Patent Publication Number: US-2019185562-A1

Title: Bispecific antigen binding molecules that bind leptin receptor and/or gp130, and methods of use thereof

Description:
This application claims the benefit of U.S. provisional patent application No. 62/607,137, filed Dec. 18, 2017 and 62/635,406, filed Feb. 26, 2018; each of which is herein incorporated by reference in its entirety. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to antigen-binding molecules, including bispecific antigen-binding molecules (e.g., bispecific antibodies) that bind human GP130 and/or human leptin receptor (LEPR), and the use of such antigen-binding molecules for the treatment of conditions and disorders related to leptin deficiency or leptin resistance. 
     SEQUENCE LISTING 
     An official copy of the sequence listing is submitted concurrently with the specification electronically via EFS-Web as an ASCII formatted sequence listing with a file name of “10397US01_SEQ_LIST_ST25.txt”, a creations date of Dec. 17, 2018, and a size of about 151 KB. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety. 
     BACKGROUND 
     Glycoprotein 130 (GP130) is a component of a receptor complex that also comprises CNTRF-alpha and LIFR-beta. Signaling through this receptor complex activates JAK/STAT signaling which, in certain biological contexts, results in reduced appetite, food intake and weight loss. 
     Leptin is a polypeptide hormone predominantly expressed by adipose tissue and is involved in the regulation of metabolism, energy balance and food intake. Leptin activity is mediated by interaction with, and signaling through, the leptin receptor. Leptin receptor, (also known as “LEPR,” “WSX,” “OB receptor,” “OB-R,” and “CD295”) is a single-pass transmembrane receptor of the class I cytokine receptor family with a large (818 amino acid) extracellular domain. Leptin deficiency, leptin resistance, and certain LEPR signaling-defective/signaling impaired mutations, are associated with obesity, type 2 diabetes, dyslipidemia, lipodystrophies, hepatic steatosis, non-alcoholic and alcoholic fatty liver diseases, severe insulin resistance, Leprechaunism/Donohue syndrome, Rabson-Mendenhall syndrome, and related complications. Therapeutic approaches to address leptin resistance, leptin deficiency, and hypoleptinemia (e.g., lipodystrophy) have mostly focused on the delivery of supplemental leptin or leptin analogues to affected individuals. Such approaches, however, have generally shown limited efficacy, particularly in leptin-resistant individuals, and are frequently associated with adverse side effects. Thus, a need exists in the art for alternative approaches to treating leptin resistance and other conditions associated with leptin deficiency or hypoleptinemia. 
     BRIEF SUMMARY OF THE INVENTION 
     The present invention relates, in part, to the concept of antibody-mediated heterodimerization of the LEPR and GP130 to activate both receptors and thereby stimulate the anorexegenic effects associated with signaling through these receptors. Accordingly, the present invention provides antigen-binding molecules (e.g., antibodies and antigen-binding fragments of antibodies) that bind human GP130 and/or human leptin receptor (LEPR). According to certain embodiments, the present invention provides bispecific antigen-binding molecules comprising a first antigen-binding domain (D1) that specifically binds human GP130, and a second antigen-binding domain (D2) that specifically binds human leptin receptor (LEPR). The present invention includes LEPR×GP130 bispecific molecules (e.g., bispecific antibodies). In certain exemplary embodiments of the invention, the anti-GP130 antigen-binding domain (D1) and the anti-LEPR (D2) antigen-binding domain each comprise different, distinct heavy chain variable regions (HCVRs) paired with the same or a different light chain variable regions (LCVRs). 
     The antigen-binding molecules (e.g., bispecific antigen-binding molecules) of the present invention are useful, inter alia, for targeting cells that express LEPR and/or cells that express GP130, or both. According to certain embodiments, the bispecific antigen-binding molecules of the present invention are useful for physically linking LEPR and GP130 to one another on the surface of a cell in order to stimulate LEPR signaling. In this manner, the bispecific antigen binding molecules of the present invention may serve as LEPR agonists in a variety of therapeutic applications where leptin and/or LEPR-mediated signaling would be beneficial (e.g., in the treatment of obesity, lipodystrophies and other diseases and disorders associated with or caused by leptin deficiency or leptin resistance). 
     Other embodiments will become apparent from a review of the ensuing detailed description. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1  shows the effects of LEPR×GP130 bispecific antibody treatment (open squares and open diamonds) on obese mice, fed a high fat diet, expressing human LEPR and human GP130, compared to treatment with isotype control antibody (closed circles). Antibodies were administered subcutaneously at 30 mg/kg on day 0 and day 7 (indicated by “Dose” and upward arrows), and the effects of antibody treatment on body weight (expressed in terms of average percent change in body weight from pre-dose) are plotted over time for each treatment group over time. Open squares represent mice treated with bsAb21236 (alternatively referred to as “H4H21236D”). Open diamonds represent mice treated with bsAb21237 (alternatively referred to as “H4H21237D”). (*) indicates P&lt;0.05 isotype control vs. bsAb21236. (#) indicates P&lt;0.05 isotype control vs. bsAb21237. 
     
    
    
     DETAILED DESCRIPTION 
     Before the present invention is described, it is to be understood that this invention is not limited to particular methods and experimental 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 particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term “about,” when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1%. For example, as used herein, the expression “about 100” includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.). 
     Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications and non-patent publications mentioned in this specification are incorporated herein by reference in their entireties. 
     GP130 Protein 
     The expressions “Glycoprotein 130,” “GP130,” “gp130,” and the like, as used herein, refer to the human GP130 protein comprising the amino acid sequence as set forth in SEQ ID NO:185 (see also UniProtKB Q17RA0). The expression “GP130” includes both monomeric and multimeric GP130 molecules. As used herein, the expression “monomeric human GP130” means a GP130 protein or portion thereof that does not contain or possess any multimerizing domains and that exists under normal conditions as a single GP130 molecule without a direct physical connection to another GP130 molecule. An exemplary monomeric GP130 molecule is the molecule referred to herein as “hGP130.mmh” comprising the amino acid sequence of SEQ ID NO:191 (see, e.g., Example 3, herein). As used herein, the expression “dimeric human GP130” means a construct comprising two GP130 molecules connected to one another through a linker, covalent bond, non-covalent bond, or through a multimerizing domain such as an antibody Fc domain. An exemplary dimeric GP130 molecule is the molecule referred to herein as “hGP130.hFc” comprising the amino acid sequence of SEQ ID NO:197 or “hGP130.mFc” comprising the amino acid sequence of SEQ ID NO:190 (see, e.g., Example 3, herein). 
     All references to proteins, polypeptides and protein fragments herein are intended to refer to the human version of the respective protein, polypeptide or protein fragment unless explicitly specified as being from a non-human species. Thus, the expression “GP130” means human GP130 unless specified as being from a non-human species, e.g., “mouse GP130,” “monkey GP130,” etc. 
     As used herein, the expression “cell surface-expressed GP130” means one or more GP130 protein(s), or the extracellular domain thereof, that is/are expressed on the surface of a cell in vitro or in vivo, such that at least a portion of a GP130 protein is exposed to the extracellular side of the cell membrane and is accessible to an antigen-binding portion of an antibody. A “cell surface-expressed GP130” can comprise or consist of a GP130 protein expressed on the surface of a cell which normally expresses GP130 protein. Alternatively, “cell surface-expressed GP130” can comprise or consist of GP130 protein expressed on the surface of a cell that normally does not express human GP130 on its surface but has been artificially engineered to express GP130 on its surface. 
     Anti-GP130 Antibodies and Antigen-Binding Fragments Thereof 
     According to one aspect of the present invention, anti-GP130 antibodies are provided (e.g., monospecific anti-GP130 antibodies). Exemplary anti-GP130 antibodies according to this aspect of the invention are listed in Tables 1 and 2 herein. Table 1 sets forth the amino acid sequence identifiers of the heavy chain variable regions (HCVRs), light chain variable regions (LCVRs), heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) of the exemplary anti-GP130 antibodies from which the bispecific antigen-binding molecules of the present invention may be derived. Table 2 sets forth the nucleic acid sequence identifiers of the HCVRs, LCVRs, HCDR1, HCDR2 HCDR3, LCDR1, LCDR2 and LCDR3 of the exemplary anti-GP130 antibodies. 
     The present invention provides antibodies or antigen-binding fragments thereof that specifically bind GP130, comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. 
     The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind GP130, comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. 
     The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind GP130, comprising an HCVR and an LCVR amino acid sequence pair (HCVR/LCVR) comprising any of the HCVR amino acid sequences listed in Table 1 paired with any of the LCVR amino acid sequences listed in Table 1. According to certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, comprising an HCVR/LCVR amino acid sequence pair contained within any of the exemplary anti-GP130 antibodies listed in Table 1. In certain embodiments, the HCVR/LCVR amino acid sequence pair is SEQ ID NOs: 154/10. 
     The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind GP130, comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. 
     The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind GP130, comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. 
     The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind GP130, comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. 
     The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind GP130, comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. 
     The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind GP130, comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. 
     The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind GP130, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. 
     The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind GP130, comprising an HCDR3 and an LCDR3 amino acid sequence pair (HCDR3/LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 1 paired with any of the LCDR3 amino acid sequences listed in Table 1. According to certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3/LCDR3 amino acid sequence pair contained within any of the exemplary anti-GP130 antibodies listed in Table 1. In certain embodiments, the HCDR3/LCDR3 amino acid sequence pair is SEQ ID NOs: 160/16. 
     The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind GP130, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any of the exemplary anti-GP130 antibodies listed in Table 1. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences set is selected from the group consisting of: SEQ ID NOs: 20-22-24-12-14-16, 28-30-32-12-14-16, 36-38-40-12-14-16, 44-46-48-12-14-16, 52-54-56-12-14-16, 60-62-64-12-14-16, 68-70-72-12-14-16, 76-78-80-12-14-16, 84-86-88-12-14-16, 92-94-96-12-14-16, 100-102-104-12-14-16, 108-110-112-12-14-16, 116-118-120-12-14-16, 124-126-128-12-14-16, 132-134-136-12-14-16, 140-142-144-12-14-16, 148-150-152-12-14-16 and 156-158-160-12-14-16. 
     In a related embodiment, the present invention provides antibodies, or antigen-binding fragments thereof that specifically bind GP130, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR amino acid sequence pair as defined by any of the exemplary anti-GP130 antibodies listed in Table 1. For example, the present invention includes antibodies or antigen-binding fragments thereof that specifically bind GP130, comprising the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences set contained within an HCVR/LCVR amino acid sequence pair of: SEQ ID NOs: 154/10. 
     Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al.,  J. Mol. Biol.  273:927-948 (1997); and Martin et al.,  Proc. Natl. Acad. Sci. USA  86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within an antibody. 
     The present invention also provides nucleic acid molecules encoding anti-GP130 antibodies or portions thereof. For example, the present invention provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. 
     The present invention also provides nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. 
     The present invention also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. 
     The present invention also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. 
     The present invention also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. 
     The present invention also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. 
     The present invention also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. 
     The present invention also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. 
     The present invention also provides nucleic acid molecules encoding an HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), wherein the HCDR1-HCDR2-HCDR3 amino acid sequence set is as defined by any of the exemplary anti-GP130 antibodies listed 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), wherein the LCDR1-LCDR2-LCDR3 amino acid sequence set is as defined by any of the exemplary anti-GP130 antibodies listed in Table 1. 
     The present invention also provides nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence of any of the HCVR amino acid sequences listed in Table 1, and wherein the LCVR comprises an amino acid sequence of any of the LCVR amino acid sequences listed in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. In certain embodiments according to this aspect of the invention, the nucleic acid molecule encodes an HCVR and LCVR, wherein the HCVR and LCVR are both derived from the same anti-GP130 antibody listed in Table 1. 
     The present invention also provides recombinant expression vectors capable of expressing a polypeptide comprising a heavy or light chain variable region of an anti-GP130 antibody. For example, the present invention includes recombinant expression vectors comprising any of the nucleic acid molecules mentioned above, i.e., nucleic acid molecules encoding any of the HCVR, LCVR, and/or CDR sequences as 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 of producing the antibodies or portions thereof by culturing the host cells under conditions permitting production of the antibodies or antibody fragments, and recovering the antibodies and antibody fragments so produced. 
     The present invention includes anti-GP130 antibodies having a modified glycosylation pattern. In some embodiments, modification to remove undesirable glycosylation sites may be useful, or an antibody lacking a fucose moiety present on the oligosaccharide chain, for example, to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, modification of galactosylation can be made in order to modify complement dependent cytotoxicity (CDC). 
     LEPR Protein 
     The expression “leptin receptor,” “LEPR,” and the like, as used herein, refers to the human leptin receptor, comprising the amino acid sequence as set forth in SEQ ID NO:186 (see also UniProtKB/Swiss-Prot Accession No. P48357). Alternative names for LEPR used in the scientific literature include “OB receptor,” “OB-R,” and “CD295.” LEPR is also referred to as “WSX” (see, e.g., U.S. Pat. No. 7,524,937). The expression “LEPR” includes both monomeric and multimeric LEPR molecules. As used herein, the expression “monomeric human LEPR” means a LEPR protein or portion thereof that does not contain or possess any multimerizing domains and that exists under normal conditions as a single LEPR molecule without a direct physical connection to another LEPR molecule. An exemplary monomeric LEPR molecule is the molecule referred to herein as “hLEPR.mmh” comprising the amino acid sequence of SEQ ID NO:187 (see, e.g., Example 10, herein). As used herein, the expression “dimeric human LEPR” means a construct comprising two LEPR molecules connected to one another through a linker, covalent bond, non-covalent bond, or through a multimerizing domain such as an antibody Fc domain. An exemplary dimeric LEPR molecule is the molecule referred to herein as “hLEPR.hFc” comprising the amino acid sequence of SEQ ID NO:189 (see, e.g., Example 10, herein). 
     All references to proteins, polypeptides and protein fragments herein are intended to refer to the human version of the respective protein, polypeptide or protein fragment unless explicitly specified as being from a non-human species. Thus, the expression “LEPR” means human LEPR unless specified as being from a non-human species, e.g., “mouse LEPR,” “monkey LEPR,” etc. 
     As used herein, the expression “cell surface-expressed LEPR” means one or more LEPR protein(s), or the extracellular domain thereof, that is/are expressed on the surface of a cell in vitro or in vivo, such that at least a portion of a LEPR protein is exposed to the extracellular side of the cell membrane and is accessible to an antigen-binding portion of an antibody. A “cell surface-expressed LEPR” can comprise or consist of a LEPR protein expressed on the surface of a cell which normally expresses LEPR protein. Alternatively, “cell surface-expressed LEPR” can comprise or consist of LEPR protein expressed on the surface of a cell that normally does not express human LEPR on its surface but has been artificially engineered to express LEPR on its surface. 
     Several isoforms of the LEPR are generated through alternative splicing, resulting in a long isoform b (LEPR-b) and several short forms, including isoform a (LEPR-a) which shows the highest and broadest expression pattern (Tartaglia L A. The leptin receptor. J Biol Chem 1997; 272: 6093-6096). LEPR-b is the predominant isoform expressed in the brain, while LEPR-a is broadly expressed in the liver. All the isoforms share the same extracellular domain, transmembrane region and a short stretch of the cytoplasmic domain, containing the Box 1 region, followed by a variable region. The long form contains intracellular sequence motifs required for mediating all the signaling capabilities of leptin whereas the short forms are lacking these regions. The extracellular domain of the short forms is identical to the signaling competent long form. 
     LEPR×GP130 Bispecific Antigen-Binding Molecules 
     The present invention is based on the concept of stimulating LEPR and GP130 signaling by bridging LEPR and GP130 on the surface of a cell. In particular, the present invention relates to the premise that a bispecific antigen-binding molecule, such as a LEPR×GP130 bispecific antibody (as described in detail elsewhere herein), is capable of stimulating LEPR-dependent signaling of STAT3 by bringing GP130 into relative proximity of the leptin receptor on the surface of cells, even in the absence leptin. In this manner, the bispecific antigen-binding molecules of the present invention may serve as LEPR agonists which may find use in therapeutic contexts where induced leptin/LEPR signaling is beneficial and/or desirable. 
     Accordingly, the present invention provides bispecific antigen binding molecules comprising a first antigen-binding domain (also referred to herein as “D1”) that binds human GP130, and a second antigen-binding domain (also referred to herein as “D2”) that binds human LEPR. According to the present invention, and as demonstrated in the working examples herein, the simultaneous binding LEPR and GP130 by the bispecific antigen-binding molecules of the invention results in stimulation of LEPR signaling. 
     The bispecific antigen-binding molecules of the present invention, may be referred to herein as “LEPR×GP130 bispecific antibodies,” or other related terminology. 
     LEPR×GP130 bispecific antigen-binding molecules of the present invention may be constructed using the antigen-binding domains derived from mono-specific (conventional) anti-LEPR antibodies and anti-GP130 antibodies. For example, a collection of monoclonal, monospecific, anti-LEPR and/or anti-GP130 antibodies may be produced using standard methods known in the art, and the antigen-binding domains thereof can be used to construct LEPR×GP130 bispecific antigen-binding molecules (e.g., bispecific antibodies) using conventional techniques known in the art. 
     Exemplary anti-LEPR antibodies that can be used in the context of the present invention to produce LEPR×GP130 bispecific antigen binding molecules include any of the anti-LEPR antibodies described in US Patent Application Publication No. 2017/0101477, the disclosure of which is incorporated herein in its entirety. Anti-LEPR antibodies that can be used to construct the LEPR×GP130 bispecific antigen-binding molecules of the present invention may be agonist antibodies, i.e., antibodies that bind human LEPR and activate LEPR signaling. In other embodiments, anti-LEPR antibodies that can be used to construct LEPR×GP130 bispecific antigen-binding molecules may be potentiating antibodies, i.e., antibodies that enhance leptin-mediated signaling through LEPR. Anti-LEPR antibodies that are useful for constructing LEPR×GP130 bispecific antigen-binding molecules may be antibodies that are able to bind LEPR in complexed with leptin. Such antibodies include those that bind LEPR and do not block the LEPR:leptin interaction. Alternatively, anti-LEPR antibodies that are useful for constructing LEPR×GP130 bispecific antigen-binding molecules may be antibodies that compete with leptin for binding to LEPR, and/or only bind LEPR in the absence of leptin. Non-limiting examples of particular anti-LEPR antibodies that can be used to construct LEPR×GP130 bispecific antigen-binding molecules include the anti-LEPR antibodies referred to herein as “mAb18445” and “mAb18446”. 
     In some embodiments, the LEPR×GP130 bispecific antigen-binding molecule is derived from an anti-LEPR antibody that potentiates leptin-mediated signaling in vitro through the LEPR-b isoform. 
     In some embodiments, the LEPR×GP130 bispecific antigen-binding molecule is derived from an anti-LEPR antibody that does not activate leptin-mediated signaling in vitro through the LEPR-a isoform. 
     Exemplary anti-GP130 antibodies that can be used in the context of the present invention to produce LEPR×GP130 bispecific antigen binding molecules include any of the anti-GP130 antibodies described elsewhere herein. Anti-GP130 antibodies that are useful for constructing LEPR×GP130 bispecific antigen-binding molecules include anti-GP130 antibodies with one or more of the following properties: binds monkey GP130, does not bind mouse or rat GP130, binds to an epitope within the FNIII domain of GP130, does not inhibit GP130 ligand-mediated signaling, and/or does not activate GP130 signaling in the absence of a GP130 ligand. GP130 ligands include, e.g., human oncostatin M (OSM), human leukemia inhibitory factor (LIF), and human ciliary neurotrophic factor (CNTF). A non-limiting example of a particular anti-GP130 antibody that can be used to construct LEPR×GP130 bispecific antigen-binding molecules include the anti-GP130 antibody referred to herein as “mAb16683”. 
     According to the present invention, a bispecific antigen-binding molecule can be a single multifunctional polypeptide, or it can be a multimeric complex of two or more polypeptides that are covalently or non-covalently associated with one another. As will be made evident by the present disclosure, any antigen binding construct which has an antigen-binding domain that specifically binds human LEPR and an antigen-binding domain that specifically binds human GP130 is regarded as a “bispecific antigen-binding molecule.” Any of the bispecific antigen-binding molecules of the invention, or variants thereof, may be constructed using standard molecular biological techniques (e.g., recombinant DNA and protein expression technology), as will be known to a person of ordinary skill in the art. 
     The bispecific antigen-binding molecules of the invention may be “isolated.” An “isolated bispecific antigen-binding molecule,” as used herein, means a bispecific antigen-binding molecule that has been identified and separated and/or recovered from at least one component of its natural environment. For example, a bispecific 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 is produced, is an “isolated bispecific antibody” for purposes of the present invention. An isolated bispecific antigen-binding molecule also includes molecules in situ within a recombinant cell. Isolated bispecific antigen-binding molecules are molecules that have been subjected to at least one purification or isolation step. According to certain embodiments, an isolated bispecific antigen-binding molecule may be substantially free of other cellular material and/or chemicals. 
     Antigen-Binding Domains 
     The bispecific antigen-binding molecules of the present invention comprise two separate antigen-binding domains (D1 and D2). As used herein, the expression “antigen-binding domain” means any peptide, polypeptide, nucleic acid molecule, scaffold-type molecule, peptide display molecule, or polypeptide-containing construct that is capable of specifically binding a particular antigen of interest (e.g., human LEPR or human GP130). The term “specifically binds” or the like, as used herein in reference to an antigen-binding domain, means that the antigen-binding domain is capable of forming a complex with a particular antigen and does not bind other unrelated antigens under ordinary test conditions. “Unrelated antigens” are proteins, peptides or polypeptides that have less than 95% amino acid identity to one another. 
     Exemplary categories of antigen-binding domains that can be used in the context of the present invention include antibodies, antigen-binding portions of antibodies, peptides that specifically interact with a particular antigen (e.g., peptibodies), receptor molecules that specifically interact with a particular antigen, proteins comprising a ligand-binding portion of a receptor that specifically binds a particular antigen, antigen-binding scaffolds (e.g., DARPins, HEAT repeat proteins, ARM repeat proteins, tetratricopeptide repeat proteins, and other scaffolds based on naturally occurring repeat proteins, etc., [see, e.g., Boersma and Pluckthun, 2011,  Curr. Opin. Biotechnol.  22:849-857, and references cited therein]), and aptamers or portions thereof. 
     Methods for determining whether two molecules specifically bind one another are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. The term “surface plasmon resonance”, as used herein, refers to an optical phenomenon that allows for the analysis of real-time interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore™ system (Biacore Life Sciences division of GE Healthcare, Piscataway, N.J.). 
     As indicated above, an “antigen-binding domain” (D1 and/or D2) may comprise or consist of an antibody or antigen-binding fragment of an antibody. The term “antibody,” as used herein, means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., human MET). The term “antibody” includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V H ) and a heavy chain constant region. The heavy chain constant region comprises three domains, C H 1, C H 2 and C H 3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V L ) and a light chain constant region. The light chain constant region comprises one domain (C L 1). The V H  and V L  regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each V H  and V L  is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the antibodies of the invention (or antigen-binding portion thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. 
     The D1 and/or D2 components of the bispecific antigen-binding molecules of the present invention may comprise or consist of antigen-binding fragments of full antibody molecules. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, 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 (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment,” as used herein. 
     An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to 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 V H  and V L  domains may be situated relative to one another in any suitable arrangement. 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, the antigen-binding fragment of an antibody may contain a monomeric V H  or V L  domain. 
     In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present invention include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-C H 3; (vi) V H -C H 2-C H 3; (vii) V H -C L ; (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -C H 3; (xi) V L -C H 1-C H 2; (xii) V L -C H 1-C H 2-C H 3; (xiii) V L -C H 2-C H 3; and (xiv) V L -C L . In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric V H  or V L  domain (e.g., by disulfide bond(s)). 
     The bispecific antigen-binding molecules of the present invention may comprise or consist of human antibodies and/or recombinant human antibodies, or fragments thereof. The term “human antibody”, as used herein, includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may nonetheless include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. 
     The bispecific antigen-binding molecules of the present invention may comprise or consist of recombinant human antibodies or antigen-binding fragments thereof. The term “recombinant human antibody”, as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V H  and V L  regions of the recombinant antibodies are sequences that, while derived from and related to human germline V H  and V L  sequences, may not naturally exist within the human antibody germline repertoire in vivo. 
     Methods for making bispecific antibodies are known in the art and may be used to construct bispecific antigen-binding molecules of the present invention. Exemplary bispecific formats that can be used in the context of the present invention include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1/IgG2, dual acting Fab (DAF)-IgG, and Mab 2  bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein, for a review of the foregoing formats). 
     Exemplary antigen-binding domains (D1 and D2) that can be included in the LEPR×GP130 bispecific antigen-binding molecules of the present invention include antigen-binding domains derived from any of the anti-LEPR and/or anti-GP130 antibodies disclosed herein or otherwise known in the art. 
     For example, the present invention includes LEPR×GP130 bispecific antigen-binding molecules comprising a D1 (GP130-binding) antigen-binding domain comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. 
     The present invention also provides LEPR×GP130 bispecific antigen-binding molecules comprising a D1 (GP130-binding) antigen-binding domain comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. 
     The present invention also provides LEPR×GP130 bispecific antigen-binding molecules comprising a D1 (GP130-binding) antigen-binding domain comprising an HCVR and an LCVR amino acid sequence pair (HCVR/LCVR) comprising any of the HCVR amino acid sequences listed in Table 1 paired with any of the LCVR amino acid sequences listed in Table 1. According to certain embodiments, the present invention provides LEPR×GP130 bispecific antigen-binding molecules comprising a D1 (GP130-binding) domain comprising an HCVR/LCVR amino acid sequence pair contained within any of the exemplary anti-MET antibodies listed in Table 1. 
     The present invention also provides LEPR×GP130 bispecific antigen-binding molecules comprising a D1 (GP130-binding) antigen-binding domain comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. 
     The present invention also provides LEPR×GP130 bispecific antigen-binding molecules comprising a D1 (GP130-binding) antigen-binding domain comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. 
     The present invention also provides LEPR×GP130 bispecific antigen-binding molecules comprising a D1 (GP130-binding) antigen-binding domain comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. 
     The present invention also provides LEPR×GP130 bispecific antigen-binding molecules comprising a D1 (GP130-binding) antigen-binding domain comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. 
     The present invention also provides LEPR×GP130 bispecific antigen-binding molecules comprising a D1 (GP130-binding) antigen-binding domain comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. 
     The present invention also provides LEPR×GP130 bispecific antigen-binding molecules comprising a D1 (GP130-binding) antigen-binding domain comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. 
     The present invention also provides LEPR×GP130 bispecific antigen-binding molecules comprising a D1 (GP130-binding) antigen-binding domain comprising an HCDR3 and an LCDR3 amino acid sequence pair (HCDR3/LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 1 paired with any of the LCDR3 amino acid sequences listed in Table 1. According to certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3/LCDR3 amino acid sequence pair contained within any of the exemplary anti-MET antibodies listed in Table 1. 
     The present invention also provides LEPR×GP130 bispecific antigen-binding molecules comprising a D1 (GP130-binding) antigen-binding domain comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any of the exemplary anti-MET antibodies listed in Table 1. 
     In a related embodiment, the present invention provides LEPR×GP130 bispecific antigen-binding molecules comprising a D1 (GP130-binding) antigen-binding domain comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR amino acid sequence pair as defined by any of the exemplary anti-MET antibodies listed in Table 1. 
     The present invention includes LEPR×GP130 bispecific antigen-binding molecules comprising a D2 (LEPR-binding) antigen-binding domain comprising a variable domain (HCVR and/or LCVR), and/or complementarity determining region (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3), derived from any of the anti-LEPR antibodies described herein, described in US Patent Application Publication No. 2017/0101477, the disclosure of which is incorporated herein in its entirety, or otherwise known in the art. 
     As non-limiting illustrative examples, the present invention includes LEPR×GP130 bispecific antigen binding molecules comprising a D1 (GP130-binding) antigen-binding domain and a D2 (LEPR-binding) antigen-binding domain, wherein the D1 antigen binding domain comprises an HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 154/10, or a set of heavy and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NOs: 156-158-160-12-14-16, and wherein the D2 (LEPR-binding) antigen-binding domain comprises an HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 170/10 or 178/10, or a set of heavy and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NOs: 172-174-176-12-14-16, or 180-182-184-12-14-16. An exemplary LEPR×GP130 bispecific antibody having these sequence characteristics is the bispecific antibody designated bsAb21236, which comprises a D1 derived from mAb16683 and a D2 derived from mAb18445. Another exemplary LEPR×GP130 bispecific antibody having these sequence characteristics is the bispecific antibody designated bsAb21237, which comprises a D1 derived from mAb16683 and a D2 derived from mAb18446. Other specific examples of bispecific antibodies of the present invention are set forth in Example 9, Table 20 herein. 
     Multimerizing Components 
     The bispecific antigen-binding molecules of the present invention, in certain embodiments, may also comprise one or more multimerizing component(s). The multimerizing components can function to maintain the association between the antigen-binding domains (D1 and D2). As used herein, a “multimerizing component” is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerizing component of the same or similar structure or constitution. For example, a multimerizing component may be a polypeptide comprising an immunoglobulin C H 3 domain. A non-limiting example of a multimerizing component is an Fc portion of an immunoglobulin, e.g., an Fc domain of an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group. In certain embodiments, the multimerizing component is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine residues. In other embodiments, the multimerizing component is a cysteine residue, or a short cysteine-containing peptide. Other multimerizing domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif. 
     In certain embodiments, the bispecific antigen-binding molecules of the present invention comprise two multimerizing domains, M1 and M2, wherein D1 is attached to M1 and D2 is attached to M2, and wherein the association of M1 with M2 facilitates the physical linkage of D1 and D2 to one another in a single bispecific antigen-binding molecule. In certain embodiments, M1 and M2 are identical to one another. For example, M1 can be an Fc domain having a particular amino acid sequence, and M2 is an Fc domain with the same amino acid sequence as M1. Alternatively, M1 and M2 may differ from one another at one or more amino acid position. For example, M1 may comprise a first immunoglobulin (Ig) C H 3 domain and M2 may comprise a second Ig C H 3 domain, wherein the first and second Ig C H 3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the targeting construct to Protein A as compared to a reference construct having identical M1 and M2 sequences. In one embodiment, the Ig C H 3 domain of M1 binds Protein A and the Ig C H 3 domain of M2 contains a mutation that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The C H 3 of M2 may further comprise a Y96F modification (by IMGT; Y436F by EU). Further modifications that may be found within the C H 3 of M2 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU) in the case of an IgG1 Fc domain; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU) in the case of an IgG2 Fc domain; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the case of an IgG4 Fc domain. 
     Variants 
     The bispecific antigen-binding molecules disclosed herein, or the antigen-binding domains thereof (D1 and/or D2) may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antigen-binding proteins or antigen-binding domains were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The present invention includes bispecific antigen-binding molecules disclosed herein, or the antigen-binding domains thereof (D1 and/or D2), which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous bispecific antigen-binding molecules, or antigen-binding domains thereof (D1 and/or D2), which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and/or CDR residues within the V H  and/or V L  domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived). Furthermore, the bispecific antigen-binding molecules, or the antigen-binding domains thereof (D1 and/or D2), of the present invention may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, bispecific antigen-binding molecules, or the antigen-binding domains thereof (D1 and/or D2), that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. bispecific antigen-binding molecules, or the antigen-binding domains thereof (D1 and/or D2), obtained in this general manner are encompassed within the present invention. 
     The present invention also includes anti-LEPR antibodies, anti-GP130 antibodies, and bispecific antigen-binding molecules comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein. Exemplary variants included within this aspect of the invention include variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes anti-LEPR antibodies, anti-GP130 antibodies, and LEPR×GP130 bispecific antigen-binding molecules having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences set herein. 
     Exemplary variants included within this aspect of the invention also include variants having substantial sequence identity to any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein. As used herein in the context of amino acid sequences, the term “substantial identity” or “substantially identical” means that two amino acid sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95%, 98% or 99% sequence identity. In certain embodiments, residue positions which are not identical differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change 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 upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, herein incorporated by reference. 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) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445, herein incorporated by reference. A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix. 
     Sequence identity between two different amino acid sequences is typically measured using sequence analysis software. Sequence analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each herein incorporated by reference. 
     LEPR×GP130 Bispecific Antigen-Binding Molecules Comprising Fc Variants 
     According to certain embodiments of the present invention, LEPR×GP130 bispecific antigen binding proteins are provided comprising an Fc domain comprising one or more mutations which enhance or diminish antibody binding to the FcRn receptor, e.g., at acidic pH as compared to neutral pH. For example, the present invention includes LEPR×GP130 bispecific antigen binding proteins comprising a mutation in the C H 2 or a C H 3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Such mutations may result in an increase in serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, e.g., a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modification comprises a 428 L (e.g., M428L) and 434S (e.g., N434S) modification; a 428 L, 2591 (e.g., V2591), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a 307 and/or 308 modification (e.g., 308F or 308P). 
     For example, the present invention includes LEPR×GP130 bispecific antigen binding proteins comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the foregoing Fc domain mutations, and other mutations within the antibody variable domains disclosed herein, are contemplated within the scope of the present invention. 
     Biological Characteristics of the LEPR×GP130 Antigen-Binding Molecules of the Invention 
     The present invention includes LEPR×GP130 bispecific antigen-binding molecules that bind human LEPR with high affinity. For example, the present invention includes LEPR×GP130 antigen-binding molecules that bind monomeric human LEPR (e.g., hLEPR.mmh) with a K D  of less than about 110 nM as measured by surface plasmon resonance at 25° C., e.g., using an assay format as defined in Example 10 herein, or a substantially similar assay. According to certain embodiments, LEPR×GP130 bispecific antigen-binding molecules are provided that bind monomeric human LEPR (e.g., hLEPR.mmh) with a dissociative half-life (t 1/2 ) of greater than about 3 minutes as measured by surface plasmon resonance at 25° C., e.g., using an assay format as defined int Example 10 herein, or a substantially similar assay. 
     The present invention includes LEPR×GP130 bispecific antigen-binding molecules that bind human GP130 with high affinity. For example, the present invention includes LEPR×GP130 antigen-binding molecules that bind monomeric human GP130 (e.g., hGP130.mmh) with a K D  of less than about 150 nM as measured by surface plasmon resonance at 25° C., e.g., using an assay format as defined in Example 10 herein, or a substantially similar assay. According to certain embodiments, LEPR×GP130 bispecific antigen-binding molecules are provided that bind monomeric human GP130 (e.g., hGP130.mmh) with a dissociative half-life (t 1/2 ) of greater than about 2.5 minutes as measured by surface plasmon resonance at 25° C., e.g., using an assay format as defined in Example 10 herein, or a substantially similar assay. 
     The present invention includes LEPR×GP130 bispecific antigen-binding molecules that are capable of binding cells that express human LEPR. In some aspects, the LEPR×GP130 bispecific antigen-binding molecules are capable of binding cells that express human LEPR, isoform b. In certain embodiments, LEPR×GP130 bispecific antigen-binding proteins are provided that bind cells expressing human LEPR in the presence and/or absence of leptin. The present invention includes LEPR×GP130 bispecific antigen-binding molecules that are capable of binding cells that express human GP130. Cell binding by a bispecific antigen-binding molecule of the present invention may be assessed by fluorescence activated cell sorting (FACS) on cells expressing LEPR and or GP130, e.g., using an assay format as defined in Example 11 herein, or a substantially similar assay. 
     The present invention includes LEPR×GP130 bispecific antigen-binding molecules that activate GP130-mediated cell signaling. In certain embodiments, the present invention includes LEPR×GP130 bispecific antigen-binding molecules that activate GP130-mediated cell signaling with a potency that is at least 20% the degree of activation observed by treatment with a GP130 ligand. For example, the present invention includes LEPR×GP130 bispecific antigen-binding molecules that activate GP130-mediated cell signaling with a potency that is at least 20% or 25% the degree of activation observed by treatment with human oncostatin M (OSM) under the same or similar experimental assay condition. Activation of GP130-mediated cell signaling by a bispecific antigen-binding molecule of the present invention may be assessed by an in vitro cell signaling assay, e.g., using an assay format as defined in Example 12 herein, or a substantially similar assay. 
     The present invention includes LEPR×GP130 bispecific antigen-binding molecules that specifically, or preferentially, activate signaling through LEPR isoform ‘b’ (long form) and do not substantially activate signaling through LEPR isoform ‘a’ (short form). According to certain embodiments, LEPR×GP130 bispecific antigen-binding molecules are provided that specifically, or preferentially, potentiate leptin signaling through LEPR isoform ‘b’ (long form) and do not substantially potentiate leptin signaling through LEPR isoform ‘a’ (short form). Activation or potentiating of signaling through LEPR isoform ‘b’ and/or LEPR isoform ‘a’ may be assessed by an in vitro assay using a reporter cell line that specifically expresses LEPR isoform ‘b’ or LEPR isoform ‘a’, e.g., using an assay format as defined in Example 14 herein, or a substantially similar assay. 
     The present invention includes LEPR×GP130 bispecific antigen-binding molecules that cause a reduction in body weight when administered to an animal. For example, the present invention includes LEPR×GP130 bispecific antigen-binding molecules that cause a 1% to 4% reduction in body weight in animals 2 to 14 days following administration of the bispecific antigen-binding molecule in a therapeutically effective dose to the animal. Weight loss induction by the bispecific antigen-binding molecules of the invention may be assessed using a genetically engineered model system, e.g., using an in vivo model as set forth in Example 13 herein, or a substantially similar model. 
     The bispecific antigen-binding molecules of the present invention may possess one or more of the aforementioned biological characteristics, or any combination thereof. The foregoing list of biological characteristics of the bispecific antigen-binding molecules of the invention is not intended to be exhaustive. Other biological characteristics of the bispecific antigen-binding molecules of the present invention will be evident to a person of ordinary skill in the art from a review of the present disclosure including the working Examples herein. 
     Epitope Mapping, Binding Domains, and Related Technologies 
     The epitope to which the antibodies and antigen-binding domains of the present invention bind may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of a LEPR or GP130 protein. Alternatively, the relevant epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) of the target protein. 
     Various techniques known to persons of ordinary skill in the art can be used to determine the epitope on LEPR and/or GP130 with which the antibodies and antigen-binding domains of the present invention interact. Exemplary techniques that can be used to determine an epitope or binding domain of a particular antibody or antigen-binding domain include, e.g., point mutagenesis (e.g., alanine scanning mutagenesis, arginine scanning mutagenesis, etc.), peptide blots analysis (Reineke, 2004, Methods Mol Biol 248:443-463), protease protection, and peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen/deuterium exchange detected by mass spectrometry. In general terms, the hydrogen/deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antibody to the deuterium-labeled protein. Next, the protein/antibody complex is transferred to water to allow hydrogen-deuterium exchange to occur at all residues except for the residues protected by the antibody (which remain deuterium-labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999)  Analytical Biochemistry  267(2):252-259; Engen and Smith (2001)  Anal. Chem.  73:256A-265A. X-ray crystal structure analysis can also be used to identify the amino acids within a polypeptide with which an antibody interacts. 
     The present invention includes LEPR×GP130 bispecific antigen-binding molecules comprising a D1 (GP130-binding) domain that binds to the same epitope as any of the specific exemplary anti-GP130 antibodies or antigen-binding domains described herein (e.g. antibodies comprising any of the amino acid sequences as set forth in Table 1 herein). The present invention includes LEPR×GP130 bispecific antigen-binding molecules comprising a D2 (LEPR-binding) domain that binds to the same epitope as any of the specific exemplary anti-LEPR antibodies or antigen-binding domains described herein (e.g. antibodies comprising any of the amino acid sequences as set forth in Table 18 herein). Likewise, the present invention also includes LEPR×GP130 bispecific antigen-binding molecules comprising a D1 (GP130-binding) domain that competes for binding to GP130 with any of the specific exemplary anti-GP130 antibodies described herein (e.g. antibodies comprising any of the amino acid sequences as set forth in Table 1 herein). Moreover, the present invention also includes LEPR×GP130 bispecific antigen-binding molecules comprising a D2 (LEPR-binding) domain that competes for binding to LEPR with any of the specific exemplary anti-LEPR antibodies described herein (e.g. antibodies comprising any of the amino acid sequences as set forth in Table 18 herein). 
     One can easily determine whether an antibody or antigen-binding domain binds to the same epitope as, or competes for binding with, a reference anti-GP130 or anti-LEPR antibody by using routine methods known in the art and exemplified herein. For example, to determine if a test antibody binds to the same epitope as a reference anti-GP130 or anti-LEPR antibody of the invention, the reference antibody is allowed to bind to a target molecule (i.e., GP130 or LEPR protein, as the case may be). Next, the ability of a test antibody to bind to the target molecule is assessed. If the test antibody is able to bind to the target molecule following saturation binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody. On the other hand, if the test antibody is not able to bind to the target molecule following saturation binding with the reference antibody, then the test antibody may bind to the same epitope as the epitope bound by the reference antibody of the invention. Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, Biacore, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art. In accordance with certain embodiments of the present invention, two antibodies bind to the same (or overlapping) epitope if, e.g., a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antibodies are deemed to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies are deemed to have “overlapping epitopes” if only a subset of the amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. 
     To determine if an antibody competes for binding (or cross-competes for binding) with a reference anti-GP130 or anti-LEPR antibody, the above-described binding methodology is performed in two orientations: In a first orientation, the reference antibody is allowed to bind to the target molecule under saturating conditions followed by assessment of binding of the test antibody to the target molecule. In a second orientation, the test antibody is allowed to bind to a target molecule under saturating conditions followed by assessment of binding of the reference antibody to the target molecule. If, in both orientations, only the first (saturating) antibody is capable of binding to the target molecule, then it is concluded that the test antibody and the reference antibody compete for binding to the target molecule. As will be appreciated by a person of ordinary skill in the art, an antibody that competes for binding with a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope. 
     The antigen-binding domains (D1 and/or D2) of the bispecific antigen-binding molecules of the present invention may be described in terms of the domains of GP130 or LEPR with which the antigen-binding domain interacts. GP130 and LEPR proteins comprise various domains referred to as D1, D2, D3 and FNIII. Accordingly, the D1 and D2 antigen-binding domains of the bispecific antigen-binding molecules of the present invention, may bind a domain of LEPR or GP130 selected from the group consisting of D1, D2, D3, or FNIII. According to certain exemplary embodiments, LEPR×GP130 bispecific antigen binding molecules are provided wherein the D1 (anti-GP130) antigen-binding domain binds to the FNIII domain of GP130, and the D2 (anti-LEPR) antigen-binding domain binds to the FNIII domain of LEPR. Other binding domain combinations are contemplated within the scope of the present invention. 
     Preparation of Human Antibodies 
     The anti-GP130, anti-LEPR antibodies, and LEPR×GP130 bispecific antibodies of the present invention can be fully human antibodies. Methods for generating 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 make human antibodies that specifically bind to human GP130 and/or human LEPR. 
     Using VELOCIMMUNE™ technology, for example, or any other similar known method for generating fully human monoclonal antibodies, high affinity chimeric antibodies to human GP130 and/or human LEPR are initially isolated having a human variable region and a mouse constant region. As in the experimental section below, the antibodies are characterized and selected for desirable characteristics, including affinity, ligand blocking activity, selectivity, epitope, etc. If necessary, mouse constant regions are replaced with a desired human constant region, for example wild-type or modified IgG1 or IgG4, to generate fully human anti-GP130 and/or anti-LEPR antibodies. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region. In certain instances, fully human anti-GP130 and/or anti-LEPR antibodies are isolated directly from antigen-positive B cells. 
     Bioequivalents 
     The present invention includes variant anti-GP130, anti-LEPR antibodies, and LEPR×GP130 bispecific antibodies having amino acid sequences that vary from those of the described antibodies but that retain the ability to bind the relevant target antigen(s) (GP130 and/or LEPR) and exert one or more of the biological function(s) of the parent antibodies from which such variants are derived. Such variant antibodies and antibody fragments comprise one or more additions, deletions, or substitutions of amino acids when compared to parent sequence, but exhibit biological activity that is essentially equivalent to that of the described antibodies. Likewise, the present invention includes DNA sequences encoding anti-GP130, anti-LEPR antibodies, and LEPR×GP130 bispecific antibodies of the present invention, wherein such DNA sequences comprise one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed parental sequence, but that encode anti-GP130, anti-LEPR antibodies, and LEPR×GP130 bispecific antibodies that are essentially bioequivalent to the exemplary antibodies disclosed herein. Examples of such variant amino acid and DNA sequences are discussed elsewhere herein. 
     Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single does or multiple dose. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied. 
     In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency. 
     In one embodiment, two antigen-binding proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching. 
     In one embodiment, two antigen-binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known. 
     Bioequivalence may be demonstrated by in vivo and in vitro methods. Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test 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) in a well-controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antibody. 
     Species Selectivity and Species Cross-Reactivity 
     The present invention, according to certain embodiments, provides anti-GP130, anti-LEPR antibodies, and LEPR×GP130 bispecific antibodies (and other antigen-binding molecules comprising anti-GP130 and/or anti-LEPR antigen-binding domains) that bind to human GP130 and human LEPR but not to the corresponding proteins from other species. The present invention also includes anti-GP130, anti-LEPR antibodies, and LEPR×GP130 bispecific antibodies (and antigen-binding molecules comprising anti-GP130 and/or anti-LEPR antigen-binding domains) that bind to human GP130 and human LEPR and to GP130 and LEPR from one or more non-human species. For example, the present invention includes bispecific antigen-binding molecules comprising a first and second antigen-binding domain, wherein the first antigen binding domain binds human and monkey (e.g.,  Macaca fascicularis ) GP130 but does not bind rodent (rat and/or mouse) GP130. The present invention includes bispecific antigen-binding molecules comprising a first and second antigen-binding domain, wherein the second antigen binding domain binds human and monkey (e.g.,  Macaca fascicularis ) LEPR but does not bind rodent (rat and/or mouse) LEPR. 
     The present invention further provides anti-GP130, anti-LEPR antibodies, and LEPR×GP130 bispecific antibodies (and other antigen-binding molecules comprising anti-GP130 and/or anti-LEPR antigen-binding domains) that bind to human GP130 and/or human LEPR, and may bind or not bind, as the case may be, to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomologous, marmoset, rhesus or chimpanzee versions of the corresponding GP130 and/or LEPR proteins. 
     Therapeutic Formulation and Administration 
     The invention provides pharmaceutical compositions comprising anti-GP130, anti-LEPR antibodies, and LEPR×GP130 bispecific antibodies (and other antigen-binding molecules comprising anti-GP130 and/or anti-LEPR antigen-binding domains) of the present invention. The pharmaceutical compositions of the invention may be formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like. 
     Therapeutic Uses of the Antibodies 
     The present invention includes methods comprising administering to a subject in need thereof (e.g., a mammal such as a human) a therapeutic composition comprising a LEPR×GP130 bispecific antigen-binding molecule (e.g., a LEPR×GP130 bispecific antigen-binding molecule comprising any of the D1 and D2 components as set forth in Table 20 herein). The therapeutic composition can comprise any of the LEPR×GP130 bispecific antigen-binding molecules disclosed herein, and a pharmaceutically acceptable carrier or diluent. 
     The LEPR×GP130 bispecific antigen-binding molecules of the invention are useful, inter alia, for the treatment, prevention and/or amelioration of any disease or disorder associated with or mediated by leptin deficiency, leptin resistance, hypoleptinemia, or otherwise treatable by stimulating or activating LEPR signaling or mimicking the natural activity of leptin in vitro or in vivo. For example, the bispecific antigen-binding molecules of the present invention are useful for treating lipodystrophy conditions. Exemplary lipodystrophy conditions that are treatable by the bispecific antigen-binding molecules of the present invention include, e.g., congenital generalized lipodystrophy, acquired generalized lipodystrophy, familial partial lipodystrophy, acquired partial lipodystrophy, centrifugal abdominal lipodystrophy, lipoatrophia annularis, localized lipodystrophy, and HIV-associated lipodystrophy. 
     The LEPR×GP130 bispecific antigen-binding molecules of the present invention are also useful for the treatment or prevention of one or more diseases or disorders selected from the group consisting of obesity, metabolic syndrome, diet-induced food craving, functional hypothalamic amenorrhea, type 1 diabetes, type 2 diabetes, insulin resistance, severe insulin resistance including severe insulin resistance due to mutation in insulin receptor, severe insulin resistance not caused by mutation in the insulin receptor, severe insulin resistance caused by a mutation in downstream signaling pathways or induced by other causes, non-alcoholic and alcoholic fatty liver diseases, Alzheimer&#39;s disease, leptin deficiency, leptin resistance, lipodystrophies, Leprechaunism/Donohue syndrome, Rabson-Mendenhall syndrome. 
     In the context of the methods of treatment described herein, the LEPR×GP130 bispecific antigen-binding molecule may be administered as a monotherapy (i.e., as the only therapeutic agent) or in combination with one or more additional therapeutic agents (examples of which are described elsewhere herein). 
     Combination Therapies and Formulations 
     The present invention includes compositions and therapeutic formulations comprising any of the LEPR×GP130 bispecific antigen-binding molecules described herein in combination with one or more additional therapeutically active components, and methods of treatment comprising administering such combinations to subjects in need thereof. 
     The LEPR×GP130 bispecific antigen-binding molecules of the present invention may be co-formulated with and/or administered in combination with one or more additional therapeutically active component(s), such as. e.g., 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, e.g., recombinant human leptin (e.g., metreleptin [MYALEPT]), PCSK9 inhibitors (e.g., anti-PCSK9 antibodies [alirocumab, evolocumab, bococizumab, lodelcizumab, ralpancizumab, etc.]), statins (atorvastatin, rosuvastatin, cerivastatin, pitavastatin, fluvastatin, simvastatin, lovastatin, pravastatin, etc.), ezetimibe, insulin, insulin variants, insulin secretagogues, metformin, sulfonylureas, sodium glucose cotransporter 2 (SGLT2) Inhibitors (e.g., dapaglifozin, canaglifozin, empagliflozin, etc.), GLP-1 agonists/analogues (e.g., extendin-4, exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, etc.), glucagon (GCG) inhibitors (e.g., anti-GCG antibodies), glucagon receptor (GCGR) inhibitors (e.g., anti-GCGR antibodies, small molecule GCGR antagonists, GCGR-specific antisense oligonucleotides, anti-GCGR aptamers [e.g., Spiegelmers], etc.), angiopoietin-like protein (ANGPTL) inhibitors (e.g., anti-ANGPTL3 antibodies, anti-ANGPTL4 antibodies, anti-ANGPTL8 antibodies, etc.), Phentermine, Orlistat, Topiramate, Bupropion, Topiramate/Phentermine, Bupropion/Naltrexone, Bupropion/Zonisamide, Pramlintide/Metrelepin, Lorcaserin, Cetilistat, Tesofensine, Velneperit, etc. 
     The additional therapeutically active component(s), e.g., any of the agents listed above or derivatives thereof, may be administered just prior to, concurrent with, or shortly after the administration of a LEPR×GP130 bispecific antigen-binding molecule of the present invention; (for purposes of the present disclosure, such administration regimens are considered the administration of a LEPR×GP130 bispecific antigen-binding molecule “in combination with” an additional therapeutically active component). The present invention includes pharmaceutical compositions in which a LEPR×GP130 bispecific antigen-binding molecule of the present invention is co-formulated with one or more of the additional therapeutically active component(s) as described elsewhere herein. 
     Administration Regimens 
     According to certain embodiments of the present invention, multiple doses of a LEPR×GP130 bispecific antigen-binding molecule (or a pharmaceutical composition comprising a combination of a LEPR×GP130 bispecific antigen-binding molecule and any of the additional therapeutically active agents mentioned herein) may be administered to a subject over a defined time course. The methods according to this aspect of the invention comprise sequentially administering to a subject multiple doses of a LEPR×GP130 bispecific antigen-binding molecule of the invention. As used herein, “sequentially administering” means that each dose of LEPR×GP130 bispecific antigen-binding molecule is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months). The present invention includes methods which comprise sequentially administering to the patient a single initial dose of a LEPR×GP130 bispecific antigen-binding molecule, followed by one or more secondary doses of the LEPR×GP130 bispecific antigen-binding molecule, and optionally followed by one or more tertiary doses of the LEPR×GP130 bispecific antigen-binding molecule. 
     The terms “initial dose,” “secondary doses,” and “tertiary doses,” refer to the temporal sequence of administration of the LEPR×GP130 bispecific antigen-binding molecule of the invention. Thus, the “initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose,” “loading dose,” “starting dose,” and the like); the “secondary doses” are the doses which are administered after the initial dose; and the “tertiary doses” are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of LEPR×GP130 bispecific antigen-binding molecule, but generally may differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of LEPR×GP130 bispecific antigen-binding molecule contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as “loading doses” followed by subsequent doses that are administered on a less frequent basis (e.g., “maintenance doses”). 
     Devices 
     The present invention also provides a vessel (e.g., a vial or chromatography column) or injection device (e.g., syringe, pre-filled syringe or autoinjector) comprising a bispecific antigen binding molecule (e.g., pharmaceutical formulation thereof) set forth herein. The vessel or injection device may be packaged into a kit. 
     An injection device is a device that introduces a substance into the body of a subject (e.g., a human) via a parenteral route, e.g., intraocular, intravitreal, intramuscular, subcutaneous or intravenous. For example, an injection device may be a syringe (e.g., pre-filled with the pharmaceutical formulation, such as an auto-injector) which, for example, includes a cylinder or barrel for holding fluid to be injected (e.g., comprising the antibody or fragment or a pharmaceutical formulation thereof), a needle for piecing skin, blood vessels or other tissue for injection of the fluid; and a plunger for pushing the fluid out of the cylinder and through the needle bore and into the body of the subject. 
     The present invention includes methods for administering a bispecific antigen binding molecule of the present invention comprising introducing e.g., injecting, the molecule into the body of the subject, e.g., with an injection device. 
     Expression Methods 
     The present invention includes recombinant methods for making a bispecific antigen binding molecule of the present invention, or an immunoglobulin chain thereof, comprising (i) introducing, into a host cell, one or more polynucleotides encoding light and/or heavy immunoglobulin chains of such a bispecific antigen binding molecule, for example, wherein the polynucleotide is in a vector; and/or integrates into the host cell chromosome and/or is operably linked to a promoter; (ii) culturing the host cell (e.g., mammalian, fungal, Chinese hamster ovary (CHO),  Pichia  or  Pichia pastoris ) under conditions favorable to expression of the polynucleotide and, (iii) optionally, isolating the bispecific antigen binding molecule or immunoglobulin chain from the host cell and/or medium in which the host cell is grown. The product of such a method also forms part of the present invention along with a pharmaceutical composition thereof. 
     In an embodiment of the invention, a method for making a bispecific antigen binding molecule includes a method of purifying the molecule, e.g., by column chromatography, precipitation and/or filtration. The product of such a method also forms part of the present invention along with a pharmaceutical composition thereof. 
     Host cells comprising a bispecific antigen binding molecule of the present invention and/or a polynucleotide encoding immunoglobulin chains of such a molecule (e.g., in a vector) are also part of the present invention. Host cells include, for example, mammalian cells such as Chinese hamster ovary (CHO) cells and fungal cells such as  Pichia  cells (e.g.,  P. pastoris ). 
     EXAMPLES 
     The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. 
     Example 1. Generation of Anti-GP130 Antibodies 
     Anti-GP130 antibodies were obtained by immunizing a genetically engineered mouse comprising DNA encoding human immunoglobulin heavy and kappa light chain variable regions with an immunogen comprising recombinant human GP130 extracellular domain. The mice used for the immunizations express a “universal light chain.” That is, the antibodies produced in this mouse have different heavy chain variable regions but essentially identical light chain variable domains. 
     The antibody immune response was monitored by a GP130-specific immunoassay. When a desired immune response was achieved splenocytes were harvested and fused with mouse myeloma cells to preserve their viability and from hybridoma cell lines. The hybridoma cell lines were screened and selected to identify cell lines that produce GP130-specific antibodies. Using this technique several anti-GP130 chimeric antibodies (i.e., antibodies possessing human variable domains and mouse constant domains) were obtained. In addition, several fully human anti-GP130 antibodies were isolated directly from antigen-positive B cells without fusion to myeloma cells, as described in US 2007/0280945A1. 
     Certain biological properties of the exemplary anti-GP130 antibodies generated in accordance with the methods of this Example, and bispecific antibodies constructed therefrom, are described in detail in the Examples set forth below. 
     Example 2. Heavy and Light Chain Variable Region Amino Acid and Nucleic Acid Sequences of Anti-GP130 
     Table 1 sets forth the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of selected anti-LEPR antibodies of the invention. (As noted above, all antibodies generated in Example 1 possess the same light chain variable region and the same light chain CDR sequences as well). The corresponding nucleic acid sequence identifiers are set forth in Table 2. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Anti-GP130 Amino Acid Sequence Identifiers 
               
            
           
           
               
               
            
               
                 Antibody 
                 SEQ ID NOs: 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Designation 
                 HCVR 
                 HCDR1 
                 HCDR2 
                 HCDR3 
                 LCVR 
                 LCDR1 
                 LCDR2 
                 LCDR3 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 MAb16614 
                 18 
                 20 
                 22 
                 24 
                 10 
                 12 
                 14 
                 16 
               
               
                 MAb16618 
                 26 
                 28 
                 30 
                 32 
                 10 
                 12 
                 14 
                 16 
               
               
                 MAb16622 
                 34 
                 36 
                 38 
                 40 
                 10 
                 12 
                 14 
                 16 
               
               
                 MAb16623 
                 42 
                 44 
                 46 
                 48 
                 10 
                 12 
                 14 
                 16 
               
               
                 MAb16636 
                 50 
                 52 
                 54 
                 56 
                 10 
                 12 
                 14 
                 16 
               
               
                 MAb16637 
                 58 
                 60 
                 62 
                 64 
                 10 
                 12 
                 14 
                 16 
               
               
                 MAb16656 
                 66 
                 68 
                 70 
                 72 
                 10 
                 12 
                 14 
                 16 
               
               
                 MAb16659 
                 74 
                 76 
                 78 
                 80 
                 10 
                 12 
                 14 
                 16 
               
               
                 MAb16662 
                 82 
                 84 
                 86 
                 88 
                 10 
                 12 
                 14 
                 16 
               
               
                 MAb16664 
                 90 
                 92 
                 94 
                 96 
                 10 
                 12 
                 14 
                 16 
               
               
                 MAb16665 
                 98 
                 100 
                 102 
                 104 
                 10 
                 12 
                 14 
                 16 
               
               
                 MAb16666 
                 106 
                 108 
                 110 
                 112 
                 10 
                 12 
                 14 
                 16 
               
               
                 MAb16669 
                 114 
                 116 
                 118 
                 120 
                 10 
                 12 
                 14 
                 16 
               
               
                 MAb16673 
                 122 
                 124 
                 126 
                 128 
                 10 
                 12 
                 14 
                 16 
               
               
                 MAb16676 
                 130 
                 132 
                 134 
                 136 
                 10 
                 12 
                 14 
                 16 
               
               
                 MAb16680 
                 138 
                 140 
                 142 
                 144 
                 10 
                 12 
                 14 
                 16 
               
               
                 MAb16682 
                 146 
                 148 
                 150 
                 152 
                 10 
                 12 
                 14 
                 16 
               
               
                 MAb16683 
                 154 
                 156 
                 158 
                 160 
                 10 
                 12 
                 14 
                 16 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Anti-GP130 Nucleic Acid Sequence Identifiers 
               
            
           
           
               
               
            
               
                 Antibody 
                 SEQ ID NOs: 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Designation 
                 HCVR 
                 HCDR1 
                 HCDR2 
                 HCDR3 
                 LCVR 
                 LCDR1 
                 LCDR2 
                 LCDR3 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 MAb16614 
                 17 
                 19 
                 21 
                 23 
                 9 
                 11 
                 13 
                 15 
               
               
                 MAb16618 
                 25 
                 27 
                 29 
                 31 
                 9 
                 11 
                 13 
                 15 
               
               
                 MAb16622 
                 33 
                 35 
                 37 
                 39 
                 9 
                 11 
                 13 
                 15 
               
               
                 MAb16623 
                 41 
                 43 
                 45 
                 47 
                 9 
                 11 
                 13 
                 15 
               
               
                 MAb16636 
                 49 
                 51 
                 53 
                 55 
                 9 
                 11 
                 13 
                 15 
               
               
                 MAb16637 
                 57 
                 59 
                 61 
                 63 
                 9 
                 11 
                 13 
                 15 
               
               
                 MAb16656 
                 65 
                 67 
                 69 
                 71 
                 9 
                 11 
                 13 
                 15 
               
               
                 MAb16659 
                 73 
                 75 
                 77 
                 79 
                 9 
                 11 
                 13 
                 15 
               
               
                 MAb16662 
                 81 
                 83 
                 85 
                 87 
                 9 
                 11 
                 13 
                 15 
               
               
                 MAb16664 
                 89 
                 91 
                 93 
                 95 
                 9 
                 11 
                 13 
                 15 
               
               
                 MAb16665 
                 97 
                 99 
                 101 
                 103 
                 9 
                 11 
                 13 
                 15 
               
               
                 MAb16666 
                 105 
                 107 
                 109 
                 111 
                 9 
                 11 
                 13 
                 15 
               
               
                 MAb16669 
                 113 
                 115 
                 117 
                 119 
                 9 
                 11 
                 13 
                 15 
               
               
                 MAb16673 
                 121 
                 123 
                 125 
                 127 
                 9 
                 11 
                 13 
                 15 
               
               
                 MAb16676 
                 129 
                 131 
                 133 
                 135 
                 9 
                 11 
                 13 
                 15 
               
               
                 MAb16680 
                 137 
                 139 
                 141 
                 143 
                 9 
                 11 
                 13 
                 15 
               
               
                 MAb16682 
                 145 
                 147 
                 149 
                 151 
                 9 
                 11 
                 13 
                 15 
               
               
                 MAb16683 
                 153 
                 155 
                 157 
                 159 
                 9 
                 11 
                 13 
                 15 
               
               
                   
               
            
           
         
       
     
     The antibodies of the present invention can be of any isotype. For example, anti-GP130 antibodies of the invention may comprise variable domain and CDR sequences as set forth in Tables 1 and 2 and a human Fc domain of isotype IgG4, IgG1, etc. For certain applications or experiments the Fc domain may be a mouse Fc domain. As will be appreciated by a person of ordinary skill in the art, an antibody having a particular Fc isotype can be converted to an antibody with a different Fc isotype (e.g., an antibody with a mouse IgG4 Fc can be converted to an antibody with a human IgG1, etc.), but in any event, the variable domains (including the CDRs)—which are indicated by the numerical identifiers shown in Tables 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. 
     Example 3. Biacore Binding Kinetics of Anti-GP130 Monoclonal Antibodies Binding to Different GP130 Reagents Measured at 25° C. and 37° C. 
     Equilibrium dissociation constant (K D ) for different GP130 reagents binding to purified anti-GP130 monoclonal antibodies were determined using a real-time surface plasmon resonance based Biacore 4000 biosensor. All binding studies were performed in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v/v Surfactant Tween-20, pH 7.4 (HBS-ET) running buffer at 25° C. and 37° C. The Biacore sensor surface was first derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (GE, # BR-1008-39) to capture anti-GP130 monoclonal antibodies. Binding studies were performed on the following monomeric and dimeric GP130 reagents: human GP130 extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (hGP130-mmH; SEQ ID NO:191),  Macaca fascicularis  GP130 extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (mfGP130-mmH; SEQ ID NO:194), human GP130 extracellular domain expressed with a C-terminal mouse IgG2a Fc tag (hGP130-hFc; SEQ ID NO:197), mouse GP130 extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (mGP130-mmH; SEQ ID NO:196) and rat GP130 extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (rGP130-mmH; SEQ ID NO:195). Reagents tagged with “mmH” are monomeric, whereas reagents tagged with “mFc” are dimeric. Thus, for example, “hGP130-mmH” is also referred to as “monomeric human GP130,” and “hGP130-mFc” is also referred to as “dimeric human GP130.” 
     Different concentrations of hGP130-mmH, mfGP130-mmH, hGP130-mFc (100 nM-3.7 nM; 3-fold serial dilution) or 100 nM of mGP130-mmH and rGP130-mmH were first prepared in HBS-ET running buffer and were injected over anti-human Fc captured anti-GP130 monoclonal antibody surface for 4 minutes at a flow rate of 304/minute, while the dissociation of monoclonal antibody bound GP130 reagent was monitored for 10 minutes in HBS-ET running buffer. The association rate (k a ) and dissociation rate (k d ) were determined by fitting the real-time binding sensorgrams to a 1:1 binding model with mass transport limitation using Scrubber 2.0c curve-fitting software. Binding dissociation equilibrium constant (K D ) and dissociative half-life (t 1/2 ) were calculated from the kinetic rates as: 
     
       
         
           
             
               
                 
                   K 
                   D 
                 
                  
                 
                   ( 
                   M 
                   ) 
                 
               
               = 
               
                 kd 
                 ka 
               
             
             , 
             
               
                 and 
                  
                 
                     
                 
                  
                 t 
                  
                 
                     
                 
                  
                 
                   1 
                   2 
                 
                  
                 
                   ( 
                   min 
                   ) 
                 
               
               = 
               
                 
                   ln 
                    
                   
                     ( 
                     2 
                     ) 
                   
                 
                 
                   60 
                   * 
                   kd 
                 
               
             
           
         
       
     
     Binding kinetics parameters for hGP130-MMH, mfGP130-MMH, hGP130.mFc, mGP130-MMH or rGP130-MMH binding to different anti-GP130 monoclonal antibodies of the invention at 25° C. and 37° C. are shown in Tables 3 through 12. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Binding kinetics parameters of hGP130-MMH binding 
               
               
                 to GP130 monoclonal antibodies at 25° C. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 100 nM 
                   
                   
                   
                   
               
               
                   
                 mAb 
                 Ag 
               
               
                   
                 Capture 
                 Bound 
                 ka 
                 kd 
                 KD 
                 t½ 
               
               
                 mAb Captured 
                 Level (RU) 
                 (RU) 
                 (1/Ms) 
                 (1/s) 
                 (M) 
                 (min) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 MAb16614 
                 143 ± 0.67 
                 18 
                 5.52E+04 
                 1.01E−02 
                 1.83E−07 
                 1.1 
               
               
                 MAb16618 
                 182 ± 0.52 
                 52 
                 3.54E+05 
                 4.25E−02 
                 1.20E−07 
                 0.3 
               
               
                 MAb16622 
                 197 ± 0.95 
                 36 
                 1.20E+05 
                 2.43E−02 
                 2.03E−07 
                 0.5 
               
               
                 MAb16623 
                 243 ± 1.48 
                 77 
                 1.80E+05 
                 3.77E−03 
                 2.10E−08 
                 3 
               
               
                 MAb16636 
                 195 ± 0.45 
                 37 
                 2.70E+04 
                 9.93E−04 
                 3.68E−08 
                 12 
               
               
                 MAb16637 
                 306 ± 1.71 
                 91 
                 7.11E+04 
                 1.10E−03 
                 1.55E−08 
                 10 
               
               
                 MAb16641 
                 154 ± 0.93 
                 111 
                 3.56E+05 
                 8.87E−04 
                 2.49E−09 
                 13 
               
               
                 MAb16646 
                 167 ± 0.45 
                 58 
                 5.88E+04 
                 7.73E−04 
                 1.31E−08 
                 15 
               
               
                 MAb16656 
                 189 ± 0.44 
                 44 
                 3.59E+04 
                 8.25E−04 
                 2.30E−08 
                 14 
               
               
                 MAb16659 
                 212 ± 0.39 
                 77 
                 6.93E+05 
                 4.59E−04 
                 6.63E−10 
                 25 
               
               
                 MAb16662 
                 215 ± 0.67 
                 22 
                 2.35E+05 
                 2.90E−02 
                 1.23E−07 
                 0.4 
               
               
                 MAb16664 
                 217 ± 0.69 
                 14 
                 6.98E+05 
                 1.64E−03 
                 2.35E−09 
                 7 
               
               
                 MAb16665 
                 294 ± 1.48 
                 20 
                 1.15E+05 
                 4.73E−02 
                 4.11E−07 
                 0.2 
               
               
                 MAb16666 
                 317 ± 2.83 
                 138 
                 3.17E+05 
                 4.56E−03 
                 1.44E−08 
                 3 
               
               
                 MAb16669 
                 152 ± 0.5 
                 17 
                 1.14E+04 
                 4.03E−04 
                 3.54E−08 
                 29 
               
               
                 MAb16673 
                 158 ± 0.32 
                 58 
                 2.15E+05 
                 1.41E−02 
                 6.55E−08 
                 0.8 
               
               
                 MAb16676 
                 176 ± 0.33 
                 51 
                 2.87E+05 
                 6.31E−04 
                 2.19E−09 
                 18 
               
               
                 MAb16680 
                 270 ± 0.91 
                 57 
                 1.47E+05 
                 4.75E−04 
                 3.24E−09 
                 24 
               
               
                 MAb16682 
                 186 ± 0.55 
                 21 
                 1.21E+05 
                 1.64E−02 
                 1.36E−07 
                 0.7 
               
               
                 MAb16683 
                 238 ± 2.72 
                 54 
                 6.97E+04 
                 3.25E−03 
                 4.67E−08 
                 4 
               
               
                 MAb16684 
                 204 ± 1.09 
                 40 
                 5.32E+04 
                 6.22E−03 
                 1.17E−07 
                 1.9 
               
               
                 MAb16687 
                 172 ± 0.55 
                 111 
                 2.04E+05 
                 4.86E−04 
                 2.38E−09 
                 24 
               
               
                 MAb16692 
                 182 ± 1.08 
                 121 
                 2.40E+05 
                 6.98E−04 
                 2.91E−09 
                 17 
               
               
                 MAb16693 
                 159 ± 0.52 
                 29 
                 1.50E+05 
                 1.51E−02 
                 1.00E−07 
                 0.8 
               
               
                 MAb16695 
                 186 ± 0.79 
                 122 
                 2.80E+05 
                 1.05E−03 
                 3.77E−09 
                 11 
               
               
                 MAb16702 
                 208 ± 0.78 
                 75 
                 1.39E+05 
                 3.74E−04 
                 2.69E−09 
                 31 
               
               
                 IgG4 Isotype 
                 229 ± 1.42 
                 2 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 Control 
               
               
                   
               
               
                 NB indicates that no binding was observed under the current experimental conditions. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Binding kinetics parameters of hGP130-MMH binding 
               
               
                 to GP130 monoclonal antibodies at 37° C. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 100 nM 
                   
                   
                   
                   
               
               
                   
                 mAb 
                 Ag 
               
               
                   
                 Capture 
                 Bound 
                 ka 
                 kd 
                 KD 
                 t½ 
               
               
                 mAb Captured 
                 Level (RU) 
                 (RU) 
                 (1/Ms) 
                 (1/s) 
                 (M) 
                 (min) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 MAb16614 
                 176 ± 2.98 
                 10 
                 2.47E+05 
                 3.00E−02 
                 1.21E−07 
                 0.4 
               
               
                 MAb16618 
                 219 ± 2.04 
                 36 
                 1.08E+06 
                 1.12E−01 
                 1.03E−07 
                 0.1 
               
               
                 MAb16622 
                 238 ± 3.05 
                 30 
                 2.55E+05 
                 5.74E−02 
                 2.25E−07 
                 0.2 
               
               
                 MAb16623 
                 285 ± 3.24 
                 68 
                 3.81E+05 
                 1.23E−02 
                 3.22E−08 
                 0.9 
               
               
                 MAb16636 
                 231 ± 2.35 
                 50 
                 6.16E+04 
                 4.41E−03 
                 7.16E−08 
                 2.6 
               
               
                 MAb16637 
                 352 ± 2.31 
                 110 
                 1.97E+05 
                 3.42E−03 
                 1.73E−08 
                 3 
               
               
                 MAb16641 
                 188 ± 3.13 
                 107 
                 7.65E+05 
                 2.49E−03 
                 3.25E−09 
                 5 
               
               
                 MAb16646 
                 212 ± 2.83 
                 66 
                 9.81E+04 
                 3.31E−03 
                 3.38E−08 
                 3.5 
               
               
                 MAb16656 
                 214 ± 1.6 
                 50 
                 7.01E+04 
                 4.26E−03 
                 6.07E−08 
                 2.7 
               
               
                 MAb16659 
                 239 ± 1.56 
                 82 
                 9.28E+05 
                 1.71E−03 
                 1.84E−09 
                 7 
               
               
                 MAb16662 
                 245 ± 0.93 
                 17 
                 4.36E+05 
                 5.85E−02 
                 1.34E−07 
                 0.2 
               
               
                 MAb16664 
                 251 ± 2.08 
                 16 
                 5.80E+05 
                 8.26E−03 
                 1.42E−08 
                 1.4 
               
               
                 MAb16665 
                 336 ± 2.56 
                 19 
                 4.23E+05 
                 6.93E−02 
                 1.64E−07 
                 0.2 
               
               
                 MAb16666 
                 350 ± 4.52 
                 104 
                 5.88E+05 
                 1.01E−02 
                 1.71E−08 
                 1.1 
               
               
                 MAb16669 
                 173 ± 1.38 
                 25 
                 5.51E+04 
                 1.06E−03 
                 1.93E−08 
                 11 
               
               
                 MAb16673 
                 180 ± 1.31 
                 40 
                 5.36E+05 
                 4.04E−02 
                 7.54E−08 
                 0.3 
               
               
                 MAb16676 
                 192 ± 1.72 
                 52 
                 3.93E+05 
                 2.36E−03 
                 6.01E−09 
                 5 
               
               
                 MAb16680 
                 291 ± 1.59 
                 58 
                 9.48E+04 
                 2.06E−03 
                 2.17E−08 
                 6 
               
               
                 MAb16682 
                 211 ± 2.32 
                 16 
                 3.01E+05 
                 3.41E−02 
                 1.13E−07 
                 0.3 
               
               
                 MAb16683 
                 262 ± 1.88 
                 38 
                 1.07E+05 
                 1.25E−02 
                 1.17E−07 
                 0.9 
               
               
                 MAb16684 
                 220 ± 1.73 
                 18 
                 1.63E+05 
                 3.60E−02 
                 2.21E−07 
                 0.3 
               
               
                 MAb16687 
                 184 ± 1.85 
                 107 
                 3.22E+05 
                 1.82E−03 
                 5.66E−09 
                 6 
               
               
                 MAb16692 
                 197 ± 1.78 
                 107 
                 7.24E+05 
                 3.05E−03 
                 4.21E−09 
                 4 
               
               
                 MAb16693 
                 180 ± 1.26 
                 22 
                 3.82E+05 
                 4.02E−02 
                 1.05E−07 
                 0.3 
               
               
                 MAb16695 
                 192 ± 1.56 
                 87 
                 4.94E+05 
                 6.27E−03 
                 1.27E−08 
                 1.8 
               
               
                 MAb16702 
                 216 ± 0.72 
                 68 
                 2.35E+05 
                 1.66E−03 
                 7.05E−09 
                 7 
               
               
                 IgG4 Isotype 
                 258 ± 1.09 
                 2 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 Control 
               
               
                   
               
               
                 NB indicates that no binding was observed under the current experimental conditions. 
               
            
           
         
       
     
     At 25° C., anti-GP130 monoclonal antibodies bound to hGP130-MMH with K D  values ranging from 663 pM to 411 nM, as shown in Table 3. At 37° C., anti-GP130 monoclonal antibodies bound to hGP130-MMH with K D  values ranging from 1.84 nM to 225 nM, as shown in Table 4. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Binding kinetics parameters of mfGP130-MMH binding 
               
               
                 to GP130 monoclonal antibodies at 25° C. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 100 nM 
                   
                   
                   
                   
               
               
                   
                 mAb 
                 Ag 
               
               
                   
                 Capture 
                 Bound 
                 ka 
                 kd 
                 KD 
                 t½ 
               
               
                 mAb Captured 
                 Level (RU) 
                 (RU) 
                 (1/Ms) 
                 (1/s) 
                 (M) 
                 (min) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 MAb16614 
                 142 ± 0.65 
                 16 
                 7.44E+04 
                 1.04E−02 
                 1.39E−07 
                 1.1 
               
               
                 MAb16618 
                 181 ± 0.48 
                 40 
                 3.74E+05 
                 6.21E−02 
                 1.66E−07 
                 0.2 
               
               
                 MAb16622 
                 195 ± 0.74 
                 34 
                 1.32E+05 
                 2.00E−02 
                 1.51E−07 
                 0.6 
               
               
                 MAb16623 
                 240 ± 0.79 
                 74 
                 1.87E+05 
                 4.76E−03 
                 2.55E−08 
                 2.4 
               
               
                 MAb16636 
                 193 ± 0.49 
                 23 
                 2.01E+04 
                 2.16E−03 
                 1.07E−07 
                 5 
               
               
                 MAb16637 
                 304 ± 3.59 
                 87 
                 6.83E+04 
                 1.09E−03 
                 1.59E−08 
                 11 
               
               
                 MAb16641 
                 153 ± 3.55 
                 109 
                 3.44E+05 
                 8.60E−04 
                 2.50E−09 
                 13 
               
               
                 MAb16646 
                 166 ± 0.29 
                 52 
                 4.79E+04 
                 7.72E−04 
                 1.61E−08 
                 15 
               
               
                 MAb16656 
                 187 ± 0.26 
                 42 
                 4.44E+04 
                 8.46E−04 
                 1.90E−08 
                 14 
               
               
                 MAb16659 
                 211 ± 0.43 
                 74 
                 5.42E+05 
                 4.40E−04 
                 8.12E−10 
                 26 
               
               
                 MAb16662 
                 214 ± 1.92 
                 15 
                 2.07E+05 
                 3.80E−02 
                 1.84E−07 
                 0.3 
               
               
                 MAb16664 
                 216 ± 1.31 
                 13 
                 5.43E+05 
                 1.75E−03 
                 3.21E−09 
                 7 
               
               
                 MAb16665 
                 294 ± 1.19 
                 16 
                 2.17E+05 
                 5.05E−02 
                 2.33E−07 
                 0.2 
               
               
                 MAb16666 
                 317 ± 2.67 
                 123 
                 2.90E+05 
                 5.30E−03 
                 1.83E−08 
                 2.2 
               
               
                 MAb16669 
                 151 ± 0.6 
                 3 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16673 
                 157 ± 0.25 
                 86 
                 3.12E+05 
                 6.93E−03 
                 2.22E−08 
                 1.7 
               
               
                 MAb16676 
                 175 ± 0.54 
                 49 
                 2.72E+05 
                 5.97E−04 
                 2.20E−09 
                 19 
               
               
                 MAb16680 
                 267 ± 0.84 
                 52 
                 1.20E+05 
                 4.89E−04 
                 4.07E−09 
                 24 
               
               
                 MAb16682 
                 184 ± 0.48 
                 20 
                 1.28E+05 
                 1.68E−02 
                 1.32E−07 
                 0.7 
               
               
                 MAb16683 
                 236 ± 1.97 
                 59 
                 6.57E+04 
                 2.46E−03 
                 3.74E−08 
                 5 
               
               
                 MAb16684 
                 203 ± 0.61 
                 4 
                 IC 
                 IC 
                 IC 
                 IC 
               
               
                 MAb16687 
                 170 ± 0.58 
                 108 
                 1.84E+05 
                 5.24E−04 
                 2.85E−09 
                 22 
               
               
                 MAb16692 
                 179 ± 0.96 
                 109 
                 2.09E+05 
                 7.45E−04 
                 3.56E−09 
                 16 
               
               
                 MAb16693 
                 159 ± 0.33 
                 6 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16695 
                 184 ± 0.5 
                 120 
                 2.67E+05 
                 9.98E−04 
                 3.74E−09 
                 12 
               
               
                 MAb16702 
                 205 ± 0.74 
                 48 
                 5.63E+04 
                 5.95E−04 
                 1.06E−08 
                 19 
               
               
                 IgG4 Isotype 
                 227 ± 2.09 
                 2 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 Control 
               
               
                   
               
               
                 NB indicates that no binding was observed under the current experimental conditions. 
               
            
           
         
       
     
     IC indicates that observed binding was inclusive and was unable to fit the real time binding data under the current experimental conditions. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Binding kinetics parameters of mfGP130-MMH binding to GP130 
               
               
                 monoclonal antibodies at 37° C. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 100 nM 
                   
                   
                   
                   
               
               
                   
                 mAb 
                 Ag 
               
               
                   
                 Capture 
                 Bound 
                 ka 
                 kd 
                 KD 
                 t½ 
               
               
                 mAb Captured 
                 Level (RU) 
                 (RU) 
                 (1/Ms) 
                 (1/s) 
                 (M) 
                 (min) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 MAb16614 
                 168 ± 1.52 
                 8 
                 3.70E+05 
                 2.67E−02 
                 7.22E−08 
                 0.4 
               
               
                 MAb16618 
                 210 ± 1.02 
                 26 
                 1.33E+06 
                 1.20E−01 
                 9.03E−08 
                 0.1 
               
               
                 MAb16622 
                 229 ± 2 
                 28 
                 2.72E+05 
                 5.37E−02 
                 1.98E−07 
                 0.2 
               
               
                 MAb16623 
                 274 ± 1.25 
                 60 
                 3.52E+05 
                 1.56E−02 
                 4.43E−08 
                 0.7 
               
               
                 MAb16636 
                 222 ± 1.09 
                 25 
                 3.95E+04 
                 9.45E−03 
                 2.39E−07 
                 1.2 
               
               
                 MAb16637 
                 342 ± 1.61 
                 104 
                 1.78E+05 
                 2.63E−03 
                 1.48E−08 
                 4 
               
               
                 MAb16641 
                 179 ± 1.7 
                 98 
                 6.94E+05 
                 2.42E−03 
                 3.48E−09 
                 5 
               
               
                 MAb16646 
                 202 ± 1.18 
                 58 
                 8.64E+04 
                 3.37E−03 
                 3.91E−08 
                 3.4 
               
               
                 MAb16656 
                 208 ± 1.42 
                 47 
                 6.70E+04 
                 4.11E−03 
                 6.13E−08 
                 2.8 
               
               
                 MAb16659 
                 232 ± 1.18 
                 78 
                 7.47E+05 
                 1.74E−03 
                 2.34E−09 
                 7 
               
               
                 MAb16662 
                 238 ± 0.85 
                 14 
                 3.55E+05 
                 5.92E−02 
                 1.67E−07 
                 0.2 
               
               
                 MAb16664 
                 243 ± 1.68 
                 16 
                 6.80E+05 
                 7.88E−03 
                 1.16E−08 
                 1.5 
               
               
                 MAb16665 
                 330 ± 0.88 
                 14 
                 4.92E+05 
                 1.01E−01 
                 2.05E−07 
                 0.1 
               
               
                 MAb16666 
                 340 ± 3.16 
                 90 
                 5.28E+05 
                 1.31E−02 
                 2.47E−08 
                 0.9 
               
               
                 MAb16669 
                 167 ± 0.83 
                 8 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16673 
                 175 ± 1.1 
                 64 
                 6.17E+05 
                 2.11E−02 
                 3.41E−08 
                 0.5 
               
               
                 MAb16676 
                 187 ± 1.43 
                 48 
                 3.41E+05 
                 2.43E−03 
                 7.12E−09 
                 5 
               
               
                 MAb16680 
                 284 ± 1.39 
                 51 
                 9.07E+04 
                 2.17E−03 
                 2.39E−08 
                 5 
               
               
                 MAb16682 
                 205 ± 0.85 
                 15 
                 3.11E+05 
                 3.86E−02 
                 1.24E−07 
                 0.3 
               
               
                 MAb16683 
                 256 ± 1.73 
                 43 
                 9.65E+04 
                 1.21E−02 
                 1.25E−07 
                 1.0 
               
               
                 MAb16684 
                 214 ± 1.12 
                 2 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16687 
                 179 ± 1.38 
                 101 
                 2.84E+05 
                 1.97E−03 
                 6.93E−09 
                 6 
               
               
                 MAb16692 
                 191 ± 1.17 
                 97 
                 6.39E+05 
                 2.97E−03 
                 4.65E−09 
                 4 
               
               
                 MAb16693 
                 175 ± 0.86 
                 8 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16695 
                 186 ± 1.27 
                 84 
                 4.33E+05 
                 5.92E−03 
                 1.37E−08 
                 2.0 
               
               
                 MAb16702 
                 211 ± 0.9 
                 46 
                 8.66E+04 
                 2.19E−03 
                 2.52E−08 
                 5 
               
               
                 IgG4 Isotype 
                 253 ± 1.16 
                 3 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 Control 
               
               
                   
               
               
                 NB indicates that no binding was observed under the current experimental conditions. 
               
            
           
         
       
     
     23 out of 26 anti-GP130 monoclonal antibodies of the invention bound to mfGP130-MMH. At 25° C., anti-GP130 monoclonal antibodies bound to mfGP130-MMH with K D  values ranging from 812 pM to 233 nM, as shown in Table 5. At 37° C., anti-GP130 monoclonal antibodies bound to mfGP130-MMH with K D  values ranging from 2.34 nM to 239 nM, as shown in Table 6. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Binding kinetics parameters of hGP130-mFc binding 
               
               
                 to GP130 monoclonal antibodies at 25° C. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 100 nM 
                   
                   
                   
                   
               
               
                   
                 mAb 
                 Ag 
               
               
                   
                 Capture 
                 Bound 
                 ka 
                 kd 
                 KD 
                 t½ 
               
               
                 mAb Captured 
                 Level (RU) 
                 (RU) 
                 (1/Ms) 
                 (1/s) 
                 (M) 
                 (min) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 MAb16614 
                 143 ± 0.19 
                 83 
                 8.57E+04 
                 3.83E−04 
                 4.47E−09 
                 30 
               
               
                 MAb16618 
                 181 ± 0.44 
                 202 
                 1.19E+06 
                 2.79E−04 
                 2.36E−10 
                 41 
               
               
                 MAb16622 
                 196 ± 0.81 
                 164 
                 4.97E+05 
                 3.06E−04 
                 6.15E−10 
                 38 
               
               
                 MAb16623 
                 242 ± 1.53 
                 184 
                 4.44E+05 
                 2.08E−04 
                 4.68E−10 
                 56 
               
               
                 MAb16636 
                 194 ± 0.62 
                 91 
                 5.88E+04 
                 1.13E−04 
                 1.92E−09 
                 102 
               
               
                 MAb16637 
                 306 ± 1.48 
                 189 
                 2.32E+05 
                 1.50E−04 
                 6.47E−10 
                 77 
               
               
                 MAb16641 
                 153 ± 1.08 
                 142 
                 7.70E+05 
                 2.97E−04 
                 3.86E−10 
                 39 
               
               
                 MAb16646 
                 167 ± 0.41 
                 77 
                 7.19E+04 
                 2.25E−04 
                 3.13E−09 
                 51 
               
               
                 MAb16656 
                 188 ± 0.42 
                 98 
                 7.81E+04 
                 1.32E−04 
                 1.68E−09 
                 88 
               
               
                 MAb16659 
                 212 ± 0.34 
                 186 
                 1.72E+06 
                 6.28E−05 
                 3.66E−11 
                 184 
               
               
                 MAb16662 
                 215 ± 1.08 
                 90 
                 6.20E+05 
                 4.79E−04 
                 7.72E−10 
                 24 
               
               
                 MAb16664 
                 217 ± 0.62 
                 26 
                 3.05E+06 
                 3.54E−04 
                 1.16E−10 
                 33 
               
               
                 MAb16665 
                 293 ± 1.36 
                 158 
                 3.99E+05 
                 1.09E−03 
                 2.72E−09 
                 11 
               
               
                 MAb16666 
                 317 ± 2.14 
                 253 
                 1.14E+06 
                 1.37E−04 
                 1.20E−10 
                 85 
               
               
                 MAb16669 
                 152 ± 0.46 
                 40 
                 2.93E+04 
                 2.41E−04 
                 8.21E−09 
                 48 
               
               
                 MAb16673 
                 158 ± 0.38 
                 169 
                 5.60E+05 
                 9.66E−05 
                 1.73E−10 
                 120 
               
               
                 MAb16676 
                 176 ± 0.22 
                 119 
                 8.53E+05 
                 7.13E−05 
                 8.36E−11 
                 162 
               
               
                 MAb16680 
                 269 ± 0.69 
                 79 
                 1.72E+05 
                 2.31E−04 
                 1.34E−09 
                 50 
               
               
                 MAb16682 
                 186 ± 0.74 
                 126 
                 1.72E+05 
                 3.61E−04 
                 2.10E−09 
                 32 
               
               
                 MAb16683 
                 236 ± 0.73 
                 107 
                 1.67E+05 
                 3.11E−04 
                 1.86E−09 
                 37 
               
               
                 MAb16684 
                 204 ± 0.27 
                 96 
                 1.28E+05 
                 4.59E−04 
                 3.60E−09 
                 25 
               
               
                 MAb16687 
                 171 ± 0.64 
                 178 
                 4.44E+05 
                 4.21E−05 
                 9.50E−11 
                 274 
               
               
                 MAb16692 
                 180 ± 0.69 
                 174 
                 6.13E+05 
                 1.18E−04 
                 1.93E−10 
                 98 
               
               
                 MAb16693 
                 159 ± 0.41 
                 123 
                 2.09E+05 
                 2.51E−04 
                 1.20E−09 
                 46 
               
               
                 MAb16695 
                 185 ± 0.29 
                 189 
                 6.83E+05 
                 2.06E−04 
                 3.01E−10 
                 56 
               
               
                 MAb16702 
                 206 ± 1.11 
                 159 
                 6.22E+05 
                 2.52E−04 
                 4.05E−10 
                 46 
               
               
                 IgG4 Isotype 
                 228 ± 1.43 
                 5 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 Control 
               
               
                   
               
               
                 NB indicates that no binding was observed under the current experimental conditions 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Binding kinetics parameters of hGP130-mFc binding 
               
               
                 to GP130 monoclonal antibodies at 37° C. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 100 nM 
                   
                   
                   
                   
               
               
                   
                 mAb 
                 Ag 
               
               
                   
                 Capture 
                 Bound 
                 ka 
                 kd 
                 KD 
                 t½ 
               
               
                 mAb Captured 
                 Level (RU) 
                 (RU) 
                 (1/Ms) 
                 (1/s) 
                 (M) 
                 (min) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 MAb16614 
                 172 ± 1.78 
                 85 
                 1.80E+05 
                 1.72E−03 
                 9.56E−09 
                  7 
               
               
                 MAb16618 
                 214 ± 1.83 
                 236 
                 1.39E+06 
                 9.34E−04 
                 6.74E−10 
                 12 
               
               
                 MAb16622 
                 232 ± 1.75 
                 201 
                 6.39E+05 
                 8.25E−04 
                 1.29E−09 
                 14 
               
               
                 MAb16623 
                 280 ± 2 
                 224 
                 5.80E+05 
                 3.25E−04 
                 5.60E−10 
                 36 
               
               
                 MAb16636 
                 225 ± 2.06 
                 136 
                 1.39E+05 
                 1.67E−04 
                 1.20E−09 
                 69 
               
               
                 MAb16637 
                 349 ± 2.73 
                 246 
                 4.96E+05 
                 3.23E−04 
                 6.50E−10 
                 36 
               
               
                 MAb16641 
                 184 ± 1.98 
                 151 
                 1.60E+06 
                 8.28E−04 
                 5.18E−10 
                 14 
               
               
                 MAb16646 
                 207 ± 1.7 
                 99 
                 1.59E+05 
                 6.56E−04 
                 4.14E−09 
                 18 
               
               
                 MAb16656 
                 212 ± 0.97 
                 123 
                 1.75E+05 
                 2.97E−04 
                 1.70E−09 
                 39 
               
               
                 MAb16659 
                 235 ± 1.2 
                 227 
                 1.91E+06 
                 4.76E−05 
                 2.49E−11 
                 243  
               
               
                 MAb16662 
                 240 ± 1.17 
                 114 
                 6.39E+05 
                 2.65E−03 
                 4.15E−09 
                  4 
               
               
                 MAb16664 
                 248 ± 1.36 
                 37 
                 1.33E+06 
                 1.09E−04 
                 8.15E−11 
                 106  
               
               
                 MAb16665 
                 334 ± 3.31 
                 172 
                 6.53E+05 
                 2.56E−03 
                 3.92E−09 
                  5 
               
               
                 MAb16666 
                 347 ± 3.54 
                 285 
                 1.21E+06 
                 3.60E−04 
                 2.98E−10 
                 32 
               
               
                 MAb16669 
                 170 ± 1.24 
                 59 
                 6.51E+04 
                 3.80E−04 
                 5.85E−09 
                 30 
               
               
                 MAb16673 
                 178 ± 1.09 
                 199 
                 6.84E+05 
                 3.13E−04 
                 4.57E−10 
                 37 
               
               
                 MAb16676 
                 190 ± 0.61 
                 140 
                 1.67E+06 
                 9.45E−05 
                 5.66E−11 
                 122  
               
               
                 MAb16680 
                 288 ± 0.8 
                 81 
                 2.43E+05 
                 4.89E−04 
                 2.01E−09 
                 24 
               
               
                 MAb16682 
                 207 ± 1.28 
                 130 
                 2.36E+05 
                 1.96E−03 
                 8.33E−09 
                  6 
               
               
                 MAb16683 
                 260 ± 1.27 
                 111 
                 1.96E+05 
                 1.14E−03 
                 5.80E−09 
                 10 
               
               
                 MAb16684 
                 217 ± 0.75 
                 103 
                 2.02E+05 
                 9.96E−04 
                 4.94E−09 
                 12 
               
               
                 MAb16687 
                 183 ± 1.22 
                 193 
                 5.89E+05 
                     1.00E−05 #   
                 1.70E−11 
                 1155 #    
               
               
                 MAb16692 
                 195 ± 0.86 
                 196 
                 1.30E+06 
                 3.29E−04 
                 2.52E−10 
                 35 
               
               
                 MAb16693 
                 178 ± 1.04 
                 140 
                 4.35E+05 
                 1.25E−03 
                 2.88E−09 
                  9 
               
               
                 MAb16695 
                 189 ± 0.93 
                 183 
                 1.32E+06 
                 7.63E−04 
                 5.80E−10 
                 15 
               
               
                 MAb16702 
                 214 ± 0.89 
                 139 
                 1.29E+06 
                 7.35E−04 
                 5.70E−10 
                 16 
               
               
                 IgG4 Isotype 
                 256 ± 0.83 
                 6 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 Control 
               
               
                   
               
               
                 NB indicates that no binding was observed under the current experimental conditions. 
               
               
                   # means no dissociation of hGP130-mFc from captured GP130 monoclonal antibody was observed and the k d  value was manually fixed at 1.00E−05 during the analysis. 
               
            
           
         
       
     
     At 25° C., anti-GP130 monoclonal antibodies bound to hGP130-mFc with K D  values ranging from 36.6 pM to 8.21 nM, as shown in Table 7. At 37° C., anti-GP130 monoclonal antibodies bound to hGP130-mFc with K D  values ranging from 17 pM to 9.56 nM, as shown in Table 8. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Binding kinetics parameters of mGP130-MMH binding 
               
               
                 to GP130 monoclonal antibodies at 25° C. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 100 nM 
                   
                   
                   
                   
               
               
                   
                 mAb 
                 Ag 
               
               
                   
                 Capture 
                 Bound 
                 ka 
                 kd 
                 KD 
                 t½ 
               
               
                 mAb Captured 
                 Level (RU) 
                 (RU) 
                 (1/Ms) 
                 (1/s) 
                 (M) 
                 (min) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 MAb16614 
                 142 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16618 
                 181 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16622 
                 195 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16623 
                 240 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16636 
                 194 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16637 
                 304 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16641 
                 152 
                 −1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16646 
                 167 
                 −1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16656 
                 187 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16659 
                 210 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16662 
                 213 
                 −1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16664 
                 216 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16665 
                 295 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16666 
                 315 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16669 
                 151 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16673 
                 157 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16676 
                 175 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16680 
                 267 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16682 
                 184 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16683 
                 235 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16684 
                 202 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16687 
                 170 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16692 
                 179 
                 −1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16693 
                 158 
                 −1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16695 
                 183 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16702 
                 206 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 IgG4 Isotype 
                 225 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 Control 
               
               
                   
               
               
                 NB indicates that no binding was observed under the current experimental conditions. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Binding kinetics parameters of mGP130-MMH binding 
               
               
                 to GP130 monoclonal antibodies at 37° C. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 100 nM 
                   
                   
                   
                   
               
               
                   
                 mAb 
                 Ag 
               
               
                   
                 Capture 
                 Bound 
                 ka 
                 kd 
                 KD 
                 t½ 
               
               
                 mAb Captured 
                 Level (RU) 
                 (RU) 
                 (1/Ms) 
                 (1/s) 
                 (M) 
                 (min) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 MAb16614 
                 167 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16618 
                 209 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16622 
                 227 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16623 
                 272 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16636 
                 220 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16637 
                 343 
                 −1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16641 
                 178 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16646 
                 200 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16656 
                 208 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16659 
                 230 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16662 
                 235 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16664 
                 246 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16665 
                 330 
                 −1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16666 
                 346 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16669 
                 166 
                 −1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16673 
                 173 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16676 
                 187 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16680 
                 283 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16682 
                 204 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16683 
                 255 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16684 
                 214 
                 −1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16687 
                 179 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16692 
                 191 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16693 
                 174 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16695 
                 185 
                 −1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16702 
                 211 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 IgG4 Isotype 
                 253 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 Control 
               
               
                   
               
               
                 NB indicates that no binding was observed under the current experimental conditions. 
               
            
           
         
       
     
     None of the anti-GP130 monoclonal antibodies of the invention bound to mGP130-MMH at 25° C. or at 37° C. as shown in Tables 9 and 10. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Binding kinetics parameters of rGP130-MMH binding 
               
               
                 to GP130 monoclonal antibodies at 25° C. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 100 nM 
                   
                   
                   
                   
               
               
                   
                 mAb 
                 Ag 
               
               
                   
                 Capture 
                 Bound 
                 ka 
                 kd 
                 KD 
                 t½ 
               
               
                 mAb Captured 
                 Level (RU) 
                 (RU) 
                 (1/Ms) 
                 (1/s) 
                 (M) 
                 (min) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 MAb16614 
                 142 ± 0.12 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16618 
                 180 ± 0.54 
                 6 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16622 
                 196 ± 0.63 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16623 
                 240 ± 0.25 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16636 
                 194 ± 0.4 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16637 
                 306 ± 2.86 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16641 
                 153 ± 0.22 
                 −2 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16646 
                 166 ± 0.18 
                 17 
                 3.84E+04 
                 1.32E−02 
                 3.44E−07 
                 0.9  
               
               
                 MAb16656 
                 188 ± 1.11 
                 −1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16659 
                 210 ± 0.3 
                 2 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16662 
                 213 ± 0.1 
                 −1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16664 
                 215 ± 0.95 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16665 
                 291 ± 0.16 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16666 
                 316 ± 1.11 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16669 
                 151 ± 0.14 
                 −2 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16673 
                 157 ± 0.13 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16676 
                 174 ± 0.16 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16680 
                 267 ± 1 
                 2 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16682 
                 184 ± 0.2 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16683 
                 233 ± 0.76 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16684 
                 202 ± 0.47 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16687 
                 170 ± 0.18 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16692 
                 180 ± 0.67 
                 8 
                 1.78E+05 
                 8.19E−02 
                 4.60E−07 
                 0.14 
               
               
                 MAb16693 
                 158 ± 0.01 
                 −1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16695 
                 183 ± 0.21 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16702 
                 206 ± 0.36 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 IgG4 Isotype 
                 226 ± 0.27 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 Control 
               
               
                   
               
               
                 NB indicates that no binding was observed under the current experimental conditions. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Binding kinetics parameters of rGP130-MMH binding 
               
               
                 to GP130 monoclonal antibodies at 37° C. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 100 nM 
                   
                   
                   
                   
               
               
                   
                 mAb 
                 Ag 
               
               
                   
                 Capture 
                 Bound 
                 ka 
                 kd 
                 KD 
                 t½ 
               
               
                 mAb Captured 
                 Level (RU) 
                 (RU) 
                 (1/Ms) 
                 (1/s) 
                 (M) 
                 (min) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 MAb16614 
                 167 ± 0.23 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16618 
                 207 ± 0.91 
                 2 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16622 
                 227 ± 0.51 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16623 
                 270 ± 0.14 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16636 
                 219 ± 0.61 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16637 
                 337 ± 1.83 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16641 
                 177 ± 0.86 
                 −1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16646 
                 199 ± 0.44 
                 7 
                 1.77E+05 
                 7.12E−02 
                 4.03E−07 
                 0.16 
               
               
                 MAb16656 
                 207 ± 0.08 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16659 
                 229 ± 0.21 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16662 
                 235 ± 0.57 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16664 
                 243 ± 0.44 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16665 
                 328 ± 1.24 
                 −1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16666 
                 341 ± 2.48 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16669 
                 166 ± 0.15 
                 −2 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16673 
                 172 ± 0.18 
                 2 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16676 
                 186 ± 0.58 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16680 
                 282 ± 0.79 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16682 
                 203 ± 0.54 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16683 
                 256 ± 0.27 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16684 
                 212 ± 1.24 
                 −2 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16687 
                 179 ± 0.18 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16692 
                 191 ± 0.49 
                 7 
                 2.39E+05 
                 2.27E−01 
                 9.50E−07 
                 0.05 
               
               
                 MAb16693 
                 174 ± 0.66 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16695 
                 184 ± 0.36 
                 0 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 MAb16702 
                 210 ± 0.16 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 IgG4 Isotype 
                 252 ± 0.45 
                 1 
                 NB 
                 NB 
                 NB 
                 NB 
               
               
                 Control 
               
               
                   
               
               
                 NB indicates that no binding was observed under the current experimental conditions. 
               
            
           
         
       
     
     As shown in Tables 11 and 12, 2 out of 26 anti-GP130 monoclonal antibodies of the invention bound to rGP130-MMH. At 25° C., anti-GP130 monoclonal antibodies bound to rGP130-MMH with K D  values ranging from 344 nM to 460 nM, as shown in Table 11. At 37° C., anti-GP130 monoclonal antibodies bound to rGP130-MMH with K D  values ranging from 403 nM to 950 as shown in Table 12. 
     Example 4: Anti-GP130 Antibody Cell Binding by FACS Analysis 
     In order to assess cell binding by anti-GP130 antibodies of the invention two cell lines were generated. One cell line generated was HEK293 cells stably over-expressing full length (FL) human gp130 (amino acids 1-918 of accession # P40189 with leucine at position 2 changed to valine, a natural variant) along with a luciferase reporter (Stat3-luciferase, Stat3-luc, SA Bioscience, #CLS-6028L). The cells were sorted twice using flow cytometry for high expression of gp130. It is known hereafter as HEK293/Stat3-luc/gp130-2X Sort. IMR-32 cells (human Neuroblastoma, ATCC), were also evaluated for cell binding as these cells express gp130 endogenously and were used for bioassays. The cells used for binding were generated to stably express a luciferase reporter (Stat3-luciferase, Stat3-luc, SA Bioscience, #CLS-6028L), and are referred to hereafter as IMR-32/STAT3-Luc cells. 
     For the FACS analysis, 10 nM of the antibodies were used to stain 0.5×106 cells/well of each cell type at 4° C. in PBS (without calcium and magnesium) containing 2% FBS for 30 minutes. IMR-32/STAT3-luc cells were incubated with 1 mg/mL mouse IgG for 30 minutes at 4° C. to block Fc receptors prior to adding the antibodies. To test whether the anti-gp130 antibody binding is specific for gp130 on the IMR-32 cells, antibodies were added to IMR-32/Stat3-luc cells with or without being pre-bound to 1000 nM of recombinant protein of the ecto-domain of human gp130 fused to myc-myc-his tag (hgp130.mmh) for 30 minutes at 25° C. After incubation with primary antibodies, the cells were stained with 8 μg/mL of Alexa Fluor®-647 conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc., anti-human #109-607-003) for 30 minutes. Cells were fixed using BD CytoFix™ (Becton Dickinson, #554655) and analyzed on an IQue® (Intellicyt®) Flow Cytometer. Unstained and secondary antibody alone controls were also tested for all cell lines. The results were analyzed using ForeCyt® (IntelliCyt®) software to determine the geometric means of fluorescence for viable cells. 
     As shown in Table 13, twenty-six anti-gp130 antibodies of the invention tested at 10 nM demonstrated binding to HEK293/Stat3-luc/gp130-2X Sort cells with binding ratios ranging from 29- to 159-fold. The anti-gp130 antibodies demonstrated binding to the HEK293 parental cells with binding ratios 4- to 45-fold. Binding ratios to the IMR-32/Stat3-luc cells ranged from 5- to 23-fold without hgp130.mmh and from 4- to 9-fold with hGP130.mmh. The isotype control antibodies and secondary antibodies alone samples demonstrated binding ratios ranging from 1- to 3-fold for HEK293 cell lines, and 1- to 8-fold for IMR-32/Stat3-luc cells. 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Binding of 10 nM anti-gp130 antibodies to HEK293/Stat3-luc/gp130-2X 
               
               
                 Sort and IMR-32/Stat3-luc cells. 
               
            
           
           
               
               
            
               
                   
                 MFI - Normalized to Unstained Control 
               
            
           
           
               
               
            
               
                   
                 IMR-32/Stat3-luc 
               
            
           
           
               
               
               
               
               
            
               
                   
                 293 
                 293/Stat3-luc/ 
                 No 
                 1 uM 
               
               
                 Antibody 
                 Parental 
                 GP130 2X sort 
                 hGP130.mmh 
                 hGP130.mmh 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 MAb16614 
                 13 
                 94 
                 7 
                 6 
               
               
                 MAb16618 
                 13 
                 105 
                 7 
                 8 
               
               
                 MAb16622 
                 9 
                 91 
                 5 
                 7 
               
               
                 MAb16623 
                 14 
                 96 
                 8 
                 7 
               
               
                 MAb16636 
                 20 
                 92 
                 8 
                 7 
               
               
                 MAb16637 
                 22 
                 121 
                 9 
                 7 
               
               
                 MAb16641 
                 13 
                 81 
                 8 
                 7 
               
               
                 MAb16646 
                 6 
                 29 
                 5 
                 6 
               
               
                 MAb16656 
                 20 
                 132 
                 12 
                 7 
               
               
                 MAb16659 
                 14 
                 81 
                 8 
                 8 
               
               
                 MAb16662 
                 7 
                 49 
                 5 
                 6 
               
               
                 MAb16664 
                 8 
                 45 
                 7 
                 8 
               
               
                 MAb16665 
                 4 
                 55 
                 5 
                 6 
               
               
                 MAb16666 
                 45 
                 159 
                 23 
                 8 
               
               
                 MAb16669 
                 9 
                 53 
                 6 
                 5 
               
               
                 MAb16673 
                 15 
                 107 
                 10 
                 7 
               
               
                 MAb16676 
                 13 
                 74 
                 9 
                 8 
               
               
                 MAb16680 
                 21 
                 99 
                 11 
                 9 
               
               
                 MAb16682 
                 5 
                 80 
                 6 
                 8 
               
               
                 MAb16683 
                 13 
                 75 
                 9 
                 7 
               
               
                 MAb16684 
                 7 
                 60 
                 5 
                 4 
               
               
                 MAb16687 
                 12 
                 71 
                 9 
                 7 
               
               
                 MAb16692 
                 23 
                 103 
                 12 
                 6 
               
               
                 MAb16693 
                 4 
                 51 
                 7 
                 7 
               
               
                 MAb16695 
                 13 
                 64 
                 10 
                 9 
               
               
                 MAb16702 
                 25 
                 111 
                 11 
                 6 
               
               
                 hIg4 Isotype 
                 3 
                 2 
                 6 
                 7 
               
               
                 Control 
               
               
                 a-h 2″ alone 
                 2 
                 2 
                 4 
                 8 
               
               
                 Unstained 
                 1 
                 1 
                 1 
                 1 
               
               
                   
               
            
           
         
       
     
     Example 5: Octet Cross-Competition Between Different Anti-GP130 Monoclonal Antibodies 
     Binding competition between a panel of anti-GP130 monoclonal antibodies was determined using a real time, label-free bio-layer interferometry assay on the Octet HTX biosensor platform (Pall ForteBio Corp.). The entire experiment was performed at 25° C. in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% v/v Surfactant Tween-20, and 1 mg/mL BSA, pH7.4 (HBS-EBT) buffer with the plate shaking at the speed of 1000 rpm. To assess whether 2 antibodies compete with one another for binding to their respective epitopes on the recombinant human GP130 (hGP130-mmH; expressed with a C-terminal myc-myc-hexahistidine tag SEQ ID:188), about 0.4-0.5 nm of hGP130-mmH was first captured onto anti-Penta-His antibody coated Octet biosensor tips (Fortebio Inc, #18-5122) by submerging the biosensor tips for 4 minutes in wells containing 40-501.1 g/mL solution of hGP130-MMH. The antigen captured biosensor tips were then saturated with the first anti-GP130 monoclonal antibody (mAb-1) by dipping into wells containing 50 μg/mL solution of mAb-1 for 4 minutes. The biosensor tips were then dipped into wells containing 50 μg/mL solution of the second anti-GP130 monoclonal antibody (mAb-2) for 3 minutes. The biosensor tips were washed in HBS-ETB buffer between every step of the experiment. The real-time binding response was monitored during the entire course of the experiment and the binding response at the end of every step was recorded. The response of mAb-2 binding to hGP130-MMH pre-complexed with mAb-1 was compared and competitive/non-competitive behavior of different anti-GP130 monoclonal antibodies was determined as shown in Table 14. 
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Cross-competition between anti-GP130 monoclonal antibodies 
               
            
           
           
               
               
               
            
               
                   
                 mAb-1 
                 mAb-2 that competes with mAb-1 
               
               
                   
                   
               
               
                   
                 MAb16659 
                 MAb16676 
               
               
                   
                   
                 MAb16695 
               
               
                   
                   
                 MAb16664 
               
               
                   
                   
                 MAb16622 
               
               
                   
                   
                 MAb16637 
               
               
                   
                 MAb16676 
                 MAb16659 
               
               
                   
                   
                 MAb16695 
               
               
                   
                   
                 MAb16664 
               
               
                   
                   
                 MAb16622 
               
               
                   
                   
                 MAb16637 
               
               
                   
                 MAb16695 
                 MAb16659 
               
               
                   
                   
                 MAb16676 
               
               
                   
                   
                 MAb16664 
               
               
                   
                   
                 MAb16622 
               
               
                   
                   
                 MAb16637 
               
               
                   
                 MAb16664 
                 MAb16659 
               
               
                   
                   
                 MAb16676 
               
               
                   
                   
                 MAb16695 
               
               
                   
                   
                 MAb16622 
               
               
                   
                   
                 MAb16637 
               
               
                   
                 MAb16622 
                 MAb16659 
               
               
                   
                   
                 MAb16676 
               
               
                   
                   
                 MAb16695 
               
               
                   
                   
                 MAb16664 
               
               
                   
                   
                 MAb16637 
               
               
                   
                   
                 MAb16665 
               
               
                   
                   
                 MAb16687 
               
               
                   
                   
                 MAb16656 
               
               
                   
                 MAb16637 
                 MAb16659 
               
               
                   
                   
                 MAb16676 
               
               
                   
                   
                 MAb16695 
               
               
                   
                   
                 MAb16664 
               
               
                   
                   
                 MAb16622 
               
               
                   
                   
                 MAb16665 
               
               
                   
                   
                 MAb16687 
               
               
                   
                   
                 MAb16656 
               
               
                   
                 MAb16665 
                 MAb16622 
               
               
                   
                   
                 MAb16637 
               
               
                   
                   
                 MAb16687 
               
               
                   
                   
                 MAb16656 
               
               
                   
                 MAb16687 
                 MAb16622 
               
               
                   
                   
                 MAb16637 
               
               
                   
                   
                 MAb16665 
               
               
                   
                   
                 MAb16656 
               
               
                   
                 MAb16656 
                 MAb16622 
               
               
                   
                   
                 MAb16637 
               
               
                   
                   
                 MAb16665 
               
               
                   
                   
                 MAb16687 
               
               
                   
                 MAb16641 
                 No mAb 
               
               
                   
                 MAb16666 
                 MAb16662 
               
               
                   
                 MAb16662 
                 MAb16666 
               
               
                   
                 MAb16618 
                 MAb16673 
               
               
                   
                   
                 MAb16636 
               
               
                   
                   
                 MAb16692 
               
               
                   
                 MAb16673 
                 MAb16618 
               
               
                   
                   
                 MAb16636 
               
               
                   
                   
                 MAb16692 
               
               
                   
                 MAb16636 
                 MAb16618 
               
               
                   
                   
                 MAb16673 
               
               
                   
                   
                 MAb16692 
               
               
                   
                   
                 MAb16614 
               
               
                   
                   
                 MAb16682 
               
               
                   
                 MAb16692 
                 MAb16618 
               
               
                   
                   
                 MAb16673 
               
               
                   
                   
                 MAb16636 
               
               
                   
                   
                 MAb16614 
               
               
                   
                   
                 MAb16682 
               
               
                   
                 MAb16614 
                 MAb16636 
               
               
                   
                   
                 MAb16692 
               
               
                   
                   
                 MAb16682 
               
               
                   
                 MAb16682 
                 MAb16636 
               
               
                   
                   
                 MAb16692 
               
               
                   
                   
                 MAb16614 
               
               
                   
                 MAb16623 
                 MAb16684 
               
               
                   
                   
                 MAb16702 
               
               
                   
                   
                 MAb16693 
               
               
                   
                   
                 MAb16669 
               
               
                   
                 MAb16684 
                 MAb16623 
               
               
                   
                   
                 MAb16702 
               
               
                   
                   
                 MAb16693 
               
               
                   
                   
                 MAb16669 
               
               
                   
                 MAb16702 
                 MAb16623 
               
               
                   
                   
                 MAb16684 
               
               
                   
                   
                 MAb16693 
               
               
                   
                   
                 MAb16669 
               
               
                   
                 MAb16693 
                 MAb16623 
               
               
                   
                   
                 MAb16684 
               
               
                   
                   
                 MAb16702 
               
               
                   
                   
                 MAb16669 
               
               
                   
                 MAb16669 
                 MAb16623 
               
               
                   
                   
                 MAb16684 
               
               
                   
                   
                 MAb16702 
               
               
                   
                   
                 MAb16693 
               
               
                   
                 MAb16646 
                 MAb16680 
               
               
                   
                 MAb16680 
                 MAb16646 
               
               
                   
                 MAb16683 
                 No mAb 
               
               
                   
                   
               
            
           
         
       
     
     Example 6: Monoclonal Antibodies Binding to GP130 Domain Proteins in Luminex-GP130 Delta D1-mmH and GP130 Delta D1-3-mmH CHO Supernatant 
     To identify the binding region of human GP130 with which anti-GP130 antibodies of the invention interact, a Luminex FLEXMAP (FM3DD, LuminexCorp) flow cytometry based analysis was utilized to characterize the interaction of recombinant human GP130 protein domains. For the assay, approximately 3 million carboxylated Microplex R  microspheres (Luminex, Cat# LC1000A), were washed, vortexed and sonicated in 0.1 M NaPO4, pH 6.2 (activation buffer) and then centrifuged to remove the supernatant. The microspheres were re-suspended in 120 μL of activation buffer and the carboxylate groups (—COOH) were activated by addition of 15 μL of 50 mg/mL of N-hydroxysuccinimide (NHS, Thermo Scientific, Cat#24500) followed by addition of 15 μL of 50 mg/mL of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC, ThermoScientific, Cat#22980) at 25° C. After 10 minutes, the pH of the reaction was reduced to 5.0 with the addition of 600 μL of 50 mM MES, pH 5 (coupling buffer), and the microspheres were vortexed, and centrifuged to remove supernatant. The activated beads were immediately mixed with 500 μL of 20 μg/mL monoclonal anti-myc antibodies with mouse IgG, in coupling buffer and incubated for two hours at 25° C. The coupling reaction was quenched by addition of 50 μL of 1M TRIS-HCl, pH 8.0 and the microspheres were rapidly vortexed, centrifuged, and washed four times with 1 mL of Dulbecco&#39;s 1× Phosphate Buffered Saline (DPBS, pH 7.2, ThermoScientific Cat#14190136), to remove uncoupled proteins and other reaction components. 
     The transiently expressed GP130 proteins, including human GP130 delta D1 expressed with a C-terminal myc-myc hexahistidine tag (human GP130 delta D1-MMH, SEQ ID NO:192) and human GP130 delta D1-D3 expressed with a C-terminal myc-myc hexahistidine tag (human GP130 delta D1-D3-MMH, SEQ ID NO:193), were suspended in serum free CHO—S-SFM II Medium (Thermo Fisher, Cat #31033020) and were then clarified by centrifugation. Purified GP130 full length extracellular domain expressed with a C-terminal myc-myc hexahistidine tag (human GP130-MMH, SEQ ID NO:191) was prepared at 10 ug/mL in PBS. Aliquots of microspheres with immobilized anti-myc monoclonal antibodies, prepared as described above, were added individually to 1 mL of the each of these protein supernatants and to 500 uL of purified GP130 protein. The microspheres were gently mixed, incubated for two hours at 25° C., washed twice with 1 mL of DBPS, centrifuged to remove the supernatant and finally resuspended in 1 mL of DPBS buffer. Forty eight μL of anti-myc IgG coupled microspheres from individual reactions with full length human GP130 and with each of the human GP130 domain proteins were withdrawn and mixed together in 3.6 mL of PBS+20 mg/mL BSA+0.05% sodium azide (blocking buffer). 
     From this mixed pool, 754 of microspheres were plated per well on a 96 well filter plate (Millipore, Cat. No: MSBVN1250) and mixed with 25 μL of individual anti-human GP130 monoclonal antibodies (0.5 or 5 μg/mL), incubated for two hours at 25° C. and then washed twice with 200 μL of DPBS with 0.05% Tween 20 (washing buffer). To detect and quantify the amounts of bound anti-GP130 antibody levels to individual microspheres, either 100 μL of 2.5 μg/mL R-Phycoerythrin conjugated goat F(ab′)2 anti-human kappa (Southern Biotech, Cat#2063-09) in blocking buffer, was added and incubated for 30 minutes at 25° C. After 30 minutes, the samples were washed twice with 200 μL of washing buffer and resuspended in 150 μL of wash buffer. The Median Fluorescence intensity (MFI) of the microspheres was measured in a Luminex Analyzer. 
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 Luminex MFI signal of anti-GP130 antibodies binding to myc tag 
               
               
                 captured full-length extracellular domain of human GP130, isolated 
               
               
                 human GP130 delta D1 and delta D1-D3 domains. 
               
            
           
           
               
               
               
               
               
            
               
                   
                 GP130 
                 GP130 
                 GP130 
                   
               
               
                   
                 Delta D1 
                 Delta D1-D3 
                 Full length 
               
               
                   
                 extracellular 
                 extracellular 
                 extracellular 
                 Interacting 
               
               
                 Antibody 
                 domain 
                 domain 
                 domain 
                 Domain(s) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 mAb16614 
                 2899 
                 56 
                 18690 
                 D2-D3 
               
               
                 mAb16618 
                 2634 
                 19 
                 18986 
                 D2-D3 
               
               
                 mAb16622 
                 391 
                 20 
                 18462 
                 D2-D3 
               
               
                 mAb16623 
                 12565 
                 16012 
                 17617 
                 FNIII 
               
               
                 mAb16636 
                 4261 
                 1067 
                 17617 
                 D2-D3 
               
               
                 mAb16637 
                 342 
                 861 
                 16899 
                 D1 
               
               
                 mAb16641 
                 51 
                 27 
                 17298 
                 D1 
               
               
                 mAb16646 
                 11329 
                 18005 
                 13957 
                 FNIII 
               
               
                 mAb16656 
                 48 
                 17 
                 15911 
                 D1 
               
               
                 mAb16659 
                 49 
                 22 
                 17092 
                 D1 
               
               
                 mAb16662 
                 3050 
                 16 
                 13926 
                 D2-D3 
               
               
                 mAb16664 
                 442 
                 419 
                 19306 
                 D1 
               
               
                 mAb16665 
                 119 
                 116 
                 16332 
                 D1 
               
               
                 mAb16666 
                 7280 
                 319 
                 16676 
                 D2-D3 
               
               
                 mAb16669 
                 12643 
                 15063 
                 17640 
                 FNIII 
               
               
                 mAb16673 
                 2344 
                 33 
                 15065 
                 D2-D3 
               
               
                 mAb16676 
                 364 
                 686 
                 18074 
                 D1 
               
               
                 mAb16680 
                 10911 
                 18713 
                 14422 
                 FNIII 
               
               
                 mAb16682 
                 1380 
                 13 
                 13825 
                 D2-D3 
               
               
                 mAb16683 
                 12026 
                 20857 
                 15220 
                 FNIII 
               
               
                 mAb16684 
                 9126 
                 11992 
                 14398 
                 FNIII 
               
               
                 mAb16687 
                 37 
                 18 
                 15485 
                 D1 
               
               
                 mAb16692 
                 3893 
                 17 
                 16202 
                 D2-D3 
               
               
                 mAb16693 
                 9449 
                 12009 
                 12943 
                 FNIII 
               
               
                 mAb16695 
                 30 
                 7 
                 14533 
                 D1 
               
               
                 mAb16702 
                 11721 
                 12500 
                 14951 
                 FNIII 
               
               
                   
               
            
           
         
       
     
     The results of the Luminex based analysis are tabulated in Table 15. Luminex MFI signal intensities indicate that the twenty six anti-GP130 antibodies of the invention bound to the human GP130 full length extracellular domain. 
     Anti-GP130 antibodies mAb16637, mAb16641, mAb16656, mAb16659, mAb16664, mAb16665, mAb16676, mAb16687 and MAb16695 lost binding to both deletion proteins, suggesting binding epitopes within the D1 domain of human GP130. Anti-GP130 antibodies mAb16614, mAb16618, mAb16622, mAb16636, mAb16662, mAb16666, mAb16673, mAb16682, mAb16692 lost binding to GP130 delta D1-D3 while retaining binding to GP130 delta D1, indicating their binding epitope is within domains D2-D3 of human GP130. 
     Anti-GP130 antibodies mAb16623, mAb16646, mAb16669, mAb16680, mAb16683, mAb16684, mAb16693, mAb16702 bound to GP130 delta D1 and GP130 delta D1-D3, indicating their binding domain is within FNIII of human GP130. 
     Example 7: Functional Cell-Based Assay with in IMR-32/Stat3-Luc Cells, without Ligands or with hOncostain M, hLIF, or hCNTF 
     In order to assess transcriptional activation or inhibition of anti-GP130 antibodies, IMR-32 cells (human Neuroblastoma ATCC) were generated to stably express a luciferase reporter (STAT3-Luc; SABiosciences, # CLS-6028L). The resulting cell line is referred to hereafter as IMR-32/STAT3-Luc (see Example 4 herein). 
     For the bioassay, IMR-32/STAT3-Luc cells were plated at 15,000 cells/well in a 96-well plate in assay buffer (0.1% FBS in Optimem with pen/strep) and incubated overnight at 37° C. in 5% CO 2 . The following day anti-gp130 antibodies or an isotype control were serially diluted from 100 nM to 24.4 pM in assay buffer (plus a sample containing buffer alone without test molecule), added to the cells and incubated at 25° C. for 30 minutes. After 30 minutes, either 100 pM human Oncostatin M (hOSM, R&amp;D System 293-OM), 20 pM human Leukemia Inhibitory Factor (hLIF, R&amp;D Systems 7734-LF), 20 pM human Ciliary Neurotrophic Factor (hCNTF, R&amp;D Systems 257-NT), or assay buffer was added to cells. hOSM, hLIF, and hCNTF were serially diluted from 10 nM to 0.17 pM in assay buffer (plus a sample containing buffer alone without test molecule) and added to cells not treated with antibodies. After 5 hours at 37° C. in 5% CO 2 , luciferase activity was measured with OneGIo™ reagent (Promega, # E6031) and Victor™ X multilabel plate reader (Perkin Elmer). The results were analyzed using nonlinear regression (4-parameter logistics) with Prism 5 software (Graph Pad) to obtain EC 50  and IC 50  values. Activation of antibodies was calculated with the maximum range of RLU achieved by the antibody over the maximum range of RLU achieved by hOSM. The percentage of inhibition was calculated with the RLU values by using the following equation: 
     
       
         
           
             
               % 
                
               
                   
               
                
               Inhibition 
             
             = 
             
               100 
               × 
               
                 
                   
                     RLU 
                     Baseline 
                   
                   - 
                   
                     RLU 
                     Inhibition 
                   
                 
                 
                   
                     RLU 
                     Baseline 
                   
                   - 
                   
                     RLU 
                     Background 
                   
                 
               
             
           
         
       
     
     In this equation “RLUBaseline” is the luminescence value from the cells treated with a constant amount of ligand (hOSM, hLIF, or hCNTF) without antibodies. “RLUInhibition” is the luminescence value with 100 nM of a particular antibody with a particular concentration of ligand, and “RLUBackground” is the luminescence value from cells without any ligands or antibodies. 
     As shown in Table 16, twenty-six anti-human gp130 antibodies of the invention were tested for their ability to either activate or inhibit activation of IMR-32/Stat3-luc cells. As shown in Table 19, in the absence of any added ligands none of the antibodies of the invention tested showed any activation of IMR-32/Stat3-luc cells. One of the 26 antibodies of the invention, MAb16692, showed complete inhibition of all three ligands tested with IC 50  values of 48 pM, 140 pM, and 230 pM for hOSM, hLIF, and hCNTF, respectively. An additional ten antibodies of the invention tested showed some inhibition of at least one of the ligands with the % inhibition ranging from 17% to 95%, with IC 50  values for the inhibiting antibodies ranging from &gt;100 nM to 88 pM. Fifteen antibodies of the invention did not show inhibition of any of ligands tested. An isotype control antibody did not demonstrate any measureable activation or inhibition of IMR-32/Stat3-luc cells. The ligands activated IMR-32/STAT3-luc cells with EC 50  values of 54 pM for hOSM, 23 pM for hLIF, and 4 pM for hCNTF. 
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 Activation and inhibition of anti-gp130 antibodies in the absence or presence of GP130 liqands in IMR-32/Stat3-luc cells. 
               
            
           
           
               
               
               
               
               
            
               
                   
                 OSM (100 pM) 
                 LIF (20 pM) 
                 CNTF (20 pM) 
                 No Ligand 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 mAb PID 
                 IC50 [M] 
                 % inhibition 
                 IC50 [M] 
                 % inhibition 
                 IC50 [M] 
                 % inhibition 
                 EC50 [M] 
               
               
                   
               
               
                 mAb16614 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Activation 
               
               
                 mAb16618 
                 &gt;5.0E−08 
                 52 
                 &gt;1.0E−07 
                 40 
                 &gt;1.0E−07 
                 33 
                 No Activation 
               
               
                 mAb16622 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Activation 
               
               
                 mAb16623 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 &gt;1.0E−07 
                 22 
                 No Activation 
               
               
                 mAb16636 
                 &gt;1.0E−08 
                 82 
                 &gt;1.0E−08 
                 76 
                 &gt;1.0E−08 
                 62 
                 No Activation 
               
               
                 mAb16637 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Activation 
               
               
                 mAb16641 
                  8.8E−11 
                 21 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Activation 
               
               
                 mAb16646 
                 &gt;1.0E−07 
                 47 
                 &gt;1.0E−07 
                 26 
                 &gt;1.0E−07 
                 30 
                 No Activation 
               
               
                 mAb16656 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Activation 
               
               
                 mAb16659 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Activation 
               
               
                 mAb16662 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Activation 
               
               
                 mAb16664 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Activation 
               
               
                 mAb16665 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Activation 
               
               
                 mAb16666 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                  7.1E−10 
                 95 
                 No Activation 
               
               
                 mAb16669 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Activation 
               
               
                 mAb16673 
                 &gt;5.0E−08 
                 62 
                  9.0E−09 
                 52 
                 &gt;1.0E−07 
                 46 
                 No Activation 
               
               
                 mAb16676 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Activation 
               
               
                 mAb16680 
                  1.8E−09 
                 71 
                  3.4E−09 
                 80 
                  9.6E−10 
                 35 
                 No Activation 
               
               
                 mAb16682 
                     &gt;1E−07 
                 23 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Activation 
               
               
                 mAb16683 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Activation 
               
               
                 mAb16684 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Activation 
               
               
                 mAb16687 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Activation 
               
               
                 mAb16692 
                  4.8E−11 
                 101  
                  1.4E−10 
                 101  
                  2.3E−10 
                 100  
                 No Activation 
               
               
                 mAb16693 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Activation 
               
               
                 mAb16695 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Activation 
               
               
                 mAb16702 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                     &gt;1E−07 
                 17 
                 No Activation 
               
               
                 Isotype Control mAb 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Inhibition 
                 No Activation 
               
               
                   
               
            
           
         
       
     
     Example 8: GP130 Purified Antibodies Blocking ELISA 
     GP130 (glycoprotein 130) is a type I cytokine receptor transmembrane protein which forms a high-affinity ternary complex with the ligand ciliary neurotropic factor (CNTF) when it is associated with CNTFRα (ciliary neurotropic factor receptor alpha subunit). The ability of anti-gp130 antibodies to block GP130 protein binding to plate bound CNTFRα/CNTF complex was measured using a competition sandwich ELISA. In this assay, various concentrations of anti-gp130 antibody were pre-mixed with a constant amount of dimeric GP130 protein and the reduction of the gp130 binding due to the presence of the antibody to the plate immobilized CNTFRα/CNTF complex was monitored. 
     The dimeric gp130 protein used in the experiments was comprised of a portion of the human gp130 extracellular domain (amino acids E23-E619 of accession number NP_002175.2) expressed with the Fc portion of the mouse IgG2a protein at the c-terminus (hGP130-mFc; SEQ ID:190, mw 94,210 daltons). The CNTFRα protein was purchased from R&amp;D Systems (amino acids Q23-P346 of accession#6992, mw 36,000 daltons). The CNTF protein was purchased from R&amp;D Systems (amino acids A2-M200 of accession#6441.1, mw 22,800 daltons). Isotype antibody control, anti-Fel d 1, and human IgG4 P  antibody were included as controls for IgG background detection. 
     The experiment was carried out using the following procedure. Human CNTFRα was coated at a concentration of 2 mg/mL in HBSS on a 96-well microtiter plate overnight at 4° C. Nonspecific binding sites were subsequently blocked using a 1% (w/v) solution of BSA in HBSS. Human CNTF at a concentration of 1 μg/ml in HBSS was added to the plate bound CNTFRα for 1 hour at room temperature. In separate dilution plates, a constant amount of 2.5 nM of human GP130-mFc protein was titrated with antibodies ranging from 3.4 pM to 200 nM in serial dilution and with no antibody present. These solutions were incubated for 1 hour at room temperature (RT) and subsequently transferred to the microtiter plates with CNTFRα/CNTF complex without washing. The plates were incubated for 2 hours at RT, washed with PBST buffer, and plate-bound hGP130-mFc was detected with an anti-mFc polyclonal antibody conjugated with horseradish peroxidase (HRP) (Jackson ImmunoResearch Inc). Samples were developed with a TMB solution (BD Biosciences, substrate A and B mixed at 1:1 ratio as per manufacturer&#39;s instructions) to produce a colorimetric reaction and then neutralized with 1M sulfuric acid before measuring absorbance at 450 nm on a Victor ×5 plate reader. 
     Data analysis was performed using a sigmoidal dose-response model within Prism™ software (GraphPad). The calculated IC 50  value, defined as the concentration of antibody required to reduce 50% of GP130 binding to CNTFRα/CNTF complex, was used as an indicator of blocking potency. Percent blockade at maximum concentration tested was calculated as an indicator of the ability of the antibodies to block binding of GP130 to CNTFRα/CNTF on the plate as determined from the dose curve. The ratio of the reduction in signal observed in the presence of the highest tested concentration of 200 nM antibody, relative to the difference between the signal with 2.5 nM GP130 with no antibody (0% blocking) and the background signal from HRP-conjugated secondary antibody alone (100% blocking), was subtracted from 100% blocking. 
     The results of the blocking ELISA are shown in Table 17 with blocking percentages in the presence of 200 nM antibody reported for all antibodies. IC 50  values are reported only for antibodies blocking &gt;30% of GP130 binding to CNTFRα/CNTF. Nineteen of twenty-six a-GP130 antibodies block &lt;30% GP130 protein binding to plate-coated CNTFRα/CNTF. Negative numbers indicate an increase of GP130 binding detected in the presence of antibody. Seven antibodies blocked GP130 protein binding to CNTFRα/CNTF &gt;30% and IC 50  values ranged from below the lower limit of quantitation for the assay of 1.25 nM to 6.5 nM, with four of them blocking 90% or more of the signal at the highest antibody concentration tested. The irrelevant blocking control antibody showed blocking of 4.5% at concentrations up to 200 nM. 
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 GP130 Purified Antibodies Blocking ELISA 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Potency of Antibody 
               
               
                   
                   
                   
                 Blocking 2.5 nM 
               
               
                   
                   
                   
                 hGP130-mFc binding 
               
               
                   
                   
                 Max Blocking 
                 to plate-bound 
               
               
                 Antibody 
                   
                 of Antibody at 
                 CNTFR/CNTF 
               
               
                 Identifier 
                 Lot # 
                 200 nM (%) 
                 complex (M) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 mAb16614 
                 MAb16614-L1 
                 −14.75 
                 — 
               
               
                 mAb16618 
                 MAb16618-L1 
                 86.65 
                 2.374E−09 
               
               
                 mAb16622 
                 MAb16622-L1 
                 −84.14 
                 — 
               
               
                 mAb16623 
                 MAb16623-L1 
                 −49.19 
                 — 
               
               
                 mAb16636 
                 MAb16636-L1 
                 94.76 
                 1.104E−09 
               
               
                 mAb16637 
                 MAb16637-L1 
                 −38.86 
                 — 
               
               
                 mAb16641 
                 MAb16641-L1 
                 14.09 
                 — 
               
               
                 mAb16646 
                 MAb16646-L1 
                 5.24 
                 — 
               
               
                 mAb16656 
                 MAb16656-L1 
                 −24.71 
                 — 
               
               
                 mAb16659 
                 MAb16659-L1 
                 −18.81 
                 — 
               
               
                 mAb16662 
                 MAb16662-L1 
                 81.93 
                 6.506E−09 
               
               
                 mAb16664 
                 MAb16664-L1 
                 −46.53 
                 — 
               
               
                 mAb16665 
                 MAb16665-L1 
                 −47.12 
                 — 
               
               
                 mAb16666 
                 MAb16666-L1 
                 103.39 
                 6.437E−10* 
               
               
                 mAb16669 
                 MAb16669-L1 
                 47.12 
                 1.232E−08 
               
               
                 mAb16673 
                 MAb16673-L1 
                 103.76 
                 2.368E−09 
               
               
                 mAb16676 
                 MAb16676-L1 
                 −15.41 
                 — 
               
               
                 mAb16680 
                 MAb16680-L1 
                 11.95 
                 — 
               
               
                 mAb16682 
                 MAb16682-L1 
                 −3.39 
                 — 
               
               
                 mAb16683 
                 MAb16683-L1 
                 −22.79 
                 — 
               
               
                 mAb16684 
                 MAb16684-L1 
                 12.91 
                 — 
               
               
                 mAb16687 
                 MAb16687-L1 
                 −17.40 
                 — 
               
               
                 mAb16692 
                 MAb16692-L1 
                 101.77 
                 7.671E−10* 
               
               
                 mAb16693 
                 MAb16693-L1 
                 4.42 
                 — 
               
               
                 mAb16695 
                 MAb16695-L1 
                 21.17 
                 — 
               
               
                 mAb16702 
                 MAb16702-L1 
                 29.13 
                 — 
               
               
                 hIgG4 Isotype 
                 07-120309 
                 4.50 
                 — 
               
               
                 Control 
               
               
                   
               
               
                 In this Example, 100% blocking is equal to OD450nm value HRP-conjugated secondary antibody with no GP130. 
               
               
                 0% blocking is OD450nm value with 2.5 nM hGP130-mFc with no antibody. 
               
               
                 Negative Max Blocking % indicates an increase of GP130 binding detected in the presence of antibody. 
               
               
                 — IC 50  values are not quantitative for antibodies blocking &lt;30% at the highest concentration tested. 
               
               
                 *Indicates IC 50  value below the lower limit of quantitation of 1.25E−09M for the assay. 
               
            
           
         
       
     
     Example 9: LEPR×GP130 Focused Approach Bispecific Screening 
     This Example describes the generation of bispecific antibodies that bind to both LEPR and GP130 for the promotion of STAT3 signaling. Such antibodies are referred to herein as “LEPR×GP130 bispecific antibodies,” or “LEPR×GP130 bsAbs,” “anti-LEPR x anti-GP130 bispecific antibodies,” or the like. In this Example, several anti-GP130 binding arms were paired with four different anti-LEPR binding arms. The anti-LEPR antibodies used to construct the bispecific antibodies of this Example are the agonistic antibodies referred to as mAb16679, mAb18445, mAb18446 and mAb18449 (see US Patent Appl. Publ. No. 2017/0101477, the disclosure of which is incorporated by reference herein in its entirety). The amino acid and nucleic acid sequences of the variable domains and CDRs of the anti-LEPR antibodies used in this Example are summarized in Tables 18 and 19, respectively. 
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 anti-LEPR Amino Acid Sequence Identifiers 
               
            
           
           
               
               
            
               
                 Antibody 
                 SEQ ID NOs: 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Designation 
                 HCVR 
                 HCDR1 
                 HCDR2 
                 HCDR3 
                 LCVR 
                 LCDR1 
                 LCDR2 
                 LCDR3 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 mAb16679 
                 2 
                 4 
                 6 
                 8 
                 10 
                 12 
                 14 
                 16 
               
               
                 mAb18449 
                 162 
                 164 
                 166 
                 168 
                 10 
                 12 
                 14 
                 16 
               
               
                 mAb18445 
                 170 
                 172 
                 174 
                 176 
                 10 
                 12 
                 14 
                 16 
               
               
                 mAb18446 
                 178 
                 180 
                 182 
                 184 
                 10 
                 12 
                 14 
                 16 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 19 
               
             
            
               
                   
               
               
                 anti-LEPR Nucleic Acid Sequence Identifiers 
               
            
           
           
               
               
            
               
                 Antibody 
                 SEQ ID NOs: 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Designation 
                 HCVR 
                 HCDR1 
                 HCDR2 
                 HCDR3 
                 LCVR 
                 LCDR1 
                 LCDR2 
                 LCDR3 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 mAb16679 
                 1 
                 3 
                 5 
                 7 
                 9 
                 11 
                 13 
                 15 
               
               
                 mAb18449 
                 161 
                 163 
                 165 
                 167 
                 9 
                 11 
                 13 
                 15 
               
               
                 mAb18445 
                 169 
                 171 
                 173 
                 175 
                 9 
                 11 
                 13 
                 15 
               
               
                 mAb18446 
                 177 
                 179 
                 181 
                 183 
                 9 
                 11 
                 13 
                 15 
               
               
                   
               
            
           
         
       
     
     Eighteen bispecific antibodies were generated through pairing of anti-LEPR binding arms from mAb16679 with binding arms from 18 different anti-GP130 antibodies. Six additional bispecific antibodies were created by pairing binding arms from anti-LEPR antibodies mAb18449, mAb18445 and mAb18446 with anti-GP130 binding arms. Standard methods were used to produce the bispecific antibodies described herein. All LEPR×GP130 bispecific antibodies shown in this example comprise the same (“common”) light chain (comprising the light chain variable region [LCVR] amino acid sequence of SEQ ID NO:10, and light chain CDR [LCDR1, LCDR2 and LCDR3] amino acid sequences of SEQ ID NOs: 12, 14 and 16). The components of the bispecific antibodies of this Example are summarized in Table 20. 
     
       
         
           
               
             
               
                 TABLE 20 
               
             
            
               
                   
               
               
                 LEPR × GP130 Bispecific Antibody Components Summary. 
               
            
           
           
               
               
            
               
                   
                 SEQ ID NOs: (Amino Acid Sequences) 
               
            
           
           
               
               
               
            
               
                 Bispecific 
                 LEPR Binding Arm Domain (D1) 
                 GP130 Binding Arm Domain (D2) 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Antibody 
                 D1-HCVR 
                 D1-HCDR1 
                 D1-HCDR2 
                 D1-HCDR3 
                 D2-HCVR 
                 D2-HCDR1 
                 D2-HCDR2 
                 D2-HCDR3 
               
               
                   
               
            
           
           
               
               
               
            
               
                 bsAb19139D 
                 mAb16679 
                 mAb 16614 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 2 
                 4 
                 6 
                 8 
                 18 
                  20 
                  22 
                 24 
               
            
           
           
               
               
               
            
               
                 bsAb19140D 
                 mAb 16679 
                 mAb 16618 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 2 
                 4 
                 6 
                 8 
                 26 
                  28 
                  30 
                 32 
               
            
           
           
               
               
               
            
               
                 bsAb19141D 
                 mAb 16679 
                 mAb 16622 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 2 
                 4 
                 6 
                 8 
                 34 
                  36 
                  38 
                 40 
               
            
           
           
               
               
               
            
               
                 bsAb19142D 
                 mAb 16679 
                 mAb 16623 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 2 
                 4 
                 6 
                 8 
                 42 
                  44 
                  46 
                 48 
               
            
           
           
               
               
               
            
               
                 bsAb19143D 
                 mAb 16679 
                 mAb 16636 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 2 
                 4 
                 6 
                 8 
                 50 
                  52 
                  54 
                 56 
               
            
           
           
               
               
               
            
               
                 bsAb19144D 
                 mAb 16679 
                 mAb 16637 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 2 
                 4 
                 6 
                 8 
                 58 
                  60 
                  62 
                 64 
               
            
           
           
               
               
               
            
               
                 bsAb19145D 
                 mAb 16679 
                 mAb 16656 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 2 
                 4 
                 6 
                 8 
                 66 
                  68 
                  70 
                 72 
               
            
           
           
               
               
               
            
               
                 bsAb19146D 
                 mAb 16679 
                 mAb 16659 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 2 
                 4 
                 6 
                 8 
                 74 
                  76 
                  78 
                 80 
               
            
           
           
               
               
               
            
               
                 bsAb19147D 
                 mAb16679 
                 mAb16662 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 2 
                 4 
                 6 
                 8 
                 82 
                  84 
                  86 
                 88 
               
            
           
           
               
               
               
            
               
                 bsAb19148D 
                 mAb16679 
                 mAb16664 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 2 
                 4 
                 6 
                 8 
                 90 
                  92 
                  94 
                 96 
               
            
           
           
               
               
               
            
               
                 bsAb19149D 
                 mAb16679 
                 mAb16665 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 2 
                 4 
                 6 
                 8 
                 98 
                 100 
                 102 
                 104 
               
            
           
           
               
               
               
            
               
                 bsAb19150D 
                 mAb16679 
                 mAb16666 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 2 
                 4 
                 6 
                 8 
                 106  
                 108 
                 110 
                 112 
               
            
           
           
               
               
               
            
               
                 bsAb19151D 
                 mAb16679 
                 mAb16669 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 2 
                 4 
                 6 
                 8 
                 114  
                 116 
                 118 
                 120 
               
            
           
           
               
               
               
            
               
                 bsAb19152D 
                 mAb16679 
                 mAb16673 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 2 
                 4 
                 6 
                 8 
                 122  
                 124 
                 126 
                 128 
               
            
           
           
               
               
               
            
               
                 bsAb19153D 
                 mAb16679 
                 mAb16676 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 2 
                 4 
                 6 
                 8 
                 130  
                 132 
                 134 
                 136 
               
            
           
           
               
               
               
            
               
                 bsAb19154D 
                 mAb16679 
                 mAb16680 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 2 
                 4 
                 6 
                 8 
                 138  
                 140 
                 142 
                 144 
               
            
           
           
               
               
               
            
               
                 bsAb19155D 
                 mAb16679 
                 mAb16682 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 2 
                 4 
                 6 
                 8 
                 146  
                 148 
                 150 
                 152 
               
            
           
           
               
               
               
            
               
                 bsAb19156D 
                 mAb16679 
                 mAb16683 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 2 
                 4 
                 6 
                 8 
                 154  
                 156 
                 158 
                 160 
               
            
           
           
               
               
               
            
               
                 bsAb19757D 
                 mAb18449 
                 mAb16622 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 162  
                 164  
                 166  
                 168  
                 34 
                  36 
                  38 
                  40 
               
            
           
           
               
               
               
            
               
                 bsAb19758D 
                 mAb18449 
                 mAb16666 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 162  
                 164  
                 166  
                 168  
                 106  
                 108 
                 110 
                 112 
               
            
           
           
               
               
               
            
               
                 bsAb21236D 
                 mAb18445 
                 mAb16683 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 170  
                 172  
                 174  
                 176  
                 154  
                 156 
                 158 
                 160 
               
            
           
           
               
               
               
            
               
                 bsAb21237D 
                 mAb18446 
                 mAb16683 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 178  
                 180  
                 182  
                 184  
                 154  
                 156 
                 158 
                 160 
               
            
           
           
               
               
               
            
               
                 bsAb27679D 
                 mAb18445 
                 mAb16683 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 170  
                 172  
                 174  
                 176  
                 154  
                 156 
                 158 
                 160 
               
            
           
           
               
               
               
            
               
                 bsAb27680D 
                 mAb18446 
                 mAb16683 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 178  
                 180  
                 182  
                 184  
                 154  
                 156 
                 158 
                 160 
               
               
                   
                   
               
            
           
         
       
     
     Example 10: Biacore Binding Kinetics of Anti-LEPR×Anti-GP130 Bispecific Antibodies Binding to Different GP130 Reagents Measured at 25° C. and 37° C. 
     Equilibrium dissociation constants (K D  values) for LEPR and GP130 binding to purified anti-LEPR/GP130 bispecific antibodies were determined using a real-time surface plasmon resonance biosensor using a Biacore 4000 instrument. All binding studies were performed in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v/v Surfactant Tween-20, pH 7.4 (HBS-ET) running buffer at 25° C. and 37° C. The Biacore sensor surface was first derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (GE, # BR-1008-39) to capture anti-LEPR/GP130 bispecific antibodies. Binding studies were performed on following reagents: human LEPR extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (hLEPR-MMH; SEQ ID NO:187),  macaca fascicularis  LEPR extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (mfLEPR-MMH; SEQ ID NO: 188), human GP130 extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (hGP130-MMH; SEQ ID NO:191), and  macaca fascicularis  GP130 extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (mfGP130-MMH; SEQ ID NO:194). Different concentrations of LEPR or GP130 reagents were first prepared in HBS-ET running buffer (100 nM-3.7 nM; 3-fold serial dilution) and were injected over anti-human Fc captured anti-LEPR/GP130 bispecific antibody surface for 4 minutes at a flow rate of 304/minute, while the dissociation of bispecific antibody bound LEPR or GP130 reagent was monitored for 10 minutes in HBS-ET running buffer. Kinetic association (k a ) and dissociation (k d ) rate constants were determined by fitting the real-time binding sensorgrams to a 1:1 binding model with mass transport limitation using Scrubber 2.0c curve-fitting software. Binding dissociation equilibrium constants (K D ) and dissociative half-lives (t 1/2 ) were calculated from the kinetic rate constants as: 
     
       
         
           
             
               
                 
                   K 
                   D 
                 
                  
                 
                   ( 
                   M 
                   ) 
                 
               
               = 
               
                 kd 
                 ka 
               
             
             , 
             
               
                 and 
                  
                 
                     
                 
                  
                 t 
                  
                 
                     
                 
                  
                 
                   1 
                   2 
                 
                  
                 
                   ( 
                   min 
                   ) 
                 
               
               = 
               
                 
                   
                     ln 
                      
                     
                       ( 
                       2 
                       ) 
                     
                   
                   
                     60 
                     * 
                     kd 
                   
                 
                 . 
               
             
           
         
       
     
     Binding kinetics parameters for hLEPR-MMH, mfLEPR-MMH, hGP130-MMH or mfGP130-MMH binding to different anti-LEPR/GP130 bispecific antibodies of the invention at 25° C. and 37° C. are shown in Tables 21 through 28. 
     
       
         
           
               
             
               
                 TABLE 21 
               
             
            
               
                   
               
               
                 Binding kinetics parameters of hLEPR-MMH binding 
               
               
                 to anti-LEPR/GP130 bispecific antibodies at 25° C. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 100 nM 
                   
                   
                   
                   
               
               
                   
                 mAb 
                 hLEPR- 
               
               
                   
                 Capture 
                 MMH 
               
               
                 Bispecific 
                 Level 
                 Bound 
                 k a   
                 k d   
                 K D   
                 t½ 
               
               
                 Antibody 
                 (RU) 
                 (RU) 
                 (1/Ms) 
                 (1/s) 
                 (M) 
                 (min) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 bsAb19139D 
                 418 ± 0.5 
                 41 
                 2.41E+04 
                 1.71E−04 
                 7.09E−09 
                 68 
               
               
                 bsAb19140D 
                 444 ± 0.7 
                 47 
                 2.45E+04 
                 1.80E−04 
                 7.36E−09 
                 64 
               
               
                 bsAb19141D 
                 458 ± 2.5 
                 45 
                 2.54E+04 
                 1.51E−04 
                 5.91E−09 
                 77 
               
               
                 bsAb19142D 
                 452 ± 2.1 
                 46 
                 2.25E+04 
                 1.75E−04 
                 7.76E−09 
                 66 
               
               
                 bsAb19143D 
                 446 ± 1.6 
                 45 
                 2.49E+04 
                 1.82E−04 
                 7.31E−09 
                 64 
               
               
                 bsAb19144D 
                 456 ± 2.6 
                 48 
                 2.46E+04 
                 1.59E−04 
                 6.48E−09 
                 73 
               
               
                 bsAb19145D 
                 437 ± 1.2 
                 41 
                 2.49E+04 
                 1.65E−04 
                 6.62E−09 
                 70 
               
               
                 bsAb19146D 
                 461 ± 1   
                 45 
                 2.20E+04 
                 1.79E−04 
                 8.16E−09 
                 64 
               
               
                 bsAb19147D 
                 439 ± 0.7 
                 42 
                 2.27E+04 
                 1.62E−04 
                 7.13E−09 
                 71 
               
               
                 bsAb19148D 
                 452 ± 1.1 
                 44 
                 2.36E+04 
                 1.72E−04 
                 7.31E−09 
                 67 
               
               
                 bsAb19149D 
                 446 ± 1.4 
                 36 
                 2.71E+04 
                 1.94E−04 
                 7.16E−09 
                 60 
               
               
                 bsAb19150D 
                 451 ± 2.9 
                 41 
                 2.40E+04 
                 1.76E−04 
                 7.35E−09 
                 66 
               
               
                 bsAb19151D 
                 419 ± 1.3 
                 46 
                 2.11E+04 
                 1.58E−04 
                 7.49E−09 
                 73 
               
               
                 bsAb19152D 
                 455 ± 2   
                 47 
                 2.45E+04 
                 1.65E−04 
                 6.75E−09 
                 70 
               
               
                 bsAb19153D 
                 441 ± 1.9 
                 40 
                 2.55E+04 
                 1.65E−04 
                 6.46E−09 
                 70 
               
               
                 bsAb19154D 
                 438 ± 0.7 
                 40 
                 2.26E+04 
                 1.80E−04 
                 7.95E−09 
                 64 
               
               
                 bsAb19155D 
                 431 ± 0.7 
                 40 
                 2.40E+04 
                 1.53E−04 
                 6.36E−09 
                 76 
               
               
                 bsAb19156D 
                 439 ± 0.8 
                 39 
                 2.31E+04 
                 1.64E−04 
                 7.11E−09 
                 70 
               
               
                 bsAb19757D 
                 432 ± 1.3 
                 57 
                 2.07E+04 
                 1.62E−04 
                 7.85E−09 
                 71 
               
               
                 bsAb19758D 
                 428 ± 4.1 
                 54 
                 1.59E+04 
                 1.63E−04 
                 1.03E−08 
                 71 
               
               
                 bsAb21236D 
                  78 ± 0.3 
                 8 
                 3.51E+04 
                 3.72E−03 
                 1.06E−07 
                 3.1 
               
               
                 bsAb21237D 
                 206 ± 0.6 
                 11 
                 1.53E+04 
                 1.21E−03 
                 7.87E−08 
                 10 
               
               
                 Isotype Control 
                 427 ± 0.8 
                 −2 
                 NB* 
                 NB* 
                 NB* 
                 NB* 
               
               
                   
               
               
                 *NB indicates that no binding was observed under the current experimental conditions. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 22 
               
             
            
               
                   
               
               
                 Binding kinetics parameters of hLEPR-MMH binding 
               
               
                 to anti-LEPR/GP130 bispecific antibodies at 37° C. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 100 nM 
                   
                   
                   
                   
               
               
                   
                 mAb 
                 hLEPR- 
               
               
                   
                 Capture 
                 MMH 
               
               
                 Bispecific 
                 Level 
                 Bound 
                 k a   
                 k d   
                 K D   
                 t½ 
               
               
                 Antibody 
                 (RU) 
                 (RU) 
                 (1/Ms) 
                 (1/s) 
                 (M) 
                 (min) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 bsAb19139D 
                 527 ± 1.6 
                 67 
                 3.58E+04 
                 7.62E−04 
                 2.12E−08 
                 15 
               
               
                 bsAb19140D 
                 559 ± 0.3 
                 75 
                 3.82E+04 
                 8.18E−04 
                 2.14E−08 
                 14 
               
               
                 bsAb19141D 
                 582 ± 2.7 
                 74 
                 3.54E+04 
                 5.89E−04 
                 1.66E−08 
                 20 
               
               
                 bsAb19142D 
                 551 ± 2.7 
                 74 
                 3.40E+04 
                 6.33E−04 
                 1.86E−08 
                 18 
               
               
                 bsAb19143D 
                 558 ± 1.5 
                 72 
                 3.55E+04 
                 7.63E−04 
                 2.15E−08 
                 15 
               
               
                 bsAb19144D 
                 568 ± 1.9 
                 78 
                 3.68E+04 
                 5.97E−04 
                 1.62E−08 
                 19 
               
               
                 bsAb19145D 
                 558 ± 1.4 
                 66 
                 3.86E+04 
                 8.05E−04 
                 2.08E−08 
                 14 
               
               
                 bsAb19146D 
                 576 ± 1   
                 69 
                 3.87E+04 
                 8.25E−04 
                 2.13E−08 
                 14 
               
               
                 bsAb19147D 
                 547 ± 2.1 
                 65 
                 3.85E+04 
                 7.91E−04 
                 2.05E−08 
                 15 
               
               
                 bsAb19148D 
                 565 ± 0.5 
                 69 
                 3.90E+04 
                 7.98E−04 
                 2.05E−08 
                 14 
               
               
                 bsAb19149D 
                 566 ± 3.2 
                 61 
                 3.38E+04 
                 6.22E−04 
                 1.84E−08 
                 19 
               
               
                 bsAb19150D 
                 544 ± 2.4 
                 63 
                 3.55E+04 
                 6.91E−04 
                 1.95E−08 
                 17 
               
               
                 bsAb19151D 
                 525 ± 1.9 
                 70 
                 4.14E+04 
                 7.60E−04 
                 1.83E−08 
                 15 
               
               
                 bsAb19152D 
                 568 ± 2   
                 74 
                 3.77E+04 
                 7.87E−04 
                 2.08E−08 
                 15 
               
               
                 bsAb19153D 
                 557 ± 1.7 
                 65 
                 4.01E+04 
                 7.91E−04 
                 1.97E−08 
                 15 
               
               
                 bsAb19154D 
                 540 ± 0.9 
                 65 
                 3.79E+04 
                 7.29E−04 
                 1.92E−08 
                 16 
               
               
                 bsAb19155D 
                 536 ± 1.5 
                 63 
                 3.97E+04 
                 7.92E−04 
                 2.00E−08 
                 15 
               
               
                 bsAb19156D 
                 539 ± 1   
                 60 
                 3.66E+04 
                 8.42E−04 
                 2.30E−08 
                 14 
               
               
                 bsAb19757D 
                 543 ± 2.4 
                 81 
                 3.28E+04 
                 6.25E−04 
                 1.91E−08 
                 18 
               
               
                 bsAb19758D 
                 519 ± 2.3 
                 73 
                 3.23E+04 
                 6.63E−04 
                 2.06E−08 
                 17 
               
               
                 bsAb21236D 
                  68 ± 0.9 
                 5 
                 1.80E+04 
                 1.31E−02 
                 7.26E−07 
                 0.9 
               
               
                 bsAb21237D 
                 269 ± 1.5 
                 15 
                 1.58E+04 
                 4.42E−03 
                 2.80E−07 
                 2.6 
               
               
                 Isotype Control mAb 
                 540 ± 1.5 
                 −1 
                 NB* 
                 NB* 
                 NB* 
                 NB* 
               
               
                   
               
               
                 *NB indicates that no binding was observed under the current experimental conditions. 
               
            
           
         
       
     
     At 25° C., anti-LEPR/GP130 bispecific antibodies bound to hLEPR-MMH with K D  values ranging from 5.91 nM to 106 nM, as shown in Table 21. At 37° C., anti-LEPR monoclonal antibodies bound to hLEPR-MMH with K D  values ranging from 16.2 nM to 726 nM, as shown in Table 22. 
     
       
         
           
               
             
               
                 TABLE 23 
               
             
            
               
                   
               
               
                 Binding kinetics parameters of mfLEPR-MMH binding 
               
               
                 to anti-LEPR/GP130 bispecific antibodies at 25° C. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 100 nM 
                   
                   
                   
                   
               
               
                   
                 mAb 
                 mfLEPR- 
               
               
                   
                 Capture 
                 MMH 
               
               
                 Bispecific 
                 Level 
                 Bound 
                 k a   
                 k d   
                 K D   
                 t½ 
               
               
                 Antibody 
                 (RU) 
                 (RU) 
                 (1/Ms) 
                 (1/s) 
                 (M) 
                 (min) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 bsAb19139D 
                 417 ± 0.9 
                 103 
                 4.36E+04 
                 1.28E−04 
                 2.93E−09 
                 90 
               
               
                 bsAb19140D 
                 442 ± 0.7 
                 112 
                 4.50E+04 
                 1.21E−04 
                 2.69E−09 
                 95 
               
               
                 bsAb19141D 
                 457 ± 0.9 
                 110 
                 4.33E+04 
                 1.24E−04 
                 2.86E−09 
                 93 
               
               
                 bsAb19142D 
                 450 ± 1.8 
                 112 
                 4.59E+04 
                 1.26E−04 
                 2.74E−09 
                 92 
               
               
                 bsAb19143D 
                 445 ± 1.7 
                 111 
                 4.48E+04 
                 1.36E−04 
                 3.04E−09 
                 85 
               
               
                 bsAb19144D 
                 455 ± 3   
                 114 
                 4.11E+04 
                 1.24E−04 
                 3.02E−09 
                 93 
               
               
                 bsAb19145D 
                 436 ± 1.2 
                 102 
                 4.49E+04 
                 1.23E−04 
                 2.75E−09 
                 94 
               
               
                 bsAb19146D 
                 460 ± 0.9 
                 107 
                 4.29E+04 
                 1.13E−04 
                 2.63E−09 
                 102 
               
               
                 bsAb19147D 
                 437 ± 0.6 
                 107 
                 4.25E+04 
                 1.19E−04 
                 2.80E−09 
                 97 
               
               
                 bsAb19148D 
                 452 ± 0.7 
                 107 
                 4.13E+04 
                 1.19E−04 
                 2.88E−09 
                 97 
               
               
                 bsAb19149D 
                 446 ± 1   
                 96 
                 4.19E+04 
                 1.11E−04 
                 2.65E−09 
                 104 
               
               
                 bsAb19150D 
                 449 ± 3.6 
                 102 
                 4.18E+04 
                 1.17E−04 
                 2.79E−09 
                 99 
               
               
                 bsAb19151D 
                 418 ± 1.5 
                 109 
                 4.25E+04 
                 1.17E−04 
                 2.74E−09 
                 99 
               
               
                 bsAb19152D 
                 454 ± 2.7 
                 113 
                 4.18E+04 
                 1.24E−04 
                 2.97E−09 
                 93 
               
               
                 bsAb19153D 
                 440 ± 1.5 
                 102 
                 4.26E+04 
                 1.26E−04 
                 2.96E−09 
                 92 
               
               
                 bsAb19154D 
                 436 ± 0.7 
                 102 
                 4.08E+04 
                 1.21E−04 
                 2.96E−09 
                 96 
               
               
                 bsAb19155D 
                 429 ± 0.9 
                 103 
                 4.20E+04 
                 1.22E−04 
                 2.90E−09 
                 95 
               
               
                 bsAb19156D 
                 439 ± 0.7 
                 97 
                 4.04E+04 
                 1.32E−04 
                 3.27E−09 
                 87 
               
               
                 bsAb19757D 
                 429 ± 1.5 
                 7 
                 IC #   
                 IC #   
                 IC #   
                 IC #   
               
               
                 bsAb19758D 
                 426 ± 3   
                 9 
                 3.86E+04 
                 2.25E−02 
                 5.83E−07 
                 0.5 
               
               
                 bsAb21236D 
                 119 ± 0.3 
                 12 
                 5.79E+04 
                 4.82E−03 
                 8.34E−08 
                 2.4 
               
               
                 bsAb21237D 
                 190 ± 0.6 
                 16 
                 3.08E+04 
                 1.16E−03 
                 3.78E−08 
                 10 
               
               
                 Isotype Control 
                 426 ± 1.1 
                 1 
                 NB* 
                 NB* 
                 NB* 
                 NB* 
               
               
                   
               
               
                 *NB indicates that no binding was observed under the current experimental conditions. 
               
               
                   # IC indicates that observed binding signal was less than three-fold above to the non-specific binding observed for isotype control antibody surface and/or the data cannot be used to measure binding kinetic parameters. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 24 
               
             
            
               
                   
               
               
                 Binding kinetics parameters of mfLEPR-MMH binding 
               
               
                 to anti-LEPR/GP130 bispecific antibodies at 37° C. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 100 nM 
                   
                   
                   
                   
               
               
                   
                 mAb 
                 mfLEPR- 
               
               
                   
                 Capture 
                 MMH 
               
               
                 Bispecific 
                 Level 
                 Bound 
                 k a   
                 k d   
                 K D   
                 t½ 
               
               
                 Antibody 
                 (RU) 
                 (RU) 
                 (1/Ms) 
                 (1/s) 
                 (M) 
                 (min) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 bsAb19139D 
                 524 ± 1.2 
                 163 
                 1.03E+05 
                 5.59E−04 
                 5.42E−09 
                 21 
               
               
                 bsAb19140D 
                 555 ± 0.8 
                 175 
                 1.02E+05 
                 5.72E−04 
                 5.60E−09 
                 20 
               
               
                 bsAb19141D 
                 579 ± 1   
                 173 
                 1.03E+05 
                 5.22E−04 
                 5.05E−09 
                 22 
               
               
                 bsAb19142D 
                 546 ± 3.5 
                 172 
                 9.49E+04 
                 5.29E−04 
                 5.57E−09 
                 22 
               
               
                 bsAb19143D 
                 555 ± 1   
                 171 
                 9.59E+04 
                 5.51E−04 
                 5.74E−09 
                 21 
               
               
                 bsAb19144D 
                 567 ± 1.5 
                 178 
                 9.91E+04 
                 5.17E−04 
                 5.22E−09 
                 22 
               
               
                 bsAb19145D 
                 554 ± 1.3 
                 162 
                 1.03E+05 
                 5.66E−04 
                 5.48E−09 
                 20 
               
               
                 bsAb19146D 
                 573 ± 1.6 
                 164 
                 1.00E+05 
                 5.68E−04 
                 5.68E−09 
                 20 
               
               
                 bsAb19147D 
                 542 ± 1.3 
                 163 
                 1.00E+05 
                 5.65E−04 
                 5.64E−09 
                 20 
               
               
                 bsAb19148D 
                 561 ± 1   
                 163 
                 9.25E+04 
                 5.71E−04 
                 6.17E−09 
                 20 
               
               
                 bsAb19149D 
                 564 ± 1.8 
                 154 
                 6.25E+04 
                 5.06E−04 
                 8.09E−09 
                 23 
               
               
                 bsAb19150D 
                 543 ± 3.4 
                 158 
                 9.89E+04 
                 5.19E−04 
                 5.25E−09 
                 22 
               
               
                 bsAb19151D 
                 521 ± 1.2 
                 167 
                 1.05E+05 
                 5.54E−04 
                 5.30E−09 
                 21 
               
               
                 bsAb19152D 
                 564 ± 1.7 
                 169 
                 9.99E+04 
                 5.89E−04 
                 5.90E−09 
                 20 
               
               
                 bsAb19153D 
                 552 ± 1.2 
                 158 
                 6.58E+04 
                 5.80E−04 
                 8.81E−09 
                 20 
               
               
                 bsAb19154D 
                 536 ± 1.2 
                 159 
                 9.60E+04 
                 5.41E−04 
                 5.64E−09 
                 21 
               
               
                 bsAb19155D 
                 532 ± 1.3 
                 155 
                 1.03E+05 
                 5.65E−04 
                 5.49E−09 
                 20 
               
               
                 bsAb19156D 
                 537 ± 1.4 
                 148 
                 9.64E+04 
                 5.67E−04 
                 5.88E−09 
                 20 
               
               
                 bsAb19757D 
                 539 ± 1.3 
                 7 
                 IC #   
                 IC #   
                 IC #   
                 IC #   
               
               
                 bsAb19758D 
                 517 ± 1.1 
                 5 
                 IC #   
                 IC #   
                 IC #   
                 IC #   
               
               
                 bsAb21236D 
                 118 ± 1.3 
                 9 
                 6.19E+04 
                 1.76E−02 
                 2.84E−07 
                 0.7 
               
               
                 bsAb21237D 
                 249 ± 0.6 
                 27 
                 2.81E+04 
                 4.24E−03 
                 1.51E−07 
                 2.7 
               
               
                 Isotype Control 
                 536 ± 1.4 
                 3 
                 NB* 
                 NB* 
                 NB* 
                 NB* 
               
               
                   
               
               
                 *NB indicates that no binding was observed under the current experimental conditions. 
               
               
                   # IC indicates that observed binding signal was less than three-fold above to the non-specific binding observed for isotype control antibody surface and/or the data cannot be used to measure binding kinetic parameters. 
               
            
           
         
       
     
     At 25° C., 21 out of 22 anti-LEPR/GP130 bispecific antibodies of the invention bound to mfLEPR-MMH with K D  values ranging from 2.63 nM to 583 nM, as shown in Table 23. At 37° C., 20 out of 22 anti-LEPR/GP130 bispecific antibodies of the invention bound to mfLEPR-MMH with K D  values ranging from 5.05 nM to 284 nM, as shown in Table 24. 
     
       
         
           
               
             
               
                 TABLE 25 
               
             
            
               
                   
               
               
                 Binding kinetics parameters of hGP130-MMH binding 
               
               
                 to anti-LEPR/GP130 bispecific antibodies at 25° C. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 100 nM 
                   
                   
                   
                   
               
               
                   
                 mAb 
                 hGP130- 
               
               
                   
                 Capture 
                 MMH 
               
               
                 Bispecific 
                 Level 
                 Bound 
                 k a   
                 k d   
                 K D   
                 t½ 
               
               
                 Antibody 
                 (RU) 
                 (RU) 
                 (1/Ms) 
                 (1/s) 
                 (M) 
                 (min) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 bsAb19139D 
                 168 ± 0.3 
                 6 
                 IC #   
                 IC #   
                 IC #   
                 IC #   
               
               
                 bsAb19140D 
                 182 ± 0.3 
                 23 
                 4.55E+05 
                 4.77E−02 
                 1.05E−07 
                 0.2 
               
               
                 bsAb19141D 
                 183 ± 0.5 
                 15 
                 1.06E+05 
                 2.32E−02 
                 2.19E−07 
                 0.5 
               
               
                 bsAb19142D 
                 184 ± 0.8 
                 31 
                 1.73E+05 
                 3.29E−03 
                 1.90E−08 
                 4 
               
               
                 bsAb19143D 
                 178 ± 0.6 
                 16 
                 3.20E+04 
                 6.47E−04 
                 2.02E−08 
                 18 
               
               
                 bsAb19144D 
                 183 ± 1   
                 27 
                 6.43E+04 
                 7.77E−04 
                 1.21E−08 
                 15 
               
               
                 bsAb19145D 
                 176 ± 0.3 
                 19 
                 3.41E+04 
                 5.98E−04 
                 1.75E−08 
                 19 
               
               
                 bsAb19146D 
                 190 ± 0.6 
                 31 
                 6.09E+05 
                 2.73E−04 
                 4.48E−10 
                 42 
               
               
                 bsAb19147D 
                 178 ± 0.3 
                 7 
                 IC #   
                 IC #   
                 IC #   
                 IC #   
               
               
                 bsAb19148D 
                 186 ± 0.6 
                 6 
                 IC #   
                 IC #   
                 IC #   
                 IC #   
               
               
                 bsAb19149D 
                 178 ± 0.5 
                 7 
                 IC #   
                 IC #   
                 IC #   
                 IC #   
               
               
                 bsAb19150D 
                 183 ± 0.8 
                 47 
                 3.42E+05 
                 4.80E−03 
                 1.40E−08 
                 2.4 
               
               
                 bsAb19151D 
                 168 ± 0.4 
                 9 
                 2.11E+04 
                 2.21E−04 
                 1.05E−08 
                 52 
               
               
                 bsAb19152D 
                 184 ± 1.1 
                 35 
                 1.96E+05 
                 1.13E−02 
                 5.74E−08 
                 1.0 
               
               
                 bsAb19153D 
                 179 ± 0.3 
                 24 
                 2.58E+05 
                 4.19E−04 
                 1.62E−09 
                 28 
               
               
                 bsAb19154D 
                 180 ± 0.5 
                 23 
                 8.02E+04 
                 2.04E−04 
                 2.54E−09 
                 57 
               
               
                 bsAb19155D 
                 176 ± 0.3 
                 7 
                 IC #   
                 IC #   
                 IC #   
                 IC #   
               
               
                 bsAb19156D 
                 182 ± 0.4 
                 27 
                 8.95E+04 
                 2.21E−03 
                 2.47E−08 
                 5 
               
               
                 bsAb19757D 
                 176 ± 0.4 
                 16 
                 1.12E+05 
                 2.18E−02 
                 1.96E−07 
                 0.5 
               
               
                 bsAb19758D 
                 177 ± 1.2 
                 51 
                 3.13E+05 
                 4.57E−03 
                 1.46E−08 
                 2.5 
               
               
                 bsAb21236D 
                 183 ± 0.4 
                 12 
                 3.03E+04 
                 4.26E−03 
                 1.40E−07 
                 2.7 
               
               
                 bsAb21237D 
                 199 ± 0.5 
                 17 
                 4.96E+04 
                 2.74E−03 
                 5.52E−08 
                 4 
               
               
                 Isotype Control 
                 175 ± 0.3 
                 1 
                 NB* 
                 NB* 
                 NB* 
                 NB* 
               
               
                   
               
               
                 *NB indicates that no binding was observed under the current experimental conditions. 
               
               
                   # IC indicates that observed binding signal was less than three-fold above to the non-specific binding observed for isotype control antibody surface and/or the data cannot be used to measure binding kinetic parameters. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 26 
               
             
            
               
                   
               
               
                 Binding kinetics parameters of hGP130-MMH binding 
               
               
                 to anti-LEPR/GP130 bispecific antibodies at 37° C. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 100 nM 
                   
                   
                   
                   
               
               
                   
                 mAb 
                 hGP130- 
               
               
                   
                 Capture 
                 MMH 
               
               
                 Bispecific 
                 Level 
                 Bound 
                 k a   
                 k d   
                 K D   
                 t½ 
               
               
                 Antibody 
                 (RU) 
                 (RU) 
                 (1/Ms) 
                 (1/s) 
                 (M) 
                 (min) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 bsAb19139D 
                 209 ± 0.4 
                 3 
                 NB* 
                 NB* 
                 NB* 
                 NB* 
               
               
                 bsAb19140D 
                 230 ± 0.5 
                 19 
                 8.29E+05 
                 1.20E−01 
                 1.44E−07 
                 0.1 
               
               
                 bsAb19141D 
                 229 ± 0.5 
                 15 
                 1.79E+05 
                 6.21E−02 
                 3.47E−07 
                 0.2 
               
               
                 bsAb19142D 
                 226 ± 0.8 
                 37 
                 3.56E+05 
                 6.22E−03 
                 1.75E−08 
                 1.9 
               
               
                 bsAb19143D 
                 219 ± 0.5 
                 27 
                 4.76E+04 
                 2.12E−03 
                 4.46E−08 
                 5 
               
               
                 bsAb19144D 
                 231 ± 1.9 
                 42 
                 1.77E+05 
                 1.88E−03 
                 1.06E−08 
                 6 
               
               
                 bsAb19145D 
                 223 ± 0.8 
                 30 
                 6.92E+04 
                 2.22E−03 
                 3.20E−08 
                 5 
               
               
                 bsAb19146D 
                 240 ± 0.6 
                 53 
                 7.12E+05 
                 6.41E−04 
                 9.01E−10 
                 18 
               
               
                 bsAb19147D 
                 217 ± 0.4 
                 5 
                 IC #   
                 IC #   
                 IC #   
                 IC #   
               
               
                 bsAb19148D 
                 231 ± 0.6 
                 8 
                 IC #   
                 IC #   
                 IC #   
                 IC #   
               
               
                 bsAb19149D 
                 222 ± 0.6 
                 8 
                 IC #   
                 IC #   
                 IC #   
                 IC #   
               
               
                 bsAb19150D 
                 223 ± 1.2 
                 52 
                 4.33E+05 
                 6.65E−03 
                 1.54E−08 
                 1.7 
               
               
                 bsAb19151D 
                 206 ± 0.4 
                 16 
                 2.24E+04 
                 3.73E−04 
                 1.66E−08 
                 31 
               
               
                 bsAb19152D 
                 234 ± 0.8 
                 28 
                 4.07E+05 
                 3.63E−02 
                 8.91E−08 
                 0.3 
               
               
                 bsAb19153D 
                 224 ± 0.3 
                 33 
                 3.19E+05 
                 1.26E−03 
                 3.95E−09 
                 9 
               
               
                 bsAb19154D 
                 223 ± 0.3 
                 37 
                 2.69E+05 
                 2.61E−04 
                 9.73E−10 
                 44 
               
               
                 bsAb19155D 
                 214 ± 0.6 
                 4 
                 NB* 
                 NB* 
                 NB* 
                 NB* 
               
               
                 bsAb19156D 
                 222 ± 0.7 
                 24 
                 8.39E+04 
                 7.97E−03 
                 9.50E−08 
                 1.4 
               
               
                 bsAb19757D 
                 216 ± 0.8 
                 16 
                 1.78E+05 
                 6.35E−02 
                 3.58E−07 
                 0.2 
               
               
                 bsAb19758D 
                 216 ± 1.3 
                 50 
                 4.48E+05 
                 7.47E−03 
                 1.67E−08 
                 1.5 
               
               
                 bsAb21236D 
                 220 ± 2.1 
                 12 
                 5.97E+04 
                 1.48E−02 
                 2.48E−07 
                 0.8 
               
               
                 bsAb21237D 
                 263 ± 1   
                 19 
                 6.11E+04 
                 8.79E−03 
                 1.44E−07 
                 1.3 
               
               
                 Isotype Control 
                 220 ± 0.7 
                 4 
                 NB* 
                 NB* 
                 NB* 
                 NB* 
               
               
                   
               
               
                 *NB indicates that no binding was observed under the current experimental conditions. 
               
               
                   # IC indicates that observed binding signal was less than three-fold above to the non-specific binding observed for isotype control antibody surface and/or the data cannot be used to measure binding kinetic parameters. 
               
            
           
         
       
     
     At 25° C., 17 out of 22 anti-LEPR/GP130 bispecific antibodies of the invention bound to hGP130-MMH with K D  values ranging from 448 pM to 219 nM, as shown in Table 25. At 37° C., 17 out of 22 anti-LEPR/GP130 bispecific antibodies bound to hGP130-MMH with K D  values ranging from 901 pM to 358 nM, as shown in Table 26. 
     
       
         
           
               
             
               
                 TABLE 27 
               
             
            
               
                   
               
               
                 Binding kinetics parameters of mfGP130-MMH binding 
               
               
                 to anti-LEPR/GP130 bispecific antibodies at 25° C. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 100 nM 
                   
                   
                   
                   
               
               
                   
                 mAb 
                 mfGP130- 
               
               
                   
                 Capture 
                 MMH 
               
               
                 Bispecific 
                 Level 
                 Bound 
                 k a   
                 k d   
                 K D   
                 t½ 
               
               
                 Antibody 
                 (RU) 
                 (RU) 
                 (1/Ms) 
                 (1/s) 
                 (M) 
                 (min) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 bsAb19139D 
                 168 ± 0.3 
                 6 
                 IC #   
                 IC #   
                 IC #   
                 IC #   
               
               
                 bsAb19140D 
                 182 ± 0.5 
                 20 
                 4.02E+05 
                 5.46E−02 
                 1.36E−07 
                 0.2 
               
               
                 bsAb19141D 
                 183 ± 0.6 
                 15 
                 1.12E+05 
                 2.07E−02 
                 1.84E−07 
                 0.6 
               
               
                 bsAb19142D 
                 183 ± 0.9 
                 28 
                 1.20E+05 
                 3.74E−03 
                 3.12E−08 
                 3.1 
               
               
                 bsAb19143D 
                 177 ± 0.7 
                 10 
                 3.01E+04 
                 1.30E−03 
                 4.30E−08 
                 9 
               
               
                 bsAb19144D 
                 183 ± 1.3 
                 27 
                 5.67E+04 
                 7.62E−04 
                 1.34E−08 
                 15 
               
               
                 bsAb19145D 
                 175 ± 0.3 
                 18 
                 3.31E+04 
                 5.76E−04 
                 1.74E−08 
                 20 
               
               
                 bsAb19146D 
                 190 ± 0.4 
                 31 
                 5.02E+05 
                 2.58E−04 
                 5.13E−10 
                 45 
               
               
                 bsAb19147D 
                 177 ± 0.3 
                 5 
                 IC #   
                 IC #   
                 IC #   
                 IC #   
               
               
                 bsAb19148D 
                 186 ± 0.6 
                 6 
                 IC #   
                 IC #   
                 IC #   
                 IC #   
               
               
                 bsAb19149D 
                 178 ± 0.8 
                 6 
                 IC #   
                 IC #   
                 IC #   
                 IC #   
               
               
                 bsAb19150D 
                 183 ± 1   
                 44 
                 3.08E+05 
                 5.19E−03 
                 1.68E−08 
                 2.2 
               
               
                 bsAb19151D 
                 168 ± 0.5 
                 3 
                 NB* 
                 NB* 
                 NB* 
                 NB* 
               
               
                 bsAb19152D 
                 184 ± 0.7 
                 52 
                 2.95E+05 
                 5.53E−03 
                 1.88E−08 
                 2.1 
               
               
                 bsAb19153D 
                 178 ± 0.4 
                 23 
                 2.29E+05 
                 3.96E−04 
                 1.73E−09 
                 29 
               
               
                 bsAb19154D 
                 180 ± 0.5 
                 22 
                 7.23E+04 
                 2.02E−04 
                 2.79E−09 
                 57 
               
               
                 bsAb19155D 
                 175 ± 0.3 
                 7 
                 9.85E+04 
                 1.84E−02 
                 1.86E−07 
                 0.6 
               
               
                 bsAb19156D 
                 182 ± 0.4 
                 30 
                 7.33E+04 
                 1.62E−03 
                 2.21E−08 
                 7 
               
               
                 bsAb19757D 
                 175 ± 0.5 
                 15 
                 1.08E+05 
                 2.04E−02 
                 1.88E−07 
                 0.6 
               
               
                 bsAb19758D 
                 176 ± 1.7 
                 46 
                 3.07E+05 
                 5.07E−03 
                 1.65E−08 
                 2.3 
               
               
                 bsAb21236D 
                 240 ± 0.6 
                 21 
                 2.94E+04 
                 3.05E−03 
                 1.04E−07 
                 3.8 
               
               
                 bsAb21237D 
                 212 ± 0.6 
                 18 
                 3.98E+04 
                 2.13E−03 
                 5.34E−08 
                 5 
               
               
                 Isotype Control 
                 175 ± 0.5 
                 1 
                 NB* 
                 NB* 
                 NB* 
                 NB* 
               
               
                   
               
               
                 *NB indicates that no binding was observed under the current experimental conditions. 
               
               
                   # IC indicates that observed binding signal was less than three-fold above to the non-specific binding observed for isotype control antibody surface and/or the data cannot be used to measure binding kinetic parameters. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 28 
               
             
            
               
                   
               
               
                 Binding kinetics parameters of mfGP130-MMH binding 
               
               
                 to anti-LEPR/GP130 bispecific antibodies at 37° C. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 100 nM 
                   
                   
                   
                   
               
               
                   
                 mAb 
                 mfGP130- 
               
               
                   
                 Capture 
                 MMH 
               
               
                 Bispecific 
                 Level 
                 Bound 
                 k a   
                 k d   
                 K D   
                 t½ 
               
               
                 Antibody 
                 (RU) 
                 (RU) 
                 (1/Ms) 
                 (1/s) 
                 (M) 
                 (min) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 bsAb19139D 
                 208 ± 0.4 
                 2 
                 NB* 
                 NB* 
                 NB* 
                 NB* 
               
               
                 bsAb19140D 
                 229 ± 0.5 
                 14 
                 7.97E+05 
                 1.46E−01 
                 1.83E−07 
                 0.1 
               
               
                 bsAb19141D 
                 228 ± 0.5 
                 15 
                 2.42E+05 
                 5.13E−02 
                 2.12E−07 
                 0.2 
               
               
                 bsAb19142D 
                 226 ± 1.4 
                 33 
                 3.32E+05 
                 7.15E−03 
                 2.16E−08 
                 1.6 
               
               
                 bsAb19143D 
                 217 ± 1.1 
                 14 
                 6.83E+04 
                 3.96E−03 
                 5.79E−08 
                 2.9 
               
               
                 bsAb19144D 
                 230 ± 1.3 
                 41 
                 1.68E+05 
                 1.83E−03 
                 1.09E−08 
                 6 
               
               
                 bsAb19145D 
                 222 ± 0.5 
                 29 
                 5.88E+04 
                 2.21E−03 
                 3.76E−08 
                 5 
               
               
                 bsAb19146D 
                 239 ± 0.5 
                 53 
                 6.53E+05 
                 6.26E−04 
                 9.59E−10 
                 18 
               
               
                 bsAb19147D 
                 216 ± 0.4 
                 3 
                 NB* 
                 NB* 
                 NB* 
                 NB* 
               
               
                 bsAb19148D 
                 231 ± 0.5 
                 8 
                 IC #   
                 IC #   
                 IC #   
                 IC #   
               
               
                 bsAb19149D 
                 221 ± 0.8 
                 8 
                 IC #   
                 IC #   
                 IC #   
                 IC #   
               
               
                 bsAb19150D 
                 222 ± 1.3 
                 46 
                 4.01E+05 
                 7.73E−03 
                 1.93E−08 
                 1.5 
               
               
                 bsAb19151D 
                 209 ± 7.1 
                 6 
                 NB* 
                 NB* 
                 NB* 
                 NB* 
               
               
                 bsAb19152D 
                 233 ± 1   
                 51 
                 3.42E+05 
                 1.77E−02 
                 5.16E−08 
                 0.7 
               
               
                 bsAb19153D 
                 223 ± 0.3 
                 32 
                 2.84E+05 
                 1.23E−03 
                 4.33E−09 
                 9 
               
               
                 bsAb19154D 
                 223 ± 0.4 
                 35 
                 2.19E+05 
                 3.43E−04 
                 1.57E−09 
                 34 
               
               
                 bsAb19155D 
                 213 ± 0.4 
                 3 
                 NB* 
                 NB* 
                 NB* 
                 NB* 
               
               
                 bsAb19156D 
                 221 ± 0.6 
                 26 
                 8.75E+04 
                 7.67E−03 
                 8.76E−08 
                 1.5 
               
               
                 bsAb19757D 
                 215 ± 0.9 
                 14 
                 1.33E+05 
                 5.98E−02 
                 4.50E−07 
                 0.2 
               
               
                 bsAb19758D 
                 215 ± 1   
                 45 
                 4.02E+05 
                 7.30E−03 
                 1.82E−08 
                 1.6 
               
               
                 bsAb21236D 
                 285 ± 2.1 
                 18 
                 6.98E+04 
                 1.25E−02 
                 1.79E−07 
                 0.9 
               
               
                 bsAb21237D 
                 270 ± 1   
                 21 
                 3.69E+04 
                 7.92E−03 
                 2.15E−07 
                 1.5 
               
               
                 Isotype Control 
                 219 ± 0.5 
                 4 
                 NB* 
                 NB* 
                 NB* 
                 NB* 
               
               
                   
               
               
                 *NB indicates that no binding was observed under the current experimental conditions. 
               
               
                   # IC indicates that observed binding signal was less than three-fold above to the non-specific binding observed for isotype control antibody surface and/or the data cannot be used to measure binding kinetic parameters. 
               
            
           
         
       
     
     At 25° C., 17 out of 22 LEPR×GP130 bispecific antibodies of the invention bound to mfGP130-MMH with K D  values ranging from 513pnM to 188 nM, as shown in Table 27. At 37° C., 16 out of 22 LEPR×GP130 bispecific antibodies of the invention bound to mfGP130-MMH with K D  values ranging from 959 pM to 450 nM, as shown in Table 28. 
     Example 11: LEPR×GP130 Bispecific Antibody Cell Binding Measured by FACS Analysis 
     In order to assess cell binding by LEPR×GP130 bispecific antibodies, HEK293 stable cell lines were generated. One cell line was generated to stably over-express full length human GP130 (amino acids 1-918 of accession # P40189 with leucine at position 2 changed to valine, a natural variant) along with a luciferase reporter (Stat3-luciferase, Stat3-luc, SA Bioscience, #CLS-6028L), and was sorted twice using flow cytometry for high expression of GP130. This cell line is referred to hereafter as “HEK293/Stat3-luc/gp130-2X Sort.” Another cell line used in this Example, known hereafter as “HEK293/hLEPR-GPI,” stably expresses the extracellular domain of human LEPR (amino acids 22-839 of accession # P48357, Isoform B) with an N-terminal myc-myc tag and C-terminal peptide sequence from human carboxypeptidase M that guides the addition of GPI (Glycosylphosphatidylinositol) such that the protein can be GPI-anchored to the membrane. 
     For the FACS analysis, 0.5×106 cells/well of HEK293 parental cells, HEK293/Stat3-luc/gp130-2X Sort cells, and HEK293/hLEPR-GPI cells, were incubated with 200 nM of the conventional antibodies against either GP130 or LEPR or with LEPR×GP130 bispecific antibodies, along with isotype control antibodies at 4° C. in PBS (without calcium and magnesium) containing 2% FBS. 
     To test whether the anti-LEPR antibody binding or LEPR×GP130 bispecific antibody binding to cells was affected by the presence of Leptin, 1 pM human Leptin (R&amp;D Systems, #398-LP) was incubated with the cells for 30 minutes, followed by the addition of anti-LEPR antibodies or isotype control antibody. After incubation with primary antibodies, the cells were stained with 8 mg/mL of Alexa Fluor®-647 conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc., #109-607-003) for 30 minutes. Cells were fixed using BD CytoFix™ (Becton Dickinson, #554655) and analyzed on an IQue® (Intellicyt) Flow Cytometer. Unstained and secondary antibody alone controls were also tested for all cell lines. The results were analyzed using ForeCyt® (IntelliCyt) and FlowJo version 10 softwares to determine the geometric means of fluorescence for viable cells. 
     As shown in Table 29, two LEPR×GP130 bispecific antibodies of the invention tested at 200 nM demonstrated binding to HEK293/gp130 2× Sort cells with binding ratios of 132- and 169-fold and binding to HEK293/hLEPR-GPI cells with binding ratios of 4423- and 6320-fold without Leptin, and 3596- and 5932-fold in the presence of 1 pM Leptin. The GP130-binding arm of the bispecific antibodies of the invention made as a conventional antibody (mAb16683) demonstrated binding to HEK293/gp130 2× Sort cells with a binding ratio of 235-fold and binding to HEK293/hLEPR-GPI cells with a binding ratio of 21-fold. The LEPR binding arms of the bispecifics (mAb18445 and mAb18446) made as a conventional antibody demonstrated binding to HEK293/hLEPR-GPI cells with binding ratios of 4711- and 7023-fold without Leptin, and 4246- and 6390-fold in the presence of 1 pM Leptin. The anti-GP130 and anti-LEPR conventional and bispecific antibodies demonstrated binding to the HEK293 parental cells with binding ratios ranging from 3- to 24-fold. The isotype control antibodies and secondary antibodies alone samples also did not demonstrate significant binding to any of the cell lines tested with or without Leptin, with binding ratios ranging from 1- to 3-fold. 
     
       
         
           
               
             
               
                 TABLE 29 
               
             
            
               
                   
               
               
                 Antibody Binding to Cells Assessed by FACS 
               
            
           
           
               
               
            
               
                   
                 Binding Ratio: 
               
               
                   
                 Normalized to Unstained Sample of Each Cell Line (FL4-A) 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 HEK293/hLEPR- 
                 HEK293/hLEPR- 
               
               
                   
                 HEK293 
                 HEK293/ 
                 GPI cells (No 
                 GPI cells (1 μM 
               
               
                 Antibody 
                 parental 
                 gp130 2X Sort 
                 Leptin) 
                 Leptin) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 bsAb21236 
                 13 
                 132 
                 4423 
                 3596 
               
               
                 (LEPR × GP130) 
               
               
                 bsAb21237 
                 24 
                 169 
                 6320 
                 5932 
               
               
                 (LEPR × GP130) 
               
               
                 mAb16683 
                 17 
                 235 
                 21 
                 Not Tested 
               
               
                 (anti-GP130 mAb) 
               
               
                 mAB18445 
                 3 
                 Not Tested 
                 4711 
                 4246 
               
               
                 (anti-LEPR mAb) 
               
               
                 mAB18446 
                 6 
                 Not Tested 
                 7023 
                 6390 
               
               
                 (anti-LEPR mAb) 
               
               
                 Isotype control 
                 2 
                 2 
                 3 
                 3 
               
               
                 antibody 
               
               
                 Secondary antibody 
                 1 
                 1 
                 3 
                 2 
               
               
                 alone 
               
               
                 No Antibody 
                 1 
                 1 
                 1 
                 Not Tested 
               
               
                   
               
            
           
         
       
     
     Example 12: Functional Cell-Based Assays 
     The cytokine receptors GP130 (amino acids 1-918 of accession # P40189) and LEPR (amino acids 1-1165 of accession # P48357) have a non-covalently associated tyrosine kinase, JAK2, bound to the membrane proximal region of their cytoplasmic domains. Treatment with cognate ligand or agonist antibody culminates in activation of JAK2, which in turn phosphorylates key tyrosine residues on the cytoplasmic region of the receptor. The phosphorylated tyrosine residues serve as docking sites for signaling complexes that upon phosphorylation lead to stimulation of signaling pathways such as STAT3 and ERK. For the LEPR, tyrosine residue Y1141 mediates STAT3 signaling and mutation of this residue to phenylalanine (Y1141F) eliminates STAT3 signaling (Carpenter et al., 1998, Proc. Natl. Acad. Sci. USA 95:6061-6066). 
     A bioassay was developed to detect the transcriptional activation of STAT3 via the promotion of GP130 and LEPR heterodimerization following treatment with LEPR×GP130 bsAbs. In particular, a reporter cell line that stably expresses mutant human LEPR (Y1141F) and wild-type human GP130, along with a STAT3 responsive luciferase reporter (STAT3-Luc; Qiagen CLS-6028L) was generated. The resulting stable cell line, referred to as HEK293.STAT3.Luc.GP130.hLEPR (Y1141F), was isolated and maintained in DME medium supplemented with 10% FBS, 1 ug/mL Puromycin, 250 ug/mL of Hygromycin B, 500 ug/mL of G418 and Penicillin/Streptomycin/L-Glutamine. Two LEPR×GP130 bispecific antibodies were identified in this bioassay, bsAb21236 and bsAb21237, which promoted STAT3 activity in the presence of leptin. 
     For the bioassay, HEK293.STAT3.Luc.GP130.hLEPR (Y1141F) cells were plated at a density of 20,000 cells/well and then the following day the media was replaced with 80 uL of Opti-MEM supplemented with 1% BSA and 0.1% FBS (Assay Buffer). Subsequently, 10 uL of fixed-concentration of 10 nM of human Leptin (hLeptin; R&amp;D Systems, #398-LP-01M) was added to the wells. Immediately following the hLeptin treatment, the bispecific antibodies were half-log serially diluted (12 points) to final concentrations ranging from 500 nM to 5 pM in Assay Buffer and were then added to the cells. The isotype control and human OSM (hOSM; R&amp;D Systems, #295-OM/CF) were half-log diluted (11 points) to final concentrations ranging from 100 nM to 1 pM in Assay Buffer and were then added to the cells. The plates were then placed in the incubator overnight at 37° C. in 5% CO 2 . One-Glo reagent (Promega, #E6051) was then added to the samples and luciferase activity was measured on Envision Multilable Plate Reader (Perkin Elmer) in Luminescent mode. The relative light units (RLU) values were obtained and the results were analyzed using nonlinear regression with GraphPad Prism software (Graph Pad). The maximum RLU value obtained from the hOSM dose response was defined as 100% activation in the HEK293.STAT3.Luc.GP130.hLepR (Y1141F) cell-based assay. 
     The ability of LEPR×GP130 bispecific antibodies to activate via GP130-mediated cell signaling was evaluated in the HEK293.STAT3.Luc.gp130.hLepR (Y1141F) cell-based assay and the resulting EC 50  values and percentage activation are shown in Table 30. The responsiveness of the cell line was confirmed using a dose response of hOSM, which demonstrated activation in the assay with an EC 50  value of 592 pM. Both LEPR×GP130 bispecific antibodies tested, bsAb21236 and bsAb21237, demonstrated activation in this assay with EC 50  values of 2.11 nM and 2.46 nM, respectively. Both bispecific antibodies have approximately 20% of maximal activation observed with hOSM. 
     
       
         
           
               
             
               
                 TABLE 30 
               
             
            
               
                   
               
               
                 Activation of HEK293.STAT3.Luc.GP130.hLEPR (Y1141F) Cell 
               
               
                 line by LEPR × GP130 Bispecific Antibodies 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Percentage activation as 
               
               
                   
                 Molecule tested 
                 EC 50  (M) 
                 compared to hOSM 
               
               
                   
                   
               
               
                   
                 bsAb21236 
                 2.109E−09 
                 25% 
               
               
                   
                 bsAb21237 
                 2.460E−09 
                 20% 
               
               
                   
                 hOSM 
                 5.919E−10 
                 100%  
               
               
                   
                 Isotype control 
                 No activation 
                 No activation 
               
               
                   
                 antibody 
               
               
                   
                   
               
            
           
         
       
     
     To confirm that the activation by LEPR×GP130 bispecific antibodies in the HEK293.STAT3.Luc.GP130.hLEPR (Y1141F) cell-based assay was due to activation through both LEPR and GP130, a competition bioassay was performed using soluble LEPR and GP130 proteins to block the activation by the bispecific antibodies. The competition bioassay utilized an excess fixed concentration, 500 nM, of the extracellular domain of human LEPR with a C-terminal hFc tag (hLEPR-hFc; SEQ ID NO:189), the extracellular domain of human LEPR with a C-terminal myc-myc-hexahistidine tag (hLEPR-MMH; SEQ ID NO:187), the extracellular domain of human GP130 with a C-terminal hFc tag (hGP130-hFc; SEQ ID NO:197), and the extracellular domain of human CNTFR with a C-terminal myc-myc-hexahistidine tag (hCNTFR-MMH; SEQ ID NO:198). 
     For the assay, HEK293.STAT3.Luc.GP130.hLEPR (Y1141F) cells were plated at the density of 20,000 cells/well and then the following day the media was replaced with 70 uL of Opti-MEM supplemented with 1% BSA and 0.1% FBS (Assay Buffer). 10 uL of fixed-concentration of 10 nM of human Leptin (hLeptin; R&amp;D Systems, #398-LP-01M) was added to the wells. Immediately following the hLeptin treatment, 10 nM of the bispecific antibodies in Assay Buffer were then added to the cells. Immediately after, an excess amount, 500 nM, of the soluble proteins hLEPR-hFc, hLEPR-MMH, hGP130-hFc, and hCNTFR-MMH were added to the appropriate designated wells. The plates were then placed in the incubator overnight at 37° C. in 5% CO2. One-Glo reagent (Promega, #E6051) was then added to the samples and luciferase activity was measured on Envision Multilable Plate Reader (Perkin Elmer) in Luminescent mode. The relative light units (RLU) values were obtained and the results were analyzed using GraphPad Prism software (GraphPad). 
     The competition assay result demonstrated that soluble hLEPR-hFc, hLEPR-MMH and hGP130-hFc were able to block the bispecific antibody activity whereas soluble hCNTFR.mmh did not block the activity of the bispecific antibodies. The activity of the bispecific antibodies alone is defined as 100% whereas the activity of isotype control represents 0% activity. 
     Table 31, below shows activation of HEK293.STAT3.Luc.GP130.hLEPR cells by bispecific antibodies in the presence of soluble human LEPR, GP130 and CNTFR. Table 32 shows RLU production in the presence of hLEPR-MMH, hLEPR-hFc, hGP130-hFc or hCNTFR-MMH. 
     
       
         
           
               
             
               
                 TABLE 31 
               
             
            
               
                   
               
               
                 Activation of HEK293.STAT3.Luc.GP130.hLEPR (Y1141F) cells by LEPR × GP130 
               
               
                 bispecific antibodies in the presence of soluble human LEPR, GP130, and CNTFR 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 RLU in the 
                 RLU in the 
                 RLU in the 
                 RLU in the 
                   
               
               
                   
                 presence of 
                 presence of 
                 presence of 
                 presence of 
               
               
                 Bispecific 
                 hLEPR-MMH 
                 hLEPR-hFc 
                 hGP130-hFc 
                 hCNTFR-MMH 
               
               
                 Antibody 
                 [500 nM] 
                 [500 nM] 
                 [500 nM] 
                 [500 nM] 
                 Assay Buffer 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 bsAb21236 
                 12840 
                 13160 
                 11640 
                 10320 
                 15520 
                 13840 
                 48560 
                 43080 
                 48920 
                 42160 
               
               
                 bsAb21237 
                 11280 
                 11800 
                 10480 
                 9480 
                 14320 
                 15680 
                 39920 
                 43400 
                 44920 
                 38080 
               
               
                 Isotype control 
                   
                   
                   
                   
                   
                   
                   
                   
                 12160 
                 12080 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 32 
               
             
            
               
                   
               
               
                 Activation of HEK293.STAT3.Luc.GP130.hLEPR (Y1141F) cells by LEPR × GP130 
               
               
                 bispecific antibodies in the presence of soluble human LEPR, GP130, and CNTFR 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 RLU in the 
                 RLU in the 
                 RLU in the 
                 RLU in the 
                   
               
               
                   
                 presence of 
                 presence of 
                 presence of 
                 presence of 
               
               
                 Bispecific 
                 hLEPR-MMH 
                 hLEPR-hFc 
                 hGP130-hFc 
                 hCNTFR-MMH 
               
               
                 Antibody 
                 [500 nM] 
                 [500 nM] 
                 [500 nM] 
                 [500 nM] 
                 Assay Buffer 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 bsAb21236 
                 3% 
                 −3% 
                 8% 
                 101% 
                 100% 
               
               
                 bsAb21237 
                 −2% 
                 −7% 
                 10% 
                 101% 
                 100% 
               
               
                 Isotype 
                   
                   
                   
                   
                 0% 
               
               
                 control 
               
               
                   
               
            
           
         
       
     
     Example 13: In Vivo Efficacy of LEPR×GP130 Bispecific Antibodies bsAb21236 and bsAb21237 in Diet-Induced Obese Mice 
     The effects of two LEPR×GP130 bispecific antibodies of the invention, bsAb21236 and bsAb21237, on body weight were determined in an in vivo model using high fat diet fed obese LEPRHu/Hu;IL6STHu/Hu mice, that express a leptin receptor composed of the human LEPR extracellular domain sequence in place of the murine LEPR extracellular domain sequence and a GP130 protein composed of the human IL6ST extracellular domain sequence in place of the murine IL6ST extracellular domain sequence. 
     On day 0, twenty-three male LEPRHu/Hu;IL6STHu/Hu mice that were fed a high fat diet for 12 weeks were randomized into three groups of 7 to 8 mice based on body weight. On day 0 and 7, each group received via subcutaneous injection a dose of either isotype control antibody at 30 mg/kg, bsAb21236 at 30 mg/kg, or bsAb21237 at 30 mg/kg. The isotype control antibody used does not bind any known mouse protein. The body weight of each mouse was measured daily for the duration of the study. The percent change in body weight from day 0 was calculated for each animal at every time point measured.  FIG. 1  summarizes the average percent change in body weight for animals in each treatment group. All results are expressed as mean±SEM. 
     As shown in  FIG. 1 , LEPRHu/Hu;IL6STHu/Hu mice treated with bsAb21236 at 30 mg/kg exhibited significant reductions in percent body weight change starting at three days post antibody treatment and at the other subsequent time points measured compared to mice injected with isotype control antibody. LEPRHu/Hu;IL6STHu/Hu mice treated with bsAb21237 at 30 mg/kg exhibited a significant reduction in percent body weight change starting at five days post antibody treatment and at the other subsequent time points measured compared to mice injected with isotype control antibody. 
     Example 14: Transcriptional Activation of STAT3 Via the Promotion of GP130 and LEPR (Short Form) Heterodimerization Following Treatment with LEPR×GP130 bsAbs 
     GP130 serves a co-receptor for multiple cytokines and is expressed broadly in human tissues (Taga T., Kishimoto T. gp130 and the interleukin-6 family of cytokines. Annu. Rev. Immunol 1997; 15:797-819). Isoforms of the LEPR are generated through alternative splicing, resulting a long isoform b (LEPR-b) and several short forms, including isoform a (LEPR-a) which shows the highest and broadest expression pattern (Tartaglia L A. The leptin receptor. J Biol Chem 1997; 272: 6093-6096). All the isoforms share the same extracellular domain, transmembrane region and a short stretch of the cytoplasmic domain, containing the Box 1 region, followed by a variable region. The long form contains intracellular sequence motifs required for mediating all the signaling capabilities of leptin whereas the short forms are lacking these regions. Since the extracellular domain of the short forms is identical to the signaling competent long form, the bispecific antibodies can bind to the short forms and generate complexes with GP130. The primary intended target tissue for LEPR agonists in general (including LEPR×GP130 bispecific antigen binding molecules) is the brain where LEPR isoform b is predominantly expressed. Given the broad expression of GP130 and isoform a of LEPR, however, there existed the potential for unwanted STAT3 activation in tissues such as the liver. 
     In order to evaluate signaling outcomes resulting from complexing of LEPR short isoform a and GP130, a bioassay was developed to detect the transcriptional activation of STAT3 via the promotion of GP130 and LEPR (short form) heterodimerization following treatment with LEPR×GP130 bsAbs. In particular, a reporter cell line that stably expresses the dominant short form of LEPR (NP_001003679.1), to be referred to as hLEPR(a), and wild-type human GP130, along with a STAT3 responsive luciferase reporter (STAT3-Luc; Qiagen CLS-6028L) was generated. The resulting stable cell line, referred to as HEK293.STAT3.Luc.GP130.hLEPR(a), was isolated and maintained in DME medium supplemented with 10% FBS, 1 ug/mL Puromycin, 250 ug/mL of Hygromycin B, 500 ug/mL of G418 and Penicillin/Streptomycin/L-Glutamine. 
     For the bioassay, HEK293.STAT3.Luc.GP130.hLEPR (a) cells were plated at a density of 20,000 cells/well and then the following day the media was replaced with 80 uL of Opti-MEM supplemented with 1% BSA and 0.1% FBS (Assay Buffer). Subsequently, 10 uL of fixed-concentration of 10 nM of human Leptin (hLeptin; R&amp;D Systems, #398-LP-01 M) was added to the wells. Immediately following the hLeptin treatment, the bispecific antibodies were half-log serially diluted (12 points) to final concentrations ranging from 500 nM to 5 pM in Assay Buffer and were then added to the cells. As controls, human leptin and OSM (hOSM; R&amp;D Systems, #295-OM/CF) were half-log diluted (11 points) to final concentrations ranging from 100 nM to 1 pM in Assay Buffer and were then added to the cells. The plates were then placed in the incubator overnight at 37° C. in 5% CO 2 . One-Glo reagent (Promega, #E6051) was then added to the samples and luciferase activity was measured on Envision Multilable Plate Reader (Perkin Elmer) in Luminescent mode. The relative light units (RLU) values were obtained and the results were analyzed using nonlinear regression with GraphPad Prism software (GraphPad). The maximum RLU value obtained from the hOSM dose response was defined as 100% activation in the HEK293.STAT3.Luc.GP130.hLEPR (a) cell-based assay. 
     The ability of LEPR×GP130 bispecific antibodies to activate via GP130-mediated cell signaling was evaluated in the HEK293.STAT3.Luc.gp130.hLEPR (a) cell-based assay and the resulting responses are shown in Table 33. The responsiveness of the cell line was confirmed using a dose response of hOSM, which demonstrated activation in the assay with an EC 50  value of 121 pM and its maximum response was designated as 100% activation. Both LEPR×GP130 bispecific antibodies tested, bsAb21236 and bsAb21237, failed to activate STAT3 signaling with LEPR(a). Similar to leptin, the bispecific antigen-binding proteins of the present invention generate productive STAT3 signaling only in cell types containing the long form of LEPR. 
     
       
         
           
               
             
               
                 TABLE 33 
               
             
            
               
                   
               
               
                 Activation of HEK293.STAT3.Luc.GP130.hLEPR (a) Cell line 
               
               
                 by LEPR × GP130 Bispecific Antibodies 
               
            
           
           
               
               
               
            
               
                   
                   
                 Percentage maximum 
               
               
                   
                   
                 activation as compared 
               
               
                 Molecule tested 
                 EC 50  (M) 
                 to hOSM 
               
               
                   
               
               
                 bsAb21236 + 10 nM 
                 No activation 
                 No activation 
               
               
                 leptin 
               
               
                 bsAb21237 + 10 nM 
                 No activation 
                 No activation 
               
               
                 leptin 
               
               
                 hOSM 
                 1.21E−10 
                 100% 
               
               
                 hLeptin 
                 No activation 
                 No activation 
               
               
                   
               
            
           
         
       
     
     In summary, the data shows that the LEPR×GP130 bispecific antibodies provided herein do not activate signaling through the “short form” of the leptin receptor (LEPR-a isoform), but do activate signaling through the “long form” of the leptin receptor (LEPR-b isoform). The relevance of this finding is that it suggests that these bispecific antibodies will exert their activity primarily in the brain where the ‘b’ isoform is predominantly expressed, but not in other tissues such as the liver where the ‘a’ form is broadly expressed. Given that these bispecific antibodies can be used to treat obesity by activating LEPR signaling in the brain, this work confirms that the bispecific antibodies provided herein are effective at targeting leptin signaling where it is needed (in the brain) while avoiding unwanted signaling elsewhere in the body (such as the liver, e.g. in inflammatory hepatocellular adenoma (see Rebouissou et al., Nature Letters, 457(8): 200-205, 2009)). 
     The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.