Antigen-binding protein that activates the leptin receptor

Abstract

To provide alternative approaches to treating leptin resistance and other conditions associated with leptin deficiency or hypoleptinemia.SOLUTION: The present invention provides antibodies and antigen-binding fragments thereof that bind human leptin receptor (LEPR). The antibodies of the present invention are agonist antibodies; i.e., binding of the anti-LEPR antibodies of the invention to LEPR causes, inter alia, activation of leptin receptor signaling in cells. 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, e.g., for mimicking, substituting for, or supplementing the normal biological activity of leptin in a subject. The antibodies and antigen-binding fragments of the present invention are therefore useful in the therapeutic treatment of diseases and disorders associated with leptin resistance and leptin deficiency.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to antibodies and antigen-binding fragments of antibodies that bind to the human leptin receptor (LEPR), as well as therapeutic and diagnostic methods using these antibodies.

[0002] Sequence Listing A formal copy of the sequence listing has been electronically submitted simultaneously with the specification via EFS-Web as a sequence listing in ASCII format with the file name 2016_10_11_10178WO01_Sequence_Listing_as_Filed_ST25.TXT, a creation date of October 11, 2016, and a size of approximately 99.6 kilobytes. The sequence listing contained in this ASCII-formatted document is part of this specification and is hereby incorporated by reference in its entirety.

Background Art

[0003] Leptin is a polypeptide hormone mainly expressed by adipose tissue and is involved in the regulation of metabolism, energy balance, and food intake. Leptin activity is mediated by its interaction with the leptin receptor and signal transduction through this 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 LEPR signal transduction deficiency / signal transduction disorder mutations are associated with obesity, type 2 diabetes, dyslipidemia, lipodystrophy, hepatic steatosis, non-alcoholic and alcoholic fatty liver diseases, severe insulin resistance, Morquio syndrome / Downahue syndrome, Rabson-Mendenhall syndrome, and related complications. Treatment approaches to address leptin resistance, leptin deficiency, and hypoleptinemia (e.g., lipodystrophy) have mainly focused on the delivery of additional leptin or leptin analogs to affected individuals. However, such approaches generally show limited efficacy, particularly in leptin-resistant individuals, and are frequently accompanied by adverse side effects. Thus, there is a need in the art for alternative approaches to treat leptin resistance and other conditions associated with leptin deficiency or hypoleptinemia. Summary of the Invention Means for Solving the Problems

[0004] The present invention provides antibodies that bind to the human leptin receptor (LEPR) and antigen-binding fragments thereof. The antibodies of the present invention are agonist antibodies; that is, when the anti-LEPR antibodies of the present invention bind to LEPR, intracellular leptin receptor signaling is activated, among other things. 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, for mimicking, substituting, or supplementing the normal biological activity of leptin in a subject. Accordingly, the antibodies and antigen-binding fragments of the present invention are useful in the therapeutic treatment of diseases and disorders associated with leptin resistance and leptin deficiency.

[0005] The antibodies of the present invention may be full-length (e.g., IgG1 or IgG4 antibodies) or may contain only the antigen-binding portion (e.g., Fab, F(ab’)2, or scFv fragments) and can be modified to affect functionality, e.g., to eliminate 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 shows the amino acid sequence identifiers of the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) of the exemplary anti-LEPR antibodies. Table 2 shows the nucleic acid sequence identifiers of the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the exemplary anti-LEPR antibodies.

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

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

[0009] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to LEPR and comprises an amino acid sequence pair (HCVR / LCVR) of HCVR and LCVR, wherein the HCVR comprises any one of the HCVR amino acid sequences listed in Table 1 that is paired with any one of the LCVR amino acid sequences listed in Table 1. According to certain embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that comprises an HCVR / LCVR amino acid sequence pair contained in any of the exemplary anti-LEPR antibodies listed in Table 1. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from the group consisting of SEQ ID 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 an antibody or an antigen-binding fragment thereof that specifically binds to LEPR and comprises a heavy chain complementarity-determining region 1 (HCDR1) comprising an amino acid sequence selected from the amino acid sequences of HCDR1s listed in Table 1, or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0011] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to LEPR and comprises a heavy chain complementarity-determining region 2 (HCDR2) comprising an amino acid sequence selected from the amino acid sequences of HCDR2s listed in Table 1, or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0012] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to LEPR and comprises a heavy chain complementarity-determining region 3 (HCDR3) comprising an amino acid sequence listed in Table 1 or a sequence substantially similar thereto and selected from any of the sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

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

[0014] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to LEPR and comprises a light chain complementarity-determining region 2 (LCDR2) comprising an amino acid sequence listed in Table 1 or a sequence substantially similar thereto and selected from any of the sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0015] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to LEPR and comprises a light chain complementarity-determining region 3 (LCDR3) comprising an amino acid sequence listed in Table 1 or a sequence substantially similar thereto and selected from any of the sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0016] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to LEPR and comprises an amino acid sequence pair of HCDR3 and LCDR3 (HCDR3 / LCDR3) including any one of the HCDR3 amino acid sequences listed in Table 1 paired with any one of the LCDR3 amino acid sequences listed in Table 1. According to certain embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that comprises an HCDR3 / LCDR3 amino acid sequence pair contained in any of the exemplary anti-LEPR antibodies listed in Table 1. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID 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 an antibody or an antigen-binding fragment thereof that specifically binds to LEPR and comprises a set of six CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) contained in any of the exemplary anti-LEPR antibodies listed in Table 1. In certain embodiments, the set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences is selected from the group consisting of SEQ ID 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 related embodiments, the invention provides an antibody or an antigen-binding fragment thereof that specifically binds to LEPR and comprises a set of six CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) contained within an HCVR / LCVR amino acid sequence pair defined by any of the exemplary anti-LEPR antibodies listed in Table 1. For example, the invention provides an antibody or an antigen-binding fragment thereof that specifically binds to LEPR and comprises a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences contained within an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 10, 26 / 10, 34 / 10, 42 / 10, 50 / 10, 58 / 66, 74 / 66, and 82 / 66. Methods and techniques for identifying CDRs in HCVR and LCVR amino acid sequences are well known in the art and can be used to identify the CDRs in the specified HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally speaking, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol., 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within antibodies.

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

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

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

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

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

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

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

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

[0027] The present invention also provides a nucleic acid molecule encoding an HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1, HCDR2, HCDR3), and the set of amino acid sequences of HCDR1, HCDR2, and HCDR3 is defined by any one of the exemplary anti-LEPR antibodies listed in Table 1.

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

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

[0030] The present invention also provides a recombinant expression vector capable of expressing a polypeptide comprising a heavy or light chain variable region of an anti-LEPR antibody. For example, the present invention includes a recombinant expression vector comprising any of the above-described nucleic acid molecules, i.e., any nucleic acid molecule encoding any of the HCVR, LCVR, and / or CDR sequences shown in Table 1. Also within the scope of the present invention are host cells into which such vectors have been introduced, a method for producing an antibody or these portions by culturing the host cells under conditions that permit the production of the antibody or antibody fragment, and a method for recovering the antibody and antibody fragment thus produced.

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

[0032] In yet another aspect, the present invention provides a method of treatment for enhancing or stimulating LEPR signaling using an anti-LEPR antibody or an antigen-binding portion of an antibody of the present invention. The method of treatment according to this aspect of the present invention includes administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising the antibody or an antigen-binding fragment of the antibody of the present invention. The disorder to be treated is any disease or condition that is improved, ameliorated, inhibited, or prevented by stimulating or activating LEPR signaling in vitro or in vivo, or by otherwise mimicking the natural activity of leptin. In a particular embodiment, for example, the following items are provided. (Item 1) (Item 1) An isolated antibody or an antigen-binding fragment thereof that binds to the human leptin receptor (LEPR) and activates LEPR signaling. (Item 2) (i) A K of less than about 150 nM as measured by surface plasmon resonance Dbinds to monomeric human LEPR at 25°C; (ii) a t greater than about 1 minute when measured by surface plasmon resonance 1 / 2 binds to monomeric human LEPR at 25°C; (iii) a K less than about 5 nM when measured by surface plasmon resonance D binds to dimeric human LEPR at 25°C; (iv) a t greater than about 15 minutes when measured by surface plasmon resonance 1 / 2 binds to dimeric human LEPR at 25°C; (v) binds to human LEPR complexed with human leptin; (vi) does not block the LEPR:leptin interaction; (vii) binds to cell surface-expressed LEPR in the presence and absence of human leptin; and (viii) an EC less than about 90 pM in a cell-based reporter assay 50 activates LEPR signaling The isolated antibody or antigen-binding fragment thereof according to item 1, which exhibits one or more properties selected from the group consisting of. (Item 3) The isolated antibody or antigen-binding fragment thereof according to item 1 or 2, which activates LEPR signaling in a cell-based reporter assay with at least 50% efficacy compared to leptin. (Item 4) The isolated antibody or antigen-binding fragment thereof according to item 3, which activates LEPR signaling in a cell-based reporter assay with at least 70% efficacy compared to leptin. (Item 5) Binds to the human leptin receptor (LEPR) and comprises (a) complementarity-determining regions (CDRs) of a heavy-chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 74, or SEQ ID NO: 82, and (b) CDRs of a light-chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 66. An isolated antibody or antigen-binding fragment thereof. (Item 6) The isolated antibody or antigen-binding fragment thereof according to item 5, comprising heavy and light chain CDRs of an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 10, 26 / 10, 34 / 10, 42 / 10, 50 / 10, 58 / 66, 74 / 66, and 82 / 66. (Item 7) The isolated antibody or antigen-binding fragment thereof according to item 5 or 6, comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 10, 26 / 10, 34 / 10, 42 / 10, 50 / 10, 58 / 66, 74 / 66, and 82 / 66. (Item 8) The isolated antibody or antigen-binding fragment thereof according to item 7, which activates LEPR signaling. (Item 9) The antibody or antigen-binding fragment thereof according to any one of items 1 to 4, which competes with a reference antibody comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 10, 26 / 10, 34 / 10, 42 / 10, 50 / 10, 58 / 66, 74 / 66, and 82 / 66 for binding to LEPR. (Item 10) The antibody or antigen-binding fragment thereof according to any one of items 1 to 4, which binds to the same epitope on LEPR as a reference antibody comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 10, 26 / 10, 34 / 10, 42 / 10, 50 / 10, 58 / 66, 74 / 66, and 82 / 66. (Item 11) A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 10, and a pharmaceutically acceptable carrier or diluent. (Item 12) A method for treating a disease or condition associated with or caused by leptin deficiency or leptin resistance, comprising administering to a subject in need thereof the pharmaceutical composition according to item 11. (Item 13) 13. The method according to item 12, wherein the disease or condition associated with or caused by leptin deficiency or leptin resistance is selected from the group consisting of lipodystrophy, obesity, metabolic syndrome, diet-induced food craving, functional hypothalamic amenorrhea, type 1 diabetes, type 2 diabetes, insulin resistance, severe insulin resistance due to mutations in the insulin receptor, Alzheimer's disease, leptin deficiency, leptin resistance, elfin syndrome / Donahue syndrome, and Rabson-Mendenhall syndrome. (Item 14) 12. A method for treating a lipodystrophic condition in a patient, comprising administering to a patient in need thereof the pharmaceutical composition according to item 11, wherein said lipodystrophic condition is selected from the group consisting of congenital generalized lipodystrophy, acquired generalized lipodystrophy, familial partial lipodystrophy, acquired partial lipodystrophy, eccentric abdominal lipodystrophy, cyclic lipodystrophy, localized lipodystrophy and HIV-associated lipodystrophy. (Item 15) A method for treating a disease or condition associated with or caused by a signal transduction-deficient or signal transduction-impaired LEPR mutation, comprising administering to a subject in need thereof the pharmaceutical composition of item 11. (Item 16) 16. The method of claim 15, wherein the signaling-deficient LEPR mutation or the signaling-impaired LEPR mutation is LEPR-A409E or LEPR-P316T. (Item 17) 17. The method according to item 15 or 16, wherein the disease or condition associated with or caused by a signaling-deficient or signaling-impaired LEPR mutation is early-onset obesity. (Item 18) Further comprising administering a second therapeutic agent to the 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 glucose cotransporter 2 (SGLT2) inhibitor, a GLP-1 agonist / analogue, a glucagon (GCG) inhibitor, a glucagon receptor (GCGR) inhibitor, an angiopoietin-like protein (ANGPTL) inhibitor, phentermine, orlistat, topiramate, bupropion, topiramate / phentermine, bupropion / naltrexone, bupropion / zonisamide, pramlintide / metreleptin, lorcaserin, cetilistat, tesofensine, and berberine, the method according to any one of items 12 to 17. (Item 19) An isolated antibody or an antigen-binding fragment thereof that binds to the human leptin receptor (LEPR) and sensitizes LEPR to an antigen. (Item 20) An isolated antibody or an antigen-binding fragment thereof according to item 19, which binds to the human leptin receptor (LEPR) and comprises (a) complementarity-determining regions (CDRs) of a heavy-chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 74, or SEQ ID NO: 82, and (b) CDRs of a light-chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 66. (Item 21) An isolated antibody or an antigen-binding fragment thereof according to item 20, comprising heavy-chain CDRs and light-chain CDRs of an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NO: 26 / 10, 34 / 10, 42 / 10, 50 / 10, 58 / 66, 74 / 66, and 82 / 66. (Item 22) An isolated antibody or an antigen-binding fragment thereof that specifically binds to human LEPR and interacts with amino acids 162-169 of SEQ ID NO: 113 and amino acids 170-181 of SEQ ID NO: 113 as determined by hydrogen / deuterium exchange. (Item 23) The isolated antibody or antigen-binding fragment according to item 22, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR2 domains selected from the group consisting of SEQ ID NOs: 4, 6, and 8 and SEQ ID NOs: 12, 14, and 16, respectively. (Item 24) The isolated monoclonal antibody or antigen-binding fragment according to item 22, comprising the HCVR / LCVR amino acid sequence pair consisting of SEQ ID NO: 2 / 10.

[0033] Other embodiments will become apparent upon review of the following detailed description.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0042] Before describing the present invention, it is to be understood that such methods and conditions are variable, and the present invention is not limited to the specific methods and experimental conditions described. Also, since the scope of the present invention is limited only by the appended claims, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0043] 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 this invention belongs. As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary by up to 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0044] Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but the preferred methods and materials are described below.

[0045] Definitions The terms "leptin receptor", "LEPR", etc. as used herein refer to the human leptin receptor comprising the amino acid sequence shown in SEQ ID NO: 113 (see also UniProtKB / Swiss-Prot accession number P48357). Alternative names for LEPR used in the scientific literature include "OB receptor", "OB-R", and "CD295". LEPR is also referred to as "WSX" (see, for example, U.S. Patent No. 7,524,937). The term "LEPR" includes both monomeric and multimeric (e.g., dimeric) LEPR molecules. As used herein, the term "monomeric human LEPR" means a LEPR protein or a portion thereof that under normal conditions exists as a single LEPR molecule that does not contain or possess any multimerization domain and is not directly physically connected to another LEPR molecule. An exemplary monomeric LEPR molecule is the molecule herein referred to as "hLEPR.mmh" that comprises the amino acid sequence of SEQ ID NO: 114 (see, for example, Example 3 herein). As used herein, the term "dimeric human LEPR" means a construct comprising two LEPR molecules connected to each other by a linker, covalent bond, non-covalent bond, or by a multimerization domain such as an antibody Fc domain. An exemplary dimeric LEPR molecule is the molecule herein referred to as "hLEPR.mFc" that comprises the amino acid sequence of SEQ ID NO: 115 (see, for example, Example 3 herein), or the molecule herein referred to as "hLEPR.hFc" that comprises the amino acid sequence of SEQ ID NO: 116. As used herein, expressions such as "anti-LEPR antibody", "antibody that specifically binds to LEPR", "LEPR-specific binding protein", etc. refer to molecules that bind to full-length human LEPR, monomeric human LEPR, dimeric human LEPR, or other constructs that comprise or consist of the LEPR extracellular domain, unless specifically indicated otherwise.

[0046] All references to proteins, polypeptides, and protein fragments in this specification are intended to refer to the human versions of the respective proteins, polypeptides, or protein fragments, unless expressly designated as being from non-human species. Thus, the expression "LEPR" means human LEPR unless designated as being from a non-human species, e.g., "mouse LEPR", "monkey LEPR", etc.

[0047] As used herein, the expression "cell surface-expressed LEPR" means an LEPR protein or extracellular domain thereof that is expressed on the surface of a cell in vitro or in vivo, such that at least a portion of the LEPR protein is exposed extracellularly on the cell membrane and accessible to the antigen-binding portion of an antibody. "Cell surface-expressed LEPR" can include, or consist of, an LEPR protein that is normally (e.g., in its native or wild-type state) expressed on the surface of a cell that expresses the LEPR protein. Alternatively, "cell surface-expressed LEPR" can include, or consist of, an LEPR protein that is normally not expressed on the surface of a cell that expresses human LEPR, but is expressed on the surface of a cell that has been artificially engineered to express LEPR on its surface.

[0048] As used herein, expressions such as "anti-LEPR antibody" or "antibody that binds to the human leptin receptor" include monospecific, monovalent antibodies, as well as bispecific antibodies that include a first arm that binds to LEPR and a second arm that binds to a second (target) antigen, wherein the anti-LEPR arm includes any of the HCVR / LCVR or CDR sequences shown in Table 1 herein.

[0049] As used herein, the term "antibody" means any antigen-binding molecule or molecular complex that specifically binds to or interacts with a particular antigen (e.g., LEPR) and contains at least one complementarity-determining region (CDR). The term "antibody" includes immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain includes a heavy chain variable region (HCVR or V H as omitted herein) and a heavy chain constant region. The heavy chain constant region includes three domains C H 1, C H 2, and C H 3. Each light chain includes a light chain variable region (LCVR or V L as omitted herein) and a light chain constant region. The light chain constant region includes one domain (C L 1). The V H and V L regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each V H and V L is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the anti-LEPR antibody (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. Amino acid consensus sequences can be defined based on a parallel analysis of two or more CDRs.

[0050] As used herein, the term "antibody" includes antigen-binding fragments of whole antibody molecules. Terms such as "antigen-binding portion of an antibody", "antigen-binding fragment of an antibody", etc. as used herein include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from whole antibody molecules using, for example, any suitable standard techniques, such as protein digestion, or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable domains and optionally constant domains. Such DNA is known and / or can be readily obtained, for example, from commercial suppliers, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques to, for example, arrange one or more variable and / or constant domains in a suitable configuration, or introduce codons, create cysteine residues, modify, add, or delete amino acids, etc.

[0051] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as CDR3 peptides), or minimal recognition units consisting of constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetra-bodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, etc. are also included within the expression "antigen-binding fragment" as used herein.

[0052] 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 generally contain at least one CDR that is adjacent to or in-frame with one or more framework sequences. V L domains and the V H domains associated with an antigen-binding fragment having V H and V L domains can be positioned relative to each other within any suitable arrangement. For example, the variable region can be a dimer and can contain a V H -V H , V H -V L or V L -V L dimer. Alternatively, an antigen-binding fragment of an antibody can contain a monomeric V H or V L domain.

[0053] In certain embodiments, an antigen-binding fragment of an antibody can contain at least one variable domain covalently linked to at least one constant domain. Exemplary configurations of variable and constant domains that can be found within antigen-binding fragments of antibodies of the present invention, non-limiting, include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-C H 3; (vi) V H -C H 2-C H 3; (vii) V H -C L ; (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -CH 3; (xi) V L -C H 1-C H 2; (xii) V L -C H 1-C H 2-C H 3; (xiii) V L -C H 2-C H 3; and (xiv) V L -C L There is. In any configuration of variable and constant domains including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other, or may be linked by a complete or partial hinge or linker region. The hinge region can consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragments of the antibodies of the present invention are non-covalently associated with each other and any of the variable and constant domain configurations listed above, and / or one or more monomeric V H or V L domains may include homodimers or heterodimers (or other multimers) (e.g., by disulfide bonds).

[0054] Similar to a complete antibody molecule, an antigen-binding fragment can be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically contain at least two different variable domains, each of which can specifically bind to a distinct 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 the antigen-binding fragments of the antibodies of the present invention using conventional techniques available in the art.

[0055] In certain embodiments of the invention, the anti-LEPR antibodies of the invention are human antibodies. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include, for example, amino acid residues not encoded by human germline immunoglobulin sequences in the CDRs, particularly CDR3 (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species such as a mouse have been grafted onto human framework sequences.

[0056] In some embodiments, the antibodies of the invention can be recombinant human antibodies. As used herein, the term "recombinant human antibody" refers to all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant combinatorial human antibody library (described further below), antibodies isolated from an animal that is transgenic for human immunoglobulin genes (e.g., a mouse) (see, e.g., Taylor et al. (1992), Nucl. Acids Res., 20:6287-6295), or antibodies prepared, expressed, produced, or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when transgenic animals for human Ig sequences are used), and thus the amino acid sequences of the V H and V L regions of the recombinant antibody are different from the human germline V H and V LA sequence that is derived from an array and is related, while it may not naturally exist in the human antibody germline repertoire in vivo.

[0057] The present invention includes antibodies having one or more mutations in, for example, the hinge, C H 2, or C H 3 regions, which may be desirable, for example, to improve the yield of the desired antibody form during production.

[0058] The antibodies of the present invention can be isolated antibodies. An "isolated antibody" as used herein means an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that is separated or recovered from at least one component of an organism in which the antibody naturally occurs or is naturally produced, or from a tissue or cell, is an "isolated antibody" for the purposes of the present invention. Isolated antibodies include antibodies in situ within recombinant cells. An isolated antibody is an antibody that has been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular materials and / or chemical substances.

[0059] The present invention includes variants of the anti-LEPR antibodies disclosed herein that contain one or more amino acid substitutions, insertions, and / or deletions within the framework and / or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies are derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein, for example, to germline sequences available from publicly available antibody sequence databases. The present invention includes antibodies and their antigen-binding fragments derived from any of the amino acid sequences disclosed herein, provided that one or more amino acids within one or more framework regions and / or within one or more CDR regions are mutated relative to the corresponding residues of the germline sequence from which the antibody is derived, or relative to the corresponding residues of another human germline sequence, or relative to conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). One of ordinary skill in the art can readily generate a large number of antibodies and antigen-binding fragments that contain one or more individual germline mutations or combinations thereof, starting from the sequences of the heavy and light chain variable regions disclosed herein. In certain embodiments, V H domain and / or V LRevert all of the framework residues and / or CDR residues within the domain to the residues found within the sequence of the original germline from which the antibody was derived. In other embodiments, only certain residues are reverted to the sequence of the original germline, for example, only the residues found within the first 8 amino acids of FR1, or within the last 8 amino acids of FR4, or only the residues found within CDR1, CDR2, or CDR3 are mutated. In other embodiments, one or more of the framework residues and / or CDR residues are mutated to the corresponding residues of the sequence of a different germline (i.e., a germline sequence different from the sequence of the germline from which the antibody originally derived). Further, the antibodies of the present invention can contain any combination of two or more germline mutations within the framework region and / or within the CDR region, in which case, for example, while a particular individual residue is mutated to the corresponding residue of the sequence of a particular germline, a particular other residue that is different from the sequence of the original germline is maintained or mutated to the corresponding residue of the sequence of a different germline. Once antibodies and antigen-binding fragments containing one or more germline mutations are obtained, these can be readily assayed for one or more desired properties, such as improvement of binding specificity, increase in binding affinity, improvement or enhancement (where appropriate) of antagonist or agonist biological properties, reduction of immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present invention.

[0060] The present invention includes anti-LEPR antibodies and antigen-binding fragments thereof that comprise amino acid sequences that are substantially similar or substantially identical to one or more variable domain or CDR amino acid sequences found in any of the exemplary anti-LEPR antibodies disclosed herein.

[0061] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences share at least 95% sequence identity, more preferably at least 98% or 99% sequence identity when optimally aligned by programs such as GAP or BESTFIT using the default gap weights. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein. If two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994), Methods Mol. Biol., Vol. 24:307-331. Examples of groups of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acid substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, conservative substitutions are any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992), Science, Vol. 256:1443-1445. A "moderately conservative" substitution is any change having a non-negative value in the PAM250 log-likelihood matrix.

[0062] The sequence similarity of a polypeptide, also called sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using a measure of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, the GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or identity between closely related polypeptides, such as homologous polypeptides from organisms of different species or between a wild-type protein and its mutant. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using programs in FASTA, GCG version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) yields an alignment and percent sequence identity for the best overlapping region between a query sequence and a search sequence (Pearson (2000), supra). Another suitable algorithm for comparing the sequences of the present invention to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, 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.

[0063] Anti-LEPR antibody comprising an Fc variant According to certain embodiments of the present invention, for example, there is provided an anti-LEPR antibody comprising an Fc domain comprising one or more mutations that enhance or decrease antibody binding to the FcRn receptor at acidic pH compared to neutral pH. For example, the present invention relates to the C of the Fc domain H 2 or C HAn anti-LEPR antibody containing a mutation within the 3 domain, where the mutation increases the affinity of the Fc domain for FcRn in an acidic environment (e.g., within an endosome where the pH ranges from about 5.5 to about 6.0). Such mutations can increase the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q); positions 250 and 428 (e.g., L or F); positions 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y); or modifications at positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K), and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P).

[0064] For example, the present invention includes an anti-LEPR antibody comprising an Fc domain containing one or more pairs or groups of mutations selected from the group consisting of 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the above Fc domain mutations, and other mutations within the antibody variable domains disclosed herein, are contemplated within the scope of the present invention.

[0065] The anti-LEPR antibody of the present invention may include a modified Fc domain with reduced effector function. As used herein, "modified Fc domain with reduced effector function" has been modified, mutated, shortened, etc. compared to the wild-type naturally occurring Fc domain, such that a molecule containing the modified Fc exhibits a reduction in the significance or degree of at least one effect selected from the group consisting of cell killing (e.g., ADCC and / or CDC), complement activation, phagocytosis, and opsonization, compared to a control agent molecule containing the wild-type naturally occurring version of the Fc portion. In certain embodiments, "modified Fc domain with reduced effector function" is an Fc domain with reduced or attenuated binding to Fc receptors (e.g., FcγR).

[0066] In certain embodiments of the present invention, the modified Fc domain is a variant IgG1 Fc or variant IgG4 Fc containing a substitution within the hinge region. For example, the modified Fc for use in connection with the present invention may include a variant IgG1 Fc, wherein at least one amino acid of the IgG1 Fc hinge region has been replaced with the corresponding amino acid from the IgG2 Fc hinge region. Alternatively, the modified Fc for use in connection with the present invention may include a variant IgG4 Fc, wherein at least one amino acid of the IgG4 Fc hinge region has been replaced with the corresponding amino acid from the IgG2 Fc hinge region. Non-limiting and exemplary modified Fc regions that can be used in connection with the present invention are shown in U.S. Patent Application Publication No. 2014 / 0243504.

[0067] Other modified Fc domains and Fc modifications that can be used in connection with the present invention include any of the modifications shown in US2014 / 0171623; US8,697,396; US2014 / 0134162; WO2014 / 043361. Methods for constructing antibodies or other antigen-binding fusion proteins containing the modified Fc domains described herein are known in the art.

[0068] Biological properties of antibodies The present invention encompasses antibodies that bind to human LEPR and activate LEPR signaling, as well as antigen-binding fragments thereof. Such antibodies may be referred to herein as "agonist antibodies." In the context of the present invention, "activation of LEPR signaling" generally means stimulation of the intracellular effects resulting from the interaction of leptin with LEPR within cells expressing LEPR. In certain embodiments, "activation of LEPR signaling" means transcriptional activation of STAT3, which can be detected using any method capable of directly or indirectly measuring or identifying STAT3 activity, for example, using a labeled version of STAT3 expressed in a reporter cell line. For example, the present invention includes antibodies and antigen-binding fragments thereof that activate LEPR signaling in a cell-based reporter assay using, for example, the cell-based assay format defined in Example 7 herein or an assay substantially similar thereto. A cell-based reporter assay for detecting LEPR activation, such as the assay shown in Example 7 herein, can generate a detectable signal that can be expressed in terms of the EC 50 value (i.e., the antibody concentration required to produce half-maximal signaling) and / or the percentage of maximal signaling observed in the presence of leptin. In certain exemplary embodiments of the present invention, anti-LEPR antibodies are provided that activate LEPR signaling with an EC 50 value of less than about 12.0 nM in a cell-based reporter assay using, for example, the assay format defined in Example 7 herein or an assay substantially similar thereto. In certain exemplary embodiments of the present invention, anti-LEPR antibodies are provided that activate LEPR signaling with a maximal percent activation compared to leptin signaling of greater than about 65% in a cell-based reporter assay using, for example, the assay format defined in Example 7 herein or an assay substantially similar thereto.

[0069] The present invention includes antibodies that bind to monomeric human LEPR with high affinity and antigen-binding fragments thereof. For example, the present invention has a K of less than about 150 nM when measured by surface plasmon resonance at 25° C. or 37° C. using, for example, the assay format defined in Example 3 herein or an assay substantially similar thereto. D It includes anti-LEPR antibodies that bind to monomeric human LEPR (e.g., hLEPR.mmh, SEQ ID NO: 114) with a K of, for example, less than about 150 nM, less than about 140 nM, less than about 130 nM, less than about 120 nM, less than about 110 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 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 than about 400 pM, or less than about 300 pM when measured by surface plasmon resonance using, for example, the assay format defined in Example 3 herein or an assay substantially similar thereto. D Anti-LEPR antibodies that bind to monomeric human LEPR at 25° C. with a K of are provided.

[0070] The present invention also includes antibodies and antigen-binding fragments thereof that bind to monomeric human LEPR (e.g., hLEPR.mmh, SEQ ID NO: 114) with a dissociation half-life (t1 / 2) of greater than about 50 minutes when measured by surface plasmon resonance at 25° C. or 37° C. using, for example, the assay format defined in Example 3 herein or an assay substantially similar thereto. According to certain embodiments, anti-LEPR antibodies that bind to monomeric human LEPR at 25° C. with a t1 / 2 of greater than about 50 minutes, greater than about 55 minutes, greater than about 60 minutes, greater than about 65 minutes, or longer are provided when measured by surface plasmon resonance using, for example, the assay format defined in Example 3 herein or an assay substantially similar thereto.

[0071] The present invention also includes antibodies that bind with high affinity to dimeric human LEPR (e.g., hLEPR.mFc, SEQ ID NO: 115) and antigen-binding fragments thereof. For example, the present invention has a K of less than about 1.5 nM when measured by surface plasmon resonance at 25°C or 37°C using, for example, the assay format defined in Example 3 herein or an assay substantially similar thereto. D The present invention includes anti-LEPR antibodies that bind to dimeric human LEPR with a K of less than about 150 nM, less than about 130 nM, less than about 110 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, or less than about 10 nM when measured by surface plasmon resonance using the assay format defined in Example 3 herein or an assay substantially similar thereto. D Anti-LEPR antibodies that bind to dimeric human LEPR at 25°C with a K of less than about 150 nM, less than about 130 nM, less than about 110 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, or less than about 10 nM are provided according to certain embodiments.

[0072] The present invention also includes antibodies that bind to dimeric human LEPR (e.g., hLEPR.mFc, SEQ ID NO: 115) with a dissociation half-life (t1 / 2) of greater than about 10 minutes when measured by surface plasmon resonance at 25°C or 37°C using, for example, the assay format defined in Example 3 herein or an assay substantially similar thereto. According to certain embodiments, anti-LEPR antibodies that bind to dimeric human LEPR at 25°C with a t1 / 2 of greater than about 10 minutes, greater than about 15 minutes, greater than about 20 minutes, greater than about 25 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, or longer are provided when measured by surface plasmon resonance using the assay format defined in Example 3 herein or an assay substantially similar thereto.

[0073] The present invention also includes antibodies and antigen-binding fragments thereof that bind to LEPR complexed with human leptin (the "LEPR complexed with human leptin" may also be represented by the expression "leptin:LEPR"). For example, the present invention includes antibodies and antigen-binding fragments thereof that can bind to a pre-formed complex comprising hLEPR and human leptin. That is, according to certain embodiments, the interaction between the anti-LEPR antibody and LEPR is not inhibited by the presence of leptin complexed with LEPR; similarly, the interaction between leptin and LEPR is not inhibited by the presence of the anti-LEPR antibody according to this aspect of the present invention. An exemplary assay format for determining whether an antibody or antigen-binding fragment thereof binds to LEPR complexed with human leptin is shown in Example 4 herein.

[0074] Similarly, the present invention also includes antibodies and antigen-binding fragments thereof that bind to LEPR and do not block the LEPR:leptin interaction. For example, the present invention includes antibodies and antigen-binding fragments thereof that can bind to LEPR, thereby forming an antibody:LEPR complex, and the resulting antibody:LEPR complex can interact with leptin to generate a ternary complex comprising the antibody, LEPR, and leptin. An exemplary assay format for determining whether an antibody or antigen-binding fragment thereof can bind to LEPR in a manner that does not block or interfere with the interaction between LEPR and leptin is shown in Example 5 herein.

[0075] The present invention also includes antibodies that bind to cell surface-expressed LEPR in the presence and / or absence of human leptin and antigen-binding fragments thereof. Cell surface-expressed LEPR means LEPR or a portion thereof (e.g., the extracellular portion of LEPR) that is expressed on the cell surface, either naturally or within an engineered cell line, such that an antibody or an antigen-binding fragment thereof can bind to the LEPR molecule. In certain embodiments, cell surface-expressed LEPR includes a recombinant complex comprising the extracellular domain of LEPR that is connected to the cell via a tag or anchor (e.g., the GPI anchor exemplified in Example 6 herein). According to this aspect of the present invention, there are provided antibodies that can bind to cell surface-expressed LEPR in the absence of leptin and can also bind to cell surface-expressed LEPR in the presence of leptin (i.e., in an environment where leptin can bind to cell surface-expressed leptin). That is, according to certain embodiments, the interaction between the anti-LEPR antibody and cell surface-expressed LEPR is not inhibited by the presence of leptin complexed with cell surface-expressed LEPR. Antibodies according to this aspect of the present invention can form a ternary complex comprising the antibody, cell surface-expressed LEPR, and leptin at the cell surface. An exemplary assay format for determining whether an antibody or an antigen-binding fragment thereof can bind to cell surface-expressed LEPR in the presence and absence of human leptin is shown in Example 6 herein.

[0076] The antibodies of the present invention can have one or more of the biological properties described above, or any combination thereof. The above list of biological properties of the antibodies of the present invention is not intended to be exhaustive. Other biological properties of the antibodies of the present invention will be apparent to those skilled in the art upon review of the present disclosure, including the examples herein.

[0077] Epitope Mapping and Related Techniques The present invention also includes anti-LEPR antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes anti-LEPR antibodies having HCVR, LCVR, and / or CDR amino acid sequences with conservative amino acid substitutions such as, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, compared to any of the HCVR, LCVR, and / or CDR amino acid sequences shown in Table 1 herein. In certain embodiments, the present invention provides anti-LEPR antibodies comprising variant HCVR, LCVR, and / or CDR amino acid sequences compared to the sequences shown in Table 1 herein (including conservative amino acid substitutions), and such variant antibodies nevertheless exhibit one or more functions and / or properties of the exemplary anti-LEPR antibodies disclosed herein.

[0078] The extracellular domain of human LEPR contains a second cytokine receptor homology domain (CRH-2) called the N-terminal cytokine receptor homology domain (CRH-1), an immunoglobulin-like (Ig) domain, and a leptin-binding domain (LBD) (Carpenter et al. (2012), Structure, 20:487-97). Further, LEPR shares the greatest homology with granulocyte colony-stimulating factor (GCSF) and glycoprotein 130 (gp13), and a similar extracellular domain size and architecture (Haniu et al. (1998), J Biol Chem, 273(44):28691-699).

[0079] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site within the variable region of an antibody molecule known as a paratope. A single antigen can have more than one epitope. Thus, different antibodies can bind to different areas on an antigen and can have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are generated by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope can include a saccharide, phosphoryl group, or sulfonyl group moiety on an antigen.

[0080] The present invention includes anti-LEPR antibodies that interact with one or more epitopes found within amino acids M1 - D839 of human LEPR (SEQ ID NO: 113). As shown in Example 11, 201 peptides derived from human LEPR had significantly reduced deuterium incorporation when bound to the antibody H4H16650P2. Peptides corresponding to amino acids 162 - 169 (LYVLPEVL in human LEPR, SEQ ID NO: 113) and 170 - 191 (EDSPLVPQKGSF in human LEPR, SEQ ID NO: 113) had slower deuterium incorporation rates when bound to H4H16650P2, indicating that this antibody binds to at least two human LEPR epitopes having the sequences LYVLPEVL or EDSPLVPQKGSF (amino acids 162 - 169 or 170 - 191 of SEQ ID NO: 113, respectively).

[0081] The epitope to which the antibody of the present invention binds can consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of the LEPR protein. Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) of LEPR. In some embodiments, the epitope is located in or near the leptin-binding domain of LEPR. In other embodiments, the epitope is in a region distinct from the leptin-binding domain of LEPR, for example, at a location on the surface of LEPR that does not interfere with leptin binding to LEPR when the antibody binds to such an epitope.

[0082] A variety of techniques known to those skilled in the art can be used to identify the amino acids within an epitope recognized by a particular antibody. Exemplary techniques include, for example, alanine scanning mutagenesis, peptide blot analysis, and peptide cleavage analysis. Additionally, methods such as epitope removal, epitope extraction, and chemical modification of the antigen can be used (Tomer, 2000, Protein Science, 9:487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. Generally speaking, the hydrogen / deuterium exchange method involves deuterium labeling of the protein of interest, followed by binding of the antibody to the deuterium-labeled protein. Next, the protein / antibody complex is transferred to water, and hydrogen-deuterium exchange is allowed to occur for all residues except those protected by the antibody (which remain deuterium-labeled). After the antibody dissociates, the target protein is subjected to protease cleavage and mass spectral analysis, thereby revealing the deuterium-labeled residues corresponding 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 crystallography of the antibody complexed with its antigen can also be used to identify the amino acids within the polypeptide with which the antibody interacts.

[0083] The present invention further includes an anti-LEPR antibody that binds to the same epitope as any of the specific exemplary antibodies described herein (e.g., an antibody comprising any of the amino acid sequences shown in Table 1 herein). Similarly, the present invention also includes an anti-LEPR antibody that competes with any of the specific exemplary antibodies described herein (e.g., an antibody comprising any of the amino acid sequences shown in Table 1 herein) for binding to LEPR.

[0084] By using conventional methods known in the art and exemplified herein, it can be determined whether an antibody binds to the same epitope as the reference anti-LEPR antibody or competes with it for binding. For example, to determine whether a test antibody binds to the same epitope as the reference anti-LEPR antibody of the present invention, the reference antibody is bound to the LEPR protein. Next, the ability of the test antibody to bind to the LEPR molecule is evaluated. If the test antibody can bind to LEPR after saturation binding with the reference anti-LEPR antibody, it can be concluded that the test antibody binds to an epitope different from the reference anti-LEPR antibody. On the other hand, if the test antibody cannot bind to the LEPR molecule after saturation binding with the reference anti-LEPR antibody, the test antibody can bind to the same epitope as the epitope to which the reference anti-LEPR antibody of the present invention binds. Additional conventional experiments (e.g., peptide mutagenesis and binding analysis) are then performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody or whether steric hindrance (or another phenomenon) is the cause of the observed lack of binding. This type of experiment can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody-binding assay available in the art. According to certain embodiments of the present invention, when measured in a competitive binding assay, for example, a 1, 5, 10, 20, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, but preferably 75%, 90%, or even 99%, the two antibodies bind to the same (or overlapping) epitope (see, e.g., Junghans et al., Cancer Res., 1990:50:1495-1502). Alternatively, if essentially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other, the two antibodies are considered to bind to the same epitope. If only a subset of the amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other, the two antibodies are considered to have "overlapping epitopes".

[0085] To determine whether an antibody competes (or cross-competes) for binding with a reference anti-LEPR antibody, the binding method described above is performed in two directions: In the first direction, the reference antibody is bound to the LEFR protein under saturating conditions, and then the binding of the test antibody to the LEPR molecule is evaluated. In the second direction, the test antibody is bound to the LEPR molecule under saturating conditions, and then the binding of the reference antibody to the LEPR molecule is evaluated. In both directions, if only the first (saturating) antibody can bind to the LEPR molecule, the test antibody and the reference antibody are concluded to compete for binding to LEPR. As will be appreciated by those skilled in the art, antibodies that compete for binding with a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0086] Preparation of Human Antibodies The anti-LEPR antibodies of the present invention can be fully human antibodies. Methods for generating monoclonal antibodies, including fully human monoclonal antibodies, are known in the art. Any such known method can be used in the context of the present invention to generate human antibodies that specifically bind to human LEPR.

[0087] For example, using the VELOCIMMUNE™ technology for generating fully human monoclonal antibodies, or any other similar known method, a high-affinity chimeric antibody against LEPR having human variable regions and murine constant regions is first isolated. As in the following experimental section, the antibodies are characterized and selected for desirable properties including affinity, ligand blocking activity, selectivity, epitope, etc. If necessary, the murine constant regions are replaced with the desired human constant regions, such as wild-type or modified IgG1 or IgG4, to generate a fully human anti-LEPR antibody. The constant region selected may vary depending on the specific use, but the high-affinity antigen-binding and target-specificity properties reside within the variable regions. In certain cases, fully human anti-LEPR antibodies are isolated directly from antigen-positive B cells.

[0088] Biological equivalents The anti-LEPR antibodies and antibody fragments of the present invention include proteins having amino acid sequences that vary from those of the described antibodies but retain the ability to bind to human LEPR. Such variant antibodies and antibody fragments contain one or more additions, deletions, or substitutions of amino acids when compared to the parental sequence, but exhibit biological activity that is essentially equivalent to that of the described antibodies. Similarly, the anti-LEPR antibody-encoding DNA sequences of the present invention include sequences that contain one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequences, but encode anti-LEPR antibodies or antibody fragments that are essentially biologically equivalent to the anti-LEPR antibodies or antibody fragments of the present invention. Examples of such variant amino acid and DNA sequences are discussed above.

[0089] Two antigen-binding proteins or antibodies are considered to be biologically equivalent if, for example, when administered at the same molar dose under the same experimental conditions, whether as a single dose or multiple doses, there is no significant difference in the rate and extent of their absorption, and they are pharmaceutical equivalents or pharmaceutical alternatives. Some antibodies are equivalent in the extent of their absorption but not in the rate of their absorption, but such differences in absorption rate are intentional and, for example, not essential for achieving an effective in vivo drug concentration during long-term use, are reflected in the labeling, and are considered not medically important for the particular drug product being studied, and can be considered equivalents or pharmaceutical alternatives if they can be considered biologically equivalent.

[0090] In one embodiment, two antigen-binding proteins are biologically equivalent if there are no clinically significant differences in their safety, purity, and potency.

[0091] In one embodiment, two antigen-binding proteins are biologically equivalent if they can be switched one or more times without an increased risk of adverse effects, including a clinically significant change in immunogenicity or a reduction in efficacy, compared to a situation where the patient is maintained on treatment without such a switch between a reference product and a biological product.

[0092] In one embodiment, two antigen-binding proteins are biologically equivalent if they both act by one or more common mechanisms of action on one or more conditions of use, to the extent that such mechanisms are known.

[0093] Biological equivalence can be demonstrated by in vivo methods and by in vitro methods. Measures of biological equivalence include, for example: (a) in vivo tests in humans or other mammals that measure the concentration of an antibody or its metabolites as a function of time in blood, plasma, serum, or other biological fluids; (b) in vitro tests that correlate with and are a valid prediction of human in vivo bioavailability data; (c) in vivo tests in humans or other mammals that measure the appropriate short-term pharmacological effects of an antibody (or its target) as a function of time; and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability of an antibody or biological equivalence.

[0094] Biologically equivalent variants of the anti-LEPR antibodies of the present invention can also be constructed, for example, by making various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unwanted or inappropriate intramolecular disulfide bridges upon reduction. In other contexts, biologically equivalent antibodies can include anti-LEPR antibody variants that contain amino acid changes that modify the glycosylation characteristics of the antibody, such as mutations that abolish or remove glycosylation.

[0095] Species selectivity and species cross-reactivity According to certain embodiments, the present invention provides anti-LEPR antibodies that bind to human LEPR but not to LEPR from other species. The present invention also includes anti-LEPR antibodies that bind to human LEPR and to LEPR from one or more non-human species. For example, the anti-LEPR antibodies of the present invention can bind to human LEPR and may or may not bind to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee LEPR. According to certain exemplary embodiments of the present invention, anti-LEPR antibodies that specifically bind to human LEPR and cynomolgus monkey (e.g., Macaca fascicularis) LEPR are provided. Other anti-LEPR antibodies of the present invention bind to human LEPR but do not bind or bind only weakly to cynomolgus monkey LEPR.

[0096] Bispecific antibodies The antibodies of the present invention can be monospecific or multispecific (e.g., bispecific). Multispecific antibodies can be specific for different epitopes of one target polypeptide or can 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 can be linked to another functional molecule, e.g., another peptide or protein, or co-expressed therewith. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or another method) to one or more other molecular entities, such as another antibody or antibody fragment, to generate a bispecific or multispecific antibody having a second binding specificity.

[0097] The present invention includes bispecific antibodies in which one arm of the immunoglobulin binds to human LEPR and the other arm of the immunoglobulin is specific for a second antigen. The LEPR-binding arm can comprise any of the HCVR / LCVR or CDR amino acid sequences shown in Table 1 herein.

[0098] Exemplary bispecific antibody formats that can be used in the context of the present invention are a first immunoglobulin (Ig) C H 3 domain and a second Ig C H 3 domain, where the first Ig C H 3 domain and the second Ig C H 3 domain differ from each other by at least one amino acid, and the at least one amino acid difference reduces the binding of the bispecific antibody to protein A as compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig C H 3 domain binds to protein A and the second Ig C H 3 domain contains a mutation, such as the H95R modification (by IMGT exon numbering; H435R by EU numbering), that reduces or abolishes binding to protein A. The second CH 3 may further comprise a Y96F modification (according to IMGT; Y436F according to EU). A second C H Additional modifications that may be found within 3 include D16E, L18M, N44S, K52N, V57M, and V82I for IgG1 antibodies (according to IMGT; D356E, L358M, N384S, K392N, V397M, and V422I according to EU); N44S, K52N, and V82I for IgG2 antibodies (IMGT; N384S, K392N, and V422I according to EU); and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I for IgG4 antibodies (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I according to EU). Variations on the bispecific antibody formats described above are contemplated to be within the scope of the present invention.

[0099] Other exemplary bispecific formats that may be used in the context of the present invention, without limitation, include, for example, scFv-based bispecific formats or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, KIH (knobs-into-holes), common light chain (e.g., common light chain with KIH (knobs-into-holes), etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1 / IgG2, dual action Fab (DAF)-IgG, and Mab 2Including bispecific formats (for a review of the formats described above, see, for example, Klein et al., 2012, mAbs, 4(6):1-11, and references cited therein). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, for example, in this case using unnatural amino acids with orthogonal chemical reactivity to create site-specific antibody-oligonucleotide conjugates, where the conjugate self-assembles into a multimeric complex with defined composition, valency, and shape (see, for example, Kazane et al., J. Am. Chem. Soc. [Epub: Dec 4, 2012]).

[0100] Therapeutic Formulations and Administration The present invention provides a pharmaceutical composition comprising an anti-LEPR antibody of the present invention or an antigen-binding fragment thereof. The pharmaceutical composition of the present invention is formulated with a suitable carrier, excipient, and other agents, etc., which bring about improved uptake, delivery, tolerance, etc. A number of suitable formulations can be found in the formulary known to all drug discovery chemists: Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (LIPOFECTIN™, Life Technologies, Carlsbad, CA, etc.), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., “Compendium of excipients for parenteral formulations”, PDA (1998), J Pharm Sci Technol, 52(238-311).

[0101] The dosage of the antibody administered to a patient can vary depending on the patient's age and size, the disease, condition, route of administration, etc. to be targeted. Suitable dosages are typically calculated by body weight or body surface area. In adult patients, it may be advantageous to administer the antibody of the present invention intravenously at a normal single dose of about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg per kg of body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. The effective dosage and schedule of administration of the anti-LEPR antibody may be determined empirically. For example, the progression of the patient can be monitored by regular evaluation and the dosage adjusted accordingly. Furthermore, interspecies scaling of dosages can be carried out using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res., 8:1351).

[0102] A variety of delivery systems are known, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis, and can be used to administer the pharmaceutical composition of the present invention (see, for example, Wu et al., 1987, J. Biol. Chem., 262:4429-4432). The introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered via any convenient route, such as injection or bolus injection, absorption through epithelial or mucocutaneous membranes (e.g., oral mucosa, rectal mucosa, and intestinal mucosa, etc.), and can be administered in combination with other bioactive agents. Administration can be systemic or local.

[0103] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using standard needles and syringes. Further, with respect to subcutaneous delivery, pen-type delivery devices are readily applicable for the delivery of the pharmaceutical composition of the present invention. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. When all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can be readily discarded and replaced with a new cartridge containing the pharmaceutical composition. In this way, the pen-type delivery device can be reused. In disposable pen-type delivery devices, there is no replaceable cartridge. Rather, disposable pen-type delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. When the reservoir of the pharmaceutical composition is empty, the entire device is discarded.

[0104] A number of reusable pen-type delivery devices and autoinjector delivery devices have application in the subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, but are not limited to, just a few examples, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen, (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany). Examples of disposable pen-type delivery devices having application in the subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, just a few examples, SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMIRA™ pen (Abbott Labs, Abbott Park, IL).

[0105] In certain situations, a pharmaceutical composition can be delivered by 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, a polymeric material can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Press, Boca Raton, Florida. In yet another embodiment, the controlled release system can be placed in proximity to the target of the composition, and thus only a portion of the systemic dose may be required (see, for example, Goodson, 1984, Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138). Other controlled release systems are discussed in a review by Langer, 1990, Science, 249:1527-1533.

[0106] Injectable preparations can include dosage forms for intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, infusion, etc. These injectable preparations can be prepared by known methods. For example, an injectable preparation can be prepared by dissolving, suspending, or emulsifying, for example, the antibody or its salt described above in a sterile aqueous medium or a sterile oily medium conventionally used for injection. Examples of aqueous media for injection include isotonic solutions containing, for example, physiological saline, glucose, and alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], and other adjuvants that can be used in combination with appropriate solubilizing agents. Examples of oily media include sesame oil, soybean oil, etc., which can be used in combination with solubilizing agents such as benzyl benzoate, benzyl alcohol. The injectable thus prepared is preferably filled into an appropriate ampoule.

[0107] The pharmaceutical composition for oral or parenteral use described above is advantageously prepared into a dosage form of a unit dose suitable for the dosage of the active ingredient. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories and the like. The amount of the antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose, and particularly, in the form of an injection, the antibody is preferably contained in an amount of about 5 to about 100 mg, and in other dosage forms, it is preferably contained in an amount of about 10 to about 250 mg.

[0108] Therapeutic use of the antibody The present invention includes a method comprising administering to a subject in need thereof a therapeutic composition comprising an anti-LEPR antibody (for example, an anti-LEPR antibody comprising any of the HCVR / LCVR or CDR sequences shown in Table 1 herein). The therapeutic composition may comprise any of the anti-LEPR antibodies disclosed herein, or antigen-binding fragments thereof, and a pharmaceutically acceptable carrier or diluent.

[0109] The antibodies of the present invention are particularly useful for the treatment, prevention, and / or amelioration of any disease or disorder associated with, mediated by, or stimulating or activating LEPR signaling in vitro or in vivo, or mimicking the natural activity of leptin, such as leptin deficiency, leptin resistance, hypoleptinemia. For example, the antibodies of the present invention and their antigen-binding fragments are useful for treating lipodystrophic conditions. Exemplary lipodystrophic conditions treatable by the antibodies and antigen-binding fragments of the present invention include, for example, congenital generalized lipodystrophy, acquired generalized lipodystrophy, familial partial lipodystrophy, acquired partial lipodystrophy, centrifugal abdominal lipodystrophy, lipoatrophia annularis, localized lipodystrophy, and HIV-associated lipodystrophy.

[0110] The present invention also includes anti-LEPR antibodies and antigen-binding fragments thereof that are useful for restoring leptin signaling to cells, tissues, and organs that express one or more LEPR mutations associated with obesity. For example, certain LEPR variants that do not exhibit signaling in the presence of leptin, or exhibit reduced signaling, have been identified and are associated with obesity and related disorders. As used herein, an LEPR variant that does not exhibit signaling in the presence of leptin is referred to as a "signaling-deficient LEPR variant". An exemplary signaling-deficient LEPR mutation is LEPR-A409E (Farooqi et al., 2007, N Engl J Med, 356(3):237-247). As used herein, an LEPR variant that exhibits reduced (compared to wild-type LEPR) signaling in the presence of leptin is referred to as a "signaling-impaired LEPR variant". An exemplary signaling-impaired LEPR mutation 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 or associated with one or more signaling-deficient (e.g., A409E) and / or signaling-impaired (e.g., P316T) LEPR variants.

[0111] The anti-LEPR antibodies of the present invention and their antigen-binding fragments are useful for the treatment or prevention of one or more diseases or disorders selected from the group consisting of obesity, metabolic syndrome, diet-induced food craving, functional hypothalamic amenorrhea, type 1 diabetes, type 2 diabetes, insulin resistance, severe insulin resistance due to mutations in the insulin receptor, severe insulin resistance not caused by mutations in the insulin receptor, severe insulin resistance caused by mutations in downstream signaling pathways or induced by other causes, non-alcoholic and alcoholic fatty liver diseases, Alzheimer's disease, leptin deficiency, leptin resistance, lipodystrophy, Prader-Willi / Donahue syndrome, Rabson-Mendenhall syndrome.

[0112] In the context of the methods of treatment described herein, the anti-LEPR antibodies can be administered as monotherapy (i.e., as the sole therapeutic agent) or in combination with one or more additional therapeutic agents (examples of which are described elsewhere herein).

[0113] Combination Therapies and Formulations The present invention includes compositions and therapeutic formulations comprising any of the anti-LEPR antibodies described herein in combination with one or more additional therapeutically active ingredients, and methods of treatment comprising administering such combinations to a subject in need thereof.

[0114] The anti-LEPR antibody of the present invention can be co-formulated and / or administered in combination with one or more additional therapeutically active ingredients, for example, pharmaceuticals formulated for treating obesity, hypercholesterolemia, hyperlipidemia, type 2 diabetes, type 1 diabetes, appetite control, infertility, etc. Examples of such additional therapeutically active ingredients include, for example, recombinant human leptin (e.g., metreleptin [MYALEPT]), PCSK9 inhibitors (e.g., anti-PCSK9 antibodies [alirocumab, evolocumab, bococizumab, lodelcizumab, ralpancizumab, etc.]), statins (atorvastatin, rosuvastatin, cerivastatin, pitavastatin, fluvastatin, simvastatin, lovastatin, pravastatin, etc.), ezetimibe, insulin, insulin variants, insulin secretagogues, metformin, sulfonylureas, sodium glucose co-transporter 2 (SGLT2) inhibitors (e.g., dapaglifozin, canaglifozin, empaglifozin, etc.), GLP-1 agonists / analogs (e.g., exendin-4, exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, etc.), glucagon (GCG) inhibitors (e.g., anti-GCG antibodies), glucagon receptor (GCGR) inhibitors (e.g., anti-GCGR antibodies, small molecule GCGR antagonists, GCGR-specific antisense oligonucleotides, anti-GCGR aptamers [e.g., Spiegelmer], etc.), angiopoietin-like protein (ANGPTL) inhibitors (e.g., anti-ANGPTL3 antibodies, anti-ANGPTL4 antibodies, anti-ANGPTL8 antibodies, etc.), phentermine, orlistat, topiramate, bupropion, topiramate / phentermine, bupropion / naltrexone, bupropion / zonisamide, pramlintide / metreleptin, lorcaserin, cetilistat, tesofensine, berneperit, etc.

[0115] An additional therapeutically active ingredient, for example, any of the agents or derivatives thereof listed above, can be administered immediately before, simultaneously with, or immediately after administration of the anti-LEPR antibody of the present invention; (for the purposes of this disclosure, such an administration regimen is considered an administration of an anti-LEPR antibody "in combination with" an additional therapeutically active ingredient). The present invention includes a pharmaceutical composition in which the anti-LEPR antibody of the present invention is co-formulated with one or more additional therapeutically active ingredients, as described elsewhere herein.

[0116] Administration regimen According to 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 therapeutic agents described herein) can be administered to a subject over a predetermined period of time. A method according to this aspect of the present invention comprises sequentially administering multiple doses of the anti-LEPR antibody of the present invention to a subject. As used herein, "sequentially administering" means that each dose of the anti-LEPR antibody is administered to the subject at different times, for example, on different days separated by a predetermined interval (e.g., several hours, several days, several weeks, or several months). The present invention includes methods comprising sequentially administering to a patient a single initial dose of an anti-LEPR antibody, followed by one or more secondary doses of the anti-LEPR antibody, and optionally one or more tertiary doses of the anti-LEPR antibody thereafter.

[0117] The terms "initial dose", "secondary dose", and "tertiary dose" refer to the chronological order of administration of the anti-LEPR antibody of the present invention. Thus, the "initial dose" is the dose administered at the beginning of the treatment regimen (also referred to as "baseline dose", "loading dose", "starting dose", etc.), the "secondary dose" is the dose administered after the initial dose, and the "tertiary dose" is the dose administered after the secondary dose. The initial, secondary, and tertiary doses can all contain the same amount of anti-LEPR antibody, but generally can differ from each other in terms of the frequency of administration. However, in certain embodiments, the amounts of anti-LEPR antibody included in the initial, secondary, and / or tertiary doses vary relative to each other during the course of treatment (e.g., are adjusted up or down as needed). In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as a "loading dose", followed by subsequent doses administered at a less frequent rate (e.g., "maintenance dose").

[0118] Diagnostic and analytical uses of the antibody The anti-LEPR antibody of the present invention can also be used, for example, for diagnostic purposes to detect and / or measure LEPR or LEPR-expressing cells in a sample. For example, the anti-LEPR antibody or a fragment thereof can be used to diagnose a condition or disease characterized by abnormal expression of LEPR (e.g., overexpression, underexpression, lack of expression, etc.). An exemplary diagnostic assay for LEPR can include contacting a sample obtained from a patient with the anti-LEPR antibody of the present invention, where the anti-LEPR antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-LEPR antibody can be used in a diagnostic application in combination with a secondary antibody that is itself detectably labeled. Detectable labels or reporter molecules include radioisotopes such as 3 H, 14 C, 32 P, 35 S, or 125Such as I; a fluorescent or chemiluminescent moiety, for example, fluorescein isothiocyanate or rhodamine, etc.; or an enzyme, for example, alkaline phosphatase, beta-galactosidase, horseradish peroxidase, or luciferase, etc. Specific exemplary assays that can be used to detect or measure LEPR in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorting (FACS), and positron emission tomography (PET) scanning.

[0119] Samples that can be used in the LEPR diagnostic assay according to the present invention include any tissue or body fluid sample obtainable from a patient that contains a detectable amount of LEPR protein or a fragment thereof under normal or pathological conditions. Generally, the level of LEPR in a specific sample obtained from a healthy patient (e.g., a patient not suffering from a disease or condition associated with abnormal LEPR levels or activity) is measured to initially establish a baseline or standard level of LEPR. This baseline level of LEPR can then be compared to the level of LEPR measured in a sample obtained from an individual suspected of having an LEPR-related disease or condition.

Example

[0120] The following examples are presented to provide a complete disclosure and description of how to make and use the methods and compositions of the present invention to those skilled in the art and are not intended to limit the scope that the inventors regard as their invention. Although efforts are made to ensure the accuracy of the numerical values used (e.g., amounts, temperatures, etc.), some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, pressures are at or near atmospheric.

[0121] (Example 1: Production of an antigen-binding protein that specifically binds to the leptin receptor (LEPR)) The anti-LEPR antibody was obtained by immunizing VELOCIMMUNE® mice (i.e., engineered mice containing DNA encoding the human immunoglobulin heavy and kappa light chain variable regions) with an immunogen comprising the extracellular domain of LEPR. The antibody immune response was monitored by a LEPR-specific immunoassay. Using previously described techniques, fully human anti-LEPR antibodies were isolated and purified.

[0122] Certain biological properties of exemplary anti-LEPR antibodies generated according to the method of this example are described in detail in the examples shown below.

[0123] (Example 2: Heavy and Light Chain Variable Region Amino Acid and Nucleic Acid Sequences) Table 1 shows the identifiers of the amino acid sequences of the heavy and light chain variable regions and CDRs of selected anti-LEPR antibodies of the present invention. The identifiers of the corresponding nucleic acid sequences are shown in Table 2. [Table 1] [Table 2]

[0124] Antibodies are typically referred to herein according to the following nomenclature: an Fc prefix (e.g., "H4H", "H1M", "H2M", etc.), followed by a numerical identifier (e.g., "16650", "16679", etc.), and then a "P" or "N" suffix. Thus, according to this nomenclature, an antibody may be referred to herein, for example, as "H4H16650P2", "H4H16679P2", etc. The Fc prefixes (H4H, H1M, and H2M) of the antibody names used herein indicate the specific Fc region isotype of the antibody. For example, the "H4H" antibody has a human IgG4 Fc, while the "H1M" antibody has a mouse IgG1 Fc (all variable regions are fully human, as represented by the initial "H" of the antibody name). As will be understood by those skilled in the art, an antibody having a specific Fc isotype can be converted to an antibody having a different Fc isotype (e.g., an antibody having a mouse IgG1 Fc can be converted to an antibody having a human IgG4, etc.), but in any case, the variable domains (including CDRs) indicated by the numerical identifiers shown in Tables 1 and 2 remain the same, and the binding properties are expected to be identical or substantially similar regardless of the nature of the Fc domain.

[0125] The "comparator mAb300D" 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.

[0126] (Example 3: Binding Affinity and Kinetic Constants of Human Monoclonal Anti - LEPR Antibodies Obtained by Surface Plasmon Resonance) Equilibrium Dissociation Constant (K for LEPR Binding to Purified Anti - LEPR Monoclonal Antibodies DThe value) was determined using a real-time surface plasmon resonance biosensor with a Biacore 4000 instrument. All binding studies were performed in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant Tween-20, pH 7.4 (HBS-ET) running buffer at 25 °C and 37 °C. The Biacore sensor surface was first derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (GE, #BR-1008-39) to capture the anti-LEPR monoclonal antibody. Binding studies were performed against the following LEPR reagents: human LEPR extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (hLEPR.mmh; SEQ ID NO: 114), macaca fascicularis LEPR extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (mfLEPR.mmh; SEQ ID NO: 117), human LEPR extracellular domain expressed with a C-terminal mouse IgG2a Fc tag (hLEPR.mFc; SEQ ID NO: 115), mouse LEPR extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (mLEPR.mmh; SEQ ID NO: 118), and rat LEPR extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (rLEPR.mmh; SEQ ID NO: 119). Different concentrations of LEPR reagents were first prepared in HBS-ET running buffer (100 nM to 3.7 nM; 3-fold serial dilution) and injected at a flow rate of 30 μL / min for 4 minutes onto the anti-human Fc capture anti-LEPR monoclonal antibody surface, while the dissociation of the monoclonal antibody-bound LEPR reagent was monitored for 10 minutes in HBS-ET running buffer. The kinetic association (k a ) and dissociation (k d ) rate constants were determined by fitting the real-time binding sensorgram to a 1:1 binding model with mass transport limitation using Scrubber 2.0c curve fitting software. The binding dissociation equilibrium constant (K D ) and dissociation half-life (t1 / 2) were

Chemical formula

[0127] Table 3 to 8 show the binding kinetic parameters for the binding of hLEPR.mmh, mfLEPR.MMH, or hLEPR.mFc to different anti-LEPR monoclonal antibodies of the present invention at 25°C and 37°C. [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8]

[0128] At 25°C, the anti-LEPR monoclonal antibody bound to hLEPR-MMH at a K D value in the range of 7.93 nM to 148 nM, as shown in Table 5. At 37°C, the anti-LEPR monoclonal antibody bound to hLEPR-MMH at a K D value in the range of 14.8 nM to 326 nM, as shown in Table 4.

[0129] Ten out of the 12 anti-LEPR monoclonal antibodies of the present invention bound to mfLEPR.MMH. At 25°C, the anti-LEPR monoclonal antibody bound to mfLEPR.MMH at a K D value in the range of 2.27 nM to 139 nM, as shown in Table 7. At 37°C, the anti-LEPR monoclonal antibody bound to mfLEPR.MMH at a K D value in the range of 5.18 nM to 264 nM, as shown in Table 8.

[0130] At 25°C, the anti-LEPR monoclonal antibodies bound to hLEPR-mFc with K values in the range of 613 pM to 5.7 nM as shown in Table 7. At 37°C, the anti-LEPR monoclonal antibodies bound to hLEPR-mFc with K values in the range of 1.16 nM to 12.8 nM as shown in Table 8. D None of the anti-LEPR monoclonal antibodies of the present invention bound to mLEPR.MMH or rLEPR.MMH at 25°C or 37°C (data not shown). D (Example 4: The anti-LEPR antibody of the present invention binds to LEPR in the presence of leptin:LEPR binding)

[0131]

[0132] The blockade of the binding of anti-LEPR antibody to LEPR by human leptin was evaluated using the real-time surface plasmon resonance biosensor of a Biacore T200 instrument. The entire study was carried out at 25 °C in 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant Tween-20 (HBS-ET running buffer). The Biacore CM5 sensor surface was first derivatized by amine-coupling human leptin (R&D Systems, #398-LP) using standard EDC / NHS surface chemistry. A complex of human LEPR and human leptin was formed by injecting 20 nM of the extracellular domain of human LEPR (hLEPR-MMH; SEQ ID NO: xx) expressed with a C-terminal myc-myc-hexahistidine tag onto the Biacore sensor surface immobilized with human leptin at a flow rate of 10 μL / min or 25 μL / min for 4 minutes to achieve a binding response of approximately 200 RU. To evaluate whether the binding of the antibody to hLEPR-MMH was blocked by human leptin, 200 nM of the anti-LEPR monoclonal antibody was injected onto the pre-formed hLEPR-MMH:human leptin complex at a flow rate of 50 μL / min or 25 μL / min for 4 - 5 minutes. All anti-LEPR antibodies of the present invention bound to the complex of hLEPR-MMH and human leptin ("leptin:LEPR"), and the signal intensities were approximately the same. The observed binding is reported in Table 9 in RU. This result indicates that human leptin does not block the binding of the tested anti-LEPR antibodies to hLEPR-MMH. [Table 9]

[0133] (Example 5: Human Leptin Receptor Blocking ELISA) For ELISA, human leptin (hLeptin; R&D Systems, #398-LP-01M) was coated onto a 96-well microtiter plate at a concentration of 5 μg / mL in PBS at 4 °C overnight. Nonspecific binding sites were subsequently blocked using a 0.5% (w / v) solution of BSA in PBS. A fixed amount of 10 nM of the extracellular domain portion of the LEPR protein (hLEPR.hFc; SEQ ID NO: 116) expressed with the C-terminal human Fc tag was titrated with anti-LEPR antibodies, hLeptin protein, or isotype control antibodies in the range of 8.5 pM to 500 nM in serial dilutions. These antibody-protein or protein-protein complexes were then incubated at room temperature (RT) for 1.5 hours. The complexes were subsequently transferred to the hLeptin-coated microtiter plate, incubated at RT for 2 hours, the wells were washed, and the hLEPR.hFc bound to the plate was detected with an anti-human IgG polyclonal antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch Inc, #109-035-098). Samples were developed with a TMB solution (BD Biosciences, #555214; substrate A and B mixed at a 1:1 ratio according to the manufacturer's instructions) to produce a colorimetric reaction, and then neutralized with 1 M sulfuric acid, after which the absorbance at 450 nm was measured with a Victor X5 plate reader.

[0134] Data analysis was performed using a sigmoidal dose-response model in Prism™ software (GraphPad). The percent block at the maximum concentration of the antibody tested was calculated as an indicator of the ability of the antibody to block the binding of 10 nM hLEPR.hFc to human leptin on the plate. In the calculation, the binding signal of 10 nM hLEPR.hFc in the absence of the antibody was referenced as 100% binding or 0% block, and the baseline signal of buffer alone in the absence of hLEPR.hFc was referenced as 0% binding or 100% block. The block data at 500 nM antibody concentration are summarized in Table 10.

[0135] As shown in Table 10, none of the anti-LEPR antibodies of the present invention demonstrated more than 28% blockade of the binding of hLEPR.hFc to the hLeptin-coated surface. However, the control antibody and hLeptin were able to block 99% of the binding of hLEPR.hFc to the hLeptin-coated surface as a positive control. The isotype control antibody did not demonstrate measurable blockade at concentrations up to 500 nM.

Table 10

[0136] (Example 6: Cell Binding by FACS Analysis Using HEK293 / Mycx2-hLepR(ecto)-GPI Anchor-Type Cells) The leptin receptor, LEPR, is a single-pass transmembrane receptor of the class I cytokine receptor family (Tartaglia et al. (1997), J Biol Chem, 272(10):6093-6). LEPR can bind to proteins mainly expressed by adipose tissue, which is involved in the regulation of leptin, food intake, and metabolism (Friedman et al. (2014), J Endocrinol, 223(1):T1-8).

[0137] To evaluate cell binding by anti-LEPR antibodies, HEK293 stable cell lines were generated. One cell line, known hereinafter as HEK293 / hLEPR-GPI, stably expressed the extracellular domain of human LEPR (amino acids 22-839 (SEQ ID NO: 113), isoform B of accession number P48357), together with an N-terminal myc-myc tag and a C-terminal peptide sequence from human carboxypeptidase M that guides the addition of GPI (glycosylphosphatidylinositol) (Deddish et al. (1990), J. Biological Chemistry, 265:25:15083-89). As a result, the protein can be tethered to the membrane by GPI. Another HEK293 cell line was generated to stably express full-length human LEPR (amino acids 1-1165, isoform B of accession number P48357 (SEQ ID NO: 113)) together with a luciferase reporter (Stat3-luciferase, Stat3-luc, SA Bioscience, #CLS-6028L). This cell line is known hereinafter as HEK293 / Stat3-luc / hLEPR-FL. HEK293 cells containing only the Stat3-luciferase reporter (HEK293 / Stat3-luc) were also generated as a control cell line.

[0138] For FACS analysis, HEK293 parental cells and HEK293 / hLEPR-GPI cells were dissociated and resuspended at 5×10 in PBS containing 2% FBS (FACS buffer) 5Cells were seeded on a 96-well V-bottom plate at a cell / well density. To test whether the ability of the anti-hLEPR antibody to bind to cells is affected by the presence of leptin, cells were incubated with FACS buffer with or without 1 μM human leptin (R&D Systems, #398-LP) at 4 °C for 30 min, followed by the addition of anti-LEPR antibody or control antibody at 10 nM in FACS buffer. Cells were subsequently incubated at 4 °C for 30 min, then washed, and then incubated with 16 μg / mL Alexa Fluor®-647-conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc., #109-547-003) at 4 °C for 30 min. Cells were subsequently fixed using BD CytoFix™ (Becton Dickinson, #554655), filtered, and analyzed on a HyperCyt Flow Cytometer (Beckman Coulter). Unstained and secondary antibody-only controls were also tested for all cell lines. Results were analyzed using ForeCyt (IntelliCyt) and FlowJo version 10 software to determine the geometric mean fluorescence of viable cells. The geometric mean fluorescence of each sample was normalized to the geometric mean of unstained cells to obtain the relative binding per condition, called the "binding ratio," and these binding ratios were recorded for each antibody tested.

[0139] As shown in Table 11, the nine anti-LEPR antibodies of the present invention tested at 10 nM were demonstrated to bind to HEK293 / hLEPR-GPI cells at binding ratios in the range of 824 to 3374-fold in the absence of leptin. The anti-LEPR antibodies also bound in the presence of 1 μM leptin at binding ratios of 398 and 4184-fold. As shown in Table 11, the control antibody tested at 10 nM demonstrated binding to HEK293 / hLEPR-GPI cells at a binding ratio of 2349-fold in the absence of leptin, but showed significantly lower binding to the cells in the presence of 1 μM leptin, with a binding ratio of 112. The anti-LEPR antibodies did not demonstrate any significant binding to HEK293 parental cells at binding ratios in the range of 1 to 9-fold with and without 1 μM leptin. Samples of isotype control antibody and secondary antibody alone also did not demonstrate significant binding to any of the cell lines, with binding ratios of 1 to 6-fold, regardless of the presence or absence of leptin.

[0140] As shown in Table 12, the four antibodies of the present invention tested at 70 nM demonstrated binding to HEK293 / hLEPR-GPI cells at binding ratios in the range of 707 to 1131-fold and binding to HEK293 / Stat3-luc / hLEPR-FL cells at binding ratios in the range of 42 to 51-fold in the absence of leptin. The anti-LEPR antibodies did not demonstrate any significant binding to HEK293 / Stat3-luc cells, with a binding ratio of 1 to 8-fold. Samples of isotype control antibody and secondary antibody alone also did not demonstrate significant binding to any of the cell lines tested, with binding ratios of 1 to 2-fold.

Table 11

Table 12

[0141] (Example 7: Anti-LEPR antibodies of the present invention activate LEPR signaling in the presence or absence of leptin) The bioassay was developed to detect the transcriptional activation of STAT3 via LEPR activation using a reporter cell line that stably expresses full-length human LEPR (hLEPR; amino acids 1 - 1165 of accession number NP_002294.2) together with a luciferase reporter (STAT3-Luc; Qiagen, #CLS-6028L) in the IMR-32 cell line, a human neuroblastoma cell line. The resulting stable cell line, designated IMR-32 / STAT3-Luc / hLEPR, was isolated and maintained in MEM-Earl medium (complete medium) supplemented with 10% FBS, NEAA, 1 μg / mL puromycin, 100 μg / mL hygromycin B, and penicillin / streptomycin / L-glutamine.

[0142] The resulting bioassay was used to measure the effect of the anti-LEPR antibody of the present invention on LEPR signaling in the presence or absence of leptin. For the bioassay, IMR-32 / STAT3-Luc / hLEPR cells were seeded in 96-well format in complete medium at a density of 20,000 cells / 100 μl / well and the next day were replaced with an appropriate volume of Opti-MEM medium supplemented with 1% BSA and 0.1% FBS (assay buffer) for 30 minutes. To measure the effect of the antibody of the present invention in the absence of leptin, the anti-LEPR antibody or isotype control antibody and human leptin (hLeptin; R&D Systems, #398-LP) were half-log serially diluted to final concentrations in the range of 100 nM to 300 fM in assay buffer, added to the cells, and incubated continuously overnight at 37 °C in 5% CO2.

[0143] To measure the effect of the antibodies of the present invention in the presence of leptin, human leptin at a fixed concentration of 200 pM in assay buffer was added to the cells, and immediately thereafter, anti-LEPR antibodies or isotype control antibodies serially diluted in a semi-logarithmic manner to a final concentration in the range of 100 nM to 300 fM were added. The samples were then incubated overnight at 37 °C in 5% CO2. Next, OneGlo reagent (Promega, #E6051) was added to the samples, and the luciferase activity was measured with an Envision Multilable plate reader (Perkin Elmer) in luminescence mode. Relative light unit (RLU) values were obtained, and the results were analyzed using non-linear regression with GraphPad Prism software (GraphPad). The maximum RLU value obtained from the hLeptin dose response was defined as 100% activation in the IMR-32 / STAT3-Luc / hLEPR assay.

[0144] As shown in Table 13, in Study 1, in the absence of hLeptin, all of the anti-LEPR antibodies tested demonstrated weak stimulation of IMR-32 / STAT3-Luc / hLEPR cells, and the EC 50 values were in the range of 134 pM to 11.9 nM, and the maximum activation was in the range of 5% to 13% of that obtained from the hLeptin dose response. In Study 2, in the absence of hLeptin, four anti-LEPR antibodies tested demonstrated stimulation of IMR-32 / STAT3-Luc / hLEPR cells, and the EC 50 values were in the range of 61.9 pM to 206.9 pM, and the maximum activation was in the range of 65% to 68% relative to the maximum activation obtained from the hLeptin dose response. In Study 1, in the presence of 200 pM hLeptin, all of the anti-LEPR antibodies tested demonstrated stimulation of IMR-32 / STAT3-Luc / hLEPR cells, and the EC 50The values were in the range of 20.2 pM to 523 pM, and the maximum activation was in the range of 66% to 107% of that of the maximum activation obtained from the hLeptin dose response. Since these antibodies enhanced leptin-induced LEPR signaling, these antibodies were classified as "enhancers" as defined herein. In Study 2, in the presence of 200 pM of hLeptin, four anti-LEPR antibodies tested demonstrated stimulation of IMR-32 / STAT3-Luc / hLEPR cells, and the EC 50 values were in the range of 51.9 pM to 257.3 pM, and the maximum activation was in the range of 76% to 88% relative to the maximum activation obtained from the hLeptin dose response. LEPR signaling was not enhanced to a measurable extent by these antibodies in the presence of leptin. Isotype control antibodies did not demonstrate any measurable stimulation of IMR-32 / STAT3-Luc / hLEPR cells in any of the assays. [Table 13]

[0145] (Example 8: Anti-LEPR antibodies of the present invention activate signaling in cells expressing signal transduction-deficient or signal transduction-disrupted LEPR mutants) LEPR mutants associated with early-onset obesity that exhibit defects or impairments in leptin-mediated signal transduction were identified. For example, LEPR-A409E is a signal transduction-deficient mutant LEPR protein that does not convert leptin signal to STAT3, and the A409E mutant was originally identified as a monogenic cause of early-onset obesity. (Farooqi et al., 2007, N Engl J Med, 356(3):237-247). LEPR-P316T is a signal transduction-disrupted mutant LEPR protein that has also been shown to be associated with early-onset obesity. (Mazen et al., 2011, Mol Genet Metab, 102:461-464).

[0146] In this example, the ability of the anti-LEPR antibodies of the present invention to stimulate LEPR signaling in cell lines expressing signal transduction-deficient or signal transduction-disrupted LEPR variants was evaluated. Specifically, reporter cell lines (HEK293) expressing wild-type LEPR, LEPR-A409E (signal transduction-deficient), or LEPR-P316T (signal transduction-disrupted) were constructed. Cells were treated with vehicle only, recombinant human leptin, control IgG, or agonist anti-LEPR antibodies of the present invention (H4H16650 or H4H16679), and the extent of LEPR signaling (measured by Western blot detection of pSTAT3-Y705 expression compared to STAT3 expression) was determined.

[0147] These experiments showed that the agonist anti-LEPR antibodies of the present invention (H4H16650 and H4H16679) stimulate LEPR signaling in cells expressing the LEPR-A409E variant or the LEPR-P316T variant in a dose-dependent manner (when measured by STAT3 expression) (Figure 2, panels B and C). In contrast, treatment with leptin induced only moderate signaling in cells expressing the LEPR-P316T variant and no signaling in cells expressing the LEPR-A409E variant (Figure 2, panel A). Furthermore, LEPR signaling was not detected in any of the cell lines treated with vehicle or IgG control antibody (data not shown). Other signal transduction-deficient or signal transduction-disrupted LEPR variants were also tested in this assay but were not activated by the anti-LEPR variants (data not shown), suggesting that this rescue effect may be variant-dependent.

[0148] The results of this example indicate that the agonist anti-LEPR antibodies of the present invention may be useful in diseases or disorders (such as early-onset obesity) caused by or associated with certain signal transduction-deficient or signal transduction-disrupted LEPR variants (e.g., LEPR-P316T or LEPR-A409E).

[0149] (Example 9: Octet Cross-Competition between Different Anti-LEPR Monoclonal Antibodies) Binding competition between panels of different anti-LEPR monoclonal antibodies was determined using a real-time label-free biolayer interferometry assay on an Octet HTX biosensor platform (Pall ForteBio Corp.). The entire experiment was performed by shaking the plate at 1000 rpm 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). To evaluate whether two antibodies can compete with each other for binding to their respective epitopes on recombinant human LEPR (hLEPR.mmh; SEQ ID NO: 114) expressed with a C-terminal myc-myc-hexahistidine tag, an anti-pentaHis antibody-coated Octet biosensor chip (Fortebio Inc, #18-5122) was first captured by immersing the biosensor chip in wells containing 20 μg / mL hLEPR-MMH with approximately 0.25 nM or 0.34 nM hLEPR-MMH for 5 minutes. Subsequently, the antigen-captured biosensor chip was saturated by immersing it in wells containing a 50 μg / mL solution of the first anti-LEPR monoclonal antibody (subsequently referred to as mAb-1) for 210 seconds. The biosensor chip was then subsequently immersed in wells containing a 50 μg / mL solution of the second anti-LEPR monoclonal antibody (subsequently referred to as mAb-2) for 150 seconds. The biosensor chip was washed with HBS-EBT buffer between every step of the experiment. The real-time binding response was monitored throughout the course of the experiment, and the binding response was recorded at the end of each step. The response of mAb-2 binding to hLEPR-MMH pre-complexed with mAb-1 was compared, and the competitive / non-competitive behavior of different anti-LEPR monoclonal antibodies was determined as shown in Tables 14 and 15.

Table 14

Table 15

[0150] (Example 10: In Vivo Potency of LEPR Agonist Antibodies H4H16650P2, H4H16679P2, H4H17319P2, and H4H17321P2 in an Inducible Mouse Model of Leptin Deficiency) The effects of the four specific agonist anti-LEPR antibodies of the invention, H4H16650P2, H4H16679P2, H4H17319P2, and H4H17321P2, on food intake, body weight, and adiposity were determined in an inducible mouse model of leptin deficiency. The leptin-deficient model was induced by hydrodynamic DNA delivery (HDD) of a plasmid encoding the murine LEPR extracellular domain with an hFc-tag (mLEPR.hFc or “leptin trap” as referred to herein; SEQ ID NO: 120). The leptin trap is secreted when expressed and binds to circulating leptin. After HDD of 50 μg of the DNA construct encoding the leptin trap, the mice exhibited increased food consumption as well as increased body fat accumulation and body weight. Hu / Hu The baseline daily food intake was measured between 7 and 4 days prior (-7 and -4 days) to the administration of the leptin trap. On day 0, 35 male LEPRs, 13 - 17 weeks old

[0151] were used. Hu / HuMice were successfully subjected to HDD using a leptin trap. Retro-orbital blood was collected on days 6 and 13 after HDD, and body composition including body fat accumulation was quantified by μCT. On day 7 after HDD, mice were randomized into five groups of 7 mice based on percent body weight change from day 0. Each group received a single dose of 3 mg / kg of isotype control antibody, 3 mg / kg of H4H16650P2, 3 mg / kg of H4H16679P2, 3 mg / kg of H4H17319P2, or 3 mg / kg of H4H17321 via subcutaneous injection. The isotype control antibody did not bind to any known mouse proteins. Food intake and body weight were measured for each animal over the duration of the study. Figure 3 summarizes the mean daily food intake for each treatment group. In Figure 3, the dotted line represents the mean baseline food intake before HDD injection. The percent change in body weight from day 0 was calculated for each animal at each time point. Figure 4 summarizes the mean percent change in body weight for animals in each antibody treatment group. Figure 5 summarizes the mean fat mass for animals in each antibody treatment group quantified by μCT 1 day before antibody treatment and 6 days after treatment. All results are expressed as mean ± SEM.

[0152] As shown in FIGS. 3 and 4, a similar increase in food intake and percent body weight change was observed among the groups of mice before antibody treatment after HDD with leptin trap. As shown in FIG. 3, mice treated with 3 mg / kg of antibody H4H16650P2 or H4H16679P2 showed a significant reduction in food intake starting from 1 day after antibody treatment (day 8 after HDD) and at subsequent measured time points compared to mice injected with the isotype control antibody. Mice treated with 3 mg / kg of antibody H4H17319P2 or H4H17321P2 showed a significant reduction in food intake 2 days after antibody treatment (day 9 after HDD) and at other subsequent measured time points compared to mice injected with the isotype control antibody. As shown in FIG. 4, mice treated with 3 mg / kg of antibody H4H16650P2 showed a significant reduction in percent body weight change 1 day after antibody treatment (day 8 after HDD) and at other subsequent measured time points compared to mice injected with the isotype control antibody. One day after antibody treatment, i.e., on day 8, mice treated with the isotype control showed an increase in body weight of 21.16 ± 1.27% from day 0, while mice treated with H4H16650P2 had an increase in body weight of 15.57 ± 0.9% from day 0. Mice treated with 3 mg / kg of antibody H4H16679P2, H4H17319P2, or H4H17321P2 showed a significant reduction in percent body weight change 2 days after antibody treatment (day 9 after HDD) and at other subsequent measured time points compared to mice injected with the isotype control antibody. On day 9, the % body weight change from day 0 was 23.18 ± 1.22, 13.17 ± 1.05, 12.95 ± 1.26, 15.98 ± 1.78, and 15.83 ± 2.01 for mice treated with isotype control, H4H16650P2, H4H16679P2, H4H17319P2, or H4H17321P2, respectively. As shown in FIG. 5, mice treated with 3 mg / kg of the isotype control antibody demonstrated a significant increase in fat mass 6 days after antibody treatment (day 13 after HDD) compared to 1 day before antibody treatment (day 6 after HDD).Mice treated with 3 mg / kg of antibody H4H16650P2, H4H16679P2, H4H17319P2, or H4H17321P2 did not show an increase in adipose mass after antibody treatment compared to before antibody treatment. Six days after treatment (day 13 after HDD), mice treated with 3 mg / kg of antibody H4H16650P2, H4H16679P2, or H4H17319P2 demonstrated a significant decrease in adipose mass compared to mice treated with 3 mg / kg of isotype control antibody.

[0153] (Example 11: Epitope Mapping of H4H16650P2 Binding to Human Leptin Receptor (hLEPR.mmh) by Hydrogen-Deuterium Exchange) Experiments were conducted to determine the amino acid residues of hLEPR.mmh (amino acids M1 - D839 of SEQ ID NO: 114) with which H4H16650P2 interacts. For this purpose, H / D exchange epitope mapping using mass spectrometry was performed. General descriptions of the H / D exchange method are shown, for example, in Ehring (1999), Analytical Biochemistry, Vol. 267, No. 2, pp. 252 - 259; and Engen and Smith (2001), Anal. Chem., Vol. 73, pp. 256A - 265A.

[0154] Experimental procedure. The HDX-MS experiment was performed on an integrated Waters HDX / MS platform consisting of a Leaptec HDX PAL system for deuterium labeling, a Waters Acquity M-Class (Auxiliary solvent manager) for sample digestion and loading, a Waters Acquity M-Class (μBinary solvent manager) for analytical column gradient, and a Synapt G2-Si mass spectrometer for peptic peptide mass measurement.

[0155] The labeled solution was prepared with 10 mM PBS buffer in D2O at pD 7.0 (equivalent to pH 6.6). For deuterium labeling, 3.8 μL of hLEPR.mmh (8 pmol / μL) or hLEPR.mmh premixed with antibody at a molar ratio of 2:1 was incubated with 56.2 μL of D2O labeling solution at various time points (e.g., non-deuterated control = 0 seconds, 1 minute, and 20 minutes of labeling). Deuteration was quenched by transferring 50 μL of the sample to 50 μL of pre-cooled quench buffer (100 mM phosphate buffer, 0.2 M TCEP in pH 2.5, 6 M guanidine chloride), and the mixed sample was incubated at 1.0 °C for 2 minutes. The quenched sample was then injected into a Waters HDX Manager for on-line pepsin / protease XIII digestion. The digested peptides were trapped at 0 °C on an ACQUITY UPLC BEH C18 1.7 μm, 2.1×5 mm VanGuard pre-column and eluted onto an analytical column ACQUITY UPLC BEH C18 1.7 μm, 1.0×50 mm for a 9-minute gradient separation of 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 at a cone voltage of 37 V, a scan time of 0.5 seconds, and a mass / charge range of 50 - 1700 Th.

[0156] To identify peptides derived from human LEPR, LC-MSE data from non-deuterated samples were processed and searched against a database containing human LEPR, pepsin, and their randomized sequences via Waters ProteinLynx Global Server (PLGS) software. The identified peptides were imported into DynamX software and filtered by two criteria: 1) minimum product per amino acid: 0.2, and 2) replicate file threshold: 3. Then, DynamX software automatically determined the deuterium incorporation of each peptide based on retention time and high mass accuracy (<10 ppm) over multiple time points, repeating three times at each time.

[0157] Results. MS EUsing an online pepsin / protease XIII column coupled with data acquisition, a total of 201 peptides derived from human LEPR were reproducibly identified in the absence or presence of antibody, showing 70% sequence coverage. Five peptides showed significantly reduced deuterium incorporation when binding to H4H16650P2 as shown in Table 16 (centroid delta value > 0.4 Dalton, p-value < 0.05). The recorded peptide masses corresponded to the average of the centroid MH+ masses from three replicates. These peptides corresponded to amino acids 162 - 169 (human LEPR; amino acids LYVLPEVL of SEQ ID NO: 113) and amino acids 170 - 181 (human LEPR; amino acids EDSPLVPQKGSF of SEQ ID NO: 113) and had a slower deuterium incorporation rate when binding to H4H16650P2. These identified residues also corresponded to residues 162 - 169 and 170 - 181 of human LEPR as defined by Uniprot entry P48357 (SEQ ID NO: 113; human leptin receptor).

Table 16

[0158] (Example 12: In Vivo Efficacy Test of LEPR Potentiator Antibodies in Humanized LEPR Mice) The effects of three specific potentiator anti-LEPR antibodies of the present invention, H4H18482P2, H4H18487P2, and H4H18492P2, on body weight and body fat accumulation were determined in genetically engineered LEPR Hu / Hu mice housed individually. This mouse expresses a leptin receptor (mLepr.hFc, SEQ ID NO: 120) composed of the human LEPR extracellular domain sequence instead of the murine LEPR extracellular domain sequence.

[0159] - On day - 19, body composition including body fat accumulation was quantified by μCT. On day 0, 48 female LEPR Hu / HuMice were randomized based on body weight into four groups of 12 mice. On day 0 and day 11, mice from each group received a single dose of 30 mg / kg of an isotype control antibody, 30 mg / kg of H4H18482P2, 30 mg / kg of H4H18487P2, or 30 mg / kg of H4H18492P2 via subcutaneous injection. The isotype control antibody does not bind to any known mouse proteins. Body weight was measured for each animal over the duration of the study. The percent change in body weight from day 0 was calculated for each animal at each time point. Figure 6 summarizes the mean percent change in body weight for animals in each treatment group. Figure 6 summarizes the mean fat mass for animals in each antibody treatment group quantified by μCT 19 days before antibody treatment and 11 days after treatment. All results are expressed as mean ± SEM.

[0160] As shown in Figure 6, a decrease in the percent change in body weight was observed after dosing with the LEPR enhancer antibody, but not after dosing with the isotype control antibody. As shown in Figure 6, mice treated with 30 mg / kg of H4H18482P2 exhibited a significant decrease in percent body weight change starting on day 2 (day 2) after treatment and at other time points compared to mice injected with the isotype control antibody. Mice treated with 30 mg / kg of H4H18487P2 exhibited a significant decrease in percent body weight change starting on day 2 and at other time points compared to mice injected with the isotype control antibody. Mice treated with 30 mg / kg of H4H18492P2 exhibited a significant decrease in percent body weight change on days 4, 5, and 17, but not at other time points, compared to mice injected with the isotype control antibody. Mice treated with 30 mg / kg of H4H18482P2 exhibited a significant decrease in percent body weight change starting on day 6 and on subsequent days, but not on days 7, 14, and 17, compared to mice injected with H4H18492P2. Mice treated with 30 mg / kg of H4H18487P2 exhibited a significant decrease in percent body weight change starting on day 3 and at other time points, but not on days 4 and 5, compared to mice injected with H4H18492P2.

[0161] As shown in Fig. 7A, there was no difference in fat mass among groups before treatment (day -19). As shown in Fig. 7B, mice treated with 30 mg / kg of antibodies H4H18482 and H4H18487 showed a statistically significant decrease in fat mass 17 days after treatment (day 12) compared to the isotype control antibody, while mice treated with H4H18492 did not.

[0162] The scope of the present invention is not limited by the specific embodiments described herein. Indeed, from the foregoing description of the invention and the accompanying drawings, various modifications will become apparent to those skilled in the art in addition to the modifications described herein. Such modifications are intended to fall within the scope of the appended claims.

[0163] (Example 13: Effect of the anti-LEPR antibody of the present invention on monkey LEPR signaling) To evaluate the transcriptional activation of the monkey leptin receptor, a stable cell line was developed. IMR-32 cells (human neuroblastoma ATCC) were generated to stably express the extracellular domain of Macaca fascicularis LEPR (MfLEPR; amino acids 22 - 837 of accession number XP_005543194.1 with threonine 827 changed to alanine) fused to the transmembrane and cytosolic domains of human LEPR (hLEPR; amino acids 840 - 1165 of accession number NP_002294.2) together with a luciferase reporter (STAT3-Luc; SABiosciences, #CLS-6028L). The resulting cell line, hereinafter referred to as IMR-32 / STAT3-Luc / MfLEPR, was isolated and maintained in MEM-Earl medium supplemented with 10% FBS, NEAA, 1 μg / mL puromycin, 100 μg / mL hygromycin B, and penicillin / streptomycin / L-glutamine.

[0164] A bioassay was performed to measure the effect of the anti-LEPR antibody of the present invention on monkey LEPR signaling in the absence of leptin. For the bioassay, IMR-32 / STAT3-Luc / MfLEPR cells were seeded at 10,000 cells / well in a 96-well plate with 0.1% FBS (assay buffer) in Optimem containing penicillin / streptomycin and incubated overnight at 37 °C in 5% CO2. The next day, human leptin (hLeptin), the anti-LEPR antibody, or an isotype control antibody was serially diluted in the assay buffer from 50 nM to 0.8 pM (samples containing buffer alone without the + test molecule) and added to the cells. After 5.5 hours at 37 °C under 5% CO2, luciferase activity was measured using OneGlo™ reagent (Promega, #E6031) and a Victor™ X multi-label plate reader (Perkin Elmer). The results were analyzed using non-linear regression (4-parameter logistics) with Prism™ 6 software (GraphPad) to obtain the EC 50 value. The percentage of antibody activation was calculated as the maximum range of RLU achieved by the antibody compared to that of the maximum range of RLU achieved by hLeptin.

[0165] As shown in Table 17, in the absence of hLeptin, all of the anti-LEPR antibodies tested showed activation of monkey LEPR signaling in IMR-32 / STAT3-Luc / mfLEPR cells, and the EC 50 values were in the range of 266 pM to 368 pM, the maximum activation was in the range of 76% to 82%, and 100% activation was obtained with hLeptin. hLeptin was activated at an EC 50 value of 333 pM. The isotype control antibody did not demonstrate any measurable stimulation of IMR-32 / STAT3-Luc / mfLEPR cells.

Table 17

[0166] (Example 14: Epitope Binding to the Full-Length Extracellular Domain of Human LEPR Using Luminex MFI Signal) To determine the epitopes of human LEPR to which the anti-LEPR antibodies of the present invention bind, the interaction of the anti-LEPR antibodies with recombinant human LEPR protein domains was characterized using an analysis based on Luminex FLEXMAP (FM3DD, Luminex Corp) flow cytometry. For the assay, approximately 3 million carboxylated Microplex R microspheres (Luminex, catalog number LC1000A) were washed, vortexed, and sonicated in 0.1 M NaPO4, pH 6.2 (activation buffer), then centrifuged to remove the supernatant. The microspheres were resuspended at 25°C in 120 μL of activation buffer, and the carboxylate groups (-COOH) were activated by adding 15 μL of 50 mg / mL N-hydroxysuccinimide (NHS, Thermo Scientific, catalog number 24500), followed by 15 μL of 50 mg / mL 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC, Thermo Scientific, catalog number 22980). After 10 minutes, the pH of the reaction was reduced to 5.0 by adding 600 μL of 50 mM MES, pH 5 (coupling buffer), the microspheres were vortexed, and centrifuged to remove the supernatant. The activated beads were immediately mixed with 500 μL of 20 μg / mL monoclonal anti-myc antibody containing mouse IgG or human IgG in coupling buffer and incubated at 25°C for 2 hours. The coupling reaction was quenched by adding 50 μL of 1 M Tris-HCl, pH 8.0, the microspheres were vortexed rapidly, centrifuged, and washed 4 times with 1 mL of DPBS to remove uncoupled protein and other reaction components.

[0167] Human LEPR extracellular domain expressed with C-terminal myc-myc hexahistidine tag (human LEPR-MMH, SEQ ID NO: 113), human LEPR CRH1(D1) expressed with C-terminal myc-myc hexahistidine tag (myc-myc hexahistidine tag, human LEPR CRH1(D1)-MMH containing amino acids 209-236, amino acids 1-208 of SEQ ID NO: 113), human LEPR CRH1(D1, D2) domain expressed with C-terminal myc-myc hexahistidine tag (myc-myc hexahistidine tag, human LEPR CRH1 (D1, D2)-MMH containing amino acids 319-346, amino acids 1-318 of SEQ ID NO: 113), human LEPR CRH1-Ig(D1, D2, D3) domain expressed with C-terminal myc-myc hexahistidine tag (myc-myc hexahistidine tag, human LEPR CRH1(D1, D2, D3)-MMH containing amino acids 279-306, amino acids 1-278 of SEQ ID NO: 113), human LEPR CRH1-Ig(D2, D3) domain expressed with C-terminal myc-myc hexahistidine tag (myc-myc hexahistidine tag, human LEPR CRH1-Ig(D2, D3)-MMH containing amino acids 199-226, amino acids 1-198 of SEQ ID NO: 113), human LEPR Ig(D3) domain expressed with C-terminal myc-myc hexahistidine tag (myc-myc hexahistidine tag, human LEPR Ig(D3)-MMH containing amino acids 89-116, amino acids 1-88 of SEQ ID NO: 113), human LEPR CRH2 domain expressed with C-terminal myc-myc hexahistidine tag (myc-myc-hexahistidine tag, human LEPR CRH2-MMH containing amino acids 208-235, amino acids 1-207 of SEQ ID NO: 113), human LEPR FNIII domain expressed with C-terminal myc-myc hexahistidine tag (myc-myc hexahistidine tag, human LEPR FNIII-MMH containing amino acids 205-232, amino acids 1-204 of SEQ ID NO: 113), and human LEPR expressed with C-terminal myc-myc hexahistidine tagThe transiently expressed LEPR protein containing the Ig-CRH2-FNIII domain (myc-myc-hexahistidine tag, human LEPR Ig-CRH2-FNIII-MMH containing amino acids 511 - 538, amino acids 1 - 510 of SEQ ID NO: 113) was suspended in serum-free CHO-S-SFM II medium (Thermo Fisher, catalog number 31033020) and then clarified by centrifugation. An aliquot of microspheres immobilized with anti-myc monoclonal antibody was prepared as described above and added individually to 1 mL of each of these protein supernatants. The microspheres were gently mixed, incubated at 25°C for 2 hours, washed twice with 1 mL of DBPS, centrifuged to remove the supernatant, and finally resuspended in 1 mL of DPBS buffer. 48 μL of anti-myc IgG-binding microspheres from each individual reaction with full-length human LEPR and with each of the human LEPR domain proteins were withdrawn and mixed together in 3.6 mL of PBS + 20 mg / mL BSA + 0.05% sodium azide (blocking buffer).

[0168] From this mixed pool, 75 μL of microspheres were seeded per well of a 96-well filter plate (Millipore, catalog number: MSBVN1250), mixed with 25 μL of individual anti-human LEPR monoclonal antibodies (0.5 or 5 μg / mL), incubated at 25°C for 2 hours, and then washed twice with 200 μL of DPBS containing 0.05% Tween20 (washing buffer). To detect and quantify the amount of anti-LEPR antibody levels bound to individual microspheres, 2.5 μg / mL R-phycoerythrin-conjugated goat F(ab’)2 anti-human kappa in blocking buffer (Southern 100 μL of R-phycoerythrin AffiniPure F(ab’)2 fragment goat anti-mouse IgG, F(ab’)2 fragment specific (Jackson Immunoresearch, Catalog No. 115-116-072) at 1.25 μg / mL in blocking buffer or Biotech, Catalog No. 2063-09) was added and incubated at 25 °C for 30 minutes. After 30 minutes, the samples were washed twice with 200 μL of wash buffer and resuspended in 150 μL of wash buffer. The median fluorescence intensity (MFI) of the microspheres was measured with a Luminex analyzer.

Table 18

[0169] The results of the Luminex-based analysis are presented in Table 18. The Luminex MFI signal intensity indicates that the 12 anti-LEPR antibodies of the present invention bind to the complete human LEPR extracellular domain. Anti-LEPR antibodies H4H18417P2, H4H18438P2, and H4H18492P2 bind to epitopes within the CRH1 D2 domain of human LEPR. Anti-LEPR antibodies H4H18449P2, H4H16650P, and H4H16679P bind to epitopes within the CRH1 (D1-2) domain of human LEPR. The anti-LEPR antibody control mAB binds to an epitope within the CRH2 domain of human LEPR. Anti-LEPR antibody H4H18445P2 binds to an epitope within the FNIII domain of human LEPR. Anti-LEPR antibodies H4H18446P2, H4H18482P2, and H4H18487P2 bind to epitopes within the Ig-CRH2-FNIII domain of human LEPR.

Claims

A method for expressing a polypeptide comprising a heavy chain variable region (HCVR) and a polypeptide comprising a light chain variable region (LCVR) of an antibody or an antigen-binding fragment thereof that specifically binds to LEPR, said method comprising: Under conditions permitting production of said antibody or fragment, the following: (i) an LCVR comprising the amino acid sequence shown in SEQ ID NO: 10 and an HCVR comprising the amino acid sequence shown in SEQ ID NO: 26, (ii) an LCVR comprising the amino acid sequence shown in SEQ ID NO: 10 and an HCVR comprising the amino acid sequence shown in SEQ ID NO: 34, (iii) an LCVR comprising the amino acid sequence shown in SEQ ID NO: 10 and an HCVR comprising the amino acid sequence shown in SEQ ID NO: 2, (iv) an LCVR comprising the amino acid sequence shown in SEQ ID NO: 10 and an HCVR comprising the amino acid sequence shown in SEQ ID NO: 18, (v) an LCVR comprising the amino acid sequence shown in SEQ ID NO: 10 and an HCVR comprising the amino acid sequence shown in SEQ ID NO: 42, (vi) an LCVR comprising the amino acid sequence shown in SEQ ID NO: 10 and an HCVR comprising the amino acid sequence shown in SEQ ID NO: 50, (vii) an LCVR comprising the amino acid sequence shown in SEQ ID NO: 10 and an HCVR comprising the amino acid sequence shown in SEQ ID NO: 58, (viii) an LCVR comprising the amino acid sequence shown in SEQ ID NO: 10 and an HCVR comprising the amino acid sequence shown in SEQ ID NO: 66, (ix) an LCVR comprising the amino acid sequence shown in SEQ ID NO: 10 and an HCVR comprising the amino acid sequence shown in SEQ ID NO: 74, (x) an LCVR comprising the amino acid sequence shown in SEQ ID NO: 90 and an HCVR comprising the amino acid sequence shown in SEQ ID NO: 82, (xi) an LCVR comprising the amino acid sequence shown in SEQ ID NO: 90 and an HCVR comprising the amino acid sequence shown in SEQ ID NO: 98, or (xii) an LCVR comprising the amino acid sequence shown in SEQ ID NO: 90 and an HCVR comprising the amino acid sequence shown in SEQ ID NO: 106 Culturing a host cell comprising a recombinant expression vector comprising a nucleic acid encoding the same, Recovering the antibody or fragment so produced A method comprising.

2. The method according to claim 1, wherein said nucleic acid encodes an HCVR as shown in SEQ ID NO: 26 and an LCVR as shown in SEQ ID NO:

10.

3. The method according to claim 1, wherein said nucleic acid encodes an HCVR as shown in SEQ ID NO: 34 and an LCVR as shown in SEQ ID NO:

10.

4. The method according to claim 1, wherein the nucleic acid encodes an HCV RNA as shown in SEQ ID NO: 2 and an LCV RNA as shown in SEQ ID NO:

10.

5. The recombinant expression vector is a nucleotide sequence as shown in SEQ ID NO: 25 and SEQ ID NO: 9; a nucleotide sequence as shown in SEQ ID NO: 33 and SEQ ID NO: 9; a nucleotide sequence as shown in SEQ ID NO: 1 and SEQ ID NO: 9; a nucleotide sequence as shown in SEQ ID NO: 17 and SEQ ID NO: 9; a nucleotide sequence as shown in SEQ ID NO: 41 and SEQ ID NO: 9; a nucleotide sequence as shown in SEQ ID NO: 49 and SEQ ID NO: 9; a nucleotide sequence as shown in SEQ ID NO: 57 and SEQ ID NO: 9; a nucleotide sequence as shown in SEQ ID NO: 65 and SEQ ID NO: 9; a nucleotide sequence as shown in SEQ ID NO: 73 and SEQ ID NO: 9; a nucleotide sequence as shown in SEQ ID NO: 81 and SEQ ID NO: 89; a nucleotide sequence as shown in SEQ ID NO: 97 and SEQ ID NO: 89; or a nucleotide sequence as shown in SEQ ID NO: 105 and SEQ ID NO: 89 comprising the method according to claim 1.

6. The method according to claim 1, wherein the recombinant expression vector comprises a nucleotide sequence as shown in SEQ ID NO: 25 and SEQ ID NO:

9.

7. The method according to claim 1, wherein the recombinant expression vector comprises a nucleotide sequence as shown in SEQ ID NO: 33 and SEQ ID NO:

9.

8. The method according to claim 1, wherein the recombinant expression vector comprises a nucleotide sequence as shown in SEQ ID NO: 1 and SEQ ID NO: 9.

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