EA041859B1 - ANTIGEN-BINDING PROTEINS THAT ACTIVATE THE LEPTIN RECEPTOR - Google Patents

Description

The field of technology to which the present invention relates

The present invention relates to antibodies and antigen-binding antibody fragments that bind the human leptin receptor (LEPR), as well as therapeutic and diagnostic uses of such antibodies.

Sequence listing

An official copy of the sequence listing is filed simultaneously with this disclosure electronically via EFS-Web as an ASCII sequence listing in a file called 2016_10_11_10178WO01_Sequence_Listing_as_Filed_ST25.TXT, created on October 11, 2016, approximately 99.6 kilobytes in size. The sequence listing contained in the ASCII document is part of the present disclosure and is incorporated herein by reference in its entirety.

Background of the Invention

Leptin is a polypeptide hormone expressed primarily in adipose tissue that is involved in the regulation of metabolism, energy balance, and food intake. The activity of leptin is mediated by interaction with the leptin receptor and signal transmission through it. The leptin receptor (also known as LEPR, WSX, OB receptor, OB-R, and CD295) is a single-pass transmembrane receptor of the class I cytokine receptor family with a large (818 amino acids) extracellular domain. Leptin deficiency, leptin resistance, and certain LEPR-defective/impaired signaling mutations are associated with obesity, type 2 diabetes, dyslipidemia, lipodystrophies, hepatic steatosis, non-alcoholic fatty liver disease and alcoholic fatty liver disease, severe insulin resistance, leprechaunism/Donoghue syndrome, Rabson-Mendenhall syndrome and related complications. Therapeutic approaches to address leptin resistance, leptin deficiency, and hypoleptinemia (eg, lipodystrophy) have predominantly focused on delivering supplemental leptin or leptin analogs to affected individuals. However, such approaches have generally shown limited efficacy, especially in leptin resistant individuals, and are often associated with unwanted side effects. Thus, there is a need in the art for alternative approaches to the treatment of leptin resistance and other conditions associated with leptin resistance or hypoleptinemia.

Brief summary of the present invention

The present invention provides antibodies and antigen-binding fragments thereof that bind the human leptin receptor (LEPR). The antibodies of the present invention are agonistic antibodies, i.e. binding of the anti-LEPR antibodies of the present invention to LEPR causes, inter alia, activation of signaling through the leptin receptor in cells. In certain embodiments, the antibodies of the present invention do not compete with leptin for binding to LEPR. The antibodies of the present invention are useful, for example, to mimic, replace, or supplement the normal biological activity of leptin in a subject. Thus, the antibodies and antigen-binding fragments of the present invention are useful in the therapeutic treatment of diseases or disorders associated with leptin resistance and leptin deficiency.

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

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

The present invention provides antibodies or antigennegative fragments thereof that specifically bind LEPR containing HCVR, containing an amino acid sequence selected from any of the HCVR amino acid sequences presented in table. 1, or a sequence actually similar to it, at least 90%, at least 95%, at least 98%, or at least 99% sequence identical to it.

The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind LEPRs containing LCVRs containing an amino acid sequence selected from any of the LCVR amino acid sequences shown in Table 1. 1, or a sequence actually similar to it, at least 90%, at least 95%, at least 98%, or at least 99% sequence identical to it.

- 1 041859

The present invention also provides antibodies, or antigen-binding fragments thereof, that specifically bind LEPR, comprising a pair of HCVR and LCVR amino acid sequences (HCVR/LCVR) comprising any of the HCVR amino acid sequences shown in Table 1 in pair with any of the LCVR amino acid sequences shown in Table 1. tab. 1. In accordance with certain variants of implementation of the present invention provides antibodies or their antigennegative fragments containing a pair of amino acid sequences HCVR/LCVR contained in any of the exemplary antibodies to LEPR presented in table. 1. In certain embodiments, a pair of HCVR/LCVR amino acid sequences is selected from the group consisting of SEQ ID NO: 2/10, 18/10, 26/10, 34/10, 42/10, 50/10, 58/ 66, 74/66 and 82/66.

The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind LEPRs containing a heavy chain CDR1 (HCDR1) containing an amino acid sequence selected from any of the HCDR1 amino acid sequences shown in Table 1. 1, or a sequence actually similar to it, at least 90%, at least 95%, at least 98%, or at least 99% sequence identical to it.

The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind LEPRs containing a heavy chain CDR2 (HCDR2) containing an amino acid sequence selected from any of the HCDR2 amino acid sequences shown in Table 1. 1, or a sequence actually similar to it, at least 90%, at least 95%, at least 98%, or at least 99% sequence identical to it.

The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind LEPRs containing a heavy chain CDR3 (HCDR3) containing an amino acid sequence selected from any of the HCDR3 amino acid sequences shown in Table 1. 1, or a sequence actually similar to it, at least 90%, at least 95%, at least 98%, or at least 99% sequence identical to it.

The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind LEPRs containing a light chain CDR1 (LCDR1) containing an amino acid sequence selected from any of the LCDR1 amino acid sequences shown in Table 1. 1, or a sequence actually similar to it, at least 90%, at least 95%, at least 98%, or at least 99% sequence identical to it.

The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind LEPRs containing a light chain CDR2 (LCDR2) containing an amino acid sequence selected from any of the LCDR2 amino acid sequences shown in Table 1. 1, or a sequence actually similar to it, at least 90%, at least 95%, at least 98%, or at least 99% sequence identical to it.

The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind LEPRs containing a light chain CDR3 (LCDR3) containing an amino acid sequence selected from any of the LCDR3 amino acid sequences shown in Table 1. 1, or a sequence actually similar to it, at least 90%, at least 95%, at least 98%, or at least 99% sequence identical to it.

The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind LEPR, comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3/LCDR3) comprising any of the HCDR3 amino acid sequences shown in Table 1 in pair with any of the LCDR3 amino acid sequences shown in Table 1. tab. 1. In accordance with certain variants of implementation of the present invention provides antibodies or antigennegative fragments containing a pair of amino acid sequences HCDR3/LCDR3 contained in any of the exemplary antibodies to LEPR presented in table. 1. In accordance with certain embodiments, a pair of HCDR3/LCDR3 amino acid sequences is selected from the group consisting of SEQ ID NO: 8/16, 24/16, 32/16, 40/16, 48/16, 56/16, 64/ 72, 80/72 and 88/72.

The present invention also provides antibodies, or antigen-binding fragments thereof, that specifically bind LEPR, comprising an array of six CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) contained in any of the exemplary anti-LEPR antibodies shown in Table 1. . 1. In accordance with certain variants of implementation of the set of amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 selected from the group consisting of SEQ ID NO: 4, 6, 8, 12, 14, 16; 20, 22, 24, 12, 14, 16; 28, 30, 32, 12, 14, 16; 36, 38, 40, 12

- 2 041859

14, 16; 44, 46, 48, 12, 14, 16; 52, 54, 56, 12, 14, 16; 60, 62, 64, 68, 70, 72; 76, 78, 80, 68, 70, 72; and 84, 86, 88, 68, 70, 72.

According to a related embodiment, the present invention also provides antibodies, or antigen-binding fragments thereof, that specifically bind LEPR, comprising an array of six CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) contained within the HCVR amino acid sequence pair. /LCVR, as determined using any of the illustrative antibodies to LEPR presented in table. 1. For example, the present invention provides antibodies or antigen-binding fragments thereof that specifically bind LEPR, comprising a plurality of amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 contained in an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2/10, 18/10, 26/10, 34/10, 42/10, 50/10, 58/66, 74/66 and 82/66. Methods and techniques for detecting CDRs in HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs in certain 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, the Kabat definition is based on sequence variability, the Chothia definition is based on the position 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 56:9268-9272 (1989). Public databases are also available for identifying CDR sequences in an antibody.

The present invention also provides nucleic acid molecules encoding anti-LEPR antibodies or portions thereof. For example, the present invention provides nucleic acid molecules encoding any of the HCVR amino acid sequences shown in Table. 1; in accordance with certain embodiments, the nucleic acid molecule contains a polynucleotide sequence selected from any of the HCVR nucleic acid sequences shown in Table. 2, or a sequence that is actually at least 90%, at least 95%, at least 98%, or at least 99% identical to it.

The present invention also provides nucleic acid molecules encoding any of the LCVR amino acid sequences shown in Table. 1; in accordance with certain embodiments, the nucleic acid molecule contains a polynucleotide sequence selected from any of the LCVR nucleic acid sequences shown in Table. 2, or a sequence that is actually at least 90%, at least 95%, at least 98%, or at least 99% identical to it.

The present invention also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences shown in Table. 1; in accordance with certain embodiments, the nucleic acid molecule contains a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences shown in Table. 2, or a sequence that is actually at least 90%, at least 95%, at least 98%, or at least 99% identical to it.

The present invention also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences shown in Table. 1; in accordance with certain embodiments, the nucleic acid molecule contains a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences shown in Table. 2, or a sequence that is actually at least 90%, at least 95%, at least 98%, or at least 99% identical to it.

The present invention also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences shown in Table. 1; in accordance with certain embodiments, the nucleic acid molecule contains a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences shown in Table. 2, or a sequence that is actually at least 90%, at least 95%, at least 98%, or at least 99% identical to it.

The present invention also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences shown in Table 1. 1; in accordance with certain embodiments, the nucleic acid molecule contains a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences shown in Table. 2, or a sequence that is actually at least 90%, at least 95%, at least 98%, or at least 99% identical to it.

The present invention also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences shown in Table 1. 1; according to definition

- 3 041859 divided embodiments, the nucleic acid molecule contains a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences shown in Table. 2, or a sequence that is actually at least 90%, at least 95%, at least 98%, or at least 99% identical to it.

The present invention also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences shown in Table 1. 1; in accordance with certain embodiments, the nucleic acid molecule contains a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences shown in Table. 2, or a sequence that is actually at least 90%, at least 95%, at least 98%, or at least 99% identical to it.

The present invention also provides nucleic acid molecules encoding HCVR, where HCVR contains a set of three CDRs (i.e. HCDR1, HCDR2, HCDR3), where the set of amino acid sequences HCDR1, HCDR2, HCDR3 is determined using any of the exemplary anti-LEPR antibodies provided in table. 1.

The present invention also provides nucleic acid molecules encoding an LCVR, wherein the LCVR comprises a set of three CDRs (i.e., LCDR1, LCDR2, LCDR3), where the set of amino acid sequences of LCDR1, LCDR2, LCDR3 is determined using any of the exemplary anti-LEPR antibodies provided in table. 1.

The present invention also provides nucleic acid molecules encoding both HCVR and LCVR, where HCVR contains the amino acid sequence of any of the HCVR amino acid sequences presented in table. 1, and where LCVR contains the amino acid sequence of any of the LCVR amino acid sequences presented in table. 1. In accordance with certain embodiments, the nucleic acid molecule contains a polynucleotide sequence selected from any of the HCVR nucleic acid sequences shown in Table. 2, or a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to it, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences represented by in table. 2, or a sequence that is actually at least 90%, at least 95%, at least 98%, or at least 99% identical to it. In certain embodiments of this aspect of the present invention, the nucleic acid molecule encodes for HCVR and LCVR, wherein both HCVR and LCVR are derived from the same anti-LEPR antibody shown in Table 1. 1.

The present invention also provides 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 recombinant expression vectors containing any of the nucleic acid molecules mentioned above, for example, nucleic acid molecules encoding any of the HCVR, LCVR and/or CDR sequences set forth in Table. 1. Also included 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 host cells under conditions conducive to the production of antibodies or antibody fragments, and recovering antibodies or antibody fragments, obtained in this way.

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

According to yet another aspect, the present invention provides therapeutic methods for enhancing or stimulating LEPR signaling with an anti-LEPR antibody or an antigen-binding portion of an antibody of the present invention. Therapeutic methods in accordance with 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 antibody fragment of the present invention to a subject in need thereof. A disorder to be treated is any disease or condition that is normalized, ameliorated, suppressed, or prevented by stimulating or activating signaling by LEPR, or otherwise mimicking natural leptin activity in vitro or in vivo.

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

Brief description of the drawings

In FIG. 1 shows the binding of dimeric human LEPR to human leptin in the presence of increasing concentrations of test anti-LEPR antibodies or control molecules measured from to

- 4 041859 by ELISA power (absorption at 450 nm).

In FIG. 2A-C show the extent of signaling by LEPR in HEK293 cells expressing either wild-type LEPR (circles), a signaling-defective LEPR mutant (A409E, squares), or a signaling-defective LEPR mutant (P316T, triangles). LEPR signaling is expressed as pSTAT3-Y705/STAT3 ratio measured by densitometry from Western blots obtained from cells treated with increasing concentrations of leptin (FIG. 2A), H4H16650 (FIG. 2B), or H4H16679 (FIG. 2C). ).

In FIG. 3 shows the mean daily food intake of leptin-deficient mice treated with either an isotype control antibody at 3 mg/kg or an anti-LEPR antibody selected from H4H16650P2, H4H16679P2, H4H17319P2, or H4H17321P2 at 3 mg/kg.

In FIG. 4 shows the mean percent change in body weight of leptin-deficient mice treated with either an isotype control antibody at 3 mg/kg or an anti-LEPR antibody selected from H4H16650P2, H4H16679P2, H4H17319P2, or H4H17321P2 at 3 mg/kg.

In FIG. 5 shows the mean body fat mass of animals in each antibody treatment group, quantified as μCT 1 day before (open bars) and 6 days after antibody treatment (solid bars), expressed as mean±SEM.

In FIG. 6 shows the mean percentage change in body weight of mice fed with 30 mg/kg of an antibody selected from H4H18482P2, H4H18487P2, H4H18492P2, or an isotype control.

Fig. 7A, B. FIG. 7A shows the adipose tissue mass of mice prior to administration of anti-LEPR antibodies H4H18482P2, H4H18487P2, or H4H18492P2. In FIG. 7B shows the adipose tissue mass of mice treated with 30 mg/kg H4H18482P2, H4H18487P2, or H4H18492P2.

Fig. 8. In FIG. 8 shows that the tested anti-LEPR antibodies activated mouse (Mf) LEPR in the IMR-32/STAT3-luc/Mf LEPR cell line.

Detailed disclosure of the present invention

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

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention belongs. As used herein, approximately, when referring to a specific numerical value being described, means that the value may differ from the value being described 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.

Definitions

The expression leptin receptor, LEPR, and the like as used herein refers to a human leptin receptor comprising the amino acid sequence set forth in SEQ ID NO:113 (see also UniProtKB/Swiss-Prot accession number P48357). Alternative names for LEPR used in the scientific literature include OB receptor, OB-R, and CD295. LEPR is also referred to as WSX (see, for example, 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 has no multimerizable domains and that exists under normal conditions as a single LEPR molecule without a direct physical association with another LEPR molecule. An exemplary monomeric LEPR molecule is referred to herein as hLEPR.mmh, containing the amino acid sequence of SEQ ID NO:114 (see, for example, Example 3 of this document). As used herein, dimeric human LEPR refers to a construct containing two LEPR molecules connected to each other by a linker, a covalent bond, a non-covalent bond, or a multimerizable 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 of SEQ ID NO:115 (see, for example, Example 3 herein) or a molecule referred to as hLEPR.hFc containing the amino acid sequence of SEQ ID NO: 116. As used herein, the terms anti-LEPR antibody, antibody that specifically binds LEPR, LEPR-specific binding protein, and the like, unless specifically stated otherwise, refer to molecules that bind full-length human LEPR, monomeric human LEPR, dimeric LEPR person or others

- 5 041859 constructs that contain or consist of an extracellular LEPR domain.

All references to proteins, polypeptides, and protein fragments herein refer to the human variant of the corresponding protein, polypeptide, or protein fragment, unless explicitly stated to be from a non-human species. Thus, the expression LEPR means human LEPR, unless it is indicated that it comes from non-human species, for example, mouse LEPR, monkey LEPR, etc.

As used herein, cell surface expressed LEPR refers to one or more LEPR proteins, or their extracellular domain, that is(are) expressed on the cell surface in vitro or in vivo such that at least a portion of the LEPR protein faces 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 normally (eg, native or wild type) 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 the human leptin receptor include both monovalent antibodies with a single specificity and bispecific antibodies containing a first fragment that binds LEPR and a second fragment that binds a second (target ) antigen, where the fragment of the antibody to LEPR contains any of the sequences HCVR/LCVR or CDR set forth in table. 1 of this document.

The term antibody as used herein means any antigen-binding molecule or molecular complex containing at least one complementarity determining region (CDR) that specifically binds to 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 linked 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, C _H 2 and C _H 3. Each light chain contains a light chain variable region (herein abbreviated LCVR or VL) 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 called complementarity determining regions (CDRs) interspersed with more conserved regions called 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. In accordance with various embodiments of the present invention, the FR of an anti-LEPR antibody (or antigen-binding site thereof) may be identical to human germline sequences, or may be naturally or artificially altered. The amino acid consensus sequence can be determined based on a side-by-side analysis of two or more CDRs.

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

Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab') ₂ fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv molecules (scFv); (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic an antibody hypervariable region (e.g., a distinct complementarity determining region (CDR), such as a CDR3 peptide), or a peptide with restricted conformational freedom.

- 6 041859 Baud FR3-CDR3-FR4. 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 antibodies, bivalent antibodies, etc.) , small molecule modular protein (SMIP) immunotherapies, and shark IgNAR variable domains are also included in the expression antigen-binding fragment used herein.

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

In accordance with 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 exemplary variable and constant domain configurations that can be found in an antigen-binding fragment of an antibody of the present invention include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) V _H -C _H 1 -C _H 2 -C _H 3; (vi) Vh-Ch2-Ch3; (vii) Vh-Cl; (viii) Vl-Ch1; (ix) Vl-Ch2; (x) Vl-Ch3; (xi) Vl-Ch1-Ch2; (xii) Vl-Ch1-Ch2-Ch3; (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 the exemplary configurations set forth above, the variable and constant domains may either be directly linked to each other, or may be linked through all or part of a hinge or linker region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that result in a flexible or semi-flexible bond between adjacent variable and/or constant domains in a single polypeptide molecule. In addition, an antigen-binding fragment of an antibody of the present invention may comprise a homodimer or heterodimer (or other multidimer) of any of the variable and constant domain configurations set forth 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 whole antibody molecules, antigen binding fragments can be monospecific or multispecific (eg, bispecific). A multispecific antigen-binding fragment of an antibody will typically contain at least two different variable domains, where each variable domain is capable of specifically binding to a separate 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 accordance with certain embodiments of the present invention, the anti-LEPR antibodies of the present invention are human antibodies. The term human antibody as used herein includes antibodies having 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-specific mutagenesis or in vivo somatic mutation), such as in CDRs and in particular CDR3. However, the term human antibody as used herein does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences.

The antibodies of the present invention may, in certain embodiments, be recombinant human antibodies. The term recombinant human antibody as used herein includes all antibodies produced, expressed, generated or isolated by a recombinant method, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described below), antibodies isolated from a combinatorial recombinant human antibody libraries (described 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 obtained, expressed, generated or isolated by any other method, including the connection of human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However

- 7 041859 according to certain embodiments, such recombinant human antibodies undergo in vitro mutagenesis (or in the case of an animal transgenic for human Ig sequences, somatic in vivo mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences which, although derived from human germline VH and V _L sequences and linked to human germline VH and V _L sequences, may not occur naturally in the in vivo human germline antibody repertoire.

The present invention encompasses antibodies with one or more mutations in the hinge, the CH2 or CH3 region may be preferred, eg under manufacturing conditions, to increase the yield of the preferred antibody form.

The antibodies of the present invention may be isolated antibodies. The term isolated antibody as used herein 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 separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody occurs naturally or is naturally formed, 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 accordance with 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 containing 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, as compared to the corresponding germline sequences from which the antibodies happened. 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 that are derived from the amino acid sequences disclosed herein, wherein one or more amino acids in one or more framework regions and/or CDR regions are mutated to the corresponding germline sequence residue(s), from which the antibody originated, or up to the corresponding germline residue(s), or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as germline mutations). One of skill in the art, starting from the heavy and light chain variable region sequences disclosed herein, can easily generate multiple antibodies and antigen-binding fragments that contain one or more individual germline mutations, or combinations thereof. In certain embodiments, all of the framework and/or CDR residues in the V _H and/or V _L domains are back-mutated to residues occurring in the original germline sequence from which the antibody originated. In other embodiments, only certain residues are back-mutated back to the original germline sequence, for example, only mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In accordance with other embodiments, one or more of the framework regions and/or one or more of the CDR residue(s) are mutated to the corresponding residue(s) of another germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody originally originated). In addition, the antibodies of the present invention may contain any combination of two or more germline mutations in the framework regions and/or CDR regions, for example, where certain individual residues mutate to the corresponding residue of the specified germline sequence, while certain other residues, that differ from the original germline sequence are retained or mutated to the corresponding residue of the other germline sequence. Once produced, antibodies and antigen-binding fragments that contain one or more germline mutations can be readily tested for one or more preferred properties, such as improved binding specificity, increased binding affinity, improved or enhanced biological antagonist or agonist properties (if necessary). ), reduced immunogenicity, and the like. Antibodies and antigen-binding fragments obtained by this general method are covered by the present invention.

The present invention provides anti-LEPR antibodies and antigen-binding fragments thereof, which contain amino acid sequences that are substantially similar or substantially identical to one or more amino acid sequences of the variable domains.

- 8 041859 or CDR as found in any of the exemplary anti-LEPR antibodies disclosed herein.

When applied to polypeptides, the term actual analogy or virtually similar means that two peptide sequences, when optimally aligned, for example, using the GAP or BESTFIT computer programs with default gap opening penalties, are at least 95% identical in sequence, even more preferably at least 98% or 99% identical in sequence. 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 with another amino acid residue having a side chain (R-group) with similar chemical properties (eg, charge or hydrophobicity). Generally, a conservative amino acid substitution will not significantly alter the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making such an adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331. Examples of groups of amino acids that have 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) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred groups of conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamine-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445. A moderately conservative substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

Sequence similarity in the case of polypeptides, also referred to as sequence identity, is usually measured using a sequence analysis computer program. A protein analysis computer program combines similar sequences using similarity scores assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, the GCG tool contains programs such as Gap and Bestfit that can be used with default parameters to determine 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, version 6.1 of the GCG. Polypeptide sequences can also be compared using FASTA using the default parameters or recommended parameters, the software tool in version 6.1 of the GCG. FASTA (eg, FASTA2 and FASTA3) provides for alignments and percent identity of the regions of best overlap between a query and a found sequence (Pearson (2000), supra). Another preferred algorithm for comparing a sequence of 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.

Anti-LEPR antibodies containing Fc variants

In accordance with certain embodiments of the present invention, anti-LEPR antibodies are provided that contain an Fc domain containing one or more mutations that increase or decrease antibody binding to the FcRn receptor, for example, at acidic pH versus neutral pH. For example, the present invention provides antibodies to LEPR containing a mutation in the CH2 or C _H 3 region of the Fc domain, where the mutation(s) increases(increases) the affinity of the Fc domain for FcRn in an acidic environment (for example, in the endosome, where the value pH ranges from about 5.5 to about 6.0). Such mutations can lead to increased serum half-life when administered to an animal. Non-limiting examples of such Fc modifications include, for example, a 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 a 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 is a modification of 428L (eg, M428L) and 434S (eg, N434S); modification 428L, 259I (for example, V259I) and 308F (for example, V308F); modification 4zzK (for example, H433K) and 434

- 9 041859 (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 in the variable domains of an antibody disclosed herein are contemplated within the scope of the present invention.

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

In accordance with certain embodiments of the present invention, the modified Fc domain is an IgG1 variant Fc or an IgG4 variant Fc containing a hinge substitution. For example, a modified Fc for use in the context of the present invention may comprise a variant IgG1 Fc wherein at least one IgG1 Fc hinge amino acid is substituted 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 wherein at least one IgG4 Fc hinge amino acid is substituted 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 set forth, for example, in US Patent Application Publication No. 2014/0243504.

Other modified Fc domains and Fc modifications that can be used in the context of the present invention include any of the modifications set forth in US 2014/0171623; US 8697396; US 2014/0134162; WO 2014/043361. Methods for constructing antibodies or other antigen-binding fusion proteins containing the modified Fc domain described herein are known in the art.

Biological characteristics of antibodies

The present invention provides antibodies and antigen-binding fragments thereof that bind human LEPR and activate signaling through LEPR. Such antibodies may be referred to as agonistic antibodies. In the context of the present invention, activation of signaling by LEPR means the stimulation of an intracellular effect that normally results from the interaction of leptin with LEPR in cells that express LEPR. In certain embodiments, activation of signaling by LEPR means STAT3 transcriptional activity that can be detected by any method that can measure or identify, directly or indirectly, STAT3 activity, such as a labeled STAT3 variant expressed in a reporter cell. cell line. For example, the present invention provides antibodies and antigen-binding fragments thereof that activate signaling by LEPR in a cellular reporter assay, eg, using the cellular assay format defined in Example 7 of this document, or a substantially similar assay. Cellular reporter assays that detect LEPR activation, such as the assay set forth in Example 7 of this document, may result in a detectable signal that can be expressed in the context of an EC ₅₀ value (e.g., antibody concentration required to obtain half-maximal transmission signal) and/or the percentage of maximum signal transmission observed in the presence of leptin. In accordance with certain illustrative embodiments of the present invention, anti-LEPR antibodies are provided that activate signaling by LEPR at an EC ₅₀ value of less than about 12.0 nM in a cellular reporter assay, for example, using the assay format defined in Example 7 of this document. , or actually similar analysis. In accordance with certain exemplary embodiments of the present invention, anti-LEPR antibodies are provided that activate signaling by LEPR at a maximum percentage of activation relative to signaling by leptin of greater than about 65% in a cellular reporter assay, e.g., using an assay format, as defined in example 7 of this document, or an actually similar analysis.

The present invention provides antibodies and antigen-binding fragments thereof, which

- 10 041859 bind monomeric human LEPR with high affinity. For example, the present invention provides anti-LEPR antibodies that bind monomeric human LEPR (e.g., hLEPR.mmh, SEQ ID NO:114) with a KD of less than about 150 nM as measured by surface plasmon resonance at 25 or 37°C. , for example, when applying the analysis format defined in example 3 of this document, or actually similar analysis. In certain embodiments, anti-LEPR antibodies are provided that bind monomeric human LEPR at 25° C. with a K _D of less than about 150 nM, less than about 140 nM, less than about 130 nM, less than about 120 nM, less than less than about 110 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM , less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, or less than about 300 pM, as measured by surface plasmon resonance, for example, using the assay format defined in Example 3 of this document, or actually similar analysis.

The present invention also provides antibodies and antigen-binding fragments thereof that bind monomeric human LEPR (e.g., hLEPR.mmh, SEQ ID NO:114) with a dissociation half-life (t1/ ₂ ) of greater than about 50 min as measured by surface plasmon resonance at 25 or 37°C, for example, using the analysis format defined in example 3 of this document, or actually similar analysis. In certain embodiments, anti-LEPR antibodies are provided that bind monomeric human LEPR at 25° C. with a t ¹ / ₂ of greater than about 50 minutes, greater than about 55 minutes, greater than about 60 minutes, greater than about 65 minutes. or longer as measured by surface plasmon resonance, for example, using the assay format defined in Example 3 of this document, or a virtually similar assay.

The present invention also provides antibodies and antigen-binding fragments thereof that bind dimeric human LEPR (eg, hLEPR.mFc, SEQ ID NO:115) with high affinity. For example, the present invention provides anti-LEPR antibodies that bind dimeric human LEPR with a K _D of less than about 1.5 nM as measured by surface plasmon resonance at 25 or 37°C, for example, using the assay format defined in example 3 of this document, or an actually similar analysis. In certain embodiments, anti-LEPR antibodies are provided that bind dimeric human LEPR at 25° C. with a K _D of less than about 150 nM, less than about 130 nM, less than about 110 nM, less than about 80 nM, less than less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, or less than about 10 nM, as measured by surface plasmon resonance, for example , when applying the analysis format defined in Example 3 of this document, or a virtually similar analysis.

The present invention also provides antibodies and antigen-binding fragments thereof that bind dimeric human LEPR (e.g., hLEPR.mFc, SEQ ID NO:115) with a dissociation half-life (VA) of greater than about 10 minutes, as measured by surface plasmon resonance at 25 or 37°C, for example, using the analysis format defined in example 3 of this document, or actually similar analysis. In certain embodiments, anti-LEPR antibodies are provided that bind dimeric human LEPR at 25° C. with a t ¹ / ₂ of greater than about 10 minutes, greater than about 15 minutes, greater than about 20 minutes, greater than about 25 minutes. , more than about 30 minutes, more than about 40 minutes, more than about 50 minutes, more than about 60 minutes, more than about 70 minutes, or longer, as measured by surface plasmon resonance, for example, using the assay format defined in example 3 of this document, or an actually similar analysis.

The present invention also provides antibodies and antigen-binding fragments thereof that bind LEPR in complex with human leptin (LEPR in complex with human leptin can also be represented as 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 containing hLEPR and human leptin. That is, in accordance with certain embodiments, the interaction between anti-LEPR and LEPR antibodies is not

- 11 041859 is suppressed by the presence of leptin in combination with LEPR; similarly, the interaction between leptin and LEPR according to this 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 set forth in Example 4 of this document.

Similarly, the present invention also provides antibodies and antigen-binding fragments thereof that bind LEPR and do not block the LEPR:leptin interaction. For example, the present invention provides antibodies and antigen-binding fragments thereof that are capable of binding to LEPR, thereby leading to the formation of an antibody:LEPR complex, where the resulting antibody:LEPR complex is able to interact with leptin to form a three-membered complex containing antibody, LEPR and leptin. An exemplary assay format for determining whether an antibody or antigen-binding fragment thereof is capable of binding LEPR without blocking or interfering with the interaction between LEPR and leptin is set forth in Example 5 of this document.

The present invention also provides antibodies and antigen-binding fragments thereof that bind cell surface expressed LEPRs in the presence and/or absence of human leptin. Cell surface expressed LEPR means LEPR or a portion thereof (e.g., the extracellular region of LEPR) expressed on the cell surface, either naturally or in an engineered cell line, such that an antibody or antigen-binding fragment thereof is capable of binding to a LEPR molecule. In certain embodiments, cell-surface expressed LEPR comprises recombinant complexes comprising an extracellular LEPR domain linked to a cell via a tag or anchor (eg, a GPI anchor, as shown in Example 6 herein). In accordance with this aspect of the present invention, antibodies are provided that are capable of binding cell surface expressed LEPR in the absence of leptin, and that are also capable of binding cell surface expressed LEPR in the presence of leptin (i.e., under conditions where leptin is able to bind to cell surface expressed LEPR). cell surface leptin receptor). That is, in accordance with 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 in accordance with this 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 cell surface-expressed LEPR in the presence or absence of human leptin is set forth in Example 6 of this document.

The antibodies of the present invention may have one or more of the aforementioned biological characteristics, or any combination thereof. The above list of biological characteristics of the antibodies of the present invention does not claim to be exhaustive. Other biological characteristics of the antibodies of the present invention will be apparent to a person skilled in the art from a review of the present disclosure, including illustrative examples of this document.

Epitope mapping and related technologies

The present invention also provides antibodies to LEPR containing 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 antibodies to LEPR with the amino acid sequences of HCVR, LCVR, and/or CDR, eg, 10 or less, 8 or less, 6 or less, 4 or less, and the like. conservative amino acid substitutions with respect to any of the amino acid sequences of HCVR, LCVR and/or CDR set forth in table. 1 of this document. In accordance with certain variants of implementation of the present invention provides antibodies to LEPR containing variant amino acid sequences of HCVR, LCVR and/or CDR in relation to the sequences set forth in table. 1 of this document (eg, containing conservative amino acid substitutions), where such variant antibodies nonetheless share one or more of the functions and/or properties of the exemplary anti-LEPR antibodies disclosed herein.

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

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. One antigen may have more than one epitope. Thus, different antibodies can bind

- 12 041859 different regions on the antigen and may have different biological effects. An epitope can be either conformational or linear. A conformational epitope is formed by spatially arranged amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope formed by adjacent amino acid residues in a polypeptide chain. Under certain conditions, the epitope may include fragments of saccharides, phosphoryl groups or sulfonyl groups of the antigen.

The present invention provides antibodies to LEPR that interact with one or more epitopes occurring in the human LEPR amino acids M1-D839 (SEQ ID NO: 113). As described in Example 11, the 201 peptide from human LEPR significantly reduced deuterium uptake when bound to the H4H16650P2 antibody. Peptides corresponding to amino acids 162-169 (human LYVLPEVL LEPR amino acids, SEQ ID NO: 113) and 170-191 (human EDSPLVPQKGSF LEPR amino acids, SEQ ID NO: 113) had slower deuterium uptake rates when bound to H4H16650P2, indicating that that this antibody binds at least two human LEPR epitopes having the sequences LYVLPEVL or EDSPLVPQKGSF (amino acids 162-169 or 170-191 respectively SEQ ID NO: 113).

The epitope to which antibodies of the present invention bind may consist of one 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 site remote from the leptin-binding domain of LEPR, for example, at a location 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 skilled in the art can be used to identify amino acids in an epitope recognized by a particular antibody. Illustrative techniques include, for example, alanine-based mutation analysis, peptide blot analysis, and peptide cleavage 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 the amino acids in the polypeptide with which the antibody interacts is hydrogen-deuterium exchange, detected using mass spectrometry. In general, the method based on hydrogen-deuterium exchange involves labeling with deuterium a protein of interest, followed by binding of the antibody to the deuterium labeled protein. The protein-antibody complex is then transferred to water to promote hydrogen-deuterium exchange in all residues except for antibody-protected residues (which remain labeled with deuterium). After dissociation of the antibody, the target protein is subjected to protease digestion and mass spectrometric analysis, thereby identifying 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 crystallographic analysis of an antibody in complex with its antigen can also be used to identify the amino acids in the polypeptide with which the antibody interacts.

The present invention further provides anti-LEPR antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein (eg, antibodies comprising any of the amino acid sequences set forth in Table 1 of this document). Similarly, the present invention also provides anti-LEPR antibodies that compete for binding to LEPR of any of the specific exemplary antibodies described herein (eg, antibodies comprising any of the amino acid sequences set forth in Table 1 of this document).

Whether an antibody binds to or competes for binding to the same epitope as a reference LEPR antibody can be determined using standard methods known in the art and exemplified 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 bound to a LEPR protein. The ability of the test antibody to bind to the LEPR molecule is then evaluated. If the test antibody is able to bind to LEPR after saturation binding to a reference anti-LEPR antibody, it can be concluded that the test antibody binds to a different epitope compared to 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 can bind to the same epitope as the epitope bound by the reference anti-LEPR antibody of the present invention. Additional routine experimental work (e.g., peptide mutation and binding assays) can be performed to confirm whether the observed lack of binding of the antibody of interest is essentially due to binding to the same epitope as the reference antibody, or spatial blocking (or other phenomenon) is reason for the absence

- 13 041859 observed binding. Experiments of this kind can be performed using ELISA, RIA, Biacore, flow cytometry, or other quantitative or qualitative antibody binding assays available in the art. In accordance with certain embodiments of the present invention, two antibodies bind to the same (or overlapping) epitope if, for example, 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, but preferably 75, 90 or even 99% as measured in a competition binding assay (see, for example, Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antibodies bind to the same epitope if substantially 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 have overlapping epitopes unless a subset of amino acid mutations that impair or abolish the binding of one antibody impair or abolish 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 bound 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 bound to the LEPR molecule under saturating conditions, followed by evaluation of the binding of the reference antibody to the LEPR molecule. If in both orientations only the first (binding) antibody is able to bind to the LEPR molecule, then it is concluded that the test antibody and the reference antibody compete for binding to LEPR. As one of skill in the art will appreciate, an antibody that competes for binding with a reference antibody may optionally bind to the same epitope as the reference antibody, but may spatially block 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 generate human antibodies that specifically bind to LEPR.

For example, using VELOCIMMUNE™ technology or any other similar known method for producing fully human monoclonal antibodies, high affinity chimeric anti-LEPR antibodies are initially isolated from a human variable region and a murine constant region. As in the experimental section below, antibodies are characterized and selected for desirable characteristics, including affinity, ligand blocking activity, selectivity, epitope, and others. wild-type or modified IgG1 or IgG4 to produce a fully human anti-LEPR antibody. While the constant region chosen may vary depending on the particular application, the variable region retains high affinity antigen binding or target specificity characteristics. 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 having amino acid sequences that differ from those of the described antibodies, but which retain the ability to bind human LEPR. Such variant antibodies and antibody fragments contain one or more additions, deletions, or substitutions of amino acids compared to the parent sequence, yet have biological activity that is substantially equivalent to that of the desired antibodies. Similarly, the DNA sequences of the present invention encoding an anti-LEPR antibody encompass sequences that contain one or more amino acid additions, deletions, or substitutions compared to the disclosed sequence, but which encode an anti-LEPR antibody or antibody fragment that is substantially equivalent to an anti-LEPR antibody or fragment. antibodies of the present invention. Examples of such variant amino acids and DNA sequences are disclosed 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 does not differ significantly when administered at the same molar dose under similar experimental conditions, either as a single dose or a multiple dose . Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in absorption but not in absorption rate, and in addition may be considered bioequivalent because such differences in absorption rate are targeted and reflected in the labeling, are not necessary to achieve effective concentrations of drugs in the body, for example, with chronic use, and are not considered

- 14 041859 medically significant for a particular investigational medicinal product.

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's treatment can be switched 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 continuous therapy. without such a switch.

In one embodiment, two antigen-binding proteins are bioequivalent if they both act according to 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. Indicators of bioequivalence include, for example, (a) an in vivo study 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 study that has been correlated with in vivo human bioequivalence data and is a reliable predictor of such; (c) an in vivo study in humans or other mammals, in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) a strictly controlled clinical trial that establishes the safety, efficacy and bioavailability or bioequivalence of the 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 not essential for biological activity can be removed or replaced with other amino acids to prevent the formation of unnecessary or erroneous intramolecular disulfide bridges during renaturation. In other contexts, bioequivalent antibodies may include anti-LEPR antibody variants containing amino acid changes 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 accordance with certain embodiments, provides anti-LEPR antibodies that bind to human LEPR but not to LEPR from other species. The present invention also provides anti-LEPR antibodies that bind to human LEPR and to LEPR from one or more non-human species. For example, the anti-LEPR antibodies of the present invention may bind to human LEPR and may or may not bind, as appropriate, to one or more of the mouse, rat, guinea pig, hamster, gerbil, porcine, cat, dog, rabbit, goats, sheep, cows, horses, camels, crabeater monkeys, marmosets, rhesus monkeys or chimpanzees. 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 human LEPR but do not bind or only slightly 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 of the same target polypeptide or may contain antigen-specific 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 form a bispecific or multispecific antibody with a second binding specificity.

The present invention provides for bispecific antibodies wherein one immunoglobulin arm binds human LEPR and the other immunoglobulin arm is specific for a second antigen. The LEPR binding arm may comprise any of the HCVR/LCVR or CDR amino acid sequences set forth in Table 1. 1 of this document.

An exemplary format for a bispecific antibody that can be used in the context of the present invention provides for the use of a first C _H 3 immunoglobulin (Ig) domain and a second C _H 3 Ig domain, wherein the first and second Ig C _H 3 domains differ from each other by at least one amino acid, and where at least one amino acid residue difference weakens the binding of the bispecific antibody to protein A compared to a bispecific antibody that does not have

- 15 041859 lychee in the amino acid residue. In one embodiment, the first Ig _C H 3 domain binds protein A and the second Ig C _H 3 domain contains a mutation that reduces or abolishes protein A binding, such as an H95R modification (according to IMGT exon numbering; H435R in according to the EU numbering). The second CH3 may additionally contain the modification Y96F (in accordance with IMGT; Y436F in accordance with EU). Additional modifications that can be found in 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. Variations in the format of the bispecific antibodies described above are within 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 diabody-based bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, overhangs-in -cavities, conventional light chain (e.g., conventional light chain with ledge-in-cavities, etc.), CrossMab, CrossFab, (SEED)-body, leucine zipper, Duobody, IgG1/IgG2, Fab (DAF)-IgG dual action and Mab ² bispecific formats (see, for example, Klein et al. 2012, mAbs 4:6, 1-11, and references cited herein for an examination of the above formats). Bispecific antibodies can also be constructed using peptide-nucleic acid conjugation, for example, where non-naturally occurring amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-nucleotide conjugates that then self-assemble into multimeric complexes with specific composition, valency, and geometry. (See, for example, Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

Therapeutic composition and administration

The present invention provides pharmaceutical compositions containing antibodies to LEPR or their antigennegative fragments of the present invention. Pharmaceutical compositions of the present invention are formulated with suitable carriers, adjuvants, and other agents that provide improved transfer, delivery, tolerability, and the like. Many suitable formulations can be found in the reference book 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 absorption pastes, emulsions such as oil in water and water in oil, carbovax emulsions (polyethylene glycols of various molecular weights), semi-liquid gels and semi-liquid mixtures containing carbovax. 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 characteristics of the patient, the target disease, conditions, route of administration, and the like. The preferred dose is usually calculated according to body weight or body surface area. In the case of an adult patient, intravenous administration of an antibody of the present invention may be preferred, typically in a single dose of from about 0.01 to about 20 mg/kg body weight, more preferably from about 0.02 to 7, from about 0.03 to about 5 or about 0.05 to about 3 mg/kg body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. Effective doses and regimens for administering anti-LEPR antibodies can be determined empirically; for example, the evolution of a patient's condition can be monitored by periodic evaluation and the dose can be adjusted accordingly. In addition, cross-species dosing can be performed using methods well known in the art (eg, Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

Various delivery systems are known and can be used to administer the pharmaceutical composition of the present invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, 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 method, for example, by infusion or bolus injection, absorption through the epithelial or mucocutaneous integument (for example, oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with other biologically active agents. The introduction can 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, in the case of subcutaneous delivery, a pen is widely used to deliver the pharmaceutical composition of the present invention.

- 16 041859

Such a syringe pen can be reusable and disposable. A reusable pen typically uses a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been entered and the cartridge is empty, the cartridge can be easily disposed of and replaced with a new cartridge that contains the pharmaceutical composition. The pen can then be reused. There is no replaceable cartridge in the disposable syringe pen. Instead, the disposable pen is delivered pre-filled with a pharmaceutical composition contained in a reservoir in the article. Once the reservoir is emptied of the pharmaceutical composition, the entire product is discarded.

Numerous reusable pens and autoinjector delivery devices are used in the subcutaneous delivery of the pharmaceutical composition of the present invention. Examples include, without limitation, AUTOPEN™ pen (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ (Disetronic Medical Systems, Burgdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, syringe - HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, Indiana), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), syringe pen BD™ (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™ and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), to name a few. Examples of reusable pens for use in the subcutaneous administration of the pharmaceutical composition of the present invention include, but are not limited to SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk) and KWIKPEN™ (Eli Lilly), SURECLICK™ autoinjector (Amgen, Towsand Oaks, CA), PENLETTM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.) and the HUMIRATM pen (Abbott Labs, Abbott Park, Illinois), to name a few.

In certain situations, the pharmaceutical composition may be delivered in a controlled release system. According to one embodiment, a pump may be used (see Langer, supra; 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 another embodiment, the controlled release system can be placed close to the target of the composition, requiring only a fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115- 138). Other controlled release systems are reviewed by Langer, 1990, Science 249:1527-1533.

Injectable forms may include intravenous, subcutaneous, intradermal and intramuscular injection dosage forms, droppers, and the like. These injection forms can be obtained by well-known methods. For example, injectable forms can be prepared, for example, by dissolving, suspending, or emulsifying an antibody or salt thereof, as described above, in a sterile aqueous or oily medium commonly used for injection. An aqueous injection medium is, for example, saline, an isotonic solution containing glucose and other excipients, and the like, for example, which can be used in combination with a suitable solvent such as an alcohol (for example, ethanol), a polyalcohol (for example, , propylene glycol, polyethylene glycol), a non-ionic surfactant (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) hydrogenated castor oil adduct)) and the like. As the oil medium, for example, sesame oil, peanut oil and the like are used, which can be used in combination with a solvent such as benzyl benzoate, benzyl alcohol and the like. The injection thus prepared is preferably filled into a suitable ampoule.

Preferably, the pharmaceutical compositions for oral or parenteral administration described above are formulated into unit dosage forms suitable to match the dosage of the active ingredients. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, and the like. especially in the form of an injection, it is preferred that the above antibody is present in an amount of from about 5 to about 100 mg, and from about 10 to about 250 mg in the case of 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 antibody (eg, an anti-LEPR antibody comprising any of the HCVR/LCVR or CDR sequences set forth in Table 1 of this document). 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, for the treatment, prevention and/or amelioration of any disease or disorder associated with or mediated by malnutrition.

- 17 041859 leptin deficiency, leptin resistance, hypoleptinemia or otherwise being treated as a result of stimulation or activation of signaling by LEPR or mimicking natural leptin activity in vitro or in vivo. For example, the antibodies and antigen-binding fragments thereof of the present invention are useful in the treatment of lipodystrophic conditions. Exemplary lipodystrophy conditions to be treated with the antibodies and antigen-binding fragments of the present invention include, for example, congenital generalized lipodystrophy, acquired generalized lipodystrophy, familial partial lipodystrophy, acquired partial lipodystrophy, centrifugal abdominal lipodystrophy, annular lipodystrophy, localized lipodystrophy, and HIV-associated lipodystrophy.

The present invention also provides anti-LEPR antibodies and antigen-binding fragments thereof, which are useful for restoring leptin signaling to cells, tissues and organs expressing one or more obesity-associated LEPR mutations. For example, certain LEPR mutants have been identified that are characterized by the absence or attenuation of signaling in the presence of leptin and are associated with obesity and related disorders. As used herein, a LEPR mutant that lacks signal transduction in the presence of leptin is referred to as a signal transduction defective LEPR mutant. An exemplary LEPR mutation with a signaling defect is LEPR-A409E (Farooqi et al., 2007, N Engl J Med 356(3): 237-247). As used herein, a LEPR mutant that exhibits impaired signaling in the presence of leptin (compared to wild type LEPR) is referred to as a signaling impaired LEPR mutant. An exemplary LEPR signal-disrupting mutation is LEPR-P316T (Mazen et al., 2011, Mol Genet Metab 102:461-464). Thus, the present invention provides anti-LEPR antibodies and antigen-binding fragments thereof, which are useful in the treatment, prevention and/or amelioration of diseases and disorders caused by or associated with one or more LEPR mutants with a signal transduction defect (e.g., A409E) and/or signal transmission (for example, P316T).

The anti-LEPR antibodies and antigen-binding fragments thereof of the present invention are also useful in the treatment or prevention of one or more diseases or disorders selected from the group consisting of obesity, metabolic syndrome, dietary cravings, functional hypothalamic amenorrhea, type 1 diabetes, type 2 diabetes, insulin resistance, severe resistance, including severe insulin resistance due to insulin receptor mutation, severe insulin resistance not due to insulin receptor mutation, severe insulin resistance due to downstream mutation or other causes , non-alcoholic and alcoholic fatty liver disease, Alzheimer's disease, leptin deficiency, leptin resistance, leprechaunism/Donoghue syndrome, and Rabson-Mendenhall syndrome.

In the context of the treatments described herein, an anti-LEPR antibody may be administered as monotherapy (eg, as a single therapeutic agent) or in combination with one or more additional therapeutic agents (examples of which are described elsewhere herein).

Combined drugs 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.

Anti-LEPR antibodies of the present invention may be formulated with and/or administered in combination with one or more additional therapeutically active ingredients such as, for example, pharmaceutical products prescribed for the treatment of obesity, hypercholesterolemia, hyperlipidemia, type 2 diabetes, type 1 diabetes, appetite control, infertility, etc. Examples of such additional therapeutically active components include, for example, recombinant human leptin (for example, metreleptin (MYALEPT)), PCSK9 inhibitors (for example, antibodies to PCSK9 (alirocumab, evolocumab, bococizumab, lodelcizumab, ralpancizumab, etc.). )), statins (atorvastatin, rosuvastatin, cerivastatin, pitavastatin, fluvastatin, simvastatin, lovastatin, pravastatin, etc.), ezetemib, insulin, insulin variants, insulin secretogens, metformin, sulfonylurea, sodium glucose cotransporter 2 (SGLT2) inhibitors (e.g. , dapagliflozin, canagliflozin, empagliflozin, etc.), GLP-1 agonists/analogs (eg, extendin-4, exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, etc.), glucagon inhibitors (GCG) (eg, anti-GCG antibodies), glucagon receptor (GCGR) inhibitors (eg, anti-GCGR antibodies), small molecule GCGR antagonists, GCGR -specific antisense oligonucleotides, aptamers to GCGR (for example, Spiegelmers), etc.), inhibitors of angiopoietin-like protein (ANGPTL) (for example, antibodies to ANGPTL3, antibodies to ANGPTL4, antibodies to ANGPTL8, etc.), phentermine, orlistat, topiramate , bupropion, topiramate/phentermine, bupropion/naltrexone, bupropion/zonisamide, pramlintide/metreleptin, lorcaserin,

- 18 041859 cetilistat, tesofensine, wellneperite, etc.

The additional therapeutically active ingredient(s), e.g., any of the agents above or derivatives thereof, may be administered immediately before, concurrently with, or shortly after administration of an anti-LEPR antibody of the present invention (for the purposes of this disclosure, such administration regimens is the administration of an anti-LEPR antibody in combination with an additional therapeutically active component). The present invention provides pharmaceutical compositions wherein an anti-LEPR antibody of the present invention is formulated with one or more additional therapeutically active ingredients as described elsewhere herein.

Modes of administration

In accordance with certain embodiments of the present invention, multiple doses of an anti-LEPR antibody (or a pharmaceutical composition comprising a combination of an anti-LEPR antibody and any of the additional therapeutically active agents mentioned herein) may be administered to a subject over a period of time. Methods in accordance with 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, sequential administration means that each dose of an anti-LEPR antibody is administered to a subject at a different point in time, 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 second doses of an anti-LEPR antibody, and then optionally one or more third doses of an anti-LEPR antibody.

The terms initial dose, second doses, and third doses refer to the time sequence of administration of an anti-LEPR antibody of the present invention. Thus, the starting dose is the dose that is administered at the start of a therapeutic regimen (also called baseline dose, loading dose, initial dose, etc.); the second doses are doses administered after the initial dose; and the third doses are doses administered after the second doses. All of the initial, second, and third doses may contain the same amount of LEPR antibody, but generally may differ from each other in frequency of administration. In certain embodiments, the amount of anti-LEPR antibody contained in the initial, second, and/or third doses varies from one another (eg, adjusted upwards or downwards as necessary) during the course of treatment. In certain embodiments, two or more (eg, 2, 3, 4, or 5) doses are administered at the start of the therapeutic regimen as bolus doses, then subsequent doses are administered on a less frequent basis (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 abnormal expression (eg, overexpression, underexpression, lack of expression, etc.) of LEPR. Exemplary diagnostic assays for LEPR may involve, 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 in diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The label or reporter molecule to be detected may be a radioisotope 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), fluorescence activated cell sorting (FACS), and positron emission tomography (PET) scanning.

Samples that can be used in LEPR diagnostic assays according to 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 specific sample obtained from a healthy patient (eg, a patient not affected by 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 level can then be compared with LEPR levels measured in samples obtained from individuals suspected of having a LEPR-related disease or condition.

Examples

The following examples are provided for the purpose of providing those skilled in the art with a complete

- 19 041859 disclosures and descriptions of how to make and use the methods and compositions of the present invention, and not for the purpose of limiting the scope of what the authors of the present invention consider as their invention. Efforts have been made to ensure accuracy with respect to the numbers used (eg quantity, temperature, etc.), however, some experimental errors and deviations must be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric or near atmospheric.

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

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

Certain biological properties of exemplary anti-LEPR antibodies prepared according to this example are detailed in the Examples section below.

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

In table. 1 sets out the amino acid sequence identifiers for the variable regions and the heavy and light chain CDRs of some of the anti-LEPR antibodies of the present invention. Identifiers of the corresponding nucleic acid sequences are set out in table. 2.

Table 1. Amino acid sequence identifiers

SEQID NO: Designation of antibodies HCVR HCDR1 HCDR2 HDDR3 LCVR LCDR1 LCDR2 LCDR3 H4H16650R2 2 4 6 8 10 12 14 16 H4H16679R2 18 20 22 24 10 12 14 16 H4H17319R2 26 28 thirty 32 10 12 14 16 H4H17321R2 34 36 38 40 10 12 14 16 H4H18417R2 42 44 46 48 10 12 14 16 H4H18438R2 50 52 54 56 10 12 14 16 H4H18445R2 58 60 62 64 10 12 14 16 H4H18446R2 66 68 70 72 10 12 14 16 H4N18449R2 74 76 78 80 10 12 14 16 H4H18482R2 82 84 86 88 90 92 94 96 H4H18487R2 98 100 102 104 90 92 94 96 H4N18492R2 106 108 BY 112 90 92 94 96

- 20 041859

Table 2. Nucleic acid sequence identifiers

SEQID NO: Designation of antibodies HCVR HCDR1 HCDR2 HDDR3 LCVR LCDR1 LCDR2 LCDR3 H4H16650R2 1 3 5 7 9 eleven 13 15 H4H16679R2 17 19 21 23 9 eleven 13 15 H4H17319R2 25 27 29 31 9 eleven 13 15 H4H17321R2 33 35 37 39 9 eleven 13 15 H4H18417R2 41 43 45 47 9 eleven 13 15 H4H18438R2 49 51 53 55 9 eleven 13 15 H4H18445R2 57 59 61 63 9 eleven 13 15 H4H18446R2 65 67 69 71 9 eleven 13 15 H4H18449R2 73 75 77 79 9 eleven 13 15 H4H18482R2 81 83 85 87 89 91 93 95 H4H18487R2 97 99 101 103 89 91 93 95 H4N18492R2 105 107 109 111 89 91 93 95

Antibodies are usually designated herein according to the following nomenclature: Fc prefix (e.g., H4H, H1M, H2M, etc.), then a numeric identifier (e.g., 16650, 16679, etc.), then a P or N suffix. Thus, in accordance with this nomenclature, an antibody may be referred to herein as, for example, H4H16650P2, H4H16679P2, etc. The Fc prefix in antibody designations used herein (H4H, H1M, and H2M) indicates the specific isotype of the Fc region of the antibody. For example, antibody H4H is a human IgG4 Fc while H1M is a mouse IgG1 Fc (all variable regions are fully human, as indicated by the first H in the antibody designation). As will be appreciated by one of skill in the art, an antibody having 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.), however, in either case, the variable domains (including CDRs), which are indicated by the numerical identifiers shown in Table. 1 and 2 will remain the same and the binding properties are expected to be identical or substantially similar regardless of the nature of the Fc domain.

The reference mAb as used in the examples herein refers to Fab9F8 as described in Fazeli et al. (2006) J Immunol Methods 312:190-200, and Carpenter et al. (2012) Structure 20(3):487-97.

Example 3 Binding affinities and reaction rate constants of human anti-LEPR monoclonal antibodies derived from surface plasmon resonance.

Equilibrium dissociation constants (K _D values) for LEPR binding to purified anti-LEPR monoclonal antibodies were determined using a real-time surface plasmon resonance biosensor using the Biacore 4000 instrument. All binding assays were performed in mM HEPES surface buffer, 150 mM NaCl, 3 mM EDTA and 0.05 vol% Tween-20 surfactant, pH 7.4 (HBS-ET) at 25 and 37°C. The Biacore touch surface was first derivatized by coupling an amine to a monoclonal mouse antibody to human Fc (GE, no. BR-1008-39) in order to capture the monoclonal antibodies to LEPR. Binding studies were performed with the following reagents for LEPR: human LEPR extracellular domain expressed with myc-myc C-terminal hexahistidine tag (hLEPR.mmh; SEQ ID NO: 114), Macaca fascicularis LEPR extracellular domain expressed with myc C-terminal hexahistidine tag -myc (mfLEPR.mmh; SEQ ID NO: 117), human LEPR extracellular domain expressed with mouse gG2a Fc C-terminal tag (hLEPR.mFc; SEQ ID NO: 115), mouse LEPR extracellular domain expressed with C-terminal hexahistidine tag myc-myc (mLEPR.mmh; SEQ ID NO: 118) and the rat LEPR extracellular domain expressed with the mycmyc C-terminal hexahistidine tag (rLEPR.mmh; SEQ ID NO: 119). Various concentrations of LEPR reagents were first prepared in HBS-ET surface buffer (100-3.7 nM; 3-fold serial dilution) and injected onto the surface of an anti-LEPR monoclonal antibody captured by anti-human Fc antibody for 4 min at a rate of flow 30 µl/min, while the dissociation associated with the monoclonal antibody

- 21 041859 LEPR reagent was monitored for 10 min in HBS-ET surface buffer. Association rate constants (ka) and dissociation rate constants (kd) were determined by fitting real-time binding sensorgrams to a mass transfer limited 1:1 binding model using Scrubber 2.0c curve fitting software. Post-binding dissociation equilibrium constants (KD) and dissociation half-lives (tV ₂ ) were calculated from the reaction rate constants as _z kd ln(2)

Kd (M) = -— , and 1½ (min) =---— ka 6G*kd

Binding kinetic parameters for binding hLEPR.mmh, mfLEPR.MMH or hLEPR.mFc to various anti-LEPR monoclonal antibodies of the present invention at 25 and 37°C are shown in Tables 3-8.

Table 3. Binding kinetic parameters for hLEPR-MMH binding to anti-LEPR monoclonal antibodies at 25°C

Captured mAb mAb capture level (RU) Linked 100 nm hLEPRMMH (RU) (1/Ms) kd (1/s) kd (M) 1½ (min) H4H16650R2 167+0.3 51 2.81E+04 2.23E-04 7.93E-09 52 H4H16679R2 192+0.7 39 2.34E+04 2D6E-04 1.05Е-08 47 H4H18417R2 163±0.4 28 6D4E+04 7.90E-03 1.29E-07 1.5 H4H18438R2 166+0.4 22 3.00E+04 2.26E-03 7.54Е-08 5D H4H18445R2 194+ 1.1 45 4.42E+04 4.78E-03 1.08E-07 2.4 H4H18446R2 163 + 2.4 16 1.81E+04 9.51Е-04 5.25E-08 12 H4N18449R2 176+1.3 54 2.91E+04 2D5E-04 8.08Е-09 49 H4H18482R2 163±0.4 47 6.31E+04 6J7E-03 1.07Е-07 1.7 H4H18487R2 190+1.2 42 4.73E+04 7, OZE-OZ 1D8E-07 1.6 H4N18492R2 167+3.1 87 8D0E+04 8.98E-04 1D1E-08 13 H4H17319R2 200±0.4 36 2.61E+04 5.29E-04 2,OSE-08 22 H4H17321R2 221+0.5 32 2.36E+04 1.96E-04 8.31Е-09 59 mAb isotype control 171+0.4 4 N.S.* N.S.* N.S.* N.S.*

*n.s. indicates that no binding was observed under current experimental conditions

- 22 041859

Table 4. Binding kinetic parameters for binding hLEPR-MMH to anti-LEPR monoclonal antibodies at 37°C

Captured mAb mAb capture level (RU) Coupled 100 nm hLEPRMMH (RU) ka (1/Ms) kd (1/s) K _D (M) 1½ (min) H4H16650R2 210±2.5 77 4.85E±04 9.58Е-04 1.98E-08 12 H4H16679R2 239±2 61 3.84Е±04 8.42E-04 2.19E-08 14 H4H18417R2 206±3.2 22 7.70E±04 1.80E-02 2,ZZE-07 0.6 H4H18438R2 206±2.4 32 3.38E±04 5.76Е-03 1.70E-07 2.0 H4H18445R2 234±2 38 5.13E±04 1.68E-02 3.26E-07 0.7 H4H18446R2 188±3.4 21 2.12E±04 2.56Е-03 1.21E-07 4.5 H4N18449R2 206±2.1 73 3.94Е±04 8.15Е-04 2.07Е-08 14 H4H18482R2 188±0.8 38 9.53Е±04 1.93E-02 2.03Е-07 0.6 H4H18487R2 219±1.7 thirty 6.51Е±04 1.86E-02 2.86E-07 0.6 H4N18492R2 192±2.2 93 1.17E±05 4.18E-03 3.59Е-08 2.8 H4H17319R2 264±0.3 44 3.54Е±04 3.41Е-03 9.63Е-08 3.4 H4H17321R2 290±0.4 61 2.95E±04 4.38E-04 1.48Е-08 26 mAb isotype control 193±1.5 6 N.S.* N.S.* N.S.* N.S.*

*n.s. indicates that no binding was observed under current experimental conditions

- 23 041859

Table 5. Binding kinetic parameters for mfLEPR.MMH binding to anti-LEPR monoclonal antibodies at 25°C

Captured mAb mAb capture level (RU) Bound 100 nm mfLEP. MMN (RU) ka (1/Ms) kd (1/s) K _D (M) 1½ (min) H4H16650R2 166±0.6 93 6.02Е±04 1.37Е-04 2.27Е-09 84 H4H16679R2 191±0.7 66 4.37E±04 1.41Е-04 3.22E-09 82 H4H18417R2 162±0.3 33 8.83Е±04 1.23E-02 1.39E-07 0.9 H4H18438R2 166±0.6 5 IC* IC* IC* IC* H4H18445R2 193±0.6 58 5.90E±04 4.86E-03 8.24Е-08 2.4 H4H18446R2 163±2.8 23 1.93Е±04 1.12E-03 5.83Е-08 10 H4N18449R2 175±0.5 6 IC* IC* IC* IC* H4H18482R2 163±0.8 63 1.01Е±05 6.74Е-03 6.66Е-08 1.7 H4H18487R2 189±0.5 59 7.37E±04 6.79Е-03 9.21Е-08 1.7 H4N18492R2 165±2.4 52 1.10E±05 1.20E-02 1.10E-07 1.0 H4H17319R2 213±0.5 83 4.00E±04 4.63E-04 1.16E-08 25 H4H17321R2 236±0.4 75 3.26E±04 1,ZZE-04 4.07Е-09 87 mAb isotype control 171±0.4 0 N.S.* N.S.* N.S.* N.S.*

*n.s. indicates that no binding was observed under current experimental conditions *IC indicates that the observed binding was inclusive and it was not possible to fit real-time binding data based on it under current experimental conditions

- 24 041859

Table 6. Binding kinetic parameters for mfLEPR.MMH binding to anti-LEPR monoclonal antibodies at 37°C

Captured mAb mAb capture level (RU) Bound 100 nm mfLEPR .MMH (RU) ka (1/Ms) kd (1/s) K _D (M) 1½ (min) H4H16650R2 204±1.7 134 1.22Е±05 7.00Е-04 5.76Е-09 16 H4H16679R2 232 ± 1.1 104 6.49Е±04 6.77Е-04 1.04Е-08 17 H4H18417R2 202±1.3 28 1.22Е±05 2.63E-02 2.17E-07 0.4 H4H18438R2 203±1.3 7 IC* IC* IC* IC* H4H18445R2 232±0.9 48 7.17E±04 1.90E-02 2.64Е-07 0.6 H4H18446R2 188±2.9 thirty 2.53Е±04 3.54Е-03 1.40E-07 3.3 H4N18449R2 202±1 6 IC* IC* IC* IC* H4H18482R2 187 ± 1.2 52 1.52Е±05 2.04Е-02 1.34Е-07 0.6 H4H18487R2 216±0.7 44 1.10E±05 1.95E-02 1.78E-07 0.6 H4N18492R2 191 ± 1.4 34 2.34Е±05 3.94Е-02 1.69Е-07 0.3 H4H17319R2 274±0.5 113 5.39E±04 3.24Е-03 6.01Е-08 3.6 H4H17321R2 304±0.7 143 4.97E±04 2.57Е-04 5.18E-09 45 mAb isotype control 190±1 1 N.S.* N.S.* N.S.* N.S.*

*n.s. indicates that no binding was observed under current experimental conditions *IC indicates that the observed binding was inclusive and it was not possible to fit real-time binding data based on it under current experimental conditions

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Table 7. Binding kinetic parameters for binding hLEPR.mFc to anti-LEPR monoclonal antibodies at 25°C

Captured mAb mAb capture level (RU) Bound 100 nm hLEPRmFc (RU) ka (1/Ms) kd (1/s) K _D (M) 1½ (min) H4H16650R2 165±0.2 102 1.06Ε±05 8.32E-05 7.85E-10 139 H4H16679R2 190±1.2 78 5.84E±04 9.68E-05 l,66E-09 119 H4H18417R2 162±0.6 90 l,40E±05 5.63E-04 4.04E-09 21 H4H18438R2 165±1.2 51 5.19E±04 2.44E-04 4.70E-09 47 H4H18445R2 192±0.4 76 l,22E±05 4.92E-04 4.03E-09 23 H4H18446R2 162±2.8 20 3.20E±04 2.08E-04 6.48E-09 56 H4N18449R2 174±0.6 116 7.05E±04 6.82E-05 9.64E-10 169 H4H18482R2 162±0.5 88 l,44E±05 4.91E-04 3.42E-09 24 H4H18487R2 188±0.6 85 l,06E±05 6.03E-04 5.70E-09 19 H4N18492R2 166±3.2 129 2.27E±05 l,39E-04 6.13E-10 83 H4H17319R2 200±0.5 69 4.77E±04 l,64E-04 3.45E-09 70 H4H17321R2 221±0.4 65 4.10E±04 8.93E-05 2.18E-09 129 mAb isotype control 170±0.7 -2 H.C.* H.C.* H.C.* H.C.*

*n.s. indicates that no binding was observed under current experimental conditions

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Table 8. Binding kinetic parameters for binding hLEPR.mFc to anti-LEPR monoclonal antibodies at 37°C

Captured mAb mAb capture level (RU) Bound 100 nm hLEPRmFc (RU) ka (1/Ms) kd (1/s) K _D (M) tvl (min) H4H16650R2 199 ± 1.9 145 1.57Ε±05 2.80E-04 1.79Е-09 41 H4H16679R2 229±2.3 116 1.21Е±05 3.10E-04 2.56Е-09 37 H4H18417R2 199±1.1 111 1.85E±05 1.05Е-03 5.64Е-09 eleven H4H18438R2 199±0.6 82 7.02E±04 5.98E-04 8.53Е-09 19 H4H18445R2 229±2 104 1.56Е±05 6.08Е-04 3.89Е-09 19 H4H18446R2 186±2.5 34 4.27E±04 5.48E-04 1.28E-08 21 H4N18449R2 198±1.6 148 1,3E±05 1.68Е-04 1.26E-09 69 H4H18482R2 185±1.3 109 1.89Е±05 7.26E-04 3.84Е-09 16 H4H18487R2 215±1.5 99 1.23Е±05 6.06Е-04 4.93Е-09 19 H4N18492R2 189±1.8 160 4,3E±05 5.00Е-04 1.16E-09 23 H4H17319R2 262±0.5 100 8.51Е±04 6.52E-04 7.66Е-09 18 H4H17321R2 289±0.4 BY 5.53Е±04 1.74Е-04 3.15E-09 66 mAb isotype control 188±0.8 1 N.S.* N.S.* N.S.* N.S.*

*n.s. indicates that no binding was observed under current experimental conditions

At 25° C., anti-LEPR monoclonal antibodies bound hLEPR-MMH with KD values ranging from 7.9 to 148 nM, as shown in Table 1. 5. At 37° C., anti-LEPR monoclonal antibodies bound hLEPR-MMH with KD values ranging from 14.8 to 326 nM, as shown in Table 5. 4.

of the 12 anti-LEPR monoclonal antibodies of the present invention bound to mfLEPR.MMH. At 25° C., anti-LEPR monoclonal antibodies bound mfLEPR.MMH with KD values ranging from 2.27 to 139 nM, as shown in Table 1. 7. At 37°C, monoclonal antibodies to LEPR contacted mfLEPR.MMH with KD values ranging from 5.18 to 264 nm, as shown in table. 8.

At 25° C., anti-LEPR monoclonal antibodies bound hLEPR-mFc with KD values ranging from 613 pM to 5.7 nM, as shown in Table 1. 7. At 37° C., anti-LEPR monoclonal antibodies bound hLEPR-mFc with KD values ranging from 1.16 to 12.8 nM as shown in Table 7. 8.

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

Example 4 Anti-LEPR antibodies of the present invention bind LEPR in the presence of leptin:LEPR complex binding.

Blocking of anti-LEPR antibodies from binding to LEPR by human leptin was assessed using a real-time plasmon surface resonance biosensor on the Biacore T200 instrument. The entire assay was performed in 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05 vol% Tween-20 surfactant (HBS-ET surface buffer) at 25°C. The Biacore CM5 sensor surface was first derivatized with amine coupling to human leptin (R&D Systems, No. 398-LP) using a standard EDC/NHS surface chemistry. The complex of human LEPR and human leptin was formed by injecting 20 nM of the extracellular domain of human LEPR expressed with the myc-myc C-terminal hexahistidine tag (hLEPR-MMH; SEQ ID NO: xx) onto a Biacore sensor surface with immobilized human leptin at a flow rate of 10 μl/min or 25 μl/min for 4 min, resulting in a binding response of approximately 200 RU. To assess whether antibody binding to hLEPR-MMH is blocked by human leptin, 200 nM anti-LEPR monoclonal antibody was injected into a preformed hLEPR-MMH:human leptin complex at a flow rate of 50 μl/min or 25 μl/min for 4-5 min. All anti-LEPR antibodies of the present invention bound to the hLEPR-MMH complex and human leptin (leptin:LEPR) with nearly similar signal strength and observed binding expressed in RU are described in Table 1. 9. This result indicated that human leptin did not block hLEPR-MMH binding to the anti-LEPR antibodies under study.

Table 9. Binding of anti-LEPR monoclonal antibodies to pre-complexed hLEPR-MMH and human leptin_____

Antibody Related hLEPR-MMH (RU) Bound 200 nM mAb (RU) H4H16650R2 196 81 H4H16679R2 195 90 H4H17319R2 196 92

Example 5 ELISA in blocking the human leptin receptor.

In an ELISA, human leptin (hLeptin; R&D Systems, no. 398-LP-O1M) was coated at 5 μg/ml in PBS in a 96-well microtiter plate overnight at 4°C. The non-specific binding sites were then blocked with a 0.5% (w/v) solution of BSA in PBS. A constant amount of a 10 nM region of the extracellular domain of the LEPR protein that was expressed with a C-terminal human Fc (hLEPR.hFc; SEQ ID NO: 116) was titrated with anti-LEPR antibodies, hLeptin protein, or an isotype control antibody ranging from 8.5 pM to 500 nM in serial breeding. These antibody-protein or protein-protein complexes were then incubated for 1.5 hours at room temperature (RT). The complexes were then transferred to microtiter plates coated with hLeptin and incubated for 2 h at RT, the wells were washed and plate-bound hLEPR.hFc was detected using a polyclonal anti-human IgG antibody conjugated with radish peroxidase (Jackson ImmunoResearch Inc, no. 109-035- 098). Samples were diluted with TMB solution (BD Biosciences, #555214; substrate A and B mixed 1:1 according to manufacturer's instructions) to give a colorimetric reaction and then neutralized with 1 M sulfuric acid before absorbance was measured at 450 nm on a plate -reader Victor X5.

Data analysis was performed using a sigmoidal dose-response function in the Prism™ computer program (GraphPad). Percent blockade at the maximum concentration of test antibody was calculated as a measure of the ability of antibodies to block the binding of 10 nM hLEPR.hFc to human leptin in the plate. When calculating the binding signal from 10 nm hLEPR.hFc without the presence of antibodies was presented as 100% binding or 0% blocking; and baseline buffer signal alone without the presence of hLEPR.hFc was represented as 0% binding or 100% blocking. Data on blocking at an antibody concentration of 500 nm summarized in table. 10.

As shown in Table. 10, none of the anti-LEPR antibodies of the present invention exhibited >28% blocking of hLEPR.hFc binding to the hLeptin coated surface. At the same time, the reference antibody and hLeptin as a positive control were able to block 99% of the binding of hLEPR.hFc to the surface coated with hLeptin. The isotype control antibody showed no measurable blocking at concentrations up to 500 nM.

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Table 10 ELISA results blocking hLEPR.hFc binding to hLeptin anti-LEPR antibodies

Antibody Blocking of 500 nM AB binding of 10 nM hLEPR.hFc to hLeptin (% blockade) H4H18487R2 5 H4H18417R2 16 H4H18482R2 25 H4N18492R2 -3 H4H18445R2 28 H4H18446R2 -5 H4N18449R2 8 H4H18438R2 15 H4H16650R2 -7 H4H16679R2 7 Н4Н173319Р2 9 H4H173321R2 6 controls Isotype control antibody -3 Human leptin 99 Reference antibody 99 Isotype control of mouse IgG2a 32

Example 6 Cell Binding in FACS Analysis of HEK293/Myx2-hLepR(ecto)-GPI Anchored Cells.

The leptin receptor, LEPR, is a single-pass transmembrane receptor of the class I cytokine receptor family (Tartaglia et al. (1997) J Biol Chem 7:272(10):6093-6). LEPR can bind to leptin, a protein predominantly expressed by adipose tissue that is involved in the regulation of food intake and metabolism (Friedman et al. (2014) J Endocrinol 223(1):T1-8).

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

When performing a FACS assay, HEK293 parental cells and HEK293/hLEPR-GPI cells were dissociated and plated in 96-well V-bottomed plates at 5x105 cells/well in PBS containing 2% FBS (FACS buffer). To investigate whether the ability of anti-hLEPR antibodies to bind to cells is affected by the presence of leptin, FACS buffer with or without 1 μM human leptin (R&D Systems, No. 398-LP) was incubated with cells for 30 min at 4°C followed by addition anti-LEPR antibodies or control antibodies at 10 nM in FACS buffer. Cells were then incubated for 30 min at 4°C followed by washing and then incubation with 16 μl/ml Alexa Fluor®-647 conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc., no. 109-547-003) for 30 minutes at 4°C. Cells were then fixed with BD CytoFix™ (Becton Dickinson, no. 554655), filtered and analyzed on a HyperCyt flow cytometer (Beckman Coulter). Controls in the form

- 29 041859 unstained and secondary antibodies separately were also examined in relation to all cell lines. The results were analyzed with the ForeCyt software (IntelliCyt) and Flow Jo, version 10, with the determination of geometric mean fluorescence for viable cells. The geometric mean of fluorescence for each sample was then normalized with respect to the geometric mean of unstained cells to give relative binding to a condition called binding coefficients, and these binding coefficients were noted for each antibody tested.

As shown in Table. 11, 9 anti-LEPR antibodies of the present invention tested at 10 nM exhibited binding to HEK293/hLEPR-GPI cells with binding ratios ranging from 824 to 3374 without leptin. Anti-LEPR antibodies also bind in the presence of 1 μM leptin with binding ratios ranging from 398 to 4184. As shown in Table. 11, the reference antibody tested at 10 nM had binding to HEK293/hLEPR-GPI cells with a binding factor of 2349 without leptin, but significantly less binding to cells in the presence of 1 μM leptin with a binding factor of 112. Anti-LEPR antibodies did not show any significant binding to parental HEK293 cells, with binding coefficients with and without 1 μM leptin ranging from 1 to 9. Isotype control and secondary antibody samples alone also did not show significant binding to either the leptin-containing or non-leptin cell line. , while the binding coefficients were in the range from 1 to 6.

As shown in Table. 12, 4 antibodies of the present invention, tested at 70 nM without leptin, were characterized by binding to HEK293/hLEPR-GPI cells with binding coefficients ranging from 707 to 1131 and to HEK293/Stat3-luc/hLEPR-FL cells with binding coefficients in the range 42 to 51. Anti-LEPR antibodies did not show any significant binding to parental HEK293/Stat3-luc cells, with binding ratios ranging from 1 to 8. Samples of isotype control antibodies and secondary antibodies alone also did not show significant binding to either which of the studied cell lines, while the binding coefficients were in the range from 1 to 2.

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Table 11. Binding of 10 nM anti-LEPR antibody to HEK293/hLEPR-GPI cells and parental HEK293 cells ± 1 µM human leptin

Binding coefficient: normalized to unstained sample of each cell line No added leptin 1 μM leptin Type of antibody Antibody parent cells HEK293/ hLEPRGPI parental cells HEK293/ hLEPR-GPI Binding coefficient: normalized to unstained sample of each cell line No added leptin 1 µM leptin Type of antibody HEK293 HEK293 H4H16650R2 5 2420 4 3124 Agonistic H4H16679R2 5 2058 8 2223 Agonistic H4H18417R2 1 1835 2 2604 Potentiating H4H18438R2 2 1486 3 2414 Potentiating H4H18445R2 2 2016 3 2488 Potentiating H4N18449R2 5 3374 9 3113 Potentiating H4H18482R2 1 1966 3 2704 Potentiating H4H18487R2 1 2422 3 2670 Potentiating H4N18492R2 3 2603 7 4184 Potentiating Antibody comparisons 6 2349 3 112 But. Isotype control antibody 1 6 2 4 But. Secondary antibody alone 1 3 2 3 But. unpainted 1 1 1 1 But.

*The classification of antibodies as agonist or potentiator is based in part on the results observed in Examples 7 and 8 of this document.

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Table 12. Binding of 70 nM anti-LEPR antibodies to HEK293/hLEPR-GPI, HEK293/Stat3-hLEPR-FL cells and HEK293/Stat3-luc parental cells

Binding coefficient: normalized to unstained sample of each cell line Type of antibody Antibody HEK293/ stat3-luc HEK293/ hLEPRGPI HEK293/Stat3-luc hLEPR-FL H4H16650R2 6 707 42 Agonistic H4H16679R2 8 1078 51 Agonistic H4H17319R2 7 1131 47 Agonistic H4H17321R2 7 1126 46 Agonistic Isotype control antibody 2 2 2 Secondary antibody alone 1 1 1 unpainted 1 1 1

Example 7 Anti-LEPR antibodies of the present invention activate LEPR signaling in the presence or absence of leptin.

A bioassay was developed to detect transcriptional activity of STAT3 as a result of LEPR activation using a reporter cell line that stably expressed full-length human LEPR (hLEPR; amino acids 1 to 1165 accession number NP_002294.2) in conjunction with a luciferase reporter (STAT3-Luc; Qiagen, #CLS-6028L) in the IMR-32 cell line, a human neuroblastoma cell line. The resulting stable cell line, termed IMR32/STAT3-Luc/hLEPR, was isolated and maintained in MEM-Earl medium supplemented with 10% FBS, NEAA, 1 μg/ml puromycin, 100 μg/ml hygromycin B, and penicillin/streptomycin/L-glutamine (full environment).

The resulting bioassay was used to measure the effect of the anti-LEPR antibodies of the present invention on LEPR signaling in the presence or absence of leptin. When using the bioassay, IMR-32/STAT3-Luc/hLEPR cells were plated at a density of 20,000 cells/100 μl/well in a 96-well format in complete medium and replaced with an appropriate volume of Opti-MEM supplemented with 1% BSA the following day. 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, an anti-LEPR antibody or an isotype control antibody and human leptin (hLeptin; R&D Systems, No. 398-LP) were serially diluted semilogarithmically to final concentrations ranging from 100 nM to 300 fM in assay buffer, which were added to the cells and then incubated overnight at 37°C in 5% CO2.

To measure the effect of the antibodies of the present invention in the presence of leptin, a fixed concentration of human leptin at 200 pM in assay buffer was added to cells, immediately followed by the addition of anti-LEPR antibodies or isotype control antibodies that were serially diluted semilogarithmically to final concentrations ranging from 100 nM to 300 fM. The samples were then incubated overnight at 37°C in 5% CO2. The OneGlo reagent (Promega, #E6051) was then added to the samples and luciferase activity was measured on an Envision multichannel plate reader (Perkin Elmer) in luminescent mode. Relative light units (RLU) values were obtained and the results were analyzed by non-linear regression using the computer program GraphPad Prism (GraphPad). The maximum RLU value derived from the dose-response curve for hLeptin was determined as 100% activation in the IMR-32/STAT3-Luc/hLEPR assay.

As shown in Table. 13, in study 1, in the absence of hLeptin, all of the tested anti-LEPR antibodies had weak stimulation on IMR-32/STAT3-Luc/hLEPR cells, with EC50 values ranging from 134 pM to 11.9 nM, and maximum activation in range from 5% to 13%, respectively, in relation to the maximum activation observed from the dose-response curve for hLeptin. In study 2, in the absence of hLeptin, 4 anti-LEPR antibodies studied stimulated IMR-32/STAT3-Luc/hLEPR cells with EC50 values ranging from 61.9 to 206.9 pM and maximum activation ranging from 65 up to 68% of the maximum activation observed from the hLeptin dose-response curve. In study 1, in the presence of 200 pM hLeptin, all of the study

- 32 041859 anti-LEPR antibodies stimulated IMR-32/STAT3-Luc/hLEPR cells with EC50 values ranging from 20.2 to 523 pM and maximum activation ranging from 66 to 107% of maximum activation observed from the dose-response curve for hLeptin. Because these antibodies enhanced leptin-induced signaling by LEPR, these antibodies were classified as potentiators as defined herein. In study 2, in the presence of hLeptin, four 200 pM anti-LEPR antibodies of interest stimulated IMR-32/STAT3-Luc/hLEPR cells with EC ₅₀ values ranging from 51.9 to 257.3 pM and maximum activation at range from 76 to 88% relative to the maximum activation observed from the dose-response curve for hLeptin. LEPR signaling was not significantly enhanced by these antibodies in the presence of leptin. The isotype control antibody did not exert any measurable stimulation on IMR-32/STAT3-Luc/hLEPR cells in any of the assays.

Table 13. Activation 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 EU ₅₀ (M) % activation Study 1 H4H18445R2 1D9E-08 5 4.10E-10 97 H4H18446R2 3.73E-10 6 3.42E-11 68 H4N18449R2 2D2E-10 13 5.23E-11 66 H4H18438R2 1.49Е-09 5 2.02E-11 76 H4H18482R2 2.69Е-10 7 1.69Е-10 94 H4H18487R2 8.01Е-10 6 4.10E-10 107 H4N18492R2 1.34Е-10 5 2.74Е-11 94 H4H18417R2 1.53Е-10 5 5.23E-10 87 Study 2 H4H16650R2 6.19E-11 68 5.19E-11 88 H4H16679R2 8.62E-11 65 7.37E-11 88 H4H17319R2 1.867E-10 68 1.914E-10 76 H4H17321R2 2.069E-10 66 2.573E-10 76

Example 8 The anti-LEPR antibodies of the present invention activate signal transduction in cells expressing LEPR mutants with a defect in signal transduction or with impaired signal transduction.

LEPR mutants were identified that were characterized by a leptin-mediated defect or signal transduction disorder and were associated with early obesity. For example, LEPR-A409E was a signaling defective mutant LEPR protein that did not signal via leptin to STAT3; the A409E mutant was initially identified as a monogenic cause of early obesity (Farooqi et al., 2007, N Engl J Med 356(3): 237-247). LEPR-P316T was a mutant LEPR protein with impaired signaling that has also been shown to be associated with early stage obesity (Mazen et al., 2011, Mol Genet Metab 102:461-464).

In this example, the ability of the anti-LEPR antibodies of the present invention to stimulate signaling by LEPR in cell lines expressing LEPR mutants with signaling defect or signal transduction was evaluated. In particular, reporter cell lines (HEK293) were constructed to express either wild-type LEPR or LEPR-A409E (signal defect) or LEPR-P316T (signal defect). Cells were treated with either base alone, recombinant human leptin, control IgG, or the anti-LEPR agonist antibodies of the present invention (H4H16650 or H4H16679) and the degree of LEPR signaling was determined (as measured by Western blot detection of pSTAT3-Y705 expression against STAT3 expression).

The LEPR agonist antibodies of the present invention (H4H16650 and H4H16679) were shown to stimulate LEPR signaling in cells expressing the LEPR-A409E mutant or the LEPR-P316T mutant (as measured by STAT3 expression) in a dose dependent manner.

- 33 041859 way (Fig. 2, panels B and C). In contrast, leptin treatment induced only negligible signaling in cells expressing the LEPR-P316T mutant and did not induce signaling in cells expressing LEPR-A409E (FIG. 2, panel A). Moreover, signaling by LEPR was not detected in any of the cell lines treated with base or control IgG antibody (data not shown). Other LEPR mutants with defective signaling or impaired signaling were explored in this assay, but were not activated with mutants to LEPR (data not shown), indicating that this accessory effect might be mutant dependent.

The results of this example indicated that the anti-LEPR agonist antibodies of the present invention may be useful in the treatment of diseases and disorders (e.g., early stage obesity) that are caused by or associated with LEPR mutants with a signal transduction defect or aberration (e.g., LEPR-P316T or LEPR-A409E).

Example 9 Octet cross-competition between different anti-LEPR monoclonal antibodies.

Competition for panel binding of different monoclonal antibodies to LEPR was determined using real-time marker-free interferometric analysis of biolayers on the Octet HTX biosensor platform (Pall ForteBio Corp.). The entire experiment was performed at 25° C. in buffer containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05 vol% Tween-20 surfactant, 1 mg/ml BSA, pH 7.4 (HBS- EBT) by shaking the plate at 1000 rpm. To assess whether two antibodies are able to compete with each other for binding to their respective epitopes on recombinant human LEPR expressed with a myc-myc C-terminal hexahistidine tag (hLEPR.mmh; SEQ ID NO: 114), about 0.25 nm or 0.34 nm hLEPR-MMH was first captured on Octet biosensor tips coated with anti-penta-His antibody (Fortebio Inc, no. 18-5122) by immersing the biosensor tips for 5 min in wells containing 20 μg/ml hLEPRMMH. Antigen-captured biosensor tips were then saturated with the first anti-LEPR monoclonal antibody (hereinafter referred to as mAb-1) by immersion in wells containing 50 μg/ml mAb-1 solution for 210 seconds. The biosensor tips were then immersed in wells containing a 50 μg/ml solution of a second anti-LEPR monoclonal antibody (hereinafter referred to as mAb-2) for 150 seconds. Biosensor tips were washed in HBS-EBT buffer between each step of the experiment. The real-time binding response was monitored throughout the course of the experiment and the binding response at the end of each step was recorded. The mAb-2 binding response to hLEPR-MMH previously complexed with mAb-1 was compared and the competitive/non-competitive behavior of anti-LEPR monoclonal antibodies was determined as shown in Table 1. 14 and 15.

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Table 14. Cross-competition between different monoclonal antibodies to LEPR

Binding of the first antibody (mAb-1) to captured hLEPR-MMH H4N18492R2 Second antibody (mAb-2) competing with mAb-1 H4H18417R2 H4H18438R2 H4H18417R2 H4N18492R2 H4H18438R2 H4H18438R2 H4N18492R2 H4H18417R2 H4H16650R2 H4H16679R2 H4H16679R2 H4H16650R2 H4H18445R2 H4H18482R2 H4H18487R2 H4H18446R2 H4H18446R2 H4H18482R2 H4H18487R2 H4H18445R2 H4H18482R2 H4H18445R2 H4H18487R2 H4H18487R2 H4H18445R2 H4H18482R2 H4N18449R2 Absent Reference antibody Absent

Table 15. Cross-competition between different monoclonal antibodies to LEPR

SHAL-1 wal-2, which competes with wal-1 H4H17319R2 H4H17321R2 H4H16650R2 H4H16679R2 H4H17321R2 H4H17319R2 H4H16650R2 H4H16679R2 H4H16650R2 H4H17319R2 H4H17321R2 H4H16679R2 H4H16679R2 H4H17319R2 H4H17321R2 H4H16650R2

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Example 10 In vivo efficacy of anti-LEPR agonist antibodies H4H16650P2, H4H16679P2, H4H17319P2 and H4H17321P2 in an inducible mouse model of leptin deficiency.

The effects of the four specific anti-LEPR agonist antibodies of the present invention, H4H16650P2, H4H16679P2, H4H17319P2 and H4H17321P2, on food intake, body weight, and fat deposition were determined in an inducible model of leptin deficiency in genetically engineered ^Hu/Hu LEPR mice that expressed the leptin receptor, which consists from a human LEPR ectodomain sequence instead of a mouse LEPR ectodomain sequence. The leptin deficiency model was induced by hydrodynamic DNA delivery (HDD) of a plasmid encoding the hFc-tagged mouse LEPR ectodomain (herein referred to as mLEPR.hFc or leptin trap; SEQ ID NO: 120). The leptin trap, when expressed, was secreted and bound circulating leptin. After HDD 50 μg of the DNA construct encoding the leptin trap, mice were characterized by increased food intake, increased fat deposition and body weight.

Daily food intake at baseline was measured between days 7 and 4 before the administration of the leptin trap (-7 and -4 days). On day 0, 35 male ^Hu/Hu LEPR mice aged 13 to 17 weeks were successfully exposed to leptin trap HDD. On days 6 and 13 after HDD, retroorbital sinus blood samples were collected and body composition, including fat deposition, was quantified by μCT. On day 7 after HDD, mice were randomized into 5 groups of 7 mice based on the percentage change in body weight since day 0. Each group received by subcutaneous injection either a single dose of isotype control antibody at 3 mg/kg, or H4H16650P2 at 3 mg/kg, or H4H16679P2 at 3 mg/kg, or H4H17319P2 at 3 mg/kg, or H4H17321 at 3 mg/kg. The isotype control antibody did not bind any known mouse protein. Food intake and body weight were measured for each animal during the study. In FIG. 3 summarizes the average daily food intake for each treatment group. In FIG. 3, the dotted line represents mean food intake at baseline before HDD injection. The percentage change in body weight from day 0 was calculated for each animal at each time point. In FIG. 4 summarizes the mean percentage change in body weight for animals in each antibody treatment group. In FIG. 5 summarizes the mean body fat mass for animals in each treatment group, quantified by µCT 1 day before and 6 days after antibody treatment. All results were expressed as mean ± SEM.

As shown in FIG. 3 and 4, after leptin trap HDD, similar increases in food intake and percentage change in body weight were observed in the groups of mice before antibody treatment. As shown in FIG. 3, mice treated with antibodies H4H16650P2 or H4H16679P2 at 3 mg/kg had significant decreases in food intake one day after antibody treatment (day 8 post-HDD) and at subsequent time points measured compared to mice injected with antibody. isotype control. Mice treated with antibodies H4H17319P2 or H4H17321P2 at 3 mg/kg had a significant decrease in food intake two days after antibody treatment (day 9 after HDD) and at other subsequent time points measured compared to mice injected with isotype antibody. control. As shown in FIG. 4, mice treated with antibody H4H16650P2 at 3 mg/kg had a significant reduction in percent body weight change one day after antibody treatment (day 8 post-HDD) and at other subsequent time points measured compared to mice injected with isotype control antibodies. One day after antibody treatment, on day 8, mice treated with isotype control showed a 21.16 ± 1.27% increase in body weight from day 0, while mice treated with H4H16650P2 showed an increase body weight by 15.57 ± 0.9% starting from day 0. Mice treated with antibodies H4H16679P2, H4H17319P2, or H4H17321P2 at 3 mg/kg showed a significant reduction in percent body weight change two days after antibody treatment (day 9 post-HDD) and at other subsequent time points measured compared to mice treated with was injected with an isotype control antibody.

On day 9, the % change in body weight since day 0 was 23.18±1.22, 13.17±1.05, 12.95±1.26, 15.98±1.78, and 15. 83 ± 2.01 for mice treated with isotopic control, H4H16650P2, H4H16679P2, H4H17319P2, or H4H17321P2, respectively. As shown in FIG. 5, mice treated with isotype control at 3 mg/kg had a significant increase in adipose tissue mass 6 days after antibody treatment (day 13 after HDD) compared to day 1 before antibody treatment (day 6 after HDD). ). Mice treated with antibodies H4H16650P2, H4H16679P2, H4H17319P2, or H4H17321P2 at 3 mg/kg did not gain in body fat after antibody treatment compared to antibody pretreatment. At 6 days post-treatment (day 13 post-HDD), mice treated with H4H16650P2, H4H16679P2, or H4H17319P2 antibodies at 3 mg/kg had significant reductions in body fat mass compared to mice treated with isotype control antibody at 3 mg/kg.

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Example 11 Epitope mapping of H4H16650P2 binding to the human leptin receptor (hLEPR.mmh) by hydrogen-deuterium exchange.

Experiments were performed to determine the amino acid residues hLEPR.mmh (amino acids M1-D839 SEQ ID NO: 114) with which H4H16650P2 interacts. For this purpose, epitope mapping was performed by mass spectrometry using H/D exchange. A general description of the H/D exchange method is set forth in, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259; and Engen and Smith (2001) Anal. Chem. 73:256A-265A.

experimental procedure.

HDX-MS experiments were performed on an integrated Waters HDX/MS platform, consisting of Leaptec's HDX PAL system for deuterium labeling, Acquity M-Class Waters (solvent accessory) for sample digestion and loading, Acquity M-Class Waters (μBinary solvent device). ) for the analytical column gradient and the Synapt G2-Si mass spectrometer for measuring the mass of peptides after peptic hydrolysis.

The labeling solution was prepared in 10 mM PBS buffer in D ₂ O at pD 7.0 (equivalent to pH 6.6). For deuterium labeling, 3.8 µl of hLEPR.mmh (8 pmol/µl) or hLEPR.mmh premixed with antibody at a 2:1 molar ratio was incubated with 56.2 µl of D2O labeling solution for various time points (e.g., non-deuterated control = 0 s, labeled for 1 min and 20 min). Deuteration was stopped by transferring 50 µl of the sample to 50 µl of pre-chilled cooling buffer (0.2 M TCEP, 6 M guanidine chloride in 100 mM phosphate buffer, pH 2.5) and the mixed sample was incubated at 1.0°C for two minutes. The paused sample was then injected into an HDX Waters online pepsin/protease XIII digester. Digested peptides were captured onto a VanGuard ACQUITY UPLC VEN C18 1.7 µm, 2.1x5 mm pre-column at 0°C and eluted onto an ACQUITY UPLC VEN C18 analytical column 1.7-µm, 1.0x50 mm, over a 9-minute gradient separation 5-40% B (mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile). The mass spectrometer was set up with a cone voltage of 37 V, a scan time of 0.5 seconds, and a mass/charge range of 50-1700 Th.

To identify peptides from human LEPR, LC-MSE data derived from a non-deuterated sample was processed and searched against a database including human LEPR, pepsin, and other randomized sequences using the Waters ProteinLynx Global Server (PLGS) computer program. The identified peptides were imported into the DynamX software and filtered using two criteria: 1) minimum products per amino acid: 0.2, and 2) file repeat limit: 3. The DynamX software then automatically determined the capture of each peptide based on retention time and high mass accuracy (<10 ppm) at multiple time points with 3 repetitions at each time.

Results. Using an online pepsin/protease XIII column with MSE data acquisition, a total of 201 peptides from LEPR were reproducibly identified in the absence or presence of an antibody representing 70% sequence coverage. Five peptides showed significantly reduced deuterium uptake (centroid delta values > 0.4 daltons at p values < 0.05) when bound to H4H16650P2, as shown in Table 1. 16. The recorded mass of peptides corresponded to the average value of the centroid MH+ mass from three repetitions. These peptides, corresponding to amino acids 162169 (human LYVLPEVL LEPR amino acids; SEQ ID NO: 113) and amino acids 170-181 (human EDSPLVPQKGSF LEPR amino acids; SEQ ID NO: 113), were characterized by a slower rate of deuterization when bound to H4H16650P2. These identified residues also corresponded to amino acid residues 162-169 and 170-181 of human LEPR as determined by the entry value in Uniprot P48357 (SEQ ID NO: 113; human leptin receptor).

Table 16. Human leptin receptor peptides with significant protection when bound to antibody H4H16650P2

Remains Deuteration for 1 minute Deuteration for 20 minutes hLEPR.m mh hLEPR.mmh + H4H16650R2 A hLEPR.mmh hLEPR.mmh + H4H16650R2 A 162-169 949.03±0.03 947.99±0.02 -1.04 949.23±0.02 948.16±0.02 -1.03 163-169 835.82±0.03 834.79±0.02 -1.03 836.03±0.02 834.94±0.02 -1.08 170-181 1310.02±0.05 1309.12±0.03 -0.89 1309.77±0.02 1309.38±0.02 -0.39

Example 12 In vivo efficacy study of anti-LEPR potentiating antibodies in mice with humanized LEPR.

The effects of the three specific anti-LEPR potentiating antibodies of the present invention, H4H18482P2, H4H18487P2, and H4H18492P2, on body weight and fat deposition were determined in single housed ^Hu/Hu genetically engineered LEPR mice that expressed leptin re

- 37 041859 receptor, which consisted of the human LEPR ectodomain sequence instead of the mouse LEPR ectodomain sequence (mLEPR.hFc, SEQ ID NO: 120).

At day -19, body composition, including fat deposition, was quantified by µCT. On day 0, 48 female LEPR ^Hu/Hu mice aged 14 to 16 weeks were randomized into four groups of 12 mice based on body weight. On days 0 and 11, mice from each group received, by subcutaneous injection, a single dose of isotype control antibody at 30 mg/kg, H4H18482P2 at 30 mg/kg, H4H18487P2 at 30 mg/kg, or H4H18492P2 at 30 mg/kg. The isotype control antibody did not bind any known mouse protein. Body weight was measured during the study for each animal. The percentage change in body weight from day 0 was calculated for each animal at each time point. In FIG. 6 summarizes the mean percentage change in body weight for animals in each treatment group. In FIG. 6 summarizes the mean body fat mass for animals in each treatment group, quantified by μCT 19 days before and 11 days after antibody treatment. All results were expressed as mean ± SEM.

As shown in FIG. 6, a reduction in percentage change in body weight was observed after administration of potentiating anti-LEPR antibodies, but not isotype control antibodies. As shown in FIG. 6, mice treated with H4H18482P2 at 30 mg/kg showed a significant reduction in percent change in body weight starting two days after treatment (Day 2) and at other time points compared to mice injected with isotype control antibody. Mice treated with H4H18487P2 at 30 mg/kg showed a significant reduction in percent change in body weight from day 2 and at other time points compared to mice injected with the isotype control antibody. Mice treated with H4H18492P2 at 30 mg/kg showed a reduction in percentage change in body weight at days 4, 5 and 17, but not at different time points, compared to mice injected with isotype control antibody. Mice treated with H4H18482P2 at 30 mg/kg showed a significant reduction in percent change in body weight from day 6 onwards, but not on days 7, 14, and 17, compared with mice treated with injection of H4H18492P2. Mice treated with H4H18487P2 at 30 mg/kg showed a significant reduction in percent change in body weight from day 3 and at other time points, but not on days 4 and 5, compared with mice injected with H4H18492P2.

As shown in FIG. 7A, there were no differences in adipose tissue mass between groups before treatment (-19 days). As shown in FIG. 7B, mice treated with antibodies H4H18482 and H4H18487, but not H4H18492, at 30 mg/kg showed a statistically significant reduction in body fat mass 17 days post-treatment (day 12) compared to the isotype control antibody.

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

Example 13 Effect of Anti-LEPR Antibodies of the Invention on Signaling Transmission by Monkey LEPR.

In order to evaluate the transcriptional activity of the monkey leptin receptor, a stable cell line was developed. IMR-32 (human neuroblastoma ATCC) cells were generated to stably express the Macaca fascicularis LEPR extracellular domain (MfLEPR; amino acids 22 to 837, accession number XP_005543194.1, with threonine at position 827 replaced by alanine) fused to the transmembrane and cytosolic human LEPR domains (hLEPR; amino acids 840 to 1165, accession number NP_002294.2) together with a luciferase reporter (STAT3-Luc; SABiosciences, no. CLS-6028L). The resulting cell line, hereinafter referred to as IMR-32/STAT3Luc/MfLEPR, was isolated and maintained in MEM-Earl medium supplemented with 10% FBS, NEAA, 1 μg/ml puromycin, 100 μg/ml hygromycin B and penicillin/strepromycin/ L-glutamine.

A bioassay was performed to measure the effect of the anti-LEPR antibodies of the present invention on signaling by monkey LEPR in the absence of leptin. When performing the assay, IMR-32/STAT3-Luc/MfLEPR cells were plated at 10,000 cells/well in a 96-well plate in 0.1% FBS in Optimem with penicillin/streptomycin (assay buffer) and incubated overnight at 37° C in 5% CO ₂ . The next day, human leptin (hLeptin), anti-LEPR antibody, or isotype control antibody was serially diluted from 50 nM to 0.8 pM in assay buffer (together with the sample containing the buffer alone without test molecule) and added to the cells. After 5.5 hours at 37° C. in 5% _CO2, luciferase activity was measured using OneGlo™ reagent (Promega, #E6031) and a Victor™ X multichannel plate reader (Perkin Elmer). The results were analyzed by non-linear regression (4-parameter logistics) using the computer program Prism™6 (GraphPad) to obtain EC ₅₀ values. Percent antibody activation was calculated as the maximum RLU range achieved by the antibody relative to the maximum RLU range achieved by hLeptin.

- 38 041859

As shown in Table. 17, in the absence of hLeptin, all of the tested anti-LEPR antibodies were characterized by signaling activation by monkey LEPR in IMR-32/STAT3-Luc/mfLEPR cells with EC50 values ranging from 266 to 368 pM and maximum activation ranging from 76 to 82%. while 100% activation was obtained as a result of the action of hLeptin. hLeptin activated at an EC50 value of 333 pM. The isotype control antibody showed no measurable stimulation of IMR-32/STAT3-Luc/mfLEPR cells.

Table 17. Activation of LEPR by Macaca fascicularis with Anti-LEPR Antibodies

Leptin or antibody EU ₅₀ (M) % activation Human leptin 3,ZZE-11 100 H4H16650R2 2.66E-10 82 H4H16679R2 2.49Е-10 80 H4H17319R2 3.65E-10 76 H4H17321R2 3.68E-10 78 Isotype control antibody Without activation Without activation

Example 14 Epitope Binding to Full Length Human LEPR Extracellular Domain by MFI Signal in Luminex.

In order to determine the human LEPR epitope to which the anti-LEPR antibodies of the present invention bind, a FLEXMAP Luminex flow cytometry assay (FM3DD, LuminexCorp) was used to characterize the interaction of anti-LEPR antibodies with the protein domains of recombinant human LEPRs. For analysis, approximately 3 million carboxylated Microplex ^R microspheres (Luminex, catalog # LC1000A) were washed, vortexed and sonicated in 0.1 M NaPO ₄ , pH 6.2 (activation buffer) and then centrifuged to remove the supernatant. The microspheres were resuspended in 120 µl of activation buffer and the carboxylate groups (-COOH) were activated by adding 15 µl of 50 mg/ml N-hydroxysuccinimide (NHS, Thermo Scientific, Cat # 24500) followed by the addition of 15 µl of 50 mg/ml 1-ethyl -3-[3dimethylaminopropyl]carbodiimide (EDC, ThermoScientific, Cat # 22980) at 25°C. After 10 minutes, the pH of the reaction mixture was lowered to 5.0 by adding 600 μl of 50 mM MES, pH 5 (binding buffer), the microspheres were vortexed and centrifuged to remove the supernatant. The activated beads were immediately mixed with 500 μl of 20 mg/ml anti-myc monoclonal antibodies with either mouse IgG or human IgG in binding buffer and incubated for 2 h at 25°C. The binding reaction was stopped by adding 50 μl 1 M Tris-HCl, pH 8.0, and the microspheres were rapidly vortexed, centrifuged and washed four times with 1 ml DPBS to remove unbound proteins and other components of the reaction mixture.

Transiently expressed LEPR proteins, including human LEPR extracellular domain expressed with C-terminal hexahistidine tag myc-myc (human LEPR-MMH, SEQ ID NO: 113), CRH1 (DI) human LEPR expressed with C-terminal hexahistidine tag myc-myc (CRH1 (DI) human MMH LEPR, amino acids 1-208 SEQ ID NO: 113 with myc-myc hexahistidine tag, amino acids 209-236), CRH1 (D1,D2) domain of human LEPR expressed from C-terminal myc-myc hexahistidine tag (CRH1 (D1,D2) human MMH LEPR, amino acids 1-318 SEQ ID NO: 113 with myc-myc hexahistidine tag, amino acids 319-346), human LEPR CRH1 domain (D1,D2,D3) -Ig expressed with C-terminal myc-myc hexahistidine tag (CRH1 (D1,D2,D3) human MMH LEPR, amino acids 1-278 SEQ ID NO: IZ with myc-myc hexahistidine tag, amino acids 279-306), domain CRH1 (D2,D3) human LEPR-Ig expressed with C-terminal myc-myc hexahistidine tag (CRH1 (D2,D3) human LEPR-MMH-Ig, amino acids 1-198 SEQ ID NO: 113 with myc-myc hexahistidine tag, amino acids 199-226), human LEPR domain (D3)-Ig expressed with myc-myc C-terminal hexahistidine tag (human LEPR (D3)-MMH-Ig, amino acids 1 -88 SEQ ID NO: IZ with myc-myc hexahistidine tag, amino acids 89-116), human CRH2 LEPR domain expressed with C-terminal myc-myc hexahistidine tag (human CRH2 LEPR MMH, amino acids 1-207 SEQ ID NO: 113c myc-myc hexahistidine tag, amino acids 208235), human FNIII LEPR domain expressed with C-terminal myc-myc hexahistidine tag (human FNIII LEPR-MMH, amino acids 1-204 SEQ ID NO: 113 with myc-myc hexahistidine tag, amino acids 205 -232), and a human-Ig CRH2-FNIII LEPR domain expressed with a myc-myc C-terminal hexahistidine tag (human CRH2-FNIII LEPR-MMH-Ig, amino acids 1-510 of SEQ ID NO: 113 with a myc-myc hexahistidine tag , amino acids 511-538) were suspended in CHO-S-SFM II serum-free medium (Thermo Fisher, cat. no. gu 31033020) and then purified by centrifugation. Aliquots of microspheres with immobilized anti-myc monoclonal antibodies obtained

-39041859 as described above was added separately to 1 ml of each of these protein supernatants. The microspheres were mixed gently, incubated for two hours at 25°C, washed twice with 1 ml DBPS, centrifuged to remove the supernatant and resuspended in 1 ml DPBS buffer. 48 µl of anti-myc IgG bound microspheres from separate reactions with full-length human LEPRs and with each of the human LEPR domain proteins were recovered 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 placed in a well of a 96-well filter plate (Millipore, catalog # 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 DPBS with 0.05% Tween 20 (wash buffer). For detection and quantitation of bound anti-LEPR antibodies with single microspheres or 100 µl 2.5 µg/ml R-phycoerythrin-conjugated goat F(ab') ₂ to the human kappa chain (Southern Biotech, cat. no. 2063-09) in blocking buffer or 100 µl 1.25 µg/ml AffiniPure R-phycoerythrin conjugated goat F(ab') ₂ fragment to mouse IgG specific for F(ab') ₂ fragment (Jackson Immunoresearch, catalog # 115-116-072) in blocking buffer was added and incubated for 30 min at 25°C. After 30 min, the samples were washed twice with 200 µl wash buffer and resuspended in 150 µl wash buffer. The mean fluorescence intensity (MFI) of the microspheres was measured in a Luminex analyzer.

Table 18. MFI signal in Luminex when anti-LEPR antibodies bind to myc-tagged full-length extracellular human LEPR domain and isolated human LEPR domains

Antibody CRH 1 (D1) CRH1 (D1,D2) CRH1-Ig (D1,D2,D3) CRH1Ig (D2,D3) ig (D3) CR H2 FNIII ig- CRH2FNIII Full length extracellular domain Probable binding site H4H18445 R2 12 thirty 22 40 19 17 230 14544 6573 FNIII H4H18446 R2 17 682 205 645 25 65 32 16852 10536 Ig-CRH2FNIII H4H18482 R2 13 40 21 52 27 23 167 15316 7311 Ig-CRH2FNIII H4H18487 R2 12 51 29 62 22 27 174 16320 7329 Ig-CRH2FNIII H4H18417 R2 10 16048 3334 5502 17 39 14 37 4887 CRH1D2 H4H18438 R2 13 18931 6572 8884 thirty 165 25 468 6251 CRH1D2 H4H18492 R2 eleven 19371 6354 8685 19 18 16 186 6382 CRH1D2 H4H18449 R2 20 2934 2056 42 24 15 13 43 7976 CRHl(Dl-2) H4H16650 R2 8 4722 2562 74 10 16 6 110 7603 CRHl(Dl-2) H4H16679 R2 12 4388 2797 34 14 33 10 42 7507 CRHl(Dl-2) H4H17319 R2 8 1246 938 14 8 91 20 8 3305 CRHl(Dl-2) H4H17321 R2 9 2649 1752 15 7 116 40 14 4696 CRH1(D1- 2) mAb comparisons -14 19 -57 27 10 9404 73 7112 3908 CRH2

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Claims (20)

The results of the analysis based on Luminex are presented in tabular form in table. 18. The MFI signal intensities in Luminex indicated that the twelve anti-LEPR antibodies of the present invention bound to the entire extracellular domain of human LEPR. Anti-LEPR antibodies H4H18417P2, H4H18438P2, and H4H18492P2 bind to epitopes in the CRH1 D2 domain of human LEPR. Anti-LEPR antibodies H4H18449P2, H4H16650P and H4H16679P bound to the CRH1(D1-2) domain of human LEPR. The anti-LEPR antibody comparison mAB bound epitopes in the human LEPR CRH2 domain. The anti-LEPR antibody H4H18445P2 bound to epitopes in the FNIII domain of human LEPR. Anti-LEPR antibodies H4H18446P2, H4H18482P2 and H4H18487P2 bind to the Ig-CRH2-FNIII domain of human LEPR. CLAIM 1. An isolated antibody or antigen-binding fragment thereof that binds the human leptin receptor (LEPR) and activates signaling through LEPR that contains a light chain variable region containing complementarity determining regions (CDRs) of a light chain variable region that contains an amino acid sequence, set forth in SEQ ID NO: 10; and a heavy chain variable region comprising the heavy chain CDRs of the heavy chain variable region which comprises the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 26, or SEQ ID NO: 34. 2. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof contains: (i) a light chain variable region that contains an LCDR1 that contains the amino acid sequence set forth in SEQ ID NO: 12; LCDR2, which contains the amino acid sequence set forth in SEQ ID NO: 14; and LCDR3, which contains the amino acid sequence set forth in SEQ ID NO: 16; and (ii) a heavy chain variable region that contains HCDR1 that contains the amino acid sequence set forth in SEQ ID NO: 4; HCDR2, which contains the amino acid sequence set forth in SEQ ID NO: 6; and HCDR3, which contains the amino acid sequence set forth in SEQ ID NO: 8; or a heavy chain variable region that contains HCDR1 that contains the amino acid sequence set forth in SEQ ID NO: 20; HCDR2, which contains the amino acid sequence set forth in SEQ ID NO: 22; and HCDR3, which contains the amino acid sequence set forth in SEQ ID NO: 24; or a heavy chain variable region that contains HCDR1 that contains the amino acid sequence set forth in SEQ ID NO: 28; HCDR2, which contains the amino acid sequence set forth in SEQ ID NO: 30; and HCDR3, which contains the amino acid sequence set forth in SEQ ID NO: 32; or a heavy chain variable region that contains HCDR1 that contains the amino acid sequence set forth in SEQ ID NO: 36; HCDR2, which contains the amino acid sequence set forth in SEQ ID NO: 38; and HCDR3 which contains the amino acid sequence set forth in SEQ ID NO: 40. 3. Selected antibody or antigennegative fragment according to claim 1 or 2, where the antibody or antigennegative fragment contains a variable region of the light chain, which contains the amino acid sequence set forth in SEQ ID NO: 10; and a heavy chain variable region that contains the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 26, or SEQ ID NO: 34. 4. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region containing complementarity-determining light chain regions of a light chain variable region that contains the amino acid sequence set forth in SEQ ID NO: 10; and a heavy chain variable region comprising the heavy chain variable region complementarity determining regions, which comprises the amino acid sequence set forth in SEQ ID NO: 26. 5. An isolated antibody or antigen-binding fragment thereof that binds the human leptin receptor (LEPR), wherein the antibody or antigen-binding fragment thereof contains a light chain variable region that contains an LCDR1 that contains the amino acid sequence set forth in SEQ ID NO: 12; LCDR2, which contains the amino acid sequence set forth in SEQ ID NO: 14; and LCDR3, which contains the amino acid sequence set forth in SEQ ID NO: 16; and the heavy chain variable region, which contains HCDR1, which contains amino acids - 41 041859 the sequence set forth in SEQ ID NO: 28; HCDR2, which contains the amino acid sequence set forth in SEQ ID NO: 30; and HCDR3 which contains the amino acid sequence set forth in SEQ ID NO: 32. 6. Selected antibody or antigennegative fragment that binds the human leptin receptor (LEPR), where the antibody or antigennegative fragment contains a light chain variable region that contains the amino acid sequence set forth in SEQ ID NO: 10; and a heavy chain variable region that contains the amino acid sequence set forth in SEQ ID NO: 26. 7. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the heavy chain variable region is linked to a human IgG1 heavy chain constant domain and the light chain variable region is linked to a human kappa light chain constant domain. 8. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the heavy chain variable region is linked to a human IgG4 heavy chain constant domain and the light chain variable region is linked to a human kappa light chain constant domain. 9. The isolated antibody or antigen-binding fragment thereof according to claim 6, which is a full-length antibody containing two heavy chains containing the specified heavy chain variable region, and two light chains containing the specified light chain variable region, interconnected by disulfide bonds. 10. The isolated antibody or antigen-binding fragment thereof according to claim 6, wherein the antibody is a recombinant human antibody. 11. Pharmaceutical composition for the treatment of a disease or condition associated with or caused by leptin deficiency or leptin resistance, or for the treatment of a disease or condition associated with a LEPR mutation as a result of a signal transduction defect or signal transduction disorder or caused by such, containing an antibody or its antigen-binding a fragment according to any one of claims 1 to 10; and a pharmaceutically acceptable carrier or diluent. 12. A method of treating a disease or condition associated with or caused by leptin deficiency or leptin resistance, the method comprising administering the pharmaceutical composition of claim 11 to a subject in need thereof. 13. The method of claim 12 wherein the disease or condition associated with or caused by leptin deficiency or leptin resistance is selected from the group consisting of lipodystrophy, obesity, metabolic syndrome, diet-induced food cravings, functional hypothalamic amenorrhea, diabetes Type 1, type 2 diabetes, insulin resistance, severe insulin resistance due to insulin receptor mutation, Alzheimer's disease, leptin deficiency, leptin resistance, leprechaunism/Donoghue syndrome, and Rabson-Mendenhall syndrome. 14. A method of treating a lipodystrophy condition in a patient, the method comprising administering the pharmaceutical composition of claim 11 to a patient in need thereof, wherein the lipodystrophy condition is selected from the group consisting of congenital generalized lipodystrophy, acquired generalized lipodystrophy, familial partial lipodystrophy, acquired partial lipodystrophy , centrifugal abdominal lipodystrophy, annular lipoatrophy, localized lipodystrophy, and HIV-associated lipodystrophy. 15. A method of treating a disease or condition associated with a LEPR mutation resulting from or caused by a signal transduction defect or signal transduction disorder, the method comprising administering the pharmaceutical composition of claim 11 to a subject in need thereof. 16. The method of claim 15, wherein the LEPR mutation resulting from a signaling defect or signaling disorder is LEPR-A409E or LEPR-P316T. 17. The method of claim 15 or 16 wherein the disease or condition associated with or caused by a LEPR mutation resulting from a signal transduction defect or signal transduction disorder is early stage obesity. 18. The method of any one of claims 12-17, further comprising administering the second therapeutic agent to the subject, wherein the second therapeutic agent is selected from the group consisting of recombinant human leptin, PCSK9 inhibitor, statin, ezetimibe, insulin, insulin variant, insulin secretogen, metformin , sulfonylurea, sodium glucose cotransporter 2 (SGLT2) inhibitor, GLP-1 agonist/analog, glucagon inhibitor (GCG), glucagon receptor inhibitor (GCGR), angiopoietin-like protein inhibitor (ANGPTL), phentermine, orlistat, topiramate, bupropion, topiramate/phentermine, bupropion/naltrexone, bupropion/zonisamide, pramlintide/metreleptin, lorcaserin, cetilistat, tesofensine, and wellneperite. 19. A pen for delivering an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 10, containing a pharmaceutical composition according to claim 11. 20. Combination containing an isolated antibody or antigen-binding fragment thereof with a second therapeutic agent for the treatment of a disease or condition associated with a mutation -