Antigen-binding protein that antagonistizes leptin receptors

Abstract

To provide alternative approaches to treating leptin resistance and other conditions associated with elevated serum leptin levels and / or excessive LEPR signaling.SOLUTION: The present invention provides antibodies and antigen-binding fragments thereof that bind to a human leptin receptor (LEPR). The antibodies of the present invention are antagonist antibodies; i.e., binding of the anti-LEPR antibodies of the invention to LEPR results in blockade or down-regulation of leptin receptor signaling in cells. Accordingly, in various embodiments, the antagonist antibodies of the present invention exhibit weak partial agonist activity.SELECTED DRAWING: None

Description

[Technical Field] 【0001】 Background of the Invention Leptin is a polypeptide hormone primarily expressed by adipose tissue, involved in regulating metabolism, energy balance, and feeding. Leptin activity is mediated by interaction with leptin receptors and signal transduction through these receptors. The leptin receptor (also known as "LEPR," "WSX," "OB receptor," "OB-R," and "CD295") is a single-pass transmembrane receptor of the class I cytokine receptor family with a large (818 amino acid) extracellular domain. Elevated expression of leptin, Ob-R leptin receptor, or both may contribute to a number of disorders, including anorexia or other psychiatric eating disorders, chronic kidney disease cachexia, other cachexias such as congestive heart failure cachexia, pulmonary cachexia, radiation cachexia, and cancer cachexia, autoimmune disorders such as inflammatory bowel disease, lupus erythematosus, multiple sclerosis, psoriasis, cardiovascular disease, hypertension, depression, non-alcoholic fatty liver disease, neurodegenerative disorders, and cancers such as hepatocellular carcinoma, melanoma, and breast cancer, but not limited to these. [Background technology] 【0002】 Proposed therapeutic approaches to address hyperleptin signaling include leptin receptor peptide antagonists and antagonist variants, e.g., soluble leptin receptor variants; competitive LEPR antagonists, e.g., antibody 9F8 (Fazeli et al. (2006) J Immunological Methods 312, pp. 190-200); or nanobodies targeting the leptin receptor (McMurphy et al., PLOS One (2014) 9 (2):e89895); the use of fibronectin III domains; appetite stimulants (e.g., ghrelin and NPY); blocking of leptin's downstream mediators (e.g., melanocortin receptor 4); and / or lifestyle modifications. However, such approaches have generally been shown to have limited efficacy. Therefore, there is a need in the art for alternative approaches to treat leptin resistance and other conditions associated with elevated serum leptin levels and / or hyperleptin signaling. Sequence List A formal copy of the sequence listing is submitted electronically via EFS-Web at the same time as this specification, as an ASCII-formatted sequence listing with the filename: 2017_11_08_10271WO01_SEQ_LIST_ST25.TXT, created on November 8, 2017, and approximately 87.4 kilobytes in size. The sequence listing contained in this ASCII-formatted document is part of this specification, and its entirety is incorporated herein by reference. [Prior art documents] [Non-patent literature] 【0003】 [Non-Patent Document 1] Fazeli et al. (2006) J Immunological Methods, Vol. 312, pp. 190-200. [Non-Patent Document 2] McMurphy et al., PLOS One (2014) Vol. 9 (Issue 2): e89895 [Overview of the project] [Means for solving the problem] 【0004】 Brief summary of the invention This invention provides an antibody that binds to the human leptin receptor (LEPR) and its antigen-binding fragment. The antibody of this invention is an antagonist antibody; that is, binding of the anti-LEPR antibody of this invention to LEPR results in blocking or downregulation of leptin receptor signaling in cells. Therefore, in various embodiments, the antagonist antibody of this invention exhibits weak partial agonist activity. Alternatively, the antibody of this invention is useful, for example, to downregulate the biological activity of leptin in a subject. Therefore, the antibody and antigen-binding fragment of this invention are useful in the treatment of diseases and disorders associated with elevated leptin signaling. 【0005】 The antibodies of the present invention may be full-length (e.g., IgG1 or IgG4 antibodies) or may consist only of antigen-binding moieties (e.g., Fab, F(ab')2, or scFv fragments), and may be modified in a manner that affects their functionality, for example, to remove residual effector function (Reddy et al., 2000, J. Immunol. Vol. 164: pp. 1925-1933). 【0006】 Exemplary LEPR antagonist antibodies of the present invention are listed in Tables 1 and 2 herein. Table 1 lists the amino acid sequence identification numbers 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 LEPR antagonist antibodies. Table 2 lists the nucleic acid sequence identification numbers of the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the exemplary LEPR antagonist antibodies. 【0007】 The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to LEPR, comprising an HCVR having an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. 【0008】 The present invention also provides an antibody or antigen-binding fragment thereof that specifically binds to LEPR, comprising an LCVR having an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. 【0009】 The present invention also provides an antibody or antigen-binding fragment thereof that specifically binds to LEPR, comprising an HCVR and LCVR amino acid sequence pair (HCVR / LCVR) containing one of the HCVR amino acid sequences listed in Table 1 paired with one of the LCVR amino acid sequences listed in Table 1. According to a particular embodiment, the present invention provides an antibody or antigen-binding fragment thereof that comprises an HCVR / LCVR amino acid sequence pair contained within one of the exemplary anti-LEPR antibodies listed in Table 1. In a particular embodiment, the HCVR / LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 10, 26 / 10, 34 / 10, 42 / 10, 50 / 10, 58 / 10, 66 / 10, and 74 / 82. 【0010】 The present invention also provides an antibody or antigen-binding fragment thereof that specifically binds to LEPR, comprising an HCDR1 having an amino acid sequence selected from any of the heavy chain CDR1 (HCDR1) sequences listed in Table 1 or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. 【0011】 The present invention also provides an antibody or antigen-binding fragment thereof that specifically binds to LEPR, comprising an HCDR2 having an amino acid sequence selected from any of the heavy chain CDR2 (HCDR2) amino acid sequences listed in Table 1 or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. 【0012】 The present invention also provides an antibody or antigen-binding fragment thereof that specifically binds to LEPR, comprising an HCDR3 having an amino acid sequence selected from any of the heavy chain CDR3 (HCDR3) amino acid sequences listed in Table 1 or substantially similar 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 antigen-binding fragment thereof that specifically binds to LEPR, comprising an LCDR1 comprising an amino acid sequence selected from any of the light chain CDR1 (LCDR1) amino acid sequences listed in Table 1 or substantially similar 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 antigen-binding fragment thereof that specifically binds to LEPR, comprising an LCDR2 having an amino acid sequence selected from any of the light chain CDR2 (LCDR2) amino acid sequences listed in Table 1 or substantially similar 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 antigen-binding fragment thereof that specifically binds to LEPR, comprising an LCDR3 having an amino acid sequence selected from any of the light chain CDR3 (LCDR3) amino acid sequences listed in Table 1 or substantially similar 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 antigen-binding fragment that specifically binds to LEPR, comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) containing one of the HCDR3 amino acid sequences listed in Table 1 paired with one of the LCDR3 amino acid sequences listed in Table 1. According to a particular embodiment, the present invention provides an antibody or antigen-binding fragment that comprises an HCDR3 / LCDR3 amino acid sequence pair contained within one of the exemplary anti-LEPR antibodies listed in Table 1. In a particular embodiment, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 8 / 16, 24 / 16, 32 / 16, 40 / 16, 48 / 16, 56 / 16, 64 / 16, 72 / 16, 80 / 16, and 80 / 88. 【0017】 The present invention also provides an antibody or its antigen-binding fragment that specifically binds to LEPR, comprising a set of six CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) contained within any of the exemplary anti-LEPR antibodies listed in Table 1. In a particular embodiment, the set of amino acid sequences for HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 is selected from the group consisting of SEQ ID NOs: 4, 6, 8, 12, 14, 16; 20, 22, 24, 12, 14, 16; 28, 30, 32, 12, 14, 16; 36, 38, 40, 12, 14, 16; 52, 54, 56, 12, 14, 16; 60, 62, 64, 12, 14, 16; 68, 70, 72, 12, 14, 16; and 76, 78, 80, 84, 86, 88. 【0018】 In related embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to LEPR, comprising a set of six CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) contained within an amino acid sequence pair of HCVR / LCVR defined by any of the exemplary anti-LEPR antibodies listed in Table 1. For example, the present invention includes an antibody or antigen-binding fragment that specifically binds to LEPR, comprising a set of amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 contained within an amino acid sequence pair of HCVR / LCVR selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 10, 26 / 10, 34 / 10, 42 / 10, 50 / 10, 58 / 10, 66 / 10, and 74 / 82. Methods and techniques for identifying CDRs within the amino acid sequences of HCVR and LCVR are well known in the art and can be used to identify CDRs within the amino acid sequences of specific HCVR and / or LCVR disclosed herein. Exemplary rules that can be used to identify CDR boundaries include, for example, the definitions of Kabat, Chothia, and AbM. Generally, Kabat's definition is based on sequence variability, Chothia's definition is based on the location of structural loop regions, and AbM's definition is a compromise between Kabat's and Chothia's approaches. See, for example, Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273: pp. 927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86: pp. 9268-9272 (1989). Public databases for identifying CDR sequences in antibodies are also available. 【0019】 The present invention also provides nucleic acid molecules encoding an anti-LEPR antibody or a portion thereof. For example, the present invention provides nucleic acid molecules encoding any of the amino acid sequences of HCVR listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the nucleic acid sequences of HCVR listed in Table 2, or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. 【0020】 The present invention also provides nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. 【0021】 The present invention also provides a nucleic acid molecule encoding any of the HCDR1 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 2, or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. 【0022】 The present invention also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 2, or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. 【0023】 The present invention also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 2, or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. 【0024】 The present invention also provides nucleic acid molecules encoding any of the amino acid sequences of LCDR1 listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the nucleic acid sequences of LCDR1 listed in Table 2, or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. 【0025】 The present invention also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 2, or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. 【0026】 The present invention also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 2, or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. 【0027】 The present invention also provides a nucleic acid molecule encoding HCVR, wherein 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 one of the exemplary anti-LEPR antibodies listed in Table 1. 【0028】 The present invention also provides a nucleic acid molecule encoding LCVR, wherein 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 one of the exemplary anti-LEPR antibodies listed in Table 1. 【0029】 The present invention also provides a nucleic acid molecule encoding both HCVR and LCVR, wherein HCVR comprises an amino acid sequence of any of the HCVR amino acid sequences listed in Table 1, and LCVR comprises an amino acid sequence of any of the LCVR amino acid sequences listed in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments of this aspect of the present invention, the nucleic acid molecule encodes both HCVR and LCVR, and both HCVR and LCVR are derived from the same anti-LEPR antibody listed in Table 1. 【0030】 The present invention also provides recombinant expression vectors capable of expressing polypeptides comprising the heavy chain or light chain variable region of an anti-LEPR antibody. For example, the present invention includes recombinant expression vectors comprising any of the above nucleic acid molecules, i.e., nucleic acid molecules encoding any of the sequences of HCVR, LCVR, and / or CDR listed in Table 1. In addition to host cells into which such vectors have been introduced, the present invention also includes methods for producing antibodies or portions thereof by culturing host cells under conditions that enable the production of antibodies or antibody fragments, and recovering the antibodies and antibody fragments thus produced. 【0031】 In another embodiment, 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 embodiment, the present invention features a composition which is a combination of an anti-LEPR antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent which is advantageously combined with the anti-LEPR antibody. 【0032】 In yet another aspect, the present invention provides a therapeutic method for downmodulating or inactivating LEPR signaling using an anti-LEPR antibody or antigen-binding moiety of the present invention. A therapeutic method according to this aspect of the present invention comprises administering a therapeutically effective amount of a pharmaceutical composition comprising the antibody or antigen-binding fragment of the present invention to a subject in need thereof. The disorder to be treated is any disease or condition that is improved, improved, inhibited or prevented by downmodulating or inactivating LEPR signaling or by blocking leptin activity. 【0033】 Other embodiments will become apparent from the detailed description below. [Brief explanation of the drawing] 【0034】 [Figure 1] Figure 1 shows the percentage change in food intake in mice treated with 30 mg / kg of anti-LEPR antibody H4H17322P2, H4H18457P2, or H4H18464, or an isotopic control antibody. 【0035】 [Figure 2] Figure 2 shows the mean percentage change in body weight of mice treated with 30 mg / kg of anti-LEPR antibody H4H17322P2, H4H18457P2, or H4H18464, or an isotopic control antibody. 【0036】 [Figure 3] Figure 3 shows the average fat mass of mice treated with 30 mg / kg of anti-LEPR antibodies H4H17322P2, H4H18457P2, or H4H18464, or their isotopic control antibodies. [Modes for carrying out the invention] 【0037】 Detailed description of the invention Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, and that such methods and conditions can be modified. It should also be understood that the scientific terms used herein are intended solely to describe specific embodiments and are not intended to be limiting, and the scope of the present invention is limited only by the appended claims. 【0038】 Unless otherwise defined, all scientific and technical terms used herein have the same meanings as those commonly understood by those skilled in the art in which this invention pertains. Where used herein, the term “about” when used in relation to a particular numerical value means that the value may vary by no more than 1% from the stated value. For example, where 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.). 【0039】 Any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of the present invention, but preferred methods and materials are described below. All patents, patent applications and non-patent literature referenced herein are incorporated herein by reference in their entirety. 【0040】 definition As used herein, the terms “leptin receptor” and “LEPR” refer to the human leptin receptor, comprising the amino acid sequence shown in Sequence ID No. 89 (see also UniProtKB / Swiss-Prot accession number P48357). Alternative names for LEPR used in 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 portion of the LEPR protein that exists under normal conditions as a single LEPR molecule without containing or possessing any multimerizing domains and without direct physical connection to another LEPR molecule. An exemplary monomeric LEPR molecule is the molecule referred to herein as “hLEPR.mmh,” comprising the amino acid sequence of Sequence ID No. 90 (see, for example, Example 3 herein). As used herein, the term “dimeric human LEPR” means a construct comprising two LEPR molecules linked to each other via a linker, covalent, non-covalent, or multimerizing domain such as an antibody Fc domain. Exemplary dimeric LEPR molecules are those referred to herein as “hLEPR.mFc” (see, for example, Example 3 herein), which comprises the amino acid sequence of SEQ ID NO: 91, or those referred to herein as “hLEPR.hFc”, which comprises the amino acid sequence of SEQ ID NO: 92. As used herein, expressions such as “anti-LEPR antibody,” “antibody that specifically binds to LEPR,” and “LEPR-specific binding protein” refer to molecules that bind to full-length human LEPR, monomeric human LEPR, dimeric human LEPR, or other constructs comprising or containing the extracellular domain of LEPR, unless specifically indicated otherwise. 【0041】 All references to proteins, polypeptides, and protein fragments in this specification are intended to refer to the human version of each protein, polypeptide, or protein fragment unless explicitly specified to be from a non-human species. Therefore, the expression "LEPR" means human LEPR unless explicitly specified to be from a non-human species, such as "mouse LEPR" or "monkey LEPR." 【0042】 As used herein, the expression “cell surface expressed LEPR” means one or more LEPR proteins, or their extracellular domains, expressed in vitro or in vivo on the surface of a cell such that at least a portion of the LEPR protein is exposed to the extracellular side of the cell membrane and is accessible to the antigen-binding portion of an antibody. “Cell surface expressed LEPR” may include or consist of LEPR proteins expressed on the surface of cells that normally express LEPR proteins (e.g., in their natural or wild-type state). Alternatively, “cell surface expressed LEPR” may include or consist of LEPR proteins expressed on the surface of cells that do not normally express human LEPR on their surface but have been artificially engineered to express LEPR on their surface. 【0043】 As used herein, expressions such as “anti-LEPR antibody” or “antibody that binds to the human leptin receptor” include both a monovalent antibody having single specificity, a first arm that binds to LEPR, and a second arm that binds to a second (target) antigen, wherein the anti-LEPR arm contains one of the HCVR / LCVR or CDR sequences listed in Table 1 herein. 【0044】 As used herein, the term "antibody" means any antigen-binding molecule or molecular complex that includes at least one complementarity-determining region (CDR) that specifically binds or interacts with a particular antigen (e.g., LEPR). The term "antibody" includes, in addition to immunoglobulin molecules that include four polypeptide chains interconnected by disulfide bonds, i.e., two heavy (H) chains and two light (L) chains, multimers thereof (e.g., IgM). Each heavy chain includes a heavy chain variable region (abbreviated herein as HCVR or V L ), and a heavy chain constant region. The heavy chain constant region includes three domains, i.e., C H 1, C H 2, and C H 3. Each light chain includes a light chain variable region (abbreviated herein as LCVR or V L ), 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 divided 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, which are arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of an anti-LEPR antibody (or antigen-binding portion thereof) may be identical to human germline sequences, or may be modified naturally or artificially. An amino acid consensus sequence can be defined based on the comparative analysis of two or more CDRs. 【0045】 As used herein, the term “antibody” also includes the antigen-binding fragment of a complete antibody molecule. As used herein, terms such as “antigen-binding portion” of an antibody and “antigen-binding fragment” of an antibody include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies may be derived from complete antibody molecules, for example, by any suitable standard technique, e.g., protein digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains and optionally constant domains. Such DNA is known and / or readily available, for example, from commercial suppliers, DNA libraries (e.g., phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated, for example, by chemical or molecular biological techniques, such as arranging one or more variable and / or constant domains into a suitable configuration, introducing codons, creating cysteine ​​residues, modifying, adding, or deleting amino acids. 【0046】 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) the hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR), e.g., the CDR3 peptide), or the smallest recognition unit consisting of amino acid residues mimicking a constrained FR3-CDR3-FR4 peptide. Other manipulated molecules, such as domain-specific antibodies, single-domain antibodies, domain deletion antibodies, chimeric antibodies, CDR graft antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunotherapy drugs (SMIPs), and shark variable IgNAR domains are also included in the expression “antigen-binding fragment” as used herein. 【0047】 The antigen-binding fragment of an antibody typically contains at least one variable domain. The variable domain may be of any size or amino acid composition and usually contains at least one CDR adjacent to or in-frame with one or more framework sequences. L V with a domain H In an antigen-binding fragment having a domain, V H and V L The domains may be positioned in any preferred arrangement relative to each other. For example, the variable region may be a dimer, V H -V H , V H -V L or V L -V L It may contain a dimer. Alternatively, the antigen-binding fragment of the antibody may contain monomer V H or V L It may contain a domain. 【0048】 In certain embodiments, the antigen-binding fragment of the antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within the antigen-binding fragment of the antibody of the present invention include: (i)V H -C H 1;(ii)V H -C H 2; (iii)V H -C H 3;(iv)V H -C H 1-C H 2;(v)V H -C H 1-C H 2-C H 3;(vi)V H -C H 2-C H 3;(vii)V H -C L ;(viii)V L -C H 1;(ix)V L -C H 2;(x)V L -C H3;(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 In any configuration of variable and constant domains, such as any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or linked by a whole or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids resulting in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody of the present invention may be linked to each other and / or one or more monomers V H Or V L The non-covalent association with the domain (e.g., via disulfide bonds) may include homodimers or heterodimers (or other polymers) of any of the variable and constant domain configurations listed above. 【0049】 Similar to complete antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody typically comprises at least two distinct variable domains, each of which can specifically bind to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, such as the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of the antigen-binding fragments of the antibody of the present invention using routine techniques available in the art. 【0050】 In certain embodiments of the present invention, the anti-LEPR antibody of the present invention is a human antibody. 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 antibody of the present invention may include, for example, amino acid residues in CDRs, particularly CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term “human antibody” is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, is grafted onto a human framework sequence. 【0051】 In some embodiments, the antibodies of the present invention may be recombinant human antibodies. As used herein, the term “recombinant human antibody” is intended to include all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into host cells (as further described below), antibodies isolated from a recombinant combinatorial human antibody library (as further described below), antibodies isolated from animals transgenic for the human immunoglobulin gene (e.g., mouse) (see, e.g., Taylor et al. (1992) Nucl. Acids Res. Vol. 20: pp. 6287-6295), or antibodies prepared, expressed, produced or isolated by any other means involving splicing of the human immunoglobulin gene sequence to another DNA sequence. Such recombinant human antibodies have variable and constant regions derived from the immunoglobulin sequence of the human germline. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, if transgenic animals are used for the human Ig sequence, in vivo somatic mutagenesis), and therefore the recombinant antibody V H and V L The amino acid sequence of the region is that of human germline V H and VL It originates from and relates to arrays, in This sequence does not necessarily need to be naturally present in the germline repertoire of human antibodies in vivo. 【0052】 This invention relates to a hinge, C H 2 or C H The antibody may contain one or more mutations in three regions, the mutations being desirable, for example, to improve the yield of a desired antibody form during manufacturing. 【0053】 The antibodies of the present invention may be isolated antibodies. As used herein, “isolated antibodies” means antibodies that have been identified and isolated and / or recovered from at least one component of their natural environment. For example, antibodies isolated or removed from at least one component of an organism, or from tissues or cells in which antibodies naturally exist or are naturally produced, are “isolated antibodies” for the purposes of the present invention. Isolated antibodies also include antibodies in situ within recombinant cells. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, isolated antibodies may be substantially free of other cellular material and / or chemicals. 【0054】 The present invention includes variants of anti-LEPR antibodies disclosed herein, comprising one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR region of the heavy and light chain variable domains compared to the corresponding germline sequence from which the antibody is derived. Such mutations can be readily identified, for example, by comparing the amino acid sequences disclosed herein to germline sequences available from public antibody sequence databases. The present invention also includes antibodies and antigen-binding fragments derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids in one or more frameworks and / or CDR regions are mutated to a corresponding residue in the germline sequence from which the antibody is derived, or to a corresponding residue in another human germline sequence, or to a conserved amino acid substitution of a residue in the corresponding germline (such sequence modifications are collectively referred to herein as “germline mutations”). Those skilled in the art can readily produce a number of antibodies and antigen-binding fragments comprising 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 and / or V LAll residues in the framework and / or CDR within the domain are mutated to revert to residues found in the original germline sequence from which the antibody originated. In other embodiments, only certain residues are mutated to revert to the original germline sequence, for example, mutated residues are found only within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or mutated residues are found only within CDR1, CDR2, or CDR3. In other embodiments, one or more residues in the framework and / or CDR are mutated to corresponding residues in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody originally originated). Furthermore, the antibody of the present invention may contain any combination of two or more germline mutations within the framework and / or CDR region, for example, certain individual residues are mutated to corresponding residues in a particular germline sequence, while certain other residues different from the original germline sequence are maintained or mutated to corresponding residues in a different germline sequence. Once antibodies and antigen-binding fragments containing one or more germline mutations are obtained, they can be easily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced biological properties of the antagonist or agonist (as in each case), or reduced immunogenicity. Antibodies and antigen-binding fragments obtained by this general method are included in the present invention. 【0055】 The present invention also includes anti-LEPR antibodies comprising a variant of any of the amino acid sequences of HCVR, LCVR, and / or CDR disclosed herein, having one or more conservative substitutions. For example, the present invention includes anti-LEPR antibodies comprising amino acid sequences of HCVR, LCVR, and / or CDR having, for example, less than 10, less than 8, less than 6, or less than 4, relative to any of the amino acid sequences of HCVR, LCVR, and / or CDR listed in Table 1 herein. In certain embodiments, the present invention provides anti-LEPR antibodies comprising a variant amino acid sequence of HCVR, LCVR, and / or CDR (including, for example, conservative substitutions) relative to the sequences listed in Table 1 herein, wherein such a variant antibody nevertheless exhibits one or more functions and / or characteristics of the exemplary anti-LEPR antibodies disclosed herein. 【0056】 The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as a paratope. A single antigen may have more than one epitope. Therefore, different antibodies may bind to different areas on an antigen and have different biological effects. Epitopes may be conformal or linear. Conformal epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope may contain a saccharide, phosphoryl group, or sulfonyl group portion on the antigen. 【0057】 The present invention comprises an anti-LEPR antibody and its antigen-binding fragment, comprising an amino acid sequence substantially similar to or substantially identical to the amino acid sequence of one or more variable domains or CDRs found in any of the exemplary anti-LEPR antibodies disclosed herein. 【0058】 When applied to polypeptides, the term “substantial similarity” or “substantially identical” means that two peptide sequences share at least 95% sequence identity, more preferably at least 98% or 99%, when optimally aligned by a program such as GAP or BESTFIT using default gap weighting. Preferably, the positions of non-identical residues differ by conservative amino acid substitutions. A “conservative amino acid substitution” is an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. If two or more amino acid sequences differ from each other by conservative substitutions, the sequence identity percentage or degree of similarity may be adjusted upward to compensate for the conservative nature of the substitutions. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. Vol. 24: pp. 307-331 (incorporated herein by reference). 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. The preferred group of conserved amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conserved substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: pp. 1443-1445 (incorporated herein by reference).A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix. 【0059】 Sequence similarity of polypeptides, also known as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, such as conserved amino acid substitutions. For example, GCG software includes programs such as Gap and Bestfit, which, when used with default parameters, can determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between wild-type proteins and their mutaines. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and sequence identity percentage of the region that best overlaps between the query sequence and the search sequence (Pearson (2000), cited above). Another preferred algorithm for comparing the sequences of the present invention against a database containing numerous 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. Vol. 215: pp. 403-410 and Altschul et al. (1997) Nucleic Acids Res. Vol. 25: pp. 3389-402 (each incorporated herein by reference). 【0060】 Anti-LEPR antibody containing Fc variant According to a particular embodiment of the present invention, an anti-LEPR antibody is provided comprising an Fc domain containing 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 provides an Fc domain with C H2 or C H The present invention relates to an anti-LEPR antibody containing a mutation in three regions, wherein the mutation increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in endosomes with a pH in the range of approximately 5.5 to 6.0). Such a mutation may result in an increased serum half-life of the antibody when administered to animals. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or 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, modifications include modifications of 428L (e.g., M428L) and 434S (e.g., N434S); modifications of 428L, 259I (e.g., V259I), and 308F (e.g., V308F); modifications of 433K (e.g., H433K) and 434 (e.g., 434Y); modifications of 252, 254, and 256 (e.g., 252Y, 254T, and 256E); modifications of 250Q and 428L (e.g., T250Q and M428L); and modifications of 307 and / or 308 (e.g., 308F or 308P). 【0061】 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 domain disclosed herein are assumed to be within the scope of the present invention. 【0062】 The anti-LEPR antibody of the present invention may include a modified Fc domain having reduced effector function. As used herein, “modified Fc domain having reduced effector function” means any Fc portion of an immunoglobulin that has been modified, mutated, cleaved, etc., compared to a wild-type, naturally occurring Fc domain, such that the molecule containing the modified Fc exhibits a reduction in the magnitude 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 comparator molecule containing a wild-type, naturally occurring version of the Fc portion. In certain embodiments, “modified Fc domain having reduced effector function” is an Fc domain having reduced or attenuated binding affinity to an Fc receptor (e.g., FcγR). 【0063】 In certain embodiments of the present invention, the modified Fc domain is a variant IgG1 Fc or variant IgG4 Fc that includes a substitution in the hinge region. For example, a modified Fc for use in the context of the present invention may include a variant IgG1 Fc in which at least one amino acid in the IgG1 Fc hinge region is replaced with a corresponding amino acid from the IgG2 Fc hinge region. Alternatively, a modified Fc for use in the context of the present invention may include a variant IgG4 Fc in which at least one amino acid in the IgG4 Fc hinge region is replaced with a corresponding amino acid from the IgG2 Fc hinge region. Non-limiting, exemplary modified Fc regions for use in the context of the present invention are described in U.S. Patent Application Publication 2014 / 0243504 (the disclosure thereof is incorporated herein by reference in its entirety), and any functionally equivalent variants of the modified Fc region are also described in the said document. 【0064】 Other modified Fc domains and Fc modifications that can be used in the context of the present invention include any of the modifications described in US2014 / 0171623;US8,697,396;US2014 / 0134162;WO2014 / 043361 (these disclosures are incorporated herein in their entirety by reference). Methods for constructing antibodies or other antigen-binding fusion proteins containing the modified Fc domains described herein are known in the art. 【0065】 Biological characteristics of antibodies The present invention comprises antibodies and antigen-binding fragments thereof that bind to human LEPR and antagonistize LEPR signaling. Such antibodies may be referred to herein as “antagonist antibodies.” In the context of the present invention, “antagonist of LEPR signaling” means an antibody or fragment thereof that binds to LEPR and inhibits the intracellular effects typically resulting from the interaction of leptin with LEPR in cells expressing LEPR. In various embodiments, the antagonist antibodies of the present invention inhibit the function of LEPR agonists and / or LEPR partial agonists. In certain embodiments, “antagonizing LEPR signaling” means inhibiting leptin-stimulated transcriptional activation of STAT3, which can be detected using any method that can directly or indirectly measure or identify STAT3 activity using, for example, a labeled version of STAT3 expressed in a reporter cell line. For example, the present invention comprises antagonist antibodies and antigen-binding fragments thereof that downmodulate LEPR signaling in a cell-based reporter assay described in Example 6, or substantially similar assays. The present invention also relates to IC2 levels ranging from 723 pM to 1.8 nM in the assay of Example 6, or substantially similar assays. 50The present invention includes antagonist antibodies and their antigen-binding fragments that demonstrate complete inhibition of leptin-inducible LEPR signaling having a value. Cell-based binding assays for detecting antibodies that bind to cells expressing LEPR, such as the assay described in Example 5 herein, demonstrated binding to HEK293 / hLEPR-GPI cells at binding ratios of 824–3187-fold in the absence of leptin and 398–3106-fold in the presence of 1 μM leptin. The antibodies of the present invention were found to bind to LEPR even in the presence of 1 μM excess leptin, indicating that the LEPR antibodies of the present invention bind to a site on hLEPR that does not overlap with the leptin binding site. The present invention also includes anti-LEPR antibodies that antagonistize leptin signaling but also partially promote LEPR signaling in the absence of leptin, such antibodies are also referred to herein as “partial agonists” or “antibodies that exhibit agonism of LEPR signaling.” 【0066】 In certain exemplary embodiments of the present invention, anti-LEPR antibodies that bind to human dimer LEPR (hLEPR.hFc, SEQ ID NO: 92) in or out of the presence of leptin are provided, none of which antibodies of the present invention demonstrate more than 40% blocking of the LEPR:leptin interaction using, for example, the assay format defined in Example 4 herein, or substantially similar assays. 【0067】 The present invention comprises an antibody that binds to monomeric human LEPR with high affinity and an antigen-binding fragment thereof. For example, the present invention can be used, for example, with an assay format defined in Example 3 herein, or a substantially similar assay, to measure K less than about 10 nM by surface plasmon resonance at 25°C. D Alternatively, measurements by surface plasmon resonance at 37°C showed a K of less than approximately 25 nM. DThe present invention includes an anti-LEPR antibody that binds to monomeric human LEPR (e.g., hLEPR.mmh, SEQ ID NO: 90). According to a particular embodiment, for example, using the assay format defined in Example 3 of this specification, or a substantially similar assay, the K levels measured by surface plasmon resonance at 25°C are less than approximately 12 nM, less than approximately 11 nM, less than approximately 10 nM, less than approximately 9 nM, less than approximately 8 nM, less than approximately 7 nM, less than approximately 6 nM, less than approximately 5 nM, less than approximately 4 nM, less than approximately 3 nM, less than approximately 2 nM, less than approximately 1 nM, less than approximately 900 pM, less than approximately 800 pM, less than approximately 700 pM, less than approximately 600 pM, less than approximately 500 pM, less than approximately 400 pM, less than approximately 300 pM, less than approximately 200 pM, or less than approximately 100 pM. D This provides an anti-LEPR antibody that binds to monomeric human LEPR. 【0068】 The present invention also relates to measuring dissociation half-lives (t) longer than approximately 5 minutes by surface plasmon resonance at 25°C or longer than approximately 1 minute by surface plasmon resonance at 37°C, using, for example, the assay format defined in Example 3 of this specification, or a substantially similar assay. 1 / 2 The present invention comprises an antibody that binds to monomeric human LEPR (e.g., hLEPR.mmh, SEQ ID NO: 90) and its antigen-binding fragment. According to a particular embodiment, for example, using the assay format defined in Example 3 herein, or a substantially similar assay, the assay is measured by surface plasmon resonance at 25°C for longer than about 5 minutes, longer than about 10 minutes, longer than about 20 minutes, longer than about 40 minutes, longer than about 50 minutes, or longer than that. 1 / 2 This provides an anti-LEPR antibody that binds to monomeric human LEPR. 【0069】 The present invention also includes an antibody that binds to a dimeric human LEPR (e.g., hLEPR.mFc, SEQ ID NO: 91) with high affinity and an antigen-binding fragment thereof. For example, the present invention relates to an antibody that binds to a dimeric human LEPR (e.g., hLEPR.mFc, SEQ ID NO: 91) with high affinity, measured by surface plasmon resonance at 25°C or 37°C, with a K content of less than approximately 4 nM, using, for example, the assay format defined in Example 3 herein, or a substantially similar assay. DThe present invention includes an anti-LEPR antibody that binds to dimeric human LEPR (e.g., hLEPR.mFc, SEQ ID NO: 91). According to certain embodiments, for example, using the assay format defined in Example 3 of this specification, or a substantially similar assay, the K levels measured by surface plasmon resonance at 25°C are less than approximately 15 nM, less than approximately 10 nM, less than approximately 9.0 nM, less than approximately 8.0 nM, less than approximately 7.0 nM, less than approximately 6.0 nM, less than approximately 5.0 nM, less than approximately 4.0 nM, less than approximately 3.0 nM, less than approximately 2.0 nM, less than approximately 1.0 nM, less than approximately 900 pM, less than approximately 800 pM, less than approximately 700 pM, less than approximately 600 pM, less than approximately 500 pM, less than approximately 400 pM, less than approximately 300 pM, less than approximately 200 pM, or less than approximately 100 pM. D This provides an anti-LEPR antibody that binds to dimeric human LEPR. 【0070】 The present invention also relates to measuring dissociation half-lives (t) longer than approximately 10 minutes by surface plasmon resonance at 25°C or 37°C, for example, using the assay format defined in Example 3 of this specification, or a substantially similar assay. 1 / 2 The assay comprises an antibody that binds to a dimeric human LEPR (e.g., hLEPR.mFc, SEQ ID NO: 91) and its antigen-binding fragment. According to a particular embodiment, for example, using the assay format defined in Example 3 of this specification, or a substantially similar assay, the assay is measured by surface plasmon resonance at 25°C for a period of time longer than about 15 minutes, longer than about 20 minutes, longer than about 30 minutes, longer than about 40 minutes, longer than 50 minutes, longer than about 60 minutes, longer than about 70 minutes, longer than 80 minutes, longer than 90 minutes, longer than 100 minutes, or longer than that. 1 / 2 This provides an anti-LEPR antibody that binds to dimeric human LEPR. 【0071】 The present invention also includes antibodies that bind to LEPR in complex with human leptin and antigen-binding fragments thereof ("LEPR in complex with human leptin" may also be expressed as "leptin:LEPR"). For example, the present invention includes antibodies that can bind to a pre-formed complex containing hLEPR and human leptin and antigen-binding fragments thereof. That is, according to certain embodiments, the interaction between an anti-LEPR antibody and LEPR is not inhibited by the presence of leptin in complex with LEPR, and similarly, according to this aspect of the present invention, the interaction between leptin and LEPR is not inhibited by the presence of an anti-LEPR antibody. An exemplary assay format for determining whether an antibody or its antigen-binding fragment binds to LEPR in complex with human leptin is described in Example 4 herein. 【0072】 The present invention also includes antibodies that bind to cell surface-expressed LEPR in the presence and / or absence of human leptin, and their antigen-binding fragments. Cell surface-expressed LEPR means LEPR or a portion thereof (e.g., the extracellular portion of LEPR) that is expressed on the surface of a cell, either naturally or in cell lines engineered to allow the antibody or its antigen-binding fragment to bind to the LEPR molecule. In certain embodiments, cell surface-expressed LEPR includes a recombinant complex comprising the extracellular domain of LEPR attached to the cell via a tag or anchor (e.g., a GPI anchor as shown in Example 6 herein). According to this aspect of the present invention, an antibody is provided that can bind to cell surface-expressed LEPR in the absence of leptin and can also bind to cell surface-expressed LEPR in the presence of leptin (i.e., under conditions in which leptin can bind to cell surface-expressed LEPR). 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 in the complex with cell surface-expressed LEPR. The antibody according to this aspect of the present invention can form a three-member complex on the surface of a cell comprising the antibody, cell surface-expressed LEPR, and leptin. An exemplary assay format for determining whether an antibody or its antigen-binding fragment can bind to cell surface-expressed LEPR in the presence and absence of human leptin is described in Example 5 of this specification. 【0073】 The antibodies of the present invention may have one or more of the biological characteristics described above, or any combination thereof. The above list of biological characteristics of the antibodies of the present invention is not intended to be exhaustive. Other biological characteristics of the antibodies of the present invention will become apparent to those skilled in the art from the examination of this disclosure, including the examples herein. 【0074】 Epitope mapping and related technologies The epitope to which the antibody of the present invention binds may consist of a single consecutive sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of the LEPR protein. Alternatively, the epitope may consist of a plurality of discontinuous amino acids (or amino acid sequences) of LEPR. In some embodiments, the epitope is located on or near the leptin-binding domain of LEPR. In other embodiments, the epitope is located in a region separate 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. 【0075】 Various techniques known to those skilled in the art can be used to identify amino acids within epitopes recognized by specific antibodies. Illustrative techniques include, for example, alanine scanning mutation analysis, peptide blot analysis, and peptide cleavage analysis. In addition, methods such as epitope removal, epitope extraction, and chemical modification of antigens can be used (Tomer (2000) Protein Science Vol. 9: pp. 487-496). Another method that can be used to identify amino acids within polypeptides that antibodies interact with is hydrogen / deuterium exchange detected by mass spectrometry. Generally, hydrogen / deuterium exchange involves deuterizing the target protein and then binding the antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water to allow hydrogen-deuterium exchange to occur at all residues except those protected by the antibody (which remain deuterium-labeled). After antibody dissociation, the target protein is subjected to protease cleavage and mass spectrometry to reveal the deuterium-labeled residues corresponding to the specific amino acids that the antibody interacts with. For example, see Ehring (1999) Analytical Biochemistry Vol. 267 (No. 2): pp. 252-259; Engen and Smith (2001) Anal. Chem. Vol. 73: pp. 256A-265A. X-ray crystallography of the antibody in complex with the antigen can also be used to identify the amino acids in the polypeptide with which the antibody interacts. 【0076】 The present invention further comprises an anti-LEPR antibody that binds to the same epitope as any of the specific exemplary antibodies described herein (for example, an antibody comprising any of the amino acid sequences listed in Table 1 of this specification). Similarly, the present invention also comprises an anti-LEPR antibody that competes for binding to LEPR with any of the specific exemplary antibodies described herein (for example, an antibody comprising any of the amino acid sequences listed in Table 1 of this specification). 【0077】 By using routine methods known in the art and illustrated herein, it is possible to determine whether an antibody binds to the same epitope as the reference anti-LEPR antibody, or whether it competes for binding to it. 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. The ability of the test antibody to bind to the LEPR molecule is then evaluated. If the test antibody can bind to LEPR following saturated binding with the reference anti-LEPR antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-LEPR antibody. On the other hand, if the test antibody cannot bind to the LEPR molecule following saturated binding with the reference anti-LEPR antibody, the test antibody may bind to the same epitope to which the reference anti-LEPR antibody of the present invention binds. Additional routine experiments (e.g., peptide mutation and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether steric hindrance (or another phenomenon) is involved in 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, for example, if one antibody in 1, 5, 10, 20, or 100-fold excess inhibits the binding of another antibody by at least 50%, preferably 75%, 90%, or even 99%, as measured in a competitive binding assay, then the two antibodies bind to the same (or overlapping) epitope (see, e.g., Junghans et al. (1990) Cancer Res. Vol. 50: pp. 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 antibody, then the two antibodies are considered to bind to the same epitope. If only some of the amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, then the two antibodies are considered to have an "overlapping epitope". 【0078】 To determine whether an antibody competes for (or cross-competes for) binding with a reference anti-LEPR antibody, the above binding methodology is performed in two directions: In the first direction, the reference antibody is bound to the LEPR protein under saturated 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 saturated conditions, and then the binding of the reference antibody to the LEPR molecule is evaluated. If, in both directions, only the first (saturated) antibody can bind to the LEPR molecule, it is concluded that the test antibody and the reference antibody compete for binding to LEPR. As will be understood by those skilled in the art, an antibody competing for binding with a reference antibody does not necessarily have to bind to the same epitope as the reference antibody, but may spatially block the binding of the reference antibody by binding to an overlapping or adjacent epitope. 【0079】 Preparation of human antibodies The anti-LEPR antibody of the present invention may be a completely human antibody. Methods for producing monoclonal antibodies, such as completely human monoclonal antibodies, are known in the art. Any such known method can be used in the context of the present invention to produce a human antibody that specifically binds to human LEPR. 【0080】 For example, using VELOCIMMUNE™ technology or any other similar known method for generating fully human monoclonal antibodies, a high-affinity chimeric antibody against LEPR having a human variable region and a mouse constant region is first isolated. As described in the experimental section below, the antibody is characterized and selected for desirable features such as affinity, ligand blocking activity, selectivity, and epitope. If necessary, the mouse constant region is replaced with a desired human constant region, e.g., wild-type or modified IgG1 or IgG4, to generate a fully human anti-LEPR antibody. The selected constant region may differ according to the specific application, but the high-affinity antigen-binding and target specificity features reside in the variable region. In a particular example, a fully human anti-LEPR antibody is isolated directly from antigen-positive B cells. Bioequivalents 【0081】 The anti-LEPR antibodies and antibody fragments of the present invention encompass proteins having amino acid sequences that are different from the described antibodies but retain the ability to bind to human LEPR. Such variant antibodies and antibody fragments include one or more additions, deletions, or substitutions of amino acids compared to the parent sequence, but exhibit biological activity essentially equivalent to that of the described antibody. Similarly, the DNA sequences encoding the anti-LEPR antibodies of the present invention include sequences encoding anti-LEPR antibodies or antibody fragments that are essentially biologically equivalent to the anti-LEPR antibodies or antibody fragments of the present invention, but include one or more additions, deletions, or substitutions of nucleotides compared to the disclosed sequence. Examples of such variant amino acid and DNA sequences are discussed above. 【0082】 Two antigen-binding proteins or antibodies are considered bioequivalents if, for example, when administered in the same molar dose under similar experimental conditions, either as a single or multiple dose, their absorption rates and extents do not show a significant difference. Some antibodies are considered equivalents or pharmacokinetic substitutes if they are equivalent in terms of absorption rate but not in terms of absorption rate. However, they may still be considered bioequivalents because such differences in absorption rate are intentional, reflected in the labeling, and are not considered essential for achieving effective drug concentrations in the body, for example, for chronic use, and are not considered medically important to the particular drug product being tested. 【0083】 In one embodiment, the two antigen-binding proteins are bioequivalent if there are no clinically significant differences in safety, purity, and titer. 【0084】 In one embodiment, two antigen-binding proteins are bioequivalent if, compared to a continuous therapy without one or more switching between a reference product and a biological product, there is no expected increase in the risk of adverse effects, such as clinically significant changes in immunogenicity or a decrease in efficacy, and the patient is able to make such a switch. 【0085】 In one embodiment, if both antigen-binding proteins act by one or more common mechanisms of action for one or more conditions of use, insofar as such mechanisms are known, they are bioequivalent. 【0086】 Bioequivalence may be demonstrated by in vivo and in vitro methods. Measures of bioequivalence include, for example, (a) in vivo studies in humans or other mammals in which the concentration of the antibody or its metabolites in blood, plasma, serum, or other bodily fluids is measured as a function of time; (b) in vitro studies that are related to and reasonably predict human in vivo bioavailability data; (c) in vivo studies in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antibody. 【0087】 Bioequivalent variants of the anti-LEPR antibody of the present invention may be constructed, for example, by various substitutions of residues or sequences, or by deletion of terminal or internal residues or sequences that are not required for biological activity. For example, deletion of cysteine ​​residues that are not essential for biological activity or replacement with other amino acids can prevent the formation of unnecessary or incorrect intramolecular disulfide crosslinks during regeneration. In other contexts, bioequivalent antibodies may include anti-LEPR antibody variants that include amino acid changes that alter the glycosylation characteristics of the antibody, such as mutations that remove or eliminate glycosylation. 【0088】 Species selectivity and species cross-reactivity According to certain exemplary embodiments of the present invention, the present invention provides an anti-LEPR antibody that binds to human LEPR but not to LEPR from other species. The present invention also includes anti-LEPR antibodies that bind to human LEPR and LEPR from one or more non-human species. For example, the anti-LEPR antibody of the present invention may bind to human LEPR and, optionally, to one or more LEPRs of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cattle, horse, camel, macaque (cynomologous), marmoset, rhesus macaque, or chimpanzee, or may not bind to them. According to certain exemplary embodiments of the present invention, an anti-LEPR antibody that specifically binds to human LEPR and macaque (e.g., cynomolgus macaque) LEPR is provided. Other anti-LEPR antibodies of the present invention bind to human LEPR but not to macaque LEPR, or only weakly. 【0089】 multispecific antibodies The antibodies of the present invention may be monospecific or multispecific (e.g., bispecific). Multispecific antibodies may be specific to different epitopes of one target polypeptide, or may contain antigen-binding domains specific to one or more target polypeptides. See, for example, Tutt et al. (1991) J. Immunol. Vol. 147: pp. 60-69; Kufer et al. (2004) Trends Biotechnol. Vol. 22: pp. 238-244. The anti-LEPR antibodies of the present invention can be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or a fragment thereof can be functionally linked (e.g., by chemical bonding, gene fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody having a second binding specificity. 【0090】 The present invention comprises a bispecific antibody in which one arm of the immunoglobulin binds to human LEPR and the other arm of the immunoglobulin is specific to a second antigen. The LEPR-binding arm may include any of the amino acid sequences of HCVR / LCVR or CDR listed in Table 1 herein. 【0091】 An exemplary bispecific antibody format usable in the context of the present invention is a first immunoglobulin (Ig) C H 3 domains and 2nd Ig C H With the use of three domains, the first and second Ig C H The three domains differ from each other by at least one amino acid, and the difference by at least one amino acid reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody lacking that amino acid difference. In one embodiment, the first Ig C H The 3 domains bind to protein A, and the second Ig C H The 3 domains contain mutations that reduce or disable protein A binding, e.g., a modification of H95R (by IMGT exon numbering; by EU numbering, H435R). Second C H 3 may further include modifications of Y96F (by IMGT; Y436F by EU). Second C H Further modifications that may be found within 3 include: for IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M, and V422I according to EU); for IgG2 antibodies, N44S, K52N, and V82I (according to IMGT; N384S, K392N, and V422I according to EU); and for IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I according to EU). Variations of the bispecific antibody formats described above are envisioned within the scope of the present invention. 【0092】 Other exemplary dual-specific formats that can be used in the context of the present invention include, for example, scFv-based or diabody dual-specific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chains (e.g., common light chains having knobs-into-holes), CrossMab, CrossFab, (SEED) bodies, leucine zippers, Duobody, IgG1 / IgG2, dual-acting Fab(DAF)-IgG, and Mab 2 Examples of bispecific formats include, but are not limited to, Klein et al. (2012), mAbs Vol. 4: No. 6, pp. 1-11, and the references cited therein, for a review of these formats. Bispecific antibodies can also be constructed using peptide / nucleic acid conjugations, for example, by using non-natural amino acids with orthogonal chemical reactivity to generate site-specific antibody-oligonucleotide conjugates, which are then self-assembled into multimeric complexes with defined composition, valency, and geometric shape (see, for example, Kazane et al., J. Am. Chem. Soc. [Epub: December 4, 2012]). 【0093】 Therapeutic formulations and administration The present invention provides pharmaceutical compositions comprising the anti-LEPR antibody or its antigen-binding fragment. The pharmaceutical compositions of the present invention are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, etc. Numerous suitable formulations can be found in prescriptions known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (LIPOFECTIN®, Life Technologies, Carlsbad, CA, etc.), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52: pp. 238-311. 【0094】 The dose of antibody administered to a patient may vary depending on the patient's age and size, target disease, condition, and route of administration. Preferred doses are typically calculated according to body weight or body surface area. In adult patients, it may be advantageous to administer the antibody of the present invention intravenously in single doses of approximately 0.01 to 20 mg / kg body weight, more preferably 0.02 to 7, 0.03 to 5, or 0.05 to 3 mg / kg body weight. The frequency and duration of treatment can be adjusted depending on the severity of the condition. Effective doses and schedules for administering anti-LEPR antibodies can be empirically determined; for example, the patient's progress can be monitored through periodic assessments, and the dose adjusted accordingly. Furthermore, interspecies scaling of doses can be performed using methods well known in the art (e.g., Mordenti et al. (1991) Pharmaceut. Res. 8: 1351). 【0095】 For example, various delivery systems are known and can be used to administer the pharmaceutical composition of the present invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, for example, Wu et al. (1987) J. Biol. Chem. Vol. 262: pp. 4429-4432). Methods of delivery include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example, by injection or bolus injection, or by absorption through epithelial or mucocutaneous lining (e.g., oral mucosa, rectal and intestinal mucosa), and may be administered together with other bioactive agents. Administration may be systemic or topical. 【0096】 The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously using standard needles and syringes. In addition, with respect to subcutaneous delivery, pen delivery devices are applied to facilitate the delivery of the pharmaceutical compositions of the present invention. Such pen delivery devices may be reusable or disposable. Reusable pen delivery devices typically utilize replaceable cartridges containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered, the cartridge becomes empty and can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. Disposable pen delivery devices do not have replaceable cartridges. Rather, disposable pen delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition is depleted from the reservoir, the entire device is discarded. 【0097】 Numerous reusable pens and automated injection devices are applicable to the subcutaneous delivery of the pharmaceutical compositions of the present invention. To name just a few examples, AUTOPEN(trademark) (Owen Examples include, but are not limited to, Mumford, Inc. (Woodstock, UK), DISETRONIC® pens (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25® pens, HUMALOG® pens, HUMALIN 70 / 30® pens (Eli Lilly and Co., Indianapolis, IN), NOVOPEN® I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR® (Novo Nordisk, Copenhagen, Denmark), BD® pens (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN®, OPTIPEN PRO®, OPTIPEN STARLET®, and OPTICLIK® (sanofi-aventis, Frankfurt, Germany). Examples of disposable pen delivery devices applied to the subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not limited to, SOLOSTAR® pen (sanofi-aventis), FLEXPEN® (Novo Nordisk), KWIKPEN® (Eli Lilly), SURECLICK® Autoinjector (Amgen, Thousand Oaks, CA), PENLET® (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA® Pen (Abbott Labs, Abbott Park, IL). 【0098】 In certain circumstances, pharmaceutical compositions can be delivered using controlled-release systems. In one embodiment, a pump may be used (see Langer, cited above; Sefton (1987) CRC Crit. Ref. Biomed. Eng. Vol. 14: p. 201). In another embodiment, polymer materials may be used (see Medical Applications of Controlled Release, Langer and Wise (eds.) (1974) CRC Pres., Boca Raton, Florida). In yet another embodiment, the controlled-release system may be placed close to the target of the composition and therefore require only a portion of the systemic dose (see, for example, Goodson (1984) Medical Applications of Controlled Release, cited above, Vol. 2, pp. 115-138). Other controlled-release systems are discussed in the review by Langer (1990) Science Vol. 249: pp. 1527-1533. 【0099】 The injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, as well as intravenous infusion. These injectable preparations can be prepared by known methods. For example, an injectable preparation can be prepared by dissolving, suspending, or emulsifying the antibody or a salt thereof in a sterile aqueous or oily medium conventionally used for injection. Examples of aqueous media for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, which can be used in combination with appropriate solubilizers, such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Examples of oily media include sesame oil and soybean oil, which can be used in combination with solubilizers, such as benzyl benzoate and benzyl alcohol. The injectable solutions thus prepared are preferably filled into appropriate ampoules. 【0100】 Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into unit dose dosage forms adapted to the dose of the active ingredient. Examples of such unit dose dosage forms include tablets, pills, capsules, injections (ampoules), and suppositories. The amount of the antibody contained is usually about 5 to about 500 mg per unit dose dosage form, and preferably about 5 to about 100 mg of the antibody in the form of an injection, and about 10 to about 250 mg for other dosage forms. 【0101】 Therapeutic use of antibodies The present invention includes a method comprising administering to a subject requiring such treatment a therapeutic composition comprising an antagonist anti-LEPR antibody (for example, an anti-LEPR antibody comprising one of the HCVR / LCVR or CDR sequences listed in Table 1 herein). The therapeutic composition may include one of the anti-LEPR antibodies disclosed herein, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier or diluent. 【0102】 The antibodies of the present invention are particularly useful for the treatment, prevention, and / or improvement of any disease or disorder associated with or mediated by elevated leptin levels (hyperleptinemia) and / or elevated expression of the OB-R leptin receptor, resulting in excessive LEPR signaling. In various embodiments, the disease or disorder is selected from, but is not limited to, anorexia or other psychiatric eating disorders, chronic kidney disease cachexia, other cachexia, e.g., congestive heart failure cachexia, pulmonary cachexia, radiation cachexia, and cancer cachexia, autoimmune disorders, e.g., inflammatory bowel disease, lupus erythematosus, multiple sclerosis, psoriasis, cardiovascular disease, hypertension, depression, neurodegenerative disorders, and cancer, e.g., hepatocellular carcinoma, melanoma, and breast cancer. 【0103】 The present invention also includes an anti-LEPR antibody and its antigen-binding fragment useful for antagonistizing LEPR signaling in cells, tissues, and organs expressing normal or high leptin levels. As used herein, a LEPR variant exhibiting enhanced signaling (compared to wild-type LEPR) in the presence of leptin is referred to as a "signal-enhancing LEPR variant." An exemplary signal-enhancing LEPR variant is LEPR-Q223R (Chagnon et al. (2009) Journal of Clinical Endocrinology & Metabolism, Vol. 85 (No. 1): pp. 29-34. Accordingly, the present invention comprises an anti-LEPR antibody and its antigen-binding fragment useful for the treatment, prevention and / or improvement of diseases and disorders caused by or associated with enhanced signaling LEPR variants. 【0104】 In the context of the therapeutic methods described herein, anti-LEPR antibodies may 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). 【0105】 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 therapeutic active ingredients, and methods of treatment comprising administering such combinations to a subject in need. 【0106】 The anti-LEPR antagonist antibody of the present invention may be formulated and / or administered together with one or more additional therapeutic active ingredients, such as pharmaceutical products prescribed for the treatment of congestive heart failure cachexia, pulmonary cachexia and cancer cachexia, autoimmune disorders such as inflammatory bowel disease, lupus erythematosus, multiple sclerosis, psoriasis, cardiovascular disease, hypertension, neurodegenerative disorders, depression, cancer such as hepatocellular carcinoma, melanoma, breast cancer, and other diseases and disorders associated with or caused by reduced leptin signaling. Examples of such additional therapeutic active ingredients include, for example, angiotensin-converting enzyme inhibitors (e.g., ACE, ACE-I, benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, trandolapril), angiotensin receptor blockers (e.g., ARBs), smooth muscle relaxants (e.g., hydralazine), and long-acting agents. Nitrates (e.g., isosorbide dinitrate with or without ACE-I and ARBs), diuretics (e.g., loop diuretics, thiazide-like diuretics and potassium-sparing diuretics), treatment of iron deficiency anemia (e.g., parenteral iron), long-acting or short-acting bronchodilators (e.g., β2 agonists, e.g., alformoterol, bufenin, clenbuterol, dopexamine, epinephrine, fenoterol (fentot) (erol), formoterol, isoetarine, isoprenaline, isoproterenol, levosalbutamol, orciprenaline, pirbuterol, procaterol, ritodrine, salbutamol, albuterol, terbutaline, tiotropium, etc.), anticholinergics (e.g., hyoscyamine, atropine, phenobarbital, scopolamine, dicyclomine, phenobarbital, mepenzolate and combinations, e.g., atropine, hyoscyamine, phenobarbital and scopolamine, etc.), corticosteroids (e.g., hydrocortisone, hydrocortisone-17-valerate, hydrocortisone-17-butyrate, prednisone, prednisolone, triamcinolone acetonide, triamcinolone alcohol, betamethasone, dexamethasone, fluocortone, flunisolide, budesonide),Long-term antibiotics (e.g., macrolides, e.g., erythromycin, azithromycin, etc., and methylxanthines, e.g., theophylline, etc.), non-steroidal anti-inflammatory drugs (NSAIDs) (e.g., aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, naproxen, oxaprozin, piroxicam, salsalate, sulindac, tolmetin, etc.), 5-aminosalicylic acids (5-ASAs) (e.g., mesalazine), immunosuppressants (e.g., For example, prednisone, TNF inhibitors, azathioprine, methotrexate, 6-mercaptopurine), relapsing-remitting multiple sclerosis therapy (e.g., interferon beta-1a, interferon beta-1b, glatiramer acetate, mitoxantrone, natalizumab, fingolimod, teriflunomide, dimethyl fumarate, alemtuzumab, daclizumab, CD20 monoclonal antibodies, e.g., rituximab, ocrelizumab, and ofatumumab), topical agents for the treatment of psoriasis (e.g., para-aminobenzoic acid, coconut oil, coal tar, dislanol) Topical agents such as corticosteroids (e.g., desoximethasone, fluocinonide, etc.), vitamin D3 and other vitamin D analogs, psoralens, etc., as well as systemic agents such as methotrexate, cyclosporine, hydroxycarbamide, fumarates such as dimethyl fumarate, and retinoids), TNF-α antagonists (e.g., infliximab, adalimumab, golimumab, and certolizumab pegol), T-cell targeting psoriasis treatments (e.g., efalizumab and alefacept), treatments for hypertension and cardiovascular disease (e.g., aspirin, Statins, e.g., atorvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin and combinations, e.g., simvastatin + ezetimibe, lovastatin + niacin, and atorvastatin + amlodipine), therapies for neurodegenerative disorders (e.g., dimevon, and proline-rich peptide (PRP)-1), antidepressants (e.g., sertraline, citalopram, fluoxetine, escitalopram, trazodone, venlafaxine, bupropion, duloxetine, paroxetine,Amitriptyline, venlafaxine, desvenlafaxine, and nortriptyline), and anticancer therapies (e.g., sorafenib, JX-594, interleukin-2, ipilimumab (Yervoy), nivolumab (Opdivo), pembrolizumab (Keytruda), vemurafenib (Zelboraf), dabrafenib (Tafinlar), trametinib (Mekinist), anthracyclines, e.g., doxol) Examples include, but are not limited to, taxanes such as Adriamycin® and Ellence®, paclitaxel (Taxol®) and docetaxel (Taxotere®), 5-fluorouracil (5-FU), cyclophosphamide (Cytoxan®), carboplatin (Paraplatin®), or combinations thereof. 【0107】 Administration regimen According to certain embodiments of the present invention, multiple doses of an anti-LEPR antagonist antibody (or a pharmaceutical composition comprising an anti-LEPR antagonist antibody and any of the additional therapeutic agents described herein) may be administered to a subject over a defined period of time. A method according to this aspect of the present invention involves sequentially administering multiple doses of the anti-LEPR antibody of the present invention to a subject. As used herein, “sequentially administered” means that each dose of the anti-LEPR antibody is administered to the subject at different points in time, for example, on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present invention includes a method comprising sequentially administering to a patient a single initial dose of anti-LEPR antibody, followed by one or more secondary doses of anti-LEPR antibody, and optionally, one or more tertiary doses of anti-LEPR antibody. 【0108】 The terms "starting dose", "secondary dose", and "tertiary dose" refer to the temporal sequence of administration of the anti-LEPR antibodies of the present invention. Thus, the "starting dose" is the dose administered at the beginning of a treatment regimen (also referred to as "baseline dose", "loading dose", "starting dose", etc.), the "secondary dose" is the dose administered after the starting dose, and the "tertiary dose" is the dose administered after the secondary dose. All of the starting, secondary, and tertiary doses may contain the same amount of anti-LEPR antibody, but usually may differ from each other in terms of the frequency of administration. However, in certain embodiments, the amount of anti-LEPR antibody contained in the starting, secondary, and / or tertiary doses may be varied from each other during the course of treatment (e.g., adjusted up or down as appropriate). In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of a treatment regimen as "loading doses", and subsequent doses are administered at a lower frequency (e.g., "maintenance doses"). 【0109】 Diagnostic and analytic uses of antibodies The anti-LEPR antibodies 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, an 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, for example, include contacting a sample obtained from a patient with an anti-LEPR antibody of the present invention, wherein the anti-LEPR antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-LEPR antibody can be used in 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 125I, for example; a fluorescent or chemiluminescent moiety, such as fluorescein isothiocyanate or rhodamine; or an enzyme, such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure LEPR in a sample include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and fluorescence-activated cell sorting (FACS). 【0110】 Samples that can be used in the LEPR diagnostic assay according to the present invention include any tissue or liquid sample that can be obtained from a patient containing 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 without a disease or condition associated with abnormal LEPR levels or activity) is first measured to 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 a disease or condition associated with LEPR. [Examples] 【0111】 The following examples are proposed to provide a complete disclosure and explanation of how the methods and compositions of the present invention are carried out or made and used, and are not intended to limit the scope of what the inventors consider to be their invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., quantity, temperature, etc.), but some experimental errors and deviations should be taken into consideration. Unless otherwise indicated, parts are parts by mass, molecular weight is average molecular weight, temperature is degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure. 【0112】 (Example 1) Production of antigen-binding proteins that specifically bind to the leptin receptor (LEPR) Anti-LEPR antibodies were obtained by immunizing VELOCIMMUNE® mice (i.e., engineered mice containing DNA encoding the variable regions of human immunoglobulin heavy chains and copper light chains) with an immunogen containing the extracellular domain of LEPR. Antibody immunoassays were monitored by LEPR-specific immunoassays. Whole human anti-LEPR antibodies were isolated and purified using previously described techniques. 【0113】 Certain biological properties of exemplary anti-LEPR antibodies produced according to the method of this embodiment will be described in detail in the following examples. 【0114】 (Example 2) Amino acid and nucleic acid sequences of the heavy chain and light chain variable regions Table 1 shows the amino acid sequence identification numbers of the heavy and light chain variable regions and CDRs of the selected anti-LEPR antibodies of the present invention. The corresponding nucleic acid sequence identification numbers are listed in Table 2. [Table 1] [Table 2] 【0115】 Antibodies are typically referred to herein according to the following nomenclature: an Fc prefix (e.g., "H4H", "H1M", "H2M", etc.), followed by an identification number (e.g., "17322", "18457", etc.), followed by a suffix of "P" or "N". Therefore, according to this nomenclature, antibodies can be referred to herein, for example, as "H4H17322P2", "H4H18457P2", etc. The Fc prefix (H4H, H1M, and H2M) in antibody names used herein indicates a specific Fc region isotype of the antibody. For example, an "H4H" antibody has a human IgG4 Fc, while an "H1M" antibody has a mouse IgG1 Fc (all variable regions are entirely human, as indicated by the initial "H" in the antibody name). As those skilled in the art will understand, antibodies having a specific Fc isotype can be converted to antibodies having a different Fc isotype (for example, an antibody having mouse IgG1 Fc can be converted to an antibody having human IgG4), but in any case the variable domain (including the CDR) indicated by the identification numbers shown in Tables 1 and 2 remains the same, and the binding properties are expected to be identical or substantially similar regardless of the properties of the Fc domain. 【0116】 Comparative antibody. The comparative antibody used in the following examples is Fab 9F8, as described in Fazeli et al. (2006) J Immunol Methods Vol. 312: pp. 190-200 and Carpenter et al. (2012) Structure Vol. 20 (No. 3): pp. 487-4897. 【0117】 (Example 3) Binding affinity and rate constants derived from surface plasmon resonance of human monoclonal anti-LEPR antibodies Equilibrium dissociation constant (K) for antigen binding to the purified anti-LEPR monoclonal antibody of the present invention DThe values ​​were determined using a Biacore 4000 instrument and a real-time surface plasmon resonance biosensor. All binding tests were performed at 25°C and 37°C in pH 7.4 running buffer (HBS-ET running buffer) containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant Tween-20. The Biacore sensor surface was initially derivatized by amine coupling with monoclonal mouse anti-human Fc antibody (GE, #BR-1008-39) to capture the anti-LEPR monoclonal antibody. The LEPR reagents tested for binding to anti-LEPR monoclonal antibodies included recombinant human LEPR extracellular domain (hLEPR.MMH; SEQ ID NO: 90) expressed with a C-terminal myc-myc-hexahistidine tag, recombinant macaque monkey LEPR extracellular domain (mfLEPR.MMH; SEQ ID NO: 93) expressed with a C-terminal myc-myc-hexahistidine tag, recombinant mouse LEPR extracellular domain (mLEPR.MMH; SEQ ID NO: 94) expressed with a C-terminal myc-myc-hexahistidine tag, recombinant rat LEPR extracellular domain (rLEPR.MMH; SEQ ID NO: 95) expressed with a C-terminal myc-myc-hexahistidine tag, and recombinant human LEPR extracellular domain (hLEPR.mFc; SEQ ID NO: 91) expressed with a C-terminal mouse IgG2a Fc tag. LEPR constructs containing an MMH tag are monomeric LEPR constructs, and LEPR constructs containing an mFc tag are dimeric LEPR constructs. Different concentrations of LEPR reagent were first prepared in HBS-ET running buffer at final concentrations ranging from 100 nM to 3.7 nM in 3-fold serial dilutions, and then injected onto anti-LEPR monoclonal antibodies captured on the surface at a flow rate of 30 μL / min for 4 minutes. Dissociation of the LEPR reagent bound to the monoclonal antibody was monitored in HBS-ET running buffer for 10 minutes. Kinetic association (k) was determined by fitting real-time binding sensograms to a 1:1 binding model with mass transport limitations using Scrubber 2.0c curve fitting software. a ) and dissociation (k d) The rate constant was determined. The association-dissociation equilibrium constant (K D ) and the dissociation half-life (t 1 / 2 ) were calculated from the kinetic rate constant as follows: 【Equation】 【0118】 Table 3 - 13 show the kinetic binding parameters for the binding of hLEPR-MMH, mfLEPR-MMH, hLEPR.mFc, mLEPR-MMH or rLEPR-MMH to different anti-LEPR monoclonal antibodies of the present invention at 25°C and 37°C. 【Table 3 - 1】 【Table 3 - 2】 【Table 4 - 1】 【Table 4 - 2】 【0119】 As shown in Table 3, at 25°C, all of the anti-LEPR monoclonal antibodies of the present invention bound to hLEPR-MMH with K D values ranging from 5.11 nM to 194 nM. As shown in Table 4, at 37°C, all of the anti-LEPR monoclonal antibodies of the present invention bound to hLEPR-MMH with K D values ranging from 11.7 nM to 248 nM. 【Table 5 - 1】 【Table 5 - 2】 【Table 6 - 1】 【Table 6 - 2】 【0120】 As shown in Table 5, at 25°C, all of the anti-LEPR monoclonal antibodies of the present invention exhibited K levels ranging from 4.16 nM to 179 nM. D The antibodies bound to mfLEPR-MMH at a specific value. As shown in Table 6, at 37°C, all of the anti-LEPR monoclonal antibodies of the present invention bound to K in the range of 7.72 nM to 261 nM. D The value was bound to mfLEPR-MMH. [Table 7] [Table 8] 【0121】 As shown in Table 7, at 25°C, all of the anti-LEPR monoclonal antibodies of the present invention exhibited K levels ranging from 443 pM to 11.9 nM. D The antibodies bound to hLEPR-mFc at a specific value. As shown in Table 8, at 37°C, all of the anti-LEPR monoclonal antibodies of the present invention bound to K in the range of 527 pM to 15.1 nM. D It bound to hLEPR-mFc by value. [Table 9] [Table 10-1] [Table 10-2] 【0122】 As shown in Table 9, at 25°C, two of the nine anti-LEPR monoclonal antibodies of the present invention exhibited K2 levels of 218 nM and 264 nM. D The antibodies bound to mLEPR-MMH at a specific value. As shown in Table 10, at 37°C, two of the nine anti-LEPR monoclonal antibodies of the present invention bound to 205 nM and 1.16 μM K. D It bound to mMEPR-MMH by value. [Table 11-1] [Table 11-2] [Table 12-1] [Table 12-2] 【0123】 As shown in Table 11, at 25°C, five of the nine anti-LEPR monoclonal antibodies of the present invention exhibited K levels ranging from 328 nM to 943 nM. D The antibodies bound to rLEPR-MMH at a specific value. As shown in Table 12, at 37°C, five of the nine anti-LEPR monoclonal antibodies of the present invention showed K levels ranging from 168 nM to 771 nM. D It bound to rLEPR-MMH by value. 【0124】 (Example 4) The anti-LEPR antibody of the present invention does not block the binding of hLEPR-hFc to hLeptin. The ability of the anti-LEPR monoclonal antibody of the present invention to block the binding of dimeric human LEPR to its natural ligand, human leptin, was measured using competitive sandwich ELISA. 【0125】 For ELISA, human leptin (hLeptin; R&D Systems, #398-LP-01M) was coated overnight at 4°C in PBS at a concentration of 5 μg / mL. Nonspecific binding sites were then blocked using a 0.5% (w / v) solution of BSA in PBS. A fixed amount (10 nM) of the extracellular domain of the LEPR protein (hLEPR.hFc; SEQ ID NO: 92), expressed with a human Fc tag at the C-terminus, was titrated with serially diluted anti-LEPR antibodies, hLeptin protein, or isotype control antibodies ranging from 8.5 pM to 500 nM. These antibody-protein or protein-protein complexes were then incubated at room temperature (RT) for 1.5 hours. Subsequently, the complex was transferred to an hLeptin-coated microtiter plate, incubated at RT for 2 hours, the wells were washed, and plate-bound hLEPR-hFc was detected using horseradish peroxidase conjugate anti-human IgG polyclonal antibody (Jackson ImmunoResearch Inc, #109-035-098). After developing the sample with TMB solution (BD Biosciences, #555214; substrates A and B mixed in a 1:1 ratio as per manufacturer's instructions) to induce a colorimetric reaction, the sample was neutralized with 1M sulfuric acid, and the absorbance was measured at 450 nm using a Victor X5 plate reader. Data analysis was performed using a sigmoid dose-response model in Prism™ software (GraphPad). The block percentage at the maximum concentration of the tested antibody was calculated as an indicator of the antibody's ability to block the binding of 10 nM hLEPR-hFc to human leptin on the plate. In the calculation, the binding signal of 10 nM hLEPR-hFc in the absence of antibody was used as a reference, representing either 100% binding or 0% block, while the baseline signal of buffer alone in the absence of hLEPR-hFc was used as a reference, representing either 0% binding or 100% block. Block data at a 500 nM antibody concentration are summarized in Table 13. [Table 13] 【0126】 As shown in Table 13, none of the anti-LEPR antibodies of the present invention demonstrated more than 40% blockage of hLEPR-hFc binding to the hLeptin-coated surface. However, the comparator antibody and hLeptin, used as positive controls, were able to block 99% of hLEPR-hFc binding to the hLeptin-coated surface. The isotype control antibody did not demonstrate measurable blockage at concentrations up to 500 nM. 【0127】 (Example 5) Cell binding by FACS analysis using HEK293 / Mycx2-hLEPR(ecto)-GPI anchored cells The leptin receptor LEPR is a single-pass transmembrane receptor belonging to the class I cytokine receptor family, possessing a large, 818-amino acid-length extracellular domain (Tartaglia (1997) J. Biol. Chem 7:272 (No. 10): pp. 6093-6096). LEPR can bind to leptin, a protein primarily expressed by adipose tissue involved in the regulation of feeding and metabolism (Friedman (2014) J Endocrinol 223 (No. 1): pp. T1-8). Different isoforms of LEPR exist, resulting in soluble or membrane-bound receptors, with the membrane-bound forms exhibiting differences in the length of their intracellular domains. The isoform with the longest intracellular domain is highly expressed in the hypothalamus, a key site of leptin action related to obesity (Friedman and Halaas (1998) Nature 395 (No. 6704): pp. 763-770). LEPR is primarily localized within cells and, to a lesser extent, on the cell surface. LEPR undergoes ligand-induced internal translocation, adding further levels of regulation to leptin signaling (Sweeney (2002) Cell Signal Vol. 14 (No. 8): pp. 655-663). 【0128】 To evaluate cell binding by the anti-LEPR antibody of the present invention, we generated the HEK293 cell line, which stably expresses the extracellular domain of human LEPR (hLEPR; amino acids 22-839 of accession number P48357, isoform B), which has an N-terminal myc-myc tag and a C-terminal peptide sequence of human carboxypeptidase M that guides the addition of GPI so that the protein can be attached to the membrane with glycosylphosphatidylinositol (GPI) (Marcic et al. (2000) J Biol Chem. vol.275(no.21):16110-1618). Since hLEPR in the cell line does not have an intracellular domain, it does not move internally upon ligand binding, greatly increasing the amount of LEPR available for antibody and / or ligand binding. After selecting cell lines in DMEM containing 10% FBS, NEAA, penicillin / streptomycin, and 500 μg / mL of G418, we screened for high receptor expression using the anti-Myc antibody. The resulting stable cell line, referred to as HEK293 / hLEPR-GPI, was maintained in DMEM containing 10% FBS, NEAA, and penicillin / streptomycin. 【0129】 For FACS analysis, HEK293 parental cells and HEK293 / hLEPR-GPI cells were dissociated and placed in PBS (FACS buffer) containing 2% FBS on a 96-well v-bottom plate in 5 × 10⁶ wells. 5 Cells were plated in wells. To test whether the binding of anti-hLEPR antibody to cells was affected by the presence of leptin, cells were incubated with FACS buffer containing or without 1 μM human leptin (R&D Systems, #398-LP) at 4°C for 30 minutes, followed by the addition of 10 nM anti-LEPR antibody or control antibody to the FACS buffer. After incubation, cells were washed and incubated with 16 μg / mL Alexa Fluor®-647 conjugate secondary antibody (Jackson ImmunoResearch Laboratories Inc., #109-547-003) at 4°C for 30 minutes. Subsequently, BD Cells were fixed using CytoFix® (Becton Dickinson, #554655), filtered, and analyzed with 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 of fluorescence in viable cells. Next, the geometric mean of fluorescence for each sample was normalized relative to the geometric mean of unstained cells to obtain the relative binding per condition, referred to as the "binding ratio," and these binding ratios for each antibody tested were recorded in Table 14. 【0130】 As shown in Table 14, the nine anti-LEPR antibodies of the present invention, tested at 10 nM, demonstrated binding to HEK293 / hLEPR-GPI cells with binding ratios ranging from 824-3187-fold in the absence of leptin and 398-3590-fold in the presence of 1 μM leptin. Based on the similarity of these binding ratios, the ability of the anti-LEPR antibodies of the present invention to bind to LEPR expressed on cells does not appear to be significantly affected by the presence of 1 μM excess leptin, suggesting that the binding sites of the anti-LEPR antibodies on LEPR do not overlap with the leptin binding sites on LEPR. The anti-LEPR antibodies of the present invention did not demonstrate any significant binding to HEK293 parental cells, with binding ratios ranging from 1 to 9-fold with and without 1 μM leptin. Binding of the comparator to cells expressing GPI-anchored LEPR was significantly reduced in the presence of leptin. Samples of isotype control antibodies and secondary antibodies alone also failed to demonstrate significant binding to either leptin-containing or leptin-absent cell lines, with binding ratios ranging from 1 to 6 times. [Table 14-1] [Table 14-2] 【0131】 (Example 6) The anti-LEPR antibody of the present invention demonstrated complete inhibition of leptin signaling in the presence of hLeptin. A bioassay was developed to detect STAT3 transcriptional activation via LEPR activation (STAT3-Luc; Qiagen, #CLS-6028L) 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 in the human neuroblastoma cell line IMR-32. The resulting stable cell line, referred to as IMR-32 / STAT3-Luc / hLEPR, was isolated and maintained in MEM-Earl medium (complete medium) supplemented with 10% FBS, NEAA, 1 ug / mL puromycin, 100 ug / mL hygromycin B, and penicillin / streptomycin / L-glutamine. 【0132】 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 plated in complete medium at a density of 20,000 cells / 100 ul / well in a 96-well format, and the medium was replaced the following day with an appropriate volume of Opti-MEM (assay buffer) supplemented with 1% BSA and 0.1% FBS 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 was serially diluted by half-log in the assay buffer to final concentrations ranging from 100 nM to 300 fM, added to the cells, and incubated overnight at 37°C in 5% CO2. To measure the effect of the antibody of the present invention in the presence of leptin, a fixed concentration of 200 pM human leptin (hLeptin; R&D Systems, #398-LP) was added to the assay buffer, followed immediately by the addition of anti-LEPR antibody or isotype control antibody, serially diluted in half-logs to final concentrations ranging from 100 nM to 300 fM. The samples were then incubated overnight at 37°C in 5% CO2. Next, OneGlo reagent (Promega, #E6051) was added to the samples, and luciferase activity was measured in Luminescent mode using an Envision Multilable Plate Reader (Perkin Elmer). Relative light units (RLU) values ​​were obtained, and the results were analyzed using nonlinear regression with GraphPad Prism software (GraphPad). The maximum RLU value obtained from the hLeptin dose response was defined as 100% activity in the IMR-32 / STAT3-Luc / hLEPR assay. 【0133】 As shown in Table 15, anti-LEPR antibodies tested in the absence of hLeptin demonstrated weak stimulation of IMR-32 / STAT3-Luc / hLEPR cells with maximal activations of 4% to 8% of the maximal activation obtained from the hLeptin dose response. One antibody exhibiting weak stimulation had an EC of 1.27 nM. 50 While one antibody, H4H18440P2, had a measurable EC value, another antibody, H4H18440P2, had a measurable EC value. 50No value was found. In the presence of 200 pM hLeptin, the three anti-LEPR antibodies tested showed IC values ​​ranging from 723 pM (antibody H4H18457P2) to 1.83 nM (antibody H4H17322P2) or 2.9 nM (antibody H4H18464P2). 50 The assay demonstrated complete inhibition of leptin in IMR-32 / STAT3-Luc / hLEPR cells. None of the six anti-LEPR antibodies tested demonstrated any measurable inhibition in the presence of 200 pM human leptin. The isotype control antibodies did not demonstrate any measurable stimulation of IMR-32 / STAT3-Luc / hLEPR cells in any of the assays. [Table 15-1] [Table 15-2] 【0134】 (Example 7) 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 the Octet HTX biosensor platform (Pall ForteBio Corp.). All experiments were performed at 25°C with plate shaking at a speed of 1000 rpm in a pH 7.4 buffer (HBS-EBT) containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant Tween-20 and 1 mg / mL BSA. 【0135】 To evaluate whether the two antibodies could compete with each other for binding to recombinant human LEPR (hLEPR.MMH; SEQ ID NO: 90) expressed with a C-terminal myc-myc-hexahistidine tag, the approximately 0.25 nm hLEPR-MMH was first captured by an Octet biosensor chip (Fortebio Inc, #18-5122) coated with an anti-pentaHis antibody by immersing the biosensor chip in a well containing 20 μg / mL of hLEPR-MMH for 5 minutes. Next, the antigen-captured biosensor chip was saturated with mAb-1 by immersing it in a well containing a solution of 50 μg / mL of the first anti-LEPR monoclonal antibody (hereinafter referred to as mAb-1) for 210 seconds. Subsequently, the biosensor chip was immersed in a well containing a solution of 50 μg / mL of the second anti-LEPR monoclonal antibody (hereinafter referred to as mAb-2) for 150 seconds. The biosensor chip was washed in HBS-EBT buffer between each step of the experiment. The binding response was monitored in real time throughout the entire experiment, and the binding response was recorded at the end of each step. The binding response of mAb-2 to hLEPR-MMH pre-complexed with mAb-1 was compared, and the competitive / non-competitive behavior of different anti-LEPR monoclonal antibodies was determined, as shown in Table 16. [Table 16] 【0136】 As shown in Table 16, antibodies H4H18439P2 and H4H18440P2 compete with each other for binding to their respective epitopes. LEPR antibodies H4H18457P2, H4H18462P2, H4H18437P2, H4H18466P2, and H4H18508P2 compete with each other for binding to their respective epitopes. Antibodies H4H17322P2 and H4H18464P2 do not compete for binding to the respective epitopes of H4H18439P2, H4H18440P2, H4H18457P2, H4H18462P2, H4H18437P2, and H4H18466P2. 【0137】 (Example 8) In vivo efficacy study of LEPR antagonist antibodies in humanized LEPR mice The effects of the three specific antagonist anti-LEPR antibodies H4H17322P2, H4H18457P2, and H4H18464P2 of the present invention on feeding, body weight, and excess fat were demonstrated in single-animal genetically modified LEPRs expressing leptin receptors composed of human LEPR ectodomain sequences instead of mouse LEPR ectodomain sequences. Hu / Hu This was determined in mice. 【0138】 Baseline daily food intake was measured between 5 days and 1 day before the procedure (-5 days and -1 days). Body composition, including excess fat, was quantified by μCT 4 days before the procedure and 6 days after the procedure (-4 days and -6 days). 32 male LEPRs aged 12-13 weeks were used on day 0. HuHu Mice were randomized into four groups of eight mice each based on their body weight from day 1 (-1). On day 0, each group received a single dose of either 30 mg / kg isotype control antibody, 30 mg / kg H4H17322P2, 30 mg / kg H4H18457P2, or 30 mg / kg H4H18464P2 via subcutaneous injection. The isotype control antibody does not bind to any known mouse protein. Food intake and body weight were measured for each animal throughout the duration of the study. Figure 1 summarizes the percentage change in food intake from baseline daily food intake for each treatment group at each time point. The percentage change in body weight from day 0 was calculated for each animal at each time point. Figure 2 summarizes the average percentage change in body weight for animals in each treatment group. Figure 3 summarizes the average fat mass for animals in each antibody treatment group, quantified by μCT 6 days prior to and 6 days after antibody treatment. All results are expressed as mean ± SEM. 【0139】 As shown in Figures 1 and 2, mice treated with anti-LEPR antagonist antibodies demonstrated increased percentage changes in food intake and body weight. These increases were not observed in mice treated with isotype control antibodies. As shown in Figure 1, mice treated with 30 mg / kg of H4H17322P2 showed a significant increase in food intake, starting one day after treatment (day 1) and at subsequent time points, compared to mice injected with isotype control antibodies. Mice treated with 30 mg / kg of H4H18457P2 showed a significant increase in food intake, starting on day 2 and at subsequent time points, compared to mice injected with isotype control antibodies. Mice treated with 30 mg / kg of H4H18464P2 showed a significant increase in food intake, starting on day 2 and at subsequent time points, except day 4, compared to mice injected with isotype control antibodies. As shown in Figure 2, mice treated with 30 mg / kg of H4H17322P2 showed a significant increase in percentage body weight change, starting 4 days after treatment (day 4) and thereafter, compared to mice injected with isotype control antibodies. Mice treated with 30 mg / kg of H4H18457P2 showed a significant increase in percentage body weight change, starting 3 days and thereafter, compared to mice injected with isotype control antibodies. Mice treated with 30 mg / kg of H4H18464P2 showed a significant increase in percentage body weight change, starting 4 days and thereafter, compared to mice injected with isotype control antibodies. As depicted in Figure 3, there was no difference in fat mass between the groups before treatment (-4 days). Mice treated with 30 mg / kg of H4H17322P2, H4H18457P2, and H4H18464P2 antibodies demonstrated a significant increase in fat mass 6 days after treatment (day 6) compared to mice treated with isotype control antibodies. In conclusion, treatment with LEPR antagonist antibodies increased food intake, body weight, and fat accumulation in mice, whereas treatment with isotype control antibodies did not. 【0140】 (Example 9) Determination of the human LEPR epitope to which the anti-LEPR antibody of the present invention binds. To determine the human LEPR epitope to which the anti-LEPR antibody of the present invention binds, the interaction of the anti-LEPR antibody with recombinant human LEPR protein domains was characterized using Luminex FLEXMAP (FM3DD, LuminexCorp) flow cytometry-based analysis. For the assay, approximately 3 million carboxylated Microplex® microspheres (Luminex, trade no. LC1000A) were washed, vortexed, sonicated in 0.1 M NaPO4 (activating buffer) at pH 6.2, and then centrifuged to remove the supernatant. Microspheres were resuspended in 120 μL of activation buffer, and the carboxyl groups (-COOH) were activated by adding 15 μL of 50 mg / mL N-hydroxysuccinimide (NHS, Thermo Scientific, t. 24500) followed by 15 μL of 50 mg / mL 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC, Thermo Scientific, t. 22980) at 25°C. After 10 minutes, 600 μL of 50 mM MES (pH 5) (binding buffer) was added to lower the pH of the reaction mixture to 5.0, the microspheres were vortexed, and the supernatant was removed by centrifugation. The activated beads were immediately mixed with 500 μL of 20 μg / mL monoclonal anti-myc antibody containing either mouse IgG or human IgG in the binding buffer and incubated at 25°C for 2 hours. The binding reaction was stopped by adding 50 μL of 1 M Tris-HCl (pH 8.0), the microspheres were rapidly vortexed, centrifuged, and washed four times with 1 mL of DPBS to remove unbound proteins and other reaction components. 【0141】 The transiently expressed LEPR protein includes the human LEPR extracellular domain (human LEPR-MMH, SEQ ID NO: 89) expressed with a C-terminal myc-myc hexahistidine tag, the human LEPR CRH1(D1) (human LEPR CRH1(D1)-MMH, amino acids 1-208 of SEQ ID NO: 89 along with the myc-myc hexahistidine tag at amino acids 209-236), the human LEPR CRH1(D1,D2) domain (human LEPR CRH1(D1,D2)-MMH, amino acids 1-318 of SEQ ID NO: 89 along with the myc-myc hexahistidine tag at amino acids 319-346), and the human LEPR expressed with a C-terminal myc-myc hexahistidine tag. CRH1-Ig(D1,D2,D3) domain (human LEPR CRH1(D1,D2,D3)-MMH, amino acids 1-278 of SEQ ID NO: 89 along with the myc-myc hexahistidine tag at amino acids 279-306), human LEPR CRH1-Ig(D2,D3) domain expressed with the C-terminal myc-myc hexahistidine tag (human LEPR CRH1-Ig(D2,Human LEPR Ig(D3)-MMH, amino acids 1-198 of SEQ ID NO: 89 along with the myc-myc hexahistidine tag at amino acids 199-226, human LEPR Ig(D3) domain expressed with the C-terminal myc-myc hexahistidine tag (human LEPR Ig(D3)-MMH, amino acids 1-88 of SEQ ID NO: 89 along with the myc-myc hexahistidine tag at amino acids 89-116), human LEPR CRH2 domain expressed with the C-terminal myc-myc hexahistidine tag (human LEPR CRH2-MMH, amino acids 1-207 of SEQ ID NO: 89 along with the myc-myc hexahistidine tag at amino acids 208-235), human LEPR FNIII domain expressed with the C-terminal myc-myc hexahistidine tag (human LEPR The microspheres contained FNIII-MMH, amino acids 1-204 of SEQ ID NO: 89 along with the myc-myc hexahistidine tag at amino acids 205-232, and the human LEPR Ig-CRH2-FNIII domain expressed with the C-terminal myc-myc hexahistidine tag (human LEPR Ig-CRH2-FNIII-MMH, amino acids 1-510 of SEQ ID NO: 89 with the myc-myc hexahistidine tag at amino acids 511-538). These were suspended in serum-free CHO-S-SFM II medium (Thermo Fisher, product no. 31033020) and then cleared by centrifugation. Aliquots of microspheres containing the immobilized anti-myc monoclonal antibody prepared as described above were individually added to each of these protein supernatants (1 mL). 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-bound microspheres were collected from individual reactions with full-length human LEPR and each human LEPR domain protein and mixed together in 3.6 mL of PBS + 20 mg / mL of BSA + 0.05% sodium azide (blocking buffer). 【0142】 From this mixed pool, 75 μL of microspheres were plated well by well onto a 96-well filter plate (Millipore, product 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 (wash buffer) containing 0.05% Tween 20. To detect and quantify the amount of anti-LEPR antibody bound to individual microspheres, 100 μL of 2.5 μg / mL R-Phycoerythrin conjugate goat F(ab')2 anti-human copper (Southern Biotech, t. 2063-09) or 100 μL of 1.25 μg / mL R-Phycoerythrin AffiniPure F(ab') or 100 μL of 1.25 μg / mL R-Phycoerythrin AffiniPure F(ab')2 Fragment Goat Anti-Mouse IgG, F(ab')2 Fragment Specific (Jackson Immunoresearch, t. 115-116-072) was added to blocking buffer and incubated at 25°C for 30 minutes. After 30 minutes, the sample was washed twice with 200 μL of washing buffer and resuspended in 150 μL of washing buffer. The median fluorescence intensity (MFI) of the microspheres was measured using a Luminex Analyzer. 【0143】 The results of the Luminex-based analysis are summarized in Table 17. The Luminex MFI signal intensity indicates that the nine anti-LEPR antibodies of the present invention bound to the complete extracellular domain of human LEPR. Anti-LEPR antibodies H4H18439P2 and H4H18440P2 bound to the epitope within the CRH1 D2 domain of human LEPR. Anti-LEPR antibodies H4H17322P2 and COMP3551 bound to the epitope within the CRH2 domain of human LEPR. Anti-LEPR antibodies H4H18437P2, H4H18457P2, H4H18508P2, H4H18466P2 and H4H18462P2 bound to the epitope within the FNIII domain of human LEPR. Anti-LEPR antibody H4H18464P2 bound to the epitope within the Ig(D3) domain of human LEPR. [Table 17] 【0144】 The scope of the present invention is not limited by the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be included within the scope of the accompanying claims. The present invention provides, for example, the following items: (Item 1) An isolated antibody or antigen-binding fragment thereof that binds to the human leptin receptor (LEPR) and antagonistizes LEPR signaling but does not block the interaction between leptin and LEPR, wherein, in measurements in an in vitro leptin binding assay, the isolated antibody or antigen-binding fragment does not block the said interaction in approximately 40% of cases. (Item 2) (i) Measurement by surface plasmon resonance showed that K was less than approximately 10 nM. D To bind to monomeric human LEPR at 25°C; (ii) Measurement by surface plasmon resonance, t longer than approximately 5 minutes 1 / 2 To bind to monomeric human LEPR at 25°C; (iii) Measurement by surface plasmon resonance showed a K of less than approximately 2.0 nM D It binds to the dimer human LEPR at 25°C; (iv) Measurement by surface plasmon resonance showed that t longer than approximately 20 minutes 1 / 2 It binds to the dimer human LEPR at 25°C; (v) To bind to human LEPR in complex with human leptin; (vi) binding to cell surface-expressed LEPR in the presence and absence of human leptin; and (vii) In cell-based reporter assays, IC25 was less than approximately 3.0 nM. 50 This inhibits leptin-induced LEPR signaling. An isolated antibody or its antigen-binding fragment according to item 1, exhibiting one or more additional properties selected from the group consisting of the following. (Item 3) An isolated antibody or its antigen-binding fragment as described in item 1 or 2, exhibiting at least partial activation of LEPR signaling in a cell-based reporter assay in the absence of human leptin. (Item 4) An isolated antibody or antigen-binding fragment thereof that binds to the human leptin receptor (LEPR), comprising (a) a complementarity-determining region (CDR) of a heavy chain variable region (HCVR) containing the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 42, and SEQ ID NO: 58, and (b) a CDR of a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 10. (Item 5) An isolated antibody or its antigen-binding fragment as described in item 4, comprising the heavy and light chain CDRs of an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 42 / 10, and 58 / 10. (Item 6) An isolated antibody or its antigen-binding fragment as described in item 5, comprising an amino acid sequence pair of HCVR / LCVR selected from the group consisting of SEQ ID NOs: 2 / 10, 42 / 10, and 58 / 10. (Item 7) An isolated antibody or its antigen-binding fragment, as described in item 6, that antagonistizes LEPR signaling. (Item 8) An isolated antibody or antigen-binding fragment according to any one of items 1 to 3, which binds to the human leptin receptor (LEPR) and comprises (a) a complementarity-determining region (CDR) of the heavy chain variable region (HCVR) containing the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 42, and SEQ ID NO: 58, and (b) a CDR of the light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 10. (Item 9) An antibody or antigen-binding fragment described in any one of items 1 to 4 that competes for binding to LEPR with a reference antibody containing an amino acid sequence pair of HCVR / LCVR selected from the group consisting of SEQ ID NOs: 2 / 10, 42 / 10, and 58 / 10. (Item 10) An antibody or antigen-binding fragment described in any one of items 1 to 9, which binds to the same epitope on the LEPR as a reference antibody containing an amino acid sequence pair of HCVR / LCVR selected from the group consisting of SEQ ID NOs: 2 / 10, 42 / 10, and 58 / 10. (Item 11) A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of items 1 to 10, and a pharmaceutically acceptable carrier or diluent. (Item 12) A method for treating a disease related to or caused by leptin signaling, comprising administering the pharmaceutical composition described in item 11 to a subject in need thereof. (Item 13) The method according to item 12, wherein the disease or condition associated with or caused by leptin signaling is selected from the group consisting of anorexia or other psychiatric eating disorders, chronic kidney disease cachexia, other cachexia, e.g., congestive heart failure cachexia, pulmonary cachexia, radiation cachexia, and cancer cachexia, autoimmune disorders, e.g., inflammatory bowel disease, lupus erythematosus, multiple sclerosis, psoriasis, cardiovascular disease, hypertension, depression, non-alcoholic fatty liver disease, neurodegenerative disorders, depression, and cancer, e.g., hepatocellular carcinoma, melanoma, and breast cancer. (Item 14) A method for treating a disease or condition associated with or caused by an activated LEPR mutation, comprising administering the pharmaceutical composition described in item 11 to a subject in need thereof. (Item 15) The method described in item 14, wherein the LEPR mutation is LEPR Q223R. (Item 16) The method according to item 14 or 15, wherein the disease or condition associated with or caused by the activated LEPR mutation is cachexia. (Item 17) The method according to any one of items 12 to 16, further comprising administering a second therapeutic agent to the subject, wherein the second therapeutic agent is selected from antidepressants, appetite stimulants, treatments for anorexia and cachexia, COX-2 inhibitors, NSAIDs, treatments for restoring loss of muscle mass, muscle function and / or muscle strength with or without cachexia, treatments for non-alcoholic fatty liver disease (NAFLD), treatments for HIV / AIDS infection, TNF-alpha production blocking agents, antioxidants, tumor necrosis factor (TNF) antagonists, B lymphocyte stimulants, angiotensin-converting enzyme (ACE) inhibitors, kinase inhibitors, and chemotherapeutic agents.

Claims

[Claim 1] A first composition for use in the production of an antibody or antigen-binding fragment thereof that binds to the human leptin receptor (LEPR), comprising an isolated polynucleotide molecule, wherein the isolated polynucleotide molecule comprises a nucleic acid sequence encoding a heavy chain variable region (HCVR) including complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3), and the amino acid sequence of the HCVR is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 42, and SEQ ID NO:

58. The first composition is characterized in that it is used in combination with a second composition comprising an isolated polynucleotide molecule comprising a nucleic acid sequence encoding a light chain variable region (LCVR) including CDRs (LCDR1, LCDR2, and LCDR3), wherein the amino acid sequence of the LCVR is described in Sequence ID No.

10. [Claim 2] A first composition for use in the production of an antibody or antigen-binding fragment thereof that binds to the human leptin receptor (LEPR), comprising an isolated polynucleotide molecule, wherein the isolated polynucleotide molecule comprises a nucleic acid sequence encoding a light chain variable region (LCVR) including CDRs (LCDR1, LCDR2, and LCDR3), and the amino acid sequence of the LCVR is as described in Sequence ID No.

10. The first composition is characterized by being used in combination with a second composition comprising an isolated polynucleotide molecule comprising a nucleic acid sequence encoding a heavy chain variable region (HCVR) including complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3), wherein the amino acid sequence of the HCVR is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 42, and SEQ ID NO:

58. [Claim 3] The composition according to claim 1 or claim 2, wherein the HCVR includes HCDR1 described in SEQ ID NO: 4, HCDR2 described in SEQ ID NO: 6, and HCDR3 described in SEQ ID NO: 8, and the LCVR includes LCDR1 described in SEQ ID NO: 12, LCDR2 described in SEQ ID NO: 14, and LCDR3 described in SEQ ID NO:

16. [Claim 4] The composition according to claim 3, wherein the HCVR comprises the amino acid sequence described in SEQ ID NO: 2, and the LCVR comprises the amino acid sequence described in SEQ ID NO:

10. [Claim 5] The composition according to claim 1 or claim 2, wherein the HCVR includes HCDR1 described in sequence number 44, HCDR2 described in sequence number 46, and HCDR3 described in sequence number 48, and the LCVR includes LCDR1 described in sequence number 12, LCDR2 described in sequence number 14, and LCDR3 described in sequence number 16. [Claim 6] The composition according to claim 5, wherein the HCVR comprises the amino acid sequence described in SEQ ID NO: 42, and the LCVR comprises the amino acid sequence described in SEQ ID NO:

10. [Claim 7] The composition according to claim 1 or claim 2, wherein the HCVR includes HCDR1 described in SEQ ID NO: 60, HCDR2 described in SEQ ID NO: 62, and HCDR3 described in SEQ ID NO: 64, and the LCVR includes LCDR1 described in SEQ ID NO: 12, LCDR2 described in SEQ ID NO: 14, and LCDR3 described in SEQ ID NO:

16. [Claim 8] The composition according to claim 7, wherein the HCVR comprises the amino acid sequence described in SEQ ID NO: 58, and the LCVR comprises the amino acid sequence described in SEQ ID NO:

10. [Claim 9] (a) The HCVR is encoded by the nucleic acid sequence described in SEQ ID NO: 1, SEQ ID NO: 41, or SEQ ID NO: 57, (b) The composition according to claim 1 or 2, wherein the LCVR is encoded by the nucleic acid sequence described in Sequence ID No.

9. [Claim 10] The composition according to claim 9, wherein HCDR1 is encoded by the nucleic acid sequence described in SEQ ID NO: 3, HCDR2 is encoded by the nucleic acid sequence described in SEQ ID NO: 5, HCDR3 is encoded by the nucleic acid sequence described in SEQ ID NO: 7, LCDR1 is encoded by the nucleic acid sequence described in SEQ ID NO: 11, LCDR2 is encoded by the nucleic acid sequence described in SEQ ID NO: 13, and LCDR3 is encoded by the nucleic acid sequence described in SEQ ID NO:

15. [Claim 11] The composition according to claim 10, wherein the HCVR is encoded by the nucleic acid sequence described in Sequence ID No. 1, and the LCVR is encoded by the nucleic acid sequence described in Sequence ID No.

9. [Claim 12] The composition according to claim 9, wherein HCDR1 is encoded by the nucleic acid sequence described in SEQ ID NO: 43, HCDR2 is encoded by the nucleic acid sequence described in SEQ ID NO: 45, HCDR3 is encoded by the nucleic acid sequence described in SEQ ID NO: 47, LCDR1 is encoded by the nucleic acid sequence described in SEQ ID NO: 11, LCDR2 is encoded by the nucleic acid sequence described in SEQ ID NO: 13, and LCDR3 is encoded by the nucleic acid sequence described in SEQ ID NO:

15. [Claim 13] The composition according to claim 12, wherein the HCVR is encoded by the nucleic acid sequence described in Sequence ID No. 41, and the LCVR is encoded by the nucleic acid sequence described in Sequence ID No.

9. [Claim 14] The composition according to claim 9, wherein HCDR1 is encoded by the nucleic acid sequence described in SEQ ID NO: 59, HCDR2 is encoded by the nucleic acid sequence described in SEQ ID NO: 61, HCDR3 is encoded by the nucleic acid sequence described in SEQ ID NO: 63, LCDR1 is encoded by the nucleic acid sequence described in SEQ ID NO: 11, LCDR2 is encoded by the nucleic acid sequence described in SEQ ID NO: 13, and LCDR3 is encoded by the nucleic acid sequence described in SEQ ID NO:

15. [Claim 15] The composition according to claim 14, wherein the HCVR is encoded by the nucleic acid sequence described in Sequence ID No. 57, and the LCVR is encoded by the nucleic acid sequence described in Sequence ID No.

9. [Claim 16] An expression vector comprising a first polynucleotide and a second polynucleotide, The first polynucleotide comprises a nucleic acid sequence encoding a heavy chain variable region (HCVR) including complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3), and the amino acid sequence of the HCVR is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 42, and SEQ ID NO:

58. An expression vector wherein the second polynucleotide comprises a nucleic acid sequence encoding a light chain variable region (LCVR) including CDRs (LCDR1, LCDR2, and LCDR3), and the amino acid sequence of the LCVR is as described in Sequence ID No.

10. [Claim 17] An isolated host cell containing the expression vector described in claim 16. [Claim 18] A method for producing an antibody or an antigen-binding fragment thereof, comprising culturing at least one host cell according to claim 17 under conditions that enable the production of the antibody or the antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thus produced. [Claim 19] A first isolated polynucleotide comprising a nucleic acid sequence encoding a heavy chain variable region (HCVR) comprising HCDR1, HCDR2, and HCDR3, wherein the HCVR comprises the amino acid sequence described in SEQ ID NO: 2, SEQ ID NO: 42, or SEQ ID NO: 58; and A second isolated polynucleotide comprising a nucleic acid sequence encoding a light chain variable region (LCVR) including LCDR1, LCDR2, and LCDR3, wherein the LCVR comprises the amino acid sequence described in SEQ ID NO:

10. A composition containing the following: [Claim 20] The nucleic acid sequence of the first isolated polynucleotide codes for HCDR1 described in SEQ ID NO: 4, HCDR2 described in SEQ ID NO: 6, and HCDR3 described in SEQ ID NO:

8. The composition according to claim 19, wherein the nucleic acid sequence of the second isolated polynucleotide codes for LCDR1 described in SEQ ID NO: 12, LCDR2 described in SEQ ID NO: 14, and LCDR3 described in SEQ ID NO:

16. [Claim 21] The composition according to claim 20, wherein the HCVR comprises the amino acid sequence described in SEQ ID NO: 2, and the LCVR comprises the amino acid sequence described in SEQ ID NO:

10. [Claim 22] The nucleic acid sequence of the first isolated polynucleotide codes for HCDR1 described in SEQ ID NO: 44, HCDR2 described in SEQ ID NO: 46, and HCDR3 described in SEQ ID NO:

48. The composition according to claim 19, wherein the nucleic acid sequence of the second isolated polynucleotide encodes LCDR1 as described in SEQ ID NO: 12, LCDR2 as described in SEQ ID NO: 14, and LCDR3 as described in SEQ ID NO:

16. [Claim 23] The composition according to claim 22, wherein the HCVR comprises the amino acid sequence described in SEQ ID NO: 42, and the LCVR comprises the amino acid sequence described in SEQ ID NO:

10. [Claim 24] The nucleic acid sequence of the first isolated polynucleotide codes for HCDR1 described in SEQ ID NO: 60, HCDR2 described in SEQ ID NO: 62, and HCDR3 described in SEQ ID NO:

64. The composition according to claim 19, wherein the nucleic acid sequence of the second isolated polynucleotide encodes LCDR1 as described in SEQ ID NO: 12, LCDR2 as described in SEQ ID NO: 14, and LCDR3 as described in SEQ ID NO:

16. [Claim 25] The composition according to claim 22, wherein the HCVR comprises the amino acid sequence described in SEQ ID NO: 58, and the LCVR comprises the amino acid sequence described in SEQ ID NO:

10. [Claim 26] The first isolated polynucleotide comprises the nucleic acid sequence described in SEQ ID NO: 1, SEQ ID NO: 41, or SEQ ID NO:

57. The composition according to claim 19, wherein the second isolated polynucleotide comprises the nucleic acid sequence described in SEQ ID NO:

9. [Claim 27] The HCDR1 is encoded by the nucleic acid sequence described in Sequence ID No. 3, the HCDR2 is encoded by the nucleic acid sequence described in Sequence ID No. 5, and the HCDR3 is encoded by the nucleic acid sequence described in Sequence ID No.

7. The composition according to claim 26, wherein LCDR1 is encoded by the nucleic acid sequence described in SEQ ID NO: 11, LCDR2 is encoded by the nucleic acid sequence described in SEQ ID NO: 13, and LCDR3 is encoded by the nucleic acid sequence described in SEQ ID NO:

15. [Claim 28] The composition according to claim 27, wherein the HCVR is encoded by the nucleic acid sequence described in Sequence ID No. 1, and the LCVR is encoded by the nucleic acid sequence described in Sequence ID No.

9. [Claim 29] The HCDR1 is encoded by the nucleic acid sequence described in Sequence ID No. 43, the HCDR2 is encoded by the nucleic acid sequence described in Sequence ID No. 45, and the HCDR3 is encoded by the nucleic acid sequence described in Sequence ID No.

47. The composition according to claim 26, wherein LCDR1 is encoded by the nucleic acid sequence described in SEQ ID NO: 11, LCDR2 is encoded by the nucleic acid sequence described in SEQ ID NO: 13, and LCDR3 is encoded by the nucleic acid sequence described in SEQ ID NO:

15. [Claim 30] The composition according to claim 29, wherein the HCVR is encoded by the nucleic acid sequence described in Sequence ID No. 41, and the LCVR is encoded by the nucleic acid sequence described in Sequence ID No.

9. [Claim 31] The HCDR1 is encoded by the nucleic acid sequence described in Sequence ID No. 59, the HCDR2 is encoded by the nucleic acid sequence described in Sequence ID No. 61, and the HCDR3 is encoded by the nucleic acid sequence described in Sequence ID No.

63. The composition according to claim 26, wherein LCDR1 is encoded by the nucleic acid sequence described in SEQ ID NO: 11, LCDR2 is encoded by the nucleic acid sequence described in SEQ ID NO: 13, and LCDR3 is encoded by the nucleic acid sequence described in SEQ ID NO:

15. [Claim 32] The composition according to claim 31, wherein the HCVR is encoded by the nucleic acid sequence described in Sequence ID No. 57, and the LCVR is encoded by the nucleic acid sequence described in Sequence ID No.

9. [Claim 33] A host cell comprising the composition according to any one of claims 19 to 32. [Claim 34] A method for producing an antibody or an antigen-binding fragment thereof, comprising culturing a host cell according to claim 33 under conditions that enable the production of the antibody or the antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thus produced.

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