EA040524B1 - ANTIGEN-BINDING PROTEINS THAT ANTAGONISM TO THE LEPTIN RECEPTOR - Google Patents

Description

Background of the Invention

Leptin is a polypeptide hormone predominantly expressed by adipose tissue and is involved in the regulation of metabolism, energy balance and food intake. Leptin activity is mediated by interaction with and signaling through the leptin receptor. The leptin receptor (also known as LEPR, WSX, OB receptor, OB-R, and CD295) is a single-pass transmembrane receptor of the class I cytokine receptor family with a large (818 amino acid) extracellular domain. Increased expression of leptin, the Ob-R leptin receptor, or both, may contribute to multiple disorders including, but not limited to, the following: anorexia or other psychiatric eating disorders, cachexia in chronic kidney disease, other cachexia such as cachexia in congestive heart failure, pulmonary cachexia, radiation cachexia and cancer cachexia, autoimmune disorders such as inflammatory bowel disease, lupus erythematosus, multiple sclerosis, psoriasis, cardiovascular disease, high blood pressure, depression, non-alcoholic hepatic steatosis, neurodegenerative disorders, depression, cancer such as renal cell carcinoma, melanoma and breast cancer.

Suggested therapeutic approaches to address high levels of leptin signaling include the use of the following: peptide leptin receptor antagonists and antagonist mutants such as soluble leptin receptor variants competitive LEPR antagonists such as the 9F8 antibody (Fazeli et al. (2006) J Immunological Methods 312, 190-200) or nanoantibodies targeting the leptin receptor (McMurphy et al., PLOS One (2014) 9(2):e89895), fibronectin III domains, orexigenic substances (eg, ghrelin and NPY), blockade of downstream mediators leptin (eg, melanocortin 4 receptor) and/or lifestyle changes. However, such approaches have generally shown limited effectiveness. Thus, there is a need in the present field for alternative approaches to the treatment of leptin resistance and other conditions associated with elevated serum leptin levels and/or excessive LEPR signaling.

Sequence listing

The official copy of the sequence listing is filed simultaneously with the electronic description of the invention via EFS-Web as an ASCII-formatted sequence listing with file name 2017_11_08_10271WO01_SEQ_LIST_ST25.TXT, creation date November 8, 2017, and file size of 87.4 KV. The sequence listing contained in this ASCII formatted document is part of the present specification and is hereby incorporated by reference in its entirety.

Brief summary of the present invention

The present invention relates to antibodies and antigen-binding fragments thereof that bind to the human leptin receptor (LEPR). The antibodies of the present invention are antagonistic antibodies; those. binding of the anti-LEPR antibodies of the present invention to LEPR results in blockade or down regulation of leptin receptor signaling in cells. Accordingly, in various embodiments, the antagonist antibodies of the present invention exhibit weak partial agonist activity. In contrast, the antibodies of the present invention are useful, for example, for down-regulating the biological activity of leptin in a subject. In this regard, the antibodies and antigen-binding fragments of the present invention are useful in the therapeutic treatment of diseases and disorders associated with increased leptin signaling.

Antibodies of the present invention may be full-length (eg, an IgG1 or IgG4 antibody) or may contain only an antigen-binding portion (eg, Fab, F(ab') ₂ or scFv fragment) and may be modified to affect functionality, eg, to eliminate residual effector functions (Reddy et al., 2000, J. Immunol. 164:1925-1933).

Illustrative antagonistic antibodies to LEPR according to the present invention are shown in table. 1 and 2 in this document. In table. 1 provides the amino acid sequence identifiers for heavy chain variable regions (HCVR), light chain variable regions (LCVR), heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) of exemplary antagonist antibodies to LEPR. In table. 2 shows the nucleic acid sequence identifiers for HCVR, LCVR, HCDR1, HCDR2 HCDR3, LCDR1, LCDR2, and LCDR3 exemplary anti-LEPR antagonist antibodies.

The present invention provides antibodies or antigen-binding fragments thereof that specifically bind to an HCVR-containing LEPR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1. 1, or a sequence substantially similar to them, which is characterized by at least 90%,

- 1 040524 at least 95%, at least 98% or at least 99% sequence identity with respect to them.

The present invention also relates to antibodies or antigen-binding fragments thereof that specifically bind to a LEPR containing an LCVR containing an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table. 1, or a sequence substantially similar to them, which has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to them.

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

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

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

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

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

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

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

The present invention also relates to antibodies or antigen-binding fragments thereof that specifically bind to a LEPR containing an HCDR3 and LCDR3 amino acid sequence pair (HCDR3/LCDR3) containing any of the HCDR3 amino acid sequences listed in Table 1. 1 paired with any of the LCDR3 amino acid sequences listed in Table. 1. In certain embodiments, the invention relates to antibodies or antigen-binding fragments thereof, comprising an HCDR3/LCDR3 amino acid sequence pair contained within any of the exemplary LEPR antibodies listed in Table 1. 1. In certain embodiments, the 2040524 amino acid sequence pair of HCDR3/LCDR3 is selected from the group consisting of SEQ ID NO: 8/16, 24/16,

32/16, 40/16, 48/16, 56/16, 64/16, 72/16, 80/16 and 80/88.

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

According to a similar embodiment, the present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to a LEPR containing a set of six CDRs (i.e. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) contained within a pair of amino acid sequences of HCVR /LCVR as defined by any of the exemplary anti-LEPR antibodies listed in Table 1. 1. For example, the present invention provides antibodies or antigen-binding fragments thereof that specifically bind to LEPR, comprising a set of amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 contained within an HCVR/LCVR amino acid sequence pair selected from the group, consisting of SEQ ID NO: 2/10, 18/10, 26/10, 34/10, 42/10, 50/10, 58/10, 66/10 and 74/82. Methods and techniques for identifying CDRs within the HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the given HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. sci. USA 86:9268-9272 (1989). Public databases are also available to identify CDR sequences within an antibody.

The present invention also relates to nucleic acid molecules encoding anti-LEPR antibodies or parts thereof. For example, the present invention relates to nucleic acid molecules encoding any of the HCVR amino acid sequences listed in table. 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 1. 2, or a sequence substantially similar to them, which has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to them.

The present invention also relates to nucleic acid molecules encoding any of the LCVR amino acid sequences listed in table. 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 1. 2, or a sequence substantially similar to them, which has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to them.

The present invention also relates to nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in table. 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 1. 2, or a sequence substantially similar to them, which has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to them.

The present invention also relates to nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in table. 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 1. 2, or a sequence substantially similar to them, which has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to them.

The present invention also relates to nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in table. 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in

- 3 040524 tab. 2, or a sequence substantially similar to them, which has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to them.

The present invention also relates to nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in table. 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 1. 2, or a sequence substantially similar to them, which has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to them.

The present invention also relates to nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in table. 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 1. 2, or a sequence substantially similar to them, which has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to them.

The present invention also relates to nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table. 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 1. 2, or a sequence substantially similar to them, which has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to them.

The present invention also relates to nucleic acid molecules encoding HCVR, and HCVR contains a set of three CDRs (i.e. HCDR1, HCDR2, HCDR3), and the set of amino acid sequences HCDR1, HCDR2, HCDR3 is as defined by any of the illustrative antibodies to LEPR listed in Table. 1.

The present invention also relates to nucleic acid molecules encoding an LCVR, wherein the LCVR contains a set of three CDRs (i.e., LCDR1, LCDR2, LCDR3), wherein the set of amino acid sequences of LCDR1, LCDR2, LCDR3 is as defined by any of the exemplary antibodies to LEPR listed in Table. 1.

The present invention also relates to nucleic acid molecules encoding both HCVR and LCVR, and HCVR contains an amino acid sequence according to any of the HCVR amino acid sequences listed in table. 1, and while LCVR contains an amino acid sequence according to any of the amino acid sequences of LCVR listed in table. 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table. 2, or a sequence substantially similar thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any from the LCVR nucleic acid sequences listed in Table. 2, or a sequence substantially similar to them, which has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to them. In certain embodiments of this aspect of the present invention, the nucleic acid molecule encodes HCVR and LCVR, with both HCVR and LCVR derived from the same anti-LEPR antibody listed in Table 1. 1.

The present invention also relates to recombinant expression vectors capable of expressing a polypeptide containing the heavy or light chain variable region of an anti-LEPR antibody. For example, the present invention provides for recombinant expression vectors containing any of the nucleic acid molecules mentioned above, ie. nucleic acid molecules encoding any of the sequences HCVR, LCVR and/or CDR, as shown in table. 1. Also provided within the scope of the present invention are host cells into which such vectors have been introduced, as well as methods for producing antibodies or parts thereof by culturing the host cells under conditions to produce antibodies or antibody fragments and isolating the antibodies and antibody fragments. antibodies thus obtained.

In another aspect, the present invention relates to a pharmaceutical composition comprising a recombinant human antibody or fragment thereof that specifically binds to LEPR and a pharmaceutically acceptable carrier. In a similar aspect, the present invention relates to a composition that is a combination of an anti-LEPR antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that combines favorably with an anti-LEPR antibody.

- 4 040524

According to another aspect, the present invention provides therapeutic methods for downregulating or abolishing LEPR signaling using an anti-LEPR antibody or an antigen-binding portion of an antibody of the present invention. Therapeutic methods according to this aspect of the present invention provide for the introduction of a therapeutically effective amount of a pharmaceutical composition containing the antibody or antigen-binding fragment of the antibody according to the present invention, in need of this subject. A therapeutic disorder is any disease or condition that is ameliorated, reduced, suppressed or prevented by down-regulating or eliminating LEPR signaling or blocking leptin activity.

Other embodiments will become apparent from a review of the following detailed description.

Brief description of the figures

In FIG. 1 shows the percentage change in food intake in mice that were treated with 30 mg/kg of anti-LEPR antibody H4H17322P2, H4H18457P2, or H4H8464 or with an isotype control antibody.

In FIG. 2 shows the mean percent change in body weight of mice that were treated with 30 mg/kg of anti-LEPR antibody H4H17322P2, H4H18457P2, or H4H18464 or with an isotype control antibody.

In FIG. 3 shows the mean adipose tissue mass of mice that were treated with 30 mg/kg of anti-LEPR antibody H4H17322P2, H4H18457P2, or H4H18464 or with an isotype control antibody.

Detailed disclosure of the present invention

Before the present invention is described, it should be understood that the present invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terminology used herein is only intended to describe specific embodiments and is not intended to be limiting, as the scope of the present invention will only be limited by the appended claims.

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as generally understood by a person skilled in the art to which the present invention pertains. As used herein, approximately, when used in relation to a particular numerical value given, means that the value may differ from the given value by no more than 1%. For example, as used herein, approximately 100 includes 99 and 101 and all values in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

While any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred methods and materials are described below. All patents, patent applications, and non-patent publications mentioned herein are incorporated herein by reference in their entirety.

Definitions

As used herein, the expression leptin receptor, LEPR and the like refers to a human leptin receptor comprising the amino acid sequence as set forth in SEQ ID NO: 89 (see also UniProtKB/Swiss-Prot accession number P48357). Alternative names for LEPR used in the scientific literature include the OB receptor, OB-R, and CD295. LEPR is also referred to as WSX (see, for example, US Pat. No. 7,524,937). The LEPR expression includes both monomeric and multimeric (eg, dimeric) LEPR molecules. As used herein, monomeric human LEPR refers to a LEPR protein or portion thereof that does not contain or possess any multimerization domains and that exists under normal conditions as a single LEPR molecule without a direct physical connection to another LEPR molecule. An exemplary monomeric LEPR molecule is a molecule, herein referred to as hLEPR.mmh, containing the amino acid sequence according to SEQ ID NO: 90 (see, for example, Example 3 herein). As used herein, the expression human dimeric LEPR refers to a construct containing two LEPR molecules connected to each other via a linker, a covalent bond, a non-covalent bond, or a multimerization domain such as the Fc domain of an antibody. An exemplary dimeric LEPR molecule is a molecule referred to herein as hLEPR.mFc containing the amino acid sequence according to SEQ ID NO: 91 (see, for example, Example 3 herein) or a molecule referred to herein as hLEPR.hFc containing the amino acid sequence according to SEQ ID NO: 92. Expressions used herein, such as an antibody to LEPR, an antibody that specifically binds to LEPR, a LEPR-specific binding protein, and the like, unless specifically indicated otherwise,

- 5 040524 refers to molecules that bind to full-length human LEPR, monomeric human LEPR, dimeric human LEPR, or other constructs that contain an extracellular domain

LEPR or consist of it.

All references to proteins, polypeptides, and protein fragments herein are intended to refer to the human version of the corresponding protein, polypeptide, or protein fragment, unless explicitly stated to be of a non-human species. Thus, the expression LEPR means human LEPR, unless it is indicated that it belongs to a non-human species, such as mouse LEPR, monkey LEPR, etc.

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

As used herein, expressions such as an antibody to LEPR or an antibody that binds to the human leptin receptor include both monovalent antibodies with a single specificity, as well as bispecific antibodies containing a first arm that binds to LEPR and a second arm that binds to the second (target) antigen, and the shoulder to LEPR contains any of the sequences HCVR/LCVR or CDR, as shown in table. 1 in this document.

As used herein, the term antibody means any antigen-binding molecule or molecular complex containing at least one complementarity determining region (CDR) that specifically binds or interacts with a particular antigen (eg, LEPR). The term antibody includes immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains and two light (L) chains connected by disulfide bonds, as well as their multimers (eg, IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2 and C _H 3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or V _L ) and a light chain constant region. The light chain constant region contains one domain (CL1). The V _H and V _L regions can be further subdivided into regions of hypervariability, referred to as complementarity-determining regions (CDRs), which alternate with regions that are more conserved, referred to as framework regions (FRs). Each V _H and V _L consists of three CDRs and four FRs, arranged from amino to carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. According to various embodiments of the present invention, the FR of an anti-LEPR antibody (or antigen-binding portion thereof) may be identical to human germline sequences, or may be naturally or synthetically modified. The amino acid consensus sequence can be determined based on parallel analysis of two or more CDRs.

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

Non-limiting examples of antigen-binding fragments include the following: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv molecules (scFv); (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR), such as a CDR3 peptide) or a FR3-CDR3-FR4 conformationally restricted peptide. Other engineered molecules such as domain specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, diabodies, tribodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), immunopharmaceuticals small modular protein (SMTP) and shark IgNAR variable domains are also contemplated by the term antigen-binding fragment as used herein.

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

In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting, illustrative variable and constant domain configurations that can be found within an antigen-binding fragment of an antibody of the present invention include the following: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) V _H Ch1-Ch2; (v) Vh-Ch1-Ch2-Ch3; (vi) Vh-Ch2-Ch3; (vii) Vh-Cl; (viii) Vl-Ch1; (ix) Vl-Ch2; (x) Vl-Ch3; (xi) VlC _h 1-C _h 2; (xii) V _l -C _h 1-C _h 2-C _h 3; (xiii) V _L -C _H 2 -C _H 3 and (xiv) V _L -C _L . In any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may either be directly linked to each other, or may be linked through a complete or partial hinge or linker region. The hinge region may be at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in a flexible or semi-flexible connection between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present invention may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other and/or with one or more V _H or V _L monomeric domains. (for example, via disulfide(s) bond(s)).

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

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

In some embodiments, the antibodies of the present invention may be recombinant human antibodies. As used herein, the term recombinant human antibody is intended to include all human antibodies that are produced, expressed, generated, or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor et al. ( 1992) Nucl Acids Res 20:6287-6295) or antibodies, semi

- 7 040524 - 7 040524 expressed, created or isolated by any other means, which include splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies contain variable and constant regions derived from a human germline immunoglobulin sequence. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, if an animal that is transgenic for human Ig sequences, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and V _L regions of the recombinant antibodies are sequences which, while derived from human germline VH and _VL sequences and related to human germline VH and VL sequences, may not naturally exist within the in vivo human germline antibody repertoire.

The present invention relates to antibodies with one or more mutations in the hinge region, CH2 or CH3 region, which may be desirable, for example, upon receipt, to improve the yield of the desired form of antibodies.

The antibodies of the present invention may be isolated antibodies. As used herein, the term isolated antibody means an antibody that has been identified and isolated and/or recovered from at least one component of its natural environment. For example, an antibody that has been isolated or removed from at least one component of an organism, or from a tissue or cell in which the antibody exists or is naturally produced, is an isolated antibody for the purposes of the present invention. An isolated antibody also includes an antibody in situ in a recombinant cell. Isolated antibodies are antibodies that have undergone at least one purification or isolation step. In certain embodiments, the isolated antibody may be substantially free of other cellular material and/or chemical compounds.

The present invention provides variants of the anti-LEPR antibodies disclosed herein comprising one or more amino acid substitutions, insertions and/or deletions in the framework regions and/or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences, of which antibodies occur. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. The present invention provides antibodies and antigen-binding fragments thereof which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework regions and/or CDR regions are mutated to the corresponding residue(s) the germline sequence from which the antibody is derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as germline mutations). lines). One of ordinary skill in the art, starting from the heavy and light chain variable region sequences disclosed herein, can readily generate various antibodies and antigen binding fragments that contain one or more individual germline mutations, or combinations thereof. In certain embodiments, all framework and/or CDR region residues within the VH and/or VL domains are back-mutated to residues occurring in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues are backmutated back to the original germline sequence, such as only mutated residues occurring within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only mutated residues occurring within CDR1, CDR2, or CDR3. In other embodiments, one or more framework and/or CDR residues are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody is derived originally). Moreover, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework regions and/or CDR regions, for example, where certain individual residues are mutated to the corresponding residue of a particular germline sequence, while certain other residues differ from the original germline sequence are retained or mutated to the corresponding residue of the differing germline sequence. Once generated, antibodies and antigen-binding fragments that contain one or more germline mutations can be readily tested for one or more of the desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (depending on the specific situations), reduced

- 8 040524 immunogenicity, etc. Antibodies and antigen-binding fragments obtained by this general method are provided by the present invention.

Also provided by the present invention are anti-LEPR antibodies comprising variants of any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present invention provides anti-LEPR antibodies with the amino acid sequences HCVR, LCVR, and/or CDR, e.g., 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions with respect to any of the set of amino acid sequences HCVR, LCVR and/or CDR presented in table. 1 in this document. In certain embodiments, the present invention provides anti-LEPR antibodies comprising variant amino acid sequences of HCVR, LCVR, and/or CDRs relative to the sequences shown in Table 1. 1 herein (eg, containing conservative amino acid substitutions), wherein such variant antibodies nonetheless exhibit one or more of the functions and/or properties of the exemplary anti-LEPR antibodies disclosed herein.

The term epitope refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may contain more than one epitope. Thus, different antibodies may bind to different regions on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is formed by spatially adjacent amino acids from different segments of a straight chain polypeptide. A linear epitope is formed by adjacent amino acid residues in a polypeptide chain. In certain instances, an epitope may include fragments of saccharides, phosphorylated groups, or sulfonyl groups on an antigen.

The present invention provides anti-LEPR antibodies and antigen binding fragments thereof, which comprise amino acid sequences that are substantially similar or substantially identical to one or more variable domain or CDR amino acid sequences found in any of the exemplary anti-LEPR antibodies disclosed. in this document.

When applied to polypeptides, the term significant similarity or substantially similar means that two peptide sequences, when optimally aligned, for example, using the GAP or BESTFIT programs using default gap penalties, have at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, positions of residues that are not identical are distinguished by conservative amino acid substitutions. A conservative amino acid substitution is one in which an amino acid residue is replaced by another amino acid residue containing a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). Typically, a conservative amino acid substitution will not substantially change the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or percent similarity can be adjusted upwards to correct for the conservative nature of the substitution. Means for effecting this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, incorporated herein by reference. Examples of groups of amino acids that contain side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine and tryptophan; (5) main side chains: lysine, arginine and histidine; (6) acid side chains: aspartate and glutamate, and (7) sulfur side chains: cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysinarginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change characterized by a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445, incorporated herein by reference. A moderately conservative substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity scores assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, the GCG software contains programs such as Gap and Bestfit that can be used with

- 9 040524 default for determining sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species, or between a wild-type protein and its mutein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA using the default or recommended parameters program in GCG version 6.1. FASTA (eg, FASTA2 and FASTA3) provides alignments and sequence identity in percentage areas of best overlap between the query sequence and search sequences (Pearson (2000) earlier). Another preferred algorithm for comparing a sequence according to the present invention with a database containing a large number of sequences from different organisms is the BLAST computer program, especially BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389-402, each of which is incorporated herein by reference.

Anti-LEPR antibodies containing Fc variants.

In certain embodiments, the present invention provides anti-LEPR antibodies comprising an Fc domain containing one or more mutations that increase or decrease antibody binding to an FcRn receptor, such as at acidic versus neutral pH. For example, according to the present invention, antibodies to LEPR are provided that contain a mutation in the CH2 or CH3 region of the Fc domain, and the mutation(s) increase(s) the affinity of the Fc domain for FcRn in an acidic environment (for example, in the endosome, where the pH is in the range from about 5.5 to about 6.0). Such mutations can lead to an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, the following: modification at position 250 (eg, E or Q); 250 and 428 (for example, L or F); 252 (eg L/Y/F/W or T), 254 (eg S or T) and 256 (eg S/R/Q/E/D or T); or a modification at position 428 and/or 433 (eg H/L/R/S/P/Q or K) and/or 434 (eg H/F or Y); or modification at position 250 and/or 428; or a modification at position 307 or 308 (eg, 308F, V308F) and 434. In one embodiment, the modification comprises the following: modification 428L (eg, M428L) and 434S (eg, N434S); modification 428L, 259I (for example, V259I) and 308F (for example, V308F); modification 433K (for example, H433K) and 434 (for example, 434Y); modification 252, 254 and 256 (for example, 252Y, 254T and 256E); modification 250Q and 428L (for example, T250Q and M428L); and modification 307 and/or 308 (eg, 308F or 308P).

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

Anti-LEPR antibodies of the present invention may contain a modified Fc domain with reduced effector function. As used herein, the term modified Fc domain with reduced effector function means any Fc portion of an immunoglobulin that has been modified, mutated, truncated, etc. with respect to a naturally occurring wild-type Fc domain such that the modified Fc-containing molecule exhibits a reduction in the severity or extent of at least one effect selected from the group consisting of the following: cell killing (e.g., ADCC and/or CDC) , complement activation, phagocytosis, and opsonization, with respect to a reference molecule containing a naturally occurring version of the wild-type Fc portion. In certain embodiments, the modified Fc domain with reduced effector function is an Fc domain with reduced or weakened Fc receptor binding (eg, FcyR).

In certain embodiments of the present invention, the modified Fc domain is an IgG1 Fc variant or an IgG4 Fc variant containing a hinge substitution. For example, a modified Fc for use in the context of the present invention may comprise an IgG1 Fc variant in which at least one IgG1 Fc hinge amino acid is replaced with a corresponding IgG2 Fc hinge amino acid. Alternatively, a modified Fc for use in the context of the present invention may comprise a variant IgG4 Fc in which at least one IgG4 Fc hinge amino acid is replaced with a corresponding IgG2 Fc hinge amino acid.

Non-limiting, illustrative modified Fc regions that can be used in the context of the present invention are provided in US Patent Application Publication No. 2014/0243504, the entire disclosure of which is incorporated herein by reference, as well as any functionally equivalent variants of the modified Fc regions, presented in it.

Other modified Fc domains and Fc modifications that can be used in context

- 10 040524 ste of the present invention, include any of the modifications presented in the application for a US patent No. US 2014/0171623; US Pat. No. US 8,697,396; US Patent Application No. US 2014/0134162; International Patent Publication No. WO 2014/043361, the entire disclosure of which is incorporated herein by reference. Methods for constructing antibodies or other antigen-binding fusion proteins containing a modified Fc domain as described herein are known in the art.

Biological characteristics of antibodies.

The present invention provides antibodies and antigen-binding fragments thereof that bind to human LEPR and antagonize LEPR signaling. Such antibodies may be referred to herein as antagonistic antibodies. In the context of the present invention, a LEPR signaling antagonist means an antibody or fragment thereof that binds to LEPR and inhibits the intracellular effect that, under normal conditions, results from the interaction of leptin with LEPR in cells that express LEPR. In various embodiments, the antagonist antibodies of the present invention inhibit LEPR agonist function and/or LEPR partial agonist function. In certain embodiments, antagonistic action against LEPR signaling means inhibition of leptin-stimulated transcriptional activation of STAT3, which can be detected using any method that can measure or identify, directly or indirectly, STAT3 activity, for example, using a labeled version of STAT3 expressed in reporter cell line. For example, the present invention provides antagonist antibodies and antigen-binding fragments thereof that negatively regulate LEPR signaling in a cellular assay using reporter genes as described in Example 6, or a substantially similar assay. The present invention also provides antagonist antibodies and antigen-binding fragments thereof that exhibit complete inhibition of leptin-induced LEPR signaling with IC ₅₀ values ranging from 723 pM to 1.8 nM in the assay of Example 6 or a substantially similar assay. Cellular binding assays that detect antibody binding to LEPR-expressing cells, such as the assay described in Example 5 herein, demonstrated binding to HEK293/hLEPR-GPI cells with binding ratios that were 824-3187 times different from leptin-free and 398-3106 times different from the value in the presence of 1 μm leptin. The antibodies of the present invention were found to bind to LEPR even in the presence of an excess of 1 μM leptin, indicating that the anti-LEPR antibodies of the present invention bind to sites on hLEPR that do not overlap with leptin binding sites. The present invention also provides anti-LEPR antibodies that antagonize leptin signaling but also stimulate partial LEPR signaling in the absence of leptin; such antibodies are also referred to herein as partial agonists or antibodies that exhibit agonism in relation to LEPR signaling.

According to certain exemplary embodiments of the present invention, anti-LEPR antibodies are provided that bind to dimeric human LEPR (hLEPR.hFc, SEQ ID NO: 92) in the presence and absence of leptin, with none of the antibodies of the present invention exhibiting more than 40 % blockade of the LEPR:leptin interaction, for example, using the assay format as defined in Example 4 herein, or a substantially similar assay.

The present invention provides antibodies and antigen-binding fragments thereof that bind to monomeric human LEPR with high affinity. For example, the present invention provides anti-LEPR antibodies that bind to monomeric human LEPR (e.g., hLEPR.mmh, SEQ ID NO: 90) with a K _D value of less than about 10 nM as measured by surface plasmon analysis at 25 °C, or less than about 25 nM, as measured by surface plasmon analysis at 37°C, for example using the assay format as defined in Example 3 herein, or a substantially similar assay. In certain embodiments, anti-LEPR antibodies are provided that bind to monomeric human LEPR at 25° C. with a K _D value of less than about 12 nM, less than about 11 nM, less than about 10 nM, less than about 9 nM, less than less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, or less than about 100 pM , as measured by surface plasmon analysis, e.g. using the analysis format as defined in Example 3 herein m document, or a substantially similar analysis.

- 11 040524

The present invention also provides antibodies and antigen-binding fragments thereof that bind to monomeric human LEPR (e.g., hLEPR.mmh, SEQ ID NO: 90) with a dissociative half-life (t ¹ / ₂ ) of greater than about 5 minutes, as measured by surface plasmon analysis at 25°C, or greater than about 1 min as measured by surface plasmon analysis at 37°C, e.g. using the assay format as defined in Example 3 herein, or essentially similar analysis. In certain embodiments, anti-LEPR antibodies are provided that bind to monomeric human LEPR at 25° C. with a t/4 of greater than about 5 minutes, greater than about 10 minutes, greater than about 20 minutes, greater than about 40 minutes, greater than about 50 minutes or longer as measured by surface plasmon analysis, for example using the assay format as defined in Example 3 herein, or a substantially similar assay.

The present invention also provides antibodies and antigen-binding fragments thereof that bind to dimeric human LEPR (eg, hLEPR.mFc, SEQ ID NO: 91) with high affinity. For example, the present invention provides anti-LEPR antibodies that bind to dimeric human LEPR (e.g., hLEPR.mFc, SEQ ID NO: 91) with a K _D value of less than about 4 nM, as measured by surface plasmon analysis at 25 or 37°C, eg using the assay format as defined in Example 3 herein, or a substantially similar assay. In certain embodiments, anti-LEPR antibodies are provided that bind to dimeric human LEPR at 25° C. with a K _D value of less than about 15 nM, less than about 10 nM, less than about 9.0 nM, less than about 8, 0 nM, less than about 7.0 nM, less than about 6.0 nM, less than about 5.0 nM, less than about 4.0 nM, less than about 3.0 nM, less than about 2.0 nM , less than about 1.0 nM, less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM , less than about 200 pM, or less than about 100 pM, as measured by surface plasmon analysis, for example using the assay format as defined in Example 3 herein, or essentially the same analysis.

The present invention also provides antibodies and antigen-binding fragments thereof that bind to dimeric human LEPR (e.g., hLEPR.mFc, SEQ ID NO: 91) with a dissociative half-life (t ¹ /2) of greater than about 10 minutes, as measured by surface plasmon analysis at 25 or 37°C, for example using the assay format as defined in Example 3 herein, or a substantially similar assay. In certain embodiments, anti-LEPR antibodies are provided that bind to dimeric human LEPR at 25°C with a t ¹ /2 value of greater than about 15 minutes, about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than greater than 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than 80 minutes, greater than 90 minutes, greater than 100 minutes, or longer, as measured by surface plasmon analysis, e.g., using the assay format as defined in example 3 herein, or a substantially similar analysis.

The present invention also provides antibodies and antigen-binding fragments thereof that bind to LEPR in complex with human leptin (the expression of LEPR in complex with human leptin can also be represented by the expression leptin:LEPR). For example, the present invention provides antibodies and antigen-binding fragments thereof that are capable of binding to a preformed complex comprising hLEPR and human leptin. In other words, according to certain embodiments, the interaction between antibodies to LEPR and LEPR is not inhibited by the presence of leptin in complex with LEPR; likewise, the interaction between leptin and LEPR according to the present aspect of the present invention is not inhibited by the presence of an anti-LEPR antibody. An exemplary assay format for determining whether an antibody, or antigen-binding fragment thereof, binds to LEPR in complex with human leptin is provided in Example 4 herein.

The present invention also provides antibodies and antigen-binding fragments thereof that bind to cell surface expressed LEPR in the presence and/or absence of human leptin. Cell surface-expressed LEPR means LEPR or a portion thereof (e.g., the extracellular portion of LEPR) expressed on the cell surface, either naturally or in an engineered cell line, such that the antibody or antigen-binding fragment thereof is capable of binding to the LEPR molecule. In certain embodiments, cell-surface-expressed LEPR includes recombinant complexes comprising an extracellular LEPR domain linked to a cell via a tag or anchor (eg, a GPI anchor, as illustrated in Example 6 herein). According to

An aspect of the present invention provides antibodies that are capable of binding to cell surface expressed LEPR in the absence of leptin, and are also capable of binding to cell surface expressed LEPR in the presence of leptin (i.e. under conditions where leptin is able to bind to expressed on the LEPR cell surface). In other words, in certain embodiments, the interaction between anti-LEPR antibodies and cell surface expressed LEPR is not inhibited by the presence of leptin in complex with cell surface expressed LEPR. Antibodies according to the present aspect of the present invention are capable of forming a three-membered complex on the cell surface containing an antibody expressed on the cell surface of LEPR and leptin. An exemplary assay format for determining whether an antibody or antigen-binding fragment thereof is capable of binding to cell surface expressed LEPR in the presence and absence of human leptin is provided in Example 5 herein.

The antibodies of the present invention may have one or more of the aforementioned biological characteristics, or any combination thereof. It is understood that the above list of biological characteristics of antibodies according to the present invention is not exhaustive. Other biological characteristics of the antibodies of the present invention will become apparent to those of ordinary skill in the art from the overview of the present disclosure, including the illustrative examples herein.

Epitope mapping and related technologies.

The epitope to which antibodies of the present invention bind may consist of a single contiguous sequence of 3 or more (e.g., 3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20 or more) amino acids of the LEPR protein. Alternatively, an epitope may be composed of a plurality of non-contiguous amino acids (or amino acid sequences) of the LEPR. In some embodiments, the epitope is located on or near the leptin-binding domain of LEPR. In other embodiments, the epitope is located at a region remote from the leptin-binding domain of LEPR, such as at a position on the surface of the LEPR where the antibody, when bound to such an epitope, does not interfere with leptin binding to LEPR.

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

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

Whether an antibody binds to the same epitope as a reference anti-LEPR antibody or competes for binding with a reference anti-LEPR antibody can be determined using conventional methods known in the art and illustrated herein. For example, to determine if an antibody of interest binds to the same epitope as a reference anti-LEPR antibody of the present invention, the reference antibody is allowed to bind to the LEPR protein. Next, the ability of the test antibody to bind to the LEPR molecule is evaluated. If the test antibody is able to bind to LEPR after saturation binding to the reference anti-LEPR antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-LEPR antibody. On the other hand, if the test antibody is unable to bind to the LEPR molecule after saturation binding to the reference anti-LEPR antibody, then the test antibody

- 13 040524 can bind to the same epitope as the epitope to which the reference anti-LEPR antibody of the present invention binds. Additional routine experiments (e.g., peptide mutation assays and binding assays) can then be performed to confirm whether the observed lack of binding of the test antibody is due to binding to the same epitope as the reference antibody, or steric blocking (or other phenomenon). ) is responsible for the lack of observed binding. Experiments of this kind can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of the present invention, two antibodies bind to the same (or overlapping) epitope if, for example, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits binding of the other by at least 50 %, but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, for example, Junghans et al. (1990) Cancer Res. 50:1495-1502). Alternatively, two antibodies are considered to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other. Two antibodies are considered to have overlapping epitopes if only a subset of amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

To determine if an antibody competes for binding (or cross-competes for binding) with a reference anti-LEPR antibody, the binding procedure described above is performed in two orientations. In the first orientation, the reference antibody is allowed to bind to the LEPR protein under saturating conditions, followed by evaluation of the binding of the test antibody to the LEPR molecule. In the second orientation, the test antibody is allowed to bind to the LEPR molecule under saturating conditions, followed by evaluation of reference antibody binding to the LEPR molecule. If, in both orientations, only the first (saturating) antibody is able to bind to the LEPR molecule, then it can be concluded that the test antibody and the reference antibody compete for binding to LEPR. One of ordinary skill in the art will appreciate that an antibody that competes for binding with a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding to an overlapping or contiguous epitope.

Obtaining human antibodies.

Anti-LEPR antibodies of the present invention may be fully human antibodies. Methods for making monoclonal antibodies, including fully human monoclonal antibodies, are known in the art. Any such known methods can be used in the context of the present invention to obtain human antibodies that specifically bind to human LEPR.

Using technology such as VELOCIMMUNE™ or any other similar known method to generate fully human monoclonal antibodies, high affinity chimeric anti-LEPR antibodies with a human variable region and a mouse constant region are first isolated. As in the experimental section below, antibodies are characterized and selected for the desired characteristics, including affinity, ligand blocking activity, selectivity, epitope, and so on. If necessary, mouse constant regions are replaced with the desired human constant region, such as wild-type IgG1 or IgG4 or modified IgG1 or IgG4 to generate a fully human anti-LEPR antibody. While the selected constant region may vary depending on the particular application, characteristics of high antigen binding affinity and target specificity are in the variable region. In certain instances, fully human anti-LEPR antibodies are isolated directly from antigen-positive B cells.

Bioequivalents.

Anti-LEPR antibodies and antibody fragments of the present invention encompass proteins with amino acid sequences that may differ from the amino acid sequences of the antibodies described, but which retain the ability to bind to human LEPR. Such variant antibodies and antibody fragments contain one or more amino acid insertions, deletions, or substitutions compared to the original sequence, but exhibit biological activity that is substantially equivalent to that of the described antibodies. Similarly, DNA sequences encoding an anti-LEPR antibody of the present invention encompass sequences that contain one or more nucleotide insertions, deletions, or substitutions compared to the disclosed sequence, but which encode an anti-LEPR antibody or antibody fragment that is substantially bioequivalent to in relation to the antibody to LEPR or antibody fragment according to the present invention. Examples of such variant amino acid and DNA sequences are discussed above.

Two antigen-binding proteins, or antibodies, are considered to be bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives, the rate and extent of absorption of which do not show a significant difference when administered in the same mo

- 14 040524 lar dose under similar experimental conditions, either as a single dose or as multiple doses. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in their extent of absorption, but not in their rate of absorption, and yet they can be considered bioequivalent because such differences in absorption rate are intentional and reflected on the label, are not significant for achieving effective concentrations of the drug in the body, for example, with chronic use, and are considered medically negligible for a particular investigational drug.

In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity, and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if the patient can switch one or more times between a reference product and a biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity or reduced efficacy, compared to continuing therapy without such a switch. .

In one embodiment, two antigen-binding proteins are bioequivalent if they both act through a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.

Bioequivalence can be demonstrated using in vivo and in vitro methods. Bioequivalence measurements include, for example, (a) an in vivo test in humans or other mammals, in which the concentration of an antibody or its metabolites is measured in blood, plasma, serum, or other body fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of in vivo human bioavailability data; (c) an in vivo test in humans or other mammals, in which the corresponding acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) a well-controlled clinical trial that establishes the safety, efficacy, or bioavailability or bioequivalence of an antibody.

Bioequivalent variants of the anti-LEPR antibodies of the present invention can be constructed, for example, by making various residue or sequence substitutions, or deleting terminal or internal residues or sequences not necessary for biological activity. For example, cysteine residues that are not essential for biological activity can be removed or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation. In other contexts, bioequivalent antibodies may include LEPR antibody variants containing amino acid substitutions that modify the glycosylation characteristics of the antibodies, such as mutations that abrogate or remove glycosylation.

Species selectivity and species cross-reactivity.

The present invention, in certain embodiments, provides anti-LEPR antibodies that bind to human LEPR but not to LEPR of other species. The present invention also provides anti-LEPR antibodies that bind to human LEPR and to LEPR of one or more non-human species. For example, the anti-LEPR antibodies of the present invention may bind to human LEPR and may or may not bind, as the case may be, to one or more of the following: mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, monkey, rhesus monkey or chimpanzee. In accordance with certain exemplary embodiments of the present invention, anti-LEPR antibodies are provided that specifically bind human LEPR and cynomolgus monkey (eg, Macaca fascicularis) LEPR. Other anti-LEPR antibodies of the present invention bind to human LEPR but do not bind or weakly bind to cynomolgus monkey LEPR.

multispecific antibodies.

The antibodies of the present invention may be monospecific or multispecific (eg, bispecific). Multispecific antibodies may be specific for different epitopes on the same target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, for example, Tutt et al. (1991) J. Immunol. 147:60-69; Kufer et al. (2004) Trends Biotechnol. 22:238-244. Anti-LEPR antibodies of the present invention may be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or antibody fragment may be operably linked (e.g., by chemical bonding, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with a second binding specificity.

According to the present invention, bispecific antibodies are provided in which one

- 15 040524 the immunoglobulin arm binds to human LEPR, the other arm of the immunoglobulin is specific for the second antigen. The LEPR binding arm may comprise any of the HCVR/LCVR or CDR amino acid sequences as shown in Table 1. 1 in this document.

An exemplary bispecific antibody format that can be used in the context of the present invention involves the use of a first C _H 3 domain of an immunoglobulin (Ig) and a second C _H 3 Ig domain, wherein the first and second C _H 3 Ig domains differ from each other by at least one amino acid. , and at the same time, at least one amino acid difference reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody in which there is no amino acid difference. In one embodiment, the first C _H 3 Ig domain binds protein A and the second Ig CH3 domain contains a mutation that reduces or abolishes protein A binding, such as an H95R modification (according to IMGT exon numbering; H435R according to EU numbering). The second CH3 may further comprise the modification Y96F (as per IMGT; Y436F as per EU). Additional modifications that can be found within the second CH3 include: D16E, L18M, N44S, K52N, V57M and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M and V422I according to EU) in the case of IgG1 antibodies; N44S, K52N and V82I (IMGT; N384S, K392N and V422I according to EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (as per IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I as per EU) for IgG4 antibodies. Variants of the bispecific antibody format described above are contemplated by the scope of the present invention.

Other illustrative bispecific formats that can be used in the context of the present invention include, without limitation, for example, scFv-based bispecific formats or bispecific formats - diabodies, IgG-scFv fusions, dual variable domain (DVD) -Ig, quadroma, ridges - in-cavities, common light chain (e.g., common light chain with ledge-in-cavities, etc.), CrossMab, CrossFab, antibody containing a chain replacement engineered domain (SEED), leucine zipper, Duobody, IgG1 /IgG2, Fab (DAF)-IgG dual action and Mab ² bispecific formats (See e.g. Klein et al. (2012) mAbs 4:6, 1-11 and references cited therein for an overview of the above formats ). Bispecific antibodies can also be constructed using peptide-nucleic acid conjugation, for example, in which unnatural amino acids with different chemical reactivity are used to create site-specific antibody-oligonucleotide conjugates, which then self-assemble into multimeric complexes of desired composition, valence, and geometry. (See, for example, Kazane et al., J. Am. Chem. Soc. (Epub: Dec. 4, 2012)).

Therapeutic composition and administration.

The present invention relates to pharmaceutical compositions containing antibodies to LEPR or their antigennegative fragments according to the present invention. Pharmaceutical compositions according to the present invention are formulated with suitable carriers, excipients and other means that provide improved transport, delivery, tolerability and the like. A variety of suitable formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, Carbowax emulsions (polyethylene glycols of various molecular weights), semi-solid gels and semi-solid mixtures containing Carbowax. See also Powell et al. Compendium of excipients for parenteral formulations PDA (1998) J Pharm Sci Technol 52:238-311.

The dose of antibody administered to a patient may vary depending on the age and size of the patient, the target disease, conditions, route of administration, and the like. The preferred dose, as a rule, is calculated depending on body weight or body surface area. In an adult patient, intravenous administration of an antibody of the present invention is advantageous, typically at a single dose of about 0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5 or about 0.05 - about 3 mg/kg of body weight. Depending on the severity of the condition, the frequency and duration of treatment may be adjusted. Effective dosages and regimens for administering anti-LEPR antibodies can be determined empirically, eg patient progression can be monitored by periodic evaluation and the dose can be adjusted accordingly. Moreover, cross-species dosing can be performed using well-known methods in the art (eg, Mordenti et al. (1991) Pharmaceut. Res. 5:1351).

Various delivery systems are known that can be used to administer the pharmaceutical composition of the present invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al. (1987) J Biol Chem 262:4429-4432). Routes of administration include, without limitation, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucous membranes (for example, through the oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with other biologically active means. Administration may be systemic or local.

The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, with respect to subcutaneous delivery, the pen delivery device easily finds use in delivering the pharmaceutical composition of the present invention. Such a pen delivery device may be reusable or disposable. A refillable pen delivery device typically uses a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been introduced and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. There is no replaceable cartridge in the disposable syringe pen delivery device. In contrast, a disposable pen delivery device comes pre-filled with a pharmaceutical composition contained in a reservoir within the device. Once the reservoir is empty of the pharmaceutical composition, the entire device is discarded.

Numerous reusable pen and autoinjector delivery devices find use in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, without limitation, the following: AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/25™ pen, pen HUMALOG™, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, Inn.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark) ), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™ and OPTICLIK™ (sanofi-Aventis, Frankfurt, Germany), to name but a few. Examples of disposable pen delivery devices that find their use in the subcutaneous delivery of a pharmaceutical composition of the present invention include, without limitation, the following: SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk) and KWIKPEN™ (Eli Lilly ), SURECLICK™ autoinjector (Amgen, Thousand Oaks, Calif.), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMIRA™ pen (Abbott Labs, Abbott Park, Ill.), to name but a few of them.

In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, formerly; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201). According to another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.) (1974) CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled release system can be placed in close proximity to the target of the composition so that only a fraction of the systemic dose is needed (see, for example, Goodson (1984) in Medical Applications of Controlled Release, formerly, vol. 2, pp. 115-138). Other controlled release systems are discussed in Langer (1990) Science 249:1527-1533.

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

The pharmaceutical compositions for oral or parenteral administration described above are advantageously prepared in unit dosage form which corresponds to the dosage of the active ingredients. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, and the like. Quantity contained

- 17 040524 of the above antibodies, as a rule, is from about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injections, it is preferred that the specified antibody is contained in an amount of from about 5 to about 100 mg and from about 10 to about 250 mg for other dosage forms.

Therapeutic applications of antibodies.

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

The antibodies of the present invention are useful, inter alia, in the treatment, prevention and/or amelioration of any disease or disorder associated with or mediated by elevated levels of leptin (hyperleptinemia) and/or overexpression of the leptin OB-R receptor, resulting in excessive signal transduction. LEPR. In various embodiments, the disease or disorder is selected without limitation from the following: anorexia or other psychiatric eating disorders, chronic kidney disease cachexia, other cachexia such as congestive heart failure cachexia, pulmonary cachexia, radiation cachexia and cancer cachexia, autoimmune disorders, such as inflammatory bowel disease, lupus erythematosus, multiple sclerosis, psoriasis, cardiovascular disease, high blood pressure, depression, neurodegenerative disorders, cancer such as renal cell carcinoma, melanoma and breast cancer.

The present invention also provides anti-LEPR antibodies and antigen-binding fragments thereof which are useful for antagonizing LEPR signaling in cells, tissues and organs expressing normal or high levels of leptin. As used herein, a LEPR mutant that exhibits enhanced signaling in the presence of leptin (compared to wild-type LEPR) is referred to as an enhanced signaling LEPR mutant. An exemplary signal-enhanced LEPR mutation is LEPR-Q223R (Chagnon et al. (2009) Journal of Clinical Endocrinology & Metabolism 85(l):29-34). Thus, according to the present invention, anti-LEPR antibodies and antigen-binding fragments thereof are provided that are useful in the treatment, prevention and/or amelioration of diseases and disorders caused by or associated with LEPR enhanced signaling mutants.

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

Types of combination therapy and formulations.

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

The anti-LEPR antagonist antibodies of the present invention can be co-formulated and/or administered in combination with one or more additional therapeutically active ingredients, such as, for example, pharmaceutical products prescribed for the treatment of the following: congestive heart failure cachexia, pulmonary cachexia, and cancer cachexia, autoimmune disorders such as inflammatory bowel disease, lupus erythematosus, multiple sclerosis, psoriasis, cardiovascular disease, high blood pressure, neurodegenerative disorders, depression, cancer such as renal cell carcinoma, melanoma, breast cancer, and other diseases and disorders, associated with or caused by decreased leptin signaling. Examples of such additional therapeutically active ingredients include, without limitation, for example, the following: angiotensin-converting enzyme inhibitors (e.g., ACE, ACE-I, benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramapril, trandolapril), blockers angiotensin receptors (eg, ARB), smooth muscle relaxants (hydralazine), long-acting nitrates (eg, isosorbide dinitrate, eg, with or without ACE-I and ARB), diuretics (eg, loop diuretics, thiazide-like diuretics, and potassium-sparing diuretics) , treatment of iron deficiency anemia (eg, parenteral iron), long- or short-acting bronchodilators (eg, β2 agonists such as arformoterol, bufenin, clenbuterol, dopexamine, epinephrine, fentotherol, formoterol, isoetarin, isoprenaline, isoproterenol, levosalbutamol, orciprenaline, pirbuterol, prokaterol, ritodrine, salbutamol, albuterol, terbutaline, tiotropium), anticholinergic drugs agents (eg, hyoscyamine, atropine, phenobarbital, scopolamine, dicyclomine, phenobarbital, mepenzolate, and combinations thereof, such

- 18 040524 as atropine, hyoscyamine, phenobarbital and scopolamine), corticosteroids (eg hydrocortisone, hydrocortisone-17-valerate, hydrocortisone-17-butyrate, prednisone, prednisolone, triamcinolone acetonide, triamcinolone alcohol, betamethasone, dexamethasone, fluocortolone, flunisolide, budesonide ), long-acting antibiotics (eg, macrolides such as erythromycin, azithromycin, and methylxanthines such as theophylline), non-steroidal anti-inflammatory drugs (NSAIDs) (eg, aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, naproxen, oxaprozin, piroxicam, salsalat, sulindac, tolmetin, etc.), 5-aminosalicylic acids (5ASA) (e.g. mesalazine), immunosuppressants (e.g. prednisone, TNF inhibitors, azathioprine, methotrexate, 6-mercaptopurine), medicines for relapsing-remitting multiple sclerosis (eg, interferon beta-1a, interferon beta-1b, glatiramer acetate, mitoxantrone, natalizumab, fingolimod, terif lunomide, dimethyl fumarate, alemtuzumab, daclizumab, anti-CD20 monoclonal antibodies such as rituximab, ocrelizumab, and ofatumumab), topical psoriasis agents (eg, topical agents such as para-aminobenzoic acid, coconut oil, coal tar, dithranol, corticosteroids such as deoxymethasone, fluocinonide, vitamin D ₃ and other vitamin D analogs, psoralen and systemic agents such as methotrexate, cyclosporine, hydroxycarbamide, fumarates such as dimethyl fumarate and retinoids), TNF-α antagonists (eg, infliximab, adalimumab, golimumab, and certolizumab pegol ), drugs for psoriasis that target T cells (eg, efalizumab and alefacept), drugs for hypertension and cardiovascular disease (eg, aspirin, statins such as atorvastatin, fluvastatin, lovastatin, mavastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin and combinations thereof such as simvastatin+ezetimibe, lovastatin+niacin and atorvastatin+am lodipine), drugs for neurodegenerative disorders (eg, dimebon and proline-rich peptide (PRP)-1), antidepressants (eg, sertraline, citalopram, fluoxetine, escitalopram, trazodone, venlafaxine, bupropion, duloxetine, paroxetine, amitriptyline, venlafacin, desvenlafaxine and nortriptyline) and anticancer drugs (eg, sorafenib, JX-594, interleukin-2, ipilimumab (Yervoy), nivolumab (Opdivo), pembrolizumab (Keytruda), vemurafenib (Zelboraf), dabrafenib (Tafinlar), trametinib (Mekinist), anthracyclines such as doxorubicin (Adriamycin®), epirubicin (Ellence®), taxanes such as paclitaxel (Taxol®) and docetaxel (Taxotere®), 5-fluorouracil (5-FU), cyclophosphamide (Cytoxan®), carboplatin (Paraplatin ®) or a combination of them).

Introduction schemes.

In certain embodiments of the present invention, multiple doses of an anti-LEPR antagonist antibody (or a pharmaceutical composition comprising combinations of an anti-LEPR antagonist antibody and any of the additional therapeutically active agents mentioned herein) may be administered to a subject over a period of time. Methods according to this aspect of the present invention provide for sequential administration of multiple doses of an anti-LEPR antibody of the present invention to a subject. As used herein, the term sequential administration means that each dose of an anti-LEPR antibody is administered to a subject at a different time point, eg, on different days separated by a predetermined interval (eg, hours, days, weeks, or months). The present invention provides methods that sequentially administer to a patient one initial dose of an anti-LEPR antibody followed by one or more secondary doses of an anti-LEPR antibody and optionally followed by one or more tertiary doses of an anti-LEPR antibody.

The terms initial dose, secondary doses, and tertiary doses refer to the time sequence of administration of an anti-LEPR antibody of the present invention. Thus, the initial dose is the dose that is administered at the beginning of the treatment regimen (also called the initial dose, loading dose, initial dose, and the like)); secondary doses are doses that are administered after the initial dose; and tertiary doses are doses that are administered after secondary doses. The initial, secondary, and tertiary doses may contain the same amount of LEPR antibody, but generally may differ from each other in terms of frequency of administration. However, in certain embodiments, the amount of anti-LEPR antibody contained in the initial, secondary, and/or tertiary doses varies relative to each other (eg, adjusted higher or lower as appropriate) over the course of treatment. In certain embodiments, two or more (eg, 2, 3, 4, or 5) doses are administered at the start of the treatment regimen as loading doses followed by subsequent, less frequently administered doses (eg, maintenance doses).

Diagnostic and analytical applications of antibodies.

Anti-LEPR antibodies of the present invention can also be used to detect and/or measure LEPR or LEPR-expressing cells in a sample, for example, for diagnostic purposes. For example, an anti-LEPR antibody, or fragment thereof, can be used to diagnose a condition or disease characterized by aberrant expression (eg, overexpression, underexpression, lack of expression, etc.) of LEPR. Illustrative diagnostic

- 19 040524 LEPR assays may include, for example, contacting a sample obtained from a patient with an anti-LEPR antibody of the present invention, wherein the anti-LEPR antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-LEPR antibody can be used for diagnostic purposes in combination with a secondary antibody that is itself labeled with a detectable label. The detectable label or reporter molecule may be a radioactive isotope such as ³ H, ¹⁴ C, ³² P, ³⁵ S, or ¹²⁵ I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase or luciferase. Specific exemplary assays that can be used to detect or measure LEPR in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in LEPR diagnostic assays in accordance with the present invention include any tissue or fluid sample obtained from a patient that contains detectable amounts of LEPR protein or fragments thereof under normal or pathological conditions. Typically, LEPR levels in a particular sample obtained from a healthy patient (eg, a patient not suffering from a disease or condition associated with abnormal LEPR levels or activity) will be measured to initially establish a baseline or standard LEPR level. This baseline LEPR content can then be compared to LEPR levels measured in samples obtained from individuals suspected of having a disease or condition associated with LEPR.

Examples

The following examples are provided so that those skilled in the art will have a full disclosure and description of how to make and use the methods and compositions of the present invention and are not intended to limit the scope of what the present inventors consider their invention. Efforts have been made to ensure accuracy with respect to the numbers used (eg quantities, temperature, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

Example 1 Design of antigen-binding proteins that specifically bind to the leptin receptor (LEPR).

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

Certain biological properties of exemplary anti-LEPR antibodies prepared according to the methods of the example are described in detail in the examples below.

Example 2 Heavy and Light Chain Variable Region Amino Acid and Nucleic Acid Sequences.

In table. 1 shows the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-LEPR antibodies of the present invention. The corresponding nucleic acid sequence identifiers are shown in Table 1. 2.

Table 1. Amino acid sequence identifiers

SEQID NO: Antibody designation HCVR HCDR1 HCDR2 HDDR3 LCVR LCDR1 LCDR2 LCDR3 H4H17322R2 2 4 6 8 10 12 14 16 H4H18437R2 18 20 22 24 10 12 14 16 H4N18439R2 26 28 thirty 32 10 12 14 16 H4H18440R2 34 36 38 40 10 12 14 16 H4H18457R2 42 44 46 48 10 12 14 16 H4H18462R2 50 52 54 56 10 12 14 16 H4H18464R2 58 60 62 64 10 12 14 16 H4H18466R2 66 68 70 72 10 12 14 16 H4N18508R2 74 76 78 80 82 84 86 88

- 20 040524

Table 2. Nucleic acid sequence identifiers

SEQID NO: Antibody designation HCVR HCDR1 HCDR2 HDDR3 LCVR LCDR1 LCDR2 LCDR3 H4H17322R2 1 3 5 7 9 eleven 13 15 H4H18437R2 17 19 21 23 9 eleven 13 15 H4N18439R2 25 27 29 31 9 eleven 13 15 H4H18440R2 33 35 37 39 9 eleven 13 15 H4H18457R2 41 43 45 47 9 eleven 13 15 H4H18462R2 49 51 53 55 9 eleven 13 15 H4H18464R2 57 59 61 63 9 eleven 13 15 H4H18466R2 65 67 69 71 9 eleven 13 15 H4N18508R2 73 75 77 79 81 83 85 87

Antibodies are generally referred to herein according to the following nomenclature: an Fc prefix (eg, H4H, H1M, H2M, etc.) followed by a numeric identifier (eg, 17322, 18457, etc.) followed by a suffix P or N. Thus, according to this nomenclature, an antibody may be referred to herein, for example, as H4H17322P2, H4H18457P2, etc. The Fc prefixes in antibody designations used herein (H4H, H1M, and H2M) indicate the specific isotype of the Fc region of the antibody. For example, the H4H antibody contains human IgG4 Fc, while the H1M antibody contains mouse IgG1 Fc (all variable regions are fully human as indicated by the first H in the antibody designation). One of ordinary skill in the art will appreciate that an antibody with a particular Fc isotype can be converted into an antibody with a different Fc isotype (e.g., a mouse IgG1 Fc antibody can be converted into a human IgG4 antibody, etc.), but in any case, the variable domains (including CDR), which are indicated using the numerical identifiers shown in the table. 1 and 2 will remain the same and the binding properties are assumed to be identical or substantially similar regardless of the nature of the Fc domain.

Reference antibody. The reference antibody used in the following examples refers to the Fab 9F8 described in Fazeli et al. (2006) J Immunol Methods 312:190-200 and Carpenter et al. (2012) Structure 20(3):487-97.

Example 3 Surface Plasma Analysis Binding Affinity Values and Kinetic Constants of Human LEPR Monoclonal Antibodies.

Dissociation equilibrium constants (KD values) for antigen binding to the purified anti-LEPR monoclonal antibodies of the present invention were determined using a real-time surface plasmon biosensor using a Biacore 4000 instrument. All binding studies were performed in running buffer containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% v/v. Surfactant Tween-20, pH 7.4 (running buffer HBS-ET) at 25 and 37°C. The Biacore sensor surface was first derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (GE, Cat # BR-1008-39) to capture anti-LEPR monoclonal antibodies. LEPR reagents tested for binding to anti-LEPR monoclonal antibodies included recombinant extracellular domain of human LEPR expressed with a C-terminal myc-myc-hexahistidine tag (hLEPR.MMH; SEQ ID NO: 90), recombinant extracellular domain of javanese LEPR macaque expressed with C-terminal myc-myc-hexahistidine tag (mfLEPR.MMH; SEQ ID NO: 93), recombinant extracellular domain of mouse LEPR expressed with C-terminal myc-myc-hexahistidine tag (mLEPR.MMH; SEQ ID NO : 94), a recombinant rat LEPR extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (rLEPR.MMH; SEQ ID NO: 95), and a recombinant human LEPR extracellular domain expressed with a mouse IgG2a Fc C-terminal tag ( hLEPR.mFc; SEQ ID NO: 91). LEPR constructs that include MMH tags are monomeric LEPR constructs, and LEPR constructs that include an mFc tag are dimeric LEPR constructs. Various concentrations of LEPR reagents were first prepared in HBS-ET running buffer at final concentrations ranging from 100 nM to 3.7 nM in 3-fold serial dilutions and then injected onto the surface captured by anti-LEPR monoclonal antibodies for 4 min at a volume velocity a flow of 30 µl/min. The dissociation of the monoclonal antibody-bound LEPR reagent was monitored for 10 min in running HBS-ET buffer. Kinetic rate constants of association (ka) and dissociation (kd) were determined by fitting real-time binding sensorgrams to a 1:1 mass transfer-limited binding model using Scrubber 2.0c curve fitting software. Dissociation equilibrium constants for binding (K _D ) and dissociative half-lives (t ¹ / ₂ ) were calculated from the kinetic rate constants as follows:

kd 1p(2)

K _D (M) = - , and 1½ (min) = - ^{v 7} ka ^{v 7} 60xkd LEPR according to the present invention at 25 and 37°C are shown in table. 3-13.

Table 3. Binding Kinetic Parameters of Anti-LEPR Antibodies Binding to hLEPR-MMH at 25°C

Captured mAb mAb capture rate (RU, response units) 100 nM bound. hLEPRMMH (EO) ka (1/mol*s) kd (1/s) kd (M) 1½ (min) H4H17322R2 178 ± 1.2 66 4.38E±04 2.24Е-04 5D1E-09 52 H4H18437R2 171±0.5 56 5.74Е±04 3.75Е-03 6.53Е-08 3.1 H4N18439R2 156±0.7 21 1.07Е±04 1.1 SE-OZ 1.06E-07 10 H4H18440R2 170±0.5 71 4.23Е±04 4.97Е-04 1D8E-08 23 H4H18457R2 166±0.9 119 2.55E±05 2.52E-03 9.89Е-09 5 H4H18462R2 175±0.6 34 6.79Е±04 7.65Е-03 1.1 SE-07 1.5 H4H18464R2 160±0.4 60 1.24Е±05 1.1 SE-OZ 9.09Е-09 10 H4H18466R2 161±0.5 24 2.64Е±04 5D1E-03 1.94Е-07 2.3 H4N18508R2 180±1.5 127 1.76E±05 1.67Е-03 9.53Е-09 7 Antibody comparisons 380±8.2 26 4.48E±04 7.46Е-05 1.66Е-09 155 isotypic control antibody 171±0.4 4 NB* NB* NB* NB*

NB* indicates that no binding was observed under the current experimental conditions.

Table 4. Binding Kinetic Parameters of Anti-LEPR Antibodies Binding to hLEPR-MMH at 37°C

Captured mAb mAb capture level (EO) 100 nM bound. hLEPRMMH (EO) ka (1/mol*s) kd (1/s) Kd (M) 1½ (min) H4H17322R2 225±3 99 6.95E±04 8D2E-04 1D7E-08 14 H4H18437R2 213±3 55 1.14Е±05 8.84Е-03 7.76Е-08 1.3 H4N18439R2 180±1.5 19 2.32E±04 4.69E-03 2.02E-07 2.5 H4H18440R2 207±1.2 95 6.34Е±04 1.73Е-03 2.73Е-08 7 H4H18457R2 198±1.6 108 3.45E±05 8.55Е-03 2.48Е-08 1.4 H4H18462R2 204±2.1 31 4.58E±04 1D4E-02 2.48Е-07 1.0 H4H18464R2 185±1.2 68 1.54Е±05 ZD5E-OZ 2.05Е-08 3.7 H4H18466R2 184 ± 1.3 26 4.55E±04 6.74Е-03 1.48Е-07 1.7 H4N18508R2 204±1.4 117 2.69Е±05 6.88Е-03 2.55Е-08 1.7 Antibody comparisons 315±7.6 37 5.39E±04 4.81E-04 8.92E-09 24 isotypic control antibody 193±1.5 6 NB* NB* NB* NB*

NB* indicates that no binding was observed under the current experimental conditions.

As shown in Table. 3, at 25° C., all anti-LEPR monoclonal antibodies of the present invention bound hLEPR-MMH with KD values ranging from 5.11 to 194 nM. As shown in Table. 4, at 37° C., all of the anti-LEPR monoclonal antibodies of the present invention bound hLEPR-MMH with KD values ranging from 11.7 to 248 nM.

Table 5. Binding Kinetic Parameters of Anti-LEPR Antibodies Binding to mfLEPR-MMH at 25°C

Captured shah Capture level mAb (EO) 100 nM bound. mfLEP R-MMH (EO) ka (1/mol*s) k _d (1/s) Kd (M) 1½ (min) H4H17322R2 177±0.5 102 7.02E±04 2.92E-04 4D6E-09 40 H4H18437R2 171±0.3 70 6.87E±04 4.05Е-03 5.89Е-08 2.9 H4N18439R2 155±0.5 22 1.57E±04 1.52E-03 9.63Е-08 8 H4H18440R2 170±0.9 67 4.95E±04 2.68E-03 5.41Е-08 4.3 H4H18457R2 165±0.4 133 2.73Е±05 3.31Е-03 1.22E-08 3.5 H4H18462R2 174±0.4 42 7.42E±04 1,ZZE-02 1.79Е-07 0.9 H4H18464R2 159±0.4 15 3.82E±04 2.31Е-03 6.04Е-08 5.0 H4H18466R2 161±0.5 thirty 4.50E±04 6.69Е-03 1.48Е-07 1.7 H4N18508R2 178±0.4 145 2.04Е±05 1.63E-03 8.00E-09 7 Antibody comparisons 359±4.8 1 NB* NB* NB* NB* Isotype control antibody 171±0.4 0 NB* NB* NB* NB*

NB* indicates that no binding was observed under the current experimental conditions.

Table 6 Binding Kinetic Parameters of Anti-LEPR Antibodies Binding to mfLEPR-MMH at 37°C

Captured mAb H4H17322R2 H4H18437R2 H4N18439R2 Capture level mAh (EO) 218 ± 1.7 207 ± 1.4 178 ± 1.4 100 nM bound. mfLEP R-MMH (EO) 142 58 14 k _a (1/mol*s) 1.43E±05 8.69E±04 4.69E±04 k _d (1/s) 1D0E-03 1.27E-02 7.92E-03 kd (M) 7.72E-09 1.47Е-07 1.69Е-07 1½ (min) 10 0.9 1.5 H4H18440R2 204±1 66 7.15E±04 7.24Е-03 1.01E-07 1.6 H4H18457R2 195±1.4 117 3.47E±05 1.04Е-02 3.00E-08 1.1 H4H18462R2 199±1.1 28 1.29Е±05 3.37E-02 2.61E-07 0.3 H4H18464R2 183±0.6 13 3.92E±04 8.41Е-03 2D5E-07 1.4 H4H18466R2 181±0.9 27 8.43Е±04 1.38E-02 1.64Е-07 0.8 H4N18508R2 200±1 125 2.85E±05 7.89Е-03 2.77Е-08 1.5 Antibody comparisons 298 ± 3.2 -1 NB* NB* NB* NB* Isotype control antibody 190±1 1 NB* NB* NB* NB*

NB* indicates that no binding was observed under the current experimental conditions.

As shown in Table. 5, at 25° C., all of the anti-LEPR monoclonal antibodies of the present invention bound mfLEPR-MMH with KD values ranging from 4.16 to 179 nM. As shown in Table. 6, at 37° C., all anti-LEPR monoclonal antibodies of the present invention bound mfLEPR-MMH with K _D values ranging from 7.72 to 261 nM.

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Table 7. Binding Kinetic Parameters of Anti-LEPR Antibodies Binding to hLEPR-mFc at 25°C

Captured shah Capture level mAb (EO) 100 nM bound. hLEPRmFc (EO) to _a (1 / mol * s) k _d (1/s) Kd (M) 1½ (min) H4H17322R2 176±0.4 74 6.45E±04 1D4E-04 1.77Е-09 102 H4H18437R2 169±1.1 70 1.02Е±05 5.36E-04 5.27Е-09 22 H4N18439R2 155±0.2 53 4.87E±04 1.80E-04 3.69Е-09 64 H4H18440R2 169±0.5 140 1.54Е±05 6.82E-05 4.43Е-10 169 H4H18457R2 165±0.8 149 4.91E±05 4.23E-04 8.63E-10 27 H4H18462R2 173±0.5 68 9.08Е±04 2.96E-04 3.26E-09 39 H4H18464R2 159±0.4 84 2.11E+05 1.88Е-04 8.91Е-10 61 H4H18466R2 160±0.2 37 3.97E±04 4.72E-04 1.19E-08 24 H4N18508R2 178±0.4 138 2.21Е±05 2.99E-04 1.35Е-09 39 Antibody comparisons 344±4.9 36 1.56Е±05 1.45Е-05 9.28E-11 798 isotypic control antibody 170±0.7 -2 NB* NB* NB* NB*

NB* indicates that no binding was observed under the current experimental conditions.

Table 8 Binding Kinetic Parameters of Anti-LEPR Antibodies Binding to hLEPR-mFc at 37°C

Captured mAb mAb capture level (EO) 100 nM bound. hLEPRmFc (EO) to _a (1 / mol * s) k _d (1/s) Kd (M) 1½ (min) H4H17322R2 214 ± 1.8 105 1,3E±05 4.40E-04 3.31E-09 26 H4H18437R2 202±1.1 88 1.29Е±05 5.56Е-04 4.30E-09 21 H4N18439R2 176±0.7 67 6.09Е±04 9.23Е-04 1.51Е-08 13 H4H18440R2 200±1.4 181 2.12E±05 1.12E-04 5.27E-10 103 H4H18457R2 193±1 166 5.56Е±05 7.04Е-04 1.27Е-09 16 H4H18462R2 196±0.9 84 1.28E±05 5.43E-04 4.26Е-09 21 H4H18464R2 182±0.4 105 4.04Е±05 5.17E-04 1.28E-09 22 H4H18466R2 179±1 52 5.87E±04 5.20E-04 8.86Е-09 22 H4N18508R2 198±1 146 4.71Е±05 1.02E-03 2.16E-09 eleven Antibody comparisons 288±2.5 50 1.5OE±O5 7.74Е-05 5.14E-10 149 Isotype control antibody 188±0.8 1 NB* NB* NB* NB*

NB* indicates that no binding was observed under the current experimental conditions.

As shown in Table. 7, at 25° C., all anti-LEPR monoclonal antibodies of the present invention bound hLEPR-mFc with K _D values ranging from 443 pM to 11.9 nM. As shown in Table. 8, at 37° C., all anti-LEPR monoclonal antibodies of the present invention bound hLEPR-mFc with K _D values ranging from 527 pM to 15.1 nM.

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Table 9. Binding Kinetic Parameters of Anti-LEPR Monoclonal Antibodies Binding to mLEPR-MMH at 25°C

Captured shah Capture level mAb (EO) 100 nM bound. mLEPRMMH (EO) ka (1/mol*s) kd (1/s) Kd (M) U/2 (min) H4H17322R2 176 0 NB* NB* NB* NB* H4H18437R2 169 6 1.58E+04 4D8E-03 2.64Е-07 2.8 H4N18439R2 155 -2 NB* NB* NB* NB* H4H18440R2 169 -3 NB* NB* NB* NB* H4H18457R2 164 1 NB* NB* NB* NB* H4H18462R2 173 2 NB* NB* NB* NB* H4H18464R2 159 -1 NB* NB* NB* NB* H4H18466R2 160 1 NB* NB* NB* NB* H4N18508R2 177 25 2.78E±05 6.07Е-02 2D8E-07 0.19 Antibody comparisons 334±2.5 2 NB* NB* NB* NB* isotypic control antibody 169 -2 NB* NB* NB* NB*

NB* indicates that no binding was observed under the current experimental conditions.

Table 10. Binding Kinetic Parameters of Anti-LEPR Antibodies Binding to mLEPR-MMH at 37°C

Captured mAb mAb capture level (EO) 100 nM bound. mLEPR-MMN (EO) to _a (1 / mol * s) k _d (1/s) Kd (M) 1½ (min) H4H17322R2 212 -1 NB* NB* NB* NB* H4H18437R2 201 6 1D5E+04 1,ZZE-02 1D6E-06 0.9 H4N18439R2 175 -1 NB* NB* NB* NB* H4H18440R2 200 -1 NB* NB* NB* NB* H4H18457R2 191 -1 NB* NB* NB* NB* H4H18462R2 194 3 IC* IC* IC* IC* H4H18464R2 181 1 NB* NB* NB* NB* H4H18466R2 178 3 NB* NB* NB* NB* H4N18508R2 196 17 5.12E±05 1.05Е-01 2.05Е-07 0.11 Antibody comparisons 283±1.1 0 NB* NB* NB* NB* isotypic control antibody 187 0 NB* NB* NB* NB*

NB* indicates that no binding was observed under the current experimental conditions,

IC* indicates that the observed binding was indeterminate and could not match the real-time binding data under the current experimental conditions.

As shown in Table. 9, at 25° C., two of the 9 anti-LEPR monoclonal antibodies of the present invention bound to mLEPR-MMH with K _D values of 218 and 264 nM. As shown in Table. 10, at 37° C., two of the 9 anti-LEPR monoclonal antibodies of the present invention bound to mLEPR-MMH with K _D values of 205 nM and 1.16 μM.

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Table 11. Binding Kinetic Parameters of Anti-LEPR Antibodies Binding to rLEPR-MMH at 25°C

Captured shah Capture level mAb (EO) 100 nM bound. rLEPRMMH (EO) ka (1/mol*s) k _d (1/s) Kd (M) 11^2 (min) H4H17322R2 176±0.9 10 IC* IC* IC* IC* H4H18437R2 169±0.2 13 4.21E±04 1.38E-02 3.28E-07 0.8 H4N18439R2 155±0.2 -1 NB* NB* NB* NB* H4H18440R2 169±0.4 -2 NB* NB* NB* NB* H4H18457R2 164±0.5 18 2.83Е±04 2.06E-02 7, ZOE-07 0.6 H4H18462R2 173±0.2 5 3.21E±04 1.08E-02 3.36E-07 1.1 H4H18464R2 159±0 -1 NB* NB* NB* NB* H4H18466R2 160±0.6 5 2.10E±04 1.98E-02 9.43Е-07 0.6 H4N18508R2 177±0.1 22 1.39Е±05 4.76E-02 3.41E-07 0.24 Antibody comparisons 328±1.6 1 NB* NB* NB* NB* Isotype control antibody 170±0.2 -1 NB* NB* NB* NB*

NB* indicates that no binding was observed under the current experimental conditions,

IC* indicates that the observed binding was indeterminate and could not match the real-time binding data under the current experimental conditions.

Table 12. Binding Kinetic Parameters of Anti-LEPR Antibodies Binding to rLEPR-MMH at 37°C

Captured shah Capture level mAb (EO) 100 nM bound. rLEPRMMH (EO) to _a (1 / mol * s) k _d (1/s) Kd (M) D/2 (min) H4H17322R2 212±0.3 9 9.32Е±04 3.28E-02 3.52E-07 0.4 H4H18437R2 200±0 10 3.07E±04 1.93E-02 6.29E-07 0.6 H4N18439R2 175±0.8 0 NB* NB* NB* NB* H4H18440R2 200±0.1 0 NB* NB* NB* NB* H4H18457R2 192±0.3 16 3.02E±04 1.75Е-02 5.80E-07 0.7 H4H18462R2 194±0.9 5 IC* IC* IC* IC* H4H18464R2 181±0.1 1 NB* NB* NB* NB* H4H18466R2 177±0.7 6 3.26E±04 2.52E-02 7.71Е-07 0.5 H4N18508R2 196 ± 1.6 17 2.71Е±05 4.57Е-02 1.68E-07 0.25 Antibody comparisons 279±0.4 -1 NB* NB* NB* NB* Isotype control antibody 186±0.1 0 NB* NB* NB* NB*

NB* indicates that no binding was observed under the current experimental conditions,

IC* indicates that the observed binding was indeterminate and could not match the real-time binding data under the current experimental conditions.

As shown in Table. 11, at 25° C., five of the 9 anti-LEPR monoclonal antibodies of the present invention bound to rLEPR-MMH with K _D values ranging from 328 nM to

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943 nM. As shown in Table. 12, at 37°C, five of the 9 anti-LEPR monoclonal antibodies of the present invention bound to rLEPR-MMH with KD values ranging from 168 to

771 nM.

Example 4 The anti-LEPR antibodies of the present invention do not block the binding of hLEPRhFc to human leptin.

The ability of the anti-LEPR monoclonal antibodies of the present invention to block the binding of dimeric human LEPR to its natural ligand, human leptin, was measured using a competitive sandwich ELISA format.

For the ELISA, human leptin (hLeptin; R&D Systems, Cat # 398-LP-01M) was applied at a concentration of 5 μg/ml in PBS to a 96-well microtiter plate overnight at 4°C. Non-specific binding sites were subsequently blocked using a 0.5% (w/v) solution of BSA in PBS. A constant amount of 10 nM portion of the LEPR protein, the extracellular domain, which was expressed with the C-terminal tag of human Fc (hLEPR.hFc; SEQ ID NO: 92), was titrated with anti-LEPR antibodies, human leptin protein (hLeptin), or an isotype control antibody ranging from 8.5 pM to 500 nM in serial dilution. These antibody-protein or protein-protein complexes were then incubated for 1.5 hours at room temperature (RT). The complexes were then transferred to human leptin-coated microtiter plates and incubated for 2 hours at RT, the wells were washed, and plate-bound hLEPR-hFc was detected using a horseradish peroxidase-conjugated anti-human IgG polyclonal antibody (Jackson ImmunoResearch Inc, cat no. 109-035-098). Samples were developed with TMB solution (BD Biosciences, cat # 555214; substrate A and B mixed 1:1 according to manufacturer's instructions) to obtain a colorimetric reaction and then neutralized with 1 M sulfuric acid until absorbance at 450 nm was measured. on the Victor X5 tablet reader. Data analysis was performed using a sigmoid dose-response model in the Prism™ software (GraphPad). Percent blockade at the maximum concentration of test antibody was calculated as an indicator of the ability of antibodies to block the binding of 10 nM hLEPRhFc to human leptin on the plate. In the calculation, the binding signal of 10 nM hLEPR-hFc without the presence of antibody was called 100% binding or 0% blocking; and the original buffer signal alone without the presence of hLEPR-hFc was called 0% binding or 100% blocking. Data regarding blocking at a concentration of 500 nm antibodies are summarized in table. 13.

Table 13 Anti-LEPR ELISA Blocking of hLEPR-hFc Binding to Human Leptin

Antibody 500 nM antibody blocking of 10 nM hLEPR-hFc binding to human leptin (% blockade) H4H18440R2 3 H4H18466R2 2 H4H18457R2 4 H4H18437R2 9 H4H18464R2 -5 H4H17322R2 13 H4N18439R2 -1 H4H18462R2 14 H4N18508R2 40 controls Isotype control antibody -3 Human leptin 99 Reference antibody 99 Isotype control mouse IgG2a 32

As shown in Table. 13, none of the anti-LEPR antibodies of the present invention exhibited >40% blockade of hLEPR-hFc binding to the surface coated with human leptin. However, the reference antibody and human leptin as a positive control were able to block 99% of hLEPR-hFc binding to the surface coated with human leptin. The isotype control antibody showed no measurable blocking at concentrations up to 500 nM.

Example 5 Binding to HEK293/Mycx2hLEPR(ecto)-GPI cells (expressing hLEPR ectodomain attached by GPI) determined by FACS analysis.

The leptin receptor, LEPR, is a single-pass transmembrane receptor of the class I cytokine receptor family with a large 818 amino acid long extracellular domain (Tartaglia (1997) J. Biol. Chem 7:272(10):6093-6). LEPR can bind to leptin, a protein predominantly expressed by adipose tissue that is involved in the regulation of food intake and metabolism (Friedman (2014) J Endocrinol 223(1):T1-8). There are various isoforms of LEPR that give rise to soluble or membrane-bound forms of the receptor, and the membrane-bound forms differ in the length of their intracellular domain. The isoform with the longest intracellular domain is highly expressed in the hypothalamus, an important site of action for leptin in relation to obesity (Friedman and Halaas (1998) Nature 395(6704):763-70). LEPR is localized predominantly in protoplasts and to a lesser extent on the cell surface. LEPR undergoes ligand-induced internalization, adding an additional layer of regulation of leptin signaling (Sweeney (2002) Cell Signal 14(8):655-63).

To evaluate cell binding of anti-LEPR antibodies of the present invention, a HEK293 cell line was generated to stably express the human LEPR extracellular domain (hLEPR; amino acids 22-839 from accession number P48357, isoform B) with a myc-myc N-terminal tag and a C-terminal peptide sequence human carboxypeptidase M, which directs the addition of glycosylphosphatidylinositol (GPI) (Marcic et al. (2000) J Biol Chem. 275(21): 16110-8) so that the protein can attach via GPI to the membrane. Because hLEPR does not have an intracellular domain in a cell line, it is not internalized upon ligand binding, greatly increasing the amount of LEPR available for antibody and/or ligand binding. The cell line was selected in DMEM containing 10% FBS, NEAA, penicillin/streptomycin and 500 μg/ml G418, then sorted for high receptor expression using anti-Myc antibody. The resulting stable cell line, which is named HEK293/hLEPR-GPI, was maintained in DMEM containing 10% FBS, NEAA and penicillin/streptomycin.

For FACS analysis, stock HEK293 cells and HEK293/hLEPR-GPI cells were separated and plated in 96-well v-bottom plates at a density of 5x105 cells/well in PBS containing 2% FBS (FACS buffer). To test whether the presence of anti-hLEPR antibodies that bind to cells depends on the presence of leptin, FACS buffer with or without 1 μM human leptin (R&D Systems, Cat # 398-LP) was incubated with cells for 30 min at 4°C. C followed by the addition of antibodies to LEPR or control antibodies at a concentration of 10 nM in FACS buffer. The cells were then incubated for 30 min at 4°C, after which they were washed and then incubated with 16 μg/ml conjugated secondary antibody Alexa Fluor®-647 (Jackson ImmunoResearch Laboratories Inc., cat no. 109-547-003) for 30 min at 4°C. Cells were then fixed using BD CytoFix™ (Becton Dickinson, Cat # 554655), filtered and analyzed on a HyperCyt flow cytometer (Beckman Coulter). Unstained and secondary control antibodies were also separately tested for all cell lines. The results were analyzed using ForeCyt (IntelliCyt) and FlowJo software version 10 to determine geometric mean fluorescence values for viable cells. The geometric mean of fluorescence for each sample was then normalized to the geometric mean of unstained cells to obtain the relative binding for each condition, referred to as binding ratios, and these binding ratios were tabulated. 14 for each test antibody.

As shown in Table. 14, 9 anti-LEPR antibodies of the present invention tested at 10 nM showed binding to HEK293/hLEPR-GPI cells with binding ratios ranging from 824-fold to 3187-fold without leptin and from 398-fold to 3590- fold in the presence of 1 μM leptin. Based on the similarity of these binding relationships, the ability of the anti-LEPR antibodies of the present invention to bind to LEPR expressed on cells does not appear to be significantly affected by the presence of an excess of 1 μM leptin, suggesting that the anti-LEPR antibody binding sites on LEPR are not overlap with leptin binding sites on LEPR. Anti-LEPR antibodies of the present invention did not show any significant binding to parental HEK293 cells with binding ratios with and without 1 μM leptin ranging from 1- to 9-fold. Binding of the reference antibody to cells expressing GPI-attached LEPR was significantly reduced in the presence of leptin. The isotype control antibody and secondary antibody samples alone also did not show significant binding to the cell line with or without leptin, with binding ratios ranging from 1- to 6-fold.

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Table 14. Anti-LEPR antibody binding at 10 nM to HEK293/hLEPR-GPI cells and stock HEK293 cells +/- 1 µM human leptin

Binding ratio: normalized to unstained sample of each cell line No added leptin 1 µM leptin Antibody HEK293 original HEK293/ hLEPR-GPI HEK293 original HEK293/ hLEPR-GPI H4H17322R2 3 1307 6 1317 H4H18457R2 2 3187 3 3106 H4H18464R2 5 2318 7 3590 H4H18437R2 1 1314 2 1441 H4N18439R2 4 1533 6 2477 H4H18440R2 3 1640 7 2557 H4H18462R2 2 1173 3 759 H4H18466R2 1 824 2 398 H4N18508R2 1 1311 2 1873 Reference antibody 6 2349 3 112 Isotype control human IgG antibody 1 6 2 4 Secondary antibody to human IgG alone 1 3 2 3 Isotype control mouse IgG antibody 2 2 3 3 Secondary anti-mouse IgG alone 2 2 2 2 Unpainted 1 1 1 1

Example 6 Anti-LEPR antibodies of the present invention demonstrated complete inhibition of leptin signaling in the presence of human leptin.

A biological assay was developed to detect transcriptional activation of STAT3 via LEPR activation using a reporter cell line that stably expresses full-length human LEPR (hLEPR; amino acids 1-1165 accession number NP_002294.2) in conjunction with a luciferase reporter (STAT3-Luc; Qiagen, Cat # . CLS-6028L) in the IMR-32 cell line, a human neuroblastoma cell line. The resulting stable cell line, termed IMR-32/STAT3-Luc/hLEPR, was isolated and maintained in MEM-Earl supplemented with 10% FBS, NEAA, 1 μg/ml puromycin, 100 μg/ml hygromycin B, and a penicillin/streptomycin mixture. /Lglutamine (complete medium).

The resulting bioassay was used to measure the effect of the anti-LEPR antibodies of the present invention on LEPR signaling in the presence and absence of leptin. For bioassay, IMR-32/STAT3-Luc/hLEPR cells were plated at 20,000 cells/100 μl/well for 96-well format in complete medium and then the next day the medium was replaced with an appropriate volume of Opti-MEM supplemented with 1% BSA and 0 .1% FBS (assay buffer) for 30 min. To measure the effect of the antibodies of the present invention in the absence of leptin, anti-LEPR antibodies or an isotype control antibody were serially diluted semi-logarithmically to final concentrations ranging from 100 nM to 300 fM in assay buffer and then added to cells and then incubated overnight at 37° C. 5% CO ₂ . To measure the effect of the antibodies of the present invention in the presence of leptin, a fixed concentration of human leptin (hLeptin; R&D Systems, Cat # 398-LP) at 200 pM in assay buffer was added to the cells, followed immediately by the addition of anti-LEPR antibodies or an isotype control. an antibody that is semi-log serially diluted to final concentrations ranging from 100 nM to 300 fM. Then the samples were incubated overnight at 37°C in 5% CO ₂ . The OneGlo reagent (Promega, Cat # E6051) was then added to the samples and luciferase activity was measured using an Envision Multilable reader.

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Plate Reader (Perkin Elmer) in luminescent mode. Relative light units (RLU) values were obtained and the results were analyzed using non-linear regression using GraphPad Prism software (GraphPad). The maximum RLU value derived from the hLeptin dose effect was defined as 100% activation in the IMR-32/STAT3-Luc/hLEPR assay.

As shown in Table. 15, in the absence of human leptin, test LEPR antibodies showed weak stimulation of IMR-32/STAT3-Luc/hLEPR cells with a maximum activation of 4% to 8%, respectively, the maximum activation derived from the dose effect of human leptin. One weakly stimulated antibody had an EC ₅₀ value of 1.27 nM, while the other of the antibodies, H4H18440P2, had no measurable EC ₅₀ value. In the presence of 200 pM human leptin, three of the tested anti-LEPR antibodies demonstrated complete inhibition of leptin in IMR-32/STAT3-Luc/hLEPR cells with IC ₅₀ values ranging from 723 pM (H4H18457P2 antibody) to 1.83 nM (H4H17322P2 antibody ) or 2.9 nM (antibody H4H18464P2). Six of the LEPR antibodies tested did not show any measurable inhibition in the presence of 200 pM human leptin. The isotype control antibody did not show any measurable stimulation of IMR-32/STAT3-Luc/hLEPR cells in any of the assays.

Table 15 Activation and inhibition of hLEPR by anti-LEPR antibodies

IMR-32/LEPR without human leptin IMR-32/LEPR with 200 pM human leptin Antibody EU ₅₀ (M) % activation 1S ₅₀ (M) % inhibition H4H18457R2 N/A N/A 7.23E-10 100 H4H17322R2 1.27Е-09 8 1.83Е-09 100 H4H18464R2 1,ZOE-10 6 2.9E-09 100 H4H18437R2 N/A* N/A* N/A* N/A* H4N18439R2 N/A* N/A* N/A* N/A* H4H18440R2 IC* 4 N/A* N/A* H4H18462R2 N/A* N/A* N/A* N/A* H4H18466R2 N/A* N/A* N/A* N/A* H4N18508R2 N/A* N/A* N/A* N/A* Antibody comparisons N/A* N/A* 1.03Е-09 100

N/A* means no measurable inhibition and/or activation in the assay, IC* means indeterminate in that the calculated EC50 value could not be determined.

Example 7 Cross-competition between various anti-LEPR monoclonal antibodies studied using Octet.

Binding competition between a panel of different anti-LEPR monoclonal antibodies was determined using real-time labelless biolayer interferometry analysis on the Octet HTX biosensor platform (Pall ForteBio Corp.). The entire experiment was carried out at 25° C. in a buffer containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% v/v. Tween-20 surfactant, 1 mg/ml BSA, pH 7.4 (HBS-EBT) with shaking the plate at 1000 rpm.

To assess whether two antibodies are able to compete with each other for binding to recombinant human LEPR expressed with a C-terminal myc-myc-hexahistidine tag (hLEPR.MMH; SEQ ID: 90), approximately 0.25 nM hLEPR-MMH at the beginning captured on Octet biosensor tips coated with antibodies to the pentagistidine tag (Fortebio Inc, Cat # 18-5122) by immersing the biosensor tips for 5 min in wells containing 20 μg/ml hLEPRMMH. The captured antigen biosensor tips were then saturated with the first anti-LEPR monoclonal antibody (then referred to as mAb-1) by dipping into wells containing 50 μg/ml mAb-1 solution for 210 s. The biosensor tips were then sequentially immersed in wells containing a 50 μg/ml solution of a second anti-LEPR monoclonal antibody (then referred to as mAb-2) for 150 seconds. The biosensor tips were washed in HBS-EBT buffer between each stage of the experiment. The real-time binding response was monitored throughout the experiment and the binding response at the end of each step was recorded. The mAb-2 binding response to hLEPRMMH previously complexed with mAb-1 was compared and the competitive/non-competitive behavior of various anti-LEPR monoclonal antibodies was determined, as shown in Table 1. 16.

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Table 16. Cross-competition between monoclonal antibodies to LEPR

Binding of the first antibody (mAb-Ι) to captured hLEPR-MMH Second antibody (mAb-2) shown to compete with mAb-Ι H4N18439R2 H4H18440R2 H4H18440R2 H4N18439R2 H4H18457R2 H4H18462R2 H4H18437R2 H4H18466R2 H4N18508R2 H4H18462R2 H4H18457R2 H4H18437R2 H4H18466R2 H4H18437R2 H4H18457R2 H4H18462R2 H4H18466R2 H4H18466R2 H4H18457R2 H4H18462R2 H4H18437R2 H4H17322R2 None H4H18464R2 None

As shown in Table. 16, antibodies H4H18439P2 and H4H18440P2 compete with each other for binding to their respective epitopes. The LEPR antibodies H4H18457P2, H4H18462P2, H4H18437P2, H4H18466P2 and H4H18508P2 compete with each other for binding to their respective epitopes. Antibodies H4H17322P2 and H4H18464P2 do not compete for binding with the corresponding epitopes H4H18439P2, H4H18440P2, H4H18457P2, H4H18462P2, H4H18437P2 and H4H18466P2.

Example 8 In Vivo Testing the Efficacy of LEPR Antagonist Antibodies in LEPR Humanized Mice.

The effects of three specific anti-LEPR antagonist antibodies of the present invention, H4H17322P2, H4H18457P2 and H4H18464P2, on food intake, body weight and obesity were determined in single caged, genetically engineered LEPR ^Hu/Hu mice that express a leptin receptor that consists of the sequence human LEPR ectodomain instead of mouse LEPR ectodomain sequence.

Baseline daily food intake was measured between day 5 and day 1 before treatment (days -5 and -1). Four days before treatment and 6 days after treatment (days -4 and -6), body tissue composition, including obesity, was quantified by microcomputed tomography. On day 0, 32 male LEPR ^Hu/Hu mice aged 12 to 13 weeks were randomized into four groups of 8 mice based on body weight 1 day before treatment (day -1). On day 0, each group received a single dose by subcutaneous injection of one of the following: isotype control antibody dose of 30 mg/kg, H4H17322P2 of 30 mg/kg, H4H18457P2 of 30 mg/kg, or H4H18464P2 of 30 mg/kg . The isotype control antibody does not bind to any known mouse protein. Food intake and body weight were measured over the duration of the study for each animal. In FIG. 1 summarizes the percentage change in food intake from mean baseline daily food intake for each treatment group at each time point. The percentage change in body weight from day 0 was calculated for each animal at each time point. In FIG. 2 summarizes the mean percent change in body weight for animals in each treatment group. In FIG. 3 summarizes the mean body fat mass for animals in each antibody treatment group, quantified by microcomputed tomography 6 days before and 6 days after antibody treatment. All results are expressed as mean±SEM.

As shown in FIG. 1 and 2, mice treated with anti-LEPR antagonist antibodies showed increases in percent change in food intake and percent change in body weight. This increase was not observed in the case of treatment with an isotype control antibody. As shown in FIG. 1, mice treated with H4H17322P2 at 30 mg/kg showed a significant increase in food intake from day 1 post-treatment (Day 1) and thereafter compared to mice injected with iso

- 31 040524 typical control antibody. Mice treated with H4H18457P2 at 30 mg/kg showed a significant increase in food intake from day 2 onwards compared to mice injected with the isotype control antibody. Mice treated with H4H18464P2 at 30 mg/kg showed a significant increase in food intake from day 2 onward, but not day 4, compared to mice injected with the isotype control antibody. As shown in FIG. 2, mice treated with H4H17322P2 at 30 mg/kg showed a significant increase in percentage change in body weight from day 4 post-treatment (Day 4) and beyond compared to mice injected with isotype control antibody. . Mice treated with H4H18457P2 at 30 mg/kg showed a significant increase in percentage change in body weight from day 3 onwards compared to mice injected with the isotype control antibody. Mice treated with H4H18464P2 at 30 mg/kg showed a significant increase in percentage change in body weight from day 4 onwards compared to mice injected with the isotype control antibody. As shown in FIG. 3, there are no differences in adipose tissue mass between groups before treatment (day -4). Mice treated with H4H17322P2, H4H18457P2 and H4H18464P2 antibody at 30 mg/kg showed a significant increase in body fat mass 6 days post-treatment (day 6) compared to the isotype control antibody. In conclusion, treatment with the LEPR antagonist antibody, but not the isotype control antibody, increased food intake, body weight, and obesity in mice.

Example 9 Determination of human LEPR epitopes to which anti-LEPR antibodies of the present invention bind.

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

Transiently Expressed LEPR Proteins Including Human LEPR Extracellular Domain Expressed with C-terminal myc-myc-hexahistidine tag (human LEPR-MMH, SEQ ID NO: 89), (D1) Human CRH1 LEPR expressed with C-terminal myc -myc-hexahistidine tag ((D1)-MMH human CRH1 LEPR, amino acids 1-208 according to SEQ ID NO: 89 with myc-mychexahistidine tag, amino acids 209-236), domain (D1,D2) of human CRH1 LEPR expressed with C -terminal myc-myc-hexahistidine tag ((D1,D2)-MMH human CRH1 LEPR, amino acids 1-318 according to SEQ ID NO: 89 with myc-myc-hexahistidine tag, amino acids 319-346), domain (D1,D2, D3) Human CRH1-Ig LEPR expressed with C-terminal myc-myc-hexahistidine tag ((D1,D2,D3)-MMH human CRH1 LEPR, amino acids 1-278 according to SEQ ID NO: 89 with myc-myc-hexahistidine tag, amino acids 279-306), domain (D2,D3) of human CRH1-Ig LEPR expressed with C-terminal myc-myc-hexahistidine tag ((D2,D3)-MMH CRH 1-Ig human LEPR, amino acids 1-198 according to SEQ ID NO: 89 with myc-myc-hexahistidine tag, amino acids 199-226), domain (D3) of human Ig LEPR expressed with C-terminal myc-myc hexahistidine tag ((D3 )-MMH Ig human LEPR, amino acids 1-88 according to SEQ ID NO: 89 with myc-myc-hexahistidine tag, amino acids 89-116), human CRH2 domain expressed with C-terminal myc-myc-hexahistidine tag (CRH2- human MMH LEPR, amino acids 1-207 according to SEQ ID NO: 89 with myc-myc-hexahistidine tag, amino acids 208-235), human FNIII LEPR domain expressed with C-terminal myc-myc-hexahistidine tag (human FNIIIMMH LEPR, amino acids 1-204 according to SEQ ID NO: 89 with a myc-myc-hexahistidine tag, amino acids 205-232), and a human Ig-CRH2-FNIII LEPR domain expressed with a C-terminal myc-myc-hexahistidine tag (Ig-CRH2-FNIII -MMH human LEPR, amino acids 1-510 according to SEQ ID NO: 89 with myc-myc-hexahistidine tag, amino acids 5 11-538) were suspended in CHO-S-SFM II serum-free medium (Thermo Fisher, cat. no. 31033020) and then clarified by centrifugation. Aliquots of microspheres with immobilized monoclonal antibodies to myc, obtained as described above, were added separately to 1 ml of each of these protein supernatants.

The microspheres were gently mixed, incubated for two hours at 25° C., washed twice with 1 ml DBPS, centrifuged to remove the supernatant, and finally resuspended in 1 ml DPBS buffer. 48 µl of anti-myc IgG-bound microspheres from separate reactions with full-length human LEPR and with each of the human LEPR domain proteins were selected and mixed together in 3.6 ml PBS + 20 mg/ml BSA + 0.05% sodium azide (blocking buffer).

From this mixed pool, 75 µl of microspheres were plated per well on a 96-well filter plate (Millipore, catalog number: MSBVN1250) and mixed with 25 µl of individual anti-human LEPR monoclonal antibodies (0.5 or 5 µg/ml), incubated for two hours at 25°C and then washed twice with 200 μl of DPBS with 0.05% Tween 20 (wash buffer). For detection and quantification of anti-LEPR antibodies bound to individual microspheres or 100 µl 2.5 µg/ml R-phycoerythrin-conjugated goat F(ab')2 anti-human kappacep antibody (Southern Biotech, Cat # 2063 -09) in blocking buffer, or 100 µl 1.25 µg/ml AffiniPure R-phycoerythrin-conjugated F(ab') in blocking buffer, or 100 µl 1.25 µg/ml R-phycoerythrin-conjugated F(ab') 2 fragments of AffiniPure goat anti-mouse IgG F(ab')2 fragment specific (Jackson Immunoresearch, Cat # 115-116-072) in blocking buffer were added and incubated for 30 minutes at 25°C. After 30 min, the samples were washed twice with 200 µl wash buffer and resuspended in 150 µl wash buffer. The median fluorescence intensity (MFI) of the microspheres was measured in a Luminex analyzer.

The results of the analysis based on Luminex are summarized in table. 17. Luminex MFI signal intensities indicate that the nine anti-LEPR antibodies of the present invention bound to the entire extracellular domain of human LEPR. Anti-LEPR antibodies, H4H18439P2 and H4H1844OP2, bind to epitopes within the D2 domain of human CRH1 LEPR. Anti-LEPR antibodies H4H17322P2 and COMP3551 associated with epitopes in the CRH2 domain of human LEPR. Anti-LEPR antibodies, H4H18437P2, H4H18457P2, H4H18508P2, H4H18466P2, and H4H18462P2, bind to epitopes within the FNIII domain of human LEPR. An anti-LEPR antibody, H4H18464P2, bound epitopes within the human LEPR Ig(D3) domain.

Table 17. Luminex-derived MFI signal of binding of anti-LEPR antibodies to the full-length extracellular domain of human LEPR and isolated myc-tagged human LEPR domains

Antibody CRH1 (D1) CRH1 (D1,D2) CRH1-Ig (D1,D2,D3) CRH1-Ig (D2,D3) ig (D3) CRH2 FNIII ig- CRH2FNIII full size extracellular domain Possible binding site H4H17322R2 14 50 31 58 26 19745 23 15097 8932 CRH2 H4H18437R2 21 82 34 43 26 25 1593 15625 8644 FNIII H4H18457R2 13 53 31 43 18 18 1187 13235 7500 FNIII H4N18508R2 10 274 217 372 24 18 741 13007 5865 FNIII H4H18466R2 15 56 209 76 26 17 457 11270 5070 FNIII H4N18439R2 eleven 21179 8021 10026 18 16 13 43 5472 CRH1D2 H4H18440R2 8 19337 8245 9165 14 17 10 32 7035 CRH1D2 H4H18464R2 13 59 5557 3742 27 24 23 15119 9385 Ig(D3) H4H18462R2 5381 5731 3961 47 21 24 581 14858 5852 FNIII H4H18462R2 * 615 665 461 25 10 8 229 11153 3610 FNIII Antibody comparisons -14 19 -57 27 10 9404 73 7112 3908 CRH2

* - antibody tested at a concentration of 0.5 µg/ml instead of 5 µg/ml.

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

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Claims (30)

CLAIM 1. An isolated antibody or antigen-binding fragment thereof that binds to the human leptin receptor (LEPR), wherein the antibody or antigen-binding fragment thereof contains the following: (a) a heavy chain variable region (HCVR) complementarity determining region (CDR) that contains an amino acid sequence, shown in SEQ ID NO selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 42 and SEQ ID NO: 58 and (b) a light chain variable region CDR (LCVR) that contains the amino acid sequence shown in SEQ ID NO : 10. 2. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment contains a light chain variable region containing: LCDR1, which contains the amino acid sequence shown in SEQ ID NO: 12; LCDR2, which contains the amino acid sequence shown in SEQ ID NO: 14; and LCDR3 which contains the amino acid sequence shown in SEQ ID NO: 16 and a heavy chain variable region containing: (i) HCDR1, which contains the amino acid sequence shown in SEQ ID NO: 4; HCDR2, which contains the amino acid sequence shown in SEQ ID NO: 6; And HCDR3, which contains the amino acid sequence shown in SEQ ID NO: 8; (ii) HCDR1, which contains the amino acid sequence shown in SEQ ID NO: 44; HCDR2, which contains the amino acid sequence shown in SEQ ID NO: 46; And HCDR3, which contains the amino acid sequence shown in SEQ ID NO: 48; or (iii) HCDR1, which contains the amino acid sequence shown in SEQ ID NO: 60; HCDR2, which contains the amino acid sequence shown in SEQ ID NO: 62; And HCDR3 which contains the amino acid sequence shown in SEQ ID NO: 64. 3. The isolated antibody or antigen-binding fragment thereof according to claim 2, which is a full-length antibody containing a heavy chain variable region that contains the amino acid sequence shown in SEQ ID NO: 2, and a light chain variable region that contains the amino acid sequence shown in SEQ ID NO: 10. 4. The isolated antibody or antigen-binding fragment thereof according to claim 2, which is a full-length antibody containing a heavy chain variable region that contains the amino acid sequence shown in SEQ ID NO: 42, and a light chain variable region that contains the amino acid sequence shown in SEQ ID NO: 10. 5. The isolated antibody or antigen-binding fragment thereof according to claim 2, which is a full-length antibody containing a heavy chain variable region that contains the amino acid sequence shown in SEQ ID NO: 58, and a light chain variable region that contains the amino acid sequence shown in SEQ ID NO: 10. 6. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier or diluent. 7. The use of a pharmaceutical composition according to claim 6 for the treatment of a disease associated with or caused by leptin signaling. 8. Use according to claim 7, wherein the disease or condition associated with or caused by leptin signaling is selected from the group consisting of the following: anorexia, psychiatric eating disorder, cachexia, cachexia in chronic kidney disease, cachexia in congestive heart failure, pulmonary cachexia, radiation cachexia, cancer cachexia, autoimmune disorder, inflammatory bowel disease, lupus erythematosus, multiple sclerosis, psoriasis, cardiovascular disease, high blood pressure, non-alcoholic hepatic steatosis, neurodegenerative disorder, depression, cancer, renal cell carcinoma, melanoma, and breast cancer glands. 9. The use of a pharmaceutical composition according to claim 6 for the treatment of a disease or condition associated with or caused by a LEPR activating mutation. 10. Use according to claim 9, wherein the LEPR mutation is LEPR Q223R. 11. Use according to claim 9 or 10 wherein the disease or condition associated with or caused by the LEPR activating mutation is cachexia. 12. Use according to any one of claims 7-11, wherein the composition is therapeutically combined with a second therapeutic agent and further comprises administering the composition to a subject, and wherein the second therapeutic agent is selected from an antidepressant, an appetite stimulant, an anorexia and cachexia drug, a COX-2 inhibitor , NSAIDs, drugs to reverse the loss of muscle mass, muscle function and / or muscle strength with or without cachexia, drugs - 34 040524 vein for non-alcoholic liver steatosis (NAFLD), drug for infection HIV/AIDS, a TNF-alpha production blocker, an antioxidant, a tumor necrosis factor (TNF) antagonist, a B-lymphocyte stimulator, an angiotensin-converting enzyme (ACE) inhibitor, a kinase inhibitor, and a chemotherapeutic agent. 13. An isolated antibody or antigen-binding fragment thereof according to claim 3, wherein the heavy chain variable region is linked to human IgG4 Fc. 14. The isolated antibody or antigen-binding fragment thereof according to claim 4, wherein the heavy chain variable region is linked to human IgG4 Fc. 15. The isolated antibody or antigen-binding fragment thereof according to claim 5, wherein the heavy chain variable region is linked to human IgG4 Fc. 16. A pharmaceutical composition containing an antibody according to claim 13 and a pharmaceutically acceptable carrier or diluent. 17. A pharmaceutical composition containing an antibody according to claim 14 and a pharmaceutically acceptable carrier or diluent. 18. A pharmaceutical composition containing an antibody according to claim 15 and a pharmaceutically acceptable carrier or diluent. 19. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment contains a light chain variable region containing: LCDR1, which contains the amino acid sequence shown in SEQ ID NO: 12; LCDR2, which contains the amino acid sequence shown in SEQ ID NO: 14; and LCDR3 which contains the amino acid sequence shown in SEQ ID NO: 16 and a heavy chain variable region containing: HCDR1, which contains the amino acid sequence shown in SEQ ID NO: 4; HCDR2, which contains the amino acid sequence shown in SEQ ID NO: 6; And HCDR3 which contains the amino acid sequence shown in SEQ ID NO: 8. 20. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment contains a light chain variable region containing: LCDR1, which contains the amino acid sequence shown in SEQ ID NO: 12; LCDR2, which contains the amino acid sequence shown in SEQ ID NO: 14; and LCDR3 which contains the amino acid sequence shown in SEQ ID NO: 16 and a heavy chain variable region containing: HCDR1, which contains the amino acid sequence shown in SEQ ID NO: 44; HCDR2, which contains the amino acid sequence shown in SEQ ID NO: 46; And HCDR3 which contains the amino acid sequence shown in SEQ ID NO: 48. 21. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment contains a light chain variable region containing: LCDR1, which contains the amino acid sequence shown in SEQ ID NO: 12; LCDR2, which contains the amino acid sequence shown in SEQ ID NO: 14; and LCDR3 which contains the amino acid sequence shown in SEQ ID NO: 16 and a heavy chain variable region containing: HCDR1, which contains the amino acid sequence shown in SEQ ID NO: 60; HCDR2, which contains the amino acid sequence shown in SEQ ID NO: 62; And HCDR3 which contains the amino acid sequence shown in SEQ ID NO: 64. 22. The isolated antibody or antigen-binding fragment thereof according to claim 13, wherein the light chain variable region is linked to a kappa light chain constant domain. 23. An isolated antibody or antigen-binding fragment thereof according to claim 14, wherein the light chain variable region is linked to a kappa light chain constant domain. 24. The isolated antibody or antigen-binding fragment thereof according to claim 15, wherein the light chain variable region is linked to a kappa light chain constant domain. 25. An isolated polynucleotide encoding a heavy chain variable region and a light chain variable region of an antibody or antigen binding fragment according to any one of claims 1-5. 26. A polynucleotide according to claim 25, containing the nucleotide sequence shown in SEQ ID NOs: 41 and 9. 27. An isolated vector containing a polynucleotide according to claim 26. 28. A host cell containing the vector of claim 27. 29. A method of treating a disease associated with or caused by leptin signaling in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of claims 1-5. 30. The method of claim 29, wherein the disease is anorexia, psychiatric eating disorder, chronic kidney disease cachexia, cachexia, congestive heart failure cachexia, pulmonary cachexia, radiation cachexia, cancer cachexia, autoimmune disorder, inflammatory bowel disease, lupus erythematosus, multiple sclerosis, -