Method for screening multispecific antibody

Abstract

Multispecific antibodies and methods of screening for such antibodies are provided.SOLUTION: The presently disclosed subject matter relates to multispecific antibodies, e.g., bispecific antibodies and biepitopic antibodies, and methods of screening for such antibodies, as well as novel antibody structures that can be used in screening for such bispecific antibodies and biepitopic antibodies. The present disclosure further provides bispecific anti-KLB antibodies and methods of treating diseases using these antibodies.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 252,549, filed November 8, 2015, which is incorporated herein in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on November 7, 2016, is titled "BB Sequence Listing.txt" and is 176,940 bytes in size.

[0003] The subject matter disclosed herein relates to multispecific antibodies, e.g., bispecific antibodies, and methods for screening for such antibodies. The subject matter disclosed herein further provides antibodies that bind to Klotho-beta, and methods for treating diseases using these antibodies. [Background technology]

[0004] Multispecific antibodies, such as bispecific antibodies, are important as research tools, diagnostic tools, and therapeutic agents. This is primarily due to the fact that such antibodies can be selected to bind with high specificity and affinity to two or more antigens or two or more epitopes present in an antigen. For example, in the case of cancer therapeutics, multispecific antibodies can be used to target cancer cells, for example, by binding antigens present on the cancer cells to immune cells, thereby eliciting an immune response. In addition, multispecific antibodies can be used as ligands for heterodimeric receptors, which are typically activated by their cognate ligands when they bind to the receptor and promote interaction between the receptor components.

[0005] Multispecific antibodies have previously been produced and identified by chemically linking fragments of antibodies, such as monoclonal antibodies, that retain the desired binding properties. This procedure requires generating and recovering specific antibody fragments, coupling the fragments using a cross-linking agent or other interacting moiety, and linking the antibody fragments to form heterodimers. Recombinant DNA techniques have also been used to coexpress two heavy-light chain pairs, in which the two heavy-light chains have different binding specificities. However, due to the random assortment of immunoglobulin heavy and light chains, this approach can produce a mixture of different antibody molecules, only a small fraction of which have the desired multispecific, e.g., bispecific, structure. For example, using these methods, homodimers can be produced, and heavy chains can pair with the wrong light chain, resulting in antibodies that do not have the desired specificity for the desired antigen or epitope. Therefore, there is a need in the art for improved methods for identifying and selecting multispecific antibodies, e.g., bispecific and bianepitopic antibodies, against a particular antigen and / or epitope of interest.

[0006] Multispecific antibodies, e.g., bispecific antibodies, have been found to be useful in functioning as receptor agonists (see, e.g., Weidle et al. (2013) Cancer Genomics Proteomics 10(1):1-18). One such receptor that can benefit from such antibodies is Klotho-beta (KLB). KLB is a 114-kDa type 1 transmembrane protein with a short intracellular domain and two extracellular glycosidase domains that lack the characteristic glutamic acid residue essential for enzymatic activity (Ito et al. (2000) Mech. Dev. 98:115-119). Among the seven major isoforms of fibroblast growth factor receptors (FGFRs) encoded by mammalian species (1b, 2b, 3b, 1c, 2c, 3c, and 4), FGFR1c, 2c, and 3c can interact with Klotho-beta, which acts as a co-receptor, to form functional receptor complexes for certain FGF ligands. For example, FGFR1c in complex with Klotho-beta appears to play a central role in mediating the metabolic effects of fibroblast growth factor 21 (FGF21) (Ogawa et al. (2007) Proc. Natl. Acad. Sci. USA 104(18):7432-37; US2010 / 0184665). FGF21 has been identified as a potent disease-modifying protein that reversed obesity and type 2 diabetes in animal disease models (Kharitonenkov et al. (2005) J. Clin. Invest. 115(6):1627-35). Recombinant FGF21 has been shown to reduce liver lipids, improve insulin sensitivity, and normalize glycemic control in leptin signaling-deficient (ob / ob or db / db) mice or mice fed a high-fat diet (HFD). Reductions in blood glucose levels and improvements in various cardiovascular risk factors have also been observed in obese and diabetic rhesus monkeys treated daily with recombinant FGF21.

[0007] Therapeutic agents targeting FGFR1c and KLB have become an area of ​​intense research. Antibody antagonists specific for FGFR1c have been reported to induce weight loss in mice and monkeys (WO 2005 / 037235), and selective activation of FGFR1c mediated by agonistic antibodies was sufficient to reproduce FGF21-induced insulin sensitization in diabetic mice (WO 2012 / 158704; Wu et al. (2011) Sci. Trans. Med. 3(113):1-10). Antibodies that bind to the KLB / FGFR1c complex have been proposed as activators / therapeutics (US Pat. No. 7,537,903; WO 2011 / 071783; WO 2012 / 158704). Other researchers have investigated two alternative approaches to selectively activate the FGFR1c / Klotho-beta complex. Foltz et al. (2012) Sci. Transl. Med. 4:162ra153 disclose a high-affinity anti-Klotho-beta antibody called mimAb1, and U.S. Patent No. 8,372,952 discloses a bispecific anti-FGFR1 / Klotho-beta avimer polypeptide C3201 linked to human serum albumin (HSA). Given the important role of Klotho-beta in glucose metabolism and metabolic diseases, there remains a need in the art to develop therapeutic antibodies and methods for modulating KLB activity. Summary of the Invention

[0008] The presently disclosed subject matter provides multispecific antibodies, e.g., bispecific and biepitopic antibodies, as well as methods for screening for such compounds. The present disclosure further provides methods for producing and analyzing such antibodies. In certain embodiments, the presently disclosed subject matter further relates to anti-KLB antibodies. Specifically, the present disclosure provides bispecific antibodies that specifically bind to at least two different epitopes present on KLB, and methods for treating disease in a subject using the bispecific antibodies, or a mixture of monospecific antibodies.

[0009] In certain embodiments, an isolated multispecific antibody, e.g., a bispecific antibody, of the present disclosure comprises a first antigen-binding polypeptide, which comprises, in an N-terminal to C-terminal direction, a VL domain, a linker, a VH domain, a CH1 domain, a CH2 domain, and a CH3 domain. In certain embodiments, the bispecific antibody is configured in a VLfH (variable light chain complete heavy chain) format. In certain embodiments, the components of the first antigen-binding polypeptide are arranged in an N-terminal to C-terminal direction in the order VL-linker-VH-CH1-CH2-CH3. In certain embodiments, the multispecific antibody does not comprise a CL domain. Alternatively, in certain embodiments, the multispecific antibody may further comprise a CL domain. In certain embodiments, the bispecific antibody is configured in a tcIgG (tri-chain IgG) format. In certain embodiments, the CL domain may be linked to the first antigen-binding polypeptide by one or more disulfide bridges. In certain embodiments, a disulfide bond between the CL domain and the first antigen-binding polypeptide links the CL domain to the CH1 domain of the first antigen-binding polypeptide.

[0010] In certain embodiments, a multispecific antibody, e.g., a bispecific antibody, of the present disclosure may further comprise a second antigen-binding polypeptide, which comprises, arranged from N- to C-terminus, a VL domain, a linker, a VH domain, a CH1 domain, a CH2 domain, and a CH3 domain. In certain embodiments, the components of the second antigen-binding polypeptide are arranged from N- to C-terminus in the following order: VL-linker-VH-CH1-CH2-CH3. In certain embodiments, the second antigen-binding polypeptide does not comprise a CL domain.

[0011] In certain embodiments, the multispecific antibody comprises two CL domains. In certain embodiments, the two CL domains are the same. In certain embodiments, one of the two CL domains is linked to a first antigen-binding polypeptide by one or more disulfide bridges, and the second of the two CL domains is linked to a second antigen-binding polypeptide by one or more disulfide bridges. In certain embodiments, disulfide bridges link the CL domain to the CH1 domains of the first and second antigen-binding polypeptides.

[0012] In certain embodiments, a multispecific antibody of the present disclosure comprises a first antigen-binding polypeptide and a second antigen-binding polypeptide, wherein the first and second antigen-binding polypeptides bind to two different epitopes on the same antigen. Alternatively, the first and second antigen-binding polypeptides bind to two different antigens. In certain embodiments, the multispecific antibody is configured in a tcBsIgG format. In certain embodiments, the multispecific antibody may be a multispecific agonist antibody or a multispecific antagonist antibody.

[0013] The antibodies disclosed herein can be multispecific agonist antibodies or multispecific antagonist antibodies. In certain embodiments, the multispecific antibodies of the presently disclosed subject matter are bispecific antibodies, such as biepitopic antibodies directed against one target or bispecific antibodies directed against two different targets. In certain embodiments, the bispecific antibodies are agonistic biepitopic antibodies. In certain embodiments, the bispecific antibodies are antagonistic biepitopic antibodies.

[0014] In certain embodiments, the presently disclosed subject matter provides an isolated biapic agonist antibody comprising a first antigen-binding polypeptide and a second antigen-binding polypeptide, each of which comprises a VL Domain, a linker, a VH Domain, a CH1 Domain, a CH2 Domain, and a CH3 Domain, arranged in an N-terminal to C-terminal direction in the following order: VL-linker-VH-CH1-CH2-CH3. In certain embodiments, the first and second antigen-binding polypeptides of the biapic agonist antibody bind to two different epitopes on the same antigen.

[0015] In certain embodiments, the biapic agonist antibody does not comprise a CL domain. In certain embodiments, the biapic agonist antibody further comprises two CL domains. In certain embodiments, the CL domain is not covalently linked to the first or second antigen-binding polypeptide. In certain embodiments, the CL domain is not covalently linked to the VL domain. In certain embodiments, the VL and CL are separated. In certain embodiments, this separation can be at any junction between the VL and CL. In certain embodiments, the junction can be at T109 (i.e., the CL can start at T109 and the VL can end at R108). In certain other embodiments, the CL can start at V110.

[0016] In certain embodiments, the two CL domains are the same. In certain embodiments, the two CL domains of a biliepitopic agonist antibody are linked to a first antigen-binding polypeptide by one or more disulfide bridges, and the second of the two CL domains is linked to a second antigen-binding polypeptide by one or more disulfide bridges. In certain embodiments, disulfide bridges link the CL domain to the CH1 domains of the first and second antigen-binding polypeptides.

[0017] In certain embodiments, a linker present in an antigen-binding polypeptide of a disclosed antibody may comprise glycine (G) and serine (S) residues. In certain embodiments, the linker has a length of about 1 to about 50 amino acids. In certain embodiments, the linker is about 20 amino acids in length. In certain embodiments, the linker comprises G4S repeats. In certain embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 68. In certain embodiments, the linker is cleavable.

[0018] In certain embodiments, the CH3 domain of a first antigen-binding polypeptide and the CH3 domain of a second antigen-binding polypeptide of the disclosed multispecific antibodies associate at an interface that has been modified to facilitate the formation of multispecific antibodies. In certain embodiments, one or more amino acid residues in the CH3 domain of the first antigen-binding polypeptide are substituted with one or more amino acid residues having a larger side chain volume, generating protrusions on the surface of the CH3 domain of the first antigen-binding polypeptide. In certain embodiments, one or more amino acid residues in the CH3 domain of the second antigen-binding polypeptide are substituted with one or more amino acid residues having a smaller side chain volume, generating cavities on the surface of the CH3 domain of the second antigen-binding polypeptide that interact with the protrusions on the surface of the CH3 domain of the first antigen-binding polypeptide. In certain embodiments, the amino acid residues having a larger side chain volume may include arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W). In certain embodiments, the amino acid residue having a smaller side chain volume may comprise alanine (A), serine (S), threonine (T), or valine (V). In certain embodiments, the knob mutation, such as a protrusion, may comprise T366W (EU numbering). In certain embodiments, the hole mutation(s), such as a cavity, may comprise at least one, at least two, or all of T366S, L368A, and Y407V (EU numbering). In certain embodiments, the multispecific or biapic antibody of the presently disclosed subject matter is of the IgG, IgA, or IgE isotype. In certain embodiments, the multispecific or biapic antibody of the presently disclosed subject matter is of the IgG isotype. In certain embodiments, the multispecific or biapic antibody is of the IgG1, IgG2, or IgG4 isotype. In certain embodiments, the biapic antibody is a biapic agonist antibody.

[0019] The presently disclosed subject matter further provides isolated nucleic acids comprising sequences encoding the disclosed multispecific or biapitopic antibodies. In certain embodiments, the biapitopic antibodies are biapitopic agonist antibodies. The presently disclosed subject matter further provides vectors comprising the disclosed nucleic acids.

[0020] The presently disclosed subject matter further provides host cells expressing the multispecific or biapitopic antibodies of the present disclosure. In certain embodiments, the present disclosure provides host cells comprising one or more nucleic acids encoding the multispecific or biapitopic antibodies. For example, and without limitation, the host cells can comprise a nucleic acid encoding a first antigen-binding polypeptide and a separate nucleic acid encoding a second antigen-binding polypeptide. Alternatively, the host cells can comprise nucleic acids encoding the first antigen-binding polypeptide and the second antigen-binding polypeptide. In certain embodiments, the host cells further comprise a separate nucleic acid, e.g., a second nucleic acid or a third nucleic acid, encoding a CL domain. In certain embodiments, the biapitopic antibody is a biapitopic agonist antibody.

[0021] The presently disclosed subject matter further provides methods for producing multispecific or biapitopic antibodies, e.g., biapitopic agonist antibodies. In certain embodiments, the methods comprise culturing host cells under conditions sufficient for the production of the multispecific or biapitopic antibodies. In certain embodiments, the methods may further comprise recovering the multispecific or biapitopic antibodies from the culture medium, e.g., by purification techniques. In certain embodiments, the biapitopic antibodies are biapitopic agonist antibodies.

[0022] In another aspect, the presently disclosed subject matter further provides a pharmaceutical composition comprising the multispecific or biapitopic antibody disclosed herein. In certain embodiments, the composition further comprises a second therapeutic agent. The presently disclosed subject matter further provides a kit comprising the multispecific or biapitopic antibody disclosed herein or a composition thereof. In certain embodiments, the biapitopic antibody is a biapitopic agonist antibody.

[0023] The presently disclosed subject matter further provides libraries comprising a plurality of the disclosed multispecific or biepitopic antibodies. In certain embodiments, the libraries can comprise a plurality of polynucleotides encoding a plurality of the disclosed multispecific or biepitopic antibodies.

[0024] In another aspect, the presently disclosed subject matter further provides a method for screening for multispecific antibodies. For example, and without limitation, the methods disclosed herein can be used to screen for biepitopic antibodies, in which the antibodies bind to two epitopes on the same antigen. In certain embodiments, the methods disclosed herein can be used to identify multispecific antibodies, e.g., bispecific or biepitopic antibodies, that exhibit agonistic or antagonistic activity. In certain embodiments, the method can include (a) obtaining a plurality of multispecific antibodies from a library; (b) assaying the plurality of multispecific antibodies for binding to a first and a second antigen or to a first and a second epitope on the same antigen; and (c) identifying a multispecific antibody that binds to the first and the second antigen or to a first and a second epitope on the same antigen.

[0025] In certain embodiments, a method of screening for a multispecific antibody may comprise: (a) expressing the multispecific antibody in a cell; (b) contacting the multispecific antibody of step (a) with a first antigen and a second antigen or a first epitope and a second epitope on the same antigen; and (c) identifying multispecific antibodies that bind to the first antigen and the second antigen or the first epitope and the second epitope on the same antigen.

[0026] In a further aspect, the presently disclosed subject matter further provides a method of screening for biapitopic agonist or antagonist antibodies. In certain embodiments, the method may include (a) obtaining a plurality of multispecific antibodies from a library, (b) assaying the plurality of multispecific antibodies for binding to a first and a second epitope on the same antigen, (c) identifying one or more biapitopic antibodies that bind to the first and second epitopes on the same antigen, and (d) distinguishing the biapitopic antibodies from one or more biapitopic antibodies that exhibit agonist or antagonist activity to obtain the biapitopic agonist or antagonist antibody. In certain embodiments, the methods may further comprise comparing the agonist or antagonist activity of the biliepitopic agonist or antagonist antibody to one or more monospecific parent antibodies, alone or in combination, from which the biliepitopic agonist or antagonist antibody was derived.

[0027] In certain embodiments, a method of screening for a biapitopic agonist or antagonist antibody may comprise: (a) expressing a multispecific antibody in a cell; (b) contacting the multispecific antibody of step (a) with a first epitope and a second epitope of the same antigen; (c) identifying multispecific antibodies that bind to the first epitope and the second epitope of the same antigen to obtain the biapitopic antibody; and (d) determining whether the biapitopic antibody exhibits agonist or antagonist activity to obtain the biapitopic agonist or antagonist antibody. In certain embodiments, the method may further comprise comparing the agonist or antagonist activity of the biapitopic agonist or antagonist antibody with one or more monospecific parent antibodies from which the biapitopic agonist or antagonist antibody was derived, either alone or in combination.

[0028] In certain embodiments, expressing the multispecific or biapitopic antibody in a cell can include introducing one or more nucleic acids encoding the multispecific or biapitopic antibody into the cell. In certain embodiments, the multispecific or biapitopic antibody is contacted with the first and second antigens or the first and second epitopes of the same antigen simultaneously in step (b). In certain embodiments, the multispecific or biapitopic antibody in step (b) is purified before contacting the multispecific or biapitopic antibody with the first and second antigens or the first and second epitopes of the same antigen. In certain embodiments, the multispecific or biapitopic antibody is purified by protein A chromatography. In certain embodiments, the antigen is present in a biocomplex. In certain embodiments, the cell is a prokaryotic cell, e.g., an Escherichia coli cell. In certain embodiments, the cell is a eukaryotic cell, e.g., a yeast cell or a mammalian cell. In certain embodiments, the mammalian cells are Chinese hamster ovary (CHO) cells. In certain embodiments, binding of the multispecific antibody to the first and / or second antigen is analyzed by ELISA. In certain embodiments, the biapic antibody is a biapic agonist antibody.

[0029] In another aspect, the present disclosure provides bispecific, e.g., biepitopic, antibodies that specifically bind to at least two epitopes present on KLB, and / or mixtures of anti-KLB antibodies that specifically bind to at least two epitopes present on KLB, and methods of treating disease in a subject using these antibodies and / or mixtures. In certain embodiments, the bispecific anti-KLB antibodies comprise a first antibody, or antigen-binding portion thereof, and a second antibody, or antigen-binding portion thereof. For example, and without limitation, the first antibody, or antigen-binding portion thereof, can comprise a heavy chain variable region and a light chain variable region, and the second antibody, or antigen-binding portion thereof, can comprise a heavy chain variable region and a light chain variable region, and the first antibody and the second antibody, or antigen-binding portion thereof, bind to different epitopes present on KLB.

[0030] In certain embodiments, a bispecific anti-KLB antibody comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 domains, and a light chain variable region comprising CDR1, CDR2, and CDR3 domains. In certain embodiments, a bispecific anti-KLB antibody, or antigen-binding portion thereof, comprises a heavy chain variable region and a light chain variable region. In certain embodiments, the heavy chain variable region comprises amino acids having a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 34, 36, 38, 40, or 42. In certain embodiments, the light chain variable region comprises amino acids having a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 33, 35, 37, 39, or 41.

[0031] In certain embodiments, a bispecific anti-KLB antibody may comprise a heavy chain variable region having a sequence about 95% identical to the sequence set forth in SEQ ID NO: 38 and a light chain variable region having a sequence about 95% identical to the sequence set forth in SEQ ID NO: 37. In certain embodiments, a bispecific anti-KLB antibody may comprise a heavy chain variable region having a sequence about 95% identical to the sequence set forth in SEQ ID NO: 42 and a light chain variable region having a sequence about 95% identical to the sequence set forth in SEQ ID NO: 41.

[0032] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising the disclosed bispecific anti-KLB antibody and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition may comprise an additional therapeutic agent.

[0033] In certain embodiments, the presently disclosed subject matter provides bispecific anti-KLB antibodies for use in methods for treating metabolic disorders, e.g., polycystic ovary syndrome (PCOS), metabolic syndrome (MetS), obesity, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), dyslipidemia, hypertension, type 2 diabetes, non-type 2 diabetes, type 1 diabetes, latent autoimmune diabetes (LAD), and maturity-onset diabetes of the young (MODY), as well as geriatric and related diseases, such as Alzheimer's disease, Parkinson's disease, and ALS. In certain embodiments, the bispecific anti-KLB antibodies are used to treat type 2 diabetes. The methods can include administering a therapeutically effective amount of an antibody of the presently disclosed subject matter to an individual. In certain embodiments, the disease is diabetes, e.g., type 2 diabetes. In certain embodiments, the methods further include administering an additional therapeutic agent to the individual.

[0034] In certain embodiments, the presently disclosed subject matter provides an isolated nucleic acid encoding a bispecific anti-KLB antibody of the presently disclosed subject matter. In certain embodiments, the presently disclosed subject matter provides a host cell comprising a nucleic acid disclosed herein. In certain embodiments, the presently disclosed subject matter further provides a hybridoma cell expressing a bispecific anti-KLB antibody or antigen-binding portion thereof disclosed herein. In certain embodiments, the presently disclosed subject matter provides a method for producing a bispecific antibody in a host cell, the method comprising transforming a host cell with a nucleic acid of the present disclosure and culturing the host cell under conditions for production of the bispecific antibody. In certain embodiments, the method further comprises recovering the antibody from the host cell.

[0035] In certain embodiments, the presently disclosed subject matter provides a method for activating the KLB-FGFR1c receptor complex in an individual, the method comprising administering to the individual an effective amount of the disclosed bispecific anti-KLB antibody. [Brief explanation of the drawings]

[0036] [Figure 1A] 1 illustrates a variable light chain complete heavy chain (VLfH) format used in an exemplary method according to one non-limiting embodiment of the disclosed subject matter. VLfH may also be referred to herein as "three-chain IgG" or "tcIgG." When the tcIgG is a bispecific antibody, it may more specifically be referred to as "tcBsIgG." [Figure 1B] 1 illustrates a variable light chain complete heavy chain (VLfH) format used in an exemplary method according to one non-limiting embodiment of the disclosed subject matter. VLfH may also be referred to herein as "three-chain IgG" or "tcIgG." When the tcIgG is a bispecific antibody, it may more specifically be referred to as "tcBsIgG." [Figure 2A] FIG. 1 depicts liquid chromatography-mass spectrometry (LC-MS) graphs showing heterodimerization of bispecific antibodies formatted into VLfH. [Figure 2B] FIG. 1 depicts liquid chromatography-mass spectrometry (LC-MS) graphs showing heterodimerization of bispecific antibodies formatted into VLfH. [Figure 3] Illustrates induction of KLB-FGFR1c receptor activity by various VLfH-formatted bispecific anti-KLB antibodies that bind to two epitopes present on KLB in a GAL4-Elk1-based luciferase assay compared to the corresponding parental monospecific VLfH-formatted antibodies. [Figure 4] Illustrates induction of KLB-FGFR1c receptor activity by various bispecific anti-KLB antibodies in IgG format that bind to two epitopes present on KLB in a GAL4-Elk1-based luciferase assay compared to the corresponding parental monospecific antibodies. [Figure 5]Illustrates induction of KLB-FGFR1c receptor activity by various bispecific anti-KLB antibodies that bind to two epitopes present on KLB in a GAL4-Elk1-based luciferase assay, compared with the corresponding parent monoclonal antibody, an anti-KLB / anti-FGFR1c bispecific antibody (BsAb2), and the isotype-matched control antibody trastuzumab. [Figure 6] Illustrates induction of KLB-FGFR1c receptor activity by various bispecific anti-KLB antibodies binding to two epitopes present on KLB in a GAL4-Elk1-based luciferase assay compared to a 1:1 mixture of the corresponding parental monospecific antibodies. [Figure 7] Illustrates induction of KLB-FGFR1c receptor activity (but not that of the closely related receptor complex, KLB-FGFR2c) by anti-KLB agonist monoclonal antibodies using a GAL4-Elk1-based luciferase reporter assay. [Figure 8] 1 illustrates that bispecific anti-KLB antibodies induced ERK phosphorylation in human primary adipocytes. [Figure 9] 1 illustrates KLB binding of monoclonal antibodies used to generate the bispecific anti-KLB antibodies disclosed herein using flow cytometry. [Figure 10] 1 illustrates induction of KLB / FGFR1c receptor activity by monoclonal antibodies used to generate bispecific anti-KLB antibodies disclosed herein using chimeric KLB and FGFR proteins. [Figure 11] (A) Summary of epitope binning for the anti-KLB monoclonal antibodies used to generate the bispecific antibody and the control anti-KLB monoclonal antibody 8C5. (B) FACS binding with HEK293T cells expressing human / rat KLB chimeric proteins. [Figure 12]An exemplary biolayer inferometry experiment used to determine epitope bins is illustrated in Figure 7. In this example, 2C12 (human IgG1) competes with 23B3 but not with 28B7 or 8C5 for binding to recombinant KLB. [Figure 13-1] 1 depicts the light and heavy chain variable region sequences for the anti-KLB antibody, clone 12B8. [Figure 13-2] 1 depicts the light and heavy chain variable region sequences for the anti-KLB antibody, clone 12B8. [Figure 14-1] 1 depicts the light and heavy chain variable region sequences for the anti-KLB antibody, clone 2C12. [Figure 14-2] 1 depicts the light and heavy chain variable region sequences for the anti-KLB antibody, clone 2C12. [Figure 15-1] 1 depicts the light and heavy chain variable region sequences for the anti-KLB antibody, clone 4H7. [Figure 15-2] 1 depicts the light and heavy chain variable region sequences for the anti-KLB antibody, clone 4H7. [Figure 16-1] 1 depicts the light and heavy chain variable region sequences for the anti-KLB antibody, clone 23B3. [Figure 16-2] 1 depicts the light and heavy chain variable region sequences for the anti-KLB antibody, clone 23B3. [Figure 17-1] 1 depicts the light and heavy chain variable region sequences for the anti-KLB antibody, clone 28B7. [Figure 17-2] 1 depicts the light and heavy chain variable region sequences for the anti-KLB antibody, clone 28B7. [Figure 18A]Figure 1 illustrates that the tcBsIgG format enables bispecific antibody production in a single cell. (A) Schematic of the tcBsIgG format. The antibody VL domain is tethered to the antibody heavy chain via a (G4S)4 linker (left). The CH1 folding defect is compensated for by CL expression in trans from a separate plasmid (right). [Figure 18B] (B) Capillary electrophoresis of the expression of the anti-KLB antibody, clone 28B7 monoepitopic tcIgG, in IgG1, 2, and 4 isotypes, and deglycosylated (N297G) versions of IgG1 and IgG4 after protein A affinity column purification. Expression as VLfH alone without CL in HECK293 cells resulted in little or no VLfH expression (lane 1); coexpression of VLfH with CL resulted in tcIgG expression (lane 2) equivalent to standard IgG expression (lane 3). [Figure 18C] The tcBsIgG format illustrates the potential for bispecific antibody generation in single cells. (C) Capillary electrophoresis of the expression of the anti-KLB antibody, clone 28B7 monoepitopic tcIgG, after Protein A affinity column purification and recovery of standard IgG1, 2, and 4, and tcIgG1, 2, and 4 counterparts, as knob half IgG (lane 1); hole half IgG (lane 2); and knob and hole co-expressed IgG in single cells. [Figure 18D] Illustrating that the tcBsIgG format enables bispecific antibody generation in a single cell, (D) Complete LC-MS / MS of the anti-KLB antibody, clone 28B7 monoepitopic tcIgG, after protein A and size exclusion chromatography. [Figure 19A] We disclose the production of anti-KLB biepitopic antibodies in the tcBsIgG format. (A) Capillary electrophoresis of co-expression of five anti-KLB tcIgG half antibodies with positive and negative expression control tcIgG in a 7x7 knob and hole combination to produce tcBsIgG in a single cell. [Figure 19B] (B) Capillary electrophoresis of parental tcIgG expression of the corresponding tcBsIgG expressed in single cells. [Figure 19C-1] (C) Analytical size exclusion chromatography (SEC) of tcIgG after ProA chromatography. [Figure 19C-2] (C) Analytical size exclusion chromatography (SEC) of tcIgG after ProA chromatography. [Figure 19C-3] (C) Analytical size exclusion chromatography (SEC) of tcIgG after ProA chromatography. [Figure 20] This paper describes the effect of antibody isotype on antibody agonist activity. (A) Luciferase reporter assay showing the activity of different isotypes. (B) The effect of different antibody isotypes on ERK1 / 2 phosphorylation in human primary adipocytes. (C) The effect of different antibody isotypes and their combinations on ERK1 / 2 phosphorylation in human primary adipocytes. [Figure 21] The difference in binding affinity between anti-KLB antibody clones 28B7 and 4H7 is disclosed. DETAILED DESCRIPTION OF THE INVENTION

[0037] For clarity, and not by way of limitation, the detailed description of the subject matter disclosed herein is divided into the following subsections. I. Definition II. Antibodies III. Screening and Production Methods IV.How to use V. Pharmaceutical Formulations VI. Products and Kits

[0038] I. Definition As used herein, an "acceptor human framework" is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence, or it may contain amino acid sequence changes. In certain embodiments, the number of amino acid changes is about 10 or less, about 9 or less, about 8 or less, about 7 or less, about 6 or less, about 5 or less, about 4 or less, about 3 or less, or about 2 or less. In certain embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0039] As used herein, "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K d ) Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0040] An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody that does not possess such alterations, which improve the affinity of the antibody for its antigen.

[0041] As used herein, "Klotho-beta," "KLB," and "beta-Klotho," unless otherwise specified, refer to any naturally occurring Klotho-beta from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed KLB, as well as any form of KLB resulting from processing within a cell. The term also encompasses mutant and naturally occurring variants of KLB, such as splice or allelic variants.

[0042] Non-limiting examples of human KLB amino acid sequences targeted by the bispecific antibodies of the present disclosure, excluding the signal sequence, are:

[0043] The term "C-terminal domain of KLB" refers to the carboxy-terminal glycosidase-like domain of KLB. For example, the C-terminal domain of the exemplary KLB protein set forth in SEQ ID NO: 1 has the amino acid sequence: FPCDFSWGVTESVLKPESVASSPQFSDPHLYVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGIS AMVTLYYPTHAHLGLPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDIYNRSGNDTYGAAHNLLVAHALAWRLYDRQFRPSQRGAVSLSLHADWAEPANPY Contains ADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFKAKSSIQFYNKVISSRGFPFENSSSR (SEQ ID NO: 2).

[0044] As referred to herein, an "antigen-binding polypeptide" is a protein or polypeptide that contains an antigen-binding region or portion that has a strong affinity for another molecule to which it binds. Antigen-binding polypeptides include antibodies or antigen-binding fragments thereof, and fusion proteins.

[0045] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, multispecific antibodies, e.g., bispecific antibodies, and antibody fragments, that exhibit the desired antigen-binding activity.

[0046] As used herein, "antibody fragment," "antigen-binding portion" of an antibody (or simply "antibody portion"), or "antigen-binding fragment" of an antibody refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen and / or epitope to which the intact antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and antibody fragments formed from multispecific, e.g., bispecific, antibodies.

[0047] In certain embodiments, the terms "agonist" and "agonistic" can refer to a molecule (e.g., an antigen-binding polypeptide and / or antibody and / or antigen-binding antibody fragment) that can interact with, e.g., bind to, a receptor (e.g., an antigen) and elicit, mimic, and / or stimulate a response or activity that is similar or the same as that elicited, mimic, and / or stimulated by the receptor's natural ligand. In certain embodiments, an agonist described herein is capable of inducing, enhancing, potentiating, and / or stimulating activation of a signaling pathway associated with the receptor.

[0048] "An antibody that competes with a reference antibody in binding" refers to an antibody that blocks the binding of the reference antibody to its antigen, such as KLB, by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen, such as KLB, by 50% or more in a competitive assay.Exemplary competitive assays are described in "Antibodies" by Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY).

[0049] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0050] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. In certain embodiments, a multispecific antibody, e.g., a bispecific antibody, of the present disclosure may be of any of the disclosed classes or subclasses (isotypes) of antibodies.

[0051] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and / or causes cell death or destruction. Cytotoxic agents include radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleases; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins (including fragments and / or variants thereof) of bacterial, fungal, plant, or animal origin; and various anti-tumor or anti-cancer agents described below.

[0052] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0053] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In certain embodiments, a human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues within the Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0054] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains, FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0055] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a heavy chain that has a structure substantially similar to a native antibody structure or that contains an Fc region as defined herein.

[0056] The terms "host cell," "host cell line," and "host cell culture," used interchangeably, refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0057] As used herein, a "human antibody" refers to an antibody that possesses an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a non-human source that utilizes the human antibody repertoire or other human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0058] As used herein, "human consensus framework" refers to a framework that represents the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as found in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda, MD (1991), vols. 1-3. In certain embodiments, for VL, the subgroup is subgroup kappa I as found in Kabat et al. (see above). In certain embodiments, for VH, the subgroup is subgroup III as found in Kabat et al. (see above).

[0059] As used herein, a "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and human FRs. In certain embodiments, a humanized antibody can comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of its HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of its FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0060] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of a natural antibody generally have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, e.g., Kindt et al., Kuby Immunology, 6 th (See, e.g., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Moreover, antibodies that bind to a specific antigen can be isolated from antigen-binding antibodies using a VH or VL domain to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0061] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contacts"). Generally, antibodies contain six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein are: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24–34 (L1), 50–56 (L2), 89–97 (L3), 31–35b (H1), 50–65 (H2), and 95–102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)); and (d) A combination of (a), (b), and / or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Unless otherwise specified, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al. (see above).

[0062] As used herein, "immunoconjugate" refers to an antibody conjugated to one or more heterologous molecule(s), including but not limited to, a cytotoxic agent.

[0063] As used interchangeably herein, "individual," "patient," or "subject" refers to a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0064] As used herein, an "isolated" antibody refers to an antibody that has been separated from the components of its natural environment. In certain embodiments, the antibody is purified to greater than 95% or 99% purity, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0065] As used herein, an "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained within a cell that ordinarily contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0066] An "isolated nucleic acid encoding an antibody" (including reference to a specific antibody, e.g., an anti-KLB antibody) refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present in one or more locations within a host cell.

[0067] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. The term includes vectors as self-replicating nucleic acid structures and vectors that have integrated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0068] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, but excludes, for example, possible variant antibodies that contain naturally occurring mutations or that arise during production of a monoclonal antibody preparation (such variants are generally present in minor amounts), or multispecific antibodies, e.g., antibodies that bind to at least two different epitopes. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0069] As used herein, a "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. The naked antibody may be present in a pharmaceutical formulation.

[0070] As used herein, "natural antibody" refers to a naturally occurring immunoglobulin molecule with a variety of structures. For example, but not limited to, a natural IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (κ) or lambda (λ), based on the amino acid sequence of its constant domain. In certain embodiments, the "CH2 domain" of a human IgG Fc region typically extends from about IgG residue 231 to about IgG residue 340. In certain embodiments, the "CH3 domain" comprises the stretch of residues from the C-terminus of the Fc region to the CH2 domain (i.e., from about amino acid residue 341 to about amino acid residue 447 of IgG).

[0071] As used herein, "hinge region" generally refers to amino acids Glu216 to Pro230 of human IgG1 (see Burton, Molec. Immunol. 22:161-206 (1985)). In certain embodiments, hinge regions of other IgG isotypes may align with the IgG1 sequence by placing the first and last cysteine ​​residues that form intra-heavy chain S-S bonds in the same positions.

[0072] The term "interface" includes "contact" amino acid residues (or other non-amino acid groups such as carbohydrate groups, NADH, biotin, FAD, or heme groups) in a first antigen-binding polypeptide that interact with one or more "contact" amino acid residues (or other non-amino acid groups) at the interface of a second antigen-binding polypeptide.

[0073] As used herein, the term "package insert" refers to instructions typically included in commercial packaging for therapeutic products that contain information about the indications, uses, dosages, administration, concomitant therapies, contraindications, and / or warnings regarding the use of such therapeutic agent.

[0074] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment across the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is written by Genentech, Inc., and its source code, together with user documentation, has been submitted to the U.S. Copyright Office, Washington, DC, 20559, and is registered under U.S. Copyright No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0075] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y where X is the number of amino acid residues scored as perfect matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0076] As used herein, the term "pharmaceutical formulation" refers to a preparation that is in a form such that the biological activity of the active ingredient contained therein is effective and that does not contain any additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0077] As used herein, "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0078] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention aimed at altering the natural history of the individual being treated and may be performed prophylactically or during the clinical pathological course. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, diminishing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or remission of disease symptoms, and remission or improved prognosis. In certain embodiments, the antibodies of the invention are used to delay the development of disease or slow the progression of disease.

[0079] A "therapeutically effective amount" or "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective to achieve a desired therapeutic or prophylactic result at the necessary dosage and for the necessary period of time. For example, without limitation, a "therapeutically effective amount" or "effective amount" can refer to the dosage of a bispecific antibody that results in a reduction of any direct or indirect pathological consequence of a disease, a slowing of disease progression, an amelioration of the condition, remission, or improvement in prognosis, prevention of the onset or recurrence of a disease, and / or alleviation of symptoms.

[0080] As used herein, the term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend to some extent on the method of measurement or determination of the value, i.e., the limitations of the measurement system. For example, "about" means within 3 or more standard deviations, as is customary in the art. Alternatively, "about" can mean within a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, more preferably within 2-fold of a value.

[0081] As used herein, any concentration range, percentage range, ratio range, or integer range is understood to include every integer value within the recited range, and fractions thereof (such as integer tenths and hundredths), where appropriate, unless otherwise specified.

[0082] II. Antibodies The presently disclosed subject matter provides multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies, e.g., bispecific antibodies, of the present disclosure have at least two different binding specificities. See, for example, U.S. Patent Nos. 5,922,845 and 5,837,243; Zeilder (1999) J. Immunol. 163:1246-1252; Somasundaram (1999) Hum. Antibodies 9:47-54; Keler (1997) Cancer Res. 57:4008-4014. In certain embodiments, the multispecific antibodies disclosed herein, e.g., biepitopic antibodies, can bind to at least two different epitopes on an antigen or at least two overlapping epitopes on an antigen. Alternatively, in certain embodiments, the multispecific antibodies of the present disclosure can bind to at least two different antigens.

[0083] As used herein, the term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes typically consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished from each other in that the binding to the former, but not the latter, is lost in the presence of denaturing solvents.

[0084] A. Multispecific antibodies The presently disclosed subject matter provides multispecific antibodies, e.g., bispecific and biapitopic antibodies, that bind to at least two epitopes present on an antigen, e.g., overlapping or non-overlapping epitopes. The presently disclosed subject matter further provides multispecific antibodies that bind to at least one epitope on a first antigen and at least one epitope on a second antigen. The multispecific antibodies disclosed herein can be agonistic or antagonistic.

[0085] In certain embodiments, a multispecific antibody, e.g., a bispecific antibody, of the present disclosure may comprise one or more antigen-binding polypeptides. For example, without limitation, a multispecific antibody of the present disclosure may comprise a first antigen-binding polypeptide and a second antigen-binding polypeptide. In certain embodiments, the first antigen-binding polypeptide and the second antigen-binding polypeptide have different binding specificities. For example, without limitation, the first antigen-binding polypeptide can bind to a first epitope on an antigen, and the second antigen-binding polypeptide can bind to a second epitope on the antigen. Alternatively, in certain embodiments, the first antigen-binding polypeptide can bind to a first antigen, and the second antigen-binding polypeptide can bind to a second antigen.

[0086] In certain embodiments, and as shown in Figure IB, the first antigen-binding polypeptide and / or the second antigen-binding polypeptide may comprise a light chain variable region (VL), a heavy chain variable region (VH), a CH1 domain, a CH2 domain, and / or a CH3 domain. In certain embodiments, the antigen-binding polypeptides of the disclosed multispecific antibodies comprise a VL, VH, a CH1 domain, a CH2 domain, and a CH3 domain, with these components positioned relative to each other in the N-terminal to C-terminal order: VL-linker-VH-CH1-CH2-CH3. In certain embodiments, the first and / or second antigen-binding polypeptide may further comprise a hinge region between the CH1 domain and the CH2 domain.

[0087] In certain embodiments, a VL domain present in an antigen-binding polypeptide (e.g., a first antigen-binding polypeptide) may be joined to a VH domain present in the same antigen-binding polypeptide by a linker. For example, and without limitation, the C-terminus of the VL domain may be linked to the N-terminus of the VH domain. In certain embodiments, where a multispecific antibody of the present disclosure comprises at least two antigen-binding polypeptides, the linkers present in each of the antigen-binding polypeptides may be the same. Alternatively, the linkers present in each of the antigen-binding polypeptides may be different.

[0088] In certain embodiments, the linker can comprise neutral, polar, or nonpolar amino acids. In certain embodiments, the linker can be about 1 to about 100 amino acids in length, e.g., about 1 to 50 amino acids in length. For example, without limitation, the linker present in the first and / or second antigen-binding polypeptide can comprise about 1 or more, about 2 or more, about 3 or more, about 4 or more, about 5 or more, about 6 or more, about 7 or more, about 8 or more, about 9 or more, about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, or about 45 or more amino acids. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO2014 / 087010, the contents of which are incorporated herein by reference in their entireties. In certain embodiments, the linker is a Gly(G)Ser(S) linker (SEQ ID NO: 70). In certain embodiments, the linker comprises a GGGGS repeat sequence, e.g., about 2 repeats ((GGGGS)2) (SEQ ID NO: 73), about 3 repeats ((GGGGS)3) (SEQ ID NO: 74), about 4 repeats ((GGGGS)4) (SEQ ID NO: 68), about 5 repeats ((GGGGS)5) (SEQ ID NO: 75), about 6 repeats ((GGGGS)6) (SEQ ID NO: 76), or about 7 repeats ((GGGGS)7) (SEQ ID NO: 77). Additional non-limiting examples of linkers are disclosed in Table 7. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 68.

[0089] In certain embodiments, the linker may be a cleavable linker, such as a self-cleavable, enzymatically cleavable, or chemically cleavable linker. See WO2011 / 034605, the entire contents of which are incorporated herein by reference. For example, and without limitation, enzymatic cleavage of the linker may involve the use of an endopeptidase or exopeptidase. Non-limiting examples of endopeptidases include urokinase, Lys-C, Asp-N, Arg-C, V8, Glu-C, chymotrypsin, trypsin, pepsin, papain, thrombin, genenase, Factor Xa, TEV (tobacco etch virus cysteine ​​protease), enterokinase, HRV C3 (human rhinovirus C3 protease), ininogenase, subtilisin-like proprotein convertase (e.g., furin (PC1), PC2, or PC3), and N-arginine dibasic convertase. Non-limiting examples of exopeptidases include carboxypeptidase A, carboxypeptidase B, carboxypeptidase D, carboxypeptidase E (also called carboxypeptidase H), carboxypeptidase M, carboxypeptidase N, or carboxypeptidase Z. In certain embodiments, chemical cleavage can be performed by using hydroxylamine, N-chlorosuccinimide, N-bromosuccinimide, or cyanogen bromide. Table 7 JPEG2026009891000001.jpg22158JPEG2026009891000002.jpg63158

[0090] In certain embodiments, for example, a multispecific antibody of the present disclosure comprises a first antigen-binding polypeptide and a second antigen-binding polypeptide, and the first antigen-binding polypeptide and the second antigen-binding polypeptide may interact by one or more disulfide bridges. For example, and without limitation, in certain embodiments, the hinge regions of the first and second antigen-binding polypeptides may interact by one or more disulfide bridges, e.g., two disulfide bridges.

[0091] In certain embodiments, the present multispecific, e.g., bispecific, antibodies may comprise a heterodimerization domain within each of the antigen-binding polypeptides of the antibodies disclosed herein. In certain embodiments, the CH3 domains of the first and second antigen-binding polypeptides of the disclosed multispecific antibodies may be modified to promote heterodimerization of the first and second antigen-binding polypeptides. For example, and without limitation, the first and / or second antigen-binding polypeptides may comprise one or more heterodimerization domains that use knob-in-hole technology (see, e.g., U.S. Patent Nos. 5,731,168 and 8,216,805, which are incorporated herein by reference in their entireties) to promote association and / or interaction between the first and second antigen-binding polypeptides.

[0092] In certain embodiments, the first and / or second antigen-binding polypeptides may be modified to generate protrusions or cavities on the surface of the antigen-binding polypeptide. For example, and without limitation, the first antigen-binding polypeptide may be modified to generate protrusions on the surface of the first antigen-binding polypeptide, and the second antigen-binding polypeptide may be modified to generate cavities on the second antigen-binding polypeptide, where the protrusions of the first antigen-binding polypeptide interact with the cavities of the second antigen-binding polypeptide to promote heterodimerization. In certain embodiments, a multispecific, e.g., bispecific, antibody of the present disclosure may comprise a first antigen-binding polypeptide and a second antigen-binding polypeptide that interact at an interface, where the first antigen-binding polypeptide has a protrusion at the interface, which can be positioned within the cavity at the interface of the second antigen-binding polypeptide. See, e.g., U.S. Patent No. 8,216,805, the entire contents of which are incorporated herein by reference.

[0093] In certain embodiments, the region of an antigen-binding polypeptide that is modified to generate a protrusion or cavity may be the CH3 domain. For example, a protrusion in the CH3 domain of a first antigen-binding polypeptide can be generated by substituting an amino acid with a small side chain, such as glycine (G), alanine (A), serine (S), threonine (T), or valine (V), with an amino acid with a large side chain, such as arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W). For example, but not limited to, a mutation that can be introduced into an antigen-binding polypeptide to generate a protrusion (e.g., a "knob") includes T366W (EU numbering). A compensatory cavity of the same or similar size as the protrusion can be generated in the CH3 domain of a second antigen-binding polypeptide, for example, by substituting an amino acid with a larger side chain for an amino acid with a smaller side chain. For example, without limitation, mutations that can be introduced into an antigen-binding polypeptide to create a cavity (e.g., a "hole") include T366S, L368A, and / or Y407V (EU numbering).

[0094] In certain embodiments, a multispecific antibody, e.g., a bispecific or biepitopic antibody, may comprise a first antigen-binding polypeptide and a second antigen-binding polypeptide, wherein one or more amino acid residues in the CH3 domain of the first antigen-binding polypeptide are substituted with one or more amino acid residues having a larger side chain volume, thereby generating a protrusion on the surface of the CH3 domain of the first antigen-binding polypeptide that interacts with the CH3 domain of the second antigen-binding polypeptide. In certain embodiments, one or more amino acid residues in the CH3 domain of the second antigen-binding polypeptide are substituted with one or more amino acid residues having a smaller side chain volume, thereby generating a cavity on the surface of the CH3 domain of the second antigen-binding polypeptide that interacts with the CH3 domain of the first antigen-binding polypeptide (see, e.g., Figure 1B).

[0095] In certain embodiments, the multispecific antibodies of the present disclosure do not comprise a light chain constant domain (CL). Alternatively, the multispecific antibodies disclosed herein may comprise one or more CL domains. For example, and without limitation, when a multispecific antibody comprises a CL domain, the CL domain interacts with the CH1 domain through one or more disulfide bridges. In certain embodiments, when a multispecific antibody of the present disclosure comprises a first antigen-binding polypeptide and a second antigen-binding polypeptide, the multispecific antibody may further comprise two CL domains. In certain embodiments, one of the CL domains interacts with the first antigen-binding polypeptide, and the other CL domain interacts with the first antigen-binding polypeptide, e.g., through one or more disulfide bridges. For example, and without limitation, the CL domains can interact with the CH1 domain of the antigen-binding polypeptide. In certain embodiments, the two CL domains can be the same or different. In certain embodiments, the CL domain is not covalently linked to the first or second antigen-binding polypeptide. In certain embodiments, the CL domain is not covalently linked to the CH1 domain. In certain embodiments, the CL domain is not covalently linked to the VL domain. In certain embodiments, the VL and CL are separated. In certain embodiments, this separation can be at any junction between the VL and CL. In certain embodiments, the junction can be at T109 (i.e., the CL can start at T109 and the VL can end at R108). In certain other embodiments, the CL starts at V110.

[0096] The presently disclosed subject matter further provides antagonistic and agonistic antibodies. For example, and without limitation, agonistic antibodies of the present disclosure are biapitopic antibodies, in which the antibodies bind to two epitopes on the same antigen. In certain embodiments, the epitopes bound by the biapitopic antibodies do not overlap or at least partially overlap on the antigen. In certain embodiments, biapitopic antibodies of the present disclosure, e.g., agonistic biapitopic antibodies, may comprise a first antigen-binding polypeptide and a second antigen-binding polypeptide as disclosed herein. In certain embodiments, the first antigen-binding polypeptide and the second antigen-binding polypeptide have different binding specificities, in which the first antigen-binding polypeptide can bind to a first epitope on the antigen and the second antigen-binding polypeptide can bind to a second epitope on the antigen. In certain embodiments, the antigen-binding polypeptides of the biapic agonistic antibodies of the presently disclosed subject matter may comprise a VL, a linker, a VH, a CH1 domain, a CH2 domain, and a CH3 domain, with these components positioned relative to each other in the N-terminal to C-terminal direction in the following order: VL-linker-VH-CH1-CH2-CH3. The antigen-binding polypeptides of the biapic agonistic antibodies of the present disclosure may further comprise heterodimerization domains within each of the polypeptides, as disclosed herein. In certain embodiments, these heterodimerization domains are generated by the knobs-in-holes technique. For example, and without limitation, a first antigen-binding polypeptide may have, e.g., a protrusion within its CH3 domain, and a second antigen-binding polypeptide may have, e.g., a cavity within its CH3 domain, which interact to promote heterodimerization.

[0097] In certain embodiments, multispecific agonist antibodies, e.g., biapitopic agonist antibodies, of the present disclosure exhibit superior agonist activity than the parent monospecific antibodies from which they are derived, either alone or in combination. For example, and without limitation, biapitopic agonist antibodies disclosed herein exhibit superior agonist activity than monospecific antibodies with identical VL and VH sequences. In certain embodiments, biapitopic agonist antibodies of the present disclosure exhibit superior agonist activity than monospecific antibodies with identical VL, VH, CH1, CH2, and / or CH3 sequences.

[0098] In certain embodiments, multispecific antagonist antibodies, e.g., biapitopic antagonist antibodies, of the present disclosure exhibit superior antagonist activity compared to the parent monospecific antibodies from which they are derived, either alone or in combination. For example, and without limitation, biapitopic antagonist antibodies disclosed herein exhibit superior antagonist activity compared to monospecific antibodies with identical VL and VH sequences. In certain embodiments, biapitopic antagonist antibodies of the present disclosure exhibit superior antagonist activity compared to monospecific antibodies with identical VL, VH, CH1, CH2, and / or CH3 sequences.

[0099] B. Bispecific anti-KLB antibody The presently disclosed subject matter further provides multispecific antibodies, e.g., bispecific and biapitopic antibodies, that can bind to Klotho-beta (KLB) and function to modulate the activity of the FGFR1c / KLB receptor complex. The disclosed anti-KLB antibodies can function as agonists of the FGFR1c / KLB receptor complex, e.g., stimulating the effect of a ligand and / or activating the FGFR1c / KLB receptor complex.

[0100] The presently disclosed subject matter provides anti-KLB antibodies or antigen-binding portions thereof, e.g., bispecific or multispecific antibodies, that bind to at least two epitopes present on a KLB protein. For example, and without limitation, the presently disclosed subject matter provides bispecific antibodies, e.g., biapitopic antibodies, having one binding site (e.g., an antigen-binding site) for a first epitope present on KLB and a second binding site for a second epitope present on KLB. In certain embodiments, the presently disclosed subject matter provides multispecific antibodies having at least three binding sites.

[0101] In certain embodiments, the disclosed bispecific antibodies of the presently disclosed subject matter bind to two epitopes present in KL1, the glycosidase-like domain of KLB. In certain embodiments, the disclosed bispecific antibodies bind to two epitopes present in KL2, the glycosidase-like domain of KLB. In certain embodiments, the disclosed bispecific antibodies bind to two epitopes present on KLB, where one of the epitopes is present in KL1 and the second epitope is present in KL2.

[0102] In certain embodiments, the disclosed bispecific antibodies of the presently disclosed subject matter bind to a fragment of KLB comprising the amino acid sequence iqfynkvissrgfpfensssrcsqtqentectvclflvqkkpliflgccffstlvlllsiaifqrqkrrkfwkaknlqhiplkkgkrvvs (SEQ ID NO: 43), or a fragment thereof.

[0103] In certain embodiments, the disclosed bispecific antibodies of the presently disclosed subject matter bind to a fragment of KLB comprising the amino acid sequence ADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDI (SEQ ID NO: 44), or a fragment thereof.

[0104] In certain embodiments, the disclosed bispecific antibodies of the presently disclosed subject matter comprise the amino acid sequence (SEQ ID NO: 45) or a fragment thereof.

[0105] In certain embodiments, the disclosed bispecific antibodies of the presently disclosed subject matter bind to one or more fragments of KLB having the amino acid sequence set forth in SEQ ID NO: 42, 43, 44, and / or 45. For example, and without limitation, a bispecific anti-KLB antibody of the present disclosure can bind to one fragment of KLB comprising the amino acid sequence set forth in SEQ ID NO: 45, or a fragment thereof, and can bind to a second fragment of KLB comprising the amino acid sequence set forth in SEQ ID NO: 44, or a fragment thereof.

[0106] In certain embodiments, the disclosed bispecific antibodies of the presently disclosed subject matter modulate KLB / FGFR1c complex activity, wherein the FGFR1c of the complex comprises the amino acid sequence KTVALGSNVEFMCKVYSDPQPHIQWLKHIEVNGSKIGPDNLPYVQILKTAGVNTTDKEMEVLHLRNVSFEDAGEYTCLAGNSIGLSHHSAWLTVLEALEERPAVMT (SEQ ID NO: 46), or a fragment thereof.

[0107] In certain embodiments, the bispecific antibodies disclosed herein may be antibody fragments or diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but are forced to pair with complementary domains on another chain, using a linker that is too short to allow pairing between the two domains on the same chain, creating two antibody, e.g., antigen, antigen-binding sites (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).

[0108] In certain embodiments, bispecific anti-KLB antibodies disclosed herein are capable of binding to KLB with sufficient affinity to render them useful as diagnostic and / or therapeutic agents in targeting KLB, hi certain embodiments, the extent to which an anti-KLB antibody binds to unrelated, non-KLB proteins is less than about 10% of the antibody's binding to KLB, as measured, for example, by radioimmunoassay (RIA).

[0109] In certain embodiments, the anti-KLB antibody disclosed herein refers to an antibody that regulates KLB / FGFR1c complex activity. For example, a bispecific anti-KLB antibody can function as an agonist and activate the KLB / FGFR1c complex. In certain embodiments, a bispecific anti-KLB antibody is an antibody that increases the activity of the KLB / FGFR1c complex by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 99.9%. In certain embodiments, a bispecific anti-KLB antibody can be an antibody that induces phosphorylation of a target downstream of the KLB / FGFR1c complex, such as MAPK and / or ERK.

[0110] In certain embodiments, a bispecific anti-KLB antibody, or antigen-binding portion thereof, comprises a heavy chain variable region and a light chain variable region. In certain embodiments, the heavy chain variable region comprises amino acids having a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 34, 36, 38, 40, or 42. In certain embodiments, the light chain variable region comprises amino acids having a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 33, 35, 37, 39, or 41.

[0111] In certain embodiments, a bispecific anti-KLB antibody may comprise a heavy chain variable region having a sequence that is about 95% identical to the sequence set forth in SEQ ID NO: 34 and a light chain variable region having a sequence that is about 95% identical to the sequence set forth in SEQ ID NO: 33.

[0112] In certain embodiments, a bispecific anti-KLB antibody may comprise a heavy chain variable region having a sequence that is about 95% identical to the sequence set forth in SEQ ID NO: 36 and a light chain variable region having a sequence that is about 95% identical to the sequence set forth in SEQ ID NO: 35.

[0113] In certain embodiments, a bispecific anti-KLB antibody may comprise a heavy chain variable region having a sequence that is about 95% identical to the sequence set forth in SEQ ID NO: 38 and a light chain variable region having a sequence that is about 95% identical to the sequence set forth in SEQ ID NO: 37.

[0114] In certain embodiments, a bispecific anti-KLB antibody may comprise a heavy chain variable region having a sequence that is about 95% identical to the sequence set forth in SEQ ID NO:40 and a light chain variable region having a sequence that is about 95% identical to the sequence set forth in SEQ ID NO:39.

[0115] In certain embodiments, a bispecific anti-KLB antibody may comprise a heavy chain variable region having a sequence that is about 95% identical to the sequence set forth in SEQ ID NO: 42 and a light chain variable region having a sequence that is about 95% identical to the sequence set forth in SEQ ID NO: 41.

[0116] In certain embodiments, a bispecific anti-KLB antibody comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 domains, and a light chain variable region comprising CDR1, CDR2, and CDR3 domains. In certain embodiments, the heavy chain variable region CDR1 domain comprises an amino acid sequence having the sequence set forth in SEQ ID NO: 3, 4, 5, 6, or 7. In certain embodiments, the heavy chain variable region CDR2 domain comprises an amino acid sequence having the sequence set forth in SEQ ID NO: 8, 9, 10, 11, or 12. In certain embodiments, the heavy chain variable region CDR3 domain comprises an amino acid sequence having the sequence set forth in SEQ ID NO: 13, 14, 15, 16, or 17. In certain embodiments, the light chain variable region CDR1 domain comprises an amino acid sequence having the sequence set forth in SEQ ID NO: 18, 19, 20, 21, and 22. In certain embodiments, the light chain variable region CDR2 domain comprises an amino acid sequence having the sequence set forth in SEQ ID NO: 23, 24, 25, 26, or 27. In certain embodiments, the light chain variable region CDR3 domain comprises an amino acid sequence having the sequence set forth in SEQ ID NO: 28, 29, 30, 31, or 32.

[0117] In certain embodiments, the bispecific anti-KLB antibody comprises a heavy chain variable region CDR1 having the sequence set forth in SEQ ID NO:7; a heavy chain variable region CDR2 having the sequence set forth in SEQ ID NO:12; a heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO:17; a light chain variable region CDR1 having the sequence set forth in SEQ ID NO:22; a light chain variable region CDR2 having the sequence set forth in SEQ ID NO:27; and a light chain variable region CDR3 having the sequence set forth in SEQ ID NO:32.

[0118] In certain embodiments, a bispecific anti-KLB antibody comprises a first antibody or antigen-binding portion thereof and a second antibody or antigen-binding portion thereof, wherein the first antibody or antigen-binding portion thereof and the second antibody or antigen-binding portion thereof bind to different epitopes present on KLB. For example, without limitation, the first antibody or antigen-binding portion thereof may comprise a heavy chain variable region and a light chain variable region, and the second antibody or antigen-binding portion thereof may comprise a heavy chain variable region and a light chain variable region. In certain embodiments, the heavy chain variable region of the first and / or second antibody or antigen-binding portion thereof comprises amino acids having a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 34, 36, 38, 40, or 42. In certain embodiments, the light chain variable region of the first and / or second antibody or antigen-binding portion thereof comprises amino acids having the sequence set forth in SEQ ID NO: 33, 35, 37, 39, or 41.

[0119] For example, and without limitation, a bispecific anti-KLB antibody may comprise a first antibody, or antigen-binding portion thereof, comprising a heavy chain variable region having an amino acid sequence at least 95% identical to the sequence set forth in SEQ ID NO: 40 and a light chain variable region having an amino acid sequence at least 95% identical to the sequence set forth in SEQ ID NO: 39, and a second antibody, or antigen-binding portion thereof, comprising a heavy chain variable region having an amino acid sequence at least 95% identical to the sequence set forth in SEQ ID NO: 42 and a light chain variable region having an amino acid sequence at least 95% identical to the sequence set forth in SEQ ID NO: 41.

[0120] In certain embodiments, a bispecific anti-KLB antibody may comprise a first antibody, or antigen-binding portion thereof, comprising a heavy chain variable region having a sequence that is about 95% identical to the sequence set forth in SEQ ID NO: 34 and a light chain variable region having a sequence that is about 95% identical to the sequence set forth in SEQ ID NO: 33, and a second antibody, or antigen-binding portion thereof, comprising a heavy chain variable region having an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 40 and a light chain variable region having an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 39.

[0121] 1. Antibody affinity In certain embodiments, the multispecific and bispecific antibodies provided herein have a dissociation constant (K d ) is about 0.001 nM to about 1 μM or about 10 -8 M~about 10 -13 For example, and without limitation, a multispecific antibody, e.g., a bispecific antibody, can be K d may be 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less. d But about 10 -8 M or less, about 10 -9 M or less, about 10 -10 M or less, about 10 -11 M or less, about 10 -12 M, or about 10 -13 It can range from M or less.

[0122] In certain embodiments, K d can be measured by radiolabeled antigen binding assay (RIA). In certain embodiments, an RIA is performed on the F(ab)2 version of a bispecific antibody of interest and its two antigens. For example, the solution binding affinity of F(ab)2 for an antigen is determined by binding F(ab)2 to a minimum concentration of ( 125I) Equilibration with labeled antigen followed by capture of bound antigen on a plate coated with anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish assay conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 [I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody Fab-12 by Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although incubation may continue for a longer period (e.g., approximately 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μL / well of scintillant (MICROSCINT-20™; Packard) is added, and the plate is counted for 10 minutes on a TOPCOUNT™ gamma counter (Packard). Concentrations of each Fab that result in 20% or less of maximal binding are selected for use in competitive binding assays.

[0123] In certain embodiments, K dcan be measured using a BIACORE® surface plasmon resonance assay. For example, assays using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) are performed at 25°C with an immobilized antigen CM5 chip at approximately 10 response units (RU). In one specific embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / mL (approximately 0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μL / min, yielding approximately 10 response units (RU) of coupled protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected at a flow rate of approximately 25 μL / min in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C. The association rate (k on ) and dissociation rate (k off The equilibrium dissociation constant (K) is calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). d ) as the ratio k off / k on See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate is 10 6 M -1 s -1If the on-rate exceeds , the on-rate can be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of increasing concentrations of antigen, as measured in a spectrometer such as a stopped-flow fitted spectrophotometer (Aviv Instruments) with a stirred cuvette or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic).

[0124] 2. Antibody fragment In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, F(ab'), diabodies, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also PCT Application No. WO 93 / 16185 and U.S. Patent Nos. 5,571,894 and 5,587,458.

[0125] Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, e.g., EP Patent Application No. 404,097; PCT Application No. WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0126] Additional non-limiting examples of antibody fragments include Fab, Fab', Fab'-SH, Fv, and scFv fragments. Single domain antibodies are antibody fragments that contain all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, single domain antibodies are human single domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0127] See US Pat. No. 5,869,046 for a discussion of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives.

[0128] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0129] 3. Chimeric and humanized antibodies In certain embodiments, the multispecific and bispecific antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In certain embodiments, a chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0130] In certain embodiments, a chimeric antibody is a humanized antibody. A non-human antibody can be humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the non-human parent antibody. A humanized antibody can contain one or more variable domains in which the HVRs, e.g., CDRs or portions thereof, are derived from a non-human antibody and the FRs or portions thereof are derived from a human antibody sequence. A humanized antibody can optionally contain at least a portion of a human constant region. In certain embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0131] Humanized antibodies and methods for making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are also described in, e.g., Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity-determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"), and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0132] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)), framework regions derived from consensus sequences of human antibodies of particular subpopulations of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al. J. Immunol., 151:2623 (1993)), human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)), and framework regions derived from screening of FR libraries (see, e.g., Baca et al. al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0133] 4. Human antibodies In certain embodiments, the multispecific and bispecific antibodies provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0134] Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus or is extrachromosomally present or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 (describing XENOMOUSE™ technology), U.S. Patent No. 5,770,429 (describing HuMab® technology), U.S. Patent No. 7,041,870 (describing KM MOUSE® technology), and U.S. Patent Application Publication No. US 2007 / 0061900 (describing VelociMouse® technology). The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining them with different human constant regions.

[0135] In certain embodiments, human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (see, e.g., Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies generated via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include those described in, for example, U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0136] In certain embodiments, human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0137] 5. Library-derived Antibodies Multispecific and bispecific antibodies of the present disclosure, e.g., bispecific anti-KLB antibodies, can be isolated by screening combinatorial libraries for antibodies with the desired activity(ies). A variety of methods are known in the art, including, but not limited to, generating phage display libraries and screening such libraries for antibodies possessing the desired binding properties. Such methods are reviewed, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and are also described, for example, by McCafferty et al., Nature 348:552-554, Clackson et al., Nature 352:624-628 (1991), Marks et al., J. Mol. Biol. 222:581-597 (1992), Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003), Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004), Lee et al. al., J. Mol. Biol. 340(5):1073-1093 (2004), Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004), and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0138] In one particular phage display method, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR), randomly recombined into phage libraries, and then screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phage typically display antibody fragments as either single-chain Fv (scFv) fragments or Fab fragments. Libraries derived from immunized sources provide high-affinity antibodies to immunogens without the need for hybridoma construction. Alternatively, as described by Griffiths et al., EMBO J., 12:725-734 (1993), naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self and self antigens without any immunization. Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 regions to achieve rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Pat. No. 5,750,373, and U.S. Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0139] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments herein.

[0140] 6. Antibody variants In certain embodiments, amino acid sequence variants of the disclosed antibodies are provided herein. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody by generating amino acid sequence variants of the antibody. Amino acid sequence variants of the multispecific antibodies, e.g., bispecific anti-KLB antibodies, of the present disclosure can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired properties, e.g., antigen binding.

[0141] a) Substitution, insertion, and deletion variants In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVRs and FRs. Non-limiting examples of conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." Non-limiting examples of more substantial changes are provided in Table 1 under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into an antibody of interest, and the products can be screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved complement-dependent cytotoxicity or antibody-dependent cell-mediated cytotoxicity.

[0142] Amino acids can be grouped according to general side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln (3) Acidic: Asp, Glu (4) Basic: His, Lys, Arg (5) Residues that affect chain orientation: Gly, Pro (6) Aromatic: Trp, Tyr, Phe

[0143] In certain embodiments, non-conservative substitutions involve exchanging a member of one of these classes for another class. TIFF2026009891000003.tif221170

[0144] In certain embodiments, one type of substitutional variant involves the substitution of one or more hypervariable region residues of a parent antibody, e.g., a humanized or human antibody. Generally, the resulting variant(s) selected for further study will have an alteration, e.g., an improvement, in a certain biological property relative to the parent antibody, such as, but not limited to, increased affinity, reduced immunogenicity, etc., and / or will have substantially retained a certain biological property of the parent antibody. A non-limiting example of a substitutional variant is an affinity matured antibody, which may be conveniently generated using, for example, phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0145] In certain embodiments, modifications (e.g., substitutions) can be made in HVRs, e.g., to improve antibody affinity. Such modifications can be made in HVR "hotspots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in antigen-contacting residues, and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries is described, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In certain embodiments of affinity maturation, diversity can be introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are often targeted in particular.

[0146] In certain embodiments, substitutions, insertions, or deletions may occur between one or more HVRs, so long as such modifications do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made within an HVR. Such modifications may, for example, be outside the antigen-contacting residues within the HVR. In certain embodiments of the above-described variant VH and VL sequences, each HVR is either unaltered or contains no more than one, two, or three amino acid substitutions.

[0147] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and antigen is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues can be targeted as candidates for substitution or eliminated. Mutants can be screened to determine whether they possess the desired properties described above.

[0148] Amino acid sequence insertions can include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. A non-limiting example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules can include the N- or C-terminal fusion of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0149] b) glycosylation variants In certain embodiments, the multispecific and bispecific antibodies provided herein may be modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence so that one or more glycosylation sites are created or removed.

[0150] In certain embodiments, where an antibody comprises an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, which are generally attached to Asn297 in the CH2 domain of the Fc region via an N-linkage. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In certain embodiments, modifications of the oligosaccharides in the antibodies of the invention may be performed to generate antibody variants with improved specific properties.

[0151] In certain embodiments, antibody variants are provided that have carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be between about 1% and about 80%, between about 1% and about 65%, between about 5% and about 65%, or between about 20% and about 40%, and values ​​therebetween.

[0152] The amount of fucose can be determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycostructures (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 of the Fc region (Eu numbering of Fc region residues). However, Asn297 can also be located approximately ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to slight sequence variations within antibodies. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Publication Nos. US 2003 / 0157108 (Presta, L.) and US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, US2004 / 0132140, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004), Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).

[0153] Defucosylated antibodies can be produced in any cell line that is deficient in protein fucosylation. Non-limiting examples of cell lines include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003 / 0157108 A1, Presta, L; and WO2004 / 056312 A1, Adams et al., especially Example 11), and knockout cell lines such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0154] Further provided are antibody variants having bisected oligosaccharides, for example, where a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Non-limiting examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.), and US 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.), WO1998 / 58964 (Raju, S.), and WO1999 / 22764 (Raju, S.).

[0155] c) Fc region variant In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0156] In certain embodiments, the present invention contemplates antibody variants that retain some but not all effector functions, making them desirable candidates for applications where in vivo antibody half-life is important, yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)), and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985), U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (e.g., the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA), and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0157] Antibodies with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0158] Certain antibody variants with improved or attenuated binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)). In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region. In certain embodiments, modifications are made in the Fc region that result in altered (i.e., improved or attenuated) C1q binding and / or complement-dependent cytotoxicity (CDC), as described, e.g., in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. 164:4178-4184 (2000).

[0159] In certain embodiments, modifications made in the Fc region of the bispecific antibodies disclosed herein can produce variant antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), as described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions that improve binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0160] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351.

[0161] d) Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine-engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues may occur at accessible sites on the antibody. By substituting these residues with cysteine, reactive thiol groups are thereby positioned at accessible sites on the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates as described herein. Cysteine-engineered antibodies can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0162] e) Antibody derivative In certain embodiments, the multispecific, e.g., bispecific, antibodies provided herein can be further modified to contain additional nonproteinaceous moieties known in the art and readily available. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde can be advantageous in manufacturing due to its stability in water. The polymers can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and when more than one polymer is attached, they can be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0163] In certain embodiments, conjugates of antibodies and nonproteinaceous moieties are provided that can be selectively heated by exposure to radiation. In certain embodiments, the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). In certain embodiments, the radiation can be of any wavelength, including but not limited to wavelengths that do not harm normal cells but heat the nonproteinaceous moiety to temperatures that kill cells adjacent to the antibody-nonproteinaceous moiety.

[0164] C. Immune complex The presently disclosed subject matter also provides immunoconjugates comprising a multispecific antibody, e.g., a bispecific antibody, as disclosed herein conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitory agent, a protein, a peptide, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioisotope. For example, an antibody or antigen-binding portion of the disclosed subject matter can be operably linked (e.g., by chemical coupling, genetic fusion, noncovalent association, etc.) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic.

[0165] In certain embodiments, the immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, such as maytansinoids (U.S. Pat. Nos. 5,208,020, 5,416,064, and EP 0 425 235 B1); auristatins (MMAE and MMAF), such as the monomethyl auristatin drug moieties DE and DF (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatins: calicheamicin or derivatives thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. Res. 58:2925-2928 (1998)), anthracyclines such as daunomycin or doxorubicin (Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al. al., J. Med. Chem. 45:4336-4343 (2002), and U.S. Patent No. 6,630,579); methotrexate; taxanes such as vindesine, docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecines; and CC1065.

[0166] In certain embodiments, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, a nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0167] In certain embodiments, the immunoconjugate comprises a multispecific antibody described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Non-limiting examples include At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 When a radioconjugate is used for detection, it can be a radioactive atom, such as tc99m or I, for scintigraphy studies. 123 , or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as again iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0168] Conjugates of antibodies and cytotoxic agents can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94 / 11026. The linker may be a "cleavable linker" that facilitates the release of the cytotoxic drug inside the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker, or disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) can be used. Non-limiting examples of linkers are disclosed above.

[0169] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, conjugates prepared with crosslinker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate), which is commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA).

[0170] IV. Screening and Production Methods The multispecific antibodies of the presently disclosed subject matter can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0171] A. Multispecific Antibody Screening Assay In certain embodiments, multispecific antibodies of the present disclosure, e.g., bispecific or biepitopic antibodies, can be identified by known methods. For example, and without limitation, bispecific or biepitopic antibodies can be identified using the "knob-into-hole" heterodimerization technique discussed above. See Ridgway et al., Protein Engineering, Vol. 9:7, p617-621 (1996); Atwell et al., J. Mol. Biol. 270, 26-35 (1997); and Spiess et al., Nat. Biotech. 31, 753-759 (2013).

[0172] The presently disclosed subject matter further provides methods for screening for multispecific antibodies, e.g., bispecific or biapitopic antibodies. In certain embodiments, the methods disclosed herein can be used to identify multispecific antibodies, e.g., bispecific or biapitopic antibodies, that exhibit agonistic or antagonistic activity.

[0173] In certain embodiments, the method comprises expressing a multispecific antibody in one or more cells. For example, and without limitation, the multispecific antibody may comprise a first antigen-binding polypeptide, a second antigen-binding polypeptide, and a CL domain. The first antigen-binding polypeptide and the second antigen-binding polypeptide may have distinct binding specificities. For example, and without limitation, the first antigen-binding polypeptide may bind to a first epitope on an antigen, and the second antigen-binding polypeptide may bind to a second epitope on the antigen. Alternatively, the first antigen-binding polypeptide may bind to a first antigen, and the second antigen-binding polypeptide may bind to a second antigen. In certain embodiments, each of the antigen-binding polypeptides comprises a VL, a VH, a CH1 domain, a CH2 domain, and a CH3 domain, and these components are arranged relative to each other in the N-terminal to C-terminal order: VL-linker-VH-CH1-CH2-CH3. In certain embodiments, the first and / or second antigen-binding polypeptide may further comprise a hinge region between the CH1 domain and the CH2 domain. In certain embodiments, the C-terminus of the VL domain may be linked to the N-terminus of the VH domain via a linker. The first and / or second antigen-binding polypeptide may further comprise a heterodimerization domain disclosed herein, e.g., a protrusion or cavity in the CH3 domain.

[0174] In certain embodiments, a nucleic acid, such as a first nucleic acid encoding a first antigen-binding polypeptide, or a vector containing the nucleic acid, can be introduced into a cell. A second nucleic acid encoding a second antigen-binding polypeptide, or a vector containing the second nucleic acid, can be introduced into a cell. In certain embodiments, a third nucleic acid encoding a CL domain, or a vector encoding the third nucleic acid, can be introduced into a cell. These nucleic acids can be introduced into a cell by any method known in the art. For example, but not limited to, the nucleic acid can be introduced into a cell by transformation or transfection.

[0175] The cells used in the disclosed methods can be any of the cells disclosed herein, such as the host cells disclosed herein. For example, without limitation, the cells can be prokaryotic cells, such as Escherichia coli (E. coli). In certain embodiments, the cells are eukaryotic cells, such as yeast cells or mammalian cells. In certain embodiments, the mammalian cells are Chinese hamster ovary (CHO) cells.

[0176] In certain embodiments, the method may further comprise contacting the multispecific antibody obtained from the one or more cells with two or more antigens, e.g., a first and a second antigen, or two or more epitopes present on the same antigen, e.g., a first and a second epitope on an antigen. In certain embodiments, the two or more epitopes may be overlapping or non-overlapping. In certain embodiments, the multispecific antibody may be contacted with two or more antigens or two or more epitopes simultaneously or sequentially. For example, and without limitation, the multispecific antibody may be contacted with one of the two or more antigens or two or more epitopes, followed by contact with the second of the two or more antigens or two or more epitopes. The multispecific antibody may be purified and / or isolated, e.g., from cells, before contacting the multispecific antibody with the antigen or epitope. For example, and without limitation, the multispecific antibody may be purified by chromatographic methods, e.g., protein A chromatography.

[0177] In certain embodiments, the method may further include identifying a multispecific antibody that binds to more than one antigen or more than one epitope. In certain embodiments, binding can be determined by any assay known in the art and disclosed herein. For example, but not limited to, binding can be determined by ELISA or by flow cytometry, such as fluorescence-activated cell sorting (FACS).

[0178] In certain embodiments, methods of screening for multispecific antibodies may include generating a library comprising a plurality of the multispecific antibodies disclosed herein. For example, and without limitation, the library may comprise a plurality of cells, each cell expressing a single multispecific antibody disclosed herein. In certain embodiments, each cell in the library expresses a first antigen-binding polypeptide, a second antigen-binding polypeptide, and a CL domain. In certain embodiments, nucleic acids encoding the first antigen-binding polypeptide, the second antigen-binding polypeptide, and / or the CL domain, or vectors comprising such nucleic acids, can be introduced into cells, for example, by transformation or transfection. In certain embodiments, each of the first antigen-binding polypeptide, the second antigen-binding polypeptide, and / or the CL domain is encoded by a separate nucleic acid or a separate vector comprising such nucleic acid. In certain embodiments, the first antigen-binding polypeptide and the second antigen-binding polypeptide are encoded by a first nucleic acid (or a first vector comprising the first nucleic acid), and the CL domain is encoded by a second nucleic acid (or a second vector comprising the second nucleic acid). For example, and without limitation, a first nucleic acid of the presently disclosed subject matter can comprise an open reading frame (ORF) encoding a first antigen-binding polypeptide and an ORF encoding a second antigen-binding polypeptide. In certain embodiments, the second nucleic acid can comprise an ORF encoding a CL domain. In certain embodiments, the CL domain is not encoded by the same nucleic acid (or vector comprising the nucleic acid) that encodes the first and / or second antigen-binding polypeptide.

[0179] The method may further include assaying a plurality of multispecific antibodies in the library for binding to two or more epitopes on the antigen, e.g., a first and a second epitope on the antigen. Alternatively, or in addition, the method may include assaying a plurality of multispecific antibodies in the library for binding to two or more antigens, e.g., a first antigen and a second antigen. In certain embodiments, binding can be determined by any assay known in the art and disclosed herein. The method may further include identifying a multispecific antibody that binds to two or more antigens or two or more epitopes on an antigen. For example, without limitation, a biepitopic antibody can be obtained using the disclosed method by identifying a multispecific antibody that binds to two epitopes on an antigen.

[0180] In certain embodiments, the screening methods for multispecific antibodies disclosed herein may further comprise determining the activity of the multispecific antibody, for example, by luciferase assay. For example, and without limitation, the method may comprise determining whether the multispecific antibody exhibits antagonist activity. In certain embodiments, the method may comprise determining whether the multispecific antibody exhibits agonist activity. In certain embodiments, the method may further comprise comparing the activity of the multispecific antibody with the activity of the monospecific parent antibody from which it was derived to identify multispecific antibodies that exhibit superior activity, e.g., antagonist activity or agonist activity, than the monospecific parent antibodies alone or in combination. The activity of a multispecific antibody can be determined by any activity assay known in the art and disclosed herein.

[0181] The presently disclosed subject matter further provides methods for identifying biapitopic antibodies using the screening methods disclosed herein. For example, and without limitation, the disclosed screening methods can be used to identify biapitopic antibodies that exhibit antagonistic or agonistic activity. In certain embodiments, the disclosed screening methods can be used to identify biapitopic antibodies that exhibit agonistic activity. In certain embodiments, biapitopic antibodies identified using the disclosed methods exhibit superior agonistic activity than the monospecific parent antibodies from which they are derived, alone or in combination. In certain embodiments, biapitopic antibodies identified using the disclosed methods exhibit superior agonistic activity than monospecific antibodies with identical VL and VH sequences. In certain embodiments, a biepitopic antibody of the present disclosure exhibits superior agonist activity than a monospecific antibody having the same VL, VH, CH1, CH2, and / or CH3 sequence, for example, as one of the antigen-binding polypeptides of the biepitopic antibody.

[0182] In certain embodiments, methods for identifying a biapitopic agonist antibody can include expressing a multispecific antibody, e.g., a bispecific or biapitopic antibody, in one or more cells. For example, without limitation, the multispecific antibody can comprise a first antigen-binding polypeptide, a second antigen-binding polypeptide, and a CL domain, as disclosed herein. In certain embodiments, the first antigen-binding polypeptide binds to a first epitope on the antigen, and the second antigen-binding polypeptide binds to a second epitope on the antigen. In certain embodiments, the first antigen-binding polypeptide has the same binding specificity and / or the same VL and VH sequences as the first monospecific antibody, and the second antigen-binding polypeptide has the same binding specificity and / or the same VL and VH sequences as the first monospecific antibody.

[0183] The method may further include contacting a multispecific antibody, e.g., a bispecific or biapitopic antibody, obtained from one or more cells with two epitopes present on the same antigen of interest, e.g., a first and a second epitope on the antigen. For example, and without limitation, the multispecific antibody can be contacted with one of the two epitopes, followed by contacting the multispecific antibody with the second of the two epitopes. As described above, the multispecific antibody, e.g., a bispecific or biapitopic antibody, can be purified before contacting with the two epitopes. In certain embodiments, the method may further include identifying a multispecific antibody that binds to the two epitopes of interest. Binding to the epitopes can be determined by any assay known in the art and disclosed herein.

[0184] In certain embodiments, the method may further include determining the activity of the biapitopic antibody that binds to the two identified epitopes. For example, without limitation, the method may include determining whether the biapitopic antibody exhibits agonist activity. The agonist activity of a biapitopic antibody can be determined by any activity assay known in the art and disclosed herein. In certain embodiments, the agonist activity of a biapitopic antibody can be determined by contacting an antigen, e.g., a receptor, with the biapitopic antibody and analyzing whether the biapitopic antibody results in activation of a signaling pathway associated with the receptor. For example, without limitation, agonist activity can be determined using an in vitro reporter-based assay, e.g., a luciferase assay, in which activation of a receptor, e.g., an antigen, results in expression of a reporter, e.g., luciferase. Alternatively, or in addition, agonist activity can be determined by in vitro cellular assays in which activation of a receptor, e.g., an antigen, results in activation of a signal transduction pathway regulated by the receptor, e.g., by analyzing downstream targets of the receptor's signal transduction pathway, e.g., protein expression, activity, and / or phosphorylation.

[0185] In certain embodiments, the method may further comprise comparing the agonist activity of the biapitopic antibody to the agonist activity of the monospecific parent antibodies (e.g., from which the biapitopic antibody was derived), alone or in combination, and identifying a biapitopic antibody that exhibits agonist activity superior to that of the monospecific parent antibodies. For example, and without limitation, the method may further comprise determining the agonist activity of the monospecific parent antibodies of the biapitopic agonist antibody (e.g., alone or in combination) using the same techniques used to determine the agonist activity of the biapitopic antibody and compare the agonist activity of the biapitopic antibody to the agonist activity of the monospecific antibodies.

[0186] In certain embodiments, the VL, VH, CH1, CH2, and / or CH3 domains of a first antigen-binding polypeptide can have the same sequence as the VL, VH, CH1, CH2, and / or CH3 domains of a first parent monoclonal antibody. In certain embodiments, the VL, VH, CH1, CH2, and / or CH3 domains of a second antigen-binding polypeptide can have the same sequence as the VL, VH, CH1, CH2, and / or CH3 domains of a second parent monoclonal antibody. The disclosed methods can enable the identification of multispecific antibodies, e.g., bispecific and biapitopic antibodies, by pairing two antigen-binding polypeptides with the binding specificities of two parent monoclonal antibodies, e.g., monospecific antibodies. In certain embodiments, multispecific antibodies identified by the disclosed screening assays exhibit higher binding affinity and / or activity, e.g., agonistic activity, than the parent antibodies, e.g., monospecific antibodies, alone or in combination. As a result, the disclosed methods provide a high-throughput method for pairwise screening of the VL and VH regions of monospecific antibodies to identify multispecific, e.g., bispecific or biapitopic, antibodies. In certain embodiments, multispecific, e.g., bispecific or biapitopic, antibodies identified by the disclosed screening methods can be formatted into different antibody formats. For example, and without limitation, multispecific, e.g., bispecific or biapitopic, antibodies identified by the disclosed methods can be reformatted into a full antibody format, e.g., of the IgG isotype.

[0187] In certain embodiments, the antigen bound by a multispecific, bispecific, or biepitopic antibody is a receptor. In certain embodiments, the antigen is a bioconjugate. For example, and without limitation, the receptor is present within a bioconjugate, e.g., in a complex with one or more co-receptors and / or proteins.

[0188] B. Binding and Other Assays In certain embodiments, antibodies of the invention can be tested for their antigen-binding activity by known methods, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or Western blot assay. Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.

[0189] In certain embodiments, antigen (e.g., KLB)-antibody complexes can be detected, for example, using an enzyme-linked antibody or antibody fragment that recognizes and specifically binds to the antigen (e.g., KLB)-antibody complexes. Alternatively, these complexes can be detected using any of a variety of other immunoassays. For example, the antibody can be radiolabeled and used in a radioimmunoassay (RIA) (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986, incorporated herein by reference). Radioisotopes can be detected by means such as the use of a Geiger counter or a scintillation counter, or by autoradiography.

[0190] In certain embodiments, a competition assay can be used to identify antibodies that compete with the multispecific antibodies, e.g., bispecific antibodies, of the presently disclosed subject matter. Detailed exemplary methods for mapping epitopes to which antibodies bind are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology, vol. 66 (Humana Press, Totowa, NJ). In a non-limiting example of an exemplary competition assay, an immobilized antigen (e.g., KLB) can be incubated in a solution containing a first labeled antibody that binds to the antigen (e.g., KLB) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to the antigen (e.g., KLB). The second antibody may be present in hybridoma supernatant. As a control, the immobilized antigen (e.g., KLB) is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to the antigen (e.g., KLB), excess unbound antibody is removed and the amount of label associated with the immobilized antigen (e.g., KLB) is measured. If the amount of label associated with the immobilized antigen (e.g., KLB) is significantly reduced in the test sample compared to the control sample, this indicates that the second antibody competes with the first antibody for binding to the antigen (e.g., KLB). See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0191] In certain embodiments, the binding of a multispecific antibody, e.g., a bispecific antibody, to cells expressing the antibody's antigen, e.g., 293T cells, can be determined using flow cytometry. For example, but not limited to, a cell line expressing an antigen or epitope (e.g., KLB) can be mixed with various concentrations of the bispecific antibody in PBS containing 0.1% BSA and 10% fetal bovine serum and incubated at 37°C for 1 hour. After washing, the cells can be reacted with a fluorescein-labeled anti-human IgG antibody under the same conditions as the primary antibody staining. The sample can then be analyzed using a FACScan instrument using light and side scatter characteristics to gate on single cells. In addition to or instead of a flow cytometry assay, an alternative assay using fluorescein microscopy can be used. Cells can be stained as described above and examined by fluorescein microscopy. This method allows visualization of individual cells and allows analysis of the localization of the antigen within the cells.

[0192] Multispecific antibodies, e.g., bispecific antibodies, of the present disclosure can be further tested for reactivity with the antibody's epitope or antigen by Western blot. For example, but not limited to, cell extracts from cells expressing the antigen of interest (e.g., KLB) can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to a nitrocellulose membrane, blocked with 10% fetal bovine serum, and probed with the monoclonal antibody to be tested. Bispecific antibody binding can be detected using anti-human IgG alkaline phosphatase and developed with BCIP / NBT substrate tablets (Sigma Chem. Co., St. Louis, MO).

[0193] C. Activity Assay The present disclosure provides assays for identifying multispecific antibodies, e.g., bispecific antibodies, with biological activity. For example, and without limitation, the present disclosure provides assays for identifying multispecific antibodies, e.g., anti-KLB antibodies, with biological activity. For example, and without limitation, biological activity for anti-KLB antibodies can include, for example, activation of the KLB / FGFR1c receptor complex. Antibodies with such biological activity in vivo and / or in vitro are also provided. In certain embodiments, the assay can include binding a bispecific antibody of the present disclosure to cells, e.g., 293T cells expressing KLB, and analyzing the activity and / or phosphorylation state of one or more downstream targets of the KLB-FGFR1 receptor complex, e.g., ERK. In certain embodiments, the assay can include administering a bispecific antibody of the present disclosure to a subject, e.g., a non-human animal, and analyzing the effect of the antibody on glucose levels in the subject.

[0194] D. Production Method The multispecific, bispecific, and biepitopic antibodies disclosed herein can be produced using any technique available or known in the art. For example, but not limited to, the antibodies can be produced using recombinant methods and compositions described, for example, in U.S. Patent No. 4,816,567. Detailed procedures for generating bispecific antibodies, e.g., anti-KLB bispecific antibodies, are described in the Examples below.

[0195] In certain embodiments, techniques for generating bispecific, biepitopic, and / or multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy-light chain pairs with different specificities, e.g., in a VLfH format (see Milstein and Cuello, Nature 305:537 (1983)), PCT Patent Application No. WO 93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), and heterodimerization domains produced using, for example, the "knobs-in-holes" technique (see, e.g., U.S. Pat. No. 5,731,168). The "knobs-in-holes" technique aims to pair two different antibody heavy chains by introducing mutations into the CH3 domain to modify the contact interface. In one heavy chain, one or more original amino acids may be replaced with amino acids with short side chains to create a "hole" or "cavity." In certain embodiments, the hole mutation(s) may include one or more of T366S, L368A, and / or Y407V (EU numbering). Conversely, one or more amino acids with large side chains were introduced into the other CH3 domain of the second antibody heavy chain to create a "knob." In certain embodiments, the knob mutation may include T366W (EU numbering). By co-expressing these two heavy chains (and two identical light chains appropriate for both heavy chains), a higher yield of heterodimer formation ("knob-hole") versus homodimer formation ("hole-hole" or "knob-knob") is observed (see Ridgway, Protein Eng. 9 (1996) 617-621, and WO 96 / 027011). The knob-in-hole technique has also been used when two heavy chains are paired with different or cognate light chains. For example, and without limitation, two heavy / light chain pairs can each be produced in separate cells, folded, purified, and assembled to form knobs-in-holes bispecific antibodies in vitro. See, e.g., WO2012 / 106587, WO2011 / 133886, and WO2013 / 055958, the contents of which are hereby incorporated by reference in their entireties. See also Example 2 of the present disclosure.Additional mutations that can be introduced into the Fc region of the disclosed antibodies to promote heterodimerization of the two heavy chains are disclosed below.

[0196] In certain embodiments, other heterodimerization domains that strongly favor heterodimer formation over homodimers can be incorporated into the disclosed multispecific antibodies, e.g., for in vitro or in vivo production of such antibodies. Non-limiting examples of such heterodimerization domains are disclosed in WO 2007 / 147901 (depicting ionic interactions), WO 2009 / 089004 (depicting electrostatic steering effects), and WO 2010 / 034605 (depicting coiled-coils), the contents of which are hereby incorporated by reference in their entireties. See also Pack and Plueckthun, Biochemistry 31:1579-1584 (1992), describing leucine zippers, and Pack et al., Bio / Technology 11:1271-1277 (1993), describing helix-turn-helix motifs. In certain embodiments, the multispecific antibodies disclosed herein may comprise one or more heterodimerization domains to generate heterodimers and facilitate, for example, the interaction between a first antigen-binding polypeptide and a second antigen-binding polypeptide of the multispecific antibody. Further non-limiting examples of techniques that can be used to generate heterodimerization domains within an antibody, e.g., within the first and / or second antigen-binding polypeptide of an antibody of the disclosure, include F405L in the first antigen-binding polypeptide chain and K409R in the second antigen-binding polypeptide chain (Labrijn et al. Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange. Proc. Nat'l. Acad. Sci. USA 110:5145-5150 (2013)); T350V, L351Y, F405A, and Y407V in the first antigen-binding polypeptide chain, and T350V, T366L, K392L, and T394W in the second antigen-binding polypeptide chain (Kreudenstein et al.Improving biophysical properties of a bispecific antibody scaffold to aid developability: quality by molecular design. MAbs 5:646-654 (2013)); K409D and K392D in the first antigen-binding polypeptide chain and D399K and E356K in the second antigen-binding polypeptide chain (Gunasekaran et al. Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG. J. Biol. Chem. 285:19637-19646 (2010)); D221E, P228E, and L368E in the first antigen-binding polypeptide chain and D221R, P228R, and K409R in the second antigen-binding polypeptide chain (Strop et al. Generating Bispecific Human IgG1 and IgG2 Antibodies from Any Antibody Pair. J Mol Biol 420:204-219 (2012)); IgG / A chimera (Davis, J. et al. SEEDbodies: fusion proteins based on strand-exchange engineered domain (SEED) CH3 heterodimers in an Fc analogue platform for asymmetric binders or immunofusions and bispecific antibodies. Protein Eng. Des. Sel 23:195-202 (2010)); H435R in one of the first or second antigen-binding polypeptide chains (US 2014 / 0248664A1); Y349C, K360E, and K409W in the first antigen-binding polypeptide chain and S354C, Q347R, D399V, and F405T in the second antigen-binding polypeptide chain (Choi et al.Crystal structures of immunoglobulin Fc heterodimers reveal the molecular basis for heterodimer formation. Mol. Immunol. 65:377-383 (2015)); and S364H and F405A in the first antigen-binding polypeptide chain, and Y349T and T394F in the second antigen-binding polypeptide chain (Moore et al. A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens. MAbs 3:546-557 (2011)).

[0197] Multispecific, biepitopic, and bispecific antibodies of the present disclosure can be synthesized by a variety of techniques, including the manipulation of electrostatic steering effects to create antibody Fc-heterodimeric molecules (WO 2009 / 089004 A1); cross-linking of two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); production of bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); use of "diabody" technology to generate bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and the use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 148(5):1547-1553 (1992)). al., J. Immunol., 152:5368 (1994)); as well as by preparation of trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

[0198] Bispecific and multispecific molecules of the present disclosure can also be produced using chemical techniques (see, e.g., Kranz (1981) Proc. Natl. Acad. Sci. USA 78:5807), "polydoma" technology (see, e.g., U.S. Pat. No. 4,474,893), or recombinant DNA techniques. Bispecific and multispecific molecules of the present invention can also be prepared by conjugating constituent binding specificities, e.g., a first epitope and a second epitope binding specificity, using methods known in the art and described herein. For example, each binding specificity of the bispecific and multispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of binding or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky (1984) J. Exp. Med. 160:1686; Liu (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described by Paulus (Behring Ins. Mitt. (1985) No. 78, 118-132; Brennan (1985) Science 229:81-83; Glennie (1987) J. Immunol. 139:2367-2375). When the binding specificities are antibodies (e.g., two humanized antibodies), they can be conjugated via sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains. This hinge region can be modified to contain an odd number of sulfhydryl residues, e.g., one, before conjugation.

[0199] In certain embodiments, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific and multispecific molecules are MAb x MAb, MAb x Fab, Fab x F(ab')2, or ligand x Fab fusion proteins. Multispecific antibodies, e.g., bispecific antibodies, of the present disclosure can be single-chain molecules, e.g., single-chain bispecific antibodies, single-chain bispecific molecules comprising one single-chain antibody and a binding determinant, or single-chain bispecific molecules comprising two binding determinants. Bispecific and multispecific molecules can also be single-chain molecules or can comprise at least two single-chain molecules. Methods for preparing bi- or multispecific molecules are described, for example, in U.S. Patent No. 5,260,203, U.S. Patent No. 5,455,030, U.S. Patent No. 4,881,175, U.S. Patent No. 5,132,405, U.S. Patent No. 5,091,513, U.S. Patent No. 5,476,786, U.S. Patent No. 5,013,653, U.S. Patent No. 5,258,498, and U.S. Patent No. 5,482,858. Engineered antibodies having three or more functional antigen-binding sites (e.g., epitope-binding sites), including "octopus antibodies," are also included herein (see, e.g., US 2006 / 0025576 A1).

[0200] In certain embodiments, an animal system can be used to produce the disclosed antibodies. One animal system for preparing hybridomas is the mouse system. Hybridoma production in mice is a well-established procedure. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known (see, e.g., Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).

[0201] The presently disclosed subject matter further provides an isolated nucleic acid encoding a multispecific antibody, e.g., a bispecific antibody, disclosed herein. For example, the isolated nucleic acid may encode a first antigen-binding polypeptide and / or a second antigen-binding polypeptide of a multispecific antibody. In certain embodiments, the first antigen-binding polypeptide and the second antigen-binding polypeptide are encoded by two separate nucleic acids. In certain embodiments, the presently disclosed subject matter further provides an isolated nucleic acid encoding a CL domain, e.g., a kappa CL domain.

[0202] In certain embodiments, the isolated nucleic acid may encode an amino acid sequence comprising an antibody VL and / or an amino acid sequence comprising an antibody VH, e.g., the light chain and / or the heavy chain of an antibody. In certain embodiments, the isolated nucleic acid may comprise a nucleotide sequence encoding a heavy chain variable region amino acid sequence having the sequence set forth in SEQ ID NOs: 34, 36, 38, 40, and 42, and / or a nucleotide sequence encoding a light chain variable region amino acid sequence having the sequence set forth in SEQ ID NOs: 33, 35, 37, 39, and 41. In certain embodiments, the nucleic acid may be present in one or more vectors, e.g., expression vectors.

[0203] As used herein, a "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thus are replicated along with the host genome. Moreover, certain vectors, expression vectors, are capable of directing the expression of genes to which they are operatively linked. In general, expression vectors utilized in recombinant DNA techniques are often in the form of plasmids (vectors). However, the disclosed subject matter is intended to include other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

[0204] In certain embodiments, nucleic acids encoding the multispecific antibody and / or one or more vectors containing the nucleic acids can be introduced into, e.g., transformed into, a host cell. In certain embodiments, the host cell can be transformed with (1) a first vector containing a nucleic acid encoding an amino acid sequence comprising a first antigen-binding polypeptide; (2) a second vector containing a nucleic acid encoding an amino acid sequence comprising a second antigen-binding polypeptide; and (3) a third vector containing a nucleic acid encoding an amino acid sequence comprising the CL domain of the antibody.

[0205] In certain embodiments, the host cell may contain, e.g., be transformed with, (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody.

[0206] In certain embodiments, methods for producing multispecific antibodies may include culturing host cells into which one or more nucleic acids encoding the antibody or specific components of the antibody, e.g., the first antigen-binding polypeptide, the second antigen-binding polypeptide, and / or the CL domain, have been introduced under conditions suitable for antibody expression, and optionally recovering the antibody from the host cells and / or host cell medium. In certain embodiments, the antibody can be recovered from the host cells by lysing the host cells. Alternatively, the antibody can be secreted from the host cells and recovered from the host cell medium. In certain embodiments, the antibody is recovered from the host cells, e.g., from a host cell lysate, or from the host cell medium by chromatographic techniques.

[0207] For recombinant production of an antibody of the invention, for example, nucleic acid encoding the above-described antibody is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the antibody).

[0208] Suitable host cells for cloning or expressing antibody-encoding vectors include the prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and antigen-binding polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing the expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and further purified. In certain embodiments, the host cell is E. coli.

[0209] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006). Suitable host cells for the expression of glycosylated antibodies can also be obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plants and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0210] In certain embodiments, plant cell cultures can be utilized as host cells. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0211] In certain embodiments, vertebrate cells may be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40 (COS-7) transformed monkey kidney CV1 lines, human embryonic kidney lines (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells described, for example, in Mather et al., Annals NYAcad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include DHFR - Examples include Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0212] IV.How to use The presently disclosed subject matter further provides methods of using the disclosed multispecific and bispecific antibodies. In certain embodiments, the disclosed methods are directed to therapeutic uses of the disclosed antibodies. In certain embodiments, the disclosed methods are directed to the use of the disclosed antibodies in diagnostic methods.

[0213] A. Treatment method In certain embodiments, one or more antibodies of the presently disclosed subject matter can be used to treat a disease in a subject. In certain embodiments, a method of treating an individual comprises administering to the individual a therapeutically effective amount of an antibody disclosed herein. In certain embodiments, the method can further comprise administering to the subject a therapeutically effective amount of at least one additional therapeutic agent. Non-limiting examples of additional therapeutic agents, e.g., second therapeutic agents, are described below.

[0214] In certain embodiments, disease can be metabolic disorder.Non-limiting examples of metabolic disorder include but are not limited to polycystic ovarian syndrome (PCOS), metabolic syndrome (MetS), obesity, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), dyslipidemia, hypertension, type 2 diabetes, non-type 2 diabetes, type 1 diabetes, latent autoimmune diabetes (LAD), maturity-onset diabetes of the young (MODY), and geriatric disease and related diseases such as Alzheimer's disease, Parkinson's disease and ALS.

[0215] In certain embodiments, the presently disclosed subject matter provides methods for treating immune-related diseases or disorders, such as autoimmune diseases or disorders and inflammatory diseases. In certain embodiments, the methods comprise administering to a subject in need thereof an antibody of the present disclosure, or a pharmaceutical composition comprising the antibody. Non-limiting examples of immune-related diseases or disorders include systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis, idiopathic inflammatory myopathies, Sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, thyroiditis, diabetes mellitus, type I or II diabetes mellitus, immune-mediated kidney disease, demyelinating diseases of the central and peripheral nervous system, such as multiple sclerosis, idiopathic demyelinating polyneuropathy, or Guillain-Barré syndrome, and chronic inflammatory demyelinating polyneuropathy. These include: hepatic and biliary disorders, such as infectious autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis; inflammatory bowel disease, ulcerative colitis, Crohn's disease, gluten-sensitive enteropathy and Whipple's disease; bullous dermatoses, erythema multiforme and contact dermatitis; psoriasis; allergic diseases, such as asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity, and urticaria; immune diseases of the lung, such as eosinophilic pneumonia, idiopathic pulmonary fibrosis, and hypersensitivity pneumonitis; transplant-related diseases, including graft rejection and graft-versus-host disease. In certain other embodiments, the immune-related disease is asthma, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, lupus erythematosus, psoriasis, chronic obstructive pulmonary disease, or idiopathic pulmonary fibrosis.

[0216] In certain embodiments, the presently disclosed subject matter provides a method for treating a cell proliferation-associated disease or disorder, comprising administering to a subject in need thereof an antibody of the present disclosure or a pharmaceutical composition comprising the antibody. In certain embodiments, the cell proliferation-associated disease may be, but is not limited to, colorectal cancer, renal cell carcinoma (e.g., renal cell carcinoma), melanoma, bladder cancer, ovarian cancer, breast cancer (e.g., triple-negative breast cancer, HER2-positive breast cancer, or hormone receptor-positive cancer), and non-small cell lung cancer (e.g., squamous non-small cell lung cancer or non-squamous non-small cell lung cancer). In certain embodiments, cancers treated by the methods of the present disclosure include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. In certain embodiments, cancers to be treated by the methods of the present disclosure include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), melanoma, renal cell carcinoma, cancer of the peritoneum, hepatocellular carcinoma, gastric cancer or cancer of the stomach (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, and various types of head and neck cancer, as well as B-cell lymphomas (low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, and the like). Cancers include, but are not limited to, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phagocytosis, edema (such as that associated with brain tumors), and Meigs syndrome. In certain embodiments, the cancer may be an early-stage or late-stage cancer. In certain embodiments, the cancer may be a primary tumor. In certain embodiments, the cancer may be a metastatic tumor at a second site derived from any of the above types of cancer.

[0217] In certain embodiments, a multispecific antibody, e.g., a bispecific anti-KLB antibody, for use in the disclosed methods may be present in a pharmaceutical composition. In certain embodiments, the pharmaceutical composition may include a pharmaceutically acceptable carrier. Additionally or alternatively, the pharmaceutical composition may include a second therapeutic agent. When the antibody is administered together with another therapeutic agent, the two can be administered in either order or simultaneously.

[0218] For the prevention or treatment of disease, the appropriate dosage, e.g., a therapeutically effective amount, of an antibody of the presently disclosed subject matter, when used alone or in combination with one or more other additional therapeutic agents, will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is being administered for prophylactic or therapeutic purposes, previous therapy, the patient's medical history and response to the antibody, and the discretion of the attending physician.

[0219] In certain embodiments, the antibody is administered to a patient at one time or over a series of treatments. For example, without limitation, the antibody and / or pharmaceutical formulation containing the antibody disclosed herein can be administered to a subject twice a day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks, once a month, once every two months, once every three months, once every six months, or once a year. In certain embodiments, the antibody and / or pharmaceutical formulation containing the antibody disclosed herein can be administered to a subject once a week or once a month.

[0220] In certain embodiments, depending on the type and severity of the disease, an initial candidate dosage for administration to a patient may be about 1 μg / kg to about 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg), whether by one or more separate administrations or by continuous infusion. A typical daily dose may range from about 1 μg / kg to 100 mg / kg, depending on the factors discussed above. In certain embodiments, the daily dose may be greater than about 100 mg / kg. In some methods, dosage is adjusted to achieve a plasma antibody concentration of 1 to 1000 μg / mL, and in some methods, a plasma antibody concentration of 25 to 300 μg / mL. Alternatively, the antibody may be administered as a sustained-release formulation, in which case less frequent administration is required. Dosage and frequency may vary based on the half-life of the antibody in the patient.

[0221] For repeated administrations over several days or longer, depending on the condition, treatment may generally be continued until a desired suppression of disease symptoms occurs. In certain embodiments, the dosage of the antibody may range from about 0.05 mg / kg to about 10 mg / kg. For example, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, weekly or once every three weeks (e.g., so that the patient receives about two to about 20, or, for example, about six, doses of the antibody). A higher initial loading dose, followed by one or more lower doses, may be administered.

[0222] In certain embodiments, the method can further include monitoring the subject and determining the effectiveness of the treatment. For example, the progress of this therapy can be easily monitored by conventional techniques and assays.

[0223] B. Diagnostic and Detection Methods The presently disclosed subject matter provides methods for diagnosing and / or detecting diseases using the disclosed antibodies disclosed herein. In a further aspect, methods for detecting the presence and / or level of an antigen in a biological sample are provided. As used herein, the term "detecting" encompasses quantitative and / or qualitative detection.

[0224] Non-limiting examples of samples include, but are not limited to, cultured cells, cell supernatants, cell lysates, serum, plasma, biological fluids (e.g., blood, plasma, serum, stool, urine, lymph, ascites, ductal lavage, nipple aspirate, saliva, bronchoalveolar lavage, tears, and cerebrospinal fluid), and tissue samples. The source of the sample can be solid tissue from an individual (e.g., a freshly collected, frozen, and / or preserved organ, tissue sample, biopsy, or aspirate), blood or any blood component, biological fluid (e.g., urine, lymph, cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid, etc.), or cells. In certain embodiments, the biological sample is tissue and / or cells from the liver.

[0225] In certain embodiments, a diagnostic or detection method comprises contacting a biological sample with a bispecific anti-KLB antibody described herein under conditions that allow binding of the bispecific anti-KLB antibody to KLB, and detecting whether a complex is formed between the bispecific anti-KLB antibody and KLB. Such methods may be in vitro or in vivo methods, such as immunofluorescence or Western blot. In certain embodiments, the bispecific anti-KLB antibodies disclosed herein are used to select subjects eligible for anti-KLB antibody therapy, for example, when KLB is a biomarker for patient selection.

[0226] In certain embodiments, the disclosed antibodies for use in the present methods can be labeled. Labels include, but are not limited to, labels or moieties that are directly detected, such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels, as well as moieties that are indirectly detected, such as through an enzymatic reaction or molecular interaction, such as an enzyme or ligand. Non-limiting examples of labels include radioisotopes. 32 P, 14 C. 125 I, 3 H, and 131 Examples of suitable dye oxidizers include, but are not limited to, I, rare earth chelates or fluorophores such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases such as firefly luciferase and bacterial luciferase (see U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, enzymes that oxidize dye precursors using hydrogen peroxide, such as heterocyclic oxidases such as uricase and xanthine oxidase coupled with HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0227] V. Pharmaceutical Formulations The presently disclosed subject matter further provides pharmaceutical formulations containing the multispecific, e.g., bispecific, antibodies described herein, together with a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical compositions may include a combination of multiple (e.g., two or more) isolated multispecific, e.g., bispecific, antibodies of the presently disclosed subject matter, and / or antigen-binding portions thereof.

[0228] In certain embodiments, the disclosed pharmaceutical formulations can be prepared by combining the disclosed multispecific, e.g., bispecific anti-KLB antibodies having a desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized formulation or aqueous solution. For example, but not limited to, lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. In certain embodiments, aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO 2006 / 044908, the latter formulations comprising a histidine-acetate buffer.

[0229] In certain embodiments, antibodies of the disclosure may be greater than about 80%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, greater than about 99.1%, greater than about 99.2%, greater than about 99.3%, greater than about 99.4%, greater than about 99.5%, greater than about 99.6%, greater than about 99.7%, greater than about 99.8%, or greater than about 99.9% pure.

[0230] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; Examples of pharmaceutically acceptable carriers include, but are not limited to, proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Further non-limiting examples of pharmaceutically acceptable carriers further include intercalating drug dispersants, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968, the contents of which are incorporated herein by reference in their entireties. In certain embodiments, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0231] The carrier can be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., bispecific antibody, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0232] The pharmaceutical compositions of the present disclosure can also be administered in combination therapy, i.e., in combination with other drugs. In certain embodiments, the pharmaceutical compositions disclosed herein can also contain two or more active ingredients, as needed for the specific indication to be treated, for example, active ingredients with complementary activities that do not adversely affect each other. In certain embodiments, the pharmaceutical preparation can contain a second active ingredient for treating the same disease as the disease treated by the first therapeutic agent. Such active ingredients are preferably present in combination in amounts effective for the intended purpose.

[0233] The compositions of the present disclosure can be administered by a variety of methods known in the art. The route and / or mode of administration varies depending on the desired results. The active compound can be prepared with a carrier that protects the compound from rapid release, such as a controlled-release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are described, for example, in *Sustained and Controlled Release Drug Delivery Systems*, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978. In certain embodiments, the pharmaceutical composition is manufactured under the Good Manufacturing Practice (GMP) conditions of the U.S. Food and Drug Administration.

[0234] Sustained-release formulations containing the disclosed bispecific anti-KLB antibodies can also be prepared. Suitable examples of sustained-release preparations include, but are not limited to, semipermeable matrices of solid hydrophobic polymers containing the antibodies, which matrices are in the form of shaped articles, e.g., films, or microcapsules. In certain embodiments, the active ingredient can be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacylate) microcapsules, respectively, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or microemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0235] To administer an antibody of the present disclosure via certain routes of administration, it may be necessary to coat the compound with, or co-administer with, a material to prevent its inactivation. For example, the compound may be administered to a subject in an appropriate carrier, such as a liposome, or diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions and conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

[0236] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injections or dispersions.The use of such media and agents for pharmaceutical active substances is well known in the art.Except for the case where any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical composition of the present invention is contemplated.A supplementary active ingredient can also be incorporated into the composition.

[0237] Therapeutic compositions typically must be sterile, substantially isotonic, and stable under the conditions of manufacture and storage. The compositions can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0238] Sterile injectable solutions can be prepared by incorporating the required amount of the bispecific antibody in an appropriate solvent with one or a combination of the ingredients listed above, as required, followed by microfiltration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization), which yield a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof.

[0239] Therapeutic compositions can be administered by medical devices known in the art. For example, therapeutic compositions of the present disclosure can be administered by needleless hypodermic injection devices, such as those disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of implants and modules useful in the present invention include U.S. Pat. No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for transdermal administration of drugs; U.S. Pat. No. 4,447,233, which discloses a drug infusion pump for delivering drugs at precise infusion rates; U.S. Pat. No. 4,447,224, which discloses a variable flow rate implantable infusion device for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multiple chambers; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known.

[0240] Formulations of the present invention for therapeutic compositions include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of bispecific antibody that can be combined with a carrier material to produce a single dosage form will vary depending upon the subject treated and the particular mode of administration. The amount of bispecific antibody that can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition that produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about 99 percent active ingredient, from about 0.1 percent to about 70 percent, or from about 1 percent to about 30 percent active ingredient.

[0241] In addition, the formulation of the subject matter disclosed herein suitable for vaginal administration includes pessaries, tampons, creams, gels, pastes, foams, or spray formulations containing carriers known in the art to be appropriate.Dosage forms for topical or transdermal administration of the compound of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants.The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0242] The phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0243] These pharmaceutical compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured both by the above-mentioned sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like, in the compositions. Furthermore, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0244] When the antibodies of the present invention are administered to humans and animals as pharmaceuticals, they can be given as they are, or as a pharmaceutical composition comprising, for example, about 0.01% to about 99.5% (or about 0.1 to about 90%) of the bispecific antibody in combination with a pharmaceutically acceptable carrier.

[0245] VI. Products and Kits The presently disclosed subject matter further relates to articles of manufacture and kits. For example, without limitation, articles of manufacture and / or kits of the present disclosure can include one or more multispecific antibodies, e.g., bispecific antibodies, disclosed herein.

[0246] In certain embodiments, the article of manufacture comprises a container and a label or package insert on or associated with the container. Non-limiting examples of suitable containers include bottles, vials, syringes, and IV solution bags. The container may be formed from a variety of materials, such as glass or plastic. The container may hold a composition that is effective for treating, preventing, and / or diagnosing a condition, by itself or in combination with another composition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle).

[0247] In certain embodiments, the disclosed articles of manufacture and / or kits contain materials useful for the treatment, prevention, and / or diagnosis of the above-mentioned disorders. In certain embodiments, at least one active agent in the composition is an antibody of the presently disclosed subject matter. The label or package insert may indicate that the composition is used to treat the selected condition.

[0248] In certain embodiments, the article of manufacture may comprise (a) a first container having therein a composition comprising a bispecific antibody disclosed herein of the presently disclosed subject matter, and (b) a second container having therein a composition comprising an additional cytotoxic or therapeutic agent. In certain embodiments, the article of manufacture may further comprise a package insert indicating that the composition can be used to treat a particular condition.

[0249] In certain embodiments, the article of manufacture may further comprise an additional container, such as a second or third container comprising a pharmaceutically acceptable buffer, such as, but not limited to, bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0250] The following examples are merely illustrative of the subject matter disclosed herein and should not be construed as limiting in any way. [Example]

[0251] Example 1: Development and characterization of anti-KLB antibodies This example describes the generation of anti-KLB monoclonal antibodies. To generate anti-KLB monoclonal antibodies, KLB knockout (KLB.ko) mice (Genentech, South San Francisco, CA) were immunized with 50 μg of either pRK or pCMV vectors expressing either KLB or FGFR1c, or the pCMV.hKLB.IRES.hFGFR1c vector expressing human KLB and FGFR1c with or without mFlt3 ligand (DNA) and mGM-CSF (DNA) (Genentech) diluted in lactated Ringer's solution, via hydrodynamic tail vein (HTV) injection at 1-4 week intervals for a total of 3-13 injections, or with 5 million 300.19 cells stably transfected with human KLB and FGFR1c diluted in PBS via intraperitoneal (ip) injection weekly for a total of 12 injections. Mice received either KLB and FGFR1c plasmid DNA via HTV or 5 million 300.19-KLB / FGFR1c transfected cells with 2 μg each of human KLB protein and cynomolgus monkey KLB protein via i.p. injection, each at 50 μg, as a prefusion boost.

[0252] Spleens were harvested 3 days after the final immunization. Splenocytes from mice whose sera showed strong binding to 293 cells overexpressing human KLB and / or the KLB / FGFR1c complex by FACS were fused with P3X63-Ag8U.1 mouse myeloma cells (American Type Culture Collection, Manassas, VA) by electrofusion (Cyto Pulse CEEF-50 apparatus, BTX Harvard Apparatus, Holliston, MA). After washing twice with Cytofusion Medium C (BTX Harvard Apparatus 47-0001), the isolated splenocytes and myeloma cells were mixed at a 1:1 ratio and then resuspended in Cytofusion Medium C at 10 million cells / ml. Electrofusion was performed according to the manufacturer's instructions. The fused cells were cultured overnight in ClonaCell-HY Medium C (Stemcell Technologies, catalog no. 03803) at 37°C in a 7% CO2 incubator. The next day, the fused cells were centrifuged and resuspended in 10 ml of ClonaCell-HY Medium C with anti-mouse IgG-FITC (Jackson Immunoresearch, West Grove, PA), then gently mixed with 90 ml of methylcellulose-based ClonaCell-HY Medium D (StemCell Technologies, Cat. No. 03804) containing HAT components. Cells were plated in OmniTray plates (Thermo Fisher Scientific, Rochester, NY) and grown at 37°C in a 7% CO2 incubator. After 6–7 days of culture, fluorescent colonies were selected and transferred to 96-well plates (Becton Dickinson, Cat. No. 353075) containing 200 μL / well of ClonaCell-HY Medium E (StemCell Technologies, Cat. No. 03805) using a Clonepix FL (Molecular Devices, Sunnyvale, CA). Hybridoma medium was replaced 3 days before ELISA screening.Seven days after picking, supernatants were screened for anti-mouse IgG by ELISA. Hybridomas showing mouse IgG expression by ELISA were expanded and screened for binding to 293 cells overexpressing human KLβ, mouse KLB, cynomolgus KLB, human KLB / FGFR1c complex, cynomolgus KLB / FGFR1c complex, human FGFR1c, and / or human KLα / KLB by cell-based ELISA and / or FACS. Supernatants were also screened for binding competition with anti-KLB 8C5 antibody (Genentech) by FACS. All IgG-positive supernatants were also screened for agonist activity in a GAL / Elk1 luciferase assay using 293 cells overexpressing human FGFR1c and the KLβ / FGFR1c complex. RNA was extracted from FACS-positive and agonistic hybridoma cell lines using the RNeasy kit (Qiagen, Hilden, Germany), and cDNA was generated and amplified for sequencing. Heavy and light chain variable region genes were inserted into pRK plasmid vectors (Genentech, Inc.) for expression. Plasmid DNA from unique clones that demonstrated FACS-binding and agonistic activity was recombinantly expressed in 293 cells. Supernatants were then purified by protein A affinity chromatography as previously described (Hongo et al., Hybridoma 19:303, 2000).

[0253] Using the methods described above, approximately four different hybridomas, 2C12, 4H7, 23B3, and 28B7, were identified that produce monoclonal anti-KLB antibodies that bind to a single epitope on KLB.

[0254] The fifth monoclonal antibody, 12B8, was identified using the following experimental method. Balb / c mice were immunized with HEK293 cells stably expressing hFGFR1c and hKLB proteins. Twelve weeks later, the pancreas was harvested and hybridomas were generated. Anti-hKLB antibody-producing hybridomas were identified by FACS analysis using the HEK293 cells used for immunization. Briefly, 293 cells expressing hKLB alone, hFGFR1 alone, or both were stained with diluted hybridoma supernatants in FACS buffer (0.5% BSA in PBS) and PE-conjugated goat anti-mouse IgG antibody (Jackson Labs). The stained cells were washed using the same FACS buffer. The stained cells were analyzed using a FACScan (Becton Dickinson) and FlowJo FACS analysis software (Tree Star). cDNAs encoding the IgG heavy and light chains were cloned into expression vectors.

[0255] These five anti-KLB antibodies, 12B8, 2C12, 4H7, 23B3, and 28B7, were shown to function as KLB / FGFR1c agonist antibodies (Figures 4 and 5). The heavy chain variable region and light chain variable region CDR sequences for these five antibodies are shown in Tables 2 and 3, respectively. The full-length heavy chain and light chain sequences of these anti-KLB antibodies are shown in Table 4 and Figures 13 to 17. These antibodies are referred to herein as parent antibodies. Table 2. Heavy chain variable region CDR sequences for anti-KLB antibodies JPEG2026009891000004.jpg53158Table 3. Light chain variable region CDR sequences for anti-KLB antibodies JPEG2026009891000005.jpg53158Table 4. Heavy and light chain variable regions of identified anti-KLB antibodies JPEG2026009891000006.jpg156158

[0256] Example 2: Screening for bispecific anti-KLB antibodies In this example, bispecific anti-KLB antibodies were screened and the KLB / FGFR1c agonist activity of the bispecific anti-KLB antibodies was tested in 293T cells.

[0257] Bispecific antibodies that bind to two binding sites, e.g., epitopes, on KLB were generated using the parent anti-KLB antibody sequences listed above in Tables 1 and 2. See Table 5 for a summary of the bispecific antibodies generated. The bispecific antibodies were produced using a "knob-into-hole" heterodimerization technique using a single light chain variable region linked to a complete heavy chain in a VLfH (variable light full heavy) format (see Figure 1). See also Ridgway et al., Protein Engineering, Vol. 9:7, p617-621 (1996); Atwell et al., J. Mol. Biol. 270, 26-35 (1997); Spiess et al., Nat. Biotech. 31, 753-759 (2013).

[0258] To screen for bispecific antibodies that exhibited KLB / FGFR1c agonist activity, each of the light and heavy chains was expressed in a VLfH format. As shown in Figure 1, the VLfH format contains a full-length heavy chain linked to a variable light chain domain via a linker containing the amino acid sequence set forth in SEQ ID NO: 68. To generate a polypeptide containing a light chain variable domain and a full-length heavy chain in the VLfH format, a cDNA encoding ATGAYA (SEQ ID NO: 83) was commercially synthesized, containing the light chain variable domain up to R108 (according to the EU numbering system) - GGGGSGGGGSGGGGSGGGGS (linker, SEQ ID NO: 68) - CH1 residue F126 (according to the EU numbering system), a BsiWI restriction site at the 5' end, and a PspOMI / ApaI restriction site at the 3' end. The cDNA was digested with the restriction enzymes BsiWI and PspOMI for subcloning into a standard pRK vector encoding complete heavy chains with various isotypes and hinge variants containing a knob-hole mutation in CH3. The resulting translated polypeptide comprises the light chain variable domain of the form MGWSCIILFLVATATGAYA (signal peptide, SEQ ID NO: 69)-R108 (according to the EU numbering system)-GGGGSGGGGSGGGGSGGGGS (linker, SEQ ID NO: 68)-complete heavy chain.

[0259] The same light chain constant (CL) domain was used for each pair of VLfH polypeptides. The CL domain was amplified by polymerase chain reaction (PCR) from a plasmid containing a cDNA for human kappa (κ) light chain, comprising the following nucleotide sequence set forth in SEQ ID NO: 66, using primers: 5'-TTTCCCTTTATCGATTGAATTCCACCATGGGATGGTCATGTATC ATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAACTGTGGCTGCACCATCTGTCTTC-3' (SEQ ID NO: 63), 5'-TTTCCCTTTAAGCTTAACACTCTCCCCTGTTGAAGC TCTTTGT-3' (SEQ ID NO: 64). SEQ ID NO: 66 is provided below. CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCTTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCGTGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAG TGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 66) SEQ ID NO:66 encodes the amino acid sequence set forth in SEQ ID NO:65, provided below: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:65)

[0260] The amplified DNA was then digested with the restriction enzymes ClaI and HindIII for subcloning into a standard pRK vector containing the same restriction enzyme sites as the unique sites before the start of the open reading frame (ORF) and after the stop codon, resulting in a polypeptide having the amino acid sequence set forth in SEQ ID NO:67, provided below: MGWSCIILFLVATATGVHSTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 67). The kappa constant light chain (CL) begins at, but is not limited to, T109 (according to the EU numbering system), including R108 and the preceding elbow residues. This constant light (CL) DNA was cloned once and reused for pairing with the VLfH DNA disclosed above.

[0261] Equal amounts of VLfH and CL pRK DNA were mixed for expression in transiently transfected cultures of CHO (Wong et al. (2010) Biotechnol. Bioeng., 106:751-763) or HEK293T (Bos et al. (2014) Journal of Biotechnology, 180:10-16) cells, as previously described, to promote heterodimerization of the bispecific antibody. The CL domain was expressed as a separate polypeptide from the VLfH polypeptide. It has previously been shown that the CL domain folds autonomously and does not interact with BiP during folding (Hellman et al. (1999) J. Cell Biol., 144:21-30). In addition, the CL domain can be efficiently secreted by itself because it does not have an ER retention signal that would stall secretion until paired with a heavy chain (HC). The light chain (LC) and surrogate light chain (λ5) (Bankovich et al. (2007) Science 316:291-294) can interact with HC through two domains. In addition to the CL:CH1 interface, the VL (for LC) and the first beta chain (for λ5) provide additional stability during the interaction. It is currently unknown whether the association kinetics for isolated CL successfully (i) displaces champerones such as BiP on CH1, (ii) stably pairs with CH1, and (iii) stabilizes CH1, allowing for final proline isomerization in CH1 and tightening quickly enough to enter the final folded state (Feige et al. (2009) Mol Cell. 34:569-579). Without the contribution of variable domain interactions, the CL:CH1 interaction may allow proper folding of CH1, but may be too transient to reverse the folding defect of VL-HC fusions.

[0262] Mass spectrometry was performed to determine whether co-expression of two VLfH polypeptides resulted in heterodimerization. Mass spectrometry data were acquired using an Agilent 6230 TOF LC-MS system and a 1290 Infinity HPLC (Agilent Technology, Santa Clara, CA, USA). The bispecific antibody was purified using a 4.6 mm × 50 mm PLRP-S reversed-phase column (Agilent Technology). The complete mass was obtained by Maximum Entropy Deconvolution using MassHunter software (Qualitative Analysis B.04.00). As shown in Figures 2A-B, co-expression of VLfH resulted in heterodimerization of the two VLfH polypeptides. The disclosed method resulted in greater than 95% heterodimerization and less than 5% homodimerization. Table 5. Bispecific anti-KLB antibodies TIFF2026009891000007.tif44170

[0263] To determine the agonist activity of the bispecific antibodies in the VLfH format, a luciferase assay was performed. HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum (FBS), GlutaMax (Life Technologies), and Antibiotic-Antimycotic (Life Technologies) and transiently transfected using FuGENE® HD Transfection Reagent (Roche) with an expression vector encoding Renilla luciferase (pRL-SV40, Promega), FGFR1c, a transcriptional activator (pFA2-Elk1, Stratagene), and a firefly luciferase reporter driven by a GAL4 binding site (pFR-Luc, Stratagene). The following day, transfected cells were cultured in serum-free medium for an additional 6–8 h and tested with increasing concentrations of the bispecific anti-KLB antibodies and the corresponding parental monoclonal antibodies. Recombinant human FGF21 (R&D) was used as a reference in some luciferase experiments. Cells were lysed with Passive Lysis Buffer (Promega) and incubated either at 4°C for 1 hour or at -20°C overnight. Cellular luciferase activity was determined using the DUAL-GLO® Luciferase Assay System (Promega) and an EnVision® Multilabel Reader (PerkinElmer). Firefly luciferase activity was normalized to coexpressed Renilla luciferase activity, and results were plotted in relative light units (RLU) as a function of ligand concentration.

[0264] The FGFR1 gene was inactivated in HEK293TdelFGFR1 cells using CRISPR / Cas9 technology with the guide RNAs aacttcactgtcttggcagccgg and gatctccaggtacaggggcgagg. Cells were transfected with a Cas9 expression plasmid driven by a CMV promoter and a plasmid expressing each guide RNA under the control of a U6 promoter. Three days after transfection, colonies were sorted at one cell per well into 96-well plates containing conditioned medium composed of 75% DMEM and 25% supernatant from the transfected culture, which had been previously filtered through a 22-micron filter. Colonies were screened for the absence of FGFR1c surface expression by FACS. Genomic DNA PCR using primers ATGCTCTCCCCTCCTCGG (SEQ ID NO: 84) and AGGCCCCTGTGCAATAGATG (SEQ ID NO: 85) was used to further confirm that the FGFR1-deficient clone carried a deletion in the FGFR1 locus. One FGFR1-negative clone was expanded and used in luciferase reporter assays.

[0265] As shown in Figure 3, bispecific antibodies (tcBsIgG) in the VLfH format exhibited agonistic activity. For example, the bispecific antibody 12B8 / 23B3 in the VLfH format exhibited higher agonistic activity than the parent antibodies 12B8 and 23B3 in the VLfH format alone (Figure 3). In addition, the bispecific antibody 12B8 / 23B7 in the VLfH format exhibited higher agonistic activity than the parent antibodies 12B8 and 23B7 in the VLfH format alone (Figure 3). These results indicate that the VLfH-based screening method for identifying bispecific antibodies does not significantly affect agonistic activity and can be used as a general screen for bispecific antibodies. Five biepitopic antibodies and the parent antibodies were sequentially produced in the same tcBsIgG format but using two-column purification to remove HMW species and other potential impurities. These monodisperse tcBsIgG preparations retained superior agonist activity compared to the monospecific parent (data not shown), confirming the need for further purification during the initial screening process.

[0266] Example 3: Characterization of bispecific anti-KLB antibodies The bispecific antibodies identified in Example 2 were reformatted into bispecific IgG antibodies and analyzed for KLB / FGFR1c agonist activity using luciferase as described in Example 2. As shown in Figures 4 and 5, the VLfH-formatted bispecific antibodies (tcBsIgG) identified to exhibit agonist activity also exhibited activity when reformatted into IgG antibodies, indicating that the screening method described in Example 2 did not result in any false positives. For example, the bispecific antibody 12B8 / 23B3 exhibited higher agonist activity than the parent antibodies 12B8 and 23B3 alone. Similar results were observed for the bispecific anti-KLB antibodies 12B8 / 2C12, 23B3 / 28B7, 2C12 / 28B7, and 4H7 / 28B7 (Figures 4 and 5). These results indicate that the disclosed bispecific antibodies exhibit higher agonist activity than their corresponding parent monospecific antibodies alone.

[0267] The efficacy of bispecific anti-KLB antibodies was analyzed by comparing the activity of a 1:1 mixture of the corresponding parent antibody pairs. For example, the activity of the bispecific antibody 23B3-28B7 was compared to the activity of a 1:1 mixture of 23B3 and 28B7. As shown in Figure 6, the complex-specific anti-KLB bispecific antibody pair 23B3 / 28B7 required locked bispecificity. That is, the agonistic effect of the 23B3 and 28B7 binding sites is superior when these variable regions are part of the same bispecific antibody than when they act as two separate monoclonal antibodies.

[0268] Example 4: FGFR1c is required for the agonistic activity of KLB bispecific antibodies In this example, the disclosed bispecific antibodies were further analyzed to determine whether their agonistic activity was dependent on expression of FGFR1c and / or KLB.

[0269] When tested in a GAL4-Elk1 luciferase assay in HEK293T cells expressing FGFR1c with KLB or FGFR2c with KLB, we observed that the agonistic activity of the anti-KLB antibodies required the expression of KLB and FGFR1c (Figure 7). The bispecific antibodies induced luciferase activity in a dose-dependent manner in cells expressing recombinant FGFR1c and hKLB, but not in cells coexpressing KLB with FGFR2c, a receptor closely related to FGFR1c, indicating that these bispecific antibodies act as KLB-dependent FGFR agonists (Figure 7).

[0270] Furthermore, the bispecific anti-KLB antibody exhibited the activity of inducing the phosphorylation of MAPK signaling mediators such as ERK in primary human adipocytes, which represent a relevant cell type for the antidiabetic activity of FGF21 (Figure 8). FGF21, anti-KLB / anti-FGFR1c bispecific antibody (BsAb2), a bispecific antibody containing both FGFR1c and KLB arms (primary antibody), and the isotype-matched antibody trastuzumab were used as controls.

[0271] To further understand the mode of action of the bispecific anti-KLB antibodies, their ability to trigger the MAPK phosphorylation cascade in HEK293T cells expressing KLB and FGFR1c was quantified using a luciferase-based reporter assay.

[0272] Antibody binding was determined by flow cytometry. Cells were plated at 2 × 10 in 96-well tissue culture plates. 4 Cells were plated at 100 ng / well and grown overnight. The next day, each well was transfected with 100 ng of plasmid DNA using FugeneHD transfection reagent (Roche) according to the manufacturer's specifications. After 48 hours, cells were dissociated, washed in cold PBS supplemented with 3% fetal calf serum (FCS), incubated on ice for 45 minutes with 2 μg / ml of the indicated anti-KLB antibody, washed again, and then incubated on ice for 45 minutes with 4 μg / ml of anti-human IgG-Alexa 488 (Life Technologies). Cells were analyzed by flow cytometry using a FACSCalibur analyzer (Becton Dickinson). As shown in Table 6 below, this bispecific antibody exhibited higher EC values ​​compared to the primary anti-KLB / anti-FGFR1c bispecific antibody, BsAb2. 50 For example, the bispecific anti-KLB antibody 23B3 / 28B7 exhibited a significantly higher EC of 0.63 nM compared to BsAb2 using a luciferase-based reporter assay. 50 Values ​​were presented (see Table 6). Table 6 JPEG2026009891000008.jpg43158

[0273] Example 5: Epitope mapping of anti-KLB antibodies In this example, epitope mapping of the anti-KLB antibodies identified in Example 1 was performed by FACS (FIG. 9), luciferase-based reporter assay (FIG. 10), and epitope binning (FIGS. 11A-11B).

[0274] The chimeric human / rat KLB proteins (Figures 9 and 10) or chimeric human FGFR1c / FGFR2c proteins (Figure 10) described below were transiently expressed on the surface of HEK293T or HEK293TdelFGFR1 cells, stained with the indicated antibodies, and analyzed by flow cytometry (Figure 9) or luciferase-based reporter assays (Figure 10). In Figures 9 and 10, KLB and FGFR chimeras are depicted pictorially, with the human KLB or FGFR1c sequence shown in black and the rat KLB or human FGFR2c sequence shown in white.

[0275] Based on these assays, monoclonal antibody 12B8 has been shown to bind to the amino acid sequence of KLB. It can be inferred that the antibody binds to at least a portion of IQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFLGCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRVVS (sequence number 43).

[0276] Monoclonal antibodies 2C12, 23B3, and 4H7 have the amino acid sequence It binds to at least a portion of ADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDI (SEQ ID NO: 44).

[0277] The monoclonal antibody 28B7 has the amino acid sequence (SEQ ID NO: 45).

[0278] Similarly, the FGFR1c sequence KTVALGSNVEFMCKVYSDPQPHIQWLKHIEVNGSKIGPDNLPYVQILKTAGVNTTDKEMEVLHLRNVSFEDAGEYTCLAGNSIGLSHHSAWLTVLEALEERPAVMT (SEQ ID NO: 46) is required for monoclonal antibodies 12B8, 23B3, 4H7, and 28B7 to exert agonist activity.

[0279] The following human / rat KLB and human FGFR1c / FGFR2c proteins and chimeras were cloned into vectors and expressed on the surface of HEK293T cells for FACS analysis or HEK293TdelFGFR1 for luciferase assays. The human KLB sequence is shown in bold, and the homologous sequence near the junction of the human / rat sequences is italicized. Similarly, FGFR2c is shown in bold, and the junction sequence homologous between FGFR1c and FGFR2c is italicized.

[0280] Human KLB: JPEG2026009891000009.jpg144160JPEG2026009891000010.jpg554(SEQ ID NO:47).

[0281] Homologous rat region corresponding to KLB protein A: (SEQ ID NO: 48)

[0282] Human / rat KLB chimera: JPEG2026009891000011.jpg90161JPEG2026009891000012.jpg44157JPEG2026009891000013.jpg6145(sequence number 49)

[0283] Human / rat KLB chimera: JPEG2026009891000014.jpg145161JPEG2026009891000015.jpg520 (sequence number 50)

[0284] Human / rat KLB chimera: JPEG2026009891000016.jpg22160JPEG2026009891000017.jpg121160JPEG2026009891000018.jpg623 (sequence number 51)

[0285] Human / rat KLB chimera: JPEG2026009891000019.jpg113161JPEG2026009891000020.jpg20157JPEG2026009891000021.jpg691 (sequence number 52)

[0286] Human / rat KLB chimera: JPEG2026009891000022.jpg136160JPEG2026009891000023.jpg586 (sequence number 53)

[0287] Human / rat KLB chimera: JPEG2026009891000024.jpg52160JPEG2026009891000025.jpg82160JPEG2026009891000026.jpg572 (sequence number 54)

[0288] Human / rat KLB chimera: JPEG2026009891000027.jpg136161JPEG2026009891000028.jpg566 (sequence number 55)

[0289] Human / rat KLB chimera: JPEG2026009891000029.jpg136161JPEG2026009891000030.jpg557 (sequence number 56)

[0290] Human FGFR1c: JPEG2026009891000031.jpg90160JPEG2026009891000032.jpg21160JPEG2026009891000033.jpg694 (sequence number 57)

[0291] Human FGFR2c: (SEQ ID NO: 58)

[0292] FGFR1c / FGFR2c chimera: JPEG2026009891000034.jpg75160JPEG2026009891000035.jpg37160JPEG2026009891000036.jpg617 (sequence number 59)

[0293] FGFR1c / FGFR2c chimera: JPEG2026009891000037.jpg113160JPEG2026009891000038.jpg667 (sequence number 60)

[0294] FGFR1c / FGFR2c chimera: JPEG2026009891000039.jpg59160JPEG2026009891000040.jpg52161JPEG2026009891000041.jpg516 (sequence number 61).

[0295] FGFR1c / FGFR2c chimera: JPEG2026009891000042.jpg111157JPEG2026009891000043.jpg575 (sequence number 62).

[0296] Epitope binning experiments (Figures 11A-11B) were performed by BioLayer Inferometry in 8- or 16-channel mode on an Octet Red384 system (Pall Life Sciences, Menlo Park, CA) using anti-mouse Fc or anti-human Fc capture biosensors. The assay consisted of a 7-step binding cycle: 1) immersion of the anti-mouse Fc biosensor in running buffer (1x kinetic buffer, ForteBio 18-5032) for 1 min to establish a baseline, 2) capture of 20 μg / ml mouse IgG2a (reference) antibody for 10 min, 3) establishment of another baseline for 1.5 min, 4) loading of 100 nM human KLB for 10 min and establishment of a third baseline for 1.5 min, 6) association of 5 μg / ml human IgG1 (test) antibody for 10 min, and 7) dissociation for 10 min. Epitope binning using an anti-human Fc capture biosensor was performed similarly, and the summary in Figure 11 shows the bins determined for each antibody.

[0297] Figure 12 shows an exemplary biolayer inferometry experiment, in which 2C12 (human IgG1) competes with 23B3 but not with 28B7 or 8C5 (mouse IgG) for binding to recombinant KLB.

[0298] Example 6: Three-chain bispecific IgG: an antibody platform for rapid bispecific antibody screening Materials and Methods Characterization of purified antibodies by capillary electrophoresis Samples were analyzed on a Caliper GX II microfluidic system (PerkinElmer Biotechnology, Waltham, MA, USA). All samples were prepared as previously described (Kim, 2016). Chips were prepared according to the manufacturer's instructions provided in the LabChip GXII User Guide.

[0299] Characterization of purified antibodies by LC-MS / MS Mass spectrometry data was acquired using an Agilent 6230 TOF LC-MS system and a 1290 Infinity HPLC (Agilent Technology, Santa Clara, CA). IgG was separated on a 4.6 mm × 50 mm PLRP-S reversed-phase column (Agilent Technology, Santa Clara, CA). Complete masses were obtained by Maximum Entropy Deconvolution using MassHunter software (Qualitative Analysis B.04.00).

[0300] Biacore Kinetics analysis To determine the binding affinity of tcIgG and conventional IgG, surface plasmon resonance (SPR) measurements were performed using a BIAcore T200 and a protein A sensor chip. Dilutions of monomeric human KLB were injected as the analyte onto immobilized tcIgG or conventional IgG at 25°C to determine monovalent affinity. The association rate (k) and dissociation rate (k) were calculated using a simple one-to-one Langmuir binding model. The equilibrium dissociation constant (K) was calculated as the ratio k / k.

[0301] Western blot Human primary subcutaneous preadipocytes were obtained from Lonza (Walkersville, MD). Cells were grown and differentiated according to the supplier's protocol. Briefly, cells were grown in preadipocyte basal medium-2 containing FBS, L-glutamine, and GA-1000. After reaching confluence, cells were differentiated in growth medium containing dexamethasone, indomethacin, and 3-isobutyl-1-methylxanthine (IBMX). For ERK signaling analysis, cells were differentiated for 10 days, grown in serum-free medium for 3 hours, and then further cultured with the indicated antibodies for another hour. Cells were lysed in 2x LDS buffer (Invitrogen, USA) containing protease and phosphatase inhibitor tablets (Roche, USA) to generate cell extracts. Samples were used for Western blot analysis by standard methods. Antibodies used for Western blot analysis were from Cell Signaling Technology (Danvers, MA): pERK1 / 2 (T202 / 204), (catalog no. 4370), ERK1 / 2 (catalog no. 4695), and HSP90 (catalog no. 4874).

[0302] result Transformer V L -HC fusion and C L Expression resolves the cognate light chain pairing problem for BsIgG A schematic diagram of the novel three-chain BsIgG (tcBsIgG) format is shown in Figure 18A. In this novel platform, heavy chain heterodimerization was achieved by previously described "knob-into-hole" mutations (Ridgway, 1996; Atwell, 1997). L A VLfH-HC fusion was designed, using a short (Gly4Ser)4-linker to obtain correct heavy-light chain pairing, similar to the design of antibody scFvs. L The C-terminal part of the domain was tethered to the N-terminus of the heavy chain (Figure 18A, left). L Expression of VLfH alone without the C1 domain resulted in little or no VLfH expression based on capillary electrophoresis (CE-SDS) after protein A affinity column purification and recovery (Fig. 18B, lane 1). L This result led to the previous observation that the C domain requires pairing (Feige, 2009). L It has been proposed that pairing with the C domain acts as a final quality control step, ensuring that properly folded antibodies are secreted (Feige, 2014). To test this, isolated C L We sought to determine whether the CH1 domain could complement the folding of the CH1 domain and allow productive secretion of the VLfH hybrid protein (Figure 18A, right) to yield a three-chain IgG (tcIgG). The anti-KLB antibody, clone 28B7 monoepitopic tcIgG, was synthesized with IgG1, 2, and 4 isotypes, as well as deglycosylated (N) IgG1 and IgG4. 297 G) version. LAfter cotransfection of separate plasmids encoding the domains, we successfully recovered antibodies with yields comparable to those of the intact parent antibody (Figure 18B). While BsIgG, a human IgG1 isotype, is the predominant antibody class currently in clinical development, other isotypes are also commonly utilized as monospecific antibodies to modulate antibody effector function and activity. Therefore, we evaluated whether the tcIgG technology could be applied to other therapeutically relevant human isotypes, IgG2 and IgG4. L The domain requirements were consistent across VLfH expression in IgG2 and IgG4, and the behavior of the common deglycosylated versions of IgG1 and IgG4 carrying the N297G mutation was similar (Figure 18B). L The overall yield after co-expression was comparable to that of the corresponding wild-type IgG isotype control. L It was shown that the interchain disulfide bond between V and CH1 was efficiently formed. Therefore, antibody V was required for productive folding and secretion of the antibody. L and C L The domains need not be covalently connected as a single polypeptide chain.

[0303] Next, we assessed whether the tcIgG format was compatible with knob-into-hole mutations for potential bispecific antibody production. Expression and assembly of knob and hole half IgGs, either individually or coexpressed, were compared. As previously observed, expression of either knob or hole alone resulted primarily in half antibodies and some covalent homodimers (Figure 18C). No significant differences in expression or assembly were observed between standard IgG and tcIgG formats. Coexpression of knob and hole half antibodies in the same cells resulted in efficient assembly of 150 KDa species of all three major human isotypes: IgG1, IgG2, and IgG4 (Figure 18C). The observed yields and product quality of human tcIgG2 and tcIgG4 as monospecific bivalent tcIgG and bispecific tcIgG were comparable to their corresponding IgG isotypes.

[0304] To ensure that single-cell expression of the tcIgG knob and tcIgG hole did not affect heterodimer formation of the antibody heavy chains, the purified antibody was analyzed by mass spectrometry (Figure 18D). Only negligible homodimer contaminants were detected. The isolated C16 antibody, referred to herein as the three-chain IgG (tcIgG) format, L We conclude that the domain-tethered VLfH format does not adversely affect heterodimer formation, as their abundance is comparable to that of conventional BsIgG.

[0305] Production of anti-KLB biepitope antibodies of the tcBsIgG format To generate the biapitopic antibodies from which the five anti-KLB monoclonal antibodies described above were derived, each antibody was first cloned into a VLfH knob vector and a VLfH hole vector. This allowed for the production of 25 different antibodies (Table 8) in the tcIgG format, containing 10 possible biapitopic combinations in both heavy chain orientations (i.e., knob / hole and hole / knob). Having both knob-hole orientations for each antibody combination provided independent replicates. In addition, parental monospecific antibodies were generated in tcIgG knob and tcIgG.hole co-expression formats (Table 8, gray cells) to serve as benchmarks, and positive (Table 8, (+) expression control) and negative (Table 8, (-) expression control) knob and hole tcIgG expression controls were included to pair with the five anti-KLB monoclonal antibodies. Table 8. tc after expression in HEK293T and purification by MabSelectSure IgG recovery yield (mg / L) TIFF2026009891000044.tif129170

[0306] All 25 tcIgG antibodies were characterized by caliper electrophoresis and showed comparable assembly efficiency. The predominant band for all antibodies was approximately 150 kDa, indicating correct domain pairing and disulfide bond formation (Figure 19A). We also noticed that inefficient expression of the parent antibody limited the expression yield of the resulting biapitopic antibody. This is a consequence of efficient heterodimerization due to knob-into-hole mutations. For example, the negative expression control tcIgG had reduced yields when expressed as a bivalent and when coexpressed with any other antibody (Figures 19A and 19B). Together, this ensured minimal homodimer formation, as confirmed by mass spectrometry.

[0307] To further characterize the tcIgG, the tcIgG was analyzed by analytical size-exclusion chromatography (SEC). After HTP expression and single-column purification by protein A chromatography, the tcIgG contained high-molecular-weight (HMW) species ranging from 1% to 70%, which appears to be related to the properties of the parent tcIgG and also correlates with their parent standard IgG. The positive expression control tcIgG coexpressed as knob and hole tcIgG had no HMW species, whereas two relatively low-expressing anti-KLB tcIgGs, 12B8 and 23B3, had much higher amounts of HMW species (Figure 19C). Interestingly, the amount of HMW species averaged out when the well-behaved tcIgG half antibodies were coexpressed with the poorly behaved tcIgG half antibodies, as well as when the positive expression control tcIgG was coexpressed with 12B8 tcIgG and when the poorly behaved 12B8 tcIgG was coexpressed with the slightly better behaved 23B3 tcIgG (Figure 19C). Although the HMW may affect the activity of each antibody, the inventors decided to proceed with characterization of 25 antibodies in a luciferase reporter assay. Because additional column steps would reduce throughput, the inventors chose to verify activity with more purified material.

[0308] In vitro screening of anti-KLB tcBsIgG biepitopic antibodies identifies five superior pairs that translate into linker-less BsIgG. In vitro screening of anti-KLB tcBsIgG biepitopic antibodies was described in Example 2. Five excellent pairs that translated into linker-free BsIgG were identified: 12B8 / 23B3, 12B8 / 2C12, 23B3 / 28B7, 2C12 / 28B7, and 4H7 / 28B7 (Figures 3-6).

[0309] To ensure that the linker in the tcIgG format did not interfere with the binding affinity of the bicepitopic tcIgG during screening, the binding kinetics of the antibodies with the highest relative agonistic activity, 4H7 and 28B7, were assessed by Biacore as both tcIgG1 and standard IgG1. No significant differences in binding kinetics for these two formats were observed, indicating that the tcIgG format preserves the binding kinetics of the parent antibody (Table 9). Table 9. Binding kinetics of anti-KLB antibodies 28B7 and 4H7 as IgG and tcIgG formats. Values ​​are the mean ± range of two independent Biacore experiments. TIFF2026009891000045.tif66170

[0310] Influence of antibody isotype on antibody agonist activity It has previously been reported that antibody isotype can modulate the agonist and ligand-mimetic activity of antibodies (White, 2014; Sampei, 2014). Based on this, we investigated whether antibody isotype could affect the activity of the 4H7 / 28B7 biapic antibody, which had the most agonist properties compared to other biapic pairs. Human IgG1, IgG2, and IgG4 isotypes were produced (in vitro assembly) and the activity of the different isotypes was compared in a luciferase reporter assay (Figure 20A). For the human IgG2 construct, the second hinge cysteine ​​(C233 in Kabat numbering, equivalent to C220S in EU numbering) was mutated to serine to enable the most efficient in vitro assembly. In this assay, the activity of the 4H7 / 28B7 biapic antibody as an IgG1 isotype was approximately twice that of the IgG2 isotype, while the IgG4 isotype had intermediate levels of activity. The EC50 values ​​for these three antibodies were similar, consistent with the notion that antibody isotype does not affect binding affinity.

[0311] Next, we expanded the assay to assess ERK1 / 2 phosphorylation in human primary adipocytes. For this assay, differentiated primary human adipocytes were treated with various monoclonal antibodies, and ERK1 / 2 phosphorylation levels were assessed by Western blot. The results of this assay using primary cells were consistent with the luciferase reporter assay. The 4H7 / 28B7 biatopic antibody acts as an agonist in all IgG1, IgG2, and IgG4 isotype backgrounds (Figures 20B and 20C). Furthermore, the 4H7 and 28B7 antibodies, when combined as a 1:1 mixture or as a single molecule biatopic antibody, exhibited superior activity compared to the single parent molecule activity.

[0312] Consideration Previous studies have shown that bispecific and biapitopic antibodies can act as potent ligand-mimetic pairs. However, finding these antibodies is often challenging, requiring the screening of up to thousands of combinations to identify unique functional combinations (Zhang, 2012; Kitazawa, 2012; Kolumam, 2015). The tcBsIgG format described herein simplifies the expression and production of BsIgGs and provides a novel system that enables high-throughput screening of large panels of BsIgG combinations. Using this strategy, we demonstrated that the activity of agonist antibodies can be further enhanced by co-formulation with or combination with biapitopic antibodies. The biapitopic combinations that exhibited enhanced activity were not entirely predictable from characterization of the parent antibodies, demonstrating the utility of the tcBsIgG system. These observations may extend to other agonist antibodies, such as those targeting OX40, CD27, or GITR, resulting in immunostimulatory activity.

[0313] The tcBsIgG format involves modifications to the antibody structure, particularly the addition of a linker and neoepitope at the N-terminus of the CL, which may stimulate an immunogenic response in vivo. However, these issues are not relevant for in vitro screening activities. Once several promising antibody pairs are identified, a select number of BsIgGs can be reformatted in the traditional BsIgG format for subsequent preclinical and clinical development. At this point, it is essential for any screening format that the results obtained can be replicated in the traditional IgG format. The tcIgG format described herein preserves the overall architecture of the antibody and allows the results to be converted to a linker-less BsIgG, which is more desirable for clinical use. This has advantages over other techniques, such as fusion of scFvs to heterodimeric Fc (Moore, 2011), which offer similar throughput in producing bispecific molecules as the tcBsIgG format but can alter the geometry and distance between the targeting arms. Additionally, another advantage of the tcBsIgG format compared to other BsIgG formats is that fewer total plasmids need to be cloned to produce the bispecific antibody matrix (i.e., only one plasmid for each half-antibody). Beyond antibody screening in drug development, the tcIgG format has the potential to produce bispecific antibodies for diagnostic applications. The ability to produce tcIgG in a single cell provides a cost-effective method for producing BsIgG.

[0314] Another advantage of the tcIgG format is its applicability to IgG2 and IgG4 isotypes. Antibody isotype-specific activity has been reported. For example, it has been previously reported that human IgG2 antibodies against CD40 exhibit superagonistic activity compared with human IgG1 and IgG4 isotypes (White, 2014). Additionally, it has been reported that the amplitude of factor VIII ligand mimetic activity of a bispecific antibody directed against factor IXa / X was antibody class dependent (Sampei, 2014). Based on these observations, we compared the agonistic activity of the 4H7 / 28B7 lead-biepitope IgG as human IgG1, IgG2, and IgG4 isotypes. Significant differences were observed between antibody isotypes, with human IgG1 demonstrating the best agonistic activity. This may be partially attributable to the initial screening of anti-KLB pairs as human IgG1. If initial screening had been performed using BsIgG of all isotypes, different pairs and isotypes may have been selected. The compatibility of the tsIgG format with other human isotypes may enable this screening. Additionally, this allows for the formation of ternary complexes with different potencies of engagement with Fc gamma receptors, which is irrelevant in the present anti-KLB model system but may be important for the discovery of primary targeting agonist antibodies.

[0315] In conclusion, the tcBsIgG format offers an excellent strategy for BsIgG screening for a wide range of applications.

[0316] Example 7: Binding affinity studies In this example, the disclosed bispecific antibodies were further analyzed to determine their binding affinities. The binding affinity parameters (k(1 / Ms), k(1 / s), and K(nM)) of anti-KLB antibody clones 28B7 and 4H7 were determined in both IgG and tcIgG formats (Figure 21). Within each antibody clone, the difference in binding affinity between the conventional IgG and tcIgG formats was minimal. For example, the K(s) of clone 28B7 in the IgG and tcIgG formats were 2.65 nM and 2.05 nM, respectively. The K(s) of clone 4H7 in the IgG and tcIgG formats were 0.78 nM and 1.29 nM, respectively.

[0317] References Atwell, S., Ridgway, JB, Wells, JA and Carter, P. (1997) J Mol Biol, 270 26-35. Feige, MJand Buchner, J. (2014) Biochim. Biophys. Acta, 1844 2024-2031. Feige, MJ, Groscurth, S., Marcinowski, M., Shimizu, Y., Kessler, H., Hendershot, LMand Buchner, J. (2009) Mol Cell, 34 569-579. Kim, HS. (2016) MAbs, 8 1536-1547. Kitazawa, T. et al. (2012) Nat Medicine, 18 1570-1574. Kolumam,G.et al.(2015)EBioMedicine,2 730-743. Moore, GLet al. (2011) MAbs, 3 546-557. Ridgway, JB, Presta, LGand Carter, P. (1996) Protein Eng., 9 617-621. Sampei, Z. et al. (2014) MAbs 0-00. White, AL et al. (2014) Cancer Cell 1-25. Zhang, H., Wilson, IA and Lerner, RA (2012) Proc. Natl. Acad. Sci. USa, 109 15728-15733.

[0318] In addition to the various embodiments shown and claimed, the disclosed subject matter is directed to other embodiments having other combinations of the features disclosed and claimed herein. Thus, specific features presented herein can be combined with each other in other ways within the scope of the disclosed subject matter, such that the disclosed subject matter includes any suitable combination of features disclosed herein. The foregoing descriptions of specific embodiments of the disclosed subject matter have been presented for purposes of illustration and description and are not intended to be exhaustive or to limit the disclosed subject matter to the disclosed embodiments.

[0319] It will be apparent to those skilled in the art that various modifications and variations can be made to the compositions and methods of the disclosed subject matter without departing from the spirit or scope of the disclosed subject matter. Accordingly, it is intended that the disclosed subject matter include modifications and variations that come within the scope of the appended claims and their equivalents. Various publications, patents, and patent applications are cited herein, the contents of which are incorporated herein by reference in their entireties.

Claims

1. 1. An isolated multispecific antibody comprising a first antigen-binding polypeptide, wherein said first antigen-binding polypeptide comprises a VL Domain, a linker, a VH Domain, a CH1 Domain, a CH2 Domain, and a CH3 Domain, which are positioned in an N-terminal to C-terminal direction in the following order: VL-linker-VH-CH1-CH2-CH3.

2. The multispecific antibody of claim 1 , wherein the multispecific antibody does not comprise a CL domain.

3. The multispecific antibody of claim 1 , further comprising a CL domain.

4. 4. The multispecific antibody of claim 3, wherein the CL domain is linked to the first antigen-binding polypeptide by one or more disulfide bridges.

5. 5. The multispecific antibody of claim 4, wherein the disulfide bridge links the CL domain to the CH1 domain of the first antigen-binding polypeptide.

6. 6. The multispecific antibody of any one of claims 1 to 5, further comprising a second antigen-binding polypeptide, said second antigen-binding polypeptide comprising a VL Domain, a linker, a VH Domain, a CH1 Domain, a CH2 Domain, and a CH3 Domain positioned in the N-terminal to C-terminal direction in the order: VL-linker-VH-CH1-CH2-CH3.

7. The multispecific antibody of claim 6, wherein the second antigen-binding polypeptide does not comprise a CL domain.

8. The multispecific antibody of claim 6, wherein the multispecific antibody comprises two CL domains.

9. The multispecific antibody of claim 8, wherein the two CL domains are the same.

10. 10. The multispecific antibody of claim 8 or 9, wherein one of the two CL domains is linked to the first antigen-binding polypeptide by one or more disulfide bridges and the second of the two CL domains is linked to the second antigen-binding polypeptide by one or more disulfide bridges.

11. 11. The multispecific antibody of claim 10, wherein the disulfide bridge links the CL domain to the CH1 domain of the first antigen-binding polypeptide and the second antigen-binding polypeptide.

12. The multispecific antibody of any one of claims 6 to 11, wherein the first and second antigen-binding polypeptides bind to two different epitopes of the same antigen.

13. The multispecific antibody of any one of claims 6 to 11, wherein the first and second antigen-binding polypeptides bind to two different antigens.

14. The multispecific antibody of any one of claims 1 to 13, wherein the linker comprises one or more glycine (G) and serine (S) residues.

15. The multispecific antibody of any one of claims 1 to 14, wherein the linker has a length of from about 1 to about 50 amino acids.

16. The multispecific antibody of any one of claims 1 to 15, wherein the linker is about 20 amino acids in length.

17. The linker is G ４ The multispecific antibody of any one of claims 1 to 16, comprising S repeats.

18. The multispecific antibody of any one of claims 1 to 17, wherein the linker comprises the amino acid sequence of SEQ ID NO:

68.

19. The multispecific antibody of any one of claims 1 to 16, wherein the linker is cleavable.

20. 20. The multispecific antibody of any one of claims 6 to 19, wherein the CH3 domain of the first antigen-binding polypeptide and the CH3 domain of the second antigen-binding polypeptide associate at an interface that is modified to facilitate formation of the multispecific antibody.

21. (a) one or more amino acid residues in the CH3 domain of the first antigen-binding polypeptide are replaced with one or more amino acid residues having a larger side chain volume to generate a protrusion on the surface of the CH3 domain of the first antigen-binding polypeptide that interacts with the CH3 domain of the second antigen-binding polypeptide; 21. The multispecific antibody of claim 20, wherein (b) one or more amino acid residues of the CH3 domain of said second antigen-binding polypeptide are substituted with one or more amino acid residues having a smaller side chain volume, generating a cavity on the surface of the CH3 domain of said second antigen-binding polypeptide that interacts with the CH3 domain of said first antigen-binding polypeptide.

22. 22. The multispecific antibody of claim 21 , wherein the amino acid residue with the larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

23. 22. The multispecific antibody of claim 21 , wherein the amino acid residue with the smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

24. The multispecific antibody of any one of claims 1 to 23, wherein the antibody is of the IgG, IgA, or IgE isotype.

25. The multispecific antibody of any one of claims 1 to 24, wherein the antibody is of the IgG isotype.

26. The antibody is IgG １ , IgG ２ , or IgG ４ 26. The multispecific antibody of claim 25, which is an isotype.

27. The multispecific antibody of any one of claims 1 to 26, wherein the multispecific antibody is an agonist multispecific antibody or an antagonist multispecific antibody.

28. The multispecific antibody of any one of claims 1 to 27, wherein the multispecific antibody is a bispecific antibody.

29. 29. The multispecific antibody of claim 28, wherein the bispecific antibody is an agonistic biepitopic antibody.

30. 1. An isolated biapic agonist antibody comprising a first antigen-binding polypeptide and a second antigen-binding polypeptide, wherein each of the first and second antigen-binding polypeptides comprises a VL Domain, a linker, a VH Domain, a CH1 Domain, a CH2 Domain, and a CH3 Domain, arranged in an N-terminal to C-terminal direction in the following order: VL-linker-VH-CH1-CH2-CH3.

31. 31. The biepitopic agonist antibody of claim 30, wherein the first and second antigen-binding polypeptides bind to two different epitopes on the same antigen.

32. The biapitopic antibody of claim 30 or 31, wherein the biapitopic antibody does not contain a CL domain.

33. The biepitopic agonist antibody of claim 30 or 31, further comprising two CL domains.

34. The biepitopic agonist antibody of claim 33, wherein the two CL domains are the same.

35. 35. The biepitopic agonist antibody of claim 33 or 34, wherein one of the two CL domains is linked to the first antigen-binding polypeptide by one or more disulfide bridges and the second of the two CL domains is linked to the second antigen-binding polypeptide by one or more disulfide bridges.

36. 36. The biepitopic agonist antibody of claim 35, wherein the disulfide bridge links the CL domain to the CH1 domain of the first antigen-binding polypeptide and the second antigen-binding polypeptide.

37. The biepitopic agonist antibody of any one of claims 30 to 36, wherein the linker comprises one or more glycine (G) and serine (S) residues.

38. The biepitopic agonist antibody of any one of claims 30 to 37, wherein the linker has a length of about 1 to about 50 amino acids.

39. The biepitopic agonist antibody of any one of claims 30 to 38, wherein the linker is about 20 amino acids in length.

40. The linker is G ４ 40. The biepitopic agonist antibody of any one of claims 30 to 39, comprising S repeats.

41. The biepitopic agonist antibody of any one of claims 30 to 40, wherein the linker comprises the amino acid sequence of SEQ ID NO:

68.

42. The biepitopic agonist antibody of any one of claims 30 to 39, wherein the linker is cleavable.

43. 43. The biepitopic agonist antibody of any one of claims 30-42, wherein the CH3 domain of the first antigen-binding polypeptide and the CH3 domain of the second antigen-binding polypeptide associate at an interface that is modified to facilitate the formation of multispecific antibodies.

44. (a) one or more amino acid residues in the CH3 domain of the first antigen-binding polypeptide are replaced with one or more amino acid residues having a larger side chain volume to generate a protrusion on the surface of the CH3 domain of the first antigen-binding polypeptide that interacts with the CH3 domain of the second antigen-binding polypeptide; (b) one or more amino acid residues of the CH3 domain of the second antigen-binding polypeptide are substituted with one or more amino acid residues having a smaller side chain volume to generate a cavity on the surface of the CH3 domain of the second antigen-binding polypeptide that interacts with the CH3 domain of the first antigen-binding polypeptide.

45. 45. The biepitopic agonist antibody of claim 44, wherein the amino acid residue having the larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

46. 45. The biepitopic agonist antibody of claim 44, wherein the amino acid residue having the smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

47. The biepitopic agonist antibody of any one of claims 30 to 46, wherein the antibody is of the IgG, IgA, or IgE isotype.

48. The biepitopic agonist antibody of any one of claims 30 to 47, wherein the antibody is of the IgG isotype.

49. The antibody is IgG １ , IgG ２ , or IgG ４ The biepitopic agonist antibody of claims 30 to 48, which is an isotype.

50. 50. An isolated nucleic acid comprising a sequence encoding the multispecific antibody of any one of claims 1 to 29 or the biepitopic agonistic antibody of any one of claims 30 to 49.

51. A vector comprising the nucleic acid of claim 50.

52. A host cell expressing the multispecific antibody of any one of claims 1 to 29 or the biapitopic agonist antibody of any one of claims 30 to 49.

53. 52. A host cell comprising the nucleic acid of claim 50 or the vector of claim 51.

54. 54. The host cell of claim 53, further comprising a nucleic acid encoding a CL domain.

55. 55. A method for producing a multispecific antibody, the method comprising culturing a host cell of claim 52, 53, or 54 under conditions sufficient for the production of said multispecific antibody.

56. 55. A method for producing a biapitopic agonist antibody, comprising culturing a host cell of claim 52, 53, or 54 under conditions sufficient for the production of the multispecific antibody or the biapitopic agonist antibody.

57. 56. The method of claim 55, further comprising recovering the multispecific antibody or the biepitopic agonist antibody from the culture.

58. 57. The method of claim 56, further comprising recovering the multispecific antibody or the biepitopic agonist antibody from the culture.

59. 58. A biepitopic agonist antibody produced by the method of claim 56 or 57.

60. 60. A pharmaceutical composition comprising the multispecific antibody of any one of claims 1 to 29 or 58 or the biepitopic agonist antibody of any one of claims 30 to 49 or 59, and a pharmaceutically acceptable carrier.

61. 61. The composition of claim 60, further comprising a second therapeutic agent.

62. A kit comprising the multispecific antibody of any one of claims 1 to 29 or the biepitopic agonist antibody of any one of claims 30 to 49.

63. A library comprising a plurality of multispecific antibodies according to any one of claims 1 to 29.

64. A library comprising a plurality of polynucleotides encoding a plurality of multispecific antibodies according to any one of claims 1 to 29.

65. A method for screening a multispecific antibody, comprising: (a) obtaining a plurality of multispecific antibodies from the library of claim 63 or 64; and (b) assaying the plurality of multispecific antibodies for binding to a first and a second antigen or a first and a second epitope of the same antigen; (c) identifying a multispecific antibody that binds to the first and second antigens or to the first and second epitopes of the same antigen.

66. 1. A method for identifying a multispecific antibody, comprising: (a) expressing in a cell the multispecific antibody of any one of claims 1 to 29; (b) contacting the multispecific antibody of step (a) with a first antigen and a second antigen or a first epitope and a second epitope of the same antigen; (c) identifying a multispecific antibody that binds to the first antigen and the second antigen or to the first epitope and the second epitope of the same antigen.

67. A method for screening for a biepitope agonist antibody, comprising: (a) obtaining a plurality of multispecific antibodies from the library of claim 63 or 64; and (b) assaying the plurality of multispecific antibodies for binding to first and second epitopes of the same antigen; (c) distinguishing one or more biepitopic agonist antibodies from the plurality of multispecific antibodies that bind to the first and second epitopes of the same antigen; (d) distinguishing the biapitopic antibody from the one or more biapitopic agonist antibodies that exhibit agonist activity to obtain a biapitopic agonist antibody.

68. A method for screening for a biepitope agonist antibody, comprising: (a) expressing in a cell the multispecific antibody of any one of claims 1 to 29; (b) contacting the multispecific antibody of step (a) with a first epitope and a second epitope of the same antigen; (c) identifying multispecific antibodies that bind to the first epitope and the second epitope of the same antigen to obtain biepitopic antibodies; (d) determining whether the biapitopic antibody exhibits agonist activity to obtain a biapitopic agonist antibody.

69. 69. The method of claim 67 or 68, further comprising comparing the agonist activity of the biepitopic agonist antibody with the monospecific antibody from which the biepitopic agonist antibody was derived.

70. 70. The method of any one of claims 66 or 68-69, wherein expressing the multispecific antibody comprises introducing into the cell one or more nucleic acids encoding the multispecific antibody.

71. 71. The multispecific antibody of any one of claims 66 or 68 to 70, wherein the multispecific antibody is contacted with the first and second antigens or the first and second epitopes of the same antigen simultaneously in step (b).

72. 72. The multispecific antibody of any one of claims 66 or 68 to 71, wherein the multispecific antibody of step (b) is purified before contacting the multispecific antibody with the first and second antigens or the first and second epitopes of the same antigen.

73. 73. The method of claim 72, wherein the multispecific antibody is purified by Protein A chromatography.

74. 74. The method of any one of claims 65 to 73, wherein the antigen is in a bioconjugate.

75. 75. The method of any one of claims 66 or 68-74, wherein the cell is a prokaryotic cell.

76. 76. The method of claim 75, wherein the prokaryotic cell is an Escherichia coli cell.

77. 75. The method of any one of claims 66 or 68 to 74, wherein the cell is a eukaryotic cell.

78. 78. The method of claim 77, wherein the eukaryotic cell is a yeast cell or a mammalian cell.

79. 79. The method of claim 78, wherein the mammalian cell is a Chinese hamster ovary cell.

80. 80. The method of any one of claims 65 to 79, wherein binding of the multispecific antibody to the first and / or second antigen or first and / or second epitope is analyzed by ELISA.

81. 81. An isolated biepitopic agonist antibody identified and / or produced using the method of any of claims 65 to 80.

82. An isolated bispecific antibody or antigen-binding portion thereof that binds to two epitopes of KLB.

83. 1. An isolated bispecific antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is selected from the group consisting of SEQ ID NOs: 34, 36, 38, 40, and 42, and the light chain variable region is selected from the group consisting of SEQ ID NOs: 33, 35, 37, 39, and 41.

84. 1. An isolated bispecific anti-KLB antibody or antigen-binding portion thereof, comprising: (a) a heavy chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-7 and conservative substitutions thereof; (b) a heavy chain variable region CDR2 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-12 and conservative substitutions thereof; (c) a heavy chain variable region CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-17 and conservative substitutions thereof; (d) a light chain variable region CDR1 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22 and conservative substitutions thereof; (e) a light chain variable region CDR2 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-27 and conservative substitutions thereof; (f) a light chain variable region CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 28 to 32 and conservative substitutions thereof.

85. 1. An isolated bispecific anti-KLB antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are: (a) a heavy chain variable region comprising an amino acid sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 34, and a light chain variable region comprising an amino acid sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 33; (b) a heavy chain variable region comprising an amino acid sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 36, and a light chain variable region comprising an amino acid sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 35; (c) a heavy chain variable region comprising an amino acid sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 38, and a light chain variable region comprising an amino acid sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 37; (d) a heavy chain variable region comprising an amino acid sequence having at least 95% identity to the sequence set forth in SEQ ID NO:40, and a light chain variable region comprising an amino acid sequence having at least 95% identity to the sequence set forth in SEQ ID NO:39; and (e) the isolated bispecific anti-KLB antibody or antigen-binding portion thereof, wherein the antibody is selected from the group consisting of a heavy chain variable region comprising an amino acid sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 42, and a light chain variable region comprising an amino acid sequence having at least 95% identity to the sequence set forth in SEQ ID NO:

41.

86. 1. An isolated bispecific anti-KLB antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are: (a) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 34, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 33; (b) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 36, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 35; (c) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 38, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 37; (d) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 40, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 39; and (e) the isolated bispecific anti-KLB antibody or antigen-binding portion thereof, wherein the antibody is selected from the group consisting of a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 42, and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:

41.

87. An isolated bispecific anti-KLB antibody or antigen-binding portion thereof, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 domains, and a light chain variable region comprising CDR1, CDR2, and CDR3 domains.

88. 88. The bispecific antibody of claim 87, wherein the heavy chain variable region CDR1 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-7.

89. 89. The bispecific antibody of claim 87 or claim 88, wherein the heavy chain variable region CDR2 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 12.

90. 90. The bispecific antibody of any one of claims 87 to 89, wherein the heavy chain variable region CDR3 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 17.

91. 91. The bispecific antibody of any one of claims 87 to 90, wherein the light chain variable region CDR1 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 22.

92. 92. The bispecific antibody of any one of claims 87 to 91, wherein the light chain variable region CDR2 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 27.

93. 93. The bispecific antibody of any one of claims 87 to 92, wherein the light chain variable region CDR3 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 28 to 32.

94. 1. An isolated bispecific anti-KLB antibody, or antigen-binding portion thereof, comprising: a heavy chain variable region comprising CDR1, CDR2, and CDR3 domains; and a light chain variable region comprising CDR1, CDR2, and CDR3 domains, wherein the heavy chain variable region and light chain variable region CDR1, CDR2, and CDR3 domains are: (a) a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:3; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:8; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:13; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:18; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:23; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:28; (b) a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:4; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:9; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:14; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:19; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:24; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:29; (c) a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:5; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:10; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:15; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:20; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:25; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:30; (d) a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:6; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:11; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:16; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:21; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:26; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:31; and (e) the isolated bispecific anti-KLB antibody or antigen-binding portion thereof, wherein the antibody is selected from the group consisting of a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:7; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:12; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:17; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:22; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:27; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:

32.

95. 1. An isolated bispecific anti-KLB antibody or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 34 and conservative substitutions thereof, and the light chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 33 and conservative substitutions thereof.

96. 1. An isolated bispecific anti-KLB antibody or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 36 and conservative substitutions thereof, and the light chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 35 and conservative substitutions thereof.

97. 1. An isolated bispecific anti-KLB antibody or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 38 and conservative substitutions thereof, and the light chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 37 and conservative substitutions thereof.

98. 1. An isolated bispecific anti-KLB antibody or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO:40 and conservative substitutions thereof, and the light chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO:39 and conservative substitutions thereof.

99. 1. An isolated bispecific anti-KLB antibody or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 42 and conservative substitutions thereof, and the light chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 41 and conservative substitutions thereof.

100. 1. An isolated bispecific anti-KLB antibody or antigen-binding portion thereof, comprising: a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:3; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:8; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:13; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:18; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:23; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:

28.

101. 1. An isolated bispecific anti-KLB antibody or antigen-binding portion thereof, comprising: a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:4; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:9; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:14; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:19; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:24; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:

29.

102. 1. An isolated bispecific anti-KLB antibody or antigen-binding portion thereof, comprising: a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:5; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:10; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:15; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:20; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:25; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:

30.

103. 1. An isolated bispecific anti-KLB antibody or antigen-binding portion thereof, comprising: a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:6; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:11; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:16; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:21; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:26; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:

31.

104. 1. An isolated bispecific anti-KLB antibody or antigen-binding portion thereof, comprising: a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:7; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:12; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:17; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:22; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:27; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:

32.

105. 1. An isolated bispecific anti-KLB antibody, comprising: (a) a first antibody or antigen-binding portion thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is selected from the group consisting of SEQ ID NOs: 34, 36, 38, 40, and 42, and conservative substitutions thereof, and the light chain variable region is selected from the group consisting of SEQ ID NOs: 33, 35, 37, 39, and 41, and conservative substitutions thereof; (b) a second antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is selected from the group consisting of SEQ ID NOs: 34, 36, 38, 40, and 42, and conservative substitutions thereof, and the light chain variable region is selected from the group consisting of SEQ ID NOs: 33, 35, 37, 39, and 41, and conservative substitutions thereof; The isolated bispecific anti-KLB antibody, wherein the first antibody or antigen-binding portion thereof and the second antibody or antigen-binding portion thereof bind to different epitopes present on KLB.

106. 1. An isolated bispecific anti-KLB antibody, comprising: (a) a first antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 34, and the light chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 33; and (b) a second antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 36, and the light chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO:

35.

107. 1. An isolated bispecific anti-KLB antibody, comprising: (a) a first antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 34, and the light chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 33; and (b) a second antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 38, and the light chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO:

37.

108. 1. An isolated bispecific anti-KLB antibody, comprising: (a) a first antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 34, and the light chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 33; and (b) a second antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO:40, and the light chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO:

39.

109. 1. An isolated bispecific anti-KLB antibody, comprising: (a) a first antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 34, and the light chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 33; and (b) a second antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 42 and the light chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO:

41.

110. 1. An isolated bispecific anti-KLB antibody, comprising: (a) a first antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 38, and the light chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 37; and (b) a second antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 36, and the light chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO:

35.

111. 1. An isolated bispecific anti-KLB antibody, comprising: (a) a first antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 42, and the light chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 41; and (b) a second antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 36, and the light chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO:

35.

112. 1. An isolated bispecific anti-KLB antibody, comprising: (a) a first antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO:40, and the light chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO:39; and (b) a second antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 38, and the light chain variable region comprises amino acids having a sequence at least 85% identical to the sequence set forth in SEQ ID NO:

37.

113. 1. An isolated bispecific anti-KLB antibody, comprising: (a) a first antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 85% identical to the sequence set forth in SEQ ID NO: 42, and the light chain variable region comprises amino acids having a sequence at least 85% identical to the sequence set forth in SEQ ID NO: 41; and (b) a second antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 85% identical to the sequence set forth in SEQ ID NO: 38, and the light chain variable region comprises amino acids having a sequence at least 85% identical to the sequence set forth in SEQ ID NO:

37.

114. 1. An isolated bispecific anti-KLB antibody, comprising: (a) a first antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 85% identical to the sequence set forth in SEQ ID NO: 36, and the light chain variable region comprises amino acids having a sequence at least 85% identical to the sequence set forth in SEQ ID NO: 35; and (b) a second antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 85% identical to the sequence set forth in SEQ ID NO:40, and the light chain variable region comprises amino acids having a sequence at least 85% identical to the sequence set forth in SEQ ID NO:

39.

115. 1. An isolated bispecific anti-KLB antibody, comprising: (a) a first antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 85% identical to the sequence set forth in SEQ ID NO:40, and the light chain variable region comprises amino acids having a sequence at least 85% identical to the sequence set forth in SEQ ID NO:39; and (b) a second antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acids having a sequence at least 85% identical to the sequence set forth in SEQ ID NO: 42, and the light chain variable region comprises amino acids having a sequence at least 85% identical to the sequence set forth in SEQ ID NO:

41.

116. 1. An isolated bispecific anti-KLB antibody, comprising: (a) a first antibody or antigen-binding portion thereof, comprising: a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:3; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:8; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:13; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:18; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:23; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:28; (b) a second antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:4; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:9; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:14; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:19; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:24; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:

29.

117. 1. An isolated bispecific anti-KLB antibody, comprising: (a) a first antibody or antigen-binding portion thereof, comprising: a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:3; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:8; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:13; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:18; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:23; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:28; (b) a second antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:5; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:10; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:15; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:20; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:25; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:

30.

118. 1. An isolated bispecific anti-KLB antibody, comprising: (a) a first antibody or antigen-binding portion thereof, comprising: a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:3; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:8; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:13; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:18; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:23; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:28; (b) a second antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:6; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:11; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:16; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:21; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:26; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:

31.

119. 1. An isolated bispecific anti-KLB antibody, comprising: (a) a first antibody or antigen-binding portion thereof, comprising: a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:3; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:8; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:13; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:18; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:23; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:28; (b) a second antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:7; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:12; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:17; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:22; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:27; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:

32.

120. 1. An isolated bispecific anti-KLB antibody, comprising: (a) a first antibody or antigen-binding portion thereof, comprising: a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:5; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:10; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:15; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:20; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:25; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:30; (b) a second antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:4; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:9; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:14; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:19; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:24; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:

29.

121. 1. An isolated bispecific anti-KLB antibody, comprising: (a) a first antibody or antigen-binding portion thereof, comprising: a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:6; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:11; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:16; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:21; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:26; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:31; (b) a second antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:4; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:9; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:14; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:19; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:24; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:

29.

122. 1. An isolated bispecific anti-KLB antibody, comprising: (a) a first antibody or antigen-binding portion thereof, comprising: a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:7; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:12; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:17; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:22; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:27; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:32; (b) a second antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:4; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:9; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:14; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:19; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:24; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:

29.

123. 1. An isolated bispecific anti-KLB antibody, comprising: (a) a first antibody or antigen-binding portion thereof, comprising: a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:5; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:10; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:15; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:20; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:25; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:30; (b) a second antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:6; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:11; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:16; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:21; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:26; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:

31.

124. 1. An isolated bispecific anti-KLB antibody, comprising: (a) a first antibody or antigen-binding portion thereof, comprising: a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:5; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:10; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:15; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:20; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:25; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:30; (b) a second antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:7; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:12; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:17; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:22; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:27; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:

32.

125. 1. An isolated bispecific anti-KLB antibody, comprising: (a) a first antibody or antigen-binding portion thereof, comprising: a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:7; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:12; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:17; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:22; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:27; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:32; (b) a second antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:6; a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:11; a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:16; a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO:21; a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO:26; and a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:

31.

126. 1. An isolated bispecific antibody, comprising: (a) a first polypeptide comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is selected from the group consisting of SEQ ID NOs: 34, 36, 38, 40, and 42, and conservative substitutions thereof, and the light chain variable region is selected from the group consisting of SEQ ID NOs: 33, 35, 37, 39, and 41, and conservative substitutions thereof; (b) a second polypeptide comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is selected from the group consisting of SEQ ID NOs: 34, 36, 38, 40, and 42, and conservative substitutions thereof, and the light chain variable region is selected from the group consisting of SEQ ID NOs: 33, 35, 37, 39, and 41, and conservative substitutions thereof; The isolated bispecific antibody, wherein the first and second polypeptides each bind to a different epitope present on KLB.

127. An isolated bispecific antibody or antigen-binding portion thereof that binds to two epitopes present on KLB, said two epitopes being located within the KL2 domain of said KLB.

128. An isolated bispecific antibody or antigen-binding portion thereof that binds to two epitopes present on KLB, wherein the two epitopes of KLB are located within the KL1 domain of KLB.

129. An isolated bispecific antibody or antigen-binding portion thereof that binds to two epitopes present on KLB, one of the two epitopes of KLB being located within the KL2 domain of KLB.

130. 130. The isolated bispecific antibody of claim 129, wherein the second of the two epitopes present on said KLB is located within the KL2 domain of said KLB.

131. An isolated bispecific antibody or antigen-binding portion thereof that binds to an epitope on KLB, comprising the amino acid sequence set forth in SEQ ID NO:

43.

132. An isolated bispecific antibody or antigen-binding portion thereof that binds to an epitope on KLB, comprising the amino acid sequence set forth in SEQ ID NO:

44.

133. An isolated bispecific antibody or antigen-binding portion thereof that binds to an epitope on KLB, comprising the amino acid sequence set forth in SEQ ID NO:

45.

134. 134. The isolated bispecific antibody or antigen-binding portion thereof of any one of claims 82 to 133, wherein the antibody or antigen-binding portion thereof has KLB / FGFR1c agonist activity.

135. 135. The isolated bispecific antibody or antigen-binding portion thereof of any one of claims 82 to 134, which is a chimeric antibody or antigen-binding portion thereof.

136. 136. The isolated bispecific antibody or antigen-binding portion thereof of any one of claims 82 to 135, wherein said antibody is a full-length IgG antibody.

137. 135. The isolated bispecific antibody or antigen-binding portion thereof of any one of claims 82 to 134, which is a humanized antibody or antigen-binding portion thereof.

138. The antigen-binding portion is F(ab'). ２ or chemically bonded F(ab') ２ 138. The isolated bispecific antibody or antigen-binding portion thereof of any one of claims 82 to 135 or 137, wherein

139. 139. The isolated bispecific antibody or antigen-binding portion thereof of any one of claims 82 to 138, wherein the antibody or antigen-binding portion thereof activates the KLB / FGFR1c complex.

140. 140. The isolated bispecific antibody or antigen-binding portion thereof of any one of claims 82 to 139, wherein the antibody or antigen-binding portion thereof lowers blood glucose levels in vivo.

141. 141. The isolated bispecific antibody or antigen-binding portion thereof of any one of claims 81 to 140, wherein the antibody or antigen-binding portion thereof does not significantly affect bone mineral density.

142. The antibody or antigen-binding portion thereof is －８ 142. The isolated bispecific antibody or antigen-binding portion thereof of any one of claims 82 to 141, which binds to KLB with a binding affinity of M or greater.

143. 143. Use of the isolated bispecific antibody of any one of claims 82 to 142 in the manufacture of a pharmaceutical composition for treating a metabolic disorder, wherein the metabolic disorder is selected from the group consisting of polycystic ovary syndrome, metabolic syndrome, obesity, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, dyslipidemia, hypertension, type 2 diabetes, non-type 2 diabetes, type 1 diabetes, latent autoimmune diabetes, and maturity-onset diabetes of the young, as well as geriatric and related diseases such as Alzheimer's disease, Parkinson's disease, and ALS.

144. 143. A method of treating an individual having a metabolic disorder, the method comprising administering to said individual a therapeutically effective amount of the isolated bispecific antibody of any one of claims 82-142.

145. 145. The method of claim 144, further comprising administering a second therapeutic agent to the individual.

146. 146. The method of claim 144 or 145, wherein the metabolic disorder is selected from the group consisting of polycystic ovary syndrome, metabolic syndrome, obesity, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, dyslipidemia, hypertension, type 2 diabetes, non-type 2 diabetes, type 1 diabetes, latent autoimmune diabetes, and maturity-onset diabetes of the young, as well as geriatric and related diseases such as Alzheimer's disease, Parkinson's disease, and ALS.

147. 147. The method of any one of claims 144 to 146, wherein the metabolic disorder is diabetes.

148. 143. A pharmaceutical composition comprising the isolated bispecific antibody or antigen-binding portion thereof of any one of claims 82 to 142 and a pharmaceutically acceptable carrier.

149. 149. The composition of claim 148, further comprising a second therapeutic agent.

150. 143. A method of activating the KLB-FGFR1 receptor complex in an individual, the method comprising administering to said individual a therapeutically effective amount of the isolated bispecific antibody of any one of claims 82 to 142.

151. 143. An isolated nucleic acid comprising a sequence encoding the isolated bispecific antibody or antigen-binding portion thereof of any one of claims 82 to 142.

152. A host cell expressing the isolated bispecific antibody or antigen-binding portion thereof of any one of claims 82 to 142.

153. A hybridoma cell expressing the isolated bispecific antibody or antigen-binding portion thereof of any one of claims 82 to 142.

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