Antigen-binding protein that activates the leptin receptor

Abstract

To provide alternative approaches to treating leptin resistance and other conditions associated with leptin deficiency or hypoleptinemia.SOLUTION: The present invention provides antibodies and antigen-binding fragments thereof that bind human leptin receptor (LEPR). The antibodies of the present invention are agonist antibodies; i.e., binding of the anti-LEPR antibodies of the invention to LEPR causes, inter alia, activation of leptin receptor signaling in cells. In certain embodiments, the antibodies of the present invention do not compete with leptin for binding to LEPR. The antibodies of the present invention are useful, e.g., for mimicking, substituting for, or supplementing the normal biological activity of leptin in a subject. The antibodies and antigen-binding fragments of the present invention are therefore useful in the therapeutic treatment of diseases and disorders associated with leptin resistance and leptin deficiency.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Sequence Listing An official copy of the Sequence Listing has been submitted contemporaneously with the specification electronically via EFS-Web as an ASCII Sequence Listing with the filename 2016_10_11_10178WO01_Sequence_Listing_as_Filed_ST25.TXT, a creation date of October 11, 2016, and a size of approximately 99.6 kilobytes. The Sequence Listing contained in this ASCII document is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

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

[0004] The present invention provides antibodies and antigen-binding fragments thereof that bind to the human leptin receptor (LEPR). The antibodies of the present invention are agonistic antibodies; that is, when the anti-LEPR antibodies of the present invention bind to the LEPR, they activate, among other things, intracellular leptin receptor signaling. In certain embodiments, the antibodies of the present invention do not compete with leptin for binding to the LEPR. The antibodies of the present invention are useful, for example, to mimic, replace, or supplement the normal biological activity of leptin in a subject. Thus, the antibodies and antigen-binding fragments of the present invention are useful in the therapeutic treatment of diseases and disorders associated with leptin resistance and leptin deficiency.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0021] The present invention also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1, although in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 2, or a sequence substantially similar thereto, 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, although in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 2, or a sequence substantially similar thereto, 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, although in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 2, or a sequence substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0024] The present invention also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1, although in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 2, or sequences substantially similar thereto, 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, although 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 sequences substantially similar thereto, 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, although 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 sequences substantially similar thereto, 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 an HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1, HCDR2, and HCDR3), and the set of amino acid sequences of HCDR1, HCDR2, and HCDR3 are defined by any of the exemplary anti-LEPR antibodies listed in Table 1.

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

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

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

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

[0032] In yet another aspect, the present invention provides a method of treatment for enhancing or stimulating LEPR signaling using an anti-LEPR antibody or an antigen-binding portion of an antibody of the present invention. The treatment method according to this aspect of the present invention comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an antibody or antigen-binding fragment of the antibody of the present invention. The disorder to be treated is Any disease or condition that is ameliorated, ameliorated, inhibited, or prevented by stimulating or activating LEPR signaling, or by otherwise mimicking the natural activity of leptin, either in vitro or in vivo. In certain embodiments, for example, the following items are provided: (Item 1) An isolated antibody or antigen-binding fragment thereof that binds to the human leptin receptor (LEPR) and activates LEPR signaling. (Item 2) (i) a K of less than about 150 nM as measured by surface plasmon resonance Dbinds to monomeric human LEPR at 25°C; (ii) a t of greater than about 1 minute as measured by surface plasmon resonance 1 / 2 binds to monomeric human LEPR at 25°C; (iii) a K of less than about 5 nM as measured by surface plasmon resonance D binds to dimeric human LEPR at 25°C; (iv) a t of greater than about 15 minutes as measured by surface plasmon resonance 1 / 2 binds to dimeric human LEPR at 25°C; (v) binds to human LEPR complexed with human leptin; (vi) LEPR:leptin interaction is not blocked; (vii) binds to cell surface-expressed LEPR in the presence and absence of human leptin; and (viii) an EC of less than about 90 pM in a cell-based reporter assay 50 Activate LEPR signaling with 2. The isolated antibody or antigen-binding fragment thereof according to item 1, wherein the antibody or antigen-binding fragment exhibits one or more properties selected from the group consisting of: (Item 3) 3. The isolated antibody or antigen-binding fragment thereof of item 1 or 2, which activates LEPR signaling in a cell-based reporter assay at least 50% as effectively as leptin. (Item 4) 4. The isolated antibody or antigen-binding fragment thereof of item 3, which activates LEPR signaling in a cell-based reporter assay with at least 70% efficacy compared to leptin. (Item 5) An isolated antibody or antigen-binding fragment thereof that binds to the human leptin receptor (LEPR) and comprises (a) a complementarity-determining region (CDR) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 74, or SEQ ID NO: 82, and (b) a CDR of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 66. (Item 6) 6. The isolated antibody or antigen-binding fragment thereof of item 5, comprising the heavy chain CDRs and light chain CDRs of an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 10, 26 / 10, 34 / 10, 42 / 10, 50 / 10, 58 / 66, 74 / 66, and 82 / 66. (Item 7) Item 8. The isolated antibody or antigen-binding fragment thereof according to Item 5 or 6, comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 10, 26 / 10, 34 / 10, 42 / 10, 50 / 10, 58 / 66, 74 / 66, and 82 / 66. 8. The isolated antibody or antigen-binding fragment thereof of item 7, which activates LEPR signaling. (Item 9) 5. The antibody or antigen-binding fragment thereof of any one of items 1 to 4, which competes for binding to LEPR with a reference antibody comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 10, 26 / 10, 34 / 10, 42 / 10, 50 / 10, 58 / 66, 74 / 66 and 82 / 66. (Item 10) 5. The antibody or antigen-binding fragment thereof according to any one of items 1 to 4, which binds to the same epitope on LEPR as a reference antibody comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 10, 26 / 10, 34 / 10, 42 / 10, 50 / 10, 58 / 66, 74 / 66, and 82 / 66. (Item 11) 11. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 10, and a pharmaceutically acceptable carrier or diluent. (Item 12) 12. A method for treating a disease or condition associated with or caused by leptin deficiency or leptin resistance, comprising administering to a subject in need thereof the pharmaceutical composition of item 11. (Item 13) Item 13. The method of item 12, wherein the disease or condition associated with or caused by leptin deficiency or leptin resistance is selected from the group consisting of lipodystrophy, obesity, metabolic syndrome, diet-induced food craving, functional hypothalamic amenorrhea, type 1 diabetes, type 2 diabetes, insulin resistance, severe insulin resistance due to mutations in the insulin receptor, Alzheimer's disease, leptin deficiency, leptin resistance, leprechaunism / Donahue syndrome, and Rabson-Mendenhall syndrome. (Item 14) 12. A method for treating a lipodystrophic condition in a patient, comprising administering to a patient in need thereof the pharmaceutical composition of item 11, wherein the lipodystrophic condition is selected from the group consisting of congenital generalized lipodystrophy, acquired generalized lipodystrophy, familial partial lipodystrophy, acquired partial lipodystrophy, centrifugal abdominal lipodystrophy, cyclic lipodystrophy, localized lipodystrophy, and HIV-associated lipodystrophy. (Item 15) 12. A method for treating a disease or condition associated with or caused by a signaling-deficient or signaling-impaired LEPR mutation, comprising administering to a subject in need thereof the pharmaceutical composition of item 11. (Item 16) 16. The method of item 15, wherein the signaling-deficient LEPR mutation or the signaling-impaired LEPR mutation is LEPR-A409E or LEPR-P316T. (Item 17) 17. The method of item 15 or 16, wherein the disease or condition associated with or caused by a signaling-deficient or signaling-impaired LEPR mutation is early-onset obesity. (Item 18) 18. The method of any one of items 12 to 17, further comprising administering to the subject a second therapeutic agent, wherein the second therapeutic agent is selected from the group consisting of recombinant human leptin, PCSK9 inhibitors, statins, ezetimibe, insulin, insulin variants, insulin secretagogues, metformin, sulfonylureas, sodium glucose co-transporter 2 (SGLT2) inhibitors, GLP-1 agonists / analogs, glucagon (GCG) inhibitors, glucagon receptor (GCGR) inhibitors, angiopoietin-like protein (ANGPTL) inhibitors, phentermine, orlistat, topiramate, bupropion, topiramate / phentermine, bupropion / naltrexone, bupropion / zonisamide, pramlintide / metreleptin, lorcaserin, cetilistat, tesofensine, and velneperit. (Item 19) An isolated antibody or antigen-binding fragment thereof that binds to the human leptin receptor (LEPR) and sensitizes the LEPR to an antigen. (Item 20) 20. The isolated antibody of item 19, or an antigen-binding fragment thereof, which binds to the human leptin receptor (LEPR) and comprises (a) a complementarity-determining region (CDR) of the heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 74, or SEQ ID NO: 82, and (b) a CDR of the light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 66. (Item 21) 22. The isolated antibody or antigen-binding fragment thereof according to claim 20, comprising a heavy chain CDR and a light chain CDR of an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 26 / 10, 34 / 10, 42 / 10, 50 / 10, 58 / 66, 74 / 66, and 82 / 66. An isolated antibody or antigen-binding fragment thereof that specifically binds human LEPR and interacts with amino acids 162-169 of SEQ ID NO: 113 and amino acids 170-181 of SEQ ID NO: 113 as determined by hydrogen / deuterium exchange. (Item 23) 23. The isolated antibody or antigen-binding fragment of item 22, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR2 domains selected from the group consisting of SEQ ID NOs: 4, 6, and 8 and SEQ ID NOs: 12, 14, and 16, respectively. (Item 24) 23. The isolated monoclonal antibody or antigen-binding fragment of item 22, comprising the HCVR / LCVR amino acid sequence pair consisting of SEQ ID NO: 2 / 10.

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

[0034] [Figure 1] FIG. 1 depicts the binding of dimeric human LEPR to human leptin in the presence of increasing concentrations of test anti-LEPR antibodies or control molecules, as measured by ELISA (absorbance at 450 nm).

[0035] [Figure 2] Figures 2A-2C illustrate the extent of LEPR signaling in HEK293 cells expressing wild-type LEPR (circles), a signaling-deficient LEPR mutant (A409E, squares), or a signaling-impaired LEPR mutant (P316T, triangles). LEPR signaling is expressed as the ratio of pSTAT3-Y705 / STAT3 measured by densitometry from Western blots prepared from cells treated with increasing concentrations of leptin (Figure 2A), H4H16650 (Figure 2B), or H4H16679 (Figure 2C).

[0036] [Figure 3] FIG. 3 shows the mean daily food intake of leptin-deficient mice dosed with either 3 mg / kg of an isotype control antibody or 3 mg / kg of a LEPR antibody selected from H4H16650P2, H4H16679P2, H4H17319P2, or H4H17321P2.

[0037] [Figure 4] FIG. 4 shows the mean percent change in body weight for mice dosed with either an isotype control antibody at 3 mg / kg or a LEPR antibody selected from H4H16650P2, H4H16679P2, H4H17319P2, or H4H17321P2 at 3 mg / kg.

[0038] [Figure 5] Figure 5 shows the mean fat mass of animals in each antibody treatment group quantified by μCT 1 day before (unshaded bars) and 6 days after (shaded bars) antibody treatment, expressed as mean ± SEM.

[0039] [Figure 6] FIG. 6 shows the percent change in body weight of mice fed 30 mg / kg of an antibody selected from H4H18482P2, H4H18487P2, H4H18492P2, or an isotype control.

[0040] [Figure 7] Figure 7A shows the fat mass of mice before dosing with anti-LEPR antibodies H4H18482P2, H4H18487P2, or H4H18492P2. Figure 7B shows the fat mass of mice treated with 30 mg / kg of H4H18482P2, H4H18487P2, or H4H18492P2.

[0041] [Figure 8] FIG. 8 shows that the anti-LEPR antibodies tested activated monkey (Mf) LEPR in the IMR-32 / STAT3-luc / Mf LEPR cell line. DETAILED DESCRIPTION OF THE INVENTION

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

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.In this specification, the term "about" when used in reference to a specific stated value means that the value can vary by 1% or less from the stated value.For example, in this specification, the expression "about 100" includes 99 and 101 and all values ​​therebetween (for example, 99.1, 99.2, 99.3, 99.4, etc.).

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

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

[0046] All references herein to proteins, polypeptides, and protein fragments are intended to refer to the human version of the respective protein, polypeptide, or protein fragment, unless expressly specified as being from a non-human species. Thus, the expression "LEPR" refers to human LEPR unless specified as being from a non-human species, e.g., "mouse LEPR," "monkey LEPR," etc.

[0047] As used herein, the phrase "cell surface-expressed LEPR" refers to one or more LEPR proteins, or extracellular domains thereof, that are expressed on the surface of a cell in vitro or in vivo, such that at least a portion of the LEPR protein is exposed to the extracellular side of the cell membrane and is accessible to the antigen-binding portion of an antibody. A "cell surface-expressed LEPR" may include or consist of a LEPR protein that is expressed on the surface of a cell that normally (e.g., in a natural or wild-type state) expresses the LEPR protein. Alternatively, a "cell surface-expressed LEPR" may include or consist of a LEPR protein that is expressed on the surface of a cell that does not normally express human LEPR on its surface but has been artificially engineered to express LEPR on its surface.

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

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

[0050] The term "antibody," as used herein, also includes antigen-binding fragments of a complete antibody molecule. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from complete antibody molecules using, for example, any suitable standard technique, such as proteolytic 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 sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains in the appropriate configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

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

[0052] An antigen-binding fragment of an antibody will typically contain at least one variable domain, which may be of any size or amino acid composition and generally contains at least one CDR adjacent to or in-frame with one or more framework sequences. L Domain and associated V H In the antigen-binding fragment containing the V domain, H and V L The domains can be positioned relative to each other in any suitable configuration. For example, the variable region can be a dimer, with the V H -V H , V H -V L , or V L -V L Alternatively, the antigen-binding fragment of an antibody may comprise a dimer of monomeric V H or V L It may contain domains.

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

[0054] Like intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies will typically comprise at least two different variable domains, each capable of specifically binding to a separate antigen or to a different epitope of the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of the antigen-binding fragments of antibodies of the present invention using routine techniques available in the art.

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

[0056] In some embodiments, antibodies of the present invention may be recombinant human antibodies. The term "recombinant human antibody," as used herein, is intended to include all human antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (described further below), antibodies isolated from recombinant combinatorial human antibody libraries (described further below), antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, created, or isolated by any other means involving splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), thereby modifying the V H and V L The amino acid sequence of the region is human germline V H and V LThese are sequences that, while derived from and related to the sequence, may not naturally occur within the human antibody germline repertoire in vivo.

[0057] The present invention provides a method for the preparation of a polypeptide comprising a hinge, C, or C-terminal fragment thereof, which may be desirable, for example, to improve the yield of a desired antibody form during production. H 2, or C H It encompasses antibodies with one or more mutations in three regions.

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

[0059] The present invention includes variants of the anti-LEPR antibodies disclosed herein that contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibody was derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof that are derived from any of the amino acid sequences disclosed herein, but in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody was derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. H Domain and / or V LAll of the framework and / or CDR residues within a domain are backmutated to the residue found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are backmutated to the original germline sequence, e.g., only residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4 are mutated, or only residues found in CDR1, CDR2, or CDR3 are mutated. In other embodiments, one or more framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived). Furthermore, antibodies of the present invention can contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once antibodies and antigen-binding fragments containing one or more germline mutations are obtained, they can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed by the present invention.

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

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

[0062] Sequence similarity of polypeptides, also known as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and its mutein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) generates alignments and percent sequence identity for the regions of best overlap between the query and search sequences (Pearson (2000) supra). Another suitable algorithm for comparing the sequences of the present invention to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

[0063] Anti-LEPR antibodies containing Fc variants According to certain embodiments of the present invention, there are provided anti-LEPR antibodies comprising an Fc domain comprising one or more mutations that enhance or decrease antibody binding to the FcRn receptor at, for example, acidic pH compared to neutral pH. For example, the present invention provides an anti-LEPR antibody comprising an Fc domain comprising one or more mutations that enhance or decrease antibody binding to the FcRn receptor at, for example, acidic pH compared to neutral pH. H 2 or C HAnti-LEPR antibodies include those containing mutations in the FcRn region that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., endosomes, where the pH ranges from about 5.5 to about 6.0). Such mutations can increase the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q); 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, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K), and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P).

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

[0065] The anti-LEPR antibodies of the present invention may comprise a modified Fc domain with reduced effector function. As used herein, a "modified Fc domain with reduced effector function" refers to any Fc portion of an immunoglobulin that has been modified, mutated, shortened, etc., relative to a wild-type, naturally occurring Fc domain, such that a molecule comprising the modified Fc exhibits a reduced severity or extent of at least one effect selected from the group consisting of cell killing (e.g., ADCC and / or CDC), complement activation, phagocytosis, and opsonization, relative to a control molecule comprising a wild-type, naturally occurring version of the Fc portion. In certain embodiments, a "modified Fc domain with reduced effector function" is an Fc domain with reduced or attenuated binding to an Fc receptor (e.g., FcγR).

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

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

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

[0069] The present invention includes antibodies and antigen-binding fragments thereof that bind to monomeric human LEPR with high affinity. For example, the present invention includes antibodies and antigen-binding fragments thereof that have a K of less than about 150 nM as measured by surface plasmon resonance at 25° C. or 37° C., e.g., using the assay format set forth in Example 3 herein, or a substantially similar assay. D and less than about 150 nM, less than about 140 nM, less than about 130 nM, less than about 120 nM, less than about 110 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 100 nM, ...100 nM, less than about 100 nM, less than about 150 nM, less than about 140 nM, less than about 130 nM, less than about 120 nM, less than about 110 nM, less than about 100 nM, less than about 100 nM, less than about 150 nM, less than about 150 nM, less than about 150 nM, less than about 160 nM, less than about 160 nM, less than about 170 nM, less than about 170 nM, less than about 180 nM, less than about 180 nM, less than about 190 nM, less than about 200 nM, less than about 210 nM, less K less than 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, or less than about 300 pM D Anti-LEPR antibodies are provided that bind to monomeric human LEPR at 25°C.

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

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

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

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

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

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

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

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

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

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

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

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

[0082] Various techniques known to those skilled in the art can be used to identify the amino acids within the epitope recognized by a particular antibody. Exemplary techniques include, for example, alanine scanning mutation analysis, peptide blot analysis, and peptide cleavage analysis. Additionally, methods such as epitope removal, epitope extraction, and chemical modification of antigens can be used (Tomer, 2000, Protein Science, 9:487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange, detected by mass spectrometry. Generally, the hydrogen / deuterium exchange method involves deuterium labeling of the protein of interest, followed by binding of an antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, allowing hydrogen-deuterium exchange to occur at all residues except those protected by the antibody (which remain deuterium-labeled). After the antibody dissociates, the target protein is subjected to protease cleavage and mass spectrometry analysis, revealing the deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999), Analytical Biochemistry 267(2):252-259; Engen and Smith (2001), Anal. Chem. 73:256A-265A. X-ray crystallography of an antibody complexed with its antigen can also be used to identify the amino acids within the polypeptide with which the antibody interacts.

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

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

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

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

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

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

[0089] Two antigen-binding proteins or antibodies are considered bioequivalent if they are pharmaceutical equivalents or pharmaceutical substitutes if, when administered at the same molar dose under similar experimental conditions, e.g., in a single dose or multiple doses, their rate and extent of absorption do not differ significantly. Some antibodies are equivalent in their extent of absorption but not in their rate of absorption, but would be considered bioequivalent if such differences in absorption rate are intentional, not essential to achieving effective body drug concentrations, e.g., during chronic use, are reflected in the labeling, and are not considered medically significant for the particular drug product being studied.

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

[0091] In one embodiment, two antigen binding proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity or diminished efficacy, compared to continuing treatment without such a switch.

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

[0093] Bioequivalence can be supported by in vivo and in vitro methods. Measures of bioequivalence include, for example, (a) in vivo studies in humans or other mammals that measure the concentration of an antibody or its metabolites in blood, plasma, serum, or other biological fluid as a function of time; (b) in vitro studies that correlate with and are reasonably predictive of in vivo bioavailability data in humans; (c) in vivo studies in humans or other mammals that measure the relevant short-term pharmacological effect of the antibody (or its target) as a function of time; and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability, or bioequivalence, of an antibody.

[0094] Biologically equivalent variants of the anti-LEPR antibodies of the invention can be constructed, for example, by making various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexts, biologically equivalent antibodies can include anti-LEPR antibody variants containing amino acid changes that modify the glycosylation characteristics of the antibody, for example, mutations that ablate or eliminate glycosylation.

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

[0096] multispecific antibody Antibodies of the present invention may be monospecific or multispecific (e.g., bispecific). Multispecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al., 1991, J. Immunol., 147:60-69; Kufer et al., 2004, Trends Biotechnol., 22:238-244. Anti-LEPR antibodies of the present invention may be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof may be operatively linked (e.g., by chemical coupling, genetic fusion, noncovalent association, or another method) to one or more other molecular entities, such as another antibody or antibody fragment, to generate a bispecific or multispecific antibody having a second binding specificity.

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

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

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

[0100] Therapeutic Formulations and Administration The present invention provides pharmaceutical compositions comprising the anti-LEPR antibodies of the present invention or antigen-binding fragments thereof. The pharmaceutical compositions of the present invention are formulated with suitable carriers, excipients, and other agents that provide improved entry, delivery, tolerance, and the like. Many suitable formulations can be found in formularies known to all medicinal chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (LIPOFECTIN™, Life Technologies, Carlsbad, CA, etc.), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998), J. See also Pharm Sci Technol, 52:238-311.

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

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

[0103] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. Furthermore, for subcutaneous delivery, a pen delivery device is easily adapted to deliver the pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. In this way, the pen delivery device can be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, the disposable pen delivery device is pre-filled with the pharmaceutical composition, which is held in a reservoir within the device. Once the reservoir of the pharmaceutical composition is empty, the entire device is discarded.

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

[0105] In certain circumstances, pharmaceutical compositions can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng., Vol. 14:201). In another embodiment, a polymeric material can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Press, Boca Raton, Florida. In yet another embodiment, the controlled release system can be placed in proximity to the target of the composition, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science, Vol. 249:1527-1533.

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

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

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

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

[0110] The present invention also includes anti-LEPR antibodies and their antigen-binding fragments that are useful for restoring leptin signaling to cells, tissues, and organs that express one or more LEPR mutations associated with obesity. For example, certain LEPR mutants have been identified that exhibit no signaling or reduced signaling in the presence of leptin and are associated with obesity and related disorders. LEPR mutants that do not exhibit signaling in the presence of leptin are referred to herein as "signaling-deficient LEPR mutants." An exemplary signaling-deficient LEPR mutant is LEPR-A409E (Farooqi et al., 2007, N Engl J Med, 356(3):237-247). LEPR mutants that exhibit reduced signaling (compared to wild-type LEPR) in the presence of leptin are referred to herein as "signaling-impaired LEPR mutants." An exemplary signaling-impaired LEPR mutation is LEPR-P316T (Mazen et al., 2011, Mol Genet Metab 102:461-464). Accordingly, the present invention includes anti-LEPR antibodies and antigen-binding fragments thereof that are useful for treating, preventing, and / or ameliorating diseases and disorders caused by or associated with one or more signaling-deficient (e.g., A409E) and / or signaling-impaired (e.g., P316T) LEPR mutants.

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

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

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

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

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

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

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

[0118] Diagnostic and Analytical 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 (e.g., overexpression, underexpression, lack of expression, etc.) of LEPR. An exemplary diagnostic assay for LEPR can include contacting a sample obtained from a patient with an anti-LEPR antibody of the present invention, where the anti-LEPR antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-LEPR antibody can be used in diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be a radioisotope, e.g., 3 H, 14 C. 32 P, 35 S, or 1251, etc.; a fluorescent or chemiluminescent moiety, such as fluorescein isothiocyanate or rhodamine; or an enzyme, such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase, or luciferase, etc. Specific exemplary assays that can be used to detect or measure LEPR in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorting (FACS), and positron emission tomography (PET) scanning.

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

[0120] The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

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

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

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

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

[0125] As used in the Examples herein, "comparator mAb300D" refers to Fab9F8, described in Fazeli et al. (2006), J Immunol Methods 312:190-200 and Carpenter et al. (2012), Structure 20(3):487-97.

[0126] Example 3: Binding Affinity and Kinetic Constants of Human Monoclonal Anti-LEPR Antibodies Obtained by Surface Plasmon Resonance The equilibrium dissociation constant (K) for LEPR binding to purified anti-LEPR monoclonal antibodies DThe binding activity (RI) was determined using a real-time surface plasmon resonance biosensor with a Biacore4000 instrument. All binding studies were performed at 25°C and 37°C in a running buffer of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant Tween-20, pH 7.4 (HBS-ET). The Biacore sensor surface was first derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (GE, #BR-1008-39) to capture the anti-LEPR monoclonal antibody. Binding studies were performed on the following LEPR reagents: human LEPR extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (hLEPR.mmh; SEQ ID NO: 114), macaca fascicularis LEPR extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (mfLEPR.mmh; SEQ ID NO: 117), human LEPR extracellular domain expressed with a C-terminal mouse IgG2a Fc tag (hLEPR.mFc; SEQ ID NO: 115), mouse LEPR extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (mLEPR.mmh; SEQ ID NO: 118), and rat LEPR extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (rLEPR.mmh; SEQ ID NO: 119). Different concentrations of LEPR reagent were first prepared in HBS-ET running buffer (100 nM to 3.7 nM; 3-fold serial dilutions) and injected over the anti-human Fc-captured anti-LEPR monoclonal antibody surface at a flow rate of 30 μL / min for 4 min, while the dissociation of the monoclonal antibody-bound LEPR reagent was monitored for 10 min in HBS-ET running buffer. a ) and dissociation (k d The binding-dissociation equilibrium constant (K) was determined by fitting the real-time binding sensorgrams to a 1:1 binding model with mass transport limitation using Scrubber 2.0c curve fitting software. D ) and dissociation half-life (t1 / 2), [ka] was calculated from the kinetic rate constant as

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

[0128] At 25°C, anti-LEPR monoclonal antibodies exhibited K values ​​ranging from 7.93 nM to 148 nM, as shown in Table 5. D At 37°C, the anti-LEPR monoclonal antibodies bound to hLEPR-MMH with K values ​​ranging from 14.8 nM to 326 nM, as shown in Table 4. D The values ​​were those of hLEPR-MMH.

[0129] Ten of the twelve anti-LEPR monoclonal antibodies of the present invention bound to mfLEPR.MMH. At 25°C, the anti-LEPR monoclonal antibodies bound to mfLEPR.MMH with K values ​​ranging from 2.27 nM to 139 nM, as shown in Table 7. D At 37°C, the anti-LEPR monoclonal antibodies bound to mfLEPR.MMH with K values ​​ranging from 5.18 nM to 264 nM, as shown in Table 8. D It bound to mfLEPR.MMH at the value.

[0130] At 25°C, anti-LEPR monoclonal antibodies exhibited K values ​​ranging from 613 pM to 5.7 nM, as shown in Table 7. D At 37°C, the anti-LEPR monoclonal antibodies bound to hLEPR-mFc with K values ​​ranging from 1.16 nM to 12.8 nM, as shown in Table 8. D The values ​​were consistent with those of the control.

[0131] None of the anti-LEPR monoclonal antibodies of the invention bound to mLEPR.MMH or rLEPR.MMH at 25° C. or 37° C. (data not shown).

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

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

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

[0135] As shown in Table 10, none of the anti-LEPR antibodies of the invention demonstrated greater than 28% blocking of hLEPR.hFc binding to hLeptin-coated surfaces. However, the control antibody and hLeptin, as positive controls, were able to block 99% of hLEPR.hFc binding to hLeptin-coated surfaces. The isotype control antibody demonstrated no measurable blocking at concentrations up to 500 nM. [Table 10]

[0136] Example 6: Cell binding by FACS analysis using HEK293 / Mycx2-hLepR(ecto)-GPI anchored cells The leptin receptor, LEPR, is a single-pass transmembrane receptor of the class I cytokine receptor family (Tartaglia et al. (1997), J Biol Chem 7:272(10):6093-6). LEPR can bind to leptin, a protein expressed primarily by adipose tissue that is involved in regulating food intake and metabolism (Friedman et al. (2014), J Endocrinol 223(1):T1-8).

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

[0138] For FACS analysis, HEK293 parental cells and HEK293 / hLEPR-GPI cells were dissociated and cultured at 5 × 10 in 2% FBS-containing PBS (FACS buffer). 5Cells were plated at 1000 cells / well in a 96-well V-bottom plate. To test whether the ability of anti-hLEPR antibodies to bind to cells was affected by the presence of leptin, FACS buffer containing or not 1 μM human leptin (R&D Systems, #398-LP) was incubated with the cells for 30 minutes at 4°C, followed by the addition of 10 nM anti-LEPR or control antibodies in FACS buffer. Cells were subsequently incubated for 30 minutes at 4°C, washed, and then incubated with 16 μg / mL Alexa Fluor®-647-conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc., #109-547-003) for 30 minutes at 4°C. Cells were subsequently fixed using BD CytoFix™ (Becton Dickinson, #554655), filtered, and analyzed on a HyperCyt Flow Cytometer (Beckman Coulter). Unstained and secondary antibody-only controls were also tested for all cell lines. Results were analyzed using ForeCyt (IntelliCyt) and FlowJo version 10 software to determine the geometric mean of viable cell fluorescence. The geometric mean of fluorescence for each sample was normalized to the geometric mean of unstained cells to obtain relative binding per condition, referred to as the "binding ratio." These binding ratios were recorded for each antibody tested.

[0139] As shown in Table 11, nine anti-LEPR antibodies of the invention tested at 10 nM demonstrated binding to HEK293 / hLEPR-GPI cells without leptin at binding ratios ranging from 824- to 3374-fold. The anti-LEPR antibodies also bound in the presence of 1 μM leptin at binding ratios of 398 and 4184-fold. As shown in Table 11, a control antibody tested at 10 nM demonstrated binding to HEK293 / hLEPR-GPI cells without leptin at a binding ratio of 2349-fold, but showed significantly lower binding to the cells in the presence of 1 μM leptin, with a binding ratio of 112. The anti-LEPR antibodies did not demonstrate any significant binding to HEK293 parental cells at binding ratios ranging from 1- to 9-fold, with and without 1 μM leptin. Isotype control antibody and secondary antibody alone samples also demonstrated no significant binding to any of the cell lines, with or without leptin, with binding ratios ranging from 1-6 fold.

[0140] As shown in Table 12, the four antibodies of the invention tested at 70 nM without leptin demonstrated binding to HEK293 / hLEPR-GPI cells with binding ratios ranging from 707 to 1131-fold, and to HEK293 / Stat3-luc / hLEPR-FL cells with binding ratios ranging from 42 to 51. The anti-LEPR antibodies did not demonstrate any significant binding to HEK293 / Stat3-luc cells, with binding ratios ranging from 1 to 8-fold. The isotype control antibody and secondary antibody-alone samples also did not demonstrate significant binding to any of the cell lines tested, with binding ratios ranging from 1 to 2-fold. [Table 11] [Table 12]

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

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

[0143] To measure the effect of antibodies of the present invention in the presence of leptin, a fixed concentration of 200 pM human leptin in assay buffer was added to cells, followed immediately by the addition of anti-LEPR or isotype control antibodies in half-log serial dilutions to final concentrations ranging from 100 nM to 300 fM. Samples were then incubated overnight at 37°C in 5% CO2. OneGlo reagent (Promega, #E6051) was then added to the samples, and luciferase activity was measured in luminescence mode on an Envision Multilable plate reader (Perkin Elmer). Relative light unit (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% activation in the IMR-32 / STAT3-Luc / hLEPR assay.

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

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

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

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

[0148] The results of this example indicate that the agonist anti-LEPR antibodies of the invention may be useful in diseases or disorders (e.g., early-onset obesity) caused by or associated with certain signaling defects or impaired LEPR mutants (e.g., LEPR-P316T or LEPR-A409E).

[0149] Example 9: Octet cross-competition between different anti-LEPR monoclonal antibodies Binding competition between a panel of different anti-LEPR monoclonal antibodies was determined using a real-time label-free biolayer interferometry assay on an Octet HTX biosensor platform (Pall ForteBio Corp.). The entire experiment was performed at 25°C with plate shaking at 1000 rpm in a buffer containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant Tween-20, 1 mg / mL BSA, pH 7.4 (HBS-EBT). To assess whether the two antibodies could compete with each other for binding to their respective epitopes on recombinant human LEPR (hLEPR.mmh; SEQ ID NO: 114) expressed with a C-terminal myc-myc-hexahistidine tag, approximately 0.25 nM or 0.34 nM hLEPR-MMH was first captured onto an anti-pentaHis antibody-coated Octet biosensor chip (Fortebio Inc, #18-5122) by immersing the biosensor chip in a well containing 20 μg / mL hLEPR-MMH for 5 minutes. The antigen-captured biosensor chip was then saturated by immersing the first anti-LEPR monoclonal antibody (hereafter referred to as mAb-1) in a well containing a 50 μg / mL solution of mAb-1 for 210 seconds. The biosensor chip was then subsequently immersed in a well containing a 50 μg / mL solution of the second anti-LEPR monoclonal antibody (hereafter referred to as mAb-2) for 150 seconds. The biosensor chip was washed with HBS-EBT buffer between every step of the experiment. Real-time binding responses were monitored throughout the course of the experiment, and the binding responses were recorded at the end of each step. The binding responses of mAb-2 to hLEPR-MMH pre-complexed with mAb-1 were compared to determine the competitive / non-competitive behavior of different anti-LEPR monoclonal antibodies, as shown in Tables 14 and 15. [Table 14] [Table 15]

[0150] Example 10: In vivo efficacy of LEPR agonist antibodies H4H16650P2, H4H16679P2, H4H17319P2, and H4H17321P2 in an inducible mouse model of leptin deficiency The effects of four specific agonist anti-LEPR antibodies of the present invention, H4H16650P2, H4H16679P2, H4H17319P2, and H4H17321P2, on food intake, body weight, and adiposity were examined in a genetically engineered LEPR mouse expressing a leptin receptor composed of the human LEPR ectodomain sequence instead of the murine LEPR ectodomain sequence. Hu / Hu This was determined in an inducible model of leptin deficiency in mice. The model of leptin deficiency was induced by hydrodynamic DNA delivery (HDD) of a plasmid encoding hFc-tagged mouse LEPR ectodomain (referred to herein as mLEPR.hFc or "leptin trap"; SEQ ID NO: 120). When expressed, leptin trap binds to secreted and circulating leptin. After HDD of 50 μg of a DNA construct encoding leptin trap, mice showed increased food consumption and increased body fat accumulation and body weight.

[0151] Baseline daily food intake was measured 7 and 4 days (days -7 and -4) before administration of leptin trap. On day 0, 35 13- to 17-week-old male LEP rats were administered 100 mg of LEP trap. Hu / HuMice were successfully subjected to HDD using the leptin trap. Retroorbital blood was collected on days 6 and 13 after HDD, and body composition, including adiposity, was quantified by μCT. On day 7 after HDD, mice were randomized into five groups of seven mice based on percent body weight change from day 0. Each group received a single dose of 3 mg / kg isotype control antibody, 3 mg / kg H4H16650P2, 3 mg / kg H4H16679P2, 3 mg / kg H4H17319P2, or 3 mg / kg H4H17321 via subcutaneous injection. The isotype control antibody did not bind to any known mouse protein. Food intake and body weight were measured for each animal over the duration of the study. Figure 3 summarizes the average daily food intake for each treatment group. In Figure 3, the dotted line represents the average baseline food intake before HDD injection. The percent change in body weight from day 0 was calculated for each animal at each time point. Figure 4 summarizes the mean percent change in body weight for animals in each antibody treatment group. Figure 5 summarizes the mean fat mass for animals in each antibody treatment group as quantified by μCT 1 day before and 6 days after antibody treatment. All results are expressed as mean ± SEM.

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

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

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

[0155] Labeling solutions were prepared in 10 mM PBS buffer in DO at pH 7.0 (equivalent to pH 6.6). For deuterium labeling, 3.8 μL of hLEPR.mmh (8 pmol / μL) or hLEPR.mmh premixed with antibody at a 2:1 molar ratio was incubated with 56.2 μL of DO labeling solution for various time points (e.g., non-deuterated control = 0 s, 1 min, and 20 min labeled). Deuteration was quenched by transferring 50 μL of sample to 50 μL of pre-chilled quench buffer (0.2 M TCEP, 6 M guanidine chloride in 100 mM phosphate buffer, pH 2.5), and the mixed sample was incubated at 1.0°C for 2 min. The quenched sample was then injected into a Waters HDX Manager for online pepsin / protease XIII digestion. Digested peptides were trapped on an ACQUITY UPLC BEH C18 1.7 μm, 2.1 × 5 mm VanGuard precolumn at 0 °C and eluted onto an analytical column, ACQUITY UPLC BEH C18 1.7 μm, 1.0 × 50 mm, for a 9-minute gradient separation of 5% to 40% B (mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile). The mass spectrometer was set to a cone voltage of 37 V, a scan time of 0.5 s, and a mass / charge range of 50 to 1700 Th.

[0156] To identify peptides derived from human LEPR, LC-MSE data from undeuterated samples were processed and searched against a database containing human LEPR, pepsin, and their randomized sequences via Waters ProteinLynx Global Server (PLGS) software. Identified peptides were imported into DynamX software and filtered by two criteria: 1) a minimum number of products per amino acid of 0.2, and 2) a duplicate file threshold of 3. DynamX software then automatically determined the deuterium incorporation of each peptide based on retention time and high mass accuracy (<10 ppm) across multiple time points, with three replicates per time point.

[0157] result. M.S. EUsing an online pepsin / protease XIII column coupled with data acquisition, a total of 201 peptides from human LEPR were reproducibly identified in the absence or presence of antibodies, demonstrating 70% sequence coverage. Five peptides showed significantly reduced deuterium incorporation when bound to H4H16650P2 (centroid delta values ​​>0.4 Daltons, p-values ​​<0.05), as shown in Table 16. The reported peptide masses correspond to the average centroid MH+ masses from three replicates. These peptides, corresponding to amino acids 162-169 (human LEPR; amino acids LYVLPEVL of SEQ ID NO: 113) and 170-181 (human LEPR; amino acids EDSPLVPQKGSF of SEQ ID NO: 113), had slower deuteration rates when bound to H4H16650P2. These identified residues also correspond to residues 162-169 and 170-181 of human LEPR, defined as Uniprot entry P48357 (SEQ ID NO: 113; human leptin receptor). [Table 16]

[0158] Example 12: In vivo efficacy testing of LEPR enhancer antibodies in humanized LEPR mice The effects of the three specific potentiator anti-LEPR antibodies of the present invention, H4H18482P2, H4H18487P2, and H4H18492P2, on body weight and body fat accumulation were examined using a single-harbored genetically engineered LEPR antibody. Hu / Hu This was determined in mice, which express a leptin receptor composed of the human LEPR ectodomain sequence in place of the murine LEPR ectodomain sequence (mLEPR.hFc, SEQ ID NO: 120).

[0159] On day -19, body composition, including body fat depots, was quantified by μCT. On day 0, 48 14- to 16-week-old female LEPR mice were analyzed. Hu / HuMice were randomized based on body weight into four groups of 12 mice. On days 0 and 11, mice from each group received a single dose of 30 mg / kg of isotype control antibody, 30 mg / kg of H4H18482P2, 30 mg / kg of H4H18487P2, or 30 mg / kg of H4H18492P2 via subcutaneous injection. The isotype control antibody does not bind to any known mouse protein. Body weight was measured for each animal over the duration of the study. The percent change in body weight from day 0 was calculated for each animal at each time point. Figure 6 summarizes the average percent change in body weight for animals in each treatment group. Figure 6 summarizes the average fat mass for animals in each antibody treatment group, quantified by μCT 19 days before and 11 days after antibody treatment. All results are expressed as mean ± SEM.

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

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

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

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

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

[0165] As shown in Table 17, in the absence of hLeptin, all of the tested anti-LEPR antibodies demonstrated activation of monkey LEPR signaling in IMR-32 / STAT3-Luc / mfLEPR cells, and EC 50 The values ​​ranged from 266 pM to 368 pM, and the maximal activation ranged from 76% to 82%, with 100% activation being obtained with hLeptin, which had an EC of 333 pM. 50 The isotype control antibody did not demonstrate any measurable stimulation of IMR-32 / STAT3-Luc / mfLEPR cells. [Table 17]

[0166] Example 14: Epitope binding to the full-length extracellular domain of human LEPR using Luminex MFI signal To determine the epitope of human LEPR that is bound by the anti-LEPR antibodies of the present invention, Luminex FLEXMAP (FM3DD, LuminexCorp) flow cytometry-based analysis was used to characterize the interaction of anti-LEPR antibodies with recombinant human LEPR protein domains. For the assay, approximately 3 million carboxylated Microplex R Microspheres (Luminex, catalog no. LC1000A) were washed, vortexed, and sonicated in 0.1 M NaPO4, pH 6.2 (activation buffer), then centrifuged to remove the supernatant. The microspheres were resuspended in 120 μL of activation buffer at 25°C, and the carboxylate groups (-COOH) were activated by adding 15 μL of 50 mg / mL N-hydroxysuccinimide (NHS, Thermo Scientific, catalog no. 24500), followed by 15 μL of 50 mg / mL 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC, Thermo Scientific, catalog no. 22980). After 10 minutes, the pH of the reaction was reduced to 5.0 by adding 600 μL of 50 mM MES, pH 5 (coupling buffer), and the microspheres were vortexed and centrifuged to remove the supernatant. The activated beads were immediately mixed with 500 μL of 20 μg / mL monoclonal anti-myc antibody containing mouse IgG or human IgG in coupling buffer and incubated for 2 hours at 25° C. The coupling reaction was quenched by adding 50 μL of 1 M Tris-HCl, pH 8.0, and the microspheres were rapidly vortexed, centrifuged, and washed four times with 1 mL of DPBS to remove uncoupled proteins and other reaction components.

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

[0168] From this mixed pool, 75 μL of microspheres were plated per well of a 96-well filter plate (Millipore, catalog number: MSBVN1250), mixed with 25 μL of individual anti-human LEPR monoclonal antibodies (0.5 or 5 μg / mL), incubated at 25°C for 2 hours, and then washed twice with 200 μL of DPBS (wash buffer) containing 0.05% Tween 20. To detect and quantify the levels of anti-LEPR antibodies bound to individual microspheres, 2.5 μg / mL R-phycoerythrin-conjugated goat F(ab')2 anti-human kappa (Southern 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, Catalog No. 115-116-072) in blocking buffer was added and incubated for 30 minutes at 25°C. After 30 minutes, samples were washed twice with 200 μL of wash buffer and resuspended in 150 μL of wash buffer. The median fluorescence intensity (MFI) of the microspheres was measured using a Luminex analyzer. [Table 18]

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

Claims

1. A first composition for use in producing an antibody or antigen-binding fragment thereof that specifically binds to a human leptin receptor, the first composition comprising an isolated polynucleotide molecule comprising a nucleotide sequence encoding a heavy chain variable region of the antibody or antigen-binding fragment thereof that specifically binds to a human leptin receptor, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:26, and the first composition is used in combination with a second composition comprising an isolated polynucleotide molecule comprising a nucleotide sequence encoding a light chain variable region of the antibody or antigen-binding fragment thereof that specifically binds to a human leptin receptor, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

10.

2. The first composition described in claim 1, wherein the isolated polynucleotide molecule comprising a nucleotide sequence encoding a heavy chain variable region of an antibody or its antigen-binding fragment that specifically binds to a human leptin receptor comprises the nucleotide sequence set forth in SEQ ID NO:

25.

3. A first composition for use in producing an antibody or antigen-binding fragment thereof that specifically binds to a human leptin receptor, the first composition comprising an isolated polynucleotide molecule comprising a nucleotide sequence encoding a light chain variable region of the antibody or antigen-binding fragment thereof that specifically binds to a human leptin receptor, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10, and the first composition is used in combination with a second composition comprising an isolated polynucleotide molecule comprising a nucleotide sequence encoding a heavy chain variable region of the antibody or antigen-binding fragment thereof that specifically binds to a human leptin receptor, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

26.

4. The first composition described in claim 3, wherein the isolated polynucleotide molecule comprising a nucleotide sequence encoding a light chain variable region of an antibody or its antigen-binding fragment that specifically binds to a human leptin receptor comprises the nucleotide sequence set forth in SEQ ID NO:

9.

5. (a) a first polynucleotide molecule comprising a nucleotide sequence encoding a heavy chain variable region of an antibody or antigen-binding fragment thereof that specifically binds to a human leptin receptor, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:26; and (b) a second polynucleotide molecule comprising a nucleotide sequence encoding a light chain variable region of an antibody or antigen-binding fragment thereof that specifically binds to a human leptin receptor, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10; and A composition comprising:

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