Leptin receptor agonistic antibodies as mono or combo therapies to treating obesity

Abstract

To provide an antibody having agonistic activity to leptin receptor (LEPR) and usable as a therapeutic agent for treating obesity.SOLUTION: Provided is an antibody that binds to leptin receptor (LEPR) and stimulates it. In one embodiment, the disclosed antibody comprises at least one of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having specific sequences, or a sequence having at least 90%, having at least 95%, having at least 97%, or having at least 99% sequence identity thereto.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to leptin receptor agonist antibodies and related therapies. [Background technology]

[0002] Leptin is a circulating hormone produced and released by adipocytes. Leptin signals through its receptor (LEPR (also called OB-R)), which is expressed by neurons in the hypothalamus, to regulate metabolism and appetite. LEPR is a cell surface single-pass transmembrane receptor of the class I cytokine receptor family. Activation of LEPR by leptin binding activates intracellular Janus kinase (JAK), leading to the phosphorylation of downstream effector proteins.

[0003] Loss-of-function mutations in the leptin or LEPR genes cause severe, early-life onset obesity. Recombinant leptin protein (metreleptin) has been approved to treat symptoms in patients with leptin deficiency and lipodystrophy. Metreleptin treatment has also been shown to reduce body weight in individuals with low circulating leptin levels (Depaoli et al., Diabetes. 2018 Jul 67:296-LB) and improve liver pathology in patients with nonalcoholic steatohepatitis (Akinci et al., Med. 2021 Jul 9;2(7):814-835). However, its short half-life, adverse side effects, and the development of anti-leptin autoantibodies limit metreleptin's clinical application. Furthermore, patients with common obesity do not respond to metreleptin treatment due to leptin resistance. (Farr et al.Curr Opinion Endo Diab Obes.2015;22(5):353-359).

[0004] Additionally, LEPR antibodies have been reported in WO 2017066204(A1) and WO 2019195796(A1). However, there remains a need for LEPR agonist antibodies that bind to LEPR in some species with desirable binding affinity, activate LEPR in some species, exhibit beneficial properties with respect to Fcγ receptor binding, do not desensitize LEPR signaling, activate signaling-deficient LEPR, and / or can be advantageously combined with second therapeutic agents.

[0005] Therefore, new therapeutic agents for stimulating the LEPR pathway would significantly broaden clinical applications. Summary of the Invention

[0006] The present disclosure provides antibodies that bind to and stimulate the leptin receptor (LEPR).

[0007] In one embodiment, an antibody of the present disclosure comprises at least one of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 selected from at least one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity thereto.

[0008] In one embodiment, the antibody comprises an HCDR1 having SEQ ID NO: 7, an HCDR2 having SEQ ID NO: 8, an HCDR3 having SEQ ID NO: 9, an LCDR1 having SEQ ID NO: 1 or 2, an LCDR2 having SEQ ID NO: 3, and an LCDR3 having SEQ ID NO: 4, 5, or 6.

[0009] In one embodiment, the antibody comprises an LCDR1 having SEQ ID NO: 1 and an LCDR3 having SEQ ID NO: 5. In one embodiment, the antibody comprises an LCDR1 having SEQ ID NO: 1 and an LCDR3 having SEQ ID NO: 4. In one embodiment, the antibody comprises an LCDR1 having SEQ ID NO: 2 and an LCDR3 having SEQ ID NO: 5. In one embodiment, the antibody comprises an LCDR1 having SEQ ID NO: 1 and an LCDR3 having SEQ ID NO: 6.

[0010] In one embodiment, the antibody comprises a VH and VL sequence selected from SEQ ID NOs: 10, 12, 14, 16, and 18, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity thereto.

[0011] In one embodiment, the disclosure provides an antibody having a VH set forth in SEQ ID NO: 18 and a VL set forth in SEQ ID NO: 10. In one embodiment, the disclosure provides an antibody having a VH set forth in SEQ ID NO: 18 and a VL set forth in SEQ ID NO: 12. In one embodiment, the disclosure provides an antibody having a VH set forth in SEQ ID NO: 18 and a VL set forth in SEQ ID NO: 14. In one embodiment, the disclosure provides an antibody having a VH set forth in SEQ ID NO: 18 and a VL set forth in SEQ ID NO: 16.

[0012] In some embodiments, the antibodies or antigen-binding fragments disclosed herein are of the human IgG1 or IgG4 subclass. In some embodiments, the antibodies or antigen-binding fragments disclosed herein are of the human IgG1 subclass.

[0013] In one embodiment, the antibody comprises an HC and LC sequence selected from SEQ ID NOs: 11, 13, 15, 17, and 19, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity thereto.

[0014] In one embodiment, the disclosure provides an antibody having a HC set forth in SEQ ID NO: 19 and a LC set forth in SEQ ID NO: 11. In one embodiment, the disclosure provides an antibody having a HC set forth in SEQ ID NO: 19 and a LC set forth in SEQ ID NO: 13. In one embodiment, the disclosure provides an antibody having a HC set forth in SEQ ID NO: 19 and a LC set forth in SEQ ID NO: 15. In one embodiment, the disclosure provides an antibody having a HC set forth in SEQ ID NO: 19 and a LC set forth in SEQ ID NO: 17.

[0015] In another aspect, the present disclosure provides a pharmaceutical composition comprising an antibody of the present disclosure. In one embodiment, the pharmaceutical composition comprises an antibody of the present disclosure and a second therapeutic agent. The second therapeutic agent is selected from a drug having GLP-1 receptor agonist activity, a drug having amylin receptor agonist activity, a drug having calcitonin receptor agonist activity, and a drug having glucagon receptor agonist activity. In one embodiment, the second therapeutic agent has GLP-1 receptor agonist activity and GIP receptor agonist activity. In another embodiment, the second therapeutic agent has GLP-1 receptor agonist activity, GIP receptor agonist activity, and glucagon receptor agonist activity.

[0016] In another aspect, the present disclosure provides a method of treating a disease or disorder associated with the leptin receptor. In one embodiment, the disease or disorder is associated with or mediated by leptin deficiency, leptin resistance, or is otherwise treatable by stimulating the LEPR. In another embodiment, the disease or disorder comprises obesity. The method according to this aspect comprises administering an antibody of the present disclosure as a monotherapy or combination therapy.

[0017] In combination therapy, the antibody of the present disclosure is administered simultaneously, separately, or sequentially in combination with a second therapeutic agent. The second therapeutic agent comprises an agent having GLP-1 receptor agonist activity, amylin receptor agonist activity, or glucagon receptor agonist activity. In one embodiment, the second therapeutic agent has GLP-1 receptor agonist activity and GIP receptor agonist activity. In another embodiment, the second therapeutic agent has GLP-1 receptor agonist activity, GIP receptor agonist activity, and glucagon receptor agonist activity.

[0018] In some embodiments, the second therapeutic agent is selected from SEQ ID NOs: 39-57. [Brief explanation of the drawings]

[0019] [Figure 1] 1 shows binding of exemplary antibodies of the disclosure to human LEPR. [Figure 2] Binding of exemplary antibodies of the disclosure to hLepR in the presence of leptin protein is shown as a percentage of the control. [Figure 3] 1 shows C1q binding of exemplary antibodies of the present disclosure. [Figure 4] 1 shows the percent body weight of diet-induced obese mice administered a GLP-1 receptor agonist in combination with a leptin receptor agonist compared to controls. [Figure 5] FIG. 1 shows the percent body weight of diet-induced obese mice treated with a dual GLP1R / GIPR agonist in combination with a leptin receptor agonist compared to controls. [Figure 6] Figure 1 shows the percent body weight of diet-induced obese mice treated with a GLP1R / GIPR / GCGR triple agonist in combination with a leptin receptor agonist compared to controls. [Figure 7] 1 shows the percent body weight of diet-induced obese mice administered a glucagon receptor agonist in combination with a leptin receptor agonist compared to controls. [Figure 8]1 shows the percent body weight of diet-induced obese mice administered an amylin receptor agonist in combination with a leptin receptor agonist compared to controls. [Figure 9] 1 shows the percent body weight compared to controls for diet-induced obese mice treated with a leptin receptor agonist in combination with either an amylin receptor agonist or an amylin and calcitonin receptor co-agonist. [Figure 10] 1 shows the percent body weight of cynomolgus monkeys administered exemplary antibodies disclosed herein compared to controls. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present disclosure provides antibodies that bind to and stimulate the leptin receptor (LEPR). As used herein, unless otherwise specified, LEPR refers to the human leptin receptor (hLepR) as set forth in SEQ ID NO: 28. The extracellular domain of human LEPR is set forth in SEQ ID NO: 29.

[0021] As used herein, the term "antibody" refers to an immunoglobulin molecule that binds to an antigen. Antibody embodiments include monoclonal antibodies, polyclonal antibodies, single-chain fragment variable (scFv), antibody fragments, human antibodies, humanized antibodies, chimeric antibodies, bispecific or multispecific antibodies, or conjugated antibodies. The antibody may be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4).

[0022] An exemplary antibody of the present disclosure is an immunoglobulin G (IgG) antibody composed of four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked via interchain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains contains a variable region of about 100 to 125 amino acids or more, which is primarily responsible for antigen recognition. The carboxyl-terminal portion of each of the four polypeptide chains contains a constant region, which is primarily responsible for effector function and antibody recycling. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. IgG isotypes may be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0023] In some embodiments, the antibodies or antigen-binding fragments disclosed herein are of the human IgG1 or IgG4 subclass.

[0024] In some embodiments, the antibodies or antigen-binding fragments disclosed herein are of the human IgG1 subclass.

[0025] The VH and VL regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). The CDRs are exposed on the surface of the protein and are critical regions of the antibody for antigen-binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Herein, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3," and the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3." The CDRs contain most of the residues that form specific interactions with the antigen.The assignment of amino acid residues to CDRs can be performed using the methods of Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins," Journal of Molecular Biology 1987, 196:901-917; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins," Journal of Molecular Biology 1997, 273:927-948), North (North et al., "A New Clustering of Antibody CDR Loop Conformations," Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (the international ImMunoGeneTics database, available at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999;27:209-212. Unless otherwise specified, the North CDR definitions are used for antibodies that bind human LEPR as described herein.

[0026] Embodiments of the present disclosure also include antibody fragments or antigen-binding fragments, as used herein, that comprise at least a portion of an antibody that retains the ability to specifically interact with an antigen or an epitope of an antigen, such as a Fab, Fab', F(ab')2, Fv fragment, scFv antibody fragment, scFab, disulfide-linked Fv (sdFv), or Fd fragment.

[0027] As used herein, the term "antigen-binding domain" refers to a part of a molecule that binds to an antigen or an epitope of an antigen.

[0028] As used herein, the term "bispecific" refers to a molecule that contains two different antigen-binding domains. A bispecific binding molecule can bind to two different antigens or two different epitopes of the same antigen. Embodiments of the present disclosure also include bispecific antibodies that bind to LEPR.

[0029] The present disclosure provides a VH comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, which in turn comprise the heavy chain (HC).

[0030] In some embodiments, the disclosure provides a VH comprising at least one of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0031] In some embodiments, antibodies of the present disclosure provide a VH comprising at least one of HCDR1 (SEQ ID NO: 7), HCDR2 (SEQ ID NO: 8), and HCDR3 (SEQ ID NO: 9). Exemplary VHs comprise a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 18.

[0032] Exemplary HCs having HCDR1 (SEQ ID NO:7), HCDR2 (SEQ ID NO:8), and HCDR3 (SEQ ID NO:9) include sequences that are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:19.

[0033] In some embodiments, the disclosure provides an antibody having an LCDR1 set forth in SEQ ID NO: 1 and an LCDR3 set forth in SEQ ID NO: 5. In some embodiments, the antibody further comprises an LCDR2 set forth in SEQ ID NO: 3.

[0034] In some embodiments, the disclosure provides a VL comprising SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5. An exemplary VL is set forth in SEQ ID NO: 16. In some embodiments, the VL comprises a sequence that is at least 75%, at least 80%, at least 85%, 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 16.

[0035] Exemplary LCs having SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5 include sequences that are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:17.

[0036] In one embodiment, an antibody of the disclosure comprises a heavy chain (HC) comprising SEQ ID NO:19 and a light chain (LC) comprising SEQ ID NO:17.

[0037] In one embodiment, an antibody of the disclosure comprises a heavy chain (HC) consisting of SEQ ID NO:19 and a light chain (LC) consisting of SEQ ID NO:17.

[0038] In some embodiments, the disclosure provides antibodies having SEQ ID NO: 1 and SEQ ID NO: 4. In some embodiments, the antibody further comprises an LCDR2 as set forth in SEQ ID NO: 3.

[0039] In some embodiments, the disclosure provides a VL comprising SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 4. An exemplary VL is set forth in SEQ ID NO: 10. In some embodiments, the VL comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 10.

[0040] Exemplary LCs having SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:4 include sequences that are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:11.

[0041] In one embodiment, an antibody of the disclosure comprises a heavy chain (HC) comprising SEQ ID NO:19 and a light chain (LC) comprising SEQ ID NO:11.

[0042] In one embodiment, an antibody of the disclosure comprises a heavy chain (HC) consisting of SEQ ID NO:19 and a light chain (LC) consisting of SEQ ID NO:11.

[0043] In some embodiments, the disclosure provides antibodies having SEQ ID NO: 2 and SEQ ID NO: 5. In some embodiments, the antibody further comprises an LCDR2 as set forth in SEQ ID NO: 3.

[0044] In some embodiments, the disclosure provides a VL comprising SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 5. An exemplary VL is set forth in SEQ ID NO: 12. In some embodiments, the VL comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 12.

[0045] Exemplary LCs having SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:5 include sequences that are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:13.

[0046] In one embodiment, an antibody of the disclosure comprises a heavy chain (HC) comprising SEQ ID NO:19 and a light chain (LC) comprising SEQ ID NO:13.

[0047] In one embodiment, an antibody of the disclosure comprises a heavy chain (HC) consisting of SEQ ID NO:19 and a light chain (LC) consisting of SEQ ID NO:13.

[0048] In some embodiments, the disclosure provides antibodies having SEQ ID NO: 1 and SEQ ID NO: 6. In some embodiments, the antibody further comprises an LCDR2 as set forth in SEQ ID NO:3.

[0049] In some embodiments, the disclosure provides a VL comprising SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 6. An exemplary VL is set forth in SEQ ID NO: 14. In some embodiments, the VL comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 97%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 14.

[0050] Exemplary LCs having SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5 include sequences that are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:15.

[0051] In some embodiments, an antibody of the present disclosure comprises an HCDR1 comprising SEQ ID NO:7, an HCDR2 comprising SEQ ID NO:8, an HCDR3 comprising SEQ ID NO:9, an LCDR1 comprising SEQ ID NO:1 or SEQ ID NO:2, an LCDR2 comprising SEQ ID NO:3, and an LCDR3 comprising SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0052] In one embodiment, an antibody of the disclosure comprises a heavy chain (HC) comprising SEQ ID NO:19 and a light chain (LC) comprising SEQ ID NO:15.

[0053] In one embodiment, an antibody of the disclosure comprises a heavy chain (HC) consisting of SEQ ID NO:19 and a light chain (LC) consisting of SEQ ID NO:15.

[0054] Exemplary CDRs of the Antibodies of the Disclosure [Table 1]

[0055] In certain embodiments, the set of amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0056] Exemplary antibodies of the present disclosure include an antibody having a LC set forth in SEQ ID NO: 11 and a HC set forth in SEQ ID NO: 19. Another exemplary antibody of the present disclosure includes an antibody having a LC set forth in SEQ ID NO: 13 and a HC set forth in SEQ ID NO: 19. Another exemplary antibody of the present disclosure includes an antibody having a LC set forth in SEQ ID NO: 15 and a HC set forth in SEQ ID NO: 19. Another exemplary antibody of the present disclosure includes an antibody having a LC set forth in SEQ ID NO: 17 and a HC set forth in SEQ ID NO: 19.

[0057] In some embodiments, the LC and / or HC comprise a signal peptide. Exemplary signal peptides include SEQ ID NO:20, and the corresponding cDNA is shown in SEQ ID NO:21.

[0058] As used herein in reference to two or more amino acid sequences, percent identity refers to two or more sequences that have a specified percentage of the same amino acid residues when compared and aligned for maximum correspondence using a sequence comparison algorithm (e.g., BLASTP and BLASTN, or other algorithms available to those skilled in the art) or by visual inspection. Depending on the application, percent identity can exist over a region of the sequences being compared, e.g., a functional domain, or over the entire length of the two sequences being compared. As an example, percent sequence identity can be compared to a reference sequence. For example, when using a sequence comparison algorithm, test and reference sequences can be input into a computer (and, if necessary, subsequence coordinates can be further specified, along with sequence algorithm program parameters). The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters. Exemplary sequence alignment and / or homology / homology algorithms are available through Smith & Waterman, Adv. Appl. Math. 2:482 (1981), Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), GAP, BESTFIT, FASTA, and TFASTA (the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Ausubel et al., infra). One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0059] The present disclosure provides antibodies that bind to and stimulate human LEPR. Certain antibodies of the present disclosure that bind to and stimulate LEPR have one or more of the following properties: bind to LEPR of some species with desirable binding affinity, activate LEPR of some species, have beneficial properties with respect to Fcγ receptor binding, do not desensitize LEPR signaling, activate signaling-deficient LEPR, and can be advantageously combined with secondary therapeutic agents.

[0060] The term "agonize," as used herein, refers to the ability of an antibody, antibody fragment, or binding molecule to induce or increase one or more activities or functions associated with an antigen. An antigen is a molecule to which an antibody, antibody fragment, or binding molecule binds. An antigen can be a receptor (e.g., LEPR).

[0061] As used herein, agonism and percent agonism are determined by methods known in the art and / or essentially as described herein, including, for example, Example 4 below.

[0062] The term "bind," as used herein, unless otherwise specified, refers to the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, bringing the two proteins or molecules into proximity as determined by common methods known in the art.

[0063] The terms "selectively bind" or "specifically bind" mean that an antibody of the present disclosure interacts with human and / or cynomolgus monkey and / or canine LEPR more frequently, more rapidly, for a longer period of time, with higher affinity, or some combination of the above, than with other substances. An antibody may be said to specifically bind an antigen if it binds at least 25% greater, at least 50% greater, at least 100% greater, at least 200% greater, or at least 500% greater than it binds to a different antigen or non-antigen target, as measured by techniques known in the art. Exemplary techniques include competitive ELISA or K by SPR at 25°C or 30°C. D Measurements include:

[0064] In some embodiments, the antibodies of the disclosure have a K of 5.0E-7M or less. D In another embodiment, the antibodies of the disclosure bind to human LEPR with a K of 5.0E-8M or less. D In another embodiment, the antibodies of the disclosure bind to human LEPR with a K of 2.0E-8M or less. D It binds to human LEPR.

[0065] In some embodiments, antibodies of the disclosure have a K of 1E-10 M to 5.0E-7 M. D In some embodiments, the antibodies of the disclosure bind to human LEPR with a K of 1E-10 M to 5.0E-8 M. D In some embodiments, the antibodies of the disclosure bind to human LEPR with a K of 1E-10 M to 3.0E-8 M. D In some embodiments, the antibodies of the disclosure bind to human LEPR with a K of 1E-10 M to 2.0E-8 M. D In some embodiments, the antibodies of the disclosure bind to human LEPR with a K of 1E-9M to 2.5E-8M. D In some embodiments, the antibodies of the disclosure bind to human LEPR with a K of 1E-9M to 2.0E-8M. D It binds to human LEPR.

[0066] In some embodiments, the antibodies of the disclosure have a K of 5.0E-7M or less.D In another embodiment, the antibodies of the disclosure bind to cynomolgus monkey LEPR with a K of 5.0E-8M or less. D In another embodiment, the antibodies of the disclosure bind to cynomolgus monkey LEPR with a K of 2.0E-8M or less. D binds to cynomolgus monkey LEPR.

[0067] In some embodiments, antibodies of the disclosure have a K of 1E-10 to 5.0E-7 M. D In some embodiments, the antibodies of the disclosure bind to cynomolgus monkey LEPR with a K of 1E-10 M to 5.0E-8 M. D In some embodiments, the antibodies of the disclosure bind to cynomolgus monkey LEPR with a K of 1E-10 to 2.0E-8 M. D In some embodiments, the antibodies of the disclosure bind to cynomolgus monkey LEPR with a K of 1E-9 to 2.0E-8 M. D binds to cynomolgus monkey LEPR.

[0068] In some embodiments, the antibodies of the disclosure have a K of 5.0E-6M or less. D In another embodiment, the antibodies of the disclosure bind to canine LEPR with a K of 5.0E-7M or less. D In another embodiment, the antibodies of the disclosure bind to canine LEPR with a K of 2.0E-7M or less. D In another embodiment, the antibodies of the disclosure bind to canine LEPR with a K of 1.5E-7M or less. D binds to canine LEPR.

[0069] In some embodiments, antibodies of the disclosure have a K of 1E-10 M to 5.0E-6 M. D In some embodiments, antibodies of the disclosure bind to canine LEPR with a K of 1E-10 M to 5.0E-7 M. D In some embodiments, antibodies of the disclosure bind to canine LEPR with a K of 1E-10 M to 3.5E-7 M. D In some embodiments, antibodies of the disclosure bind to canine LEPR with a K of 1E-10 M to 1.5E-7 M. DIn some embodiments, antibodies of the disclosure bind to canine LEPR with a K of 1E-8M to 1.5E-7M. D and binds to canine LEPR.

[0070] In some embodiments, the antibodies of the disclosure have a K of 1E-9M to 2.0E-8M. D (human), K of 1E-9 to 2.0E-8M D (cynomolgus monkey) and K of 1E-9M to 1.5E-7M D (Dog) is provided.

[0071] As used herein, K D is determined by methods known in the art and / or methods essentially as described herein, including surface plasmon resonance (SPR) at 25° C. or 30° C. Exemplary methods are provided in Example 3 below.

[0072] In some embodiments, the antibodies of the disclosure have an EC 50 In another embodiment, the antibodies of the disclosure have an EC 50 (human). In another embodiment, the antibodies of the disclosure have an EC 50 (human). In another embodiment, the antibodies of the disclosure have an EC 50 (human). In another embodiment, the antibodies of the disclosure have an EC 50 (Human).

[0073] In some embodiments, the antibodies of the disclosure have an EC 50 In some embodiments, the antibodies of the disclosure have an EC 50 In some embodiments, the antibodies of the disclosure have an EC 50 In some embodiments, the antibodies of the disclosure have an EC 50In some embodiments, the antibodies of the disclosure have an EC 50 (Human).

[0074] In some embodiments, the antibodies of the disclosure have an EC 50 In another embodiment, the antibodies of the disclosure have an EC 50 (cynomolgus monkeys). In another embodiment, the antibodies of the disclosure have an EC 50 (cynomolgus monkeys). In another embodiment, the antibodies of the disclosure have an EC 50 (cynomolgus monkeys). In another embodiment, the antibodies of the disclosure have an EC 50 (cynomolgus monkey).

[0075] In some embodiments, the antibodies of the disclosure have an EC 50 In some embodiments, the antibodies of the disclosure have an EC 50 In some embodiments, the antibodies of the disclosure have an EC 50 In some embodiments, the antibodies of the disclosure have an EC 50 (cynomolgus monkey).

[0076] In some embodiments, the antibodies of the disclosure have an EC 50 In another embodiment, the antibodies of the disclosure have an EC 50 In another embodiment, the antibodies of the disclosure have an EC 50 In another embodiment, the antibodies of the disclosure have an EC 50 (dog). In another embodiment, the antibodies of the disclosure have an EC 50 In another embodiment, the antibodies of the disclosure have an EC 50In another embodiment, the antibodies of the disclosure have an EC 50 In another embodiment, the antibodies of the disclosure have an EC 50 (Dog).

[0077] In some embodiments, the antibodies of the disclosure have an EC 50 In some embodiments, the antibodies of the disclosure have an EC 50 In some embodiments, the antibodies of the disclosure have an EC 50 In some embodiments, the antibodies of the disclosure have an EC 50 (dog). In some embodiments, the antibodies of the disclosure have an EC 50 In some embodiments, the antibodies of the disclosure have an EC 50 (Dog).

[0078] In one embodiment, the antibodies of the disclosure have an EC 50 (human), EC of 1.0 nM to 0.01 nM 50 (cynomolgus monkeys) and EC 50 (Dog) is provided.

[0079] As used herein, EC 50 is determined by methods known in the art and / or methods essentially as described herein, including, for example, phosphorylation of STAT3 in cells assays. An exemplary method is provided in Example 4 below.

[0080] In one embodiment, an antibody of the present disclosure provides at least 45%, at least 50%, at least 55%, or at least 60% agonism of human LEPR compared to human leptin. In one embodiment, an antibody of the present disclosure provides 45%-75% agonism of human LEPR compared to human leptin. In one embodiment, an antibody of the present disclosure provides 50%-70% agonism of human LEPR compared to human leptin. In one embodiment, an antibody of the present disclosure provides 55%-65% agonism of human LEPR compared to human leptin.

[0081] In one embodiment, an antibody of the present disclosure provides at least 20%, at least 25%, at least 30%, or at least 35% agonism of the cynomolgus LEPR compared to human leptin. In one embodiment, an antibody of the present disclosure provides 20%-50% agonism of the cynomolgus LEPR compared to human leptin. In one embodiment, an antibody of the present disclosure provides 20%-40% agonism of the cynomolgus LEPR compared to human leptin. In one embodiment, an antibody of the present disclosure provides 20%-30% agonism of the cynomolgus LEPR compared to human leptin.

[0082] In one embodiment, an antibody of the disclosure provides at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, or at least 85% agonism of canine LEPR compared to human leptin.

[0083] In one embodiment, an antibody of the present disclosure provides 45% to 85% agonism of the canine LEPR compared to human leptin. In one embodiment, an antibody of the present disclosure provides 50% to 85% agonism of the canine LEPR compared to human leptin. In one embodiment, an antibody of the present disclosure provides 65% to 85% agonism of the canine LEPR compared to human leptin. In one embodiment, an antibody of the present disclosure provides 65% to 80% agonism of the canine LEPR compared to human leptin.

[0084] In one embodiment, an antibody of the disclosure provides 50-60% agonism of the human LEPR, 20% or more agonism of the cynomolgus monkey LEPR, and 70-80% agonism of the canine LEPR compared to human leptin.

[0085] The present disclosure provides antibodies that selectively bind to human, cynomolgus monkey (Macaca fascicularis), and canine (dingo (Canis lupus dingo)) LEPR. In some embodiments, the present disclosure provides antibodies that selectively bind to human and canine LEPR.

[0086] The present disclosure provides antibodies that do not desensitize LEPR signaling.

[0087] The present disclosure provides nucleic acids encoding heavy or light chains of antibodies that bind to and stimulate the leptin receptor (LEPR), or vectors containing such nucleic acids.

[0088] Exemplary nucleic acids include nucleic acid sequences that encode the polypeptide sequences set forth in any of SEQ ID NOs: 11, 13, 15, 17, and 19. Exemplary nucleic acids include SEQ ID NOs: 22, 23, 24, 25, and 26.

[0089] The terms "nucleic acid" or "polynucleotide," as used interchangeably herein, refer to a polymer of nucleotides, including single- and / or double-stranded nucleotide-containing molecules, such as DNA, cDNA, and RNA molecules, that incorporate naturally occurring nucleotides, modified nucleotides, and / or nucleotide analogs. A polynucleotide of the present disclosure can also include material incorporated therein, for example, by a DNA or RNA polymerase or a synthetic reaction.

[0090] The polynucleotides of the present disclosure can be expressed in host cells, for example, after the polynucleotide is operably linked to an expression control sequence. Expression control sequences capable of expressing an operably linked polynucleotide are well known in the art. For example, an expression vector can include a sequence encoding one or more signal peptides that facilitate secretion of the polypeptide from a host cell. The signal peptide can be, for example, an immunoglobulin signal peptide or a heterologous signal peptide. An expression vector containing a polynucleotide of interest (e.g., a polynucleotide encoding an antibody polypeptide) can be transferred into host cells by well-known methods. Additionally, the expression vector can include one or more selectable markers, such as, for example, tetracycline, neomycin, and dihydrofolate reductase, to facilitate detection of host cells transformed with the desired polynucleotide sequence.

[0091] Some embodiments of the present disclosure provide vectors comprising a nucleic acid sequence encoding a heavy or light chain of an antibody that specifically binds to and stimulates a leptin receptor. In certain embodiments, the vector comprises a nucleic acid according to at least one of SEQ ID NOs: 11, 13, 15, 17, 19, 22, 23, 24, 25, and 26.

[0092] In another aspect, the disclosure provides a host cell comprising one or more of SEQ ID NOs: 11, 13, 15, 17, 19, 22, 23, 24, 25, and 26.

[0093] In some embodiments, a cell, e.g., a host cell, comprises a vector having a first nucleic acid sequence encoding at least one of SEQ ID NOs: 11, 13, 15, 17, 19, 22, 23, 24, 25, and 26, and a second nucleic acid sequence encoding at least one of SEQ ID NOs: 11, 13, 15, 17, 19, 22, 23, 24, 25, and 26.

[0094] Host cells include cells stably or transiently transfected, transformed, transduced, or infected with one or more expression vectors expressing all or a portion of an antibody of the present disclosure. According to some embodiments, host cells can be stably or transiently transfected, transformed, transduced, or infected with an expression vector expressing the HC polypeptide and an expression vector expressing the LC polypeptide of an antibody of the present disclosure. In some embodiments, host cells can be stably or transiently transfected, transformed, transduced, or infected with expression vectors expressing the HC and LC polypeptides of an antibody of the present disclosure. Antibodies of the present disclosure can be produced in mammalian cells, such as CHO, NS0, HEK293, or COS cells, according to techniques well known in the art.

[0095] The present disclosure further provides a process for producing an antibody or antigen-binding fragment thereof that specifically binds to a leptin receptor described herein by culturing the above-described host cells, e.g., mammalian host cells, under conditions such that the antibody is expressed, and recovering the expressed antibody from the culture medium.

[0096] The medium into which the antibodies of the present disclosure are secreted can be purified by conventional techniques, such as mixed-mode methods of ion exchange and hydrophobic interaction chromatography. For example, the medium can be applied to and eluted from a Protein A or Protein G column using conventional methods; mixed-mode methods of ion exchange and hydrophobic interaction chromatography can also be used. Soluble aggregates and multimers can be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The product can be immediately frozen, for example, at -70°C, refrigerated, or lyophilized. Various methods of protein purification can be used, and such methods are known in the art, see, for example, Deutscher, Methods in Enzymology 182:83-89 (1990), and Scopes, Protein Purification: Principles and Practice, 3 (2003). rdEdition, Springer, NY (1994).

[0097] The present disclosure further provides an antibody or antigen-binding fragment thereof produced by any of the processes described herein.

[0098] Mammalian antibody expression typically results in glycosylation. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of a sugar, such as N-acetylgalactosamine, galactose, or xylose, to a serine or threonine. Typically, glycosylation occurs in the Fc region of antibodies at a highly conserved N-glycosylation site (e.g., position 297 in IgG1 according to the IMGT or EU index numbering). Glycosylation sites can be modified to alter glycosylation (e.g., to block or reduce glycosylation, or to alter the amino acid sequence to generate additional or diverse glycosylation).

[0099] Mammalian expression of antibodies from the IgG subclass can result in processing of the C-terminal amino acids from one or both heavy chains. For example, in the case of IgG1 antibodies, one or two C-terminal amino acids can be removed. For example, IgG1 antibodies, under certain circumstances, can have the C-terminal lysine, if present, truncated or trimmed from the heavy chain during expression. Additionally, the penultimate glycine can be truncated or trimmed from the heavy chain as well.

[0100] Expression of an antibody in a mammal can also result in modification of the N-terminal amino acid, for example, if the most N-terminal amino acid of a heavy or light chain is glutamine, it can be changed to pyroglutamic acid.

[0101] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an antibody disclosed herein and above. The pharmaceutical composition may further comprise at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0102] In some embodiments, the present disclosure provides pharmaceutical compositions comprising an antibody that binds to and stimulates LEPR (disclosed herein) and a second therapeutic agent.

[0103] In some embodiments, the second therapeutic agent restores leptin sensitivity.

[0104] In some embodiments, the second therapeutic agent comprises a drug with amylin receptor agonist activity. Exemplary drugs with amylin receptor agonist activity include amylin or amylin analogs. Examples of drugs with amylin receptor agonist activity include petrelintide, caglilintide, and pramlintide.

[0105] In another embodiment, the second therapeutic agent has amylin receptor agonist activity and calcitonin receptor agonist activity.

[0106] In some embodiments, the second therapeutic agent comprises an agent selected from SEQ ID NOs: 39-47.

[0107] In some embodiments, the second therapeutic agent comprises an agent selected from SEQ ID NOs: 48-49.

[0108] In some embodiments, the second therapeutic agent comprises an agent selected from SEQ ID NOs: 50-51.

[0109] In some embodiments, the second therapeutic agent has GLP-1 (glucagon-like peptide-1) receptor agonist activity. In some embodiments, the second therapeutic agent with GLP-1 receptor agonist activity is an antibody, a small molecule, a protein, a polypeptide, a peptide, or an aptamer. In some embodiments, the second therapeutic agent having GLP-1 receptor agonist activity is selected from exenatide, exenatide extended-release, dulaglutide, liraglutide, lixisenatide, semaglutide, cotadutide, neuglutide, oxyntomodulin, mazdutide, retatortide, tirzepatide, albiglutide, veinaglutide PEG-loxenatide, pembidutide, albiglutide, extenzin-4, fluvodutide, pembidutide, taspoglutide, efpegluatide, rotigliplon, danugliplon, brorenatide, dapiglutide, cinchonine, efosipegtrazide, efinopeglutide, econoglutide, orforgliplon, rotigliplon, and danugliplon.

[0110] In some embodiments, the second therapeutic agent is a compound of the formula [ka] or a pharmaceutically acceptable salt thereof, wherein R1 is a 5-10-membered aryl group containing 1-2 heteroatoms selected from -C(=O)(OZ1), -P(=O)(X)(Y), and N, O, and S optionally substituted with 1-2 R7 independently selected from halogen, C1-6 alkyl, haloC1-6 alkyl, haloC1-6 alkoxy, -OR5, C3-10 cycloalkyl, C6-10 aryl, 5-10-membered heteroaryl, and 5-10-membered heterocyclyl. heteroaryl, wherein R2 is selected from the group consisting of -C(=O)(OZ2), -P(=O)(X)(Y), and a 5-10 membered heteroaryl containing 1-2 heteroatoms selected from N, O, and S optionally substituted with 1-2 R7 independently selected from halogen, C1-6 alkyl, haloC1-6 alkyl, haloC1-6 alkoxy, -OR5, C3-10 cycloalkyl, C6-10 aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl; each R7 is independently selected from the group consisting of halogen, C1-6 alkyl, haloC1-6 alkyl, haloC1-6 alkoxy, C1-6 alkoxy, C3-10 cycloalkyl, C6-10 aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl; X and Y are each independently selected from the group consisting of -OR4, NR5R6, C1-6 alkyl, and haloC1-6 alkyl; each R4 is independently selected from the group consisting of hydrogen, C1-6 alkyl, each R is independently hydrogen or C alkyl; each R is independently hydrogen or C alkyl; and Z and Z are each independently selected from the group consisting of hydrogen, C alkyl, haloC alkyl, haloC alkoxy, C alkoxy, C cycloalkyl, and C aryl. In some embodiments, at least one of Z and Z is not hydrogen.

[0111] In some embodiments, the second therapeutic agent is a compound of the formula [ka] or a pharmaceutically acceptable salt thereof, wherein: Aib is 2-aniinoisobutynic acid; and each instance of J1, J2, and J3 is independently an amino acid selected from Aib, naturally occurring amino acids, and unnatural amino acids; U1 is -(J4)n1-(J5)n2-(J6)n3-(J7)n4-, U2 is -(J8)n5-(J9)n6-(J10)n7-(J11)n8-, each instance of J4, J5, J6, J7, J8, J9, J10, and J11 is independently a naturally occurring amino acid or a non-natural amino acid; and each of n1, n2, n3, n4, n5, n6, n7, and n8 is independently 0 or 1, with the proviso that the sum n1 + n2 + n3 + n4 + n5 + n6 + n7 + n8 is 4; R1 is selected from the group consisting of -C(=O)(OZ1), -P(=O)(X)(Y) and a 5-10 membered heteroaryl containing 1-2 heteroatoms selected from N, O and S, wherein the heteroaryl is optionally substituted with 1-2 R7 independently selected from halogen, C1-6 alkyl, haloC1-6 alkyl, haloC1-6 alkoxy, OR5, C3-10 cycloalkyl, C6-10 aryl, a 5-10 membered heteroaryl and a 5-10 membered heterocyclyl; and R2 is -C(=O)(OZ2), -P(=O)(X)(Y) and a 5-10 membered heteroaryl containing 1-2 heteroatoms selected from N, O and S. 10-membered heteroaryl, wherein the heteroaryl is optionally substituted with 1-2 R7 independently selected from halogen, C1-6 alkyl, haloC1-6 alkyl, haloC1-6 alkoxy, -OR3, C3-10 cycloalkyl, C6-10 aryl, 5-10-membered heteroaryl, and 5-10-membered heterocyclyl, and each Rz is independently selected from the group consisting of halogen, C1-6 alkyl, haloC1-6 alkyl, haloC1-6 alkoxy, C1-6 alkoxy, C3-10 cycloalkyl, C6-10 aryl, 5-10-membered heteroaryl, and 5-10-membered heterocyclyl; X and Y are each independently selected from the group consisting of -OR, NR, R, C alkyl, and haloC alkyl; each R is independently selected from the group consisting of hydrogen, C alkyl, haloC alkyl, C aryl, and C aryl alkyl; each R is independently hydrogen or C alkyl; and each R is independently hydrogen or C alkyl; Z1 and Z2 are each independently selected from the group consisting of hydrogen, C1-6 alkyl, haloC1-6 alkyl, haloC1-6 alkoxy, C1-6 alkoxy, C3-10 cycloalkyl, and C6-10 aryl.

[0112] In some embodiments, the second therapeutic agent is a compound of the formula YX1EGTFTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS, Including, During the ceremony, X1 is Aib, X2 is Aib, K at position 20 is chemically modified by conjugation of ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)a-CO—(CH2)b-CO2H, where a is 1-2 and b is 10-20, to the ε-amino group of the K side chain; X3 is Phe or 1-Nal; The C-terminal amino acid is optionally amidated as a C-terminal primary amide.

[0113] "Aib" is alpha-aminoisobutyric acid, and "1-Nal" is 1-naphthylalanine.

[0114] In some embodiments, the second therapeutic agent having GLP-1 receptor agonist activity is selected from SEQ ID NOs: 52-56.

[0115] In some embodiments, the second therapeutic agent is an agent that exhibits one or more of amylin receptor agonist activity, GLP-1 receptor agonist activity, and glucagon receptor agonist activity.

[0116] In some embodiments, the second therapeutic agent having GLP-1 receptor agonist activity is a dual agonist of a GLP-1 receptor agonist and a GIP receptor agonist. In some embodiments, the second therapeutic agent having GLP-1 receptor agonist activity is a dual agonist of a GLP-1 receptor agonist and a GCG receptor agonist. In some embodiments, the second therapeutic agent comprises a dual agonist of the GLP-1 and GIP receptors (GLP1R / GIPR). Examples of dual agonists of the GLP-1 and GIP receptors include the compounds set forth in SEQ ID NO: 53 or SEQ ID NO: 54.

[0117] In some embodiments, the second therapeutic agent having GLP-1 receptor agonist activity is a triagonist of the GLP-1, GIP (glucose-dependent insulinotropic polypeptide), and glucagon receptors (GIPR / GLP1R / GCGR). Examples of second therapeutic agents that are triagonists of the GIP / GLP-1 / glucagon receptor include the compounds set forth in SEQ ID NO:55 or SEQ ID NO:56.

[0118] In some embodiments, the second therapeutic agent has glucagon receptor agonist activity. Exemplary agents include glucagon or glucagon analogs. An example of an agent with glucagon receptor agonist activity is SEQ ID NO: 57.

[0119] In another aspect, the present disclosure provides a method for treating a disease or disorder associated with or mediated by leptin deficiency, leptin resistance, or otherwise treatable by stimulating LEPR, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or composition of the present disclosure.

[0120] As used herein, "treatment" or "treating" refers to any process that may slow, control, retard, or halt the progression of a disorder or disease symptom disclosed herein, but does not necessarily indicate the complete disappearance of all disorder or disease symptoms. Treatment includes the administration of a protein or nucleic acid or vector or composition to treat a disease or disorder in a patient, particularly a human.

[0121] As used herein, a "therapeutically effective amount" refers to the amount of a compound or composition needed to achieve a desired result.

[0122] In one embodiment, the present disclosure provides a method of treating a disorder or disease associated with a signaling-deficient LEPR, including a LEPR with an A409E mutation, by administering an antibody or composition of the present disclosure to a subject in need thereof.

[0123] In some embodiments, the present disclosure provides methods of treating a disease or disorder associated with or mediated by leptin deficiency by administering an antibody or composition of the present disclosure to a subject in need thereof, wherein the disease or disorder comprises a lipodystrophic disorder, examples of which include congenital generalized lipodystrophy, acquired generalized lipodystrophy, familial partial lipodystrophy, acquired partial lipodystrophy, centrifugal abdominal lipodystrophy, cyclic lipodystrophy, and focal lipodystrophy.

[0124] In one embodiment, the present disclosure provides a method of inducing weight loss, weight control, chronic weight control, or treating obesity by administering to a subject an antibody or composition of the present disclosure, the method comprising administering to a subject in need thereof an antibody or composition of the present disclosure.

[0125] As used herein, the term "obesity" refers to a disease or disorder associated with excess body fat that increases the risk of health problems. In one embodiment, the term "obese" includes adult subjects with a body mass index (BMI) of 30 kg / m or greater. In another embodiment, the term "obese" includes adult subjects with a BMI of 25 kg / m or greater. "Chronic weight management" refers to the desired loss of weight or maintenance of weight.

[0126] In one embodiment, the present disclosure provides a method for chronic weight management in a subject in need thereof, wherein the subject has obesity or is overweight, wherein the method of chronic weight management comprises administering to a subject in need thereof an antibody or composition of the present disclosure.

[0127] In one embodiment, the term "overweight" includes adult subjects with a BMI of 27 kg / m or greater. In another embodiment, the term "overweight" includes adult subjects with a BMI of 23 kg / m or greater. In another embodiment, "overweight" includes adult subjects with a BMI of 23-30, 23-27, 25-30, 27-30, 23-25, or 25-27 kg / m.

[0128] In one embodiment, "chronic weight management" refers to a method of maintaining weight loss as an adjunct to a reduced calorie diet and increased physical activity in a person currently or previously characterized as having obesity or who is overweight, wherein the method of chronic weight management comprises administering to a subject in need thereof an antibody or composition of the disclosure.

[0129] In one embodiment, the disclosure provides a method of inducing weight loss in a subject having a BMI of 23-30, 23-27, 25-30, 27-30, 23-25, or 25-27 kg / m2, the method comprising administering to the subject an antibody or composition of the disclosure.

[0130] The antibodies or compositions of the present disclosure can be administered as monotherapy or as combination therapy. In one embodiment, the combination therapy comprises simultaneous, separate, or sequential combination of an antibody disclosed herein with a second therapeutic agent to induce weight loss, weight control, chronic weight control, or treat obesity. In one embodiment, the combination therapy comprises simultaneous, separate, or sequential combination of an antibody disclosed herein with a second therapeutic agent to treat a disease or disorder associated with or mediated by leptin deficiency, leptin resistance, or a disease or disorder otherwise treatable by stimulating LEPR.

[0131] Disclosed herein are second therapeutic agents administered in combination with a LEPR agonist antibody. In some embodiments, the second therapeutic agent comprises a therapeutic agent that restores leptin sensitivity.

[0132] In one embodiment, the disclosure provides an antibody or composition of the disclosure for use in therapy, chronic weight management, or treatment of obesity.

[0133] In one embodiment, the disclosure provides for the use of an antibody of an antibody disclosed herein in the manufacture of a medicament for the treatment, chronic weight management, or treatment of obesity.

[0134] In some embodiments, the second therapeutic agent is an agent that exhibits one or more of amylin receptor agonist activity, GLP-1 receptor agonist activity, GIP receptor agonist activity, and glucagon receptor agonist activity. Exemplary second therapeutic agents are set forth in SEQ ID NOS: 39-57.

[0135] As used herein, the words "a," "an," and "the" mean "one or more."

[0136] As used herein, an antibody, small molecule, protein, peptide, or aptamer of the disclosure includes the respective free base form and pharmaceutically acceptable salts thereof. [Example]

[0137] Example 1. Antibody Expression and Purification Antibodies A, B, C, and D were each produced in a mammalian cell expression system using a CHOK1 cell derivative (Lonza Biologics Inc.). The cDNA sequences encoding the antibodies were subcloned into a GS-containing expression plasmid backbone (pEE12.4-based plasmid; Lonza Biologics Inc.).

[0138] [Table 2]

[0139] The cDNA sequence was fused in frame to the coding sequence of the signal peptide sequence METDTLLLWVLLLWVPGSTG (SEQ ID NO: 20) to enhance secretion of the antibody into tissue culture medium. Expression was driven by the viral CMV promoter.

[0140] To produce antibodies by transient transfection, CHOK1-derived cells were cultured in a bioreactor to a density of 35-50 e6 vc / mL. A PEI-based method was used to transfect the cells with an equal stoichiometric ratio of recombinant expression plasmids. Briefly, an appropriate volume of CHOK1-derived suspension cells at a density of 20 e6 cells / mL was transferred to a shake flask, and both PEI and recombinant plasmid DNA were added to the cells. The cells were incubated in suspension culture at 32 °C for 7 days. At the end of the production period, the cells were removed by centrifugation, and the antibody was purified from the clarified medium.

[0141] Alternatively, to generate antibodies via stable transfection, CHOK1-derived cells were stably transfected using a lipid-based transfection reagent and an appropriate amount of recombinant expression plasmid. The transfected cells were maintained in suspension culture at an appropriate cell density. Selection of transfected cells was achieved by growth in chemically defined, serum-free medium containing 25 μM MSX and incubated at 32–37°C and 5–7% CO2. The cells were then removed by centrifugation, and the antibody was purified from the clarified medium.

[0142] Antibodies were secreted from CHO cells into the culture medium and subsequently purified by Protein A affinity chromatography followed by cation exchange chromatography. Specifically, antibodies from the collected culture medium were captured on MabSelect PrismA Protein A resin (Cytiva). The resin was then briefly washed with a wash buffer, such as phosphate-buffered saline (PBS, pH 7.4) or a Tris-containing buffer, to remove nonspecifically bound material. The protein was eluted from the resin with a low pH solution, such as 10 mM citric acid, pH 3. Fractions containing the antibody were pooled. The pH can then be adjusted to approximately 5 by adding a base, such as 3 M sodium acetate, pH 5.0. The antibody can be further purified by ion exchange chromatography using a resin such as POROS 50 HS (ThermoFisher). The antibody can be eluted from the column using a 0 to 1 M NaCl gradient in 20 mM sodium acetate, pH 5.0, over 20 column volumes.

[0143] The purified antibody can then be concentrated and / or buffer exchanged into phosphate buffered saline or other suitable formulation buffer by ultrafiltration / diafiltration methods.

[0144] Example 2. Binding of Antibody A, Antibody B, Antibody C, and Antibody D to Human LEPR Antibodies were generated according to Example 1 and tested for their ability to bind to human LEPR by ELISA.

[0145] A 96-well high-binding ELISA plate (Greiner catalog number 650061) was coated with 50 μL / well of human LEPR ECD (SEQ ID NO: 30) at 2 μg / mL in carbonate buffer (50 mM NaHCO3) overnight at 4°C. The plate was washed with an automated plate washer and blocked with 200 μL / well of casein (Thermo Fisher, catalog number 37528) for 1 hour at RT. The plate was then washed. The antibody was titrated, starting at 50 μg / mL in casein, serially diluted 1:3 across the plate. The plate was incubated at room temperature for 1 hour. The plate was then washed.

[0146] 50 μL / well of 1 μg / mL goat anti-human kappa AP conjugate in casein (Southern Biotech, Catalog No. 2060-04) was added to each well. The mixture was incubated at room temperature for 1 hour. The plate was then washed.

[0147] Develop with 100 μL / well PMP / AMP 0.5 mL in 15 mL water (development reagent) and read at 560 nM.

[0148] The data are shown in Figure 1. The curves show the relative binding of IgG antibodies to human LEPR. LEPR is bound to an ELISA plate and the antibodies are titrated in 3-fold increments starting at 50 μg / mL.

[0149] Summary: Figure 1 demonstrates that antibodies A, B, C, and D bind to human LEPR with varying affinities.

[0150] Example 3: Binding affinity and kinetics of antibodies A, B, and C to LEPR of various species Antibodies A, B, and C are IgG1 mAbs that bind to the leptin receptor (LEPR). Surface plasmon resonance (SPR) on a Biacore T200 (Cytiva) was used to measure the binding affinity and kinetics of antibodies A, B, and C to human, cynomolgus monkey, mouse, rat, dog, and rabbit LEPR-ECD. His-tagged ECDs were expressed and purified for each species and used for affinity analysis. This experiment utilized a Protein A / G chip (Xantec, catalog number PAGHC200M) with HBS-EP + 0.01% BSA as the running buffer. Antibodies were diluted to 1 μg / mL in running buffer and captured onto a single flow cell on the chip surface. Each LEPR-ECD species was diluted to 1000 nM in running buffer, followed by a two-fold serial dilution. Each ligand concentration was injected over all flow cells at 100 μL / min for 150 seconds, followed by a 600-second dissociation phase. Regeneration was performed by injecting 10 mM glycine pH 1.5 at 10 μL / min for 30 seconds. Data were analyzed in Biacore T200 Evaluation Software 3.1 by flow cell 1 reference subtraction and 0 nM blank subtraction. Data were globally fitted using a "1:1 binding" binding model to determine the on-rate (k) of each ligand. a ) and off-rate (k d The affinity (K D ) is K D =k d / k a The binding kinetics were calculated according to the relationship: Data are reported as the mean ± standard deviation of experimental replicates.

[0151] [Table 3]

[0152] Summary: Table 3 demonstrates that antibodies A, B, and C bind to human, cynomolgus monkey, and dog LEPR with varying affinities. Antibodies A, B, and C do not bind to mouse, rat, or rabbit LEPR with appreciable affinity.

[0153] Example 4. Anti-LEPR antibodies A, B, and C activate LEPR in several species LEPR is a cell surface single-pass transmembrane receptor. Activation of LEPR leads to phosphorylation of intracellular STAT3 (pSTAT3) and subsequent target gene transcription. To measure LEPR activation by anti-LEPR antibodies in this disclosure, five independent STAT-luciferase reporter cell lines were established by transfecting HEK293 cells with DNA plasmids encoding full-length LEPR and STAT3-luciferase reporter genes from different species (Table 4). These cell lines stably expressing full-length LEPR and STAT3-luciferase were maintained in DMEM medium (Gibco, #12430-054) containing 10% fetal bovine serum (FBS, Gibco, #10082-147), antibiotics (Gibco, #15240-062), 1x sodium pyruvate (Gibco, #113060-070), 1x GlutaMax supplement (Gibco, #35050-061), and resistance selection drugs, as shown in Table 4.

[0154] Before seeding cells for the luciferase assay, poly-D-lysine-coated 96-well plates (Corning Inc., #354651) were rinsed once with phosphate-buffered saline (PBS). 20,000–24,000 STAT3-luciferase reporter cells were then seeded onto the poly-D-lysine 96-well plates in 100 μL / well of plating medium consisting of DMEM / F12 (3:1, Gibco, #93-0152DK), 10% fetal bovine serum (FBS, Gibco, #10082-147), 1× GlutaMax supplement (Gibco, #35050061), 20 mM HEPES (Gibco, #15630-080), and antibiotics (Gibco, #15240-062). After overnight incubation at 37°C and 5% CO2, the culture medium was replaced with 75 μL of assay medium containing OPTI-MEM (Gibco, #31985-070), 1% BSA (Gibco, BSA Fraction V (7.5%), #15260-037), and 0.1% dialyzed fetal bovine serum (FBS, Gibco, #26400-044). Test articles were diluted in 25 μL of assay medium and added to each well. The cells were further incubated at 37°C with 5% CO2 for 20 hours. Cell lysates and One-Glo luciferase substrate solution (Promega, #E6120) were then added to each well, and relative luciferase units (RLU) were measured using a ClarioStar plate reader. For each reporter cell line, the positive control LEPR activation was calculated as the mean RLU stimulated by 20 nM recombinant human leptin protein (SEQ ID NO: 27) minus the mean RLU of unstimulated cells, which was taken as the maximum LEPR activation (100%).All test article-induced LEPR activations were normalized to this positive control value for the same reporter cell line.Concentration-response curves were then plotted as the percentage of maximum LEPR activation versus the log drug concentration function.

[0155] As shown in Table 5, recombinant human leptin protein (SEQ ID NO: 27) activated human, cynomolgus monkey, dog, rat, and mouse LEPR and increased EC 50The EC values ​​were 0.0491 ± 0.0044 nM, 0.6743 ± 0.1098 nM, 0.5815 ± 0.1723 nM, 6.293 ± 0.6202 nM, and 0.0788 ± 0.017 nM, respectively. Anti-LEPR antibodies A, B, and C showed EC values ​​ranging from 0.1604 nM to 0.3576 nM in HEK293-STAT3Luc-hLepR1 cells. 50 Anti-LEPR antibodies A, B, and C also activated cynomolgus monkey (Macaca fascicularis) LEPR (cynomolgus monkey LEPR) and increased EC in HEK293-STAT3Luc-cmLepR2 cells. 50 The values ​​ranged from 0.8786 nM to 2.0187 nM, with maximum LEPR activation ranging from 20.00% to 21.30%. Furthermore, anti-LEPR antibodies A, B, and C activated gray wolf (Canis lupus) LEPR (canine-LEPR) and reduced EC2 activity in HEK293-STAT3Luc-dLepR7 cells. 50 The values ​​ranged from 0.4751 nM to 0.8543 nM, with maximum LEPR activation of 76.47% to 76.70%. Anti-LEPR antibodies A, B, and C did not activate the LEPR of the house mouse (Mus musculus) or the rat (Rattus rattus) at concentrations up to 90 nM.

[0156] [Table 4]

[0157] Table 5. Potency and maximal activation of LEPR by antibodies Recombinant human leptin protein (SEQ ID NO: 27) was used to generate dose-response data and subsequent nonlinear fit curves using Prizm software. The top of the nonlinear fit curve for each receptor species was compared to the response to 20 nM recombinant human leptin protein (SEQ ID NO: 27) and expressed as percent agonism.

[0158] [Table 5]

[0159] Antibody CO is a comparative LEPR antibody having the HC sequence shown as SEQ ID NO:36 and the LC sequence shown as SEQ ID NO:37.

[0160] Summary: Antibodies A, B, and C activated human, cynomolgus monkey, and dog LEPR in a pSTAT3-luciferase reporter assay. Antibodies A, B, and C did not activate mouse or rat LEPR.

[0161] Example 5: Antibodies A, B and C did not compete with leptin for binding to LEPR. To test whether binding of anti-LEPR antibodies to LEPR competes with leptin-LEPR interaction, an ELISA-based competitive binding assay was performed.

[0162] Recombinant LEPR extracellular domain (SEQ ID NO: 30) fused to hIgG1-Fc (LEPR-Fc) was diluted in phosphate buffer (PBS, Gibco, #20012-027) and applied to a 96-well Costar assay plate (Corning, #3690) at 5 μg / mL and 50 μL / well. The solution was incubated at 4°C for 20 hours to allow for sufficient protein coating. The plate was then rinsed three times with 160 μL / well of 1x wash buffer (R&D, #WA126, 25x), blocked with casein blocking solution (ThermoFisher, #37582) for 1 hour at room temperature, and rinsed twice with wash buffer. A series of concentrations of anti-LEPR antibodies (antibodies A, B, and C) were then diluted in 60 μL of casein blocking solution and added to each well. Serial concentrations of LEPR-Fc (SEQ ID NO: 30) were used as a positive control to compete with the plate-bound receptor for leptin. Casein blocking solution was used as a negative control. Plates were incubated with either anti-LEPR antibody, positive control, or negative control for 90 minutes at room temperature. Next, 20 μL of recombinant human leptin protein (R&D, #398-LP-05M) diluted in casein blocking buffer was added to each plate to a final concentration of 2 nM. The plates were then mixed with gentle shaking for 30 seconds, followed by an additional 90-minute incubation at room temperature. The plates were rinsed three times with wash buffer. 50 μL / well of 0.5 μg / mL biotinylated anti-hLeptin antibody (Abcam, #AB271278) diluted in casein blocking solution was added to each well and incubated for 1 hour at room temperature. The plates were then washed three times with wash buffer. 50 μL of detection antibody streptavidin-HRP (Life Technologies, #SNN2004), diluted 1:5,000 in casein blocking solution, was applied to each well and incubated at room temperature for 20 minutes. The plate was then washed three times, and 50 μL of 1-Step-Ultra TMB-ELISA substrate (ThermoFischer, #34029) was added to each well and incubated at room temperature for 5 minutes. The reaction was stopped by adding 25 μL of stop solution (R&D, #DY994) to each well.Data were obtained using a microplate reader with absorbance set at 450 nm (OD450). The average OD450 reading in the casein-coated wells (without LEPR-Fc (SEQ ID NO: 30) coating) was subtracted from all raw OD450 readings to generate normalized OD450 readings. The average normalized OD450 reading from the negative control (2 nM leptin-incubated wells without competitor) was taken as 100 percent (100%) binding. All normalized OD450 readings were compared to this OD450 reading and expressed as a percentage of binding. GraphPad Prism software was used to plot the top and bottom of the dose-response curves and IC. 50 As shown in Table 6 and Figure 2, recombinant hLEPR-TEV-hIgG1Fc competed with leptin with an IC of 2.575 μg / mL. 50 The leptin-LEPR antibodies bound to the LEPR on the plate. None of the anti-LEPR antibodies (A, B, and C) competed with the leptin-LEPR interaction.

[0163] [Table 6]

[0164] Figure 2 shows a competitive binding assay of antibodies to LEPR and leptin, showing the binding of exemplary antibodies of the present disclosure to hLepR in the presence of leptin protein as a percentage of the control. Summary: Antibodies A, B, and C did not compete with recombinant leptin protein for binding to its receptor LEPR in an ELISA binding assay.

[0165] Example 6. Cell surface LEPR binding of antibodies A, B, and C To test whether antibodies A, B, and C bind to human and cynomolgus monkey LEPR on live cells, flow cytometry was used to measure cell surface-bound antibodies with a fluorescently labeled secondary anti-human IgG. To ensure the specificity of antibodies A, B, and C binding to LEPR, each run included an isotype IgG control, a secondary-only control, and two channel compensation controls. The aforementioned controls are well known in the art. For example, in the case of the IgG control, it is a non-binding IgG containing an Fc mutation that eliminates FcγR binding. The IgG control does not contain the YTE mutation.

[0166] Binding of antibodies A, B, and C to human and cynomolgus monkey LEPR on the surface of live cells was assessed in HEK293-STAT3Luc-hLepR1 and HEK293-STAT3Luc-cmLepR2 cells (cell lines used are listed in Table 4). Exponential-phase cells were dissociated with 1x Accutase™ (Invitrogen, #00-4555-56) according to the manufacturer's guidelines and strained by gravity flow onto a 40 μm cell strainer (Fisherbrand, #22363547). Cells were counted, pelleted, and resuspended in 0.2–0.26 ml of blocking buffer containing PBS (Gibco, #20012027), 0.3% BSA (Gibco, #15260-037), 4 mM EDTA, 0.01% sodium azide (Sigma, #S202-100G), 25 μg / mL human Fc block (BD, #564219), and 100 μg / mL goat gamma globulin (JacksonImmuno, #005-000-002). e6 The cells were resuspended in 20 μL per cell. The cells were incubated in blocking buffer at room temperature for 10 minutes. After rinsing once with PBS, the cells were transferred to a U-bottom 96-well plate in 200 μL and 0.2-0.26 μL of PBS. e6Cells were transferred at 1000 x g / well. ArC™ Viability beads (Invitrogen, #A10346) and UltraComp eBeads Plus™ (ThermoFisher, #01-333-342) were added to assess cell viability and allow for compensation of fluorophore emission spectra. Cells were pelleted and stained with 30 μL / well of LIVE / DEAD™ Zombie Violet diluted 1:500 in PBS stain (BioLegend, #423113) for 10 minutes at room temperature. Stained cells were washed with flow buffer consisting of 1x PBS (Gibco, #20012027), 0.3% BSA (Gibco, #15260-037), 4 mM EDTA, and 0.01% sodium azide (Sigma, #S202-100G). Cells were then incubated with 30 μL / well of antibody diluted in flow buffer at 1000 nM as the high dose, followed by a 9-point dose response including 5-fold dilutions (total of 9 concentrations for each antibody) for 30 minutes at 4° C. in the dark. After rinsing three times with flow buffer, cells were incubated with 30 μL / well of AlexaFluor® 647-F(ab')2 goat anti-human IgG, Fcγ (JacksonImmuno, #109-606-170) 1:1000 diluted in cold flow buffer for 20 minutes at 4° C. Cells were resuspended in 120 μL / well of cold flow buffer and subjected to flow cytometry on a BD Biosciences LSRFortessa X20 flow cytometer for acquisition in the following channels: FSC, SSC, BV421 (purple 405 nm 450 / 50), and APC / AF647 (red 633 nm 670 / 30), with a limit of 30,000 events per well. Sample raw data was compensated and processed using FlowJo v10 software, and EC values ​​were calculated for each antibody using GraphPad Prism nonlinear regression asymmetric sigmoidal curves (plotting concentration vs. gMFI). 50 This assay, which included both human and cynomolgus LEPR cell lines, was performed a total of four times on four different days, and the means are shown in Tables 7 and 8.

[0167] [Table 7]

[0168] [Table 8]

[0169] Summary: The data in Tables 7 and 8 demonstrate that antibodies A, B, and C bind to cell surface human and cynomolgus LEPR with varying affinities.

[0170] Example 7. Binding of Antibody A to Fcγ Receptors To determine whether antibody Fc affects the Fcγ receptor binding properties of Antibody A, binding to the human FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa receptor extracellular domains (ECDs) was measured by SPR at 25°C. Antibody A and Antibody A-IgG1 share the same CDRs. Antibody A differs from Antibody A-IgG1 by having L234A, L235A, D265S, M252Y, S254T, and T256E mutations in the heavy chain (HC). An IgG1 positive control and an IgG1 non-binding control were used to confirm the suitability of the assay.

[0171] SPR analysis of antibody binding was performed using a Biacore T200 instrument (Cytiva) with Biacore reagents and Scrubber2 Biacore evaluation software (Biologics 2008). A Series-S CM5 chip (Cytiva P / N BR-1005-30) was prepared using the manufacturer's EDC / NHS amine coupling method (Cytiva P / N BR-1000-50). Briefly, the surfaces of all four flow cells were activated by injecting a 1:1 mixture of EDC / NHS at 10 μL / min for 7 minutes. Protein A (Calbiochem P / N 539202) was diluted to 100 μg / mL in 10 mM acetate, pH 4.5 buffer, and immobilized to approximately 4000 RU on all four flow cells by injecting at a flow rate of 10 μL / min for 7 minutes. Unreacted sites were blocked with a 7-minute injection of ethanolamine at 10 μL / min. Non-covalently bound proteins were removed using 2×10 μL injections of glycine, pH 1.5.

[0172] FcγR ECD-FcγRI (CD64), FcγRIIA_131R, and FcγRIIA_131H (CD32a), FcγRIIIA_158V, FcγRIIIA_158F (CD16a), and FcγRIIb (CD32b, inhibitory receptor) (see, e.g., Bruhns et al., Blood. 2009 Apr 16;113(16):3716-25) were produced from stable CHO cell expression according to methods well known in the art and purified using IgG Sepharose and size exclusion chromatography.

[0173] For FcγRI binding, antibodies were diluted to 2.5 μg / mL in running buffer (1x HBS-EP+, Teknova P / N H8022), and approximately 150 RU of each antibody was captured on flow cells 2–4 (RU capture). FC1 was the reference flow cell; therefore, no antibody was captured on FC1. FcγRI ECD was diluted to 200 nM in running buffer and then serially diluted 2-fold in running buffer to 0.78 nM. Duplicate injections of each concentration were injected for 120 seconds at 40 μL / min on all FCs, followed by a 1200-second dissociation phase. Regeneration was performed by injecting 15 μL of 10 mM glycine, pH 1.5, at 30 μL / min on all FCs. Reference-subtracted data were collected as FC2-FC1, FC3-FC1, and FC4-FC1. Measurements were performed at 25°C. Affinity (K D ) were calculated using either steady-state equilibrium analysis with Scrubber 2 Biacore® evaluation software or the "1:1 (Langmuir) binding" model of BIA evaluation.

[0174] For FcγRIIa, FcγRIIb, and FcγRIIIa binding, antibodies were diluted to 5 μg / mL in running buffer, and approximately 500 RU were captured on flow cells 2–4 (RU capture). FC1 was the reference flow cell. Fcγ receptor ECD was diluted to 10 μM in running buffer and then serially diluted 2-fold to 39 nM in running buffer. Duplicate injections of each concentration were injected into all FCs at 40 μL / min for 60 s, followed by a 120 s dissociation phase. Regeneration was performed by injecting 15 μL of 10 mM glycine, pH 1.5, into all FCs at 30 μL / min.

[0175] Reference-subtracted data were collected as FC2-FC1, FC3-FC1, and FC4-FC1. Measurements were taken at 25°C. Affinity (K D ) was calculated using steady-state equilibrium analysis with Scrubber2 Biacore® evaluation software.

[0176] Following procedures essentially as described above, the following data, as shown in Table 9, was obtained.

[0177] [Table 9] Assays were performed three times independently. * Standard deviations were not determined for measurements >10 μM.

[0178] Summary: SPR binding data for Antibody A and various controls to FcγRI, FcγRIIA_131H, FcγRIIA_131R, FcγRIIb, FcγRIIIA_158V and FcγRIIIA_158F. Antibody A shows no detectable binding to Fcγ receptors.

[0179] Example 8. Antibodies A, B, and C did not desensitize LEPR signaling in vitro. Activation of LEPR by leptin leads to its internalization from the cell surface. As a result, LEPR becomes unavailable to extracellular ligands and becomes unresponsive to subsequent ligand stimulation. The lack of response to repeated agonist exposure is called receptor desensitization. To test whether antibodies A, B, and C desensitize LEPR, we measured the phosphorylation of STAT3 protein in HEK293-STAT3Luc-hLepR1 cells following an exposure, washout, and re-exposure protocol.

[0180] HEK293-STAT3Luc-hLepR1 cells were seeded into poly-D-lysine 96-well plates (approximately 80,000 cells / well) in growth medium consisting of DMEM (Gibco, #2430-054), 10% fetal bovine serum (FBS, Gibco, #10082-147), 1x sodium pyruvate (Gibco, #113060-070, 100x), 1x GlutaMAX supplement (Gibco, #3Ag5050-061), and antibiotics (Gibco, #15240-062). After overnight incubation at 37°C and 5% CO2, the culture medium was removed and the cells were washed once with serum-free medium. Then, 50 μL / well serum-free medium (DMEM / F12, Gibco, #93-0152DK; 0.3% BSA, BSA Fraction V (7.5%), (Gibco, #15260-037) was added. The cells were incubated in 50 μL of serum-free medium for 1 hour. The cells were then divided into a control group and an antibody exposure group. In the negative control group, 50 μL of serum-free medium was added to each well. In the antibody exposure group, anti-LEPR antibodies were diluted with 50 μL of serum-free medium and added to the cells to reach a final concentration of 30 nM for each antibody. The 30 nM concentration was determined based on the EC20 concentration of all anti-LEPR antibodies in this disclosure. 99Because the concentration exceeded the saturating amount of anti-LEPR antibody, cells were exposed to saturating amounts of the anti-LEPR antibody, reaching maximum LEPR activation by the antibody during exposure. Cells from all groups were then incubated at 37°C and 5% CO2 for 1 hour to allow for LEPR activation and potential internalization induced by the anti-LEPR antibody. After 1 hour of incubation, antibody-exposed cells and control-exposed cells were rinsed three times with 150 μL / well of serum-free medium for 10 minutes each (at 37°C and 5% CO2). Cells were then given growth medium (50 μL / well) and allowed to recover for 3 hours at 37°C and 5% CO2. After washing once with serum-free medium, the growth medium was replaced with serum-free medium (50 μL / well), and the cells were incubated for an additional hour at 37°C and 5% CO2. Serial dilutions of each anti-LEPR antibody in the present disclosure were then added to cells previously exposed to the same antibody or control-treated cells to allow for LEPR activation upon antibody re-exposure. After 15 minutes of incubation, cells were lysed with 50 μL / well of lysis buffer (PerkinElmer, #ALSU-pST3-A10K) and mixed by shaking at room temperature for 10 minutes. Phosphorylated STAT3 (pSTAT3) was measured using the AlphaLISA-SureFire-Ultra p-Stat (Tyr705) Assay Kit (PerkinElmer, #ALSU-pST3-A10K) according to the manufacturer's protocol. The average pSTAT3α signal from cells treated with 30 nM recombinant human leptin protein (SEQ ID NO: 27) was subtracted by the average pSTAT3α signal from the negative control, which was set as 100% and defined as the maximum STAT3 phosphorylation. The pSTAT3α signal readings of the test article treatments were then normalized to the maximum STAT3 phosphorylation value. Concentration-response curves were then plotted as the percentage of pSTAT3α signal as a function of the log drug concentration for each antibody.

[0181] As shown in Table 10, anti-LEPR antibody treatment resulted in very similar concentration-response curves in cells pre-exposed to antibody compared to vehicle-exposed cells. Antibody A had an EC of 0.3309 nM. 50and an EC of 0.3601 nM compared to pSTAT3 induction by Antibody A in vehicle-exposed cells with a maximal activation of 68.93%. 50 Antibody A induced pSTAT3 signaling in pre-exposed and washed-out cells with a maximal activation of 71.92%. Similarly, Antibody B induced pSTAT3 signaling in pre-exposed and washed-out cells with an EC of 0.9862 nM. 50 and pSTAT3 induction by antibody B in vehicle-exposed cells with a maximal activation of 70.48%. 50 Antibody B induced pSTAT3 signaling in pre-exposed and washed-out cells with an EC of 1.136 nM, compared to pSTAT3 induction by Antibody C in vehicle-exposed cells with an EC of 1.038 nM and 71.69% maximal activation. 50 and induced pSTAT3 signaling in antibody C pre-exposed and washed-out cells with a maximum activation of 73.63%.

[0182] [Table 10]

[0183] Summary: Pre-exposure of LEPR-expressing HEK293 cells to 30 nM of antibody A, B, or C for 1 hour followed by a 3-hour washout did not affect the subsequent concentration-response curves of antibody A, B, or C. These data demonstrated that anti-LEPR antibodies A, B, and C do not desensitize the human LEPR signaling pathway in vitro.

[0184] Example 9. Activation of signaling-deficient LEPR by antibodies A, B, and C The missense mutation A409E in the LEPR gene was associated with autosomal recessive early-onset obesity in humans (Farooqi et al., 2007, N Engl J Med 356(3):237-247). Mutant human LEPR-A409E receptors did not respond to leptin stimulation. To test whether anti-LEPR antibodies could activate these mutant human LEPR receptors, we established a STAT3-luciferase reporter assay using HEK293 cells expressing hLEPR-A409E. These cells were cultured in growth medium DMEM / F12 (Gibco, #12430-054), 10% fetal bovine serum (FBS, Gibco, #10082-147), antibiotics (Gibco, #15240-062), sodium pyruvate (Gibco, #11360-070), and GlutaMax supplement (Gibco, #35050-061) in the presence of drug selection 250 μg / mL hygromycin (Invitrogen, #10687010) and 800 μg / mL Geneticin (Corning, #30-234-Cl).

[0185] Before seeding cells for the luciferase assay, poly-D-lysine-coated 96-well plates (Corning Inc., BioCoat, #354651, poly-D-dissolving coating) were rinsed once with phosphate-buffered saline (PBS) and once with plating medium. 20,000–24,000 STAT3-luciferase reporter cells were then seeded onto the poly-D-lysine 96-well plates in 100 μL / well of plating medium consisting of DMEM / F12 (3:1, Gibco, #93-0152DK), 10% fetal bovine serum (FBS, Gibco, #10082-147), 1× GlutaMax supplement (Gibco, #35050061), 20 mM HEPES (Gibco, #15630-080), and antibiotics (Gibco, #15240-062). After overnight incubation at 37°C and 5% CO2, the culture medium was replaced with 75 μL assay medium containing OPTI-MEM (Gibco, #31985-070) with 1% BSA (BSA Fraction V (7.5%), Gibco, #15260-037), 0.1% dialyzed fetal bovine serum (FBS, Gibco, #26400-044) for 1 hour at 37°C and 5% CO2. Test articles were diluted in 25 μL assay medium and added to each well. Cells were further incubated at 37°C with 5% CO2 for 20 hours. Cell lysate and One-Glo luciferase substrate solution (Promega, #E6120) were then added to each well, and relative luciferase units (RLU) were measured using a ClarioStar plate reader. Concentration-response curves were then plotted as raw RLU readings versus the log drug concentration. EC 50 was calculated using GraphPad Prism software.

[0186] As shown in Table 11, antibodies A, B, and C had EC values ​​of 0.08811 nM, 0.272 nM, and 0.2532 nM, respectively. 50activated mutant LEPR-A409E. In contrast, recombinant human leptin protein (SEQ ID NO: 27) did not activate LEPR-A409E. These results suggest that the anti-LEPR antibodies of the present disclosure may be applied to treat patients with rare missense mutations in the LEPR gene.

[0187] [Table 11] * Upper and EC of the curve of SEQ ID NO: 27 50 The results were projected by GraphPad Prism software. 50 The values ​​demonstrated that SEQ ID NO:27 was inactive in this assay.

[0188] Summary: Antibodies A, B, and C activated the signaling-impaired mutant LEPR-A409E. In contrast, recombinant human leptin protein (SEQ ID NO: 27) did not activate the signaling-deficient mutant LEPR-A409E.

[0189] Example 10. Antibodies A, B, and C induced pERK in human LEPR-expressing HEK293 cells. Antibodies were tested for their ability to induce extracellular signal-related kinase (pERK) phosphorylation in HEK293-STAT3Luc-hLepR1 cells. To measure pERK induction, cells were first cultured overnight at 37°C in 96-well plates containing DMEM medium supplemented with 10% FBS. On day 2, the medium was removed, and the cells were washed in serum-free DMEM and incubated for an additional 24 hours in serum-free DMEM. On day 3, the medium was removed, and 1:4 serial dilutions of antibodies A, B, and C at a maximum concentration of 200 nM in serum-free DMEM were added to the cells for 15 minutes. Tests were performed in duplicate. Recombinant human leptin protein (SEQ ID NO: 27) was used as a positive control in this assay. IgG1 with Fc modifications to abolish binding to Fcγ receptors was used as a negative control (IgG isotype control). Phospho-ERK1 / 2 in whole cell lysates was assessed by measuring amplified luminescence using the AlphaLISA SureFire Ultra p-ERK1 / 2 (Thr202 / Tyr204) kit (PerkinElmer, #ALSU-PERK-A-HV). Data were calculated using a four-parameter sigmoidal fit of the data (SigmaPlot software). Results, presented in Table 12, represent the average EC of two independent experiments. 50 is.

[0190] [Table 12]

[0191] Summary: Antibodies A, B, and C induced ERK phosphorylation in human LEPR-expressing HEK293 cells.

[0192] Example 11. Complement component C1q binding of antibody A by ELISA A 96-well microplate was coated with 100 μL / well of each antibody diluted in DPBS (Dulbecco's HyClone) at concentrations ranging from 10 μg / mL to 0.19 μg / mL. Tests were performed in duplicate wells. The plate was sealed and incubated overnight at 4°C. The coating reagent was removed from each well, and 200 μL / well of casein blocking reagent (Thermo) was added. The plate was sealed and incubated for 2 hours at room temperature (RT). Each well was washed three times with wash buffer (1x TBE containing 0.05% Tween 20). 100 microliters / well of 10 μg / mL human C1q (MS Biomedical) diluted in casein blocking reagent was added, and the plate was incubated for 3 hours at room temperature. The plate was then washed three times with wash buffer, after which 100 μL / well of a 1:800 dilution of sheep anti-human C1q-HRP (Abcam #ab46191) in casein blocker was added and incubated for 1 hour at room temperature. The plate was washed six times with wash buffer, and 100 μL / well of TMB substrate (Pierce) was added to each well and incubated for 7 minutes. The reaction was stopped by adding 100 microliters of 1N HCl to each well. The optical density was immediately measured using a colorimetric microplate reader set at 450 nm.

[0193] Figure 3 shows C1q binding to Antibody A. Results (mean ± SD) are shown for one of three experiments performed. (A) is an IgG1 isotype positive control, (B) is an IgG1 isotype non-binding control used to verify assay suitability, (C) is Antibody A, and (D) is an Antibody A-IgG1 control. Curve fit: 4-parameter logistic in GraphPad Prism. Conditions B and C are overlapping and essentially at baseline.

[0194] Summary: Antibody A does not bind complement component C1q by ELISA. Related control molecules and Antibody A-IgG1 functioned as expected.

[0195] Example 12. Relative FcRn binding of antibody A by FcRn-HPLC IgG binding to the neonatal Fc receptor (FcRn) is an important mechanism for antibody recycling and a determinant of antibody pharmacokinetics (PK). Modifications of the Fc region of antibodies have been shown to modulate antibody PK. Antibody A is an IgG1 mAb with L234A, L235A, and D265S mutations to reduce FcγR and C1q binding, and M252Y, S254T, and T256E mutations to modulate the antibody's clearance profile. The effects of these combined mutation sets were assessed by FcRn-HPLC. Stock antibody solutions were diluted to 1 mg / mL in 20 mM MES, 140 mM NaCl, pH 5.5. An Agilent 1290 HPLC equipped with an FcRn Affinity Column Gen2 (Roche, catalog number 09430857001) was pre-equilibrated with 20 mM MES, 140 mM NaCl, pH 5.5 at a flow rate of 0.2 mL / min. After a 20 μL antibody injection, the column was washed with 20 mM MES, 140 mM NaCl, pH 5.5 for 10 minutes. The antibody was then eluted by a pH gradient elution to 20 mM Tris, 140 mM NaCl, pH 8.8 over 90 minutes. Chromatograms were blank-subtracted to remove baseline drift. The retention times and widths of the major peaks at 50% peak height are shown in Table 13.

[0196] Briakinumab is an IgG1 control with impaired PK due to ineffective FcRn recycling, whereas ustekinumab displays an FcRn profile typical of IgG. (Schoch A et al. Charge-mediated influence of the antibody variable domain on FcRn-dependent pharmacokinetics. Proc Natl Acad Sci US A. 2015 May 12;112(19):5997-6002.) A panel of comparative antibodies with matched VH and VL domains were generated for this study, differing only in their Fc regions: 1) Antibody A with unmodified IgG1 (Antibody A IgG1); 2) Antibody A with M252Y, S254T, and T256E, but without L234A, L235A, and D265S (Antibody A IgG1-YTE); and 3) Antibody A grafted onto IgG4 S228P, F234A, and L235A (Antibody A IgG4PAA).

[0197] Ustekinumab showed a relatively sharp peak with a retention time of 69.4 minutes and a peak width of 1.9 minutes, while burkinumab eluted at 88.9 minutes with a broad 10-minute peak width and a higher retention time. The FcRn binding profile of Antibody A IgG1 was very similar to that of ustekinumab, with nearly identical retention times and peak widths. All YTE-containing samples showed similar FcRn binding profiles, with increased elution pH (retention time) and moderately broadened peak widths compared to Antibody A IgG1.

[0198] [Table 13]

[0199] Summary: Antibody binding to FcRn was tested by FcRn-HPLC. Antibody A eluted the FcRn-HPLC column with a peak width and retention time of 2.7 minutes, which was delayed relative to its variable domain-matched IgG1 control and ustekinumab.

[0200] Example 14. Combined treatment with a LEPR agonist and a GLP-1 agonist resulted in significant weight loss in diet-induced obese mice Four-week-old male Black 6 (BL6) mice were fed a high-fat diet (Research Diet #D12492) for 16 weeks. These 20-week-old diet-induced obese (DIO) male mice were used for the study described below. Antibodies A, B, C, and D of the present disclosure are not cross-reactive in mice. A leptin receptor agonist comprising a leptin protein fused to Fc (Fc-leptin) (SEQ ID NO: 38) is used as a surrogate for a LEPR agonist antibody. SEQ ID NO: 38 binds to and stimulates the leptin receptor. At the start of the study (day 1), the diet was changed to a normal chow diet (Research Diets #D2014). On day 7, mice were subcutaneously injected daily with vehicle (40 mM Tris, pH 8.0) (FIG. 4, condition A), 40 nmol / kg of LEPR agonist (Fc-leptin (SEQ ID NO: 38)) (FIG. 4, condition B), 30 nmol / kg of GLP-1 receptor agonist (SEQ ID NO: 52) (FIG. 4, condition C), or a combination of LEPR agonist (SEQ ID NO: 38) (40 nmol / kg) and GLP-1 receptor agonist (SEQ ID NO: 52) (30 nmol / kg) (FIG. 4, condition D). As shown in FIG. 4, combined treatment with leptin receptor agonist and GLP-1 receptor agonist (SEQ ID NO: 52) resulted in greater weight loss compared to treatment with either GLP-1 receptor agonist or LEPR agonist (SEQ ID NO: 38) alone.

[0201] FIG. 4 shows that treatment with a GLP-1 receptor agonist in combination with a leptin receptor agonist resulted in further weight loss in obese mice. Diet-induced obese mice were switched from a high-fat diet (HFD) to normal chow for 6 days. These mice were then given daily subcutaneous injections of compounds (indicated by arrows) starting on the 7th day. Body weight was measured daily. The percentage change in body weight from baseline was plotted against time and shown in the graph. Conditions included: (A) vehicle control; (B) leptin receptor agonist (SEQ ID NO: 38); (C) GLP-1 receptor agonist (SEQ ID NO: 52); (D) GLP-1 receptor agonist (SEQ ID NO: 52) combined with leptin receptor agonist (SEQ ID NO: 38).

[0202] Example 15. Combined treatment with a LEPR agonist and a GLP1R / GIPR dual agonist resulted in significant weight loss in diet-induced obese mice Four-week-old male Black 6 (BL6) mice were fed a high-fat diet (Research Diet #D12492) for 16 weeks. These 20-week-old diet-induced obese (DIO) male mice were used for the study described below. At the start of the study (day 1), the diet was changed to a normal chow diet (Research Diets #D2014). On day 7, the mice received daily subcutaneous injections of vehicle (40 mM Tris, pH 8.0) (FIG. 5, condition A), 3 nmol / kg of GLP1R / GIPR dual agonist (SEQ ID NO: 53) (FIG. 5, condition B), or a combination of leptin receptor agonist (SEQ ID NO: 38) (40 nmol / kg) and GLP1R / GIPR dual agonist (3 nmol / kg) (FIG. 5, condition C). As shown in Figure 5, combined treatment with a leptin receptor agonist and a GLP1R / GIPR dual agonist resulted in greater weight loss compared to treatment with a GLP1R / GIPR dual agonist alone.

[0203] FIG. 5 shows that treatment with a dual GLP1R / GIPR agonist in combination with a leptin receptor agonist resulted in further weight loss in obese mice. Diet-induced obese mice were switched from a high-fat diet (HFD) to normal chow for 6 days. These mice were then given daily subcutaneous injections of compounds (indicated by arrows) starting on the 7th day. Body weight was measured daily. The percentage change in body weight from baseline was plotted against time and shown in the graph. Conditions included: (A) vehicle control; (B) dual GLP1R / GIPR agonist (SEQ ID NO: 53); (C) a combination of dual GLP1R / GIPR agonist (SEQ ID NO: 53) and leptin receptor agonist (SEQ ID NO: 38).

[0204] Example 16. Combined treatment with a LEPR agonist and a GLP1R / GIPR / GCGR triple agonist resulted in significant weight loss in diet-induced obese mice Four-week-old male Black 6 (BL6) mice were fed a high-fat diet (Research Diet #D12492) for 16 weeks. These 20-week-old diet-induced obese (DIO) male mice were used for the study described below. At the start of the study (day 1), the diet was changed to a normal chow diet (Research Diets #D2014). On day 7, the mice received daily subcutaneous injections of vehicle (40 mM Tris, pH 8.0) (FIG. 6, condition A), leptin receptor agonist (SEQ ID NO: 38) at 40 nmol / kg (FIG. 6, condition B), GLP1R / GIPR / GCGR triple agonist (SEQ ID NO: 55) at 3 nmol / kg (FIG. 6, condition C), or a combination of leptin receptor agonist (40 nmol / kg) and GLP1R / GIPR / GCGR triple agonist (3 nmol / kg) (FIG. 6, condition D). As shown in Figure 6, combined treatment with a leptin receptor agonist and a GLP1R / GIPR / GCGR triple agonist resulted in greater weight loss compared to GLP1R / GIPR / GCGR triple agonist or leptin receptor agonist treatment alone.

[0205] FIG. 6 shows that treatment with a GLP1R / GIPR / GCGR triple agonist in combination with a leptin receptor agonist resulted in further weight loss in obese mice. Diet-induced obese mice were switched from a high-fat diet (HFD) to normal chow for 6 days. These mice were then given daily subcutaneous injections of compounds (indicated by arrows) starting on the 7th day. Body weight was measured daily. The percentage change in body weight from baseline was plotted against time and shown in the graph. Vehicle control compounds included: (A) vehicle control; (B) leptin receptor agonist (SEQ ID NO: 38); (C) GLP1R / GIPR / GCGR triple agonist (SEQ ID NO: 55); (D) a combination of GLP1R / GIPR / GCGR triple agonist (SEQ ID NO: 55) and leptin receptor agonist (SEQ ID NO: 38).

[0206] Example 17. Combined treatment with a LEPR agonist and a glucagon receptor agonist resulted in significant weight loss in diet-induced obese mice Four-week-old male Black 6 (BL6) mice were fed a high-fat diet (Research Diet #D12492) for 16 weeks. These 20-week-old diet-induced obese (DIO) male mice were used for the study described below. At the start of the study (day 1), the diet was changed to a normal chow diet (Research Diets #D2014). On day 7, the mice were subcutaneously injected daily with vehicle (40 mM Tris, pH 8.0) (Figure 7, condition A), 6 nmol / kg of glucagon receptor agonist (SEQ ID NO: 57) (Figure 7, condition B), or a combination of leptin receptor agonist (40 nmol / kg) and glucagon receptor agonist (6 nmol / kg) (Figure 7, condition C). On day 21, the mice were switched to receiving subcutaneous compound administration every other day (Q2D) until the end of the study. As shown in Figure 7, combined leptin receptor agonist and glucagon receptor agonist treatment resulted in more weight loss compared to glucagon receptor agonist treatment alone.

[0207] FIG. 7 shows that glucagon receptor agonist treatment in combination with leptin receptor agonist resulted in further weight loss in obese mice. Diet-induced obese mice were switched from a high-fat diet (HFD) to normal chow for 6 days. These mice were then given daily subcutaneous injections of compound (indicated by arrows) starting on day 7. Starting on day 21 (Q2D), mice were switched to receiving subcutaneous compound injections every other day. Body weight was measured daily. The percentage change in body weight from baseline was plotted against time and shown in the graph. Conditions included: (A) vehicle control; (B) glucagon receptor agonist (SEQ ID NO: 57); (C) a combination of glucagon receptor agonist (SEQ ID NO: 57) and leptin receptor agonist (SEQ ID NO: 38).

[0208] Summary: When a surrogate LEPR agonist for an antibody of the present disclosure was combined with either an agent with GLP-1 receptor agonist activity, an agent with dual GLP1 / GIP receptor agonist activity, or an agent with triple GLP1 / GIP / glucagon receptor agonist activity, significantly greater weight loss was observed in DIO mice receiving the combination treatment than with either agent alone. Furthermore, a leptin receptor agonist in combination with a glucagon receptor agonist caused significantly greater weight loss in DIO mice than the glucagon receptor agonist alone.

[0209] Example 18. Combined treatment with an amylin receptor agonist and an LEPR agonist resulted in significant weight loss in diet-induced obese rats Eight-week-old male Long-Evans rats were fed a high-fat diet (Teklad Custom Diet #95217) for 12 weeks. The resulting 20-week-old diet-induced obese (DIO) rats were used in the study. These DIO rats were then subcutaneously injected daily with an amylin receptor agonist (SEQ ID NO: 39) (10 nmol / kg), an LEPR agonist (SEQ ID NO: 38) (100 nmol / kg), a combination of an amylin receptor agonist (SEQ ID NO: 39) (10 nmol / kg) and an LEPR agonist (100 nmol / kg) (sequential administration), or a placebo. The DIO rats were weighed daily. As shown in Figure 8, combined treatment with an amylin receptor agonist and a leptin receptor agonist resulted in significantly greater weight loss than either monotherapy alone.

[0210] FIG. 8 shows that treatment with an amylin receptor agonist (SEQ ID NO: 39) in combination with a leptin receptor agonist (SEQ ID NO: 38) resulted in further weight loss in diet-induced obese rats. Obese rats fed a high-fat diet were given daily subcutaneous injections of drugs as indicated in the graph legend. Body weight was measured daily. The percentage change in body weight from baseline was plotted against time and shown in the graph. Conditions included: (A) vehicle control; (B) 100 nmol / kg leptin receptor agonist (SEQ ID NO: 38); (C) 10 nmol / kg amylin receptor agonist (SEQ ID NO: 39); (D) a combination of 10 nmol / kg amylin receptor agonist (SEQ ID NO: 39) and 100 nmol / kg leptin receptor agonist (SEQ ID NO: 38).

[0211] Summary: Combined treatment with a leptin receptor agonist and an amylin receptor agonist resulted in greater weight loss in DIO rats than either monotherapy alone.

[0212] Example 19. LEPR agonists in combination with either amylin receptor agonists or amylin and calcitonin receptor coagonists resulted in significant weight loss in diet-induced obese rats Eight-week-old male Long-Evans rats were fed a high-fat diet (Teklad Custom Diet #95217) for 14 weeks. The resulting 22-week-old diet-induced obese (DIO) rats were used in the study. These DIO rats were then given daily subcutaneous injections of an amylin receptor agonist (SEQ ID NO: 41) (6 nmol / kg), an amylin and calcitonin receptor co-agonist (SEQ ID NO: 51) (5 nmol / kg), an LEPR agonist (SEQ ID NO: 38) (90 nmol / kg), or a combination thereof. The DIO rats were weighed daily. As shown in Figure 9, combined treatment with an amylin receptor agonist and a leptin receptor agonist resulted in significantly greater weight loss than either monotherapy alone. Similarly, combined treatment with an amylin receptor, a calcitonin receptor co-agonist, and a leptin receptor agonist also resulted in significantly greater weight loss than either monotherapy alone.

[0213] 9 shows that treatment with a leptin receptor agonist (SEQ ID NO: 38) in combination with either an amylin receptor agonist (SEQ ID NO: 41) or an amylin and calcitonin receptor co-agonist (SEQ ID NO: 51) resulted in further weight loss in diet-induced obese rats. Obese rats fed a high-fat diet were given daily subcutaneous injections of drug as indicated in the graph legend. Body weight was measured daily. The percent weight change from baseline was plotted against time and is shown in the graph. Conditions included: (A) vehicle control; (B) leptin receptor agonist (SEQ ID NO: 38); (C) amylin receptor agonist (SEQ ID NO: 41); (D) amylin and calcitonin receptor co-agonist (SEQ ID NO: 51); (E) a combination of leptin receptor agonist (SEQ ID NO: 38) and amylin receptor agonist (SEQ ID NO: 41); (F) a combination of leptin receptor agonist (SEQ ID NO: 38) and amylin and calcitonin receptor co-agonist (SEQ ID NO: 51).

[0214] Summary: Combined treatment with a leptin receptor agonist and either an amylin receptor agonist or an amylin and calcitonin receptor coagonist caused greater weight loss in DIO rats than either monotherapy alone.

[0215] Example 20. Treatment of monkeys with Antibody A resulted in weight loss. The efficacy of Antibody A was tested in non-human primates. Fourteen non-obese male cynomolgus monkeys (Macaca fascicularis), aged 2-5 years, weighing 3-5 kg, were group-housed in European cages (7 monkeys per cage) for 45 days. The monkeys were then randomized according to their body weight into two groups to receive subcutaneous administration of either Antibody A (10 mg / kg, N=7) or placebo (phosphate-buffered saline, N=7) once a week for 6 weeks. The monkeys were fed food (Purina 4059) twice daily and had free access to water throughout the study. Body weight was measured twice a week during the treatment period and for an additional 3 weeks after the final treatment. The percentage of weight change was plotted as a function of time. As shown in Figure 10 and Table 14, monkeys treated with Antibody A lost significantly more weight than those receiving placebo.

[0216] FIG. 10 shows that Antibody A treatment resulted in significant weight loss in cynomolgus monkeys. Male monkeys (cynomolgus monkeys (Macaca fascicularis)) were administered placebo (A) or 10 mg / kg antibody A (B) subcutaneously once weekly for 6 weeks. Body weight was measured for a total of 115 days. The mean percent weight change from baseline on day 0 was plotted at each time point. Error bars represent the standard error of the mean (SEM). Monkeys treated with antibody A showed significant weight loss compared to the placebo group through day 101 of the study. A multiple unpaired t-test showed a significant difference compared to the placebo group at all time points except day 0. * P<0.005, * P<0.05, ns: P>0.05.

[0217] [Table 14]

[0218] Summary: Antibody A treatment resulted in significant weight loss compared with placebo in non-obese cynomolgus monkeys.

[0219] Sequence Listing SEQ ID NO: 1 LCDR1 of antibodies A, C and D RASQGISSSLA

[0220] SEQ ID NO: 2 LCDR1 of antibody B RVSQGISSSLA

[0221] SEQ ID NO: 3: LCDR2 of antibodies A, B, C and D YTASTLQS

[0222] SEQ ID NO: 4: LCDR3 of antibody A QQLIYYPFT

[0223] SEQ ID NO: 5 LCDR3 of antibodies B and D QQLNYYPFT

[0224] SEQ ID NO: 6: LCDR3 of antibody C QQLNYYPFS

[0225] SEQ ID NO: 7 HCDR1 of antibodies A, B, C and D AASGFTFSSFAMS

[0226] SEQ ID NO: 8 HCDR2 of antibodies A, B, C and D AISGSGGSTY

[0227] SEQ ID NO: 9 HCDR3 of antibodies A, B, C and D AKDQGDWDFLFDY

[0228] SEQ ID NO: 10 VL of antibody A

Table 15

[0229] SEQ ID NO: 11 LC of antibody A Table 16

[0230] SEQ ID NO: 12 VL of antibody B

Table 17

[0231] SEQ ID NO: 13 LC of antibody B

Table 18

[0232] SEQ ID NO: 14 VL of antibody C

Table 19

[0233] SEQ ID NO: 15 LC of antibody C Table 20

[0234] SEQ ID NO: 16; antibody D VL

Table 21

[0235] SEQ ID NO: 17; Antibody D LC

Table 22

[0236] SEQ ID NO: 18 VH of antibodies A, B, C and D EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFAMSWVRQAPGKGLEWVSAISGSGGSTYSADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDQGDWDFLFDYWGQGTLVTVSS

[0237] SEQ ID NO: 19 HC of antibodies A, B, C and D EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFAMSWVRQAPGKGLEWVSAISGSGGSTYSADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDQGDWDFLFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0238] SEQ ID NO: 20 signal peptide sequence METDTLLLWVLLLWVPGSTG

[0239] SEQ ID NO: 21: Signal peptide cDNA ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGATCTACCGGT

[0240] SEQ ID NO: 22 LCA, cDNA GACATCCAGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGGGCATTAGCAGTTCTTTAGCCTGGTATCAGCAAAAACCAGGAAAAGCCCCTAAGCTCCTGATCTATACTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGCTTATTTATTACCCTTTCACTTTCGGCCAGGGGACCAAGGTGGAAATCAAAAGAACTGTGGCGGCGCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCCGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC

[0241] SEQ ID NO: 23 LC B, cDNA GACATCCAGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCCGGGTCAGTCAGGGCATTAGCAGTTCTTTAGCCTGGTATCAGCAAAAACCAGGAAAAGCCCCTAAGCTCCTGATCTATACTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGCTTAATTATTACCCTTTCACTTTCGGCCAGGGGACCAAGGTGGAAATCAAAAGAACTGTGGCGGCGCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCCGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC

[0242] SEQ ID NO: 24;LC C, cDNA GACATCCAGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGGGCATTAGCAGTTCTTTAGCCTGGTATCAGCAAAAACCAGGAAAAGCCCCTAAGCTCCTGATCTATACTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGCTTAATTATTACCCTTTCTCTTTCGGCCAGGGGACCAAGGTGGAAATCAAAAGAACTGTGGCGGCGCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCCGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC

[0243] SEQ ID NO: 25; LCD, cDNA GACATCCAGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGGGCATTAGCAGTTCTTTAGCCTGGTATCAGCAAAAACCAGGAAAAGCCCCTAAGCTCCTGATCTATACTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGCTTAATTATTACCCTTTCACTTTCGGCCAGGGGACCAAGGTGGAAATCAAAAGAACTGTGGCGGCGCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCCGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC

[0244] SEQ ID NO: 26 HC, cDNA

[0245] SEQ ID NO: 27 recombinant human leptin variant VPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPAASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC

[0246] SEQ ID NO: 28; Human LEPR, full length sequence (NP_002294.2)

[0247] SEQ ID NO: 29; Human LEPR-ECD-His FNLSYPITPWRFKLSCMPPNSTYDYFLLPAGLSKNTSNSNGHYETAVEPKFNSSGTHFSNLSKTTFHCCFRSEQDRNCSLCADNIEGKTFVSTVNSLVFQQIDANWNIQCWLKGDLKLFICYVESLFKNLFRNYNYKVHLLYVLPEVLEDSPLVPQKGSFQMVHCNCSVHECCECLVPVPTAKLNDTLLMCLKITSGGVIFQSPLMSVQPINMVKPDPPLGLHMEITDDGNLKISWSSPPLVPFPLQYQVKYSENSTTVIREADKIVSATSLLVDSILPGSSYEVQVRGKRLDGPGIWSDWSTPRVFTTQDVIYFPPKILTSVGSNVSFHCIYKKENKIVPSKEIVWWMNLAEKIPQSQYDVVSDHVSKVTFFNLNETKPRGKFTYDAVYCCNEHECHHRYAELYVIDVNINISCETDGYLTKMTCRWSTSTIQSLAESTLQLRYHRSSLYCSDIPSIHPISEPKDCYLQSDGFYECIFQPIFLLSGYTMWIRINHSLGSLDSPPTCVLPDSVVKPLPPSSVKAEITINIGLLKISWEKPVFPENNLQFQIRYGLSGKEVQWKMYEVYDAKSKSVSLPVPDLCAVYAVQVRCKRLDGLGYWSNWSNPAYTVVMDIKVPMRGPEFWRIINGDTMKKEKNVTLLWKPLMKNDSLCSVQRYVINHHTSCNGTWSEDVGNHTKFTFLWTEQAHTVTVLAINSIGASVANFNLTFSWPMSKVNIVQSLSAYPLNSSCVIVSWILSPSDYKLMYFIIEWKNLNEDGEIKWLRISSSVKKYYIHDHFIPIEKYQFSLYPIFMEGVGKPKIINSFTQDDIEKHQSDAGAAAHHHHHH

[0248] SEQ ID NO: 30; human LEPR ECD-TEV-Fc

[0249] SEQ ID NO: 31; cynomolgus monkey LEPR (>XP_005543194.1(Macaca fascicularis))

[0250] SEQ ID NO: 32; canine LEPR; (>XP_025283785.1 Dingo (Canis lupus dingo))

[0251] SEQ ID NO: 33; rat LEPR; (>XP_032757577.1 Leptin receptor [Rattus rattus])

[0252] SEQ ID NO: 34; mouse LEPR; (XP_036019643.1 House mouse (Mus musculus))

[0253] SEQ ID NO: 35; Rabbit LEPR-ECD-His FNLAYPVTPWRFKLSCMPANATHDYFLLPAGISKNTSNSSGHYEAIIEDKFNSSDTYFSNLSQTTFYCCFWSEQDTNCSVRADNIEGKTFVSTVNSLVFQQVGANWDIQCQMKGDLKLFICYMESLLKNPFKNVGLKVHLLYVLPEMLEDSLLVPQKGTFQMVQCNCSVHERCECHVPVPAAKLNYTLLMYFKVTSGGVFLQSPLMSVQLIDAVKPDPPLGLRMEITDKGNLKISWSNPAQVPFPLQYQVKYSENSTTIIREVAEIVSATFLLVDSVLPGSSYKAQVRGRRLDGPGTWSDWSTPQIFVTQDVIYFPPKILTSVGSNVSFHCIYKNEHKIVSSKQIVWWMNLAEKIPQSQYTVVNDRVSKVTFPNLNATKPRGKFTYDAVYCCRDHECHHRYAELYVVDVNINISCETDGYLTKMTCRWSANTIQSLVGSTLQLRYHRSSLYCSDIPSIHPISEPKECHLQRDGFYECIFQPIFLLSGYTMWIRVNHSLGSLDSPPTCVLPDSVVKPLPPSIVKAEITVNIGLLKLSWEKPVFPENNLQFQIRYGLSGNEKQWKVFEVHDSKSKSANLSVPDLCAVYAAQVRCKRLDGLGYWSNWSKPAYTVVKDVKVPVRGPEFWRIIDGDVTKKERNVTLLWKPLMKSDSLCSVSRYVVNHYTSHNGTWSEDVGNHTRFTFIWAEQVHTVTVVAINSIGASSSNFNLTFSWPVSKVNTVQSLSAYPLNSSCVILSWIPLPSDYNLMYFIIEWKNLNEDNEIKWLRIPSSVKKYSIHDNFIPIEKYQFSLYPIFMEGVGKPKIINSFTQDNDEKLQNDAGAAAHHHHHH

[0254] SEQ ID NO: 36, antibody CO HC QVQLVESGGSVVQPGRSLRLSCAASGFTFSTYAMYWVRQTPGKGLEWVAVLYSDGSNKYYIDSVKGRFTISRDTSTNTLYLQMSSLRADDSALYYCARLNWDYWYFDLWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0255] SEQ ID NO: 37, antibody CO LC DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0256] SEQ ID NO: 38, mouse Fc-leptin (LEPR Ag Ab surrogate) VPRDSGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYS KLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGGGGSVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC

[0257] SEQ ID NO: 39 Amylin receptor agonist [Table 23]

[0258] SEQ ID NO: 40 Amylin receptor agonist γE-CNTATCATG-Orn-LAE-αMeF-LVRSSN-NMeN-FGPKLPPTEVGSNTY-NH2 There is a thioacetal bridge between the cysteines at positions 2 and 7. [ka]

[0259] SEQ ID NO: 41 Amylin receptor agonist γE-CNTATCATG-Orn-LAE-αMeF-LVRSSN-NMeN-FGPKLPPTEVGSNTY-NH2 There is a thioacetal bridge between the cysteines at positions 2 and 7. The lysine at position 26 has the formula (γE)2-CO-(CH2) 18 -CO2H is attached to the fatty acid linker moiety. [ka]

[0260] SEQ ID NO: 42 Amylin receptor agonist activity [Table 24] There is a thioacetal bridge between the cysteines at positions 2 and 7. [ka]

[0261] SEQ ID NO: 43 Amylin receptor agonist γE-CNTATCATG-Orn-LAE-αMeF-LVRSSN-NMeD-FGPKLPPTEVGSNTY-NH2 There is a thioacetal bridge between the cysteines at positions 2 and 7. The lysine at position 26 has the formula (γE)2-CO-(CH2) 18 -CO2H is attached to the fatty acid linker moiety. [ka]

[0262] SEQ ID NO: 44 Amylin receptor agonist KCETATCATG-Orn-LAE-αMeF-LVRSSN-NMeD-FGPILPPTEVGSNTY-NH2 There is a thioacetal bridge between the cysteines at positions 2 and 7. [ka]

[0263] SEQ ID NO: 45 Amylin receptor agonist activity KCETATCATG-Orn-LAE-αMeF-LVRSSN-NMeD-FGPILPPTEVGSNTY-NH2 There is a thioacetal bridge between the cysteines at positions 2 and 7. The lysine at position 1 has the formula (γE)2-CO-(CH2) 18 -CO2H is attached to the fatty acid linker moiety. [ka]

[0264] SEQ ID NO: 46 Amylin receptor agonist KCETATCATG-Orn-αMeL-AEFLVRSSHNFGPILPPTEVGSNTY-NH2 There is a thioacetal bridge between the cysteines at positions 2 and 7. [ka]

[0265] SEQ ID NO: 47 Amylin receptor agonist KCETATCATG-Orn-αMeL-AEFLVRSSHNFGPILPPTEVGSNTY-NH2 There is a thioacetal bridge between the cysteines at positions 2 and 7. The lysine at position 1 has the formula (γE)2-CO-(CH2) 18 -CO2H is attached to the fatty acid linker moiety. [ka]

[0266] SEQ ID NO: 48 Dual amylin and calcitonin receptor agonist Acetyl-ASHLSTAVLGKLS-Aib-ELHKLEDYPRTDVGAESP-NH2

[0267] SEQ ID NO: 49 Dual amylin and calcitonin receptor agonist Acetyl-ASHLSTAVLGK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO—(CH2) 18 -CO2H)LS-Aib-ELHKLEDYPRTDVGAESP-NH2

[0268] SEQ ID NO: 50 Amylin receptor agonist KCNTATCATQRLANFLVHSSNNFGPILPTNVGSNTY-NH2 In this formula, there are disulfide bonds at positions 2 and 7.

[0269] SEQ ID NO: 51 Amylin receptor agonist CO2H-(CH2) 18 -CO-γE-KCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTP-NH2 Sequence modifications to h-amylin: 14E, 17R, 25P, 28P, 29P, 37P

[0270] SEQ ID NO: 52 GLP-1 receptor agonist H-Aib-EGTFTSDVSSYLEGQAAKEFIAWLVRGRG In the formula, K at position 20 represents the ε-amino group of the K side chain and (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γE)-CO-(CH2) 16 It is chemically modified by conjugation with -CO2H. [ka]

[0271] SEQ ID NO: 53, a dual agonist of the GLP-1 and GIP receptors Y-Aib-EGTFTSDYSI-Aib-LDKIAQK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-γE2-CO-(CH2) 18 -CO2H)A-1Nal-VQWLIAGGPSSGAPPPS-NH2

[0272] SEQ ID NO: 54 Dual agonist of GLP-1 and GIP receptors Y-Aib-EGTFTSDYSI-Aib-LDKIAQK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γE)-CO-(CH2) 18 -CO2H)AFVQWLIAGGPSSGAPPPS-NH2 K at position 20 is (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γE)1-CO-(CH2) 18 It is chemically modified through conjugation of the K side chain with -CO2H to the ε-amino group, and the C-terminal amino acid is amidated as a C-terminal primary amide. [ka] L-Tyrosyl-2-methylalanyl-L-α-glutamylglycyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-α-aspartyl-L-tyrosyl-L-seryl-L-isoleucyl-2-methylalanyl-L-leucyl-L-α-aspartyl-L-lysyl-L-isoleucyl-L-alanyl-L-glutaminyl-N 6 -[(22S)-22,42-Dicarboxy-1,10,19,24-tetraoxo-3,6,12,15-tetraoxa-9,18,23-triazadtetracon-1-yl]-L-lysyl-L-alanyl-L-phenylalanyl-L-valyl-L-glutaminyl-L-tryptophyl-L-leucyl-L-isoleucyl-L-alanylglycylglycyl-L-prolyl-L-seryl-L-serylglycyl-L-alanyl-L-prolyl-L-prolyl-L-prolyl-L-serinamide

[0273] SEQ ID NO: 55, a triagonist of the GIP / GLP-1 / glucagon receptor [Table 25]

[0274] SEQ ID NO: 56 Triagonist GLP1R / GIPR / GCGR Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)-(γE)-CO-(CH2) 18 -CO2H)AQ-Aib-AFIEYLLEGGPSSGAPPPS-NH2 In the formula, K at position 17 corresponds to the ε-amino group of the K side chain and (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)-(γE)-CO-(CH2) 18It is chemically modified by conjugation with -CO2H, and the C-terminal amino acid is amidated as a C-terminal primary amide. [ka]

[0275] SEQ ID NO: 57; Glucagon receptor agonist Y-Aib-QGTFTSDYSKYLD-Aib-KKAK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO-(CH2) 16 -CO2H)EFVEWLLETGPSSGAPPPS-NH2

[0276] SEQ ID NO: 58; Cynomolgus LEPR-ECD-His FNLSYPITPWRFKLSCMPPNSTYDYFLLPAGLSKNTSNLNGHYETAVEFNSSDTHFSNLSKTTFHCCFRSEQDRNCSLCADNIEGKTFVSTVNSSVFQQMGANWNIQCWLKGDLKLFICYVESLFKNPFKNYKHKVHLLYVLPEVLEDSPLVPQKGSFQMVHCNCSVHERCECLVPVPTAKLNDTLLMCLKITSGGVIFQSPLMSVQPINMVKPDPPLGLRMEITDDGNLKISWSSPPLVPFPLQYEVKYSENSTTVIREADKIVSATSLLVDGILPGSSYEVQVRGKRLDGPGIWSDWSTPHVFTTQDVIYFPPKILTSVGSNVSFHCIYKNENKIVSSKKIVWWMNLAEKIPQSQYDVVSDHVSKVTFFNLNETKPRGKFTYDAVYCCNEHECHHRYAELYVIDVNINISCETDGHLTKMTCRWSTNTIQSLAGSTLQLRYRRSSLYCFDIPSIHPISKPKDCYLQSDGFYECVFQPIFLLSGYTMWIRINHPLGSLDSPPTCVLPDSVVKPLPPSSVKAEIIKNIGLLKISWEKPVFPENNLQFQIRYGLSGKEIQWKMYDVYDAKSKSVSLPVPDFCAVYAVQVRCKRSDGLGLWSNWSNPAYTVVMDIKVPMRGPEFWRIINGDTMKKEKNVTLLWKPLMKNDSLCSVQRYVINHHTSCNGTWSEDVGNHTKFTFLWTEQAHTVTVLAINSIGASVANFNLTFSWPMSKVNIVQSLSAYPLNSSCVILSWILSPSDYKLMYFIIEWKNLNEDGEIKWLRISSSVKKYYIHDHFIPIEKYQFSLYPIFMEGVGKPKIINSFTQDNTEKHQNDGHHHHHH

[0277] SEQ ID NO: 59; Canine LEPR-ECD-His FNLAYPITPWRFKLSCMPPNTTYDFLLPAGISRNTSNLNEHYEAVVEAKLNSSSTYISNLSSKTTFHCCFWSKEDKNCSVHADNMEGKAFVSTVSSLVFQQIGANWNIQCWMKEDLKLFICYMESLFKNPFKTYDLKVHLLYVLPEVLEESPPVPQKGGFQIVPCNCSVHDSCECHVPVPTAELNHTLLMYLKITLGGINFQSPLMSVKPINVVKPDPPLGLHMEITDTGNLKISWSSPTLVPFQLQYQVRYSENSSTNVRKANEIVSATSLLIDSVLPGSSYEVQVRGKKLDGPGIWGDWSTPLIFITQDVIYFPPKILTSVGSNVSFHCIYKSENKIVSSKKIVWWLNLAEKIPQSQYNMVGDRVSKVTFPNLNATKPRGKFTYDAVYCCKEQECHHRYAELYVIDVNINISCETDGYLTKMTCRWSTNAIQSLEGSTLQLRYHRSSLYCSDVPSIHPISEPKDCHLRRDGFYECIFQPIFLLSGYTMWIKINHSLGSLDSSPTCVVPDSVVKPLPPSSVKAEITVKIGLLKISWEKPVFPENNLKFQIRYGLNGKEVQWKIYEVYDTKLKSTSLPVPDLCAVYAVQVRCKRLDGLGYWSNWSTPAYTVVTDVKVPTRGPEFWRMIDEDTSRKERNVTLLWKPLMKNDSLCSVRKYVVKHHTSRNGTWSEDVGNHTKFTFLWTEQAHSVTVLAVNSIGASSVNFNLTFSWPMSKVNTVQSLSAYPLNSTCVLLSWTLTPSDYYLTYFITEWKILNEDSEIKWLRIPPSVKKYYIHDHFIPIEKYQFSLYPVFMEGVGKPKTINSFTQDDIEKHQNDHHHHHH

[0278] SEQ ID NO: 60; Rat LEPR-ECD-His LNLAYPTSPWRFKLFCAPPSTTDDSFLSPAGVPNNTSSLKGASEALVEAKFNSTGIYVSELSKTIFHCCFGNEQGQNCSALTGNTEGKTLASVVKPLVFRQLGVNWDIECWMKGDLTLFICHMEPLLKNPFKNYDSKVHLLYDLPEVIDDLPLPPLKDSFQTVQCNCSVRECECHVPVPRAKVNYALLMYLEITSAGVSFQSPLMSLQPMLVVKPDPPLGLRMEVTDDGNLKISWDSQTKAPFPLQYQVKYLENSTIVREAAEIVSDTSLLVDSVLPGSSYEVQVRSKRLDGSGVWSDWSLPQLFTTQDVMYFPPKILTSVGSNASFCCIYKNENQTISSKQIVWWMNLAEKIPETQYNTVSDHISKVTFSNLKATRPRGKFTYDAVYCCNEQACHHRYAELYVIDVNINISCETDGYLTKMTCRWSPSTIQSLVGSTVQLRYHRRSLYCPDNPSIRPTSELKNCVLQTDGFYECVFQPIFLLSGYTMWIRINHSLGSLDSPPTCVLPDSVVKPLPPSNVKAEITINTGLLKVSWEKPVFPENNLQFQIRYGLNGKEIQWKTHEVFDAKSKSASLPVSDLCAVYVVQVRCRRLDGLGYWSNWSSPAYTLVMDVKVPMRGPEFWRIMDGDITKKERNVTLLWKPLMKNDSLCSVRRYVVKHRTAHNGTWSQDVGNQTNLTFLWAESAHTVTVLAINSIGASLVNFNLTFSWPMSKVNAVQSLSAYPLSSSCVILSWTLSPNDYSLLYLVIEWKNLNDDDGMKWLRIPSNVNKYYIHDNFIPIEKYQFSLYPVFMEGVGKPKIINGFTKDDIAKQQNDAGAAAHHHHHH

[0279] SEQ ID NO: 61; Mouse LEPR-ECD-His LNLAYPISPWKFKLFCGPPNTTDDSFLSPAGAPNNASALKGASEAIVEAKFNSSGIYVPELSKTVFHCCFGNEQGQNCSALTDNTEGKTLASVVKASVFRQLGVNWDIECWMKGDLTLFICHMEPLPKNPFKNYDSKVHLLYDLPEVIDDSPLPPLKDSFQTVQCNCSLRGCECHVPVPRAKLNYALLMYLEITSAGVSFQSPLMSLQPMLVVKPDPPLGLHMEVTDDGNLKISWDSQTMAPFPLQYQVKYLENSTIVREAAEIVSATSLLVDSVLPGSSYEVQVRSKRLDGSGVWSDWSSPQVFTTQDVVYFPPKILTSVGSNASFHCIYKNENQIISSKQIVWWRNLAEKIPEIQYSIVSDRVSKVTFSNLKATRPRGKFTYDAVYCCNEQACHHRYAELYVIDVNINISCETDGYLTKMTCRWSPSTIQSLVGSTVQLRYHRRSLYCPDSPSIHPTSEPKNCVLQRDGFYECVFQPIFLLSGYTMWIRINHSLGSLDSPPTCVLPDSVVKPLPPSNVKAEITVNTGLLKVSWEKPVFPENNLQFQIRYGLSGKEIQWKTHEVFDAKSKSASLLVSDLCAVYVVQVRCRRLDGLGYWSNWSSPAYTLVMDVKVPMRGPEFWRKMDGDVTKKERNVTLLWKPLTKNDSLCSVRRYVVKHRTAHNGTWSEDVGNRTNLTFLWTEPAHTVTVLAVNSLGASLVNFNLTFSWPMSKVSAVESLSAYPLSSSCVILSWTLSPDDYSLLYLVIEWKILNEDDGMKWLRIPSNVKKFYIHDNFIPIEKYQFSLYPVFMEGVGKPKIINGFTKDAIDKQQNDAGAAAHHHHHHSGS

Claims

1. An antibody that binds to a leptin receptor (LEPR), the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprising light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3; a) said HCDR1 comprises SEQ ID NO:7; b) said HCDR2 comprises SEQ ID NO:8; c) said HCDR3 comprises SEQ ID NO: 9; d) said LCDR1 comprises SEQ ID NO: 1 or 2; e) said LCDR2 comprises SEQ ID NO:3; f) the LCDR3 comprises SEQ ID NO: 4, 5 or 6; An antibody that binds to the leptin receptor (LEPR).

2. a) said LCDR1 comprises SEQ ID NO: 1 and said LCDR3 comprises SEQ ID NO: 5; b) said LCDR1 comprises SEQ ID NO: 1 and said LCDR3 comprises SEQ ID NO: 4; c) said LCDR1 comprises SEQ ID NO:2 and said LCDR3 comprises SEQ ID NO:5; d) said LCDR1 comprises SEQ ID NO: 1 and said LCDR3 comprises SEQ ID NO: 6; The antibody described in claim 1.

3. The antibody of any one of claims 1 to 2, wherein the antibody has a human IgG1 or IgG4 subclass.

4. The antibody of any one of claims 1 to 3, wherein the antibody has a human IgG1 subclass.

5. The antibody of any one of claims 1 to 4, wherein the VH comprises SEQ ID NO: 18 and the VL comprises SEQ ID NO:

16.

6. The antibody of any one of claims 1 to 5, wherein the antibody comprises a heavy chain (HC) comprising SEQ ID NO: 19 and a light chain (LC) comprising SEQ ID NO:

17.

7. The antibody of any one of claims 1 to 5, wherein the antibody comprises a heavy chain (HC) consisting of SEQ ID NO: 19 and a light chain (LC) consisting of SEQ ID NO:

17.

8. The antibody of any one of claims 1 to 4, wherein the VH comprises SEQ ID NO: 18 and the VL comprises SEQ ID NO:

10.

9. The antibody of any one of claims 1 to 4 and 8, wherein the antibody comprises a heavy chain (HC) comprising SEQ ID NO: 19 and a light chain (LC) comprising SEQ ID NO:

11.

10. The antibody of any one of claims 1 to 4 and 8, wherein the antibody comprises a heavy chain (HC) consisting of SEQ ID NO: 19 and a light chain (LC) consisting of SEQ ID NO:

11.

11. The antibody of any one of claims 1 to 4, wherein the VH comprises SEQ ID NO: 18 and the VL comprises SEQ ID NO:

12.

12. The antibody of any one of claims 1 to 4 and 11, wherein the antibody comprises a heavy chain (HC) comprising SEQ ID NO: 19 and a light chain (LC) comprising SEQ ID NO:

13.

13. The antibody of any one of claims 1 to 4 and 11, wherein the antibody comprises a heavy chain (HC) consisting of SEQ ID NO: 19 and a light chain (LC) consisting of SEQ ID NO:

13.

14. The antibody of any one of claims 1 to 4, wherein the VH comprises SEQ ID NO: 18 and the VL comprises SEQ ID NO:

14.

15. The antibody of any one of claims 1 to 4 and 14, wherein the antibody comprises a heavy chain (HC) comprising SEQ ID NO: 19 and a light chain (LC) comprising SEQ ID NO:

15.

16. The antibody of any one of claims 1 to 4 and 14, wherein the antibody comprises a heavy chain (HC) consisting of SEQ ID NO: 19 and a light chain (LC) consisting of SEQ ID NO:

15.

17. The antibody has a dissociation equilibrium constant (K Ｄ The antibody of any one of claims 1 to 16, which binds to human LEPR at

18. The antibody has a dissociation equilibrium constant (K Ｄ The antibody of any one of claims 1 to 17, which binds to cynomolgus monkey LEPR at

19. The antibody has a dissociation equilibrium constant (K Ｄ The antibody of any one of claims 1 to 18, which binds to canine LEPR at

20. The above K Ｄ The antibody of any one of claims 17 to 19, wherein is determined by a surface plasmon resonance (SPR) assay.

21. The antibody has an EC ５０ The antibody according to any one of claims 1 to 20, having a (human)

22. The antibody has an EC ５０ The antibody according to any one of claims 1 to 21, having a cynomolgus monkey (Macaca fascicularis).

23. The antibody has an EC ５０ The antibody according to any one of claims 1 to 22, having a (dog)

24. The EC ５０ The antibody of any one of claims 21 to 23, wherein is determined by a pSTAT3 assay.

25. The antibody of any one of claims 1 to 24, wherein the antibody provides 55% to 65% agonism of human LEPR compared to human leptin.

26. 26. The antibody of any one of claims 1 to 25, wherein the antibody provides 20% to 30% agonism of cynomolgus monkey LEPR compared to human leptin.

27. The antibody of any one of claims 1 to 26, wherein the antibody provides 65% to 85% agonism of canine LEPR compared to human leptin.

28. The antibody of any one of claims 25 to 27, wherein the agonism is determined by a pSTAT3 assay.

29. A nucleic acid comprising a sequence encoding at least one of SEQ ID NOs: 1-20.

30. A vector comprising the nucleic acid of claim 29.

31. A cell comprising the vector of claim 30.

32. 32. The cell of claim 31 , wherein the cell is a mammalian cell.

33. A pharmaceutical composition comprising the antibody of any one of claims 1 to 28 and a pharmaceutically acceptable excipient, diluent, or carrier.

34. A pharmaceutical composition comprising the antibody of any one of claims 1 to 28 and a second therapeutic agent.

35. The pharmaceutical composition of claim 34, wherein the second therapeutic agent comprises an agent set forth in any one of SEQ ID NOs: 40-47.

36. The pharmaceutical composition of claim 34, wherein the second therapeutic agent comprises an agent set forth in any one of SEQ ID NOs: 50-51.

37. 35. The pharmaceutical composition of claim 34, wherein the second therapeutic agent has GLP-1 agonist activity.

38. 38. The pharmaceutical composition of claim 37, wherein the second therapeutic agent is a dual agonist of the GLP-1 receptor and the GIP receptor.

39. 39. The pharmaceutical composition of claim 38, wherein the dual agonist of the GLP-1 receptor and the GIP receptor comprises SEQ ID NO:

54.

40. 38. The pharmaceutical composition of claim 37, wherein the second therapeutic agent comprises SEQ ID NO:

56.

41. 29. The antibody of any one of claims 1 to 28 for simultaneous, separate or sequential use in combination with a second therapeutic agent selected from SEQ ID NOs: 39 to 57 in the treatment of obesity.

42. A therapeutic agent selected from SEQ ID NOs: 39 to 57 for simultaneous, separate or sequential use in combination with an antibody according to any one of claims 1 to 28 in the treatment of obesity.

43. 41. A method of treating obesity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody or composition of any one of claims 1-28 and 33-40.

44. An antibody or composition according to any one of claims 1 to 28 and 33 to 40 for use in therapy.

45. An antibody or composition according to any one of claims 1 to 28 and 33 to 40 for use in the treatment of obesity.

46. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 28 for use in the treatment of obesity.

47. Use of an antibody according to any one of claims 1 to 28 in the manufacture of a medicament for treating obesity.

48. 29. A method of treating a lipodystrophic disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody according to any one of claims 1 to 28.

49. The antibody of any one of claims 1 to 28 for use in the treatment of a lipodystrophic disorder.

50. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 28 for use in the treatment of a lipodystrophic disorder.

51. Use of an antibody according to any one of claims 1 to 28 in the manufacture of a medicament for the treatment of a lipodystrophic disorder.

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