performed during follow-up. Following the treatment period, subjects will be observed for a 16-week drug-free period. During the study, safety assessments will include vital signs, physical examination, ECG, laboratory tests, and AE monitoring. Drug concentrations, markers of target interaction (sLEPR) and investigational biomarkers will also be measured throughout the study.

Duration of the study

[0338] The duration of Part A of the study per subject is approximately 19 weeks, including the screening period. The duration of Part B of the study per subject is approximately 35 weeks, including the pre-screening/screening/baseline assessment period. The end of the study is considered to be the last visit of the last subject in part B.

Dose/route/schedule of administration of treatment(s)

[0339] H4H17319P2 will be supplied as a lyophilized powder in a sterile 20 mL single-use glass vial for IV or SC administration. The corresponding placebo for H4H17319P2 is prepared in the same composition without the addition of protein. For Part A, single doses will be given IV and SC. For Part B, selection of dose, dosing interval, and route of administration (IV or SC) will be made based on the safety data, PK, and, if available, PK/PD from Part A.

Procedures and assessments

[0340] Safety will be assessed by monitoring/evaluating TEAE, vital signs, physical examination, electrocardiogram (ECG), and laboratory tests. To evaluate pharmacokinetics, samples will be collected in both frequent and sparse sampling to measure serum concentrations of H4H17319P2 at predetermined time points. Pharmacodynamics will be assessed by measuring body weight and waist circumference, assessing food intake and appetite, and body composition using DXA and MRI.

[0341] Basic criteria for evaluating effectiveness

1. Number of treatment-emergent adverse events (TEAE) [Time frame: week 12 (end of treatment period)]

[0342] Secondary Performance Criteria

1. Serum concentrations of H4H17319P2 over time [Time frame: up to week 27 (end of study)]

2. Percentage change in body weight in overweight or obese subjects [Time Frame: Baseline to Week 12]

3. Absolute change in body weight in overweight or obese subjects [Time frame: baseline to week 12]

4. Change from Baseline in Caloric Intake in Response to a Standardized Diet in Overweight or Obese Subjects [Time Frame: Baseline to Week 12]

5. Change in Lipid Regulatory Protein Levels Over Time Following Single Doses of H4H17319R2 [Time Frame: Up to Week 16]

6. Change in Lipid Regulatory Protein Levels Over Time Following Repeated Doses of H4H17319R2 [Time Frame: Up to Week 27]

7. Incidence of antibodies to drug H4H17319R2 over time after single doses of H4H17319R2 [Time frame: up to week 16]

8. Incidence of antibodies to drug H4H17319R2 over time after repeated doses of H4H17319R2 [Time frame: up to week 27]

Pharmacokinetic Variables

[0343] In addition to time, concentrations of total H4H17319P2 will be measured. Pharmacokinetic parameters may include, without limitation, the following:

• AUC _last - area under the curve (AUC), calculated from time zero to the moment of the last positive concentration

• AUC _0-τ - AUC calculated for a dose interval of length t

• C _max - peak concentration

• t _max - time to C _max

• CL - ground clearance

• C _trough - minimum concentration

[0344] Note that for Part B, the choice of these (and other) parameters depends on the final sampling schedule chosen as well as the resulting data.

Drug Antibody Variables

[0345] Anti-drug antibody (ADA) variables include the ADA response and its titer as follows:

• Treatment-emergent response, defined as any positive response in the post-dose ADA assay if results at baseline are negative.

• Treatment-stimulated ADA response, defined as any positive response in the post-dose ADA assay that is 9 or more times the baseline titer levels if the baseline is positive in the ADA assay

• Title values

• Title category

- Low (titer < 1000)

- Moderate (1000 ≤ titer ≤ 10000)

- High (title > 10000)

Pharmacodynamic and other biomarker variables

[0346] Pharmacodynamic and biomarker variables are: body weight, ad lib food intake estimates, appetite estimates, serum/plasma glycemic parameters (e.g., fasting glucose, insulin, HbAlc) and lipid parameters (e.g., total cholesterol, TG , LDL-C, HDL-C), DXA measurements of total and body fat and lean mass, MRI quantification of regional SC and visceral fat, ANGPTL3, leptin and sLEPR levels. Additional investigational biomarkers will include the effects of H4H17319P2 on thyroid hormones (T3, T4, TSH), luteinizing hormone (LH), testosterone, estradiol, cortisol and adiponectin.

Performance Evaluation Procedures

[0347] Body weight will be assessed at screening and throughout the study at scheduled study visits. Body weight will be assessed in triplicate using high-precision calibrated digital scales before other assessments in the study are performed. Before assessing body weight, subjects are required to empty their bladder (until the bladder is empty). During body weight assessments, subjects must wear underwear and no shoes. Body weight values will be recorded to the nearest 0.1 kg.

[0348] All anthropometric measurements should be performed in triplicate, with the final value reported being the average. Triceps, subscapularis, supiliac, and thigh skinfold thicknesses should be measured on the right side of the body on dry, intact skin, unless deformity or missing limb requires otherwise. To measure waist circumference, the subject is instructed to stand upright and relaxed with his arms at his sides and his feet pointing forward. The iliac crest and inferior edges of the ribs are identified by palpation, and the skin overlying these areas is marked with a pen. Then, using a tape measure, determine the midpoint between these marks on the skin and mark it with a pen. Then measure the waist circumference at the midline mark using a tape measure at the end of a light exhalation. Height is measured in a fully erect standing position at the end of inspiration using a calibrated stadiometer and recorded to the nearest 0.1 cm. For Part B and optional cohorts in Part A, height will be measured as a one-time measurement at the prescreening or screening visit, respectively. Body mass index will be calculated as the average of your weight (in kilograms) divided by the square of your height (in meters). In Part A (except for optional cohorts), the average height value must be used for calculation. In Part B and optional cohorts in Part A, height (one-time prescreening or screening measurement, respectively) and weight (average of 3 measurements) at each visit are used to calculate BMI.

[0349] Food intake will be assessed ad libitum. Subjects will be excluded from screening if they have abnormal eating behaviors or food aversions used in assessing food intake. Energy intake (breakfast + lunch + dinner) using the ad lib food intake assessment will be quantified in Part B at baseline, after 4 weeks of treatment, and at the end of the treatment period (12 weeks). Subjects will be asked to avoid exercise and alcohol consumption 1 day prior to clinic visits when nutritional assessments will be conducted.

[0350] Patients will be admitted to the clinic on an empty stomach, arriving at the clinic in the morning. During inpatient food intake assessments, subjects will receive their assigned treatment (study drug or placebo) during the 4-week treatment period. They will then be provided with a standardized breakfast, lunch and dinner with fixed amounts of calories and macronutrients, and then fasted overnight. The next day they will be provided with breakfast ad lib, lunch ad lib and dinner ad lib. During the ad lib test meal, subjects will be provided with an amount of food significantly larger (4-5 times) than a typical serving. Meals will be formulated by a nutritionist based on standardized macronutrient content and known calorie content. All meals and serving items will be discreetly weighed using special calibrated scales before and after test meals to estimate food intake to the nearest 0.1 g. Subjects will eat all meals in their own designated rooms that do not have a clock. , radios, mobile phones and televisions to eliminate temporary or social cues that may influence food intake. Meals will be presented in a manner that disguises the amount of food consumed so that eating is not influenced by visual/social cues of overeating, such as the amount of food available. Subjects will be asked to eat as much or as little as they wish and will not be disturbed during the test meal and will not be allowed to engage in leisure activities such as reading, listening to music, talking on the phone or watching videos. Subjects will be allowed to leave the room for the test meal when they are sufficiently full, and if they have not done so after 1 hour, the meal will be stopped by study staff. Clinic Appetite Assessment and Daily Appetite Questionnaire (Part B only)

[0351] Appetite will be assessed in the clinic using a checklist (Flint, 2000), with subjects instructed to record their response on a visual analogue scale. Subjects will complete an appetite assessment in the clinic approximately 30 minutes before and after a standardized dinner during the first day of the clinic, and will also complete a daily appetite questionnaire during the baseline, treatment, and follow-up assessment periods. Care will be taken to ensure that research subjects do not share survey results with other research participants. Details of the questions used in the clinic appetite assessment and in the daily appetite questionnaire for Part B of the study will be described in the study manual, and the questions (once final approval) will be submitted to the ethics committee for review prior to the start of Part B.

Body composition according to DXA (Part B only)

[0352] DXA is widely used in clinical whole-body skeletal densitometry. The total examination time is short (~6 to 7 minutes) and ionizing radiation doses are minimal at ~0.1 mGy. Dual-energy X-ray absorptiometry provides estimates of lean body mass (LBM) and body composition and has been used to measure LBM in clinical research settings. The clinical validity of DXA for measuring LBM is supported by longitudinal studies demonstrating a significant association between changes in LBM and decline in physical function (Goodpaster, 2006). Dual-energy X-ray absorptiometry will be performed twice during the baseline assessment period, at the end-of-treatment visit, and at the end of the study. With regard to individual preparation of subjects for a DXA scan, efforts should be made to maintain appropriate hydration, appropriate food intake, appropriate caffeine intake, appropriate activity (no physical activity 24 hours prior to scanning), appropriate clothing, and appropriate positioning of the subject on the DXA scan table. throughout the study. In addition, DXA scans should be performed at the same time of day for any given subject throughout the study.

Body composition according to MRI (part B only)

[0353] Magnetic resonance imaging is used to measure the fat fraction of the liver. A multiple gradient-reconstructed echo sequence is recommended that provides axial images to cover the entire liver. The times of occurrence of the echo signal must be such that at successive times of appearance of the echo signal (TE), fat and water alternate in antiphase and in phase. To minimize motion artifacts, data should be acquired during short breath-hold periods. Complete data collection will take from 3 to 5 minutes. Fat scanning of the thighs requires careful patient positioning and immobilization using molded foam supports and localizers. Complete hip MRI data acquisition may take up to 10 minutes. Subjects may be required to fast for 10 to 12 hours before their morning scan.

Schedule of Study Activities and Notes

[0354] Study assessments and procedures are presented by period and visit in Figures 25, 26, and 27 with the following notes.

1. Discharge from the clinic for non-sentinel SC dose groups, but the study site has the option for the subject to stay overnight with discharge the next day.

2. Discharge from the clinic for the IV dose group and SC safety signal group.

3. On Day 1 for Cohorts IV in Part A, vital signs and AE monitoring must also be taken before dosing, at 30 minutes, at the end of the study drug infusion, and at 1 hour, 2 hours, 4 hours, and 8 hours after infusion. . On Day 1 for the SC cohorts in Part A, vital signs should also be taken and AE monitored before dosing, 30 minutes after injection, and at 1 hour, 2 hours, 4 hours, and 8 hours after injection. For Part B dose administration on Day 1, vital signs and AE monitoring must also be taken before dosing, at 30 minutes, at the end of the study drug infusion for IV doses, and at 1 hour, 2 hours, 4 hours, and 8 hours. after infusion or injection for IV or SC doses, respectively. On subsequent dosing days, vital signs should also be taken and AE monitored before dosing, at 30 minutes, at the end of the study drug infusion for IV doses, and at 1 hour, 2 hours, and 4 hours after infusion or injection for IV and SC. doses accordingly. Vital signs also include orthostatic blood pressure assessments at the screening visit (Parts A and B) and Day 1 (Part A only). For orthostatic assessments, blood pressure and heart rate are measured while the subject is lying supine for about 10 minutes, after standing for about 1 minute, and after standing for about 3 minutes.

4. Blood is drawn after fasting for at least 8 hours. On dosing days, only the predose sample should be collected on an empty stomach.

5. Body weight must be measured in triplicate on an empty stomach, after urinating (with an empty bladder), without shoes and only in underwear using special calibrated scales.

6. Skinfold thickness should be determined in triplicate in the following areas: triceps, subscapularis, suprailiac, and thigh to provide an adequate description of body fat distribution.

7. Sample collection for drug concentration, sLEPR concentration and ANGPTL3 concentration on day 1 will occur before infusion/injection, after infusion/injection ± 15 minutes and after 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 after infusion/injection. sLEPR and ANGPTL3 can only be analyzed at the subset of time points at which drug concentrations are measured.

8. DNA can be collected at any visit.

9. Possibility to stay overnight with discharge the next day. For Part B, subjects will also enter the clinic the day before Day 1 (on Day -1) and stay overnight. Day 1 procedures (except for assessments that must be performed after an overnight fast, ECG, PK measurements, and study drug administration), such as drug screening, urinalysis, and urine pregnancy tests, can be performed on day -1.

10. The visit on day 30 should occur exactly 1 day after the visit on day 29.

11. The visit on day 85 should occur 1 day after the visit on day 84.

12. The frequency of dosing of the study drug in Part B will depend on the results obtained in Part A.

13. Two baseline images (DXA and MRI) will be acquired from day -29 to day -14. Both DXA and MRI can be performed on the same day or on separate days; however, 1st and 2nd DXA and 1st and 2nd MRI should be performed on separate days.

14. DXA and MRI procedures for Visit 18 can be performed for up to 5 days before visit or for up to 5 days after Visit 18, but DXA and MRI should not be performed <24 hours after administration of the study drug dose. DXA and MRI procedures for Visit 24 can be performed 7 days before Visit 24.

15. For Part B, the actual sample collection schedule will depend on the interim review of the RK data from Part A. However, since this is a repeat dose, frequent samples will be collected after the first dose, and trough samples will be collected at other time points drug.

16. Appetite assessments in the clinic will be performed before and after a standardized dinner.

17. The Center will monitor the subject's compliance with completing the daily appetite questionnaire at each scheduled visit.

18. An ECG will be performed prior to dosing.

Safety

[0355] Vital signs, including temperature, blood pressure, pulse rate, and respiratory rate, will be collected after the subject has been at rest in a sitting or lying position for at least 10 minutes prior to dosing at the time points indicated in figures 25-27.

[0356] A thorough physical examination will be performed at the time points indicated in Figures 25-27. Any abnormalities that may be present should be carefully examined and assessed, as indicated by the subject's medical history.

[0357] An electrocardiogram should be performed prior to blood collection during visits requiring blood collection. A standard 12-lead ECG will be performed at the time points indicated in Figures 25-27. A 12-lead ECG should be performed in the supine position after the subject has been at rest for at least 10 minutes. The ECG will be interpreted by a researcher on site. The heart rate will be recorded according to the ventricular rhythm, and the PR, QRS, RR, QTcB and QTcF intervals will also be recorded. Any clinically significant abnormality should be documented as an AE/SAE, if applicable. Each ECG recording will be analyzed in comparison with the screening recording. ECG strips or reports will be stored with primary documents.

[0358] Samples will be analyzed for hematology, biochemistry, urinalysis, drug screening, and pregnancy tests (serum or urine). Detailed instructions for collecting blood samples can be found in the laboratory manual provided to the study sites. Samples for laboratory testing will be collected at the visits indicated in Figures 25-27. Tests include: Blood chemistry: sodium, potassium, chloride, bicarbonate, calcium, glucose, albumin, serum total protein, creatinine, blood urea nitrogen (BUN), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, lactate dehydrogenase (LDH), gamma-glutamyltransferase (GGT), total bilirubin, total cholesterol (low-density lipoprotein [LDL] and high-density lipoprotein [HDL]), triglycerides, uric acid, creatine phosphokinase (CPK), LDL-C, HDL-C; hematology: hemoglobin, hematocrit, red blood cell count (RBC), white blood cell count (WBC), red blood cell indices (mean corpuscular volume [MCV], mean corpuscular hemoglobin [MCH], mean corpuscular hemoglobin concentration [MCHC], red blood cell distribution by volume [RDW ]), platelet count, leukogram (*additional flow cytometry testing may be performed to assess cell subpopulations if clinically significant abnormalities are observed), including neutrophils, lymphocytes, monocytes, basophils, and eosinophils; urinalysis: color, glucose, red blood cells, clarity, blood, hyaline casts and other casts, pH, bilirubin, bacteria, specific gravity, leukocyte esterase, epithelial cells, ketone bodies, nitrites, crystals, protein, leukocytes, yeast; Other laboratory tests: leptin, insulin and endocrine hormones such as thyroid hormones (T3, T4, TSH), luteinizing hormone (LH), testosterone, estradiol, and HbAlc will be assessed in parts A and B. For part B, leptin will be measured in serum at the prescreening visit to determine subject's eligibility for inclusion in 1 of 4 cohorts. Leptin will also be measured throughout the study as indicated in Figures 25-27'. Other proteins that will be measured include adiponectin and cortisol.

Abnormal laboratory values and laboratory adverse events

[0359] All laboratory values must be verified by an investigator or authorized person. Tests with significant abnormal results that occur after initiation of treatment should be repeated to confirm the nature and extent of the abnormality. If necessary, appropriate additional studies should be initiated. If the abnormality persists or cannot be explained by events or conditions unrelated to the study drug or its administration, the medical director/study director should be consulted.

[0360] The clinical significance of an abnormal test value in the context of the disease being studied must be determined by the investigator.

Drug concentration measurements and samples

[0361] Samples to measure serum H4H17319P2 concentrations during Part A will be collected at the time points indicated in Figure 25. Formally, samples to measure serum H4H17319P2 concentrations during Part B will be collected at the time points indicated in Figure 27. However, once the Part B activity schedule is confirmed based on the interim analysis of data from Part A, samples for measurement of serum H4H17319P2 during Part B will be collected at a pool of (possibly) revised time points. Any unused samples may be used for research biomarker studies.

Anti-drug antibody levels measurements and samples

[0362] Samples to assess drug antibody levels for Parts A and B will be collected at the time points indicated in Figures 25 and 27. Any unused samples may be used for investigational biomarker studies.

Procedures using pharmacodynamic and investigative biomarkers

[0363] This study will conduct exploratory assessments to examine how H4H17319P2 may alter appetite, food intake, body weight, and circulating markers such as soluble LEPR and ANGPTL3, as well as exploratory biomarkers.

[0364] Soluble LEPR is present in the bloodstream of healthy individuals and subjects with diseases. It is a non-signaling form of the leptin receptor that is able to bind to leptin and can regulate its bioavailability. H4H17319P2 can bind to sLEPR in the circulation in a dose- and time-dependent manner. Soluble LEPR will be measured pre-dose at the Day 1 visit and at subsequent visits indicated in Figures 25 and 27. The change in sLEPR may reflect target interaction and saturation following H4H17319P2 administration.

[0365] ANGPTL3 is an endogenous lipoprotein lipase inhibitor that regulates circulating triglycerides. It is possible that H4H17319P2 regulates triglycerides through ANGPTL3; therefore, circulating concentrations of ANGPTL3 will be measured in serum predose and posttreatment at various time points after dosing to capture postprandial changes. The dose-dependent effect of H4H17319P2 on ANGPTL3 will be examined in both Part A and Part B, as indicated in Figures 25-27.

[0366] Other investigative biomarkers that can be measured in serum or plasma include leptin, adiponectin, and endocrine gland hormones, which are believed to be modulated during body weight loss. Markers will be measured according to the biomarker assessment sets shown in Figures 25-27.

Adverse reactions and adverse events

[0367] Emergency equipment and medications for the treatment of infusion reactions should be available for immediate use. All infusion reactions should be reported as AEs and assessed using appropriate grading scales. The infusion should be interrupted if any of the following AEs are observed: cough, chills/chillness, rash, scabies (itching), urticaria (rash, blisters, allergic papules), diaphoresis (sweating), hypotension, shortness of breath (lack of air), vomiting and hyperemia.

[0368] The reaction(s) should be treated symptomatically and the infusion should not be restarted. If investigators believe that there is a medical need for treatment or discontinuation of an infusion other than that described above, they must apply clinical judgment to ensure an appropriate response in accordance with typical clinical practice.

[0369] The infusion should be stopped and NOT restarted if any of the following AEs occur: anaphylaxis, laryngeal/pharyngeal edema, severe bronchospasm, chest pain, seizures, severe hypotension, other neurological symptoms (confusion, loss of consciousness, paresthesia, paralysis, etc.), any other symptoms or signs that, in the opinion of the investigator, require discontinuation of the IV infusion. The possibility of anaphylaxis should be considered if the following are observed (Sampson, 2006): acute onset of illness (minutes to hours) involving skin, mucous tissue, or both (eg, generalized urticaria, scabies or flushing, swollen lips, tongue and uvula) and at least one of the following: respiratory distress (eg, dyspnea, wheezing and bronchospasm, stridor, decreased maximum expiratory flow, hypoxemia) and decreased blood pressure or associated symptoms of end organ dysfunction (eg, hypotension [ collapse], fainting, urinary incontinence).

[0370] Emergency equipment and medications for the treatment of systemic reactions should be available for immediate use. All infusion reactions should be reported as AEs and assessed using rating scales as directed. Acute systemic reactions following study drug (SC) injection should be managed with clinical judgment to determine the appropriate response in accordance with typical clinical practice.

[0371] Local injection site reactions should be reported as AEs and assessed according to the Toxicity Rating Scale for Healthy Adult and Adolescent Volunteers Participating in Clinical Trials of Prophylactic Vaccines from the Food and Drug Administration's Industry Guidance Note ( FDA), September 2007 (cited in the Regulatory Mandatory Study Document)

[0372] An AE is any adverse medical event in a subject administered an investigational drug that may or may not have a causal relationship to the investigational drug. Therefore, an AE is any adverse and unexpected sign (including abnormal laboratory values), symptom or disease for which there is a transient association with the use of an investigational drug, regardless of whether it is considered to be related to the investigational drug or not. AE also includes any worsening (i.e., any clinically significant change in frequency and/or intensity) of a pre-existing condition for which there is a temporal association with the use of the study drug.

[0373] A serious adverse event (SAE) is any adverse medical event that, at any dose, is characterized by the following.

• Causes death - includes all deaths, even those that appear to be completely unrelated to the study drug (eg, a car accident in which the subject is a passenger).

• Life-threatening - in the opinion of the investigator, the subject is at immediate risk of death at the time of the event. This does not include AE, which, if it occurred in a more severe form, could cause death.

• Requires hospitalization or extension of current hospitalization. Inpatient admission is defined as admission to a hospital or emergency department for more than 24 hours. Extension of current hospitalization is defined as a hospital stay that is longer than initially expected for the event or prolonged due to the development of a new AE, as determined by the investigator or treating physician.

• Causes permanent or significant disability/incapacity (substantially impaired ability to perform normal life functions).

• It is a congenital anomaly/congenital defect.

• Is an important medical event - an important medical event may not be immediately life-threatening or result in death or hospitalization, but may endanger the subject or may require intervention to prevent one of the other serious outcomes listed above (eg, intensive care in a ward emergency room or at home for allergic bronchospasm; blood dyscrasias or seizures not resulting in hospitalization; or development of drug dependence or abuse).

[0374] SAE reporting criteria must be met for these events.

[0375] An adverse event of special interest (AESI; serious or non-serious) is one of the scientific and medical problems associated with a drug or a sponsor's program for which ongoing monitoring and prompt communication between the investigator and the sponsor may be appropriate. Such a phenomenon may require additional research to characterize and understand. Depending on the nature of the event, prompt communication between the trial sponsor and other parties (eg, regulatory authorities) may also be warranted.

[0376] Infusion reactions are defined as any relevant AE occurring during the infusion or within 2 hours after completion of the infusion. All infusion reactions should be reported as AEs and evaluated.

[0377] The investigator (or designee) will record all AEs that occur from the time the informed consent is signed until the end of the study. Abnormalities in laboratory values, vital signs, or ECG should be reported as AE. All SAEs, regardless of the assessment of causality with the investigational drug, must be reported to the sponsor (or designee) within 24 hours.

[0378] Information not available at the time of the initial report should be documented in a subsequent report. Supporting information such as relevant hospital or medical records and diagnostic test reports may also be requested. In the event that the investigator is informed of the SAE after the subject has completed the study, the following will apply.

[0379] Part A: SAE with onset within 30 days after the end of the study or within 112 days after the last administration of the investigational drug if the subject discontinued the study early - the SAE will be reported to the sponsor. The investigator should make every effort to obtain additional information about the outcome until the event is considered chronic and/or stable.

[0380] Part B: SAE with onset within 30 days after the end of the study or within 112 days after the last administration of the investigational drug if the subject discontinued the study early - the SAE will be reported to the sponsor. The investigator should make every effort to obtain additional information about the outcome until the event is considered chronic and/or stable.

[0381] Parts A and B: SAEs with a start date later than 30 days after the end of the study/early termination visit - only fatal SAEs and those SAEs deemed drug-related by the investigator will be reported to the sponsor. The investigator should make every effort to obtain additional information about the outcome of a drug-related SAE until the event is considered chronic and/or stable.

results

[0382] In the initial (Part A) single-ascending dose portion of the study, patients were randomized in a 3:1 ratio to receive H4H17319R2 or placebo in one of 7 cohorts to receive doses ranging from 0.3 mg/kg intravenous (IV) to 30 mg /kg IV and 300 mg subcutaneous (SC) to 600 mg SC. Fifty-six patients received a dose of H4H17319P2 or placebo, and pharmacokinetics (PK) and safety data were available for at least 85 days post-dose. A review of anonymized data did not reveal any serious or severe adverse events at any of the doses administered, and no deaths were reported. Treatment with H4H17319P2 or placebo was generally well tolerated when administered IV or SC. There were no interruptions or early cessation of treatment. Headache was the most common treatment-emergent adverse event (TEAE) reported. There were no clinically significant abnormalities or dose-related deviations from baseline in laboratory safety parameters, vital signs, or ECGs.

Example 21: Clinical Use of an Anti-LEPR Antibody for the Treatment of Congenital Leptin Deficiency in a Pediatric Patient

[0383] Briefly, at 6 months of age, the patient presented with extreme obesity, hyperphagia, hyperinsulinemia, dyslipidemia, grade 2 hepatosteatosis, and low leptin levels (0.55 ng/mL). The patient was diagnosed with leptin gene deletion (LEP ^-/- ) by polymerase chain reaction, and the diagnosis was confirmed by multiplex amplification of ligated probes according to the standards of the European Quality Assurance Network in Medical Genetics. The patient began metreleptin therapy at 24 months of age, and over the next 6 months his body weight decreased by 10 kg (from 37 kg to 27 kg). The patient then began to rapidly gain weight. The patient recently experienced respiratory failure requiring hospitalization, presumably as a result of extreme obesity and frequent infections. The physician performed a neutralizing antibody test by measuring leptin levels 1 hour after administration of metreleptin. Laboratory tests showed leptin levels <0.1 ng/mL, confirming that the patient had developed neutralizing antibodies to metreleptin. These results explain the patient's weight gain and demonstrate that metreleptin is no longer an effective therapy for treating the patient's condition.

[0384] Treatment with H4H17319P2 will be used to restore LEPR-mediated signaling in congenital leptin deficiency and to provide improvement in hyperphagia, weight gain and metabolic complications of this condition.

Inclusion/exclusion criteria for additional pediatric patients

[0385] A patient must meet the following criteria to be eligible for inclusion in this compassionate use program:

1. Congenital leptin deficiency with a confirmed genetic diagnosis of the presence of a loss-of-function LEP variant and/or gene deletion

2. Severe obesity, defined for adults as BMI≥40 kg/ ^m2 and for children as weight > ^97th percentile for the corresponding age and sex

3. Leptin levels < 1.0 ng/ml

4. Evidence of neutralizing antibodies to metreleptin, defined as:

• loss of effectiveness of metreleptin according to the treating physician with documented evidence of weight gain during leptin therapy AND

• leptin levels < 1.0 ng/mL 1 hour after leptin injection OR a positive neutralizing antibody assay by Aegerion, the manufacturer of metreleptin.

[0386] Patients eligible for an ongoing clinical trial for the treatment of congenital leptin deficiency will be excluded.

Dose selection

[0387] The dosing regimen described herein is expected to be generally well tolerated. H4H17319P2 was well tolerated in healthy adult volunteers when administered as single doses of 0.3 to 30 mg/kg IV and 300 and 600 mg SC. At an IV dose of 30 mg/kg, a maximum serum concentration of 1035 mg/L was observed, which is almost double the maximum value predicted to be observed in pediatric patients during treatment. In addition, the predicted area under the concentration-time curve (AUC) over a 1-week dosing interval at steady state is expected to be at least 8 times lower than the AUC at steady state over the same dosing interval. (no observed adverse event level [NOAEL]), calculated from a pharmacokinetic model derived from 3 preclinical toxicology studies in cynomolgus monkeys.

[0388] A loading dose of 5 mg/kg intravenous (IV) administration was selected to rapidly achieve serum H4H17319P2 concentrations of 100 mg/L or higher. The inclusion of this IV saturating dose will allow immediate assessment of the maximum concentration ( _Cmax ) of H4H17319P2 in the blood serum. A weekly maintenance dose of 250 mg subcutaneous (SC) H4H17319R2 will maintain serum trough concentrations of 100 mg/L or greater. This SC dosing schedule should begin 3 days after the loading IV dose to best maintain target serum trough concentrations.

Treatment and evaluation schedule

[0389] The initial treatment and evaluation plan for pediatric patients is presented in Table 20. Treatment and evaluation recommendations will be updated and communicated to the physician(s) as PK and PD data become available. The plan should be followed as closely as possible and any deviations should be noted. Written informed consent must be obtained before administering H4H17319R2 or performing any of the following assessments. After a single IV loading dose of 5 mg/kg on day 1, H4H17319P2 will be administered as a SC dose of 250 mg on days in the Q7 regimen (weekly). The first SC dose will be given on day 4.

[0390] H4H17319P2 will be administered as a single IV dose on Day 1 in a 1-hour infusion. Intravenous H4H17319P2 should be administered through the indicated types of IV infusion pumps (Alaris, Gemini, PC-1 or equivalent; Baxter, Flo-Gard 6201 or equivalent; Hospira, Lifecare 5000 or equivalent) and IV infusion sets (Baxter, product code 2C6571, or equivalent; Alaris, product number 2430-0500, or equivalent; Alaris, product number 11532269, or equivalent; Hospira, product number 14255-28, or equivalent; Baxter, product number 2H6480, or equivalent; Hospira, product number 12336 -05, or similar). The built-in filter and IV infusion pump should provide accurate delivery of only 1 ml/min.

[0391] The timing of serum sample collection for determination of H4H17319P2 drug concentrations and anti-H4H17319P2 antibodies, body weight estimates, metabolic parameter estimates, and sample submission to the trial sponsor are also presented in Table 20. The patient's H4H17319P2 levels and body weight measurements can be used to update recommendations for dose level and dosing frequency after day 25 and to determine whether H4H17319R2 administration shows evidence of benefit in this patient. Measuring antibody concentrations will allow us to assess whether the selected doses and dosing schedule were optimal or whether adjustments are required. Any unused serum samples collected for drug concentration and anti-drug antibody (ADA) assessments may be used for investigation of unexpected adverse events and for research purposes and may be stored for up to 15 years.

[0392] Any other disease-related clinical parameters obtained by the physician as part of the patient's routine and standard care should be reported to the trial sponsor for safety monitoring purposes. These include, but are not limited to, vital signs and laboratory values (ie, blood chemistry with liver enzymes, hematology, metabolic parameters). Such information may influence future dosing of the patient and may also help determine whether administration of H4H17319R2 shows signs of benefit or harm.

[0393] In the event of anaphylaxis or hypersensitivity, additional serum samples should be collected as soon as possible after the event. Additional labels will be provided in the event of unscheduled sample collection.

[0394] Equipment and medications should be available at the study site to provide emergency treatment for the treatment of infusion reactions following an IV infusion of H4H17319R2 or systemic reactions following an injection of H4H17319R2. Acute reactions to IV infusion or systemic reactions to injection after administration of H4H17319R2 should be managed with clinical judgment to determine the appropriate response in accordance with typical clinical practice. All safety-related findings and adverse events that become known to the treating physician should be reported to the trial sponsor as soon as possible and strictly in accordance with any local requirements regarding the use of investigational drugs from a compassionate use perspective. Such adverse events include any adverse medical event that results in death, is life-threatening, requires hospitalization or prolongation of an ongoing hospitalization, results in permanent or significant disability/incapacity, is a congenital anomaly/birth defect, or is a significant medical event (such such as intensive treatment in the emergency department or at home for allergic bronchospasm; blood dyscrasias or seizures not resulting in hospitalization; or development of drug dependence or abuse).

Preparation of H4H17319P2 for a 5 mg/kg dose administered via IV infusion bag

[0395] The volume of reduced H4H17319P2 added to the 5 mg/kg IV infusion bag is listed in Table 21 below.

[0396] The contents of each vial of H4H17319P2 will be reconstituted using 4.9 ml of sterile water for injection.

[0397] After reconstitution, each vial of H4H17319P2 will contain 4.8 ml of removal volume. When reconstituted for IV administration, the concentration of H4H17319P2 in the vial will be 50 mg/ml. The stages of recovery are as follows.

1. Obtain the required number of vials of lyophilized H4H17319P2 along with a 100 ml infusion bag with 0.9% sodium chloride.

2. Prepare H4H17319R2 by working on a hard, clean surface.

3. Remove the cap(s) from the vial(s) and wipe the top surface of the vial with an alcohol swab.

4. For each vial that will be used to administer a dose, obtain one 21-gauge needle and one 10.0 mL polypropylene syringe. With the cap on the needles, attach the 21-gauge needles to a 10.0 mL polypropylene syringe. With the cap in place, pull the plunger of the 10.0 mL syringe to the 5.5 mL mark. This is necessary to draw air into the syringes.

5. Remove the caps from the 21-gauge needles. Insert the needles into the rubber cap of the vial containing sterile water for injection. Press down on the pistons so that all the air enters the bottle. Turn the bottle upside down in one hand and make sure the tip of the needle is in the water. Fill the syringe with at least 5.5 ml of sterile water for injection. Do not place the needles on a dirty surface, touch the needles with your fingers, or breathe directly onto the needles.

6. Prime the syringes by inverting the syringes (needle side up) and pressing the plunger until air is removed from the syringes. Continue pressing the pistons until a small amount of liquid comes out and the pistons reach 4.9 ml.

7. Place the bottle with H4H17319P2 on a hard surface and insert the needle into the upper part. Add 4.9 mL of sterile water for injection to the medication vial, directing the stream of water toward the side of the vial and into the powdered medication.

8. Remove the needles from the vial by pushing all the water from the syringes into the vial. Dispose of needles and syringes in a sharps container.

9. Gently swirl the bottle in a vertical position until all the powder has dissolved.

10. Do not shake the bottle. Shaking may cause foaming.

IV-introduction N4N17319R2

[0398] The steps for administering the antibody are as follows.

1. Use standard aseptic technique to extract the required amount of H4H17319P2 according to Table 21 using an appropriately sized polypropylene syringe and a 21-gauge needle. Do not place the needle on a dirty surface, touch the needle with your fingers, or breathe directly onto the needle.

2. Before adding H4H17319R2 solutions to the 100 mL IV infusion bag, remove from the IV infusion bag a volume of 0.9% sodium chloride equal to the volume of H4H17319R2 to be added to the IV infusion bag.

3. Add the appropriate volume of H4H17319P2 to the IV infusion bag, then invert the IV infusion bag 10 times to ensure that the drug and 0.9% sodium chloride are well mixed.

4. The prepared IV infusion bag is labeled in accordance with study site-approved labeling requirements. Label should include: patient's initials, date/time of preparation, mg of H4H17319R2 per 0.9% sodium chloride 1x1 bag, instructions for intravenous infusion of the entire contents of the infusion bag and rinse for 1 hour as per protocol, administration according to date/time, and name of prescriber doctor

5. H4H17319R2 should be administered by infusion within 4 hours after recovery.

Preparation for subcutaneous administration and subcutaneous administration of N4N17319R2

[0399] The contents of each vial of H4H17319P2 will be reconstituted using 1.4 ml of sterile water for injection. Once reconstituted, each vial will contain 1.2 ml of removable volume. When reconstituted for SC administration, the concentration of H4H17319P2 is 150 mg/ml. Two vials of H4H17319P2 are required to administer the dose.

1. Prepare H4H17319R2 by working on a hard, clean surface.

2. Remove the cap(s) from the vial(s) and wipe the top surface of the vial(s) with an alcohol swab.

3. Without removing the needle cap, attach a 21-gauge needle to a 3.0 mL polypropylene syringe. With the cap in place, pull the syringe plunger back to the 2.0 mL mark to draw air into the syringe. Remove the cap from the 21-gauge needle. Insert the needle into the rubber cap of the vial containing sterile water for injection. Press down on the plunger to force all the air into the bottle. Turn the bottle upside down in one hand and make sure the tip of the needle is in the water. Fill the syringe with at least 2.0 ml of sterile water for injection. Do not place the needle on a dirty surface, touch the needle with your fingers, or breathe directly onto the needle.

4. Prime the syringe by inverting the syringe (needle up) and pressing the plunger until air is removed from the syringe. Continue pressing the plunger until a small amount of liquid comes out and the plunger reaches the 1.4 ml mark.

5. Place the bottle with H4H17319P2 on a hard surface and insert the needle into the upper part. Add 1.4 ml of sterile water for injection to the vial of H4H17319P2, directing the stream of water towards the side of the vial and into the powdered drug.

6. Remove the needle from the vial by pushing all the water from the syringe into the vial. Dispose of the needle and syringe in a sharps container.

7. Gently swirl the bottle in a vertical position until all the powder has dissolved.

8. Do not shake the bottle. Shaking may cause foaming.

SC administration of a dose of 250 mg H4H17319R2

[0400] This dose requires 2 injections of H4H17319P2. One injection is a 0.67 ml SC injection and the other is a 1.0 ml SC injection.

1. Take a 1.0ml polypropylene plastic syringe and a 3.0ml polypropylene syringe and attach a 21-gauge needle to each syringe.

2. Obtain 2 bottles of lyophilized H4H17319P2.

3. Reconstitute lyophilized H4H17319P2 in each vial with 1.4 ml of sterile water for injection as above.

4. With the needle cap on, pull back the plunger of the 1.0 ml polypropylene syringe to the 1.0 ml mark to draw air into the syringe.

5. Remove the cap from the needle and insert the needle into the rubber cap of the bottle.

6. Press the plunger and introduce all the air into the bottle.

7. While holding the needle in the vial, turn the vial upside down in one hand and ensure that the tip of the needle is in the liquid. With your other hand, pull back the plunger to draw at least 1.0 mL of medication into the syringe. Place the cap on the needle. Remove the needle and cap and place it in a sharps container.

8. Without removing the needle cap, attach a 0.5-inch 27-gauge needle to a 1.0 mL polypropylene syringe containing a minimum of 1.0 mL of drug.

9. Remove the needle cap and prime a 0.5-inch 27-gauge needle with medication. Place the cap on the needle.

10. The prepared SC syringe contains 0.67 ml = 100 mg/0.67 ml.

11. The prepared SC syringe is labeled as required by the study site's standard operating procedure.

12. With the needle cap on, pull back the plunger of the 3.0 mL polypropylene syringe to the 1.5 mL mark to draw air into the syringe.

13. Remove the cap from the needle and insert the needle into the rubber cap of the bottle.

14. Press the plunger and introduce all the air into the bottle.

15. While holding the needle in the vial, turn the vial upside down in one hand and ensure that the tip of the needle is in the liquid. With your other hand, pull back the plunger to draw at least 1.2 ml of medication into the syringe. Place the cap on the needle. Remove the needle and cap and place it in a sharps container.

16. Without removing the needle cap, attach a 0.5-inch, 27-gauge needle to a 1.0-mL polypropylene syringe containing a minimum of 1.2 mL of drug.

17. Remove the needle cap and prime a 0.5-inch 27-gauge needle with medication. Place the cap on the needle.

18. The prepared SC syringe contains 1.0 ml = 150 mg/1.0 ml.

19. The prepared SC syringe is labeled as required by the study site's standard operating procedure.

20. H4H17319P2 should be delivered within 4 hours after reconstitution.

Permanent discontinuation of H4N17319R2

It is recommended to permanently stop the administration of H4H17319R2 if the following occurs.

• Serious or severe allergic reactions thought to be related to H4H17319P2

• Specific types of liver dysfunction (eg, Hay's Law is followed ([Guidance for Industry Drug Induced Liver Injury: Premarketing Clinical Evaluation FDA 2009])

• Evidence of pregnancy

• Patient/legal representative withdraws consent

• There is no clinical benefit (ie, loss of body weight) shown to the patient after a period of time (to be discussed between the treating physician and the sponsor) of what the sponsor considers to be the optimal dose and dosing schedule of H4H17319R2.

Temporary discontinuation of N4N17319R2

It is recommended to temporarily stop administering H4H17319R2 if the following occurs.

• Neutrophil count ≤ 1.0×10 ³ /µl

• Sustained ALT/AST levels greater than 3 times the upper limit of normal (ULN), and total bilirubin > 2x ULN or AST/ALT alone > 5x ULN

• Surgical procedure

• Hospitalization

Once the condition leading to temporary discontinuation of H4H17319R2 has resolved, dosing of H4H17319R2 can be resumed. The decision to temporarily discontinue H4H17319R2 dosing and/or restart dosing of H4H17319R2 should be discussed with the sponsor's representative.

The treating physician may temporarily discontinue dosing of H4H17319R2 at any time, even without consultation with the sponsor, if the urgency of the situation requires immediate action and if it is determined to be in the best interests of the patient. However, you should contact the sponsor's representative as soon as possible. Resumption of dosing of H4H17319R2 should be discussed and agreed between the treating physician and the sponsor's representative.

[0401] Thus, H4H17319P2 will provide restoration of LEPR-mediated signaling in a patient who has congenital leptin deficiency and will provide the patient with improvement in or reversal of hyperphagia, weight gain and/or metabolic complications of this condition.

Example 22: Clinical Use of an Anti-LEPR Antibody for the Treatment of Focal Lipodystrophy in Pediatric Patients

[0402] At the age of 11 years, the patient presented with hepatosplenomegaly and high triglyceride levels (>500 mg/dL), as well as absent limb fat. She was found to have GAD (glutamic acid decarboxylase) antibodies and a commercial LMNA gene test was negative. Other features identified during evaluation for a study examining the effectiveness of metreleptin in the treatment of liver disease associated with lipodystrophy included atypical signs of arm contractures, scoliosis, and absence of adrenarche. Her baseline leptin level was 3.2 ng/dL.

[0403] Metreleptin therapy was initiated at age 13 years as part of a clinical trial protocol (ClinicalTrials.gov identifier: NCT01679197) for the treatment of high triglycerides and severe hepatic steatosis, and she continued on metreleptin for 12 months without any serious adverse events . Her metabolic parameters improved slightly after metreleptin, but her liver biopsy showed improvement. She continued metreleptin as part of a different study protocol (ClinicalTrials.gov identifier: NCT02654977). However, during the ^17th month of treatment, the patient reported fatigue, thirst, blurred vision, and polydipsia and lost 5 pounds in one month. Laboratory results showed significant hyperglycemia and hyperlipidemia with a positive anion gap and a positive ketone body test. The level of antibodies to GAD65 (the 65 kDa isoform of glutamic acid decarboxylase) was 5.39 nmol/L (retest for antibodies to GAD65: 8.38 nmol/L, two months later), and the level of leptin measured one hour after injection was could not be defined. Ultimately, the presence of antibodies to metreleptin with neutralizing activity was confirmed. This low leptin level was likely a consequence of immunogenic cross-reactivity with ongoing metreleptin therapy with the formation of metreleptin antibodies, possible ongoing loss of adipose tissue, or a mechanism that suppresses leptin production.

[0404] Positive neutralizing activity against metreleptin as well as endogenous leptin was confirmed after approximately 6 months. During this time, glucose and lipid control was significantly worse, with triglyceride levels consistently above 2000 mg/dL. The patient continued to receive high-dose insulin therapy using a basal-bolus regimen and switched to an insulin pump. She also started taking metformin. Several hospitalizations were required for diabetic ketoacidosis, pancreatitis, or prevention of pancreatitis.

[0405] The patient was then started on fenofibrate and subsequently pioglitazone due to worsening lipid control (triglycerides > 2000 mg/dL). One month after discontinuation of metreleptin, LFT values increased significantly to 10 times higher than normal. Liver biopsy showed focal portal and periportal inflammation that included plasma cell and interface damage consistent with autoimmune hepatitis. To control autoimmune hepatitis, the patient was treated with prednisone for a period of three months, which helped resolve liver function abnormalities. Both while on and after stopping prednisone, the patient suffered from multiple episodes of acute pancreatitis due to hypertriglyceridemia (up to 8000 mg/dL) combined with diabetic ketoacidosis. Due to the above metabolic complications or pancreatitis, the patient was regularly admitted to the hospital.

[0406] The patient was started on setmelanotide (ClinicalTrials.gov Identifier: NCT03262610) at a dose of 1 mg/day, and the dose was titrated upward until triglyceride levels fell below 500 mg/dL. Setmelanotide did not cause weight loss in this patient. Although there was a slight decrease in the hunger scale, the effect was not permanent. Treatment did not provide improvement in glycemic control and hypertriglyceridemia. The small reduction in total daily insulin dose was not considered clinically significant because HbAlc levels remained high. Setmelanotide had no effect on liver fat content, although visceral fat decreased slightly.

[0407] The patient's elevated triglyceride levels (>500 mg/dL and up to 2000 mg/dL) require plasma exchange therapy to prevent recurrence of pancreatitis. She is also treated with empagliflozin 10 mg daily in addition to insulin aspart, insulin glargine and metformin. Rationale

[0408] The purpose of this clinical trial protocol is to provide investigational drug (IP) H4H17319P2 to a patient who meets the required eligibility criteria and meets the following criteria set forth below.

• The patient has a serious or life-threatening disease for which the expanded access protocol is approved.

• Sufficient evidence of efficacy to expect clinically significant benefit based on the drug's proposed mechanism of action.

• There are no comparable or satisfactory alternative therapies for the treatment of this disease or condition.

• There is nothing unique about this particular patient that would suggest that there would be an unreasonable risk associated with administering H4H17319P2 to this patient.

[0409] Metreleptin antibodies with neutralizing activity have been identified in patients with generalized lipodystrophy treated with metreleptin. The effects of these neutralizing antibodies are not well understood but may include inhibition of endogenous leptin action and/or loss of metreleptin efficacy (Chan, et al. Immunogenicity associated with Metreleptin treatment in patients with obesity or lipodystrophy. Clin Endocrinol. 2016; 85(1): 137-149). Serious infections and/or deterioration of metabolic control have been reported. This patient experienced progressive metabolic deterioration and the onset of type 1 diabetes mellitus concomitant with the development of persistent neutralizing antibodies to metreleptin. It is unclear whether the disease progressed naturally or whether the development of antibodies played a role. However, the patient's lipodystrophy does not respond to proper treatment.

[0410] The purpose of this patient-specific expanded access protocol is to provide H4H17319P2 as a potential treatment for severe metabolic complications resulting from lipodystrophy in this patient with refractory hypertriglyceridemia leading to recurrent attacks of pancreatitis. Additionally, the safety and effectiveness of H4H17319R2 in this patient will be assessed. The period of treatment will depend on the degree of response in this individual patient, who is a critically ill patient with lipodystrophy, hypertriglyceridemia and recurrent pancreatitis, with the aim of assessing whether H4H17319P2 may have a therapeutic benefit in providing improvement against severe metabolic abnormalities.

[0411] Primary endpoints

• Percentage change in fasting triglyceride levels from baseline to week 24

• Achieve fasting triglyceride levels <500 mg/dL without the need for continuous plasmapheresis at week 24

• Adverse events that occurred during treatment

[0412] Secondary endpoints:

• Percentage change in fasting triglyceride levels from baseline to week 12

• Achieve fasting triglyceride levels <500 mg/dL without the need for continuous plasmapheresis at week 12

• Change in hunger score from baseline over time

• Change in fasting glucose and glycosylated hemoglobin (HbAlc) levels from baseline over time

• Incidence of hospitalizations for pancreatitis over time

• Change in average insulin dose requirement from baseline over time

• Change from baseline in other fasting lipid parameters, including fasting total cholesterol, HDL-C, LDL-C, and VLDL-C, over time.

• Percentage change from baseline in this patient's DEXA parameters and liver fat, if detectable, over time.

• Concentration of total H4H17319P2 in serum over time

• Presence of anti-drug antibodies (ADA) H4H17319P2 over time

Protocol plan

[0413] The protocol includes a 2-week screening period and 3 open-label treatment periods: 1st treatment period (weeks 1-12), 2nd treatment period (weeks 13-24) and extension treatment period (weeks 25-52 ). During the screening period, the patient will be assessed for eligibility and instructed to complete a hunger questionnaire on at least 3 separate days to better understand hunger symptoms associated with lipodystrophy and the absence or deficiency of endogenous leptin as a result of absence or deficiency of adipose tissue before treatment with H4H17319R2. Because this condition is rare, obtaining hunger scores at the time of screening will collect important details specific to the patient that will allow for a better understanding of hunger symptoms in this disorder.

[0414] During the 1st treatment period, the patient will receive H4H17319P2 according to the initial dosing schedule. At the end of treatment period 1 (week 12), efficacy (TG reduction) and drug levels (PK) of H4H17319R2 will be assessed to determine the H4H17319R2 dosing schedule for treatment period 2. The dose level and frequency of dosing in the 2nd treatment period will not exceed those in the 1st treatment period. At week 24, efficacy (TG reduction) and drug levels (PK) of H4H17319P2 will be assessed to determine the patient's eligibility to continue participation in the treatment extension period.

[0415] Thus, this patient will sequentially follow the protocol as shown in Figure 19.

[0416] Fasting triglyceride (TG) levels and the need for plasmapheresis will be the primary criteria used to assess whether a therapeutic effect has been achieved. If this patient does not demonstrate the required target therapeutic response of achieving a fasting TG <500 mg/dL by week 24, she will be removed from the protocol.

[0417] To simultaneously control hypertriglyceridemia, this patient will have the right to continue treatment with plasmapheresis if necessary at the discretion of the treating physician. Fasting TG levels to determine whether a therapeutic response to treatment with H4H17319P2 has been achieved should be measured at a time point at least 1 week after the last course of plasmapheresis treatment.

Dose selection

[0418] In the preclinical studies described in Examples 15-19 above, PD effects were associated with a wide range of H4H17319R2 exposures, while persistence of effects was observed under dosage conditions in which target mediated clearance (TMC) of H4H17319R2 was saturated. In a mouse model of lipodystrophy (described above), single doses of H4H17319R2 resulted in normalization of glucose concentrations and loss of body weight. These effects were evident at serum H4H17319P2 concentrations above 4 mg/L but were markedly more robust at concentrations saturating the TMS pathway. In the monkey studies described above, sustained effects on body weight (body weight loss or decreased body weight gain compared with control animals) were observed at steady-state concentrations above 30 mg/L. However, greater overall effects were observed when H4H17319P2 concentrations exceeded 100 mg/L, the concentration required to saturate TMS in monkeys. Given that leptin-binding receptors are widely expressed but target (signaling) receptors are limited to the central nervous system, it is suggested that preclinical PK/PD data reflect the need for saturation of TMS associated with peripheral LEPR binding to achieve adequate effects on the brain. brain. In addition, interspecies differences in the concentration required to saturate TMS and produce sustained PD effects may be a consequence of differences in loading on peripheral targets.

[0419] In the human clinical study described in Example 20, saturation of the TMS pathway was evident at serum concentrations greater than 100 mg/L based on concentration-time profiles. A population model of PK was constructed using data from this FIH study, which demonstrated the relevant pharmacokinetic characteristics of H4H17319P2 when administered as a single dose to healthy volunteers. This model was used to simulate the expected concentration profiles for different dosing schedules, assuming that the PK characteristics of H4H17319P2 observed in healthy volunteers were relevant to this patient.

[0420] H4H17319P2 will be supplied as a lyophilized powder in a sterile 20 mL single-use glass vial for IV or SC administration. Each vial contains 265 mg of H4H17319P2, which will be reconstituted with sterile water for injection for IV and SC administration.

[0421] A loading dose of 5 mg/kg intravenous (IV) administration was selected to rapidly achieve serum H4H17319P2 concentrations of 100 mg/L or higher. Inclusion of this IV loading dose will also allow immediate assessment of the maximum concentration ( _Cmax ) of H4H17319P2 in the blood serum. A weekly maintenance dose of 300 mg subcutaneous (SC) H4H17319P2 is predicted to maintain serum trough concentrations of 100 mg/L or greater. This SC dosing schedule should begin 4 days after the loading IV dose and is predicted to best maintain target serum trough concentrations. On days when the patient is undergoing plasmapheresis for clinical indications, administration of H4H17319R2 should occur after completion of plasmapheresis.

[0422] Based on data from the first part of an ongoing 2-part study involving 56 healthy subjects (Example 20), the dosing regimen described is expected to be generally well tolerated. H4H17319P2 was well tolerated by healthy adult volunteers in Example 20 when administered as single doses of 0.3 to 30 mg/kg IV and 300 and 600 mg SC. At the IV dose of 30 mg/kg, a maximum serum concentration of 1035 mg/L was observed, which is more than six times the maximum exposure predicted to be achieved in this patient over the course of the study. In addition, the predicted area under the concentration-time curve (AUC) over a 1-week dosing interval at steady state is expected to be nearly twenty-seven times lower than the AUC at steady state over the same dosing interval ( no observed adverse event level [NOAEL]), calculated from a pharmacokinetic model derived from 3 preclinical toxicology studies in cynomolgus monkeys.

[0423] Therapy of an initial loading dose of 5 mg/kg IV combined with a 300 mg weekly SC maintenance dose is expected to be tolerable and was chosen to rapidly achieve serum H4H17319P2 concentrations that saturate peripheral receptors and thereby provide stable pharmacological effects. It is possible that lower dose levels or less frequent dosing may be equally effective. Therefore, the dose level and dosing frequency may be adjusted downward after planned interim evaluations of efficacy and drug levels. In addition, because plasmapheresis may remove antibodies from the circulation, including H4H17319R2, dose adjustments following plasmapheresis may be necessary to ensure effective levels of H4H17319R2. On days when the patient is undergoing plasmapheresis, an additional dose of 300 mg of H4H17319R2 may be required to achieve target drug concentrations. Usual maximum doses (not including possible changes due to plasmapheresis) should not exceed 300 mg SC weekly.

Efficacy and drug level assessments

[0424] Interim efficacy and drug level assessments will be performed at 12 weeks and 24 weeks per protocol to evaluate whether the selected dosing regimen achieved expected drug levels and DO profiles and effects on lipid parameters. The dose level and dosing frequency may be adjusted as necessary following evaluations of efficacy and drug levels.

[0425] The data review committee will include (1) an experienced lipodystrophy specialist, (2) the patient's physician from the pediatric intensive care unit, (3) one of the subspecialists involved in her care (either a pediatric GI or hepatologist), and (4) the sponsor's representative(s) familiar with the exposure-response relationship for H4H17319P2 from preclinical and clinical studies.

Duration of patient participation

[0426] The total duration of the protocol is expected to be approximately 54 weeks, including the screening period. The patient's participation in this protocol will consist of the following periods: screening period, 1st treatment period [expected to last 12 weeks], 2nd treatment period [expected to last 12 weeks], and then if The patient meets the criteria for a long treatment extension period [28 weeks]. It is expected that if significant metabolic improvement with good safety and tolerability is demonstrated during the treatment extension period, the patient may be offered the opportunity to enroll in a future separate extension protocol to allow continued treatment. This future extended protocol will be submitted to and approved by the appropriate regulatory authorities prior to dosing a patient beyond the duration of this protocol.

[0427] The end of the trial will be considered the last visit of the last patient. The patient has the right to refuse the protocol at any time, for any reason and without any consequences. The investigator may also exclude a patient at any time due to clinical safety concerns or lack of effectiveness.

Terminating protocol execution

[0428] This protocol may be terminated early if there is a sufficiently compelling reason to do so in the opinion of the principal investigator or sponsor. The terminating party will provide written notice documenting the reason for termination of the protocol to either the investigator or the sponsor.

[0429] Circumstances that require termination of execution include, without limitation, the following.

• Determination of unexpected, significant or unacceptable risk to the patient

• Insufficient compliance with protocol requirements

• Insufficient complete and/or measurable data

• Evidence of pregnancy

• Serious or severe allergic reactions thought to be related to the drug

• Severe liver damage or dysfunction for which no other cause can be found, such as viral hepatitis A, B or C; pre-existing or acute liver disease; or other drug that may cause observable damage. Liver injury in this patient is defined as an ALT or AST level >3 times the mean baseline level AND a total bilirubin level >2 times the baseline level (or International Normalized Ratio (INR) >1.5)

• The patient withdraws consent

• Plans to change, suspend or discontinue drug development

• When reviewing the data at week 12, the data review committee concluded that there was not sufficient benefit.

• Upon review of the data at week 24, the data review committee concluded that the patient could not continuously maintain a fasting TG <500 mg/dL without monitoring with concomitant plasmapheresis.

[0430] If the data review committee unanimously believes that the patient is experiencing significant clinical benefit despite being unable to consistently maintain a TG <500 mg/dL without monitoring with concomitant plasmapheresis, dosing may be continued until the end-of-treatment visit per week 52.

Assessing adherence to treatment

[0431] To evaluate the safety, tolerability, and pharmacokinetics of a drug, it is critical that the patient receive H4H17319R2 as directed. All used H4H17319P2 will be collected to assess compliance with the protocol.

[0432] During the first 2 periods of the protocol (the initial dose period and the open-label active drug treatment period), H4H17319P2 will be administered to the patient by an experienced healthcare professional. During the open-label extension period, H4H17319R2 may be administered at the clinical site or self-administered/administered by the patient or authorized person, respectively. If the patient chooses to self-administer H4H17319R2 or has an authorized person administer H4H17319R2, training in the administration of H4H17319R2 should be provided by qualified clinical site personnel and the first instance of self-administration should be supervised by them. In addition, the patient/authorized person will be provided with a medication administration diary prior to self-administration or administration by an authorized person such as a parent or guardian. The diary must be completed each time the drug is administered. The patient and/or her caregivers will be required to keep a diary to monitor compliance with the directions for use. In addition, the timing of the dose will be recorded in the patient's diary. If the patient does not receive the full dose of medication, the amount administered will be recorded. The reason(s) for dose adjustments should be recorded in the source documents and CRF.

[0433] In addition, blood samples will be collected in accordance with the SOA to measure trough concentrations of H4H17319P2 and anti-H4H17319P2 antibodies in serum.

Previous and concomitant treatments and procedures/approved medications

[0434] If concomitant medications are not likely to pose a serious potential safety concern, then the overall goal of this protocol is to allow a patient with this extremely rare condition to participate in the protocol. Therefore, the patient will be allowed to take concomitant medications (eg, as described below) on an ongoing basis while participating in the protocol. These may include the following.

• Insulin preparations for the treatment of type 1 diabetes mellitus

• Metformin for the treatment of insulin resistance

• Vitamin D for treating deficiency

• Thyroxine or other thyroid supplements

• Other medications commonly used in patients with LD, including: endocrine therapies (eg, vitamin and calcium supplements) and other medications (eg, carnitine, coenzyme Q10, vitamins, constipation medications, allergy medications drugs)

• Neurontin along with any necessary narcotic or ketamine/lidocaine infusions, patches or tablets for pain relief.

• Effexor for mood improvement

• With the exception of low-threshold drugs (ie, anticonvulsants, digoxin, Coumadin, etc.), other drugs may be permitted if they are taken at a stable dose and deemed necessary by the treating physician.

• Plasmapheresis may be continued at the discretion of the patient’s treating physicians

[0435] There is currently no data on the interaction of H4H17319P2 with the above treatments. The patient and her caregivers should be warned about the possible side effects of drug interactions that may occur with any medications the patient will receive.

[0436] This patient will be reminded at each visit that if she needs to take any other medications during the protocol period, from screening to the final protocol visit, she must immediately inform the protocol staff and the specific(s) The drug(s) and indication(s) should be discussed with the investigator. All concomitant medications taken during the protocol must be recorded in the source documents.

Prohibited drugs and substances and related procedures

[0437] Drugs that may interfere with the evaluation of efficacy during the protocol are prohibited, such as the addition of new lipid-lowering therapies or any addition of new diabetes medications unless deemed necessary by her treating physician.

[0438] Drugs that cause anorexia, or drugs that cause anorexia as a common side effect, are prohibited during the protocol.

[0439] Concomitant procedures performed during the protocol period, including plasmapheresis and others used to treat adverse events, must be recorded in the CRF.

Grades and schedule

[0440] The schedule of assessments (SOAs) that will be conducted during execution of the protocol is shown in Figure 20 (A-B).

[0441] Although the procedures and assessments required in this protocol for a single patient are classified as "No or Minimal Risk" (except for DEXA, which may be classified as "Slight increase relative to minimal risk") according to the 2008 Guidance Document "Ethical Considerations for Clinical Trials on Medicinal Products Conducted with the Paediatric Population", includes considerations for reducing pain and distress in participants under 18 years of age.

[0442] After providing informed consent, the patient will enter the screening period. During the screening period, the patient will be assessed for eligibility and instructed to complete a hunger questionnaire on 3 separate days to better understand hunger associated with lipodystrophy prior to treatment with H4H17319P2. During screening, it is also very important to obtain any additional medical history, including a recent medical record review if necessary.

[0443] Additional medical history along with demographic data for this patient will be obtained during the screening period. Data that should be recorded in the primary document and CRF include the patient's sex, race, date of birth, and use of concomitant medications.

[0444] A recent medical history will be obtained on Day 1 before the first dose of the drug to assess the feasibility of continued participation in the protocol and compliance with the final inclusion/exclusion criteria. This recent medical history includes a review of changes from the screening results, as well as a review of the patient's recent medications and an assessment of whether any changes have occurred since the previous visit.

[0445] A complete physical examination will be performed including consideration of the peripheral lymph nodes, head, eyes (including conjunctiva), ears, nose, mouth and oropharynx, neck, heart, lungs, abdomen, musculoskeletal system including back, limbs and neurological status. This patient will also be assessed according to the Tanner scale; because she has not reached Tanner stage V. This will be done in accordance with the cSOA.

[0446] Changes from baseline in any physical examination findings determined by the investigator to be clinically significant should be recorded as AE in the appropriate CRF.

[0447] Height (cm) will be measured without shoes according to SOA using a wall-mounted stadiometer.

[0448] Review of concomitant medications will be conducted during the screening period and at each visit as per protocol. Any medications taken by the patient will be recorded in the primary documents.

[0449] The investigator is aware that dosages of concomitant antidiabetic and lipid-lowering medications may require adjustment at the start of treatment as insulin resistance and hypertriglyceridemia improve. The patient will be closely monitored while her concomitant drug(s) are adjusted. A patient receiving insulin therapy should be closely monitored during the first few weeks of treatment, as insulin doses may need to be reduced at a frequency of once a week (especially for patients receiving high doses of insulin) to avoid hypoglycemia. Likewise, the investigator should evaluate the need for lipid-lowering drug dosage adjustments at each follow-up visit during treatment of patients taking lipid-lowering drugs for hypertriglyceridemia and will consider the need for plasmapheresis. If this drug treatment is discontinued for any reason, additional adjustments to concomitant medications, such as lipid-lowering medications, may be necessary to mitigate the potential risk of pancreatitis.

[0450] Vital signs will be obtained in a sitting position after at least 5 minutes of rest each time they are measured according to the SOA. Blood pressure (BP; mmHg) and heart rate (HR; bpm) will be measured using the same technique throughout the protocol (manually or automatically). All BP and HR measurements should be taken in a sitting position after at least 5 minutes of rest. All measurements will be performed in triplicate at approximately 2-minute intervals. If possible, BP should be measured in the non-dominant arm throughout the protocol using the same technique (automatic or manual). If BP or HR increases significantly, repeated measurements and more frequent monitoring can be performed.

[0451] To ensure a minimum blood pressure reading, the patient should be instructed not to take the protocol drug on protocol days when vital signs are to be taken in the clinic, but rather to take the drug in the clinic. This is especially important during treatment period 1 visits, when blood pressure monitoring is performed more intensively during the post-dose clinic SOA period.

[0452] Body temperature (°C) and respiratory rate (breaths per minute) will be measured in a sitting position after at least 5 minutes of rest.

[0453] Single-shot 12-lead electrocardiograms will be obtained after a period of at least 10 minutes of rest in the supine position.

[0454] Laboratory tests for clinical safety must be performed by a local laboratory, and patients must fast for 8 hours. Laboratory safety tests must be performed prior to dosing.

[0455] All clinically significant laboratory abnormalities will be monitored through repeat testing and further investigated as determined by the investigator.

[0456] Liver function test abnormalities will be evaluated according to FDA guidance (2009). Samples for hematological and biochemical analysis will be collected in the fasting state. A complete blood count with platelet count and standard indices will be obtained.

• Biochemical analysis: sodium, potassium, chloride, CO ₂ , albumin, total protein, glucose, blood urea nitrogen (BUN), creatinine, uric acid, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (GGT), creatine phosphokinase (CPK), alkaline phosphatase, total bilirubin, direct bilirubin, lactate dehydrogenase (LDH), calcium and phosphorus.

• Urinalysis: pH, glucose, protein, ketone bodies, bilirubin, blood, urobilinogen, specific gravity, nitrites and leukocytes using dipstick or automated urinalysis. A microscopic examination of the urine will be performed if positive results on the test strips require further investigation.

• Coagulation profile: prothrombin time (PT) or international normalized ratio (INR) and partial thromboplastin time (PTT), also called activated partial thromboplastin time (aPTT).

[0457] Injection sites will be carefully monitored, studied and evaluated during the protocol period. Evaluation of the injection site will include identification and measurement of areas of erythema, swelling, and induration, as well as the presence of local pain, tenderness, and itching.

[0458] Unscheduled assessments may also be recorded if clinically warranted.

Measurements of drug antibody (ADA) levels

[0459] Blood samples to measure drug antibodies will be collected prior to dosing and then at time points specified in the SOA. If this patient demonstrates positive ADAs, she will be monitored until elimination.

Patient Questionnaires

[0460] Patient questionnaires will be answered by the patient and/or her caregiver after careful training.

Hunger estimates

[0461] Hunger will be assessed using a 3-part questionnaire as well as a set of 2 generic surveys administered at specific protocol visits in accordance with the SOA. Hunger will be assessed using a numerical rating scale from 0 to 10; where 0 = not hungry at all, and 10 = the hungriest possible. All results from the Daily Hunger Questionnaire will be assessed by asking the patient to rate their hunger on a Likert scale, with 0 being not at all hungry and 10 being the hungriest possible. Hunger questionnaire scores will be recorded daily prior to the patient's morning meal. The hunger questionnaire should be completed on three separate days at the time of screening and at the clinic visits before the morning dose. Patients will record their hunger ratings before their morning meal (fasting). Universal Hunger Surveys: Two universal hunger surveys will be conducted during designated visits as per protocol and assessment schedule.

Health Questionnaire SF-36

[0462] The SF-36 is a multipurpose short health survey consisting of only 36 questions. It provides an 8-scale profile of functional health and well-being scores, as well as psychometrically based physical and mental health composites and a preference-based health utility index. This is a general characteristic, as opposed to a characteristic aimed at a specific age, disease or treatment group. Accordingly, the SF-36 has proven useful in examining general and specific populations, comparing the relative burden of disease, and differentiating the health benefits provided by a wide range of different treatments.

Patient Health Questionnaire-9 (PHQ-9)

[0463] The PHQ-9 is a nine-item patient health questionnaire scale for depression. The PHQ-9 is a tool to help clinicians diagnose depression and select and monitor treatment. After the patient has completed the PHQ-9 questionnaire, protocol staff evaluate it.

[0464] If at any point during the protocol the patient's individual PHQ-9 score is ≥ 10, the patient should be referred to the MHP. PHQ-9 will be implemented in accordance with the SOA.

Columbia Suicidality Severity Rating Scale (C-SSRS)

[0465] The C-SSRS is a tool that is used not only to predict suicide attempts, but also to assess a full range of evidence-based ideas and behaviors with criteria for next steps (eg, mental health referrals (MHRs) )). In accordance with the SOA, two versions of the C-SSRS will be used in this protocol: (1) The original/screening version of the scale combines the original form with a screening form to assess suicidality over the patient's lifetime and over a predetermined period of time. This version may allow assessment of a patient's suicidality across the lifespan for data collection purposes as well as for assessing eligibility for inclusion/exclusion criteria. (2) The since last visit version of the scale assesses suicidality since the patient's last visit. This version is intended to evaluate patients who have completed at least one initial C-SSRS assessment and should be administered at each follow-up visit. The Since Last Visit version of the C-SSRS asks about any suicidal thoughts or behaviors the patient/participant may have had since the last time the C-SSRS was completed.

[0466] If at any point during the protocol the patient experiences Type 4 or 5 suicidal ideation or any suicidal behavior, the patient should be referred to the MHP.

[0467] After the screening period, which determines whether the patient meets the eligibility criteria, the patient proceeds to the 1st treatment period. In this phase, the first dose of H4H17319R2 will be administered IV on day 1 of the protocol. The first subcutaneous (SC) dose will occur on day 5 of the protocol. The patient will return to the clinic every week for her dose. Laboratory and test evaluations are described in Figure 20.

[0468] A detailed description of the safety assessments, laboratory parameters, PK and ADA that must be performed during this protocol are presented in the following sections.

[0469] The patient will be required to fast overnight the day before all visits starting with the screening visit. She will be allowed to take her regular medications with water the morning of each clinic visit.

[0470] Assessments may occur over several days during the screening period. To obtain sufficient baseline data on hunger symptoms collected on 3 separate days, the screening period should be a minimum of 3 days and up to 2 weeks.

[0471] To provide this patient and protocol staff with flexibility regarding the number of clinic visits, the actual scheduling of the clinic visits may allow flexibility in the timing of the visits. During Treatment Period 1 and Treatment Period 2, the goal will be for visits to occur within +/- 3 days. All data collected, even if outside the visit window, will be included in the endpoint analysis.

[0472] Monitoring for adverse events will continue throughout the protocol. Adverse events will be recorded in the CRF from the time of screening until the final visit as per protocol. Adverse events occurring after initiation of drug administration will be considered treatment-emergent adverse events (TEAEs). SAEs will be recorded prior to the final visit as per protocol. All adverse events should be monitored until they resolve or are clearly determined to be caused by a stable or chronic patient condition or intercurrent disease(s).

Adverse events: definitions, documentation and reporting

[0473] An adverse event (AE) is any adverse medical event associated with the use of a drug in humans, whether it is considered drug related or not. An adverse event (also called an adverse experience) can be any adverse and unintended sign (eg, laboratory abnormality), symptom, or disease for which there is a temporary association with the use of a drug, without any judgment of causation. AE may result from any use of the drug (eg, off-label use, use in combination with another drug) and any route of administration, formulation or dosage, including overdose.

[0474] H4H17319P2 was well tolerated in a first-use study in humans. Drug-related TEAEs (for which the adverse event was judged by the investigator to be possibly or likely related to the drug) were reported. Due to the very limited clinical experience to date with H4H17319R2, there may be other unknown side effects. The PI (or attending physician) will be available to protocol participants at all times in the event of a clinical emergency; both this availability and how to contact investigators in an emergency will be clearly communicated to protocol participants verbally and in writing. All interventions according to the protocol will be provided free of charge.

[0475] An AE or suspected adverse reaction is considered serious (SAE) if, in the opinion of the investigator or sponsor, it results in any of the following outcomes.

• Death.

• Life threatening. Life-threatening means that the patient was in immediate danger of dying from the reaction as it occurred, i.e. does not include a reaction that would hypothetically cause death if it occurred in a more severe form.

• Hospitalization or extension of current hospitalization. Hospitalizations and/or surgeries scheduled during the protocol period, but planned before entry into the protocol, are not considered AEs if the illness or disease existed before the patient's entry into the protocol, provided that the patient's condition did not worsen in an unexpected way during the course of the protocol. (for example, during an operation performed earlier than planned).

• Persistent or significant disability or significant impairment in the ability to perform normal life functions.

• Important medical phenomenon. An important medical event is one that may not result in death, be life-threatening, or require hospitalization, but may be considered an SAE when, based on appropriate medical judgment, it may place the patient in danger or the patient may require medical or surgical treatment. interventions to prevent one of the outcomes listed in the SAE definitions.

Examples of such medical events include allergic bronchospasm requiring intensive treatment in the emergency department or at home, blood dyscrasias or seizures that do not result in hospitalization, or the development of drug dependence or abuse.

Monitoring of adverse events and observation period

[0476] Each patient should be carefully monitored for the development of any AEs. This information should be obtained in the form of nonprompting questions (eg, “How are you feeling?”), as well as from signs and symptoms observed during each examination, observations by protocol staff, and patient self-reports.

[0477] AEs will be recorded in primary documents, starting from screening and up to and including the final visit according to the protocol. SAEs and fatalities will be recorded in the CRF SAE beginning upon signing of the ICF and continuing until the final visit as per protocol. All AEs should be monitored until they resolve or are clearly determined to be caused by a stable or chronic patient condition or intercurrent disease(s).

[0478] All AEs (serious and non-serious) spontaneously reported by the patient and/or in response to an open-ended question from protocol staff or identified during observation, physical examination or other diagnostic procedures will be recorded in the appropriate CRF. Any clinically significant deterioration in laboratory assessments or other clinical data is considered an AE and should be reported to the appropriate CRF. Whenever possible, signs and symptoms suggestive of a common underlying pathology should be noted as one complex entity.

[0479] Any SAEs occurring at any time after completion of the protocol that the investigator considers to be related to the drug must be reported to the sponsor.

[0480] All SAEs that occur during the course of the protocol must be reported to the medical supervisor within 24 hours of the investigator becoming aware of the SAE. All SAEs should be reported, regardless of whether they are causally related to the drug or not. SAE forms will be completed and the information collected will include the patient number, a narrative description (which may include relevant history, concomitant medications, and relevant laboratory and diagnostic test results) of the event, and the investigator's assessment of the severity of the event and its relationship to the drug. Sponsor or its designee may request additional information about SAE.

[0481] All correspondence regarding the SAE should be directed to the sponsor.

[0482] Close monitoring of patients will occur during the treatment period at the study site during the clinic visit, as well as during periodic telephone calls to patients by protocol staff. In the event that a patient discontinues treatment due to an AE, the patient should be encouraged to make a final protocol/early discontinuation visit to monitor the event until resolution and obtain additional protocol-defined safety assessments.

[0483] As mentioned above, a patient should be referred to an MHP if he/she has a PHQ-9 score greater than 10, any suicidal behavior, or any suicidal ideation type 4 or 5 on the C-SSRS. Referral to an MHP should also be made if, in the opinion of the investigator, it is necessary for the safety of the patient. If the patient's mental disorder can be adequately treated with psychotherapy and/or pharmacotherapy, then the patient, at the discretion of the MHP, should continue to participate in the trial.

[0484] If serious, unexpected adverse drug reactions associated with the use of a medicinal product occur, the relevant regulatory authority(ies), ethics committees (ECs) and all participating investigators will be notified on an expedited basis. The investigator is responsible for promptly notifying the Institutional Review Board (IRB)/Independent Ethics Committee (IEC), if required by the IRB/IEC, of any unexpected serious adverse drug reactions that pose a risk to human patients. An unexpected event is an event that is not reported in the IB.

[0485] For both serious and non-serious AEs, the investigator must determine both the intensity of the event and the relationship of the event to the administration of H4H17319P2. Only injection site reactions considered clinically significant by the investigator will be recorded as AEs.

[0486] The intensity of all AEs, including clinically significant treatment-emergent laboratory abnormalities, injection site reactions, and potential systemic reactions, will be assessed in accordance with CTCAE version 4.03. CTCAE grade refers to the severity of AE and ranges from grade 1 (mild AE), grade 2 (moderate AE), grade 3 (severe AE) and grade 4 (life-threatening or disabling AE) to grade 5 (death associated with AE).

[0487] Adverse events not listed in CTCAE will be assessed as follows.

• Mild: There is discomfort but no disruption to normal daily activities.

• Moderate: discomfort sufficient to reduce or alter daily activities.

• Severe: inability to work or perform normal daily activities.

• Life threatening: poses an immediate threat to life.

[0488] The relationship with the administration of H4H17319P2 will be determined by the investigator according to the following criteria.

• Absent: there is no relationship between the event and the administration of the drug. The phenomenon is associated with another etiology, such as concomitant medications or the clinical condition of the patient.

• Unlikely: The current state of knowledge indicates that an association with H4H17319P2 is unlikely, or the timing of the association is such that H4H17319P2 would not have any reasonable association with the observed phenomenon.

• Possible: a reaction that follows the likely time sequence from the administration of H4H17319P2 and is consistent with the known pattern of response to the intended drug. The reaction could be caused by the patient's clinical condition or other therapies prescribed to the patient.

• Probable: a response that follows a probable time sequence from the administration of H4H17319P2 and is consistent with a known pattern of drug response. The reaction cannot be reasonably explained by known characteristics of the patient's clinical condition or other treatments prescribed to the patient.

[0489] For the purposes of safety analyses, all AEs that are classified as possible or probable will be considered treatment-related events.

Administrative Requirements

Good Clinical Practice

[0490] The protocol will be conducted in accordance with the International Council for Harmonization (ICH) Good Clinical Practice (GCP) and related regulatory requirements. The investigator must be knowledgeable about the proper use of the drug as described in the protocol and IB. Substantial clinical documentation will be retained to demonstrate the validity of the protocol and the integrity of the data collected. Master files must be created at the beginning of the protocol, maintained throughout the protocol, and recorded according to the appropriate rules.

Ethical considerations

[0491] The protocol will be conducted in accordance with the ethical principles laid down in the Declaration of Helsinki. The IRB/IEC will review all relevant protocol documentation to ensure the rights, safety and welfare of patients. The protocol will only be conducted at study sites where IRB/IEC approval has been obtained. The protocol, IB, informed consent, advertisements (if applicable), written information provided to patients (including diary cards), safety updates, annual progress reports, and any changes to these documents will be provided to the IRB/IEC by the investigator.

[0492] H4H17319P2 will provide treatment for focal lipodystrophy (PLD) in a patient, as well as amelioration of the patient's hepatosplenomegaly, high triglyceride levels, and lack of limb fat, which are associated with PLD.

Examples 23 and 24. Effects of agonistic anti-LEPR antibodies in mouse models of congenital leptin deficiency and congenital LEPR deficiency

[0493] The effects of the specific LEPR agonist antibody of the present invention, H4H17319P2, on blood glucose levels, body weight, food intake and body composition (fat mass, lean mass and bone mass) were determined in mouse models of congenital leptin deficiency and congenital Leptin receptor deficiency due to loss of function mutation. Genetically engineered Lepr ^hu/hu Lep ^−/− mice expressing a leptin receptor that consists of the human LEPR ectodomain sequence instead of the mouse LEPR ectodomain sequence and not expressing leptin have been used as a mouse model of congenital leptin deficiency. A model of congenital LEPR deficiency is the genetically engineered Lepr ^{huA409E/huA409E} mice, which express a leptin receptor consisting of the human LEPR ectodomain sequence with the A409E missense mutation in place of the mouse LEPR ectodomain sequence.

Example 23: Effects of H4H174319P2 in a mouse model of congenital leptin deficiency

[0494] On day 0, sixteen female Lepr ^hu/hu Lep ^{−/− mice,} 27 to 32 weeks old, were divided into two groups based on body weight. Each group received a once-weekly subcutaneous injection of isotype control antibody ( ^IgG4P ) at 10 mg/kg or H4H17319P2 at 10 mg/kg on days 0, 7, 14, 21, 28, and 35. Seven female control mice Lepr ^hu/hu 27 to 32 weeks of age were treated by subcutaneous injection of once-weekly isotype control antibody ( ^IgG4P ) at 10 mg/kg on days 0, 7, 14, 21, 28, and 35. On days - 5 and 35, body composition was quantified using μST. Food intake, body weight, and blood glucose levels were measured throughout the study for each animal. Figure 21 summarizes blood glucose levels, body weight, and cumulative food intake for each treatment group. Figure 22 summarizes fat mass, lean mass, and bone mass for animals in each antibody treatment group, quantified by μST 5 days before and 35 days after antibody treatment. All results are expressed as mean ±SEM.

[0495] As shown in Figure 21A, on day 0 before antibody administration, leptin-deficient Lepr ^hu/hu Lep ^−/− mice exhibited hyperglycemia compared to control Lepr ^hu/hu mice. Leptin-deficient mice treated with H4H17319P2 at a dose of 10 mg/kg showed a significant decrease in blood glucose levels starting 2 days after antibody treatment and at other subsequent measurement time points, compared with leptin-deficient mice. which were injected with an isotype control antibody ( ^IgG4P ). Leptin-deficient Lepr ^hu/hu Lep ^−/− mice that received isotype control antibody (IgG4 ^P ) at a dose of 10 mg/kg maintained hyperglycemia at all time points measured.

[0496] As shown in Figure 21B, on day 0 before antibody administration, leptin-deficient Lepr ^hu/hu Lep ^−/− mice were significantly heavier compared to control Lepr ^hu/hu mice. Leptin-deficient mice treated with H4H17319P2 at a dose of 10 mg/kg showed a significant reduction in body weight starting 15 days after antibody treatment and at other subsequent time points of measurement, compared with leptin-deficient mice treated with by injection of an isotype control antibody ( ^IgG4P ). Leptin-deficient Lepr ^hu/hu Lep ^−/− mice that received isotype control antibody ( ^IgG4P ) at a dose of 10 mg/kg showed no reduction in body weight from baseline at all time points measured.

[0497] As shown in Figure 21C, leptin-deficient Lepr ^hu/hu Lep ^-/- mice showed a significant reduction in cumulative food intake two days after treatment with H4H17319P2 and at other subsequent measurement time points compared to leptin-deficient mice who were injected with an isotype control antibody ( ^IgG4P ).

[0498] As shown in Figure 22, prior to antibody administration on day -5, leptin-deficient Lepr ^hu/hu Lep ^−/− mice showed a significant increase in fat mass, decreased lean mass, and decreased bone mass compared to Lepr ^hu/hu mice . At day 35, leptin-deficient Lepr ^hu/hu Lep ^−/− mice treated with H4H17319P2 at 10 mg/kg showed a significant decrease in fat mass and gain in lean mass and bone mass compared to leptin-deficient mice treated by injection of isotype control antibody ( ^IgG4P ).

Example 24

[0499] On day 0, twenty male Lepr ^{huA409E/huA409E} mice between 24 and 28 weeks of age were divided into two groups based on body weight. Each group received a once-weekly subcutaneous injection of isotype control antibody ( ^IgG4P ) at 5 mg/kg or H4H17319P2 at 5 mg/kg on days 0, 7, 14, 21, 28, 35, and 42. Eight males Lepr ^hu/hu control mice, 24 to 28 weeks of age, were treated by subcutaneous injection with once-weekly isotype control antibody ( ^IgG4P ) at 5 mg/kg on days 0, 7, 14, 21, 28, 35, and 42 On days -1 and 41, body composition was quantified using μCT. Food intake, body weight, and blood glucose levels were measured throughout the study for each animal. Figure 23 summarizes blood glucose levels, body weight, and cumulative food intake for each treatment group. Figure 24 summarizes fat mass, lean mass, and bone mass for animals in each antibody treatment group, quantified by μST 1 day before and 41 days after antibody treatment. All results are expressed as mean ±SEM.

[0500] As shown in Figure 23A, on day 0 before antibody administration, leptin receptor-deficient Lepr ^{huA409E/huA409E} mice exhibited hyperglycemia compared to control Lepr ^hu/hu mice. Lepr ^{huA409E/huA409E} mice treated with H4H17319P2 at a dose of 5 mg/kg showed a significant decrease in blood glucose levels from 7 days after antibody treatment and at other subsequent measurement time points, compared to Lepr ^{huA409E/huA409E} mice who were injected with an isotype control antibody ( ^IgG4P ). Leptin receptor-deficient Lepr ^{huA409E/huA409E} mice that received isotype control antibody ( ^IgG4P ) at 5 mg/kg maintained hyperglycemia at all time points measured.

[0501] As shown in Figure 23B, on day 0 before antibody administration, leptin receptor-deficient Lepr ^{huA409E/huA409E} mice were significantly heavier compared to control Lepr ^hu/hu mice. Lepr ^{huA409E/huA409E} mice treated with H4H17319P2 at a dose of 5 mg/kg showed a significant decrease in body weight starting at 13 days after antibody treatment and at other subsequent time points of measurement, compared with leptin receptor-deficient mice. which were injected with an isotype control antibody ( ^IgG4P ). Lepr ^{huA409E/huA409E} that received isotype control antibody ( ^IgG4P ) at a dose of 5 mg/kg did not show a decrease in body weight compared to baseline at all time points measured.

[0502] As shown in Figure 23C, Lepr ^{huA409E/huA409E} showed a significant reduction in cumulative food intake 7 days after treatment with H4H17319P2 and at other subsequent measurement time points compared to leptin-deficient mice injected with isotype control antibody ( ^IgG4P ).

[0503] As shown in Figure 24, prior to antibody administration on day -1, Lepr ^{huA409E/huA409E} mice showed a significant increase in adiposity, decreased lean mass, and decreased bone mass compared to Lepr ^hu/hu mice. On day 41, Lepr ^{huA409E/huA409E} mice treated with H4H17319P2 at 5 mg/kg showed a significant decrease in fat mass and an increase in lean and bone mass compared to Lepr ^{huA409E/huA409E} mice that were injected with isotype control antibody (IgG4 ^P ).

[0504] The scope of the present invention is not limited to the specific embodiments described herein. Indeed, various modifications of the present invention other than those described herein will be apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to be within the scope of the appended claims.

--->

LIST OF SEQUENCES

<110> Regeneron Pharmaceuticals, Inc.

Gromada, Jesper

Stevis, Panagiotis

Altarejos, Judith

Murphy, Andrew J.

<120> TREATMENT METHODS USING AN AGONISTIC ANTIBODY TO THE LEPTIN RECEPTOR

<130> 9774.436WO1/10436WO01

<140> TBD

<141> 2019-04-05

<160> 120

PatentIn version 3.5

<210> 1

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 1

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaagtc cctgagactc 60

tcctgtgtag cgtctggatt caccttcagt tccgatgcca tgtactgggt ccgccaggct 120

ccaggcaagg ggctggaatg ggtggcagtt atttattatg atggaaatta tcaatactat 180

gaagactccg ttaagggtcg attcaccatc tccagagaca attcccagaa cacgctggat 240

ctgcaaatga acagcctgag agtcgacgac acggctgtat atttctgtgc gcgtctcaac 300

tgggattact ggtatctcga tctctggggc cgtggcaccc tggtcactgt ctcctca 357

<210> 2

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 2

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Lys

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Asp

20 25 30

Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Tyr Tyr Asp Gly Asn Tyr Gln Tyr Tyr Glu Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Asn Thr Leu Asp

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Val Asp Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Leu Asn Trp Asp Tyr Trp Tyr Leu Asp Leu Trp Gly Arg Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 3

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 3

ggattcacct tcagttccga tgcc 24

<210> 4

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 4

Gly Phe Thr Phe Ser Ser Asp Ala

15

<210> 5

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 5

atttattatg atggaaatta tcaa 24

<210> 6

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 6

Ile Tyr Tyr Asp Gly Asn Tyr Gln

15

<210> 7

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 7

gcgcgtctca actgggatta ctggtatctc gatctc 36

<210> 8

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 8

Ala Arg Leu Asn Trp Asp Tyr Trp Tyr Leu Asp Leu

1 5 10

<210> 9

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 9

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccgtca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccctccgat caccttcggc 300

caagggacac gactggagat taaa 324

<210> 10

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 10

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 11

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 11

cagagcatta gcagctat 18

<210> 12

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 12

Gln Ser Ile Ser Ser Tyr

15

<210> 13

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 13

gctgcatcc 9

<210> 14

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 14

Ala Ala Ser

1

<210> 15

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 15

caacagagtt acagtacccc tccgatcacc 30

<210> 16

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 16

Gln Gln Ser Tyr Ser Thr Pro Pro Ile Thr

1 5 10

<210> 17

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 17

caggtgcagc tggtggagtc cgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtacag cgtctggatt caccttcagt agttatgcca tgtactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtgtcagtt atatactatg atggaagtta taaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatgg acagcctgag agccgaggac acggctgtct attactgtgc gagttataac 300

tggaactact ggtacttcga tttctggggc cgtggcaccc tggtcactgt ctcctca 357

<210> 18

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 18

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Tyr Tyr Asp Gly Ser Tyr Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Tyr Asn Trp Asn Tyr Trp Tyr Phe Asp Phe Trp Gly Arg Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 19

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 19

ggattcacct tcagtagtta tgcc 24

<210> 20

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 20

Gly Phe Thr Phe Ser Ser Tyr Ala

15

<210> 21

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 21

atatactatg atggaagtta taaa 24

<210> 22

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 22

Ile Tyr Tyr Asp Gly Ser Tyr Lys

15

<210> 23

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 23

gcgagttata actggaacta ctggtacttc gatttc 36

<210> 24

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 24

Ala Ser Tyr Asn Trp Asn Tyr Trp Tyr Phe Asp Phe

1 5 10

<210> 25

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 25

caggtgcagc tggtggagtc tgggggaagc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cgtctggatt caccttcagt acatatgcca tgtactgggt ccgccagact 120

ccaggcaagg ggctggagtg ggtggctgtt ttatactctg atggaagtaa taaatactat 180

atagactccg tgaagggccg attcaccatc tccagagaca cttccacgaa cactctgtat 240

ctgcaaatga gcagcctgcg agccgacgac tcggctctat attactgtgc gcgtctcaac 300

tgggattact ggtacttcga tctctggggc cgtggcaccc tggtcactgt ctcctca 357

<210> 26

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 26

Gln Val Gln Leu Val Glu Ser Gly Gly Ser Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Tyr Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Leu Tyr Ser Asp Gly Ser Asn Lys Tyr Tyr Ile Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ser Thr Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ala Asp Asp Ser Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Leu Asn Trp Asp Tyr Trp Tyr Phe Asp Leu Trp Gly Arg Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 27

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 27

ggattcacct tcagtacata tgcc 24

<210> 28

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 28

Gly Phe Thr Phe Ser Thr Tyr Ala

15

<210> 29

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 29

ttatactctg atggaagtaa taaa 24

<210> 30

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 30

Leu Tyr Ser Asp Gly Ser Asn Lys

15

<210> 31

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 31

gcgcgtctca actgggatta ctggtacttc gatctc 36

<210> 32

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 32

Ala Arg Leu Asn Trp Asp Tyr Trp Tyr Phe Asp Leu

1 5 10

<210> 33

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 33

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgaag cgtctggatt cagcagcagt gacaatgcca tgtactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtgtcagtt atatatcatg atggaagtta taaatactat 180

gaagactccg tgaagggccg attcaccatc gccagagaca attccaagaa cacgctttat 240

ttgcaaatga acagcctgag agccgaggac acggctgtat attactgtgc gaggtataac 300

tggaaccact ggtacttcga tgtctggggc cgtggcaccc tggtcactgt ctcctca 357

<210> 34

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 34

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Ser Ser Ser Asp Asn

20 25 30

Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Tyr His Asp Gly Ser Tyr Lys Tyr Tyr Glu Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ala Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asn Trp Asn His Trp Tyr Phe Asp Val Trp Gly Arg Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 35

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 35

ggattcagca gcagtgacaa tgcc 24

<210> 36

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 36

Gly Phe Ser Ser Ser Asp Asn Ala

15

<210> 37

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 37

atatatcatg atggaagtta taaa 24

<210> 38

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 38

Ile Tyr His Asp Gly Ser Tyr Lys

15

<210> 39

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 39

gcgaggtata actggaacca ctggtacttc gatgtc 36

<210> 40

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 40

Ala Arg Tyr Asn Trp Asn His Trp Tyr Phe Asp Val

1 5 10

<210> 41

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 41

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt acctatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtgtcagtt atatcatatg acgaaagtaa taagtactat 180

gcagactccg tgaagggccg attcaccatt tctagagaca attccaagaa cgcgctgtat 240

ttacaaatga acagcctgag aaatgaggac acggctgtgt attactgtgc gagagatcgg 300

ccttttggat tggttaccgg atggttcgac ccctggggcc agggaaccct ggtcaccgtc 360

tcctca 366

<210> 42

<211> 122

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 42

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Ser Tyr Asp Glu Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ala Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Asn Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Pro Phe Gly Leu Val Thr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 43

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 43

ggattcacct tcagtaccta tggc 24

<210> 44

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 44

Gly Phe Thr Phe Ser Thr Tyr Gly

15

<210> 45

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 45

atatcatatg acgaaagtaa taag 24

<210> 46

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 46

Ile Ser Tyr Asp Glu Ser Asn Lys

15

<210> 47

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 47

gcgagagatc ggccttttgg attggttacc ggatggttcg acccc 45

<210> 48

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 48

Ala Arg Asp Arg Pro Phe Gly Leu Val Thr Gly Trp Phe Asp Pro

1 5 10 15

<210> 49

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 49

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cgtctggatt cagtttcaat acctatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtgacaatt atatggtatg atggaagtat taaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggctgtgt attattgtgc gagaggtgga 300

tatagtggct acctctactt tgactactgg ggccagggaa ccctggtcac cgtctcctca 360

<210> 50

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 50

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Asn Thr Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Thr Ile Ile Trp Tyr Asp Gly Ser Ile Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Ser Gly Tyr Leu Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 51

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 51

ggattcagtt tcaataccta tggc 24

<210> 52

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 52

Gly Phe Ser Phe Asn Thr Tyr Gly

15

<210> 53

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 53

atatggtatg atggaagtat taaa 24

<210> 54

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 54

Ile Trp Tyr Asp Gly Ser Ile Lys

15

<210> 55

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 55

gcgagaggtg gatatagtgg ctacctctac tttgactac 39

<210> 56

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 56

Ala Arg Gly Gly Tyr Ser Gly Tyr Leu Tyr Phe Asp Tyr

1 5 10

<210> 57

<211> 372

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 57

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agcggtggtg actactggag ctggatccgc 120

cagctcccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcgcctac 180

tataatccgt ccctcaagag tcgaggtacc atatcaatag acacgtctaa gaaccagttc 240

tccctgaagc tgacctctgt gactgccgcg gacacggccg tatatttctg tgtgaaatta 300

cgatttttgg agtggttctt ggggggctgg ttcggcccct ggggccaggt aaccctggtc 360

accgtctcct ca 372

<210> 58

<211> 124

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 58

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly Asp Tyr Trp Ser Trp Ile Arg Gln Leu Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Ala Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Gly Thr Ile Ser Ile Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe

85 90 95

Cys Val Lys Leu Arg Phe Leu Glu Trp Phe Leu Gly Gly Trp Phe Gly

100 105 110

Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 59

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 59

ggtggctcca tcagcagcgg tggtgactac 30

<210> 60

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 60

Gly Gly Ser Ile Ser Ser Gly Gly Asp Tyr

1 5 10

<210> 61

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 61

atctattaca gtgggagcgc from 21

<210> 62

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 62

Ile Tyr Tyr Ser Gly Ser Ala

15

<210> 63

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 63

gtgaaattac gatttttgga gtggttcttg gggggctggt tcggcccc 48

<210> 64

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 64

Val Lys Leu Arg Phe Leu Glu Trp Phe Leu Gly Gly Trp Phe Gly Pro

1 5 10 15

<210> 65

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 65

gaggtgcagc tggtggagtc tgggggaggc ttggtacagc cggggggggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttagc aactatggca tgacctgggt ccgccaggct 120

ccagggaagg gcctggaatg ggtctcagct attactggtg gtggtggtag cacatactac 180

tcaaactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacggtgtat 240

ctgcgaatga acagtgtgag agccgaggac acggccgtat attactgtgc gaaatataag 300

tggaacttcg tggacgactg gggccaggga accacggtca ccgtctcctc a 351

<210> 66

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 66

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Thr Gly Gly Gly Gly Ser Thr Tyr Tyr Ser Asn Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Arg Met Asn Ser Val Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Tyr Lys Trp Asn Phe Val Asp Asp Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 67

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 67

ggattcacct ttagcaacta tggc 24

<210> 68

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 68

Gly Phe Thr Phe Ser Asn Tyr Gly

15

<210> 69

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 69

attactggtg gtggtggtag caca 24

<210> 70

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 70

Ile Thr Gly Gly Gly Gly Ser Thr

15

<210> 71

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 71

gcgaaatata agtggaactt cgtggacgac 30

<210> 72

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 72

Ala Lys Tyr Lys Trp Asn Phe Val Asp Asp

1 5 10

<210> 73

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 73

gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgttg cctctggatt caccttcaat aaatacgaca tgcactgggt ccgccaaact 120

actggaaaag gtctagagtg ggtctcaggt attgatactg atggtgacac atactatcca 180

ggctccgtga agggccgatt caccatctcc agagaaaatg ccgagaactc cctgtatctt 240

caaatgaacg gcctgagagt cggggacacg gctgtgtatt actgtgcaag atggccttgg 300

agtggtttct atggtgcttt tgatatctgg ggccaaggga caatggtcac cgtctcttca 360

<210> 74

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 74

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asn Lys Tyr

20 25 30

Asp Met His Trp Val Arg Gln Thr Thr Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Asp Thr Asp Gly Asp Thr Tyr Tyr Pro Gly Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Glu Asn Ser Leu Tyr Leu

65 70 75 80

Gln Met Asn Gly Leu Arg Val Gly Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Trp Pro Trp Ser Gly Phe Tyr Gly Ala Phe Asp Ile Trp Gly Gln

100 105 110

Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 75

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 75

ggattcacct tcaataaata cgac 24

<210> 76

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 76

Gly Phe Thr Phe Asn Lys Tyr Asp

15

<210> 77

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 77

attgatactg atggtgacac a 21

<210> 78

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 78

Ile Asp Thr Asp Gly Asp Thr

15

<210> 79

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 79

gcaagatggc cttggagtgg tttctatggt gcttttgata tc 42

<210> 80

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 80

Ala Arg Trp Pro Trp Ser Gly Phe Tyr Gly Ala Phe Asp Ile

1 5 10

<210> 81

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 81

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtaatt actactggaa ctggatccgc 120

caacagccag gagaggggcct ggagtggatt gcttacatct atcacaatgg ggtcaccaac 180

ttcaatccgt ccctcaagag tcgacttact atatcagtag acacgtctaa gactcagttc 240

tccctgaagt tgaggtctgt gactgccgcg gacacggccg tttattactg tgcgagatca 300

ggcagctggt tcgagaactg gtacttcgat ctctggggcc gtggcaccct ggtcactgtc 360

tcctca 366

<210> 82

<211> 122

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 82

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asn Tyr Tyr Trp Asn Trp Ile Arg Gln Gln Pro Gly Glu Gly Leu Glu

35 40 45

Trp Ile Ala Tyr Ile Tyr His Asn Gly Val Thr Asn Phe Asn Pro Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Thr Gln Phe

65 70 75 80

Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ser Gly Ser Trp Phe Glu Asn Trp Tyr Phe Asp Leu Trp

100 105 110

Gly Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 83

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 83

ggtggctcca tcagcagtgg taattactac 30

<210> 84

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 84

Gly Gly Ser Ile Ser Ser Gly Asn Tyr Tyr

1 5 10

<210> 85

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 85

atctatcaca atggggtcac from 21

<210> 86

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 86

Ile Tyr His Asn Gly Val Thr

15

<210> 87

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 87

gcgagatcag gcagctggtt cgagaactgg tacttcgatc tc 42

<210> 88

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 88

Ala Arg Ser Gly Ser Trp Phe Glu Asn Trp Tyr Phe Asp Leu

1 5 10

<210> 89

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 89

gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcaggggccac tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcaccttg gacgttcggc 300

caagggacca aggtggaaat caaa 324

<210> 90

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 90

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 91

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 91

cagagtgtta gcagcagcta from 21

<210> 92

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 92

Gln Ser Val Ser Ser Ser Tyr

15

<210> 93

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 93

ggtgcatcc 9

<210> 94

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 94

Gly Ala Ser

1

<210> 95

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 95

cagcagtatg gtagctcacc ttggacg 27

<210> 96

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 96

Gln Gln Tyr Gly Ser Ser Pro Trp Thr

15

<210> 97

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 97

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagt aattcctact ggagctggat ccggcagccc 120

ccagggaagg gactggagtg gattggatat gtctattccc gtgggaacac caagtacaac 180

ccctccctca cgagtcgagt caccatgtca tttgacacgt ccaagaacca gttctccctg 240

aaactgaggt ctgtgaccgc cgcagacacg gccgtgtatt actgtgcgag aagcagcagc 300

tggtacgagg actggtactt cgatctctgg ggccgtggca ccctggtcac tgtctcctca 360

<210> 98

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 98

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Asn Ser

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Val Tyr Ser Arg Gly Asn Thr Lys Tyr Asn Pro Ser Leu Thr

50 55 60

Ser Arg Val Thr Met Ser Phe Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ser Ser Ser Trp Tyr Glu Asp Trp Tyr Phe Asp Leu Trp Gly Arg

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 99

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 99

ggtggctcca tcagtaattc ctac 24

<210> 100

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 100

Gly Gly Ser Ile Ser Asn Ser Tyr

15

<210> 101

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 101

gtctattccc gtgggaacac c 21

<210> 102

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 102

Val Tyr Ser Arg Gly Asn Thr

15

<210> 103

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 103

gcgagaagca gcagctggta cgaggactgg tacttcgatc tc 42

<210> 104

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 104

Ala Arg Ser Ser Ser Trp Tyr Glu Asp Trp Tyr Phe Asp Leu

1 5 10

<210> 105

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 105

caggtgcagc tacagcagtg gggcgcaggg ctgtttaagc cttcggagac cctgtccctc 60

acctgcgatg tctatggtgg gtccttcaga ggttattatt ggagttggat ccgccagccc 120

ccagggaagg ggctggagtg gattggggaa atcagttata gtggtttcac caattacaac 180

ccgtccctca agagtcgagt catcatatca atagatacgt ccaagaacca gttctccctg 240

aagatgagct ctgtgaccgc cgcggacacg gctgtttatt actgtgcgag agttacctat 300

ggttatggga cctttgatta ttggggccag ggaaccctgg tcaccgtctc ctca 354

<210> 106

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 106

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Phe Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Asp Val Tyr Gly Gly Ser Phe Arg Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Ser Tyr Ser Gly Phe Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Ile Ile Ser Ile Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Val Thr Tyr Gly Tyr Gly Thr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 107

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 107

ggtgggtcct tcagaggtta ttat 24

<210> 108

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 108

Gly Gly Ser Phe Arg Gly Tyr Tyr

15

<210> 109

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 109

atcagttata gtggtttcac from 21

<210> 110

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 110

Ile Ser Tyr Ser Gly Phe Thr

15

<210> 111

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 111

gcgagagtta cctatggtta tgggaccttt gattat 36

<210> 112

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 112

Ala Arg Val Thr Tyr Gly Tyr Gly Thr Phe Asp Tyr

1 5 10

<210> 113

<211> 1165

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hLEPR under accession number P48357

<400> 113

Met Ile Cys Gln Lys Phe Cys Val Val Leu Leu His Trp Glu Phe Ile

1 5 10 15

Tyr Val Ile Thr Ala Phe Asn Leu Ser Tyr Pro Ile Thr Pro Trp Arg

20 25 30

Phe Lys Leu Ser Cys Met Pro Pro Asn Ser Thr Tyr Asp Tyr Phe Leu

35 40 45

Leu Pro Ala Gly Leu Ser Lys Asn Thr Ser Asn Ser Asn Gly His Tyr

50 55 60

Glu Thr Ala Val Glu Pro Lys Phe Asn Ser Ser Gly Thr His Phe Ser

65 70 75 80

Asn Leu Ser Lys Thr Thr Phe His Cys Cys Phe Arg Ser Glu Gln Asp

85 90 95

Arg Asn Cys Ser Leu Cys Ala Asp Asn Ile Glu Gly Lys Thr Phe Val

100 105 110

Ser Thr Val Asn Ser Leu Val Phe Gln Gln Ile Asp Ala Asn Trp Asn

115 120 125

Ile Gln Cys Trp Leu Lys Gly Asp Leu Lys Leu Phe Ile Cys Tyr Val

130 135 140

Glu Ser Leu Phe Lys Asn Leu Phe Arg Asn Tyr Asn Tyr Lys Val His

145 150 155 160

Leu Leu Tyr Val Leu Pro Glu Val Leu Glu Asp Ser Pro Leu Val Pro

165 170 175

Gln Lys Gly Ser Phe Gln Met Val His Cys Asn Cys Ser Val His Glu

180 185 190

Cys Cys Glu Cys Leu Val Pro Val Pro Thr Ala Lys Leu Asn Asp Thr

195 200 205

Leu Leu Met Cys Leu Lys Ile Thr Ser Gly Gly Val Ile Phe Gln Ser

210 215 220

Pro Leu Met Ser Val Gln Pro Ile Asn Met Val Lys Pro Asp Pro Pro

225 230 235 240

Leu Gly Leu His Met Glu Ile Thr Asp Asp Gly Asn Leu Lys Ile Ser

245 250 255

Trp Ser Ser Pro Pro Leu Val Pro Phe Pro Leu Gln Tyr Gln Val Lys

260 265 270

Tyr Ser Glu Asn Ser Thr Thr Val Ile Arg Glu Ala Asp Lys Ile Val

275 280 285

Ser Ala Thr Ser Leu Leu Val Asp Ser Ile Leu Pro Gly Ser Ser Tyr

290 295 300

Glu Val Gln Val Arg Gly Lys Arg Leu Asp Gly Pro Gly Ile Trp Ser

305 310 315 320

Asp Trp Ser Thr Pro Arg Val Phe Thr Thr Gln Asp Val Ile Tyr Phe

325 330 335

Pro Pro Lys Ile Leu Thr Ser Val Gly Ser Asn Val Ser Phe His Cys

340 345 350

Ile Tyr Lys Lys Glu Asn Lys Ile Val Pro Ser Lys Glu Ile Val Trp

355 360 365

Trp Met Asn Leu Ala Glu Lys Ile Pro Gln Ser Gln Tyr Asp Val Val

370 375 380

Ser Asp His Val Ser Lys Val Thr Phe Phe Asn Leu Asn Glu Thr Lys

385 390 395 400

Pro Arg Gly Lys Phe Thr Tyr Asp Ala Val Tyr Cys Cys Asn Glu His

405 410 415

Glu Cys His His Arg Tyr Ala Glu Leu Tyr Val Ile Asp Val Asn Ile

420 425 430

Asn Ile Ser Cys Glu Thr Asp Gly Tyr Leu Thr Lys Met Thr Cys Arg

435 440 445

Trp Ser Thr Ser Thr Ile Gln Ser Leu Ala Glu Ser Thr Leu Gln Leu

450 455 460

Arg Tyr His Arg Ser Ser Leu Tyr Cys Ser Asp Ile Pro Ser Ile His

465 470 475 480

Pro Ile Ser Glu Pro Lys Asp Cys Tyr Leu Gln Ser Asp Gly Phe Tyr

485 490 495

Glu Cys Ile Phe Gln Pro Ile Phe Leu Leu Ser Gly Tyr Thr Met Trp

500 505 510

Ile Arg Ile Asn His Ser Leu Gly Ser Leu Asp Ser Pro Pro Thr Cys

515 520 525

Val Leu Pro Asp Ser Val Val Lys Pro Leu Pro Pro Ser Ser Val Lys

530 535 540

Ala Glu Ile Thr Ile Asn Ile Gly Leu Leu Lys Ile Ser Trp Glu Lys

545 550 555 560

Pro Val Phe Pro Glu Asn Asn Leu Gln Phe Gln Ile Arg Tyr Gly Leu

565 570 575

Ser Gly Lys Glu Val Gln Trp Lys Met Tyr Glu Val Tyr Asp Ala Lys

580 585 590

Ser Lys Ser Val Ser Leu Pro Val Pro Asp Leu Cys Ala Val Tyr Ala

595 600 605

Val Gln Val Arg Cys Lys Arg Leu Asp Gly Leu Gly Tyr Trp Ser Asn

610 615 620

Trp Ser Asn Pro Ala Tyr Thr Val Val Met Asp Ile Lys Val Pro Met

625 630 635 640

Arg Gly Pro Glu Phe Trp Arg Ile Ile Asn Gly Asp Thr Met Lys Lys

645 650 655

Glu Lys Asn Val Thr Leu Leu Trp Lys Pro Leu Met Lys Asn Asp Ser

660 665 670

Leu Cys Ser Val Gln Arg Tyr Val Ile Asn His His Thr Ser Cys Asn

675 680 685

Gly Thr Trp Ser Glu Asp Val Gly Asn His Thr Lys Phe Thr Phe Leu

690 695 700

Trp Thr Glu Gln Ala His Thr Val Thr Val Leu Ala Ile Asn Ser Ile

705 710 715 720

Gly Ala Ser Val Ala Asn Phe Asn Leu Thr Phe Ser Trp Pro Met Ser

725 730 735

Lys Val Asn Ile Val Gln Ser Leu Ser Ala Tyr Pro Leu Asn Ser Ser

740 745 750

Cys Val Ile Val Ser Trp Ile Leu Ser Pro Ser Asp Tyr Lys Leu Met

755 760 765

Tyr Phe Ile Ile Glu Trp Lys Asn Leu Asn Glu Asp Gly Glu Ile Lys

770 775 780

Trp Leu Arg Ile Ser Ser Ser Val Lys Lys Tyr Tyr Ile His Asp His

785 790 795 800

Phe Ile Pro Ile Glu Lys Tyr Gln Phe Ser Leu Tyr Pro Ile Phe Met

805 810 815

Glu Gly Val Gly Lys Pro Lys Ile Ile Asn Ser Phe Thr Gln Asp Asp

820 825 830

Ile Glu Lys His Gln Ser Asp Ala Gly Leu Tyr Val Ile Val Pro Val

835 840 845

Ile Ile Ser Ser Ser Ile Leu Leu Leu Gly Thr Leu Leu Ile Ser His

850 855 860

Gln Arg Met Lys Lys Leu Phe Trp Glu Asp Val Pro Asn Pro Lys Asn

865 870 875 880

Cys Ser Trp Ala Gln Gly Leu Asn Phe Gln Lys Pro Glu Thr Phe Glu

885 890 895

His Leu Phe Ile Lys His Thr Ala Ser Val Thr Cys Gly Pro Leu Leu

900 905 910

Leu Glu Pro Glu Thr Ile Ser Glu Asp Ile Ser Val Asp Thr Ser Trp

915 920 925

Lys Asn Lys Asp Glu Met Met Pro Thr Thr Val Val Ser Leu Leu Ser

930 935 940

Thr Thr Asp Leu Glu Lys Gly Ser Val Cys Ile Ser Asp Gln Phe Asn

945 950 955 960

Ser Val Asn Phe Ser Glu Ala Glu Gly Thr Glu Val Thr Tyr Glu Asp

965 970 975

Glu Ser Gln Arg Gln Pro Phe Val Lys Tyr Ala Thr Leu Ile Ser Asn

980 985 990

Ser Lys Pro Ser Glu Thr Gly Glu Glu Gln Gly Leu Ile Asn Ser Ser

995 1000 1005

Val Thr Lys Cys Phe Ser Ser Lys Asn Ser Pro Leu Lys Asp Ser

1010 1015 1020

Phe Ser Asn Ser Ser Trp Glu Ile Glu Ala Gln Ala Phe Phe Ile

1025 1030 1035

Leu Ser Asp Gln His Pro Asn Ile Ile Ser Pro His Leu Thr Phe

1040 1045 1050

Ser Glu Gly Leu Asp Glu Leu Leu Lys Leu Glu Gly Asn Phe Pro

1055 1060 1065

Glu Glu Asn Asn Asp Lys Lys Ser Ile Tyr Tyr Leu Gly Val Thr

1070 1075 1080

Ser Ile Lys Lys Arg Glu Ser Gly Val Leu Leu Thr Asp Lys Ser

1085 1090 1095

Arg Val Ser Cys Pro Phe Pro Ala Pro Cys Leu Phe Thr Asp Ile

1100 1105 1110

Arg Val Leu Gln Asp Ser Cys Ser His Phe Val Glu Asn Asn Ile

1115 1120 1125

Asn Leu Gly Thr Ser Ser Lys Lys Thr Phe Ala Ser Tyr Met Pro

1130 1135 1140

Gln Phe Gln Thr Cys Ser Thr Gln Thr His Lys Ile Met Glu Asn

1145 1150 1155

Lys Met Cys Asp Leu Thr Val

1160 1165

<210> 114

<211> 846

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hLEPR.mmh aa 1—818: F22-D839 from P48357 aa 819-846:

myc-myc-hexahistidine tag

<400> 114

Phe Asn Leu Ser Tyr Pro Ile Thr Pro Trp Arg Phe Lys Leu Ser Cys

1 5 10 15

Met Pro Pro Asn Ser Thr Tyr Asp Tyr Phe Leu Leu Pro Ala Gly Leu

20 25 30

Ser Lys Asn Thr Ser Asn Ser Asn Gly His Tyr Glu Thr Ala Val Glu

35 40 45

Pro Lys Phe Asn Ser Ser Gly Thr His Phe Ser Asn Leu Ser Lys Thr

50 55 60

Thr Phe His Cys Cys Phe Arg Ser Glu Gln Asp Arg Asn Cys Ser Leu

65 70 75 80

Cys Ala Asp Asn Ile Glu Gly Lys Thr Phe Val Ser Thr Val Asn Ser

85 90 95

Leu Val Phe Gln Gln Ile Asp Ala Asn Trp Asn Ile Gln Cys Trp Leu

100 105 110

Lys Gly Asp Leu Lys Leu Phe Ile Cys Tyr Val Glu Ser Leu Phe Lys

115 120 125

Asn Leu Phe Arg Asn Tyr Asn Tyr Lys Val His Leu Leu Tyr Val Leu

130 135 140

Pro Glu Val Leu Glu Asp Ser Pro Leu Val Pro Gln Lys Gly Ser Phe

145 150 155 160

Gln Met Val His Cys Asn Cys Ser Val His Glu Cys Cys Glu Cys Leu

165 170 175

Val Pro Val Pro Thr Ala Lys Leu Asn Asp Thr Leu Leu Met Cys Leu

180 185 190

Lys Ile Thr Ser Gly Gly Val Ile Phe Gln Ser Pro Leu Met Ser Val

195 200 205

Gln Pro Ile Asn Met Val Lys Pro Asp Pro Pro Leu Gly Leu His Met

210 215 220

Glu Ile Thr Asp Asp Gly Asn Leu Lys Ile Ser Trp Ser Ser Pro Pro

225 230 235 240

Leu Val Pro Phe Pro Leu Gln Tyr Gln Val Lys Tyr Ser Glu Asn Ser

245 250 255

Thr Thr Val Ile Arg Glu Ala Asp Lys Ile Val Ser Ala Thr Ser Leu

260 265 270

Leu Val Asp Ser Ile Leu Pro Gly Ser Ser Tyr Glu Val Gln Val Arg

275 280 285

Gly Lys Arg Leu Asp Gly Pro Gly Ile Trp Ser Asp Trp Ser Thr Pro

290 295 300

Arg Val Phe Thr Thr Gln Asp Val Ile Tyr Phe Pro Pro Lys Ile Leu

305 310 315 320

Thr Ser Val Gly Ser Asn Val Ser Phe His Cys Ile Tyr Lys Lys Glu

325 330 335

Asn Lys Ile Val Pro Ser Lys Glu Ile Val Trp Trp Met Asn Leu Ala

340 345 350

Glu Lys Ile Pro Gln Ser Gln Tyr Asp Val Val Ser Asp His Val Ser

355 360 365

Lys Val Thr Phe Phe Asn Leu Asn Glu Thr Lys Pro Arg Gly Lys Phe

370 375 380

Thr Tyr Asp Ala Val Tyr Cys Cys Asn Glu His Glu Cys His His Arg

385 390 395 400

Tyr Ala Glu Leu Tyr Val Ile Asp Val Asn Ile Asn Ile Ser Cys Glu

405 410 415

Thr Asp Gly Tyr Leu Thr Lys Met Thr Cys Arg Trp Ser Thr Ser Thr

420 425 430

Ile Gln Ser Leu Ala Glu Ser Thr Leu Gln Leu Arg Tyr His Arg Ser

435 440 445

Ser Leu Tyr Cys Ser Asp Ile Pro Ser Ile His Pro Ile Ser Glu Pro

450 455 460

Lys Asp Cys Tyr Leu Gln Ser Asp Gly Phe Tyr Glu Cys Ile Phe Gln

465 470 475 480

Pro Ile Phe Leu Leu Ser Gly Tyr Thr Met Trp Ile Arg Ile Asn His

485 490 495

Ser Leu Gly Ser Leu Asp Ser Pro Pro Thr Cys Val Leu Pro Asp Ser

500 505 510

Val Val Lys Pro Leu Pro Pro Ser Ser Val Lys Ala Glu Ile Thr Ile

515 520 525

Asn Ile Gly Leu Leu Lys Ile Ser Trp Glu Lys Pro Val Phe Pro Glu

530 535 540

Asn Asn Leu Gln Phe Gln Ile Arg Tyr Gly Leu Ser Gly Lys Glu Val

545 550 555 560

Gln Trp Lys Met Tyr Glu Val Tyr Asp Ala Lys Ser Lys Ser Val Ser

565 570 575

Leu Pro Val Pro Asp Leu Cys Ala Val Tyr Ala Val Gln Val Arg Cys

580 585 590

Lys Arg Leu Asp Gly Leu Gly Tyr Trp Ser Asn Trp Ser Asn Pro Ala

595 600 605

Tyr Thr Val Val Met Asp Ile Lys Val Pro Met Arg Gly Pro Glu Phe

610 615 620

Trp Arg Ile Ile Asn Gly Asp Thr Met Lys Lys Glu Lys Asn Val Thr

625 630 635 640

Leu Leu Trp Lys Pro Leu Met Lys Asn Asp Ser Leu Cys Ser Val Gln

645 650 655

Arg Tyr Val Ile Asn His His Thr Ser Cys Asn Gly Thr Trp Ser Glu

660 665 670

Asp Val Gly Asn His Thr Lys Phe Thr Phe Leu Trp Thr Glu Gln Ala

675 680 685

His Thr Val Thr Val Leu Ala Ile Asn Ser Ile Gly Ala Ser Val Ala

690 695 700

Asn Phe Asn Leu Thr Phe Ser Trp Pro Met Ser Lys Val Asn Ile Val

705 710 715 720

Gln Ser Leu Ser Ala Tyr Pro Leu Asn Ser Ser Cys Val Ile Val Ser

725 730 735

Trp Ile Leu Ser Pro Ser Asp Tyr Lys Leu Met Tyr Phe Ile Ile Glu

740 745 750

Trp Lys Asn Leu Asn Glu Asp Gly Glu Ile Lys Trp Leu Arg Ile Ser

755 760 765

Ser Ser Val Lys Lys Tyr Tyr Ile His Asp His Phe Ile Pro Ile Glu

770 775 780

Lys Tyr Gln Phe Ser Leu Tyr Pro Ile Phe Met Glu Gly Val Gly Lys

785 790 795 800

Pro Lys Ile Ile Asn Ser Phe Thr Gln Asp Asp Ile Glu Lys His Gln

805 810 815

Ser Asp Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Gly Gly Glu Gln

820 825 830

Lys Leu Ile Ser Glu Glu Asp Leu His His His His His

835 840 845

<210> 115

<211> 1051

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hLEPR.mFc aa 1–818: F22–D839 from P48357 aa 819–1051: mouse IgG2a

(E98—K330 from P01863)

<400> 115

Phe Asn Leu Ser Tyr Pro Ile Thr Pro Trp Arg Phe Lys Leu Ser Cys

1 5 10 15

Met Pro Pro Asn Ser Thr Tyr Asp Tyr Phe Leu Leu Pro Ala Gly Leu

20 25 30

Ser Lys Asn Thr Ser Asn Ser Asn Gly His Tyr Glu Thr Ala Val Glu

35 40 45

Pro Lys Phe Asn Ser Ser Gly Thr His Phe Ser Asn Leu Ser Lys Thr

50 55 60

Thr Phe His Cys Cys Phe Arg Ser Glu Gln Asp Arg Asn Cys Ser Leu

65 70 75 80

Cys Ala Asp Asn Ile Glu Gly Lys Thr Phe Val Ser Thr Val Asn Ser

85 90 95

Leu Val Phe Gln Gln Ile Asp Ala Asn Trp Asn Ile Gln Cys Trp Leu

100 105 110

Lys Gly Asp Leu Lys Leu Phe Ile Cys Tyr Val Glu Ser Leu Phe Lys

115 120 125

Asn Leu Phe Arg Asn Tyr Asn Tyr Lys Val His Leu Leu Tyr Val Leu

130 135 140

Pro Glu Val Leu Glu Asp Ser Pro Leu Val Pro Gln Lys Gly Ser Phe

145 150 155 160

Gln Met Val His Cys Asn Cys Ser Val His Glu Cys Cys Glu Cys Leu

165 170 175

Val Pro Val Pro Thr Ala Lys Leu Asn Asp Thr Leu Leu Met Cys Leu

180 185 190

Lys Ile Thr Ser Gly Gly Val Ile Phe Gln Ser Pro Leu Met Ser Val

195 200 205

Gln Pro Ile Asn Met Val Lys Pro Asp Pro Pro Leu Gly Leu His Met

210 215 220

Glu Ile Thr Asp Asp Gly Asn Leu Lys Ile Ser Trp Ser Ser Pro Pro

225 230 235 240

Leu Val Pro Phe Pro Leu Gln Tyr Gln Val Lys Tyr Ser Glu Asn Ser

245 250 255

Thr Thr Val Ile Arg Glu Ala Asp Lys Ile Val Ser Ala Thr Ser Leu

260 265 270

Leu Val Asp Ser Ile Leu Pro Gly Ser Ser Tyr Glu Val Gln Val Arg

275 280 285

Gly Lys Arg Leu Asp Gly Pro Gly Ile Trp Ser Asp Trp Ser Thr Pro

290 295 300

Arg Val Phe Thr Thr Gln Asp Val Ile Tyr Phe Pro Pro Lys Ile Leu

305 310 315 320

Thr Ser Val Gly Ser Asn Val Ser Phe His Cys Ile Tyr Lys Lys Glu

325 330 335

Asn Lys Ile Val Pro Ser Lys Glu Ile Val Trp Trp Met Asn Leu Ala

340 345 350

Glu Lys Ile Pro Gln Ser Gln Tyr Asp Val Val Ser Asp His Val Ser

355 360 365

Lys Val Thr Phe Phe Asn Leu Asn Glu Thr Lys Pro Arg Gly Lys Phe

370 375 380

Thr Tyr Asp Ala Val Tyr Cys Cys Asn Glu His Glu Cys His His Arg

385 390 395 400

Tyr Ala Glu Leu Tyr Val Ile Asp Val Asn Ile Asn Ile Ser Cys Glu

405 410 415

Thr Asp Gly Tyr Leu Thr Lys Met Thr Cys Arg Trp Ser Thr Ser Thr

420 425 430

Ile Gln Ser Leu Ala Glu Ser Thr Leu Gln Leu Arg Tyr His Arg Ser

435 440 445

Ser Leu Tyr Cys Ser Asp Ile Pro Ser Ile His Pro Ile Ser Glu Pro

450 455 460

Lys Asp Cys Tyr Leu Gln Ser Asp Gly Phe Tyr Glu Cys Ile Phe Gln

465 470 475 480

Pro Ile Phe Leu Leu Ser Gly Tyr Thr Met Trp Ile Arg Ile Asn His

485 490 495

Ser Leu Gly Ser Leu Asp Ser Pro Pro Thr Cys Val Leu Pro Asp Ser

500 505 510

Val Val Lys Pro Leu Pro Pro Ser Ser Val Lys Ala Glu Ile Thr Ile

515 520 525

Asn Ile Gly Leu Leu Lys Ile Ser Trp Glu Lys Pro Val Phe Pro Glu

530 535 540

Asn Asn Leu Gln Phe Gln Ile Arg Tyr Gly Leu Ser Gly Lys Glu Val

545 550 555 560

Gln Trp Lys Met Tyr Glu Val Tyr Asp Ala Lys Ser Lys Ser Val Ser

565 570 575

Leu Pro Val Pro Asp Leu Cys Ala Val Tyr Ala Val Gln Val Arg Cys

580 585 590

Lys Arg Leu Asp Gly Leu Gly Tyr Trp Ser Asn Trp Ser Asn Pro Ala

595 600 605

Tyr Thr Val Val Met Asp Ile Lys Val Pro Met Arg Gly Pro Glu Phe

610 615 620

Trp Arg Ile Ile Asn Gly Asp Thr Met Lys Lys Glu Lys Asn Val Thr

625 630 635 640

Leu Leu Trp Lys Pro Leu Met Lys Asn Asp Ser Leu Cys Ser Val Gln

645 650 655

Arg Tyr Val Ile Asn His His Thr Ser Cys Asn Gly Thr Trp Ser Glu

660 665 670

Asp Val Gly Asn His Thr Lys Phe Thr Phe Leu Trp Thr Glu Gln Ala

675 680 685

His Thr Val Thr Val Leu Ala Ile Asn Ser Ile Gly Ala Ser Val Ala

690 695 700

Asn Phe Asn Leu Thr Phe Ser Trp Pro Met Ser Lys Val Asn Ile Val

705 710 715 720

Gln Ser Leu Ser Ala Tyr Pro Leu Asn Ser Ser Cys Val Ile Val Ser

725 730 735

Trp Ile Leu Ser Pro Ser Asp Tyr Lys Leu Met Tyr Phe Ile Ile Glu

740 745 750

Trp Lys Asn Leu Asn Glu Asp Gly Glu Ile Lys Trp Leu Arg Ile Ser

755 760 765

Ser Ser Val Lys Lys Tyr Tyr Ile His Asp His Phe Ile Pro Ile Glu

770 775 780

Lys Tyr Gln Phe Ser Leu Tyr Pro Ile Phe Met Glu Gly Val Gly Lys

785 790 795 800

Pro Lys Ile Ile Asn Ser Phe Thr Gln Asp Asp Ile Glu Lys His Gln

805 810 815

Ser Asp Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys

820 825 830

Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro

835 840 845

Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr

850 855 860

Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser

865 870 875 880

Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His

885 890 895

Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile

900 905 910

Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn

915 920 925

Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys

930 935 940

Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu

945 950 955 960

Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe

965 970 975

Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu

980 985 990

Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr

995 1000 1005

Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu

1010 1015 1020

Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn

1025 1030 1035

His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

1040 1045 1050

<210> 116

<211> 1045

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hLEPR.hFc aa 1-818: F22-D839 from P48357 aa 819-1045: label representing

human IgG1 (D104—K330 from P01857)

<400> 116

Phe Asn Leu Ser Tyr Pro Ile Thr Pro Trp Arg Phe Lys Leu Ser Cys

1 5 10 15

Met Pro Pro Asn Ser Thr Tyr Asp Tyr Phe Leu Leu Pro Ala Gly Leu

20 25 30

Ser Lys Asn Thr Ser Asn Ser Asn Gly His Tyr Glu Thr Ala Val Glu

35 40 45

Pro Lys Phe Asn Ser Ser Gly Thr His Phe Ser Asn Leu Ser Lys Thr

50 55 60

Thr Phe His Cys Cys Phe Arg Ser Glu Gln Asp Arg Asn Cys Ser Leu

65 70 75 80

Cys Ala Asp Asn Ile Glu Gly Lys Thr Phe Val Ser Thr Val Asn Ser

85 90 95

Leu Val Phe Gln Gln Ile Asp Ala Asn Trp Asn Ile Gln Cys Trp Leu

100 105 110

Lys Gly Asp Leu Lys Leu Phe Ile Cys Tyr Val Glu Ser Leu Phe Lys

115 120 125

Asn Leu Phe Arg Asn Tyr Asn Tyr Lys Val His Leu Leu Tyr Val Leu

130 135 140

Pro Glu Val Leu Glu Asp Ser Pro Leu Val Pro Gln Lys Gly Ser Phe

145 150 155 160

Gln Met Val His Cys Asn Cys Ser Val His Glu Cys Cys Glu Cys Leu

165 170 175

Val Pro Val Pro Thr Ala Lys Leu Asn Asp Thr Leu Leu Met Cys Leu

180 185 190

Lys Ile Thr Ser Gly Gly Val Ile Phe Gln Ser Pro Leu Met Ser Val

195 200 205

Gln Pro Ile Asn Met Val Lys Pro Asp Pro Pro Leu Gly Leu His Met

210 215 220

Glu Ile Thr Asp Asp Gly Asn Leu Lys Ile Ser Trp Ser Ser Pro Pro

225 230 235 240

Leu Val Pro Phe Pro Leu Gln Tyr Gln Val Lys Tyr Ser Glu Asn Ser

245 250 255

Thr Thr Val Ile Arg Glu Ala Asp Lys Ile Val Ser Ala Thr Ser Leu

260 265 270

Leu Val Asp Ser Ile Leu Pro Gly Ser Ser Tyr Glu Val Gln Val Arg

275 280 285

Gly Lys Arg Leu Asp Gly Pro Gly Ile Trp Ser Asp Trp Ser Thr Pro

290 295 300

Arg Val Phe Thr Thr Gln Asp Val Ile Tyr Phe Pro Pro Lys Ile Leu

305 310 315 320

Thr Ser Val Gly Ser Asn Val Ser Phe His Cys Ile Tyr Lys Lys Glu

325 330 335

Asn Lys Ile Val Pro Ser Lys Glu Ile Val Trp Trp Met Asn Leu Ala

340 345 350

Glu Lys Ile Pro Gln Ser Gln Tyr Asp Val Val Ser Asp His Val Ser

355 360 365

Lys Val Thr Phe Phe Asn Leu Asn Glu Thr Lys Pro Arg Gly Lys Phe

370 375 380

Thr Tyr Asp Ala Val Tyr Cys Cys Asn Glu His Glu Cys His His Arg

385 390 395 400

Tyr Ala Glu Leu Tyr Val Ile Asp Val Asn Ile Asn Ile Ser Cys Glu

405 410 415

Thr Asp Gly Tyr Leu Thr Lys Met Thr Cys Arg Trp Ser Thr Ser Thr

420 425 430

Ile Gln Ser Leu Ala Glu Ser Thr Leu Gln Leu Arg Tyr His Arg Ser

435 440 445

Ser Leu Tyr Cys Ser Asp Ile Pro Ser Ile His Pro Ile Ser Glu Pro

450 455 460

Lys Asp Cys Tyr Leu Gln Ser Asp Gly Phe Tyr Glu Cys Ile Phe Gln

465 470 475 480

Pro Ile Phe Leu Leu Ser Gly Tyr Thr Met Trp Ile Arg Ile Asn His

485 490 495

Ser Leu Gly Ser Leu Asp Ser Pro Pro Thr Cys Val Leu Pro Asp Ser

500 505 510

Val Val Lys Pro Leu Pro Pro Ser Ser Val Lys Ala Glu Ile Thr Ile

515 520 525

Asn Ile Gly Leu Leu Lys Ile Ser Trp Glu Lys Pro Val Phe Pro Glu

530 535 540

Asn Asn Leu Gln Phe Gln Ile Arg Tyr Gly Leu Ser Gly Lys Glu Val

545 550 555 560

Gln Trp Lys Met Tyr Glu Val Tyr Asp Ala Lys Ser Lys Ser Val Ser

565 570 575

Leu Pro Val Pro Asp Leu Cys Ala Val Tyr Ala Val Gln Val Arg Cys

580 585 590

Lys Arg Leu Asp Gly Leu Gly Tyr Trp Ser Asn Trp Ser Asn Pro Ala

595 600 605

Tyr Thr Val Val Met Asp Ile Lys Val Pro Met Arg Gly Pro Glu Phe

610 615 620

Trp Arg Ile Ile Asn Gly Asp Thr Met Lys Lys Glu Lys Asn Val Thr

625 630 635 640

Leu Leu Trp Lys Pro Leu Met Lys Asn Asp Ser Leu Cys Ser Val Gln

645 650 655

Arg Tyr Val Ile Asn His His Thr Ser Cys Asn Gly Thr Trp Ser Glu

660 665 670

Asp Val Gly Asn His Thr Lys Phe Thr Phe Leu Trp Thr Glu Gln Ala

675 680 685

His Thr Val Thr Val Leu Ala Ile Asn Ser Ile Gly Ala Ser Val Ala

690 695 700

Asn Phe Asn Leu Thr Phe Ser Trp Pro Met Ser Lys Val Asn Ile Val

705 710 715 720

Gln Ser Leu Ser Ala Tyr Pro Leu Asn Ser Ser Cys Val Ile Val Ser

725 730 735

Trp Ile Leu Ser Pro Ser Asp Tyr Lys Leu Met Tyr Phe Ile Ile Glu

740 745 750

Trp Lys Asn Leu Asn Glu Asp Gly Glu Ile Lys Trp Leu Arg Ile Ser

755 760 765

Ser Ser Val Lys Lys Tyr Tyr Ile His Asp His Phe Ile Pro Ile Glu

770 775 780

Lys Tyr Gln Phe Ser Leu Tyr Pro Ile Phe Met Glu Gly Val Gly Lys

785 790 795 800

Pro Lys Ile Ile Asn Ser Phe Thr Gln Asp Asp Ile Glu Lys His Gln

805 810 815

Ser Asp Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

820 825 830

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

835 840 845

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

850 855 860

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

865 870 875 880

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

885 890 895

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

900 905 910

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

915 920 925

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

930 935 940

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

945 950 955 960

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

965 970 975

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

980 985 990

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

995 1000 1005

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

1010 1015 1020

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

1025 1030 1035

Leu Ser Leu Ser Pro Gly Lys

1040 1045

<210> 117

<211> 844

<212> PROTEIN

<213> Artificial sequence

<220>

<223> MFlePR.mmh aa 1—816: F22—D837 from Macaca fascicularis with T827A substitution

from XP_005543194.1 aa 817—844: myc-myc-hexahistidine tag

<400> 117

Phe Asn Leu Ser Tyr Pro Ile Thr Pro Trp Arg Phe Lys Leu Ser Cys

1 5 10 15

Met Pro Pro Asn Ser Thr Tyr Asp Tyr Phe Leu Leu Pro Ala Gly Leu

20 25 30

Ser Lys Asn Thr Ser Asn Leu Asn Gly His Tyr Glu Thr Ala Val Glu

35 40 45

Phe Asn Ser Ser Asp Thr His Phe Ser Asn Leu Ser Lys Thr Thr Phe

50 55 60

His Cys Cys Phe Arg Ser Glu Gln Asp Arg Asn Cys Ser Leu Cys Ala

65 70 75 80

Asp Asn Ile Glu Gly Lys Thr Phe Val Ser Thr Val Asn Ser Ser Val

85 90 95

Phe Gln Gln Met Gly Ala Asn Trp Asn Ile Gln Cys Trp Leu Lys Gly

100 105 110

Asp Leu Lys Leu Phe Ile Cys Tyr Val Glu Ser Leu Phe Lys Asn Pro

115 120 125

Phe Lys Asn Tyr Lys His Lys Val His Leu Leu Tyr Val Leu Pro Glu

130 135 140

Val Leu Glu Asp Ser Pro Leu Val Pro Gln Lys Gly Ser Phe Gln Met

145 150 155 160

Val His Cys Asn Cys Ser Val His Glu Arg Cys Glu Cys Leu Val Pro

165 170 175

Val Pro Thr Ala Lys Leu Asn Asp Thr Leu Leu Met Cys Leu Lys Ile

180 185 190

Thr Ser Gly Gly Val Ile Phe Gln Ser Pro Leu Met Ser Val Gln Pro

195 200 205

Ile Asn Met Val Lys Pro Asp Pro Pro Leu Gly Leu Arg Met Glu Ile

210 215 220

Thr Asp Asp Gly Asn Leu Lys Ile Ser Trp Ser Ser Pro Pro Leu Val

225 230 235 240

Pro Phe Pro Leu Gln Tyr Glu Val Lys Tyr Ser Glu Asn Ser Thr Thr

245 250 255

Val Ile Arg Glu Ala Asp Lys Ile Val Ser Ala Thr Ser Leu Leu Val

260 265 270

Asp Gly Ile Leu Pro Gly Ser Ser Tyr Glu Val Gln Val Arg Gly Lys

275 280 285

Arg Leu Asp Gly Pro Gly Ile Trp Ser Asp Trp Ser Thr Pro His Val

290 295 300

Phe Thr Thr Gln Asp Val Ile Tyr Phe Pro Pro Lys Ile Leu Thr Ser

305 310 315 320

Val Gly Ser Asn Val Ser Phe His Cys Ile Tyr Lys Asn Glu Asn Lys

325 330 335

Ile Val Ser Ser Lys Lys Ile Val Trp Trp Met Asn Leu Ala Glu Lys

340 345 350

Ile Pro Gln Ser Gln Tyr Asp Val Val Ser Asp His Val Ser Lys Val

355 360 365

Thr Phe Phe Asn Leu Asn Glu Thr Lys Pro Arg Gly Lys Phe Thr Tyr

370 375 380

Asp Ala Val Tyr Cys Cys Asn Glu His Glu Cys His His Arg Tyr Ala

385 390 395 400

Glu Leu Tyr Val Ile Asp Val Asn Ile Asn Ile Ser Cys Glu Thr Asp

405 410 415

Gly His Leu Thr Lys Met Thr Cys Arg Trp Ser Thr Asn Thr Ile Gln

420 425 430

Ser Leu Ala Gly Ser Thr Leu Gln Leu Arg Tyr Arg Arg Ser Ser Leu

435 440 445

Tyr Cys Phe Asp Ile Pro Ser Ile His Pro Ile Ser Lys Pro Lys Asp

450 455 460

Cys Tyr Leu Gln Ser Asp Gly Phe Tyr Glu Cys Val Phe Gln Pro Ile

465 470 475 480

Phe Leu Leu Ser Gly Tyr Thr Met Trp Ile Arg Ile Asn His Pro Leu

485 490 495

Gly Ser Leu Asp Ser Pro Pro Thr Cys Val Leu Pro Asp Ser Val Val

500 505 510

Lys Pro Leu Pro Pro Ser Ser Val Lys Ala Glu Ile Ile Lys Asn Ile

515 520 525

Gly Leu Leu Lys Ile Ser Trp Glu Lys Pro Val Phe Pro Glu Asn Asn

530 535 540

Leu Gln Phe Gln Ile Arg Tyr Gly Leu Ser Gly Lys Glu Ile Gln Trp

545 550 555 560

Lys Met Tyr Asp Val Tyr Asp Ala Lys Ser Lys Ser Val Ser Leu Pro

565 570 575

Val Pro Asp Phe Cys Ala Val Tyr Ala Val Gln Val Arg Cys Lys Arg

580 585 590

Ser Asp Gly Leu Gly Leu Trp Ser Asn Trp Ser Asn Pro Ala Tyr Thr

595 600 605

Val Val Met Asp Ile Lys Val Pro Met Arg Gly Pro Glu Phe Trp Arg

610 615 620

Ile Ile Asn Gly Asp Thr Met Lys Lys Glu Lys Asn Val Thr Leu Leu

625 630 635 640

Trp Lys Pro Leu Met Lys Asn Asp Ser Leu Cys Ser Val Gln Arg Tyr

645 650 655

Val Ile Asn His His Thr Ser Cys Asn Gly Thr Trp Ser Glu Asp Val

660 665 670

Gly Asn His Thr Lys Phe Thr Phe Leu Trp Thr Glu Gln Ala His Thr

675 680 685

Val Thr Val Leu Ala Ile Asn Ser Ile Gly Ala Ser Val Ala Asn Phe

690 695 700

Asn Leu Thr Phe Ser Trp Pro Met Ser Lys Val Asn Ile Val Gln Ser

705 710 715 720

Leu Ser Ala Tyr Pro Leu Asn Ser Ser Cys Val Ile Leu Ser Trp Ile

725 730 735

Leu Ser Pro Ser Asp Tyr Lys Leu Met Tyr Phe Ile Ile Glu Trp Lys

740 745 750

Asn Leu Asn Glu Asp Gly Glu Ile Lys Trp Leu Arg Ile Ser Ser Ser

755 760 765

Val Lys Lys Tyr Tyr Ile His Asp His Phe Ile Pro Ile Glu Lys Tyr

770 775 780

Gln Phe Ser Leu Tyr Pro Ile Phe Met Glu Gly Val Gly Lys Pro Lys

785 790 795 800

Ile Ile Asn Ser Phe Ala Gln Asp Asn Thr Glu Lys His Gln Asn Asp

805 810 815

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Gly Gly Glu Gln Lys Leu

820 825 830

Ile Ser Glu Glu Asp Leu His His His His His

835 840

<210> 118

<211> 846

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mLEPR.mmh aa 1—818: mouse LEPR (L22—G839 NP_666258.2) aa

817–846: myc-myc-hexahistidine tag

<400> 118

Leu Asn Leu Ala Tyr Pro Ile Ser Pro Trp Lys Phe Lys Leu Phe Cys

1 5 10 15

Gly Pro Pro Asn Thr Thr Asp Asp Ser Phe Leu Ser Pro Ala Gly Ala

20 25 30

Pro Asn Asn Ala Ser Ala Leu Lys Gly Ala Ser Glu Ala Ile Val Glu

35 40 45

Ala Lys Phe Asn Ser Ser Gly Ile Tyr Val Pro Glu Leu Ser Lys Thr

50 55 60

Val Phe His Cys Cys Phe Gly Asn Glu Gln Gly Gln Asn Cys Ser Ala

65 70 75 80

Leu Thr Asp Asn Thr Glu Gly Lys Thr Leu Ala Ser Val Val Lys Ala

85 90 95

Ser Val Phe Arg Gln Leu Gly Val Asn Trp Asp Ile Glu Cys Trp Met

100 105 110

Lys Gly Asp Leu Thr Leu Phe Ile Cys His Met Glu Pro Leu Pro Lys

115 120 125

Asn Pro Phe Lys Asn Tyr Asp Ser Lys Val His Leu Leu Tyr Asp Leu

130 135 140

Pro Glu Val Ile Asp Asp Ser Pro Leu Pro Pro Leu Lys Asp Ser Phe

145 150 155 160

Gln Thr Val Gln Cys Asn Cys Ser Leu Arg Gly Cys Glu Cys His Val

165 170 175

Pro Val Pro Arg Ala Lys Leu Asn Tyr Ala Leu Leu Met Tyr Leu Glu

180 185 190

Ile Thr Ser Ala Gly Val Ser Phe Gln Ser Pro Leu Met Ser Leu Gln

195 200 205

Pro Met Leu Val Val Lys Pro Asp Pro Pro Leu Gly Leu His Met Glu

210 215 220

Val Thr Asp Asp Gly Asn Leu Lys Ile Ser Trp Asp Ser Gln Thr Met

225 230 235 240

Ala Pro Phe Pro Leu Gln Tyr Gln Val Lys Tyr Leu Glu Asn Ser Thr

245 250 255

Ile Val Arg Glu Ala Ala Glu Ile Val Ser Ala Thr Ser Leu Leu Val

260 265 270

Asp Ser Val Leu Pro Gly Ser Ser Tyr Glu Val Gln Val Arg Ser Lys

275 280 285

Arg Leu Asp Gly Ser Gly Val Trp Ser Asp Trp Ser Ser Pro Gln Val

290 295 300

Phe Thr Thr Gln Asp Val Val Tyr Phe Pro Pro Lys Ile Leu Thr Ser

305 310 315 320

Val Gly Ser Asn Ala Ser Phe His Cys Ile Tyr Lys Asn Glu Asn Gln

325 330 335

Ile Ile Ser Ser Lys Gln Ile Val Trp Trp Arg Asn Leu Ala Glu Lys

340 345 350

Ile Pro Glu Ile Gln Tyr Ser Ile Val Ser Asp Arg Val Ser Lys Val

355 360 365

Thr Phe Ser Asn Leu Lys Ala Thr Arg Pro Arg Gly Lys Phe Thr Tyr

370 375 380

Asp Ala Val Tyr Cys Cys Asn Glu Gln Ala Cys His His Arg Tyr Ala

385 390 395 400

Glu Leu Tyr Val Ile Asp Val Asn Ile Asn Ile Ser Cys Glu Thr Asp

405 410 415

Gly Tyr Leu Thr Lys Met Thr Cys Arg Trp Ser Pro Ser Thr Ile Gln

420 425 430

Ser Leu Val Gly Ser Thr Val Gln Leu Arg Tyr His Arg Arg Ser Leu

435 440 445

Tyr Cys Pro Asp Ser Pro Ser Ile His Pro Thr Ser Glu Pro Lys Asn

450 455 460

Cys Val Leu Gln Arg Asp Gly Phe Tyr Glu Cys Val Phe Gln Pro Ile

465 470 475 480

Phe Leu Leu Ser Gly Tyr Thr Met Trp Ile Arg Ile Asn His Ser Leu

485 490 495

Gly Ser Leu Asp Ser Pro Pro Thr Cys Val Leu Pro Asp Ser Val Val

500 505 510

Lys Pro Leu Pro Pro Ser Asn Val Lys Ala Glu Ile Thr Val Asn Thr

515 520 525

Gly Leu Leu Lys Val Ser Trp Glu Lys Pro Val Phe Pro Glu Asn Asn

530 535 540

Leu Gln Phe Gln Ile Arg Tyr Gly Leu Ser Gly Lys Glu Ile Gln Trp

545 550 555 560

Lys Thr His Glu Val Phe Asp Ala Lys Ser Lys Ser Ala Ser Leu Leu

565 570 575

Val Ser Asp Leu Cys Ala Val Tyr Val Val Gln Val Arg Cys Arg Arg

580 585 590

Leu Asp Gly Leu Gly Tyr Trp Ser Asn Trp Ser Ser Pro Ala Tyr Thr

595 600 605

Leu Val Met Asp Val Lys Val Pro Met Arg Gly Pro Glu Phe Trp Arg

610 615 620

Lys Met Asp Gly Asp Val Thr Lys Lys Glu Arg Asn Val Thr Leu Leu

625 630 635 640

Trp Lys Pro Leu Thr Lys Asn Asp Ser Leu Cys Ser Val Arg Arg Tyr

645 650 655

Val Val Lys His Arg Thr Ala His Asn Gly Thr Trp Ser Glu Asp Val

660 665 670

Gly Asn Arg Thr Asn Leu Thr Phe Leu Trp Thr Glu Pro Ala His Thr

675 680 685

Val Thr Val Leu Ala Val Asn Ser Leu Gly Ala Ser Leu Val Asn Phe

690 695 700

Asn Leu Thr Phe Ser Trp Pro Met Ser Lys Val Ser Ala Val Glu Ser

705 710 715 720

Leu Ser Ala Tyr Pro Leu Ser Ser Ser Cys Val Ile Leu Ser Trp Thr

725 730 735

Leu Ser Pro Asp Asp Tyr Ser Leu Leu Tyr Leu Val Ile Glu Trp Lys

740 745 750

Ile Leu Asn Glu Asp Asp Gly Met Lys Trp Leu Arg Ile Pro Ser Asn

755 760 765

Val Lys Lys Phe Tyr Ile His Asp Asn Phe Ile Pro Ile Glu Lys Tyr

770 775 780

Gln Phe Ser Leu Tyr Pro Val Phe Met Glu Gly Val Gly Lys Pro Lys

785 790 795 800

Ile Ile Asn Gly Phe Thr Lys Asp Ala Ile Asp Lys Gln Gln Asn Asp

805 810 815

Ala Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Gly Gly Glu Gln

820 825 830

Lys Leu Ile Ser Glu Glu Asp Leu His His His His His

835 840 845

<210> 119

<211> 846

<212> PROTEIN

<213> Artificial sequence

<220>

<223> rLEPR.mmh aa 1—818: rat LEPR (L22-G839 NP_036728.1) aa

819–846: myc-myc-hexahistidine tag

<400> 119

Leu Asn Leu Ala Tyr Pro Thr Ser Pro Trp Arg Phe Lys Leu Phe Cys

1 5 10 15

Ala Pro Pro Ser Thr Thr Asp Asp Ser Phe Leu Ser Pro Ala Gly Val

20 25 30

Pro Asn Asn Thr Ser Ser Leu Lys Gly Ala Ser Glu Ala Leu Val Glu

35 40 45

Ala Lys Phe Asn Ser Thr Gly Ile Tyr Val Ser Glu Leu Ser Lys Thr

50 55 60

Ile Phe His Cys Cys Phe Gly Asn Glu Gln Gly Gln Asn Cys Ser Ala

65 70 75 80

Leu Thr Gly Asn Thr Glu Gly Lys Thr Leu Ala Ser Val Val Lys Pro

85 90 95

Leu Val Phe Arg Gln Leu Gly Val Asn Trp Asp Ile Glu Cys Trp Met

100 105 110

Lys Gly Asp Leu Thr Leu Phe Ile Cys His Met Glu Pro Leu Leu Lys

115 120 125

Asn Pro Phe Lys Asn Tyr Asp Ser Lys Val His Leu Leu Tyr Asp Leu

130 135 140

Pro Glu Val Ile Asp Asp Leu Pro Leu Pro Pro Leu Lys Asp Ser Phe

145 150 155 160

Gln Thr Val Gln Cys Asn Cys Ser Val Arg Glu Cys Glu Cys His Val

165 170 175

Pro Val Pro Arg Ala Lys Val Asn Tyr Ala Leu Leu Met Tyr Leu Glu

180 185 190

Ile Thr Ser Ala Gly Val Ser Phe Gln Ser Pro Leu Met Ser Leu Gln

195 200 205

Pro Met Leu Val Val Lys Pro Asp Pro Pro Leu Gly Leu Arg Met Glu

210 215 220

Val Thr Asp Asp Gly Asn Leu Lys Ile Ser Trp Asp Ser Gln Thr Lys

225 230 235 240

Ala Pro Phe Pro Leu Gln Tyr Gln Val Lys Tyr Leu Glu Asn Ser Thr

245 250 255

Ile Val Arg Glu Ala Ala Glu Ile Val Ser Asp Thr Ser Leu Leu Val

260 265 270

Asp Ser Val Leu Pro Gly Ser Ser Tyr Glu Val Gln Val Arg Ser Lys

275 280 285

Arg Leu Asp Gly Ser Gly Val Trp Ser Asp Trp Ser Leu Pro Gln Leu

290 295 300

Phe Thr Thr Gln Asp Val Met Tyr Phe Pro Pro Lys Ile Leu Thr Ser

305 310 315 320

Val Gly Ser Asn Ala Ser Phe Cys Cys Ile Tyr Lys Asn Glu Asn Gln

325 330 335

Thr Ile Ser Ser Lys Gln Ile Val Trp Trp Met Asn Leu Ala Glu Lys

340 345 350

Ile Pro Glu Thr Gln Tyr Asn Thr Val Ser Asp His Ile Ser Lys Val

355 360 365

Thr Phe Ser Asn Leu Lys Ala Thr Arg Pro Arg Gly Lys Phe Thr Tyr

370 375 380

Asp Ala Val Tyr Cys Cys Asn Glu Gln Ala Cys His His Arg Tyr Ala

385 390 395 400

Glu Leu Tyr Val Ile Asp Val Asn Ile Asn Ile Ser Cys Glu Thr Asp

405 410 415

Gly Tyr Leu Thr Lys Met Thr Cys Arg Trp Ser Pro Ser Thr Ile Gln

420 425 430

Ser Leu Val Gly Ser Thr Val Gln Leu Arg Tyr His Arg Arg Ser Leu

435 440 445

Tyr Cys Pro Asp Asn Pro Ser Ile Arg Pro Thr Ser Glu Leu Lys Asn

450 455 460

Cys Val Leu Gln Thr Asp Gly Phe Tyr Glu Cys Val Phe Gln Pro Ile

465 470 475 480

Phe Leu Leu Ser Gly Tyr Thr Met Trp Ile Arg Ile Asn His Ser Leu

485 490 495

Gly Ser Leu Asp Ser Pro Pro Thr Cys Val Leu Pro Asp Ser Val Val

500 505 510

Lys Pro Leu Pro Pro Ser Asn Val Lys Ala Glu Ile Thr Ile Asn Thr

515 520 525

Gly Leu Leu Lys Val Ser Trp Glu Lys Pro Val Phe Pro Glu Asn Asn

530 535 540

Leu Gln Phe Gln Ile Arg Tyr Gly Leu Asn Gly Lys Glu Ile Gln Trp

545 550 555 560

Lys Thr His Glu Val Phe Asp Ala Lys Ser Lys Ser Ala Ser Leu Pro

565 570 575

Val Ser Asp Leu Cys Ala Val Tyr Val Val Gln Val Arg Cys Arg Arg

580 585 590

Leu Asp Gly Leu Gly Tyr Trp Ser Asn Trp Ser Ser Pro Ala Tyr Thr

595 600 605

Leu Val Met Asp Val Lys Val Pro Met Arg Gly Pro Glu Phe Trp Arg

610 615 620

Ile Met Asp Gly Asp Ile Thr Lys Lys Glu Arg Asn Val Thr Leu Leu

625 630 635 640

Trp Lys Pro Leu Met Lys Asn Asp Ser Leu Cys Ser Val Arg Arg Tyr

645 650 655

Val Val Lys His Arg Thr Ala His Asn Gly Thr Trp Ser Gln Asp Val

660 665 670

Gly Asn Gln Thr Asn Leu Thr Phe Leu Trp Ala Glu Ser Ala His Thr

675 680 685

Val Thr Val Leu Ala Ile Asn Ser Ile Gly Ala Ser Leu Val Asn Phe

690 695 700

Asn Leu Thr Phe Ser Trp Pro Met Ser Lys Val Asn Ala Val Gln Ser

705 710 715 720

Leu Ser Ala Tyr Pro Leu Ser Ser Ser Cys Val Ile Leu Ser Trp Thr

725 730 735

Leu Ser Pro Asn Asp Tyr Ser Leu Leu Tyr Leu Val Ile Glu Trp Lys

740 745 750

Asn Leu Asn Asp Asp Asp Gly Met Lys Trp Leu Arg Ile Pro Ser Asn

755 760 765

Val Asn Lys Tyr Tyr Ile His Asp Asn Phe Ile Pro Ile Glu Lys Tyr

770 775 780

Gln Phe Ser Leu Tyr Pro Val Phe Met Glu Gly Val Gly Lys Pro Lys

785 790 795 800

Ile Ile Asn Gly Phe Thr Lys Asp Asp Ile Ala Lys Gln Gln Asn Asp

805 810 815

Ala Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Gly Gly Glu Gln

820 825 830

Lys Leu Ile Ser Glu Glu Asp Leu His His His His His

835 840 845

<210> 120

<211> 1045

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mLEPR.hFc aa 1–818: mouse LEPR (L22–G839 NP_666258.2) aa

819-1045: tag representing human IgG1

(D104—K330 from P01857)

<400> 120

Leu Asn Leu Ala Tyr Pro Ile Ser Pro Trp Lys Phe Lys Leu Phe Cys

1 5 10 15

Gly Pro Pro Asn Thr Thr Asp Asp Ser Phe Leu Ser Pro Ala Gly Ala

20 25 30

Pro Asn Asn Ala Ser Ala Leu Lys Gly Ala Ser Glu Ala Ile Val Glu

35 40 45

Ala Lys Phe Asn Ser Ser Gly Ile Tyr Val Pro Glu Leu Ser Lys Thr

50 55 60

Val Phe His Cys Cys Phe Gly Asn Glu Gln Gly Gln Asn Cys Ser Ala

65 70 75 80

Leu Thr Asp Asn Thr Glu Gly Lys Thr Leu Ala Ser Val Val Lys Ala

85 90 95

Ser Val Phe Arg Gln Leu Gly Val Asn Trp Asp Ile Glu Cys Trp Met

100 105 110

Lys Gly Asp Leu Thr Leu Phe Ile Cys His Met Glu Pro Leu Pro Lys

115 120 125

Asn Pro Phe Lys Asn Tyr Asp Ser Lys Val His Leu Leu Tyr Asp Leu

130 135 140

Pro Glu Val Ile Asp Asp Ser Pro Leu Pro Pro Leu Lys Asp Ser Phe

145 150 155 160

Gln Thr Val Gln Cys Asn Cys Ser Leu Arg Gly Cys Glu Cys His Val

165 170 175

Pro Val Pro Arg Ala Lys Leu Asn Tyr Ala Leu Leu Met Tyr Leu Glu

180 185 190

Ile Thr Ser Ala Gly Val Ser Phe Gln Ser Pro Leu Met Ser Leu Gln

195 200 205

Pro Met Leu Val Val Lys Pro Asp Pro Pro Leu Gly Leu His Met Glu

210 215 220

Val Thr Asp Asp Gly Asn Leu Lys Ile Ser Trp Asp Ser Gln Thr Met

225 230 235 240

Ala Pro Phe Pro Leu Gln Tyr Gln Val Lys Tyr Leu Glu Asn Ser Thr

245 250 255

Ile Val Arg Glu Ala Ala Glu Ile Val Ser Ala Thr Ser Leu Leu Val

260 265 270

Asp Ser Val Leu Pro Gly Ser Ser Tyr Glu Val Gln Val Arg Ser Lys

275 280 285

Arg Leu Asp Gly Ser Gly Val Trp Ser Asp Trp Ser Ser Pro Gln Val

290 295 300

Phe Thr Thr Gln Asp Val Val Tyr Phe Pro Pro Lys Ile Leu Thr Ser

305 310 315 320

Val Gly Ser Asn Ala Ser Phe His Cys Ile Tyr Lys Asn Glu Asn Gln

325 330 335

Ile Ile Ser Ser Lys Gln Ile Val Trp Trp Arg Asn Leu Ala Glu Lys

340 345 350

Ile Pro Glu Ile Gln Tyr Ser Ile Val Ser Asp Arg Val Ser Lys Val

355 360 365

Thr Phe Ser Asn Leu Lys Ala Thr Arg Pro Arg Gly Lys Phe Thr Tyr

370 375 380

Asp Ala Val Tyr Cys Cys Asn Glu Gln Ala Cys His His Arg Tyr Ala

385 390 395 400

Glu Leu Tyr Val Ile Asp Val Asn Ile Asn Ile Ser Cys Glu Thr Asp

405 410 415

Gly Tyr Leu Thr Lys Met Thr Cys Arg Trp Ser Pro Ser Thr Ile Gln

420 425 430

Ser Leu Val Gly Ser Thr Val Gln Leu Arg Tyr His Arg Arg Ser Leu

435 440 445

Tyr Cys Pro Asp Ser Pro Ser Ile His Pro Thr Ser Glu Pro Lys Asn

450 455 460

Cys Val Leu Gln Arg Asp Gly Phe Tyr Glu Cys Val Phe Gln Pro Ile

465 470 475 480

Phe Leu Leu Ser Gly Tyr Thr Met Trp Ile Arg Ile Asn His Ser Leu

485 490 495

Gly Ser Leu Asp Ser Pro Pro Thr Cys Val Leu Pro Asp Ser Val Val

500 505 510

Lys Pro Leu Pro Pro Ser Asn Val Lys Ala Glu Ile Thr Val Asn Thr

515 520 525

Gly Leu Leu Lys Val Ser Trp Glu Lys Pro Val Phe Pro Glu Asn Asn

530 535 540

Leu Gln Phe Gln Ile Arg Tyr Gly Leu Ser Gly Lys Glu Ile Gln Trp

545 550 555 560

Lys Thr His Glu Val Phe Asp Ala Lys Ser Lys Ser Ala Ser Leu Leu

565 570 575

Val Ser Asp Leu Cys Ala Val Tyr Val Val Gln Val Arg Cys Arg Arg

580 585 590

Leu Asp Gly Leu Gly Tyr Trp Ser Asn Trp Ser Ser Pro Ala Tyr Thr

595 600 605

Leu Val Met Asp Val Lys Val Pro Met Arg Gly Pro Glu Phe Trp Arg

610 615 620

Lys Met Asp Gly Asp Val Thr Lys Lys Glu Arg Asn Val Thr Leu Leu

625 630 635 640

Trp Lys Pro Leu Thr Lys Asn Asp Ser Leu Cys Ser Val Arg Arg Tyr

645 650 655

Val Val Lys His Arg Thr Ala His Asn Gly Thr Trp Ser Glu Asp Val

660 665 670

Gly Asn Arg Thr Asn Leu Thr Phe Leu Trp Thr Glu Pro Ala His Thr

675 680 685

Val Thr Val Leu Ala Val Asn Ser Leu Gly Ala Ser Leu Val Asn Phe

690 695 700

Asn Leu Thr Phe Ser Trp Pro Met Ser Lys Val Ser Ala Val Glu Ser

705 710 715 720

Leu Ser Ala Tyr Pro Leu Ser Ser Ser Cys Val Ile Leu Ser Trp Thr

725 730 735

Leu Ser Pro Asp Asp Tyr Ser Leu Leu Tyr Leu Val Ile Glu Trp Lys

740 745 750

Ile Leu Asn Glu Asp Asp Gly Met Lys Trp Leu Arg Ile Pro Ser Asn

755 760 765

Val Lys Lys Phe Tyr Ile His Asp Asn Phe Ile Pro Ile Glu Lys Tyr

770 775 780

Gln Phe Ser Leu Tyr Pro Val Phe Met Glu Gly Val Gly Lys Pro Lys

785 790 795 800

Ile Ile Asn Gly Phe Thr Lys Asp Ala Ile Asp Lys Gln Gln Asn Asp

805 810 815

Ala Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

820 825 830

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

835 840 845

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

850 855 860

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

865 870 875 880

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

885 890 895

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

900 905 910

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

915 920 925

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

930 935 940

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

945 950 955 960

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

965 970 975

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

980 985 990

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

995 1000 1005

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

1010 1015 1020

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

1025 1030 1035

Leu Ser Leu Ser Pro Gly Lys

1040 1045

<---

Claims (4)

1. A method of increasing bone mass in a subject having low bone mass, which is a symptom of congenital leptin deficiency from which the subject suffers, comprising administering a pharmaceutical composition containing an antibody or antigen-binding fragment thereof that binds the human leptin receptor (LEPR) and activates LEPR-mediated signal transduction containing a heavy chain variable region (HCVR), which contains HCDR1, HCDR2 and HCDR3 HCVR containing the amino acid sequence of SEQ ID NO: 26; and a light chain variable region (LCVR) which contains LCDR1, LCDR2 and LCDR3 LCVR containing the amino acid sequence of SEQ ID NO: 10, and a pharmaceutically acceptable carrier or diluent, to a subject in need thereof. 2. The method 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 method according to claim 1, where the antibody or its antigen-binding fragment contains a combination of amino acid sequences HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 under SEQ ID NO: 28/30/32/12/14/16. 4. Method according to any one of paragraphs. 1-3, further comprising administering a second therapeutic agent to a subject, wherein the second therapeutic agent is 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 inhibitor -glucose cotransporter 2 (SGLT2), GLP-1 agonist/analog, glucagon inhibitor (GCG), glucagon receptor inhibitor (GCGR), angiopoietin-like protein inhibitor (ANGPTL), phentermine, orlistat, topiramate, bupropion, topiramate/phentermine, bupropion /naltrexone, bupropion/zonisamide, pramlintide/metreleptin, lorcaserin, cetilistat, tesofensine, wellneperite, anticonvulsant, digoxin, coumadin, vitamin D, thyroxine, thyroid supplement, vitamin supplement, calcium supplement, carnitine, coenzyme Q10, medicinal anti-constipation drug, anti-allergy drugs, gabapentin, narcotic drug, ketamine, lidocaine and venlafaxine hydrochloride.